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Pharmaciana ,,n e ,,[q t&_rr7 a4//4,_
Vol.x, No.x, Bulan 201x, Hal. xx-xx 8to .,(,i.
*.y^r 6o4 lL^.,
ISSN: 2088 4559; e-ISSN: 2477 0256
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DOI : 10. 1"2928lpharmacian a.xxxx.xxxx q-^crzc*31 , (,\ I la,i 61,-r*
ta"
Combination of cisplatin with extracts pearl grzss (Hedyotis carymbosa L.), and extract ->
{w
't
brotowali {Tinospora crispa) as new therapy on 4Tl breast cancer cells with induction and
I cell cycle modulation in vitro assays,

Rollando Rollando*r, Muhammad Hilmi Afthonii

'Plrrr*o"y, Faculty of Science ancl technology, Ma C'hung Universiy,, Malang

ABSTRACT

Cisplatin is a chemotherapy agent that had any side effects. Conrbination with pearl grass was
cornpound pearl grass and berberine found .in brotowali sterns that have been shown to have
cytotoxic activity in cancer cells. This study airns to examine the effects the combination of
cisplatin. ethanolic extract of pearl grass (EEPG) and Ethanolic extract of brotowali rod (EEBR) on
enhanced breast cancer cell sensitivity confirrned by apoptotic induction and cell cycle modulation.
Cy,totoxic effects were tested using the MTT assay in 4Tl cells. The combination test of cisplatin
with the extract aims to Deterrnine the combination index (Cl) and cell viability. Combination
in inducing apoptosis and cell cycle modulation was observed by florv cytometry rnethod.
eft-ect
The results of the cytotoxic test in cornbination showed the value of the combination index below
one at EEPG concentration of 1 mg / rnl, EEBR 6 mg / mL, and cisplatin is 1.68 mg/ml. The
combination of EEPG, EEBR. and cisplatin causes an accumulation of cells in S phase (29.98%)
and induces apoptosis in breast cancer cells.4Tl. Results PROVE that EEPG and EEBR can be
developed as co-agent chemotherapy with cisplatin to improve the effectiveness of breast cancer
treatment.

Key wo rds : Cisp latin, cy tot oxi city, Hedy otis co ry mb os a L., Tinosp o ru crisp a.

* {l$a'<'va t\c^t;\ t lu7

EJAIb,4-
LT^A ,{*''u, c \

Corre.spgnd i ng a ut ho r:
Nar(e. Rlllando Rollando
PharrVcy. Facully of Science and technology, Ma Chung University, Malang
Email: ro.llando@machung.ac.id
No Hp:

tourno I homepage : http ://jou rn a l. u ad.a c.id/i nd ex. ph p/PH ARM ACI ANA
BBarmaciana ISSN: 2088 4559; e-ISSN: 2477 0256
INTRODUCTION (llpt)
Cancer one cause of death in the world, in 2008 about 7.6 million deaths (around 13% of
all deaths), and expected to increase to reach 13.1 million deaths in 2030 (WHO, 2013). Breast
cancer ranked first cases of cancer in women worldwide, with the incidence rate of 1,676,633
(IARC,2012).

selecti
2002).
multiple drug resistance and adverse side effects (Minami 2010). Chemotherapy drugs,
mostly in the treatment of ', is cisplatin. effects include neuroloxicity.
nephrotoxicity (Milosavl t 0), bone marrow suppression. The high doses of cisplatin
cause side effects (Florea 20ll). Therefore, it is necessary to research to find a
cure for breast cancer methods are active and selective.
Treatment strategy cancer cells not only on one target mechanism but need to
for rnultiple target mechanism, especially for be proliferate cells and migrate et al. t4)
4Tl cells are one of cancer cells in metastatic phase, and these cells
and invasion t2).
Indonesia, had many resources, had potential to developed as a chemotherapeutic
agent in breast cancer such as grass pearl and brotowali. Pearl grass contains Ursolic acid is known
to possess anti-proliferation activities and antiangiogenetic, there by preventing the spread of
cancer cells to other organs. Also, Ursolic acid can inhibit the expression regulation of
proinflammatory cytokines by inhibiting the activation of NF-rB (Foo and Nolan, 1999). Besides,
the plant contains alkaloids brotowali berberine known to have cytotoxic activify in
cells HCT-S cells NUGC gastric cancer and nasopharyngeal cancer cells Hone-l
2009).
Doses of chemotherapeutic agents can be reduced by combining a chemotherapeutic agent

,t*t\g compound of natural ingredients to produce the same therapeutic effect on target cells (Zhao
q9: 2004). Based on these studies, seaweed extract and extract pearls brotowali potential
developed as a combination chemotherapy agent cisplatin for breast cancer treatment. The
combination of pearls grass and extract seaweed extract brotowali with cisplatin is expected to
reduce the dose of cisplatin and minimize the side effects of cisplatin. This study was conducted to
see the cy'totoxic effects caused by a combination of ethanolic extracts of pearl grass (EEPG) and
ethanolic extract brotowali (EEBR) with cisplatin via modulation of cell cycle and apoptosis
induction.

IVIATERIALS AND LETHOD (llpt)

Materials
Materials used in this research are HCoL and TCa powder obtained from UPT Materi Materia
Medica Batu, Malang. Dirnethyl sulfoxide (wako chernical USA), Cisplatin (wako chemical USA),
DMEM (Gibco, Invitrogen USA), Fetal Bovine Serum (Gibco, Invitrogen USA),1.5% penicillin-
streptomycin (v/v) (Gibco, lnvitrogen USA), 0.57o Fungizone (v/v) (Gibco, lnvitrogen USA).
Tissue culture dish (TCD) (IWAKI), Trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco,
Invitrogen Canada), reagent used was 3-(4,5-dimethylthiazol-2-il)-O 2018 The Authors. Published

Combination of cisplotin extrocts of pearl gross (Hedyotis corymboso L.), ond extroct brotowoli {Tinosporo
crispo) os new theropy on 4T1 breost cancer cellsin vitro assoys (Rollondo Rollondo)
 Pharmaciana ISSN: 2088 4559; e-ISSN: 7477 0256 33
by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license
(http://creativecommons. org/licenses/by14. 0l),2,5-diphenyltetrazolium bromide (MTT) (Sigma-
Aldrich, USA), sodium dodecyl sulfate (Merck-Schuchardt, Germany), and propidium iodide (PI)
solution in phosphate-buffered saline (PBS) which contains 1 mgAni (minimum 95% (high-
performance liquid chromatography), Sigma-Aldrich Co., St Louise, MO, 63178, triton X-100 for
GC, (E. Merck, 64271, Darmstadt, Germany), and annexin V-FLOUS Apoptosis Detection Kit
(Roche, USA)

Methods
Materials Research. Plants and grass pearl brotowali obtained from UPT (Technical
Implementation Unit) Materia Medica Batu, East Java. 4Tl breast cancer cells obtained fi'om the
collection of the Laboratory of Parasitology, Faculty Medicine, University of Gadjah Mada.

1. Extraction Rod Brotowali and Grass Pearl.

Brotowali and grass pollen pearl * each weighed 250 grams and macerated with I liter of
ethanol 960/o for one day. Furthermore, screened with flannel and sediment macerated again. The
resulting extract is evaporated to separate it with a solvent so that the extract thickens (Rollando,
2018)

2. Identification of Chemical Compounds in Extracts

Both extracts weighed 2 mg and dissolved in I mL ethanol. Extract spotted on TLC plates
were then inserted into the chamber containing the mobile phase chloroform-ethanol 9: 1. Once the
mobile phase reaches the limit mark, the plate removed. The plates were sprayed with sulfuric
Cerium reagents and reagent Dragendroff. The plates dried in an oven. band fonned identified its
value hRF,

3. Preparation and Harvest cell.

4Tl cells were transferred to a conical tube containing high glucose DMEM and
centrifuged at 600 rpm for 5 minutes. The supernatant was discarded and new media added to the
flask and the cells were suspended until homogeneous. Suspension cells grown in Tissue Culture
Dish (TCD) and incubated in CO2 incubator at 37 oC. Cells were observed with a microscope and
incubated in a 5o/o C02 incubator. After confluent cel1s (*80%), harvesting the cells by removing
the media and cells were washed with PBS 3 mL 2 times, then added 0.25Yo trypsin-EDTA, and
in a C02 incubator.
3 mL media add into TCD contains cells then resuspended cells using a micropipette
) on and bottom ofTCD so that the cells do not gather. The cell suspension transferred to a
i new sterile conical tube. The number of cells was counted with a hemocytometer and cell counter.
Cells then made a cell suspension with a concentration as needed. Cy.totoxic test of single and
combination performed by using density 8xl0a cells/wells.
A stock solution of each extract was diluted with the culture medium to obtain a
concentration of 1, 1A,25,50, 75, 100,200 mg / mL. These solutions are used as a single cytotoxic
test solution. The cisplatin test solution is diluted with the culture medium with a concentration of'
0.5,1,2,4, 8 pg/ml (Rollando, 2018)

Cambinatian of cisplotin extrocts of pearl gross (Hedyotis corymbosa L.), and extroct brotowali (Tinosporo
crispo) os new theropy on 477 breost csncer cellsin vitro ossoys (Rollando Rollando)
Bharmaciana ISSN: 2088 4559; e-ISSN: 2477 A256
4. Cytotoxic Test Single and Combination Using MTT Assay
Cells were harvested and then diluted with the culture were implanted
into 96-well microplate as much as 100 mliwells and incubated 24 hours ina5oh CO2 incubator.
Before featment, the media in a plate removed and washed with lx PBS as much as 100
ml/pitting, PBS was removed, and the cell was given a test solution of 100 rnl/pitting.
Furthermore, these cells incubated 24 hours. After incubation, cells washed with PBS, and
MTT reagent added about 100 mllpitting. Cells were then incubated 3-4 hours at 37 o C. After
that, the stopper solution (10% SDS in 0.01 N HCI) as much as 100 mliwells and cells were
incubated overnight at room temperature in the dark, then read with an ELISA reader at l. 595 nm
and obtained absorption equivalent to absorbance 4Tl cells living.
treatment data is converled into percent viability were used to calculate
obtained then tested the cytotoxicity of the combination of pearl
ic extracts, extract brotowali, and cisplatin at various levels as shown in
Table I

Table l. Comparison of Content Combination Grass Extract Pearl Extract Brotowali, and Cisplatin
Comparison of Treatment Single treatment Single treatment Single treatment
Combination
1. EEPG (tC561/12); EEPG (l/12 tc5o) EEBR (li 12IC50) C (1/12 tC50)
EEBR (t/12ICso);
c (1/l2IC5o)
2. EEPG (l16IC5s); EEPC (l/6 iC5e); EEBR (l/6ICso) C (l/6 IC50)
EEBR (l/6IC56);
c (t/6IC5o)
3. eePG (l/3 tcs); EEPG (l/3IC50) EEBR (l/3 ICso) C (li3 ICso)
EEBR (l/3 rCso);
c (1/3 IC5o)
4. KS KS KM KM
Description: EEPG: Grass Pearls ethanolic extract; EEBR : ethanolic extract Brotowali Root; C :
Cisplatin; KS: Control Cells; KM: Control Media

5. Cell Preparation for-Observation of Apoptosis by Flow cytometry method.

Atotal of 5xl0)cells/wellsplantedina6-well plateeach l000mLandincubatedfor24
hours. Given cell and EEBR EEPG single solution, as weil as its combination with cisplatin at
concentrations of selected series. In an only treatment included 900 mL concentration series EEPC,
EEBR, and cisplatin in wells. In control, cells are added 900 ml of the culture medium in wells.
After treatment, the cells were incubated for 24 hours.
The combination, treatment included 300 mL EEPG and EEBR and added 300 mL of
cisplatin concentration series. After incubation, the cell media transferred to conical tubes, and
wells washed with 500 mL of PBS. Harvesting is done by adding 0.25% trypsin-EDTA as much as
200 mI-/wells and incubated 3 minutes to allow the cells apart. Cells resuspended again to be a
single cell and then transferred into the same conical tube. Sel-centrifuged at 2000 rpm for 5
minutes, then media removed. The cell sediment formed was placed in a conical tube were covered
with aluminum foil and dissolved in buffer kit annexin V-FLOUS plus 2 mL of 2 mL Pl, and
annexin V. The cell suspension is further homogenized and incubated for 10 min at room
temperature in an aluminum foil wrapped conical, Furthermore, (a{ a
Combination of cisplotin extrdcts of pearl grass (Hedyotis corymbosa L.), and extroct brotowali (Tinospora
crispo) os new theropy on 4T1- breost concer cellsin vitro ossays (Rollondo Rotlondo)
Pharrnaciana ISSN: 2088 4559; e-lSSNr 2477 0256 35

6. Observation Cycle Sel.

Total of 5x I 05 cells/wells is transferred into a 6-we11 plate each I 000 mL and incubatecl. Next, add
the reagent florv cl.tometry (25 ltropidium lodide+ 1 mL of RNase + 0.5 mL of Triton-X + 500 mL
of PBS for 1 sample) and allou'ed to stand 10 minutes. The cell suspension was homogenized and
transferred to flow-tube and analyzed by FACS Calibur flow cytometer for cell cycle profile. Data
were analyzed using flow to look at the distribution of cells in each phase. ( I
\I IZUU
-T ,/\
Data Analysis
Analysis of Cytotoxic Agents / Material I Substance Single.
Data in the form of absorbance,conyerted to cell.viability with the calculation:
;:'ls e."lser;-*rl --i'g-q:t:eelc i
.:t,:i.:e rer.
- ;:i $: cs ji f,ililtr.si ;.?s :iIed:it'--n;iil'nl
r[]::t'f,i
-
Cell viability was analyzed ra'ith software Microsofl Excel 2007 to obtain the value of linearity (r)
ancl IC5p of the picture between the log concentration curve u'ith the percentage of cell viability. R r
value compared rvith the table (p <0.05). The,effectiveness of the test compound is determined by
the perrcentage reduction in cell viability. (pU.tr I )
Comparative Analysis of Combination of Cell Viability EEPG, EEBR, and The combination
with cisplatin.
Testing the cytotoxic effect ol the mixture was conducted by MTT assay. Combined eff'ects
determined by calculating combination index based on Chou and drug reduction index
(DRl)(Reynolds and Maurer, 2005) for EIIPG, EEBR and cisplatin using software Compu Syn.

',-'"*l:+'lj
'' -;i1.,:1 ' lI;rl' :l,r,j
Description: (Dx) 1, (Dx) 2. and (Dx) 3: Concenlrations of singie compounds obtained from
intraplate percent of cell viability caused by the combination treatment (x) in the regression
equation which generates IC56 values. Dl, D2, and D3: J'he concentration of each test con-lpound is
used to test the combination

Table 2. Interpretation Value Combination Index (CI) (Holly 17)

Clvalue Interpretation
<0.1 Powertul synergistic effect
0.1-0.3 Strong synergi stic eff'ect
0.3-0.7 The synergistic effect
0.7-t.9 The synergistic effect mild-moderate
0.9-1.I Approaching the addictive effect
l.t-1.45 Mild-moderate antagoriistic effect
1.45-3.3 antagonistic efl-ect
> 3.3 Ef€ strong antagonist

Combination of cisplotin extracts af pearl gross (Hedyotis corymboso L-), and extroct brotowoli {Tinospora
crispo) as new theropy on 4TL breost cancer cellsin vitra ossays {Rollando Rollando)
9Earmaciana ISSN:2088 4559; e-ISSN: 2477 A756
Analysis of Apoptosis and Cycle Sel.
Data flor,v cyometry shows the percentage of cells in tbur quadrants, namely LL (lower
left) which means the percent of live cells, LR (lower right), which means the percent of cell early
apoptosis, UL (upper left), which means the percent of cell necrosis, and UR (upper right ) which
tneans the percent of late apoptotic cells. Induction of apoptosis is known to compare the treatment
effect compounds and combinations rvith control ceils.
Data were analyzed using flow c).tometry flowing to look at the distribution of ceils in each phase
presentation Gl, S and G2 / M. Inhibition of cell cycle can be determined by comparing the
treatment effect of the test solution with control cells.

Result and Discussion

Examination Test Resultso Chemical Compounds Extract.
Examination of the chemical content and EEBR EEPG done to ensure that the extract
and terpenoids, which have been scientifically proven to have cytotoxic activity
2002). Test results using a reagent Cerium sulfate showed that the value of HRF bv 5 in
at EEPG shows the amount of HRF 16 and 25 (Figure I ).

-l **- i
'r!

_!
IJ
-t
-j
l
J
ahcd
Figure l. Test Results Dragendorff, (a) in EEPG, (c) the EEBR. Test Cerium Sulfate, (b) at EEBR,
(d) the EEPG. Elution was carried out with the stationar,v phase silica gel 60 F254 and a mobile
phase mixture of chlorofonn: methanol (9: l). The detection was made in visible light.
This indicates that the plant extracts are both carbon atorns (Harborne, 1987). When the
plates are sprayed rvith reagent DragendrolT produce one stainwith a value of 25 FIRI reddish-
orange in EEBR, rvhile at EEPG not found stains. Stain positive for alkaloids when orange-brown
stains after being sprayed with reageilt Dragendroff (MOH, 1985). Thus, EEBR contains alkaloids
and not alkaloids. Other compounds in EEPG has a content of triterpenoids
(Y 2017), containing 2 triperpenoid compound cycloeucalenol and
aqas r6)

Cytotoxic Test Results Single Ef,PG, EBBR, and Cisplatin in Breast Cancer Cells
4Tt,

Combination of cisplotin extracts of pearl grass {Hedyotis corymbaso L.), ond extract brotowoli (Tinospors
crispo) as new therapy on 4T1 breast concer cellsin vitro ossays (Rollando Rollondo)
Pharmaciana ISSN: 2088 4559; e-ISSN: 2477 0256 37
Cyotoxic test was conducted to determine the potential of ethanolic exlracts of pearl grass
(EEPG). ethanolic extract brotowali (EEBR) and cisplatin in inhibiting breast cancer cell 4T1.
Parameters used cytotoxic test is IC56, namely the concentration of a substance that triggers cell
death of 509/o of the population.

T'able 3. IC56 value EEPC, EEBR and Cisplatin in 4Ti Cells

samples ICso

EEBR 14.35 * 0.54 pg i mL

Ciplatin 5.04 + 0.75 mg / mL

Treatment with EEPG (Figure 2b), EEBR (F-igure 2c) and cisplatin (Figure 2d) shows a r(.-1r
decrease in live-cell population compared to controls (Figure 2a). Look rounded, and fragmented pt-U,'l'+; " -,

cells indicate changes in cell morphology (Florea and Busselberg,20l l), but the difference is not
known due to cell death due to necrosis or apoptosis process, and the process of inhibition of
n
27'fv' \)
proliferation. or4rt
frr,no
Jr,K ol,L
fr&.;1r,r
r/X

.*,-re I

A B

CD
Figure 2. Effects J'reatment of 4TI cells. A total of'8000 cells/wells in 96-well plates, incubated for
24 hours in Hi-glucose DMEM media. Observations were carried out under an inverted rnicroscope
with a magnification of 100x. (A) control cell; (B) EEFG5.98pg / mL; (C) EEBl4.35 ug / mL" (d)
Cisplatin5.04 ug I mL.
Combination of cisplatin extrocts of pearl grass (Hedyotis corymbosa L.), and extract brotowoli {Tinospora
crispo) os new therapy on 4T1" breast cancer cellsin vitro assoys (Rallondo Rollando)
 EBarmaciana ISSN: 2088 4559; e-ISSN: 2477 0256
Cytotoxic Test Results combination EEPG, EEBR, and Cisplatin in Breast Cancer Cells 4Tl.
Concentration series combination in a row EEPC 0.5; 1; 2 mg lmL, EEBR of 2.5; 5; l0
mg / mL and cisplatin is L25; 2.5; 5 lm. The combination EEPG, EEB& and cisplatin on the third
series show the concentration of CI values <l (Table 4), so that their combination has a synergistic
effect.

Table 4. Value Combination Index (CI) Cisplatin Combination with EEBR and EEPG on 4T1 cells.

concentration Cell viability CI

ratio ("/o)
lll2lcso 52.86 0.43
1/6lcso 45.76 0.58
1/3ICs{} 39.87 0.86

Modulation of Cell Cycle combination EEPG, EEBR and Cisplatin in Breast Cancer Cells
4Tl-
The process of DNA synthesis in cancer cells through the cell cycle similar to healthy cells
(Foster, 2008). One of the main targets in inhibiting proliferation of cancer cells inthe modulation
of cell cycle can be observed using flow cytometry (King, 2000). Flow cytometry was able to
detect each phase of the cell cycle based on the number of chromosomes in each phase (G1, S, and
GrlM)(F Propidium iodide was used to color each phase being able to interact with
DNA 2003 ). Observation of cell cycle profile done at the24th hour (King,2000). The
of the cell cycle in detail in Table 5.

Table 5. Percentage Distribution of Cell Cycle After Treatment With Flowing Program
$- Samples Gl (%) S (%) G2 t}/d(%) % cY
Control 48,98 11.62 24.68 8.87
A (EEPG 1 ug / ml) 42.82 17.92 16.83 9.81
B (EEBR (6 ug / ml) 34.83 13.82 24.83 8.98
C (Cisplatin 1.68 m) 43.81 t1.24 29.93 9.81

The combination of A, B, and C 24.82 29.98 15.98 7,87

The test results showed the treatment with EEPG accumulation in S phase, whereas in thd'
accumulation phase EEBR S and Gz / M. Cisplatin causes accumulation in S phase combination

fr* VL.,t". )
EEPG, EEBR and cisplatin causes a collection of cells in S phase when compared with control
cells. In the combination treatment of the distribution percentage in S phase cell cycle by 29.48%
higher than the single cisplatin amounted to 11.24o/o. Accumulation of cells in S phase
combinations increased compared to the untreated cells (control cells) of 11.62%to 29.98oh. Cell
accumulation possible because of a cell cycle arrest in the phase.

Obserryation of Apoptosis combination EEPG, EEB& and cisplatin.

Combinotion of cisplotin extrqcts of peorl gross (Hedyotis corymbosa L.), and extroct brotowoli (Tinosporo
crispo) os new theropy on 4T1 breast cancer cellsin vitro ossays (Rollondo Rollando)
 Pharmaciana ISSN: 2088 4559; e-ISSN: 2477 0256 39
Induced apoptosis used to know the mechanism of cell death caused by treatment EEPG
EEBR, cisplatin, and their combinations on 4T1 breast cancer cells were incubated for 24 hours. ---,^.,
The combination EEPG, EEBR, and cisplatin are used in the observation of apoptosis using l/6 ( (f,'"lo@-'
IC56 concentration. The method used in this research is the method of annexin V were detected
using flow cytometry to see the induction of apoptosis that occurs in treatments cell. Annexin V is
a member of the family of phospholipid-binding protein on the cell membrane powerfully
negatively charged. Cell death caused by apoptosis or be distinguished by staining
propidium todide (PI) through intercalation with DNA 1997). The percentage ofcell
death after combination treatment EEPG,
Table 6. Percentage 4Tl cell Mortality After Treatment

Control EEPG l pg / mL, EEB Cisplatin 2.5 EEPGl ug/mL+

6mgl pM EEB6mg/mL+
mL Cis 5.3 pM
Early apoptosis (%) 1.45 2.34 1.52 2.64 10.92
Apoptosis end (%) 1.63 0.62 2.13 2.s3 1.24
Necrosis (7o) 0.86 0.65 0.98 0.87 1.62
Total 3.94 3.6r 4.63 6.04 13.78

Analysis of the percentage of cell death after treatment EEPG, EEB, cisplatin, and
combination of all three (Table 6) shows that control cell which shows the rate of cell death by
( Wb"a'-*r-
)
t3.94oh. Single cells undergoing treatment with EEPG, EEB, and cisplatin showed cell death row
amounted to 3.610A;4.63Vo; and 6.04Yo; whereas their combination resulting in cell death amounted
to 13.78Yo. It shows an increase in the percentage of cell death by 7 .74%;o in the third combination
of test material as compared to cisplatin alone.
Constraints on 4Tl cells occurred at the G2 lM phase, inhibition of phosphorylation of
[d\o
sign:altransducers qnd activators of transcription (STAT), and resistance at c-myc, the
compounds extract. STAT is a factor that required for transcriptional
+ '^-gA+
on at the molecular level causing a decrease in transcription, decreased
mitochondrial biogenesis, reduction in the biosynthesis of RNA and proteins, decreased
a
anaplerosis, increase gene inhibitors and lower glutaminolysis, at the cellular level it caused a
decrease in cell proliferate, decreased transformation metabolism and reduce the capacity of
metastasis ( Donald M. Miller, 12)

CONCLUSION
The optimum combination of pearl grass ethanolic extract 116 lc5o QVg / mL) and
ethanolic extract 1/6 brotowali lC56 (6pg / mL) with cisplatin (1.68 mg / mL) able to enhance the
cytotoxic effects of cisplatin against 4T1, and synergistic with CI 0,58, result in S arrest in 4Tl
breast cancer cells, and increassed induction ofapoptosis in 4T1 breast cancer cells.

ACKNOIVLEDGEMENT
The authors are grateful to the Laboratory of Chemistry and Pharmacy, the University of
Ma Chung,to support this research.
Combination of cisplotin extrocts of pearl gross (Hedyotis corymboso L.), and extroct brotowati (Tinospora
crispo) os new therapy on 477 breast cancer cellsin vitro assoys {Rollando Rollondo)
 \
A1
d\/

f ana ISSN: 2088 4559; e-ISSN: 2477 A256

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