Fitoterapia 83 (2012) 104–109

Contents lists available at SciVerse ScienceDirect

Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

Bioactive phenolics from the fruits of Livistona chinensis

Xiaobin Zeng a, Yihai Wang a, Qian Qiu b, Chenguang Jiang c, Yuntiao Jing c,
Guofu Qiu a, Xiangjiu He a,⁎
a
School of Pharmaceutical Sciences, Wuhan University, and Key Laboratory of combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Educa-
tion, Wuhan 430071, China
b
Department of Pharmacy Office, Renmin Hospital of Wuhan University, Wuhan 430060, China
c
Guangdong Dexin Pharmaceutical Company, Jiangmen 529100, China

a r t i c l e i n f o a b s t r a c t

Article history: This study investigated the antioxidant and cytotoxic activity of the phenolics isolated from the
Received 6 July 2011 fruits of Livistona chinensis. Four new compounds, 1-{ω-isoferul[6- (4-hydroxybutyl)pentadecanoic
Received in revised form 19 September 2011 acid]}-glycerol (1), E-[6′-(5″-hydroxypentyl)tricosyl]-4-hydroxy-3-methoxycinnamate (2), 2-(3′-
Accepted 29 September 2011 hydroxy-5′-methoxyphenyl)-3-hydroxylmethyl-7-methoxy-2,3-dihydrobenzofuran-5- carboxylic
Available online 8 October 2011
acid (3), 7-hydroxy-5,4′-dimethoxy-2-arylbenzofuran (4), together with eleven known phenolics
(5–15), were isolated and identified. Among these compounds, 1–4, 5-O-caffeoylshikimic acid
Keywords: (5), caffeic acid (7), and 3-O-caffeoylshikimic acid (8) showed potent antioxidant activity. 1–5,
Livistona chinensis and 8 showed potent antiproliferative activities with IC50 values among 5–150 μM against HepG2
1-(ω-isoferuloylalkanoyl)-glycerol
human liver cancer, HL-60 human myeloid leukemia, K562 human myeloid leukemia, and CNE-1
Long-chain trans-ferulate
human nasopharyngeal carcinoma cell lines. On the basis of these findings, it could be proposed
Benzofuran
Antioxidant activity that the fruits of L. chinensis may serve as attractive mines of powerful anticancer and antioxidant
Antiproliferative activity agents for various purposes.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction antiproliferative effects against human myeloid leukemia

As a symbol of tropical landscapes, the genus Livistona is
widely distributed in several ecosystems, including upland
hardwoods, flatwoods, and tropical hammocks over the trop-
ical zone. Its central bud has been served as a delicious food
eaten raw or cooked for swamp cabbage or heart of palm
salad. There are three species of this genus growing in
South China [1], and their fruits have traditionally been
used for analgesic and hemostatic purposes, and to treat na-
sopharyngeal carcinoma, choriocarcinoma, esophageal can-
cer, and leukemia [2–3]. It has been reported that the fruits
of Livistona chinensis showed anticancer, antibacterial and
anti-HIV-1 activities [4–5]. The fruits showed significantly
⁎ Corresponding author at: School of Pharmaceutical Sciences, Wuhan
University, Wuhan 430071 China. Tel.: + 86 27 6875 9923; fax: + 86 27
6875 9850.
E-mail address: hexiangjiu@whu.edu.cn (X. He).
cells (L1210, P388, HL-60), gastric cancer cells (SGC7901), cer-
vical cancer cells (HeLa), human liver cancer cells (HepG2,
Hele7404), melanoma cells (B16), colon cancer cells (HT-29),
and bladder cancer cells (T24) as well [6–11]. Earlier studies
on the chemical composition of L. chinensis fruits reported the
presence of some flavonoids, steroids, phenolics, fatty acids,
amino acids, and vitamins [12–17].
In our continuing search for natural bioactive agents from
high plants, the fruits of L. chinensis were fractionated and
purified. Fifteen phenolics, including four new compounds,
1-{ω-isoferul[6-(4-hydroxybutyl)pentadecanoic acid]}-glycerol
(1), E - [6′- (5″ - hydroxypentyl)tricosyl] - 4 - hydroxy - 3 -
methoxycinnamate (2), 2-(3′-hydroxy-5′-methoxyphenyl)-3 -
hydroxylmethyl - 7 - methoxy - 2,3 - dihydrobenzofuran - 5 -
carboxylic acid (3), 7-hydroxy-5,4′-dimethoxy-2-arylbenzofuran
(4), and eleven known (5–15), were isolated and identified
from the chloroform soluble fraction of a 70% EtOH extract.
Antiproliferative activities against four human tumor cell lines

0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2011.09.020
 X. Zeng et al. / Fitoterapia 83 (2012) 104–109 105

(HL-60, K562, HepG2, and CNE-1), and antioxidant assays for
scavenging ability of DPPH and superoxide anion free radicals
(O2−), were evaluated.
2. Experimental
2.1. General
Optical rotations were measured using a JASCO P-1030
automatic digital polarimeter (Tokyo, Japan). NMR spectra
were recorded on a Bruker DPX-400 spectrometer using stan-
dard Bruker pulse programs. Chemical shifts were shown as
δ-values with reference to tetramethylsilane (TMS) as an inter-
nal standard. GC–MS analysis was done using a Shimadzu GC-
14A unit coupled with a GCMS-QP 2000 instrument (Tokyo,
Japan). ESI-MS data were obtained on an Agilent 1200
HPLC/6410B TripleQuad mass spectrometer (Santa Clara, CA),
and HR-ESIMS were measured on a Bruker APEX II mass spec-
trometer (Bremen, Germany). Sephadex LH-20 (Pharmacia,
Sweden), silica gel (Qingdao Ocean Chemical Co., Ltd, Qingdao,
China), and ODS (40–63 μm, Merck, Darmstadt, Germany)
were used for column chromatography. TLC was carried out
on preparative silica gel 60 F254 and RP-18 F254 plates (Merck,
Darmstadt, Germany), and spots were visualized by spraying
the plates with 15% H2SO4, and heating them at 105 °C. Prepar-
ative HPLC was performed using an ODS column (XTerra®,
19× 250 mm, 10 μm, Waters, Milford, MA).
2.2. Plant material
combined with preparative HPLC, yielding compounds 5
(12 mg), 10 (20 mg), 11 (25 mg), and 12 (15 mg), respec-
tively. The CHCl3–MeOH (5:1) elution was subjected to an
octadecylsilanized silica gel (ODS) column, followed by a
preparative HPLC with 25% methanol (containing 0.1% CF3-
COOH, pH 3.0), yielding compound 3 (10 mg). The CHCl3–
MeOH (3:1) elution was subjected to an ODS column,
followed by a preparative Rp-HPLC with 22% methanol
(containing 0.1% CF3COOH, pH 3.0), yielding compound
13 (10 mg). The structures of compounds 1–15 are shown
in Fig. 1.
1-{ω-isoferul[6-(4-hydroxybutyl)pentadecanoic acid]}-glycerol
(1) was an amorphous powder; [α]25D +6.1 (c 0.20, CHCl3);
13
C NMR (CDCl3, 100 MHz) and 1H NMR (CDCl3, 400 MHz)
data, see Table 1; HRESIMS (positive ion mode) m/z [M+H]+
579.3939 (calcd. 579.3931).
E - [6′ - (5″ - hydroxypentyl)tricosyl] - 4 - hydroxy - 3 -
methoxycinnamate (2) was an amorphous powder; [α]25D-2.1
(c 0.20, CHCl3); 13C NMR (CDCl3, 100 MHz) and 1H NMR
(CDCl3, 400 MHz) data, see Table 1; HRESIMS (positive ion
mode) m/z [M+ H]+ 603.4996 (calcd. 603.4991).
2-(3′-hydroxy-5′-methoxyphenyl)-3-hydroxylmethyl-7-
methoxy-2,3-dihydrobenzofuran-5-carboxylic acid (3) was an
amorphous powder; [α] 25D + 8.2 (c 0.20, MeOH); 13C NMR
(DMSO-d6, 100 MHz) and 1H NMR (DMSO-d6, 400 MHz) data,
see Table 2. HRESIMS (positive ion mode) m/z [M+ Na]+
369.0940 (calcd. 369.0945).
7-Hydroxy-5,4′-dimethoxy-2-arylbenzofuran (4) was yel-
low needles; 13C NMR (CDCl3, 100 MHz) and 1H NMR (CDCl3,

The fresh fruits of L. chinensis (Jacq.) R.Br. were collected in

Jiangmen, Guangdong Province, China, in September 2008, and
were identified by Prof. Xiangjiu He of School of Pharmaceutical
Sciences at Wuhan University. A voucher specimen (no.
20090920) is available at School of Pharmaceutical Sciences,
Wuhan University in Wuhan (430071), China.

2.3. Extraction and isolation

The air-dried fruits of L. chinensis (20.0 kg) were refluxed

with 70% EtOH (150 L × 3). Evaporation of the organic solvent
under a vacuum at 55 °C yielded a crude extract (3.0 kg). The
concentrated brown syrup was resuspended in water and parti-
tioned with petroleum ether (3 L ×3), chloroform (3 L × 3),
ethyl acetate (3 L ×3) and water-saturated n-butanol (3 L ×3)
gradually to afford 90.21 g, 91.72 g, 55.05 g and 554.35 g of
dried organic extracts, respectively.
The CHCl3 fraction was fractionated over a silica gel (200–300
mesh) column by eluting gradually with MeOH in CHCl3. This
process yielded 19 fractions. The CHCl3–MeOH (50:1) elution
was separated on a silica gel column, using cyclohexane/ethyl
acetate (100:1→1:1) to yield compounds 8 (35 mg), 6
(15 mg), and 14 (20 mg), respectively. The CHCl3–MeOH
(30:1) elution was further purified by a silica gel column and
eluted with cyclohexane/ethyl acetate (100:1→1:1) to obtain
compounds 1 (50 mg) and 2 (65 mg), respectively. The CHCl3–
MeOH (20:1) elution was subjected to a silica gel column and
eluted with cyclohexane/ethyl acetate (100:1→1:1), yielding
compounds 4 (250 mg), 7 (350 mg), 9 (50 mg), and 15
(30 mg), respectively. The CHCl3–MeOH (10:1) elution was fur-
ther purified with silica gel column and Sephadex LH-20 column, Fig. 1. Chemical structures of compounds 1–15.
106 X. Zeng et al. / Fitoterapia 83 (2012) 104–109

Table 1 Table 3
1
H NMR and 13C NMR data of compounds 1 and 2 in CDCl3 (δ in ppm, J in Hz). 1
H NMR, 13C NMR, 1H–1H COSY and HMBC data of compound 4 in CDCl3 (δ in
ppm, J in Hz).
Position Proton Carbon
1 13 1
Position H-NMR C- H–1H HMBC
1 2 1 2
NMR COSY
1 126.9 127.0
2 156.9
2 7.04 (s, 1H) 7.04 (d, 2, 1H) 109.1 109.3
3 6.79 (s, 1H) 100.4 C(2), C(3a), C(4),
3 147.7 147.9
C(5), C(7a), C(1′)
4 146.5 146.8
4 6.60 (d, 2.4, 1H) 94.8 H(6) C(3), C(5), C(6),
5 6.92 (d, 8.0, 1H) 6.92 (d, 7.6, 1H) 115.5 115.7
C(7), C(7a)
6 7.08 (br d, 8.6, 1H) 7.08 (dd, 7.6, 2, 1H) 122.9 123.0
5 145.5
7 7.62 (d, 16.0, 1H) 7.61 (d, 16.4, 1H) 144.5 144.6
6 6.43 (d, 2.0, 1H) 97.1 H(4) C(4), C(5), C(7), C(7a)
8 6.30 (d, 16.4, 1H) 6.29 (d, 16.4, 1H) 114.5 114.6
7 157.0
9 167.3 167.4
3a 131.0
\OCH3 3.93 (s, 3H) 3.93 (s, 3H) 55.8 55.9
7a 139.4
1′ 4.18 (m, 2H) 4.19 (t, 6.8, 2H) 64.9 65.0
1′ 123.6
\CH 2.32 (m, 1H) 2.34 (m) 34.0 34.1
2′ 7.73(d, 8.4, 1H) 126.9 H(3′) C(3′), C(4′), C(5′), C(6′)
\CH2b)c) 2.32 (m, 2H) 2.34 (m) 32.8 32.8
d) 3′ 6.89(d, 8.4, 1H) 115.9 H(2′) C(1′), C(4′), C(5′)
4′ 156.3
\Ca) 3.65 (t, 6.8, 2H) 173.7 63.1
5′ 6.89(d, 8.4, 1H) 115.9 H(6′) C(1′), C(3′), C(4′)
\CH2 1.25–1.64 (m, 2H) 1.25–1.69 (m) 28.8– 28.8–
6′ 7.73(d, 8.4, 1H) 126.9 H(5′) C(2′), C(3′), C(4′), C(5′)
31.8 31.9
5-OCH3 4.00 (s, 3H) 55.3 C(5)
\CH3 0.88 (t, 6.4, 3H) 0.88 (t, 6.4, 3H) 14.0 14.1
4′-OCH3 3.85 (s, 3H) 55.1 C(4′)
1″ 4.18 (m, 2H) 65.0
2″ 4.18 (m, 1H) 68.4
3″ 3.64 (t, 6.8, 1H) 63.1 CHCl3 was to give a white solid. GC–MS was performed to elu-
4.05 (t, 6.8, 1H) cidate the structure of the white solid. Also, the H2O fraction
a–d) Carbon groups in the structure of compounds 1 and 2 (see Fig. 1). was neutralized with dilute HCl and extracted with Et2O. The
Et2O extract was identified as ferulic acid by NMR analysis.

400 MHz) data, see Table 3. HRESIMS (positive ion mode) m/z
[M+ H]+ 271.0967 (calcd. 271.0965).
2.4. Hydrolysis of compounds 1 and 2
The hydrolysis reaction was determined according to the
reported protocol [18]. Compound 1 or 2 (10 mg) was treated
with 5 mL 5% NaOH in EtOH. The mixture was refluxed for
10 h, and the solvent was removed by evaporation. The residue
was suspended in H2O and extracted with CHCl3. The dried
2.5. DPPH radical scavenging assay
The scavenging effects of the phenolics on DPPH radicals
were determined according to a previously reported method
[19]. DPPH (50 mg/L) was dissolved in MeOH. The samples
were dissolved in DMSO. The DPPH solution (995 μL) was
mixed with 5 μL of each of the samples. The mixture was shak-
en and allowed to stand at room temperature in the dark for
20 min. The absorbance of the resulting solution was measured
spectrophotometrically at 517 nm.

2.6. Superoxide anion free radical (O2−) scavenging assay

Table 2
1
H NMR, 13C NMR, 1H–1H COSY and HMBC data of compound 3 in DMSO-d6 Superoxide radical scavenging activity was assayed by the
(δ in ppm, J in Hz).
NBT reduction method according to a previously reported
Position 1
H-NMR 13
C- 1
H–1H HMBC protocol [20] with some modification. The 1000 μL reaction
NMR COSY mixture used for the O2− scavenging activity assay contained
2 5.56 (d, 6.6, 1H) 8.81 H(3) C(1′), C(4′), C(4), Tris–HCl (pH 8.1, 50 mM, 445 μL), NADH (0.15 mM, 250 μL),
C(7a), C(9) PMS (0.03 mM, 50 μL), NBT (0.10 mM, 250 μL) and sample
3 3.55 (brs, 1H) 52.2 H(2), H(9) C(1′) solution (5 μL). All samples were dissolved in Tris–HCl
4 7.55 (s, 1H) 119.1 H(6) C(3), C(6), C(7),
50 mM, pH 8.1. The reaction was conducted at 37 °C for 5 min,
C(7a), C(8)
5 123.7 and initiated by the addition of PMS. The absorbance of the
6 7.44 (brs, 1H) 113.2 H(4) C(4), C(5), C(7), resulting solution was measured spectrophotometrically at
C(7a), C(8) 560 nm.
7 143.3
8 167.1
9 3.70 (brs, 1H) 62.6 H(3)
2.7. Antiproliferative assay
3a 129.6
7a 151.6 The antiproliferative assay was performed on four human
1′ 131.6 tumor cell lines, namely human myeloid leukemia HL-60,
2′ 6.78 (s, 1H) 115.4 H(4) C(1′), C(3′), C(4′)
human myeloid leukemia K562, human liver cancer HepG2
3′ 147.6
4′ 6.93 (s, 1H) 110.5 H(2), H(6) C(2), C(5′), C(6′) and human nasopharyngeal carcinoma CNE-1, performed
5′ 146.6 according to a reported protocol [21]. All the cells were cultured
6′ 6.78 (s, 1H) 119.0 H(4) in RPMI-1640 medium (Hyclone, Logan, UT), and supplemented
3′-OCH3 3.83 (s, 3H) 55.6 C(3′) with 10% fetal bovine serum (Hyclone) and antibiotic
7-OCH3 3.75 (s, 3H) 55.6 C(7)
(100 units/mL penicillin and 100 μg/mL streptomycin) in 5%
 X. Zeng et al. / Fitoterapia 83 (2012) 104–109
CO2 at 37 °C. The cytotoxicity assay was performed according to
the MTT method in 96-well microplates. Briefly, 200 μL of ad-
herent cells was seeded into each well of 96-well cell culture
plates and allowed to adhere for 12 h before drug addition,
while suspended cells were seeded just before drug addition
with an initial density of 5 ×104 cells/mL. Each tumor cell line
was exposed to the test compound at concentrations of 50, 25,
12.5, 6.25, 3.125, and 1.56 μg/mL in quadruples for 48 h.
3. Results and discussion
3.1. Structure elucidation of new compound
Compound 1 was obtained as white amorphous powder.
The positive ESI-MS spectrum showed a molecular ion [M
+ H] + at m/z 579 and fragment ions m/z [glycerol + H] +
and [ferulate + H] + at m/z 93 and 195, respectively. In the
1
H NMR spectrum (Table 1), three signals at δH 6.92 (1H, d,
J = 8.0 Hz), 7.04 (1H, d, J = 2.4 Hz), and 7.08 (1H, dd, J = 8.0,
2.4 Hz) indicated the presence of a 1,3,4-trisubstituted ben-
zene. The signals at δH 6.30 (1H, d, J = 15.6 Hz) and 7.61
(1H, d, J = 15.6 Hz) showed a trans-double bond in the mole-
cule. The 1H NMR spectrum revealed the presence of a series
of methylene groups, assigned to a fatty acid (δH 0.88–2.37),
and seven strongly deshielded protons at 3.64 (1H, t,
J = 6.4 Hz), 4.05 (1H, t, J = 6.4 Hz) and 4.18 (5H, m). The 13C
NMR spectrum of 1 (Table 1) showed two carbonyls, eight ar-
omatic carbons, one \OCH3 group, three \CH2OH groups,
one \CHOH group and a series of signals at δC 14.1–34.1. A
comparison of the 1H and 13C NMR data of 1 with the
reported data [22] indicated that the structure of 1 is similar
to that of 1-(ω-isoferuloylalkanoyl)-glycerol. To deduce the
exact structure of the fatty acid, 1 was hydrolyzed in 5%
NaOH. After the usual workup, the nonpolar organic extract
was analyzed with GC/MS and NMR spectroscopy, and
the structure of the fatty acid was identified to be 6-(4-
hydroxybutyl)pentadecanoic acid. Therefore, compound 1
was elucidated to be 1-{ω-isoferul[6-(4-hydroxybutyl)
pentadecanoic acid]}-glycerol, which was a new compound.
Compound 2 was obtained as a white amorphous powder.
The negative ESI-MS spectrum showed a molecular ion [M–
H]− at m/z 601, and a fragment ion [ferulate]− at m/z 193. In
the 1H NMR spectrum (Table 1), three signals at δH 6.92 (1H,
d, J = 8.0 Hz ), 7.04 ( 1H, d, J = 2.4 Hz), and 7.08 (1H, dd,
J = 7.6, 2.0 Hz), two signals at δH 6.30 (1H, d, J = 15.6 Hz) and
7.61 (1H, d, J = 15.6 Hz) and one signal at δH 3.93 (3H, s) indi-
cated the presence of a ferulic acid moiety. The 1H NMR also
revealed a series of methylene groups assigned to a long
chain hydrocarbon (δH 0.88–1.69). Furthermore, four strongly
deshielded protons at 3.64 (2H, t, J = 6.8 Hz) and 4.19 (2H, t,
J = 6.8 Hz) revealed two methylene groups attached to oxygen.
The 13C NMR spectrum of 2 (Table 1) showed the presence of a
carbonyl carbon, eight aromatic carbons, a \OCH3 group, two
\CH2OH groups and a series of signals at δC 14.1–34.1. In
order to determine the lengths of the long chain hydrocarbon
and the position of the methyne, 2 was hydrolyzed in 5%
NaOH. A series of long chain hydrocarbons and ferulic acid
was obtained from the hydrolysis. The long chain hydrocarbons
were identified as 6-octadecylundecane-1,11-diol by means of
GC/MS and NMR analysis. Thus, compound 2 was elucidated to
107
be E-[6′-(5″-hydroxypentyl)tricosyl]-4-hydroxy-3-methoxy-
cinnamate, which was a new compound.
Compound 3 was obtained as amorphous powder. Posi-
tive electrospray ionization mass spectrometry (ESI-MS) pro-
duced the ion [M + Na] + at m/z 369, indicating a molecular
mass of 346, which is compatible with the molecular formula
C18H18O7. Its molecular formula was confirmed by HR-ESIMS,
which showed the ion [M + Na] + at m/z 369.0940 (calcd.
369.0950). The 1H NMR, 13C NMR, DEPT and HMQC data for
3 (Table 2) indicated the presence of a 1,3,5-trisubstituted
benzene moiety [δH 6.78 (2H, s, H-2′, 6′), 6.93 (1H, s, H-4′)],
a 1,2,3,5-tetrasubstituted benzene ring [δH 7.55 (1H, s, H-2),
7.44 (1H, brs, H-6)], two methoxy groups [δH 3.83 (3H, s,
3′-OCH3), 3.75 (3H, s, 7-OCH3)], one oxymethine [δH 5.56
(1H, d, J = 6.6 Hz, H-2)], one oxymethylene [δH 3.70 (1H,
brs, H-9)] and one methene [δH 3.55 (1H, brs, H-3)]. From
the 1H– 1H COSY spectrum, a partial structure (\OCHCHCH2-
OH) was feasibly deduced. From above, it was deduced that 3
contains a benzofuran fragment. In the HMBC spectrum, the
correlations of H-2 (δH 5.56) to C-9 (δC 62.6), C-4′ (δC
110.5), C-2′ (δC 115.4), C-1′ (δC 131.6) and C-7a (δC 151.6),
H-3 (δH 3.55) to C-3a (δC 129.6), 3′-OCH3 (δH 3.83) to C-3′
(δC 147.6), 7-OCH3 (δH 3.75) to C-7 (δC 143.3), and \COOH
(δC 167.1) to H-4 (δH 7.55) and H-6 (δH 7.44) suggested 3
could be 2-(3′-hydroxy-5′-methoxyphenyl)-3-hydroxylmethyl-
7-methoxy-2,3-dihydrobenzofuran-5-carboxylic acid. Since
the coupling constant of H-2 was 6.6 Hz, the relative config-
uration of C-2 and C-3 was determined to be trans [23]. This
configuration was opposite to that of the compound isolated
from Tarenna attenuate [24]. The optical rotation value of 3
([α] 20D + 8.2 (c 0.20, MeOH)), which was different from
that of balanophonin [23], suggested the absolute configura-
tions of C-2 and C-3 to be 2S and 3R by comparing with the
literature [25], respectively. Therefore, the structure of com-
pound 3 was established as 2-(3′-hydroxy-5′-methoxyphenyl)-
3 - hydroxylmethyl - 7 - methoxy - 2,3 -dihydrobenzofuran-5-
carboxylic acid, which was a new compound.
Compound 4 was obtained as yellow needles, and the ESI-
MS spectrum showed an ion [M + H] + at m/z 271, consisted
with the molecular formula C16H14O4. The 1H and 1H- 1H
COSY spectrum (Table 3) indicated the presence of one AA′
BB′ spin system comprised of protons resonating at δ 6.89
(2H, d, J = 8.4 Hz) and δ 7.73 (2H, d, J = 8.4 Hz). Additionally,
the 1H NMR and 1H– 1H COSY spectrum of 4 displayed a
1,2,3,5-tetrasubstituted benzene unit [δH 6.60 (1H, d,
J = 2.0 Hz), and δH 6.43 (1H, d, J = 2.0 Hz)]. The 13C NMR
spectrum (Table 3) included 14 nonequivalent carbon sig-
nals, implying the presence of a 6-2-6 system indicative of
an arylbenzofuran skeleton. This was further confirmed by
the 1H NMR spectrum, which showed a doublet at δ 6.79
(1H, s) assignable to H-3. These data suggested that 4 was
an isomeride of livistone C, a compound isolated previously
from the fruits of L. chinensis [15]. The NMR spectra data of
4 and livistone C were very similar, with the exception of
one methoxy signal. On the basis of HMBC correlations
(Table 3), the singlet signals at δ 3.85 (3H, s) and δ 4.00
(3H, s), which typically represent methoxy protons, showed
correlations with C-4′ (δ 156.3) and C-5 (δ 145.5), thus con-
firming the assignment of the methoxy groups at C-4′ and
C-5, respectively. From the data above, compound 4 was
identified as 7-hydroxy-5,4′-dimethoxy-2-arylbenzofuran.
108 X. Zeng et al. / Fitoterapia 83 (2012) 104–109

The known compounds were identified, by comparing their Table 5

spectroscopic data with the data previously reported, as 5-O- Antiproliferative activities of the phenolics (1–9) against HL-60, Mata,
HepG2 and CNE-1 cell lines.
caffeoylshikimic acid (5) [26], (E)-3-(4-hydroxyphenyl)acrylic
acid (6) [27], caffeic acid (7) [28], 3-O-caffeoylshikimic Compound IC50 (μM)
acid (8) [29], 6,7-dihydroxy-2H-chromen-2-one (9) [30],
HL-60 Mata HepG2 CNE-1
4-hydroxybenzaldehyde (10), 3-hydroxy-4-methoxybenzoic
1 26.06 ± 2.09 33.68 ± 3.22 45.96 ± 4.18 37.73 ± 2.35
acid (11), 4 - hydroxybenzoic acid (12), 4 - hydroxy - 3 -
2 28.44 ± 1.96 40.58 ± 3.72 43.16 ± 3.23 40.53 ± 3.35
methoxybenzoic acid (13), 3-hydroxy-4-methoxybenzaldehyde 3 73.20 ± 4.19 82.68 ± 3.12 62.76 ± 6.13 79.73 ± 6.25
(14) and 3,4-dihydroxybenzoic acid (15), respectively. 4 7.63 ± 0.49 10.68 ± 0.82 9.76 ± 0.73 7.73 ± 0.65
5 121.91 ± 133.96 ± 109.54 ± 92.3 ± 7.93
3.2. Antioxidant and antiproliferative activity of the phenolics 10.07 12.16 2.04
6 N150 N 150 N 150 N150
7 N150 N 150 N 150 N150
The DPPH scavenging and superoxide anion free radical 8 126.24 ± 135.79 ± N 150 N150
(O2−) scavenging abilities of the phenolics (1–9) isolated 11.55 13.34
from L. chinensis were shown in Table 4. 1 showed the high- 9 N150 N 150 N 150 113.73
± 9.78
est antioxidant activity based on the results of these two
Cisplatin 12.60 ± 0.89 15.70 ± 0.63 15.50 ± 1.21 14.54 ± 1.06
antioxidant screening models, followed by 5. Table 4 demon-
strates that 1, 2, 5 and 8 had higher antioxidant capacity than
that of caffeic acid (7) or (E)-3-(4-hydroxyphenyl)acrylic 2 and HL-60, which showed an antiproliferative efficacy of
acid (6) on an equivalent molar basis. Furthermore, the ester- 67.3% and 71.5% at the highest concentration of 100 μg/mL, re-
ification that occurred at position 6 of shikimic acid was more spectively. From the dose–response curve of antiproliferative
effective in boosting antioxidant activity than the esterifica- activity against HL-60 cells among 1, 2 and 4 (Fig. 2), 1 and 2
tion that occurred at position 4 among two caffeoylshikimic showed higher dose–response efficacy than 4. Maybe, 1 and 2,
acids. whose structures have both hydrophilic and hydrophobic
The antiproliferative activities of the phenolics against four groups, are beneficial to permeate that the complex cell system
human tumor cell lines were shown in Table 5. 4 exhibited sig- of the molecules.
nificant inhibition of proliferation against the four cell lines, and
showed stronger inhibition of the proliferation of these selected 4. Conclusions
cell lines than cisplatin. It should be noted that the analogs of 3
and 4 have been reported to exhibit significant cytotoxic activ- In current research, the phytochemical analysis of the 70%
ity against p-388, HB16, A549 and HT-29 cells [31–32]. 1 and 2 EtOH extract of the fruits of L. chinensis led to the isolation of
exhibited significant effects on these cancer cell lines. This find- fifteen phenolics, four of them were new compounds, and ten
ing was in accordance with the former research that ferulate were isolated for the first time from the genus Livistona. Sev-
and caffeic alkyl esters demonstrate antitumor proliferation ac- eral constituents of L. chinensis fruits have been shown to
tivity with colon breast, lung, and gastric human cell lines [33]. possess strong antioxidant and antiproliferative activities.
Moreover, the results showed that inhibitory activity increased Depending on the method used, 1, 2 and 4 proved to be effec-
greatly when caffeic acid was esterified with shikimic acid. 4 tive radical scavengers and cancer preventing agents in vitro.
seemed to cause complete inhibition of the viability four target Free radical induced oxidative stress has been hypothesized
cell lines, resulting in an antiproliferative efficacy of about 90% to be a major factor in the development of several degenerative
at the concentration 12.5 μg/mL. Also, 1 showed complete anti- chronic diseases. Oxidative stress can cause oxidative damage
proliferation of HL-60 at the highest concentration tested with
the antiproliferative efficacy of 75.2%. Good reducing capacity
for the tetrazolium salt was also observed for 2 against HepG
100
Cell Proliferation Inhibition (%)

Table 4 80
Antioxidant activities of the isolated compounds (1–9).

Compound IC50 (μM)

60
DPPH radical Superoxide anion
free radical (O2−)
Compound 1
1 2.26 ± 0.21 1.91 ± 0.13 Compound 2
2 2.72 ± 0.22 2.50 ± 0.23 40 Compound 4
3 4.90 ± 0.32 4.50 ± 0.31
4 4.30 ± 0.42 3.50 ± 0.33
5 2.60 ± 0.17 2.40 ± 0.09
6 7.54 ± 0.67 6.53 ± 0.49 20
0 10 20 30 40 50 60
7 3.76 ± 0.23 3.51 ± 0.25
Concentration (µg/ml )
8 2.87 ± 0.16 2.54 ± 0.11
9 6.55 ± 0.45 5.32 ± 0.33
Fig. 2. Dose–response curve of antiproliferative activity against HL-60 of
Quercetin 5.40 ± 0.30 4.60 ± 0.20
compounds 1, 2 and 4 (mean ± SD, n = 3).
 X. Zeng et al. / Fitoterapia 83 (2012) 104–109
to large biomolecules, such as lipids, proteins, and DNA, result-
ing in an increased risk for inflammatory diseases, cardiovascu-
lar disease, cancer, diabetes, Alzheimer's disease, cataracts, and
age-related functional decline [34–35]. The DPPH radical assay
was known to give reliable information concerning the antiox-
idant ability of the tested compounds and extracts. At the same
time, the superoxide anion free radical assay has been widely
used to evaluate the free radical scavenging ability. And this
radical was generated by a chemical system composed of
PMS, NADH and oxygen. This assay was known to give reliable
information concerning the antioxidant ability of the tested
compounds. The phenolics isolated from the fruits of cabbage
palm showed potential free radical and superoxide anion free
radical (O2−) scavenging activities.
These results indicated the fruits of L. chinensis could be
served as cancer preventing agents, which were in accordance
with their traditional uses. It can serve as potent anticancer and
antioxidant agents. The mechanisms of those compounds in
cancer prevention are worthy of further investigation.
Acknowledgments
This research was partly supported by the Start Fund of
Wuhan University. The HR-ESIMS and optical rotations were
measured by Dr. Jinshan Tang, Institute of Traditional Chinese
Medicine & Natural Products, Jinan University, China. The ESI-
MS were kindly measured by Professor Naili Wang, Shenzhen
Research Center of Traditional Chinese medicine & Natural
Products. Special thanks were given to Miss Lillian Nordahl,
Department of Pharmacology at University of Cambridge, for
her kind help in manuscript preparation.
References
[1] Pei SJ, Cheng SY, Tong SJ. Flora of China. Beijing: Science Press; 1991.
p. 25.
[2] Healthy Ministry of Guangzhou Force Logistics. Common Chinese Herbal
Medicine Handbook. Beijing: People's Health Publishing House; 1969.
p. 772–3.
109
[3] Zhao GP, Dai S, Chen ES. Dictionary of Traditional Chinese Medicine.
Shanghai: Shanghai Science and Technology Press; 2001. p. 2459–60.
[4] Kaur G, Singh RP. Food Chem Toxicol 2008;46:2429–34.
[5] Li CY, Zeng YB, Peng F, Dai HF, Zheng YT. Chin Trad Herb Drugs 2008;39:
1833–8.
[6] Wang H, Li A, Dong XP, Xu XY. J Chin Med Mat 2008;31:718–22.
[7] Chen Y, Lin XH, Li SG, Weng SM, Yao H. J Fujian Med Univ 2008;42:
93–5.
[8] Zhu YL, Chen SL, Peng LL, Tang L. Chem Bioeng 2007;24:35–7.
[9] Sartippour MR, Liu CH, Shao ZM, Go VL, Herber D, Nguyen M. Oncol Rep
2001;8:1355–7.
[10] Cheung S, Tai J. Oncol Rep 2005;14:1331–6.
[11] Huang WC, Hsu RM, Chi LM, Leu YL, Chang YS, Yu JS. Cancer Lett
2007;248:137–46.
[12] Chen P, Yang JS. Chin Trad Herb Drugs 2007;8:665–7.
[13] Chen P, Yang JS. Chin Pharm J 2008;43:1669–70.
[14] Liu ZP, Cui JG, Liu HX. Cereal Oil Proc 2009;2:43–4.
[15] Tao Y, Yang SP, Zhang HY, Liao SG, Wei W, Yan W, et al. J Asian Nat Prod
Res 2009;11:243–9.
[16] Mala V, Dahot MU. Sci Int (Lahore) 1994;6:231–5.
[17] Zeng XB, Qiu Q, Jiang CG, Jing YT, Qiu GF, He XJ. Fitoterpia 2011;82:
609–14.
[18] Boonyaratavej S, Tantayanontha S, Kitchanachai P, Chaichantipyuth C,
Chittawong V, Miles DH. J Nat Prod 1992;55:1761–3.
[19] Yen GC, Chen HY. J Agric Food Chem 1995;43:27–32.
[20] Valentao P, Fernandes E, Carvalho F, Andrade PB, Seabra RM, Bastos
MD. Biol Pharm Bull 2002;25:1324–7.
[21] He XJ, Liu RH. J Agric Food Chem 2007;55:4366–70.
[22] Kawanishi K, Hashimoto Y. Phytochemistry 1987;26:749–52.
[23] Sy LK, Brown GD. Phytochemistry 1999;50:781–5.
[24] Yang XW, Zhao PJ, Ma YL, Xiao HT, Zuo YQ, He HP, et al. J Nat Prod
2007;70:521–5.
[25] Zhao XH, Chen DH, Si JY, Pan RL, Shen LG. Acta Pharmacol Sin 2002;37:
535–8.
[26] Veit M, Weidner C, Strack D, Wray V, Witte L, Czygan FC. Phytochemistry
1992;31:3483–5.
[27] Chiang YM, Liu HK, Lo JM, Chien SC, Chan YF, Lee TH, et al. J Chin Chem
Soc 2003;50:161–6.
[28] Deyama T, Ikawa T, Kitagawa S, Nishibe S. Chem Pharm Bull 1987;35:
1785–9.
[29] Ozawa T, Imagawa H. Agric Biol Chem 1988;52:595–7.
[30] Cussans NJ, Huckerby TN. Tetrahedron 1975;31:2719–26.
[31] Tsai IL, Hsieh CF, Duh CY, Chen IS. Phytochemistry 1996;43:1261–3.
[32] Sandro M, Lauro ESB, Young GS, Patrick FJ, Chai HB, Eun JP, et al.
Phytochemistry 2002;59:635–41.
[33] Jayaprakasam B, Vanisree M, Zhang Y, Dewitt DL, Nair MG. J Agric Food
Chem 2006;54:5375–81.
[34] Ames BN, Gold LS. Mutat Res 1991;250:3–16.
[35] Ames BN, Shigenaga MK, Gold LS. Environ Health Perspect 1993;101:
35–44.