ARTIKEL HASIL PENELITIAN

Antitumor Activity of Soursop (Annona muricata L.) Leaves

On Prostate Cancer Cell Line

*Ade Zuhrotun, Rizky Abdullah, Meiliana Thamrin, Resti Febriliza

Faculty of Pharmacy, Universitas Padjadjaran,
Jalan Raya Bandung-Sumedang KM 21,5 Jatinangor 45363 West Java, Indonesia

Abstract
Soursop (Annona muricata L.) leaves were used as a traditional drug for many diseases such as cancer. Statistical data from
one cancer hospital in Jakarta in the period of 2005 and 2007 showed that prostate cancer has become one of ten common
cancer cases in ambulatory patients. This research was conducted to investigate the potency of an herbal drug for prostate
cancer. The soursop leaves were extracted by maceration and fractionated by liquid-liquid extraction. The activity of all
samples was then tested by Brine Shrimp Lethality Test (BSLT) and MTT assay using prostate cell line, LNCaP. The result
showed that soursop leaves extract and all fractions had a cytotoxic activity to Artemia salina L. larvae (LC50<1000 µg/ml).
The MTT assay showed that soursop leaves extract, n-hexane and ethyl acetate fractions had a proliferative effect to LNCaP
cell line (IC50<350 µg/ml). Whereas, water fraction had no effect up to 400 µg/ml. Ethyl acetate fraction showed the best
antitumor activity because its LC50 and IC50 values were the lowest. It can be suggested that soursop leaves showed antitumor
activity and had a prospective to be developed as herbal drug for prostate cancer therapy.

Keywords: Soursop leaves, Annona muricata L., Brine Shrimp Lethality Test (BSLT), LNCaP

Abstrak
Daun sirsak (Annona muricata L.) banyak digunakan dalam pengobatan tradisional untuk berbagai macam penyakit seperti
kanker. Berdasarkan data statistik dari salah satu rumah sakit kanker di Jakarta pada periode 2005 hingga 2007, menunjukkan
bahwa kanker prostat telah menjadi 1 dari 10 macam kasus kanker yang umum diderita oleh pasien ambulatori. Penelitian ini
dilakukan untuk menyelidiki potensi dari obat herbal untuk kanker prostat. Daun sirsak diekstraksi dengan cara maserasi dan
fraksinasi menggunakan ekstraksi cair-cair. Aktifitas dari keseluruhan sampel kemudian diuji menggunakan Brine Shrimp
Lethality Test (BSLT) dan uji MTT dengan kultur sel kanker, LNCaP. Hasil penelitian menunjukkan bahwa ekstrak daun
sirsak dan seluruh fraksinya memiliki aktifitas sitotoksik terhadap larva Artemia salina L. dengan LC50<1000 µg/ml. Hasil dari
pengujian MTT menunjukkan bahwa ekstrak daun sirsak dari fraksi n-heksana dan etil asetat memiliki efek proliferatif pada
kultur sel LNCaP (IC50 < 350 µg/ml). Sedangkan fraksi air dari daun sirsak hingga konsentrasi 400 µg/ml tidak menunjukkan
adanya efek yang sama. Fraksi etil asetat menunjukkan aktivitas antitumor yang paling baik karena memiliki nilai LC50 dan
IC50 yang paling rendah. Sehingga dapat disimpulkan bahwa daun sirsak menunjukkan adanya aktivitas antitumor dan
berpotensi untuk dikembangkan sebagai obat herbal untuk terapi kanker prostat.

Kata kunci : Daun sirsak, Annona muricata L., Brine Shrimp Lethality Test (BSLT), LNCaP

Introduction from Taxus brevifolia, and camptothecin from

Camptotheca acuminata (Nwafor et al. 2001). This
Cancer was a leading cause of death and accounted research was conducted to investigate the potency of
for 7.6 million deaths in the world (around 13% of all soursop leaves (Annona muricata L.) for prostate
deaths) in 2008. Lung, breast, colorectal, stomach and cancer.
prostate cancers cause the majority of cancer deaths
(WHO 2013). In Indonesia, cancer was the sixth Soursop leaves were empirically used as a traditional
major cause of death (5.7% of 4,552 death cases) drug. In America, this is use for treatment of
according to health survey period July 2006-August hypertension, colon parasite, tumor, and cancer; in
2008. Heart, lung, cervix, breast, ovary and prostate Peru it is use for treatment of digestion, inflammation,
cancers cause the majority of cancer deaths parasite and tumor (Zuhud 2011) and in Indonesia it is
(Balitbangkes 2008). Statistical data from one cancer used for treatment of Cyst, hypertension, diabetic,
hospital in Jakarta in the period of 2005-2007 showed tumor, and cancer (prostate, pancreatic and lung)
that prostate cancer has become one of ten common (Radi 1998).
cancer cases in ambulatory patients (National Cancer
Center 2013). Scientific research has been showed that soursop
leaves were able to kill various cancer cells
A lot of anticancer agents were derived from plants (McLaughlin and Sastrodihardjo 1995), inhibited the
such as podophyllin from Podophyllum rhizome, growth of bile cancer cell line (Rieser et al. 1996),
vinca alkaloid from Vinca rosea, paclitaxel/taxol and cytotoxic agent of tumor cells (Chang and Wu

*Corresponding author. e-mail: airkusaja@yahoo.com

Acta Pharmaceutica Indonesia, Vol. XXXVIII, No. 2, 2013 - 43

Zuhrotun et al.

2001). From this data, we assumed that soursoup 1000 ppm or (µg/mL) in vial containing of 5 mL
leaves have the potency for prostate cancer therapy. artificial sea water (38 g sea salt per liter of water)
and 10 brine shrimp in each of three replicates.
Alternatively, material may be dissolved in dimethyl
Materials and Methods sulfoxide up to 50 µL. Survivors of Artemia salina
Leach are counted after 24 hours. These data are
Materials processed in simple program for probit analysis on
Soursop leaves, shrimp egg (Artemia salina Leach), personal computer to estimate LC50 values with 95%
aquadest, ethanol 95%, ethyl acetate, n-hexane, confidence intervals for statistically significant
toluene, chloroform, ether, amyl alcohol, hydrochloric comparison of potencies. In cases where data were
acid, sulfuric acid, acetic acid glacial, ammonia, insufficient for this technique, the dose-response data
bismuth-subnitric, mercury(II)chloride, Ferri (III) were transformed into a straight line by means of log
chloride, chloral hydrate, sodium hydroxide, transformation; the LD50 was derived from the best fit
potassium Iodide, sodium chloride, sodium line obtained by linear regression analysis.
tiosulphate anhydrate, gelatin powder, vanillin
powder, magnesium powder. LNCaP cell line, RPMI- Antitumor activity
1640 media, antibiotic Penicillin-Streptomycin, Fetal Antitumor activity was performed using LNCaP
Bovine Serum. human prostate cancer cell lines in the presence of
various concentrations of extract or fractions by a
Tools colorimetric tetrazolium salts (3(4,5-dimethyl-thiazol-
Oven, grinder, macerator, rotavapor (Buchi Rotava- 2-yl)-2,5 diphenyltetrazolium bromide) or MTT
por R-3000), waterbath, electric dryer (MITSEDA assay. This methods has described previously in
HD 350), separation funnel, picnometer, crucible Octaviani et al. (2013). Briefly, LNCaP cell lines (2 ×
porcelain, aerator (RESUN air-pump), neon lamp, 104 in 50 µl/well) were plated in 96-well plates. After
micropipette, volume pipette, 96-well plate, the initial cell seeding, different concentrations of
distillation apparatus, analytical balance, common extract or fractions were added and incubated for 24
glassware in laboratory, microscope (OLYMPUS h. Ten microliters of water-soluble tetrazolium
CX31), furnace, spectrophotometry UV-Vis, micro- (WST)-8 assay. Cell-counting solution was added to
plate reader. each well and incubated at 37°C for 3h. After the
addition of 100 µl/well of 1 NHCl, the cell
Sample preparation proliferation rate was then determined by measuring
the absorbance at 450 nm, with a reference
Soursop leaves were collected from Rancaekek, West wavelength of 650 nm. The absorbance was read
Java-Indonesia in February 2012. The plant was using a microtiter plate reader. Results were derived
identified in Plant Taxonomy Laboratory, from triplicate experiments.
Departement of Biology, Faculty of Mathematic and
Natural Science Universitas Padjadjaran that belong
to Annonaceae, species Annona muricata L. Result and Discussion
Powdered crude drug was extracted by maceration
using 96% ethanol. The filtrate was condensed using Macroscopic and Microscopic analysis
vacuum rotavaporator then electric dryer.
Concentrated extract was then fractionated by liquid- Macroscopic analysis of soursop leaves showed that
liquid extraction (LLE) using n-hexane and ethyl soursop leaves appear as simple mild brown leaves,
acetate, respectively, where the extract was dissolved obtuse apices, attenuate or obtuse bases, ciliate
in aquadest previously. Macroscopic and microscopic margin, laevis texture, and ovale or obovate shape
analysis, extract quality characterization and with long 6-18 cm wide 2-6 cm. By microscopic
phytochemical screening have been done using analysis fragments identified were epidermis with
standard procedures (Ditjen POM 1987; 1979). anomositic stomates, vascular trace with trachea, and
filament. These result has appropriate with
characteristic of soursop leaves in reference standard.
Bioactivity screening
Bioactivity of extract and all LLE fractions was Extract quality characterization
monitored by Brine Shrimp Lethality Test (BSLT)
using nauplii Artemia salina Leach. Procedures were Extract quality characterization is beneficial to
developed by McLaughlin et al. (1998) and Meyer et indicate identity and quality of soursop leaves extract
al. (1982) where natural product extract and fractions that was done by determination of specific and non-
are tested at initial concentrations of 10, 100, and specific parameters. This was one of requirement of
natural product (extract or fractions) that will

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 Zuhrotun et al

developed as Obat Herbal Terstandar (Indonesian (Voight 1984). Ash contents are figure out the amount
Standardized Herbal Drug). of external and internal mineral (Depkes RI 2000).

Specific parameters are describe extract efficacy that Table 1. Result of Phytochemical Screening
associates with secondary metabolite content or active Secondary
substance in extract. This was reflected by Crude Drug Extract
Metabolites
organoleptic analysis, total of extractive matters and
phytochemical screening (Depkes RI 2000). Alkaloid - -
According to organoleptic analysis, soursop leaves Flavonoid + +
extract appeared as viscous mass, sticky, dark green
with spesific odor. Table 1 referred to phytochemical Tannin - -
screening results, showed that both crude drug and
extract consisted of flavonoid, poliphenolic, mono and Poliphenolic + +
sesquiterpenoid, steroid and quinon. This also pointed Mono &
+ +
out that extraction solvent was appropriated as it was seskuiterpenoid
able carried all metabolites from initial sample (crude
Steroid + +
drug). Extractive matters of maceration process and
LLE are shown in Table 2. Triterpenoid - -

Non-specific parameters i.e specific weight, water Quinon + +

content, total ash content and acid-insoluble ash
content are describes safety and stability of soursop Saponin + +
leaves extract (Table 3). Specific weight associates
with purity. Concentrated extract must have water
content under 30% that kept from fungi growth

Table 2. Results of extraction and fractionation

Rendement
Sample Initial Mass(g) Extractive Matters (g)
(%)
1349.93
Soursop leaves 225.25 16.68
(powdered crude drug)
n-hexane fraction 25.42 42.36
60.00
Ethyl acetate fraction 11.99 19.98
(concentrated extract)
Water fraction 20.15 33.58

100

80
Mortality (%)

60
Extract

40 n-hexane fraction

20 ethyl acetate
fraction

0
0 200 400 600 800 1000
Concentration (µg/mL)
Figure 1. First stage % mortality values at different concentration.

Acta Pharmaceutica Indonesia, Vol. XXXVIII, No. 2, 2013 - 45

Zuhrotun et al.

100

Cell Proliferation Inhibition

Extract

n-hexane
(%) 50 Fraction
Ethyl Acetate
Fraction
Water Fraction
0
0.8
1.6
3.1
6.3
12.5
25
50
100
200
400
Concentration (µg/mL)
Figure 2. Activity of samples against LNCaP human prostate cancer cell line

Activity test: b. MTT assay result

a. BSLT results Figure 2 displayed the activity of samples against
BSLT was done in two stages. First stage applied with LNCaP human prostate cancer cell line. The figure
initiated doses 1000 µg/mL, 100 µg/mL, 10 µg/mL, showed that extract, n-hexane fraction, and ethyl
and 1 µg/mL. Figure 1 showed % mortality of each acetate fraction inhibited the LNCaP cell and reach
extract, n-hexane fraction, ethyl acetate fraction and 50% cell proliferation inhibition at different
water fraction at different concentration. concentration. IC50 value of each samples were 29.6
µg/mL; 328.2 µg/mL; and 18.2 µg/mL respectively.
Table 3. Characteristic of quality On the contrary, water fraction until the highest
concentration tested failed to reach 50% cell
Parameter Results proliferation inhibition, indicated low anticancer
activity against LNCaP human prostate cancer cell
Specific weight 0.766 line.
Water content (% v/b) 18.000
In this research, correlation between BSLT study with
Total ash content (% b/b) 5.778
its in vitro study (MTT assay) were proven to be true
Acid-insoluble ash content (Meyer et al. 1982). In our BSLT results, ethyl acetate
0.822
(% b/b) fraction that showed the most toxic agent with LC50
Water soluble extractive value 6.08 µg/mL, in an in vitro study against LNCaP
27.667
matters (% b/b) cancer cell lines also showed the best antitumor
Ethanol soluble extractive activity with IC50 value 18.2 µg/mL.
82.333
matters (% b/b)
Important finding of our research to support and
verify empiric or Indonesian traditional usage of
At the second stages, variation of doses were made in soursop leaves (Annona muricata L.) for treatment of
narrow range approached to 50% mortality value of prostate cancer. So soursop leaves can be developed
each extract, n-hexane fraction, ethyl acetate fraction as herbal anticancer for prostate cancer therapy, where
and water fraction. After that LC50 value of each extract and ethyl acetate fractions showed strong
samples were calculated, that respectively was 70 activity.
µg/mL; 64.62 µg/mL; 6.08 µg/mL and 124.98 µg/mL.
These results showed that extract and all fractions
demonstrated cytotoxic activity (LC50 < 1000 µg/ml)
Conclusions
to Artemia salina L. larvae (Meyer et al. 1982).
Cytotoxic activity of soursop leaves extract was a The BSLT result showed that soursop leaves extract
synergistic effect of its metabolite constituents, and all fractions demonstrated cytotoxic activity
reflected by LC50 value of each fractions. Ethyl (LC50<1000 µg/ml) to Artemia salina L. larvae. The
acetate fraction have the best cytotoxic activity MTT assay showed that soursop leaves extract, n-
because its LC50 values were the lowest. hexane and ethyl acetate fractions had proliferative
effect to LNCaP cell line (IC50<350 µg/ml). Whereas,
water fraction had no effect up to 400 µg/ml. Ethyl
acetate fraction showed the best anticancer activity

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 Zuhrotun et al

because its LC50 and IC50 values were the lowest. It aquatica, A. graveolens, and M. utilisima to LNCaP
can be suggested that soursop leaves showed prostate cancer cell lines, J. Nat. Pharm. 4(1): 67-70.
antitumor activity and prospective to be developed as
herbal drug for prostate cancer therapy. Radi J, 1998, Sirsak, Budidaya dan Pemanfaatan-nya,
Kanisius, Yogyakarta.
Acknowledgment
Rieser MJ, Gu ZM, Fang XP, Zeng L, Wood KV
We thank to Direktorat Jenderal Perguruan Tinggi Mclauglin JL, 1996, Five Novel Monotetrahydrofuran
(DIKTI) that funded research by Program Hibah Ring Acetogenins from the Seeds of Annona
Bersaing Desentralisasi Universitas Padjadjaran 2012. muricata, Agr, Evo Research Center, Purdue
University, J. Nat. Prod. 59(2): 100-108.
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