W. Li et al. / Fitoterapia 98 (2014) 248–253
249
Fig. 1. Structures of compounds 1 and 2.
macroporous resin D101 (Qingdao Haiyang Chemical Co. Ltd.,
China), Sephadex LH-20 (Pharmacia, U.S.A.), and RP-C18 (40–
63 μm, YMC, Japan) were used for column chromatography. The
pre-coated silica gel GF254 plates (Qingdao Marine Chemical
Plant, China) were used for TLC. Spots were visualized by
heating silica gel plates immersed in vanillin-H2SO4 in ethanol.
All solvents used were of analytical grade (Jiangsu Hanbon
Science and Technology Co. Ltd., China).
16.5 min) and 2 (24 mg, tR = 13.5 min) using MeOH:H2O
(90:10, v/v).
2.3.1. Salvianolic acid T (1)
Yellowish amorphous powder; [α]2D5 + 196.6 (c = 0.25,
MeOH); UV (MeOH) λ max nm: 220, 326; IR (KBr) νmax cm−1
:
3150, 2878, 2817, 1693, 1600, 1519, 1445, 1360, 1250, 1115,
968, 865, 812; negative HR-ESI-MS [M–H]− m/z: 537.1027
(Calcd for C27H21O1−2, 537.1038); 1H and 13C NMR data, see
Table 1.
2.2. Plant material
The roots of S. miltiorrhiza (batch number: 20100301)
were acquired in March 2010 from Shaanxi Tasly Plant
Pharmaceutical Co., Ltd. (Shaanxi, China), which is Good
Agricultural Practices (GAP) base for the agriculture and
process of S. miltiorrhiza licensed by the State Food and Drug
Administration, P. R. China (SFDA). A voucher specimen (No:
20100318) has been deposited in the Modern TCM Depart-
ment, Tasly R&D Institute.
2.3.2. Salvianolic acid U (2)
Yellowish amorphous powder; [α]2D5 − 157.6 (c = 0.16,
MeOH); UV (MeOH) λ max nm: 225, 325; IR (KBr) νmax cm−1
:
3306, 2881, 2822, 1687, 1602, 1519, 1445, 1360, 1250, 1113,
969, 865, 812; negative HR-ESI-MS [M–H]− m/z: 537.1034
(Calcd for C27H21O1−2, 537.1038); 1H and 13C NMR data, see
Table 1.
2.4. Hydrolysis reaction
2.3. Extraction and isolation
Compounds 1 and 2 (10 mg) are respectively stirred in
hydrochloric acid (5 ml, 6 M), at water bath (40 °C). The
reaction is stirred until completion (high performance liquid
chromatography, 12 h) then quenched with water, extracted 3
times with an equal volume of ethyl acetate, the combined
organic phases, concentration in rotary evaporator is done at
30 °C.
Chromatographic conditions: use Agilent Eclipse XDB-C18
(4.6 mm × 250 mm, 5 μm) as the stationary phase and 0.2%
formic acid-acetonitrile as mobile phase as specified in the
following table gradient elution; flow rate: 1 ml/min; The wave
length of the detection is 280 nm and the column temperature
is 25 °C; injection volume 20 μl.
Air-dried roots of the plant material (20 kg) were
extracted with 95% EtOH under reflux for 3 h. The EtOH
extract (2 kg) was suspended in water and partitioned with
CHCl3 (210 g), EtOAc (450 g) and n-BuOH (190 g). The EtOAc
extract was chromatographed over a D101 macroporous
resin column (15 × 60 cm), eluted successively with H2O, 30%,
50%, 80%, and 95% EtOH in H2O. The 80% EtOH fraction was
chromatographed on silica gel column eluted with CHCl3:
MeOH (100: 0–60: 40, v/v) to obtain eight fractions (A–H).
Fraction D (20 g) was applied to a silica gel column eluted with
CHCl3:MeOH:HCOOH (95:5:0–60:38:2, v/v/v) to give four
fractions (DA–DD). Fraction DC (2.5 g) was separated over silica
gel column eluted with CHCl3:MeOH:HCOOH (90:10:3–
40:10:0.5, v/v/v) to afford three fractions (DCA–DCC). Fraction
2.5. Preparation of fragment I by hydrolysis degradation
DCB (200 mg) was separated over RP-C18 column eluted with
MeOH:H2O:HCOOH (60:40:0.3–90:10:0.5, v/v/v) to afford
three fractions (DCBA–DCBD). Fraction DCBD (60 mg) was
purified by Sephadex LH-20 with CHCl3:MeOH (50:50, v/v)
To determine the hydrolysis reaction mixture, major peaks
in the chromatogram attribution were scanned by mass
spectrometry. Centralized collection of degraded fragment I
(m/z = 197) eluent, and extracted three times with an equal
and preparative HPLC to afford compounds 1 (11 mg, tR
=