Plant materials
Tung tree fruits in each of the four developmental stages (Fig. 1) were harvested from a farm in Sanuki City, Kagawa Prefecture, in September and October of 2018. The fruits contain pericarp and four or five seeds, which consist of a hard seed coat and an oily endosperm (Fig. 2). The seed coat and endosperm were separated from tung seeds in the fourth development stage and used to quantify the lignans in the tissues.
Synthesis of isoamericanol A
Isoamericanol A, as a standard compound, was synthesized from the radical coupling reaction of caffeyl alcohol [6]. Caffeyl alcohol (892 mg, 5.37 mmol) was dissolved in an acetone–toluene mixture (50 mL, 1:2, v/v) and Ag2CO3 (1.427 g) was added to the solution at room temperature. After stirring for 20 h, the solid was filtered off and the solvent was concentrated under reduced pressure. The product was purified by silica gel column chromatography and a Sep-Pak C18 cartridge (Waters), followed by recrystallization, to obtain isoamericanol A (21.8 mg) as the 7′-8′ trans-isomer.
Isoamericanol A. colourless solid. LC–ESI–MS m/z: 329 [M–H]−. 1H NMR (CD3OD): δ 3.47 (1H, dd, J = 12.0, 4.6 Hz, H-9b), 3.67 (1H, dd, J = 12.0, 2.3 Hz, H-9a), 3.99 (1H, ddd, J = 7.4, 4.6, 2.3 Hz, H-8), 4.18 (2H, dd, J = 6.3, 1.7 Hz, H-9’), 4.80 (1H, d, J = 8.0 Hz, H-7), 6.20 (1H, dt, J = 16.0, 5.7 Hz, H-8’), 6.49 (1H, d, J = 16.0 Hz, H-7’), 6.76 (1H, dd, J = 8.0, 2.3 Hz, H-6), 6.80 (1H, d, J = 8.0 Hz, H-5), 6.85 (1H, d, J = 2.3 Hz, H-2), 6.89 (1H, d, J = 8.0 Hz, H-5’), 6.92 (1H, dd, J = 8.6, 1.7 Hz, H-6’), 6.96 (1H, d, J = 1.7 Hz, H-2’). 13C NMR (CD3OD): δ 61.9 (C-9), 63.6 (C-9’), 77.4 (C-7), 79.9 (C-8), 115.4 (C-2), 115.3 (C-2’), 116.1 (C-5), 117.8 (C-5’), 120.6 (C-6), 120.2 (C-6’), 129.3 (C-1), 131.2 (C-7’), 127.9 (C-8’), 131.8 (C-1’), 144.4 (C-3’), 145.1 (C-4’), 146.5 (C-3), 147.0 (C-4).
Preparation of samples
Each sample (ca. 8 g) of whole seed, seed coat, and endosperm were ground with a mortar and pestle, defatted with n-hexane (30 mL × 3), and extracted with methanol (30 mL × 3). The methanol extracts were concentrated under reduced pressure, and the oily extracts were purified on a Sep-Pak silica cartridge, eluting with 50% ethyl acetate/hexane (30 mL) and then 100% ethyl acetate (30 mL). The 100% ethyl acetate fraction was concentrated to dryness, and the concentrate was dissolved in 10 mL of MeOH, filtered through a 0.45-μm membrane filter, and then analysed by HPLC.
Quantitative HPLC analysis of catechol lignans
Quantitative analysis was performed using a JASCO LC-2000 plus system equipped with JASCO UV-2075 plus, a UV–VIS detector (254 nm), and Chromato Pro software (ver. 5.0). Separation was performed on a reversed-phase column (ϕ 4.6 × 250 mm, COSMOSIL, 5C18-MS-II) with a guard column. The mobile phase consisted of 20% MeOH (containing 0.1% acetic acid) that was increased to 70% MeOH (containing 0.1% acetic acid) over 40 min using a linear gradient, followed by isocratic elution for 10 min. The flow rate was adjusted to 1.0 mL/min, and the column temperature was maintained at 40 °C. The injection volume of the samples and standards was 10 μL each. Standards of isoamericanol A, americanol A, and (+)-3,3′-bisdemethylpinoresinol were obtained from defatted tung tree seeds [5]. A calibration curve was constructed by plotting the peak area versus the concentration of the synthesized isoamericanol A (0.02–0.4 μg/μL). The limit of detection of isoamericanol A was found to be 0.4 ng/μL, tenfold the signal-to-noise ratio. The amounts of catechol lignan and neolignans were expressed as isoamericanol equivalents.
Oil content and fatty acid composition
Endosperm tissues (ca. 3 g) collected at the different developmental stages were milled into a fine powder. The powder was extracted with diethyl ether (120 mL) using a Soxhlet extractor. The extract was washed with saturated NaCl solution, evaporated in vacuo, and weighed. The fatty acid composition was determined using the methanolysis method [3]. The oil was added to KOH and methanol and heated at 60 °C for 2 h. The reaction mixture was cooled, and the ester layer was separated using a funnel, washed with water, and then run through a silica gel column. The fatty acid methyl esters (FAMEs) were analysed by GC–MS (Shimadzu, GCMS-QP2010 SE) equipped with a capillary column (GL Sciences, IntertCap 225, 0.25 mm I.D. × 30 m, df 0.25 μm) using helium as the carrier gas. The oven temperature was set to 180 °C, and increased to 230 °C at a rate of 1 °C/min. The mass spectrometer was operated at an ionization energy of 70 eV and scanned from m/z 50 to 600. The FAMEs were identified by comparison with the standards and Wiley library data.