Pharmaceutical Biology, 2012; 50(10): 1276–1280

© 2012 Informa Healthcare USA, Inc.

ISSN 1388-0209 print/ISSN 1744-5116 online
DOI: 10.3109/13880209.2012.673628

Research article

 valuation of cytotoxic activity of patriscabratine, tetracosane

E
and various flavonoids isolated from the Bangladeshi
medicinal plant Acrostichum aureum
Shaikh Jamal Uddin1,2, Darren Grice3, and Evelin Tiralongo1,4
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1
School of Pharmacy, Griffith University, Gold Coast campus, Queensland, Australia, 2Pharmacy Discipline, Khulna
University, Khulna, Bangladesh, 3Institute for Glycomics and School of Medical Sciences, Griffith University, Gold Coast
campus, Queensland, Australia, and 4Griffith Health Institute, Griffith University, Gold Coast campus, Queensland, Australia

Abstract
Context: Acrostichum aureum L. (Pteridaceae), a mangrove fern, has been used as a Bangladeshi traditional medicine
for a variety of diseases including peptic ulcer.
Objective: Isolation and structural elucidation of cytotoxic secondary metabolites from the methanol extract of the
aerial parts of A. aureum.
For personal use only.

Materials and methods: Compounds were isolated using HPLC. The compound structures were elucidated by 1D and
2D NMR, MS and other spectroscopic methods using published data. The compounds were tested for their cytotoxic
activity against healthy and cancer cells using the MTT assay. Active compounds were further evaluated for apoptosis –
and necrosis-inducing potential against gastric cancer cells (AGS) using the FITC Annexin V apoptosis assay.
Results and discussion: Seven known compounds, patriscabratine, tetracosane and 5 flavonoids (quercetin-3-O-β-
d-glucoside, quercetin-3-O-β-d-glucosyl-(6→1)-α-l-rhamnoside, quercetin-3-O-α-l-rhamnoside, quercetin-3-O-α-
l-rhamnosyl-7-O-β-d-glucoside and kaempferol) were isolated. Patriscabratine was found moderately cytotoxic
against AGS, MDA-MB-231 and MCF-7 cells with IC50 values ranging from 69.8 to 197.3 μM. Tetracosane showed some
cytotoxic activity against AGS, MDA-MB-231, HT-29 and NIH 3T3 cells with IC50 values ranging from 128.7 to >250 μM.
Patriscabratine and tetracosane displayed an apoptotic effect (10%) on AGS cells within 24 h which was increased
(20%) after 48 h, and was comparable to, if not greater, than the positive control, cycloheximide.
Conclusion: Except for quercetin-3-O-β-d-glucoside and kaempferol; compounds were isolated for the first time from
this plant and evaluated for their cytotoxic activity. The results highlight the potential of this plant as a source of
bioactive compounds and provide a rationale for its traditional use in peptic ulcer treatment.
Keywords: Pteridaceae, Mangrove fern, cytotoxicity, apoptosis and necrosis

Introduction
Acrostichum aureum L. (Pteridaceae) is a mangrove
fern that occurs in tropical and subtropical areas
worldwide (Momtaz, 2008). In Bangladesh, it is found
in the Sundarban and other coastal belts as ‘Tiger fern’,
because it provides a suitable hiding place for the famous
carnivorous Royal Bengal Tiger of the Sundarban (Zafrul,
2000). In Bangladesh, preparations from rhizomes and
leaves of A. aureum are used to treat wounds, peptic ulcers
and boils (Momtaz, 2008). In China, the rhizome is used
to treat worm infections (Momtaz, 2008), whereas, Fijian
people use it to treat asthma, constipation, elephantiasis,
febrifuge, and chest pain (Coambie & Ash, 1994). The
native people of Costa Rica use leaves as emollients,
whereas, the Cuna people (Panama and Colombia)
use the young fiddleheads to extract fish bones from
the throat and as a medicinal bath for infants (Natural
Resources Conservation Service, 2010). The crude extract
of a Japanese A. aureum specimen is reported to possess
anti-oxidant, tyrosinase inhibiting activity (Lai et al.,

Address for Correspondence: Dr Evelin Tiralongo, School of Pharmacy, Griffith University, Gold Coast campus, 4222, Qld, Australia.
Tel: + 61 7 5552 7098. Fax: + 61 7 5552 8804. E-mail: E.Tiralongo@griffith.edu.au
(Received 15 October 2011; revised 13 January 2012; accepted 05 March 2012)

1276
 Patriscabratine and tetracosane from Acrostichum aureum
2009), while a Hainan specimen reported anti-tumor
activity against cervical cancer cell line (Dai et al., 2005).
Recently, we reported the cytotoxic effect of water and
methanol extracts from a Bangladeshi specimen of A.
aureum on gastric, colon and breast cancer cells (Uddin
et al., 2011). So far, a total of 19 compounds have been
isolated from A. aureum belonging to different natural
product classes, such as sterols, flavonoids, fatty acids and
long-chain hydrocarbons (Mei et al., 2006; Nobutoshi et
al., 1981; Sultana et al., 1986).
Here we report on the isolation and cytotoxic activ-
ity evaluation of seven known compounds (1-7), five of
which were isolated for the first time from A. aureum.
Two compounds, namely tetracosane and patriscabra-
tine, were further evaluated for their apoptosis – and
necrosis-inducing potential on gastric adenocarcinoma
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cells (AGS).
Materials and methods
General experimental procedures
Optical rotations were measured on a Jasco P-1010
polarimeter. IR spectra were recorded on a Bruker
Optics alpha-QuickSnap FT-IR spectrophotometer.
NMR spectra were recorded on either a Bruker Avance
300 or 600 MHz spectrometer in CD3OD. LR-MS were
obtained on a Bruker Daltonics esquire 3000 mass spec-
For personal use only.
trometer. Analytical HPLC was performed on a Varian
Prostar instrument with a 335 DAD using a RP (Luna
C18, 5 μm, 250 × 4.6 mm) column. Preparative HPLC
was performed on a Waters instrument equipped with
a Waters 600E pump, Rheodyne 7725i injector, using a
RP (Luna C18, 5 μm, 150 × 21.2 mm) column. SPE car-
tridges (Alltech, 10 g, RP-C18) were used to fractionate
the extract.
Plant material
The aerial parts of A. aureum were collected from tidal
forests in the coastal Sundarbans (a swamp region in
the Ganges delta) of Bangladesh in February, 2007.
The plant material was identified by Dr. Momtaz Mahal
Mirza, Principle Scientific Officer, Bangladesh National
Herbarium, Dhaka, and shade-dried. A specimen was
deposited in the Bangladesh National Herbarium, Dhaka
(Voucher no.: DACB 31538).
Preparation of plant extract and isolation of cytotoxic
compounds
The dried and pulverized whole plant material of A.
aureum (150 g) was extracted with methanol (1 L) by
soaking it overnight at room temperature with continu-
ous stirring. The extract was filtered and the residue was
further extracted with methanol (3 × 1 L) for 1 h under
sonication. All extracts were combined and concentrated
under reduced pressure to give 7.57 g extract (5.04% w/w).
The resulting methanol extract was partitioned between
n-hexane and methanol (1:1) to give two fractions. The
methanol fraction (6.70 g) was further sub-fractionated
© 2012 Informa Healthcare USA, Inc.
1277
into four fractions using reverse-phase SPE columns
with a H2O:MeOH stepwise gradient (100:0 (SPE1), 80:20
(SPE2), 60:40 (SPE3) and 0:100 (SPE4)).
The SPE3 (40% methanol) and SPE4 (100% methanol)
fractions were further analyzed by preparative RP-HPLC
using a H2O:MeOH gradient containing 0.05% TFA. The
SPE3 fraction (0.42 g) yielded compound 1 (40 mg) and SPE4
fraction (0.60 g) yielded compounds 2 (20.0 mg), 3 (5.0 mg),
4 (0.60 mg), 5/6 as mixture (2.90 mg) and 7 (9.0 mg).
Spectroscopic properties of isolated compounds
The structures of the isolated compounds were eluci-
dated by the comparison of 1D and 2D NMR, MS and
other spectroscopic data with published data.
In vitro assay for cytotoxic activity
Cell lines
All cell lines (NIH 3T3, ATCC: CRL-1658; AGS, ATCC:
CRL-1739; HT-29, ATCC: HTB-38; MCF-7, ATCC: HTB-
22; and MDA-MB-231, ATCC: HTB-26) were purchased
from ATCC, Manassas, VA 20108, USA. Cell lines were
cultured in Advanced Dulbecco’s modified Eagle’s
medium supplemented with 10% inactivated newborn
calf serum and 5 mM l-glutamine, and grown at 37°C in a
humidified atmosphere of 5% CO2 in air.
MTT cytotoxicity assay
The cytotoxicity of the compounds was tested against
normal mouse fibroblast cells and four human cancer
cell lines using the MTT assay according to the method
described by Uddin et al. (2011) and references therein.
The IC50 values were calculated with Probit analysis soft-
ware (Bakr, 2010). Cycloheximide was used as a positive
control generating IC50 values of 1.1, 3.6, 12.8, 1.2, and
218.2 μM against NIH 3T3, AGS, HT29, estrogen receptor
negative (ER-) MDA-MB-231 and estrogen receptor posi-
tive (ER+) MCF-7 cells, respectively.
Annexin V-FITC apoptosis measurement
The FITC Annexin V apoptosis assay was used to mea-
sure apoptosis of the isolated cytotoxic compounds from
A. aureum against a human gastric adenocarcinoma
(AGS) cell line according to Jason et al. (2008). Cells were
treated with tetracosane (500 μg/mL) and patriscabra-
tine (100 μg/mL) for 24 and 48 h and analyzed with the
BD FACS Calibur Flow Cytometer (BD Bioscience, San
Jose, California, USA). Cells with no treatment served as a
negative control and cycloheximide (150 μg/mL) served
as a positive control.
Results and discussion
A total of seven compounds (1-7), namely, tetracosane
(1) (Yamaji et al., 2010); quercetin-3-O-β-d-glucoside
or hyperoside (2) (Matsuura et al., 2002); quercetin-
3-O-β-d-glucosyl-(6→1)-α-l-rhamnoside or rutin
(3) (Abdullah et al., 2008); quercetin-3-O-α-l-
rhamnoside (4) (Fossen et al., 1999; Iorizzi et al., 2001);
 1278 S. J. Uddin et al.
quercetin-3-O-α-l-rhamnosyl-7-O-β-d-glucoside
(5); kaempferol (6) (Imperato, 2008; Xiao et al., 2006);
and patriscabratine (7) (Gu et al., 2002) (Figure 1),
were isolated from the methanol extract of A. aureum
following subfractionation using SPE C18 cartridges.
Only compound 2 and 6 have been reported from this
plant previously.
Tetracosane (1) showed significant cytotoxicity (IC50
128.7 μM) only against HT-29 colon cancer cells. It (1)
showed some toxicity against the estrogen-dependent
breast cancer (MDA-MB-231) cells (IC50 >250 μM) and
gastric cancer cells (AGS; IC50 >250 μM) but no toxicity
against estrogen-independent breast cancer (MCF-7)
cells. Tetracosane has been identified as a component in
many volatile oils from plants, however to-date there are
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For personal use only.
Figure 1. Chemical structures of isolated compounds 1–7.
 Pharmaceutical Biology
no reports on the pharmacological activity of tetracosane
itself. Although a variety of pharmacological activities,
including cytotoxicity, have been reported for volatile
oils containing tetracosane (Al Ashaal et al., 2010; Kansoh
et al., 2009) this study is the first report on the cytotoxicity
of tetracosane itself.
Patriscabratine (7) showed significant cytotoxicity
against gastric (AGS) (IC50 133.6 μM) and breast can-
cer cells (MDA-MB-231; IC50 69.8 μM and MCF-7; IC50
197.3 μM), but no cytotoxicity against normal mouse
fibroblasts (NIH3T3) and colon cancer cells (HT-29).
Previous research showed that synthetic patriscabratine
has cytotoxic activity against breast carcinoma cell lines
(ER- MDA-MB-231) (Yuan et al., 2010), with the IC50 value
for MDA-MB-231 being slightly higher (IC50 >100 μM)
 Patriscabratine and tetracosane from Acrostichum aureum
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Figure 2. Flow cytometry results for AGS cells following treatment
with and without tetracosane (1) (500 μg/mL) and patriscabratine
(7) (100 μg/mL) in comparison to cycloheximide (150 μg/mL)
For personal use only.
after 24 and 48 h incubation. A represents AV+/PI- (early apoptosis)
and B represents AV+/PI+ (late apoptosis or necrosis) after 24 and
48 h treatment, respectively.
than in our study (IC50 69.8 μM). This is, however, the
first report of patriscabratine’s (7) effects on ER+ MCF-7
breast cancer cells, as well as colon cancer cells.
None of the five known flavonoids (2, 3, 4, 5 and 6)
isolated from A. aureum showed detectable cytotoxic
activity (IC50 >500 μM) against the cell lines tested in this
study confirming no or low cytotoxicity against various
cancer cell lines in previous studies for the flavonoids (2,
3, 4 and 6) (Afifi et al., 2009; Li et al., 2009; Mei et al., 2006;
Yang & Liu, 2009). However, our study is the first report
on the evaluation of cytotoxicity for quercetin-3-O-α-l-
rhamnosyl-7-O-β-d-glucoside (5).
No report exists to-date on the apoptotic potential of
pure tetracosane and patriscabratine in human cancer
cells. In this study, tetracosane (1) and patriscabratine
(7) caused early apoptosis (AV+/PI-) to a similar degree
and with similar time dependence. Both compounds
showed approximately 10 and 20% apoptosis follow-
ing 24 and 48 h of treatment, respectively (Figure 2A).
For both compounds early apoptosis was induced to a
similar degree as cycloheximide at 24 and 48 h, however
both compounds showed greater late apoptosis/necrosis
effects than the positive control cycloheximide following
48 h treatment.
Although the concentration of tetracosane (1) was
approximately 3× higher than cycloheximide (150 μg/mL)
the apoptosis inducing potential of patriscabratine (7)
can certainly be seen as stronger than the positive
© 2012 Informa Healthcare USA, Inc.
1279
control, having shown apoptosis at lower concentration
(100 μg/mL) than cycloheximide.
In conclusion, this study describes for the first time
the isolation of tetracosane, patriscabratine and five
flavonoids from A. aureum. The cytotoxic potential of
isolated tetracosane (1), quercetin-3-O-α-l-rhamnosyl-
7-O-β-d-glucoside (5) and patriscabratine (7) against
different cancer cell lines was evaluated for the first
time, and the mode of cytotoxicity for (1) and (7) against
gastric cancer (AGS) cells was revealed as induction of
apoptosis.
A. aureum is traditionally used against peptic ulcer,
which is a risk factor for the development of gastric
cancer (Rayburn et al., 2009). Interestingly, patriscabra-
tine (7), isolated from this plant, had significant effects
against gastric cancer cells which could contribute to the
reported traditional medicinal effects of the plant.
Declaration of interest
Shaikh J. Uddin was funded by a Griffith University
Postgraduate Research Scholarship (GUPRS) and a
Griffith University International Postgraduate Research
Scholarship (GUIPRS). The authors report no conflicts of
interest.
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