50
C.C.S. Santos et al. / Phytochemistry 140 (2017) 45e51
absorbance at 734 nm was calculated for each concentration rela-
tive to a blank absorbance (methanol) and was plotted as a function
of concentration of compound or standard, 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid (Trolox, Aldrich Chemical
Co., Gillingham, Dorset, UK). The antioxidant activities are
expressed as TEAC values in comparison with TEAC activity of
quercetin 3-O-glucopyranoside, used as reference compound. The
TEAC value is defined as the concentration of a standard Trolox
solution with the same antioxidant capacity as a 1 mg/mL of the
tested extract.
semipreparative HPLC using MeOH-H
(flow rate 2.5 mL/min) to yield compounds 12 (5.2 mg,
2
O (3:7) as mobile phase
t
R
¼ 26.0 min) 13 (5.9 mg, t
¼ 36.2 min), Fractions 90e91 (65.9 mg) were chromatographed
O (3:7) as mobile phase
(flow rate 2.5 mL/min) to yield compound (10.1 mg,
¼ 10.0 min).
R
¼ 30.0 min) and 15 (5.7 mg,
t
R
by semipreparative HPLC using MeOH-H
2
1
t
R
0
3.8. Vanillyl alcohol 4-O-b-D-(6 -O-galloyl) glucopyranoside (8)
KBr
ꢀ1
Amorphous white solid; C21
1785, 1660; H and C NMR (methanol-d , 600 MHz) data, see
H
24
O
12; IR max cm : 3450, 2930,
n
1
13
3.6. LC-ESI(Orbitrap)MS analysis
4
-
23 12
Table 2; HRESIMS m/z 467.1190 [M-H] (calcd for C21H O ,
Qualitative LC-MS was performed using a Thermo Scientific
467.1173).
Accela HPLC system (Thermo Scientific, Germany) equipped with
a C18 reversed-phase (RP) column (2.1 ꢁ 250 mm; X-Terra MS C18
0
3.9. 2-Hydroxy-4-methoxyphenol 1-O-
glucopyranoside (10)
b
-D-(6 -O-galloyl)
5
lm; Waters, Milford, MA) at a flow rate of 0.2
m
l/min and
coupled to a LTQ-Orbitrap XL mass spectrometer. The gradient
elution was carried out by using 0.1% formic acid as eluent A and
acetonitrile as B. The HPLC gradient started at 5% (B) hold for
KBr
ꢀ1
Amorphous white solid; C20
H
22
O12; IR
n
max cm : 3430, 2945,
, 600 MHz) data, see
1
13
1765, 1675; H and C NMR (methanol-d
4
-
5
min and after 50 min, %B was at 100% holding it for 5 min
21 12
Table 2; HRESIMS m/z 453.1033 [M-H] (calcd for C20H O ,
before returning back to the starting percentage. The instrument
was calibrated using the manufacturer's calibration standards.
The scan was collected in the Orbitrap at a resolution of 30 000
in a m/z range of 230e1500 amu. The m/z of each identified
compound was calculated to 4 decimal places and measured with
a mass accuracy <3.5 ppm. The source voltage was 4.0 kV and
capillary voltage ꢀ35 kV, the tube lens offset ꢀ126 V and the
453.2500).
0
3.10. Dihydrosinapyl alcohol 4-O-(6 -O-galloyl)-
glucopyranoside (11)
b
-D-
KBr
ꢀ1
Amorphous white solid; C24
1750,1685,1130; H and C NMR (methanol-d
Table 2; HRESIMS m/z 525.1608 [M-H] (calcd for C24H O ,
29 13
H
30
O
13; IR
n
max cm : 3420, 2950,
, 600 MHz) data, see
1
13
4
ꢂ
-
capillary temperature was set at 280 C, the auxiliary gas was set
at 10 (arbitrary units) and the sheath gas at 20 (arbitrary units).
In full LC-ESIMS experiments Total Ion Current (TIC) profile was
produced by monitoring the intensity of all the ions produced
and acquired in every scan during the chromatographic run. In
order to get structural information, Data Dependent experiments
525.1587).
0
3.11. 4,9-Dihydroxypropiophenone-9-O-(6 -O-galloyl)-
glucopyranoside (12)
b
-D-
2
KBr
ꢀ1
were performed by acquiring MS spectra of the most intense
ions produced during the acquisition.
Amorphous white solid; C22
1770, 1715, 1680, 1200; H and C NMR (methanol-d
data, see Table 2; HRESIMS m/z 479.1190 [M-H] (calcd for
C H O12, 479.1173).
22 23
24
H O12; IR
13
n
max cm : 3410, 2930,
, 600 MHz)
1
4
-
3.7. Isolation procedure
0
The hydroalcoholic extract was dried under vacuum and 3 g
3.12. 3,4,5- trimethoxybenzyl alcohol 7-O-(6 -O-galloyl)-b-D-
were fractionated on a Sephadex LH-20 (Pharmacia) column
glucopyranoside (14)
(
(
100 ꢁ 5 cm), using MeOH as mobile phase, affording 91 fractions
KBr
ꢀ1
8 mL), monitored by TLC.
Amorphous white solid; C23
28
H O
13
13; IR
n
max cm : 3400, 2925,
, 600 MHz)
1
Fraction 14e18 (111.0 mg) were chromatographed by semi-
1775, 1720, 1685, 1135; H and C NMR (methanol-d
data, see Table 2; HRESIMS m/z 511.1452 [M-H] (calcd for
C H O13, 511.1434).
23 27
4
-
preparative HPLC using MeOH-H
.5 mL/min) to yield compounds 3 (6.3 mg, t
5.8 mg, t ¼ 48.0 min). Fractions
¼ 10.0 min) and 16 (8.2 mg, t
2
O (3:7) as mobile phase (flow rate
2
(
1
R
¼ 4.8 min), 11
R
R
9e25 (134.3 mg) were chromatographed by semipreparative HPLC
3.13. Acid hydrolysis
using MeOH-H
2
O (3:7) as mobile phase (flow rate 2.5 mL/min) to
¼ 10.5 min) and 14 (10.2 mg,
¼ 46.0 min). Fractions 26e31 (130.9 mg) were chromatographed
by semipreparative HPLC using MeOH-H O (1:3) as mobile phase
flow rate 2.5 mL/min) to yield compounds (6.6 mg,
¼ 16.2 min) and 6 (5.7 mg,
¼ 15.8 min). Fractions 31e32 (103.5 mg) were chromatographed
O (3:7) as mobile phase
flow rate 2.5 mL/min) to yield compound 10 (4.8 mg,
¼ 11.5 min). Fractions 37e39 (31.8 mg) were chromatographed
O (3:7) as mobile phase
flow rate 2.5 mL/min) to yield compound 9 (5.0 mg, t
¼ 10.4 min).
Fraction 48 (32.4 mg) were chromatographed by semipreparative
HPLC using MeOH-H O (3:7) as mobile phase (flow rate 2.5 mL/
min) to yield compounds 2 (5.5 mg, t
¼ 9.0 min) and 7 (9.4 mg,
¼ 43.0 min).
Fractions 64e74 (105.9 mg) were chromatographed by
yield compounds 8 (8.6 mg, t
R
A mixture of compounds 8 (5 mg),10 (3 mg),11 (3 mg),12 (3 mg)
and 14 (5 mg) was heated at 60 C with 1:10.5 N HCl-dioxane (3 mL)
for 2 h, and then evaporated in vacuo. The solution was partitioned
ꢂ
t
R
2
(
t
t
4
with CH
MB-3. The H
silica gel column, using CHCl
2
Cl
2
-H
O layer was then concentrated and passed through a
-MeOH-H O (7:1:1.2, lower layer) as
2 2
O, and the H O layer was neutralized with Amberlite
R
R
¼ 10.0 min), 5 (5.1 mg, t
R
2
3
2
by semipreparative HPLC using MeOH-H
2
eluting solvent to afford glucose. The D configuration of glucose was
established by comparison of its optical rotation value with those
reported in the literature (Horo et al., 2015). The optical rotation was
(
t
R
by semipreparative HPLC using MeOH-H
(
2
determined after dissolving the sugars in H
2
O and allowing them to
2
2
R
stand for 24 h; D-glucose [a]
D
þ47.9 (c 0.1).
2
3.14. Cancer cell lines
R
t
R
Human alveolar basal carcinoma (A549), obtained from the
European Collection of Cell Cultures (ECACC), and HeLa cells