Effects of Labisia pumila plant extract on the rate of growth of Human Skin Fibroblasts Cells (HSFof growth of Human Skin Fibroblasts Cells (HSF

1184)

Mohd Mukrish, H.1, Rohana, A.1, Fadzilah Adibah, A. M.1, Sarmidi, M.R.21 Faculty of Chemical and Natural Resources Engineering, Universiti

Teknologi Malaysia (UTM) 81310 Skudai Johor MalaysiaTeknologi Malaysia (UTM), 81310, Skudai, Johor, Malaysia 2 Chemical Engineering Pilot Plant (CEPP), Faculty of Chemical and Natural

Resources Engineering, Universiti Teknologi Malaysia (UTM), 81310, Skudai, Johor MalaysiaJohor, Malaysia

ABSTRACTABSTRACT

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 plant ( ) pextract was determined by an efficacy test using spectrophotometry method involving MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) This step is very important as a non-diphenyltetrazolium bromide). This step is very important as a nonsuitable 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 Datagrowth is defined as the growth of the cells monitored on daily basis. Data showed that the plant extract has caused significant growth promotion of the Human Skin Fibroblasts (HSF1184) cells.

CHAPTER 1: INTRODUCTION

INTRODUCTIONINTRODUCTION

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

i l id i i i ff hi iproven potential to provide moisturizing effect, whitening, sunscreen, antioxidant etc [1]

LABISIA PUMILALABISIA 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 i i d f i d i l h l i ldetermine its mode of action and potential pharmacological application

Two studies have shown that the plant exhibit oestrogenic Two studies have shown that the plant exhibit oestrogenicproperties [2]

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

HUMAN SKIN FIBROBLASTSHUMAN 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 subculturescell lines can continue growing through many subcultures The cells is available from ECACC United Kingdom Human skin fibroblasts cells are the major component of the skin dermis j p

which are involved in the organization and production of ECM product Involves in maintaining the integrity of the human skin

Wid l d i ll l i Widely used in cell culture environment They are a well established system for in vitro analysis of fibroblast

growth, migration and collagen metabolism [3]g , g g [ ] been previously used to study skin aging [4], wound healing [5], genetic

disorder [6], evaluating cosmetic formulations toxicity [7][8], and chemical cytotoxicity [9]cytotoxicity [9]

CELL REGENERATIONCELL REGENERATION

commonly known as cell growth is defined as the increase in cell population

i t t t i t i th i t it f t i t f very important to maintain the integrity of a certain type of cells

constant and direct exposure to the environment can induce constant and direct exposure to the environment can induce adaptive or degenerative pathways and influence ageing

Skin regeneration is very important because skin can repairSkin regeneration is very important because skin can repair itself and function normally

SKIN PHYSIOLOGYSKIN PHYSIOLOGY

Physiology of the skin.. The skin is the largest organ of the body, weighing about four kilo-grams

d band covering about two square metres Skin is made up of several layers namely:

Stratum corneumStratum 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 particularly infectivehuman body from the harmful environmental agents, particularly infective organisms (bacteria or viruses germs), dirt, dust and sunlight

CROSS SECTION OF SKINCROSS SECTION OF SKIN

CHAPTER 2 : METHODS &CHAPTER 2 : METHODS & MATERIALS

PREPARATIONPREPARATION

Samples of dried, grounded L. pumila were extracted with a laboratory scale extractor in water at 100 C for 4 hTh t ti ti b t th d i d d d The extraction ratio between the dried, grounded raw material and water was 1:10 by mass

the solid part was removed by filtration and the liquid part 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 andThe inlet temperature of the spray dryer was set at 180 C and the outlet was set at 103 C [4]

CELL CULTURECELL 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) andmedium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics

All cells were maintained at 37C in a humidified atmosphereAll cells were maintained at 37 C in a humidified atmosphere of 5% CO2.

normal Human skin Fibroblasts (HSF1184) cells from passage ( ) p g8-10 were used.

MTT BIOASSAYMTT BIOASSAY

laboratory test and standard colorimetric assays used to determine cytotoxicity of potential medicinal agents

d th t i t i l i th t ld lt iand other toxic materials, since those agents would result in cell toxicity and therefore metabolic dysfunction and therefore decreased performance in the assaytherefore decreased performance in the assay

Following treatment with L. pumila extract, culture medium was removed and MTT (0.33 g/L) solution was added for 90 ( g/ )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

TREATMENTTREATMENT5 HSF 1184 cells were seeded at a density of 1105 cells per well

human skin fibroblasts cells were cultured in fifteen 6-well l tplates

All 6-well plates will represent the growth of the cells from day 0 until day 14day 0 until day 14

The cells were treated with the Labisia pumila plant extract of 1 x 104 ug/ml concentration at day 01 x 10 ug/ml concentration at day 0

Cultured media without plant extract (control)

Cultured media with the addition of plant extract

CELL COUNTINGCELL COUNTING

Daily growth of the cells were monitored and cells population were counted using haemocytometer from day 0 until day 14T bl l i t t i h t t d Trypan blue exclusion test using a haemocytometer was used to do cell counting

Trypan blue is excluded by live cells but accumulates in dead Trypan blue is excluded by live cells but accumulates in dead cells

The number of stained (non-viable) cells and non-stainedThe number of stained (non viable) cells and non stained (viable) cells are counted under a light microscope

The numbers of cells counted daily using haemocytometerThe numbers of cells counted daily using haemocytometer were plotted in a graph

TRYPAN BLUE EXCLUSION TESTTRYPAN BLUE EXCLUSION TEST

STATISTICAL ANALYSISSTATISTICAL ANALYSIS

Statistical analyses are performed using Sigma Plot Values are expressed as mean SE with three independent

i texperiments The results are represented as the meansS.E.M. All p values

of less than 0 05 were considered statistically significantof less than 0.05 were considered statistically significant

CHAPTER 3: RESULT ANDCHAPTER 3: RESULT AND DISCUSSION

EFFICACY STUDIESEFFICACY STUDIESEffect of FBS on cell growth at different concentration of Labisia pumila

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Concentration (ug/ml)

Control 1e-4 1e-3 0.01 0.1 1 10 100 1000 100000.0

With FBS Without FBS

that Labisia pumila has a very significant effect on the growth of HSF 1184 cell lineThi i t i b th diti i th di ith F t l B i This is true in both conditions, in the media with Fetal Bovine Serum (FBS) and in the media without Fetal Bovine Serum

A medium alone without FBS will not provide a complete 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 theNormally 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 14th ff ti t ti f th l t t t the effective concentration of the plant extract was determined and the concentration being chosen is 1 x 10-4ug/mlug/ml

At this concentration, the growth stimulation of the cells was almost doubled and it did not show any toxic effect to the ycells.

RATE OF GROWTHRATE OF GROWTH10080

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Day

Control Labisia pumila Viability for control (%) Viability for Labisia (%)

the addition of Labisia pumila extract has caused a significant increase in the growth of the cellsN ll th ll t k b t 14 d t b l t l di d Normally, the cells took about 14 days to be completely died, however after the addition of the plant extract; live cells were still available on day 14still available on day 14

Even though the number of cells declined on the same day (day 7) for both conditions, this could happen due to the ( y ) , ppaccumulation 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 direct effect on the population doubling time p p g(PDT) of the cells

CONCLUSIONCONCLUSION

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 stimulatory effect of Labisia pumilay p

REFERENCESREFERENCES

1. Blum P, Schurch C, Zulli F (2007) Topische Anwendung von dedifferenzierten Pflanzenzellen fur den Schutz und dieErneuerung von Hautstammzellen. Patent pending

2. Institute for Medical Research (IMR), 2002. Kacip Fatimah. Malaysian Herbal Medicine Res. Center "Estrogenic and Androgenic Activities of Kacip Fatimah (Labisia Pumila)", Kuala Lumpur (Online).

3. Booth, A. B. Et al. Biochem, Biophys. Acta 607;145 (1980).4. Hyun-kyung Choi, Dong-hyun Kim, Jin Wook Kim, Sulaiman Ngadiran, Mohamad Roji Sarmidi, and Chang Seo Park.

Labisia pumila Extract Protects Skin Cells from Photoaging Caused by UVB Irradiation. Journal of Bioscience and Bioengineering VOL. 109 No. 3, 291296, 2010.

5. Saltzman, W. M. (2004). Tissue Engineering: Principles for the Design of Replacement Organ and Tissues. New York: Oxford University Press.

6. Paradisi, M., McClintock, D., Boguslavsky, R. L., Pedicelli, C., Worman, H. J and Djabali, K. (2005). Dermal Fibroblast in Hutchinson-Gilford Progeria Syndrome with the Lamin A G608G Mutation have Dysmorphic Nuclei and are Hypersensitive to Heat Stress Cell Biology 6 27Hypersensitive to Heat Stress. Cell Biology. 6,27.

7. Losio, N., Bertasi, B., DAbrosca, F., Ferrari, M., Avalle, N., and Fishbach, M. (1999). In Vitro Product Safety Evaluation: A screening Study on a series of Finished Cosmetic Products. Alternatives to Laboratory Animals. 27, 351.

8. Gerhard J. Nohynek, Eric Antignac, Thomas Re, Herve Toutain. (2009). Safety Assessment of personal care products/cosmetics and their ingredients. Toxicology and Applied Pharmacology. 243(2010) 239-259.p g gy pp gy ( )

9. Shrivastava, H. Y., Ravikumar, T., Shanmugasundaram, N., Babu, M. and Nair, B. U. (2005). Cytotoxicity Studies of Chromium (III) Complexes on Human Dermal Fibroblasts. Free Radical Biology and Medicine. 38(1), 58-69.

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