dry extract (462 g), a portion (400 g) of which was suspended in H O and fractionated using solvent extraction by hexane and
2
®
n-BuOH. The BuOH fraction was concentrated to dryness (215 g), placed on a column with Amberlite XAD1180N (800 g),
and eluted with H O and EtOH (40 and 90%) to afford fractions PEXAD-01 (142 g), PEXAD-02 (48 g), and PEXAD-03
2
TM
(19 g), respectively. Fraction PEXAD-01 (140 g) was placed on a column with Superlite DAX-8 (600 g) and eluted with
H O to afford fraction PEXAD-01-1 (92 g), which was separated over a column of Sepabeads SP-20SS (500 g) using an
®
2
H O–Me CO gradient (100:0ꢄ40:60). Fractions eluted by H O and Me CO (10%) were placed on Sephadex LH-20
2
2
2
2
®
(CC, 2 ꢂ 90 cm, H O–Me CO eluent, 100:0ꢄ30:70) and on Toyopearl HW-75F (CC, 500 mL, H O–Me CO eluent,
2
2
2
2
100:0ꢄ50:50) to isolate 1 (93 mg), 2 (54 mg), mucic acid 2-O-gallate (7, 437 mg), mucic acid 2-O-gallate 1,4-lactone
(8, 118 mg), mucic acid 5-O-gallate 1,4-lactone (9, 127 mg) [4], gallic acid (3, 10.47 g) [9], glucogallin (4, 1-O-galloyl-ꢀ-D-
glucose, 1.64 g), 1,6-di-O-galloyl-ꢀ-D-glucose (5, 14 mg), and 3,4,6-tri-O-galloyl-ꢀ-D-glucose (6, 52 mg) [10]. Fraction
PEXAD-02 (40 g) was separated over Sephadex LH-20 (CC, 2 ꢂ 50 cm, H O–Me CO eluent, 100:0ꢄ30:70) and RP-SiO
2
2
2
(CC, 2 ꢂ 40 cm, H O–MeCN eluent, 100:0ꢄ0:100) and by preparative HPLC (conditions 1) to isolate chebulic acid (10,
2
29 mg), chebulanin (11, 118 mg), chebulagic acid (12, 153 mg), chebulinic acid (13, 34 mg) [11], corilagin (14, 37 mg) [12],
ellagic acid (15, 573 mg) [13], and isoquercitrin (16, 9 mg) [14]. Fraction PEXAD-03 contained polymeric phenolic constituents
(probably procyanidins) and was not investigated further.
Mucic Acid 2,5-Di-O-gallate (1). C H O . UV spectrum (ÌåÎÍ, ꢇ , nm): 209, 275. FAB-ÌÑ, m/z: 513
20 18 16
max
–
–
–
– 1
[M – H] , 361 [(M – galloyl) – H] , 209 [(M – galloyl ꢂ 2) – H] , 191 [(M – galloyl ꢂ 2 – H O) – H] . Í NMR spectrum
2
(500 MHz, ÌåÎÍ-d , ꢁ, ppm, J/Hz): mucoyl: 4.39 (1H, dd, J = 2.1, 9.7, Í-4), 4.81 (1H, dd, J = 2.0, 9.7, Í-3), 5.60 (1H, d, J = 2.1,
4
13
Í-5), 5.87 (1H, d, J = 2.0, Í-2); 2-Î-galloyl: 7.15 (2Í, s, Í-2ꢈ, 6ꢈ); 5-Î-galloyl: 7.27 (2Í, s, Í-2ꢈꢈ, 6ꢈꢈ). C NMR spectrum
(125 MHz, ÌåÎÍ-d , ꢁ, ppm): mucoyl: 71.7 (C-3), 72.1 (C-4), 72.9 (C-5), 74.5 (C-2), 170.3 (C-6), 170.8 (C-1); 2-Î-galloyl:
4
110.2 (C-2ꢈ, 6ꢈ), 121.0 (C-1ꢈ), 139.7 (C-4ꢈ), 146.3 (C-3ꢈ, 5ꢈ), 167.0 (C-7ꢈ); 5-Î-galloyl: 110.2 (C-2ꢈꢈ, 6ꢈꢈ), 121.5 (C-1ꢈꢈ), 139.3
(C-4ꢈꢈ), 146.0 (C-3ꢈꢈ, 5ꢈꢈ), 166.4 (C-7ꢈꢈ).
Hydrolysis of 1. A solution of 1 (10 mg) in H O (1 mL) was incubated with tannase (10 U, from Aspergillus ficuum,
2
Sigma, 150 U/g) at 37°C for 10 h. The resulting mixture was concentrated to dryness in vacuo, dissolved in MeOH (1 mL),
and analyzed by HPLC (conditions 1). Gallic acid (t 3.12 min) was detected in the hydrolysate. Then, the solution was
R
treated with MeI—Na CO (0.5 mL/200 ꢅg) at 25°C for 30 min. The reaction mixture was placed on an SiO column (1 ꢂ 15 cm)
2
3
2
and eluted by CHCl –MeOH (100:0ꢄ50:50) to afford mucic acid dimethyl ester (2.5 mg) that was identified by PMR and
3
13
C NMR data and comparison with a sample prepared by methylation of mucic acid (Aldrich) in an analogous manner.
1
Mucic Acid Dimethyl Ester. Í NMR spectrum (500 MHz, Py-d , ꢁ, ppm): 3.61 (6Í, s, ÎÑÍ ), 5.12 (2H, s, Í-2, 5),
5
3
13
5.44 (2Í, s, Í-3, 4). C NMR spectrum (125 MHz, Py-d , ꢁ, ppm): 49.6 (ÎÑÍ ), 70.4 (Ñ-2, 5), 71.9 (Ñ-3, 4).
5
3
Mucic Acid 2,5-Di-O-gallate 1,4-Lactone (2). C H O . UV spectrum (ÌåÎÍ, ꢇ , nm): 210, 274. FAB-ÌÑ,
20 16 15
max
–
– 1
m/z: 495 [M – H] , 343 [(M – galloyl) – H] . Í NMR spectrum (500 MHz, ÌåÎÍ-d , ꢁ, ppm, J/Hz): mucoyl: 4.96 (1H, dd, J = 9.0,
4
8.4, Í-3), 5.03 (1H, dd, J = 2.0, 8.4, Í-4), 5.69 (1H, d, J = 2.0, Í-5), 6.02 (1H, d, J = 9.0, Í-2); 2-Î-galloyl: 7.19 (2Í, s, Í-2ꢈ, 6ꢈ);
13
5-Î-galloyl: 7.32 (2Í, s, Í-2ꢈꢈ, 6ꢈꢈ). C NMR spectrum (125 MHz, ÌåÎÍ-d , ꢁ, ppm): mucoyl: 72.4 (C-3), 73.4 (C-5), 75.9 (C-2),
4
79.5 (C-4), 169.8 (C-6), 171.5 (C-1); 2-Î-galloyl: 110.4 (C-2ꢈ, 6ꢈ), 121.0 (C-1ꢈ), 139.5 (C-4ꢈ), 146.5 (C-3ꢈ, 5ꢈ), 167.1 (C-7ꢈ);
5-Î-galloyl: 110.4 (C-2ꢈꢈ, 6ꢈꢈ), 121.4 (C-1ꢈꢈ), 139.1 (C-4ꢈꢈ), 146.2 (C-3ꢈꢈ, 5ꢈꢈ), 166.5 (C-7ꢈꢈ).
HPLC. Conditions 1: Summit liquid chromatograph (Dionex), LiChrospher PR-18 column (10 ꢂ 250 mm, # 10 ꢅm,
Merck), mobile phase H O (A) and MeCN (B), gradient mode (% B): 0–100 min, 0–70%; flow rate 2 mL/min, column
2
temperature 30°C, UV detector at ꢇ 280 nm. Conditions 2: A-02 Milikhrom microcolumn liquid chromatograph (Ekonova),
ProntoSIL-120-5-C18AQ column (2 ꢂ 75 mm, # 5 ꢅm, Metrohm AG), mobile phase LiClO (0.2 M) in HClO (0.01 M) (A)
4
4
and MeCN (B), gradient mode (% B): 0–10 min, 5–15, flow rate 150 ꢅL/min, column temperature 35°C, UV detector at ꢇ 274 nm.
Plant material was quantitatively analyzed on an HPLC-UV microcolumn. For this, plant material (40 mg) was
transferred to an Eppendorf tube (2 mL), treated with EtOH (60%, 1 mL), sonicated (50 kHz, 30 min, 40°C), and centrifuged
(6,000 g, 20 min). The resulting extract was filtered through a membrane filter (0.45 ꢅm) and used for the analysis (1 ꢅL).
Conditions: A-02 Milikhrom microcolumn liquid chromatograph (Ekonova), ProntoSIL-120-5-C18AQ column (2 ꢂ 75 mm,
# 5 ꢅm, Metrohm AG), mobile phase LiClO (0.2 M) in HClO (0.006 M) (A) and MeCN (B), gradient mode (% B):
4
4
(0–5 min, 2–5; 5–15 min, 5–7; 15–20 min, 7–15; 20–30 min, 15–30; 30–35 min, 30–50), flow rate 150 ꢅL/min, column
temperature 35°C, UV detector at ꢇ 254 nm.
The contents of pure constituents were calculated from calibration curves that were constructed using commercial
standards (gallic acid, ellagic acid, corilagin, all Sigma-Aldrich), isolated compounds ꢉ95% pure (glucogallin, 1,6-di-O-
galloyl-ꢀ-D-glucose, 3,4,6-tri-O-galloyl-ꢀ-D-glucose, chebulic acid, chebulanin, chebulagic acid, chebulinic acid, mucic acid
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