The Activity of Virgin Coconut Oil to Increase Proliferation and

COX-2 Expression towards NIH 3T3 Cell Line

Jansen Silalahi

, Dian Ika Perbina Meliala

, Yuandani

, Linda Margata

and Denny Satria

Faculty of Pharmacy, Universitas Sumatera Utara, Medan, Indonesia

Keywords: VCO, Acid Value, %FFA, NIH 3T3, Proliferation Cell, Percent of Wound Closed, Expression of COX-2.

Abstract: This research aims to investigate the effect of VCO (Virgin Coconut Oil) to increase the activity of NIH

3T3 cell proliferation and COX-2 expression. The sample used were VCO, and acid value was determined.

Proliferation was appraised using the MTT method. Furthermore, wound healing activity assays were

established with a microscopic system, and expression of COX-2 was determined using RT-PCR. Acid

value and % FFA of VCO are 1.07±0.01 and 0.51±0.02. VCO 62.5µg/mL with viability cells were

107.758±0.45%; 104.45±0.48% and 104.45±0.48% after 24h, 48h, and 72 h incubation, cell migration in the

wound healing assay after 24 h and 48 h incubation (49.11±0.09% and 74.82±0.22%), and increase in

expression of COX-2 (Control=1; and VCO=1.21). The results explain that VCO supply potent proliferation

output. This study is planned to appraise of wound closure activity of VCO on the scratched monolayer of

NIH 3T3 cell line.

1 INTRODUCTION

Wound healing is a complicated process involving

many cells consisting of four phases namely the

phases of hemostasis, inflammation, proliferation,

and remodeling (Stamm, et al., 2016). The

hemostasis phase is the beginning of the wound

healing process by involving platelets (Rodrigues, et

al., 2016). During the inflammatory phase,

fibroblasts function as cytokine secretions, and

growth factors to activate the body's defense system

(Ridiandries, et al., 2018). During the proliferation

and remodeling phases, fibroblasts are important for

granulating and reorganizing tissues of the

extracellular matrix (Ariffin and Hasham, 2016).

Wound healing is associated with bacterial

contamination in the wound area (Ariffin and

Hasham, 2016).

The ultimate aim of wound healing

is to restore the functional properties of the leather

and prevent infection (Ariffin and Hasham, 2016).

The COX enzyme consists of 2 isoenzymes such

as COX-1, COX-2 and COX-3 (COX-1 variants)

(Chandrasekharan, et al., 2002). COX-2 plays a role

in the process of angiogenesis The expression of

COX-2 affects the process of migration,

angiogenesis, and proliferation of fibroblasts

(Futagami, et al., 2002).The process of angiogenesis,

proliferation, and migration of fibroblasts is very

important in wound healing. The expression of

COX-2 affects the process of proliferation,

angiogenesis, and migration of fibroblasts

(Futagami, et al., 2002).

This research aims to evaluate the effect of VCO

(Virgin Coconut Oil) in increasing the activity of

cell proliferation, COX-2 expression, and wound

healing migration NIH 3T3 cells.

2 MATERIALS AND METHODS

2.1 Materials

VCO (Palem Mustika®, Indonesia) and all

chemicals and reagents that were used in this study

were of analytical grade. NIH 3T3 cells were gotten

from Institute of Pharmacy, Gajah Mada University.

NIH 3T3 cells were modified in Dulbecco’s

modified Eagle’s medium added with 10% Fetal

bovine serum and saved 37

C with a CO

provide of

5%.

778

Silalahi, J., Meliala, D., Yuandani, ., Margata, L. and Satria, D.

The Activity of Virgin Coconut Oil to Increase Proliferation and COX-2 Expression towards NIH 3T3 Cell Line.

DOI: 10.5220/0010088907780781

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

778-781

ISBN: 978-989-758-449-7

2.2 Methods

2.2.1 Acid Value Determination

Five (5) g VCO was weighed and procedure titration

carried out, then the acid value and free fatty acid

(FFA) percentage of HVCO was calculated as

previously described

(Margata, et al., 2018; Silalahi,

et al., 2016).

2.2.2 Proliferative Activity

HVCO (1000 μg/mL to 15.625 μg/mL in co-solvent

DMSO (Sigma) was submitted for proliferative test.

In that way, NIH3T3 cell line (58.5 x10

cells/mL)

was grown in DMEM complete medium. After 24;

48 and 72 h treatment, MTT assay was performed

and cell viability was counted to determine the

proliferative activity (Harahap, et al., 2018).

2.2.3 Wound Healing Migration Assay

The migration assay was carried out with NIH3T3

cells were seeded at 5x104cells/well in 24-well

plates and saved for 24 h at 37

C. Cultured cells

were washed up with PBS and added culture media

which containing 0.5% FBS and saved for 24 h.

Scratch was done in the bottom center of the well

within cell layer using yellow tip. Cell residues in

the plate were washed up with PBS and treated with

HVCO and incubated for 48 h at 37

C and

documented under the inverted microscope against

cell migration rapidity after 0, 24, and 48 h. The

space from scratch treatment between control and

treatment cultur cell was measured using Image J

software and defined as cell migration area

(Freiesleben, et al., 2017; Harahap, et al., 2018).

2.2.4 Expression of COX-2

NIH3T3 cells (5x10

cells/well) were planted into

6-well plate and saved for 24 h and RNA extraction

followed Harahap, et al., 2018. The supernatant was

divided and used for RNA extraction (Genaid, USA)

and RNA concentration was determined by

spectrophotometric method (Nanodrop) and stored at

-80

C until used. Complementary DNA (cDNA) was

synthesized from 3.0 μg total RNA using RT-PCR

kit (Toyobo, Japan) to make final volume of 20 μL

using random primers based on the manufacturer’s

instructions. RT-PCR was carried out in AB 7500

Fast (ABI, USA). The reaction mixture consisted of

GoTaq Green (12.5 μL) (Promega), 1.0 μL of cDNA

1 μL forward primers, 1 μL reverse primers, and 9.5

μL ddH

O to make a total volume of 25 μL. β-actin

was used as internal reference control. The PCR

primers were used for β-actin (F: 5’-gtc gta cca ctg

gca ttg t-3’; R: 5’-cag ctg tgg tga agc t-3’), Cox-2 (F:

5’-cca gca ctt cac gca tca gt-3’; R: 5’-acg ctg tct agc

cag agt ttc ag-3’). The PCR condition were

comprised of first incubation at 95

C for 2 minutes,

C for 30 sec, annealing at 55

C 30 sec, extension

at 72

for 1 minute, and 35 cycles. The PCR

products were detected by electrophoresis in 2%

agarose gels, and added gel red 10 μL. Then, they

were visualized with gel doc (Li, et al., 2017;

Harahap, et al., 2018).

2.2.5 Statistic Analysis

The results were served as means ± SD. The

statistical analysis was carried out by using SPSS

edition 21.

3 RESULT

3.1 Acid Value and %FFA of VCO

Acid value is 1.07±0.01 mg NaOH/g oil and free

fatty acids (FFA) is 0.51±0.02%.

3.2 Proliferative Activity

To appraise the effect of VCO to increase the

quantity of cells by stimulating cell proliferation.

The percentage of viable cells after treatment and

incubation for 24h, 48h, and 72h (107.76±0.45;

107.94±0.45; 104.45±0.48) showed the stimulation

effect of VCO towards proliferation of NIH3T3

cells. The effect of VCO is given in Figure 1.

100

101

102

103

104

105

106

107

108

109

110

24h 48h 72h

ViableCells

Hours

Figure 1: Percentage of Viable Cells of NIH3T3 Cells

were Treated by VCO 62.5 µg/mL for 24; 48 and 72 h and

Measured Viable Cells.

The Activity of Virgin Coconut Oil to Increase Proliferation and COX-2 Expression towards NIH 3T3 Cell Line

779

3.3 Wound Healing Migration

The scratch wound healing assay was done to

evaluate the influence of VCO on NIH3T3

migration. The wound healing migration of VCO is

given in Figure 2. A little wound repair was

observed in wells with VCO at 62.5 μg/mL after 24

and 48h incubation with 49.11 ± 0.09% and 74.82 ±

0.22% respectively closure area.



Figure 2: Wound Healing Migration Assay. NIH3T3 cells

were treated by VCO for 0; 24 and 48h and measured the

closure area. (a) control cells; (b) VCO 62.5 μg/mL.

3.4 COX-2 Expression

Two steps RT-PCR were used to evaluate COX-2

expression in NIH3T3 cells after the treatment with

VCO. VCO showed a significant up-regulatory

effect on the expression of COX-2. The COX-2

expression is given in Figure 3.



4 DISCUSSION

Acid value is defined as mg NaOH used to

neutralize FFA contained in 1 g of fats or oils to

indicate the amount of FFA in one gram fats or oils

(Silalahi, et al., 2016).

Lauric acid is antibacterial and anti-

inflammatory agent that able to overcome skin

problems (Ariffin and Hasham, 2016). Lauric acid

can decrease the time for complete epithelialization,

because lauric acid can increase proliferation cells

and migration cells (Ariffin and Hasham, 2016).

0.2

0.4

0.6

0.8

1.2

1.4

Contro lCells VCO

COX‐2Expression

COX ‐2

Figure 3: COX-2 Expression (a) control cells; (b)VCO

62.5 µg/mL.

Thourghout wound healing process, cells at the

wound side migrate, and proliferate, caused to re-

epithelialization of the wound side. Migration of

NIH 3T3 fibroblasts was evaluate with wound

healing assay. Lauric acid can increase

proliferation cells and migration cells (Ariffin and

Hasham, 2016). Cell migration activity in the VCO

group is faster than control group. Lauric acid can

increase proliferation cells and migration cells.

Lauric acid is found in VCO that stimulate cells to

migrate. So the percentage of VCO group cell

migration is faster than control group.

Cyclooxygenase-2 (COX-2) is an inducible

enzyme which plays a critical role in multiple

pathophysiological processes including

atherosclerosis, inflammation, tumorigenesis, tissue

injury, and angiogenesis (Futagami, et al., 2002).

COX-2 protein and mRNA were expressed primarily

in the head and basal layers of the epidermal wound

edges, which are arranged of proliferative and

migratory cells (Futagami, et al., 2002). In the

Ebeling, et al study, COX-2 is one of the wound

healing parameters. Pentacyclic triterpene and

botulin are active compounds of birch bark extract.

They influence the inflammatory phase of wound

healing by upregulating chemokines,

proinflammatory cytokines, and cyclooxygenase-2

(COX-2) in human primary keratinocytes. COX-2

and IL-6 that their mRNA enlargement is due to a

mRNA stabilizing effect, a process in which p38

MAPK and HuR (human antigen R) are primarily

involved (Ebeling, et al., 2014). In this study, Lauric

acid increase of expression COX-2 which mediated

angiogenesis and migration NIH 3T3 cell Two steps

RT-PCR were used to evaluated COX-2 expression

in NIH3T3 cells after the treatment with VCO.

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

780

5 CONCLUSION

VCO can increase proliferation cells, COX-2

expression, and accelerate wound closure. So that

VCO has wound healing activities.

ACKNOWLEDGEMENTS

This work was supported by University of Sumatera

Utara through “Hibah Penelitian Guru Besar”

funding 2018.

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