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Studies on the Antioxidant and Cytotoxic Potentials of the Peel
Extract of Dacryodes rostrata
Rahma Micho Widyanto1*, Lulu Safira1, Nur Faadiyah Sofian1, Syifa Aulia Mardhiyati1, Primaridiana Pradiptasari1,
Cleonara Yanuar Dini2, Wahyu Diah Proborini3
1School of Nutrition, Faculty of Medicine, Universitas Brawijaya, Jl. Veteran, Malang
2Department of Nutrition, Universitas Negeri Surabaya, Jl. Lidah Wetan, Lidah Wetan, Surabaya
3Chemical Engineering Department, Faculty of Engineering, Universitas Tribhuwana Tunggadewi, Jl. Telaga Warna Tlogomas
Malang
Abstract: Breast cancer is one type of cancer that causes the highest death in women in Indonesia.
Alternative herbal-based cancer treatments have been developed, one of which is using fruits. Dacryodes
rostrata, a fruit commonly consumed by residents in Kalimantan, is rich in antioxidants such as flavonoids
and phenolics. The purpose of this study was to determine the value of antioxidant and cytotoxic activity
of water and ethanol extract of D. rostrata peel against T-47D breast cancer cell lines. The fruit extraction
was carried out by using maceration method. Antioxidant activity test using 2,2-diphenyl-1-picrylhydrazyl
(DPPH) method and cytotoxic test using MTT method (3-(4,5-dimethylthiazole-2-yl)-2,5-
diphenitetrazolium bromide) assay. The results showed that D. rostrata water and ethanol extract had
antioxidant activity with IC50 values 121.7 ppm and 59.27 ppm, respectively. While cytotoxic effect on T-
47D cells with IC50 values of 322.55 ppm and 143.02 ppm, respectively. This study showed that D.
rostrata peel water extract had moderate antioxidant activity and moderate cytotoxic effect against T-47D
breast cancer cells in vitro which could be used as a chemo preventive to prevent and inhibit cancer cell
growth.
Keywords: Dacryodes rostrata, Antioxidant, Cytotoxic, Breast cancer, T-47D cell line
*Corresponding author: micho@ub.ac.id
1 Introduction
Breast cancer is a condition of the emergence of malignant
tumours originating from the breast glands. In breast
cancer, there is an abnormal proliferation of cells until the
cells get out of control and form a lump in the breast.
Breast cancer is one type of cancer that causes the highest
death in women in Indonesia. Breast cancer in Indonesia
was ranked first in the incidence of new cases and ranked
second in deaths from cancer where the prevalence was
highest among females (30.9%) [1].
Cancer treatment efforts can be done through radiation,
surgery, chemotherapy, and hormone therapy. Among
these efforts, the therapy that is considered the most
effective for breast cancer is chemotherapy. However, this
therapy has side effects that do not only kill cancer cells
but also kill normal cells in the body, causing nausea and
vomiting, and also trigger cachexia [2]. To overcome these
problems, cancer treatment can also be pursued with herbal
treatment, a treatment carried out by utilizing materials
from plants [3].
D. rostrata is one of Indonesia's endemic fruits
originating from Kalimantan and can be found in all
districts in West Kalimantan. This fruit is usually
consumed by soaking it in hot water for a few minutes
until the flesh is edible. The flesh and skin of D. rostrata
fruit can be an important source of energy and minerals for
humans. In addition, D. rostrata has a rich content of
protein, minerals, and oils. D. rostrata also has antioxidant
compounds such as flavonoids and phenols that can
prevent oxidative stress and can work against cancer
through the mechanism of activating the apoptotic pathway
of cancer cells [4, 5, 6, 7].
This study aims to evaluate the potential antioxidant
and anti-cancer activity of D. rostrata extract.
Measurement of antioxidant activity using the 2,2-
diphenyl-1-picrylhydrazyl (DPPH) method and anti-cancer
assay using the in vitro 3-(4,5-Dimethylthiazol-2-yl)-2,5-
Diphenyltetrazolium Bromide (MTT) assay method of T-
47D breast cancer cell line.
2 Research Method
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
(http://creativecommons.org/licenses/by/4.0/).
BIO Web of Conferences 41, 07005 (2021) https://doi.org/10.1051/bioconf/20214107005
BioMIC 2021
2.1 Preparation of extract
The fruit of D. rostrata purchased in Malawi Regency,
West Kalimantan. The sample used is peel flesh and
extracted with water and ethanol as a solvent. The extract
was then tested for antioxidant activity using the DPPH
method with concentration range from 62.5; 125; 250; and
500 ppm. And cytotoxic assay using MTT with
concentration range from 62.5; 125; 500; and 1000 ppm.
Controls for antioxidant and cytotoxic assay were ascorbic
acid and cisplatin, respectively. The test results will be
analyzed to determine the IC50 value.
The making of D. rostrata water and ethanol 50%
extract was based on a modified method [8]. A total of 50
g peel flesh dissolved into 250 ml of water or ethanol 50%.
For water extraction, after 40 minutes the results of the
immersion were heated by the water bath method for two
hours with a temperature range of 70-80 °C. Then the
heating results were separated between the liquid and the
solid by centrifugation at a speed of 10000 rpm for 15
minutes. For ethanol 50% extraction, sample were
separated using centrifuge at 1000 rpm for 15 minutes. The
supernatant is then evaporated by the water bath method in
the range of 90-100 ° C for 7 hours until the liquid
becomes concentrated. After evaporating, a vacuum oven
process is carried out until peel extracts are obtained.
2.2 Antioxidant Activity
Antioxidant activity assay was based on modified method
[9]. 50 ppm DPPH solution [Sigma-Aldrich] was prepared
by weighing 5 mg of DPPH dissolved with 100 mL of
methanol in a volumetric flask. The stock solution of D.
rostrata peel water extract was made at 1500 ppm by
weighing 150 mg of D. rostrata peel and dissolved with 1
mL DMSO [Merck] and methanol to 100 mL. From the
stock solution, the test solution was made into 4
concentrations (62.5; 125; 250; 500 ppm). The comparison
solutions were also made. A stock solution of 100 ppm was
prepared by weighing 1 mg of ascorbic acid and diluted
with methanol until it was homogeneous and the volume
was made up to 10 mL. Concentration variations were
made to 0, 5, 1, and 2 ppm.
D. rostrata peel water extract and ethanol 50% extract
of D. rostrata peel and ascorbic acid were taken 6 mL from
each concentration and on each were added 4 mL of
DPPH. The solution was then vortexed and incubated in
the darkroom at 37°C. Absorbance measurements were
carried out at a wavelength of 517 nm. The antioxidant
activity was conducted using 4 series of triplicate
concentration with ascorbic acid as a positive control.
2.3 Cell Culture and Cytotoxic Assay
Cell culture and cytotoxic assay were based on modified
method [10]. T-47D breast cancer cells were obtained from
the Cell Culture Laboratory of BPPT-LAPTIAB Puspiptek
Serpong. T-47D breast cancer cells were thawed with
gentle stirring on an air bath at 37°C for 2 minutes. After
the dilution, T-47D breast cancer cells were removed and
decontaminated using 70% ethanol under strict aseptic
conditions. The T-47D breast cancer cells were then
transferred to a centrifuge tube containing 9 mL of
complete culture medium, then centrifuged [Hettich] about
125 x g for 5-7 minutes. The centrifuged supernatant was
discarded and the cells were dissolved in the new medium.
Subsequently, T-47D breast cancer cells were incubated at
37°C in a 5% CO2 incubator [Memmert] and cultured for 3
– 7 days to achieve 80% confluency.
The cells (5x104 cells/100 ȝl) were regenerated, then
distributed into wells on 96-well plates, the homogeneity
of cell suspensions was maintained by resuspension them
periodically, while three wells were left for media control.
The cells were incubated in a CO2 incubator overnight.
The cell medium was removed with a 180° plate. Each
well was filled with 100 PBS and discharged again with a
180° plate. The concentration series of water extract of D.
rostrata peel and ethanol 50% extract of D. rostrata peel
(62.5; 250; 500; 1000 ppm) and cisplatin (3; 6; 9; 12 ppm)
were inserted into the well (triplicate). The cells were then
incubated in a CO2 incubator for 24 hours.
100 ȝl MTT [Promega] was added to the RPMI culture
medium [Gibco] (0.5 mg/ml) and then incubated in a CO2
incubator for 3-4 hours. The cell conditions were examined
using an inverted microscope. The MTT reaction was
stopped when formazan was formed clearly by adding 100
l stopper in 0.1 N HCl in the medium containing MTT. The
incubation was carried out at room temperature in the dark.
After incubation, the plates were shaken on a shaker. Then
the absorbance was measured at a wavelength of 570 nm
using an ELISA reader [BioTek]. The cytotoxic assay was
applied post-test only control group design on 4 series of
triplicate concentrations with Cisplatin as a positive
control.
2.4 Data Analysis
Data analysis to determine IC50 values using Microsoft
Excel 2010 and IBM SPSS Statistics for Windows,
Version 20 (IBM Corp., Armonk, NY, USA) SPSS.
3 Results and Discussions
3.1 Antioxidant activity
Tests of antioxidant activity on water extract of D. rostrata
peel, ethanol 50% extract of D. rostrata peel, and ascorbic
acid as a comparison with the DPPH method showed
changes in each concentration. The results of absorbance
and percent inhibition shown in Table 1. are the average of
the 3 replications that have been carried out.
Antioxidant activity testing is used to measure the
ability of antioxidants to reduce free radicals. The results
of the antioxidant activity test using ethanol 50% extract
and water extract of D. rostrata peel have antioxidant
activity with IC50 values of 121.7 ppm and 59.27 ppm
respectively. The strength of antioxidant activity is divided
into 5 categories, namely very strong if the IC50 value is
<50 ppm, strong if the IC50 value is 50-100 ppm, moderate
if the IC50 value is 101-250 ppm, weak if the IC50 value is
251-500 ppm, and inactive if the IC50 value is >500 ppm
[11]. Based on these categories, the antioxidant activity of
the ethanol 50% extract of D. rostrata peel can be
categorized as moderate, while the antioxidant activity of
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BIO Web of Conferences 41, 07005 (2021) https://doi.org/10.1051/bioconf/20214107005
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the water extract of D. rostrata peel is categorized as
strong antioxidant activity.
Several factors can affect the decrease in antioxidant
activity, one of which is the heating process when making
extracts. Phenol compounds can undergo changes caused
by the heating process [12]. The higher the temperature
used during the heating process, the phenolic compounds
that act as antioxidants decreased, hence the strength of
antioxidant activity decreased as well [13]. A long heating
process with high temperatures can cause a decrease in
antioxidant activity [14]. In this study, water extract and
ethanol 50% extract of D. rostrata peel were heated using
an oven and a vacuum oven to remove solvents. The
existence of the heating process is thought to be one of the
factors that cause a decrease in the ability of phenolic and
flavonoid compounds that function as free radical
scavengers, resulting in a decrease in the strength of
antioxidant activity.
Another factor that affects the antioxidant activity in
this study is the solvent used. Different solvents can affect
the IC50 antioxidant activity results. A very significant
difference in the solvent used in the same sample can result
in a higher or lower IC50 [15]. The results of the research
on the antioxidant activity of water extract and ethanol
50% extract of D. rostrata peel showed that water solvent
produced higher antioxidant activity. This result is in line
with the previous research that the antioxidant activity of
D. rostrata peel pulp that is produced from various
solvents such as hexane, ethyl acetate, butanol, ethanol
50%, and water, had different results [16]. In this study, it
was seen that water produced a higher antioxidant activity
than ethanol 50%. The effect of the type of solvent on the
samples tested resulted in different results. This is due to
the different nature of the polarity of the bioactive
compounds of each fruit so that the solvent with similar
polarity to the fruit being tested will attract a lot of the
bioactive components present in the fruit.
Table 1. DPPH activity of Extract of D. rostrata
y
Data were presented as mean ± standard deviation. Each sample
was performed in triplicate
3.2 Cytotoxic activity
In the cytotoxicity test, the MTT method was used using
cisplatin as a positive control. The absorbance used to
calculate the percent inhibition of proliferation in T-47D
cancer cells which can be seen in Table 2 is the average of
the 3 replications that have been carried out.
In this test, water extract and ethanol 50% extract of D.
rostrata peel respectively produced IC50 of 322.55 ppm
and 143.02 ppm. If the value is 100 ppm < IC50 < 1000
ppm, the strength can be categorized as moderate
cytotoxic, hence the second extract has a cytotoxic effect
and is included in the moderate category [17]. Based on
this classification, it is known that the ethanol 50% extract
of D. rostrata peel is moderately cytotoxic.
In a previous study regarding the antioxidant content
found in the flesh of the D. rostrata peel air solvents and
ethanol 50%, it shows that the highest antioxidant content
comes from phenols and flavonoids [16]. Flavonoids can
activate p53 protein by inhibiting tumor growth through
cell cycle and apoptosis [18]. Meanwhile, the content of
phenolic compounds can increase the inhibition of cancer
cells [19]
Table 2. Cytotoxic of Extract of D. rostrata in T-47D cell line
Data were presented as mean ± standard deviation. Each sample
was performed in triplicate
Fig. 1. Linear Regression Curve of Water Extract of D.
rostrata Concentration with Percentage of Inhibition of T-47D
Cell Proliferation.
Fig. 2. Linear Regression Curve of Ethanol 50% Extract of D.
rostrata Concentration with Percentage of Inhibition of T-47D
Cell Proliferation.
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Fig. 3. T-47 D Cell line Treated with Water Extract of D.
rostrata. (A) 0 ppm; (B) 62.5 ppm; (C) 125 ppm; (D) 250 ppm;
(E) 500 ppm.
Fig. 4. T-47 D Cell line Treated with Ethanol 50% Extract of D.
rostrata. (A) 0 ppm; (B) 62.5 ppm; (C) 125 ppm; (D) 250 ppm;
(E) 500 ppm.
4 Conclusions
It can be concluded that the water and ethanol 50% extract
of D. rostrata peel had moderate antioxidant activity and
cytotoxic effects on T-47D breast cancer cells in vitro so
that they can be used as chemo preventives to prevent and
inhibit the growth of cancer cells.
This research is fully supported by the "Tim Riset Kemayau".
Thank you also conveyed to the Chemistry Laboratory of
Universitas Tribhuwana Tunggadewi and BPPT LAPTIAB Cell
Culture Laboratory Puspiptek Serpong.
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