HETEROCYCLES

Short Paper | Regular issue | Vol. 85, No. 12, 2012, pp. 3015-3019
Received, 19th September, 2012, Accepted, 17th October, 2012, Published online, 23rd October, 2012.
DOI: 10.3987/COM-12-12589

■ Nigelactone, New Phthalide Glucoside from the Seeds Nigella glandulifera

Yu Feng, Yu-Ming Liu,* Qing-Hua Liu, and Ying-Jie Lei

Department of Pharmacy Engineering, Tianjin University of Technology, No.391, Bin Shui Xi Road, Xi Qing District, Tianjin , China

Abstract

Chemical investigations of the seeds of Nigella glandulifera have resulted in the isolation of a new phthalide derivative, nigelactone (1), together with two known metabolites. Their structures were established by detailed analysis of their 1D and 2D NMR spectra and mass spectroscopic data. Nigelactone (1) possesses a rare structural skeleton found in nature.

Nigella glandulifera FREYN et SINT. is an annual erect herbaceous plant, found widely in the southwest and western part of China. Its seeds are commonly eaten in many food preparations by Uigur and are believed to have diuretic, analgesic, spasmolytic, galactagogue and bronchodilator properties.1 In our previous work, a number of bioactive compounds, such as alkaloids,2 triterpenoid saponins3 and flavonol glycosides4 were isolated from the N. glandulifera. Recently we obtained a new phthalide derivative (1), named nigelactone, together with two known compounds, glycerol (2) and 1-linoleoyl glycerol (3), from the seeds of N. glandulifera. This paper reports the isolation and structural elucidation of nigelactone (1) (Figure 1), which possesses a rare structural skeleton found in nature.

Compound 1 was obtained as a pale yellow solid. Its molecular formula was determined as C19H24O9 according to the [M+Na]+ at 419.1324 (calculated for C19H24O9Na, 419.1318) in the positive HR-ESI-MS. On acid hydrolysis, 1 gave D-glucose. Strong absorption bands accounting for hydroxyl group (3396 cm-1), α, β-unsaturated γ-lactone group (1730 cm-1) and aromatic group (1614, 1507 cm-1) could be observed in its IR spectrum. Its negative ESI-MS spectrum showed a quasimolecular ion at m/z 395.3 [M-H]-. The resulting MS-MS spectrum showed fragment ion at m/z 233.2 [(M-H)-162]-, derived from losses of terminal glucose. In the positive ESI-MS spectrum its pseudomolecular ion [M+H]+ was observed at m/z 397.2. The adducts [M+Na]+ at m/z 419.3 and [M+K]+ at m/z 435.3 were also observed. In the ESI-MS+2 (m/z 419.3, [M+Na]+) spectrum 1 showed fragment at m/z 256.9 [M+Na-162]+, also corresponding to the losses of the glucose.
The 13C NMR and DEPT spectra of 1 contained 19 carbon resonances, including two resulting from methyl groups, two from sp2 methines, and five from sp3 methines, whereas further three signals resulted from methylene groups, and seven resonances were assigned to quaternary carbons. The 1H NMR spectrum of 1 showed characteristic signals for an aromatic proton at δH 6.65 (1H, s) and 3-methyl-2-butenyl group (δH 5.12 t, 3.24 d, 1.73 s, 1.68 s). The presence of the glucose moiety was also suggested by the observation of seven protons at δH 3.39, 3.45, 3.47, 3.55, 3.70, 3.92, 4.92. Furthermore, β-configuration at the anomeric proton δH 4.92 was determined from its relatively large 3JH1″-H2″ coupling constant (8.0 Hz). Additionally, glycosidation position located on the C-6 was derived from the HMBC correlation between δH 4.92 (H-1″) and δC 155.3 (C-6). The HMBC correlation from δH 6.65 (H-7) to the carbonyl carbon δC 171.3 (C-1) and the NOESY correlation from δH 6.65 (H-7) to the anomeric proton δH 4.92 (H-1″) indicated that the carbonyl group and the glucose existed on the two sides of H-7 position; the HMBC correlations from δH 5.17 (H-3) to the carbonyl carbon δC 171.3 (C-1) and from δH 5.17 (H-3) to three aromatic carbons [δC 149.7 (C-3a), δC 115.8 (C-4) and δC 104.1 (C-7a)] revealed that the γ-lactone was connected with the benzene ring in parallel; the NOESY correlation from δH 5.17 (H-3) to δH 3.24 (H-1′) confirmed that the 3-methyl-2-butenyl group was located close to the γ-lactone moiety. Analysis of the 1H-1H DQF-COSY, HSQC, HMBC and NOESY spectra led to the proposed structure 1 and enabled the complete assignment of the 1H and 13C NMR spectra (Table 1). The important NOESY interactions of compound 1 are shown in Figure 2. So compound 1 was identified as 5-hydroxy-4-(3-methyl-2-butenyl)-6-O-β-D-glucopyranosyl-1(3H)-isobenzofuranone, named nigelactone.

Nigelactone (1), a prenylated phthalide glycoside, is one of the acyclic hemiterpenoid phthalides. To the best of our knowledge, only six compounds of the acyclic hemiterpenoid phthalides (O-prenylated form not included) were found in nature,5-8 and their carbonyl groups are all positioned peri to the aromatic methoxy or hydroxy moiety. So nigelactone (1) is the first acyclic hemiterpenoid phthalide in nature, which carbonyl group is placed peri to the aromatic hydrogen. Since phthalide derivatives are compounds of the polyketide metabolism,9 this type of phthalides such as in nigelactone (1) could be formed owing to distinct ring-closure position in polyketides, and then this type of phthalides might differ significantly from the other in terms of its biosynthesis.9

EXPERIMENTAL
General. Optical rotation was measured on a Rudolph Autopol IV polarimeter. UV spectrum was taken in MeOH using a Hitachi U-3310 spectrophotometer. IR spectrum was recorded in KBr discs on a Nicolet FT-IR AVATAR 370 spectrophotometer. ESI-MS and HR-ESI-MS were obtained on a Finnigan Surveyor LC-LCQ Advantage Max and a Bruker micrOTOF-Q II spectrometers, respectively. 1H- and 13C-NMR spectra were acquired on a Bruker Avance III 400 spectrometer with the residual solvent signals as an internal standard (CD3OD δH 3.30 ppm, δC 47.6 ppm). 1H-1H DQF-COSY, NOESY, HSQC and HMBC spectra were recorded using conventional pulse sequences. Silica gel 60H (400-500 mesh) and Silica gel GF254 sheets (0.20-0.25 mm) (both from Qingdao Haiyang Chemical Group Co., China) were used for column chromatography and TLC, respectively.
Plant material. The seeds of Nigella glandulifera FREYN et SINT. were collected from Ürümuqi in Xinjiang Uigur autonomy, China, in February 2011, and identified by Prof. Qing-Hua Liu, Xinjiang Institute of Materia Medica.
Extraction and isolation. The oil-free seeds (18 kg) of N. glandulifera were extracted three times with 95% EtOH for 2 h under reflux and then extracted three times with 50% EtOH for 2 h under reflux. After combination and removal of the solvent in vacuo, the EtOH extract was then suspended in distilled water and partitioned successively with petroleum ether, EtOAc and n-BuOH. The EtOAc fraction (80 g) was chromatographed over silica gel and eluted with CHCl3-MeOH gradient solvent (20:1~1:1). Combination of similar fractions on the basis of TLC analysis afforded 9 fractions. Fraction 5 was subjected to silica gel column chromatography and eluted with CHCl3–MeOH (10:1) to yield 1 (25 mg), 2 (17 mg) and 3 (89 mg).
Nigelactone (1): pale yellow solid, mp 113-115 ˚C; [α] -80.0 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 225 (4.05), 261 (2.69), 291 (3.31) nm; IR (KBr) νmax 3396, 2919, 1730, 1614, 1507, 1363, 1237, 1061 cm-1; 1H-NMR (400 MHz, CD3OD) and 13C-NMR (100 MHz, CD3OD) data provided in Table 1; 1H-NMR (400 MHz, DMSO-d6, standard TMS) δ 1.64 (3H, s, 4′-CH3), 1.70 (3H, s, 5′-CH3), 3.16 (2H, d, J = 6.8 Hz, H-1′), 3.25 (1H, m, H-4″), 3.29 (1H, m, H-5″), 3.28 (1H, m, H-3″), 3.30 (1H, m, H-2″), 3.53 (1H, dd, J = 5.6, 11.6 Hz, H-6A″), 3.69 (1H, d, J = 11.6 Hz, H-6B″), 4.94 (1H, d, J = 6.8 Hz, H-1″), 5.11 (1H, t, J = 7.0 Hz, H-2′), 5.16 (2H, s, H-3), 6.70 (1H, s, H-7), 10.68 (1H, s, 5-OH); ESI-MS (negative mode) m/z 395.3 [M-H]-; ESI-MS-2 (negative mode, M′ = 395.3 [M-H]-) m/z 233.2 [(M-H)-162]- ; ESI-MS (positive mode) m/z 397.2 [M+H]+, 419.3 [M+Na]+, 435.3 [M+K]+; ESI-MS+2 (positive mode, M′ = 419.3 [M+Na]+) m/z 256.9 [M+Na-162]+; HR-ESI-MS (positive mode) m/z 419.1324 [M+Na]+ (calcd 419.1318 for C19H24O9Na).
Acid Hydrolysis of (1): Compound 1 (6 mg) was refluxed with 10% HCl in 75% EtOH (9 mL) for 6 h. The reaction mixture was diluted with H2O and extracted with CHCl3. The H2O layer was neutralized with Ag2CO3 and analyzed by TLC [EtOAc-MeOH-AcOH-H2O (12:3:3:2)]. The Rf value (0.43) of the sample was identical to that of standard glucose. Next, the H2O layer was evaporated to dryness under reduce pressure and the residue was separated on silica gel by CC. Fractions were detected by TLC. The fraction which contained the pure sugar was concertrated, then was measured on the Autopol IV polarimeter, The positive optical rotation value was showed, thus the sugar moiety of 1 was identified as D-glucose.

ACKNOWLEDGEMENT
This work was supported by the National Natural Science Foundation of People’s Republic of China, under grant no. 81073153.

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