Illicaborins A–C, three prenylated C6–C3 compounds from the fruits of Illicium arborescens

Food Chemistry 123 (2010) 1105–1111

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Illicaborins A–C, three prenylated C6–C3 compounds from the fruits of Illicium arborescens Ya-Xu Lin a, Ahmed Eid Fazary a, Shun-Ying Chen b, Ching-Te Chien b, Yao-Haur Kuo c, Shiow-Yunn Sheu d, Ya-Ching Shen a,* a

School of Pharmacy, College of Medicine, National Taiwan University, Jen-Ai Rd., Sec. 1, Taipei 100, Taiwan, ROC Division of Silviculture, Taiwan Forestry Research Institute, Taipei, Taiwan, ROC National Research Institute of Chinese Medicine, Taipei, Taiwan, ROC d School of Pharmacy, Taipei Medical University, Taipei, Taiwan, ROC b c

a r t i c l e

i n f o

Article history: Received 11 January 2010 Received in revised form 6 April 2010 Accepted 13 May 2010

Keywords: Illicium arborescens Illicaborins Cytotoxic activities

a b s t r a c t Three new prenylated C6–C3 compounds, illicaborins A–C (1–3), were isolated and characterised from the fruits of Illicium arborescens Hayata. Their structures were determined by extensive spectroscopic analyses (UV, CD, IR, 1H NMR, 13C NMR, 1H–1H COSY, HMQC, HMBC, and NOESY) including molecular modelling. Compound 1 possesses a new carbon skeleton having a prenylated C6–C3 skeleton with an additional CH2OH group at the C-2 position. The cytotoxic activities of compounds 1–3 were tested and evaluated against Hep-2, Daoy, MCF-7 and WiDr tumour cell lines. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Illicium is a genus of flowering plants containing 42 species of evergreen shrubs and small trees, and is the sole genus in family Illiciaceae (Zomlefer, 1994). Plants of the genus are native to the tropical and subtropical regions of eastern and southeastern Asia, southeastern North America, and the West Indies. The fruits of Illicium verum, known as Chinese star anise, were used to treat infant colic, and as a seasoning in Chinese and south east-Asian cooking. Japanese star anise (Illicium religiosum), which contains shikimic acid, neurotoxin anisatin and neoanisatin, was used to produce incense (Aronson, 2006a). The fruits of some Illicium plants are locally taken to dispel wind and cold and relieve pain (treating rheumatism, and skin inflammation) in traditional Chinese medicine (Aronson et al., 2004). Because of their biological activities (cancer chemoprevention) (Aronson, 2006b; Taylor, Taylor, & Krings, 2009) and toxicities, a wide variety of bioactive chemical constituents such as sesquiterpenes (Jian, Kousuke, Mamiko, Chun, & Yoshiyasu, 2002b; Jian et al., 2002a; Miyako, Jian, Chun, Miwa, & Kenichi, 2008), neolignans (Miyako et al., 2007; Thomas & Jorg, 2000; Wen, Yun, Shi, Jing, & Dong, 2007), neoanisatines (Chun et al., 1990), prenylated C6–C3 compounds (Yoshiyasu, Naomi, Toshinori, & Mitsuaki, 1992; Yoshiyasu, Yuko, & Mitsuaki, 1997), phenylpropanoids (Lai & Geoffery, 1998a; Qin et al., 2009), pheno* Corresponding author. Tel.: +886 2 23123456x62226; fax: +886 2 23919098. E-mail address: [email protected] (Y.-C. Shen). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.05.069

lates (Yakushijin et al., 1983), diterpenes (Lai & Geoffery, 1998b), have been well studied recently. Several members of the neuro-toxic Illicium species have been the subjects of phytochemical investigation mainly for the purpose of clarifying the relationship between structure and toxicity, thus resulting in the isolation of a number of natural products. Recently, we reported seven new phytoquinoids and secoprezizaane-type sesquiterpenes from the aerial parts of Illicium arborescens Hayata (Chang et al., 2010). In a continuation of our research programs oriented toward bioactive natural products from endemic plants of Taiwan, the current work is the phytochemical research of the fruits of I. arborescens collected in different years. As the EtOAc/ CH2Cl2 extracts contain a complex mixture of substances, the examination of different chromatographic fractions of an EtOAc/ CH2Cl2 extract from the fruits of this species has led to the isolation of three new prenylated C6–C3 compounds, illicaborins A–C (1–3) (Fig. 1), in addition to nine known compounds, 1-allyl-2,3-(methylenedioxy)-5-methoxybenzene (4), apiol (5), illicinone E (6), 11epi-illicinone E (7), 2,3-dehydroillifunone C (8), illifunone A (9), illifunone B (10), illifunone C (11) and illifunone D (12). The structures of these compounds were elucidated through detailed spectroscopic analyses, mainly 2D-NMR experiments (1H–1HCOSY, HQMC, and HMBC). The configuration at the chiral centres and the geometry of the double bonds were deduced from NOESY spectra and by virtue of molecular modelling. Further study of the cytotoxic activities of the three new illicaborins A–C (1–3) was undertaken.

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Fig. 1. Compounds 1–3 isolated from the fruits of Illicium arborescens Hayata.

2. Materials and methods 2.1. General Column chromatography (CC); silica gel 60 (Merck) and Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden). FC = flash chromatography. Prep. TLC: pre-coated silica gel plates (Merck; silica gel 60 F-254, 1 mm). Optical rotations: Jasco DIP1000 polarimeter. UV Spectra: Hitachi U-3210 spectrometer; kmax (log e) in nm. IR spectra: Hitachi T-2001 spectrometer; in cm 1. 1 H-, 13C NMR, COSY, HMQC, HMBC, and NOESY experiments: Bruker AV-400 spectrometer, SiMe4 as internal standard; d in ppm, coupling constants J in Hz. HR-ESI-MS and ESI-MS: Bruker TOF mass spectrometer; in m/z (rel.%). 2.2. Plant material

(35 mg). By applying Si gel open column chromatography on fraction 10, and eluting with a gradient of n-hexane–CH2Cl2–MeOH (80:50:0–0:20:1) three fractions (fraction 10a (3.91 g); fraction 10b (268 mg); fraction 10c (80 mg)) were obtained. Fraction 10a (3.91 g) was subjected to a Si gel column chromatography eluting with n-hexane/CH2Cl2/MeOH (80:50:1–0:20:1) to give two fractions (fraction 10a-1, 2.0 g and fraction 10a-2, 1.26 g). Fraction 10a-1 was applied on a NP HPLC column using n-hexane/EtOAc/ MeOH (42:10:1) to give compound 9 (153 mg) and fraction 10a11 (123 mg). Fraction 10a-11 was further subjected to RP HPLC (MeOH/H2O/CAN, 40:10:1) yielded 6 (9.2 mg) and 7 (41.8 mg). Fraction 10a-2 (1.26 g) applied to NP HPLC (n-hexane/EtOAc/ MeOH, 43:40:1) furnished 1 (16.4 mg), 11 (193 mg) and 12 Table 1 1 H NMR spectroscopic data for compounds 1–3. No.

The fruits of I. arborescens were collected from Ping-tong County in July 2007, and were identified by one of the authors (C.-T. Chien). A voucher specimen was deposited in the School of Pharmacy, College of Medicine, National Taiwan University, Taipei.

1 2 3 4 5 6 7

2.3. Extraction and isolation The dried fruits of I. arborescens (1.98 kg) were powdered and extracted with a mixture of CH2Cl2/EtOAc and partitioned between ethyl acetate and water (1:1) leading to a EtOAc-soluble layer and an aqueous layer. The EtOAc-soluble layer was concentrated to a dark green of viscous residue (39 g), which was followed by Si gel column chromatography (flash column) eluted with n-hexane, ethyl acetate and MeOH (100% n-hexane: 100% MeOH), to yield 10 fractions. Fraction 1 (612 mg) was subjected to a normal-phase HPLC with a gradient n-hexane–CH2Cl2 (4:1) to yield compound 3 (61 mg) and fraction 1a (376 mg). The latter was applied to a Si gel column chromatography and eluted with n-hexane–EtOAc (12:1) to give fraction 1a-1 (268 mg). Further separation of 1a-1 on a normal-phase HPLC column, developed with gradient n-hexane–EtOAc (90:1), afforded compounds 4 (97.2 mg) and 5

8 9 10 11 12 13 14 15

dH (mult, J, Hz) 1a

2a

3b

2.24 (overlap)

1.75 m 2.36 dd (13.8, 4.5) 2.62 m

6.44 s

5.43 1.96 2.41 5.52 5.09

5.34 2.20 2.65 5.69 5.02 5.03 2.05 2.20 4.67

s m dd (13.4, 5.7) m (overlap)

1.96 m 2.26 (overlap) 4.74 dd (10.4, 4.6) 1.17 1.38 3.43 4.03

s s d (10.6) d (10.6)

s m m m dd (1.4, 11.2) dd (1.4, 16.6) m m dd (10.3, 5.0)

1.15 s 1.34 s

16 a b

Data were recorded in CDCl3 at 200 MHz. Data were recorded in CDCl3 at 400 MHz.

5.89 s 3.30 d (6.3) 5.95 m 5.05 (overlap) 3.30 d (6.3) 5.05 (overlap) 1.70 s 1.78 s 3.97 s

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(40.7 mg). Fraction 10b (268 mg) was subjected to RP HPLC with a gradient MeOH/H2O/ACN (20:43:4) yielding two compounds (compound 8 (73 mg) and compound 10 (70 mg)). Fraction 10c (80 mg) was applied to RP HPLC (MeOH/H2O/CAN, 40:51:10) to yield compound 2 (5.9 mg). Illicaborin A (1): colourless oil. [a]D23 +30 (c 0.1, CH2Cl2). UV (CH2Cl2) kmax (log e): 252 nm (2.38). CD (CH2Cl2) [h]206 197, [h]210 223, [h]214 597, [h]255 271, [h]318 249. IR (film) vmax: 3293, 1636, 1376, 1189, 954 cm 1. 1H and 13C NMR (CDCl3): see Tables 1 and 2, respectively. HR-ESI-MS m/z 305.1368 [M+Na]+ (calcd for C15H22O5Na, 305.1365). Illicaborin B (2): colourless oil. [a]D22 20 (c 0.1, CH2Cl2). UV (CH2Cl2) kmax (log e): 250 nm (2.41). CD (CH2Cl2) [h]210 156, [h]218 593, [h]266 428, [h]315 445. IR (film) vmax 3385, 1643, 1188, 954 cm 1. 1H and 13C NMR (CDCl3): see Tables 1 and 2, respectively. HR-ESI-MS m/z 275.1258 [M+Na]+ (calcd for C14H20O4Na, 275.1259). Illicaborin C (3): colourless oil. [a]D22 +102 (c 0.1, CH2Cl2); UV (CH2Cl2) kmax (log e): 250 nm (2.29) 281 (2.15) nm. IR (film) vmax 2925, 1619, 1476, 1052, 979 cm 1. 1H and 13C NMR (CDCl3): see Tables 1 and 2, respectively. HR-ESI-MS m/z 283.1309 [M+Na]+ (calcd for C16H20O3Na, 283.1310).

Table 2 13 C NMR spectroscopic data for compounds 1–3.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 a

6 5

15 2 7 8

3

4

OH

12

147.1 (C) 135.2 (C) 104.2 (CH) 125.4 (C) 141.6 (C) 131.8 (C) 100.6 (CH2) 25.1 (CH2) 137.2 (CH) 115.5 (CH2) 37.2 (CH2) 123.4 (CH) 130.8 (C) 25.7 (CH3) 17.8 (CH3) 59.6 (CH3)

Extensive Si gel column and preparative HPLC chromatographic separations using a gradient solvent system of EtOAc/CH2Cl2 afforded three new prenylated C6–C3 compounds, illicaborins A–C (1–3) from the fruits of I. arborescens. In the following, the structural elucidation, cytotoxic activities and a plausible biogenetic pathway are discussed. Illicaborin A (1), [a] +30 (CH2Cl2), was obtained as a colourless oily substance with an assigned molecular formula C15H22O5, as deduced from its HR-ESI-MS spectrum that gave a molecular ion

O

6

1

11

OH

12 2

7

OH

4

2

9 15 3

13 11 12

2

O

4 7

O

5

1

6

16

O

8 9

10

3 Fig. 2. Selected key COSY (

), HMBC (

10

3

8

1

14

13

O

5

OH

14

10

3

199.7 (C) 76.1 (C) 38.7 (CH2) 40.0 (CH) 178.7 (C) 100.6 (CH) 34.1 (CH2) 135.8 (CH) 117.1 (CH2) 38.7 (CH2) 90.9 (CH) 70.4 (C) 24.3 (CH3) 27.2 (CH3) 199.7 (C) 76.1 (C) 38.7 (CH2)

Data were recorded in CDCl3 at 100 MHz.

13

11

HO

9

O

2

202.1 (C) 49.4 (C) 41.9 (CH2) 73.6 (C) 181.8 (C) 102.1 (CH) 41.2 (CH2) 132.5 (CH) 119.4 (CH2) 38.9 (CH2) 91.3 (CH) 70.3 (C) 24.4 (CH3) 27.2 (CH3) 70.9 (CH2)

3. Results and discussions

Cytotoxicity was tested against Hep-2 (human laryngeal carcinoma), Daoy (human medulloblastoma), MCF-7 (human breast adenocarcinoma), and WiDr (human colon adenocarcinoma) tumour cell lines. The assay procedure using MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] was carried out as previously described (Shen et al., 2005). The cells were cultured in RPMI-1640 medium. After seeding of cells in a 96-well microplate for 4 h, 20 ll of sample was placed into each well and incubated at 37 °C for 3 days, and then 20 ll MTT was added for 5 h. After removing the medium and putting DMSO (200 ll/well) into the microplate with shaking for 10 min, the formazan crystals were

1

1

redissolved and their absorbance was measured on a microtiter plate reader (Dynatech, MR 7000) at a wavelength of 550 nm.

2.4. Cytotoxicity assay

O

d Ca

No.

) of 1–3.

14

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peak at m/z 305.1368 [M+Na]+. The IR spectrum revealed absorption bands attributed to a hydroxyl group (3293 cm 1) and an a,b-conjugated ketone (1636 cm 1) groups. The 1H spectroscopic data of compound 1 displayed 22 protons and the 13C NMR spectroscopic data indicated 15 carbon atoms (Tables 1 and 2). The 1H NMR spectrum showed one singlet signal at dH 5.43 due to one olefinic proton of an a,b-conjugated ketone, two proton signals at dH 5.52 (m, H-8) and dH 5.09 (overlap, H-9) due to a propylene-based sp2 hybrid orbital, two singlet signals at dH 1.17 (H13) and dH 1.38 (H-14) suggesting two neighbouring methyl groups, five methylene groups at dH 2.24 (overlap, H-3), dH 1.96 (m, H-7), dH 2.41 (dd, J = 13.4, 5.7 Hz, H-7), dH 1.96 (m, H-10), 2.26 (overlap, H-10), dH 5.09 (overlap, H-9), dH 1.96 (m, H-9), dH 3.43 (d, J = 10.6 Hz, H-15), dH 4.03 (d, J = 10.6 Hz, H-15), a series of signals at dH 1.96 (m, H-10), 2.26 (overlap, H-10), dH 3.43 (d, J = 10.6 Hz, H-15), dH 4.03 (d, J = 10.6 Hz, H-15) and 4.74 (dd, J = 10.4, 4.6 Hz, H-11) typical of the tetrahydrofurano-ring system present in illicinone (Yakushijin et al., 1984), and illifunone. Based on 13C NMR and DEPT spectroscopic data (Table 2), the presence of this furano-ring was supported. The 13C NMR spectrum showed two methyl carbons (dC 24.4 and 27.2), three olefinic carbons (dC 202.1, 102.1, and 181.8) indicating an a,b-conjugated ketone, four oxygenated carbons (dC 70.9, 73.6, 70.3 and 91.3) supporting the presence of the illifunone skeleton (Yakushijin et al., 1983), two olefinic carbons (dC 132.5 and 119.4) indicating the presence of the propylene-based sp2 hybrid orbital, as well as three methylene carbons (dC 41.9, 41.2, and 38.9), and one quarternary carbon (dC 49.4, C-2). The 1H–1H COSY spectrum revealed two sets of correlations (H-7/H-8/H-9 and H-10/H-11), the former indicating an acrylic base group. HMBC correlations of H-3/C-1, C-2, C-4, C-5, H6/C-1, C-2, C-4, C-5 and C-1, C-6, C-5 showed the presence of a six-membered ring, with an a,b-conjugated carbonyl fragment (Fig. 2). These findings pointed out a furano-ring system. Based on the NMR data in the literature (Isao, Toshihiko, & Nobusuke, 1988; Yoshiyasu, Naomi, & Mitsuaki, 1995; Yoshiyasu et al., 1992) regarding the relative configurations of illifunones A–D at C-4 and C-11, we assumed that compound 1 contains an a-hydroxy group at C-4. This was supported by NOESY experiments. NOESY correlations between H-11/H-10a (dH 2.20, m) and H-13, H-14/H10b (dH 2.05, m) suggested that H-11 is a-oriented (Fig. 3). NOESY

H

H

O

H

O

Hb

10 3 15

H HO

2 Fig. 4. Computer-generated perspective models of compounds 1 and 2 using MM2 force field calculation.

correlations between H-10b/Me-13, Me-14; H-10b/H-3b; H-3b/H7; H-3a/H-15 assigned the configuration of H-7 on the b-face and

13

Hb

7 8

1

Ha

OH

H

OH

11

4

Ha

Hb

O

14

HO

H

2

H

8

H

3

Ha

H

1

O

Ha H

2 15 11

13 14

3

4

2

12

O 7

5 1

O

6

16

O

8 9

10

3 Fig. 3. Selected key NOESY (

) correlations of 1–3.

11

10

4

13

Hb

H

14

OH

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the C-2 hydroxymethylene group at the a-disposition. A molecular model of structure 1 was generated by CS Chem 3D version 9.0 using MM2 force field calculation for energy minimisation (ChemBioUltra Calculation program) as shown in Fig. 4. The MM2 was calculated for C-7 allyl a and b positions. It was found that the molecular energy is 21.0 kcal/mol for the b-allyl position while it is 24.4 kcal/mol for the a-allyl position. Also, the results were consistent with the R configuration at C-2 and C-11, and S configuration at C-4 in 1 as established by NOESY experiments (Fig. 3). The CD spectrum of 1 (CD (CH2Cl2) [h]206 197, [h]210 223, [h]214 597, [h]255 271, [h]318 249) showed positive Cotton effects at 219, 229, and 318 nm of the a,b-conjugated ketone, and negative Cotton effects at 214, and 255 nm, and absolute configuration at C2 and C4 as R (Iida & Ito, 1983; Stephens & Lowe, 1985). The above spectroscopic data of 1 exhibited the typical feature of prenylated C6 C3 compounds connected with an additional CH2OH group at C-2. Thus, the structure of 1 was established as shown in Fig. 1 and the name illicaborin A was given. Illicaborin B (2), [a] 20 (CH2Cl2), was obtained as a colourless oily substance with a molecular formula C14H20O4 as deduced from a pseudomolecular ion peak at m/z 275.1258 [M+Na]+ in its HRESI-MS spectrum. The UV and IR spectra were similar to those of 1 suggesting an analogue of the prenylated C6–C3 compound. The 1 H NMR spectroscopic data of 2 displayed 20 protons, while the 13 C NMR spectrum indicated 14 carbons (Tables 1 and 2). The 1H NMR spectrum showed an olefinic proton at dH 5.43 (a,b-conjugated ketone), three propylene groups at dH 5.69 (m, H-8), dH 5.02 (dd, J = 1.4, 11.2 Hz, H-9), dH 5.03 (dd, J = 1.4, 16.6 Hz, H-9),

Table 3 Cytotoxic activities of compounds 1–3 against human tumour cells. Compound/cancer cell linea

Illicaborin A (1) Illicaborin B (2) Illicaborin C (3)

Hep-2

Daoy

MCF-7

WiDr

(–)b 10.32 (–)

(–) 14.52 (–)

(–) 16.82 (–)

(–) 17.16 (–)

a Cell lines: Hep-2 (human laryngeal carcinoma), Daoy (human medulloblastoma), MCF-7 (human breast adenocarcinoma), and WiDr (human colon adenocarcinoma) tumour cell lines. b (–): Inactive.

two methyl groups at dH 1.15 (H-13), dH 1.34 (H-14) and an oxygenated methine proton at dH 4.67 (dd, J = 10.3, 5.0 Hz, H-11), indicating the presence of a tetrahydrofurano-ring system (Stephens & Lowe, 1985; Yakushijin et al., 1984). The 13C NMR spectrum exhibited an a,b-unsaturated ketone (dC 199.7, 100.6, and 178.7), two methyl carbons (dC 24.4 and 27.2), three oxygenated carbons (dC 76.1, 70.4, and 90.9) similar to 1 (Stephens & Lowe, 1985; Yakushijin et al., 1984). However, COSY NMR correlations between H-3/H4/H-10/H-11 in 2 indicated that the hydroxyl group at C-4 in 1 was replaced by a proton in 2. In the HMBC spectrum, long-range correlations from H-3 and H-6 to C-2, C-4, and C-6 confirmed that 2 possesses an illifunone skeleton in the six-membered ring, from H-3 to C-7, and from H-7 to C-1, C-2, C-3, C-8, and C-9 speculating a six-membered ring in which C-2 is connected with a propylene side chain, different from 1 (Miyako et al., 2007). The additional

3 2 2

2

2

IC50 (lg/ml)

2

2

H

2

2

2 2

Scheme 1. Plausible biogenetic pathway of illicaborins A and B (1 and 2).

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2

2

2

2

2

2

2

2

2

2

Scheme 2. Plausible biogenetic pathway of illicaborin C (3).

HMBC correlations established unambiguously the typical skeleton of illifunone (Fig. 2). The relative configuration of 2 was determined from the NOESY experiment (Fig. 3) in which all the observed correlations were in agreement with the interatomic distances measured in a 3D molecular model. A computer-generated model for 2 using MM2 force field calculation is shown in Fig. 4. The MM2 was calculated for C-2-OH a/b positions. It was found that the molecular energy is 18.8 kcal/mol for the a-OH position while it is 18.1 kcal/mol for b-OH position. Also, this result agreed with the stereochemistry of 2 as established by NOESY experiments, in which compound 2 may have the configuration of 2R, 4S and 11R. The CD spectrum of compound 2 (CD (CH2Cl2) [h]206 197, [h]210 223, [h]214 597, [h]255 271, [h]318 249) showed positive Cotton effects at 214 and 225 nm, and negative Cotton effects at 210, and 218 nm, suggesting that the absolute configuration of C2, C4, and C11 should be R, S, and R respectively. Illicaborin C (3), [a] +102 (CH2Cl2), was obtained as a light yellow transparent oil with a molecular formula C16H20O3 as deduced from its HR-ESI-MS spectrum at m/z 283.1309 [M+Na]+. The IR (1619, 1476 cm 1, aromatic ring), and UV (250 and 281 nm) spectra suggested that compound 3 belongs to a phytoquinoid skeleton (Yoshiyasu et al., 1997). The 1H NMR spectroscopic data of 3 displayed 20 protons while 13C NMR spectrum 16 carbons (Tables 1 and 2). The 1H NMR spectrum revealed that 3 contains an aromatic ring (dH 6.44, H-3), a methoxy group (dH 3.97, H-16), and a dioxygenated methylene (dH 5.89, H-7). Signals including a methylene group, dH 3.30 (d, J = 6.3 Hz, H-8), and an alkenyl sp2 hybrid proton at dH 5.95 (m, H-9), and at dH 5.05 (H-10) indicated the presence of an acrylic base with a 1,1-dimethyl-propenyl group while signals at dH 1.70 (H-14), dH 1.78 (H-15), dH 5.05 (overlap, H-12), and dH 3.30 (d, J = 6.3 Hz, H-11) accounted for the presence of propenyl side chain. 13C NMR spectroscopic data (Table 2) showed two methyl (dC 17.8, 25.7), a methoxy (dC 59.6), a dioxygenated methylene carbon (dC 100.6), two methylene (dC 25.1 and 37.2), a terminal olefinic methylene carbon (dC 115.5), and six aromatic carbons (dC 125.4, 130.8, 131.8, 135.2, 141.6, 147.1). The 1H–1H COSY spectrum (Fig. 2) revealed correlations of H-11/H-12, and H-8/H-9/H10. HMBC correlations of 3 assigned the benzene ring connected with a 1,1-dimethyl-propenyl at C-4 and an acrylic side chain at C-6. Based on the above 2D-NMR analysis (Figs. 2 and 3), the structure of 3 was established and the name illicaborin C was given. Cancer chemopreventive activities of phenylpropanoids and phytoquinoids isolated from species of Illicium have been evalu-

ated recently (Itoigawa et al., 2004). In the present work, in vitro cytotoxic activities of 1–3 were investigated against Hep-2 (human laryngeal carcinoma), Daoy (human medulloblastoma), MCF-7 (human breast adenocarcinoma), and WiDr (human colon adenocarcinoma) tumour cell lines. The results are shown in Table 3. Among the compounds tested, 2 exhibited a moderate cytotoxic activity against all four tumour cell lines. Compounds 1–3 are proposed to be biogenetically derived from phenylalanine, which is the source of the prenylated C6–C3 fragment. The plausible biogenetic pathways for compounds 1–3 are illustrated in Schemes 1 and 2, respectively. The structural elucidation of the other nine compounds 4–12 are in agreement with those found in the literature, 1-allyl-2,3(methylenedioxy)-5-methoxybenzene (Kenichi et al., 1983), apiol (Giesbrecht, Franca, Gottlieb, & Rocha, 1974), illicinone E (Isao, Shigeko, Zhi, & Takashi, 1997), 11-epi-illicinone E (Lai & Geoffery 1998c), 2,3-dehydroillifunone C (Yoshiyasu, Naomi, Yuuko, & Mitsuaki, 1994), illifunones A–D (Kenichi et al., 1983). Acknowledgement The authors thank the National Science Council, Republic of China, for financial support (NSC96-2323-B-002-018). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.foodchem.2010.05.069. References Aronson, J. (2006a). Illiciaceae. Meyler’s Side Effects of Drugs: The International Encyclopedia of Adverse Drug Reactions and Interactions, 1715–1716. Aronson, J. (2006b). Herbal medicines. Meyler’s Side Effects of Drugs: The International Encyclopedia of Adverse Drug Reactions and Interactions, 1609–1625. Aronson, J., Masataka, A., Itoigawa, C., Tokuda, H., Fumio, E., Nishino, H., et al. (2004). Cancer chemopreventive activity of phenylpropanoids and phytoquinoids from Illicium plants. Cancer Letters, 214, 165–169. Chang, J.-Y., Abd El-Razek, M. H., Chen, Y.-H., Cheng, Y.-B., Chen, S.-Y., Chien, C.-T., et al. (2010). Phytoquinoids and secoprezizaane-type sesquiterpenes from Illicium arborescens. Helvetica Chimica Acta, 93, 123–132. Chun, Y., Miwa, H., Naosuke, B., Masakatsu, T., Hiroshi, K., Nobusuke, K., et al. (1990). A new toxic neoanisatin derivative from the pericarps of Illicium majus. Chemical Pharmaceutical Bulletin, 38, 291–292. Giesbrecht, A., Franca, N., Gottlieb, O., & Rocha, A. (1974). The neolignans of Licaria canella. Phytochemistry, 13, 2285–2293.

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