Bioactive phenolics from the fruits of Livistona chinensis

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 Education, Wuhan 430071, China Department of Pharmacy Office, Renmin Hospital of Wuhan University, Wuhan 430060, China c Guangdong Dexin Pharmaceutical Company, Jiangmen 529100, China b

a r t i c l e

i n f o

Article history: Received 6 July 2011 Received in revised form 19 September 2011 Accepted 29 September 2011 Available online 8 October 2011 Keywords: Livistona chinensis 1-(ω-isoferuloylalkanoyl)-glycerol Long-chain trans-ferulate Benzofuran Antioxidant activity Antiproliferative activity

a b s t r a c t This study investigated the antioxidant and cytotoxic activity of the phenolics isolated from the fruits of Livistona chinensis. 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), together with eleven known phenolics (5–15), were isolated and identified. Among these compounds, 1–4, 5-O-caffeoylshikimic acid (5), caffeic acid (7), and 3-O-caffeoylshikimic acid (8) showed potent antioxidant activity. 1–5, and 8 showed potent antiproliferative activities with IC50 values among 5–150 μM against HepG2 human liver cancer, HL-60 human myeloid leukemia, K562 human myeloid leukemia, and CNE-1 human nasopharyngeal carcinoma cell lines. On the basis of these findings, it could be proposed that the fruits of L. chinensis may serve as attractive mines of powerful anticancer and antioxidant agents for various purposes. © 2011 Elsevier B.V. All rights reserved.

1. Introduction As a symbol of tropical landscapes, the genus Livistona is widely distributed in several ecosystems, including upland hardwoods, flatwoods, and tropical hammocks over the tropical 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 nasopharyngeal carcinoma, choriocarcinoma, esophageal cancer, 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: [email protected] (X. He). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.09.020

antiproliferative effects against human myeloid leukemia cells (L1210, P388, HL-60), gastric cancer cells (SGC7901), cervical 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

X. Zeng et al. / Fitoterapia 83 (2012) 104–109

(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 standard Bruker pulse programs. Chemical shifts were shown as δ-values with reference to tetramethylsilane (TMS) as an internal standard. GC–MS analysis was done using a Shimadzu GC14A 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 spectrometer (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. Preparative 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), respectively. 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% CF3COOH, 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-7methoxy-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 yellow 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 partitioned 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 further purified with silica gel column and Sephadex LH-20 column,

105

Fig. 1. Chemical structures of compounds 1–15.

106

X. Zeng et al. / Fitoterapia 83 (2012) 104–109

Table 1 1 H NMR and 13C NMR data of compounds 1 and 2 in CDCl3 (δ in ppm, J in Hz). Position

Proton 1

1 2 3 4 5 6 7 8 9 \OCH3 1′ \CH \CH2b)c)

Carbon 2

1

2

126.9 109.1 147.7 146.5 6.92 (d, 8.0, 1H) 6.92 (d, 7.6, 1H) 115.5 7.08 (br d, 8.6, 1H) 7.08 (dd, 7.6, 2, 1H) 122.9 7.62 (d, 16.0, 1H) 7.61 (d, 16.4, 1H) 144.5 6.30 (d, 16.4, 1H) 6.29 (d, 16.4, 1H) 114.5 167.3 3.93 (s, 3H) 3.93 (s, 3H) 55.8 4.18 (m, 2H) 4.19 (t, 6.8, 2H) 64.9 2.32 (m, 1H) 2.34 (m) 34.0 2.32 (m, 2H) 2.34 (m) 32.8

127.0 109.3 147.9 146.8 115.7 123.0 144.6 114.6 167.4 55.9 65.0 34.1 32.8

\Ca) \CH2

3.65 (t, 6.8, 2H) 1.25–1.64 (m, 2H) 1.25–1.69 (m)

\CH3 1″ 2″ 3″

0.88 4.18 4.18 3.64 4.05

63.1 28.8– 31.9 14.1

7.04 (s, 1H)

7.04 (d, 2, 1H)

d)

(t, 6.4, 3H) (m, 2H) (m, 1H) (t, 6.8, 1H) (t, 6.8, 1H)

0.88 (t, 6.4, 3H)

173.7 28.8– 31.8 14.0 65.0 68.4 63.1

a–d) Carbon groups in the structure of compounds 1 and 2 (see Fig. 1).

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

Table 3 1 H NMR, 13C NMR, 1H–1H COSY and HMBC data of compound 4 in CDCl3 (δ in ppm, J in Hz). Position

1

H-NMR

2 3

6.79 (s, 1H)

4

6.60 (d, 2.4, 1H)

5 6 7 3a 7a 1′ 2′ 3′ 4′ 5′ 6′ 5-OCH3 4′-OCH3

6.43 (d, 2.0, 1H)

7.73(d, 8.4, 1H) 6.89(d, 8.4, 1H) 6.89(d, 8.4, 1H) 7.73(d, 8.4, 1H) 4.00 (s, 3H) 3.85 (s, 3H)

13 CNMR

1 H–1H COSY

156.9 100.4 94.8 145.5 97.1 157.0 131.0 139.4 123.6 126.9 115.9 156.3 115.9 126.9 55.3 55.1

H(6)

HMBC

C(2), C(5), C(3), C(7),

C(3a), C(4), C(7a), C(1′) C(5), C(6), C(7a)

H(4)

C(4), C(5), C(7), C(7a)

H(3′) H(2′)

C(3′), C(4′), C(5′), C(6′) C(1′), C(4′), C(5′)

H(6′) H(5′)

C(1′), C(3′), C(4′) C(2′), C(3′), C(4′), C(5′) C(5) C(4′)

CHCl3 was to give a white solid. GC–MS was performed to elucidate the structure of the white solid. Also, the H2O fraction was neutralized with dilute HCl and extracted with Et2O. The Et2O extract was identified as ferulic acid by NMR analysis. 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 shaken 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 (δ in ppm, J in Hz). Position

1

2

5.56 (d, 6.6, 1H)

3 4

3.55 (brs, 1H) 7.55 (s, 1H)

52.2 119.1

H(2), H(9) H(6)

5 6

7.44 (brs, 1H)

123.7 113.2

H(4)

7 8 9 3a 7a 1′ 2′ 3′ 4′ 5′ 6′ 3′-OCH3 7-OCH3

H-NMR

3.70 (brs, 1H)

6.78 (s, 1H) 6.93 (s, 1H) 6.78 (s, 1H) 3.83 (s, 3H) 3.75 (s, 3H)

13 CNMR

8.81

143.3 167.1 62.6 129.6 151.6 131.6 115.4 147.6 110.5 146.6 119.0 55.6 55.6

1 H–1H COSY

HMBC

H(3)

C(1′), C(4′), C(4), C(7a), C(9) C(1′) C(3), C(6), C(7), C(7a), C(8) C(4), C(5), C(7), C(7a), C(8)

Superoxide radical scavenging activity was assayed by the NBT reduction method according to a previously reported protocol [20] with some modification. The 1000 μL reaction mixture used for the O2− scavenging activity assay contained Tris–HCl (pH 8.1, 50 mM, 445 μL), NADH (0.15 mM, 250 μL), PMS (0.03 mM, 50 μL), NBT (0.10 mM, 250 μL) and sample solution (5 μL). All samples were dissolved in Tris–HCl 50 mM, pH 8.1. The reaction was conducted at 37 °C for 5 min, and initiated by the addition of PMS. The absorbance of the resulting solution was measured spectrophotometrically at 560 nm. 2.7. Antiproliferative assay

H(3)

H(4)

C(1′), C(3′), C(4′)

H(2), H(6)

C(2), C(5′), C(6′)

H(4) C(3′) C(7)

The antiproliferative assay was performed on four human tumor cell lines, namely human myeloid leukemia HL-60, human myeloid leukemia K562, human liver cancer HepG2 and human nasopharyngeal carcinoma CNE-1, performed according to a reported protocol [21]. All the cells were cultured in RPMI-1640 medium (Hyclone, Logan, UT), and supplemented with 10% fetal bovine serum (Hyclone) and antibiotic (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 adherent 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 benzene. 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 molecule. 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 aromatic 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-(4hydroxybutyl)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) indicated 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-methoxycinnamate, which was a new compound. Compound 3 was obtained as amorphous powder. Positive electrospray ionization mass spectrometry (ESI-MS) produced 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 (\OCHCHCH2OH) 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-hydroxylmethyl7-methoxy-2,3-dihydrobenzofuran-5-carboxylic acid. Since the coupling constant of H-2 was 6.6 Hz, the relative configuration 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 configurations of C-2 and C-3 to be 2S and 3R by comparing with the literature [25], respectively. Therefore, the structure of compound 3 was established as 2-(3′-hydroxy-5′-methoxyphenyl)3 - hydroxylmethyl - 7 - methoxy - 2,3 -dihydrobenzofuran-5carboxylic acid, which was a new compound. Compound 4 was obtained as yellow needles, and the ESIMS 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 signals, 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 confirming 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 spectroscopic data with the data previously reported, as 5-Ocaffeoylshikimic acid (5) [26], (E)-3-(4-hydroxyphenyl)acrylic acid (6) [27], caffeic acid (7) [28], 3-O-caffeoylshikimic acid (8) [29], 6,7-dihydroxy-2H-chromen-2-one (9) [30], 4-hydroxybenzaldehyde (10), 3-hydroxy-4-methoxybenzoic acid (11), 4 - hydroxybenzoic acid (12), 4 - hydroxy - 3 methoxybenzoic acid (13), 3-hydroxy-4-methoxybenzaldehyde (14) and 3,4-dihydroxybenzoic acid (15), respectively. 3.2. Antioxidant and antiproliferative activity of the phenolics The DPPH scavenging and superoxide anion free radical (O2−) scavenging abilities of the phenolics (1–9) isolated from L. chinensis were shown in Table 4. 1 showed the highest antioxidant activity based on the results of these two antioxidant screening models, followed by 5. Table 4 demonstrates that 1, 2, 5 and 8 had higher antioxidant capacity than that of caffeic acid (7) or (E)-3-(4-hydroxyphenyl)acrylic acid (6) on an equivalent molar basis. Furthermore, the esterification that occurred at position 6 of shikimic acid was more effective in boosting antioxidant activity than the esterification that occurred at position 4 among two caffeoylshikimic acids. The antiproliferative activities of the phenolics against four human tumor cell lines were shown in Table 5. 4 exhibited significant inhibition of proliferation against the four cell lines, and showed stronger inhibition of the proliferation of these selected cell lines than cisplatin. It should be noted that the analogs of 3 and 4 have been reported to exhibit significant cytotoxic activity against p-388, HB16, A549 and HT-29 cells [31–32]. 1 and 2 exhibited significant effects on these cancer cell lines. This finding was in accordance with the former research that ferulate and caffeic alkyl esters demonstrate antitumor proliferation activity with colon breast, lung, and gastric human cell lines [33]. Moreover, the results showed that inhibitory activity increased greatly when caffeic acid was esterified with shikimic acid. 4 seemed to cause complete inhibition of the viability four target cell lines, resulting in an antiproliferative efficacy of about 90% at the concentration 12.5 μg/mL. Also, 1 showed complete antiproliferation 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

Table 5 Antiproliferative activities of the phenolics (1–9) against HL-60, Mata, HepG2 and CNE-1 cell lines. Compound IC50 (μM) HL-60

Mata

HepG2

CNE-1

33.68 ± 3.22 40.58 ± 3.72 82.68 ± 3.12 10.68 ± 0.82 133.96 ± 12.16 N 150 N 150 135.79 ± 13.34 N 150

45.96 ± 4.18 43.16 ± 3.23 62.76 ± 6.13 9.76 ± 0.73 109.54 ± 2.04 N 150 N 150 N 150

37.73 ± 2.35 40.53 ± 3.35 79.73 ± 6.25 7.73 ± 0.65 92.3 ± 7.93

9

26.06 ± 2.09 28.44 ± 1.96 73.20 ± 4.19 7.63 ± 0.49 121.91 ± 10.07 N150 N150 126.24 ± 11.55 N150

N 150

Cisplatin

12.60 ± 0.89

15.70 ± 0.63

15.50 ± 1.21

113.73 ± 9.78 14.54 ± 1.06

1 2 3 4 5 6 7 8

N150 N150 N150

2 and HL-60, which showed an antiproliferative efficacy of 67.3% and 71.5% at the highest concentration of 100 μg/mL, respectively. From the dose–response curve of antiproliferative activity against HL-60 cells among 1, 2 and 4 (Fig. 2), 1 and 2 showed higher dose–response efficacy than 4. Maybe, 1 and 2, whose structures have both hydrophilic and hydrophobic groups, are beneficial to permeate that the complex cell system of the molecules. 4. Conclusions In current research, the phytochemical analysis of the 70% EtOH extract of the fruits of L. chinensis led to the isolation of fifteen phenolics, four of them were new compounds, and ten were isolated for the first time from the genus Livistona. Several constituents of L. chinensis fruits have been shown to possess strong antioxidant and antiproliferative activities. Depending on the method used, 1, 2 and 4 proved to be effective radical scavengers and cancer preventing agents in vitro. Free radical induced oxidative stress has been hypothesized to be a major factor in the development of several degenerative chronic diseases. Oxidative stress can cause oxidative damage

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

1 2 3 4 5 6 7 8 9 Quercetin

IC50 (μM) DPPH radical

Superoxide anion free radical (O2−)

2.26 ± 0.21 2.72 ± 0.22 4.90 ± 0.32 4.30 ± 0.42 2.60 ± 0.17 7.54 ± 0.67 3.76 ± 0.23 2.87 ± 0.16 6.55 ± 0.45 5.40 ± 0.30

1.91 ± 0.13 2.50 ± 0.23 4.50 ± 0.31 3.50 ± 0.33 2.40 ± 0.09 6.53 ± 0.49 3.51 ± 0.25 2.54 ± 0.11 5.32 ± 0.33 4.60 ± 0.20

Cell Proliferation Inhibition (%)

100

80

60

Compound 1 Compound 2 Compound 4

40

20 0

10

20

30

40

50

60

Concentration (µg/ml ) Fig. 2. Dose–response curve of antiproliferative activity against HL-60 of 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, resulting in an increased risk for inflammatory diseases, cardiovascular 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 antioxidant 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 ESIMS 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.