9514 J. Agric. Food Chem., Vol. 54, No. 25, 2006
Stark and Hofmann
MHz, MeOD, HMQC, HMBC) δ 28.2 [C-4], 60.6 [C-6′′′/C-6′′], 60.8
[C-6′′/C-6′′′], 66.0 [C-3], 69.7 [C-4′′′/4′′], 70.0 [C-4′′/C-4′′′], 72.1
[C-2′′′], 73.3 [C-2′′], 75.4 [C-1′′], 76.0 [C-1′′′], 78.08 [C-3′′/C-3′′′],
78.12 [C-3′′′/C-3′′], 78.6 [C-2], 80.8 [C-5′′/C-5′′′], 81.1 [C-5′′′/C-5′′],
99.9 [C-4a], 103.5 [C-8], 104.8 [C-6], 113.7 [C-2′], 114.6 [C-5′], 117.6
[C-6′], 130.8 [C-1′], 144.3 [C-3′/C-4′], 144.6 [C-4′/C-3′], 153.1 [C-8a],
154.1 [C-7], 154.6 [C-5].
Hz, H-C(6b′′)], 3.95-4.00 [m, 2H, H-C(3, 4′′)], 4.01 [dd, 1H, J )
9.6 Hz, H-C(2′′)], 4.57 [d, 1H, J ) 7.2 Hz, H-C(2)], 4.81 [d, 1H, J
) 10.0 Hz, H-C(1′′)], 5.94 [s, 1H, H-C(8)], 6.70 [dd, 1H, J ) 2.0,
8.4 Hz, H-C(6′′)], 6.76 [d, 1H, J ) 8.4 Hz, H-C(5′′)], 6.82 [d, 1H,
J ) 2.0 Hz, H-C(2′′)]; 13C NMR (100 MHz, MeOD, HMQC, HMBC)
δ 27.0 [C-4], 61.6 [C-6′′], 67.3 [C-3], 69.2 [C-4′′], 70.7 [C-2′′], 74.6
[C-3′′], 75.8 [C-1′′], 79.3 [C-5′′], 81.4 [C-2], 94.5 [C-8], 100.4 [C-4a],
104.6 [C-6], 113.8 [C-2′], 114.7 [C-5′], 118.6 [C-6′], 130.7 [C-1′],
144.85 [C-3′/C-4′], 144.86 [C-4′/C-3′], 154.5 [C-8a], 154.8 [C-7/C-5],
154.9 [C-5/C-7].
(-)-Epicatechin-6-C-â-D-glucopyranoside, 6 (Figure 3): UV-vis
(MeOH/0.1% HCOOH, pH 2.5; 2:8, v/v) λmax ) 218, 231, 280 nm;
1
MS (ESI-), m/z 451 (100%, [M - 1]-), 331 (20%, [M - 121]-); H
(-)-Epicatechin-8-C-â-D-galactopyranoside, 10 (Figure 3): UV-
vis (MeOH/0.1% HCOOH, pH 2.5; 2:8, v/v) λmax ) 218, 231, 280
nm; MS (ESI-), m/z 451 (100%, [M - 1]-), 331 (33%, [M - 121]-);
1H NMR (400 MHz, MeOD, COSY) δ 2.88 [ddd, 2H, J ) 2.0, 4.0,
16.4 Hz, H-C(4a,b)], 3.60 [ddd, 1H, J ) 1.6, 6.0 Hz, H-C(5′′)], 3.63
[ddd, 1H, Jae ) 3.0, 9.4 (aa) Hz, H-C(3′′)], 3.70 [dd, 1H, J ) 5.0, 11.4
Hz, H-C(6a′′)], 3.76 [dd, 1H, J ) 7.0, 11.4 Hz, H-C(6b′′)], 3.95 [d,
1H, J ) 2.8 Hz, H-C(4′′)], 4.15 [m, 1H, H-C(3)], 4.16 [dd, 1H, J )
8.8, Hz, H-C(2′′)], 4.89 [s, 1H, H-C(2)], 4.90 [d, 1H, J ) 8.8 Hz,
H-C(1′′)], 6.01 [s, 1H, H-C(6)], 6.78 [d, 1H, J ) 8.0 Hz, H-C(5′)],
6.80 [dd, 1H, J ) 2.0, 8.0 Hz, H-C(6′)], 7.03 [d, 1H, J ) 2.0 Hz,
H-C(2′)]; 13C NMR (100 MHz, MeOD, HMQC, HMBC) δ 28.3 [C-4],
61.6 [C-6′′], 66.0 [C-3], 69.1 [C-4′′], 70.9 [C-2′′′], 74.8 [C-3′′], 75.5
[C-1′′], 78.6 [C-2], 78.8 [C-5′′], 95.9 [C-6], 98.2 [C-4a], 103.5 [C-8],
113.7 [C-2′], 114.6 [C-5′], 117.5 [C-6′], 131.0 [C-1′], 144.3 [C-3′],
144.6 [C-4′], 153.2 [C-8a], 155.7 [C-7], 156.7 [C-5].
(-)-Epicatechin-6-C-â-D,8-C-â-D-digalactopyranoside, 11 (Figure
3): UV-vis (MeOH/0.1% HCOOH, pH 2.5; 2:8, v/v) λmax ) 218,
231, 280 nm; MS (ESI-), m/z 613 (100%, [M - 1]-), 493 (29%, [M
- 121]-), 331 (11%, [M - 283]-); 1H NMR (400 MHz, MeOD,
COSY) δ 2.77 [ddd, 2H, J ) 3.2, 4.4, 17.2 Hz, H-C(4a,b)], 3.51 [m,
4H, H-C(3′′, 3′′′, 5′′, 5′′′)], 3.63 [m, 4H, H-C(6′′a,b, 6′′′a,b)], 3.85
[d, 1H, J ) 2.8 Hz, H-C(4′′′)], 3.88 [dd, 1H, J ) 9.2, 9.6 Hz,
H-C(2′′)], 3.88 [d, 1H, J ) 2.8 Hz, H-C(4′′)], 4.06 [m, 1H, H-C(3)],
4.07 [dd, 1H, J ) 9.2 Hz, H-C(2′′′)], 4.76 [d, 1H, J ) 9.6 Hz,
H-C(1′′′)], 4.78 [s, 1H, H-C(2)], 4.78 [d, 1H, J ) 9.2 Hz, H-C(1′′′)],
6.67 [d, 1H, J ) 8.4 Hz, H-C(5′)], 6.70 [dd, 1H, J ) 1.6, 8.0 Hz,
H-C(6′)], 6.92 [d, 1H, J ) 1.6 Hz, H-C(2′)]; 13C NMR (100 MHz,
MeOD, HMQC, HMBC) δ 28.0 [C-4], 61.4 [C-6′′, C-6′′′], 65.9 [C-3],
69.0 [C-4′′/4′′′], 69.2 [C-4′′′/C-4′′], 70.6 [C-2′′′], 71.4 [C-2′′], 74.7
[C-3′′, C-3′′′], 75.5 [C-1′′, C-1′′′], 78.5 [C-2], 79.2 [C-5′′′, C-5′′], 99.2
[C-4a], 103.1 [C-8], 105.0 [C-6], 113.6 [C-2′], 114.6 [C-5′], 117.4
[C-6′], 130.8 [C-1′], 144.2 [C-3′/C-4′], 144.4 [C-4′/C-3′], 153.1 [C-8a],
153.8 [C-7, C-5].
(-)-Catechin: CD (MeOH) λmax(∆ꢀ) ) 232 (+1.91), 280 (+0.51);
UV-vis (MeOH/0.1% HCOOH, pH 2.5; 2:8, v/v) λmax ) 218, 231,
280 nm.
(+)-Catechin: CD (MeOH) λmax(∆ꢀ) ) 230 (-0.85), 280 (-0.26);
UV-vis (MeOH/0.1% HCOOH, pH 2.5; 2:8, v/v) λmax ) 218, 231,
280 nm.
(-)-Epicatechin: CD (MeOH) λmax(∆ꢀ) ) 239 (-1.41), 279 (-0.63);
UV-vis (MeOH/0.1% HCOOH, pH 2.5; 2:8, v/v) λmax ) 218, 231,
280 nm.
Identification of Flavan-3-ol-C-glycosides 1-11 in Cocoa Powder.
Nonalkalized or alkalized roasted cocoa powder (5.0 g) was extracted
with n-pentane (5 × 30 mL) at room temperature for 30 min. The
residual cocoa material was then extracted five times with acetone/
water (70:30, v/v; 30 mL each) for 45 min at room temperature with
stirring. After centrifugation, the liquid layer was freed from acetone
under reduced pressure at 30 °C and then freeze-dried to give the
acetone/water extract. Aliquots (∼50 mg) of the acetone/water extract
were taken up in a methanol/water mixture (1:1, v/v; 10 mL), which
was acidified to pH 2.5 with formic acid (0.1% in water). After
membrane filtration, aliquots (5 µL) were analyzed by means of HPLC-
MS/MS, which was equipped with a 150 × 2 mm i.d., 5 µm, RP
phenylhexyl column (Phenomenex) operated with a flow rate of 0.2
mL/min. Chromatography was performed starting with aqueous formic
acid (0.1%, pH 2.5), held for 10 min, and the methanol content was
increased to 40% in 40 min, increased to 100% within 10 min, and,
finally, held at 100% for 10 min. By means of the multiple-reaction
monitoring (MRM) mode, the individual flavan-3-ol-C-glycosides 1,
NMR (400 MHz, MeOD, COSY) δ 2.76 [dd, 1H, J ) 3.2, 16.4 Hz,
H-C(4a)], 2.89 [dd, 1H, J ) 4.4, 16.4 Hz, H-C(4b)], 3.43 [m, 1H,
H-C(5′′)], 3.50 [dd, 1H, J ) 8.4, 8.8 Hz, H-C(3′′)], 3.54 [dd, 1H, J
) 8.0, 8.8 Hz, H-C(4′′)], 3.69 [dd, 1H, J ) 8.8, 9.6 Hz, H-C(2′′)],
3.81 [dd, 1H, J ) 4.6, 12.2 Hz, H-C(6a′′)], 3.88 [dd, 1H, J ) 2.2,
12.2 Hz, H-C(6b′′)], 4.21 [m, 1H, J ) 3.2, 4.4 Hz, H-C(3)], 4.86 [s,
1H, H-C(2)], 4.88 [d, 1H, J ) 9.6 Hz, H-C(1′′)], 6.01 [s, 1H,
H-C(8)], 6.77 [d, 1H, J ) 8.4 Hz, H-C(5′)], 6.81 [dd, 1H, J ) 1.6,
8.4 Hz, H-C(6′)], 6.98 [d, 1H, J ) 1.6 Hz, H-C(2′)]; 13C NMR (100
MHz, MeOD, HMQC, HMBC) δ 27.8 [C-4], 60.7 [C-6′′], 66.1 [C-3],
69.8 [C-4′′], 73.2 [C-2′′], 76.1 [C-1′′], 78.2 [C-3′′], 78.5 [C-2], 81.1
[C-5′′], 95.2 [C-8], 100.0 [C-4a], 103.8 [C-6], 114.0 [C-2′], 114.5 [C-5′],
118.0 [C-6′], 130.7 [C-1′], 144.5 [C-3′/C-4′], 144.6 [C-4′/C-3′], 154.5
[C-8a], 155.1 [C-7/C-5], 155.1 [C-5/C-7].
(-)-Catechin-8-C-â-D-galactopyranoside, 7 (Figure 3): CD (MeOH)
max(∆ꢀ) ) 239 (+1.94), 283 (+1.15), 301 (-0.30); UV-vis (MeOH/
λ
0.1% HCOOH, pH 2.5; 2:8, v/v) λmax ) 218, 231, 280 nm; MS (ESI-),
m/z 451 (100%, [M - 1]-), 331 (35%, [M-121]-); 1H NMR (400 MHz,
MeOD, COSY) δ 2.56 [dd, 1H, J ) 6.6, 16.4 Hz, H-C(4a)], 2.70 [dd,
1H, J ) 5.2, 16.4 Hz, H-C(4b)], 3.51 [dd, 1H, Jae ) 3.0, 9.4 (aa) Hz,
H-C(3′′)], 3.58 [ddd, 1H, J ) 1.6, 5.2 Hz, H-C(5′′)], 3.68 [dd, 1H,
J ) 4.8, 11.2 Hz, H-C(6a′′)], 3.75 [dd, 1H, J ) 7.0, 11.2 Hz,
H-C(6b′′)], 3.90 [d, 1H, J ) 2.8 Hz, H-C(4′′)], 4.02 [ddd, 1H, J )
5.2, 6.4 Hz, H-C(3)], 4.10 [dd, 1H, J ) 9.2, 9.6, Hz, H-C(2′′)], 4.79
[d, 1H, J ) 6.0 Hz, H-C(2)], 4.82 [d, 1H, J ) 9.6 Hz, H-C(1′′)],
5.98 [s, 1H, H-C(6)], 6.67 [dd, 1H, J ) 2.0, 8.4 Hz, H-C(6′)], 6.74
[d, 1H, J ) 8.4 Hz, H-C(5′)], 6.80 [d, 1H, J ) 2.0 Hz, H-C(2′)]; 13
C
NMR (100 MHz, MeOD, HMQC, HMBC) δ 26.0 [C-4], 61.7 [C-6′′],
67.0 [C-3], 69.3 [C-4′′], 70.6 [C-2′′], 74.8 [C-3′′], 75.5 [C-1′′], 79.1
[C-5′′], 80.9 [C-2], 95.7 [C-6], 99.2 [C-4a], 103.4 [C-8], 113.3 [C-2′],
114.8 [C-5′], 118.0 [C-6′], 131.0 [C-1′], 144.7 [C-3′], 144.9 [C-4′],
152.9 [C-8a], 155.7 [C-7], 156.1 [C-5].
(-)-Catechin-6-C-â-D,8-C-â-D-digalactopyranoside, 8 (Figure 3):
CD (MeOH) λmax(∆ꢀ) ) 239 (-1.91), 286 (+0.61), 301 (-0.20); UV-
vis (MeOH/0.1% HCOOH, pH 2.5; 2:8, v/v): λmax ) 218, 231, 280
nm; MS (ESI-), m/z 613 (100%, [M - 1]-), 451 (35%, [M - 163]-),
493 (31%, [M - 121]-), 373 (15%, [M - 241]-), 331 (11%, [M -
283]-); 1H NMR (400 MHz, MeOD, COSY) δ 2.57 [dd, 1H, J ) 6.8,
16.4 Hz, H-C(4a)], 2.81 [dd, 1H, J ) 5.0, 16.4 Hz, H-C(4b)], 3.55
[dd, 1H, Jae ) 2.8, 9.6 (aa) Hz, H-C(3′′′)], 3.60-3,67 [m, 3H, H-C(3′′,
5′′, 5′′′)], 3.70-3,82 [m, 4H, H-C(6′′a,b, 6′′′a,b)], 3.94 [d, 1H, J )
2.8 Hz, H-C(4′′′)], 3.98-4.03 [m, 3H, H-C(3, 2′′, 4′′)], 4.09 [dd,
1H, J ) 9.6 Hz, H-C(2′′′)], 4.74 [d, 1H, J ) 6.4 Hz, H-C(2)], 4.84
[d, 1H, J ) 9.6 Hz, H-C(1′′′)], 4.87 [d, 1H, J ) 9.6 Hz, H-C(1′′)],
6.70 [dd, 1H, J ) 2.0, 8.0 Hz, H-C(6′)], 6.76 [d, 1H, J ) 8.0 Hz,
H-C(5′)], 6.82 [d, 1H, J ) 2.0 Hz, H-C(2′)]; 13C NMR (100 MHz,
MeOD, HMQC, HMBC) δ 26.4 [C-4], 61.6 [C-6′′/C-6′′′], 61.7 [C-6′′′/
C-6′′], 67.1 [C-3], 69.21 [C-4′′/4′′′], 69.25 [C-4′′′/C-4′′], 69.9 [C-2′′],
70.8 [C-2′′′], 74.4 [C-3′′′], 74.7 [C-3′′], 75.5 [C-1′′/C-1′′′], 75.6 [C-1′′′/
C-1′′], 79.3 [C-5′′/C-5′′′], 79.4 [C-5′′′/C-5′′], 81.2 [C-2], 100.2 [C-4a],
103.1 [C-8], 104.9 [C-6], 113.4 [C-2′], 114.7 [C-5′], 118.2 [C-6′], 130.8
[C-1′], 144.8 [C-3′/C-4′], 144.9 [C-4′/C-3′], 152.4 [C-8a], 153.4 [C-7/
C-5], 154.6 [C-5/C-7].
(-)-Catechin-6-C-â-D-galactopyranoside, 9 (Figure 3): CD (MeOH)
λ
max(∆ꢀ) ) 240 (-1.40), 283 (+0.33), 301 (-0.18); UV-vis (MeOH/
0.1% HCOOH, pH 2.5; 2:8, v/v) λmax ) 218, 231, 280 nm; MS (ESI-),
1
m/z 451 (100%, [M - 1]-), 331 (30%, [M - 121]-); H NMR (400
MHz, MeOD, COSY) δ 2.50 [dd, 1H, J ) 7.8, 16.2 Hz, H-C(4a)],
2.86 [dd, 1H, J ) 5.4, 16.2 Hz, H-C(4b)], 3.58 [dd, 1H, Jae ) 2.8, 9.6
(aa) Hz, H-C(3′′)], 3.64 [ddd, 1H, J ) 5.6, 6.4 Hz, H-C(5′′)], 3.72
[dd, 1H, J ) 4.8, 11.6 Hz, H-C(6a′′)], 3.79 [dd, 1H, J ) 7.0, 11.6