CYTOTOXICITY OF COMBINATION OF DOXORUBISIN AND THE

CHARACTERIZED ETANOLIC EXTRACT OF SESOOT (Garcinia picrorrhiza Miq.)

GRAPE FRUIT (GpKar) ON HUMAN FIBROBLAST CELL

Sri Utami1, Susi Endrini1, Said Nafik2, Ervi Afifah3, Seila Arumwardana3, Hayatun Nufus3,
Dwi D. Rihibiha3, Hanna Sari W. Kusuma3, Putri Ramadani3, Wahyu Widowati4

1
Faculty of Medicine, YARSI University, Jl. Letjend Suprapto, Cempaka Putih, Jakarta
Pusat, 10510, Indonesia.
2
Directorate General of Intellectual Property, Ministry of Law and Human Rights, Republic
of Indonesia, Jl. H.R. Rasuna Said, Kuningan, Jakarta Selatan.
3
Aretha Medika Utama, Biomolecular and Biomedical Research Center, Jl. Babakan Jeruk 2
No. 9, Bandung 40163, West Java. Indonesia.
4
Faculty of Medicine, Maranatha Christian University, Jl. Prof. Drg. Surya Sumantri No. 65
Bandung 40164, West Java, Indonesia.

*Corresponding author:

Dr. Sri Utami, S.Si., M.Si., S.H.

Faculty of Medicine, YARSI University, Jl. Letjend Suprapto, Cempaka Putih, Jakarta Pusat,
10510, Indonesia.
e-mail address: uutsuyono@yahoo.com
 ABSTRACT
Cancer is a disease indicated by loss of control in cycle cell regulation and homeostatis
in multicellular organism. The highest cancer suffered by women in Indonesia is breast
cancer. Breast cancer has been commonly treated with chemotherapeutic agents, doxorubicin,
yet it generates adverse effects. Therefore, combination with herbal products or other
compounds that can enhance cytotoxicity without side effects are required. The combination
of doxorubicin and extract is expected to be spesifically toxic to cancer cell. Thus, we aimed
to observe cytotoxicity of GpKar and doxorubicin on human fibroblast cells, BJ. Cells
number and viability were measured with 20 µL (3-(4,5-dimethylthiazol-2-yl)-5-(3
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). Treatments consisted of
combination of doxorubicin (0.02 μg/mL) and GpKar 66.47 µg/mL (DES1), 132.94 µg/mL
(DES2) and 265.89 μg/mL (DES3). DES3 showed lowest viability among treatments
(89.32%). DES1 and DES2 showed high viability (>90%) which were 97.93% and 95.08%
respectively. However, DES1 was the only treatment which was comparable to control,
whilst DES2 showed significant difference compared to control. Thus, combination of
doxorubicin (0.02 μg/mL) and GpKar 66.47 µg/mL was considered safe to be further used in
the next assay. The result of the present study showed that combination of Doxorubicin and
GpKar showed high viability on normal fibroblast cells. These results indicate that
combination of both compounds can be used in cancer therapy without affecting normal cell.

Keywords: Garcinia picrorrizha, doxorubicin, cytotoxic, human fibroblast

ABSTRAK
Kanker adalah penyakit yang ditandai dengan kehilangan kontrol dalam regulasi siklus
sel pada organisme multiselular. Kanker payudara merupakan kanker tertinggi yang diderita
wanita di Indonesia. Kanker payudara umumnya diobati dengan agen kemoterapi, yaitu
doksorubisin. Akan tetapi, doksorubisin sering menimbulkan efek samping. Oleh karena itu,
dibutuhkan kombinasi doksorubisin dengan senyawa herbal yang dapat menigkatkan
sitotoksik doksorubisin. Kombinasi doksorubisin dengan ekstrak tanaman diharapkan dapat
membunuh sel kanker tanpa berefek pada sel normal. Tujuan dari penelitian ini adalah untuk
menguji sitotoksik GpKar dan doxorubicin pada sel fibroblas manusia, BJ. Jumlah sel dan
viabilitas diukur dengan 20 µL (3-(4,5-dimethylthiazol-2-yl)-5-(3 carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium (MTS). Perlakuan terdiri dari kombinasi doksorubisin (0.02
μg/mL) dan GpKar 66.47 µg/mL (DES1), 132.94 µg/mL (DES2) and 265.89 μg/mL (DES3).
DES3 menunjukkan viabilitas yang paling rendah dibandingkan perlakuan lain (89.32%).
DES1 dan DES2 menunjukkan viabilitas yang tinggi (>90%) yaitu sebesar maisng-masing
97.93% dan 95.08%. DES1 merupakan satu-satunya perlakuan yang tidak berbeda signifikan
secara statistik dengan kontrrol, sedangkan DES2 menunjukkan beda signifikan terhada
kontrol. Hasil ini menunjukkan bahwa kombinasi tersebut tidak berbahaya terhadap sel
normal. Oleh sebab itu, doksorubisin (0.02 μg/mL) dan GpKar 66.47 µg/mL dinggap aman
digunakan untuk tahap selanjutnya.

Kata Kunci: Garcinia picrorrizha, doxorubicin, sitotoksik, fibroblas manusia

BACKGROUND
Cancer is a diease indicated by loss of control in cycle cell regulation and homeostasis
in multicellular organism. Cancer cells gradually develop and they are insensitive to anti-
growth signals (1). According to International Union Againsts Cancer (2), cancer contributes
about 12% to overall death worldwide and ranked the second killer after cardiovascular
disease. World Health Organization (WHO) predicted that there are 12 million people each
year to suffer cancer that leads to death of 7.6 million. The incidence is thought to elevate in
2030 to 26 million people and 17 miilion death due to the disease. This grows faster in poor
and developing countries. Riset Kesehatan Dasar (Riskesdas) reported in 2007, prevalency of
cancer in Indonesia was 4.3 of 1000 people. The highest cancer suffered by women in
Indonesia is breast cancer with number of incidence of 26 cases per 100.000 women,
followed by cervix cancer that scored 16 cases per 100.000 women.
Breast cancer has been commonly treated with chemotherapeutic agents, doxorubicin,
yet it generates adverse effects such as dizzyness, vomiting, and
cardiac arrhythmias. Therefore, combination with herbal products or other compounds that
can enhance cytotoxicity without side effects are required. Several studies utilized
combination between garcinol and doxorubicin that showed better performance compared to
the compound alone.
Previous study showed that aceton and n-hexane extract of G. picrorrhiza Miq. bark
exhibited antimutagenic effect on standard mutans, which is potential to be used as anticancer
(3). Combination of mandarin orange (Citrus reticulata) pericarp ethanolic extract–
doxorubicin showed antiproliferative effects on MCF-7 (4). Combination of butanol fraction
of endofit mushroom secondary metabolite–doxorubicin showed synergistic effect in
antiproliferative, cell cycle, and apoptotic induction properties MCF-7 and T47D (5).
However, it remains unclear the effect of GpKar on normal cell except on Vero cell.
The combination of doxorubicin and GpKart is expected to be spesifically toxic to cancer
cell. Thus, we aimed to observe cytotoxicity of GpKar and doxorubicin on human fibroblast
cells, BJ.

MATERIALS AND METHOD

Extraction
Garcinia picrorrhiza Miq. fruit were obtained from Kebun Raya Bogor, LIPI. The rape fruit
is shown from the color of its seed that is brown. The rape fruits were extracted using
maceration technique with distillated ethanol 70% for 24 h and then it was filtered. The
filtration was repeated until the colorless filtrate, and further evaporated. The extract was
stored at a temperature of -20o C.

Cell Culture
BJ cells [ATCC®CRL-2522] were provided by Biomolecular and Biomedical Research
Center, Aretha Medika Utama. Cells were grown in α-Minimum Essential Medium Eagle (α-
MEM) [Biowest L0475], 10% Fetal Bovine Serum (FBS) [Biowest S181H], 1% Pennicilin
Streptomycin [Biowest L0022], and maintained at 37 °C in humidified atmosphere and 5%
CO2 until the cells were 80-90% confluence. Growth medium was removed and washed with
phosphate buffer saline (PBS) [Gibco 14200075]. Cells were then added with trypsin-EDTA
[Biowest L0931-500], incubated at 37 °C for 3 min. Tripsynization was stopped by adding
growth medium in equal volume. Cells were suspended and replaced into tube, centrifuged at
500 xg for 4 min. Supernatant was removed and pellet were resuspended with 4-5 mL growth
medium. Cell suspension was aliquoted into T-flask containing growth medium with density
of 8000 cell/cm2. Medium was replaced every two days. Cells were incubated at 37 °C, 5%
CO2.

Cells viability
Cells number and viability were measured with 20 µL (3-(4,5-dimethylthiazol-2-yl)-5-(3
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) [Promega, Madison, WI,
USA], and incubated at 37 °C for 3 hours. Briefly, 100 μL cells in were plated (5×103 cells
per well) and incubated for 24 h at 37 °C in a humidified atmosphere and 5% CO2. The
medium then discarded and added with 90 μL of new medium and 10 μL of combination of
doxorubicin (0.02 μg/mL) and GpKar (66.47, 132.94 and 265.89 μg/mL) in DMSO in
different plate in triplicate then incubated for 24 h. Untreated cells were served as the control.
The 20 μL MTS was added to each well. The plate was incubated in 5% CO2 at 37 °C
incubator for 4 h. The absorbance was measured at 490 nm on a microplate reader
[MultiSkan Go Thermoscientific]. The data is presented as the percentage of viable cells (%).
The viability assay was performed to determine the safe and nontoxic concentration for the
next assay.

RESULTS
Table 1. Fibroblast cells treated with GpKar
Treatments Cytotoxicity
Cells Viability Inhibition
Control 6192.00 ± 184.09a 100.00 ± 0.00a 0.00 ± 0.00a
DMSO 6109.58 ± 139.19a 98.69 ± 1.05ab 1.31 ± 1.05ab
DES1 6061.75 ± 106.73a 97.93 ± 1.41b 2.07 ± 1.41b
DES2 5883.33 ± 56.43b 95.08 ± 2.66c 4.92 ± 2.66c
DES3 5529.42 ± 116.76c 89.32 ± 1.24d 10.68 ± 1.24d
Data is presented as mean±standard deviation. Statistical analysis was performed with
ANOVA and Duncan post hoc.

As shown in Table 1, combination of Doxorubicin and Sesoot extract were toxic to BJ

cells compared to control. Combination of doxorubicin 0.02 μg/mL and extract of 265.89
μg/mL showed lowest viability among treatments (89.32%). Extract of 66.47 μg/mL (DES1)
and 132.94 μg/mL (DES2) showed high viability (>90%) which were 97.93% and 95.08%
resepectively. However, DES1 was the only treatment which was comparable to control,
whilst DES2 showed significant difference compared to control. Thus, DES1 was considered
safe to be further used in the next assay.

DISCUSSION
Breast cancer has been commonly treated with chemotherapeutic agents, doxorubicin,
yet it generates adverse effects. Combination with herbal products or other compounds that
can enhance cytotoxicity without side effects are then suggested. Plants extracts have been
widely used in anticancer therapy due to its high toxicity, thus, they are important to also
observe their toxicity on normal cells. Recent study shows some medicinal plant extracts
from Bangladesh exhibited high toxicity on pancreatic cancer cell and low toxicity on normal
fibroblast cell Hs68 (6).
The result of the present study showed that combination of doxorubicin and G.
picrorrhiza extract showed high viability on normal fibroblast cells. These results indicate
that combination of both compounds can be used in cancer therapy without affecting normal
cell. Several studies utilized combination between garcinol and doxorubicin that showed
better performance compared to the compound alone. Adverse effects of doxorubicin was
basically reduced by garcinol (7). Garcinol or camboginol, a derivative of benzofenon, can be
obtained from G. indica (8) and G. picrorrhiza Miq. (9).
Pyhtochemical compounds of Garcinia have been studied and showed presence of
prenilated xanthones, biflavonoids, and benzophenones (8). Garcinol on MCF-7 that has
positive estrogen receptor and MDA-MB 123 with negative receptor, showed growth
inhibition and induced apoptosis on cancer-specific cell. Thus, garcinol alone can provide
beneficial effect as chemopreventive agent, mainly in breast cancer (10)

CONCLUSION
Combination of doxorubicin and GpKar showed high viability on normal fibroblast
cells. These results indicate that combination of both compounds can be used in cancer
therapy without affecting normal cell.

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