Indonesian Journal of Cancer Chemoprevention, June 2019

ISSN: 2088–0197
e-ISSN: 2355-8989

Selectivity Index of Alpinia galanga Extract and

1’-Acetoxychavicol Acetate on Cancer Cell Lines
Muhammad Da’i1,*, Khairunnisa Azani Meilinasary1, Andi Suhendi1, Sari Haryanti2

1
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Universitas Muhammadiyah Surakarta, Surakarta, Indonesia
2
The Center for Research and Development of Traditional Medicinal Plants and Medicines (B2P2TOOT), Karanganyar,
Indonesia

Abstract

Previous research stated that galangal (Alpinia galanga) extract has a potential as
cytotoxic agent with active compound of 1’-Acetoxychavicol Acetate (ACA). The objective
of this study was to determine the selectivity of ethanol extract, ethyl acetate fraction,
and methanol fraction of of galangal, and ACA on cancer cell lines. Cytotoxic activity was
carried out using the MTT method on T47D breast cancer, WiDr colon cancer, HeLa cervical
cancer, and Vero normal cell lines. The results showed that galangal ethanol extract and its
fractions had selectivity index equal to or less than 2 on cancer cells. Meanwhile, ACA had
selectivity index more than 3 on T47D cell and HeLa cell. ACA showed a strong cytotoxic
activity against cancer cells T47D, HeLa, and WiDr with IC50 values of 3.14, 7.26, and 12.49
μg/ml, respectively. Based on data, it could be concluded that ACA was the most selective
to inhibit T47D cell with a selectivity index of 6.6.

Keywords: 1’-Acetoxychavicol acetate, galangal (Alpinia galanga), selective index,

cytotoxic

Introduction ing cancer is galangal (Alpinia galanga) (Kuntorini,

2005).
Cancer is a disease that causes high mor- Zaeoung, et al., (2005) stated that Alpinia
tality in the world. In 2012, 8.2 million deaths have galanga as an antioxidant and free radicals scav-
been caused by this disease (Pusat Data dan In- enger has cytotoxic activity against MCF7 (breast
formasi, 2015). Chemotherapy, surgery, radiation, adeno-carcinoma) and LS174T (intestinal adeno-
and hormonal therapy are the number of common carcinoma) cells. Galangal extract at the dose of
therapies for cancer patients; however, they require 225, 450, and 750 mg/kgBW/day could increase the
high costs as well as a number of side effects due to apoptosis process and reduce proliferative activity
the low selectivity of therapy. Researchers therefore in breast cancer cells (Hartono, 2009). Cytotoxic
continue to conduct research to obtain more selec-
tive anticancer drugs. One of the potential ingredi- Submitted: June 17, 2019
ents to be developed as anti-cancer with better se- Revised: June 27, 2019
Accepted: June 27, 2019
lectivity is herbal medicines. One source of herbal
medicines that has been traditionally used for cur- *Corresponding author: muhammad.dai@ums.ac.id

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Indones. J. Cancer Chemoprevent., 10(2), 95-100

activity, described as IC50 value, on HeLa cervical toxychavicol acetate (LKT Laboratories Inc.), Ros-
cancer cell line of galangal extract from three lo- well Park Memorial Institute (RPMI 1640, Gibco),
cal markets were 13.26, 36.32, and >100 µg/mL in dimethyl sulfoxide (DMSO) (Merck), Fetal Bovine
order. Meanwhile, Alpinia galangal extract (AGE) Serum 10% (FBS, Gibco), penicillin-streptomy-
from Pasar Legi (Surakarta, Indonesia) on MCF7 cin 1% (Gibco), tripsin (Gibco), sodium dodecyl
and T47D breast cancer cell lines have IC50 value sulfate (SDS, Gibco), MTT (Sigma), fungizone
of 15.80 and 12.50 µg/mL, respectively (Suhendi (Gibco). Cell lines (T47D, HeLa, and WiDr) were
et al., 2017). Galangal contains several phenylpro- obtained from Laboratorium Balai Besar Penelitian
panoid compounds, including 1'-acetoxychavicol dan Pengembangan Tanaman Obat dan Obat Tra-
acetate (ACA), 1'-acetoxyeugenol acetate, trans-p- disional Tawangmangu, Karanganyar, Indonesia
coumaril diacetate, 1'hydroxyccapsol acetate, and (B2P2TOOT).
trans-coumaryl alcohol (Matsuda, et al., 2005).
ACA is the main composition of Alpinia galanga Extraction and fractionation
(Baradwaj, et al., 2017; Hasima, et al., 2010). Extraction was conducted by maceration
ACA has cytotoxic against various cancer process with ethanol 95% as solvent within 3 days.
cell lines such as A549 cancer cells (lung cancer), Liquid extract was then evaporated to obtain the
SNU638 (stomach cancer), HCT116 (colon can- thick extract. Ten milligrams of thick extract were
cer), HT1080 (fibrosarcoma), and HL60 (leuke- dissolved in 10 mL of distilled water and 10 mL of
mia) with IC50 values of 8.14, 1.27, 1.77, 1.20, and ethyl acetate in a separating funnel. The top layer
2.39μg/mL, respectively (Nam, et al., 2005). An- (ethyl acetate partition) was taken and evaporated
other research by Zeng, et al., (2015) revealed that in a water bath covered in aluminum foil to form the
ACA showed cytotoxic activity on HeLa (cervical ethyl acetate fraction. Subsequently, the ethyl ace-
cancer), A549 (lung cancer), HepG-2 (liver cancer) tate fraction was dissolved using methanol (metha-
and SMMC-7721 (liver cancer) with IC50 values of nol fraction). The fractionation was done in tripli-
85.1, 64.44, 74.51, and 61.27μg/mL, respectively. cates.
This study was conducted to determine the selec-
tivity of galangal extract and ACA in breast cancer MTT Assay
cells (T47D), cervical cancer cells (HeLa) and co- The MTT reagent (0.5 mg/mL) was pre-
lon cancer cells (WiDr) compared to normal cells pared by taking 1 mL of stock solution of MTT in
(Vero). The finding would serve as a basic for the PBS (50 mg/10 mL) and diluted it by media up to
further development targeted on cytotoxic research. 10 mL (for 1 well plate). Once disposed, the cell
was washed with PBS and 100 µL MTT reagent
Method was added to each well, including media control
(without cells). The cell, following this, was incu-
Materials that were used in this study in- bated for 2-4 hours in a CO2 incubator. The cell was
clude evaporator (Heidolph), waterbath (Chang- then examined with an inverted microscope. After
zhou Nuohai XMTD-204), analytical balance formazan was clearly formed, a stopper reagent
(Sartorius), micro pipette (Soccorex), LAF (Nu- (SDS 10%) was added in 0.1 N HCl. The plate was
aire), hemocytometer (Marienfield Germany), wrapped with paper or aluminum foil and incubated
96-well-plate (Iwaki), conical tube, ELISA reader in a dark place at room temperature for one night.
(BioTek), incubator (Binder), microscope (Olym- Absorbance of each well was then read by an ELISA
pus), galangal rhizome (Laboratorium Balai Besar reader at λ = 595 nm. IC50 was calculated based on
Penelitian dan Pengembangan Tanaman Obat dan linier regression equation between viability cells
Obat Tradisional, Tawangmangu, Indonesia ), Ace- and concentration of samples (Mosmann,1983).

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Indonesian Journal of Cancer Chemoprevention, June 2019
ISSN: 2088–0197
e-ISSN: 2355-8989

Selectivity Index Analysis uid extraction. Grouping chemical constituent was

Selectivity Index (SI) is obtained from the based on distribution constanta of chemicals in
IC50 value of a compound against normal cells di- non-immiscible solvent (Berthod and Carda-Broch,
vided by the IC50 value of cancer cells (Aljewari, 2004). The target of fractionation was to obtain the
et al., 2010; Badisa, et al., 2006). Compounds are rich fraction of active constituent. One of active
classified as high selectivity if the SI value is >3 compounds of galangal extract is ACA, which has
and less selective if the SI value is <3 (Sutejo, et al., potential as an anticancer (Asri and Winarko, 2016).
2016). Fractionation results showed the yield of ethyl ace-
tate fraction obtained as 7.43 gram, higher than the
Results and Discussion methanol fraction as 1.30 gram.
The cytotoxicity determination of galan-
The material used in this study is galangal gal ethanol extract, ethyl acetate fraction, meth-
rhizomes obtained from the Center for Research anol fraction and ACA compounds were carried
and Development of Traditional Medicinal Plants out using the MTT method on breast cancer cells
and Medicines (B2P2TOOT) Tawangmangu, (T47D), cervical cancer cells (HeLa), colon cancer
Karanganyar Regency, Central Java. Morphologi- cells (WiDr), and normal cells (Vero). Morphology
cally, the fresh galangal rhizomes are characterized of cancer cells after treated by samples is shown in
with small and thick, fleshy, cylindrical about 2-4 Figure 1.
cm in diameter, and branched. The outer parts are The value of IC50 of samples were cal-
rather brown, reddish or pale greenish yellow with culated based on regression equation (Figure 2)
white and reddish, hard glossy scales, and while of % viability cell percentage vs concentration.
the inside part is white. The flesh of old rhizomes The value of IC50 of samples on three cancer cells
is rough fibrous. When getting dried, the rhizomes showed a strong cytotoxic activity (Table 1). Due to
turn somewhat greenish, and the fibers become IC50 values, the ACA is viewed to have the most ac-
hard and tough. Dried rhizome was then extracted tive cytotoxic activity against all cancer cells tested.
by maceration using 96% ethanol as solvent. The Ethyl acetate fraction has better cytotoxic activity
result of thick extract was 73.09 g with yield of compared to ethanol extract and methanol fraction.
8.12%. As ACA compounds are semi-polar, they could dis-
To group chemical constituents in extract, solve more in ethyl acetate. The compounds have
fractionation was conducted. The method used in the most active cytotoxic activity in T47D cells fol-
fractionation of galangal extract was a liquid-liq- lowed by HeLa cells and WiDr cells. IC50 values of

Vero T47D HeLa WiDr

Figure 1. Morphology of cancer cells after administration of ACA at a dose of 10μg/mL. The cells were observed
after 24 h of treatments under an inverted microscope with magnification of 100x.

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Indones. J. Cancer Chemoprevent., 10(2), 95-100

Table 1. Selectivity index of extract, ethyl acetate, methanol fractions and ACA on T47D, HeLa, WiDr, and Vero
cell lines.
T47D HeLa WiDr Vero
Samples
IC50 (μg/mL) SI IC50 (μg/mL) SI IC50 (μg/mL) SI IC50 (μg/mL) SI
Ethanol extract 44.93 ± 1.08 1.6 40.00 ± 0.55 1.9 66.11 ± 3.15 1.1 74.06 ± 7.13 1
Ethyl acetate fraction 42.29 ± 3.28 1.3 35.54 ± 0.44 1.5 48.81 ± 1.68 1.1 53.66 ± 2.57 1
Methanol fraction 40.49 ± 2.13 1.8 35.76 ± 1.53 2 55.24 ± 1.62 1.3 72.77 ± 0.35 1
ACA 3.14 ± 0.14 6.6 7.26 ± 0.12 3.5 12.49 ± 1.09 2 25.25 ± 0.92 1

ACA in T47D, HeLa, and WiDr were found in 3.14; IC50 value <50 μg/mL, and iweak if the IC50 value >
7.26, and 12.49μg/mL, respectively. The potential 50μg/mL (Ellithey, et al., 2014) (Table 1). From the
of a compound is classified as strong cytotoxic results obtained, it can be concluded that ACA has
agent if the IC50 value <20µg/mL, moderate if the strong cytotoxic properties.

A B

C D

Figure 2. Graphics of correlation of viability cell percentage vs concentration of ACA, ethanol extract of Alpinia
galangal, and its fractions. Percentages of viable cells were obtained based on colorimetric reaction of MTT
that reduced by reductase enzyme resulting formazan and the absorbance was measured on wavelength 550
nm. Ethanolic extract showed negative slope that indicated the cytitoxic effect and ACA most potent to inhibit
the cell growth on Vero, T47D, HeLa, and WiDr cell lines. A: ethanol extract of Alpinia galangal; B: methanol
fraction Alpinia galangal; C: ethyl acetate fraction Alpinia galangal; D: 1’-acetoxychavicol acetate (ACA).

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ISSN: 2088–0197
e-ISSN: 2355-8989

The selectivity of chemopreventive agents References

means that only cells identified as cancer cells are
attacked. The higher the selectivity index of a com- Aljewari, H., AL-Faisal, A.H.M. and Nader, M., 2010,
pound to cells, the more selective the compounds In vitro Cytotoxic Activity of the L-asparaginase
to kill or inhibit the growth of a cancer cell with Extracted and Purified from Pathogenic Escher-
the smaller effect on normal cells. The small se- ichia coli Against four Leukemic cell lines. Pre-
lectivity index indicates that the compound is less sented at the 2nd Annual International Confer-
toxic to normal cells rather than to cancer cells. ence of Northest Pharmacy Research, Faculty of
Compounds with high SI values offer the potential Pharmacy, Mahasarkham University, Thailand,
for safer and more effective therapy in cancer ther- pp. 21–23.
apy (Segun, et al., 2019). Based on the test results Asri, A. and Winarko, S., 2016, Antiproliferative Ac-
galangal ethanol extract, ethyl acetate fraction, and tivity by Ethanolic Extract of Red Alpinia ga-
methanol fraction are found to be less selective for langa (L) Willd in Inoculated Breast Carcinoma
all tested cells (SI<3). Meanwhile, ACA is selective Cells of C3H Mice, J. Adv. Med. Pharm. Sci., 5,
for T47D and HeLa cells with SI>3 and less selec- 1–9.
tive for WiDr cells. The results of this research need Badisa, R.B., Ayuk-Takem, L.T., Ikediobi, C.O. and
a further study to determine the anticancer potential Walker, E.H., 2006, Selective Anticancer Activ-
in animal models. Due to the lack of selectivity and ity of Pure Licamichauxiioic-B Acid in Cultured
many side effects (such as fatigue, nausea, alopecia Cell Lines, Pharm. Biol., 44, 141–145.
and others) of cancer chemotherapy, these finding Baradwaj, R.G., Rao, M.V. and Kumar, T.S., 2017,
promising to develop as selective anticancer agent Novel purification of 1’S-1’-Acetoxychavicol
(González-Arriagada, et al., 2013; Ihbe-Heffinger, acetate from Alpinia galanga and its cytotoxic
et al., 2013). plus antiproliferative activity in colorectal ade-
nocarcinoma cell line SW480, Biomed. Pharma-
Conclusion cother., 91, 485–493.
Berthod, A. and Carda-Broch, S., 2004, Determina-
The extract and fractions of Alpinia ga- tion of liquid–liquid partition coefficients by
langal ethanol extract have potential cytotoxic separation methods, J. Chromatogr. A, Esti-
activity on T47D, HeLa and WiDr. Compound mation of Physicochemical Properties by Chro-
1’-acetoxychavicol acetate has a strong cyto- matographic and Electrophoretic Techniques,
toxic activity against cancer cells T47D, HeLa 1037, 3–14.
and WiDr with IC50 values of 3.14, 7.26 and Ellithey, M.S., Lall, N., Hussein, A.A. and Meyer, D.,
12.49μg/ml, respectively and selective for T47D 2014, Cytotoxic and HIV-1 enzyme inhibitory ac-
breast cancer cells with selectivity index of 6.6. tivities of Red Sea marine organisms. BMC Com-
plement, Altern. Med., 14, 77.
Acknowledgment González-Arriagada, W.A., de Andrade, M.A.C.,
Ramos, L.M.A., Bezerra, J.R.S., Santos-Silva,
This work has been funded by Universitas A.R. and Lopes, M.A., 2013, Evaluation of an
Muhammadiyah Surakarta by PINPRU research educational video to improve the understanding
scheme. of radiotherapy side effects on head and neck

 99
Da’i, et al., 2019
Indones. J. Cancer Chemoprevent., 10(2), 95-100

cancer patients, Support. Care Cancer Off. J. eration and cytotoxicity assays, J. of Immunol.
Multinatl. Assoc. Support. Care Cancer, 21, Methods, 65(1–2), 55-63.
2007–2015. Nam, J.-W., Kim, S.-J., Han, A.-R., Lee, S.-K. and
Hartono, N.W.B., 2009, Pengaruh Alpinia galanga Seo, E.-K., 2005, Cytotoxic phenylpropanoids
(Lengkuas) Terhadap Aktivitas Proliferasi Sel from the rhizomes of Alpinia galanga, J. Appl.
dan Indeks Apoptosis Pada Adenokarsinoma Pharmacol., 13, 263–266.
Mencit C3H (Thesis masters). UNIVERSITAS Pusat Data dan Informasi, K.R., 2015, Infodatin_Stop
DIPONEGORO. Kanker.
Hasima, N., Aun, L.I.L., Azmi, M.N., Aziz, A.N., Thir- Segun, P.A., Ogbole, O.O., Ismail, F.M.D., Nahar, L.,
thagiri, E., Ibrahim, H. and Awang, K., 2010, Evans, A.R., Ajaiyeoba, E.O. and Sarker, S.D.,
1′S-1′-Acetoxyeugenol acetate: A new chemo- 2019, Resveratrol derivatives from Commiphora
therapeutic natural compound against MCF-7 africana (A. Rich.) Endl. display cytotoxicity
human breast cancer cells, Phytomedicine, 17, and selectivity against several human cancer
935–939. cell lines, Phytother. Res., 33, 159–166.
Ihbe-Heffinger, A., Paessens, B., Berger, K., Shlaen, Suhendi, A., Wikantyasning, E.R., Setyadi, G.,
M., Bernard, R., von Schilling, C. and Peschel, Wahyuni, A.S. and Da’i, M., 2017, Acetoxy Chav-
C., 2013, The impact of chemotherapy-induced icol Acetate (ACA) Concentration and Cytotoxic
side effects on medical care usage and cost in Activity of Alpinia galanga Extract on HeLa,
German hospital care--an observational analy- MCF7 and T47D Cancer Cell Lines, Indones. J.
sis on non-small-cell lung cancer patients, Sup- Cancer Chemoprevention, 8, 81–84.
port. Care Cancer Off. J. Multinatl. Assoc. Sup- Sutejo, I.R., Putri, H. and Meiyanto, E., 2016, The Se-
port. Care Cancer, 21, 1665–1675. lectivity of Ethanolic Extract of Buah Makassar
Kuntorini, E.M., 2005, Botani Ekonomi Suku Zingibe- (Brucea javanica) on Metastatic Breast Cancer
raceae Sebagai Obat Tradisional oleh Masyara- Cells, J. Agromedicine Med. Sci., 2, 1–6.
kat di Kotamadya Banjarbaru, BIOSCIENTIAE, 2. Zaeoung, S., Plubrukarn, A. and Keawpradub, N.,
Matsuda, H., Ando, S., Morikawa, T., Kataoka, S. 2005, Cytotoxic and free radical scavenging
and Yoshikawa, M., 2005, Structure-activity activities of zingiberaceous rhizomes, Warasan
relationships of 1’S-1’-acetoxychavicol ace- Songkhla Nakharin Sakha Witthayasat Lae Tech-
tate for inhibitory effect on NO production in nol.
lipopolysaccharide-activated mouse peritoneal Zeng, Q., Lu, C.-L., Zhang, X. and Jiang, J.-G., 2015,
macrophages, Bioorg. Med. Chem. Lett., 15, Isolation and identification of ingredients in-
1949–1953. ducing cancer cell death from the seeds of Al-
Mosmann, T, 1983, Rapid colorimetric assay for cel- pinia galanga, a Chinese spice, Food Funct., 6,
lular growth and survival: Application to prolif- 431–443.

100