Anticancer Activity of Fibraurea Tinctoria with DLD1 Celline

Cytotoxicity Assay

Riski Sulistiarini

1,2*

, Andreanus A. Soemardji

, Elfahmi

, Maria Immaculata Iwo

, Danang Waluyo

Dian Japany Puspitasari

School

of Pharmacy, Bandung Institute of Technology, Jl. Ganesha 10 Bandung, Indonesia, 40132

Pharmacy Faculty of Mulawarman University, Jl. Gn. Kelua, Samarinda, East Borneo, Indonesia, 75243

Biotech Center BPPT, Building 630 PUSPIPTEK Area, Setu, Tangerang Selatan, Banten 15314

Key Words: Anticancer, DLD1 Cel line, Cytotoxicity Assay

Abstract: This study aims to determine the cytotoxic effects of Fibraurea tinctorial plants on DLD1 colon cancer cell

lines in vitro and determine the IC50 values of these plants. The test begins with cell-line culture in the DMEM

medium. Cultures that were ready with an estimated concentration in fresh media of 2.5x 104cells / 200uL

were exposed to 0.4uL test material in 0.1% DMSO with concentrations starting at 6.25 ppm; 12.5 ppm; 25

ppm; 50 ppm; 100 ppm; 200ppm; 400ppm; 800ppm; and 1600ppm which was then incubated for 48 hours.

The results are from methanol extract, hexane fraction, ethyl acetate fraction and HA and HB subfraction

from Fibraurea tinctorial plants, only HB subfaction which shows inhibitory activity against DLD1 colon

cancer cell line with 28% at 1600 ppm concentration.

1 INTRODUCTION

The International Agency for Research on Cancer

estimates the mortality and prevalence of cancer for

184 countries in the world at 14.1 million new cancer

cases, with 8.2 million cancer deaths, and 32.6

million people living with cancer (within five years

of diagnosis ) in 2012. By 2030, it is projected that

there will be 26 million new cancer cases and 17

million cancer deaths per year (Solowey et al. 2014;

Thun et al. 2010). Colorectal cancer is a disease that

significantly attacks millions of people every year

worldwide and is considered cancer with the third

most common occurrence rate in men and number

two in women. In the western world, with 655,000

deaths per year, colorectal cancer ranks third in

cancer-causing deaths (Granlund et al. 2011; Selek et

al. 2018).

Natural products have played a major role in

cancer chemotherapy. Anti-cancer drugs were

introduced into therapy in Western countries for

around 70 years, around 49% were either obtained

from direct organisms or derived from natural

material products (Gurnani et al. 2014). Actinomycin

D, several anthracycline D derivatives (including

daunorubicin, doxorubicin, epirubicin, idarubicin,

and valrubicin), bleomycin, valcicin, cyclic and

valrubicin derived from microbes that have been

tested for cancer chemotherapy (Cragg, Grothaus,

and Newman 2009) (Kinghorn et al. 2009). At

present 4 ingredients that become anti-cancer drugs

derived from plants are used clinically in the US and

Europe, namely vinca (Catharanthus) bisindole

alkaloids (vinblastin, vincristine, vinorelbine,

vinflunine), semi-synthetic epipodophyllotoxins

(etoposide, teniposide, and etoposide phosphate) ),

taxel (paclitaxel and paclitaxel albumin-stable

nanoparticle formulations, docetaxel, cabazitaxel),

and campotekin derivatives (irinotecan and

topotecan) (Kinghorn et al. 2016).

In Indonesia itself, the use of medicines from

natural ingredients is a culture and hereditary habits.

Kayu kuning is a plant that is used as an anti-cancer

therapy and also in conditions of digestive tract

infections. One type of yellow wood is tinctorial

Fibraurea (Wahyudi, Ratnadewi, and Siswoyo 2016).

This plant was reported to have inhibitory activity

against MCF-7 cell line from chloroform extract with

IC50 value as significant as 11.2 ppm, although

methanol and water extracts did not show significant

activity to inhibit cell line extracted from breast

cancer (Keawpradub and Dej-adisai 2005)

Sulistiarini, R., Soemardji, A., Elfahmi, ., Iwo, M., Waluyo, D. and Puspitasari, D.

Anticancer Activity of Fibraurea Tinctoria with DLD1 Celline Cytotoxicity Assay.

DOI: 10.5220/0009126401710175

In Proceedings of the 2nd Health Science International Conference (HSIC 2019), pages 171-175

ISBN: 978-989-758-462-6

171

Due to the presence of empirical information

about its ability to treat conditions in gastrointestinal

infections, a search of the colonic cell line to see the

potential of this plant as an agent for colon-rectal

therapy.

2 METHODS

Material

The research material in the form of Fibraurea

tinctorial plants was obtained from the Wiranto Kadri

Forest of Samboja East Kalimantan. Plant parts used

are the stem. Parts of this plant are then dried, cut and

mashed into simplicia powder.

Extract, Fraction and Isolate Preparation

Simplisia powder was extracted by the reflux method.

The extract obtained is then concentrated. The extract

was subsequently fractionated using organic solvents

with multi-level polarity. The obtained fractions were

then separated by the classical column method to

produce several nearly pure Fibraurea tinctorial

isolates.

Cancer Colon Cancer Cell Preparation DLD1

The DLD-1 cell line was obtained from the

Biotechnology Center BPPT Serpong, Indonesia.

Cells were grown in Dulbecco modified Eagle's

medium (DMEM) with 4 mM L-glutamine and 10%

heat-inactivated fetal calf serum. Cells were cultured

at 37 ° C in a humidified atmosphere of 95% water

and 5% CO 2, refed every two days and passaged

weekly. Cells were allowed to grow for 72–96 hours

to confluence before use.

Test Material Preparation

Extracts, fractions and isolates of tinctorial Fibraurea

plants were dissolved using 100% DMSO with a final

concentration of DMSO of 1% on the test material.

The UI material is placed in the microwell plate with

the test material template located in the second

column with dilution pointing to the right column up

to column 11. The 12th column contains positive

control of staurosporine, while column 1 contains

100% DMSO. All test materials are Duplo.

Testing the Test Material against Cancer Colon

Cancer Cells DL1

Count the cell number of remaining cell suspension

from passage (Calculate average of cell number from

3 different squares, Calculate the concentration of cell

suspension using formula below, concentration =

(average of cell number from 3 different squares x

104 x dilution rate) cells/mL, Do not forget to

multiple by the dilution rate). Prepare cell suspension

with the following concentration using fresh medium

(Cell number for DLD1 : 1.25 x 105cells/mL (2.5x

104cells/200mL)). Put 100 µL of the cell suspension

to each well of 96 well plates. Place the plate in 37°C

incubator for 24 hours (overnight). Add 0.4 µL of

each extract (dissolved in 100% DMSO) for each

well. Place the plate in incubator (37°C, 5% CO2)

incubator for 48 hours. Typical 96-well plate layout

for cytotoxicity assay showed figure 1.

Figure 1: Typical 96-well plate layout for cytotoxicity assay

HSIC 2019 - The Health Science International Conference

172

Calculate Result

We have removed the medium from the plate by

aspirator (using 1 mL serology pipette with yellow

tip). Change the tip for each sample. Wash the cell by

100 µL of PBS for each well. Aspirate the PBS. Add

1 mL of CCK-8 into the tray, then add 10 mL of

DMEM medium on it. Mix well. Add 100 µL of

DMEM containing CCK-8 into each well. Place the

plate in 37°C incubator for 3 hours. Measure the

absorbance of each well at 450 nm by a plate reader.

Calculate the survival rate as follow for each extract

and their medium











–





–



 100% (1)

As : Abs of sample well;

Ac : Abs of control well (DMSO);

Ab : Abs of Positive control well (Staurosporine)

3 RESULTS AND DISCUSSION

This test aims to determine the ability of Fibraurea

tinctorial plants to inhibit dld1 cell line as a picture of

inhibition of rectal colon cancer that occurs in

humans. The study began by collecting Fibraurea

tinctorial plants, extracting and separating active

compounds based on the polarity of the compounds

contained in these plants.

Extracts, fractions and compounds of the

separated product were tested on cell line dld1 cell

culture in the DEMB media according to the

procedure stated. The test material was presented

with cell cultures in varying concentrations ranging

from 6.25 ppm to 1600 ppm. The test results show the

value of% survival rate, which can be seen in Table

Seen from Table 1 above, the% survival rate of all

test materials and concentration variations are at

values above 90% or equal to DMSO (negative

control) which means there is no specific

concentration of extract, fraction or isolate material

which results in inhibition of cell line dld1 growth.

HB isolates showed inhibition to the percentage of

cell lines that grew by only 24.48%, but this only

occurred at a concentration of 1600 ppm. The value

of% survival if presented in the form of a curve can

be seen in Figure 1.

Tabel 1: Percentage survival rate Fibraurea tinctoria pada cell line dld1

Consentration

(ppm)

Survival rate (%)

Isolat HA Isolat HB Hexan extract Etilasetat extract Methanol extract

1600.00 108.44 ± 0.030 24.48 ± 1.415 83.069 ± 0.017 99.58 ± 0.006 111.61 ± 0.034

800.00 90.89 ± 0.153 111.32 ± 0.009 95.92 ± 0.495 112.62 ± 0.001 110.93 ± 0.005

400.00 105.24 ± 0.012 110.16 ± 0.057 98.13 ± 0.149 107.867 ± 0.056 116.51 ± 0.065

200.00 107.27 ± 0.068 103.86 ± 0.166 90.55 ± 0.010 110.42 ± 0.125 110.27 ± 0.053

100.00 110.39 ± 0.085 109.42 ± 0.040 108.96 ± 0.032 105.99 ± 0.265 117.28 ± 0.041

50.00 111.87 ± 0.087 98.45 ± 0.995 94.14 ± 0.338 107.77 ± 0.003 115.22 ± 0.020

25.00 102.20 ± 0.100 107.49 ± 0.034 102.7 ± 0.245 110.93 ± 0.048 113.01 ± 0.08

12.50 104.17 ± 0.040 101.98 ± 0.077 90.80 ± 0.098 97.36 ± 0.048 107.84 ± 0.09

6.25 107.99 ± 0.012 102.99 ± 0.089 90.61 ± 0.015 104.00 ± 0.1 110.1 ± 0.040

3.13 110.97 ± 0.023 108.74 ± 0.120 81.76 ± 0.021 98.87 ± 0.13 109.16 ± 0.042

Positife

control

(straurosporin)

1.89 ± 0.82

Negative

control

(DMSO)

Anticancer Activity of Fibraurea Tinctoria with DLD1 Celline Cytotoxicity Assay

173

Figure 2: Chart of survival rate Fibraurea tinctorial

From the overall results of the test and the value

of% survival rate, the IC50 value is calculated by the

linear regression equation, the IC50 value obtained is

shown in table 2 below:

Table 2: IC

of Fibraurea tinctorial.

Sample

(ppm)

Isolat HA > 100

Isolat HB >100

Hexan extract > 100

Etilasetat extract > 100

Methanol extract > 100

From the table above, if correlated with the

literature which states that the extract has potential as

an anticancer if the IC50 value <30ppm (Istiqomah,

Muti’ah, and Hayati 2015) and pure isolate if the

IC50 value is at a concentration of 2-4 ppm

(Heliawati et al. 2015) then the entire material above

has no potential as an anticancer in dld1 cell line cell

culture. This test does not mean to be useless, but it

can be information that if used as other medicinal

ingredients, extracts, fractions and isolates from

Fibraurea tinctorial plants have an extensive

therapeutic range to no toxic effect on body cells both

normal and abnormal (Cancer).

From this research, it can be concluded that the

extract of methanol, n-hexane fraction, ethyl acetate

fraction, and Fibraurea tinctoria isolate did not have

anticancer activity with the testing model of dld1 cell

line.

ACKNOWLEDGMENTS

Thank you to the Samboja East Kalimantan Natural

Resources Conservation Center for plant

identification, Pharmacy of Faculty Mulawarman

University for learning opportunities, Biotechnology

centre of BPPT Serpog for activity testing facilities

for cell line dld1, Indonesian Ministry of Finance

Ministry of Education Funding Scholarship

Management Institute for financial assistance, and for

the Pharmacy School of the Bandung Institute of

Technology for research facilities.

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