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the deacetate derivative of the aglycone 1b (1.5 mg). The H2O layer was con-
centrated under reduced pressure to dryness, to give a residue of the sugar
fraction. The residue was dissolved in pyridine (0.1 ml), to which 0.08 M L-
cysteine methyl ester hydrochloride in pyridine (0.15 ml) was added. The
mixture was kept at 60 °C for 1.5 h. After the reaction mixture was dried in
ferent position compared to 1 (Fig. 1). The HMBC correla-
tions from H2-2 (dH 2.54, 2.35), H2-6 (dH 2.88, 2.60), Me-14
(dH 1.06) and H-10 (dH 1.74) to C-1 (dC 80.8) were ob-
served, respectively, indicating the hydroxy group was as-
cribed to C-1. The b-D-glucopyranosyl moiety located at C- vacuo, the residue was trimethylsilylated with 1-trimethylsilylimidazole
(0.1 ml) for 2 h. The mixture was partitioned between n-hexane and H2O
(0.3 ml each) and then the n-hexane extract was analyzed by GC-MS under
11 in 2 was determined by the HMBC correlation between
Glc-H-1 (dH 4.53) and C-11 (dC 82.0) along with the down-
shift anomeric carbon signal at dC 98.6.20) From the above
data, the gross planar structure of 2 was deduced as 1-hy-
droxytorilolone 11-O-b-D-glucopyranoside. The stereochem-
istry of H-7, H-8 and H-10 was determined to be the same as
that of 1, on the basis of the NOESY spectrum (Fig. 2) and
coupling constants. The b-configuration of OH-1 was con-
firmed by the signals of Me-14 and H-10 at dH 1.06 and 1.74
the following conditions: capillary column, EQUITYTM-1 (30 mꢄ0.25
mmꢄ0.25 mm, Supelco); column temperature, 230 °C; injection tempera-
ture, 250 °C; carrier N2 gas; detection in EI mode, ionization potential,
70 eV; ion-source temperature, 280 °C.16,19) D-Glucose in 1 was confirmed by
comparison of the retention times of its derivatives with those of standard D-
glucose and L-glucose derivatives prepared in a similar way which showed
retention times of 11.25 and 10.79 min, respectively. Sugar in 2 (1.0 mg) was
also identified by the same method.
Enzymatic Hydrolysis A solution of 1 (3.5 mg) in 0.1 M acetate buffer
(pH 4.0, 1.0 ml) was treated with naringinase (Sigma Chemical Co., 2 units),
and then the reaction mixture was stirred at 40 °C for 72 h. The reaction
mixture was passed through a Sep-Pak ODS cartridge (Waters) and washed
with H2O and CH3OH to give the aglycone 1a (1.8 mg). Through a similar
procedure, enzymatic hydrolysis of 2 (3.5 mg) was carried out to afford the
aglycone 2a (1.6 mg).
(1b,7b,8b,10b)-11-O-Acetyl-8,11-dihydroxy-4-guaien-3-one (1a): Color-
less oil. [a]D22 ꢀ20.6° (cꢁ0.16, CHCl3 : MeOHꢁ1 : 1); positive-ion HR-ESI-
MS m/z 317.1720 (Calcd for C17H26O4Na, 317.1729); 1H-NMR (C5D5N,
500 MHz) d: 5.65 (1H, m, H-8), 3.22 (1H, d, Jꢁ12.5 Hz, H-6), 2.95 (1H, m,
H-2), 2.88 (1H, dd, Jꢁ12.5, 10.0 Hz, H-6), 2.60 (1H, m, H-2), 2.29 (1H, m,
H-9), 2.28 (1H, m, H-1), 2.12 (1H, m, H-9), 2.10 (3H, s, Me-1ꢃ), 2.06 (1H,
m, H-7), 1.99 (3H, s, Me-15), 1.86 (1H, m, H-10), 1.52 (3H, s, Me-13), 1.48
(3H, s, Me-12), 1.19 (3H, d, Jꢁ7.0 Hz, Me-14); 13C-NMR (C5D5N,
1
in the H-NMR spectrum, since the values of the chemical
shifts of Me-14 and H-10 are dH 1.07, 1.74 for b-configura-
tion and dH 0.77, 2.31 for a-orientation.22,24) It was also sup-
ported by comparison of the optical rotation of the aglycone
of 2 (2a, [a]D22 ꢀ5.1°) with those of the similar compounds
([a]D20 ꢀ13.7° for 1b-hydroxytorilin, whereas ꢂ24.4° for
1a-hydroxytorilin).24) Thus, the structure of 2 was estab-
lished as 1b-hydroxytorilolone 11-O-b-D-glucopyranoside,
namely (1b,7b,8b,10b)-1,8,11-trihydroxy-4-guaien-3-one
11-O-b-D-glucopyranoside.
Experimental
General Experimental Procedures Optical rotations were measured 125 MHz) d: 207.5 (C-3), 175.6 (C-5), 171.1 (C-1ꢃ), 134.8 (C-4), 73.1 (C-
using a Rudolph Autopol IV digital polarimeter with a 0.5 dm length cell.
8), 72.0 (C-11), 51.3 (C-1), 48.7 (C-7), 41.6 (C-2), 40.9 (C-9), 33.9 (C-10),
HR-ESI-MS was taken on a Bruker Daltonics Apex III mass spectrometer. 28.6 (C-12), 27.0 (C-13), 25.9 (C-6), 23.2 (C-14), 21.5 (C-2ꢃ), 8.1 (C-15).
All NMR spectra were recorded on a Bruker ARX-500 and ARX-125 MHz
(1b,7b,8b,10b)-1,8,11-Trihydroxy-4-guaien-3-one (2a): Colorless oil.
NMR spectrometer equipped with a CH dual 5f probe. Samples were dis- [a]D22 ꢀ5.1° (cꢁ0.14, CHCl3 : MeOHꢁ1 : 4); positive-ion HR-ESI-MS m/z
1
solved in 0.6 ml CD3OD or C5D5N and transferred into a 5 mm NMR tube.
291.1579 (Calcd for C15H24O4Na, 291.1572); H-NMR (C5D5N, 500 MHz)
All chemical shifts are expressed as d (ppm) relative to the internal standard d: 4.72 (1H, dt, Jꢁ7.1, 3.0 Hz, H-8), 3.17 (1H, dd, Jꢁ13.5, 10.0 Hz, H-6),
trimethylsilyl (TMS) (dꢁ0 ppm), and scalar coupling constants are reported
in Hz. Silica gel (200—300 mesh, Qingdao Haiyang Chemical Co. Ltd.,
3.10 (1H, d, Jꢁ13.5 Hz, H-6), 2.95 (1H, m, H-9), 2.92 (1H, d, Jꢁ18.0 Hz,
H-2), 2.79 (1H, d, Jꢁ18.0 Hz, H-2), 2.10 (1H, ddd, Jꢁ14.5, 7.1, 1.5 Hz, H-
China), Sephadex LH-20 (Ammersham Pharmacia Biotech) and octadecyl 9), 1.87 (3H, s, Me-15), 1.86 (1H, m, H-10), 1.76 (3H, s, Me-13), 1.72 (1H,
silica (ODS) (35—50 mm, Alltech) were used for column chromatography. m, H-7), 1.62 (3H, s, Me-12), 1.34 (3H, d, Jꢁ7.0 Hz, Me-14); 13C-NMR
Preparative HPLC was performed using ODS column (Waters Sunfire ODS- (C5D5N, 125 MHz) d: 206.7 (C-3), 176.0 (C-5), 133.7 (C-4), 79.0 (C-1),
C18, 10 mm i.d.ꢄ250 mm).
73.3 (C-11), 70.4 (C-8), 50.6 (C-2), 50.6 (C-7), 39.4 (C-9), 38.1 (C-10), 29.4
Plant Material The fruits of Daucus carota L. were purchased in Sep- (C-12), 29.2 (C-13), 22.4 (C-6), 19.1 (C-14), 8.1 (C-15).
tember 2007 from Hangzhou, Zhejiang Province, P. R. of China, and identi-
fied by one of the authors (Lin Zhang). A voucher specimen was deposited
in the Herbarium of the College of Biomedical Engineering and Instrument
Sciences, Zhejiang University, People’s Republic of China.
Acknowledgements This research was partially supported by Zhejiang
Provincial Natural Science Foundation of China (Y2090436).
Extraction and Isolation The air-dried fruits of D. carota L. (3 kg)
were refluxed two times with 95% aqueous EtOH. The combined EtOH ex-
tracts were concentrated, suspended in H2O, and then partitioned with petro-
leum ether, CHCl3, EtOAc and n-BuOH successively to give four different
polar parts. The n-BuOH layer (10.2 g) was subjected to silica gel CC with a
gradient of CHCl3/MeOH (15 : 1—8 : 1) to give eight fractions (1—8). Frac-
tion 2 was loaded on silica gel CC with CHCl3/MeOH (8 : 1) to give three
fractions (A1—A3). Fraction A2 (0.35 g) was purified by Sephadex LH-20
CC with CHCl3/MeOH (1 : 1), followed by silica gel CC with CHCl3/
(CH3)2CO (1 : 1) to obtain 1 (9.2 mg). Fraction 5 (0.2 g) was chro-
matographed on silica gel CC with CHCl3/MeOH (9 : 1) and then further
separated by repeated HPLC purification with 20% aqueous MeOH to afford
2 (7.0 mg).
11-O-Acetyl-torilolone 8-O-b-D-Glucopyranoside (1): Amorphous pow-
der; [a]D22 ꢀ25.4° (cꢁ1.0, MeOH); UV (MeOH) lmax: 244 nm; 1H-NMR
(CD3OD, 500 MHz) and 13C-NMR (CD3OD, 125 MHz), see Table 1; posi-
tive-ion HR-ESI-MS m/z 479.2243 (Calcd for C23H36O9Na, 479.2252).
1b-Hydroxytorilolone 11-O-b-D-Glucopyranoside (2): Amorphous pow-
der; [a]D22 ꢀ24.9° (cꢁ0.4, MeOH); UV (MeOH) lmax: 240 nm; 1H-NMR
(CD3OD, 500 MHz) and 13C-NMR (CD3OD, 125 MHz), see Table 1; posi-
tive-ion HR-ESI-MS m/z 453.2078 (Calcd for C21H34O9Na, 453.2095).
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