Cubelin

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 Chemical structure of cubelin (1) and its partial structures A, B and C. The numeration was assigned based on the ¹³C-NMR spectroscopic data.












 A new diterpene, identified as (+)-6-(4-hydroxy-4-methyl-2-pentenoyl)-4,6-dimethyl-5-(3-methyl-2-butenyl)-1,3-cyclohexadienecarbaldehyde (1, cubelin), was isolated from a methanol extract of Litsea cubeba fruits by normal phase column chromatography and purified by preparative HPLC. The structure elucidation was conducted by spectroscopic methods (UV, IR, ESI-TOF-MS, 1-D and 2-D NMR). Cubelin exhibited activity against HeLa cell viability and proliferation. The cells also exhibited changes in nuclear morphology which are hallmarks of apoptotic cell death. The presence of cleaved caspase-3/-7, caspase-8 and caspase-9 in the cubelin treated population indicated the potential of the compound to induce apoptosis in HeLa cells via both intrinsic and extrinsic pathways.

 http://www.mdpi.com/1420-3049/19/5/6838/htm

Molecules 2014, 19(5), 6838-6850; doi:10.3390/molecules19056838

A New Diterpene from Litsea cubeba Fruits: Structure Elucidation and Capability to Induce Apoptosis in HeLa Cells

Piyapat Trisonthi ^1,*, Akihiko Sato ¹, Hisashi Nishiwaki ¹ and Hirotoshi Tamura ^1,2,*

¹

The United Graduate School of Agricultural Science (UGAS), Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime Prefecture 790-8566, Japan

²

The Graduate School of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa Prefecture 761-0795, Japan

*

Authors to whom correspondence should be addressed; E-Mails: pyp.trisonthi@gmail.com (P.T.); tamura@ag.kagawa-u.ac.jp (H.T.); Tel.: +81-87-891-3104 (H.T.).

 

Structure Elucidation

Compound 1, (+)-6-(4-hydroxy-4-methyl-2-pentenoyl)-4,6-dimethyl-5-(3-methyl-2-butenyl)-1,3-cyclohexadienecarbaldehyde, was obtained as a pale yellowish oil with a specific optical rotation [α]20 D of +274.025 (c = 0.02, CHCl₃) and maximum UV absorptions at 216 and 324 nm. The molecular weight minus a hydroxyl group was determined by HRESIMS as C₂₀H₂₈O₂ (obsd. at m/z 299.2017 [M−OH+H]⁺, cald. m/z 299.2011) and another peak was observed as C₂₀H₂₇O₃Na⁺ (obsd. at m/z 339.1911 [M+Na]⁺, cald. m/z 339.1936). The data suggested that the molecular formula of 1 is C₂₀H₂₈O₃ (m/z 316)_, which indicated seven degrees of unsaturation. The IR spectrum showed hydroxyl group (3516–3393 cm⁻¹, broad), unsaturated carbons (3034 cm⁻¹), saturated carbons (2978 cm⁻¹) and overlapping bands of conjugated aldehyde and ketone (1670 cm⁻¹) absorption bands.
The structure elucidation was explained based on NMR spectroscopic data (Table 1). Chemical shifts (δ) were reported in part per million (ppm), standardized by chemical shift of TMS and residue of CDCl₃. In total, 20 signals were observed in the ¹³C-NMR spectrum. Signals interpreted by the DEPT experiment suggested the presence of six aliphatic methyls at δ 16.46, 17.85, 24.04, 25.81, 29.26 and 29.47, one aliphatic methylene at δ 25.57, one aliphatic methine at δ 46.27, one aliphatic quaternary carbon at δ 52.99, one tertiary oxygenated carbon at δ 71.20, five olefinic methine carbons at δ 118.94, 121.83, 123.09, 148.45 and 153.04, three tertiary olefinic carbons at δ 132.60, 136.37 and 156.38, one aldehyde carbon at δ 193.06 and one ketone carbon at δ 200.28. These data supported the molecular formula as C₂₀H₂₈O_3. The ¹H-NMR spectrum revealed one aldehyde proton at δ 9.46 (1H, s) and six methyl groups at δ 1.28, 1.30, 1.39, 1.52, 1.62 and 1.95 (3H, s). According to the correlation based on the ¹H-¹H COSY spectrum, a methylene group at δ 2.08 (2H, dd, J = 6.50, 7.56 Hz) has spin coupling with two adjacent protons which are an aliphatic methine proton at δ 2.73 (1H, t, J = 6.50 Hz) and a vinyl methine proton at δ 5.04 (1H, t, J = 1.38, 7.56 Hz). This datum indicated the existence of a [C=C₍₁₃₎H-C₍₄₎H₂-C₍₈₎H-] fragment. In addition, two vinyl methine protons at δ 6.54 and δ 6.88 (1H, d, J = 15.12 Hz), and two methine protons at δ 5.87 and δ 6.83 (1H, d, J = 5.52 Hz) were also correlated to be a [-C₍₁₂₎H=C₍₁₇₎H-] fragment, and a [C=C₍₁₁₎H-C₍₁₆₎H=C] fragment, respectively.

 

Extraction, Isolation and Identification of (+)-6-(4-Hydroxy-4-methyl-2-pentenoyl)-4,6-dimethyl-5-(3-methyl-2-butenyl)-1,3-cyclohexadienecarbaldehyde (Cubelin, 1)

Air dried L. cubeba fruits (2 kg) were ground into a coarse powder and then extracted with hexane (3× 4L). The remaining fruit residue was extracted again with methanol (3× 4L). After the methanol extract was filtered and the solvent was removed under vacuum, the extract (300 g) was dissolved in diethyl ether (2 L). The diethyl ether soluble filtrate (50 g) was then subjected to silica gel column chromatography (Wakogel C-300, 200 × 50 mm, 45–75 μm particle size) after the solvent was removed. Elution was done with mixtures of hexane and diethyl ether (1:0, 3:1, 1:1, 1:3 and 0:1, 1L each) to give 20 fractions (250 mL/fraction). Fraction 11 and 12, which were eluted by 1:1 mobile phase, were subjected to preparative RP-HPLC (Mightysil RP-18GP, 250 × 10 mm, isocratic: 54% Acetonitrile, 0.1% TFA in H₂O, flow rate: 0.6 mL/min, UV-VIS detector: 230 nm) to afford cubelin (retention time: 120–125 min). The amount of cubelin obtained from 2 kg of fresh fruit was 64 mg. Pale yellowish oil; [α]20 D +274.025 (c = 0.02, CHCl₃); UV λ_max (logε): 216 (2.99), 324 (2.69) nm; IR (KBr): υ_max 3597, 3516–3393 (broad), 3034, 3010, 2978, 1670, 1629, 1561 cm⁻¹; HRESIMS: m/z 299.2017 [M−OH+H]⁺ (calcd. 299.2011 for C₂₀H₂₈O₂), m/z 339.1911 [M+Na]⁺ (cald. m/z 339.1936 for C₂₀H₂₈O₃Na⁺). ¹H and ¹³C-NMR data are shown in Table 1.

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