EFFECTS OF GAMMA-STERILIZATION ON

PHYSICOCHEMICAL PROPERTIES AND

STABILITY OF Nigella sativa OIL AND
GENTAMICIN FORMULATION

BY

NOR LIYANA BINTI JAAFAR

INTERNATIONAL ISLAMIC UNIVERSITY

MALAYSIA

2015

EFFECTS OF GAMMA-STERILIZATION ON
PHYSICOCHEMICAL PROPERTIES AND
STABILITY OF Nigella sativa OIL AND
GENTAMICIN FORMULATION

BY
NOR LIYANA BINTI JAAFAR

A thesis submitted in partial fulfilment of the requirement

for the degree of Bachelor of Biomedical Science

Kulliyyah of Allied Health Science

International Islamic University Malaysia

MAY 2015

ABSTRACT

Sterilization of medical products for systemic application is mandatory before the

products can be marketed. Gamma irradiation is one of the widely used method for
sterilization. This study aims to determine the effects of gamma-sterilization on the
stability and physicochemical properties of Nigella sativa-gentamicin emulsion. Four
emulsions (A, B, C and D) with different concentration of N. sativa oil in each
samples had been formulated, whereas the concentration of gentamicin was made
constant at 0.1% (w/v) in all samples. All of the samples were stored at three different
storage conditions (8C, 25C and 50C). 120 mL from each samples were sent for
gamma-sterilization. Several stability tests were performed on the samples before and
after gamma-sterilization. The stability evaluations include organoleptic
characteristics, centrifugation test (5000rpm, 5 minutes), freeze-thaw cycle, pH
determination, particle size measurement and zeta-potential analysis. The emulsions of
both pre- and post-gamma sterilization had been tested on biofilm-producing bacteria
of S. aureus, P. aeroginosa and S. epidermidis. Results showed no colour changes and
phase separation were presented to all of the samples that kept at 8C at all-time
points. At 25C storage conditions, phase separation was formed at day 14 in all four
formulations for both pre- and post-gamma-sterilization samples. Samples kept 50C
were the most unstable since colour changes and phase separation formed at day 7
onwards. The particle size of the samples showed significant difference between preand post-gamma sterilization. Whereas, no significant different in term of zetapotential before and after gamma-sterilization. Results showed that samples kept at
8C demonstrated to be the most stable throughout the study.

ABSTRAK

Pensterilan produk perubatan untuk aplikasi sistemik adalah wajib sebelum produk
boleh dipasarkan. Sinaran gamma adalah salah satu kaedah yang digunakan secara
meluas untuk tujuan pensterilan. Kajian ini bertujuan untuk menentukan kesan
gamma-pensterilan pada kestabilan dan sifat fizikokimia emulsi Nigella sativagentamicin. Empat emulsi (A, B, C dan D) dengan kepekatan minyak N. sativa yang
berbeza dalam setiap sampel telah dirumuskan, manakala kepekatan gentamicin dibuat
tetap pada 0.1% (w / v) dalam semua sampel. Semua sampel disimpan pada tiga
keadaan penyimpanan yang berbeza (8C, 25C dan 50C). 120 mL daripada setiap
sampel telah dihantar untuk gamma-pensterilan. Beberapa ujian kestabilan telah
dilakukan ke atas sampel sebelum dan selepas gamma-pensterilan. Penilaian
kestabilan termasuk ciri-ciri organoleptik, ujian centrifugasi (5000 rpm, 5 minit),
kitaran beku-cair, penentuan pH, pengukuran saiz zarah dan analisis zeta-berpotensi.
Emulsi untuk sebelum dan selepas gamma-pensterilan telah diuji ke atas bakteria yang
menghasilkan biofilem iaitu S. aureus, P. aeroginosa dan S. epidermidis. Keputusan
kajian menunjukkan tiada perubahan warna dan pemisahan fasa yang berlaku kepada
semua sampel yang disimpan pada 8C. Pada keadaan penyimpanan 25C, pemisahan
fasa telah berlaku pada hari ke-14 dalam keempat-empat formulasi untuk pra- dan
pasca-gamma-pensterilan. Sampel yang disimpan pada suhu 50C adalah yang paling
tidak stabil kerana perubahan warna dan pemisahan fasa terbentuk pada hari ke-7 dan
seterusnya. Saiz zarah menunjukkan perbezaan yang signifikan antara sampel pra dan
pasca-gamma pensterilan. Sebaliknya, tidak ada perbezaan yang signifikan dari segi
zeta-potensi sebelum dan selepas gamma-pensterilan. Hasil kajian menunjukkan
bahawa sampel-sampel yang disimpan pada suhu 8C menunjukkan tahap kestabilan
yang paling baik sepanjang eksperimen ini berjalan.

APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms
to acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a thesis for the degree of Bachelor of Biomedical Science
..
Asst. Prof. Dr. Mohd Affendi Bin.
Mohd Shafri
Supervisor
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
thesis for the degree of Bachelor of Biomedical Science
..
Name:
Examiner 1
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
thesis for the degree of Bachelor of Biomedical Science
..
Name:
Examiner 2
This thesis was submitted to the Department of Biomedical Science and is accepted as
a partial fulfilment of the requirements for the degree of Bachelor of Biomedical
Science
..
Asst. Prof. Dr. Ibrahim Adham
Bin. Taib
Head, Department of Biomedical
Science

DECLARATION

I hereby declare that this thesis is the result of my own investigation, except where
otherwise stated. I also declare that it has not been previously or concurrently
submitted as a whole for any other degrees at IIUM or other institutions.

Nor Liyana Binti Jaafar

Signature.

Date ..................

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION
OF FAIR USE OF UNPUBLISHED RESEARCH
Copyright 2015 by International Islamic University Malaysia. All rights reserved.
EFFECTS OF STERILIZATION ON PHYSICOCHEMICAL PROPERTIES
AND STABILITY OF Nigella sativa AND GENTAMICIN FORMULATION
No part of this unpublished research may be reproduced, stored in a retrieval
system, or transmitted, in any form or by any means, electronic, mechanical,
photocopying, recording or otherwise without prior written permission of the
copyright holder except as provided below.
1.

Any material contained in or derived from this unpublished research

may be used by others in their writing with due acknowledgement.

2.

IIUM or its library will have the right to make and transmit copies (print
or electronic) for institutional and academic purposes.

3.

The IIUM library will have the right to make, store in a retrieval system
and supply copies of this unpublished research if requested by other
universities and research libraries.

Affirmed by
Nor Liyana Binti Jaafar
....
Signature

..
Date

ACKNOWLEDGEMENT

In The Name of Allah, the Most Gracious, the Most Merciful

Alhamdulillah, praised to Allah T for His blessing and mercy, this thesis is completed
with given strength and wisdom granted by Him.
First and foremost, I would like to express my heartfelt gratitude to my
supervisor Asst. Prof. Dr. Mohd Affendi b. Mohd Shafri for his constant guidance and
valuable advice throughout my project.

His enthusiasm in scientific research really

inspired me in completing this study.

A million thanks to postgraduate students Sr. Fathin Athirah Yusof and Br
Khairul Ikhwan Yaakob for their constant source of encouragement, endless support and
valuable advice. Their willingness to help me has motivated me wholly throughout my
project. I really do appreciate all their willingness to guide me in order to let me
understand, manage the lab operation systems and accomplish the understanding of the
project.
Besides, I would like express my appreciation to all lab technicians and science
officer for their kindness and help in providing me with a good environment and
facilities to complete this study.
Finally, an honorable mention goes to my families and friends for their boundless
understandings and supports. Without helps of the particular that mentioned above, I
would face many difficulties in completing my final year project.
Last but not least, I would like to thank all those who have involved directly and
indirectly throughout my final year project. May the blessing of Allah be upon them.

TABLE OF CONTENTS

bAstrac
AbnstracMily
oApvralge
Declartion
CopyrightPea
Ackndowmglet
TableonfCts
ListbofTlea
ListgofFure
ListofSymbl/Equna

i
i
iv
v
vi
vi
vi
x
xi
vxi

HACPTENR1:IODU
1
1.1 RESEARCH BACKGROUND1
1.2 AIMS AND OBJECTIVES..3
1.3 HYPOTHESIS..3
HACPTER2:LIUVW
4
2.1 LITERATURE REVIEW.4
2.1.1 Definition of Sterilization......4
2.1.2 Advantages of Gamma-Sterilization..5
2.1.3 Disadvantages of Gamma-Sterilization.6
2.1.4 Emulsion...7
2.1.5 Stability Testing of Emulsion....9
HACPTER3:MODLGY
12
3.1MATERIALS...12
3.1.1 Apparatus and Equipment13
3.1.2 Chemicals and Reagents...13
3.1.3 Glassware and Disposable/Consumable Items.13
3.1.4 Microorganisms....14
3.2 METHODS..14
3.2.1 Emulsification Process..14
3.2.2 Stability Tests...16
3.2.2.1 Organoleptic Characteristics..16
3.2.2.2 Centrifugation Test....16
3.2.2.3 Freeze-Thaw Cycle ...16
3.2.2.4 pH Determination ..17
3.2.2.5 Particle Size Measurement.17
3.2.2.6 Zeta Potential Analysis .17
3.2.3 Gamma-sterilization.18
3.2.4 Antimicrobial Susceptibility Assays18
3.2.4.1 Preparation of Disc... 18

3.2.4.2 Preparation of Inoculum ...18

3.3.4.3 Disc Diffusion Assay 18
3.2.5 Statistical Analysis.. .19
HACPTER4:SULNDFIG
20
4.1 RESULTS...21
4.1.1 Organoleptic Characteristics22
4.1.2 Centrifugation Test..21
4.1.3 Particle Size Measurement..24
4.1.4 Zeta-Potential Analysis26
4.1.5 pH Measurement 28
4.1.6 Freeze-Thaw Cycle Test..30
4.1.7 Antimicrobial Susceptibility Assay.31
HACPTER5:DISUONL
35
5.1 DISCUSSION.35
5.1.1 Introduction..36
5.1.2 Organoleptic Characteristics34
5.1.2.1 Color.35
5.1.2.2 Phase Separation...37
5.1.3 Centrifugation Test..38
5.1.4 Particle Size Measurement..38
5.1.5 Zeta Potential Analysis....39
5.1.6 pH Measurement..40
5.1.7 Freeze-Thaw Cycle Test..42
5.1.8 Antimicrobial Susceptibility Assay.42
5.2 LIMITATIONS..45
5.2 CONCLUSION..45
5.3 FUTURE WORK...47
REFNCS

48

APXENDI

53

LIST OF TABLES

Table No.
Table 3.1

Apparatus and equipment list with model type and brand/

manufacturer

Page No.
24

Table 3.2

List of chemicals and reagents with brand/ manufacture

25

Table 3.3

Glassware and disposable/consumable items with

brand/manufacturer

25

Table 3.4

The formulation of emulsion A, B, C and D with different

concentration of Nigella sativa oil and constant concentration
of gentamicin

27

Table 4.1

Organoleptic characteristics of emulsion a for pre-gamma and

post-gamma sterilization kept in three different storage
temperature

32

Table 4.2

Organoleptic characteristics of emulsion b for pre-gamma and

post-gamma sterilization kept in three different storage
temperature

32

Table 4.3

Organoleptic characteristics of emulsion c for pre-gamma and

post-gamma sterilization kept in three different storage
temperature

33

Table 4.4

Organoleptic characteristics of emulsion d for pre-gamma and

post-gamma sterilization kept in three different storage
temperature

33

Table 4.5

Centrifugation result on the emulsion A of pre- and postgamma- sterilization kept in three different storage conditions.

34

Table 4.6

Centrifugation result on the emulsion B of pre- and post-

34

10

gamma- sterilization kept in three different storage conditions

Table 4.7

Centrifugation result on the emulsion C of pre- and postgamma- sterilization kept in three different storage conditions

34

Table 4.8

Centrifugation result on the emulsion D of pre- and postgamma- sterilization kept in three different storage conditions.

35

Table 4.9

Freeze-thaw cycle results of gentamicin-N.sativa emulsion (A,

B, C and D) for three cycles before and after gammasterilization

42

11

LIST OF FIGURES

Figure No.
Figure 2.1

Breakdown process of the emulsion (Adamczyk, 2013)

Page No.
24

Figure 3.1

Schematic diagram of gentamicin-N.sativa emulsion

formulation (Emulsion A, B, C and D). The diagram was
taken from Fathin et al., (2014).

28

Figure 3.2

Summary of the methods

32

Figure 4.1

Mean particle size of emulsion A for pre and post-gamma

sterilization kept in variable storage temperature

36

Figure 4.2

Mean particle size of emulsion B for pre and post-gamma

sterilization kept in variable storage temperature
Mean particle size of emulsion C for pre and post-gamma
sterilization kept in variable storage temperature
Mean particle size of emulsion D for pre and post-gamma
sterilization kept in variable storage temperature
Mean zeta-potential of emulsion A for pre and post-gamma
sterilization kept in variable storage temperature

37

Mean zeta-potential of emulsion B for pre and post-gamma

sterilization kept in variable storage temperature
Mean zeta-potential of emulsion C for pre and post-gamma
sterilization kept in variable storage temperature

39

Mean zeta-potential of emulsion D for pre and post-gamma

sterilization kept in variable storage temperature
Mean pH value of emulsion A for pre and post-gamma
sterilization kept in variable storage temperature
Mean pH value of emulsion B for pre and post-gamma
sterilization kept in variable storage temperature
Mean pH value of emulsion C for pre and post-gamma
sterilization kept in variable storage temperature
Mean pH value of emulsion D for pre and post-gamma
sterilization kept in variable storage temperature
The mean of inhibition zone of P. aeroginosa OS004

40

Figure 4.3
Figure 4.4
Figure 4.5

Figure 4.6
Figure 4.7

Figure 4.8
Figure 4.9
Figure 4.10
Figure 4.11
Figure 4.12
Figure 4.13

Figure 4.14 The mean of inhibition zone of P. aeroginosa ATCC

12

37
38
38

39

40
41
41
42
43
44

Figure 4.15 The mean of inhibition zone S. aureus OS001

44

Figure 4.16 The mean of inhibition zone S. aureus OS003

45

Figure 4.17 The mean of inhibition zone S. aureus ATCC

45

Figure 4.18 The mean of inhibition zone of S. epidermidis OS01

(Resistant group)

46

Figure 4.19 The mean of inhibition zone of S.epidermidis ATCC (Control

group

46

13

LIST OF SYMBOLS / ABBREVIATION

Symbols/ Abbreviation
O/W

Oil-in-Water

W/O

Water-in-Oil

F/T

Freeze-Thaw

nm

nanometer

degrees of Celcius

rpm

Revolutions per minute

NSO

Nigella sativa oil

mV

millivolt

cfu

colony forming unit

v/v

volume per volume

14