Analysis of the linoleic acid was performed on Finnigan GC-MS in the electronic ionization mode, and helium
(
99.9999%) was used as carrier gas with flow rate of 1.2 mL/min. The GC separation was performed using a Rt×25 MS
capillary column (15 m × 0. 25 mm × 0. 25 μm) obtained from the USA. The following temperature program was maintained:
0° for 1 min, 4°/min from 70° to 200°, and maintained for 6 min, then 15°/min from 200° to 280°. The injections were
7
performed in the splitless mode at 280°. The mass range of scan was 60–360 Da.
Plant Material. Balanophora involucrate Hook. f. was collected from Muyu town of the Shennongjia Forest District
in July 2005 and identified by Dr. Fa-Ju Chen. A voucher specimen was deposited at the Hubei Key Laboratory of Natural
Products Research and Development, China Three Gorges University, Yichang, P. R. China.
Extraction and Isolation. The air-dried whole plant (1.8 kg) was exhaustively extracted with 95% ethanol
(
5 L × 5) on refluxing at 60°C. The extract was evaporated in vacuo to yield a residue and freeze-dried, affording a powder,
which was divided into petroleum-ether (64.3 g), ethyl acetate (289.1 g), n-butanol (197.6 g), and water-soluble fractions. The
ethyl acetate–soluble extract (100 g out of 289.1 g) was chromatographed with gradient elution (100% petroleum-ether→100%
ethyl acetate→100% methanol). Repeated silica gel column and Sephadex LH-20 chromatography afforded compounds 1–4,
1
(20 mg), 2 (500 mg), 3 (30 mg), 4 (15 mg).
Base Hydrolysis. Ten mg of 1 was dissolved in a mixture of methanol and tetrahydrofuran at 1:1 volume ratio, and a
N NaOH solution was added to the mixture, which was stirred at 50° for 2 hours. After the base hydrolysis was finished, 10 mL
2
of water was added, the precipitation was filtrated and recrystallizated with CHCl and methanol (2:1), and compound 1a was
3
obtained. The filtrate was acidified by adding 2 N HCl unless the value of pH was less than 7, extracted with CHCl three
3
times, evaporated in vacuo, and dissolved in 1 mL CHCl to test for GC-MS.
3
Compound 1, coloreess oil. HR-ESI-MS at m/z 837.2770 (calcd 837.2775). The NMR data, see Table 1.
1
Compound 2, white powder, mp 149–151°C. H NMR (CD OD, 400 MHz, δ, ppm, J/Hz): 3.42–3.90 (6H, m), 3.89
3
(
7
3H, s), 4.22 (2H, d, J = 5.5), 4.90 (1H, d, J = 7.2), 6.28 (1H, dt, J = 15.9, 5.5), 6.56 (1H, d, J = 15.9), 6.96 (1H, dd, J = 8.4, 1.4),
.08 (1H, d, J = 1.4), 7.12 (1H, d, J = 8.4). C NMR (MeOD, 100 MHz, δ, ppm): 133.8 (C-1), 111.5 (C-2), 150.9 (C-3), 147.7
13
(
C-4), 118.0 (C-5), 120.8 (C-6), 131.3 (C-7), 128.9 (C-8), 63.8 (C-9), 56.8 (OCH ), 102.8 (C-1′), 74.9 (C-2′), 77.9 (C-3′), 71.4
3
(
C-4′), 78.2 (C-5′), 62.6 (C-6′).
1
Compound 3, white powder, mp 193–195°C. H NMR (CD OD, 400 MHz, δ, ppm, J/Hz): 3.20–3.90 (6H, m), 3.30
3
(
3H, s), 3.83 (3H, s), 4.17 (2H, d, J = 5.2), 4.8 (1H, d, J = 7.3), 6.24 (1H, dt, J = 14.6, 5.2), 6.49 (1H, d, J = 14.6), 6.92 (1H, dd,
13
J = 8.4, 1.2), 7.02 (1H, d, J = 1.2), 7.05 (1H, d, J = 8.4). C NMR (CD OD, 100 MHz, δ, ppm): 134.0 (C-1), 111.7 (C-2), 150.9
3
(
C-3), 147.7 (C-4), 117.9 (C-5), 121.3 (C-6), 131.7 (C-7), 129.3 (C-8), 64.1 (C-9), 50.3 (C-10), 57.2 (OCH ), 102.9 (C-1′), 75.1
3
(
C-2′), 77.9 (C-3′), 71.6 (C-4′), 78.4 (C-5′), 62.7 (C-6′).
1
Compound 4, white powder, mp 212–213°C. H NMR (DMSO-d , 400 MHz, δ, ppm, J/Hz): 3.20–3.90 (6H, m),
6
3
.78 (3H, s), 4.98 (1H, d, J = 6.8), 6.69 (1H, dd, J = 16.0, 8.0), 7.11 (1H, d, J = 8.4), 7.25 (1H, d, J = 8.4), 7.30 (1H, s), 7.63 (1H,
1
3
d, J = 16.0), 9.50 (1H, d, J = 8.0). C NMR (DMSO-d , 100 MHz, δ, ppm): 129.4 (C-1), 112.6 (C-2), 155.5 (C-3), 149.9
6
(
C-4), 115.0 (C-5), 116.2 (C-6), 127.8 (C-7), 124.5 (C-8), 197.1 (C-9), 56.9 (OCH ), 100.5 (C-1′), 73.9 (C-2′), 77.0 (C-3′),
3
7
0.5 (C-4′), 77.5 (C-5′), 61.6 (C-6′).
ACKNOWLEDGMENT
This research was financially supported by the Opening Fund from the Key Laboratory of NuclearAnalysis Techniques,
ChineseAcademy of Sciences (K115), and the Startup Scientific Research Fund for Doctor from China Three Gorges University
Structures and Activities of Constituents Inhibiting COX-2 from Balanophora involucrata).
(
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