Antioxidant and Anti-inflammatory Activity of Salacca zalacca

(Gaertn.) Voss Peel Ethanolic Extract

on Lead Induced Fibroblast Cells

Ermi Girsang

, I Nyoman Ehrich Lister

, Chrismis Novalinda Ginting

, Sri Lestari Nasution

Suhartina Suhartina

, Ubaydillah Zedd Munshy

, Rizal Rizal

, Wahyu Widowati

Universitas Prima Indonesia, Jl. Belanga No. 1, Medan 20118, North Sumatera, Indonesia

Biomolecular and Biomedical Research Center, Aretha Medika Utama, Jl. Babakan Jeruk II No. 9, Bandung 40163, West

Java, Indonesia

Faculty of Medicine, Maranatha Christian University, Jl. Surya Sumantri No. 65, Bandung 40164, West Java, Indonesia

Keywords: Lead, intracellular ROS, Cytotoxic, Salacca zalacca.

Abstract: Lead toxicity is a serious environmental disease and its effects on the human body are overwhelming. Lead

can increase reactive oxygen species (ROS) levels in the body which results in oxidative stress. Elevated ROS

levels can stimulate inflammation and cellular aging. Plants extract have the abilities as antioxidant and anti-

inflammatory agent to prevent aging and toxicity including Salacca zalacca peels extract (SPE). Cytotoxicity

assay of SPE towards fibroblast cells (BJ) was handle using MTS (3-4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium). Intracellular ROS levels were observed by flow

cytometry using DCF-DA fluorescent probe. Fibroblast cells were incubated at 37

C, 5% CO

, treated by 25

and 100 µg/ml SPE for 4 hours and followed by 400 µM Pb for 72 hours. Anti-inflammatory capacity was

conducted using ELISA to measure IL-10 and TNF-α. SPE at 3.13-100 µg/ml were nontoxic to the BJ cells.

Accumulation of intracellular ROS levels in lead-induced BJ cells were decreased by treatment using SPE at

25 and 100 µg/ml. SPE at 25 and 100 µg/ml elevated IL-10 and decreased TNF-α related to positive control

(lead-induced cells). This research shows that S. zalacca peels extract has the ability as protective effect

related to Pb poisoning.

1 INTRODUCTION

Lead (Pb) toxicity is a serious environmental disease

and its effects on the human body are overwhelming

(Wani et al., 2015). Lead can cause an increase in

reactive oxygen species (ROS) levels in the body

which results in oxidative stress. Oxidative stress is a

condition where there is an inequality between the

antioxidant defences and level of ROS, and causes

oxidative damage (Redza-Dutordoir and Averill-

Bates, 2016). The generation of ROS in cells is in

equilibrium with the defence system against free

radicals. Excessive formation of ROS can cause stress

response which then causes an increase in the aging

process of cells (Kuilman et al., 2010). Conversely,

low ROS levels affect the lifetime of an organism

(Davalli et al., 2016). Several disease and aging

process affected by accumulation of ROS which

induces apoptosis and can drive even skin cancer

(Widowati et al., 2016). Furthermore, ROS

production have a crucial role to the elevate of many

inflammatory disorder (Mittal et al., 2014).

Salacca zalacca known as snake fruit is a plant

species of the palm tree family (Arecaceae) that

native to Indonesia. Snake fruit peel are the major

waste of the consumption because is hard and

inedible, even so the previous study discover that

snake fruit peel contains important phenolic

compound such as rutin, chlorogenic acid, caffeic

acid, and protocatechuic acid (Girsang et al., 2019).

Moreover, phytochemical compounds discovered in

snake fruit peel were known to be active as an

antioxidant (Gulcin, 2006; Kikuzaki et al., 2002;

Liang and Kitts, 2015; Yang et al., 2008). In this

study, we investigated the antioxidant and anti-

inflammatory effects of SPE on lead-induced

fibroblast cells (BJ) as aging role model.

Girsang, E., Lister, I., Ginting, C., Nasution, S., Suhartina, S., Munshy, U., Rizal, R. and Widowati, W.

Antioxidant and Anti-inﬂammatory Activity of Salacca zalacca (Gaertn.) Voss Peel Ethanolic Extract on Lead Induced Fibroblast Cells.

DOI: 10.5220/0009588200680073

In Proceedings of the 6th International Conference on Advanced Molecular Bioscience and Biomedical Engineering (ICAMBBE 2019) - Bio-Prospecting Natural Biological Compounds for

Seeds Vaccine and Drug Discovery, pages 68-73

ISBN: 978-989-758-483-1

2 MATERIALS AND METHODS

2.1 Cytotoxicity Assay

Human fibroblast cell line (BJ) (ATCC

CRL-

2522

) was received from Aretha Medika Utama,

Biomolecular and Biomedical Research Center,

Bandung, Indonesia. We detected to determine the

maximum tolerance concentration of SPE on BJ cells

and to determine the optimal oxidative damage

concentration of lead (Pb) for the following

experiments. BJ Cells were cultured in MEM

(Biowest, L0416-500) suplemented with 10% fetal

bovine serum (FBS) (Biowest, S1810-500), 1%

Antibiotic/antimycotic (ABAM, Biowest,

L0010100), 1% Nanomycopulitine (Biowest, L-X16-

100), 1% Amphotericin B (Gibco, 1%), 0.1%

Gentamicin (Gibco, 15750045). Cells were incubated

at 37

C in a humidified atmosphere with 5% CO

After that, 80% of cells confluency, 5 x 10

cells were

seeded in each well of 96-well plate. After 24 h

incubation, the cells were treated with SPE at various

concentrations (3.13, 6.25, 12.5, 25, 50, and 100

µg/ml) for 24 h. To elect cell viability, 3-(4,5-

dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-

2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay

(Promega, Madison, WI, USA) was used. MTS was

added to each well at a ratio of 1:10 (Novilla et al.,

2017; Widowati et al., 2016) The plate was incubated

in 5% CO

at 37

C for 4 h. Absorbance was measured

at 490 nm on a microplate reader. The data are given

as the percentage of viable cells (%) and data were

analyzed using ANOVA and continued by Tukey post

hoc test.

2.2 Intracellular Reactive Oxygen

Species Analysis

The intracellular ROS levels were detected by flow

cytometry using a DCF-DA fluorescent probe

(invitrogen) in accordance with the method of Jie et

al (Jie et al., 2006; Prahastuti et al., 2019; Widowati

et al., 2014) with slight modification. After 7 days of

culture, BJ cells were digested with trypsin-EDTA

and 10

cells were incubated with 10 µM DCF-DA at

C for 30 min, after that incubated with SPE (25

and 100 µg/ml) for 4 h and followed by 400 µM Pb

for 3 days. The intracellular ROS levels were

analyzed using Milteny Flow Cytometer (MAQS

quant). BJ Cells treated with Pb without SPE

treatment showed as controls. The analyzed

fluorescence values were expressed as a percentage

of control.

2.3 IL-10 and TNF-α Evaluation

Evaluation of Interleukin 10 (IL-10) and Tumour

necrosis factor-α (TNF-α) were conducted using

ELISA Kit, IL-10 (Elabsci, E-EL-H0103) and TNF-α

(Elabsci, E-EL-H0109), conditioned medium (CM)

was used as a sample. Conditioned medium was

collected after culture of fibroblast cells and treatment

using SPE (25 and 100 µg/ml for 4 hours) following

by inducted using Pb (400 µM for 72 hours). Cells

were incubated at 37

C in a humidified atmosphere

with 5% CO

. The method was in accordance with

manufacturer protocol. Sample absorbances were

read at 450 nm using spectrophotometer (Multiskan

GO, Thermo Scientific). Colour changes of samples

are evaluated then read immediately at 450 nm

wavelength and the IL-10, TNF-α concentration can

be determined based on a protein standard curve

(Noverina et al., 2019; Widowati et al., 2018, 2017).

3 RESULTS AND DISCUSSION

Skin aging is a perplexing natural phenomenon

defined by continuous loss of structural stability and

function of the skin, beside skin aging can be induced

by environmental factors such as lead exposure.

Continuous lead exposure can lead to increased

physical changes in the skin and connective tissue

over intracellular ROS and cell contents (Widowati et

al., 2016).

3.1 Cytotoxicity Assay

Many cell biological studies using fibroblast for

standard cell line. Fibroblasts are liable for the

metabolism and synthesis of most connective-tissue

components and also play an effective part in the

body’s natural immune responses. Fibroblasts are the

major cells in granulation tissue and scar forming

over inflammation. In this research we define the

cytotoxicity of SPE toward fibroblast cells, depend on

the results SPE decreased cell growth only at the

highest concentration (100 µg/ml).

To employ this natural compound to health

concern, toxicity assay was conducted to assure

safety. Tetrazolium compound 3-(4,5-

dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-

2-(4-sulfophenyl)-2H-tetrazolium (MTS) has been

used for this assay. The MTS compound is bio-

reduced by cells into a colored formazan product due

to conversion by dehydrogenase enzymes in

metabolically active cells (Novilla et al., 2017).

Antioxidant and Anti-inﬂammatory Activity of Salacca zalacca (Gaertn.) Voss Peel Ethanolic Extract on Lead Induced Fibroblast Cells

Figure 1: Effect of S. zalacca peels ethanolic extract (SPE)

on cytotoxicity of fibroblast cells. *The histograms are

presented as mean ± standard deviation. The data were

analyzed with ANOVA and continued with Tukey post hoc

test. Different letters (a, ab, bc, c) indicate significant

differences among treatment. Control: cells without any

treatments; Vehicle Control: cells with DMSO 10%

treatment.

The total of each concentration of SPE were

found non-cytotoxic and safe for fibroblast cell (3.13,

6.25, 12.5, 25, 50, and 100 µg/ml) (Figure 1), as the

viability of the cells was more than 75%

(Kanlayavattanakul et al., 2012). The lower number

of cell viability was obtained from the highest

concentration of SPE but not cytotoxic. The data of

viable cell number were presented in percentage of

viable cell. Depend on the post hoc test results, the

safe concentration that can be used for further

experiments and not harmful for cells are found in

SPE concentrations 25 and 100 µg/ml.

3.2 Intracellular ROS Analysis

The accretion of ROS which generate apoptosis is

then an main contributor to several disorders and

aging (Orr and Sohal, 1994). The accretion of

intracellular ROS in aging process can drives to loss

of skin elasticity and cause formation of wrinkle,

brown spots, uneven pigmentation, and even skin

cancer (Widowati et al., 2016). Our outcome in

accordance with previous research which express that

lead exposure can elevate the ROS levels (Lopes et

al., 2016). Salacca zalacca peel extract has natural

compound such as chlorogenic acid (Liang and Kitts,

2015). Addition of chlorogenic acid can decrease

ROS levels, in accordance with Hoelzl et al (Hoelzl

et al., 2010), chlorogenic acid can decrease levels of

ROS approximately 20.3% after hydrogen peroxide

exposure.

Fluorescence intensity used as an indicator of

ROS production levels. Figure 2 shows significant

increase of ROS levels (relatively 25%) in cells

induced lead (400 µM) for 72 hours compared to

negative control (Cells stained using DCF-DA). The

outcome of treatment using SPE with concentration

of 25 and 100 µg/ml can significantly decrease ROS

levels (relatively 15% and 23%). The better optimal

SPE concentration in decreasing ROS levels was 100

µg/ml, but the concentration did not contrast

significantly from SPE concentration of 100 µg/ml

based on Tukey post hoc test.

Figure 2: Effect SPE toward ROS production on lead-

poisoned fibroblast cell.

The histograms are presented as

mean ± standard deviation. The data were analyzed with

ANOVA and continued with Tukey post hoc test (p<0.05).

Different letters (a, ab, b, c) indicate significant differences

among treatment. Negative control: cells without any

treatments; Vehicle control: cells with DMSO 10%

treatment; Positive control: cells with Pb induced; SPE 25:

cells treated Pb + SPE 25 µg/ml treatment; SPE 100: cells

treated Pb + SPE 100 µg/ml treatment.

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

Figure 3: The representative of dot blot of various

concentrations of SPE treatment on lead-poisoned

fibroblast cell toward ROS level. TBHP = tert-butyl

hydroperoxide, DCFDA = 2’,7’-dichlorofluorescin

diacetate. a: Negative control (Cell only + DCFDA)

(0.16%); b: Positive control (Cell only + Pb) (35.63%); c:

S. zalacca Peels Extract induced 25 µg/ml (4.55%); d: S.

zalacca Peels Extract induced 100 µg/ml (0.75%).

3.3 IL-10 and TNF-α Evaluation

Accumulation of intracellular ROS production can

improve an increase the expression of NF-kB, leading

to the upregulation of factors elaborated in

inflammation. Tumour Necrosis Factor-α (TNF-α)

effective releasers of IL-6 which is one of the basic

cytokines to be related to the aging process (Morley

and Baumgartner, 2011). The effect of TNF-α can

turn triggers effects that elevate inflammation that can

indicate with elevated ROS levels (B. Marcu et al.,

2010). Contrary, IL-10 is a cytokine which has a role

as an anti-inflammatory and manage by stimulating

antagonist proteins against TNF-α as pro-

inflammatory cytokine (Wojdasiewicz et al., 2014).

During inflammation, IL-10 and TNF-α are

cytokines that responsible. TNF-α and IL-10 has

contrary role, TNF-α has a role as pro-inflammatory

cytokine (Laksmitawati et al., 2016), while IL-10 has

a role of anti-inflammatory cytokine (Morley and

Baumgartner, 2011). Evaluation of IL-10 and TNF-α

have been conducted using ELISA method.

Fibroblast cells induced with lead has the highest

levels of TNF-α among others, although SPE

inclusion shows significant decrease in TNF-α.

Besides, fibroblast cells lead-induced has the lowest

levels of IL-10 and inclusion of SPE can elevate the

IL-10 levels. SPE at 25 and 100 µg/ml can reduce the

TNF-α levels but not significant, as well as IL-10

levels, both SPE concentration can elevate IL-10

levels but the results are not significant based on post

hoc test.

Figure 4: Effect of SPE toward TNF-α level (a) and IL-

10 level (b) on lead-poisoned cells model. The

histograms are presented as mean ± standard deviation. The

data were analyzed with ANOVA and continued with

Tukey post hoc test (P<0.05). Different letters (a, b) on

figure A and (a, ab, b) on figure B indicate significant

differences among treatment.

Negative control: cells

without any treatments; Vehicle Control: cells with DMSO

10% treatment; Positive control: cells with Pb induced; SPE

25: cells treated Pb + SPE 25 µg/ml treatment; SPE 100:

cells treated Pb + SPE 100 µg/ml treatment.

4 CONCLUSIONS

This current study exhibit the compatibility of S.

zalacca peels ethanolic extract as a promising

antioxidant and anti-inflammatory agent through

decrease of intracellular ROS levels and anti-

inflammatory abilities to decrease pro-inflammatory

cytokine (TNF-α) and increase anti-inflammatory

cytokine (IL-10) on lead-poisoned human fibroblast

cells.

Antioxidant and Anti-inﬂammatory Activity of Salacca zalacca (Gaertn.) Voss Peel Ethanolic Extract on Lead Induced Fibroblast Cells

ACKNOWLEDGEMENTS

We are gratefully acknowledge the financial support

of the Research Center and Service Community,

Universitas Prima Indonesia, Medan, North

Sumatera, Indonesia for research grant 2018. This

study also was funded, facilitated and supported by

Biomolecular and Biomedical Research Center,

Aretha Medika Utama, Bandung, West Java,

Indonesia. We are thankful to Hanna Sari Widya

Kusuma, Ika Adhani Sholihah, Dewani Tediana

Yusepany, Dwi Surya Artie, Anisa Siwianti from

Biomolecular and Biomedical Research Center,

Aretha Medika Utama, Bandung, West Java,

Indonesia for their valuable assistance.

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Antioxidant and Anti-inﬂammatory Activity of Salacca zalacca (Gaertn.) Voss Peel Ethanolic Extract on Lead Induced Fibroblast Cells