A sulphated flavone glycoside from Livistona australis and its antioxidant and cytotoxic activity

Article abstract of DOI:10.1080/14786419.2011.587188

A new flavone glycoside tricin 7-O - glucopyranoside-2-sulphate sodium salt along with 14 known flavonoid compounds were isolated and identified from the aqueous methanol extract of Livistona australis leaves. Their structures were established on the basis of extensive NMR (¹H, ¹³C, HSQC and H-H COSY) and ESIMS data. Antioxidant and cytotoxicity properties of the methanol extract of the leaves as well as the new compound were investigated.

Full text of DOI:10.1080/14786419.2011.587188

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A sulphated flavone glycoside from
Livistona australis and its antioxidant
and cytotoxic activity
a
b
a
Mona E.S. Kassem , Soha Shoela , Mona M. Marzouk & Amany
c
A. Sleem
a
Department of Phytochemistry and Plant Systematics, National
Research Centre, El Tahrir Street, Dokki 12311, Cairo, Egypt
b
Department of Pharmacognosy, Faculty of Pharmacy, Al Azhar
University, Nasr City 1137, Cairo, Egypt
c
Department of Pharmacognosy, National Research Centre, El
Tahrir Street, Dokki 12311, Cairo, Egypt
Published online: 28 Jul 2011.
To cite this article: Mona E.S. Kassem , Soha Shoela , Mona M. Marzouk & Amany A. Sleem
(
2012) A sulphated flavone glycoside from Livistona australis and its antioxidant and cytotoxic
activity, Natural Product Research: Formerly Natural Product Letters, 26:15, 1381-1387, DOI:
0.1080/14786419.2011.587188
1
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Natural Product Research
Vol. 26, No. 15, August 2012, 1381–1387
A sulphated flavone glycoside from Livistona australis and its antioxidant
and cytotoxic activity
a
b
a
c
Mona E.S. Kassem *, Soha Shoela , Mona M. Marzouk and Amany A. Sleem
a
Department of Phytochemistry and Plant Systematics, National Research Centre, El Tahrir Street,
Dokki 12311, Cairo, Egypt; Department of Pharmacognosy, Faculty of Pharmacy, Al Azhar
b
c
University, Nasr City 1137, Cairo, Egypt; Department of Pharmacognosy, National Research
Centre, El Tahrir Street, Dokki 12311, Cairo, Egypt
(
Received 19 August 2010; final version received 14 April 2011)
00
A new flavone glycoside tricin 7-O-ꢀ-glucopyranoside-2 -sulphate sodium salt
along with 14 known flavonoid compounds were isolated and identified from the
aqueous methanol extract of Livistona australis leaves. Their structures were
1
13
established on the basis of extensive NMR ( H, C, HSQC and H-H COSY) and
ESIMS data. Antioxidant and cytotoxicity properties of the methanol extract of
the leaves as well as the new compound were investigated.
Keywords: Livistona australis; Arecaceae (¼Palmae); sulphated flavone glycoside;
in vivo antioxidant effect; in vitro cytotoxicity
1
. Introduction
Arecaceae, also called Palmae, are among the best known and extensively cultivated plant
families. Livistona australis (R. Br.) Mart. or cabbage-tree palm belongs to the Palmae
family. It is a tall, slender palm growing up to about 25 m in height and 0.35 m in diameter.
It has leaves plaited like a fan; the cabbage of these is small but sweet and edible (Boland
et al., 1984). Some species of the same genus (i.e. Livistona chinenses) are used in folk
medicine in Southern China for treating various tumours (Ah Lok, 1998). A previous
chemical investigation on the leaves of L. australis resulted in the isolation of a
new pyranone derivative, 3-hydroxy-2-(4-hydroyphenyl)-6-methyl-4-H-pyran-4-one
(
El-Desouky, Kassem, Al-Fifi, & Gamal El-Deen, 2009). In the course of our phytochem-
ical investigation, a new sulphated tricin glycoside together with 14 known flavonoids were
isolated and identified. In vivo antioxidant activities of a new glycoside as well as the
methanol extract of L. australis leaves were evaluated by determination of blood
glutathione (GSH) level (Beutler, Duron, & Kellely, 1963). Furthermore, their in vitro
cytotoxic activities were tested against three human carcinoma cell lines: colon carcinoma
HCT116, breast carcinoma MCF7 and liver carcinoma HEPG2 (Skehan et al., 1990).
2
2
. Results and discussion
.1. Identification of isolated compounds
0
0
A new flavone glycoside, tricin 7-O-ꢀ-glucopyranoside-2 -sulphate sodium salt (1),
was isolated and identified, together with 14 known flavonoid compounds;
*
Corresponding author. Email: monakassem111@hotmail.com
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2011.587188
http://www.tandfonline.com

1
382
M.E.S. Kassem et al.
genkwanin-6-C-ꢀ-glucopyranoside (2), genkwanin 8-C-ꢀ-glucopyranoside (3), isovitexin
0
(
4), isoorientin (5), orientin (6) tricin 7-O-ꢀ-glucopyranoside (7), tricin 4 -O-ꢀ-glucopyr-
anoside (8), luteolin 7-O-ꢀ-glucopyranoside (9), quercetin 3-O-ꢀ-glucopyranoside (10),
qurecetin 3-O-ꢀ-galactopyanoside (11), tricin (12), quercetin (11), apigenin (14) and
luteolin (15). The chemical structures of the isolated compounds were elucidated by
extensive UV, NMR and MS spectral data (Figure 1). The identification of known
flavonoids 2–15 has been determined by comparing their spectral data with those
previously published ones (Ahmed & Shahat, 2006; Guvenalp & Irenzer, 2005; Hasegawa,
Tanaka, Hosoda, Takano, & Ohta, 2008; Kim et al., 2010; Kwon, E. Kim, W.J. Kim,
W.K. Kim, & C. Kim, 2002).
Compound 1 was obtained as a yellow amorphous powder that exhibits an anionic
character on paper electrophoretic analysis. The negative ESIMS spectrum of 1 exhibited
ꢀ
ion peak [M – H] at 571.14 m/z (100%), corresponding to the molecular formula
ꢀ
C O H S, other significant ion peaks at m/z 593.24 [M þ Na-H] and 329.4 [M-H-
2
3
15 23
ꢀ
glucose-SO ] confirmed the presence of sodium and sulphate group in the sample
3
molecule. UV spectral data with diagnostic shift reagents indicated a flavone with
0
substituted –OH group at C-7, while those at C-5 and C-4 are free (Mabry, Markham, &
1
Thomas, 1970; Markham, 1982). H-NMR spectrum revealed two equivalent aromatic
0
0
0
0
protons (ꢁ 7.33, s, H-2 , H-6 ) and two methoxy groups (ꢁ 3.85, s, 6H), typical of the 3 , 4 ,
0
5
trisubstituted ring B of the flavonoid unit. Ring A showed two meta coupled protons
resonated at ꢁ 6.84 and 6.40 (J ¼ 1.8 Hz), each integrated to one-proton, and assigned for
C-8 and C-6, respectively. Moreover, one-proton singlet at ꢁ 7.0 assigned for H-3
confirmed the flavone nucleus of compound 1. The presence of anomeric proton of the
sugar moiety at a downfield chemical shift (ꢁ 5.23, d, J ¼ 7.5 Hz) compared to that of tricin
7
-O-ꢀ-glucopyranoside data suggested that the ꢀ-glucose unit should be substituted
(
Mabry, Markham, & Chari, 1982). The situation of substitution was recognised by the
presence of a triplet sugar signal located downfield at ꢁ 4.03 experienced by the proton
0
0
attached to carbon bearing the sodium sulphate substituent (H-2 ). The assignment was
Figure 1. Chemical structures of compounds 1–15 isolated from L. australis leaves.

Natural Product Research
1383
1
also confirmed by H– H COSY experiment, whereby a cross-peak correlated the
1
00
00
13
anomeric glucose (ꢁ 5.23, H-1 ) to (ꢁ 4.03, H-2 ). The C-NMR spectra displayed 23
carbon resonances, 17 of which were assigned to tricin as the aglycone moiety and six for
the glucose moiety (Agrawal & Bansal, 1989; Yoon, Kim, & Huh, 2000). Resonances of
ring A carbons showed the typical shifts due to the presence of a sugar unit linked to the
C-7 hydroxyl group, 1.4 ppm upfield for C-7 and 1 ppm downfield for ortho carbons (C-6
and C-8, respectively) compared to tricin aglycone (Mabry et al., 1982). The presence of a
00
sulphate group linked to the C-2 of glucose moiety was followed from the upfield shift of
the anomeric carbon of glucose moiety at ꢁ 97.9 (4.5) and the corresponding downfield
00
shift of C-2 signal to 78.98 ppm (3.1) (Nawwar & Buddrus, 1981). The sugar moiety was
identified in the pyranose form by comparing its chemical shift values with previously
published ones (Agrawal & Bansal, 1989). The assignment of the various sugar carbons
was made by 2D-HMQC experiment. The structure was also confirmed by acid hydrolysis;
ꢁ
on controlled acid hydrolysis (10% aqueous AcOH, 10 min, 100 C), compound 1 yielded
an intermediate which was separated by PPC and identified as tricin 7-O-ꢀ-glucopyrano-
1
side (CoPC, UV, H-NMR) (Kwon et al., 2002). Complete acid hydrolysis (2N HCl, 2 h,
ꢁ
1
00 C) afforded tricin, glucose (CoPC with authentic samples) and sulphate detected by
precipitation with barium chloride. The presence of sodium salt was also confirmed by the
golden yellow flame test.
Based on the above information, compound 1 was characterised as tricin 7-O-ꢀ-
00
glucopyranoside-2 -sulphate sodium salt (Figure 1).
2
.2. In vivo antioxidant activity
Free radical formation and oxidative stress may act as a common pathway to diabetes
itself and to its later complications. Hyperglycaemia is accompanied with decrease in GSH
level. GSH is a tripeptide normally present at high concentrations intracellularly and
constitutes the major reducing capacity of the cytoplasm. It is known to protect the
cellular system against toxic effects of lipid peroxidation (Lu, 1999). In this study, the
reduced levels of GSH in the diabetic rats were significantly restored by compound 1 and
methanol extract of L. australis leaves (the percent of change was 3.1% and 18.4%,
respectively, compared to 37.46% for the untreated diabetic rats group). Compound 1 is
the most potent; it showed activity comparable to that of vitamin E and the plant
methanol extract at the tested dose levels (Table 1). This indicates that compound 1 and
the methanol extract can either increase the biosynthesis of GSH or reduce the oxidative
stress leading to less degradation of GSH, or have both effects (Maritim, Sanders, &
Watkins, 2003; Rajasekaran, Sivagnanam, & Subramanian, 2005).
Table 1. Effect of methanol extract of L. australis leaves, compound 1 and
reference drug (vitamin E) on the blood GSH of experimental rats (n ¼ 6).
Group
Blood GSH (mg%)
% Change
Control (1 mL saline)
Diabetic
36.3 ꢂ 1.2
–
22.7 ꢂ 0.4*
35.9 ꢂ 0.8**
29.6 ꢂ 0.6**
37.46
1.1
18.4
ꢀ
1
Diabetic þ vitamine E (7.5 mg kg
)
Diabetic þ methanol extract of L. australis
ꢀ1
leaves (100 mg kg
)
ꢀ
1
)
Diabetic þ compound 1 (0.1 mg kg
35.2 ꢂ 0.9**
3.1
Notes: Data are expressed as mean ꢂ SEM of 6 rats/group. *Statistically signifi-
cant difference from control at p 5 0.01. **Statistically significant difference from
diabetic at p 5 0.01.

1
384
M.E.S. Kassem et al.
2
.3. In vitro cytotoxic activity
In vitro cytotoxicities (see Section 3.7) of compound 1 and the methanol extract were
assayed against liver carcinoma HEPG2, breast carcinoma MCF7 and colon carcinoma
HCT116 cell lines. The American National Cancer Institute assigns a significant cytotoxic
ꢀ
1
effect of extract for future bioguided studies if it exerts an IC50 value ꢃ30 mg mL
(
Stuffiness & Pezzuto, 1990). Compound 1 revealed the highest antiproliferative activity
with IC50 values of 13.5, 15.2 and 16.5 mg mL against HEPG2, MCF7 and HCT116,
respectively, while the methanol extract exhibited less activity against HEPG2 and MCF7
ꢀ1
ꢀ1
cell lines with IC50 values of 21.9 and 22 mg mL , respectively, and the weakest against
ꢀ
1
colon carcinoma HCT116 cell line with IC50 values of 45.8 mg mL
.
3
3
1
. Experimental
.1. General experimental procedure
1 13
-D and 2-D NMR experiments ( H, C, H-H COSY and HMQC) were recorded using a
1
13
Jeol EX-500 spectrometer: 500 MHz ( H-NMR), 125 MHz ( C-NMR). The chemical
shifts are expressed in (ppm) and the coupling constants in Hz. The mass spectra (ESIMS)
were measured on a LCQ Advantage Thermo Finnigan spectrometer; m/z (rel.%) and the
UV spectra recorded on UV spectrophotometer (Shimadzu model 2401 PC). Column
chromatography was carried out on a Polyamide 6S (Riedel-De-Haen AG, Seelze Haen
AG, Seelze Hanver, Germany) and Sephadex LH-20 (Pharmacia). The paper chromatog-
raphy (Whatman No. 1 and 3 MM) was performed using solvent systems: (1) H O, (2)
2
1
and (4) BBPW (C H –n-BuOH–pyridine-H O 1 : 5 : 3 : 3, upper layer). Solvents 3 and 4
5% HOAc (H O–HOAc 85:15), (3) BAW (n-BuOH–HOAc–H O 4 : 1 : 5, upper layer),
2 2
6
6
2
were used for sugar analysis. The compounds were detected by their UV absorption at a
wavelength of 254 nm.
3
.2. Plant material
Fresh plant material was collected from Orman garden, Giza, Cairo, in October 2009.
Authentication was performed by Dr M. El-Gibali, former Researcher of Botany at the
National Research Centre (NRC). A voucher specimen (no. 124) was deposited in the
Herbarium of NRC (CAIRC).
3
.3. Animals
Adult male albino rats of Sprague-Dawley strain weighing 130–150 g were obtained from
the breeding colony of (NRC). The animals were kept under the same hygienic condition,
well-balanced diet and water supplied ad libitum.
3
.4. Biochemical kit
Alloxan was purchased from Sigma Co. Cairo, Egypt, vitamin E (dl ꢂ-tocopheryl acetate)
from Pharco Pharmaceutical Co. Egypt, GSH kit from Wak Co., Germany, and glucose
´
reagent kit was from BioMerieux, France.
3
.5. Extraction and isolation
Two kilograms of the dried powdered L. australis leaves were extracted with 70 : 30
MeOH: water (3 ꢄ 6 L), and the extract was concentrated to dryness under vacuum at
ꢁ
7
0 C. The residue (200 g) was dissolved in water (500 mL). The aqueous solution was
successively extracted with n-hexane (2 ꢄ 500 mL) for defatting. The defatted methanol
2
extract was subjected to polyamide 6S column chromatography (125 ꢄ 5 cm ), eluting with

Natural Product Research
1385
MeOH/H O mixtures of decreasing polarities to yield five main fractions. Fraction I (20%
2
MeOH/H O), chromatographed on PC using BAW (double solvent) two times and then
2
purified on a Sephadex LH-20 column using methanol, yielded compound 1 (35 mg).
Fractions II–V afforded compounds 2–15. Their isolation was achieved by a combination
2
of smaller polyamide column (75 ꢄ 2.5 cm ) and PPC using H O, 15% HOAc and BAW as
2
eluents and then purified on Sephadex LH-20 column using methanol as eluent.
00
.5.1. Tricin 7-O-ꢀ-glucopyranoside-2 -sulphate sodium salt (1)
3
Yellow amorphous powder, Rf-values: 0.92 (H O), 0.87 (HOAc), 0.45 (BAW).
2
Electrophoretic mobility: 1.8 cm, on Whatman no. 3 MM paper, buffer solution of pH
ꢀ
1
–
2
5
5
, H O–AcOH–HCOOH (89.5 : 8 : 2.5), 90 min, 50 V cm . Negative ESIMS m/z: [M ꢀ H]
2
ꢀ
71.14 (100%) corresponding to a molecular formula C O H S and [M þ Na-H]
2
3
15 23
93.24 (C O H S Na). UV ꢃmax nm in MeOH: 283, 349; MeOH/NaOMe: 280, 425;
2
3
15 23
AlCl : 287, 318, 362, 404; AlCl /HCl: 289, 316, 362, 403; NaOAc: 283, 349, 419; NaOAc/
3
3
1
H BO : 281, 360. H-NMR, 500 MHz in DMSO-d : ꢁ 7.33 (2H, s, H-2 , H-6 ), 7.00 (1H, s,
0
0
3
3
6
H-3), 6.84 (1H, d, J ¼ 1.5 Hz; H-8), 6.40 (1H, d, J ¼ 1.5 Hz; H-6), 3.85 (6H, s, 2 [OCH ]),
3
0
0
00
00
00
5
3
(
(
(
.23 (1H, d, J ¼ 7.5 Hz; H-1 ), 4.03 (1H, t, H-2 ), 3.70 (1H, m, H-6 ), 3.60 (1H, t, H-3 ),
00
00 13
.42 (1H, m, H-5 ), and 3.22 (1H, m, H-4 ). C-NMR, 125 MHz in DMSO-d : ꢁ 182.6
6
0 0 0
C-4), 164.7 (C-2), 163.2 (C-7), 161.7 (C-5), 157.3 (C-9), 148.8 (C-3 ,5 ), 140.6 (C-4 ), 120.7
0
0
0
00
C-1 ), 105.9 (C-3), 105.0 (C-2 ,6 ), 104.3 (C-10), 99.8 (C-6), 97.9 (C-1 ), 96.1 (C-8), 79.0
00
00
00
00
00
C-2 ),77.5 (C-5 ), 76.5 (C-3 ), 70.1 (C-4 ), ꢁ 61.1 (C-6 ) and ꢁ 56.9 (2 OCH ).
3
3
.6. Determination of antioxidant activity
The antioxidant activities of the methanolic extract of L. australis and compound 1 were
determined by measuring the GSH level in blood of alloxan-induced diabetic rats upon
administration of extract and compound 1 for one week (Beutler et al., 1963; Eliasson &
Samet, 1969). Thirty rats were divided into five groups (six animals each). One group was
kept as a negative control while diabetes was induced in the other groups by a single intra-
ꢀ
1
peritoneal administration of alloxan at a dose of 150 mg kg bodyweight (BW) followed
by an overnight fast. Blood samples were collected from the retro-orbital venous plexus of
each rat and the blood glucose level was measured to confirm induction of diabetes using
´
BioMerieux kits (Trinder, 1969). Diabetic rats were divided into four groups. The first
group was kept untreated, and the second and third groups were given the methanol
ꢀ
1
extract of L. australis and compound 1 in oral doses of 100 and 0.1 mg kg
respectively, for 7 days. The last group received the reference drug (vitamin E, 7.5 mg kg
BW,
ꢀ
1
ꢀ1
BW in an oral dose of 100 mg kg BW for 7 days.). At the end of the experiment, blood
samples were obtained and blood GSH level was measured using GSH kit [Elman’s
reagent, 5, 5-dithio bis-(2-nitrobenzoic acid), to yield a stable yellow colour which can be
measured colorimetrically at 412 nm]. The intensity of the yellow colour developed is
related to the amount of GSH in blood. Results were presented as the mean ꢂ SEM.
Student’s t test was used to analyse statistical significance (Table 1).
3
.7. Determination of cytotoxic activity
Compound 1 as well as the methanol extract of L. australis were screened for cytotoxicity
using the method of Skehan (Skehan et al., 1990). Cells were plated in 96-multiwell plate
(
the wall of the plate. Different concentrations of the tested substance (0, 1, 2.5, 5 and
104 cells/well) for 24 h before treatment with the extract to allow attachment of the cell to
ꢀ
1
1
individual dose. Monolayer cells were incubated with compounds for 48 h at 37 C in an
0 mg mL ) were added to the cell monolayer. Triplicate wells were prepared for each
ꢁ

1
386
M.E.S. Kassem et al.
atmosphere of 5% CO . After 48 h, cells were fixed, washed and stained with
2
sulphorhodamine B stain. Excess stain was washed with acetic acid and the attached
stain recovered with Tris EDTA buffer. Colour intensity was measured in an ELISA
reader. The relation between surviving fraction and drug concentration was plotted to
obtain the survival curve of each tumour cell line as compared with doxorubicin (the
control anticancer drug).
4
. Conclusion
In this study, a new sulphated flavone glycoside 1, along with 14 known flavonoid
compounds, were characterised for the first time from L. australis leaves. Compound 1 and
the methanol extract of the plant leaves revealed a remarkable biological activity in
restoring the reduced GSH levels in diabetic rats, which could be attributed to their
antioxidant activity or their role in increasing the biosynthesis of GSH. Compound 1 also
exhibited a more potent cytotoxic activity against HEPG2, MCF7 and HCT116 than the
methanol extract of the plant leaves.
Acknowledgements
This study was financially supported by the Phytochemistry and Plant Systematics Department,
National Research Centre, Egypt. The authors are grateful to the Cancer Institute, Cairo University,
for carrying out the cytotoxicity screening.
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