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H. Zhang et al. / Phytochemistry Letters 10 (2014) 164–167
3. Experimental
3.1. General experimental procedure
Optical rotation was determined on a Rudolph Research
Analytical Autopol II automatic polarimeter. IR spectra were
recorded on a Bruker Tensor 27 spectrometer using KBr disks. NMR
spectra were recorded on a Bruker AVANCE DRX-500 spectrometer
and a Varian INOVA 500 MHz spectrometer. HRMS spectra were
made on a Varian IonSpec FT 7.0 T mass spectrometer. All solvents
used were of analytical grade (Tianjin Jiangtian Chemical
Technology Co., Ltd.). Silica gel (100–200 and 200–300 mesh,
Qingdao Haiyang Chemical Co., Ltd.), D101 macroporous resin
Fig. 3. Selected HMBC correlations for compound 2.
Asano et al., 1993). Its 1H NMR and 13C NMR spectra revealed the
presence of two angular methyl groups at dH 0.94 (s), 1.27 (s) and
(Tianjin Haiguang Chemical Co., Ltd.), ODS Silica gel (40–63 mm,
d
C 16.1, 14.6. Evidence for the presence of a methyl ketone and two
double bonds at C-5 and C-16 came from a three-proton singlet at dH
2.20 and two vinylic proton signals at H 5.60 and
H 6.56 in the 1H
NMR of 3, which showed a close similarity to that of l ,3
LiChroprep RP-18, Merck KGaA) and Sephadex LH-20 (Amersham
Pharmacia Biotech AB) were used for open-column chromatogra-
phy. TLC was carried out using precoated plates with GF254 silica
gel (Qingdao Haiyang Chemical Co., Ltd.). Spots on TLC were
visually observed under UV light and by spraying with 5% sulfuric
acid in alcohol reagent followed by heating. Preparative HPLC was
performed using ODS columns (Agilent ZORBAX SB-C18,
d
d
b
b-
dihydroxy-5,16-pregnadien-20-one (Gamboa-Angulo et al., 1996),
suggesting that 3 had a pregnane skeleton. Full assignments of the
protons and carbons (Table 2) of 3 were accomplished by analysis of
1H NMR, 13C NMR, COSY, HSQC, HMBC and NOESY spectra. The
obviousHMBCcorrelation between H-19and C-1confirmed thatthe
21.2 mm ꢁ 250 mm, 7
mm).
carbon signal at
d
C 84.9 was designated to C-1 in the ring A, and the
H 4.75 was assigned to H-1 by its HSQC correlation
C 84.9 (C-1) of 3 was 7.3 ppm higher than dC
-dihydroxy-5,16-pregnadien-20-one (Gamboa-
Angulo et al., 1996), but similar to dC 85.5 (C-1) of ruscogenin 1-
sulfate (Asano et al., 1993), C 84.1 (C-1) of spirost-5-en-1 ,3 -diol
1-sulfate (Oulad-Ali et al., 1996), and dC 85.1 (C-1) of spirost-
5,25(27)-dien-1 ,3 -diol 1-sulfate from the title plant (Yang et al.,
2010b), confirming the sulfate group at C-1. In the NOESY plot, the
intensecorrelationsbetweenH-1( H 4.75) and H-3 ( H 3.90) verified
the -configurations of the oxygenated substituents at C-1 and C-3.
The 1H and 13C NMR signals of A/B ring and Me-19 were identical to
those of spirost-5-en-1 ,3 -diol 1-sulfate (Oulad-Ali et al., 1996)
and spirost-5,25(27)-dien-1 ,3 -diol 1-sulfate from the title plant
(Yang et al., 2010b), further verifying one sulfate connected to C-1,
one hydroxy at C-3 and their -configurations. Based on the above
evidence, compound 3 was indicated to be 3 -hydroxyl-pregna-
5,16-diene-20-one-1 -yl sulfate.
3.2. Plant material
proton signal at
d
with C-1. The signal at
77.6 (C-1) of l ,3
d
The roots and rhizomes of H. thibetanus were collected from Mei
County, Shaanxi Province in the People’s Republic of China in
September 2007 and were authenticated by Prof. Zhen Hai Wu. A
voucher specimen (S200609002) is deposited in School of
Pharmaceutical Science and Technology, Tianjin University, Tianjin
300072, People’s Republic of China.
b
b
d
b b
b
b
d
d
3.3. Extraction and isolation
b
The dried roots and rhizomes of H. thibetanus (8.0 kg) were
pulverized and refluxed with 95% (v/v) EtOH twice (each time 6 l)
and then 60% (v/v) EtOH once (6 l). The combined extracts were
concentrated to give a residue (1.5 kg) which was suspended in
water to a final volume of 5 l and then partitioned sequentially
with petroleum ether (PE) (60–90 8C), CHCl3, EtOAc, and n-BuOH.
The CHCl3 extract (34.7 g) was applied to silica gel column
chromatography with a step gradient of PE–Me2CO (9:1 to 6:4, v/
v), producing fractions 75–76 repeatedly followed by silica gel
column chromatography (CHCl3–Me2CO, 7:3 to 5:5, v/v and
CHCl3–MeOH, 98:2 to 9:1, v/v) and purified by Sephadex LH-20
(MeOH), compound 1 (30 mg) was obtained by further recrystalli-
zation with Me2CO.
b
b
b
b
b
b
b
In addition to the three new steroids (compounds 1–3), one
previously known compound (4) was also isolated and identified
by the spectroscopic data and physical data as pregna-5,16-diene-
20-one-3
b
-yl O-
a
-L-rhamnopyranosyl-(1!2)-O-[
b-D-glucopyra-
nosyl-(1!4)]-
b
-D-glucopyranoside, which was identical to the
structure reported in the patent (Li and Shen, 2012).
Table 2
1H (500 MHz), 13C (125 MHz) NMR spectroscopic data of compound 3 (pyridine-d5)a
No.
dH
dC
No.
dH
dC
1
4.75, dd (12.0, 4.5)
84.9
11ax
11eq
12ax
12eq
13
1.74
23.6
2axb
2eqc
3
2.24, ddd (12.0, 11.5, 11.5)
39.6
–
3.02, m
1.60
–
3.43, m
35.8
–
3.99, dddd (5.0,5.0,10.5,12.5)
67.8
43.6
–
2.50, m
–
4ax
4eq
5
2.56, m
45.9
56.5
32.4
144.3
155.7
16.1
14.6
196.2
27.1
2.65, m
14
1.37, m
2.10, m; 1.89
6.56, brs
–
–
139.0
125.3
31.4
31.6
50.4
43.2
15
6
5.60, brd (5.5)
16
7
1.85,1.56
1.53
17
8
18
0.94, s
1.27, s
–
9
1.73
19
10
–
20
21
2.20, s
a
Full assignments of the protons and carbons were accomplished by analysis of COSY, HSQC and HMBC spectra, and coupling pattern and coupling constants (J in Hz) are in
parentheses. Overlapped signals were given without designating multiplicity.
b
ax = axial.
c
eq = equatorial.