THE PHOTOPROTECTIVE AND COLLAGEN STIMULATORY EFFECTS OF
LABISIA PUMILA VAR PUMILA EXTRACT ON UVB
IRRADIATED HUMAN SKIN FIBROBLAST (HSF1184) CELLS
MOHD MUKRISH BIN MOHD HANAFI @ HANAFIAH
UNIVERSITI TEKNOLOGI MALAYSIA
i
THE PHOTOPROTECTIVE AND COLLAGEN STIMULATORY EFFECTS OF
LABISIA PUMILA VAR PUMILA EXTRACT ON UVB
IRRADIATED HUMAN SKIN FIBROBLAST (HSF1184) CELLS
MOHD MUKRISH BIN MOHD HANAFI @ HANAFIAH
A thesis submitted in fulfilment of the
requirements for the award of the degree of
Master of Engineering (Bioprocess)
Faculty of Chemical Engineering
Universiti Teknologi Malaysia
MARCH 2012
iii
To my beloved family
iv
ACKNOWLEDGEMENT
First and foremost, Alhamdulillah, thanks to Allah s.w.t., for giving me the
strengths, guidance and patience in completing this project. With His blessing, this
project is finally accomplished.
In preparing this project, I have learned more and realize that, this is only part
of my learning process. Here, I would like to express my sincere appreciation to Prof
Dr. Mohamad Roji Sarmidi, my project’s supervisor, for his continuous believe in
me, encouragement, guidance, and willingness to give me a helping hand and
advices. I would also want to express my appreciation to my co-supervisor,
Associate Professor Dr Fadzilah Adibah Abdul Majid for her advice and guidance
throughout the course of the research.
Special appreciation is extended to Wan Norazrina Wan Rusli for her efforts,
time and motivation. Her contribution towards the making of this project is priceless.
Without her help, this project would not have been the same. I would also like to
thank all Tissue Culture Engineering Research Group (TCERG) members, for their
support during the course of this study.
Last but not least, I am pleased to thank my dear family; for their love,
prayers and support. Special thanks to all my fellow friends, who were directly and
indirectly involved in the making of this thesis. Thanks a lot for everything. May
Allah repay all your kind deeds in the future.
v
ABSTRACT
This study mainly concentrates on the investigation of the photoprotective
effect of Labisia pumila extract on the growth of Human Skin Fibroblasts cells
(HSF1184) and collagen synthesis by the HSF1184 cells. In this study, the water
extract of Labisia pumila and the methanolic extract of Labisia pumila showed
growth stimulatory effect on HSF1184 cell line at the same concentration throughout
the study, at 1x 10-5 µg/ml. At higher concentrations, both Labisia pumila plant
extracts showed inhibitory effect on the growth of the cells, after 24 hours of
treatment. After 48 hours of treatment, all concentrations of the water extract of
Labisia pumila showed growth stimulatory effect on the HSF1184 cells in both nonultraviolets B (UVB) irradiated and UVB irradiated cells cultured in media with fetal
bovine serum (FBS) or without FBS. The growth stimulatory effect was more
significant in HSF1184 cells culture media without FBS. These results demonstrate
the time dependent effect of the water extract of Labisia pumila on the HSF1184
cells. Both plant extracts caused significant increase in the collagen production of the
HSF1184 cells in both UVB and non UVB irradiated HSF1184 cells. At
concentration of 1 x 10-5 µg/ml, the water extract of Labisia pumila was able to
increase the collagen synthesis in the HSF1184 UVB irradiated cells up to 70%
whereas the methanolic extract of Labisia pumila has increased the collagen
synthesis only by 20%, this concentration has proven to be the optimum
concentration for both extracts of Labisia pumila throughout the course of this study
and this result clearly indicate that the water extract of Labisia pumila is a better
extract than the methanolic extract of Labisia pumila. In conclusion, the Labisia
pumila plant extracts showed a significant photoprotective effect and has great
potential to be developed and integrated into cosmeceutical product in the future.
vi
ABSTRAK
Kajian ini memberikan perhatian utama kepada kesan perlindungan cahaya
Labisia pumila terhadap pertumbuhan sel fibroblast kulit manusia (HSF1184) dan
penghasilan kolagen oleh sel HSF1184. Dalam kajian ini, Kedua-dua ekstrak akues
Labisia pumila dan ekstrak methanol Labisia pumila menunjukkan kesan
pertumbuhan pada sel HSF1184 pada kepekatan yang sama sepanjang kajian, iaitu
pada 1x 10-5µg / ml. Pada kepekatan yang lebih tinggi, kedua-dua ekstrak tumbuhan
Labisia pumila menunjukkan kesan yg menghalang pertumbuhan sel, setelah 24 jam
rawatan. Selepas 48 jam rawatan, semua kepekatan yang digunakan menunjukkan
kesan petumbuhan pada sel HSF1184 yang dibiakkan dalam medium dengan serum
tumbesaran ataupun dalam medium tanpa serum tumbesaran dalam kedua-dua
keadaan sama ada terdedah kepada sinaran ultra-lembayung (UVB) ataupun tidak.
Kesan pertumbuhan ini lebih ketara bagi sel HSF1184 yang dibiakkan di dalam
medium tanpa serum tumbesaran. Ini menunjukkan kebergantungan kesan ekstrak
akues dan ekstrak methanol Labisia pumila kepada tempoh pendedahan terhadap sel
HSF1184. Kedua-dua ekstrak menyebabkan peningkatan ketara dalam pengeluaran
kolagen sel HSF1184 dalam kedua-dua keadaan kajian. Pada kepekatan 1 x 10-5µg /
ml, ekstrak akues Labisia pumila mampu untuk meningkatkan penghasilan kolagen
dalam sel HSF1184 yang terdedah kepada UVB sehingga 70% manakala ekstrak
methanol Labisia pumila hanya menyebabkan kenaikan sebanyak 20%, ini juga
menunjukkan kesan ekstrak akues Labisia pumila lebih efektif daripada kesan yang
dihasilkan daripada ekstrak methanol Labisia pumila. Kesimpulannya, ekstrak
tumbuhan Labisia pumila menunjukkan kesan perlindungan terhadap cahaya ultralembayung dan mempunyai potensi besar untuk dibangunkan dan diintegrasikan ke
dalam produk kosmetik pada masa akan datang.
vii
TABLE OF CONTENTS
CHAPTER
1
2
TITLE
PAGE
TITLE
i
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENTS
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
xi
LIST OF FIGURES
xii
LIST OF ABBREVATIONS
xv
INTRODUCTION
1
1.1 Introduction
1
1.2 Research Background
4
1.3 Problem Statement
6
1.4 Hypothesis
6
1.5 Objectives of the study
6
1.6 Scopes of the study
7
LITERATURE REVIEW
8
2.1 Skin
8
2.2 Physiology of The Skin
9
viii
2.2.1 Epidermis
10
2.2.2 Dermis
11
2.2.3 Hypodermis
13
2.3 Labisia pumila Extract
13
2.3.1 The Traditional Use
13
2.3.2 Components and Compounds
14
2.3.3 Phytoestrogenic Properties in Labisia pumila
17
Leading to Skin Perfection
2.3.4 Labisia pumila Content of Phytoestrogen as
21
a Potent Compound in Cosmeceutical Product
2.3.5 Labisia pumila Content as a Potent Agent
26
Against Photoaging Skin
2.4 Human Skin Fibroblasts
29
2.5 Collagen
31
2.6 Collagen Synthesis
33
2.7 Collagen Degradation
37
2.8 Collagenase Assay
38
2.9 Ultraviolet (UV) Radiation
39
2.10 Cellular Senescence
44
2.11 Possible Contribution of Cellular Senescence to Skin
45
Aging
2.12 Cell Culture
3
47
2.12.1 Cell Growth and Maintenance
47
2.12.2 Inoculation
48
2.12.3 Subculture
48
2.13 Phases in Cell Culture
49
MATERIALS AND METHODS
52
3.0 Introduction
52
3.1 Research Design
53
3.2 Materials
54
3.2.1 Chemicals
54
3.2.2 Cell culture
54
3.2.3 Extract preparation
55
ix
3.3 Cell Culture Protocols
4
56
3.3.1 Subculture and Routine maintenances
56
3.3.2 Cells counting and Cells viability
56
3.3.3 Cell Splitting
58
3.3.4 Cell Cryopreservation
58
3.3.5 Cell Recovery
59
3.4 Proliferation Analysis
60
3.4.1 MTT Assay
60
3.4.2 MTT Standard Curve
62
3.4.3 Growth profile
62
3.5 Sircol Collagen Assay
64
3.6 UVB Irradiation
65
3.7 Statistical Analysis
66
RESULTS AND DISCUSSION
67
4.1 Human Skin Fibroblast Cell Line (HSF1184)
67
4.1.1 The Growth Curve of the normal HSF1184 cells
4.1.1.1 The complete Growth Curve (Profile)
68
70
of the HSF1184 cell line
4.1.2 Morphological observation
4.2 Cell-based assay – treatment of Labisia pumila
72
74
extracts
4.2.1 The Effect of Labisia pumila aqueous extract on
74
HSF1184 cell growth (Cytotoxicity Studies)
4.2.1.1 Cultured medium supplemented
74
with FBS
4.2.1.2 Cultured medium without FBS
4.2.2 The Effect of Labisia pumila aqueous extract on
76
80
the growth curve (profile) of HSF1184 cell line
4.2.2.1 The Effect of Labisia pumila aqueous
83
Extract on the growth curve (profile)
of HSF1184 cell line in the absence
of FBS (Serum free media)
4.3 The Photoprotective effect of Labisia pumila extracts
87
x
Against UVB irradiation
4.3.1 The effect of UVB irradiation on the Growth
88
Curve of HSF1184 cell line
4.3.2 The Photoprotective effect of the water extract
90
Of Labisia pumila on UVB irradiated HSF1184
cell line
4.3.3 Time dependent effect of the water extract of
94
Labisia pumila on UVB irradiated HSF1184
cell line
4.3.4 The Photoprotective effect of the water extract
96
Of Labisia pumila on UVB irradiated HSF1184
cell line in serum free media.
4.3.5 The Photoprotective effect of the methanolic
99
extract of Labisia pumila on the UVB
irradiated HSF1184 cells
4.4 The effect of the Labisia pumila plant extracts on
104
the synthesis of collagen in HSF1184 cell line.
5
CONCLUSIONS AND RECCOMENDATION
111
5.1 Summaries
111
5.2 Recommendation
114
REFERENCES
116
xi
LIST OF TABLES
TABLE NO.
4.1
TITLE
Typical characteristics of bovine serum
PAGE
77
xii
LIST OF FIGURES
FIGURE NO.
TITLE
PAGE
2.1
Schematic of the skin
9
2.2
The location of collagen in human skin
31
2.3
Visible light
39
2.4
UVA and UVB penetrating effect on the human skin
40
2.5
Phases of cell culture
51
3.1
The design of the overall experimental procedures
53
3.2
Haemocytometer (improved Neubauer), magnified
view of the total area of the grid showingviable cells
as unstained and clear, with a refractile ring around
them and non-viable cells are dark and have no
refractile ring
57
3.3
Cell’s growth curve
63
4.1
The normal growth curve of HSF1184 cells
68
4.2
The complete growth profile of HSF1184 cell line
71
4.3
The morphology of HSF1184 cell line on (a) day 1
and (b) day 5 using 10X magnification of light
microscopy
4.4
73
Aqueous extract of Labisia pumila at different
concentration treated to HSF1184 cell line (with
FBS).
4.5
74
Aqueous extract of Labisia pumila at different
concentration treated to HSF1184 cell line (without
FBS).
76
xiii
4.6
Comparative studies of the effect of the water extract
of Labisia pumila on the growth of HSF1184 cell line
in culture media supplemented with FBS and serum
free media.
4.7
The effect of the water extract of Labisia pumila on
the growth curve of HSF1184 cell line.
4.8
79
81
The effect of the water extract of Labisia pumila on
the growth curve of HSF1184 cell line in serum free
media.
4.9
83
Comparative studies of the effect of the water extract
of Labisia pumila on the growth curve of HSF1184
cell line in culture media supplemented with FBS and
serum free media.
4.10
85
Comparative studies of cells viability in media
supplemented with FBS and serum free media after
the addition of the water extract of Labisia pumila.
4.11
The effect of UVB (80 mJ/cm2) irradiation the growth
curve of HSF1184 cell line.
4.12
90
The efficacy studies for the water extract of Labisia
pumila.
4.14
88
The effect of water extract of Labisia pumila on UVB
irradiated HSF1184 cell line
4.13
86
92
Comparative studies of Water extract of Labisia
pumila on non-irradiated and UVB irradiated
HSF1184 cells (80mJ/cm2)
4.15
93
Time-Dependent Effect of the Water Extract of
Labisia pumila on the growth of UVB Irradiated
HSF1184 cells
4.16
94
Growth Stimulatory effect the water extract of
Labisia pumila on UVB-Irradiated HSF1184 cells
after 48 hours without Growth Serum
4.17
Comparative studies of the effect of the water extract
of Labisia pumila on UVB irradiated cells in media
96
xiv
with FBS and serum free media.
4.18
97
The effect of the methanolic extract of Labisia pumila
on the growth of UVB irradiated HSF1184 cells in
culture media supplemented with FBS.
4.19
99
The effect of the methanolic extract of Labisia pumila
on the growth of UVB irradiated HSF1184 cells in
serum free media.
4.20
100
Comparative studies of the effect of the methanolic
extract of Labisia pumila on UVB irradiated cells in
media with FBS and serum free media.
4.21
101
Comparative studies of the effect of 2 different
extracts of Labisia pumila on UVB irradiated cells in
media with FBS and serum free media.
4.22
The effect of water extract of Labisia pumila on the
collagen synthesis in HSF1184 cell line.
4.23
103
105
The effect of water extract of Labisia pumila on the
collagen synthesis in UVB irradiated HSF1184 cell
line.
4.24
The effect of methanolic extract of Labisia pumila on
the collagen synthesis in HSF1184 cell line.
4.25
106
108
The effect of methanolic extract of Labisia pumila on
the collagen synthesis in UVB irradiated HSF1184
cell line.
109
xv
LIST OF ABBREVIATIONS
APC
AHA
CO2
CDKs
DPBS
DMEM
DMSO
DNA
EB
ECM
HSF
FBS
FSH
GSG
HRT
IR
KF
LH
MHT
MMP
mRNA
OA
PBS
PDT
PICP
-
PINP
-
ROS
SCA
SR
TPC
TIMP
rER
UV
-
Antigen Presenting Cells
Alpha-hydroxy acids
Carbon dioxide
Cyclin Dependent Kinases
Dulbecco Phosphate-buffered Saline
Dulbecco Minimum Essential Medium
Dimethyl Sulfoxide
Deoxyribonucleic Acid
Ethidium Bromide
Extra Cellular Matrix
Human Skin Fibroblasts
Fetal Bovine Serum
Follicle Stimulating Hormone
Glycosaminoglycans
Hormone Replacement Therapy
Infrared
Kacip Fatimah
Luitenizing Hormone
Menopausal Hormone Therapy
Matrix Metalloproteinases
Messenger RNA
Acridine Orange
Phosphate-Buffered Salines
Population Doubling Time
Procollagen C-End Terminal Pro
Peptide
Procollagen N-End Terminal Pro
Peptide
Reactive Oxygen Species
Sircol Collagen Assay
Sirius red
Total Phenolic Content
Tissue Inhibitor Metalloproteinase
Rough Endoplasmic Reticulum
Ultraviolet
CHAPTER 1
INTRODUCTION
1.1
Introduction
Plant extract has been used since ancient time for cosmetic and pharmaceutical
applications. Different parts of plant including leaves, fruits flowers, stem, barks, buds,
and roots were used accordingly. Cosmetically, plant and plant extracts were used to
provide moisturizing, whitening, tanning, colour cosmetics, sunscreen, free radicalscavenging, anti-oxidant, immunostimulant, washings, preservatives, and thickening
effects (Blum et al. 2007). A lot of research has been done on different plant extract in
order to get as much understanding as possible so that a detail chemical profile of the
plant can be obtained. This fundamental procedure has been proven to be very important
to the scientific community to determine the potentials and benefits of a plant extracts in
cosmetic or pharmaceutical applications (Andrea et al. 2004).
Despite various use and benefits, the use of plants and plant materials is bounded
by certain limitation: 1) the availability of the plant might be restricted through seasons,
limited stocks, protection of the plant, problem in cultivation, and bad harvest . 2) the
quality of the plants produced differ due to seasonal changes, methods in cultivation,
2
geography and clone types. These limitations should be overcome to realize the full
potential of the plant extracts in health promotion (Blum et al. 2007). The advancement
in technology has given us a solution to address these issues. For example, cell culture
technology is used in cell based assay which is crucial in exploratory work in assessing
bioactivities of plant extracts.
Cell culture technology is defined as a series of complex process by which cells
are grown under controlled environment. The availability of this method developed in
mid 1900s enables us to carry out a detail study on every possible aspect of cell
properties, characteristics and metabolism that is affected by the addition of plant
extracts in any cultured media or environment. In this particular study, human skin
fibroblasts were being used as the subject for analysis as it is a well-established system
for in vitro analysis of cell growth (Yamada et al. 2004), migration, and collagen
metabolism (Nawrat et al. 2005). Human skin fibroblasts has been previously used to
study skin aging (Hyun-Kyung Choi et al. 2009), wound healing (Saltzman, 2004),
genetic disorder (Paradisi et al. 2005; Jones et al. 2004), evaluating cosmetic
formulations toxicity (Losio et al. 1999; Gerhard et al 2009) and chemical cytotoxicity
(Shrivastava et al. 2005).
The integration of cell culture technology and plant extract studies is expected to
play a significant role in the advancement of the field of ethnopharmacology (Andrea et
al. 2004). This field involves the study of cosmetics, cosmeceuticals and remedies to
heal skin problems and diseases using traditionally used herbs and plant extracts
(Aburjai et al 2003). Cell culture technology enables us to investigate the effect of plant
extract on cell properties and characteristics. One of the most important properties and
characteristics of cells are their ability to regenerate themselves as part of repair
mechanism.
3
Cell regeneration or commonly known as cell growth is defined as the increase in
cell population which is very important to maintain the integrity of a certain type of
cells. Human skin for examples consists mainly of three major types of cells, namely
keratinocytes, melanocytes and fibroblast (Kanitakis 2002). Human skin functions as an
integument which simply means “covering” and acts more than an external body
covering. Due to constant and direct exposure to the environment, the three components
of the skin including important cell types experience stress conditions that can induce
adaptive or degenerative pathways and influence ageing (Francois et al. 2005). One of
the stress conditions that are very harmful to our skin is the chronic exposure to
ultraviolet (UV) irradiation. UV irradiation is thought to be the real cause for skin
damage that leads to premature ageing of the skin called photoaging (Kang et al. 2003;
Jenkins, 2002).
Direct exposure to ultraviolet (UV) light especially UVB can interfere with
normal molecular and biological functions of the fundamental components of human
skin which is keratinocytes, melanocytes and fibroblasts cells (Xu et al. 2005; Rittie et
al. 2002). These major types of cell are very important to maintain the physiological and
histological functions of the skin (Chuong et al. 2002; Kanitakis 2002), therefore it is
vital to protect the integrity of these cells. Constant exposure to UV light might also
cause significant damage to skin cell directly that can lead to cell death. This might
occur due to the increase in the number of reactive oxygen species (ROS) which is very
damaging to the skin cells (Francois et al. 2005). These are the main reason why UV
light exposure can speed up ageing process in human skin.
Skin regeneration is very important in this condition so that the skin can repair
itself and function normally. However, in most situations full regeneration is not
achieved as the skin cells have lost their ability to regenerate on their own capacity.
Finding of chemical compounds or the use of plant extracts that can stimulate cell
4
growth after rigorous exposure to stressful environment might provide the solution for
this problematic condition. The discovery of a natural product that address this issue
might also become the alternative source of growth promoter that can be integrated into
culture media to replace the expensive synthetic growth serum currently used in most
culture media. In this study, an extract from a plant called Labisia pumila var pumila
was used to investigate its effect on the growth of human fibroblast cells (HSF) and
collagen synthesis stimulation when the plant extract is introduced to the cell in a tissue
culture environment.
1.2
Research Background
Labisia pumila is one of the popular herbs in Malaysia and is popularly known as
"Kacip Fatimah" domestically. The Malay people have been using Kacip Fatimah for
many generations. It has been used extensively by the Malay women in childbirth due to
its ability to induce and facilitate childbirth. Apart from that, Kacip Fatimah is also part
of the traditional medication used as post-partum therapies (Burkill, 1935). There are
three types of Labisia pumila that can be found in Malaysia namely, Labisia pumila var.
alata, Labisia pumila var. pumila and Labisia pumila var. lanceolata (Stone, 1988).
Differentiation of the three varieties of Labisia pumila is very important in terms of their
chemical and physical characteristics as well as biological activity so that proper
bioactive ingredients can be identified in order to produce a quality herbal product
without jeopardising its safety and efficacy.
5
As mentioned in the earlier part of section 1.2, Kacip Fatimah has been the most
favoured herbs among the indigenous people to treat menstrual irregularities and
postpartum. Traditionally, certain parts of the plant are boiled and the water extract will
be taken as a drink. Even though there are no scientific evidence on the efficacy of this
traditional method of preparation, Kacip Fatimah has been continually used in traditional
medicine. Recently a lot of interest has been shown in herbal medicine as it is said to be
the natural way of healing. Due to the advances in technology, nowadays, people can get
as much information as they want and be able to pick and choose, leading them to opt
for herbal medicines as to their perception it is natural to the human body unlike the
conventional drugs used in modern medicines. Therefore, a lot of studies have been
carried out to determine the potential pharmalogical application of herbal preparation.
However, a lot of products that exploit the use of traditional herbs has been marketed
commercially even without the proper knowledge of its mode of action. This is a very
unhealthy practice but it shows the general acceptance of the public towards herbalbased products. Several studies of Labisia pumila (Jamal et al,1999, Ayida et al, 2007,
Husniza, 2002) were carried out and the results of these studies clearly indicate the
phytoestrogenic properties of Labisia pumila. One of the studies which used the
ethanolic extract of the roots of Labisia pumila var. alata was tested on the human
endometrial adenocarcinoma cells of the Ishikawa-Var I line, indicated that the plant
extract has a weak but specific estrogenic effect on the cells by enhancing the secretion
of alkaline phosphatase. Another recent studies conducted by the Institute of Medical
Research (IMR) has showed that Labisia pumila plant extract was able to displace
estradiol binding to antibodies raised against estradiol, making it similar to other
estrogens such as estrone and estriol(Jamal et al,1999)
This study was carried out to evaluate the photoprotective effect of Labisia
pumila on the growth of human skin fibroblast with and without exposure to UV
irradiation and also to investigate the effect of the plant extract on the stimulation of
collagen synthesis which is vital to skin health.
6
1.3
Problem Statement
Premature skin ageing caused by ultraviolet irradiation from the sun has
deleterious effects in human skin. These includes disruption in the growth of human skin
cells (human skin fibroblast cells), and the impairment of collagen synthesis that play a
very important role in maintaining the structural integrity of the human skin.
1.4
Hypothesis
Labisia pumila (Kacip Fatimah) extracts promote the growth of human skin
fibroblast cells (HSF1184) previously exposed to UVB irradiation and stimulate
collagen synthesis in order to maintain the structural integrity of the human skin so that
the effect of extrinsic ageing can be minimized.
1.5
Objectives of the study
The objective of this study was to study the photo-protective effect of Labisia pumila
extracts on UVB irradiated human skin fibroblast (HSF1184) cells. Its potential in
promoting the growth of human skin fibroblast cells exposed to UVB irradiation and the
stimulatory effect on collagen synthesis were investigated
7
1.6
Scopes of the study
This study consists of three main scopes.
1. To investigate the effect of Labisia pumila extracts on the growth of UVB
irradiated human skin fibroblast (HSF1184) cells
2. To identify the difference between the effect of Labisia pumila extracts on
normal HSF 1184 cells growth and UVB irradiated HSF 1184 cells
3. To investigate the potential of Labisia pumila extracts on the stimulation of
collagen synthesis in HSF 1184 cells
CHAPTER 2
LITERATURE REVIEW
2.1
Skin
The development of skin as an organ commences during the foetal stage,
however, its final development only occur postnatally. Skin is the integument organ
which simply means the outer “covering”. However, physiologically, skin is much more
than just an external body covering of the human body. As well as serving as a
protective barrier against injury (abrasions, cuts, burns), infectious pathogens and
ultraviolet radiation, skin assists in body temperature regulation, vitamin D synthesis,
ion excretion, and sensory reception (touch and pain), and it has remarkable regenerative
capacity (William et al, 2008).
These vital functions are achieved due to the presence of skin’s capillary network
and sweat glands which allow skin to act as a mini-excretory system. The cutaneous
sensory receptors that are part of the component of the complex human nervous system,
are also located in the skin. These complex array of sensors include touch, pressure,
temperature and pain receptors provide a great deal of external environment information
(Marieb, 1997). Human skin also has the ability to transmit important emotional signals
9
to the environment, such paleness or blushing of the face and production of scents
(pheromones) (Yannas, 2000).
2.2
Physiology of the skin
The integument, the largest organ of the body, is composed of skin and skin
appendages- nails, hair, sweat glands, and sebaceous glands. The total weight and
overall surface area of the skin in the adult are 3-5 kg and 1.5-2 m2, respectively. Skin
thickness, between 0.5 and 3 mm, varies regionally (William et al, 2008). Skin is
thickest on the back and thinnest on the eyelid. Human skin consists of three important
layers, the stratified squamous keratinized epithelium on its outer part, called the
epidermis, and an inner layer of fibrous connective tissue, called the dermis. These two
layer are separated by structure called basal lamina. Collagen VII is an important
component of the basal lamina (Palsson and Bhatia, 2004). The third layer, which is a
loose layer of subcutaneous connective tissue called the hypodermis, attaches skin to the
underlying structures and permits movement over most body parts (Rashmi et al, 2008).
Figure 2.1
Schematic of the skin (Human Histology 3rd Edition)
10
2.2.1 Epidermis
The epidermis is avascular because it does not have blood supply of its own
(Marieb, 1997) the epidermis is good at holding water, which helps make the skin elastic
and maintains the body’s balance of fluid and electrolytes (Turkington and Dover,
1996). Epidermis is made up of about 95% keratinocytes or dead skin cells that serves as
a barrier in keeping harmful substances from injuring the skin and preventing water and
other essential substances from escaping the body (Turkington and Dover, 1996). In
addition to the keratinocytes, other specialised cells present in the basal layer are
melanocytes, Langerhans cells and Merkel cells. Melanocytes secrete melanosomes
containing melanin (eumelanin or phaeomelanin) that provide the protection required by
the human body from ultraviolet radiations and free radicals (Rashmi et al, 2008).
Langerhan cells which are derived from the bone marrow and as part of the immune
system function as antigen presenting cells (APC) of the skin. Merkel cells working in
tandem with nerve endings are present in the dermis and are responsible for cutaneous
sensation. As mentioned earlier, normal epidermis also contains appendages such as hair
follicles and sweat glands (Lam, 1999).
Epidermis is comprised of stratum corneum (horny layer) which is the outermost
layer exposed to the environment, stratum granulosum (granular layer), stratum
spinosum (prickle cell layer) and stratum basale (basal layer) (Turkington and Dover,
1996). The stratum corneum is a nonviable epidermal cell layer 10-15 layers thick. This
epidermal part accounts for about three quarters of the epidermal thickness (Marieb,
1997). The stratum corneum is made up of keratin enriched dead cells (corified or horny
cells), entirely surrounded by crystalline intercellular lipid domains. This anucleated
keratinocytes (corneocytes) oriented like bricks in the surrounding lipid (that serve as a
mortar) and forms the prime barriers to the transdermal delivery of actives (Bouwstra et
al, 2000). After providing a brief protective function to the human skin, the stratum
corneum is imperceptibly sloughed off. This process is called keratinisation (Marieb,
1997).
11
In spite of being a viable epidermal layer, the next layer, the stratum granulosum
(1-3 cell layers thick) contains enzymes. These enzymes have the potential to cause
degradation of vital cells organelles such as nuclei. By synthesizing keratin and
degrading cells organelles, the keratinocytes in this layer gradually differentiate into the
corneocytes of stratum corneum. Apart from that, the keratinocytes are also responsible
is synthesizing membrane coating granule that carry the precursors for intercellular lipid
lamellae of the stratum corneum (Rashmi et al, 2008).
The next epidermal layer is known as stratum spinosum (2-6 layers of columnar
keratinocytes). In this layer, keratin aggregates to form filaments called tonofilaments
that on further condensation produce cell membrane connecting structure called
desmosomes. The stratum spinosum together with stratum basale is known as the
Malphigian layer. The stratum basale is the layer with all the typical cell organelles and
is the only layer that is capable of cell division (Turkington and Dover, 1996).
The net proliferative rate of skin varies according to the region of the body. The
turnover of the skin could be in the order of a few weeks, which regenerates a large
amount of flakes over time. Skin is the body’s third most prolific tissue (Palsson and
Bhatia, 2004).
2.2.2
Dermis
The dermis or corium (true skin) is 3-5 mm thick and is composed of numerous
connective tissue especially collagen fibrils and elastic tissues that respectively provide
support and flexibility to the dermis (Rashmi et al, 2008). Collagen is the important
component of the skin that provides the structural integrity of the human skin. Dermis
12
also contains amorphous ground substance which is a highly organized hydrated
semisolid gel (glycosaminoglycans and glycoproteins) (Rashmi et al, 2008). Fibronectin
and hyaluronic acid are example of glycoproteins and glycosaminoglycans respectively
(David, 1993). Embedded in this layer are systems and structures common to other
organs such as lymph channels, blood vessels, nerve fibres, and muscle cells. Structures
that are unique to the dermis are hair follicles, sebaceous glands and sweat glands
(Rashmi et al, 2008).
The connective tissue making up the epidermis is comprised of two major
components, the papillary and the reticular areas. Similar to the epidermal layer, the
thickness of the dermal layer varies according to regions and locations on the human
body. The papillary layer makes up the upper dermal region. This component is uneven
and has finger like projections from its superior surface, called dermal papillae, which
provide the indentation to the above epidermal layer. Many dermal papillae contain
capillary loops, which furnish nutrients to the epidermis (Marieb, 1997).
Another layer of the dermis is known as the reticular layer. This layer is located
in the deepest skin part of the skin and contains blood vessels, sweat and oil glands and
deep pressure receptors. Phagocytes which are part of the human immune system can be
found in abundance in this layer. These large number of phagocytic cells act to prevent
bacteria that have managed to break through the epidermis from penetrating any deeper
into human body (Marieb 1997).
13
2.2.3
Hypodermis
The hypodermis (or the subcutaneous fat layer) acts as a bridge that connects the
dermis and the underlying organs. It will also provide insulatory effect to the body and
protects it from mechanical shock (Rashmi et al, 2008).
2.3
Labisia pumila Extract
2.3.1
The Traditional Use
Labisia pumila belongs to Myrsinaceae family, locally known as Kacip Fatimah,
is a herb that has been widely used as in South East Asian communities for a variety of
illnesses and also used as health supplements. It is an indigenous medicinal herb of
Malaysia and sometimes also referred locally as Akar Fatimah, Selusoh Fatimah, Tadah
Matahari, Rumput Siti Fatimah, Bunga Belangkas Hutan and Pokok Pinggang (Jamal et
al., 2003). There are three varieties of Labisia pumila, i.e. Labisia pumila var. alata,
Labisia pumila var. pumila and Labisia pumila var. lanceolata (Arifin, 2005). Each
variety has their respective use and local healers tend to use var alata and var pumila
traditionally (Ibrahim & Jaafar, 2011). This herb’s extract is prepared by boiling the
roots, leaves or the whole plant with water and the extract is taken orally (Burkill, 1935;
Zainon et al, 1999; Runi, 2001). The decoction of the roots also given to pregnant
women between one or two months before delivery, as this is believed to induce and
expedite labour (Jamal et al, 2003). It has been also widely used with a long history by
women in Malaysia to treat post-partum illnesses, to assist contraction of the birth
channel (Jamal, 2005), shrink the uterus, improve menstrual cycle, weight loss (Ariffin,
14
2005), It was also reported that Labisia pumila can be used for delaying fertility and to
regain body strength; while some other folkloric uses include treatment of flatulence,
dysentery, dysmenorrhoea, gonorrhoea and “sickness in the bones” (Jamia et al, 2003).
Therefore Labisia pumila is known as “queen of plants” of all Malaysian herbs
(Zaizuhana et al, 2006).
2.3.2
Components and Compounds
Few phytochemical components have been identified in Labisia pumila var. alata
(Norhaiza et al, 2009) such as anthocyanins and antioxidant components which are also
present in fruits and plants. A study by Zhang and Ye (2008) suggest that both phenolic
acids and flavanoids are believed to be responsible for the wide spectrum of
pharmacological activities attributed to the herb. The antioxidant activity of the aqueous
extract has been reported as providing significant protection to human dermal fibroblast,
from cell damage caused by UV irradiation (Choi et al, 2010), most likely due to the
presence of flavanoids (Norhaiza et al., 2009). Labisia pumila var. alata root and leaves
were found contain two novel benzoquinod compounds 1,2 as major components
(Norhaiza et al, 2009).
A preliminary screening of total phenolic content (TPC) was done by Chua et al.
(2011). The study shows that MeOH extract of this herb contained the highest amount of
phenolic phytochemicals, followed by the 60% MeOH extract. Flavonoids constituted a
large portion of the phytochemicals in the 60% MeOH leaf extract. The 40% MeOH
fraction was found to have the highest DPPH scavenging activity. Nine flavonols, two
flavanols and nine phenolic acids were detected in this fraction using UPLC–ESIMS/MS coupled with powerful software for data processing and interpretation. With the
aid of principal component analysis (PCA), the complex MS data were mined for the
15
similarities and differences in phytochemical composition between the fractions. This
information is essential for future studies in biomarker discovery (Chua et al., 2011).
Various extracts of Labisia pumila have antioxidative properties comparable to
Silymarin which is an extract of the well-known European milk thistle plant Silybum
marianum (Pinnell, 2003, Norhaiza, 2009). A study was conducted to examine the
presence of antioxidative activities of both Labisia pumila var. alata and Labisia pumila
var. pumila using DPPH, FRAP and β-carotene bleaching methods. In addition, ascorbic
acid, β-carotene, anthocyanin, total flavonoid and total phenolic content were also
analyzed. Results of the studies showed six of them to have high activities of antioxidant
in Labisia pumila var. alata compared to that of Labisia pumila var. pumila. The results
obtained showed that Labisia pumila var. alata contained higher antioxidative activities
in the three methods applied compared to Labisia pumila var. pumila. In DPPH, FRAP
and β-carotene bleaching methods, Labisia pumila var. alata had high antioxidant
activities with 299.84 µM trolox/g db, 164.16 µM trolox/g db and 89.22%, respectively.
The same pattern of antioxidant activities also can be observed in ascorbic acid, βcarotene and anthocyanin in Labisia pumila var. alata compared to Labisia pumila var.
pumila with 0.022, 3.175 and 0.328 mg/g FW, respectively. Labisia pumila var. pumila
had higher total flavonoid content than Labisia pumila var. alata with 1.281 mg/g FW.
For total phenolic content, no significant different was observed because the amount of
total phenolic content ranging from 2.53 to 2.55 mg/g FW. There is a positive
correlation between antioxidant capacities and individual antioxidative compounds in
the following order β-carotene>flavonoid>vitamin C>total anthocyanins >phenolics
(Norhaiza et al., 2009).
16
This identification of components such as β-carotene and flavanoid which
contribute to the highest antioxidative compounds present in this herb has lead Choi et
al. (2010) to see the potential of this herb in skin protection. Choi et al. (2010)
demonstrate the efficacy of Labisia pumila extract for specifically protecting skin
against photoaging. In this study, 50% of free radical scavenging activity (FSC50) to be
equal to that produced by 156 µM ascorbic acid, inhibition of TNF-α production and the
expression of COX-2 and also collagen restoration was back to normal after treatment
with this herb. On the other hand, the enhanced MMP-1expression upon UVB
irradiation was down regulated by Labisia pumila extract in a dose-dependent manner.
Treatment of normal keratinocytes with Labisia pumila extract attenuated UVB-induced
MMP-9 expression. The results collectively suggest Labisia pumila extract has
tremendous potential as an anti-photoaging cosmetic ingredient (Choi et al., 2010).
Karimi et al. (2011) used Labisia pumila to determine the phytochemical
compound and antimicrobial activities of methanolic extract of this plant. In this study,
various types of the plant were used namely Labisia pumila var. alata, Labisia pumila
var. pumila and Labisia pumila var. lanceolata. The result shows that high content of
flavanoid present in this herb especially in Labisia pumila var. pumila, while gallic acid
and caffeic acid also present in all three varieties, in fact Labisia pumila var. alata has
the highest gallic acid content as compare to the other 2 varieties. The latter also has
higher content of gallic acid compared to which had been previously found in fresh
Mauritian black tea leaves (Luximon-Ramma et al., 2005) and three other medicinal
plant species (i.e., Tectona grandis, Shilajit spp., and Valeriana wallachi). Labisia
pumila var. pumila on the other hand has higher kaempherol value as compared to ginko
biloba (Repollés et al., 2006). The leaf of Labisia pumila var. pumila also has higher
quercetin content compared to onion and garlic (Nuutila et al., 2003), and also high
content of saponin (Karimi et al., 2011).
17
2.3.3 Phytoestrogenic Properties in Labisia pumila Leading to Skin Perfection
As mentioned in section 2.3.1, Labisia pumila is a very popular herb that has
been traditionally in women’s health especially in accommodating childbirth and postpartum medication. Its exclusive use in women has led to the belief that it contains
phytoestrogens, a compound with similar chemical structure to estrogen, in quite a
significant amount, (Jamal et al., 2003). Theoretically, phytoestrogen can act as an antiestrogenic agent by blocking the estrogen receptors and exerting weaker estrogenic
effect compared with the hormone (Institute of Food Science and Technology [IFST],
2001; Al Wahaibi et al., 2008). The water extract of Labisia pumila var. alata inhibits
estradiol binding to antibodies against estradiol, suggesting the presence of estrogen-like
compounds (Husniza, 2002). Furthermore, Labisia pumila var. alata increased uterine
weight in ovariectomized and dihydrotestosterone-induced polycystic ovarian syndrome
rats, thus exhibiting estrogenic properties (Manneras et al., 2010). Labisia pumila var.
alata was also able to initiate lipolysis in adipose tissue in a manner similar to that
reported for estrogen (Ayida et al., 2007).
Most known phytoestrogen (plant oestrogen) are also responsible in abortion and
contraception. During later stages of pregnancy and labour, it is believed that estrogen
plays an important role in causing uterine contraction (Jamal et al., 1998). Therefore, in
this experiment two varieties of Labisia pumila var. alata and Labisia pumila var. pumila
were assessed for their estrogenic activities by performing an in vitro recombinant yeastbased estrogen assay in 96-well microtitre plates (Routledge and Sumpter, 1996).
However, ethanol and water extracts of both Labisia pumila var. alata and Labisia
pumila var. pumila roots and leaves did not show any significant estrogenic activity. All
extracts were tested over a concentration of 1 to 1000 μg/ml and compared to a standard
17β-estradiol (17β-E2). The negative result of estrogenicity indicate that the
phytoestrogen could either be absent in all the extract or require microbial metabolic
transformation in the intestine in order to become active (Jamal et al., 1998). However
18
this result by Jamal et al. (1998) has been contradicted to present studies that show the
presence of estrogenic effect in several experiments (Manneras et al., 2009).
Aqueous, acid hydrolysed and ethanolic extracts of the roots and leaves of
Labisia pumila var. alata and Labisia pumila var. pumila were investigated by Jamal et
al. (2003) for their estrogenic and cytotoxic effects using Ishikawa cell line by
performing an in vitro Ishikawa alkaline phosphatase assay and an in vitro protein assay,
respectively. Among them, only the ethanol extract of the root of Labisia pumila var.
alata exhibited a weak oestrogenic activity at 10-50 mg/ml. The samples that exhibited
significant cytotoxic effect were the ethanol extract of the roots of Labisia pumila var.
alata (IC50 433 mg/ml), and the aqueous extracts of the roots of Labisia pumila var. alata
(IC50 433 mg/ml) and the leaves of Labisia pumila var. pumila (IC50 458 mg/ml). From
the in vitro studies, the ethanol extract of the roots of Labisia pumila var. alata was
found to be weakly oestrogenic but the ethanol root extract of Labisia pumila var. alata
and Labisia pumila var. pumila and the water extracts of the roots Labisia pumila var.
alata and Labisia pumila var. pumila were found to be cytotoxic (Jamal et al., 2003).
The presence of phytoestrogens in this herb could be used to relieve menopausal
syndromes (Bhathena and Velasquez, 2002). Results showed that water extract of
Labisia pumila var. alata maintained the elastic lamellae architecture of the
ovariectomized rat aortae in a manner comparable to that of the normal rats. The
comparison between aortic wall thicknesses was reported for both Labisia pumila var.
alata extracts and estrogen replacement therapy. Treatment with Labisia pumila var.
alata extracts showed a significantly thicker wall as compared to estrogen treatment. A
more elastic aorta is advantageous because it will allow blood to flow smoothly from the
heart and put less pressure on the other organs. The results of this study are consistent
with previous research that demonstrated the effect of estrogen and phytoestrogen
treatments. Phytoestrogen intake was also reported to be associated with lower aortic
stiffness (van der Schouw et al., 2002) thus it would be possible to apply the same
19
concept to Labisia pumila var. alata, based on the estrogenic activity of the extract. As
estrogen is known to have cardio-protective effect in post-menopausal women (Skafar et
al., 1997), it is possible to hypothesize that Labisia pumila var. alata may have similar
cardio protective effects. Knowing that Labisia pumila var. alata has estrogenic activity,
it is logical that this herb may also potentiate the contractile responses to vasopressin.
Results implied a possible role for Labisia pumila var. alata in modulating
postmenopausal cardiovascular risks (Al-Wahaibi et al., 2008).
With such a massive research about phytoestrogen present in Labisia pumila, it is
possible for this herb to have a potential towards skin perfection. Nowadays, women
especially are getting relentlessly obsessed with skin care product and their promising
effects. They are now more careful in the selection of beauty products and they are more
concerned with the nature-based products compared with chemical-based-products. At
present, traditional methods for skin care has been recognised by many around the
world. Researchers are now actively looking for the advantages available to the
ingredients used in making herbal-based cosmetics.
Phytoestrogen could be categorized into 3 categories which are isoflavons,
coumenstans and lignans. Isoflavons are the most thoroughly examined among those
three which displays similarity to the mammal estrogen molecule and are found in soy
beans, lentils and red clover. The most important isoflavones are genistein and daidecin.
The structural similarity to 17β-estradiol explains the estrogen-like effects, which may
be traced back to the interaction of these substances with the estrogen receptor (Wang et
al., 1996; Sator, 2006). Nutrition in Asian countries, with its large phytoestrogen
content, is thought to be the reason why Asian women rarely suffer from climacteric
symptoms. The biological potency of isoflavonoids is significantly inferior to that of
synthetic estrogens (Sator, 2006). When phytoestrogens are topically applied, they
behave like estrogens by causing a proliferation of the epidermis, supporting collagen
synthesis and reducing enzymatic collagen degradation (Sator, 2006).
20
A controlled open European multicentre study examined the effect of a cosmetic
cream preparation including isoflavone (Novadiol®) on 234 women: maximum age 65
years, at least 3 years since menopause, no HRT or other substances affecting the skin
aging process (Bayerl and Keil, 2002; Sator, 2006). Skin dryness and roughness were
significantly improved at the treated areas by 32.9% and 22%, respectively, in
comparison with the untreated skin areas. Facial wrinkles were significantly reduced by
22% and skin looseness was significantly reduced by 24% (Sator, 2006).
The famous phytoestrogen was actively observed in soy. In fact, the benefits of
soy in skin health centered entirely on the isoflavones towards skin aging. Extrinsic skin
aging has been attributing to the damaging effect of solar ultra violet (UV) radiation.
Some early research demonstrated that 10 µM genistein treatment of UVA-exposed
human epidermoid carcinoma cell suppressed the production of 8-hydroxy-2’deoxyguanosine (8OHdG), a formofoxidative DNA damage (Liu et al., 1999; Blair and
Tabor, 2009). The ability of genistein to reduce oxidative DNA damage in the system
was suggested to be due to its ability to scavenge free oxygen radicals. Isoflavones from
soy also has been reported to support skin health by additional mechanism which may or
may not be related to the antioxidant properties. Sudel et al. (2005) reported that in vitro
treatment of human fibroblasts with purified genistein resulted in increased collagen
synthesis (Blair and Tabor, 2009).
21
2.3.4 Labisia pumila Content of Phytoestrogen as a Potent Compound in
Cosmeceutical Product.
Aging, especially in women is a continuous process from birth to its final stage,
menopause. The decrease in estrogen production is the hallmark of menopause which
follows gradual changes in the menstrual cycle accompanied by gonadotropin hormone
changes by increased of follicle stimulating hormone (FSH)(O’Connor et al., 2001). The
number of ovarian premature follicles is fixed at birth and gradually decreases each time
follicles mature and release oocytes. At perimenopause, the follicular number has
decreased substantially, and those present respond poorly to FSH and LH, resulting in
cycle irregularity and erratic ovulation (O’Connor et al., 2001). There is gradual
decrease in progesterone and estrogen, but hormone fluctuations are common. This
period can last from 3-10 years before menopause. Irregular menstrual cycles are the
most common first sign of perimenopause, along with increasing levels of FSH. As the
number of follicles keeps decreasing, estrogen production continues to fall. At some
point, the estrogen-based feedback mechanisms associated with LH secretion are
disrupted, leading to non-ovulatory cycles. At the onset of menopause, when ovulation
ceases entirely, LH starts rising again.
Menopause is due mainly to declining ovarian function, as the pool of primordial
follicles is exhausted. The feedback control mechanisms of the hypothalamic-pituitaryovarian axis are disrupted, leading to increased levels of follicle stimulating hormone,
FSH, but with unchanged levels of hypothalamic gonadotrophin releasing hormone,
GnRH. Estradiol and progesterone production is sharply reduced due to cessation of the
menstrual cycle and mature follicular development (TeVelde et al., 1998). The increased
levels of FSH have no effect on estrogen production, because there is limited expression
of FSH receptors in the follicles, rendering them insensitive to FSH. Ovarian
steroidogenesis during menopause is restricted to androgen production. It is established
that menopausal theca cells are responsive to luitenizing hormone, LH and produce
androstenedione and testosterone (O’Connor et al., 2001). Most of the circulating
22
androgens come from the adrenal gland. During menopause, estrone is exclusively
produced at remote sites (mainly adipose tissue) by the conversion of androstenedione.
The rate of this conversion correlates to body size (amount of adipose tissue) (O’Connor
et al., 2001).
A research by Tsavachidou and Liebman (2002) suggest that during
perimenopause, both estradiol and progesterone levels decrease. In menopause, estradiol
levels drop dramatically, below basal levels, and stay there throughout the cycle. The
estradiol peaks around ovulation and during the second half of the cycle do not exist,
being consistent with the lack of ovulation and the follicular death in the menopausal
ovaries.
The skin is a target of many hormones, especially estrogens. Estrogen and other
hormone receptors have been detected, inter alia, in keratinocytes, fibroblast, sebaceous
glands, hair follicles, endocrine glands, and blood vessel (Schmidt et al., 1990). This is
one of the main reasons of postmenopausal women having aged skin – they are lacked of
estrogens hormone. In clinical terms, many females experience a sudden onset of skin
aging symptoms several months after menopause. Several studies have been proof that
the lack of estrogens in the system is associated with aging effects, especially for
women who experience menopause.
One of the first signs which women experience is increasing skin dryness
followed by a loss of skin firmness and elasticity and leads to wrinkles. All these
symptoms correspond to changes in collagenous and elastic fibres due to the lack of
estrogen (Schmidt et al., 1990). Skin collagen contents in adults decrease by 1% every
year (Shuster et al., 1985). This process is more evident in women than men.
Approximately, 30% of skin collagen is lost in the first five years after menopause, with
an average decline of 2.1% per postmenaopausal year over a period of 20 years.
23
Estrogens reverse this trend and increase skin collagen (Zouboulis, 2000). Estrogens also
enhance the synthesis of hyaluronic acid and promote water retention (Epstein and
Munderloh, (1975). Animal studies indicate that estrogens induce several changes in the
connective tissue of the dermis, including increased mucopulysaccharide incorporation,
hydroxyl-proline turnover, and alterations in the extracellular matrix (Holland et al.,
1994; Sator, 2006). Researchers are actively searching for suitable candidate in
combating aging skin towards these women and several hormone-based products have
been tested to be positive in reducing aging effect.
Menopause causes hypoestrogenism that adds to the effect of age and causes
further deleterious skin changes. The role of Hormone Replacement Therapy in such
circumstances is not surprisingly, an important one. Both estrogen and androgen
receptors have been identified on dermal fibroblasts and epidermal keratinocytes
(MacLean et al, 1990). In a study to identify specific estrogen-sensitive structures
normal human skin was examined for the binding of the ER D5 antibody which is
associated with p29, a 29 kD protein found in the cytoplasm of normal estrogensensitive cells (Jamec & Wojnarowska, 1987). Strong and specific staining was seen in
the epidermis, with a gradient showing the most intense staining in the granular layer.
Similar positive staining was seen in the hair follicles and sebaceous glands. Variable
staining was seen in the endocrine glands and vessels. These findings demonstrate the
receptor p29 to be present in these structures, and hence suggest that estrogens may exert
a specific effect on these tissues (Jamec & Wojnarowska, 1987).
Estrogen could increase the rate of collagen production also influencing the
degree of polymerisation of GAG’s (glycosaminoglycans). Estrogens increase dermal
hydroscopic qualities (Danforth et al., 1974), probably through enhanced synthesis of
dermal hyaluronic acid (Grosman et al., 1971). Collagenous fibrils were found to be less
fragmented in the dermis in women treated with estrogens.
24
The decline in skin collagen content after the menopause occurs at a much more
rapid rate in the initial postmenopausal years than in the later ones. Some 30% of skin
collagen is lost in the first 5 years after the menopause with an average decline of 2.1%
per postmenopausal year over a period of 20 years (Brincat et al., 1983). The increase in
skin collagen content after 6 months of sex hormone therapy depends on the collagen
content at the start of treatment (Brincat et al., 1985). In women with a low skin collagen
content, estrogens are initially of therapeutic and later of prophylactic value while in
those with mild loss of collagen content in the early menopausal years estrogens are of
prophylactic value only. Thus a deficiency in skin collagen can be corrected but not
overcorrected (Brincat et al., 1987). Following the menopause, skin collagen content and
skin thickness are increased in women on estrogen replacement therapy compared to age
matched women on no treatment (Brincat et al., 1985; Castelo-Branco et al., 1992).
Prospective studies have shown that skin thickness, skin collagen and bone mass
increase in postmenopausal women who start estrogen replacement. Beneficial changes
using both topical (Varila et al., 1995) as well as with oestradiol implants can be
obtained. Topical oestradiol gel has also been shown to increase skin collagen content as
measured by skin hydroxy proline. Skin blister fluids were assayed and an increase of
both Procollagen C-End Terminal Pro Peptide (PICP) and Procollagen N-End Terminal
Pro Peptide (PINP) characterised with the gel. Pro Collagen C-End Terminal
polypeptide protein and Pro Collagen N-End Terminal polypeptide are released by
enzymatic cleavage when the pro collagen molecule is released extracellularly (Varila et
al., 1995). This is therefore a post translational event and the presence of free PICP and
PINP serves to indicate collagen production. The I in PICP and PINP refer to Type I
Collagen.
A study done by Partrairca et al. (2007) evaluates the additional benefits of
topical estrogen on the dermal collagen of the facial skin in postmenopausal women
using oral hormone therapy. Estrogen receptors have been detected in skin, and recent
studies suggest that estrogens exert their effect in skin through the same molecular
pathways used in other non-reproductive tissues. Although systemic menopausal
25
hormone therapy (MHT) has been used for many years, recent trials have reported a
significant increased risk of breast cancer and other pathologies with this treatment. This
has led to reconsider the risks and benefits of MHT (Verdier-Sevrain et al., 2006). For
this reason, high doses of systemic MHT cannot be recommended to treat skin aging.
Therefore, the topical estrogen treatment may be an alternative for ameliorating the skin,
specially the collagen amount. Partriarca et al. (2007) showed that the addition of
estrogen gel may be useful for increasing the dermal collagen.
Partriarca et al. (2007) also suggests that topical estrogen could act on the skin
without increase its systemic level by the fact that topical estrogen may increase the
collagen amount and prevent the cutaneous aging (Whitmore, 1997). From this study,
the systemic estrogen alone did not seem to have a positive effect in skin collagen.
Otherwise, some studies showed that the decrease in skin collagen in women of
postmenopausal age may not be an exclusive age-related phenomenon, but could be
related to hypoestrogenism (Castelo-Branco et al., 1992; Affinito et al., 1999; Partriaca
et al., 2007).
Estrogen loss at menopause has a profound influence on skin. Estrogen treatment
in postmenopausal women has been repeatedly shown to increase collagen content,
dermal thickness and elasticity, and data on the effect of estrogen on skin water content
are also promising. Further, physiologic studies on estrogen and wound healing suggest
that hormone replacement therapy (HRT) may play a beneficial role in cutaneous injury
repair. Results on the effect of HRT on other physiologic characteristics of skin, such as
elastin content, sebaceous secretions, wrinkling and blood flow, are discordant. Given
the responsiveness of skin to estrogen, the effects of HRT on aging skin require further
examination, and careful molecular studies will likely clarify estrogen's effects at the
cellular level (Brincat et al., 2005).
26
2.3.5 Labisia pumila Content as a Potent Agent against Photoaging Skin
Skin suffers progressive morphologic and physiologic decrement with increasing
age and provides the first obvious evidence of the aging process. Skin aging can be
classified into light-induced aging (photoaging, exogenous aging) and endogenous aging
(Makrantonaki et al, 2007). The latter occurs in non-exposed areas, which are not in
direct contact with environmental factors such as ultraviolet (UV) and infrared (IR)
irradiation (e.g., the inner side of the upper arm) (Makrantonaki et al, 2007), and is
mainly attributed to genetic factors and alterations of the endocrine environment. In
contrast to photoaging, endogenously aged skin reflects degradation processes of the
entire organism. Among all environmental factors, solar UV radiation is the most
important in premature skin aging, a process accordingly termed photoaging. Over
recent years, substantial progress has been made in elucidating the underlying molecular
mechanisms. From these studies, it is now clear that both UVB (290–320 nm) and UVA
(320– 400 nm) radiations contribute to photoaging. UV induced alterations at the level
of the dermis are best studied and appear to be largely responsible for the phenotype of
photoaged skin. It is also generally agreed that UVB acts preferentially on the epidermis
where it not only damages DNA in keratinocytes and melanocytes, but also causes the
production of soluble factors including proteolytic enzymes, which in a second step
affect the dermis; in contrast, UVA radiation penetrates far more deeply on average and
hence exerts direct effects on both the epidermal and the dermal compartments
(Makrantonaki et al, 2007). UVA is also 10–100 times more abundant in sunlight than
UVB, depending on the season and time of the day. Therefore, it has been proposed that,
although UVA photons are individually far less biologically active than UVB photons,
UVA radiation may be at least as important as UVB radiation in the pathogenesis of
photoaging (Berneburg et al., 2000). The exact mechanisms by which UV radiation
causes premature skin aging is not yet clear, but a number of molecular pathways
explaining one or more of the key features of photoaged skin have been described. Some
of these models are based on irradiation protocols, which use single or few UV
exposures, whereas others take into account the fact that photoaging results from chronic
27
UV damage, and as a consequence employ chronic repetitive irradiation protocols. Still
others rely on largely theoretical constructs rather than experimental observations.
In the tropic a major concern is skin damage due to the exposure to ultraviolet
irradiation that leads to premature skin ageing. There are several products in the market
that address this condition. The most popular topical product is based on retinoic acid or
retinol. These products are available as prescription products as they are known to cause
severe irritation and promote skin sensitivity to sun light and other non-irritating
cosmetic materials. In addition retinoic acid and retinol are not stable, easily oxidized in
the presence of oxygen and require special delivery such as encapsulation.
Other
product is based on Alpha-hydroxy acids (AHA). AHA based products unfortunately
require very low pH environment to be active. A very low pH formulation is known to
cause irritation on skin.
Anti-ageing products continue to become very popular and show very high
market value throughout the world regardless of the climate condition as people starts to
believe the potential of this product to enhance their skin health and to some extend
delay aging. Instead of using chemical derived ingredients, consumers are increasingly
concerned about the safety and purity of the products they use, as well as their effect on
the environment. Products that claim to be "natural", "organic" or eco-friendly are
growing in popularity across regions, and eventually becoming one of the mainstream
products. However, the truth behind the real ingredients used to formulate the products
remain unanswered as most product developers tend to claim of using natural product as
the main ingredient but in reality the concentration of bioactive ingredient derived from
the plant is not in significant amount.
28
Previous study being done on the Labisia pumila plant extract clearly displayed
the potential of this plant extract to be used as a photo protective agent against extrinsic
ageing (Choi et al., 2010). Recent studies have shown the ability of this plant extract to
stimulate growth of the human skin fibroblast cells in vitro after exposure to ultraviolet
(UV) radiation. Apart from that, Labisia pumila could also up regulate the synthesis of
collagen in human dermal fibroblast cells which is responsible for the integrity and
mechanical strength of the human skin (Choi et al., 2010). Labisia pumila plant extract
also has the ability the protect the human skin from continuous attack of the reactive
oxygen species (ROS) generated by critical UVB exposure, this is due to the present of
bioflavonoids and phenolic acids contents in the plant extract (Siavash et al., 2011).
Exposure of skin to UV radiation (UVR) results in skin modification including
erythema, tanning, immunomodulation and possibly cancerogenesis (Michael et al.,
2004). These clinical responses are dose-dependent, they are not necessarily linked to
one another by a direct cause-effect relationship, but may be the consequence of the
pleiotropic behaviour of UV radiation and have been described in detail in the literature
(Berneburg et al., 2000). Biochemical analysis of biological material exposed to UVR
has allowed one to identify some of its major targets such as DNA, proteins and
component of the extracellular matrix (ECM) including cellular lipids (Punnonen et al,.
1991). Results from experiments performed in vivo as well as in vitro seem to indicate
that
UV
damage
triggers
erythema,
immunodepression,
melanogenesis
and
carcinogenesis as well (Michael et al., 2004). The histological analysis of skin subjected
to ultraviolet radiation allowed one to point out that some cells in the epidermis undergo
dramatic modifications consisting of periplasmic edema, shrunken cytoplasm and
pyknotic nucleus. Chronic exposure to UV radiation also could cause significant
morphological modifications including cell retraction, surface blebbing, apoptosis and
cytoskeleton rearrangement (Michael et al., 2004).
29
From the results of these studies, it indicates that Labisia pumila has a major
potential to be used as one of the active ingredients in cosmeceutical products due to its
phytoestrogenic properties and the ability to delay photoaging. The results presented in
this thesis will further support the initial claim and will lead to a more advanced research
on Labisia pumila plant extract.
2.4
Human Skin Fibroblasts
Fibroblasts (L. Fibra, fiber; Gk. blastos, germ), a spindle-shaped cell is the
primary cell type in the dermis of the skin. Dermal fibroblasts are an essential
component of the skin; they not only produce and organize the extracellular matrix of
the dermis but they also communicate with each other and other cell types, playing a
crucial role in regulating skin physiology. Other resident cells include epidermal ,
vascular, and neural cells (Ansel et al, 1996; Detmar, 1996; Werner and Simola, 2001).
In addition, skin also contain various cells of hematopoietic origin that includes
monocytes/macrophages, neutrophils, and lymphocytes (Nestle and Nickoloff, 1995;
Gonzalez-Ramos et al, 1996; Lugovic et al, 2001). All parts of the fibroblasts cells
surface have the ability to release extracellular matrix (ECM) constituents, which
include procollagen, tropoelastin, proteoglycan and the glycoprotein that forms the
microfibrillar scaffolding of the elastic fibres. Fibroblasts also produce collagenase for
their own internal use in breaking down collagen that they ingest (David. 1993).
During wound healing, fibroblasts, monocytes, macrophages, lymphocytes and
endothelial cells are co-ordinately regulated in a cascade of cellular responses (Minuth,
2005; Martin, 1997). These type of cells are attracted to the wound sites by the localized
release of growth factors or known as cytokines such as platelet-derived growth factor
(Pierce et al, 1991). After migrating to the site, fibroblasts will synthesize and reorganize
30
the ECM components. Large quantities of collagen are produced by fibroblasts (Lam,
1999). In addition, fibroblasts are capable of secreting factors for growth promotion
(Goulet et al, 1996), differentiation of keratinocytes cells and on the deposition of
basement proteins (El Ghalbzouri, 2002).
The use of the term “fibroblasts” is very common when describing a cell types
regardless of its tissue origin. This is because the presence of fibroblasts cell is not only
confined to the human skin, but it can also be found in various tissues. However, the
fibroblast-like cell populations derived from other types of tissues will show a difference
characteristics when cultured in vitro. Tissue-specific differences were found between
skin fibroblasts and non-skin fibroblasts (Lemonnier et al, 1980). In this particular study,
human skin fibroblast is used as a mean to complete the research. There are two types of
cells used for in vitro studies, the primary cell and the human skin fibroblast cells that
are obtained from a specific cell lines. In this study, the human skin fibroblast used is
obtained from a cell line known as HSF 1184. Most primary cells have limited lifespan
unlike cell line that has acquired the ability to proliferate indefinitely either through
random mutation or deliberate modification, such as artificial expression of the
telomerase gene.
When fibroblasts first dissociated from the tissue, the cells are spherical but
elongate to spindle shape on attachment to a solid surface. Fibroblasts have excellence
growth characteristics and that is one of the most important reason why fibroblasts have
been the most favoured cells to be used in cell cultures or in vitro studies. Fibroblasts
can adapt easily in the culture environment and have growth rates with a doubling time
of 18-24 hours (Butler, 2004).
31
2.5
Collagen
Collagen is found in all of our connective tissues, such as dermis, bones, tendons,
and ligaments, and also provides for the structural integrity of all of our internal organs
(Burgeson, 1987; Prockop, 1998). Therefore, because of its wide distribution throughout
our bodies, it represents one of the most abundant naturally occurring proteins on earth
(Van der Rest et al, 1991). In addition to its natural abundance, there are well over 1,000
commercial products on the market today that contain collagen and collagen enhancers.
These products are represented by body and hand lotions, nail treatments, firming gels,
wrinkle injections, eye pads, and even anti-cancer treatments to name but a few. In
recent years, new high-tech wound dressing materials and skin substitutes have become
available for the treatment of partial-thickness injuries as well as full-thickness and
chronic dermal ulcers.
Figure 2.2
The location of collagen in human skin (Elizabeth et al, 2011)
32
There are close to 20 different types of collagen found in our bodies (Prockop et
al, 1995; Miller and Gay, 1987). Each one of these collagens is encoded by a specific
gene. The predominant form is Type I collagen (K. A. Piez., 1985). This fibrillar form of
collagen represents over 90 percent of our total collagen and is composed of three very
long protein chains. Each protein chain is referred to as an "Alpha" chain. Two of the
Alpha chains are identical and are called Alpha-1 chains, whereas the third chain is
slightly different and is called Alpha-2. The three chains are wrapped around each other
to form a triple helical structure called a collagen monomer(K. A. Piez., 1985). This
configuration imparts tremendous strength to the protein. To understand the overall
structure of the collagen molecule, think of it as the reinforcement rods called re-bar that
are used in concrete construction. Indeed if one converts the molecular dimensions of
the collagen molecule to measurements that we can relate to, the molecule when scaled
up would measure one inch in diameter to approximately 17 feet long(K. A. Piez.,
1985). Therefore, collagen is indeed nature's re-bar, because it is responsible for the
strength and integrity of all of our connective tissues and organ structures (M.E Nimni
and Harkness al 1988). Basically all of the collagens share this triple-helical molecular
structure as described above. However, the various other types of collagens have slightly
different amino acid compositions and provide other specific functions in our bodies
(E.J, Kucharz, 1992, and E.J Miller, 1988). Type II collagen is the form that is found
exclusively in cartilaginous tissues. It is usually associated with proteoglycans or
"ground substance" and therefore functions as a shock absorber in our joints and
vertebrae. Type III collagen is also found in our skin as well as in blood vessels and
internal organs. In the adult, the skin contains about 80-percent Type I and 20-percent
Type III collagen (K. A. Piez., 1985). In newborns, the Type III content is greater than
that found in the adult. It is thought that the supple nature of the newborn skin as well as
the flexibility of blood vessels is due in part to the presence of Type III collagen. During
the initial period of wound healing, there is an increased expression of Type III collagen
(Clore et al, 1979).
33
Type IV collagen is found in basement membranes and basal lamina structures
and functions as a filtration system. Because of the complex interactions between the
Type IV collagen and the noncollagenous components of the basement membrane, a
meshwork is formed that filters cells as well as molecules and light (E.J. Miller 1984).
For example, in the lens capsule of the eye, the basement membrane plays a role in light
filtration. In the kidney, the glomerulus basement membrane is responsible for filtration
of the blood to remove waste products. The basement membrane in the walls of blood
vessels controls the movement of oxygen and nutrients out of the circulation and into the
tissues. Likewise, the basal lamina in the skin delineates the dermis from the epidermis
and controls the movement of materials in and out of the dermis (Wolfgang Friess,
1997).
Type V collagen is found in essentially all tissues and is associated with Types I
and III. In addition it is often found around the perimeter of many cells and functions as
a cytoskeleton. It is of interest to note that there appears to be a particular abundance of
Type V collagen in the intestine compared to other tissues (Graham et al, 1988).
2.6
Collagen Synthesis
The biosynthetic pathway responsible for collagen production is a very complex
one (Prockop et al, 1979; Kivirikko and Risteli, 1976). Each specific collagen type is
encoded by a specific gene; the genes for all of the collagen types are found on a variety
of chromosomes. As the messenger RNA (mRNA) for each collagen type is transcribed
from the gene, or DNA "blueprint," it undergoes many processing steps to produce a
final code for that specific collagen type. This step is called mRNA processing. Once the
final pro-alpha chain mRNA is produced, it attaches to the site of actual protein
synthesis. This step of the synthesis is called translation. This site of pro-alpha chain
34
mRNA translation is found on the membrane-bound ribosomes also called the rough
endoplasmic reticulum or rER. Like most other proteins that are destined for function in
the extracellular environment, collagen is also synthesized on the rER.
A precursor form of collagen called procollagen is produced initially (Bellamy and
Bornstein, 1971). Procollagen contains extension proteins on each end called amino and carboxy
procollagen extension propeptides. These nonhelical portions of the procollagen molecule make
it very soluble and therefore easy to move within the cell as it undergoes further modifications.
As the collagen molecule is produced, it undergoes many changes, termed post-translational
modifications (Prockop et al, 1979; Kivirikko and Risteli, 1976). These modifications take place
in the Golgi compartment of the ER.
Collagen, like most proteins that are destined for transport to the extracellular spaces for
their function or activity, is produced initially as a larger precursor molecule called procollagen
(Bellamy and Bornstein, 1971). Procollagen contains additional peptides at both ends that are
unlike collagen. On one end of the molecule, called the amino terminal end, special bonds called
disulfide bonds are formed among three procollagen chains and insure that the chains line up in
the proper alignment. This step is called registration. Once registration occurs, the three chains
wrap around each other forming a string-like structure.
One of the first modifications to take place is the very critical step of hydroxylation of
selected proline and lysine amino acids in the newly synthesized procollagen protein. Specific
enzymes called hydroxylases are responsible for these important reactions needed to form
hydroxyproline and hydroxylysine. The hydroxylase enzymes require Vitamin C and Iron as
cofactors (Mussini et al, 1967). If a patient is Vitamin C deficient, then this reaction will not
occur. In the absence of hydroxyproline, the collagen chains cannot form a proper helical
structure, and the resultant molecule is weak and quickly destroyed (Bienkowski et al, 1978).
The end result is poor wound healing, and the clinical condition is called scurvy (Hirschmann
and Raugi, 1999).
35
Some of the newly formed hydroxylysine amino acids are glycosylated by the
addition of sugars, such as galactose and glucose (Anttinen et al, 1978). The enzymes
that catalyze the glycosylation step, galactosyl and glucosyl transferases, require the
trace metal manganese (Mn+2). The glycosylation step imparts unique chemical and
structural characteristics to the newly formed collagen molecule and may influence fibril
size (Kivirikko and Myllyla, 1979). It is of interest to note that the glycosylation
enzymes are found with the highest activities in the very young and decrease as we age
(Anttinen et al, 1978).
While inside the cell and when the procollagen peptides are intact, the molecule
is about 1,000 times more soluble than it is at a later stage when the extension peptides
are removed (Prockop et al, 1979). This high degree of solubility allows the procollagen
molecule to be transported easily within the cell where it is moved by means of
specialized structures called microtubules to the cell surface where it is secreted into the
extracellular spaces (Diegelmann and Peterkofsky, 1972). As the procollagen is secreted
from the cell, it is acted upon by specialized enzymes called procollagen proteinases that
remove both of the extension peptides from the ends of the molecule (Lapiere et al,
1971). Portions of these digested end pieces are thought to re-enter the cell and regulate
the amount of collagen synthesis by a feed-back type of mechanism (Lichtenstein et al,
1973; Wiestner et al, 1979). The processed molecule is referred to as collagen and now
begins to be involved in the important process of fiber formation.
In the extracellular spaces, another post-translational modification takes place as
the triple helical collagen molecules line up and begin to form fibrils and then fibers.
This step is called crosslink formation and is promoted by another specialized enzyme
called lysyl oxidase (Bailey et al, 1974). This reaction places stable crosslinks within
(intramolecular crosslinks) and between the molecules (intermolecular crosslinks). This
is the critical step that gives the collagen fibers such tremendous strength. On a per
weight basis, the strength of collagen approaches the tensile strength of steel. One can
36
visualize the ultrastructure of collagen by thinking of the individual molecules as a piece
of sewing thread. Many of these threads are wrapped around one another to form a string
(fibrils). These strings then form cords; the cords associate to form a rope, and the ropes
interact to form cables(Bailey et al, 1974). The structure is just like the steel rope cables
on the Golden Gate bridge. This highly organized structure is what is responsible for the
strength of tendons, ligaments, bones, and dermis.
When the normal collagen in our tissues is injured and replaced by scar collagen,
the connective tissue does not regain this highly organized structure. That is why scar
collagen is always weaker than the original collagen. The maximum regain in tensile
strength of scar collagen is about 70 to 80 percent of the original (Schilling, 1968).
Collagen synthesis and remoulding continue at the wound site long after the injury. The
body is constantly trying to remodel the scar collagen to achieve the original collagen
ultrastructure that was present before the injury. This remodelling involves on-going
collagen synthesis and collagen degradation. Anything that interferes with protein
synthesis will cause the equilibrium to shift, and collagen degradation will be greater
than collagen synthesis. For example, patients who are malnourished or patients
receiving chemotherapy may experience wound dehiscence, because the wound site will
become weak due to a shift in the balance toward collagen degradation. It is of interest
to note that when wounds in the fetus heal, they do so in such a manner that the original
collagen ultrastructure is achieved (Mast et al, 1991). If only we understood more about
the biology and mechanisms responsible for the rapid and optimal wound healing
response seen in the fetus, we would have greater insight into the management of adult
wounds (Mast et al, 1992).
37
2.7
Collagen Degradation
Of equal importance in the total picture of collagen metabolism is the complex
process of collagen degradation. Normally, the collagen in our connective tissues turns
over at a very slow and controlled rate. However, during rapid growth and in disease
states, such as arthritis, cancer, and chronic non healing ulcers, the extent of collagen
degradation can be quite extensive. In normal healthy tissues where the collagen is fully
hydroxylated and in a triple helical structure, the molecule is resistant to attack by most
proteases. Under these normal healthy conditions, only specialized enzymes called
collagenases can attack the collagen molecule (Gross et al, 1974). The group of
collagenases belong to a family of enzymes called matrix metalloproteinases or MMPs.
Many cells in our bodies can synthesize and release collagenase including
fibroblasts, macrophages, neutrophils, osteoclasts, and tumor cells. One of the reasons
that some neoplastic cells can be so invasive is because they release potent collagenases
and can break down the collagen around them. Then they can break down the basement
membranes of blood vessels and spread throughout the body. In chronic pressure ulcers,
there is a massive invasion of neutrophils, and they release a very potent collagenase
called MMP-8 that is responsible for connective tissue breakdown (Yager et al, 1996).
Some exciting new research suggests that members of the tetracycline family of
antibiotics, such as doxycycline, when given systemically, may be useful to treat
pressure ulcers, because at low doses, they inhibit MMP-8 (Golub et al, 1998).
38
2.8
Collagenase Assay
In this study, Sircol collagenase assay was used to investigate the effect of the
Labisia pumila plant extract on collagen synthesis of the HSF1184 cells. Sircol
collagenase assay is a type of biological assay currently being recommended to use for
collagen synthesis analysis in cell culture studies. Bioassay or also known as biological
standardization is a type of scientific experiment conducted to measure the effect of a
substance on a living organism (Levine, 1973., Jamall et al, 1981). Bioassays are
normally used to determine the concentration or biological activity of a substance or
metabolites such as vitamin, hormone, proteins and plant growth factor.
Collagen contains the unique amino acid hydroxyproline (HyPro) which is
involved in the stabilization of the triple helical molecule (Ricky et al, 2010). In order to
calculate the total collagen content in a cell culture environment, normally the
concentration of HyPro is frequently used. Previously, several methods of calculating
the collagen content in cell culture environment has been developed, those methods
include, radiolabelling, chromatographic and calorimetric assays (Leroy et al, 1966,
Light ND, 1985). All these methods are developed based on HyPro estimation to ensure
accurate determination of collagen content. Recently, the Sircol Collagen Assay (SCA;
Biocolor Ltd., Northern Ireland) has been introduced. This method is a more convenient
method for quantification of collagen content in cell culture environment. The
development of SCA is based on the amino acid binding property of Sirius red (SR F3B;
CI 35782), an anionic dye with sulphonated acid side-chain groups that react with the
side-chain groups of basic amino acids (Sweat et al, 1964, Constantine and Mowry,
1968). Nowadays, SR is widely used as a selective histochemical stain for collagen in
normal and pathological tissue analysis (Kratky et al, 1996, Brotchie et al, 1999, Borges
et al, 2007, Choi et al, 2010). Although there are some debates on the reliability of the
SR method as discussed by Ricky et al, 2010, this method has continued to be the most
popular method for collagen content quantification.
39
2.9
Ultraviolet (UV) Radiation
Ultraviolet radiation (UV) is part of the electromagnetic (light) spectrum that
originates from the sun. it is not visible to the naked eye because it has shorter
wavelengths than the visible light. There are mainly three types of UV radiation namely
UVA, UVB and UVC. UVA has the longest wavelength (320-400nm) and is further
divided into two subtypes, UVA I and UVA II. The wavelength of UVB ranges from
290-320nm while UVC has the shortest wavelength. Therefore most UVC will be
absorbed by the ozone layer and rarely reach the surface of the earth (Berneburg et al,
2000).
Figure 2.3
Visible light (Skin Cancer Foundation, 2011)
40
Both UVA and UVB can penetrate the ozone layer and play a very important
role in conditions such as premature skin aging, eye damage (including cataracts), and
skin related cancers (Brash et al, 1999). They could also cause immune system
suppression which will reduce the ability of our body defence mechanism to fight these
and other maladies. UVA is normally related to the occurrence of skin cancer (Michael
et al, 2004) whereas UVB is known to be one of the cause of photoaging that leads to
premature skin aging in human (Gail, 2002)
Figure 2.4
UVA and UVB penetrating effect on the human skin (Skin Cancer
Foundation, 2011)
41
According to figure 2.4, UVA has better penetrating effect compared to UVB.
Therefore UVA can penetrate deeper in the skin than UVB. Although UVA is able to
penetrate deep until the dermis of the skin, most studies over the past two decades has
showed that UVA normally cause damages to the keratinocytes cells located at the
epidermis of the skin, and this where most skin cancer occurs (Michael et al, 2004,
Brash et al, 1999). UVB as displayed in figure 2.4, is only able to penetrate until the
epidermis layer of the skin, thus it tends to damage the skin’s more superficial part.
Some of the most popular symptoms of the skin damage caused by UVB exposure
include skin reddening, and sunburn. Chronic exposure to UVB radiation could cause
photoaging which accelerates the normal skin ageing. This is one of the most important
problem that plagued people who lives in tropical climate as they are exposed to sunlight
longer than any other people living in different part of the world (Helen, 2008)
As mentioned in chapter 1, extrinsic ageing is primarily caused by chronic
exposure to ultraviolet radiation especially UVB radiation. According to Gilchrest, 1996,
almost 80% of facial ageing is attributable to sun exposure. Photoaged skin is clinically
characterized by loss of elasticity, increased roughness and dryness, irregular
pigmentation that cause the formation of black spots, and deep wrinkling (Kligman and
Kligman, 1986). The exact mechanisms by which UV radiation causes premature skin
aging is not yet clear, but a number of molecular pathways explaining one or more of the
key features of photoaged skin have been described. Some of these models are based on
irradiation protocols, which use single or few UV exposures, whereas others take into
account the fact that photoaging results from chronic UV damage, and as a consequence
employ chronic repetitive irradiation protocols. Still others rely on largely theoretical
constructs rather than experimental observations. UVB could cause damage to the three
principle components of the skin namely, collagen fibres, the elastic fibre network and
the glycosaminoglycans (Oxlund and Andreassen, 1980). The disruption of these
structures will cause the loss of integrity to the mechanical strength of the skin thus
leading to skin ageing.
42
In principle it is reasonable to assume that UV radiation leads to an enhanced and
accelerated degradation and/or a decreased synthesis of collagen fibers, and current
knowledge indicates that both mechanisms may be involved (Rittie and Fisher, 2002). A
large number of studies unambiguously demonstrate that the induction of matrix
metalloproteinases (MMPs) plays a major role in the pathogenesis of photoaging. As
indicated by their name, these zinc-dependent endopeptidases show proteolytic activity
in their ability to degrade matrix proteins such as collagen and elastin. There are
different types of collagen (proteins) available in the skin and each MMP degrades
different dermal matrix proteins; for example, MMP-1 cleaves collagen types I, II, and
III, whereasMMP-9, which is also called gelatinase, degrades collagen types IV and V
and gelatin (Fisher et al, 1998). MMPs are regulated by their endogenous inhibitor called
tissue inhibitor metalloproteinase (TIMPs). UV radiation will stimulate overproduction
of MMPs, therefore it can cause imbalance between MMPs and TIMPs. As more MMPs
are available in the extracellular matrix (ECM) of the cells, degradation of certain types
of collagens will be increased. TIMPs production are not influenced by the exposure to
UV radiation, therefore while MMPs production increase in response to UV radiation
the production of TIMPs remain the same thus leading to the imbalance which proves to
be very damaging to the skin (Fisher et al, 1998). In a very simplified scheme, UVA
radiation would mostly act indirectly through the generation of reactive oxygen
species(ROS), in particular, singlet oxygen, which can subsequently exert a multitude of
effects such as lipid peroxidation, activation of transcription factors, and generation of
DNA strand breaks (Fisher et al, 1998).While UVB radiation induced MMP induction
has been shown to involve the generation of ROS as well (Scharffetter-Kochanek et al,
2000), the main mechanism of UVB is by inducing DNA damage. Recent studies have
indeed provided evidence that enhanced repair of UVB-induced cyclobutane pyrimidine
dimers in the DNA of epidermal keratinocytes through topical application of liposomally
encapsulated DNA repair enzymes on UVB-irradiated human skin prevents UVB
radiation-induced epidermal MMP expression. (Wenk et al, 2001). The activity of
MMPs is tightly regulated by transcriptional regulation and elegant in vivo studies by
Fisher et al (2000) has demonstrated that exposure of human skin to UVB radiation
leads to the activation of the respective transcription factors (Dong et al, 2008).
43
Accordingly, UV exposure of human skin not only leads to the induction of
MMPs within hours after irradiation, but already within minutes, transcription factors
AP-1 and NF-kB, which are known stimulatory factors of MMP genes, are induced
(Rittie and Fisher, 2002). These effects can be observed at low UVB dose levels,
because transcription factor activation and MMP-1 induction can be achieved by
exposing human skin to one tenth of the dose necessary for skin reddening (0.1 minimal
erythema dose). Subsequent work by the same group clarified the major components of
the molecular pathway by which UVB exposure leads to the degradation of matrix
proteins in human skin (Rittie and Fisher, 2002). Low-dose UVB irradiation induces a
signalling cascade, which involves up regulation of epidermal growth factor receptors
(EGFR), the GTP-binding regulatory protein p21Ras, extracellular signal-regulated
kinase (ERK), c-jun aminoterminal kinase (JNK), and p38. Elevated c-jun together with
constitutively expressed c-fos increases activation of AP-1. Identification of this UVBinduced signaling pathway not only unravels the complexity of the molecular basis,
which underlies UVB radiation-induced gene expression in human skin, but also
provides a rationale for the efficacy of tretinoin (all trans- retinoic acid) in the treatment
of photoaged skin. Accordingly, topical pre-treatment with tretinoin inhibits the
induction and activity of MMPs in UVB-irradiated skin through prevention of AP-1
activation. In addition to destruction of existing collagen through activation of MMPs,
failure to replace damaged collagen is thought to contribute to photoaging as well.
Accordingly, in chronically photo damaged skin, collagen synthesis is down regulated as
compared to sun-protected skin (Fisher et al, 1997). The mechanism by which UV
radiation interferes with collagen synthesis is not yet known, but a recent study has
provided evidence that fibroblasts in severely (photo) damaged skin have less interaction
with intact collagen and are thus exposed to less mechanical tension, and it has been
proposed that this situation might lead to decreased collagen synthesis (Fisher et al,
2000).
44
2.10
Cellular Senescence
Most if not all human cells irreversibly arrest growth with a peculiar large and
flat cell morphology after a limited number of cells divisions in culture (Dimri, 2005;
Itahana et al, 2004).This process known as cellular senescence was first described by
Hayflick and colleagues in cultured human fibroblasts (Hayflick and Moorhead, 1961).
Since the time of Hayflick’s discovery, cellular senescence has been described in other
cell types such as melanocytes, epithelial cells, keratinocytes, and endothelial cells. It is
likely that in higher organisms, all cell types capable of undergoing mitotic divisions
undergo cellular senescence in culture and possibly in vivo. Since the predominant cause
of cellular senescence in culture appears to be mitosis or repetitive cell divisions,
cellular senescence is also described as replicative senescence (Dimri, 2005; Itahana et
al, 2004).
It is thought that cellular senescence in culture reflect an aging process in vivo,
and hence is also known as cellular aging (Itahana et al, 2004). Human cells have linear
chromosomes, each chromosomes shortens from its ends, or telomeres, during every
round of cell division. Because human somatic cells lack enzyme telomerase, which
rebuilds telomere ends, human chromosomes keep shortening, eventually sending a
DNA damage signal to cells and withdrawing permanently from the cell cycle, leading
to replicative senescence (Dimri, 2005). Recent evidence suggests that cells also
undergo senescence in response to various stress signals (Dimri, 2005), such as
inappropriate activation of oncogenes, strong mitogenic signals, direct DNA damage
caused by genotoxic agents and radiation, and chromatin remodelling agents (Itahana,
2004). Cellular senescence induced by stress signals is known as premature or
accelerated senescence. Thus cellular senescence refers to replicative and premature
senescence, the former type of senescence is caused by primarily by stress causing
agents that might include UV irradiation. Both type of senescence display overlapping
phenotypes and may be equally important for tissue or organ aging in organism.
45
Different proteins are involved in the maintenance and generation of senescence
phenotype. One such protein is p53, a tumor suppressor that is mutated in large number
of human cancers. It is a transcription factor and acts as a tumor suppressor, in part by
inducing its target such as p21 protein, which is an inhibitor of cell cycle progression. In
human fibroblasts, dysfunctional telomeres signal via tumor suppressor p53 and its
target p21 to stop cell proliferation and set up the early stage of cellular senescence. The
late stage of cellular senescence is maintained by retinoblastoma tumor suppressor pRb,
which is also mutated in human cancers. pRb acts via another cell cycle regulatory
protein p16 which blocks activity of cyclin dependent kinases (CDKs), and allows cells
to permanently withdraw from the cell cycle. Stress signals also induce senescence via
p53-p21 and/or pRb-p16 pathways (Dimri, 2005).
As described above, various cell types such as fibroblasts, keratinocytes,
melanocytes, and Langerhans cells are integral part of the human skin, and they all
undergo cellular senescence when cultured in vitro. It is very likely that cellular
senescence of these different cell types contributes to overall deterioration seen in aging
skin.
2.11
Possible Contribution of Cellular Senescence to Skin Aging
Cellular senescence is accompanied by several undesirable changes in gene
expression, which can directly or indirectly influence and exacerbate skin aging. Skin
aging has two components, extrinsic and intrinsic aging (Gilchrest, 1996; Jenkins, 2002).
The major cause of extrinsic aging is UV induced damage to skin during exposure to
sun. Because of its relation to sun exposure, extrinsic aging is also known as photoaging.
On the other hand, intrinsic or chronological aging is caused by progressive time
dependent changes in skin tissues. This type of aging is complex and has multiple
46
genetic and epigenetic components, which are common to other bodily tissues. In
mitotically active somatic cells, these genetic and epigenetic changes occurring over
time may results in cellular senescence. Since skin contains mitotically active or mitosis
competent cells, it is very likely that cellular senescence and gene expression changes
associated with it directly contribute to aging of skin.
Cellular senescence-associated genes contribute to intrinsic aging of skin
primarily by effecting extracellular matrix (ECM) structure. As described above, activity
of two key matrix metalloproteinases MMP1 (Collagenase) and MMP3 (Stromelysin) is
substantially up regulated in senescence fibroblasts, while activity of TIMP1 and TIMP3
is reduced due to down regulation of genes encoding these proteins. This imbalance of
TIMPs and MMPs, coupled with low collagen and low elastin biosynthesis by senescent
cells can cause dermal thinning and increased wrinkling as seen in aging skin.
This differential gene expression pattern of senescent cells can also augment the
effects of photoaging by further increasing the activities of MMPs that are also induced
by UV irradiation. In fact, it has been shown that chronic UV irradiation induces
premature senescence accompanied by upregulation of MMP1 in dermal fibroblasts.
Thus, accelerated senescence in skin cells by UV irradiation may also directly contribute
to photoaging (Naru et al, 2005). Furthermore, the reduced proliferative capacity of
senescent cells due to down regulation of cell cycle regulatory genes ensures that dermal
repair is impaired and cells that are lost due to excessive photoaging are not replenished.
Progressive depletion of skin stem cell pool, which has been postulated to occur
with advanced age possibly due to induction of cellular senescence, may also affect skin
healing and exacerbate intrinsic and extrinsic aging.
47
2.12
Cell Culture
Cells, when removed from the human body will continue to grow if supplied
with nutrients and growth factors. The growth of human cells outside human body is
commonly known as cell culture. The cells are capable of division by mitosis and the
cell population can continue to grow. The growth of cells in cell culture is limited by
specific parameters such as nutrient depletion and availability of growth factor. Cell
culture is quite different from culture of the whole organ. Organ culture is the
maintenance of the whole organs or fragments of tissue with the retention of a balance
relationship between the associated cell types that exist in vivo (Butler, 2004).
Cell cultures are performed for various purposes including:
x
To investigate the normal physiology and biochemistry of a specific cell. For
example, metabolic pathways can be investigated by applying radioactively
labelled substrates and subsequently looking at product formation.
x
To investigate the effect of a certain compound of the physiology of the cells.
This could be a natural product like Labisia pumila that is used for this study.
x
To produce artificial tissue by combining specific cell types in sequence. This
method has been applied in the production of artificial human skin.
x
To synthesize valuable biological products from large scale cell cultures.
2.12.1 Cell Growth and Maintenance
Quantification of cell growth is important in routine cell culture maintenance in
order to monitor the consistency of the culture and to determine the best to perform
subculture, optimum dilution and the estimated plating efficiency at different cell
densities (Freshney, 2000).
48
2.12.2 Inoculation
Once cells are isolated from tissue, a culture can be initiated by inoculating the
cells into sterile growth environment. This method is true for primary cell. For cell line
which is used for the purpose of this study, the method for inoculation is easier as
extraction of cells from the a primary donor is not required to be done. Nowadays, cell
line can be directly obtained from authorised supplier as a lot of cell lines are available
for research purposes.
Human skin fibroblast from cell line HSF1184 is an anchorage dependent cell
type which will attach to an available growth surface within a few hours of inoculation.
The attachment process involves the flattening and spreading of cells into a
characteristic shape (Butler, 2004).
2.12.3 Subculture
When cells stop growing in culture, new culture can be established by
inoculating some of the cells into fresh medium. This method is called subculturing or
passaging. Cultures are given a passage number which indicates the number of
subcultures performed since the cells were obtained or isolated (Butler, 2004).
Subculture should not be performed when cells are still within the lag period, and cells
should always be taken between the middle of the log phase and the time before which
they have entered the stationary phase (Freshney, 2004). The viability of the cells will be
seriously disrupted if cells are left in the culture environment for too long (Butler,
2004).s
49
The subculture of an anchorage dependent cell involves detachment of the cells
from the growth surface of one culture flask and reinnoculation of the cells into a new
culture flask containing newly fresh medium.
2.13
Phases in Cell Culture
The lag phase is an adaptation period of cells to the new environment after
subculture or reseeding, where there is no apparent increase in cell concentration or cell
density. This phase is associated with the cellular synthesis of growth factors which may
be required to reach a critical concentration before growth can actually take place
(Butler, 2004), replacing elements of the cell surface and extracellular matrix; attaches
to the substrate and spread out (Freshney, 2004). The length of this phase is dependent
on the culture medium formulation as well as the initial concentration and state of the
cells (Butler, 2004).
The log phase is the period of exponential increase in the cell number and
terminating in one or two population doubling time (PDT) after confluency is reached.
The length of this phase depends on the seeding concentration, the growth rate of the
cells, and the concentration that inhibits cell proliferation. PDT is the time taken for the
culture to increase by twofold in the middle of the log phase growth period. It is used to
quantify the response of the cells to different inhibitory or stimulatory culture conditions
(Freshney, 2004).
50
The stationary phase occurs when there is no further increase in cell
concentration. Death rate and growth rate are similar during this phase. Even though the
net growth is zero, cells may still be metabolically active. The reason for limited growth
at this phase may be due to either depletion of nutrients or accumulation of waste
products that are toxic to the cells (Butler, 2004).
The death phase follows the stationary phase, and occurs as a result of cell death.
The measured viable cell concentration decreases as the cells lyse and their intracellular
metabolites are released into the growth medium. There are two possible mechanisms of
cell death in culture, apoptosis or necrosis (Butler, 2004).
Knowledge of the growth state of a culture is vital when designing cell culture
study. Cultures vary significantly in many of their properties between the phases. The
status of the culture during the initial part of the experiment and at the time of sampling
should be taken into account. Generally, cell cultures are mostly consistent and grow
uniformly during the log phase and sampling at the end of the log phase will give the
highest yield and greatest reproducibility. Adding a drug in the middle of the log phase
and assaying later may give different results, depending on whether the culture is still sin
log phase or has entered stationary phase (Freshney, 2004).
51
Figure 2.5
Phases of cell culture (Freshney, 2004)
CHAPTER 3
MATERIALS AND METHODS
3.0
Introduction
This chapter describes the materials and the methodology used while
implementing the studies. All methods carried out during the experiments are explained
in greater details in this chapter.
Figure 3.1
Cell line (Human Skin
Fibroblast)
HSF 1184
Preparation
3.1 Research Design
The design of the overall experimental procedures
3. Potential of Labisia pumilaon
the stimulation of collagen
synthesis in HSF1184 cells
Addition of Labisia
pumila extract on the
cultured HSF1184 cells
2. The difference effect of
Labisia pumila extract on
normal HSF1184 cells and
UVB irradiated HSF1184 cells
Methanolic extract of
Labisia pumila
Standardized water extract
of Labisia pumila
UVB Irradiation
Cytotoxicity studies
x To identify the
optimum
concentration of
Labisia pumila
extract
1. The effect of Labisia pumila
extract on the growth of UVB
irradiated HSF1184 cells
SIRCOL Collagenase
Assay
Exposure to UVB or
without UVB
Growth curve generated
x Compare the effect of
Labisia pumila extract on
UVB irradiated non UVB
irradiated HSF1184 cells
No UVB Irradiation
Exposure to UVB irradiation
Addition of Labisia pumila
extract on the cultured
HSF1184 cells
53
3.2
3.2.1
Materials
Chemicals
Antibiotic-antimycotic, penicillin-streptomycin, trypsin-EDTA (0.25% trpysin,
EDTA 4Na), phosphate-buffered salines (PBS), Collegenase assay kit, fetal bovine
serum (FBS) and Dulbeco’s Modified Eagle Medium: (DMEM) were purchased from
GIBCO®, USA. All other chemicals were obtained from Sigma-Aldrich®, USA, unless
stated otherwise. Cell culture grade chemicals and analytical grade chemicals were used
in this study depending on their appropriate application.
3.2.2
Cell culture
Cell culture assays are used extensively in biocompatibility assay and this
includes the use of cell culture systems to identify cytotoxicity.
HSF 1184 cells
(catalogue no. 90011883) (Human Skin Fibroblasts) (available from ECACC, United
Kingdom) derived from normal Caucasian human skin cells are used for the purpose of
this study.
3.2.3 Extract preparation
54
The plant extract, Labisia pumila var pumila, used in this study was procured
from Forest Research Institute Malaysia (FRIM). There are two types of Labisia pumila
extracts used in this study, the water extract and the methanolic extract. To prepare the
water extract of Labisia pumila; samples of dried, grounded Labisia pumila var pumila
were extracted with a laboratory scale extractor in water at 100 °C for 4 hours. The
extraction ratio between the dried, grounded raw material and water was 1:10 by mass.
Following extraction, the solid part was removed by filtration and the liquid part was
directly spray dried (Production Minor GEA NIRO). The inlet temperature of the spray
dryer was set at 180 °C and the outlet was set at 103 °C. To prepare the methanolic
extract of Labisia pumila; samples of dried, grounded Labisia pumila var pumila were
extracted with a laboratory scale extractor in methanol at 100 °C for 4 hours. The
extraction ratio between the dried, grounded raw material and water was 1:10 by mass.
Following extraction, the solid part was removed by filtration and the liquid part was
directly spray dried (Production Minor GEA NIRO). The inlet temperature of the spray
dryer was set at 180 °C and the outlet was set at 103 °C. The samples were then stored at
-20°C until further analysis. Both extracts were prepared from the same source. The
source and the type of preparation for the plant extracts remain consistent at every stage
of the study (Lee et al, 2011). 9 different concentrations of the water extract of Labisia
pumila and methanolic extract of Labisia pumila were used in this study.
3.3
Cell Culture Protocols
55
3.3.1
Subculture and Routine Maintenance
HSF1184 were cultured in Dulbecco’s modified essential medium (DMEM)
containing 10% fetal bovine serum (FBS) and 1% antibiotics. All cells were maintained
at 37ºC in a humidified atmosphere of 5% CO2. Cells took up to 96 hours until reaching
confluency (Freshney, 2005).
3.3.2
Cell Counting and Cell Viability
The dye exclusion test is normally used in cell culture to determine the number
of viable cells present in a cell suspension. This method is based on the principle that
live cells possess intact cell membrane which will exclude certain dyes whereas dead
cells do not. In this study, Trypan blue exclusion test using Neubauer improved brightline haemocytometer (FORTUNA®, Germany) was used to do cell counting. Trypan
blue is excluded by live cells but accumulates in dead cells. A clear coverslip was placed
on a haemocytometer slide. 50 µL of cell suspension was mixed with 50 µL trypan blue.
The cell suspension mixture was carefully transferred to the edge of the coverslip. The
number of stained (non-viable) cells and non-stained (viable) cells were counted under a
light microscope. Concentration of viable cells and the percentage of viable cells are
calculated using the formula;
Cells/ml = the average count per square x dilution factor x 10 4(3.1)
56
Total cells = cell/ml x original volume from which sample is removed (3.2)
The percentage viability is calculated as follow:
Percentage viability =
Number of unstained cells × 100
(3.3)
Total number of stained and unstained
This test was used to count cells for experimental setup and also for proliferation
analysis of the HSF 1184 cells culture in 25 cm2 T- flask (Freshney, 2005).
Figure 3.2
Haemocytometer (improved Neubauer), magnified view of the total area
of the grid showing viable cells as unstained and clear, with a refractile ring around them
and non-viable cells are dark and have no refractile ring (Freshney, 2005).
3.3.3
Cell Splitting
57
The HSF1184 cells were subcultured when the monolayer culture of the cells has
reached almost 80-90% confluency. The medium (DMEM) used in this monolayer
culture was aspirated before the t-flasks were rinsed with PBS (0.2ml/cm2). These
procedures were done in order to remove traces of serum which could cause inhibition to
the action of tyrpsin. After that, the wash solution was removed and 0.25% trypsin
EDTA (0.1ml/cm2) was added. The t-flask containing the HSF1184 cells was incubated
for 5 minutes at 37ºC. When the cells were ready, the cells began to change and
transform into rounded shapes and detached from the surface of the t-flasks. This
observation can be made by using an inverted microscope. Then, equal volume of fresh
medium was added to neutralize the trypsin. The cells were washed with PBS and cell
viability was checked with trypan blue exclusion test. Fibroblasts seeding density was
prepared by diluting the cells with the culture medium to required volume. Cells were
then incubated at 37ºC in a humidified atmosphere of 5% CO2 (Freshney, 2005).
3.3.4 Cell Cryopreservation
Healthy HSF1184 cells at log phase (exponential phase) were used for the
freezing procedure. The healthy cells needed to be trypsinized centrifuged properly
before adding the freezing medium. The freezing medium was prepared manually and
contained 90% FBS and 10% DMSO. The freezing medium was slowly added to the cell
suspension at the concentration approximately 106–107 cells/ml. of cells concentration.
Aliquots of c Cells were then aliqouted into at 1.2 ml in cryogenic vial were labelled
cryogenic vials. Before putting the HSF1184 cells into a liquid nitrogen tank, the cells
must be were kept inside a freezing container (Nalge Nunc) with the temperature of -
58
70ºC, for 24 hours for adaptation process. After 24 hours the HSF1184 cells were
transferred into a liquid nitrogen storage (Freshney, 2005).
3.3.5 Cell Recovery
HSF1184 labelled cryogenic vial containing the cells were slowly and carefully
removed from the liquid nitrogen storage in order to revive and produce a cell culture.
The HSF1184 cells were then thawed by gently agitating the vial in a water bath set at
37ºC temperature until it was completely thawed and ready to be transferred into a tflask containing DMEM as the medium. The cells were immediately transferred into a
preheated medium by using pipettes and then centrifuged. After that, the supernatant
was discarded from the centrifuge tube leaving a cell pellet. The cell pellet was
suspended in a fresh medium and the number of viable cells was calculated by using the
tryphan blue exclusion test as explained in section 3.2.2. The HSF1184 cells were then
incubated at 37ºC in a humidified atmosphere with 5% CO2 (Freshney, 2005).
59
3.4 Proliferation Analysis
3.4.1
MTT Assay
Cell proliferation is the measurement of the number of cells that are dividing in a
culture. One way of measuring this parameter is by performing MTT assay. This assay is
useful in the measurement of cell proliferation in response to a particular stimulus or
toxin and in the derivation of cell growth curves. The yellow tetrazolium dye, MTT (3(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) is reduced by metabolically
active cells, in part by the action of mitochondrial dehydrogenase enzymes, to generate
reducing equivalents such as NADH and NADPH. The resulting intracellular purple
formazan can be dissolved in acidified isopropanol and quantified by spectrophometric
means (ELISA reader). The amount of formazan formed is directly proportional to the
number of metabolically active cells.
Normally MTT assay is used for cytotoxicity studies of a drug or an extract on a
certain types of cell and in this case, the HSF1184 cell line. A monolayer culture was
trypsinized and cells in growth medium containing serum were collected. The
suspension was centrifuged at 3300 rpm for 10 minutes until cells pellet were formed.
The cells pellets were resuspended in growth medium and cells were counted. Cells
were diluted to a density of 1 x 105 cell/ml and were transferred into a 10 ml petri dish.
A 100 µl of cell suspension was then added to each well of the flat-bottomed 96-well
plate. The plates were incubated in a humidified atmosphere at 37°C for 48 hours to
ensure that the cells were in the exponential growth phase (log phase) before the
addition of the Labisia pumila extracts (Freshney, 2005).
60
In order to determine the effect of the plant extract concentration on cells
cytotoxicity, the medium from the well-plates was removed and a serial dilution was
performed on the Labisia pumila extracts starting from the highest concentration (10
mg/ml and 1 mg/ml) and the last well were left with cells and growth medium only
without the addition of the plant extracts. This well was acted as a negative control for
the experiment. Plates were then incubated for another 48 hours (Freshney, 2005).
After 48 hours, 10 µl of the MTT solution (5 mg/ml dissolved in PBS) was added
into all wells and incubated again for 4 hours in a humidified atmosphere at 37°C. After
4 hours, both the medium and the MTT solution were removed from the wells and 200µl
of DMSO was added in each wells in order to allow dissolution of the purple MTTformazan crystals.
The absorbency was measured at wavelength of 570nm and reference
wavelength 630nm with a microplate reader within one hour after adding MTT solvent.
The relative difference to control was determined by (Ahmad et al, 2005);
Relative difference to control =
Sample O. D
(3.4)
Control O. D
The MTT assay procedure for 24-well plate is similar to the 96-well plate, the
only difference is the volume of MTT solution and MTT solvent added to the well. The
volume of the solvent was 0.4ml and 0.2ml respectively. The absorbance measurement
was done by transferring 100 µl of the solution to 96-well plate and later measured by a
microplate reader.
61
3.4.2
MTT Standard Curve
Standard curve for HSF1184 cells was obtained by preparing serial dilution of
cells in culture medium starting at 3x102 to 9x104 cells/well in duplicate, incubating the
culture overnight and performing MTT assay. Cell seeding density (cells/cm2) was
plotted against absorbance.
3.4.3
Growth Profile
A growth curve or a growth profile is an empirical model normally used to
evaluate the evolution in term of quantity of a certain cell line over time. Generation of a
growth curve can be very useful as it can show the characteristics of a cell line.
Information such as the lag time, population doubling time, and saturation density could
be easily determined directly from the growth curve.
62
Figure 3.3
Cell’s growth curve (Freshney, 2000).
Figure 3.3 shows common phases involved in the development of a cell line. In
this study, the human skin fibroblast cell line which has been used as the main
experimental subject share the same model of development. There are 4 main phases of
cell developments namely, the lag phase, the exponential phase, the stationary phase and
the death phase.
In order to achieve this purpose, fifteen 25cm2 t-flasks were prepared. In each tflask, 1x104 cells were seeded to ensure all t-flask will have the same number of cells at
the start of the experiment. All the t-flasks were stored in the CO2 incubator. After 24
hours, the first t-flask was taken out of the incubator so that the number of cells in day 1
63
of the study could be determined. This procedure includes the basic cell handling
methods such as trypsinization and centrifuging the cells so that a cell pellet will be
obtained. Cell counting was done by using haemocytometer as explained in section 3.2.2
of this chapter. These procedures will be repeated for all the remaining 14 t-flasks every
day until day 15.
Initially, the growth curve was done without adding any plant extract to the cells
so that a standard growth curve of the human skin fibroblast cells could be obtained. In
order to study the effect of the plant extract on the rate of growth of the cells, the same
method was used but two batches of cells were prepared. One batch of cells was added
with the plant extract and the other batch served as the control of the study. Therefore, at
the end of the study, two growth curves were generated and compared.
3.5
Sircol Collagen Assay
The Sircol Collagen Assay is a type of biological assay used in cell culture
environment to evaluate the amount of acid and pepsin-soluble collagens and this
procedure was based on a dye-binding method. The Sircol Collagen Assay is suitable
test to be used to monitor and evaluate the amount of collagen produced in cell culture
(in-vitro). This particular method is suitable in order to detect collagen, soluble in cold
acid or pepsin, released into cell culture medium during cell growth and cell
maintenance, and collagen, soluble in cold acid or pepsin, recovered from newly formed
extracellular matrix that has been deposited onto cell culture treated plastic surfaces, (Tflasks and microwell plates).
64
Cultured HSF1184 cells, standards and reagent blanks (100µl) were mixed with
Sircol Dye reagent (1ml) in microcentrifuge tubes for 30 minutes. The collagen-dye
complex formed precipitates out of solution. The complex was packed firmly to the
bottom of the tubes by centrifugation at 12,000g for 10 minutes. Unbound dye was
removed by inverting and draining tubes. Ice-cold acid-salt wash (750µl) was gently
added to remove unbound dye from pellet surface and microcentrifuge tube. The tubes
were centrifuged at 12,000rpm for 10 minutes and the wash drained off. Bound dye is
released and dissolved via addition of alkali reagent (250µl or 1000µl depending on
pellet size) and vortex mixing for 10 minutes. Released dye is measured
spectrophotometrically at 555nm or colorimetrically with a blue-green filter using a
multiwell plate reader (Choi et al, 2010).
3.6
UVB Irradiation
Human skin fibroblast cells (HSF1184) were seeded at a density of 2×105
cells/well in 60 mm culture dishes and cultured in DMEM to 70% confluence. Cells
were then starved in serum free DMEM for 24 hours and rinsed with phosphate-buffered
saline (PBS). Exposure to UVB irradiation was performed at 312 nm, 25 or 52 mJ/cm2
by using a Philips F20T12/UV-B source (270–390 nm; containing2.6% UVC, 43.6%
UVB, 53.8% UVA), as measured with a SX-312 research radiometer (Uvitec, USA).
UVB-irradiated cells were cultured in serum free DMEM with or without Labisia
pumila extract or ascorbic acid (Sigma, USA) for 48 hours (Choi et al, 2010).
65
3.7
Statistical Analysis
Statistical analyses were performed using Sigma Plot10.0. Values were
expressed as means ± SE with three independent experiments. Statistical significance of
treatments was determined using the paired Student’s t test.
The t statistic to test whether the means are different can be calculated as
follows:
Sx1x2 is the standard deviation, 1 is group one and 2 is group two. The
denominator of t is the standard error of the difference between two means. Once a t
value is determined, a p-value can be found using a table of values from Student's tdistribution. If the calculated p-value is below the threshold chosen for statistical
significance (usually the 0.10, the 0.05, or 0.01level), the null hypothesis is rejected in
favour of the alternative hypothesis.
CHAPTER 4
RESULTS AND DISCUSSION
This chapter presents the results and discussion of the experimental work.
4.1
Human Skin Fibroblast Cell Line (HSF1184)
Human Skin Fibroblast cell line or HSF1184 was used throughout the course of
this study. This section describes the normal growth curve of the cell line, morphology
and cell handling.
68
4.1.1 The Growth Curve of the normal HSF1184 cells
The experiment was conducted using eight 25cm2 T-Flask and monitored for 8
days period without medium replenishment. The cell seeding used in this experiment
was 1x105cells/ml. The number of cells that indicated the growth of cells was calculated
daily by using tryphan blue exclusion and the result of HSF1184 growth curve showed a
typical pattern of cell growth (Freshney, 2000).
According to the normal growth pattern suggested by Freshney, 2000, there are
several growth phases that can be identified in the growth curve or also known as the
complete growth profile of the cell. Based on the graph shown in Figure 4.1, the growth
curve of a normal HSF1184 cells can be divided into 3 phases namely, the lag phase, the
log phase and the stationary phase.
Cell Concentration (cells/ml), x 105
4
Saturation density
3.5
3
2.5
2
1.5
Lag phase
1
Log phase
0.5
Stationary phase
0
0
1
2
3
4
Day
Figure 4.1
The normal growth curve of HSF1184 cells
5
6
7
8
69
According to Figure 4.1, the lag phase occurred for 3 days. The start of the lag
phase was obtained by extrapolating a line drawn through the points of the log phase
until it intersects with the seeding density (1 x 105 cells/ml). The elapsed time was read
from the intercept. The cell goes through lag phase as they need to adapt themselves to
the growth conditions which include the nutrient content and the growth serum (Fetal
Bovine Serum, FBS) available for the cells to grow. In this period the HSF1184 cells are
maturing and not yet able to duplicate. The graph shows a small downward projection as
some of the cells unable to cope with the new environment causing the reduction in the
cell number. However, the remaining cells that were able to cope better started to
duplicate upon maturity giving an upward projection of the graph at later stage of the lag
phase.
The cells started to enter the log phase on day 3 and continued increasing
exponentially until day 5. This phase is mainly characterised by the doubling of the cells
population. The doubling of the cell population is known as the population doubling
time (PDT) which is a very important information in cell culture analysis. The
population PDT, times taken for the culture to increase by twofold in the middle of the
log phase was about 1 day (24 hours). According to Scheneider et al, 1977, the
population doubling time for human skin fibroblast cells taken from human fetal tissue is
approximately 23 hours. Therefore it is worth mentioning that the data obtained from
this experiment is comparable to the experiment being done before by Scheneider et al.
Cell growth started to decelerate after day 5 and reached stationary phase at day
7. Saturation density, the concentration of cells in the stationary phase was estimated to
be 3.78 x 105cells/ml. the death phase occurred after stationary phase (after day 7). It
was difficult to measure accurately, as a steady state is not easily achieved in the
stationary phase (Freshney, 2000). However, most cultures with regular medium
replenishment will be able to continue proliferate (although at reduced rate) well beyond
confluency, resulting in multilayers of cells. Adult skin fibroblasts, which expressed
70
contact inhibition of movement, will continue to proliferate as well, laying down layers
of collagen between the cells layers until multilayers of six or more cells can be reached
under optimal condition (Freshney 2000).
This experiment was also conducted to determine the growth pattern of the cell
line in normal growth condition. The growth profile of the cell is very important in order
to identify and determine the number of days required for the cell to multiply and grow
to produce sufficient cell number for further analysis. According to Freshney (2000),
adding a drug or a bioactive extract in the middle of the exponential phase may provide
a different result. Therefore, in this study which involved the addition of aqueous and
methanolic extracts of Labisia pumila, a complete understanding of the growth profile
was crucial as the expected effect of the extracts depends on the phases of the cell
culture. The results depend on whether the culture is still in the exponential growth
phase when it is harvested or whether it has entered the stationary phase.
4.1.1.1 The complete Growth Curve (Profile) of the HSF1184 cell line
The growth curve displayed in section 4.1.1 explains the three main phases in
cell culture and in this case, the three phases that occur in the growth of HSF1184 cells.
However, it does not show the complete growth profile of the cells. This piece of
information is very important, as a complete growth profile shows the full lifespan of the
cell line in cultured medium without replenishment.
71
The experiment was conducted in 12 days so that a complete growth profile of
HSF 1184 cell line can be fully obtained and understood. This experiment was carried
out by using 13 six-well-plates. All the six-well-plates were labelled from 0 until 12
indicating the day number of the sample. Each six-well-plate were seeded with the same
number of cells at the beginning of the experiment (1 x 105 cells/ml).The number of
cells were monitored and calculated by using the tryphan blue exclusion test as
explained in section 3.2.2. The graph in Figure 4.3 presents the result of the 12 days
growth profile of the cells.
3.0E+06
100
90
Cells Density (cells/ml)
2.5E+06
80
70
2.0E+06
60
50
1.5E+06
40
1.0E+06
30
20
5.0E+05
10
0.0E+00
0
0
1
2
3
4
5
6
7
8
9
10
Day
HSF1184
Figure 4.2
Cell Viability (%)
The complete growth profile of HSF1184 cell line
11
12
13
14
72
From Figure 4.2, the HSF 1184 cell line took about 12 days to enter the death
phase. The trend of growth similar to Figure 4.1 was observed in this experiment. This
was a good indicator to show the consistency in data collection during experimental
works. In 6 days, the cell reached the plateau for maximum growth. After day 6 the
viability of the cell declined significantly. Therefore, the cells density also showed
significant reduction after day 6.
The complete growth profile of the cell is very important to identify and
determine the number of days it will take for the cell to multiply and grow to produce
the required number of cells in any experiment and analysis. The construction of a
growth profile of any cell line is the very basic step before any further experiment or
analysis can be done because cell physiology and the pattern of growth of the cell can be
fully understood from the growth profile (Freshney, 2000).
4.1.2 Morphological observation
Human Skin Fibroblast Cell Line (HSF1184) was cultured as described in
chapter 3 section 3.2.1. Figure 4.3 shows the morphological appearance of HSF1184 cell
line observed under a light microscopy at day 1 and day 5 upon reaching confluency.
HSF1184 cell line is a monolayer cells, which grow on an artificial substrate and
in this study, the flask surface made of disposable plastic (polystyrene). The substrate
was charged to allow cell adhesion and spreading. This cell line requires attachment
surface in order to grow, therefore it is classified as ‘anchorage dependent’ cells
(Freshney 2000). In Figure 4.3, the cells are observed as spindle-shaped cells at day 1.
This is the normal shape of the HSF1184 cells. Any irregularities in the cell shape
73
indicate cell ageing or contamination of the medium contents used in the experiment.
The number of HSF1184 cells at day 1 was low as the cells were still in the lag phase.
At day 5, the cells were clumped together but still maintain the regular spindle-shaped.
At this stage, the cells have reached almost 100% confluency and were ready to be
harvested for further analysis. According to Freshney (2000), this type of cells
morphology can be regarded as epithelial.
(a)
(b)
Figure 4.3
The morphology of HSF1184 cell line on (a) day 1 and (b) day 5 using
10X magnification of light microscopy
74
4.2
Cell-based assay – treatment of Labisia pumila extracts
This section describes the cell based assay studies implemented on HSF1184 cell
line by introducing Labisia pumila plant extracts on the cultured medium.
4.2.1 The Effect of Labisia pumila aquoeus extract on HSF1184 cell growth
Absorbance ,Relative difference to control
(optical density, OD)
4.2.1.1 Cultured medium supplemented with FBS
1.6
1.4
*
*
*
1.2
**
1
0.8
0.6
0.4
0.2
**
**
0
Concentration (µg/ml)
Labisia pumila aqueous extract
Figure 4.4
Aqueous extract of Labisia pumila at different concentration treated to
HSF1184 cell line (with FBS). Assays were performed in 3 replicates from 3
independent experiments. Values are means± SEM (*,p<0.01;**,p<0.05).
75
Figure 4.5 shows the effect of different concentration of the water extract of
Labisia pumila on the HSF1184 cells. The experiment was carried out based on the
methodology explained in section 3.3.1. These studies involved the use of a biological
assay known as the MTT assay. 9 different concentrations of the water extract of Labisia
pumila were prepared using serial dilution. This is very important because the HSF1184
cells reacted differently depending on the concentration of the plant extract. This study
was also called the cytotoxicity studies and it was carried out to get the suitable
concentration of the water extract of Labisia pumila so that it can be used for further
analysis.
This experiment was implemented in the normal cultured medium supplemented
with FBS as the growth factor. The graph in Figure 4.4 shows that the water extract of
Labisia pumila stimulates cell growth at lower concentration. Significant cell growth
promotion of the HSF1184 cells was observed at 1 x 10-4µg/ml and 1 x 10-3µg/ml. The
cell growth increased to almost 30% as compared to the HSF1184 cells not treated with
the water extract of Labisia pumila, which acted as the negative control. The relative
difference of the growth compared to the control was calculated by using the formula 3.4
stated in section 3.3.1. The stimulation of HSF1184 cells growth caused by the water
extract of Labisia pumila might be due to the defence mechanism exerted by the cells as
a precaution to the introduction of a new foreign object to the system. However, there is
a possibility that the introduction of the water extract of Labisia pumila at certain
concentrations might directly intervene with the growth pathway of the cells resulting in
growth stimulation.
The cells growth was suppressed at two different concentrations according to
Figure 4.5. These concentrations of the water extract of Labisia pumila were 1 x
103µg/ml and 1 x 104µg/ml. This result clearly indicated that inhibition of cell
proliferation occurred at high concentration of the water extract of Labisia pumila.
Therefore, it can be concluded that at 1 x 10-4µg/ml, the aqueous extract of Labisia
76
pumila was suitable for this study and this result is consistent with the earlier study done
by Choi et al, 2010.
Absorbance ,Relative difference to control (
optical density, OD)
4.2.1.2 Cultured medium without FBS
3
2.5
*
*
*
2
1.5
**
1
0.5
**
**
0
Concentration (µg/ml)
Labisia pumila aqueous extract
Figure 4.5
Aqueous extract of Labisia pumila at different concentration treated to
HSF1184 cell line (without FBS). Assays were performed in 3 replicates from 3
independent experiments. Values are means± SEM (*,p<0.01;**,p<0.05).
Figure 4.5 shows the effect of different concentration of the water extract of
Labisia pumila on the growth of HSF1184 cell line without the presence of FBS in the
cultured medium. Under normal condition, a cell culture medium must contain nutrient
required for cell survival and growth serum which is essential for cell proliferation.
Serum is added to the culture media at a concentration of 10% (v/v) to promote cell
growth. Serum is the supernatant of clotted blood which contains undefined materials
77
essential for cell proliferation. The use of growth serum in media depends on the type of
cells being cultured, but mostly, serum are made of cow (bovine) or horse (equine)
which are the most effective because of its high content of embryonic growth factors
(Butler, 2004). Table 4.1 shows the typical characteristics of bovine serum.
Table 4.1
pH
6.85-7.05
Typical characteristics of bovine serum (Butler, 2004)
Osmolarity
Protein content
Albumin content
(mOsm/l)
(mg/ml)
(mg/ml)
250-295
60-80
30-50
Although the use of serum is very important in cell culture media, a lot of studies
have been done in searching for a suitable replacement to the current commercially used
growth serum in culture media (Michaela et al, 2010, J. van der Valk et al, 2010). There
are a number of widely recognized disadvantages connected with its use;
a. It is chemically undefined and variation between batches can result in
inconsistent promotion of cell growth.
b. It is expensive, fetal calf serum (FCS) for example account for 70-80% of the
cost of some formulations. This is an important consideration in large scale
cultures.
c. The proteins in serum can compromise the extraction and purification procedures
for cell-secreted proteins.
d. Serum is vulnerable to contamination with infectious agents such as viruses and
prions.
78
Therefore, this study was conducted to assess the potential of the water extract of
Labisia pumila to be used in serum free media. The experiment was carried out by using
the same methodology as the experiment explained in section 4.2.1.1 with the only
difference in the absence of FBS in the cell culture media.
Figure 4.5 shows an almost identical trend of growth stimulation when compared
with figure 4.4. The growth of HSF1184 cells was significantly stimulated at lower
concentration of the water extract of Labisia pumila. The concentration that exhibited
the most prominent and significant growth promotion were 1 x 10-4µg/ml and 1 x 103
µg/ml and this result was consistent with the earlier study explained in section 4.2.1.1.
In this study, the growth of HSF1184 cells was stimulated more than twofold (2.4x) at
these concentration. This growth stimulation was even higher than the addition of the
water extract of Labisia pumila in cell culture media with FBS at the same
concentration. This can be clearly seen when the results of the two experiments were
compared side by side as displayed in Figure 4.6.
79
Absorbance ,Relative difference to control ( optical density,
OD)
3
2.5
2
1.5
1
0.5
0
Concentration (µg/ml)
with FBS
Figure 4.6
without FBS
Comparative studies of the effect of the water extract of Labisia pumila
on the growth of HSF1184 cell line in culture media supplemented with FBS and serum
free media.
At higher concentration of the water extract of Labisia pumila, the growth of
HSF1184 cells were inhibited. The concentrations at which the plant extract was toxic to
the cells were identical to the previous result in section 4.2.1.1 (1 x 10 3µg/ml and 1 x
104µg/ml). Therefore, based on the result of this study, the water extract of Labisia
pumila has a great potential to be used in serum free media as growth stimulation was
significant at 1 x 10-4µg/ml concentration of the extract even without the presence of a
growth serum. As mentioned in the earlier part of section 4.2.1.2, normal cell culture
media would require the presence of a growth serum, however, a lot of studies has been
conducted in searching for a suitable replacement for the current commercially used
80
growth serum in culture media (Michaela et al, 2010, J. van der Valk et al, 2010). The
ability of the water extract of Labisia pumila to stimulate growth even without the
presence of a growth serum in the cell culture media could mean that the bioactive
compounds available in the water extract of Labisia pumila could become an option to
the current commercially used growth serum.
4.2.2 The Effect of Labisia pumila aqueous extract on the growth curve (profile)
of HSF1184 cell line.
In sections 4.2.1 and 4.2.2 the effect of the water extract Labisia pumila on the
growth of HSF1184 cell lines by using MTT cell-based assay were discussed. As
mentioned in section 4.2.1, these studies were carried out to identify and determine the
suitable concentration of the water extract of Labisia pumila. In this section we are
going to discuss about the effect of plant extract on the complete growth curve (profile)
of HSF1184 cell line as mentioned in section 4.1.1.1.
From the growth profile the effect of the plant extract on the rate of growth of the
HSF1184 cell line was determined. The rate of growth of the cells can be observed from
the changes that occur to the gradient of the graph (Figure 4.7) mostly during the
exponential growth phase (lag phase). We can also observe any improvement in cells
viability upon the addition of the water extract of Labisia pumila throughout the course
of the experiment.
81
100
30
90
Cells Density (x104 cells/ml)
25
80
70
20
60
50
15
40
10
30
20
5
10
0
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Days
Figure 4.7
Labisia pumila
Viability for Labisia pumila (%)
Control
Viability for Control (%)
The effect of the water extract of Labisia pumila on the growth curve of
HSF1184 cell line.
Figure 4.7 shows the effect of the water extract of Labisia pumila on the growth
curve of the HSF1184 cell line. From the graph, HSF1184 cells took about 14 days to be
completely dead once cultured in the media without the addition of the plant extract.
However, when the water extract of Labisia pumila with concentration of 1 x 10-4 µg/ml
were added at the beginning of the experiment, the HSF1184 cells took more than 14
days to be completely dead. Both cell batches started with the same cells concentration
(1 x 104 cells/ml) to ensure the result of this experiment was due to the effect of the plant
extract.
82
This experiment was carried out in a normal culture media supplemented with
FBS. The growth curve for HSF1184 cells with the addition of the water extract of
Labisia pumila in the culture media showed an upward projection when compared to the
control. The growth stimulation was very significant especially during the log phase and
the cell number has increased almost four-fold at every phases of the growth. However,
despite the significant increase in the cells number (cells concentration), this cell batch
reached the stationary (plateau) phase of the growth on day 7 which was identical to the
negative control batch. After day 7, both cell batches underwent the declining phase
where the number of cells reduced significantly until all the cells were dead.
As mentioned in section 3.2.2, cells viability is the percentage of the viable cell
over the total number of cells counted using Trypan exclusion assay with
haemocytometer under a light microscope. According to Figure 4.7, the HSF1184 cells
viability improved slightly in the culture media with the addition of the plant extract
when compared to the negative control. This was true for most part of the log phase and
the entire part of the declining (death) phase of the cell’s growth. Improvement in cells
viability means that the addition of the water extract of Labisia pumila was able to
promote growth and enhance the survival of the cells.
83
4.2.2.1 The Effect of Labisia pumila aqueous extract on the growth curve (profile)
Cell Density (x 104 cells/ml)
of HSF1184 cell line in the absence of FBS (Serum free media)
8
100
7
90
80
6
70
5
60
4
50
3
40
30
2
20
1
10
0
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Day
Figure 4.8
Control
Labisia pumila
Viability for Control (%)
Viability for Labisia pumila (%)
The effect of the water extract of Labisia pumila on the growth curve of
HSF1184 cell line in serum free media.
This study was carried out to investigate the effect of the water extract of Labisia
pumila on the growth of HSF1184 cell line in serum free media. The HSF1184 cells
which acted as the negative control in this experiment was also cultured in a serum free
media without being supplemented with FBS. As mentioned in section 4.2.1.2, the result
of this study could lead to the use of the plant extract in serum free media in the future.
84
The concentration of the water extract of Labisia pumila used in this experiment was the
same as in the section 4.2.2 (1 x 10-4 µg/ml).
According to figure 4.8, the addition of the water extract of Labisia pumila into
the culture media has increased the number of days before the cells were completely
dead when compared to the negative control which took about 14 days to be completely
dead. The addition of the plant extract also caused upward projection to the growth
curve and this has increased the number of cells significantly at every phases of growth
of the HSF1184 cell line. However, both cell batches took about 6 days to reach the
stationary (plateau) phase and this trend was identical as explained in section 4.2.2.
In Figure 4.8, it is clear that water extract of Labisia pumila has caused
significant improvement to the cells viability compared to the negative control. This can
be observed at every phases of growth and most prominently during the declining phase.
85
Cells Density (x104 cells/ml)
25
20
15
10
5
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Days
Labisia pumila
Figure 4.9
Labisia pumila (without FBS)
Comparative studies of the effect of the water extract of Labisia pumila
on the growth curve of HSF1184 cell line in culture media supplemented with FBS and
serum free media.
Figure 4.9 combines both graphs so that we can easily analyse the growth pattern
in both conditions. The trend of growth was identical in both media supplemented with
FBS and serum free media. However, the number of cells was significantly reduced in
the culture media without the presence of FBS. Although the water extract of Labisia
pumila has caused growth promotion in serum free media compared to the negative
control also in serum free media, but the overall cells concentration were greatly
reduced. This situation might indicate the lack of certain growth factors in the water
extract of Labisia pumila despite showing the growth promoter characteristics.
Therefore in the future, Labisia pumila could be enhanced with some growth factors that
address this condition and still be considered as a better option to the current
commercially used growth serum.
86
120
Cells Viability (%)
100
80
60
40
20
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Day
Viability for Labisia pumila with FBS (%)
Figure 4.10
Viability for Labisia pumila without FBS
Comparative studies of cells viability in media supplemented with FBS
and serum free media after the addition of the water extract of Labisia pumila.
Figure 4.10 shows the effect of the water extract of Labisia pumila on the cells
viability in media supplemented with FBS and serum free media. As you can see from
the graph, the effect of the plant extract was more significant in the serum free media
compared to the serum supplemented with FBS. This was true at most of the growth
phases of HSF1184 cells. Although the number of cells reduced significantly in serum
free media, the greater improvement in cells viability gave us an indication that the
presence of serum and in this case, FBS, could hinder the total effect of the water extract
of Labisia pumila. Therefore, this result, again, clearly proved that the water extract of
Labisia pumila could certainly be used in serum free media and is a good candidate for
serum
replacement
due
to
its
growth
promotory
effect.
87
4.3
The Photoprotective effect of Labisia pumila extracts against UVB
irradiation
Extrinsic aging occur due to the chronic exposure to UVB radiation. This is one
of the most important problem that affect people living in the tropical region of the
world as they are exposed to sunlight for the most part of the day compared to the other
people living in part of the world which far from the equator. The effect of chronic
exposure to UVB radiation has been explained in greater details in section 2.9.
This experiment was conducted to evaluate the photoprotective effect of the
Labisia pumila extracts on the growth of HSF1184 cell line after exposure to UVB
source. The photoprotective characteristics of Labisia pumila extracts include;
I.
The ability of the plant extracts to protect the HSF1184 cells against the
damaging effect of the UVB radiation.
II.
The ability of the plant extracts to cause cell regeneration (growth promoting)
after exposure to UVB radiation.
The methodology used in this part of the study was explained in section 3.5 in
chapter 3.
88
4.3.1 The effect of UVB irradiation on the Growth Curve of HSF1184 cell line
80
Cell Concentration ^104 (cells/ml)
70
60
50
40
30
UVB irradiation
20
10
0
0
1
2
3
4
5
6
7
8
9
Day
Figure 4.11
UVB Irradiated
Control
Total Cells (UVB Irradiated)
Total Cells (Control)
The effect of UVB (80 mJ/cm2) irradiation the growth curve of HSF1184
cell line.
This experiment was carried out in a normal culture media supplemented with
FBS but without the addition of the Labisia pumila plant extracts. Figure 4.11 shows the
effect of UVB irradiation on the growth profile of HSF1184 cell line. From the graph,
the negative control shows a similar growth pattern to the previous result explained in
section 4.2.2. This batch of cells reached the plateau (stationary) phase on day 6 and
started the declining phase after day 6. In this graph, the total cells which was calculated
using equation 4.1:
89
Total cells = Viable cells + non-Viable cells (4.1)
The total cells number for the negative control was significantly higher than the
cells batch which was exposed to 80 mJ/cm2 UVB irradiation. The total number of cells
was the same for both cell batches for the first 2 days, but the cell concentrations started
to decline significantly after day 2 for the cell batch exposed with UVB irradiation. This
occurred because the cells batch was exposed to UVB on day 2. Without exposure to
UVB irradiation, the HSF1184 cells were able to grow normally showing its typical
growth pattern. However, exposure to UVB has caused severe disruption to the cells
growth due to the damaging effect of the UVB exposure. The possible damaging
mechanism of UVB irradiation was explained in section 2.9. One of the possible
damaging mechanisms includes the production of free radicals such as superoxide
particles which can severely harm and kill the HSF1184 cells (Michael et al, 2004,
Brash et al, 1999). The cell batch which was exposed to UVB was completely dead in 8
days while the other batch (not exposed to UVB) continued to grow according to its
typical growth curve pattern.
90
4.3.2 The Photoprotective effect of the water extract of Labisia pumila on UVB
irradiated HSF1184 cell line.
Relative Optical Density ,OD ( Relative to control)
1.4
*
1.2
**
1
**
**
*
0.8
*
*
0.6
0.4
0.2
0
Concentration (µg/ml)
Figure 4.12
The effect of water extract of Labisia pumila on UVB irradiated
HSF1184 cell line. (Control – HSF1184 cells exposed to UVB irradiation but without
the addition of the water extract of Labisia pumila). Assays were performed in 3
replicates
from
3
(*,p<0.01;**,p<0.05).
independent
experiments.
Values
are
means±
SEM
91
Figure 4.12 shows the effect of the water extract of Labisia pumila on UVB
irradiated HSF1184 cell line. In this study the cells were exposed to 80 mJ/cm2 of UVB
irradiation. The methodology of this experiment was explained in section 3.5 and
involved the use of MTT assay. In this experiment, the water extract of Labisia pumila
were added into the cell batch after exposure to UVB irradiation and were left for 24
hours before MTT assay was done to calculate the number of cells.
From Figure 4.12, only one concentration was observed to have photoprotective
effect on the growth of HSF1184 cells after UVB irradiation, and this occurred at
concentration of 1 x 10-5 µg/ml. In section 4.2.1, the most effective concentration of the
water extract of Labisia pumila which has stimulatory growth effect on the HSF1184
cells was at 1 x 10-4 µg/ml. However, in this study, that concentration was unable to
cause cells regeneration after exposure to UVB irradiation. Hence, another experiment to
evaluate the efficacy of the water extract of Labisia pumila was carried out to see the
effect of the plant extract at the concentration of 1 x 10-5 µg/ml and the result of the
study can be seen in Figure 4.13.
92
Relative Optical Density ,OD ( Relative to control)
1.4
*
1.2
*
*
**
1
**
**
0.8
0.6
0.4
0.2
0
Concentration (µg/ml)
Figure 4.13
The efficacy studies for the water extract of Labisia pumila. (Control –
HSF1184 cells without the addition of water extract of Labisia pumila) Assays were
performed in 3 replicates from 3 independent experiments. Values are means± SEM
(*,p<0.01;**,p<0.05).
According to Figure 4.13, at concentration of 1 x 10-5 µg/ml of the water extract
of Labisia pumila, the growth stimulatory effect on the HSF1184 cells was higher than
at concentration of 1 x 10-4 µg/ml. this result has further supported the result observed in
Figure 4.12. Figure 4.13 also shows that at concentration higher than 1 x 10-5 µg/ml of
the water extract of Labisia pumila, the cells regeneration did not occur as the index
values were lower than that of negative control. Therefore based on the results obtained
from these studies, the water extract of Labisia pumila showed the photoprotective
effects against UVB irradiation at lower concentration. This can be clearly seen when
93
both graphs were put side by side so that easier comparison can be made. This can be
observed in Figure 4.14.
1.4
Relative Optical Density ,OD ( Relative to control)
1.2
1
0.8
0.6
0.4
0.2
0
Concentration (µg/ml)
Without UV
Figure 4.14
With UV
Comparative studies of Water extract of Labisia pumila on non-irradiated
and UVB irradiated HSF1184 cells (80mJ/cm2)
94
4.3.3 Time dependent effect of the water extract of Labisia pumila on UVB
irradiated HSF1184 cell line
In section 4.3.2, after exposure to UVB irradiation, the HSF1184 cells were
treated with the water extract of Labisia pumila and were left for 24 hours for the plant
extract to manifest its effect. Choi et al 2010, has adapted this methodology when
analysing the effect of a plant extract on the growth of HSF1184 cell line. However, 24
hours might not be the ideal time duration for the plant extract to show its full ability.
Therefore this study was carried out to see the time dependent effect of the water extract
Cell concentrations, relative optical difference (OD)
of Labisia pumila on UVB irradiated HSF1184 cell line.
2.5
*
2.0
*
*
**
*
**
1.5
*
1.0
*
*
**
**
*
0.5
0.0
Concentration (µg/ml)
24 hours
Figure 4.15
48 hours
Time-Dependent Effect of the Water Extract of Labisia pumila on the
growth of UVB Irradiated HSF1184 cells. (Control – HSF1184 cells exposed to UVB
irradiation but without the addition of water extract of Labisia pumila). Assays were
performed in 3 replicates from 3 independent experiments. Values are means± SEM
(*,p<0.01;**,p<0.05).
95
Figure 4.15 shows the time-dependent effect of the water extract of Labisia
pumila on the growth of UVB irradiated HSF1184 cell line. The concentrations of the
plant extract used in this experiment were the same as explained in section 4.3.2.
According to the graph in Figure 4.15, the effect of the water extract of Labisia pumila
on the growth of UVB irradiated HSF1184 cells was very significant after 48 hours. The
concentration of the plant extract of 1 x 10-5 µg/ml showed the most prominent effect as
it has caused the increased in the growth of the HSF1184 cells up to two-fold. But this
study also showed that all 9 different concentrations of the water extract of Labisia
pumila (after 48 hours of treatment) had stimulated the growth of the cells after exposure
to UVB irradiation, this has not occurred after 24 hours of treatment with the plant
extract. In fact, after 24 hours of treatment only one concentration caused growth
stimulation of the cells while the remaining 8 concentration which was higher than the
effective concentration (1 x 10-5 µg/ml) caused significant growth inhibition. The results
of this study might indicate that continuous exposure to the water extract of Labisia
pumila could give better photoprotective effect for the HSF1184 cells as the plant extract
requires more time to manifest its full effect.
Therefore, this study has showed time dependent effect was important when
treating the UVB irradiated HSF1184 cell line with the water extract of Labisia pumila
as different length of time gave different effect.
96
4.3.4 The Photoprotective effect of the water extract of Labisia pumila on UVB
Cells Concentration , relative optical difference (OD)
irradiated HSF1184 cell line in serum free media.
2.0
*
1.8
1.6
*
**
**
1.4
**
*
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Concentration (µg/ml)
Water Extract
Figure 4.16
Growth Stimulatory effect of the water extract of Labisia pumila on
UVB-Irradiated HSF1184 cells after 48 hours without Growth Serum. Assays were
performed in 3 replicates from 3 independent experiments. Values are means± SEM
(*,p<0.01;**,p<0.05).
Cells Concentration , relative optical difference (OD)
97
2.5
*
2.0
*
*
*
1.5
*
*
**
**
**
**
**
**
1.0
0.5
0.0
Concentration (µg/ml)
Without FBS
Figure 4.17
With FBS
Comparative studies of the effect of the water extract of Labisia pumila
on UVB irradiated cells in media with FBS and serum free media. Assays were
performed in 3 replicates from 3 independent experiments. Values are means± SEM
(*,p<0.01;**,p<0.05).
Figure 4.16 shows the growth stimulatory effect of the water extract of Labisia
pumila on UVB-Irradiated HSF1184 cells after 48 hours of treatment, without growth
serum. Based on the graph, at the concentration of 1 x 10-5 µg/ml of the water extract of
Labisia pumila, the growth of HSF1184 cells increased significantly when compared to
the negative control (HSF1184 cells exposed to UVB irradiation but without the addition
of the water extract of Labisia pumila) and the other 8 concentrations. All concentrations
of the plant aqueous extract showed growth stimulatory effect on the UVB irradiated
HSF1184 cells after 48 hours of treatment with the plant extract. This was evidenced
when the number of cells concentration reflected by the relative optical difference to the
negative control, was higher. None of the 9 concentrations used in this experiment
showed growth inhibitory effect on the cells.
98
Figure 4.17 shows the comparative studies of the effect of the water extract of
Labisia pumila on UVB irradiated cells in media with FBS and serum free media after
48 hours of treatment with the plant extract. Based on the graph in Figure 4.6, the
growth stimulatory effect of the plant aqueous extract was more significant in culture
media supplemented with FBS at all 9 concentrations used in this experiment. However,
at concentration of 1 x 10-5 µg/ml, the effect of the water extract of Labisia pumila was
almost identical in both culture media supplemented with FBS and serum free media
because both condition showed almost two-fold increase in the growth of HSF1184
cells. Once again these results have proven that the water extract of Labisia pumila
showed significant photoprotective effect in both culture media supplemented with FBS
and serum free media by inducing cells regeneration after exposure to the 80 mJ/cm2 of
UVB irradiation. The effect of the plant aqueous extract was more prominent when the
UVB irradiated HSF1184 cells were left treated for 48 hours instead of 24 hours.
99
4.3.5
The Photoprotective effect of the methanolic extract of Labisia pumila on
Cells Concentration , relative optical difference
(OD)
the UVB irradiated HSF1184 cells (after 48 hours of treatment)
1.6
*
1.4
1.2
1.0
0.8
*
0.6
*
*
**
0.4
0.2
0.0
control 1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+00 1.00E+01 1.00E+02
Concentration (µg/ml)
Figure 4.18
The effect of the methanolic extract of Labisia pumila on the growth of
UVB irradiated HSF1184 cells in culture media supplemented with FBS. Assays were
performed in 3 replicates from 3 independent experiments. Values are means± SEM
(*,p<0.01;**,p<0.05).
100
Cells Concentration , relative optical difference (OD)
1.2
1.0
0.8
0.6
0.4
*
**
*
**
*
0.2
*
0.0
control 1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+00 1.00E+01 1.00E+02
Concentration (µg/ml)
Figure 4.19
The effect of the methanolic extract of Labisia pumila on the growth of
UVB irradiated HSF1184 cells in serum free media. Assays were performed in 3
replicates
from
3
(*,p<0.01;**,p<0.05).
independent
experiments.
Values
are
means±
SEM
Cells Concentration , relative optical difference
(OD)
101
1.6
*
1.4
1.2
1.0
0.8
*
0.6
0.4
*
0.2
**
*
*
**
*
**
*
*
0.0
control 1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+001.00E+011.00E+02
Concentration (µg/ml)
Methanol (without FBS)
Figure 4.20
Methanol (with FBS)
Comparative studies of the effect of the methanolic extract of Labisia
pumila on UVB irradiated cells in media with FBS and serum free media. Assays were
performed in 3 replicates from 3 independent experiments. Values are means± SEM
(*,p<0.01;**,p<0.05).
In this study the methanolic extract of Labisia pumila was used to treat the UVB
irradiated HSF1184 cell line. Figure 4.18 shows the effect of the methanolic extract of
Labisia pumila on the growth of UVB irradiated HSF1184 cells in culture media
supplemented with FBS. The effective concentration for the methanolic extract was
identical to the effective concentration for the water extract of Labisia pumila which is 1
x 10-5 µg/ml. At this concentration, the methanolic extract of the plant increased cell
growth by almost 40% compared to the negative control (HSF1184 cells exposed to
UVB irradiation but without the addition of the methanolic extract of Labisia pumila).
However, at concentration higher than this, the methanolic extract of Labisia pumila
102
showed inhibitory effect to the growth of the UVB irradiated HSF1184 cells. Therefore,
cells regeneration only occurs at concentration of 1 x 10-5 µg/ml.
Figure 4.19 shows the effect of the methanolic extract of Labisia pumila on the
growth of UVB irradiated HSF1184 cell line in serum free media. In this study,
inhibitory effect to the growth of the UVB irradiated cells was observed at all
concentrations of the methanolic extract of the plant. Therefore 100% methanol was a
less suitable solvent to extract the appropriate phytochemical of from the Labisia pumila
plant. This might be due to the absence of some important bioactive components which
was not readily dissolved in 100% methanol. This result proved that the appropriate
bioactive ingredient including some growth factors are present in the aqueous extract of
Labisia pumila.
Figure 4.20 shows both results involving the use of the methanolic extract of
Labisia pumila in both culture media supplemented with FBS and serum free media. In
this graph, it is clear that the methanolic extract only induce growth stimulation in
culture media supplemented with FBS at only one concentration which was 1x 10-5
µg/ml while higher concentrations result inhibitory effect to the growth of UVB
irradiated HSF1184 cell line in both media conditions.
Cells Concentration , relative optical difference (OD)
103
2.5
2.0
1.5
1.0
0.5
0.0
control 1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+00 1.00E+01 1.00E+02
Concentration (µg/ml)
Figure 4.21
Water Extract (with FBS)
Methanol (with FBS)
Water Extract (without FBS)
Methanol (without FBS)
Comparative studies of the effect of 2 different extracts of Labisia pumila
on UVB irradiated cells in media with FBS and serum free media. (Control – HSF1184
cells exposed to UVB irradiation but without the addition of different extracts of Labisia
pumila in cell culture media supplemented with FBS and serum free media).
The comparative studies of the effect of 2 different extracts of Labisia pumila on
UVB irradiated HSF1184 cells in cultured media supplemented with FBS and serum
free media is shown in Figure 4.21. The effect of the water extract of Labisia pumila
was more significant at any concentrations in both media condition. This again,
demonstrates the effectiveness of the water extract of the plant compared to the
methanolic extract.
104
4.4
The effect of the Labisia pumila plant extracts on the synthesis of collagen in
HSF1184 cell line.
Collagen is one the most important structural protein in the human body.
Collagen in the skin involved in maintaining the physical integrity of the skin and its
mechanical strength. As we age, collagen synthesis in the human skin fibroblast cells is
reduced and causes the skin to lose its physical integrity and eventually leading to the
formation of wrinkles and saggy looking skin. One of the reason that could lead to
accelerated aging or commonly known as extrinsic aging is due to chronic exposure to
ultraviolet radiation. The mechanism in which the UV light could cause accelerated
aging was explained in greater details in section 2.9. It is believed that chronic exposure
to UVB irradiation could cause increase production of matrix metalloproteinases
(MMPs) which plays a major role in the pathogenesis of photoaging. This will result in
the increase degradation of different types of collagen in the human skin.
In this study, the Sircol™ Collagen Assay was used to evaluate the collagen
synthesis in the normal HSF1184 cells and UVB irradiated HSF1184 cells. The
methodology of the experiment was explained in section 3.4.
Collagen concentrations, relative optical difference to
control (OD)
105
1.8
*
1.6
*
1.4
*
1.2
1
0.8
0.6
0.4
0.2
0
control
1.0E-05
1.0E-04
1.0E-03
Concentration (µg/ml)
Figure 4.22
The effect of the water extract of Labisia pumila on the collagen
synthesis in HSF1184 cell line. (Control – HSF1184 cells without the addition of water
extract of Labisia pumila). Assays were performed in 3 replicates from 3 independent
experiments. Values are means± SEM (*,p<0.01;**,p<0.05).
106
Collagen concentrations, relative optical difference to
control (OD)
2
*
1.8
1.6
*
1.4
*
1.2
1
0.8
0.6
0.4
0.2
0
control
1.0E-05
1.0E-04
1.0E-03
Concentration (µg/ml)
Figure 4.23
The effect of the water extract of Labisia pumila on the collagen
synthesis in UVB irradiated HSF1184 cell line. (Control – HSF1184 cells exposed to
UVB irradiation but without the addition of water extract of Labisia pumila).Assays
were performed in 3 replicates from 3 independent experiments. Values are means±
SEM (*,p<0.01;**,p<0.05).
Figure 4.22 shows the effect of water extract of Labisia pumila on the collagen
synthesis in HSF1184 cells. In this experiment only 3 concentrations were chosen as
these 3 concentrations proved to be consistent in stimulating growth of the HSF1184
cells in the previous studies. According to the graph in Figure 4.22 all three
concentration caused stimulation in collagen synthesis in the HSF1184 cells. The
concentration that showed the most significant collagen stimulatory effect was at 1x 10-5
µg/ml. At this concentration the collagen synthesis increased up to 50% compared to the
negative control (Control – HSF1184 cells without the addition of the water extract of
107
Labisia pumila). The results in this experiment were collected after 48 hours of
treatment with the water extract of Labisia pumila. The results were consistent with the
previous study done by Choi et al 2010 involving the same plant extract.
Figure 4.23 shows the effect of the water extract of Labisia pumila on the
collagen synthesis in UVB irradiated HSF1184 cells. In this experiment, the HSF1184
cells were cultured in media supplemented with FBS and were exposed to 80 mJ/cm2 of
UVB radiation. A similar trend was observed in this experiment as in Figure 4.22,
however at the same concentration of 1 x 10-5 µg/ml of the water extract of Labisia
pumila, the collagen production increased up to 60%, which was higher than the
experiment without UVB irradiation. This observation has further supported the earlier
finding that the water extract of Labisia pumila was photoprotective against the
damaging effect of the UVB irradiation as well as promote the collagen synthesis which
is essential to avoid photoaging.
108
Collagen concentrations, relative optical difference to
control (OD)
1.4
*
1.2
1
0.8
*
0.6
*
0.4
0.2
0
control
1.0E-05
1.0E-04
1.0E-03
Concentration (µg/ml)
Figure 4.24
The effect of methanolic extract of Labisia pumila on the collagen
synthesis in HSF1184 cell line. (Control – HSF1184 cells without the addition of
methanolic extract of Labisia pumila).Assays were performed in 3 replicates from 3
independent experiments. Values are means± SEM (*,p<0.01;**,p<0.05).
109
Collagen concentrations, relative optical difference
to control (OD)
1.4
*
1.2
1
0.8
0.6
*
*
0.4
0.2
0
control
1.0E-05
1.0E-04
1.0E-03
Concentration (µg/ml)
Figure 4.25
The effect of methanolic extract of Labisia pumila on the collagen
synthesis in UVB irradiated HSF1184 cell line. (Control – HSF1184 cells exposed to
UVB irradiation but without the addition of methanolic extract of Labisia
pumila).Assays were performed in 3 replicates from 3 independent experiments. Values
are means± SEM (*,p<0.01;**,p<0.05).
Figure 4.24 shows the effect of methanolic extract of Labisia pumila on the
collagen synthesis in HSF1184 cell line. Collagen synthesis was increased at only one
particular concentration of the methanolic extract of Labisia pumila, which is at 1 x 10-5
µg/ml. At 1 x 10-4 µg/ml and 1 x 10-3 µg/ml, the collagen concentration was reduced
significantly compared to the negative control (Control – HSF1184 cells without the
addition of methanolic extract of Labisia pumila). This might indicate that, at higher
concentration, the methanolic extract of Labisia pumila could have inhibitory effect to
the HSF1184 cells leading to the significant reduction in the collagen synthesis.
110
Collagen synthesis in the skin normally occur in the human skin fibroblasts cells,
therefore reduction in the number of cells could also be the reason to the reduced
number of collagen concentration. This result was consistent with the previous study
explained in section 4.35, because at higher concentration the methanolic extract of the
plant showed inhibitory effect to the growth of HSF1184 cells.
Figure 4.25 shows the effect of methanolic extract of Labisia pumila on the
collagen synthesis in UVB irradiated HSF1184 cell line. Again, the collagen synthesis
stimulatory effect was only observed at concentration of 1 x 10-5 µg/ml of the
methanolic extract of Labisia pumila, whereas collagen synthesis was greatly reduced at
the other two higher concentrations.
The water extract of the Labisia pumila proved to have greater collagen synthesis
stimulatory effect compared to the methanolic extract of Labisia pumila in both
experimental conditions.
CHAPTER 5
CONCLUSIONS AND RECOMMENDATIONS
5.1
Summaries
The objective of this master thesis was to investigate the photoprotective
effect of the water extract of Labisia pumila and the methanolic extract of Labisia
pumila. The photoprotective effects include; 1) the ability of the Labisia pumila plant
extract to stimulate HSF1184 cells regeneration after exposure to the UVB irradiation,
and 2) the ability of the Labisia pumila plant extracts to stimulate collagen synthesis in
HSF1184 cells after exposure to UVB as collagen is the main structural proteins that
give the mechanical integrity of the skin.
In the first part of the study, a complete growth profile of the HSF1184 cells
was obtained. According to this result, the HSF1184 cell line took about 12 days to be
completely died. Within the 12 days, the HSF1184 cell line has undergone
several growth phases including the lag phase, log phase, stationary phase and death
phase. The results collected throughout the course of this thesis have proven that both
Labisia pumila plant extracts used in this study were able to accomplish those three
main objectives at certain concentration of the Labisia pumila plant extracts. Both the
112
water extract of Labisia pumila and the methanolic extract of Labisia pumila showed
growth stimulatory effect on HSF1184 cell line at the same concentration, which was
consistent for each and every analysis done in this study, at 1x 10 -5 µ g/ml. At
concentration higher than this, both Labisia pumila plant extracts showed inhibitory
effect on the growth of the cells, if the HSF1184 cells were treated with the
plant extracts for 24 hours. This was true for both non-UVB irradiated and UVB
irradiated cells.
After 48 hours of treatment, the concentration of the Labisia pumila
plant extracts, which displayed the most significant stimulatory effect, was at 1x 10-5
µ g/ml (up to two-fold growth improvement), and this has been consistent throughout the
course of the study. Therefore, it can be concluded that the effect of the water extract of
Labisia pumila on the growth of the cells also depends on the amount of time the
extracts were allowed to interact with the HSF1184 cells. The time-dependent effect of
the Labisia pumila plant extract was only done for the water extract and not the
methanolic extract. In this study, the HSF1184 cells treated with the methanolic
extract of Labisia pumila was exposed for 48 hours to allow the interaction between the
cells and the plant extract. The experiment was not repeated at 24 hours of time
exposure because it might not be enough for the methanolic extract of Labisia pumila
to fully interact with the HSF1184 cells within the 24 hours’ time period. However, in
the future, this comparative study should be done so that a clearer picture of how the
methanolic extract of Labisia pumila interact with the HSF1184 cells could be outlined.
Apart from that, these results also prove that both Labisia pumila extracts were able to
stimulate HSF1184 cells regeneration after being exposed to UVB despite the
damaging effect of the UVB irradiation. This is one of the main sought after
characteristics in the development of any cosmeceutical product.
113
In this study, the effect of the Labisia pumila plant extracts on the HSF1184
cells was investigated in 2 different culture media conditions namely; a complete
culture media supplemented with growth serum (FBS) and culture media without the
existence of growth serum (FBS). As explained in section 4.2, the presence of a growth
serum in a cell culture media is important for the growth of any types of cell. Normally
without the presence of a growth serum, cells will cease to grow. However, in this
particular study, the experiment was repeated on HSF1184 cell culture media without
FBS because the presence of a growth serum might intervene with the interaction of
the Labisia pumila plant extracts and HSF1184 cells. This has proven to be true as the
effect of Labisia pumila plant extract on the HSF1184 cells which were cultured in
the medium without FBS showed more significant cells regeneration at 1 x 10 -4µ g/ml
of the water extract of Labisia pumila compared to its effect on HSF1184 cells cultured
in a normal medium supplemented with FBS. This was indeed a very good finding,
as this result could be used in future study to investigate the potential of the plant
extract to become the replacement for the current commercially used growth serum
such as FCS and FBS which have a lot of disadvantages.
In the final part of the master thesis, the effect of the water and methanolic
extracts of Labisia pumila on the synthesis of collagen in the HSF1184 cells were
explained and discussed. Besides being able to stimulate HSF1184 cells regeneration,
both plant extracts caused significant increase in the collagen concentration of
the HSF1184 cells in both UVB non-irradiated and UVB irradiated cells. However,
the effect of the water extract of Labisia pumila was greater than the methanolic
extract of Labisia pumila. At concentration of 1 x 10-5µ g/ml, the water extract of
Labisia pumila was able to increase the collagen synthesis in the HSF1184 UVB
irradiated cells up to 70% whereas the methanolic extract of Labisia pumila has
increased the collagen synthesis only by 20%. Higher concentration of the methanolic
extract of the plant showed inhibitory effect to the collagen synthesis, but this was not
the case for the water extract of Labisia pumila at both 1 x 10-4 µ g/ml and 1 x 10-3
114
µ g/ml, as the collagen synthesis increased slightly in both concentrations.
In conclusion, the objective of this master thesis as described in section 1.5 was
achieved. The Labisia pumila plant extracts showed a significant photoprotective effect
and has great potential to be developed and integrated into cosmeceutical product in
the future as it can address the three most important criteria in skin cosmetics.
5.2
Recommendation
Further studies should be done focusing on the identification of the possible
bioactive compounds or phytochemicals that are responsible for the photoprotective
effect of the Labisia pumila plant extract.
Apart from that, future studies should also try to identify the mechanism on
how the Labisia pumila plant extract could cause growth stimulation and also
increase collagen synthesis. The growth stimulatory effect on this study might occur due
to the adaptive mechanism of the HSF1184 cells or it could occur due to the presents
of some bioactive compounds in the plant that acted as the precursor to both growth and
collagen synthesis pathways. Therefore, this study is important to get the confirmation
on the actual mechanism on how the extract shows its photoprotective effects.
Culture media used in this study was supposed to imitate the blood circulatory
system in the human body. In reality, our blood circulatory system has a mechanism to
excrete metabolic waste products that are harmful to cell growth. This study was done
115
without medium replenishment and this means, the metabolic waste products
available in the media could cause cell death and eventually affect the result of
the study. Therefore, further studies should be done with medium replenishment so
that cell death does not happen due to the toxicity of the metabolic waste products.
115
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