Introduction

Mangiferin is a xanthone derivative and xanthones are some of the most potent antioxidants
known, they are thought to be more potent than both vitamin C or vitamin E and are sometimes
unofficially referred to as “super antioxidants.” Xanthones are heat stable molecules. Mangiferin
is widely distributed as C-glucoside in higher plants, where it provides protection to producer
plants against different forms of static and dynamic stresses including ingress of pathogenic
microorganisms. It is a pharmacologically active phytochemical and a natural polyphenolic
antioxidant present in the bark, fruits, roots, and leaves of Mangifera indica Linn, and other
medicinal plants recommended in the Indian system of medicine for treatment of a number of
immunodeficiency diseases 1,2,3 (8, 9, 24).
Mangiferin is the the primary active compound found in mangoes (Mangifera indica L.), a
flavonoid compound that contains diphenylpropane (C6-C3-C6). These compounds are known to
have iron-blocking characteristic, antioxidant, antimutagenic, anti-cancer, anti-inflammatory, and
antialergi. 4 23
Mangiferin sebagai senyawa aktif utama yang terdapat dalam mangga (Mangifera indica L.),
merupakan senyawa flavonoid dengan kerangka diphenylpropane (C6-C3-C6). Senyawa ini
diketahui memiliki sifat pencekal besi, antioksidan, antimutagenik, anti-kanker, anti-inflamasi,
dan antialergi.23

Gambar 1. Chemistry structure of mangiferin (1,3,6,7-tetrahydroxyxanthone-C2-

beta-d-glucoside) (Masibo M).3

Mangiferin are flavonoid compounds, such as the flavonoids similar to the others, suspected of
working inside the cell nucleus. The flavonoid compound quercetin has been known to work in
the cell and used as a comparison.5,6 12, 55 This research was conducted to determine the
viability of cultured HepG2 cells treated with flavonoid compounds mangiferin and quercetin
Mangiferin merupakan senyawa flavonoid, sama seperti senyawa flavonoid yang lain, diduga
bekerja didalam inti sel. Senyawa Flavonoid kuersetin diketahui bekerja di dalam sel dan
digunakan sebagai pembanding. (12, 55) Tujuan penelitian untuk menentukan viabilitas lini sel
HepG2 yang diberi dengan senyawa falavonoid mangiferin dan kuersetin.

Material and Methods

HepG2 Cell Cultured

Confluent 70-80%

Optimizing
mangiferin &
kuersetin (25-200μM)

Cell viability by
(kualitative) 40CPL
Axiovert microscope

Optimal dose 25-100 μM

Test of cell viability
for mangiferin and
by MTS
kuersetin

This study is an experimental research laboratory, using the HepG2 cell line obtained from cell
culture laboratories Eijkman Institute for Molecular Biology. The place of research, laboratory
FKGUI Oral Biology. Cells were cultured in RPMI 1640 medium with glutamine on
supplementation with 10% FBS, 1% penicillin and streptomycin and gentamicin 0.5%. Cells
were cultured in an incubator with 5% CO2 conditions at 37 ° C. Change of medium done 2-3
times per week. Cells were cultured up to confluent.

Penelitian ini adalah penelitian eksperimental laboratorium, menggunakan Lini sel HepG2 yang
diperoleh dari laboratorium kultur sel Lembaga Biologi Molekuler Eijkman. Tempat penelitian,
laboratorium Oral Biologi FKGUI. Sel dikultur dalam medium RPMI 1640 dengan glutamin
yang di suplementasi dengan FBS 10%, penicilin dan streptomisin 1% dan gentamisin 0,5%. Sel
dikultur dalam inkubator dengan kondisi CO2 5% pada suhu 37oC. Pergantian medium dilakukan
2-3 kali perminggu. Sel dikultur sampai konfluent.

Preparation for cell cultures

HepG2 cell lines were cultured in RPMI 1640 medium with glutamine on supplementation with
10% FBS, 1% penicillin and streptomycin and gentamicin 0.5%. Cells were cultured in an
incubator with 5% CO2 conditions at 37 ° C. The number of cells used in each well is
approximately 2x104 cells / mL for a microplate wells 96. Cells were cultured in the incubator
with 5% CO2 conditions at 37 ° C up to 70-80% confluent, then treated active compound, next
were cultured in an incubator for 24 hours. In this case after the cells were harvested and
dissolved in the medium, the culture taken as many as 10 mL and 10 mL trypan blue is added.
Cells were counted with a hemocytometer Neubauer Chamber. Cells were counted are living
cells, the cells can not be stained by trypan blue. Cells were counted in 5 field of view by using
the formula calculation of the number of cells as follows:

Cell count was within a five the visual field x 2 x 10 4 x volume solvent medium) / 5

Persiapan kultur sel

Sel HepG2 dikultur dalam medium RPMI 1640 dengan glutamin yang di suplementasi
dengan FBS 10%, penicilin dan streptomisin 1% dan gentamisin 0,5%. Sel dikultur dalam
inkubator dengan kondisi CO2 5% pada suhu 37oC. Jumlah sel yang dimasukkan ke dalam tiap
sumur adalah sekitar 2x104 sel/mL untuk sumuran mikroplat 96. Sel dikultur dalam inkubator
dengan kondisi CO2 5% pada suhu 37oC sampai 70-80% konfluensi baru diberi senyawa aktif,
kemudian dikultur dalam inkubator selama 24 jam. Dalam hal ini setelah sel dipanen dan
dilarutkan dalam medium kemudian kultur diambil sebanyak 10 μL dan ditambahkan 10 μL
trypan blue. Sel dihitung dengan hemocytometer Neubauer Chamber. Sel yang dihitung adalah
sel yang hidup, yaitu sel yang tidak dapat diwarnai oleh trypan blue. Sel dihitung dalam 5
lapangan pandang dengan menggunakan rumus perhitungan jumlah sel sebagai berikut:

Jumlah sel dalamlima lapangan pandang x 2 x 10 4 x volume medium pelarut

5

Viability Test Cell Line HepG2 for mangiferin and qucetin treatment.

After a 70-80% confluent cells, the cells were treated for 24 hours with mangiferin and
quercetin. Mangiferin and quercetin stock solution is prepared in a concentration of 10 mM in
dimethyl sulfoxide (DMSO). HepG2 cell lines were incubated with each active ingredient
concentration of 25 μM, 50 μM, 100μM and 200μM. The negative control used HepG2 cell line
without any intervention and treated with DMSO (solvent DMSO is mangiferin and quercetin)
with variations of the same concentration of active ingredient.

Uji Viabilitas Lini Sel HepG2 terhadap pemberian mangiferin dan kuersetin.
Setelah sel konfluen 70-80%. Selanjutnya sel diberi perlakuan selama 24 jam dengan mangiferin
dan kuersetin. Larutan stok mangiferin dibuat dalam kosentrasi 10 mM dalam dimethyl sulfoxide
(DMSO). Lini sel HepG2 diinkubasi dengan masing-masing bahan aktif pada kosentrasi 25 μM,
50 μM, 100μM dan 200 μM. Sebagai kontrol negatif digunakan sel HepG2 tanpa intervensi
apapun dan yang diberi perlakuan DMSO (DMSO adalah pelarut mangiferin dan kuersetin)
dengan variasi konsentrasi yang sama dengan bahan aktif.

Picture of Microscopic Cell Line HepG2 after treatment Mangiferin, Quercetin

Mangiferin and quercetin treatment with variations in concentration of 25 μM, 50 μM, 100 μM
and 200 μM, in cells that have 70-80% confluent, carried out by changing the medium matches
mangiferin and quercetin concentration , next in culture for 24 hours. Microscopic appearance of
cells was observed under a microscope Axiovert 40 CPL with 10x magnification
Gambaran Mikroskopis Lini Sel HepG2 dengan pemberian Mangiferin, Kuersetin
Pemberian mangiferin dan kuersetin dengan variasi konsentrasi 25 µM, 50 µM, 100 µM, dan
200 µM, pada sel yang telah konfluen 70-80%, dilakukan penggantian medium sesuai dengan
konsentrasi mangiferin, kuersetin, CuCl2 dan DFO yang digunakan selanjutnya di kultur selama
24 jam. Gambaran mikroskopis sel diamati dengan menggunakan mikroskop Axiovert 40 CPL
dengan pembesaran 10x,

Cell viability using MTS assay (The Cell Titer 96® Aqueous Assay, Promega)
Quantitative analysis of cell viability by MTS Assay kit that contains tetrazolium compound ([3-
(4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium,
inner salt , MTS (a)] and reagent electron coupling (phenazine methosulfate, PMS or phenazine
ethosulfate, PES). MTS is reduced by the cells into a product formazan that is soluble in the
culture medium. Changes MTS into formazan catalyzed by dehydrogenase enzyme found in the
cell active metabolic. in the cells to be tested are added 20 mL solution of MTS in each well and
then incubated for 3 hours. After the visible color change subsequent measurement of the
intensity of the colors on an ELISA reader at a wavelength of 490 nm. the amount of product
formazan was measured at a wavelength of 490 nm is directly proportional to the proportion of
the number of living cells in culture.

Cell viability using MTS assay (The Cell Titer 96® Aqueous Assay, Promega)
Kuantitatif viabilitas sel dianalisis menggunakan kit MTS Assay yang mengandung senyawa
tetrazolium ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium, inner salt, MTS (a)] dan reagen electron coupling (phenazine methosulfate, PMS
atau phenazine ethosulfate, PES). MTS direduksi oleh sel menjadi produk formazan yang larut
dalam medium kultur. Perubahan MTS menjadi formazan dikatalisis oleh enzim dehidrogenase
yang ditemukan dalam sel yang aktif secara metabolik. Pada Sel yang akan diuji ditambahkan 20
µL larutan MTS pada masing-masing sumur dan selanjutnya diinkubasi selama 3 jam. Setelah
terlihat perubahan warna selanjutnya dilakukan pengukuran intensitas warna pada ELISA reader
pada panjang gelombang 490 nm. Jumlah produk formazan diukur pada panjang gelombang 490
nm berbanding langsung dengan proporsi jumlah sel hidup dalam kultur.

Results:

50µM 100µM 200µM A Mangiferein 25µM

Cell Control

B Kuersetin 25 µM 50 µM 100 µM 200 µM

Fig. 2.
A.B. Cell Viability with Microscop Axiovert 40 CPL, cell control, treatment of cells with
mangiferin, quercetin, between the concentration of 25 μM, 50 μM, 100 μM, and 200 μM. At
concentration for between 25-100 μM cell was still viable, but at a concentration of 200 μM cell
lysis

Fig. 3. Cell viability using MTS assay

After exposure at 25 – 100 µM concentrations of mangiferin, quercetin and combined mangiferin and
quercetin (50% + 50%) was above 85%. Viability of untreated control cells was set to 100%. The results
are presented as means ± SD.

DISCUSSION
This research was a laboratory experimental study designed for analyze the viability of HepG2
cell line treated with mangiferin flavonoids in vitro. In this study as a negative control cell line
HepG2 was without conducting any intervention. The positive control are flavonoid quercetin
because the structure is similar to mangiferin flavonoid compound. 5, 6

PEMBAHASAN
Penelitian ini merupakan studi eksperimental laboratorium yang dirancang untuk menganalisis
viabilitas lini sel HepG2 dengan pemberian flavonoid mangiferin secara in vitro.
Pada penelitian ini sebagai kontrol negatif adalah lini sel HepG2 yang dibiarkan tumbuh, tanpa
dilakukan intervensi apapun. Kontrol positif dari mangiferin, adalah kuersetin karena stukturnya
mirip flavonoid.

Cell Viability with Microscop Axiovert 40 CPL,

Microscopic appearance of cells with the mangiferin and quercetin at concentrations between 25-
100 μM revealed no difference with the control cells, but in the treatment of mangiferin 200 μM
visible difference in the cells, when compared to control cells, seen a lot of cell lysis and
detached (arrows), indicates the possibility of death cell. For ensure this required a quantitative
examination of cell death such as the with the measurement of cell viability with the MTS assay
performed on continued research. (Figure 2)

Gambaran mikroskopik sel dengan mangiferin pada konsentrasi diantara 25-100 µM tidak
menunjukkan perbedaan dengan sel kontrol, tetapi pada perlakuan mangiferin 200 µM terlihat
perbedaan sel, bila dibandingkan dengan sel kontrol, terlihat banyak sel yang lisis dan terlepas
(tanda panah), kemungkinan menunjukan kematian sel. Untuk memastikan hal tersebut
diperlukan pemeriksaan kematian sel secara kwantitatif seperti dengan pengukuran viabilitas sel
dengan tes MTS yang dilakukan pada penelitian lanjut.

Viability of cell line HepG2 with the mangiferin treatment using MTS test

The viability test conducted to prove whether test substance is treated in the study may affect the
growth of HepG2 cell line. Tests carried out by adding an electron coupling reagent (phenazine
methosulfate, PMS or phenazine ethosulfate, PES), and tetrazolium MTS. MTS reduced by cells
into formazan product that is soluble in the culture medium. Changes MTS into formazan
catalyzed by dehydrogenase enzymes found in cells aktif. 7 33
(Figure 3)

Uji viabilitas lini sel HepG2 terhadap mangiferin using MTS assay
Uji viabilitas dilakukan untuk membuktikan apakah zat uji yang diberikan pada penelitian dapat
mempengaruhi pertumbuhan lini sel HepG2. Pengujian dilakukan dengan menambahkan suatu
pereaksi electron coupling (phenazine methosulfate, PMS atau phenazine ethosulfate, PES), dan
tetrazolium MTS. MTS direduksi oleh sel menjadi produk formazan yang larut dalam medium
kultur. Perubahan MTS menjadi formazan dikatalisis oleh enzim dehidrogenase yang ditemukan
dalam sel yang aktif.33 (Gambar 4)
Figure 4. The intermediate electron acceptor pheazine ethyl sulfite (PES), there is a transfer of electrons
from NADH (the enzyme dehydrogenase) in the cytoplasm that reduces tetrazolium MTS into formazan
product (arrows), in the medium kultur. 8,9 67, 68

Gambar 4. Intermediat akseptor elektron pheazine etil sulfit (PES), ada transfer elektron dari
NADH (enzim dehydrogenase) dalam sitoplasma yang mereduksi tetrazolium MTS menjadi
produk formazan (garis panah ), dalam medium kultur.67, 68

The results of this study showed that mangiferin is not toxic, because cell viability after exposure
at all concentrations of mangiferin and quercetin was above 85%. Viability of untreated control
10
cells was set to 100%. The test results are similar to results reported by Rao et al 69 using cell
line mesensefalon. Results of in vitro studies have also explained that the concentration
mangiferin 25-100μM secure enough and does not inhibit cell growth.
Hasil penelitian ini menunjukkan bahwa mangiferin tidak bersifat toksis, karena sel HepG2 yang
diberi konsentrasi mangiferin 25-100 µM, tetap viable diatas 85%, sebanding dengan kontrol
negatif. Hasil uji ini sama seperti hasil yang dilaporkan oleh Rao dkk69 menggunakan lini sel
mesensefalon. Hasil penelitian in vitro ini juga menjelaskan bahwa pada konsentrasi mangiferin
25-100µM cukup aman dan tidak menghambat pertumbuhan sel.