Cytotoxicity, Antiproliferative and Apoptotic Effect of Lime Prasite Subandrate, et al.

MOLEKUL Articles
www.jmolekul.com https://doi.org/10.20884/1.jm.2019.14.1.442

Cytotoxicity, Antiproliferative and Apoptotic Effect of n-Hexane Fraction of Lime Parasite

(Dendrophtoe pentandra)

Subandrate1, Masayu Farah Diba2*, Salni3, Triwani4, Sri Nita4

1
Department of Biochemistry, Faculty of Medicine, Universitas Sriwijaya, Palembang
2
Biomedical Program, Faculty of Medicine, Universitas Sriwijaya, Palembang
3
Department of Biology, Faculty Mathematics and Natural Sciences, Universitas Sriwijaya, Palembang
4
Department of Medical of Biology, Faculty of Medicine, Universitas Sriwijaya, Palembang

*
Corresponding author email: farahdiba.unsri2011@gmail.com

Received: 16 Sep 2018; Accepted: 30 Jan 2019; Available online: 5 Jun 2019

ABSTRACT. Breast cancer is one of the biggest causes of death in women in the world. Lime parasite (Dendrophtoe
pentandra (L.) Miq.), a folk remedy used by Indonesian people, is believed to be efficacious as anticancer drug. This
research aims to know the activity of n-hexane fractions of lime parasite in inhibiting the proliferation and apoptosis of
T47D cells in vitro. Cytotoxic test with MTT method assay from n-hexane fractions used a multilevel concentration.
Antiproliferative test was carried out by the method of MTT assay and cell doubling time was calculated at the time of
duplication. Apoptotic test was done with concentration of 1 IC50 and ½ IC50 which was analyzed by flow cytometry. The
results reveals that fractions of lime parasite have cytotoxic activity with concentration of IC50 is included in moderate
cytotoxic level. The result of the doubling time of the optimum fraction of n-hexane is in 31 hours with the concentration of
¼IC50. Results for the flow cytometry shows the fraction of n-hexane does not induce apoptosis in cells of T47D. Those
results show that the active fraction of lime parasite has cytotoxic activity which is able to inhibit proliferation, but does not
induce apoptosis of T47D cell.

Keywords: antiproliferative, apoptotic, cytotoxic, lime parasite, Dendrophtoe pentandra

INTRODUCTION activity. Some other studies support this statement though

Breast cancer is a physical health problem causing more researches must be performed concerning with the
morbidity and mortality (Torre, Bray, Siegel, Ferlay, effects of the extract and n-hexane fraction of lime
Tieulent, and Jemal, 2015). Breast cancer is one of the parasite (D. petandra (L.) Miq.) as antiproliferative cells
biggest causes of death in women each year. In Indonesia, inducing apoptosis of breast cancer of T47D line but there
the prevalence of breast cancer in the year 2013 amounted has been no research found up till now.
to 0.5% with an estimated population of 61,682 cases
(Depkes RI, 2015). WHO data in 2018 stated that cancer EXPERIMENTAL SECTION
is the leading cause of death in the world with estimated Tools and Materials
627,000 women died from breast cancer – that is A tool for extraction and fractionation are aluminum
approximately 15% of all cancer deaths among women. foil, paper filter, autoclave, bottle jam, bottle vial,
One of the countries that still utilizes traditional medicine separating funnel, Erlenmeyer flask, beaker, flask split,
by making use of medicinal plants is Indonesia. Some blander, blow dryer, Rotary evaporator, analytical scales.
interviews, though not structured, were carried out in Tools for cytotoxic, antiproliferative, and apoptosis test
Samarinda, East Kalimantan province and showed that the are 5% CO2 incubator with the temperature of 37 ºC,
General area of Samarinda utilized lime parasite plant as micropipette, blue tip, yellow tip, analytic scales, conical,
an anticancer drug against breast cancer and cervical microtube, hemocytometer, counter, microscope, inverted
cancer. vortex, LAF (Laminar air flow), ELISA reader, flow
In principle the extract and active fraction of lime cytometry, and camera.
parasite is believed to have potential as herbal anticancer The chemical used for the extraction is ethanol 96%,
agent. The results on the research of Elsyana, Bintang, ethyl acetate, n-hexane. Whereas for testing cytotoxicity,
and Priosoeryanto (2016), n-hexane fraction, exhibited antiproliferative, and apoptosis, it is needed dimethyl
cytotoxic activity and also possessed antiproliferative sulfoxide (DMSO), 10% H2SO4, Saline Buffer 1 x
activity on K562 and MCM-B2 cancer cell lines. Phosphate, Media culture (MK) (RPMI), MTT. 5 mg/mL
Therefore, it was suggested that clove mistletoe (D. PBS (50 mg 10 mL PBS and MTT), SDS 10% in 0.1 N
petandra (L.) Miq.) had a potent natural anticancer HCl, Foetal Bovine Serum (FBS), diphenyl tetrazolium
1
Molekul, Vol. 14. No. 1, Mei 2019: 1 – 5

bromide, sodium dodecyl sulfate (SDS), HCl, NaOH, (0.125; 0.25; 0.5; 0.0625; 0.03125 μg/mL). The cells were
sodium bicarbonate, (N-2, N-acetyl hydro piperaziln3- also treated with culture media (1% of DMSO) used as
ethanol sulfanic acid (Hepus), distillate, aqua function 1% negative control. After 24 hours of extracts, active
penicillin-streptomycin, 3% acridine, Orange-ethidium fraction and drugs exposure and culture medium were
bromide. removed and 10 µl of MTT reactant is added. After the
incubation process for 4 hours, MTT/media was removed
Research Procedures
and DMSO (100 µL) was added to dissolve crystals of
Fractionation
formazan with SDS of 10%. Absorbance was measured
Fractionation was done by the method of the FCC
by the ELISA reader.
(Fractionation liquid-Liquid) with solvent n-hexane, ethyl
The entire experiment was carried out in triplicate.
acetate, and ethanol. The extract was dissolved into 200
The concentration of substance required for 50% of
mL of ethanol and aquades with the comparison of 1:1
growth inhibition (IC50) is estimated as that gives a 50%
and put into a separating funnel, after that 200 mL of n-
decrease of absorbance in comparison with control
hexane was added and shuffled slowly and kept until a
absorbance compared with the control incubated
separate solution layer was visible. The fraction solution
simultaneously without substance (Mutalib, Ali, Othman,
was then kept and separated into jam bottles and the
Ramasamy, and Ramat, 2016; Yee, Fauzi, Najihah, Daud,
process was repeated in the same way several times until
and Sulain, 2017).
the solution color turned clear.
Fractionation was continued using ethyl acetate with Antiproliferative Test with the Technique of Doubling
the same volume and way to get the fraction of n-hexane. Time
The fraction of n-hexane was evaporated with a Rotary Doubling time is the time taken for the cancer cells to
evaporator until it was thick and continued with the grow twice as large. The cells were starved for 24 h in
drying process using hairdryer (Salni, Marisa, and Mukti. culture media containing 0.5% FBS. Next, the cells were
2011). The Fractionation of n-hexane yielded was then grown in 96 multiple dishes, at the same time samples
brought to the anticancer assay activity. were given with the concentrations that are not deadly
(scores of IC50, 1/2 IC50, 1/4 IC50, and 1/8 IC50) Sampling
Harvesting Cells and Calculation of Cells
was observed at 0, 24, and 48 hours. The amount of the
Take the cell from the incubator of CO2, observe the
living cells in each well was calculated with the MTT
condition of cells. Harvesting Cells was done after the
method and made a curve absorbance was made versus
cells reached 80% confluence. The next step is discarding
the length of incubation (CCRC, 2014).
media using a micropipette, because PBS on media
culture can turn trypsin non-active. Washing the cell is Apoptosis Assay
repeated twice with PBS volume ± 5 mL. Add trypsin- The number of cells required for apoptosis assay is
EDTA (the 0.25% trypsin 0.25%) evenly and incubate in 5xl05-lxl06 cells which were then planted in a microplate
the incubator for 3 minutes. Add media ± 500 µL to of 6 wells, and incubated for 24 hours. The next day the
enable trypsin. Observe the state of the cell in the cells were given a solution of the test and then incubated
microscope. Apply resuspension if there is still a cell clot. again for 24 hours. The media from each well was then
Transfer cells that have broken off into the new sterile taken in each concentration and then put in a 15 mL
conical tube. With a counter count the cells on the conical tube and then washed with PBS once and then
hemocytometer, observe under a microscope. Calculation accommodated on the same conical tube. Trypsin of 250
of the cells is done with the following formula: the µL was added on the wells then incubated for 3 minutes at
number of calculated cells/ml = ( ∑ cell room A+∑ cell the temperature of 37 °C (under the microscope make
room B+∑ cell room C+∑ cell rooms D/4) x 104. sure the cells were not lumpy to get maximum results).
After that 1 mL of culture media is added and then the
Cytotoxic Test by the Method MTT
media was accommodated in 15 mL conical tubes.
T47D cell culture derived from a collection of
Centrifuged at the speed of 2000 rpm for 5 minutes then
Laboratory Parasitology Faculty of Medicine University
the supernatant was removed. After added 1 mL of PBS
of Gajah Mada, Yogyakarta. The cells were routinely
media then the media moved into 1.5 mL conical tube and
grown with RPMI-1640 medium supplemented with 10%
centrifuged again with a speed of 2000 rpm for 3 minutes,
fetal calf serum and 1% of penicillin/Streptomycin and
then supernatant is thrown. Next annexin was added and
incubated at humidity of 37 °C, 5% CO2 in T75 (75 cm2)
measured with flow cytometer (Hostanska, Nisslein,
pumpkin tubes. The potential effects on the viability of
Freudenstein, Reichling, and Saller, 2004; Hasibuan, and
the cells are investigated using the MTT assay as the
Chrestella 2015).
indicator of active cells on metabolic basis. Cells were
diluted in medium culture into a concentration of 1×105
RESULTS AND DISCUSSION
cells/mL and squeezed into 96 holes of wells, and
incubated at 37 ºC with humidity of 5% of CO2 for 24 Fractionation of The Extract of Lime parasite
hours. 24.68 grams of lime parasite extract as the results of
The cells were then given treatment with the extract of extraction, furthermore 20.75 grams of condensed extract
ethanol the active fraction of Lime parasite (D. of lime parasite (D. pentandra (L.) Miq) used for liquid
pentandra) (1000; 500; 250; 125; 67.5 μg/mL) via double solvent fractionation method (FCC). The fraction of 20.75
dilution series As the positive cell controls treatment with grams of the extract of lime parasite on fraction of
doxorubicin was given with terraced concentration n-hexane has a larger weight of 3.1 grams (14.93%).

2
Cytotoxicity, Antiproliferative and Apoptotic Effect of Lime Prasite Subandrate, et al.

The percentage yield of the fraction of the extract of based on the research of Weerapreeyakul, Nonpunya,
lime parasite was 14.93% for fraction of n-hexane. Barusrux, and Thitimetharoch, (2012) they stated that the
Percentage yield results in this study differ from the IC50 has very powerful cytotoxicity if it is < 10 μg/mL,
results on the research of Elsyana, et al. (2016), with the the powerful cytoxicity is present if IC50 is between 10 to
percentage of yield in the fraction of n-hexane of 0.44%, 100 μg/mL, the IC50 score is cytotoxic if among 100-500
the fraction of ethyl acetate 2.45% and fraction of ethanol μg/mL.
of 4.65% of clove parasite (D. pentandra), where the
Double Time Antiproliferative Test
percentage of n-hexane fraction obtained was less than
Antiproliferative test is used towards fraction of n-
this research. The differences of results obtained may be
hexane with 4 concentration namely 1 IC50, ½ IC50, ¼
due to different host types and methods used in
IC50, and ⅛ IC50. Antiproliferative test is carried out using
fractionation.
MTT method. Data obtained is in the absorbance of living
Test of Cytotoxic for n-Fraction of Lime parasite cells. Antiproliferative test proves that the number of
Cytotoxic test of the extracts and fractions of lime living cells at 0, 24, 48 hours which can be described in a
parasite against cells of T47D cancer was done with linear regression equation is then made between the log of
MTT method Assay. Variation of test concentration the number of living cells by the duration of incubation to
namely 1000; 500; 250; 125; 67.5 µg/ml. For positive obtain the optimum time of cells in proliferative conduct
control doxorubicin was used in concentrations of 0.5; after being given the treatment.
0.25; 0.125; 0.0625; 0.03125 µg/mL, whereas the The calculation result of the duration of doubling time
negative control used media and control cell. The density (Table 2.) reveals that the inhibitory property of the test
of T47D cells used in the microplate was 1x104 cells/well. substance against the velocity of cell to conduct
Based on the percentage of viability using excel proliferation by comparing the duration of the doubling
calculation the score of IC50 for each treatment group can time of cell control with the duration of doubling time
be determined. For IC50 the fraction of n-hexane is of the fraction of n-hexane. The fraction of n-hexane of
158.280 μg/mL. It shows that the fraction of n-hexane has lime parasite has a doubling time of IC 50 ± 28 hours
fairly active activities that T47D cell is able to inhibit (Table 2.), which means at the hour of 28 the cells will
50% on the concentration. So it can be stated that the divide themselves into two. For ½ IC50, ¼ IC50, and 1/8
fraction of n-hexane is toxic against T47D cells. It can be IC50 the length of time needed for the cells to multiply
believed that the toxic active compound in T47D cells are themselves is in 30 hours, 31 hours and 30 hours. The cell
present in the fraction of n-hexane. control has a doubling time approximately 28 hours. The
Cytotoxic activity fraction of n-hexane of lime doubling time of the fraction of n-hexane does not differ
parasite indicates a toxicity towards T47D cells too much when compared with cell control. However,
because the score of IC50 is less than 500 μg/mL. The IC the faction still provides inhibition in the proliferation of
score of the sample belongs to the cytotoxic group are T47D cells.

Table 1. Results of cytotoxic test of n-hexane fraction of lime parasite

% (life
Material Absorbance ± SD IC50 (µg/mL)
cell)
1000 0.155 ± 0.0097 7.241
500 0.125± 0.0062 3.699
n -hexane 250 0.224 ± 0.0073 15.348 158.280
fraction (µg/mL)
125 0.698 ± 0.0017 71.271
62.5 0.783 ± 0.0278 81.307
0.5 0.116 ± 0.00094 2.558
0.25 0.114 ± 0.0054 2.322
Doxorubicin 0.125 0.237 ± 0.0095 16.844 0.074
(µg/mL)
0.0625 0.639 ±0.0137 64.384
0.03125 0.785 ± 0.0612 81.621

Table 2. Doubling Time of the test of antiproliferation of n-hexane fraction of lime parasite
N-hexane fraction
Material Control
IC50 1/2 IC50 1/4 IC50 1/8 IC50
Doubled cell 10000 7.240 8.380 9.000 9.180
Log number of cells 4 3.860 3.923 3.954 3.962
Doubling time
28 28 30 31 30
(hour)

3
Molekul, Vol. 14. No. 1, Mei 2019: 1 – 5

The results of the doubling time show that the used to distinguish the living cells, apoptosis, and
concentration of ¼IC50 is the optimum concentration of in necrosis. The percentage is calculated based on the score
inhibiting the growth of T47D cells when compared with of apoptosis of 1 IC50 and ½ IC50 of fraction n-hexane is
other concentrations (Table 2.). If the duration of 158, 28 μg/mL and 79.57 μg/mL, with the negative
doubling time of the Concentration of 1-¼ IC50 is control used is the control of the cell control T47D of
described in a curve, it will form an upside down U curve. whereas the positive control is that doxorubicin of the
According to Calabrese (2008), in the science of concentration of 0,1 μg/mL (Fig. 1).
Toxicology, hormesis is a phenomenon of dose reaction Based on Table 3, It is noted that no occurrence of
that small doses may cause stimulation effect while large the increase of the percentage of the apoptosis of T47D
doses can cause inhibition effect, which is described as a cell after providing 1 IC50 and ½IC50 of fraction of n-
form of the response of an upside down U curve or hexane when compared with the cell control . But after
inverted form of J. giving the fraction of n-hexane, the cells undergo necrosis
with the percentage 98.83% and 97.1%. So it can be
Result of Apoptosis Induction Using Flowcytometry
deduced that the active fraction of n-hexane of lime
The principle of flowcytometry is using reagent of
parasite cannot induce apoptosis in cancer cells of T47D
Annexin V to bind phosphatidylserine on the surface of
cancer.
cells that undergo apoptosis and Propidium iodide (PI) is

Table 3. Observation the results of flowcytometry of lime parasite against T47D Cells in inducing apoptosis

Living Early Late Total

Concentration Necrosis
cells Apoptosis Apoptosis apoptosis
(µg/mL) (%)
(%) (%) (%) (%)
Control Cell 96.4 0.47 1.13 1.6 2
IC50 158.28 0.7 0.03 0.46 0.49 98.83
1/2 IC50 79.14 1.81 0.15 1 1.15 97.1

Figure 1. Result of apoptosis induction using flowcytometry. (a) Control cells, (b) cells was given treatment 1 IC50, (c)
cells was given treatment ½ IC50. Note: R1= live cells, R2= early apoptosis, R3= late apoptosis and R4= necrosis.

4
Cytotoxicity, Antiproliferative and Apoptotic Effect of Lime Prasite Subandrate, et al.

CONCLUSIONS Hostanska, K., Nisslein, T., Freudenstein, J., Reichling, J.,

Lime parasite have toxic properties against cells of and Saller, R. (2004). Evaluation of Cell Death
T47D witch the score is 158.28 μg/mL as IC50 value. n- Caused by Triterpene Glycosides and Phenolic
hexane fraction of Lime parasite with doubling time Substances from Cimifuga racemosa Extract in
method affect the proliferation of T47D cells in 31 hour Human MCF-7 Breast Cancer Cells. Biol. Pharm.
with 39.57 μg/mL IC50 concentration and has no effect to Bull. 27(12): 1970-1975.
induce apoptosis on T47D cells. Mutalib, M., A., Ali, F., Othman, F., Ramasamy, R.,
Ramat, A. (2016). Phenolics profile and anti-
ACKNOWLEDGEMENTS proliferative activity of Cyphomandra betacea
The authors say thankful to Universitas Sriwijaya which Fruit in Breast and Liver Cancer Cells.
support this research by grant Sateks 2018 for financial SpringerPlus. 5:2105.
support. Salni, Hanifa M., Ratna W. (2011). Isolasi Senyawa
Antibakteri dari Daun Jengkol (Pithecolobium
REFERENCES lobatum Benth) dan Penentuan Nilai KHM-nya.
Calabrese, E. J. (2008). Hormesis: Why It Is Important to Jurnal Penelitian Sains. 14 (1(D)) 14109.
Toxicology and Toxicologists. Environmental Torre L.A, Bray F, Siegel, R.L, Ferlay, J., Tieulent, J.L.,
Toxicology and Chemistry. 27(7): 1451-1474. and Jemal, A. (2015). Global cancer statistics,
CCRC (Cancer Chemoprevention Research Center). 2012. CA: a Cancer Journal for Clinicians.
(2014). Prosedur Tetap Uji Pengamatan Proliferasi 65(2):87-108.
Sel (Doubling Time). Fakultas Farmasi Universitas Weerapreeyakul, N., Nonpunya, A., Barusrux, S.,
Gadjah Mada. Yogyakarta. Thitimetharoch, T. (2012). Evaluation of The
Depkes RI. (2015). InfoDATIN Pusat Data dan Informasi Anticancer Potential Of Six Herbs Against A
Kementerian Kesehatan RI. Jakarta: Kementrian Hepatoma Cell Line. Chinese Medicine. 7(15): 1-
Kesehatan RI. 7.
Elsyana, V., Bintang, M., Priosoeryanto, B. (2016). WHO. (2018). Breast Cancer.
Cytotoxicity and Antiproliferative Activity Assay https://www.who.int/cancer/prevention/diagnosis-
of Clove Mistletoe (Dendrophtoe pentandra (L.) screening/breast-cancer/en/. (Accessed on
Miq.) Leaves Extracts. Research Article. 6 pages. December 2018).
Hasibuan, P. A. Z. and Chrestella, J. (2014). Efek Yee, S. L., Fauzi, N. F. M., Najihah, N. N. Daud, N., M.,
Kokemoterapi Ekstrak n-Heksan dan Etilasetat Sulain, M. D. (2017). Study of Dendrophtoe
Daun BangunBangun (Plectranthus amboinicus, pentandra Ethyl Acetate Extract as Portential
(Lour.) Spreng.) Terhadap Doksorubisin pada Sel Anticancer Candidate on Safety and Toxicity
Kanker Payudara MCF7 dan T47D. Report Aspects. Journal of Analytical & Pharmaceutical
research Universitas Sumatera Utara. Research. 6(1): 00167.