LOADING CAPACITY AND RELEASE PROPERTY OF
PIPERINE LOADED SILICA AEROGEL AND SILICA XEROGEL
NURUL HIDAYAH MOHD YUNOS
UNIVERSITI TEKNOLOGI MALAYSIA
ii
LOADING CAPACITY AND RELEASE PROPERTY OF
PIPERINE LOADED SILICA AEROGEL AND SILICA XEROGEL
NURUL HIDAYAH MOHD YUNOS
A thesis submitted in fulfilment of the
requirements for the award of the degree of
Master of Science (Chemistry)
Faculty of Science
Universiti Teknologi Malaysia
JUNE 2010
iii
To my beloved mother and father
iv
ACKNOWLEDGEMENTS
In the name of Allah the Almighty Lord of the world. Thanks to Him for
giving me the opportunity and will to finish this thesis. I would like to warmly thank
my supervisor, Professor Dr. Halimaton Hamdan for giving me the opportunity for
this work, for her optimism, patience and generosity. I am extraordinarily thankful
to Dr. Lee Siew Ling, who always been very kind and supportive, for her guidance
and evaluation throughout the undertaking of this research. I would like to thank her
contribution during my research and write up. I am very thankful to Assoc. Prof. Dr.
Farediah Ahmad and Mohd Farriz Kammil for their generosity to supply the natural
product, piperine. I would like to express my sincere gratitude to my research team
friends, Zeolite and Nanostructured Materials Research members, for their
knowledge, opinion, information, assistance, and sharing almost everyday labworries and –joys.
I am also very grateful to all the staffs of Faculty of Science and Ibnu Sina
Institute for Fundamental Science Studies, Universiti Teknologi Malaysia; for their
technical assistance and for making my experiments possible. Not forgetting my
colleagues, who have been very helpful, thank you for their friendliness. I also wish
to thank all other staff and colleagues who are not mentioned here. A special
appreciation to my parents, family and husband. My constant inspiration and drive
come from this wonderful support group. I would like to dedicate my work to them.
A billion thanks would not be enough. Thank you.
v
ABSTRACT
The feasibility of silica matrix as an oral drug delivery carrier for natural
product was explored. Piperine was loaded into silica aerogel and xerogel via three
different methods; impregnation, physical mixing and direct synthesis. Fouriertransform infrared (FTIR) and ultraviolet-visible (UV-Vis) spectroscopies results
strongly indicated that no detectable drug degradation had occurred during the
loading procedure. Brunauer-Emmett-Teller surface analysis (BET), X-ray
diffraction (XRD), and field-emission scanning electron microscopy (FESEM)
results indicated the successful loading of piperine into silica matrices. XRD results
show the amorphization of piperine crystals after loading process with silica aerogel
or xerogel, indicating the increment in the specific surface area of the drug. The
degree of crystallinity of piperine loaded silica aerogel is extremely low compared to
piperine-xerogel formulations. UV-Vis spectroscopy analysis revealed that the
amount of piperine loaded was higher in silica aerogel than silica xerogel. This was
due to the larger pore and higher surface area of silica aerogel compared to silica
xerogel. Investigation on the release profile of piperine from loaded silica matrices in
simulated gastric and intestinal fluids found that piperine loaded silica matrices
dissolve faster than crystalline drug due to increase in specific surface area and non
crystallinity state of the system. Piperine loaded silica aerogel gave the fastest
dissolution (up to 100%), followed by piperine-xerogel (up to 45%) and crystalline
piperine (< 5%). Formulations prepared via direct synthesis showed the fastest
release, followed by impregnated and physically mixed systems. The ease in collapse
of the silica matrices structure in water was observed to favor a faster release.
vi
ABSTRAK
Kajian telah dijalankan terhadap keupayaan matriks silika sebagai pembawa
dalam sistem penyampaian ubat oral bagi hasilan semulajadi. Piperina dimuatkan ke
dalam silika aerogel dan xerogel menggunakan tiga kaedah; pengisitepuan,
pencampuran fizik dan sintesis langsung. Data analisis spektroskopi inframerah
transformasi Fourier (FTIR) dan ultralembayung-nampak (UV-Vis) menunjukkan
tiada degradasi ubat berlaku semasa prosedur pemuatan. Data analisis permukaan
Brunauer-Emmett-Teller (BET), pembelauan sinar-X (XRD) dan mikroskopi
imbasan elektron pancaran medan (FESEM) menunjukkan piperina telah berjaya
dimuatkan ke dalam matriks silika. Kajian XRD menunjukkan pengamorfusan hablur
piperina selepas proses pemuatan terhadap silika aerogel dan xerogel yang
menunjukkan peningkatan dalam luas permukaan spesifik ubat tersebut. Darjah
kehabluran silika aerogel termuat piperina adalah sangat rendah berbanding
formulasi piperina-xerogel. Analisis spektroskopi UV-Vis menjelaskan bahawa
kuantiti piperina adalah lebih tinggi di dalam silika aerogel berbanding silika
xerogel. Ini disebabkan saiz liang yang lebih besar dan luas permukaan yang lebih
tinggi dalam silika aerogel berbanding silika xerogel. Kajian terhadap profil
pelepasan piperina daripada matriks silika di dalam simulasi bendalir gastrik dan
usus menunjukkan bahawa matriks silika termuat piperina larut lebih pantas
berbanding hablur ubat disebabkan peningkatan dalam luas permukaan dan sifat
bukan hablur sistem tersebut. Silika aerogel termuat piperina memberikan pelarutan
paling pantas (sehingga 100%), diikuti oleh piperina-xerogel (sehingga 45%) dan
hablur piperina (<5%). Formulasi yang disediakan dengan kaedah pemuatan
langsung menunjukkan pelepasan paling cepat, diikuti oleh kaedah pengisitepuan
dan pencampuran fizik. Struktur matriks silika yang mudah terurai di dalam air
menggalakkan pelepasan.
vii
TABLE OF CONTENTS
CHAPTER
1
TITLE
PAGE
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENTS
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
xi
LIST OF FIGURES
xiii
LIST OF ABBREVIATIONS
xix
LIST OF SYMBOLS
xxi
LIST OF APPENDICES
xxii
INTRODUCTION
1.1
General
1
1.2
Natural Product as Modern Medicine
2
1.3
Drug Delivery Carriers
3
1.4
Nanotechnology in Drug Delivery System
4
1.5
Problem Statement
6
1.6
Objectives of Research
9
1.7
Scope of Research
10
viii
2
LITERATURE REVIEW
2.1
2.2
Material
11
2.1.1 Silica Matrix
12
2.1.1.1 Silica Aerogel
14
2.1.1.2 Silica Xerogel
16
2.1.2 Piperine
17
Synthesis of Silica Matrices
19
2.2.1 Rice Husk Ash as Silica Source
20
2.2.2 Sol-Gel Method
21
2.2.2.1 Silica Gel Drying
2.3
3
21
2.2.3 Dissolution Enhancement of Drug
23
Survey on Characterization Techniques
25
RESEARCH METHODOLOGY
3.1
Synthesis of Silica Matrices
28
3.2
Drug (Piperine)
30
3.3
Drug Loaded Silica Matrices
31
3.3.1 Physical Mixing
31
3.3.2 Impregnation
31
3.3.3 Direct Synthesis
33
3.3.4 Sample Codes
35
Characterization
36
3.4.1 UV-Vis Spectroscopy
36
3.4.2 FTIR Spectroscopy
37
3.4.3 BET Surface Area Analysis
39
3.4.4 X-Ray Diffraction
41
3.4.5 Field Emission Scanning Electron Microscopy
43
Investigation on Drug Dissolution Profile
44
3.4
3.5
ix
4
RESULTS AND DISCUSSION
4.1
Synthesis of Silica Aerogel and Silica Xerogel
45
4.2
Characterization of Silica Matrices and Piperine
46
4.2.1 FTIR Spectroscopy Analysis of Silica Matrices
46
4.2.2 FTIR Spectroscopy Analysis of Piperine
49
4.2.3 Morphology and Crystallinity Studies of Silica
50
Aerogel, Silica Xerogel and Piperine
4.3
Preparation of Piperine-Silica Aerogel and Piperine-
53
Silica Xerogel Formulations
4.4
Degradation Study of Piperine Loaded Silica Matrices
54
4.5
Drug Loading Capacity
58
4.6
Physically Mixed Piperine-Silica Matrices
59
4.6.1 Morphology and Crystallinity Studies of
61
Physically Mixed Piperine-Silica Matrices
4.7
Piperine Impregnated Silica Matrices
67
4.7.1 Effect of Solvent Volume on the Drug Loading
68
Capacity
4.7.2 Morphology Study of Piperine Impregnated
71
Silica Matrices
4.7.3 Crystallinity Study of Piperine Impregnated
75
Silica Matrices
4.8
Piperine Loaded Silica Matrices Via Direct Synthesis
78
4.8.1 Effect of Aging Conditions
81
4.8.2 Efficiency of Loading
83
4.8.3 Crystallinity and Morphology Studies of
84
Piperine Loaded Silica Matrices via Direct
Synthesis
4.9
Comparison of Degree of Crystallinity of Piperine
87
Loaded Silica Matrices via Different Methods
4.10
Drug Dissolution Study
91
4.10.1 Physically Mixed Piperine-Silica Matrices
99
4.10.2 Piperine Impregnated Silica Matrices
101
x
4.10.3 Piperine Loaded Silica Matrices via Direct
102
Synthesis
4.10.4 Comparison of Drug Dissolution Profile of
104
Formulations Prepared via Different Methods
5
CONCLUSIONS
5.1
REFERENCES
Appendices
Conclusions
107
110
125-127
xi
LIST OF TABLES
TABLE NO.
TITLE
PAGE
1.1
Tablet excipients and their uses
4
2.1
Properties of silica aerogel
15
2.2
Critical conditions of several substances
23
3.1
Temperature increment steps for supercritical drying
30
3.2
Properties of model drug (Piperine)
30
3.3
Sample codes for various formulations
35
4.1
Physical properties of silica aerogel and silica xerogel
45
4.2
FTIR absorption bands of silica aerogel and silica
48
xerogel
4.3
FTIR absorption bands of piperine
50
4.4
Drug loading capacity and surface area of piperine-
60
silica aerogel and piperine-silica xerogel formulations
via physical mixing
4.5
Degree of crystallinity of physically mixed piperine-
66
silica matrices
4.6
Drug loading capacity and surface area of piperine-
69
silica aerogel and piperine-silica xerogel formulations
via impregnation
4.7
Degree of crystallinity of piperine impregnated silica
78
matrices
4.8
Effect of aging period on the loading efficiency and
surface area of piperine-silica matrices formulations
82
xii
4.9
Comparison on crystallinity degree of piperine-silica
matrices formulations prepared via different methods
90
xi
LIST OF FIGURES
FIGURE NO.
TITLE
PAGE
1.1
Typical contents of a conventional tablet
3
1.2
Flow diagram of research activities
10
2.1
Different forms of hydroxyl group that can occur on
13
the surface of silica: (a) single hydroxyl group, (b)
double or geminal hydroxyl group (c) triple hydroxyl
group
2.2
Comparison of gel network of wet solgel, xerogel and
16
aerogel
2.3
Chemical structure of piperine
2.4
Schematic phase diagram for pure carbon dioxide and
water
2.5
18
22
Effect of particle size reduction towards the
increment of surface area
24
3.1
Synthesis of silica aerogel and silica xerogel
28
3.2
Flow diagram of drug loading procedure via
32
impregnation and physical mixing methods
3.3
Flow diagram of drug loading procedure via direct
33
synthesis method
3.4
Schematic diagram of supercritical carbon dioxide
34
3.5
Typical BET plot
40
3.6
Illustration of the geometry used for the simplified
42
derivation of Bragg’s law
xii
3.7
Schematic diagram of dissolution testing apparatus
44
assembly
4.1
FTIR spectra of silica aerogel and silica xerogel
47
4.2
The presence of OH groups due to the silanol groups,
48
physically and chemically adsorbed water.
4.3
FTIR spectrum of piperine
49
4.4
FESEM micrograph of synthesized silica aerogel
51
4.5
FESEM micrograph of synthesized silica xerogel
51
4.6
X-ray diffractograms of (a) silica aerogel, and (b)
52
silica xerogel
4.7
FESEM micrograph of pure piperine (250 X
52
magnification)
4.8
X-ray diffractogram of crystalline piperine
53
4.9
UV-Visible spectra of pure piperine and piperine
55
loaded silica matrices
4.10
FTIR spectra of piperine loaded silica aerogel
56
formulations
4.11
FTIR spectra of piperine loaded silica xerogel
57
formulations
4.12
Possible hydrogen bonding in piperine
58
4.13
Diagrammatic procedure of physical mixing (co-
59
grinding) and expected loading type.
4.14
FESEM micrograph of 20 wt % physically-mixed
61
piperine-aerogel
4.15
FESEM micrograph of 20 wt % physically-mixed
62
piperine-xerogel
4.16
FESEM micrograph of 50 % physically-mixed
63
piperine-aerogel
4.17
FESEM micrograph of 50 wt % physically-mixed
63
piperine-xerogel
4.18
X-ray diffractograms of physically-mixed piperineaerogel with different loading capacity
64
xiii
4.19
X-ray diffractograms of physical mixed piperine-
65
xerogel with different loading capacity
4.20
Diagram of drug loading procedure and expected
67
drug loading via impregnation
4.21
Effect of solvent volume to the loading of 20 wt %
68
piperine into silica matrices.
4.22
Drug loading and SBET of piperine impregnated silica
70
aerogel
4.23
Drug loading and SBET of piperine impregnated silica
71
xerogel
4.24
FESEM micrograph of 50 wt% piperine impregnated
72
silica aerogel prepared with excessive ethanol
4.25
FESEM micrograph of 50 wt % piperine impregnated
72
silica aerogel prepared with minimum ethanol
4.26
FESEM micrograph of 50 wt % piperine impregnated
73
silica xerogel prepared with excessive ethanol
magnification 1,500 X
4.27
FESEM micrograph of 50 wt % piperine impregnated
74
silica xerogel prepared with excessive ethanol
magnification 20,000 X
4.28
FESEM micrograph of 50 wt % piperine impregnated
74
silica xerogel prepared with minimum ethanol
magnification 20,000 X
4.29
X-ray diffractograms of piperine impregnated aerogel
76
with different loading capacity
4.30
X-ray diffractograms of piperine impregnated silica
77
xerogel with different loading capacity
4.31
Diagrammatic procedure of piperine loaded silica
79
xerogel via direct synthesis
4.32
FESEM micrograph of piperine loaded silica xerogel
via direct synthesis using ethanolic solution of
piperine
80
xiv
4.33
FESEM micrograph of piperine loaded silica xerogel
80
via direct synthesis using ground piperine
4.34
Effect of aging period on loading efficiency and
82
surface area of piperine-silica matrices formulations
4.35
Loading efficiency of piperine loaded silica matrices
84
via direct synthesis
4.36
X-ray diffractograms of (a) piperine loaded silica
85
aerogel (10 wt%), (b) piperine loaded silica aerogel
(20 wt%), (c) piperine loaded silica xerogel (10 wt%)
and (d) piperine loaded silica xerogel (20 wt%),
synthesized via direct synthesis
4.37
FESEM micrograph of 20 wt % piperine loaded silica
86
aerogel via direct synthesis
4.38
FESEM micrograph of 20 wt % piperine loaded silica
87
xerogel via direct synthesis
4.39
X-ray diffractograms of piperine loaded silica aerogel
88
synthesized via different methods: (a) crystalline
piperine (b) physical mixed piperine-aerogel (20 wt
%) (c) piperine impregnated aerogel (20 wt%) and
(d) direct synthesized piperine loaded aerogel (20 wt
%)
4.40
X-ray diffractograms of piperine loaded silica
89
xerogel synthesized via different methods: (a)
crystalline piperine, (b) physical mixed piperinexerogel (20 wt %), (c) piperine impregnated xerogel
(20 wt %) and (d) direct synthesized piperine loaded
xerogel (20 wt %)
4.41
Dissolution profiles of crystalline piperine in 0.1M
92
hydrochloric acid and phosphate buffer saline
4.42
Effect of different loading methods on the dissolution
rate
of
physically
formulations in 0.1M HCl
mixed
piperine-aerogel
93
xv
4.43
Effect of different loading methods on the dissolution
rate
of
physically
mixed
93
piperine-aerogel
formulations in 0.05 mM PBS
4.44
Effect of different loading methods on the dissolution
rate
of
physically
mixed
94
piperine-xerogel
formulations in 0.1M HCl
4.45
Effect of different loading methods on the dissolution
rate
of
physically
mixed
94
piperine-xerogel
formulations in 0.05 mM PBS
4.46
Effect of different loading methods on the dissolution
97
rate of piperine impregnated aerogel formulations in
0.1M HCl
4.47
Effect of different loading methods on the dissolution
96
rate of piperine impregnated aerogel formulations in
0.05 mM PBS
4.48
Effect of different loading methods on the dissolution
96
rate of piperine impregnated xerogel formulations in
0.1M HCl
4.49
Effect of different loading methods on the dissolution
97
rate of piperine impregnated xerogel formulations in
0.05 mM PBS
4.50
Effect of different loading methods on the dissolution
98
rate of directly synthesized piperine loaded silica
matrices formulations in 0.1M HCl
4.51
Effect of different loading methods on the dissolution
98
rate of directly synthesized piperine loaded silica
matrices formulations in 0.05 mM PBS
4.52
Dissolution profile of 20 wt % physically mixed
100
piperine-silica matrices formulations in 0.1 M HCl
4.53
Dissolution profile of 20 wt % physically mixed
piperine-silica matrices formulations in 0.05 mM
PBS
100
xvi
4.54
Dissolution profile of 20 wt % piperine impregnated
101
silica matrices (a) crystalline piperine, (b) piperinexerogel (c) piperine-aerogel (d) piperine-xerogel
(minimum
ethanol),
and
(e)
piperine-aerogel
(minimum ethanol) in 0.1 M HCl
4.55
Dissolution profile of 20 wt % piperine impregnated
102
silica matrices (a) crystalline piperine, (b) piperinexerogel (c) piperine-aerogel (d) piperine-xerogel
(minimum
ethanol),
and
(e)
piperine-aerogel
(minimum ethanol) in 0.05 mM PBS
4.56
Dissolution profiles of directly synthesized 20 wt %
103
piperine loaded silica matrices in 0.1 M HCl
4.57
Dissolution profiles of directly synthesized 20 wt %
104
piperine loaded silica matrices in 0.05 mM PBS
4.58
Effect of different loading methods on the dissolution
105
rate of 20 wt % piperine-aerogel formulations in
0.1M HCl
4.59
Effect of different loading methods on the dissolution
106
rate of 20 wt % piperine-xerogel formulations in 0.1
M HCl
5.1
Outcome of research activities
109
xix
LIST OF ABBREVIATIONS
BET
-
Brunauer Emmett and Teller
DDC
-
drug delivery carrier
DDS
-
drug delivery system
FESEM
-
field emission-scanning electron microscopy
FTIR
-
Fourier transform infrared
GRAS
-
Generally Recognised as Safe
H2SO4
-
sulphuric acid
KBr
-
potassium bromide
MCM
-
Mobil Crystalline of Materials
N2
-
nitrogen gas
Na2SiO3
-
sodium silicate
NaOH
-
sodium hydroxide
NCPE
-
nanocomposite polymer electrolytes
NOAEL
-
no observed adverse effect level
OTC
-
over-the-counter
RHA
-
rice husk ash
RH
-
rice husk
SA
-
silica aerogel
SBA
-
Santa Barbara Amorphous type materials
SCF
-
supercritical fluid
SiO2
-
silicon dioxide, silica
SX
-
silica xerogel
TEOS
-
tetraethyl orthosilicate
TMOS
-
tetramethyl orthosilicate
TUD-1
-
Technische Universiteit Delft mesoporous silica materials
xx
USA
-
United States of America
US$
-
United States Dollar
US FDA
-
United States’ Food and Drug Administration
UV-Vis
-
ultraviolet-visible
WHO
-
World Health Organization
XRD
-
X-ray diffraction
Rpm
-
round per minute
PGA
-
phenylglycine amide enzymes
xxi
LIST OF SYMBOLS
C
-
concentration
D
-
diffusion coefficient
g
-
gram
h
-
thickness of diffusion layer
h
-
Planck’s constant
k
-
force constant
k'
-
dielectric constant
K
-
Kelvin
m
-
meter
n
-
diffraction order
λ
-
wavelength
ºC
-
degree Celsius
Pa
-
Pascal
Pc
-
critical pressure
SBET
-
BET surface area
T
-
temperature
t
-
time
Tc
-
critical temperature
v
-
vibrational energy level
W
-
Watt
θ
-
diffraction angle
xxii
LIST OF APPENDICES
APPENDIX
TITLE
PAGE
A
Calibration curve of piperine in ethanol
125
B
Calibration curve of piperine in 0.05 M
126
potassium
phosphate
buffer
(PBS)
solution, pH 7
C
Calibration curve of piperine in 0.1 M
hydrochloric acid (HCl), pH 1
127
CHAPTER I
INTRODUCTION
1.1
General
The word medicine is derived from the Latin ars medicina, meaning the art
of healing. When it comes to art, it may represent different approaches, sources,
process, and belief in diagnosing, preventing and curing illness. Generally, medicine
is described as a discipline devoted to understanding and treating disease, often
referring to physical and chemical mechanisms [1]. Specifically, medicine means a
substance or mixture of substances used in restoring or preserving health. Early
records on medicine have been discovered from early traditional Chinese medicine,
Indian Ayurveda, Arab Unani medicine, the Americas and ancient Greek medicine
[2]. Historically, these ancient treatments include plants, animal parts, minerals and
non-medication therapies. The revelation of the mysteries and mystiques in these
ancient treatments through science and technology has led to the discoveries and
innovations in modern drug delivery system.
According to World Health Organization (WHO), about three-quarters of the
world population relies on the traditional remedies for their primary health needs. In
Malaysia, an estimated US$ 500 million is spent annually on traditional medicine
2
compared to about US$ 300 million on allopathic medicine [2]. However, plantderived products are preferred and have dominated the human pharmacopoeia for
thousand of years almost unchallenged [3]. About 25% of modern medicines are
made from plants, first used traditionally [4]. The rising concern on the safety and
efficacy of plant-derived drug has ignited massive volume of research in this field.
1.2
Natural Product as Modern Medicine
A resurgence of interest in the study of medicinal plants has been taking
place during the last decades. The world market for herbal medicines based on
traditional knowledge is estimated at US$ 60 thousand million in 2005 [5].
Traditional herbal remedies also remain a very important part of life in Asian
markets such as China, Japan, and India. In Japan, 60-70% of allopathic doctors
prescribe kampo medicines (Japanese herbal medicines) for their patients [2, 6]. In
Malaysia, traditional forms of Malay, Chinese and Indian medicine are used
extensively.
Despite of fast pace in medical advances, consumer in West are turning to
alternative remedies such as herbals in increasing numbers, due to factors such as
ageing populations, escalating healthcare costs, and trend towards self care [7, 8].
For example, in United States of America (USA) alone, herbal sales increased by
101% in mainstream markets between May 1996 and May 1998. Natural ingredients
are also being incorporated increasingly into conventional prescription and over-thecounter (OTC) medicines as research continues into the safety and efficacy of new
and known substances [9, 10].
3
1.3
Drug Delivery Carriers
In pharmaceutical field, the drug delivery carriers (DDC) serve as
mechanisms to improve the delivery and effectiveness of drugs. It is advantageous
to use drug carrier than pure drugs since DDC can decrease drug metabolism and
reduce the toxicity of drugs [11]. DDC can also increase drug absorption and
improve its release profile. As well as the active ingredient, the conventional tablet
may contain inactive ingredients, or excipients, which are used in the delivery of
each therapeutic product, such as filler, binder, colour and flavour to ensure that the
tablet is easy to use and of a high quality [12]. The following pie chart (1.1) shows
the typical contents of a tablet, while Table 1.1 describes their function in a drug
formulation [13].
Filler / Diluent
Active Ingredient
Figure 1.1
Flavourings & Colourings
Binder
Lubricant, Glidant &
Antiadherent
Disintegrant
Typical contents of a conventional tablet
4
Table 1.1:
Excipients
Tablet excipients and their uses [13]
Function
Filler (or
Filler, such as sucrose or lactose, is included to increase the size of
diluent)
the tablet. This is necessary as often the amount of 'active' is so tiny
that the tablet would be too small to handle without it.
Disintegrant
Disintegrants help the tablet to break down into small fragments,
when it is ingested. This helps the medicine to dissolve and be taken
up by the body so that it can act more quickly. Disintegrants may
include potato or cocoa butter.
Binder
A binder, such as glucose or sucrose, is added to hold the tablet
together after it has been compressed, stopping it from breaking
down into its separate ingredients.
Glidant
The glidant helps to keep the powder making up the tablet flowing
as the tablet is being made, stopping it from forming lumps.
Lubricant
Lubricants ensure that the tablet has a smooth surface and that the
powder does not stick to the equipment used to make the tablet.
Antiadherent
The antiadherent also stops the powder from sticking to the
equipment as the tablet is being made.
Flavour
Flavouring agents help to make the tablet taste better.
Colourant
Colours are added to help you to recognize your tablet and to make
it easier to take your medicine correctly.
1.4
Nanotechnology in Drug Delivery System
Nanotechnology has impacted and led significant advances in many fields. It
is defined as that area of science and technology where dimensions and tolerance are
in the range of 1.0 nm to 100 nm [14, 15]. It is also defined by Bawa et al. as the
design, characterization, production, and application of structures, devices, and
systems by controlled manipulation of size and shape at the nanometer scale (atomic,
5
molecular, and macromolecular scale) that produces structures, devices, and systems
with at least one novel/superior characteristic or property [16]. Nanotechnology
based tools and techniques are expected to create innovations and play critical role in
mainstream biomedical applications including in drug delivery, imaging, and novel
drug discovery techniques [17].
One of the important areas of nanotechnology is nanomedicine, which refers
to highly specific medical intervention at the molecular scale for diagnosis,
prevention, and treatment of disease [18]. The overall goal of nanomedicine is the
same as it always has been in medicine; to diagnose as accurately and early as
possible, treat as effectively as possible without side effects, and evaluate the
efficacy of treatment invasively. By manipulating drugs and other materials at the
nanometre scale, the fundamental properties and bioactivity of materials can be
altered.
Nano-enabled drug delivery system (DDS) has led the development in
medicine for improving the efficiency of drug and targeting aimed point of diseases.
This is demonstrated by the fact that approximately 13% of the current global
pharmaceutical market is related to the products incorporating a drug delivery
system [19, 20]. The global pharmaceutical market was estimated at about US$406
billion in 2002 and expected to grow more than double in value to US$ 1.3 trillion
by 2020 [21, 22].
There has been considerable interest in developing biodegradable
nanoparticles as effective drug delivery devices. Nanoparticles are solid, colloidal
particle consisting of macromolecular substances that vary in size from 10 nm to
1000 nm [23].
In nano-enabled drug delivery system, the drug of interest is
dissolved, entrapped, adsorbed, attached, or encapsulated into/onto the nanoparticles
matrix. The advantages of using nanoparticles for drug delivery are due to their
three main properties:
6
•
Nano-scale: because of their small size, they can penetrate through
smaller capillaries and are taken up by cells, which allow efficient drug
accumulation at the target sites.
•
High surface area: the dissolution rate of drug depends on its surface
area and solubility. Higher surface area allows more contact with the
dissolution medium.
•
Biodegradable: allows sustained drug release within target site over a
period of days or even weeks.
Nanoparticulate drug delivery carriers allow for faster drug absorption,
controlled dosage releases, and shielding from the body’s immune system which
enhance the effectiveness of already existing drugs [16, 24]. Silica aerogel and silica
xerogel, silica matrices made by the sol gel process have emerged as ideal carrier
materials for drug delivery system. The use of silica in delivery system can provide
several benefits:
•
High surface areas provide the possibility of high drug loadings in the
matrix and thus increase the rate of reaction;
•
The open pore morphology of silica matrices allow substrates to quickly
move into the interior regions of the particle;
•
Solvents
used
in
the
processing
of
the
silica
materials
are
environmentally friendly;
•
1.5
Silica has been widely used as excipients in pharmaceutical industry.
Problem Statement
Since last decades, most drugs have been prepared in designed form to ensure
for accurate dosage and convenience for drug administration. There has been a
7
dramatic increase in the awareness of continuous, prolonged, controlled-release
medication. This is due to many of original controlled-released system is
administered in high dose at a given time only and have to repeat that dose several
hours or days later. This is not economical and sometimes results in damaging side
effect [25]. Furthermore, the drug release rate is hard to control due to different
properties of the drugs. Normally, the release rate is whether too slow for crystalline
and/or too fast for amorphous drug [26-27]. Amorphous form due to absence of an
ordered crystal lattice requires minimal energy and thus provides maximal solubility
advantage as compared to the crystalline and hydrated form of drug.
In this research, piperine (5-benzo[1,3]dioxol-5-yl-1-piperidin-1-yl-penta2,4-dien-1-one) is chosen as model drug due to its availability and commercial value
in Malaysia. Piperine is naturally occurring in black pepper plant, Piper nigrum Linn
with a yield of 35-55 % of its oleoresin [28]. Piperine has been evaluated by Food
and Agriculture Organization of United Nations and World Health Organization
(WHO) which indicated that piperine is safe to be used as food and flavouring agents
[29]. It has been used as Ayurvedic and Chinese traditional medicine for a long time.
Besides, there has been strong growth in sales of product that all-natural or a high
proportion of pure, natural ingredients, especially in food, pharmaceutical and
cosmetic products [7].
In comparison to ground spice, piperine is hygienic, concentrated and can be
standardized for acceptable dosage [28]. However, piperine is a poor soluble drug
and practically insoluble in water resulting in a poor absorption from gastrointestinal
tract [30, 31]. It also exhibits sensitivity to light and oxygen that can undergo
hydrolysis to piperidine and piperinic acid and also photolysis to iso-chavicine which
lacks the pepper characteristics. Besides, if piperine were to remain captive in the
form of raw black pepper, it will take time for its bioavailability enhancing property
to be released [32]. In market, Bioperine®, a standardized piperine extract has been
patented and claimed to exhibit bioavailability enhancer property to other nutrients
[33]. However, the formulation of this supplement may contain several excipients,
such as protein, carbohydrates, sugar, calcium carbonate, talc, magnesium stearate,
8
starch-gelatine paste, and diluents. The use of too many components has involved
several different steps that could subsequently lead into troublesome production.
Excipients package active ingredient into discrete amounts that are easy to
handle, give medications a specific look and colour for branding purposes, or imbue
unpleasant-tasting medication to help with patient compliance, among hundreds of
other uses [34]. On the other hand, some consumers or patients have suffered of
being allergic to these additives that commonly appear in many medications [35].
For example, people who have difficulty digesting starch might react to tablets and
capsules in which starch is the filler or binder. Others may be allergic to certain
artificial colours used in producing medicine preparation. In more serious case,
inappropriate use of talc or magnesium silicate as filler in drug tablets can lead to
severe pulmonary toxicological responses [36]. Increasing number of allergy
incidences along with rising concerns over ingredient safety in medicinal products
must be taken into account in designing drug delivery system.
In order to overcome these problems, silica aerogel and xerogel maybe
loaded with piperine via different methods. To the best of our knowledge, there is no
report on silica matrices loaded with piperine. This may avoid the use of other
excipients as these silica matrices could act as the filler and binder themselves. The
loading of drug with silica matrices is expected to increase the dissolution rate and
stability of piperine by increasing its surface area and wettability, while protecting
piperine against destructive changes [28, 37]. In drug delivery system, the
dissolution rate and solubility of poorly soluble drugs should be improved for
desired overall performance of the system because a low dissolution rate resulting in
poor absorption from the gastrointestinal tract [38]. Estimates by the pharmaceutical
companies are that about 40% of the potential drugs are poorly soluble in water [39].
In previous studies, the use of nanoparticles as the drug delivery carriers has proven
that they could accelerate the release rate of many kind of drugs [40-44].
9
The use of silica matrices such as silica aerogel, and silica xerogel as the drug
delivery carriers have been explored recently. The feasibility of silica aerogels as
drug delivery system was recently reported [45-47].
It was found that no
degradation occurred during loading process via adsorption method using a low
temperature supercritical extraction unit [46]. The enhancement in dissolution rate
of drug loaded silica aerogel compared to crystalline drug was reported [45-47]. On
the other hand, the drug loading is limited. Previous studies showed the potential of
silica xerogel as the delivery system for the controlled release of various kinds of
drugs [48-51]. However, the high density and limited surface area of silica xerogel
has somehow limited its potential. Besides, in most of the previous studies, an
orthosilicate such as tetramethyl orthosilicate (TMOS) or tetraethyl orthosilicate
(TEOS) was used as silica source in preparing silica monoliths containing loaded
drugs [47-51]. On the other hand, a new aqueous route for synthesis of silica
monoliths that used commercially available sodium silicate as a precursor has been
developed. However, alkoxysilanes and the commercially available sodium silicate
are usually too expensive that silica aerogel production in an industrial scale is not
economically practical. In order to overcome these problems, a modified aqueous
route for synthesis of silica monoliths using rice husk ash as the cheap silicon source
for production of pure silicate solution as a precursor was developed.
1.6
Objectives of Research
The objectives of this research are:
1. To synthesize and characterize piperine loaded silica matrices.
2. To determine the loading capacity and the drug release rate of the newly
designed nano-enabled of drug delivery system.
3. To investigate the influences of synthesis conditions on the loading and
release property of piperine.
10
1.7
Scope of Research
The silica matrices (silica aerogel and silica xerogel) were synthesized via
sol-gel process using rice husk ash as silica source, followed by the loading of drug
via different methods which are:
•
Physical mixing (co-grinding)
•
Impregnation
•
Direct synthesis
Then, all samples are characterized for structural analysis using Fourier transform
infrared spectroscopy (FTIR), Ultra-Visible spectroscopy (UV-Vis), and X-ray
diffraction (XRD). The morphology and pore characteristics of the samples are
analysed using field emission scanning electron microscopy (FESEM) and BET
surface area analyzer, respectively. Meanwhile, the drug loading and its release rate
investigation is carried out using UV-Vis spectroscopy. The research programme is
designed according to the methodology outlined in the scheme as shown in Figure
1.2.
Synthesis of silica aerogel
and silica xerogel
Synthesis of drug loaded
silica aerogel and drug
loaded silica xerogel
Characterizations
1. UV-Vis spectroscopy
2. FTIR spectroscopy
3. BET surface area analysis
4. X-Ray diffraction
5. FESEM
Drug loading capacity
UV-Vis Spectroscopy
Drug release rate study
Optimization of synthesis
conditions
Figure 1.2
Flow diagram of research activities
CHAPTER II
LITERATURE REVIEW
2.1
Material
Drug delivery systems (DDS) are polymeric or lipid carrier systems that
transport drugs to their target sites in a manner that provides their maximum
therapeutic activity, prevent their degradation or inactivation during transit to the
target sites, and protect the body from adverse reactions due to inappropriate
disposition [52]. The goal of DDS is to release the drugs to simultaneously provide
safety, effectiveness, and reliability. Recently, studies on the application of solid
carriers as drug delivery tool have gained significant interests. Solid carriers can be
microporous inorganic substances, high surface area colloidal inorganic adsorbent
substances, cross-linked polymers or nanoparticle adsorbents, for example, silica,
silicates, magnesium trisilicate, magnesium hydroxide, talcum, and cross-linked
sodium carboxymethyl cellulose [46, 53]. Nanoparticles are solid, colloidal particle
consisting of macromolecular substances that vary in size from 10 nm to 1000 nm.
Nanoparticles, particularly polymeric nanoparticles, have been investigated since
1970s as an alternative to liposome which suffered from inherent problems such as
low encapsulation, rapid leakage, poor stability and production difficulties [54-56].
12
2.1.1
Silica Matrix
Silica (silicon dioxide, SiO2) occurs widely in nature. Silica exists in three
different crystalline forms which are quartz, tridymite and cristobalite. Quartz also
exists in different colours due to presence of impurities such as agate, opal (white)
and amethyst (purple) [57]. It also occurs in bamboo, rice husk, and feathers of some
birds [58-59]. Silica and silicates are used extensively in pharmacy as pharmaceutical
aids because of their suitable physical properties and limited reactivity. Some
applications of silica are grinding aid for pharmaceutical formulations and stabilizing
agent for organic compounds subject to temperature-humidity degradation conditions
[60-61]. Besides, colloidal silica is claimed useful for treating subcutaneous wounds,
tuberculosis and many circulation problems such as hardening of the arteries [62].
An evaluation on the airborne allergen inactivation effect of colloidal silica showed
almost 100 % inactivation of Japanese cedar pollen allergen, which bring up
colloidal silica as a promising material for allergen inactivation [63]. In addition,
silica is widely used as a source to develop mesoporous materials due to a number of
advantages:
•
Excellent thermal stability, having a glass transition temperature of 900
ºC and no measured weight loss at 450 ºC
•
The pore size may be controlled and easily limited to 10 nm
Interest on the application of silica particles as host matrix for biomaterial has
intensified in the last decade. Many reports have emerged on the application of
synthetic porous silica based materials as potential drug delivery system.
Pharmaceutical controlling delivery systems offer numerous advantages compared to
conventionally administrated drugs in dosage forms, such as improved efficiency and
reduced toxicity. The presence of silanol groups in silica matrices provides possible
hydrogen bonding with the drug compounds [60]. Figure 2.1 shows three forms of
hydroxyl group that occur on the surface of silica matrix [59]. They are single
hydroxyl group (O(Si-OH)O2), double or geminal hydroxyl group and triple
hydroxyl group, based on the number of hydroxyl groups attached to silicon.
13
(c)
OH
(a)
Si
HO
OH
(b)
Si
OH
O
O
Si
O
Si
HO
Figure 2.1:
OH
OH
OH
O
Si
O
OH
OH
Different forms of hydroxyl group that can occur on the surface of
silica: (a) single hydroxyl group, (b) double or geminal hydroxyl group (c) triple
hydroxyl group [59].
Investigation on drug loading and release property of ibuprofen loaded
MCM-41 proved that pharmaceutically active compounds could be loaded with great
efficiency in mesoporous silica materials [64-65]. This material contains nanosized
pores that allow for inclusion of drug into the pores.
It also exhibits exciting
structural features of large specific surface area (up to 1000 m2g-1) and ordered
cylindrical mesopores with a narrow size distribution.
However, the synthesis
process includes the presence and removal of surfactants, which is not cost-effective
and troublesome.
The feasibility of mesoporous silica material TUD-1 (Technische Universiteit
Delft) for drug delivery was also reported. TUD-1 was synthesized by aging, drying,
and calcining a homogeneous mixture; consisted of a silicon alkoxide source such as
tetraethyl orthosilicate (TEOS) [66]. The results of the study demonstrated the
successful inclusion and release of ibuprofen in the silica mesoporous silica such as
MCM-41 and SBA-15 [67]. However, the use of TEOS is unfavourable as the
alkoxysilanes are expensive and toxic.
14
2.1.1.1 Silica Aerogel
Silica aerogel is a low-density solid material derived from gel, in which the
liquid component of the gel has been replaced with air. It is the lightest and lowest
density solid known to exist [68]. It can be hydrophilic or hydrophobic based on the
synthesis condition and surface modification method. Basically, the surface
hydrophilicity-hydrophobicity of aerogel is tailored to fulfil various desired
applications. Rao et al. [69] reported the application of hydrophobic silica aerogel
for the transportation of liquids. It has been shown that small quantity of liquid can
be transported without mass loss, very efficiently on a surface coated with
superhydrophobic silica aerogel. Silica aerogel is also well-known as an excellent
thermal insulator. Insulation flame penetration test carried out by Hansen and Frame
[70] proved that silica aerogel is the best insulation material yet. The physical
properties of silica aerogel are shown in Table 2.1.
Table 2.1:
Properties
Surface area
Properties of Silica Aerogel
Value
400 - 1000 m2/g
Porosity
80 - 99.8 %
Density
~0.003 g/cm3
Thermal insulation
0.005 W/m K
Dielectric constant
k’= 1.0 - 2.0
Refraction index
~1.05
Silica aerogel has been used as a support for various catalysts since it exhibits
a very high surface area (400-1000 m2/g). Titanium dioxide supported on silica
aerogel has been extensively studied as catalyst and optic application, due to its high
thermal stability and high chemical durability [71]. Recently, it was reported that the
addition of silica aerogel powder into nanocomposite polymer electrolytes (NCPE)
significantly increased the polymer segmental motion, fraction of free lithium ions,
extra conductive pathway, transporting sites and free volume [72]. Consequently,
the mobility of lithium ions was increased and ionic conductivity was enhanced by
15
threefold higher than the system prepared without silica aerogel, resulted in the asprepared NCPE a potential candidate in batteries.
The application of silica aerogel as carrier material is not limited to inorganic
materials only but applicable to biomaterials such as enzymes, bacteria, and
biopolymer such as chitosan and cellulose [73-74]. Maury and Pierre [75] reported
the increment of the enzyme catalytic activity of lipase encapsulated silica aerogel
compared to free lipase.
Silica aerogel offers protection for the enzyme from
deterioration brought about by the solvent.
The immobilization of three other
enzymes (PGA, thermolysin, and chymotrypsin) in silica aerogel was also
demonstrated by Basso et al. [76].
Silica aerogel has emerged as ideal drug delivery carrier due to its
biocompatibility. The chemical composition of silica aerogel is identical with that of
amorphous silicon oxide (Aerosil), which has been used in the pharmaceutical
industry since 1940 [45]. It has been shown that orally administrated Aerosil passes
through the gastrointestinal tract without being resorbed in detectable quantities.
Thus, it is expected that silica aerogel, would have similar characteristics. Silica
aerogel has much larger internal surface area, compared to that of Aerosil (200 m2/g).
This allows silica aerogel to exhibit superior properties to Aerosil in drug delivery
system [46-47]. Furthermore, the production of Aerosil includes the pyrogenically
prepared synthetic silicon dioxide glass that involves heating at a temperature of 9501200 ºC [77], hence making the technique energy and cost consuming.
Being chemically inert and non-harmful to human body, silica aerogel may
easily find an application in pharmaceutical industry.
This nanomaterial also
exhibits exciting structural features like open pore structure, very low density and
extremely high porosity. Thus, application of aerogel may potentially improve the
adsorption and dissolution of drugs. Furthermore, silica aerogel possesses higher
mechanical strength, enhanced thermal stability and negligible swelling in organic
solvents compared to most organic polymers [78].
16
The feasibility of silica aerogels as drug delivery carrier was reported. It was
found that no degradation occurred during loading process and the drugs (ketoprofen
and griseofulvin) adsorbed on silica aerogels dissolved faster than the crystalline
drugs [45, 47]. An extremely fast release – even compared to the nanocrystals - of
drugs was achieved by loading the drug into hydrophilic aerogel. Meanwhile,
hydrophobic aerogels exhibited slower drug release rate; that is governed by
diffusion. However, low drug loading (dithranol and niclosamid) was also reported
[46].
2.1.1.2 Silica Xerogel
Silica xerogel is normally synthesized by sol-gel process and is formed once
the gel is dried under ambient condition [48-51]. Conventional gel drying in the air,
however, resulted in considerable shrinkage of the gel.
The phenomenon was
explained by the formation of liquid-vapour interfaces within the gel network [40,
48, 79]. Thus, silica xerogel possesses lower surface area (less than 300 m2/g) than
silica aerogel. The following figure (Figure 2.2) shows comparison of the networks
of wet solgel, xerogel and aerogel.
Wet Solgel
(Hydrogel)
Gel Particles
Figure 2.2:
Xerogel
Aerogel
Solvent
Comparison of gel network of wet solgel, xerogel and aerogel.
17
Previous studies on silica xerogel as a carrier material in controlled delivery
indicate that silica xerogels are biocompatible and non-toxic materials. Since the
incorporation of various biological molecules such as drug and proteins into silica
xerogel can be carried out at room temperature, silica xerogel has been explored for
various biomedical applications, including oral and implantable drug delivery
systems [48-51, 79]. The application of silica xerogel for the controlled release of
heparin showed that the released heparin from different xerogels studied retained
about 90 % of its biological activity [51]. In addition, the synthesis of silica xerogel
is considerably easy, safe and inexpensive. By taking the chemical and physical
parameters into account while preparing silica xerogels, different matrices with
different properties can be produced.
2.1.2
Drug (Piperine)
Black pepper, Piper nigrum Linn (Piperaceae), is a well-known spice and
widely available in Asia countries. It has been used as Ayurvedic and Chinese
traditional medicine for a long time. It is commonly used as a good remedy for
treating gonorrhoea, menstrual pain, tuberculosis, sleeping problems and arthritic
conditions [80-81]. In Malaysia, black pepper is used traditionally as a medicinal
herb. The seeds with garlic, wrapped in banana leaves and heated over fire, are used
to treat asthma [82]. Black pepper, quoted as ‘King of Spices’ is also described as a
drug which increases digestive power, improves appetite, cures cold, cough,
dyspnoea, disease of the throat, intermittent fever, colic, dysentery, worms and piles
[83]. It is listed by US Food and Drug Administration (US FDA) as Generally
Recognised as Safe (GRAS) and contains 5-9 % of the active compound, piperine
[84]. Piperine (1-peroyl-piperidine) has also been reported to occur in other Piper
species for example, Piper longum, Piper betle, and Piper aurantiacum.
chemical structure of piperine is shown in the following Figure 2.3.
The
18
Figure 2.3:
Chemical structure of Piperine.
Piperine has been demonstrated in ‘in vitro’ studies to protect against
oxidative damage by inhibiting or quenching free radicals and reactive oxygen
species [85-86]. Reactive oxygen species and organic free radical intermediates
formed from many carcinogens are suggested to be involved in the initiation of
carcinogenic transformations. It also reported the immunomodulatory and antitumor
activity of piperine [87-88].
Work by Pradeep and Kuttan [89] demonstrated the antimetastatic activity of
piperine. It was found that piperine stimulates the replication of melanocytes [90].
Piperine and its analogues or derivatives inhibit the proliferation of melanoma cells,
thus, may be used in the treatment of skin cancer. Besides, it may also be helpful in
reducing inflammation, improving digestion, and relieving pain and asthma [91].
Piperine was also claimed to exhibit anti-depressant effect [92]. Previous researchers
showed that piperine may be used as structural template to develop anti-diarrhoeal
agents [87].
Among other uses, the ability of piperine in improving the bioavailability of
other nutrients becomes its most significant characteristic [93-94]. There is a great
interest and medical need for the improvement of bioavailability of large number of
drugs which are poorly bioavailable, given for long period, toxic and expensive.
Bioavailability enhancement helps to lower dosage levels and shorten the course.
The effect of piperine on the bioavailability of propanolol has been studied and
proven to enhance the bioavailability of this drug. Thus, large doses that frequently
19
causes side effect can be avoided [95].
In other report, the problem of poor
availability of curcumin can be overcome by adding a low dose of piperine, which
increases the uptake of curcumin by 2000 % in human [96].
Piperine is also
compatible and can be co-administered with various nutritional materials. Schmitt
[97] claimed that piperine can increase, improve or accelerate the absorption of
minerals and trace elements such as vitamin D, calcium, selenium, copper, zinc, and
chromium.
Study on the immunotoxicological effect of piperine shows that the lowest
dose of 1.12 mg piperine per kg body weight had no immunotoxic effect. Therefore,
it may be considered as immunologically safe “no observed adverse effect level
(NOAEL)” dose [98]. Unfortunately, piperine was very sensitive towards light and
oxygen. Moreover, the suggested drug dosage for a person was not more than 20 mg
per day [99]. Thus, the development of a suitable drug delivery carrier is desired in
order to protect as well as to dilute the piperine into a proper dosage. Up to date,
however, the research on drug delivery carrier for piperine is limited.
2.2
Synthesis of Silica Matrices
Research and development upon drug and pharmaceutical industries is a
tedious, arduous and expensive. It is necessary to analyze the entire elements and
processes and identify steps where changes can be made to increase efficiency and
save time. In order to do this, several considerations must be taken into account in
the production of silica aerogel and silica xerogel, such as production sources, cost of
starting materials, scalability, potential environmental and toxicity issues and time.
20
2.2.1
Rice Husk Ash as Silica Source
Rice husk is a waste product of the agriculture activity in most countries in
Asia and particularly in Malaysia. Rice husk has posed a major problem of disposal
to the rice milling industry in Malaysia and elsewhere in the world. Waste products
from the early stages of rice handling can be used in a number of applications including
biofertilizers, substrates for high value mushroom culture e.g. Ganoderma (Lin Zhi),
bicycle tyres (husks with resins), fertilizers, charcoal, deodorizers and pest control agents
(carbonized husks) [100-101]. Rice husk has also emerged as one of biomass
resources for electricity production other than palm oil waste, wood industry residue,
baggase, and agricultural waste [102].
Burning of rice husk at high temperature (about 700 ºC) leaves about 20 %
rice husk ash of its weight. Rice husk ash (RHA) is one of the most silica rich raw
materials containing about 90-98 % silica after complete combustion [103-104]. The
large amount of silica freely obtained from RHA provides an abundant and cheap
alternative source of silica which is useful for many applications particularly as a
support for heterogeneous catalysis [105-106]. The stabilization of Malaysian soil
by mixing with rice husk ash to improve its engineering properties is described by
Ali et al. [107].
The application of RHA also includes as the source for the production of
value-added materials such as zeolite, mesoporous MCM-48, MCM-41 and glass
[108-111]. In tropical countries where rice husk are abundant and considered as
waste materials, use of RHA as the silica source is particularly attractive, because
this would generally lead to cheaper production costs while help alleviate disposal
costs and environmental damage.
21
2.2.2
Sol-Gel Method
Sol gel technology has attracted considerable attention due to possibility to
obtaining submicron and nano-sized materials. The sol-gel process, as the name
implies, involves evolution of inorganic networks through formation of a colloidal
suspension (sol) and gelation of the sol to form a network in a continuous liquid
phase (gel) [112-113].
A sol-gel technology that was developed during the last two decades offers
new possibilities for incorporating active ingredients within silica matrix [114]. Sol
gel derived silica matrices, which are typically biodegradable can be used in drug
encapsulation and delivery applications [115]. The most essential features of the
technique are:
•
Ultra homogeneity – incorporated molecules can be separated at a nanoscale
level;
•
Low processing temperature – also temperature-sensitive molecules can be
processed;
•
Non-toxicity of silica gel matrix – final composites can be implemented as
sustained-release systems or biocompatible implantable materials.
2.2.2.1 Silica Gel Drying
Silica xerogel could be easily obtained by drying the wet gel at ambient
temperature.
The drying temperature can also be controlled for the drying of
temperature-sensitive drug-xerogel hybrids.
However, in the synthesis of silica
aerogel, the wet gel needs to be dried supercritically to avoid the gel shrinking.
Supercritical fluid processing is becoming a popular focus of research because of the
wide range of application for which it is suitable [116]. Two of the main advantages
22
of supercritical fluid (SCF) technology are that it requires few or no organic solvents
and little or no heating to produce the fundamental particles [117].
In pharmaceutical applications, a common solvent for supercritical drying is
liquid carbon dioxide due to it being relatively inert and having a workable critical
point conditions (31.1 ºC, 73.8 bar). Popularity of carbon dioxide stems from the
fact that it is non-toxic and non-flammable, also the second least expensive solvent
after water [118].
Carbon dioxide has emerged as an environmentally friendly
substitute for many halogenated and other organic solvents thereby reducing
atmospheric pollution and eliminating solvent residues in products [119].
The
schematic phase diagram of pure CO2 and water is shown in Figure 2.4.
Figure 2.4:
Schematic phase diagram for a pure CO2 and water.
The use of binary and multicomponent liquids has gained attention as it
provide the possibility to manipulate the critical temperature of the mixture, or to
introduce polar or non polar features to regulate interactions of the fluid with a
23
specific compounds [118]. The critical temperature assume-value of binary mixtures
often takes values between the critical temperatures of the components while for the
critical pressure, it is usually higher than the critical pressures of pure components.
For example, addition of about 7 mole % ethanol to carbon dioxide results in mixture
with a critical temperature about 52 ºC, but a critical pressure of 97 bar, compared to
31.1 ºC and 73.8 bar for carbon dioxide and 240.9 ºC and 61.4 bar for ethanol. In
Table 2.2, the critical properties of some compounds which are commonly used as
supercritical fluids are shown [120].
Table 2.2:
Critical conditions of several substances [120]
Solvent
Tc (K) Pc (MPa) Solvent
Tc (K) Pc (MPa)
Acetone
508.1
4.70
Hexafluoroethane
293.0
3.06
Ammonia
405.6
11.3
Ethanol
513.9
6.14
Carbon dioxide
304.1
7.38
Methanol
512.6
8.09
Cyclohexane
553.5
4.07
n-hexane
507.5
3.01
Diethyl ether
466.7
3.64
Propane
369.8
4.25
Difluoromethane
351.6
5.83
Propylene
364.9
4.60
Difluoroethane
386.7
4.50
Sulfur hexafluoride
318.7
3.76
Dimethyl ether
400.0
5.24
Tetrafluoromethane
227.6
3.74
Ethane
305.3
4.87
Toluene
591.8
41.1
Ethylene
282.4
5.04
Trifluoromethane
299.3
4.86
Ethyne
308.3
6.14
Water
647.3
22.1
2.2.3
Dissolution Enhancement of Drug
Bioavailability of poorly water-soluble drug is limited by their solubility and
dissolution rate. Insolubility will lead to poor dissolution and subsequently decrease
drug absorption from gastrointestinal tract, while the drug absorption is the critical
point to measure the efficiency of some therapeutic drugs [121]. According to
24
Noyes-Whitney equation, factors such as solubility and surface area can affect the
dissolution rate [122]:
dM
=
DS (Cs-Cb)
dt
(Equation 2.1)
h
where:
dM/dt = the dissolution rate
M
= the amount of drug (material) dissolved (usually in mg or mmol)
t
= time (seconds)
D
= the diffusion coefficient of the drug in the solution (cm2/s)
S
= the surface area of exposed solid (cm2)
h
= the thickness of diffusion layer
Cs
= the concentration of the drug in the diffusion layer
Cb
= the drug concentration in bulk solution at time t.
where the dissolution rate of the drug is based upon the surface area of the particle
which is exposed to the solubilizing liquid, high surface area allows more contact to
the dissolution medium; body fluid such as saliva, gastric juice or intestinal fluid. It
is easier to increase the surface area by reducing the particle size of drug crystals
than to increase the drug solubility. Figure 2.5 below shows the relationship between
the particle size and its surface area.
2x
x
2x
x
6 ( 2x X 2x ) = 24 x2
Figure 2.5:
8 X 6 X (x X x ) = 48 x2
Effect of particle size reduction towards the increment of surface area
25
Several studies were carried out in order to increase the dissolution rate of
drug through nano- or micronization of drug particles [123-125]. Several methods of
reducing particle size have been suggested. Physical method such as milling and
grinding are successful in particle size reduction. However, the ground crystals tend
to agglomerate, thereby creating a surface with higher energy than that of original
crystals, and reducing the effective surface area for dissolution [126]. The
agglomeration may be due to van der Waals attraction or hydrophobicity [127].
Higher surface area can also be achieved either by decreasing crystallinity or by
using nanoparticles in the drug formulation.
Nanoparticles act as the inclusion compound between the drug molecule and
a host particle. To be effective, the host/guest inclusion compound must have a
higher solubility than the individual drug molecule [128]. An inclusion complex of a
drug is usually not crystalline and thus should have higher solubility than a
crystalline material. Theoretically, non-crystalline (amorphous) solids are generally
more soluble than crystalline solids. Amorphous form, due to absence of an ordered
crystal lattice requires minimal energy and thus provides maximal solubility
advantage as compared to the crystalline forms of drug [129].
2.3
Survey on Characterization Techniques
A wide variety of instruments are capable for the characterization in
pharmaceutical research. Structures of samples were identified by employing powder
X-ray diffractometer, FTIR spectroscopy, UV-Vis spectroscopy, and FESEM.
Besides that, surface area of catalysts was measured with nitrogen adsorption method
by BET surface area analyzer.
26
The applications of infrared (IR) absorption spectroscopy are one of the most
fundamental and useful spectroscopic techniques available in the arsenal of a
pharmaceutical scientist. The IR spectroscopy is routinely used as an identification
assay method for various intermediate compounds, active pharmaceutical
ingredients, excipients, and formulated drug products and the methodology can be
also be developed as a quantitative techniques for any of this. Generally, this method
is used in the determination the functional group present in a molecule to reveal the
information on the types of bonding and classification of chemical compounds by
producing an infrared absorption spectrum that is like a molecular ‘finger print’
[130].
Previous work by Ternes and Krause [131] used infrared spectroscopy for the
characterization and determination of piperine and piperine isomers in egg. Piperine
and its isomers were determined by observing their trans-configuration bond at 960970 cm-1, which is shifted to about 1000 cm-1 by conjugated carbonylic groups.
FTIR spectrum of piperine indicates the characteristic peak of amide, conjugated
double bond, aromatic ring and presence of –C-O- bond [132]. FTIR spectroscopy is
also commonly used to determine the change of chemical nature of drugs after
loading procedure [46-47, 133].
The other technique to determine the degradation of drug is the ultravioletvisible spectroscopy. The ultraviolet-visible (UV-Vis) spectroscopy is an instrument
commonly used in the laboratory that analyzes compounds in the ultraviolet and
visible regions (180 to 820 nm) of the electromagnetic spectrum. UV-Vis absorption
spectroscopy can be applied in qualitative and quantitative drug analysis. This
technique is one of the spectroscopic methods based on the interaction of
electromagnetic radiation with the material. It allows one to determine the
wavelength and maximum absorbance of compounds. UV-Vis spectroscopy was
used extensively in the assessment and detection of piperine. Pure piperine appears
as yellowish crystals and its UV-Vis spectrum was detected with absorption maxima
at 340 nm which reflects that the compound contains highly conjugated aromatic ring
27
[131, 134]. UV-Vis spectroscopy is also use for confirmation of identity purpose of
piperine isolated from natural product [135].
The Brunauer-Emmet-Teller (BET) theory was originally elaborated by
Brunauer, Emmet, and Teller [136]. This technique is crucial to study the effect of
surface area of drug particles, drug carriers, or formulations on the dissolution profile
of studied drug. Higher surface area provides wider contact with the dissolution
medium consequently increase the wettability of the product. Various preparation
techniques applied in order to achieve higher surface area compared to original drugs
which includes micronization [137], cogrinding [123-125], use of nanoparticles [46,
55], and generation of drug nanoparticles via spray drying [138] and supercritical
fluid extraction [43, 139]. Increase of surface area in these techniques was proven to
improve the dissolution rate of drugs of interest.
It is important for evaluating the structure of piperine, silica matrices, and
piperine-silica formulations to observe its morphology and microstructure that are
the most basic physical properties. Field emission-scanning electron microscope
(FESEM) generally is used for observation of morphology and microstructure and
can capture the composite structure in the sight directly [140]. Work by Sanganwar
et. al. [127] used microscopic observation to study the size and deagglomeration of
itraconazole crystals after mixing with silica nanoparticles. Electron microscopy was
also used to determine the presence and formation of nanoparticles in drug delivery
system [141].
X-ray powder diffraction (XRD) is a rapid analytical technique primarily
used for phased identification of a crystalline material and can provide information
on unit cell dimensions. In most previous studies, XRD was used to determine the
crystallinity reduction or generation of amorphous state of new formulations [142].
The generation of amorphous state translates into a shift in the angle of diffraction
and/or broadening and subsequent splitting of the peaks [143]. XRD can also be used
to study crystal habit before and after addition of drug carrier or additives [144-145].
28
CHAPTER III
RESEARCH METHODOLOGY
3.1
Synthesis of Silica Matrices
In this study, silica aerogel (SA) and silica xerogel (SX) were synthesized via
modified aqueous colloidal sol-gel process. Generally, the production of these silica
matrices consists of three stages, which are (I) the preparation of sodium silicate, (II)
synthesis of wet gel and (III) the gel drying. The general procedure of the synthesis
process is shown in Figure 3.1.
Stage I
Stage II
Rice Husk
Alcogel
Combustion
Extraction
with
alcohol
Silica Ash
NaOH
Hydrolysis
Figure 3.1
Gelation
Stage III
Supercritical
drying
Silica Aerogel
Ambient
temperature
drying
Aquagel
Synthesis of silica aerogel and silica xerogel
Silica Xerogel
29
The synthesis of silica aerogel involves two major steps: (1) the preparation
of the alcogel by sol-gel process and (2) the supercritical drying of the wet gel to
remove the solvent. Rice husk ash (RHA) and the sodium hydroxide (NaOH)
(Merck; 99 %) were used to prepare sodium silicate solution. The mass ratio applied
was 39.13 g RHA: 14.55 g NaOH: 450 g H2O. The mixture was stirred for two days
at 90 ºC and then filtered to separate the filtrate from undissolved residue. This low
cost sodium silicate (Na2SiO3) precursor was used to prepare silica aquagel through
hydrothermal process using concentrated sulphuric acid (H2SO4) (Merck; 96 %).
The silica content of the as-prepared sodium silicate was determined by
adding concentrated sulphuric acid dropwise into 100 g sodium silicate solution until
gelation. Then, the aquagel was filtered and washed with warm distilled water (60
ºC) to remove the trapped sodium sulphate salt from the gel until pH 7. The gel was
dried at 120 ºC overnight, cooled and weighed. The silicate solution was then diluted
to obtain sodium silicate with 4 % silica.
% Silica content =
Dry gel
100 g
x
100 %
15 mL H2SO4 was added dropwise into 250 mL Na2SiO3 and aged for 2 day
to strengthen the silica network.
Then, the aquagel obtained was washed with
distilled water to remove the sodium sulfate salt resulted from neutralization, until
pH 7. Silica alcogel is prepared after the condensation of ethanol for at least 12
hours. Then, the gels were dried supercritically with N2 in the autoclave.
Supercritical drying of the alcogel was conducted in a high temperature
supercritical system.
To be dried supercritically, about 350 mL alcogel and
additional 500 mL ethanol was placed in 2L extraction autoclave. The temperature
of the autoclave was slowly raised stepwise as shown in Table 3.1, until the critical
temperature and pressure were reached. Then, the whole system was held for 1 h,
while the pressure was maintained at 1500 psi. Afterwards, the ethanol was slowly
30
released from the system until the pressure drop to 30 psi followed by dynamic
drying with flowing N2 gas for 15 min at constant pressure (36 psi). Then, the
system was left to cool.
Table 3.1:
Temperature increment steps for supercritical drying
Duration (minute)
60
60
60
60
60
60
Temperature (ºC)
100
150
200
225
250
275
Silica xerogel (SX) was also synthesized, following the similar route as the
synthesis of silica aerogel except for the gel drying. For SX, the aquagel was dried
at ambient temperature. Firstly, sufficient amount of sulphuric acid (96 %) was
added dropwise under vigorous stirring into 250 mL sodium silicate solution until
gelation. The gel was aged at room temperature in distilled water for 24 hours.
Finally, it was dried in an oven at 100 ºC for several days until a constant weight was
obtained.
3.2
Drug (Piperine)
The model drug used for the experiment is piperine (piperinoyl-piperidine)
and its properties are summarized in Table 3.2. The drug was prepared in Natural
Product Laboratory, Department of Chemistry, Universiti Teknologi Malaysia and
was used as received.
Table 3.2:
Structure
Properties of model drug (Piperine)
Physical Appearance
TMelting (ºC)
Pale yellow, needle 131-134
like crystals
MW (g mol-1)
285.34
31
3.3
Drug Loaded Silica Matrices
Three methods were used in order to load the silica matrices with piperine,
which are physical mixing (co-grinding), impregnation, and direct synthesis. Brief
procedure is described in the following section.
3.3.1
Physical Mixing
Firstly, the coarse piperine crystals were ground to obtain fine powder.
Sufficient amount of piperine was later co-ground with silica aerogel and silica
xerogel, respectively, using porcelain mortar until homogeneous. Lastly, the mixed
powders were sieved through 212 μm screen and stored in air-tight sample bottles.
3.3.2
Impregnation
Drug loading via impregnation was carried out by stirring the silica aerogel in
ethanolic piperine solution. For this purpose, aerogel was firstly calcined at 150 ºC
for 5 hours to obtain hydrophilic silica aerogel. Meanwhile, sufficient amount of
piperine was dissolved in ethanol (Hayman; 99.7% v/v). Then, weighed amount of
silica aerogel was added into the piperine solution, and the mixture was stirred
vigorously until homogeneous. The piperine:carrier weight ratio applied in this
study were 1:4, 2:3, 1:1 and 3:2. Lastly, the wet mixture was dried at 40 ºC until a
constant weight was obtained. Similar procedure was done to load the silica xerogel
with piperine.
32
The piperine-carrier formulations were also prepared by adding minimum
amount of ethanol. In this case, ethanol acted as the binding agent between piperine
and silica matrices. Firstly, the physical mixtures of piperine-silica matrix were
prepared following the procedure described in section 3.3.1 above. Then, ethanol
was added dropwise until moist mixture was obtained. The mixture was then dried at
40 ºC in oven, ground and sieved. Figure 3.2 shows the flow diagram of drug
loading procedure via impregnation and physical mixing methods.
(I) Coarse
piperine crystals
Grinding
Fine powder
+
Silica
matrices
(II)
Mixed powder
Grinding &
sieving
Sieving
Dry granules
Grinding, drying, sieving
Compression
+ Minimum
EtOH
(III)
Impregnated
with minimum
EtOH
Dissolved in
EtOH
Piperine solution
Drying
TABLET
+ Silica
matrices
(IV) Piperine
impregnated silica
matrices
Figure 3.2:
Flow diagram of drug loading procedure via impregnation and
physical mixing methods.
33
3.3.3
Direct Synthesis
The piperine-loaded silica aerogel was also prepared through chemical
reaction during the sol-gel process. The piperine was loaded at pH ~7, which was
slightly before the gelation, in order to minimize the denaturation of drug. Firstly,
0.2 g piperine was dissolved completely in 10 mL ethanol to ensure homogeneous
distribution of piperine in gel. Meanwhile, concentrated sulphuric acid (H2SO4) was
added dropwise into 20 g of sodium silicate (4 % silica) solution under mild stirring
until the gelation was about to occur (pH ~7). The ethanolic piperine solution was
added into the mixture and was left to gel. The formed gel was aged for 2 days,
followed by washing with distilled water. Then, the gel was aged in ethanol for 2
days. Similar samples were also prepared with different drug loading and aging
period. Figure 3.3 shows the flow diagram of drug loading procedure via direct
synthesis method.
Na2SiO3
(4 % silica)
H2SO4
dropwise
Sol pH ~7
20 mg/mL
ethanolic
solution of
piperine
Piperine-loaded wet gel
Aging (2 days)
Washing with
distilled water
Aging in ethanol
Drying
Figure 3.3:
Flow diagram of drug loading procedure via direct synthesis method.
34
In order to get piperine-loaded silica aerogel, the aged gel was dried
supercritically by using supercritical carbon dioxide (SC-CO2) extractor. In brief, the
prepared silica gel sample was carefully placed in 1 L extraction autoclave, which
was firstly chilled to 25 ºC. The trapped air was then flushed out with carbon
dioxide gas before the system was closed for the drying process. Liquid CO2 was
vented in until the autoclave pressure reached to 50 bar.
The autoclave was
pressurized with purified CO2 gas up to 80 bar at 25 ºC for 6 hours. During this
period, the ethanol in the silica gel was replaced by liquid CO2. Then, the autoclave
was heated to 40 ºC with constant pressure at 80 bar for 4 hours. Dynamic drying
was performed by using high pressure purified CO2 for 10 minutes at 40 ºC and 80
bar. Afterwards, the autoclave was slowly depressurized to atmosphere at 40 ºC.
Finally, dried silica aerogel was obtained. The schematic diagram of supercritical
carbon dioxide extraction unit is shown in Figure 3.4. Piperine-loaded silica xerogel
formulations were prepared by drying the drug-loaded gel at 40 ºC.
Chiller
Figure 3.4:
Flow meter
Schematic diagram of supercritical carbon dioxide
Autoclave
CO2
Collector
CO2
Pump
35
3.3.4
Sample Codes
The prepared samples were coded according to the details in Table 3.3.
Table 3.3: Sample codes for various formulations
Sample Code
Carrier
Method
Piperine
(wt %)
SA
Silica aerogel
-
SX
Silica xerogel
-
PAPM 20
Silica aerogel
Physical mixing
20
PAPM 40
Silica aerogel
Physical mixing
40
PAPM 50
Silica aerogel
Physical mixing
50
PAPM 60
Silica aerogel
Physical mixing
60
PXPM 20
Silica xerogel
Physical mixing
20
PXPM 40
Silica xerogel
Physical mixing
40
PXPM 50
Silica xerogel
Physical mixing
50
PXPM 60
Silica xerogel
Physical mixing
60
PAIM 20
Silica aerogel
Impregnation
20
PAIM 40
Silica aerogel
Impregnation
40
PAIM 50
Silica aerogel
Impregnation
50
PAIM 60
Silica aerogel
Impregnation
60
PXIM 20
Silica xerogel
Impregnation
20
PXIM 40
Silica xerogel
Impregnation
40
PXIM 50
Silica xerogel
Impregnation
50
PXIM 60
Silica xerogel
Impregnation
60
PADS 10
Silica aerogel
Direct synthesis
10
PADS 20
Silica aerogel
Direct synthesis
20
PXDS 10
Silica xerogel
Direct synthesis
10
PXDS 20
Silica xerogel
Direct synthesis
20
36
3.4
Characterization
All
the
samples
were
characterized
using
Ultraviolet-Visible
spectrophotometry (UV-Vis), Fourier transform infrared spectroscopy (FTIR), BET
surface area analysis, X-Ray diffraction (XRD), and field emission scanning electron
microscopy (FESEM).
3.4.1
UV-Vis Spectroscopy
Ultraviolet and visible spectra were used to determine the chemical nature of
the drug before and after the loading process. The degradation of piperine was
determined by comparing the characteristic peaks for drug and drug loaded ones.
The method that is most often used in a quantitative way to determine concentrations
of an absorbing species in solutions is by using Beer-Lambert Law as shown in
following equation [146].
A = -log10 ( I / I0) = ε c L
Where A
(Equation 3.1)
= measured absorbance
I0
= intensity of the incident light at a given wavelength
I
= transmitted intensity
L
= path length through the cell
c
= concentration of absorbing species
ε
= molar extinction coefficient
A spectroscopy can be either single beam or double beam. In single beam
instrument, all of the light passes through the sample cell. The I0 must be measured
by removing the sample. On the other hand, in a double beam instrument, the light is
37
split into two beams before it reach the sample. One beam is used as the reference;
the other beam passes through the sample. In UV-Vis spectroscopy, an
approximately monochromatic light beam is generally employed. This is selected
with the aid of a monochromator from the total emission spectrum of a light source.
In this region of electromagnetic spectrum, the nature of a radiation is expressed as
wavelength (nm) [147]. Samples for UV-Vis spectroscopy are most often liquid and
are typically placed in a transparent cell, known as cuvette.
For the measurement of absorption in UV-Vis region, the loaded carriers
were dispersed in ethanol. The UV-Vis spectra were measured on Perkin Elmer
Lambda 25 spectrophotometer under ambient conditions using quartz test cuvettes.
The absorbance value reading at maximum peak for piperine was used for
quantitative analysis on the base of calibration curve.
Five solutions were prepared with different concentrations. The slope of
absorbance readings produced a linear calibration curve. In order to determine the
drug concentration in the sample, 10 ppm solution of the samples were prepared by
dissolving the sample powders in ethanol (99.7 %). The solution was stirred for at
least 60 minutes to ensure the complete dissolution of the drug. The concentration of
the drug in ethanol was determined using UV-Vis spectrophotometry.
3.4.2
FTIR Spectroscopy
All the samples were characterized by FTIR spectroscopy in order to identify
the chemical bonding of the samples as well as to determine the degradation
occurrence of piperine. Based on Hooke’s law, the vibrational frequency between
two atoms might be approximated as:
38
v=
1
2π
k
µ
(Equation 3.2)
where μ is the reduced mass of the two atoms, such that μ= (m1m2)/(m1 + m2), and k
is the force constant of the bond (dynes/cm). Quantum mechanical analysis of the
harmonic oscillator model reveals a series of equally spaced vibrational energy levels
(defined by the vibrational quantum number v, where v = 1, 2, 3 …) that are
expressed as:
1

Ev =  v + hv 0
2

(Equation 3.3)
where Ev is the energy of the vth level, h is Planck’s constant, and v0 is the
fundamental vibrational frequency [148].
In the infrared spectroscopy, frequencies are ranged from 400 to 4000cm-1
but the IR spectra of silica based materials show typical absorption band in certain
regions. The assignment of each region is as follow [45, 149-152]:
i.
Stretching vibrations of hydroxyl groups (3000-3700 cm-1). This region
associated with H-bridging hydroxyl (-Si – OH ….O – Si-) groups, isolated
silanol (-Si – OH) and adsorbed molecular water.
ii.
Si – O – CH3 symmetric stretching and C-H stretching at 2960 cm-1
iii.
Si – O – Si and vibration of SiO2 network at 1860 and 800 cm-1
iv.
H – O – H for adsorbed molecular water around 1650 cm-1
v.
≡ Si – O , vibration of silica network at around 1000 cm-1
vi.
Si – O – Si vibrational mode around 800 cm-1
vii.
Si – O – Si deformation at 948, 460 cm-1
39
For FTIR measurements, the samples were powdered and compressed with
potassium bromide (KBr) and placed in the sample holder. The absorption spectra
were scanned over the wave number range between 4000 and 400 cm-1 by FTIR
spectrometer Perkin-Elmer Spectrum One at ambient temperature. A vibrational
transition might be approximated by the consideration of atoms bonded together
within a molecule as a harmonic oscillator [153].
3.4.3 BET Surface Area Analysis
The BET specific surface measurement based on the gas adsorption is often
applied as a simple method to evaluate the surface area of a porous material. Such a
material which is surrounded by and in equilibrium with a certain gas which has a
certain temperature, T, and relative vapour pressure, p/po, adsorbed physically a
certain amount of gas. The amount of adsorbed gas is dependent on its relative
vapour pressure and is proportional to the external and internal surface of the
material. The connection between relative vapour pressure and amount of adsorbed
gas at a constant temperature is called an adsorption isotherm.
The determination of surface areas from the BET theory is a straight forward
application of equation. The BET equation is generally expressed as follow [154]:
1
1
=W C
m
W [(P / Po) - 1
Where W
Wm
+
C–1
Wm
P
Po
(Equation 3.4)
= volume of gas adsorbed at pressure P
= volume of gas adsorbed in monolayer, same units as V
40
P0
= saturation pressure of adsorbate gas at the experimental temperature
C
= a constant related to the enthalphy of adsorption in the first
adsorption layer.
A plot of 1/ [W (P0/P) – 1] versus P/P0, as shown in Figure 3.5, yields a straight line
1/ [W (P0/P) – 1 ]
usually in the range of 0.05 ≤ ≤ 0.35.
A
i
P/P0
Figure 3.5: Typical BET plot.
After calculation of Wm and C, the surface area (ABET) can be calculated by:
ABET =
WmNAs
v
(Equation 3.5)
where NA is Avogadro number (6.023 x 1023 mol-1), s is adsorption cross section, and
V is molar volume of adsorbent gas.
41
Any condensable inert vapour can be used in the BET method, but for the
most reliable measurements, the molecules should be small. Krypton, argon, and
nitrogen are suitable choices in view of their commercial availability.
Liquid
nitrogen is readily available coolant, but argon and krypton are expensive relative to
nitrogen and must be highly purified [155]. Consequently, nitrogen is usually used
since it relatively cheap and readily available in high purity.
The BET specific surface area and total pore volume (measured at P/Po=
0.95) of the silica aerogel, silica xerogel, piperine, and drug-loaded silica matrices
was measured using ThermoFinnigan Qsurf Surface Area Analyzer M3 series.
Approximately 0.025 g of sample powder was put into a sample tube holder followed
by preheating in the heating mantle attached to the instrument and connected to a
supply of gas mixture (nitrogen and helium). This step was done to eliminate the
contaminants such as moisture and volatile compounds which were physically
adsorbed on the sample surface. Specific surface area was calculated following the
BET procedure.
3.4.4
X-Ray Diffraction
. X-ray diffraction involves the measurement of the intensity of X-rays
scattered from electron bound to atoms [156]. Waves scattered at atoms at different
positions arrive at the detector with a relative phase shift. Therefore, the measured
intensities yield information about the relative atomic positions. The easiest access to
the structural information in powder diffraction is via the well-known Bragg
equation, which describes the principle of X-ray diffraction in terms of a reflection of
X-rays by sets of lattice plane [157]. Lattice planes are crystallographic planes,
characterized by the index triplet hkl, the so-called Miller indices. Bragg analysis
treats X-rays like visible light being reflected by the surface of a mirror, with the Xray being specularly reflected at the lattice planes.
42
The derivation of Bragg’s Law is shown in Figure 3.6. Diffraction generally
occurs when the wavelength of the wave motion is of the same order of magnitude as
the repeat distance between scattering center with:
nλ = 2d sin θ
(Equation 3.6)
where d = interplanar spacing of parallel lattice planes
n = diffraction order (1, 2, 3…)
λ = wavelength of the X-rays
θ = diffraction angle, angle between the incoming and outgoing X-rays beams
Incoming beam
Diffracted beam
θ
θ
d
2θ
Figure 3.6: Illustration of the geometry used for the simplified derivation of Bragg’s
law [157].
X-ray diffractometers consist of three basic elements: an X-ray tube, a sample
holder, and as X-ray detector [158]. X-ray is generated in anode ray tube by heating
a filament to produce electrons, accelerating the electrons toward a target by
applying a voltage, and bombarding the target material with electrons.
When
electrons have sufficient energy to dislodge inner shell electrons of the target
material, characteristic X-ray spectra are produced.
43
X-ray powder diffraction patterns of piperine, silica aerogel, silica xerogel
and drug loaded silica matrices were recorded using Bruker D8 Advance
diffractometer to investigate the crystallinity of samples. Samples were irradiated
with monochromatized Cu Kα (λ = 1.5405Å) radiation and analyzed at 2θ between
5º and 45º. XRD analysis was carried out at a step of 0.05º and step time 1s. The
voltage and current used were 40 kV and 40 mA, respectively. The XRD analysis
was carried out at Ibnu Sina Institute for Fundamental Science Studies, UTM.
3.4.5
Field Emission Scanning Electron Microscopy (FESEM)
SEM is a method of irradiating the electron beam to the sample, and
obtaining the observation image based on the generated second beam [159]. For
SEM observation, conductivity is necessary for the sample, and the conductive thin
film of gold etc. is formed by sputtering on the surface of insulation material. There
is a little shape restriction in the sample, and an easy and effective evaluation is
possible. The field emission scanning electron microscope (FESEM) is similarly
configured to a conventional SEM, except that a cold field emission electron source
is used, which permits higher image resolution to be attained, increased signal to
noise ratio, and increased depth of field [160]. Thus, FESEM can observe at high
magnification of about 300,000 times, and can observe nanosize microstructure.
The morphology of all samples were observed at different magnification
using field emission scanning electron microscope (FE-SEM JSM-6701F, JEOL)
operated with acceleration voltage of 2.0 kV and working distance about 3 mm. The
samples were scattered onto metal cylinders with conductive carbon tape, dried in a
vacuum chamber and coated with a platinum layer in sputter coater unit (JFC-1600,
JEOL). This analysis was carried out at Ibnu Sina Institute for Fundamental Science
Studies, UTM.
44
3.5 Investigation on Drug Dissolution Profile
The in vitro study release of drugs from the carrier was performed in two
simulated fluids: gastric fluid (0.1 M HCl or pH ~1) and intestinal fluid (phosphate
buffer saline, pH ~7), using dissolution in a flow through cell [161]. The samples
were compacted into 0.1 g disks using two flat face punch and die (d = 13 mm) under
pressure of 1500 psi. One disk of samples was immersed in into 500 ml dissolution
medium at 37 ºC, and the solution was continually stirred at 100 rpm. The drug
dissolution study was done using custom-made apparatus assembly following USP
IV test apparatus (flow through cell) recommended by United States Pharmacopoeia
(USP) [162]. Triplicate samples (5 ml) were withdrawn from the dissolution vessels
at selected time interval (10 minutes) and replaced with fresh dissolution medium to
maintain the volume. Each sample was analyzed for drug concentration at maximum
absorbance of piperine at a wavelength of 340 nm on an UV-Vis spectrophotometer
(Perkin-Elmer Lambda).
Thermostat
Flow through cell
Magnetic
stirrer
Figure 3.7:
Tablet
Schematic diagram of dissolution testing apparatus assembly
CHAPTER IV
RESULTS AND DISCUSSION
4.1
Synthesis of Silica Aerogel and Silica Xerogel
Silica aerogel and silica xerogel were synthesized via sol-gel method. The
resulting silica aerogel appears as fluffy semi-transparent white powder, whilst silica
xerogel as hard, dense white powder. The chemical equation of the synthesis of
silica matrices from sodium silicate is illustrated below.
Na2SiO3
+
H2SO4
SiO2
+
Na2SO4
(Equation 4.1)
Table 4.1 lists the physical properties of synthesized silica aerogel and silica
xerogel.
Table 4.1:
Properties
Physical appearance
Density (g/cm3)
Surface area (m2/g)
3
Pore volume (cm /g)
Physical properties of silica aerogel and silica xerogel
Silica Aerogel
Silica Xerogel
White, fluffy powder
White, dense, hard gel
0.064
0.622
405
116
0.23
0.96
46
Silica aerogel and silica xerogel have surface areas of 405 m2/g and 116 m2/g,
respectively. The relatively low surface area of silica xerogel as compared to silica
aerogel may be explained by the gel shrinkage due to capillary pressure during
ambient pressure drying. It is dense, hard gel. These results are consistent with
those of other studies, whereby the supercritical drying technique applied in the
production of silica aerogel may have avoided the gel network shrinkage [71, 150,
163-165]. This type of technique prevents the formation of liquid-vapour meniscus
from receding during the emptying of pores in the wet gel. Thus the liquid surface
tension and capillary pressure may be eliminated which consequently avoid collapse
of the gel pore volume and results in high porosity and remarkably large surface area.
4.2
Characterization of Silica Matrices and Piperine
The model drug (piperine) and as-synthesized silica aerogel and silica xerogel
were characterized by using X-ray diffraction analysis (XRD), Fourier-transformed
infrared spectroscopy (FTIR) and field emission scanning electron microscopy
(FESEM).
4.2.1
FTIR Spectroscopy Analysis of Silica Matrices
Figure 4.1 shows the FTIR spectra of synthesized silica aerogel and silica
xerogel. In both spectra, a broad peak appears around 3440 cm-1 due to the O-H
stretching; caused by physically adsorbed water. The presence of this absorption
band may also be associated to H-bridging hydroxyl (-Si – OH …O – Si-) groups and
isolated silanol (-Si – OH). Weaker absorption band shown in Figure 4.1 clearly
indicates the hydrophobic nature of the aerogels. The hydrophobicity of silica
47
aerogel is supported by the presence of peaks around 2980 cm-1 corresponding to CH bond. Meanwhile, the peak at 1628 cm-1 corresponds to the bending mode of O-H
of water. The spectrum also shows strong peaks at around 1100 cm-1 and 800 cm-1,
and 470 cm-1 due to asymmetric, symmetric, and bending modes of SiO2 respectively
which normally appear in any silica product. The peaks at around 1200 and 960 cm-1
are related to the Si-C bonds.
Relative Transmittance, %
(b)
(a)
4000.0
3000
2000
1500
1000
400.0
Wavenumber, cm-1
Figure 4.1:
FTIR spectra of (a) silica aerogel and (b) silica xerogel
In Figure 4.1(b), the symmetric and asymmetric stretching vibrations of Si-OSi and a bending of Si-O-Si mode correspond to the peaks at 1250 cm-1, 990 cm-1,
802 cm-1 and 466 cm-1, respectively. The absorption band at 960 cm-1 is related to
the vibration of Si-OH bonding. The OH bands at around 3440 and 1635 cm-1
indicate the presence of absorbed water. The characteristic peaks of both silica
aerogel and xerogel are summarized in Table 4.2.
48
Table 4.2: FTIR absorption bands of silica aerogel and silica xerogel
Type of Bonding
Frequency (cm-1)
Type of Vibration
Silica Aerogel
Silica Xerogel
3440
–O–H
Stretching
3440
–C–H
Stretching
2980
Si–O–Si
asymmetric stretching
1100
1119
Si–O–Si
symmetric stretching
800
802
Si–O–Si
Bending
470
466
H–O–H
Bending
1627
1635
Si–C
Stretching
967
960
Silica matrices present an extremely large number of accessible hydroxyl
groups, and consequently show strong hydrogen-bonding effects. Figure 4.2 shows
the presence of hydroxyl groups due to the silanol groups, physically adsorbed water
and chemically adsorbed water [166].
Physically adsorbed H2O
H
H
O
Chemically adsorbed water
H
H
H
Hydrogen
Bond
HO
Figure 4.2:
H
H
O
H
O
O
H
H
H
H
H
Silanol
group
O
O
O
O
O
O
Si
Si
Si
Si
Si
Si
O
O
O
O
O
H
OH
The presence of OH groups due to the silanol groups, physically and
chemically adsorbed water.
49
4.2.2
FTIR Spectroscopy Analysis of Piperine
The FTIR spectroscopy was used to determine the characteristic absorption
peaks for piperine. Figure 4.3 shows the spectrum of piperine. As shown at 960-970
cm-1, there is a typical trans-configuration bond, shifted to about 1000 cm-1 by
conjugated carbonylic group. There is an observable band at 1005 cm-1. Apart from
the intense -C-H aromatic bands between 2800 and 3100 cm-1, main FTIR signals
occur in the fingerprint range between 1000 and 1700 cm-1. The spectrum presents
very good resolution of the aromatic and aliphatic –C=C– as well as O=C–N–
stretching vibrations detected between 1500 and 1640 cm-1. The signal observed at
1450 cm-1 is assigned to CH2– bending vibration whereas the other bands in the
range of 1100 and 1400 cm-1 are mainly due to –C–C– stretching and –C–H bending
vibrations of the piperine molecule.
30
26
Transmittance, %
22
18
14
10
6
4000.0
3000
2000
1500
Wavenumber, cm-1
Figure 4.3: FTIR spectrum of piperine.
1000
400.0
50
Table 4.3: FTIR absorption bands of piperine
Type of Bonding
Type of Vibration
Frequency (cm-1)
–C–H
Stretching
2937, 2797
=C–H aromatic
Stretching
3100
=C–H aromatic
Bending
846
–C–H
Bending
1447
C=C aromatic
Stretching
1583 and 1447
C=C aliphatic
Stretching
1609
–N–C=O
Stretching
1631
1000
Trans-configuration
C–N
4.2.3
Stretching
1247
Morphology and Crystallinity Studies of Silica Aerogel, Silica Xerogel
and Piperine
The morphology of drug particles and silica matrices was investigated and
FESEM micrographs are illustrated in the following figures. The synthesized silica
aerogel exhibits porous network structure, which contains solid clusters of 10-60 nm
and pores below 100 nm in between them (Figure 4.4). As can be seen in Figure 4.5,
silica xerogel has a highly dense morphology, which appears as tiny spherical
nanoparticles, approximately 30-60 nm in diameter. The micrograph also shows the
aggregates of particles in silica xerogel. Besides that, the diffractograms reveal that
the silica aerogel and silica xerogel are amorphous (Figure 4.6). These findings
correlate the BET surface area analysis result shown in Table 4.1, and suggest the
connection between porosity and the surface area. Surface area of silica aerogel is
about three times higher than silica xerogel.
51
Figure 4.4: FESEM micrograph of synthesized silica aerogel.
Figure 4.5: FESEM micrograph of synthesized silica xerogel.
Relative Intensity
52
(a) Silica aerogel
(b) Silica xerogel
5
10
Figure 4.6:
15
20
25
30
2-Theta - Scale
35
40
45
50
X-ray diffractograms of (a) silica aerogel, and (b) silica xerogel.
In contrary, the drug substance exists in a crystal form, which has a needleshaped appearance with smooth surface. The drug crystals have a low specific
surface area due to their large particle size (Figure 4.7). The high crystallinity of
piperine as shown in this figure also explains the high intensity peaks in its X-ray
diffractogram as illustrated in Figure 4.8. Pure piperine shows several diffraction
peaks typical of crystalline powder.
Figure 4.7: FESEM micrograph of pure piperine (250 X magnification)
53
2100
1800
Intensity (a.u)
1500
1200
900
600
300
0
5
10
20
30
40
50
2-Theta - Scale
Figure 4.8:
X-ray diffractogram of crystalline piperine.
Highly crystalline drug with very low surface area (SBET ~ 3 m2/g) explains
the poor dissolution rate of this drug. Thus, the generation of amorphous state to
increase its surface area is essential for drug dissolution enhancement. The
amorphous nature of silica aerogel and silica xerogel is expected to allow therapeutic
and diagnostic agents to be encapsulated, covalently attached or adsorbed onto such
carriers, creating formulations with higher surface area to overcome solubility issues.
4.3
Preparation of Piperine-Aerogel and Piperine-Xerogel Formulations
Piperine-silica aerogel and piperine-silica xerogel formulations were
successfully prepared via three methods which are physical mixing, impregnation
54
and direct synthesis. Yellowish, free flowing powders obtained were ground and
stored in air-tight sample bottles. The formulations were characterized using FTIR,
FESEM, XRD and BET surface area analysis.
4.4
Degradation Study of Piperine Loaded Silica Matrices
Taking into consideration the different conditions underwent by each drug
under each preparation method, such as grinding pressure, use of solvents and
supercritical-CO2 drying, UV-Vis spectroscopy was used to determine whether the
chemical nature of piperine was changed during the loading process. The maximum
absorbance was located at wavelength 340 nm, which is identical to literature value
by Ternes et. al.[131] and Kanaki et. al. [135]. In UV-Vis spectroscopy, a light
source emits white light which is made monochromatic and then columnated to pass
through the sample to a detector. When white light passes through or is reflected by
a coloured substance, a characteristic portion of the mixed wavelengths is absorbed.
As all the piperine-silica formulations appear as yellowish free-flow powders, the
absorption should be detected at 420-430 nm. Absorption at shorter wavelength (340
nm) is due to the presence of aromatic systems which contain p electrons, absorb
strongly in the ultraviolet [160]. Figure 4.9 indicates that characteristic peaks of
piperine were detected at the same positions for both pure piperine and piperinesilica matrices formulations. Therefore, it is concluded that the loading procedure
does not influence the chemical nature of the drug investigated in the experiments.
Relative Absorbance (A)
55
(a)Piperine
(b)PAIM
(c)PXPM
(d)PAIM
(e)PXIM
(f)PADS
(g)PXDS
270.0
280
300
320
340
Wavelength, λ (nm)
360
380
400.0
Figure 4.9: UV-Visible spectra of pure piperine and piperine loaded silica matrices
(a) Crystalline piperine, (b) physical mixed piperine-aerogel (PAPM), (c) physical
mixed piperine-xerogel (PXPM) (d) piperine impregnated aerogel (PAIM), (e)
piperine impregnated xerogel (PXIM), (f) direct synthesis of piperine-aerogel
(PADS), and (g) direct synthesized piperine-xerogel (PXDS) in 0.1 M HCl.
The UV-Vis spectrum of directly synthesized piperine-aerogel (Figure 4.9f)
showed a blue shift, where the maximum absorbance was detected at 335 nm. The n
electrons in a molecule are highly affected by hydrogen bond formation. The energy
levels of n electrons decrease significantly in a solvent that has the ability to form
hydrogen bonds. This causes a shift in the maximum of an n →л* transition to
shorter wavelength. Thus, shifting of directly synthesized piperine-aerogel spectrum
showed the presence of hydrogen bonding between drug molecule and other
molecules containing O-H and N-H functional groups. This may be due to the
presence of silanol groups in silica aerogel or the presence of ethanol used in the
sample preparation.
56
The FTIR spectra of loaded aerogels were also recorded and compared with
that of the original piperine in its crystalline form. After loading of piperine, it is
clearly shown that the location of the characteristic peaks was found to be identical
to that of pure silica aerogel and the corresponding drug (Figure 4.10). This strongly
suggests that the drug has been successfully loaded into/onto silica aerogel via
physical mixing, impregnation and direct synthesis methods.
(a) Silica aerogel
(b) PADS 20 wt %
Transmittance, %
(c) PAIM 50 wt %
(d) PAPM 50 wt %
(e) Piperine
4000.0
3000
2000
1500
1000
400.0
Wavenumber, cm-1
Figure 4.10: FTIR spectra of piperine loaded silica aerogel formulations.
57
For all formulations, the drug loaded silica aerogels have shown stronger
adsorption peak at around 3400 cm-1, which implies that the loading process has
affected the hydrophilicity-hydrophobicity properties of the sample. Since there was
no difference in characteristic peaks observed between impregnated, directly
synthesized and physically mixed samples, therefore no drug degradation occurred
during the loading process.
(a)Silica Xerogel
Transmittance, %
(b) PXDS 20 wt %
(c) PXIM 50 wt %
(d) PXPM 50 wt %
(e) Piperine
4000.0
3000
2000
1500
1000
400.0
Wavenumber, cm-1
Figure 4.12: FTIR spectra of piperine loaded silica xerogel formulations.
58
The spectra of silica xerogel, drug loaded silica xerogel, and corresponding
drug are shown in Figure 4.11. After the drug loading, additional absorption peaks
that characterized the drug are observed in the spectra. Absorption bands at 2980
cm-1 and fingerprint range indicate that piperine was successfully loaded into/onto
silica matrices (Figures 4.10 to 4.11). Besides that, the presence of broad peak
around 3400 cm-1 corresponding to O-H bond may be contributed by hydrogen
bonding between piperine and silica matrices.
Figure 4.12 shows the possible
hydrogen bonding in piperine due to the presence of lone pairs that can interact with
the hydroxyl groups in silica matrices.
Hydrogen bond
Figure 4.12: Possible hydrogen bonding in piperine
4.5
Drug Loading Capacity
The concentration of drug in the formulations was determined using UV-Vis
spectrophotometer using calibration data. The calibration curve of ethanolic solution
of piperine with different concentrations is presented Appendix A.
59
4.6
Physically Mixed Piperine-Silica Matrices
Physical mixing is the simplest method of preparation of drug formulations.
Figure 4.13 shows the diagram of expected coating of piperine with silica matrices.
The size of drug particles could be easily reduced by grinding and milling. However,
without silica matrices or other excipients, ground drug tends to agglomerate,
creating a surface with higher energy than that of original crystals.
The
agglomeration will then reduce effective surface area essential for faster drug
dissolution. The interactions between drug and carrier such as electrostatic bonds,
Van der Waals forces and hydrogen bonding may retard self association of drug
molecules, thus inhibit crystallization and increase solubility.
Piperine
Cogrinding
+
Agglomeration of piperine particles
Piperine
particles
Silica matrices
Figure 4.13: Diagrammatic procedure of physical mixing (co-grinding) and expected
loading type.
The concentration of piperine in silica matrices and their single point BET
data are presented in Table 4.4. The loading efficiency for physically mixed piperinesilica aerogel is up to 101 %. The loading capacity exceeded the experimental data,
may be due to the losses of silica aerogel during co-grinding process because of its
60
lightweight. The difference between theoretical and experimental results may also
be due to the loss of drug substance by degradation of drug during loading process.
The most important finding is the loading capacity of drug in silica aerogel is
higher than silica xerogel. The higher surface area of silica aerogel evidently allows
more piperine to be loaded. In addition, due to its low density, silica aerogel having
a larger volume than silica xerogel allows more efficient coating of the drug by
nanoparticles.
Table 4.4: Drug loading capacity and surface area of piperine-silica aerogel and
piperine-silica xerogel formulations via physical mixing
Formulation / Description
PAPM20 / Physically mixed
Attempted Loading
Actual Loading
SBET
(wt % piperine)
(wt % piperine)
(m2/g)
20
16.5
128
40
36.5
88
50
47.4
74
60
61.0
59
20
14.0
45
40
23.7
23
50
36.6
13
60
44.1
9
piperine-aerogel
PAPM40 / Physically mixed
piperine-aerogel
PAPM50 / Physically mixed
piperine-aerogel
PAPM60 / Physically mixed
piperine-aerogel
PXPM20 / Physically mixed
piperine-xerogel
PXPM40 / Physically mixed
piperine-xerogel
PXPM50 / Physically mixed
piperine-xerogel
PXPM 60 / Physically
mixed piperine-xerogel
61
4.6.1
Morphology and Crystallinity Studies of Physically Mixed PiperineSilica Matrices
From FESEM micrograph of physically mixed piperine-aerogel with low
loading (20 wt %), a homogeneous mixture was obtained (Figure 4.14). No large
crystal was observed, indicating that low drug loading resulted in successful
micronization of piperine particles. It is interesting to find out that the morphology
of drug particles changed dramatically by the addition of silica xerogel during cogrinding process. Figure 4.15 shows that piperine appears as porous particles.
Although the micronization of piperine was incomplete, the surface area of silica
xerogel loaded with 20 wt % piperine is higher (45 m2/g) than silica xerogel loaded
with 60 wt % piperine (9 m2/g).
Figure 4.14: FESEM micrograph of 20 wt % physically mixed piperine-aerogel
(PAPM 20).
62
Mixture of piperine
and silica xerogel
Piperine coated with
silica xerogel
Figure 4.15: FESEM micrograph of 20 wt % physically mixed piperine-xerogel
(PXPM 20).
Figures 4.16 and 4.17 show relatively smaller particles compared to
crystalline drug obtained after higher drug loading in both matrices.
The
microscopic observation revealed that of silica nanoparticles coated the coarse
particles of piperine, which subsequently avoid the agglomeration between drug
particles. However, Table 4.4 shows that these samples have lower surface area
compared to samples with lower drug loading, maybe due to larger particle size of
piperine and agglomeration of silica matrices as shown in Figures 4.16 and 4.17,
which may subsequently lead to the decrease in surface area.
63
Agglomerated silica aerogel
Silica aerogel coated
piperine particles
Figure 4.16: FESEM micrograph of 50 wt % physically mixed piperine-aerogel
(PAPM 50).
Piperine particles
Agglomerated
silica xerogel
Figure 4.17: FESEM micrograph of 50 wt % physically mixed piperine-xerogel
(PXPM 50).
64
The X-ray diffraction patterns of physically mixed piperine-aerogel and
piperine-xerogel are shown in Figures 4.18 to 4.19. Silica matrices loaded with
piperine show several peaks corresponding to piperine but differ in intensity,
implying that the major component in the formulations is partly crystalline.
Relative Intensity
(a)
(b)
(c)
(d)
(e)
10
5
20
30
40
50
2-Theta-Scale (Degree)
Figure 4.18: X-ray diffractograms of physical mixed piperine-aerogel with different
loading capacity: (a) crystalline piperine, (b) 60 wt %, (c) 50 wt %, (d) 40 wt %, and
(e) 20 wt %
..
65
Relative Intensity
(a)
(b)
(c)
(d)
(e)
5
10
20
30
40
50
2-Theta-Scale (Degree)
Figure 4.19: X-ray diffractograms of physical mixed piperine-xerogel with different
loading capacity: (a) crystalline piperine, (b) 60 wt %, (c) 50 wt %, (d) 40 wt %, and
(e) 20 wt %.
66
In general, the peak intensity of piperine-loaded silica aerogels are extremely
low compared to the pure piperine, which suggests that complete amorphization of
then drug crystals, is achieved. Furthermore, it shows the strong interaction between
the drug and silica aerogel surface, compared to the drug-loaded silica xerogels,
which show more intense peaks. The degree of crystallinity of physically mixed
piperine-silica matrices formulations as compared to pure crystalline piperine is
summarized in Table 4.5.
Table 4.5: Degree of crystallinity of physically mixed piperine-silica matrices.
Sample
14.7˚ 2θ
Degree of
25.8˚ 2θ
Degree of
(Intensity)
Crystallinity
(Intensity)
Crystallinity
(%)
Piperine
PAPM 20 (20 wt %
(%)
1775
100
1900
100
72
4.1
114
6.0
219
12.3
175
9.2
321
18.1
401
21.1
400
22.5
400
21.1
126
7.1
130
6.8
207
11.7
274
14.4
339
19.1
549
28.9
1625
91.5
385
20.3
Piperine-Aerogel)
PAPM 40 (40 wt %
Piperine-Aerogel)
PAPM 50 (50 wt %
Piperine-Aerogel)
PAPM 60 (60 wt %
Piperine-Aerogel)
PXPM 20 (20 wt %
Piperine-Xerogel)
PXPM 40 (40 wt %
Piperine-Xerogel)
PXPM 50 (50 wt %
Piperine- Xerogel)
PXPM 60 (60 wt %
Piperine-Xerogel)
67
4.7
Piperine Impregnated Silica Matrices
There are several parameters that must be taken into account in the
preparation of piperine impregnated silica matrices, such as type and volume of
solvent. Ethanol was chosen as the solvent as it fulfils the pharmaceutical
requirements which are safe (non toxic), may dissolve model drug in large quantity
and does not react with the drug itself. It is theoretically expected that, through
impregnation, the drug solution would enter the porous matrix of silica, as well as
adsorb onto the outer space of silica particles (Figure 4.20). Drug degradation may
occur due to repetitive soaking and drying procedure.
+
Piperine Solution
Dry mixture of
silica matrix and
piperine
+
Minimum
solvent
Drug solution
enter the pore
Silica
nanoparticles
Drug solution
coat the outer
surface of
silica matrices
Silica matrix
avoid drug
recrystallization
Figure 4.20: Diagram of drug loading procedure and expected drug loading via
impregnation.
68
4.7.1
Effect of Solvent Volume on the Drug Loading Capacity
The presence of solvent during impregnation may decompose the silica
matrix network especially silica xerogel due to hydrophilicity. Thus, the effect of
solvent volume on the loading capacity was investigated in order to determine the
optimum amount of solvent needed for the best loading without collapsing the silica
polymeric network.
The following bar chart (Figure 4.21) shows the effect of
solvent volume on the loading of 20 wt % of piperine into silica aerogel and silica
xerogel.
Loading Efficiency (%)
140
Piperine-Aerogel
Piperine Xerogel
120
100
80
60
40
20
0
10
20
30
40
Volume (mL)
Figure 4.21: Effect of solvent volume to the loading of 20 wt % piperine into silica
matrices.
From the chart, it can be seen that the amount of solvent (ethanol) did not
really affect the loading efficiency in silica aerogel. This finding may be due to the
slightly hydrophobic nature of silica aerogel that avoid significant collapse of its
matrix. In contrary, the drug loading in silica xerogel exceeded the expected loading,
indicated by the degradation of silica xerogel. The use of larger amount of solvent
resulted in the presence of observable piperine crystals in piperine-xerogel
formulations, which can not be claimed as loaded drug. Taking all considerations,
69
the optimum volume of solvent needed for impregnation is 10 mL/g of total weight
of piperine and silica matrices. The use of lesser amount of ethanol is insufficient to
wet the mixture.
In some piperine loaded silica matrices samples, fine crystals could be
observed in the dried powders. The powder was quickly washed on a filter with 5 or
10 mL of ethanol in order to remove the excess piperine molecules coating the outer
surface. The loading capacities of washed samples were determined using UV-Vis
spectrophotometer and listed in Table 4.5.
Table 4.6: Drug loading capacity and surface area of piperine-silica aerogel and
piperine-silica xerogel formulations via impregnation
Formulation / Description
PAIM20 / Piperine
Attempted Loading
Actual Loading
SBET
(wt % piperine)
(wt % piperine)
(m2/g)
20
21.5
124
40
31.6
83
50
48.0
74
60
55.4
53
20
20.8
47
40
32.2
40
50
36.5
30
60
43.2
29
impregnated silica aerogel
PAIM40 / Piperine
impregnated silica aerogel
PAIM50 / Piperine
impregnated silica aerogel
PAIM60 / Piperine
impregnated silica aerogel
PXIM20 / Piperine
impregnated silica xerogel
PXIM40 / Piperine
impregnated silica xerogel
PXIM50 / Piperine
impregnated silica xerogel
PXIM 60 / Piperine
impregnated silica xerogel
70
The concentration of piperine in silica aerogel is higher compared to piperinesilica xerogel formulations. High surface area of silica aerogel in addition to its
porosity and hydrophobicity, contribute to this findings. It is assumed that there was
no reduction in silica aerogel volume due to collapse of framework in ethanol. As
discussed previously, the impregnation process has affected the hydrophobicityhydrophilicity of silica aerogel, where the piperine impregnated silica aerogel
formulations show hydrophilic properties. The hydrophilicity of these samples is
expected to favour the dissolution rate of piperine.
The surface area values determined dropped significantly after loading of
drug compared to raw silica matrices (Figures 4.22 and 4.23). There are several
possible explanations for this finding. The decrease of surface area value may
suggest that piperine is successfully loaded within the pores.
Indeed, piperine
molecules diffused with the solvent, drawn into the pores by capillary action and
remain trapped after solvent removal.
100
140
90
120
100
70
60
80
50
60
40
30
SBET m2/g
Loading (w/w %)
80
40
20
20
10
0
0
PAIM20
Loading Attempt
PAIM40
PAIM50
Actual Loading
PAIM60
SBET (m2/g)
Figure 4.22: Drug loading and SBET of piperine impregnated silica aerogel (PAIM).
71
100
140
90
120
100
70
60
80
50
60
40
30
40
SBET (m2/g)
Loading (w/w %)
80
20
20
10
0
0
PXIM20
PXIM40
Loading Attempt
PXIM50
Actual Loading
PXIM60
SBET (m2/g)
Figure 4.23: Drug loading and SBET of piperine impregnated silica xerogel (PXIM).
Besides, the lower surface area within the samples indicates the blocked
pores caused by staking of drug over the surface. On the other hand, this result
might be caused by the collapse of the pore structure of the carriers due to absorption
of ethanol during impregnation.
It is noteworthy that, the surface areas of
synthesized piperine loaded silica matrices are significantly higher than that of pure
crystalline piperine.
4.7.2
Morphology Study of Piperine Impregnated Silica Matrices
The microscopic observation revealed the presence of nanoparticles of the
carriers coating the coarse particles of piperine. In case of piperine-loaded silica
aerogel, the drug crystals are fully covered with silica aerogel and do not have such a
smooth surface as the pure piperine (Figure 4.24 and 4.25).
72
Figure 4.24: FESEM micrograph of 50 wt% piperine impregnated silica aerogel
prepared with excessive ethanol
Figure 4.25: FESEM micrograph of 50 wt % piperine impregnated silica aerogel
prepared with minimum ethanol
73
The coating of drug crystals with silica aerogel may form a protective layer
that prevents the recrystallization of drug particles. It is observed that the piperine
has lost its crystalline characteristics, as there is no observable crystal particles
corresponded to piperine. Denser morphology could be observed in Figure 4.24 if
excessive ethanol was used for the impregnation process compared to formulations
prepared with minimum ethanol as shown in Figure 4.25.
Besides, by using
minimum amount of ethanol, the degradation of silica matrix could be avoided as
shorter drying period was needed while ethanol just acted as the binding agent.
On the other hand, the crystal particle of piperine with smooth surface was
observed in the micrograph of drug-loaded silica xerogel (Figures 4.26 and 4.27),
may be due to recrystallization of drug crystals during the loading procedure. The
collapse of silica xerogel due to its hydrophilicity may reduce its volume and was
insufficient to avoid the nucleation of drug particles.
Piperine particles
Figure 4.26: FESEM micrograph of 50 wt % piperine impregnated silica xerogel
prepared with excessive ethanol magnification 1,500 X
74
Crystal surface
Silica xerogel
Figure 4.27: FESEM micrograph of 50 wt % piperine impregnated silica xerogel
prepared with excessive ethanol magnification 20,000 X
Figure 4.28: FESEM micrograph of 50 wt % piperine impregnated silica xerogel
prepared with minimum ethanol magnification 20,000 X.
75
In order to prevent the degradation of silica xerogel, minimum amount of
solvent (10 mL) was used during drug loading.
Figure 4.28 shows that the
formulation powder appears as homogeneously distributed spherical nanoparticles,
suggesting higher surface area compared to samples prepared by soaking silica
xerogel in excessive ethanol.
BET surface area analysis proved that 50 wt %
piperine impregnated xerogel prepared with minimum ethanol has higher surface
area (30 m2/g) than silica xerogel soaked in ethanolic piperine solution (11 m2/g).
Thus, the degradation of silica matrices could be avoided by limiting the amount of
solvent.
4.7.3
Crystallinity Study of Piperine Impregnated Silica Matrices
Figures 4.29 and 4.30 show the diffractograms of piperine impregnated silica
aerogel and silica xerogel, respectively. Crystallinity study on impregnated samples
revealed that the intensity of characteristic peaks of piperine decreased with
increasing amount of silica matrices, as summarized in Table 4.7. This proved that
the inclusion of amorphous silica matrix may reduce the crystallinity of piperine,
consequently lead to faster dissolution.
The diffractograms of piperine impregnated silica xerogels shows more
intense peaks compared to piperine impregnated silica aerogels, suggesting higher
degree of crystallinity in those samples. This finding well agrees with the BET
analysis result which indicates that piperine xerogel formulations possess lower
surface area than piperine-aerogel. Basically, higher crystallinity leads to lower
surface area, as described by Noyes-Whitney equation [122].
76
Relative Intensity
(a)
(b)
(c)
(d)
5
10
20
30
40
50
2-Theta-Scale (Degree)
Figure 4.29: X-ray diffractograms of piperine impregnated aerogel with different
loading capacity: (a) crystalline piperine, (b) 60 wt %, (c) 40 wt % and (d) 20 wt %.
Relative Intensity
(a)
(b)
(c)
(d)
5
10
20
30
40
50
2-Theta-Scale (Degree)
Figure 4.30: X-ray diffractograms of piperine impregnated silica xerogel with
different loading capacity: (a) crystalline piperine, (b) 60 wt %, (c) 40 wt % and (d)
20 wt %.
12
Table 4.7: Degree of crystallinity of piperine impregnated silica matrices.
Sample
Piperine
PAIM 20 (20 wt %
14.7˚ 2θ
Crystallinity
25.8˚ 2θ
Crystallinity
(Intensity)
(%)
(Intensity)
(%)
1775
100
1900
100
70
3.9
80
4.2
887
50.0
233
12.3
553
31.2
371
19.5
165
9.3
122
6.4
353
19.9
301
15.8
717
40.4
557
29.3
Piperine-Aerogel)
PAIM 40 (40 wt %
Piperine-Aerogel)
PAIM 60 (60 wt %
Piperine-Aerogel)
PXIM 20 (20 wt %
Piperine-Xerogel)
PXIM 40 (40 wt %
Piperine-Xerogel)
PXIM 60 (60 wt %
Piperine-Xerogel)
4.8
Piperine Loaded Silica Matrices via Direct Synthesis
Piperine loaded silica matrices were also prepared via direct synthesis
method. In this method, different synthesis conditions were studied. Drug was
loaded just before the occurrence of gelation. The loading of powdered-piperine into
the sol resulted in non homogeneous drug distribution in wet gel. To ensure the
homogeneity of drug distribution, piperine was first dissolved completely in ethanol
before loading into the sol. Minimum amount of solvent (ethanol) was used to
minimize the possible reaction between sodium silicate and the solvent. The diagram
below shows the distribution of piperine in the dry xerogel (Figure 4.31). This
theory was confirmed by microscopic observation using FESEM.
13
+
Ground
piperine
Piperine loaded silica
xerogel
Sol pH~7
+
Piperine
dissolved in
ethanol
Piperine loaded silica
xerogel
Figure 4.31: Diagrammatic procedure of piperine loaded silica xerogel via direct
synthesis.
The micrographs show that directly synthesized piperine loaded silica xerogel
using ethanolic solution of piperine appears as homogenous mixture (Figure 4.32),
while the presence of piperine crystals could be observed if ground piperine was used
during the loading (Figure 4.33). By dissolving in ethanol, piperine is molecularly
dispersed within the matrix, thus creating monolithic drug delivery device. In the
case of poorly water-soluble drug like piperine, homogeneously distributed system
can be used to increase drug release rate in the human body compared to
conventional dosage forms.
14
Figure 4.32: FESEM micrograph of piperine loaded silica xerogel via direct
synthesis using ethanolic solution of piperine.
Piperine particles
Figure 4.33: FESEM micrograph of piperine loaded silica xerogel via direct
synthesis using ground piperine.
15
4.8.1
Effect of Aging Conditions
Aging of wet gel is a crucial procedure as it helps to strengthen the gel
network in order to avoid collapse of polymer framework. Normally, silica aquagel
was aged in water before drying. However, in this study, use of water as the aging
medium resulted in extended period of drying for drug loaded silica xerogel. The
exposure of heat to the samples in long period may decompose the drug. Meanwhile,
in the synthesis of drug loaded silica aerogel, higher pressure and temperature was
needed to achieve the supercritical condition in the presence of water. So, water
must be replaced with other media having milder supercritical conditions, whereby
alcohol is the best choice. In addition, alcohol has a lower surface tension than water
[167].
The surface tension is related to shrinkage pressure which leads to the
collapse of polymeric network. This is explained by the following equation.
P=
2γ
r
(Equation 4.2)
where P is shrinkage pressure, γ is surface tension and r is pore radius. Pore radius
is directly related to particle size.
However, solvents can trigger and help the
recrystallization of drug crystals. Direct aging in ethanol after gelation resulted in
low drug loading as it washes out the drug from the aquagel. Besides, drug tends to
crystallize at the gel surface if aged for a longer period in ethanol. Thus, the aging
conditions must be optimized in order to ensure the best result.
In order to overcome these problems, the piperine loaded wet gels were aged
for two days in water, followed by aging in ethanol to replace the water. The effect
of aging period to the drug loading and surface area is summarized in Table 4.8 and
Figure 4.34.
16
Table 4.8: Effect of aging period on the loading efficiency and surface area of
directly synthesized 20 wt % piperine-silica matrices formulations.
Formulations
Acronyms
Aging Period
Loading
SBET
(days)
Efficiency
(m2/g)
Water
Ethanol
(%)
Piperine- silica
PA-2W
2
0
79
167
aerogel
PA-2W2E
2
2
68
329
PA-2W4E
2
4
56
350
PA-2W6E
2
6
43
364
Piperine- silica
PX-2W
2
0
59
60
xerogel
PX-2W2E
2
2
52
155
PX-2W4E
2
4
28
103
PX-2W6E
2
6
24
124
400
100
Loading Efficiency
SBET
80
350
300
70
250
60
50
200
40
150
30
SBET (m2/g)
Loading Efficiency (%)
90
100
20
50
PX-2W6E
PX-2W4E
PX-2W2E
PX-2W
PA-2W6E
PA-2W4E
PA-2W2E
0
PA-2W
10
0
Figure 4.34: Effect of aging period on loading efficiency and surface area of
piperine-silica matrices formulations.
17
During the aging process, the piperine-loaded gel was washed with fresh
ethanol for every 2 hours (three times) in order to completely remove the water
before soaking in ethanol for two days. The ethanol-soaked gels (alcogel) were then
dried in ambient pressure and supercritical-CO2 to produce piperine loaded silica
xerogel and piperine loaded silica aerogel, respectively.
From the data, the loading efficiencies were quite low and decreased with
longer aging period. This indicated the loss of drug due to washing, aging and
drying procedure of the gels. Some of the drug might have leached out from the
silica matrix or washed out by supercritical CO2. From the bar chart, the optimum
aging period to get the best loading and the highest surface area is two days in water
continued by two days in ethanol.
4.8.2
Efficiency of Loading
An attempt to load different amount of drug via direct synthesis was carried
out to determine the drug loading limit. Formulations with 0.1 g (11 wt %), 0.2 g (20
wt %), 0.3 g (27 wt %) and 0.4 g (33 wt %) drug were successfully prepared and the
efficiency of loading is presented in Figure 4.35. The results indicate that the sol did
not gel when drug loading is >0.5 g (38.5 wt %).
Homogeneous yellowish powders were obtained from all directly synthesized
piperine-aerogel and piperine-xerogel samples with low drug loading. However, a
thin layer of drug crystals appear at the outer surface of silica xerogel after loading of
0.4 g piperine. This layer was removed and flash-washed with a little amount of
ethanol, dried and ground before the drug concentration in the formulation was
determined. Generally, silica aerogel gave a better loading efficiency than xerogel
by 15-20 %. This indicates that supercritical-CO2 procedure efficiently avoided the
18
gel network shrinkage in piperine-aerogel samples, consequently preserved the gel
volume and surface area, allowing more piperine to be loaded.
100
Silica Aerogel
Silica Xerogel
90
Loading Efficiency (%)
80
70
60
50
40
30
20
10
0
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Loading attempt (g)
Figure 4.35: Loading efficiency of piperine loaded silica matrices via direct
synthesis.
4.8.3
Crystallinity and Morphology Studies of Piperine Loaded Silica Matrices
via Direct Synthesis
Figure 4.36 shows the diffraction patterns of piperine loaded silica matrices
via direct synthesis. Observations on directly synthesized samples showed no
diffraction peaks of piperine, indicating highly amorphous nature of the formulations
which explains the high surface area of these samples as shown in Table 4.8.
19
Relative Intensity
(d)
(c)
(b)
(a)
5
10
20
30
40
50
2-Theta-Scale (Degree)
Figure 4.36: X-ray diffractograms of piperine loaded silica via direct synthesis (a)
aerogel with 10 wt % loading, (b) aerogel with 20 wt % loading, (c) xerogel 10 wt %
loading and (d) xerogel with 20 wt % loading.
Samples with different piperine loading gave similar diffractograms that also
illustrate amorphous materials (Figure 4.36). On processing via direct synthesis, the
entrapment of drug inside nanosize pores thermodynamically prevents the formation
of critical nuclei needed for crystallization. Low drug loading in directly synthesized
formulations also contributes to this finding as there is limited crystalline species in
those samples.
20
Morphology studies on piperine-loaded silica matrices revealed that the
synthesized samples appear as spherical particles, where the size is larger in piperinexerogel formulations compared to piperine-aerogel ones (Figures 4.37 and 4.38).
There are no crystals observed in respective micrographs, which correlate with XRD
analysis whereby all directly synthesized samples are amorphous. Therefore, it can
be concluded that amorphization of piperine could be achieved via direct drug
loading before the occurrence of gelation.
Figure 4.37: FESEM micrograph of 20 wt % piperine loaded silica aerogel via direct
synthesis (PADS 20).
21
Figure 4.38: FESEM micrograph of 20 wt % piperine loaded silica xerogel via direct
synthesis (PXDS 20).
4.9 Comparison of Degree of Crystallinity of Piperine Loaded Silica Matrices
via Different Methods
Comparison on degree of crystallinity of piperine-aerogel with same loading
prepared via different methods showed that directly synthesized piperine-aerogel had
the lowest crystallinity, followed by impregnated and physically mixed formulations
(Figure 4.39). This sequence does not apply to piperine-xerogel formulations which
showed that piperine impregnated silica xerogel had the highest crystallinity
compared to directly synthesized and physically mixed piperine-xerogel (Figure
4.40). It is obvious that the conventional grinding applied in the preparation of
physical mixture of piperine-silica matrices could only micronize the drug but not
22
eliminate the crystallinity of piperine.
The degree of crystallinity of directly
Relative Intensity
synthesized piperine-silica matrices formulations is summarized in Table 4.9.
(a) Piperine
(b) PAPM
(c) PAIM
(d) PADS
5
10
20
30
40
50
2-Theta-Scale (Degree)
Figure 4.39: X-ray diffractograms of crystalline piperine and 20 wt % piperine
loaded silica aerogel synthesized via different methods: (a) crystalline piperine, (b)
physically mixed piperine-aerogel (PAPM), (c) piperine impregnated aerogel
(PAIM), and (d) directly synthesized piperine loaded aerogel (PADS).
Relative Intensity
23
(a) Piperine
(b) PXPM
(c) PXIM
(d) PXDS
5
10
20
30
40
50
2-Theta-Scale (Degree)
Figure 4.40: X-ray diffractograms of piperine and 20 wt % piperine loaded silica
xerogel synthesized via different methods: (a) crystalline piperine, (b) physically
mixed piperine-xerogel (PXPM), (c) piperine impregnated xerogel (PAIM) and (d)
directly synthesized piperine loaded xerogel (PXDS).
24
Table 4.9: Comparison on degree of crystallinity of piperine-silica matrices
formulations prepared via different methods
Samples
14.7˚ 2θ
Degree of
(Intensity)
Crystallinity
25.8˚ 2θ
(Intensity) Crystallinity
(%)
Crystalline piperine
PAPM/Physically mixed
Degree of
(%)
1775
100
1900
100
72
4.1
114
6.0
70
3.9
80
4.2
piperine-aerogel
PAIM/Piperine
impregnated aerogel
PADS/piperine-aerogel
via direct synthesis
PXPM/Physically mixed
Amorphous
126
7.1
130
6.8
165
9.3
122
6.4
piperine-xerogel
PXIM/Piperine
impregnated xerogel
PXDS/piperine-xerogel
via direct synthesis
Amorphous
Silica Aerogel
Amorphous
Silica Xerogel
Amorphous
On the other hand, impregnation process offers better interaction between
drug and its carriers, thus increases uniformity of drug distribution in the product. In
addition, recrystallization might occur due to the presence of solvent and insufficient
particle coating by silica matrices.
Due to its high density compared to silica
aerogel, silica xerogel possesses limited volume that is not sufficient to prevent drug
recrystallization. This explains the high intensity of peaks in piperine impregnated
silica xerogel samples.
25
In the case of directly synthesized piperine-silica matrices, drug loading
procedure which took place before gelation and drying processes ensures
homogeneous distribution of drug in the samples. The drug particles were
encapsulated in silica network upon gelation, thus securely avoided the nucleation of
piperine crystals. This finding is consistent with FESEM micrographs in Figures
4.37 and 4.38 which show that directly synthesized piperine-silica aerogel and
piperine-silica xerogel appear as spherical particles without any definite structure.
The generation of amorphous phase will increase the surface area of piperine
particles, proven by the BET data which was discussed previously. In this case, the
generation of amorphous phase is due to the reduction in particle size and loss of
crystallinity as shown in Figures 4.37 and 4.38. Amorphous form due to absence of
an ordered crystal lattice requires minimal energy and thus provides maximal
solubility advantage as compared to the crystalline and hydrated form of drug. Thus,
the formulations are expected to improve the solubility and to enhance the
dissolution rate of crystalline piperine.
4.10
Drug Dissolution Study
The concentration in the vessel was calculated using calibration curves. The
release curves are presented as time versus concentration of piperine in the vessel.
The calibration curves of piperine in 0.05 mM potassium phosphate buffer (intestinal
fluid) and 0.1 M hydrochloric acid (gastric juice) are illustrated in Appendix B.
Figure 4.41 shows the dissolution profiles of crystalline piperine in hydrochloric acid
and potassium phosphate buffer. The dissolution rates of piperine in both dissolution
media were very poor and during 180 min dissolution testing, a maximum of 5 %
drug was released. The reason for poor dissolution of poor drug could be poor
wettability, high crystallinity, and/or agglomeration of particles.
26
Cumulative Release (%)
6
Dissolution Medium:
Hydrochloric Acid
Phosphate Buffer Saline
5
4
3
2
1
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minute)
Figure 4.41: Dissolution profiles of crystalline piperine in 0.1M hydrochloric acid
and phosphate buffer saline.
The drug release profiles of physically mixed piperine-silica matrices
formulations with different drug loading in both simulated body fluid are presented
in Figures 4.42 to 4.45. Addition of silica matrices may profoundly affect the
dissolution rate of piperine. Both piperine-aerogel and piperine-xerogel formulations
showed the same trend, which was drug dissolution rate increased with increased
amount of carriers. A prerequisite of fast dissolution from the formulations was
apparently due to rapid dissolution of the hydrophilic carrier particles dissolved
rapidly, delivering a fine particulate suspension of drug particles. As an effect of
swelling and collapse of silica matrices, the wetted surface of carrier increased and
promoted wettability and dispersibility of piperine. Thus, the desired release was
controllable by adjusting the amount of carrier or drug itself. However, the release
rate of piperine-aerogel formulations is about two times faster than piperine xerogel,
as it has a larger surface area which provides a wider contactable area with the
dissolution medium. Different dissolution media did not affect the rate of drug
release.
27
100
20 wt %
Cumulative Release (%)
90
40 wt %
50 wt %
60 wt %
Piperine
60
100
140
180
80
70
60
50
40
30
20
10
0
0
20
40
80
120
160
200
Time (Minutes)
Figure 4.42: Effect of different loading methods on the dissolution rate of physically
mixed piperine-aerogel formulations in 0.1M HCl.
Cumulative Release (%)
100
20 wt %
90
40 wt %
50 wt %
60 wt %
100
140
Piperine
80
70
60
50
40
30
20
10
0
0
20
40
60
80
120
160
180
200
Time (Minutes)
Figure 4.43: Effect of different loading methods on the dissolution rate of physically
mixed piperine-aerogel formulations in 0.05 mM PBS.
28
100
20 wt %
Cumulative Release (%)
90
40 wt %
50 wt %
Piperine
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
Figure 4.44: Effect of different loading methods on the dissolution rate of physically
mixed piperine-xerogel formulations in 0.1M HCl.
100
20 wt %
Cumulative Release (%)
90
40 wt %
50 wt %
Piperine
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
Figure 4.45: Effect of different loading methods on the dissolution rate of physically
mixed piperine-xerogel formulations in 0.05 mM PBS.
29
Figures 4.46 to 4.49 show the dissolution rate of impregnated piperine-silica
matrices formulations. Piperine impregnated silica matrices show very good
dissolutions which are up to 80% for piperine-aerogel and up to 40% for piperinexerogel samples. This may be due to the loading procedure that avoided degradation
of silica by minimizing the amount of solvent.
Figure 4.46 and 4.47 show that piperine impregnated aerogel, which
possesses higher surface area shows faster dissolution than piperine impregnated
xerogel (Figures 4.48 and 4.49). Release rates of impregnated formulations also
show similar profile in both simulated gastric and intestinal fluids, indicating that
these formulations can be released and absorbed effectively in stomach and intestine.
100
Piperine
20 wt %
40 wt %
50 wt %
90
Cumulative Release (%)
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
Figure 4.46: Effect of different loading methods on the dissolution rate of piperine
impregnated aerogel formulations in 0.1M HCl.
30
100
20 wt %
40 wt %
50 wt %
Piperine
Cumulative Mass (%)
90
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
Figure 4.47: Effect of different loading methods on the dissolution rate of piperine
impregnated aerogel formulations in 0.05 mM PBS.
100
Piperine
Cumulative Release (%)
90
20 wt %
40 wt %
50 wt %
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
Figure 4.48: Effect of different loading methods on the dissolution rate of piperine
impregnated xerogel formulations in 0.1M HCl.
31
100
20 wt %
Cumulative Release (%)
90
40 wt %
50 wt %
Piperine
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
Figure 4.49: Effect of different loading methods on the dissolution rate of piperine
impregnated xerogel formulations in 0.05 mM PBS.
Figures 4.50 and 4.51 show the dissolution profiles of directly synthesized
piperine-silica matrices with different drug loading in simulated gastric and intestinal
fluids. Formulations with higher drug loading show slower release. Piperine-aerogel
shows extremely faster release rate as compared to piperine-xerogel.
Through
supercritical-CO2 drying, the collapse and shrinkage of gel network were minimized,
thus piperine-aerogel with remarkable high surface area were produced. As drug
dissolution rate is strongly correlated with surface area, directly-synthesized
piperine-aerogel presents the best conditions for rapid drug release.
Faster
dissolution of piperine-aerogel formulations may also be due their low degree of
crystallinity. Different dissolution media with different pH did not give any impact
on the dissolution as it shows similar profiles in both simulated body fluid.
32
PX10 wt %
PX 20 wt %
PA 10 wt %
PA 20 wt %
100
Cumulative Release (%)
90
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
Figure 4.50: Effect of different loading capacity on the dissolution rate of directly
synthesized piperine loaded silica matrices formulations in 0.1M HCl.
PA 10 wt %
PA 20 wt %
PX 20 wt %
PX 10 wt %
100
Cumulative Release (%)
90
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
Figure 4.51: Effect of different loading capacity on the dissolution rate of directly
synthesized piperine loaded silica matrices formulations in 0.05 mM PBS.
33
Faster drug release in lower drug loading samples suggests the relationship
between dissolution rate and surface area of those formulations (Figures 4.46 to
4.51). This is due to higher drug loading resulted in lower surface area. Indeed,
higher percentage of silica matrices contributed to the decrease of crystallinity degree
of piperine. This was in good agreement with XRD results which suggest that more
drug was converted to amorphous form with lower drug loading.
Besides that, all formulations show improved release in both simulated body
fluids (Figures 4.46 to 4.51). This also suggest that bioavailability of piperine could
be benefited in both gastric and intestinal fluids, which is valuable for nutrients
absorption. As piperine is well-known for its bioavailability-enhancer property,
therefore the formulations may be co-administered with other nutritional materials
for different target sites.
4.10.1`Physically Mixed Piperine-Silica Matrices
The dissolution profiles of physically mixed samples in Figures 4.52 and 4.53
show similar releases except for the first two hours, where piperine-aerogel showed
faster release than piperine xerogel formulations. This may be due to the larger
volume of silica aerogel which provided more contactable surface for wetting with
dissolution medium. The dissolution of physically mixed formulations depends on
the efficiency of drug particles micronization through grinding process. Evidently,
using silica matrix as the carrier, the drug particles were successfully micronized
while agglomeration of ground crystals was reduced, resulted in improved drug
release rate.
34
Cumulative Release (%)
100
PAPM
90
PXPM
Crystalline
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
Figure 4.52: Dissolution profile of 20 wt % physically mixed piperine-silica
matrices formulations in 0.1 M HCl
100
Cumulative Release (%)
90
Crystalline
PAPM
PXPM
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
Figure 4.53: Dissolution profile of 20 wt % physically mixed piperine-silica
matrices formulations in 0.05 mM PBS
35
4.10.2 Piperine Impregnated Silica Matrices
In the case of piperine impregnated silica matrices, the effect of different
carrier and the volume of the solvent used in the impregnation process was studied.
Samples prepared with minimum amount of solvent showed faster release than the
ones prepared with excessive solvent (Figures 4.54 and 4.55). This was due to the
collapse of silica network in ethanol because of its hydrophilicity that destructed the
surface area.
100
PXIMME
PAIMME
PAIM
PXIM
Piperine
Cumulative Release (%)
90
80
(e)
70
60
50
40
(d)
30
(c)
20
(b)
(a)
10
0
0
50
100
150
200
Time (Minutes)
Figure 4.54: Dissolution profile of 20 wt % piperine impregnated silica matrices (a)
crystalline piperine, (b) piperine impregnated xerogel (PXIM) (c) piperine
impregnated aerogel (PAIM) (d) piperine-xerogel with minimum ethanol (PXIMME)
and (e) piperine-aerogel with minimum ethanol (PAIMME), in 0.1 M HCl.
The collapse of silica matrices was minimized by using minimum ethanol
which consequently restored their porous nature to favour rapid dissolution. As
discussed before, the use of minimum ethanol also prevented the recrystallization of
36
piperine, which may also affect the dissolution profile. Amorphous drug substances
are at a higher energy state, therefore, in general have higher solubility dissolution
rate compared to crystalline materials. Thus, prevention of recrystallization by
limiting the amount of ethanol enhanced the dissolution of piperine.
100
Piperine
PAIMME
PXIMME
PAIM
PXIM
Cumulative Release (%)
90
80
(e)
70
60
50
(d)
(e)
40
30
(b)
20
10
(a)
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
Figure 4.55: Dissolution profile of 20 wt % piperine impregnated silica matrices (a)
crystalline piperine, (b) piperine impregnated xerogel (PXIM) (c) piperine
impregnated aerogel (PAIM) (d) piperine-xerogel with minimum ethanol (PXIMME)
and (e) piperine-aerogel with minimum ethanol (PAIMME), in 0.05 mM PBS.
4.10.3 Piperine Loaded Silica Matrices via Direct Synthesis
Dissolution study on 20 wt % piperine formulations prepared via direct
synthesis revealed a strong relation between the surface area and the dissolution rate
of sample. Figure 4.56 shows that piperine-xerogel formulation showed an extremely
37
slow release (~20 %) compared to piperine-aerogel sample which showed 100 %
release in 0.1M HCl during 3 hours dissolution.
Similar release profiles were obtained for these formulations by using PBS as
dissolution medium. Thus, it may be concluded that the higher is the surface area,
the faster is the dissolution.
In addition, slow release of directly synthesized
piperine-xerogel may be resulted from successful encapsulation of piperine particles
within silica xerogel matrix. Thus, the use of silica xerogel may be desirable in
sustained release drug delivery system. This finding corroborates with previous
research by Ahola et. al. who used silica xerogel for prolonged release of heparin
[168].
(c) PADS
100
Cumulative Release (%)
90
80
70
60
50
40
(b) PXDS
30
20
(a)Piperine
10
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
Figure 4.56: Dissolution profiles of directly synthesized 20 wt % piperine loaded
silica matrices in 0.1 M HCl (a) crystalline piperine, (b) directly synthesized
piperine-xerogel (PXDS) and (c) directly synthesized piperine-aerogel (PADS).
38
(c) PADS
100
Cumulative Release (%)
90
80
70
60
50
40
30
20
(b)PXDS
10
(a)Piperine
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
Figure 4.57: Dissolution profiles of directly synthesized 20 wt % piperine loaded
silica matrices in 0.05 mM PBS (a) crystalline piperine, (b) directly synthesized
piperine-xerogel (PXDS) and directly synthesized piperine-aerogel (PADS).
4.10.4 Comparison of Drug Dissolution Profile of Formulations Prepared via
Different Methods
Figure 4.58 shows the comparative release profile of pure piperine, physical
mixture of piperine-aerogel (20 wt %) (PAPM), piperine impregnated aerogel (20 wt
%) (PAIM) and directly synthesized piperine-aerogel (20 wt %) (PADS). It was
observed that the dissolution rates of drug on all piperine-aerogel formulations were
increased compared to pure drug. The best dissolution rate was achieved from
directly synthesized piperine-aerogel, followed by impregnated and physically mixed
formulations. Significant improvement in dissolution rate by directly synthesized
piperine-aerogel may be explained by its high surface area in the formulation
prepared using supercritical-CO2 (SCCO2). Besides that, SCCO2 may also catalyze
the micronization of drug particles and promote fast dissolution.
39
100
PADS
90
PAIM
Cumulative Release (%)
80
70
60
50
40
PAPM
30
20
10
Piperine
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
PADS – Directly synthesized piperine-silica aerogel
PAIM – Piperine impregnated silica aerogel
PAPM – Physically mixed piperine-silica aerogel
Piperine
Figure 4.58: Effect of different loading methods on the dissolution rate of 20 wt %
piperine-aerogel formulations in 0.1M HCl
Although directly synthesized piperine-xerogel possesses higher surface area
compared to piperine impregnated xerogel, it showed slower release, as illustrated in
Figure 4.59. This may be due to successful entrapment of piperine in silica xerogel
matrix. Thus, dissolution was controlled by rate of diffusion of drug through the
pore. Meanwhile, the drug release in physically mixed and impregnated piperinexerogel was triggered by direct wetting with dissolution medium and collapse of
silica xerogel.
40
100
90
Cumulative Release (%)
80
70
60
50
PXIM
40
PXDS
PXPM
30
20
10
Piperine
0
0
20
40
60
80
100
120
140
160
180
200
Time (Minutes)
PXDS- Directly synthesized piperine-xerogel
PXPM - Physically mixed piperine-xerogel
PXIM - Piperine impregnated xerogel
Crystalline Piperine
Figure 4.59: Effect of different loading methods on the dissolution rate of 20 wt %
piperine-xerogel formulations in 0.1 M HCl.
Generally, piperine-aerogel formulations give faster release than piperinexerogels. This proves that piperine release rate is dependent on particle size and
surface area of drug formulation. A decrease in drug particle size increased the
surface area, and hence dissolution. As silica aerogel readily possesses higher surface
area compared to silica xerogel, its application evidently enhanced the dissolution
profile of water-insoluble drug.
CHAPTER V
CONCLUSIONS
5.1
Conclusions
Silica aerogel and silica xerogel were synthesized via high temperature
supercritical extraction and ambient pressure drying. Piperine, as the model drug, is
loaded on the silica matrices via physical mixing, impregnation and direct synthesis.
UV-Visible and FTIR spectroscopy analyses indicate that no degradation occurred
during drug loading process. BET surface area analysis showed significant drop in
surface area after drug loading indicating successful loading of piperine.
FESEM reveals that the size of piperine crystals is reduced in physically
mixed and impregnated systems; subsequently contributes to the increment in the
surface area.
Meanwhile, directly synthesized piperine loaded silica matrices
appeared as spherical nanoparticles. The finding coincides with the XRD results
which indicate a decrease in drug crystallinity in piperine-loaded silica matrices.
XRD analysis also indicates that complete amorphization of drug crystal is achieved
via direct synthesis.
108
Results of the study indicate that aerogel give better drug loading (up to 100
% efficiency) compared to silica xerogel. Impregnation method gives the best
loading, followed by physical mixing and direct synthesis. Relatively low drug
loading (up to 27 wt %) is observed in the formulations prepared via direct synthesis;
the extent of which is influenced by losses of drug during aging, washing and drying
procedures.
The release profiles of piperine loaded silica matrices in simulated gastric
and intestine fluids apparently show that the rate of dissolution is faster from the
piperine-silica matrices formulations compared to that of pure crystalline piperine.
This finding is influenced by the increase in surface area and amorphization of drug
crystals. In piperine-aerogel formulations, the best dissolution rate with 100 %
release within 3 hours is achieved from formulation prepared via direct synthesis,
followed by impregnation and physical mixing.
Rapid release of directly synthesized piperine-aerogel is governed by high
surface area and porosity that enable rapid wetting of drug with dissolution
mediums. Impregnation using minimum amount of ethanol enables drug to enter the
silica matrices through the pores and avoids the denaturation of the silica matrix. As
the drug dissolution rate is strongly dependent on the effective surface area of the
formulations, this technique preserves the porosity, resulting in better dissolution
profiles over crystalline drug.
In piperine-xerogels dissolution study, relatively slower releases are observed
from directly synthesized piperine-xerogel formulations. In case of formulations
prepared via physical mixing and impregnation, the silica gels are rapidly wetted,
such that the drug molecules are surrounded by the dissolution medium, allowing
fast dissolution. On the other hand, the encapsulated drug molecules were firstly
diffused from the pore of silica matrix, before the matrix itself collapses due to its
hydrophilic property. The ease of collapse of the silica matrices structure in water
evidently favours a faster release.
109
The dissolution rate of poorly water-soluble piperine is successfully
increased, consequently improves its bioavailability. Dissolution study also revealed
that all formulations show similar releases either in simulated gastric juice or
intestinal fluid.
Thus, the formulations can be released and absorbed in both
stomach and intestine. As piperine is well recognized as bioavailability-enhancer,
thus the newly designed nano-enabled piperine loaded silica matrices can be coadministered with other nutrients for different target sites.
While overcoming
solubility issue, nano-enabled delivery system using silica matrices synthesized from
rice husk ash promises versatile applications in pharmaceutical industry. The overall
outcome of synthesis and analysis of piperine loaded silica matrices is summarized
in following Figure 5.1.
I) Efficiency of Loading
•
Silica aerogel gives better drug loading compared to silica
xerogel.
II) Technique of Loading
•
Impregnation > Physical Mixing > Direct Synthesis
III) Dissolution Rate
•
Direct Synthesis > Impregnation > Physical Mixing
Figure 5.1: Outcome of research activities
110
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1.2
1
y = 0.0958x
2
R = 0.9974
Absorbance (A)
0.8
0.6
0.4
0.2
0
0
2
4
6
8
10
Concentration (ppm)
Appendix A: Calibration curve of piperine in ethanol
12
126
1.2
y = 0.01x
1
2
R = 0.9993
Absorbance (A)
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
120
Concentration (ppm)
Appendix B: Calibration curve of piperine in 0.05 M potassium phosphate buffer
(PBS) solution, pH 7.
127
0.9
Absorbance (A)
0.8
0.7
0.6
0.5
y = 0.0084x
0.4
2
R = 0.9991
0.3
0.2
0.1
0
0
20
40
60
80
100
120
Concentration (mg/L)
Appendix C: Calibration curve of piperine in 0.1 M hydrochloric acid (HCl), pH 1.