Journal of Ethnopharmacology 193 (2016) 362–367

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Antitumor activity of Annona squamosa seed oil

Yong Chen n, Yayun Chen, Yeye Shi, Chengyao Ma, Xunan Wang, Yue Li, Yunjie Miao,
Jianwei Chen, Xiang Li n
College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China

art ic l e i nf o a b s t r a c t

Article history: Context: Custard apple (Annona squamosa Linn.) is an edible tropical fruit, and its seeds have been used
Received 12 May 2016 to treat “malignant sore” (cancer) and other usage as insecticide. A comparison of extraction processes,
Received in revised form chemical composition analysis and antitumor activity of A. squamosa seed oil (ASO) were investigated.
8 August 2016
Materials and methods: The optimal extraction parameters of ASO were established by comparing per-
Accepted 22 August 2016
Available online 23 August 2016
colation, soxhlet, ultrasonic and SFE-CO2 extraction methods. The chemical composition of fatty acid and
content of total annonaceous acetogenins (ACGs) of ASO was investigated by GC-MS and colorimetric
Keywords: assay, and anti-tumor activity of ASO was tested using H22 xenografts bearing mice.
Annona squamosa seed oil Results: The optimal extraction parameters of ASO were obtained as follows: using soxhlet extraction
Soxhlet extraction
method with extraction solvent of petroleum ether, temperature of 80 °C, and extraction time of 90 min.
Unsaturated fatty acid
Under these conditions, the yield of ASO was 22.65%. GC-MS analysis results showed that the main
Annonaceous acetogenin
Anti-tumor chemical compositions of fatty acid of ASO were palmitic acid (9.92%), linoleic acid (20.49%), oleic acid
(56.50%) and stearic acid (9.14%). The total ACGs content in ASO was 41.00 mg/g. ASO inhibited the
growth of H22 tumor cells in mice with a maximum inhibitory rate of 53.54% by oral administration.
Furthermore, it was found that ASO exerted an antitumor effect via decreasing interleukin-6 (IL-6), janus
kinase (Jak) and phosphorylated signal transducers and activators of transcription (p-Stat3) expression.
Discussion and conclusion: The results demonstrated that ASO suppressed the H22 solid tumor devel-
opment may due to its main chemical constituents unsaturated fatty acid and ACGs via IL-6/Jak/Stat3
pathway. ASO may be a potential candidate for the treatment of cancer.
& 2016 Published by Elsevier Ireland Ltd.

1. Introduction Kusmenoglu, 2003), anti-inflammatory (Delmastro-Greenwood

Custard apple (Annona squamosa L.) is an edible tropical fruit,
and is also called sugar apple or sweetsop. As a famous tropical
fruit, it is consumed in lots of countries. Botanical extracts have
been used as a source of medicinal agents for thousands of years.
The seed extract of A. squamosa was used in the south of China as a
folk remedy to treat “malignant sores” (cancer) (Guangdong Food
and Drug Administration, 2004). Phytochemical and pharmacolo-
gical studies of the seeds have focused on annonaceous acet-
ogenins (ACGs), which have a strong antitumor activity (Bermejo
et al., 2005; Chen et al., 2012a; Liaw et al., 2010; Sun et al., 2014).
ACGs binding to mitochondrial respiratory chain complex I de-
pends on a very complex combination of structural factors, mainly
involving the tetrahydrofuran group and the α, β-unsaturated γ-
lactone ring moieties (Bermejo et al., 2005). Vegetable oils are
known to have antibacterial, antifungal (Erdemoglu and
n
Corresponding authors.
E-mail addresses: achenyong@gmail.com (Y. Chen),
lixiang_8182@163.com (X. Li).
et al., 2014), antioxidant (Wang et al., 2011) and antineoplastic
(Jiang et al., 2008; Muzio et al., 2006) properties attributed to their
most abundant component fatty acids. As a class of nonpolar
compounds, A. squamosa seed oil (ASO) may contain a certain
amount of ACGs.
Treating cancer with botanical extracts has been shown to be
an easy and effective approach in China. Coix lacryma-jobi (adlay)
seed oil could enhance the antitumor activity of Ganoderma luci-
dum triterpene-loaded microemulsions (Qu et al., 2014). Brucea
javanica oil (BJO) (Yan et al., 2015) has been widely used for
treatment of lung cancer (Nie et al., 2012). The fatty acid methyl
esters obtained from A. cornifolia (another Annona species) showed
significant antifungal, antioxidant and cytotoxic potential activities
(Lima et al., 2011, 2012). Our previous study also indicated that
ASO showed selective cytotoxic activity against HepG2 cell line
(Qiu et al., 2014). Experiments with cells in monolayer or sus-
pension do not provide sufficient information on the activity of
compounds on clinically important solid tumors. Nevertheless,
information about the contents of ACGs and anti-tumor activity of
ASO in vivo is nonexistent.
In the present work, the optimal extraction parameters of ASO

http://dx.doi.org/10.1016/j.jep.2016.08.036
0378-8741/& 2016 Published by Elsevier Ireland Ltd.
 Y. Chen et al. / Journal of Ethnopharmacology 193 (2016) 362–367 363

were established. The chemical compositions of fatty acid and Table 1

content of total ACGs of ASO were investigated by GC-MS and ASO yields of different extraction methods.
colorimetric assay, and anti-tumor activity of ASO was tested using
Extraction Extraction Extraction time Yield (%)
H22 xenografts bearing mice. method solvents (min) (Mean7 SD)

Percolation – – 8.377 0.07

Soxhlet – – 17.00 70.01
2. Materials and methods
Ultrasonic – – 13.68 70.75
SFE-CO2 – – 11.87 7 0.06
2.1. Materials
Soxhlet Petroleum ether – 17.137 0.18
Ether – 15.78 7 0.21
Motrilin (Cortes et al., 1993) (Fig. 1S) was isolated from the Petroleum ether 30 17.00 70.01
seeds of A. squamosa L. in our laboratory. Molecular structure was 60 20.25 70.67
characterized based on spectroscopic analysis and the purity of 90 22.65 70.63
120 20.87 70.61
motrilin reached above 98% determined by HPLC-DAD-ELSD ana-
lysis. 5-Fluorouracil (5-Fu) used as anti-tumor agent was pur-
chased from Sigma (Shanghai, China). Analytical grade petroleum
Table 2
ether (60–90 °C) and ether were purchased from Guanghua Sci- Chemical composition from the FAMEs of ASO.
ence and Technology Company (Guangdong, China). The deionized
water was obtained from the Milli-Q direct-8 system (Millipore, No. Retention time Compounds Molecular formula Peak area %
Bedford, MA, USA).
1 21.888 Palmitic acid C16H32O2 9.92
2 27.004 Linoleic acid C18H32O2 20.49
2.2. Extraction Process of ASO 3 27.187 Oleic acid C18H34O2 56.50
4 27.971 Stearic acid C18H36O2 9.14
Seeds of A. squamosa were collected from Yunnan Province in 5 32.697 Tricosane C23H48 0.42
6 33.601 Eicosanoic acid C20H40O2 0.38
January 2014 and identified by Prof. Jian-Wei Chen (Nanjing Uni-
7 35.363 Tetracosane C24H50 0.85
versity of Chinese Medicine, Jiangsu, China). The sample was au- 8 37.927 Pentacosane C25H52 1.12
thenticated and deposited at the herbarium of the Pharmaceutical 9 40.616 Hexacosane C26H54 1.18
College of Nanjing University of Chinese Medicine, Jiangsu (No.
Peak area %: relative percentage (peak area relative to the total peak area).
FLZ20140101).
Dried seeds (20 g, 40-mesh) were extracted by percolation
extraction (extraction solvent of 200 mL petroleum ether and Table 3
Total ACGs content of ASO.
temperature of 25 °C), soxhlet extraction (extraction solvent of
petroleum ether, temperature of 80 °C, and extraction time of No. ASO concentra- Absorbance (A) Total ACGs con- Total ACGs con-
30 min), ultrasonic extraction (extraction solvent of 200 mL pet- tion (mg/mL) centration (mol/L) tent (mg/g,
roleum ether, temperature of 30 °C, and extraction time of mean7 SD)
30 min), and SFE-CO2 extraction (extraction pressure of 30 MPa,
1 5.60 0.372 3.71
10  4 41.007 0.62
temperature of 35 °C, extraction time of 60 min and modifier of 2 5.37 0.349 3.47
10  4
20 mL 95% ethanol) method. Different extraction solvents (petro- 3 6.02 0.402 4.01
10  4
leum ether and ether) and times (30–120 min) were investigated
to optimize the extraction parameters of soxhlet extraction
method. Table 4
Effect of ASO and 5-Fu on H22 solid tumor growth.

2.3. GC-MS Analysis of ASO Sample (dose) Mice number Tumor weight (g, mean Inhibition (%)
Start/End 7 SD)
ASO (150 mg) was hydrolyzed with 0.5 M NaOH-CH3OH (5 mL)
at 65 °C for 40 min. The reaction mixture was methylated with 14% Control 10/10 0.667 0.19 –
5-Fu (15 mg/kg) 10/10 0.37 70.06n 44.73
PF3-CH3OH (3 mL) at 65 °C for 5 min, and then extracted with
ASO (0.5 mL/kg) 10/10 0.45 7 0.20 31.97
n-hexane (5 mL) and NaCl (3 mL). 300 μL of the n-hexane ex- ASO (1.0 mL/kg) 10/10 0.317 0.12nn 53.54
traction was diluted to 5 mL, and centrifuged at 12,000 rpm for
10 min to get fatty acid methyl esters (FAMEs) of ASO. The Values are presented as mean7 SD.
n
The analysis of the FAMEs was performed on an Agilent GC-MS P o 0.05 compared with control.
nn
(GC6890N-MSD5973, Palo Alto, CA, USA), equipped with a HP-5MS Po 0.01 compared.

capillary column (30 m
250 mm, 0.25 mm, Agilent, USA). The
FAMEs (1 μL) was injected using helium as the carrier gas, and the
split ratio was 1:10. The temperature was held at 130 °C for 5 min
and programmed to 230 °C at 3 °C/min, and held for 5 min. The
flow rate was 1.0 mL/min. The mass scan parameters included
electron impact ionization voltage of 70 eV and mass range of
50.0–500.0 amu. Chemical compositions were identified by com-
parison of their mass spectra to the NIST 02 mass spectral library
(Giuliani et al., 2007).
2.4. Colorimetric analysis of total ACGs
The color reaction of compounds contain α, β-unsaturated γ-
lactone ring moieties with alkaline picrate (Baljet reaction) follows
Lambert-Beer's law and has been used in many quantitative studies
(Neuwald, 1950). The absorption maximum is at 490 nm (Kurkela,
1958). Total ACGs content was determined by colorimetric method
using alkaline picrate reagent with modification. ASO (5 mg/mL),
picric acid (2 mg/mL) and NaOH (5 mg/mL) were dissolved in
ethanol (10 mL). Stock standard solutions of motrilin (622.48 g/mol)
was prepared in methanol at a concentration of 0.5 mg/mL. Work-
ing standard solutions were prepared by diluting standard solution
with methanol to give five different concentrations in the range of
1.61
10  4– 4.82
10  4 mol/L for calibration curves. Then all the
test tubes were kept in 70 °C water for 10 min, centrifuged at
5000 rpm for 10 min for all solution. The absorbance of the super-
natant was measured by using 752 UV-spectrophotometer
364 Y. Chen et al. / Journal of Ethnopharmacology 193 (2016) 362–367
Fig. 1. Liver, spleen and thymus indexes of H22 bearing mice after administration of ASO and 5-Fu. For each group, organs from 10 mice were analyzed. Organ index ¼(organ
weight/final bodyweight)
100. *P o 0.05 compared with control. **P o0.01 compared with control.
(Shanghai Spectrum Instruments Co., Ltd., Shanghai, China) at
constant wavelength 490 nm. Quantification was performed by
absorbance using the external standard method. The estimation of
total ACGs content in ASO was performed at room temperature and
in triplicates.
2.5. Animals
Male Kunming mice [SCXK (Shanghai) 2007–0005] weighting
20.0 72.0 g were purchased from Slac laboratory animal Co. Ltd.
(Shanghai, China). All the procedures were approved by Animal
Ethical Council of Nanjing University of Chinese Medicine. The
mice were maintained under laboratory conditions at 25 °C under
a normal 12 h/12 h light/dark cycle with humidity of 55% and fed
with food and water ad libitum. The mice were allowed, at least,
5 days to adapt to the laboratory environment before experiments.
2.6. In vivo anti-tumor activity
Anti-tumor effect on H22 was observed in normal male
Kunming mice. The test was performed by observing the effect on
the growth of the tumor as described previously (Yang et al., 2013,
2015). A dose of 0.2 mL of aseptic H22 cells (about 1.0
107/mL)
was implanted subcutaneously at the right groin of mice (Qin
et al., 2004; Chen et al., 2012b).
Twenty-four hours after the tumor inoculation of the H22 cells,
forty male Kunming were grouped (n ¼10) as follows: model
control group, 5-Fu group, a high dose ASO group, and a low dose
ASO group. LD50 of the ASO was found to be 10.0 mL/kg body
weight on male Kunming in our preliminary experiment (data not
shown). ASO and 5-Fu dissolved in 0.5% CMC-Na were used as test
samples. High and low dose ASO groups received intra-gastric
perfusion of ASO at the dose of 1.0 mL and 0.5 mL/kg, respectively.
In the 5-Fu group, 5-Fu (15 mg/kg) was given intragastrically to the
mice as positive control group. The mice in the model control
group were given the same volume of 0.5% CMC-Na instead of the
test solution. All these treatments were administered in-
tragastrically one time daily for 7 days in a dose of 0.1 mL/10 g.
Twenty-four hours after the last administration, all mice were
sacrificed by cervical dislocation, and the tumor and main organs
(liver, kidney, spleen and thymus) were dissected and weighed.
The tumor growth inhibition rate was calculated as: Inhibition rate
(%)¼ 100
(C  T)/C, where C is the average tumor weight of the
control group and T is the tumor weight of the treated sample
group. Organ index¼ (organ weight/final bodyweight)
100.
2.7. Histological and immunohistochemical examination
The mice were sacrificed at day 7 after treatments, the speci-
mens of tumor were taken and fixed in 10% formalin and analyzed
for pathological changes with hematoxylin and eosin.
Immunohistochemical detection of interleukin-6 (IL-6), janus
kinase (Jak) and phosphorylated signal transducers and activators
of transcription (p-Stat3) was performed by standard im-
munohistochemical techniques with primary antibodies against
IL-6, Jak and p-Stat3 (Bioworld, Shanghai, China), and horseradish
peroxidase (HRP)-Polymer anti-Mouse/Rabbit IHC Kit (Mainxin-
Bio, Fuzhou, China). Average density was measured with Motic
Image Advanced 3.2 software (Motic China Group Co., LTD.).
2.8. Statistical analysis
All results were presented as mean 7SD. Data was analyzed by
student's t-test, two-way or one-way ANOVA followed by Dun-
nett's t-test. Results were regarded statistically significant if the P-
values were less than 0.05.
3. Results
3.1. Optimization of extraction conditions and GC-MS analysis of
ASO
As shown in Table 1, the maximum efficiency of the chosen
extraction method for ASO was soxhlet extraction method with
yields of 17.00%. In single factor experiments, results showed that
the optimal extraction parameters of ASO was using soxhlet ex-
traction method with extraction solvent of petroleum ether, tem-
perature of 80 °C, and extraction time of 90 min. Under these
conditions, the yield of ASO was 22.65%. Four compounds in-
cluding palmitic acid (9.92%), linoleic acid (20.49%), oleic acid
(56.50%) and stearic acid (9.14%) (Table 2) were identified from the
FAMEs of ASO by GC-MS (Fig. 2S).
3.2. Colorimetric analysis of total ACGs
The colorimetric reaction of ACGs with alkaline picrate pro-
duced orange color, as the presence of α, β-unsaturated γ-lactone
ring moieties in ACGs. The total ACGs content of ASO determined
by colorimetric method was reported as motrilin equivalents. The
calibration curve for motrilin was: y¼932.4c þ 0.025 (R2 ¼0.9980)
 Y. Chen et al. / Journal of Ethnopharmacology 193 (2016) 362–367 365

Fig. 2. Immunohistochemistry of IL-6, JAK and p-STAT3 in H22 solid tumor taken from mice after receiving repeated i.g. of 0.5 and 1.0 mL/kg body weight ASO, or 0.1 mL/10 g
0.5% CMC-Na alone (control group) for 7 days. Representative sections from one mouse are given for each group. Magnification, 200
. Ten mice were used for each group
and tissues from three different mice in each group and 3 randomly selected areas from each slide were analyzed. Quantitative data (mean 7 SD.) are presented using the
average density values of IL-6, JAK and p-STAT3 positive cells. *P o 0.05 compared with control. **P o 0.01 compared with control.

(Fig. 1S). The total ACGs content in ASO was 41.00 70.62 mg/g the Jak and p-Stat3 expression was lower in tumor tissues taken
(Table 3). from ASO-treated mice than control mice. IL-6 expression in tumor
tissues of 1.0 mL/kg ASO-treated mice was lower than that of
3.3. In vivo anti-tumor activity control mice (P o0.05).

The results in Table 4 showed that ASO exhibited an anti-tumor

effect against H22 in a dose-dependent manner when adminis-
tered by oral. The inhibition rate of ASO reached 53.54% at a dose
of 1.0 mL/kg/day. The spleen index of 5-Fu-treated mice, and the
spleen and thymus indices of 5-Fu- and ASO-treated mice were
lower than that of control mice (Fig. 1).
3.4. Effect of ASO treatment on IL-6, Jak and p-Stat3 production
Cells positive for IL-6, Jak and p-Stat3 were found in tumor
tissues (Fig. 2). The immunohistochemistry analysis showed that
4. Discussion
Cancer diseases are major problems which still remain un-
resolved. Treating cancer with botanical extracts has been shown
to be an easy and effective approach in China. BJO (Yan et al.,
2015), contains oleic acid, linoleic acid, stearic acid, palmitic acid,
arachidonic acid, and other unsaturated fatty acids (Ma et al.,
2013), has been widely used for treatment of lung cancer as one of
Chinese patent drugs (Nie et al., 2012). Our previous phytochem-
ical study showed that ASO contained palmitic acid (17.1%), stearic
366 Y. Chen et al. / Journal of Ethnopharmacology 193 (2016) 362–367
acid (12.7%), oleic acid (41.7%) and linoleic acid (9.4%) (Qiu et al.,
2014). In this study, the optimal extraction method improved the
content of unsaturated fatty acids (UFA). UFA are known to have
antineoplastic activity (Muzio et al., 2006; Jiang et al., 2008).
In present study, colorimetric analysis result exhibited that ASO
contains 4.1% ACGs as motrilin equivalents. ACGs are major
bioactive compounds in A. squamosa seeds, which have a strong
antitumor activity (Bermejo et al., 2005; Chen et al., 2013). The
most active ACGs exhibited in-vitro activity (ED50) in the range of
10  9–10  12 μg/mL (Mangal et al., 2015). Our previous pharmaco-
logical study showed that ASO exhibited selective cytotoxic ac-
tivity against HepG2 cell line (Qiu et al., 2014). Experiments with
cells in monolayer or suspension do not provide sufficient in-
formation on the activity of compounds on clinically important
solid tumors. Nowadays, researchers all over the world are at-
tempting to explore novel anti-tumor drugs. Numerous murine
models have been developed to study human cancer. Although
some components of the immune system are missing, these
models have been developed to investigate the factors involved in
malignant transformation, invasion and metastasis, as well as to
examine response to therapy (Richmond et al., 2008). Further-
more, the anti-tumor effect of ASO was investigated by mice
bearing H22 hepatoma cells transplantation tumor model, which is
one of the most common tumor models employed in the anti-
tumor research (Wu et al., 2010). ASO inhibited the growth of H22
tumor cells in mice with a maximum inhibitory rate of 53.54%.
α, β-unsaturated γ-lactone moieties are known to be Michael
reaction acceptors (Powers et al., 2002). Michael reaction acceptor
molecules are important inhibitors of Stat3 activation (Zhao and
Cong, 2012). IL-6 is produced by a variety of cell types during in-
fection, trauma, and immunological challenge (Kishimoto et al.,
1995). Cellular activation by IL-6 requires binding to its cognate
soluble interleukin-6 receptor (sIL-6R) and the resulting dimer-
ization of gp130. This mediates phosphorylation of gp130-asso-
ciated Jaks, which facilitates docking of Stat1/Stat3 factors to
gp130, and their phosphorylation (Kishimoto et al., 1995). IL-6/Jak/
Stat3 pathway serve to maintain cells progressing towards neo-
plastic growth, protecting them from cellular apoptotic deletion
and chemotherapeutic drugs (Hodge et al., 2005). Persistent sig-
naling of specific Stats, in particular Stat3 and Stat5, has been
demonstrated to directly contribute to oncogenesis by stimulating
cell proliferation and preventing apoptosis, so activated Stat sig-
naling in human tumors provides novel molecular targets for
therapeutic intervention (Buettner et al., 2002; Kastritis et al.,
2009). However, despite this potential as a therapeutic target, and
the extensive attempts by many laboratories and pharmaceutical
companies to develop an effective Stat3 inhibitor for use in the
clinic, no direct Stat3 inhibitor has been approved for clinical use
(Wake and Watson, 2015). ASO suppressed the H22 solid tumor
development may due to its chemical constituents unsaturated
fatty acid and ACGs, and immunohistochemical examination re-
sults showed that ASO exerted an antitumor effect via IL-6/Jak/
Stat3 pathway.
5. Conclusion
The results demonstrated that ASO contains unsaturated fatty
acid and ACGs, and effectively suppressed the H22 solid tumor
development. This beneficial effect was related to IL-6/Jak/Stat3
pathway. Further studies on the mechanisms of action and active
principles of ASO may contribute to the development of novel
drugs for cancer therapy in the future.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article. This
work was supported by grants from National Natural Science
Foundation of China (81274057, 81403082 and 81573577) and the
Project Funded by the Priority Academic Program Development of
Jiangsu Higher Education Institutions (PAPD). We acknowledge the
technical support provided by Dr. Shan MQ (GC-MS) at the Uni-
versity of Chinese Medicine.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.jep.2016.08.036.
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