6
D.Y. Lee et al. / Phytochemistry xxx (2017) 1e8
(25:75, 50:50, 75:25, 100:0, each 8 L) to give four sub-fractions
(B1eB4).
with CH3CN:H2O with 0.1% HCO2H (25:75, v/v), to afford 1 (25 mg)
and 2 (9 mg), respectively. Subfr. 6 was applied to Sephadex LH-20
column eluted with MeOH:H2O (1:1, v/v) to acetone-H2O (8:2, v/v,
linear gradient) to give 7 subfractions. Subfr. 6-5 and 6-6 were
purified by semi-preparative HPLC, eluted with CH3CN/H2O with
0.1% HCO2H (21:79, v/v), to give 7 (65 mg) and 5 (9 mg) and 8
(13 mg), respectively.
B1 fraction (40 g out of 75 g) was fractionated via an MPLC,
eluted with CH3CN:H2O with 0.1% HCO2H (0:100 / 25:75, v/v,
linear gradient), to yield 6 subfractions. Subfr. 1 (2.7 g) was sepa-
rated on a semi-preparative HPLC column (CH3CN:H2O with 0.1%
HCO2H (1:99)) to give 22 (150 mg). Subfr. 2 and 5 were recrystal-
lized using H2O to afford 25 (2.0 g) and 31 (800 mg), respectively.
Subfr. 3 (3.2 g) was separated on semi-preparative HPLC column
(CH3CN/H2O with 0.1% HCO2H (5:95)) to give 35 (4 mg), 40 (15 mg),
24 (33 mg), and 19 (29 mg). Subfr. 4 (6.3 g) was further fractionated
via an MPLC, eluted with CH3CN:H2O with 0.1% HCO2H
(3:97 / 25:75, v/v, linear gradient), to yield 4 subfractions. Subfr.
4-1 was separated on semi-preparative HPLC column using
CH3CN:H2O with 0.1% HCO2H (8:92) to give 42 (4 mg), 15 (29 mg),
and 44 mg (10 mg). Subfr. 4-2 and 4-3 were recrystallized using
H2O to afford 48 (55 mg) and 30 (30 mg), respectively. Subfr. 4-4
was separated on a Sephadex LH-20 column using MeOH:H2O (1:1,
v/v) to acetone:H2O (8:2, v/v, linear gradient) and purified by semi-
preparative HPLC (CH3CN:H2O with 0.1% HCO2H (7:93 / 11:89, v/v,
linear gradient)) to give 9 (14 mg), 20 (70 mg). 37 (150 mg), 38
(700 mg), and 41 (10 mg). Subfr. 6 (20.3 g) was subjected to an ODS
C18 MPLC column, eluted with CH3CN:H2O with 0.1% HCO2H
(15:85 / 25:75, v/v, linear gradient), to 3 subfractions. Subfr. 6-1
was purified by semi-preparative HPLC using MeOH/H2O with 0.1%
HCO2H (79:21, v/v) to give 33 (14 mg) and 34 (11 mg). Recrystalli-
zation of subfr. 6-2 and 6-3 from H2O yielded 34 (140 mg) and 21
(10 g), respectively.
The B2 fraction (50 g out of 250 g) was subjected to MPLC eluted
with a CH3CN:H2O with 0.1% HCO2H linear gradient (5:95 to 30:70,
v/v) to yield 6 subfractions. Subfr. 2 (8.2 g) was further fractionated
via an MPLC eluted with CH3CN/H2O with 0.1% HCO2H
(5:95 / 25:75, v/v), to yield 5 subfractions. Subfr. 2-1 was sepa-
rated on semi-preparative HPLC column using CH3CN:H2O with
0.1% HCO2H (6:94, v/v) to give 27 (51 mg) and 28 (32 mg). Subfr. 2-2
was separated on semi-preparative HPLC column using CH3CN:H2O
with 0.1% HCO2H (7:93, v/v) to afford 29 (20 mg) and 36 (22 mg).
Subfr. 2-3 yielded 23 (8 mg) and 11 (110 mg) after separation on
semi-preparative HPLC column using CH3CN:H2O with 0.1% HCO2H
(9:91, v/v). Subfr. 2-4 yielded 45 (90 mg), 46 (9 mg), and 47
(120 mg) after separation on semi-preparative HPLC column using
CH3CN:H2O with 0.1% HCO2H (6:94 to 10:90, v/v, linear gradient).
Subfr. 2-5 was purified by semi-preparative HPLC, eluted with a
CH3CN/H2O with 0.1% HCO2H (12:88), to afford 13 (14 mg). Subfr. 4
(2.5 g) was separated on semi-preparative HPLC column using
CH3CN:H2O with 0.1% HCO2H (11:89, v/v) to afford 18 (70 mg) and
39 (18 mg). Subfr. 5 (6.7 g) was further fractionated by Sephadex
LH-20 CC (MeOH-H2O 1:1 to acetone-H2O 8:2, v/v, linear gradient)
to give 7 subfractions. Of these, subfr. 5-3 gave 26 (50 mg). Subfr. 5-
4.3.1. 1,2,3-Tri-O-galloyl-6-O-cinnamoyl-
b
-
D
-glucose (1)
Brown amorphous powder; ½a D20
ꢁ
þ52.4 (c 0.10, MeOH); UV
(MeOH) lmax (log ε) 217 (4.9) and 280 (4.7) nm; IR nmax: 3412, 1707,
1617, 1532, 1455, 1321, 1205, 1031, 760, 674 cmꢂ1; For 1H NMR
(acetone-d6, 800 MHz) and 13C NMR (acetone-d6, 200 MHz) spec-
troscopic data, see Table 1; HRMS (ESI-TOF) m/z [M e H]- 765.1323
(calcd. for C36H29O19, 765.1303).
4.3.2. 1,2,3,6-Tetra-O-galloyl-4-O-cinnamoyl-b-D-glucose (2)
Brown amorphous powder; ½a D20
þ28.4 (c 0.10, MeOH); UV
ꢁ
(MeOH) lmax (log ε) 218 (4.9) and 281 (4.7) nm; IR nmax: 3411, 1708,
1617, 1532, 1455, 1321, 1208, 1031, 759, 670 cmꢂ1; For 1H NMR
(acetone-d6:D2O
¼
9:1, 600 MHz) and 13C NMR (acetone-
d6:D2O ¼ 9:1, 150 MHz) spectroscopic data, see Table 1; HRMS (ESI-
TOF) m/z [M e H]- 917.1425 (calcd. for C43H33O23, 917.1413).
4.3.3. 4-O-(200,400-di-O-galloyl-
a-L-rhamnosyl)ellagic acid (5)
Brown amorphous powder; ½a D20
ꢁ
-37.4 (c 0.10, MeOH); UV
(MeOH) lmax (log ε) 219 (4.6) 258 (4.5) and 356 (3.9) nm; IR nmax
:
3364, 2945, 1708, 1615, 1448, 1347, 1213, 1028, 758, 677 cmꢂ1; For
1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz)
spectroscopic data, see Table 1; HRMS (ESI-TOF) m/z [M e H]-
751.0786 (calcd. for C34H23O20, 751.0782).
4.4. Acid hydrolysis of 13 and 14
A solution of 13 in 1N H2SO4 (2 mg in 10 ml) was heated under
reflux for 12 h. After cooling, the resulting solution was neutralized
with saturated aqueous Na2CO3 solution. The precipitates formed
were collected by filtration and dissolved in MeOH. The product
was identified as ellagic acid (43) by TLC comparison under UV light
(254 nm) with an authentic sample on a silica gel TLC plate, and the
plate was developed with a mobile phase of EtOAc-toluene-HCO2H
(5: 5: 2.5, v/v, Rf 0.30). The filtrate was analyzed by analytical HPLC
(solvent system: aqueous 0.1% HCO2H (A) and MeCN (B), gradient
condition: 1%e10% B in 20 min, 1 ml/min) to detect chebulic acid
(22, tR 7.6 min) and gallic acid (25, tR 14.0 min), which were iden-
tified by comparing the retention time with authentic samples.
Parallel studies were carried out with 14 (Supplementary
information, Fig. S12).
4
was separated on semi-preparative HPLC column using
CH3CN:H2O with 0.1% HCO2H (15:85, v/v) to give 14 (55 mg) and 16
(50 mg). Subfr. 5-4 yielded 12 (17 mg), 10 (16 mg), and 17 (15 mg)
after separation on semi-preparative HPLC column using
CH3CN:H2O with 0.1% HCO2H (15:85 to 17:83, v/v, linear gradient).
Subfr. 5-5 was recrystallized using MeOH:H2O (1:3, v/v) to afford 6
(6 mg).
B3 fraction (20 g) was fractionated via MPLC, eluted with
MeOH:H2O with 0.1% HCO2H (20:80 / 50:50, v/v, linear gradient),
to yield 7 subfractions. Recrystallization of subfr. 4 (1.5 g) from
MeOH yielded 43 (1.2 g) and its filtrate yielded 3 (14 mg) and 4
(11 mg) after separation on semi-preparative HPLC column using
CH3CN/H2O with 0.1% HCO2H (17:83, v/v). Subfr. 5 was passed
through a Sephadex LH-20 column eluted with MeOH:H2O (1:1, v/
v) to acetone:H2O (8:2, v/v, linear gradient), to afford 9 subfractions.
Subfr. 5-7 and 5-8 was purified by semi-preparative HPLC, eluted
4.5. Absolute configuration determination of monosaccharides for
1, 2, 5, 9, 10, 13, 14, 16, and 17
Compounds 1, 2, 5, 9, 10, 13, 14, 16, and 17 (each 0.5 mg) were
individually hydrolyzed by 1 N H2SO4 (1 ml) heated at 100 ꢃC for 2 h
and neutralized with saturated aqueous Na2CO3 solution. After the
solution was dried in vacuo, each residue was dissolved in pyridine
(0.2 ml) containing
L
-cysteine methyl ester hydrochloride (1.0 mg)
and heated at 60 ꢃC for 1 h. 1
mL (1.1 mg) of o-torylisothiocyanate
was added to the mixtures, which were heated at 60 ꢃC for 1 h. Each
final mixture was directly analyzed by analytical RP-HPLC (solvent
system: aqueous 0.1% HCO2H (A) and MeCN (B), gradient condition:
20%e40% B in 40 min, 1 mL/min). The tR of the peak at 21.0 and
27.4 min coincided with that of the thiocarbamoyl thiazolidine
derivatives of D-glucose and L-rhamnose, respectively.
Please cite this article in press as: Lee, D.Y., et al., Hydrolyzable tannins from the fruits of Terminalia chebula Retz and their a-glucosidase