Isolation and characterisation of the sesquiterpene lactones from Lactuca sativa L var. anagustata

Food Chemistry 120 (2010) 1083–1088

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Isolation and characterisation of the sesquiterpene lactones from Lactuca sativa L var. anagustata Yi-Feng Han *, Gui-Xiu Cao, Xiao-Jing Gao, Min Xia Department of Chemistry, Zhejiang Sci-Tech University, Hangzhou 310018, PR China

a r t i c l e

i n f o

Article history: Received 7 May 2009 Received in revised form 9 October 2009 Accepted 24 November 2009

Keywords: Lactuca sativa L var. anagustata Compositae Sesquiterpene lactone Cytotoxicity Antioxidation

a b s t r a c t Lactuca sativa L var. anagustata is widely used as both a delicious vegetable and a traditional medicine in China. Chemical investigation of the methanolic extract of L. sativa L var. anagustata has now led to the isolation of three new sesquiterpenes, together with eight known ones. All the compounds were isolated by chromatography on a silica gel column and preparative thin-layer chromatography (PTLC). Their structures were elucidated by means of spectroscopic methods, including 2D-NMR experiments. Cytotoxicity of the new compounds was assayed against selected cancer cell lines, including the human epithelial carcinoma (HeLa) and human colon carcinoma (HCT-116) cell lines. Radical-scavenging activities of the seven compounds were determined by DPPH radical-scavenging assay. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Some plants are used as both delicious vegetables and important folk medicines. Thus, studies on the chemical constituents of these plants are of great importance. Lactuca sativa L var. anagustata, which belongs to the genus of Lactuca, Compositae, is just such a plant. L. sativa L var. anagustata (celtuce), also called ‘‘Chinese lettuce” or ‘‘wosun”, is a cultivar of lettuce in China grown as a ‘‘stem-used type” vegetable (Editorial Committee of Flora of China, 1989). The whole plant has also been used as a traditional medicine for the treatment of stomach problems, stimulate digestion, and to enhance appetite and relieve inflammation. Despite its importance, this plant has not been a subject of phytochemical analysis until now. Interestingly, its kindred plant, L. sativa L (lettuce), a ‘‘leafused” vegetable popular in western countries, has received more chemical and biochemical attention, focused on sesquiterpene lactones (Mahmoud, Kassem, Abdel-Salam, & Zdero, 1986), phytols (Bang et al., 2002), carotenoids (Kim, Fonseca, Choi, & Kubota, 2007), polyphenol oxidase and phenols (Altunkaya & Gökmen, 2008; Gawlik-Dziki, Złotek, & Swieca, 2008), micronutrients (Nicolle et al., 2004) and proteins (Piero, Puglisi, & Petrone, 2002). Our interest in chemical constituents of plants that can be used as medicine and food materials prompted us to investigate the phytochemical analysis of this plant, which resulted in the isolation of three new sesquiterpenes (1–3), together with eight known ones (4–11) (Fig. 1). Their structures were elucidated by HR–ESI– * Corresponding author. Tel.: +86 571 86843230; fax: +86 571 86843600. E-mail address: [email protected] (Y.-F. Han). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.11.056

MS, IR and 1D- and 2D-NMR experiments. In addition, the cytotoxicities of compounds 1–3 were evaluated against selected cancer cell lines, including the human epithelial carcinoma (HeLa) and human colon carcinoma (HCT-116) cell lines. Compounds 2, 3, 6–9 and 11 were tested by DPPH radical-scavenging assay, in which compound 9 exhibits weak antioxidant activity. 2. Materials and methods 2.1. General procedures Optical rotations were measured on a Perkin–Elmer Model 341 polarimeter. IR spectra were recorded on a Nicolet NEXUS 670 FTIR instrument, using KBr discs over the range 400–4000 cm1. NMR spectra were measured on a Bruker AM-400 NMR spectrometer with TMS as an internal standard. HR–ESI–MS were obtained on a Bruker Daltonics APEX-II 47e spectrometer. EI–MS was measured on an HP5988a GC-MS at 70 eV. Column chromatography was carried out on Si gel (200–300 mesh) and TLC on Si gel (GF254 10– 40 lm), both supplied by Qingdao Marine Chemical Co. 2.2. Plant material A voucher specimen (No. 070901) has been deposited in the Department of Chemistry, Zhejiang Sci-Tech University, China. The whole plant of L. sativa L var. anagustata was collected from the Carrefour supermarket in Hangzhou city, Zhejiang province of China, and was identified by Prof. Xiao-Chuan Liu, School of Life Science, Zhejiang Sci-Tech University, China.

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HO

O O

O OH

5' 4'

3'

OH

14 9

1 1'

2

2'

OH OH

10 5

4

3

R1 = O

15

6

7

11 12

H

13

GluO HO

OH H 8 7

13

6

H

O

15

OH

2

9 10 1 5

4

R2 = O

O

14

3

OMe

5''

O

O

R1

1

2

4'' 6''

O

H

8'' 7''

8

H OH O

2'' 3'' 1''

H

OH OH

OH

OH

H GluO

HO H

11 12

O 4

O

O

O

3

H

H

H

O

O

5

O O

O 6

7 OH

O OH

O

O

O

HO

O

O

GluO

8

9

OGlu

H

OH

R2

HO

H

O

O

O

O 10

11

O

Fig. 1. Structures of compounds 1–11.

2.3. Extraction and isolation of constituents The dried and powered stalks (3.5 kg) were extracted at room temperature with MeOH (12l  7d  3). The extracts were filtered and the filtrate was concentrated under reduced pressure to yield a crude extract of methanol (220 g), which was suspended in H2O (1.0 l) and extracted with petroleum ether (boiling point 60– 90 °C, 0.5 l), ethyl acetate (0.5 l) and n-butanol (0.5 l). After evaporation of the ethyl acetate and n-butanol solvents, a brown gum (8 g) and a brown–red gum (30 g) were obtained under reduced pressure, respectively. The n-BuOH extract (30 g) was subjected to repeated chromatography, eluting with a gradient of CHCl3–MeOH (20:1–0:1, v/v) and six crude fractions (A–F) were obtained. Fr. C (1.5 g) was chromatographed on a silica gel column, eluting with CHCl3–MeOH

(10:1) to give six fractions. Fraction C-4 (100 mg) was further purified by PTLC to afford 2 (15 mg, EtOAc–EtOH–H2O 25:1:0.5, Rf = 0.50), while further purification of fraction C-5 (50 mg) by PTLC, using EtOAc–EtOH–H2O (25:1:0.5), afforded 1 (15 mg, Rf = 0.45). Fr. D (2.0 g) was chromatographed on a silica gel column eluting with EtOAc–EtOH–H2O (20:1:0.5) to give seven fractions, among which, chromatography of fraction D-5 (200 mg) gave 3 (10 mg) and 11 (10 mg), and fraction D-6 (100 mg) was chromatographed on PTLC to give 7 (8 mg, EtOAc–EtOH–H2O 10:1:0.5, Rf = 0.60). Compound 9 (100 mg) was isolated from fraction E (1.2 g) by CC (EtOAc–EtOH–H2O 15:1:0.5). In the same way, the ethyl acetate extract (8 g) afforded compounds 6 (5 mg), 4 (2 mg), 5 (2 mg), 8 (0.90 g) and 10 (10 mg) by repeated column chromatography, using a CHCl3–MeOH step gradient elution and purification by PTLC as above.

Table 1 1 H NMR data for compounds 1, 3, 7 and 11 (in CD3OD-d4, 400 MHz). No.

1

3

7

11

1 2 3 5 6 7 8 9 11 13 14 15 10 20 30 40 50 60

3.76 (dd, 11.2, 5.2) 1.53 m, 1.90 m 1.61 m, 1.75 m 1.73 (d, 10.8) 4.16 (dd, 11.2, 10.8) 1.54 m 1.53 m, 1.89 m 1.33 m, 2.07 m 2.33 (dq, 7.6, 6.8) 1.23 (d, 6.8) 1.02 (s) 1.37 (s) 4.32 (d, 7.6) 3.24 m 3.52 m 3.35 m 3.89 m 3.42 (dd, 11.6, 6.4), 3.61 (dd, 11.6, 2.0)

3.09 (dd, 10.4, 6.4) 1.76 m, 1.83 m 1.57 m, 2.38 m 2.43 (dd, 10.4, 10.0) 4.03 (dd, 10.4, 10.0) 2.67 m 1.45 m, 2.47 m 2.01 m, 2.54 m – 5.42 (d, 2.8), 6.09 (d, 2.8) 4.90 (brs), 5.08 (brs) 3.72 (d, 12.0), 3.83 (d, 12.0) 4.48 (d, 6.8) 3.31 m 3.55 m 3.40 m 3.87 m 3.50 (dd, 12.0, 6.4), 3.65 (dd, 12.0, 1.6)

– 2.04 m, 2.30 m 4.59 (dd, 8.8, 4.8) 2.86 (dd, 10.0, 9.6) 4.12 (dd, 9.6, 9.6) 3.14 m 1.65 m, 2.35 m 4.56 (dd, 4.4, 3.2) – 5.53 (d, 3.2), 6.19 (d, 3.2) 5.11 (brs), 5.13 (brs) 5.40 (brs), 5.42 (brs) 4.48 (d, 7.6) 3.31 m 3.62 m 3.38 m 3.45 m 3.76 (dd, 12.0, 5.2), 3.87 (dd, 12.0, 2.8)

2.65 m 1.51 m, 1.82 m 1.84 m, 2.48 m 2.93 (dd, 10.0, 4.8) 4.02 (dd, 10.0, 10.0) 3.35 m 6.00 (dd, 10.4, 2.8) 5.42 (d, 10.4) 2.53 (dq, 11.2, 6.8) 1.24 (d, 6.8) 3.39 (d, 11.2), 3.43 (d, 11.2) 5.08 (brs), 5.15 (brs) 4.57 (d, 7.6) 3.32 m 3.61 m 3.37 m 3.55 m 3.75 (dd, 12.0, 4.8), 3.84 (dd, 12.0, 2.0)

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2.4. Cytotoxic assay The cytotoxicity assay was carried out according to the MTT [3(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] method (Hussain, Nouri, & Oliver, 1993). Compounds 1–3 and adriamycin (positive control) were dissolved in DMSO to make stock solutions, which were diluted to a working solution before use (the final DMSO concentration was 0.1% v/v). Cells were cultured at 37 °C under a humidified atmosphere of 5% CO2 and dispersed in replicates in 96-well plates with 2  103 cell/well (HeLa) and 3  103 cell/well (HCT-116) for 24 h. Compounds 1–3 and adriamycin were then added to make various final concentrations (40 lM, 10 lM, 2.5 lM, 0.63 lM, 0.16 lM, 0.04 lM). Three repli-

cate wells were used at each point in the experiment. To three parallel control wells, consisting of deionized H2O with the same DMSO concentration, were added 20 ll of MTT solution (5 mg/ ml), and the medium was then removed after 3 h of incubation and replaced by 100 ll of DMSO; the absorbance, which was detected at 550 nm using a SpectraMAX340 microplate reader (Molecular Devices, Sunnyvale, CA) with a reference wavelength at 690 nm, represents cell numbers before being treated with compounds. After being incubated for 72 h, 40 ll of MTT (5 mg/ml) were added to each well and each was incubated for 3 h; the medium was removed and 100 ll of DMSO were added to each well. The absorbance was determined as above. There are two evaluation methods of IC50:

cell viability ð%Þ ¼ A=Acon  100%; net growth rate ð%Þ ¼ ðA  Apre Þ=ðAcon  Apre Þ  100% Table 2 13 C NMR (DEPT) data for compounds 1, 3, 7 and 11 (in CD3OD-d4, 100 MHz). No.

1

3

7

11

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 20 30 40 50 60

84.1 (d) 24.3 (t) 38.0 (t) 71.4 (s) 56.2 (d) 80.9 (d) 53.2 (d) 22.6 (t) 39.2 (t) 41.4 (s) 40.7 (d) 182.5 (s) 12.3 (q) 14.4 (q) 23.9 (q) 103.8 (d) 73.4 (d) 76.5 (d) 70.5 (d) 75.7 (d) 62.1 (t)

47.1 (d) 23.3 (t) 23.8 (t) 83.5 (s) 52.9 (d) 80.8 (d) 43.5 (d) 25.9 (t) 29.5 (t) 149.6 (s) 142.6 (s) 171.5 (s) 119.3 (t) 111.1 (t) 65.8 (t) 97.6 (d) 73.8 (d) 77.6 (d) 70.1 (d) 78.2 (d) 61.7 (t)

41.5 (d) 37.1 (t) 81.0 (d) 149.5 (s) 49.7 (d) 84.5 (d) 37.6 (d) 38.8 (t) 72.1 (d) 151.4 (s) 139.9 (s) 170.5 (s) 120.7 (t) 112.9 (t) 113.4 (t) 102.4 (d) 74.0 (d) 76.7 (d) 70.5 (d) 76.2 (d) 62.1 (t)

46.2 (d) 24.9 (t) 29.1 (t) 150.6 (s) 51.8 (d) 78.2 (d) 45.9 (d) 131.0 (d) 135.5 (d) 83.6 (s) 42.2 (d) 179.7 (s) 12.6 (q) 66.4 (t) 109.5 (t) 96.8 (d) 74.0 (d) 76.4 (d) 70.3 (d) 77.5 (d) 61.9 (t)

(A is equal to absorbance of 72-h compound-treated well; Acon means absorbance of 72 h control well; Apre means absorbance before being treated with compounds). 2.5. Antioxidant capacity assay 2.5.1. Chemicals DPPH (2,20 -diphenyl-1-picrylhydrazyl, 96%) and a-tocopherol (98%) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and solvents were of analytical grade. 2.5.2. DPPH free radical-scavenging assay The DPPH radical-scavenging activity of the test samples was evaluated by the method previously reported (Mellors & Tappel, 1966). An ethanolic solution of DPPH (100 lM) was incubated with an ethanolic solution of each of the test samples (15–90 lg/ ml) for 30 min at 25 °C in the dark. The control contained all reagents without the sample while ethanol was used as a blank. The DPPH radical-scavenging activity was determined by measuring the absorbance at 517 nm using a spectrophotometer. The

Table 3 1 H and 13C NMR (DEPT) data of compounds 2, 9 and 10 (400 and 100 MHz). No.

2a 13

4.33 (dd, 8.8, 7.6) 1.78 m, 2.28 m 5.74 (brs) – 2.35 (d, 10.0) 3.40 (dd, 10.0, 10.0) 1.47 m 1.44 m, 1.98 m 1.35 m, 2.08 m – 2.31 (dq, 7.2, 6.8) – 11.5 (d, 6.8) 0.84 (s) 4.50 (d, 11.6), 4.64 (d, 11.6) – 7.17 (d, 7.6) 6.85 (d, 7.6) – 6.85 (d, 7.6) 7.17 (d, 7.6) 3.36 (brs) – 3.81 (s)

81.0 (d) 31.0 (t) 129.7 (d) 131.5 (s) 49.8 (d) 81.5 (d) 53.5 (d) 25.9 (t) 29.5 (t) 149.6 (s) 41.0 (d) 180.5 (s) 12.4 (q) 12.2 (q) 68.2 (t) 126.9 (s) 131.2 (d) 114.4 (d) 159.1 (s) 114.4 (d) 131.2 (d) 41.4 (t) 172.2 (s) 55.5 (q)

H

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 100 200 300 400 500 600 700 800 800 -OMe a b

9a

1

in CD3OD-d4. in CDCl3-d1.

C

10b

1

13

1

– – 6.57 (brs) – 3.61 (d, 10.0) 3.65 (dd, 10.0, 10.0) 2.15 (ddd, 12.0, 10.0, 9.6) 3.81 m 2.42 (dd, 13.6, 2.0), 2.78 (dd, 13.6, 10.8) – 2.59 (dq, 12.0, 7.2) – 1.43 (d, 7.2) 2.44 (brs) 4.76 (d, 17.2), 4.85 (d, 17.2) 4.40 (d, 8.0) 3.32 m 3.45 m 3.36 m 3.44 m 3.76 (dd, 12.0, 4.8), 3.89 (dd, 12.0, 2.0)

132.1 (s) 196.1 (s) 134.4 (d) 169.4 (s) 49.3 (d) 81.4 (d) 61.3 (d) 69.0 (d) 49.4 (t) 149.3 (s) 41.8 (d) 178.9 (s) 15.3 (q) 21.9 (q) 68.7 (t) 102.8 (d) 73.8 (d) 76.4 (d) 70.3 (d) 76.7 (d) 61.8 (t)

– – 6.44 (brs) – 3.67 (d, 10.0) 3.72 (dd, 10.0, 10.0) 2.35 (ddd, 12.0, 10.0, 9.6) 4.82 m 2.43 (dd, 13.6, 2.0), 2.81 (dd, 13.6, 10.0) – 2.56 (dq, 12.0, 6.8) – 1.17 (d, 6.8) 2.47 (brs) 4.37 (d, 14.4), 4.84 (d, 14.4) – 7.12 (d, 8.4) 6.78 (d, 8.4) – 6.78 (d, 8.4) 7.12 (d, 8.4) 3.37 (brs) –

H

C

H

13

C

131.6 (s) 194.9 (s) 132.0 (d) 173.9 (s) 47.6 (d) 80.0 (d) 57.3 (d) 70.1 (d) 43.4 (t) 146.4 (s) 39.6 (d) 176.9 (s) 13.4 (q) 19.9 (q) 61.1 (t) 123.4 (s) 129.5 (d) 114.5 (d) 155.7 (s) 114.5 (d) 129.5 (d) 39.8 (t) 170.6 (s)

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Table 4 1 H NMR data for compounds 4, 5, 6, 8 (in CDCl3-d1, 400 MHz). No.

4

5

6

8

1 2 3 4 5 6 7 8 9 11 13 14 15

3.15 (dd, 10.0, 8.0) 1.74 m, 2.15 m 3.77 (ddd, 10.4, 9.6, 6.0) 1.83 m 1.94 (ddd, 10.4, 10.0, 9.2) 3.88 (dd, 10.4, 9.2) 2.13 m 1.44 m, 2.26 m 4.69 (brs) 2.14 m 1.21 (d, 6.8) 4.99 (brs), 5.05 (brs) 1.24 (d, 7.6)

2.34 m 1.65 m, 1.74 m 2.41 m, 2.52 m – 2.74 (dd, 9.6, 9.2) 4.10 (dd, 10.2, 9.6) 2.32 m 1.40 m, 2.10 m 1.70 m, 1.84 m 2.23 (dq, 11.2, 7.2) 1.24 (d, 7.2) 3.37 (d, 10.8), 3.46 (d, 10.8) 4.99 (brs), 5.18 (brs)

3.97 (dd, 12.0, 6.4) 1.82 m, 2.36 m 5.33 (brs) – 2.35 (d, 11.2) 3.62 (dd, 11.2, 9.6) 2.00 m 1.50 m, 1.93 m 1.33 m, 2.01 m 2.22 m 1.24 (d, 6.8) 0.88 (s) 1.81 (s)

– – 6.44 (brs) – 3.56 (d, 10.0) 3.66 (dd, 10.0, 10.0) 2.15 (ddd, 12.0, 10.0, 9.6) 3.74 m 2.40 (dd, 13.6, 2.0), 2.76 (dd, 13.6, 10.8) 2.59 (dq, 12.0, 7.2) 1.43 (d, 7.2) 2.45 (brs) 4.43 (d, 17.6), 4.48 (d, 17.6)

Table 5 13 C NMR (DEPT) data for compounds 4, 5, 6 and 8 (in CDCl3-d1, 100 MHz). No.

4

5

6

8

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

35.1 (d) 39.5 (t) 74.1 (d) 47.1 (d) 50.4 (d) 86.0 (d) 45.9 (d) 38.0 (t) 78.5 (d) 151.2 (s) 41.9 (d) 178.5 (s) 13.0 (q) 112.1 (t) 17.8 (q)

49.3 (d) 25.2 (t) 30.0 (t) 150.5 (s) 51.9 (d) 81.5 (d) 47.6 (d) 25.6 (t) 29.7 (t) 75.8 (s) 43.8 (d) 179.9 (s) 13.2 (q) 68.9 (t) 110.1 (t)

75.6 (d) 34.5 (t) 121.3 (s) 133.8 (s) 53.9 (d) 81.5 (d) 50.8 (d) 23.1 (t) 34.7 (t) 41.3 (s) 40.9 (d) 179.8 (s) 12.6 (q) 11.3 (q) 23.5 (q)

132.3 (s) 195.8 (s) 133.1 (d) 173.4 (s) 49.2 (d) 81.2 (d) 61.4 (d) 68.0 (d) 49.3 (t) 148.3 (s) 41.7 (d) 178.6 (s) 15.3 (q) 21.8 (q) 62.2 (t)

DPPH radical-scavenging activity of a-tocopherol was also assayed for comparison. The activity (%) of the tested sample was calculated as follows:

DPPH -scavenging effect ð%Þ  
 ¼ 1  A517nm:sample A517nm:control  100 2.6. Characteristic data of compounds 1b-O-b-D-glucopyranosyl-4b-hydroxyl-5a, 6b, 11bH-eudesma-12, 6a-olide (1). Colourless gum, [a] 25D + 20 (c 0.10, MeOH); IR (KBr): mmax = 3435, 1746 cm1; HR–ESI–MS: m/z = 448.2542 [M+NH4]+ (calcd. for [C21H34O9+NH4]+: 448.2541); 1H and 13C NMR (DEPT) see Tables 1 and 2. 1b-hydroxyl-15-O-(p-methoxyphenylacetyl)-5a, 6b, 11bH-eudesma-3-en-12, 6a-olide (2). Yellow gum, [a] 25D + 15 (c 0.15, MeOH); IR (KBr): mmax = 3445, 1760, 1608, 1513, 1457 cm1; HR–ESI–MS: m/z = 437.1941 [M+NH4]+ (calcd. for [C24H36O6+Na]+: 437.1936); 1 H and 13C NMR (DEPT) see Table 3. 4a-O-b-D-glucopyranosyl-15-hydroxyl-5a, 6bH-guaiane-10(14), 11(13)-dien-12, 6a-olide (3). Colourless gum, [a] 25D + 32 (c 0.08, MeOH); IR (KBr): mmax = 3400, 1760, 1652 cm1; HR–ESI–MS: m/ z = 449.1789 [M+NH4]+ (calcd. for [C21H30O9+Na]+: 449.1782); 1H and 13C NMR (DEPT) see Tables 1 and 2. 1 H and 13C NMR (DEPT) for compounds 4–6 and 8 see Tables 4 and 5; 1H and 13C NMR (DEPT) for compounds 7 and 11 see Tables 1 and 2; 1H and 13C NMR (DEPT) for compounds 9 and 10 see Table 3.

3. Results and discussion 3.1. Phytochemical investigation Compound 1 was isolated as a colourless gum and a molecular formula of C21H34O9 was assigned by ion peak at m/z 448.2542 (calcd. 448.2541 [M+NH4]+) from HR–ESI–MS. Its IR spectrum showed the presence of hydroxyl groups at mmax 3435 cm1 and a lactone group at 1746 cm1. The 1H and 13C NMR and DEPT spectra (Tables 1 and 2) indicated that 1 contained three methyls, five methylenes, ten methines and three quaternary carbons, which was the skeleton of a sesquiterpene lactone glycoside (Yang, Shi, & Jia, 2002). Typical signals for a b-glucopyranoside were readily recognised from the NMR spectra (Yang et al., 2002), which was further confirmed as D-glucose by PC after acid hydrolysis of 1 (Rf = 0.65, EtOAc-pyridine-H2O (2:1:5); 1 (6 mg) in aqueous H2SO4 (2 M, 5 ml) and toluene (5 ml) was gently heated under reflux for 3 h). The remaining signals indicated that the aglycone was a sesquiterpene lactone and similar to the known eudesmanolide 1b-hydroxycolartin (Sanz, Falco, & Marco, 1990) with one tertiary and four secondary methyls, a c-lactone moiety, one secondary and one tertiary hydroxyl group. The attachment of glucose to the hydroxyl at C-1 was deduced from the HMBC experiment, which showed a long-range correlation between H-10 (dH 4.32, d, J = 7.6 Hz) and C-1 (dC 84.1). The doublet for H-10 (dH 4.32, d, J = 7.6 Hz) and the resonance for C-10 (dC 103.8) indicated that the glycosyl bond had the b-configuration. The large coupling constants observed for H-1 with H-2 (J1a, 2b = 11.2 Hz), H-6 with H-5 (J6b, 5a = 10.8 Hz) and H-7 (J6b, 7a = 11.2 Hz), allowed the assignment of the relative stereochemistry for H-1 as a-orientation and that of the lactone group at C-6 and C-7 as trans (6b, 7a). In the NOESY experiment, the cross-peaks observed between H-6b and H3-14, H-5a and H-1a, H-7a indicated that the A/B ring was trans-fused, and the cross-peaks between H3-15 and H-6b indicated that 15-CH3 had the b-configuration while the 4-hydroxyl group was a-orientated. In the 13C NMR spectrum, the chemical shift value of the methyl group at dC 12.3 is typical for eudesmanolides having an a-methyl group at C-11 (Gerard, Paul, & Edward, 1974). This was further confirmed by the NOESY experiment, which showed cross-peaks between H-6b and H-11b. Therefore, compound 1 was concluded to be 1b-O-b-D-glucopyranosyl-4ahydroxyl-5a, 6b, 11bH-eudesma-12, 6a-olide. Compound 2 was also isolated as a yellow gum. Its molecular formula of C24H36O6 was deduced from HR-ESI-MS spectrum which showed an [M+Na]+ ion at m/z 437.1941 (calcd. 437.1936). Its IR spectrum revealed absorption peaks for hydroxyl groups (3445 cm1), a c-lactone ring (1760 cm1) and a benzyl group (1608, 1513, 1457 cm1). The 1H NMR spectrum (Table 3) exhib-

Y.-F. Han et al. / Food Chemistry 120 (2010) 1083–1088

ited a tertiary methyl signal at dH 0.84 (s), a doublet methyl signal at dH 1.15 (J = 6.8 Hz), two oxymethine signals at dH 3.40 (dd, J = 10.0, 10.0 Hz), dH 4.33 (dd, J = 8.8, 7.6 Hz) and the characteristic signals due to a p-methoxyphenylacetate moiety [dH 3.36 (2H, brs), 3.81 (3H, s), 6.85 (2H, d, J = 7.6 Hz), 7.17 (2H, d, J = 7.6 Hz)] (Miyase & Fukushima, 1987). The 13C NMR data (Table 3) of compound 2 showed the presence of 24 carbons, including nine signals due to a p-methyoxyphenylacetate moiety [dC 55.5 (q), 41.4 (t), 126.9 (s), 131.2 (d), 114.4 (d), 159.1(s), 172.2(s)] (Miyase & Fukushima, 1987). A quaternary carbon signal was observed at dC 40.4, suggesting that this compound is an eudesmanolide type sesquiterpene (Yang et al., 2002). Furthermore, one trisubstituted double bond signal and three oxygen-bearing carbon signals were observed at dC 129.7 (d), 131.5 (s), 68.2 (t), 81.0 (d) and 81.5 (d). The remaining signals indicated that the aglycone was a sesquiterpene lactone and similar to the known eudesmanolide 11a, 13dihydrosantamain (Sanz, Gloria, & Macro, 1990). The attachment of p-methyoxyphenylacetate to the hydroxyl at C-15 was deduced from the HMBC experiment which showed the long-range correlation between H-15 (dH 4.50, d, J = 11.6 Hz) and C-800 (dC 172.2). Finally, the stereochemistry was established by NOESY experiments. Clear NOE correlations between H-6b and H3-14; H-5 and H-1, H-7 indicated that 14-CH3 had the b-configuration, while H-1, H-5, H-7 were a-orientated. Therefore, the structure of 2 was identified as 1b-hydroxyl-15-O-(p-methoxyphenylacetyl)-5a, 6b, 11bH-eudesma-3-en-12, 6a-olide. Compound 3 was obtained as a colourless gum and its molecular formula, C21H30O9, was deduced from HR-ESI-MS for the [M+NH4]+ ion peak at m/z 449.1789 (calcd. 449.1782). The IR spectrum showed that 3 contained a hydroxyl group (3400 cm1), a lactone group (1760 cm1) and double bond (1652 cm1) in its skeleton structure. The 1H and 13C NMR and DEPT spectra (Tables 1 and 2) of 3 clearly exhibited 21 carbon signals (8  CH2, 8  CH, 5  C), which was also the skeleton of a sesquiterpene lactone glycoside (Yang et al., 2002). Typical signals for a b-glucopyranoside, like 1, were still recognised from the NMR spectra (Yang et al., 2002). The remaining signals indicated that the aglycone was a sesquiterpene lactone and similar to the known guaianolide Scalesin (Spring, Heil, & Vogler, 1997) with one exocyclic methylene function, an a-methylene-c-lactone moiety, and one – CH2–O– unit. A pair of protons at dH 3.83 and 3.72, with a geminal coupling constant of J = 12.0 Hz, indicated the presence of a CH2OH group. The coupling pattern of H-5 (dH 2.43, dd, J = 10.4, 10.0 Hz) indicated only two neighbouring protons (H-6 and H-1) and a quaternary carbon at C-4. The position of the glucoside at C-4 was

% DPPH radical-scavenging

30

20

2 3 6 7 8 9 11

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established by the HMBC spectrum of a long-range correlation between H-1’ (dH 4.48, d, J = 6.8 Hz) and C-4 (dC 83.5). The stereochemistry of 3 was deduced from chemical shifts and values of the coupling constants and confirmed by the NOESY spectrum. The NOESY spectrum indicated effects between H-6 and H-15, H9b, indicating the b-orientation of the 15-CH2OH. Furthermore, clear NOEs were observed between H-5 and H-7, which showed NOE with H-1, indicating the a-orientation of H-1 and H-7. Therefore, compound 3 was concluded to be 4a-O-b-D-glucopyranosyl15-hydroxyl-5a, 6bH-guaiane-10(14), 11(13)-dien-12, 6a-olide. The known compounds were identified by comparing their physical and spectroscopic data with values reported in the literature. They are 9b-hydroxyl-4b, 11b, 13, 15-tetrahydrozaluzanin C (4) (Kisiel & Barszcz, 1996), 10b, 14-dihydroxyl-11bH-guaiane4(15)-ene-12, 6a-olide (5) (Tan, Jakupovic, Bohlmann, Jia, & Schuster, 1990), 1b-hydroxyl-5a, 6bH-eudesman-3-ene-12, 6a-olide (6) (Tan & Jia, 1992), macroclinisides A (7) (Miyase, Yamaki, & Fukushima, 1984), 11b, 13-dihydrolactucin (8) (Sarg, Omar, Khafagy, Grenz, & Bohlmann, 1982), Cichorioside B (9) (Seto et al., 1988), 11b, 13-dihydrolactucopicrin (10) (Beek et al., 1990) and 10b, 14dihydroxy-10(14), 11b(13)-tetrahydro-8, 9-didehydro-3-deoxyzaluzanin C-10-O-b-glucopyranoside (11) (Kisiel & Michalska, 2002). 3.2. Cytotoxic activity Compounds 1–3 were evaluated for their in vitro cytotoxicity against human epithelial carcinoma (HeLa) and human colon carcinoma (HCT-116) cell lines by the MTT method (Hussain et al., 1993). Unfortunately, the tested compounds exhibited no significant cytotoxicity in the two cell model systems used. Since it has been reported that sesquiterpene lactones with an a, b-unsaturated c-lactone system generally show adequate cytotoxic activity (Nosse, Chhor, Jeong, Bölhm, & Reiser, 2003), it is hard to explain why compound 3 is inactive. Maybe it is selective for cell lines or its bulky sugar moiety makes its stereoconfiguration unable to bind to target proteins. 3.3. DPPH free radical-scavenging activity Free radical-scavenging activities of seven compounds (2, 3, 6–9 and 11) were evaluated in comparison with a-tocopherol, using DPPH assay. The results of the tested compounds are presented in Fig. 2; among them, only compound 9 exhibits weak antioxidant activity. Under the same conditions, the IC50 values of the positive control, a-tocopherol, was 5.52 lg/ml. 4. Conclusions Phytochemical study of Lactuca sativa L. var anagustata has resulted in three new sesquiterpenes (1–3), together with eight known ones (4–11). To the best of our knowledge, this is the first phytochemical analysis of this plant. Taking it into consideration that this plant is a medicine and food material, our work is clearly important, revealing its chemical constituents. Moreover, to our surprise, the type of the compounds isolated from this plant is relatively singular compared with the constituents of its kindred plant Lactuca sativa L, which is a ‘‘leaf-used” vegetable popular in western countries. This remarkable difference may be duo to the different cultivation or regional disparities.

10

0 30

60

Concentration (µg/ml) 

Fig. 2. DPPH assay of compounds 2, 3, 6–9 and 11.

90

Acknowledgement This work was financially supported by the National Science Foundation of China (No. 20902082), the Talents Training

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Foundation of Key Laboratory of Advanced Textile Materials and Manufacturing Technology (Zhejiang Sci-Tech University), Ministry of Education (No. 2006QN04).

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