Effects of Labisia pumila plant extract on the rate

of growth of Human Skin Fibroblasts Cells (HSF

1184)
Mohd Mukrish, H.1, Rohana, A.1, Fadzilah Adibah, A. M.1, Sarmidi, M.R.2
1 Faculty of Chemical and Natural Resources Engineering, Universiti
Teknologi Malaysia (UTM)
(UTM), 81310
81310, Skudai,
Skudai Johor,
Johor Malaysia
2 Chemical Engineering Pilot Plant (CEPP), Faculty of Chemical and Natural
Resources Engineering, Universiti Teknologi Malaysia (UTM), 81310, Skudai,
Johor Malaysia
Johor,

ABSTRACT
Labisia pumila or commonly known as Kacip Fatimah is traditionally used to
facilitate and induce childbirth as well as post-partum medication amongst
Malaysian women. This study concentrates on the effect of a standardized
water extract of Labisia pumila on the rate of growth of Human Skin
Fibroblasts Cells ((HSF 1184).
) The effective concentration of the p
plant
extract was determined by an efficacy test using spectrophotometry
method involving MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide).
bromide) This step is very important as a nonnon
suitable concentration of the Labisia pumila extract could be toxic to the
cell culture environment and eventually lead to cell death. The rate of
growth is defined as the growth of the cells monitored on daily basis
basis. Data
showed that the plant extract has caused significant growth promotion of
the Human Skin Fibroblasts (HSF1184) cells.

CHAPTER 1: INTRODUCTION

INTRODUCTION
why plant extract
used since ancient time for cosmetic and pharmaceutical
applications
Different parts of plant including leaves, fruits flowers, stem,
barks, buds, and roots can be manipulated or use
Cosmetically, plant and plant extracts are used due to their
proven potential
i l to provide
id moisturizing
i
i i effect,
ff
whitening,
hi i
sunscreen, antioxidant etc [1]

LABISIA PUMILA
Labisia pumila
Popular herb in Malaysia, commonly known as as Kacip
Fatimah
Traditionally used to induce and facilitate childbirth as well as
post partum medication
Interest has recently been shown In the herbal preparation to
d
determine
i iits mode
d off action
i and
d potential
i l pharmacological
h
l i l
application
Two studies have shown that the plant exhibit oestrogenic
properties [2]

 Recent study has showed that the plant extract has great
potential in giving the photoprotective action to the human
skin

HUMAN SKIN FIBROBLASTS

Why choosing Human Skin Fibroblasts (HSF1184)

Human skin fibroblasts cell line is used instead of primary cells because
cell lines can continue growing through many subcultures
The cells is available from ECACC United Kingdom
Human skin fibroblasts cells are the major
j component
p
of the skin dermis
which are involved in the organization and production of ECM product
Involves in maintaining the integrity of the human skin
Wid l used
Widely
d iin cellll culture
l
environment
i
They are a well established system for in vitro analysis of fibroblast
ggrowth,, migration
g
and collagen
g metabolism [3]
[ ]
been previously used to study skin aging [4], wound healing [5], genetic
disorder [6], evaluating cosmetic formulations toxicity [7][8], and chemical
cytotoxicity [9]

CELL REGENERATION
commonly known as cell growth is defined as the increase in
cell population
very important
i
t t tto maintain
i t i th
the iintegrity
t it off a certain
t i ttype off
cells
constant and direct exposure to the environment can induce
adaptive or degenerative pathways and influence ageing
Skin regeneration is very important because skin can repair
itself and function normally

SKIN PHYSIOLOGY
Physiology of the skin..

The skin is the largest organ of the body, weighing about four kilo-grams
and
d covering about
b
two square metres
Skin is made up of several layers namely:
Stratum corneum
Epidermis
Dermis
Hypodermis
Each layers of the human skin play a very important role in protecting the
human body from the harmful environmental agents
agents, particularly infective
organisms (bacteria or viruses germs), dirt, dust and sunlight

CROSS SECTION OF SKIN

CHAPTER 2 : METHODS &

MATERIALS

PREPARATION
Samples of dried, grounded L. pumila were extracted with a
laboratory scale extractor in water at 100 C for 4 h
The
Th extraction
t ti ratio
ti b
between
t
th
the d
dried,
i d grounded
d d raw
material and water was 1:10 by mass
the solid part was removed by filtration and the liquid part
was directly spray dried
The inlet temperature of the spray dryer was set at 180 C
C and
the outlet was set at 103 C [4]

CELL CULTURE
Normal human dermal fibroblasts cells obtained from cell line
HSF1184 were cultured in Dulbeccos modified essential
medium (DMEM) containing 10% fetal bovine serum (FBS) and
1% antibiotics
37 C in a humidified atmosphere
All cells were maintained at 37C
of 5% CO2.
normal Human skin Fibroblasts ((HSF1184)) cells from p
passage
g
8-10 were used.

MTT BIOASSAY
laboratory test and standard colorimetric assays
used to determine cytotoxicity of potential medicinal agents
and
d other
th ttoxic
i materials,
t i l since
i
th
those agents
t would
ld result
lt iin
cell toxicity and therefore metabolic dysfunction and
therefore decreased performance in the assay
Following treatment with L. pumila extract, culture medium
was removed and MTT ((0.33 g/
g/L)) solution was added for 90
min at 37 C
The supernatant was discarded and isopropanol was added to
dissolve the formazan product

 The intensity was measured colorimetrically at a wavelength

of 570 nm by using ELISA reader

TREATMENT
HSF 1184 cells were seeded at a density of 1105 cells per well
human skin fibroblasts cells were cultured in fifteen 6-well
plates
l t
All 6-well plates will represent the growth of the cells from
day 0 until day 14
The cells were treated with the Labisia pumila plant extract of
1 x 104 ug/ml concentration at day 0

Cultured media without plant extract (control)

Cultured media with the addition of plant extract

CELL COUNTING
Daily growth of the cells were monitored and cells population
were counted using haemocytometer from day 0 until day 14
Trypan
T
bl exclusion
blue
l i ttestt using
i ah
haemocytometer
t
t was used
d
to do cell counting
Trypan blue is excluded by live cells but accumulates in dead
cells
The number of stained (non-viable)
(non viable) cells and non-stained
non stained
(viable) cells are counted under a light microscope
The numbers of cells counted daily using haemocytometer
were plotted in a graph

TRYPAN BLUE EXCLUSION TEST

STATISTICAL ANALYSIS
Statistical analyses are performed using Sigma Plot
Values are expressed as mean SE with three independent
experiments
i
t
The results are represented as the meansS.E.M. All p values
of less than 0
0.05
05 were considered statistically significant

CHAPTER 3: RESULT AND

DISCUSSION

EFFICACY STUDIES
Effect of FBS on cell growth at different concentration of Labisia pumila
3.0

Absorrbance (% Co
ontrol)

2.5

2.0

1.5

1.0

0.5

0.0
Control 1e-4

1e-3

0.01

0.1

10

Concentration (ug/ml)
With FBS
Without FBS

100

1000

10000

 that Labisia pumila has a very significant effect on the growth

of HSF 1184 cell line
This
Thi iis ttrue iin b
both
th conditions,
diti
iin th
the media
di with
ith Fetal
F t l Bovine
B i
Serum (FBS) and in the media without Fetal Bovine Serum
A medium alone without FBS will not provide a complete
environment for the cell to survive
Normally if a cell line is cultured in a medium without the
existence of FBS, it will only take several days before the cells
completely died
This might suggest that Labisia pumila might be a possible
substitute for a growth serum

 Daily growth of the cells were monitored and cells population

were counted using haemocytometer from day 0 until day 14
the
th effective
ff ti concentration
t ti off th
the plant
l t extract
t t was
determined and the concentration being chosen is 1 x 10-4
ug/ml
At this concentration, the growth stimulation of the cells was
almost doubled and it did not show anyy toxic effect to the
cells.

RATE OF GROWTH
80

100
90

70

80
Cells Density x10
C
05(cells/ml)

60
70
50

60

40

50
40

30

30
20
20
10

10

0
0

10

11

12

Day
Control

Labisia pumila

Viability for control (%)

Viability for Labisia (%)

13

14

15

 the addition of Labisia pumila extract has caused a significant

increase in the growth of the cells
Normally,
N
ll th
the cells
ll ttookk about
b t 14 d
days tto b
be completely
l t l di
died,
d
however after the addition of the plant extract; live cells were
still available on day 14
Even though the number of cells declined on the same day
((dayy 7)) for both conditions,, this could happen
pp due to the
accumulation of waste products in the cell culture medium
the cell number increases almost two-fold from day 1 to day 6
for cells treated with the plant extract

 this clearly indicates that Labisia pumila has a

population
p
doublingg time
direct effect on the p
(PDT) of the cells

CONCLUSION
Results clearly show that Labisia pumila has
significantly increased the growth and the rate of
growth of HSF1184 cells
Another studies should be done with constant
replenishment of the cultured media to remove the
accumulated waste products
This could eliminate the effect of waste material on
the stimulatoryy effect of Labisia p
pumila

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