Journal of Cancer Therapy, 2013, 4, 1244-1250
http://dx.doi.org/10.4236/jct.2013.47146 Published Online September 2013 (http://www.scirp.org/journal/jct)
Annonaa muricata Linn Leaf Induce Apoptosis in Cancer
Cause Virus
Okid Parama Astirin1, Anief Nur Artanti1, Meutia Srikandi Fitria1, Eva Agustina Perwitasari1,
Adi Prayitno2*
1Department of Biology, Faculty of Mathematics and Natural Science, University of Sebelas Maret, Surakarta, Indonesia; 2Depart-
ment of Dental and Oral Disease, Faculty of Medicine, University of Sebelas Maret, Surakarta, Indonesia.
Received July 18th, 2013; revised August 20th, 2013; accepted August 28th, 2013
Copyright © 2013 Okid Parama Astirin et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Introduction: Now many studies conducted on the drug substance from nature that can serve as an anticancer agent as
a potential chemoprevention agent, such as Annona muricata Linn leaf escort chemotherapy, which was flaring. The
cancer cell in humans was included the loss of p53 protein function due to mutations in the protein gene. Other causes
are that the p53 proteins are not functioning due to an increase in protein misfolding event chaperones and degradation
events ubiquitous as binding by viral protein. Method: Cytotoxicity assay performed on 24 well plate micro-cultures.
HeLa cells are as 2 × 104 cells in 100 mL in RPMI media. Created control is RPMI and solvent DMSO 0.25%. Cyto-
toxic Test performed by the method of calculation tryphan blue dye exclusion. Being fasted for 24 hours in the culture
medium, then the cells are grown in micro-plate with media plus samples with a non-lethal concentration (LC50) of
partition and fractionation Annona mu ricata Linn leaf. Sampling is performed at 24 hours. Each of these wells is calcu-
lated the number of living cells and made the curve of cell number and incubation time. Result: The results showed that
HeLa cells are being LC50 partition of leaves Annona muricata Linn in ethyl acetate his cell death rate was higher
(2000 µg/ml have 131.89%; 15.625 µg/ml have 11.37%) and in ethanol-distillate water his cell death rate was lower
(2000 µg/ml have 35.80%; 15.625 µg/ml have 3.97%). Another results showed that HeLa cells are being LC50 frac-
tionation of leaves Annona muricata Linn in chloroform his cell death rate was higher (2000 µg/ml have 91.86%;
15.625 µg/ml have 2.68%) and in ethyl acetate, his cell death rate was lower (2000 µg/ml have 23.79%; 15.625 µg/ml
have 4.69%). Figure regression LC50 of HeLa cell culture treatment with partition or fractionation looks of regression
test is the positive regression coefficien t. Conclusion: Anno na muricata Lin n leaf in chlorofo rm is a good candidate for
chemoprevention escort chemotherapy for cancer causing viruses.
Keywords: Chemoprevention; Annona muricata Linn Leaf; HeLa Cell Culture; Apoptosis
1. Introduction
Many studies conducted on the drug substance from na-
ture that can serve as an anticancer agent as a potential
chemoprevention agent’s escort chemotheraphy [1-4].
Many studies have shown the possibility of anti-cancer
compounds that have toxicity selectively kill cancer cells
without damaging normal cells, the compound derived
from acid molecule from nature like fruit, e.g. with dock-
ing methods on the component of red fruit and tested for
cancer therapy through modeling followed by molecular-
dynamics simulations [5,6]. The study on cytotoxic tests
at the cellular level has been conducted, but very limited
testing on the molecular level, especially in cancers caused
by viral infections. Now it is revealed that Annona mu-
ricata Linn members expresse d empirically to fight cancer
[7-9]. Are after being proven cellular and molecular leaf
isolate tailings will produce bioproduct that can be used
by the public as a potential chemoprevention agent’s es-
cort agency chemotheraphy?
Cancer is a disease that ranks second leading cause of
death in the world. The usual approach to cancer preven-
tion, among others by: prevention of interaction with can-
cer-causing agents, increasing defense mechanisms aga-
inst cancer and lifestyle modification s. The main focus of
this research is that cancer is caused by a viral infection
i.e. cervical cancer (CC) is the number of events, it is
*Corresponding a uthor.

Annonaa muricata Linn Leaf Induce Apoptosis in Cancer Cause Virus 1245
quite high in Indonesia is quite high. Cervical Cancer is
due to a viral infection known as the Human papilloma
virus (HPV). World prevalence rate HPV infection is
99.7% of the CC [10-12]. Cancer begins with a scene
gene expression imbalance specific role in apoptosis and
cell proliferation, and DNA repair [13]. Understanding
these processes provides the basis for chemotherapy
apoptosis through induction of cancer cell death [7,14,
15]. The process of apoptosis can occur through multiple
pathways. One point that has close links with cancer
through the indu ction of apopto sis is much p layed by p53
protein [16-18]. Failure of apoptosis regulatio n is the key
principle to the success of carcinogenesis, i.e. inhibition
of apoptosis events [19,20]. When the high incidence of
the CC in developing countries, including Indonesia, is
not addressed, it will have an impact on the rate of in-
crease in morbidity and higher mortality, and at last will
reduce the quantity and quality of human resources.
The flow of thought between chemoprevention An-
nona muricata Linn, and the incidence of apoptosis in
virus CC can be explained by the flow of the discussion
pathobiology [21,22]. Expected to chemoprevention bio-
product Annona muricata Linn leaf as Annonaceae mem-
bers can be deadly virus, which means lower stressor dy-
namics and increase of the expression of p53, so that
events can be enhanced by apoptosis [7,14,15,23,24].
Many stressors, such as hotness, spiciness, wounds
and infections, cause or trigger the expression of specific
gene. Cells infected with the virus will experience dis-
tress increases, and the expression of p53 protein, among
others aims to enhance apoptosis. The expression of p53
protein is a form of cell response to a stressor [25-27].
However, these efforts do not always succeed, even pro-
gress to cancer. Decreased expression of the p53 protein
can cause a decrease in cell apoptosis mechanism. Pro-
tein p53, Baxxl, caspase-3 is known as an inhibitor of
cancer and is a protein that plays an important role in the
regulation of apoptosis. Decrease in apoptotic cells that
are not able to offset the increase in cell proliferation
would result in the occurrence of cancer cells [28]. Cause
of p53 malfunction is clear that the control system as
chaperone as plays an important role if the folding proc-
ess fails or an erro r occurs, cau sing fold ing abnormalities
and targeted functionality to accumulate. The accumula-
tion of faulty protein folding would harm cells and can
result in apoptosis. Many data have shown, how chaper-
ones facilitate transformation towards cancer at molecu-
lar level, and support th e concept that “there are events of
protein function changes in carcinogenesis, which needs
serious attention in the development of human cancers”
[20,29]. Another cause of p53 malfunction is mutation so
the p53 proteins don’t act optimally. Approx imately 50%
of human cancer cells lose p53 function due to mutations
in the protein-coding gene [30,31]. Records of other
cause are that E6 protein of HPV-16 and 18 will resu lt in
the inactivation of the p53 gene product through binding
mechanism called the ubiquitin-dependent proteolytic
pathway (E6AP), resulting in decreased levels of p53
protein (wild-type) [32]. Returning a mutated p53 func-
tion could potentially trigger a mass apoptosis, which can
kill cancer cells effectively and prove that Actinomycin
D (chemotherapy drug) affects implantation failure of
Rattus norvegicus [33]. All were expected to explain the
role of Annona muricata Linn leaves in decreasing in-
fected virus and increasing expression of p53 protein in
the event CC patients infected with the HPV through in-
creased apoptosis.
2. Material and Method
HeLa cells were grown in culture bowls containing RP MI
1640 medium and FBS added 0.5%, Penstrep 2% and
fungizon 0.5% and incubated in a 5% CO2 incubator for
24 hours. The cells density is 2 × 104 cells/ml.
Extraction using two methods, namely an extraction
method using 96% ethanol and percolation method using
the solvent n-hexane, chloroform, ethyl acetate, and etha-
nol 96%.Results of maceration partitioned using solvents
n-hexane, chloroform, ethyl acetate, and ethanol-distil-
late water. Active extract of the results of the percolation
fractionated using vacuum liquid chromatography (VLC)
with a mobile phase n-hexane and ethyl acetate, as well
as the stationary phase silica gel 60 PF 254.
The stage to develop the test cells HeLa is by Freshney
and Gadek method with some modification [34,35]. Cy-
totoxicity assay performed on 24 well plate micro-cul-
tures. HeLa cells as 2 × 104 cells in 100 mL RPMI media.
Further isolates were given 100 mL at a concentration
series ranking triplet. Created are RPMI for medium con-
trol and solvent DMSO 0.25%. Cytotox ic Test performed
by the method of calculation tryphan blue dye exclusion
(MTT). Cells were grown for 24 hours in the culture me-
dium. The cells were grown in micro-plate with media
plus samples with a non-lethal concentration (LC50 be-
low) of partition or fractionation Annona muricata Linn
leaf. Sampling performed at 24 hours.
Each of these wells is calculated the number of living
cells. The percentage of cell death was calculated using
the modified formula Abbot [36]. Thus made curve for
the number of cells death again incubation time [37].
3. Result
The results showed (Figure 1 and Table 1) that HeLa
cells are being LC50 partition of leaves Annona muricata
Linn in ethyl acetate his cell death (apoptotic) rate was
higher (2000 µg/ml have 131.89%; 15.625 µg/ml have
11.37%) and in ethanol-distillate water his cell death
(apoptotic) rate was lower (2000 µg/ml have 35.80%;
15.625 µg/ml ha ve 3.97%)

Annonaa muricata Linn Leaf Induce Apoptosis in Cancer Cause Virus 1246
Figure1. (a) Hela cells culture treatment with DMSO as a
control (cell death/apoptotic rate: 3.80%); (b) Hela cells cul-
ture after treatment with partition of leaves Annona muri-
cata Linn in chloroform (cell death/apoptotic rate: 65.20%).
Table1. The average percentage of death cells associated
with many concentration and many solvent from partition
the leaves Annona muricata Linn. We can look that parti-
tion of leaves Annona muricata Linn in solvent ethyl acetate
make cell death/apoptotic rate is higher than other (average
percentage is 131.89% cells death in 2000.00 µg/ml and
11.37% death cells in 15.62 µg/ml concentration) but in
chloroform cell death/apoptotic rate is number 3 (average
percentage 65.20% death cells in 2000.00 µg/ml and 18.42%
death cells in 15.62 µg/ml concentration).
No. Solvent Concentration
(µg/ml) Average percentage of death cells (% )
a) 2000.00 131.89
1. Ethyl
acetate b) 15.62 11.37
a) 2000.00 106.53
2. n-heksan b) 15.62 21.41
a) 2000.00 65.20
3. Chloroform b) 15.62 18.42
a) 2000.00 35.80
4. Ethanol distillate
water b) 15.62 3.97
Figure regression LC50 of HeLa cell culture treatment
with the partition shown below in Figures 2(a)-(d). From
the looks of regression test is the positive regression co-
efficient. This value is LC50 of four-kind partition An-
nona muricata Linn leaves of HeLa cells incubated for
24 hours.
Another results showed (Figure 3 and Table 2) that
HeLa cells are being LC50 fractionation in chloroform
his cell death (apoptotic) rate was higher (2000 µg/ml
have 91.86%; 15.625 µg/ml have 2.68%) and in ethyl
acetate his cell death (apoptotic) rate was lower (2000
µg/ml have 23.79%; 15.625 µg/ml ha ve 4.69%).
Figure regression LC50 of HeLa cell culture treatment
with the fractionation shown below in Figures 4(a)-(d)).
From the looks of regression test is the positive regres-
sion coefficient. This value is LC50 of four-kind frac-
tionation Annona muricata Linn leaves of HeLa cells
incubated for 24 ho u rs.
(a) (b)
(c) (d)
Figure 2. The curve of (a) Partition Annona muricata Linn
leaves in chloroform; (b) Partition Annona muricata Linn
leaves in ethanol-distillate water; (c) Partition Annona mu-
ricata Linn leaves in n-heksan; (d) Partition Annona muri-
cata Linn leaves in ethyl acetate.
Figure 3. (a) HeLa cell culture treatment with DMSO as a
control (cell death/apoptotic rate: 3.80%); (b) HeLa cell cul-
ture after treatment with fractionation of leaves Annona
muricata Linn in chloroform (cell death/apoptotic rate:
91.86%).
4. Discussion
The incidence of cancer associated with: First, there is
increased expression or mutation of gene trigger cancer.
Second, there is a decrease in the expression or mutation
of gene cancer suppressor. Cancer suppressor gene is
normal gene that has an important function in cell ho-
meostasis. If that gene don’t work, then implicated for
occur of cancer. Third, the gene associated with cancer is
the presence of DNA-repair enzymes. Changes in the
function of these enzymes will lead to the occurrence of
cancer. Fourth, the process of apoptosis is not normal,

Annonaa muricata Linn Leaf Induce Apoptosis in Cancer Cause Virus 1247
Table 2. The average percentage of death cells associated
with many concentration and many solvent from fractiona-
tion the leaves Annona muricata Linn. We can look that
fractionation of leaves Annona muricata Linn in solvent
chloroform cell death/apoptotic rate is higher than other
(average percentage is 91.86% death cells in 2000.00 µg/ml
and 2.68% death cells in 15.62 µg/ml concentration) but in
solvent ethyl acetate cell death/apoptotic rate is lower that
other (average percentage is 23.79% death cells in 2000.00
µg/ml and 4.69% in 15.62 µg/ml concentration).
No. Solvent
Concentration
(µg/ml) Average percentage of death cells (% )
a) 2000.0091.86
1. Chloroform b) 15.62 2.68
a) 2000.0075.36
2. n-heksan b) 15.62 8.34
a) 2000.0034.77
3. Ethanol distillate
water b) 15.62 3.44
a) 2000.0023.79
4. Ethyl acetate b) 15.62 4.69
(a) (b)
(c) (d)
Figure 4. The curve of (a) Fractionation Annona muricata
Linn leaves in chloroform; (b) Fractionation Annona muri-
cata Linn leaves in ethanol-distillate water; (c) Fractiona-
tion Annona muricata Linn leaves in n-heksan; (d) Fr action-
ation Annona muricata Linn leaves in ethyl acetate.
that happen inhibition of apoptosis [13,16-18,38]. Further
developments on the definition of cancer has been sug-
gested that incidence of cancer begins in the disorder at
the level of epigenetic (methylation and/or histone modi-
fication) and continues to change at the level of genetic
(mutation) [39].
Cell distress by stressor HPV show an increase in the
expression of protein chaperones (Hsp70 and Hsp40).
Imbalance between incr eased levels of Hsp70 and Hsp40
with decreased ATP production by the mitochondria so
made possible for error events of protein folding of pro-
tein denatured or protein newly translated, so that the
protein can’t function normally. Abnormal function of
these proteins results in the inhibition of apoptosis and
increased cell proliferation that triggers carcinogenesis
[29,40]. In the state of distress caused by HPV, the cells
will express Hsp for the purpose of homeostasis, an im-
balance will develop into cancer [41,42].
Various compounds present in Annonaceae familia,
including potential anti-cancer compounds. Three com-
pounds in Annona muricata Linn a potential anti-cancer
namely are monotetrahydrofuran acetogenins, muricin H,
muricin I, and cis-annomontacin [43]. Acetogenin com-
pounds (squamocin A, B, C, and D) and annotemoyin-1
and -2 the Annonaceae have cytotoxic effects [44], pla-
telet agregation inhibitor [45], inhibitor of HIV replica-
tion [46], antidiabetic agents (antihiperglikemik) and anti-
oxidants [47,48], pesticide [49], and can be used in the
treatment of Neisseria gonorrhea [50]. Squamocin serves
as insecticides, while the ascimicin ha ve an antileukemia
effect [51]. Caryophyllene oxide on the bark has analge-
sic and anti-inflammatory activity [52], and cyclosqua-
mosin D on seed proved showed inhibition of proinflam-
matory cytokines in macrophages [45]. Further treatment
of cancer with natural ingredients is not always the spe-
cific target. Anti-cancer herbal remedies white turmeric
(Curcuma zedoaria) affects organs such as the always do
mitosis ovarian follicles in mice [53]. Acetogen in the An-
nonaceae are composed of fatty acids C32 or C34 long
chain fatty acids. Bioactivity acetogenin was diverse as
anticancer, immunosuppressive, pesticide, antiprotozoal,
and antimicrobial. Acetogenin wall membrane inhibited
mitochondrial ATP production, resulting in the produc-
tion of energy in cancer cells and stops cancer cells even-
tually die [54]. Acetogenin was very selective, only at-
tacks cancer cells that have excess ATP. These com-
pounds do not attack other cells are normal in the body,
disrupting the circulation of cancer cells by reducing the
amount of ATP [55]. Soursop leaf cell-killing colon can-
cer cells to 10,000 times stronger than Adriamycin and
other chemotherapy. Some derivatives in different types
of structures and some isomers showed significant selec-
tivity cancer cell line, for example, the fight against pro-
state cancer (PC-3). The main mode of action is aceto-
genin inhibitor of NADH: oxidoreductase uniquinone,
the enzyme complex is important in oxidative phospho-
rylation in the mitochondria and inhibits NADH oxidase
uniquinone the plasma membrane of cancer cells [56].

Annonaa muricata Linn Leaf Induce Apoptosis in Cancer Cause Virus 1248
5. Conclusion
Annona muricata Linn leaf in chloroform is a good ca-
didate for chemoprevention escort chemotherapy for can-
cer cause virus.
6. Acknowledgements
We thank to acknowledge High er Education Competitive
Research Project Ministry of Education and Culture Re-
public of Indonesia for Grand Featured Research Univer-
sities 2013, LPPT of Gajah Mada University and special
thanks to acknowledge, Prof Dr Rafik Karsidi, MSc as a
rector of Sebelas Maret University Surakarta Indonesia,
Prof Ir Ari Handoko Ramelan, MSc (Hons), PhD as a
dean of Faculty of Mathematics and Natural Science of
Sebelas Maret University Surakarta Indonesia and thanks
to acknowledge Prof Dr Suhartono Taat Putra MS for
much inspirations to wrote this article.
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