Thermal Transformation Products of Ellagitannins
J. Agric. Food Chem., Vol. 55, No. 10, 2007 4111
Figure 2. RP-HPLC chromatogram of native castalagin (A) and vescalagin
(C) and after thermal treatment (B, D) for 60 min at 180 °C.
Figure 3. Excerpt of 1H NMR spectrum (400 MHz, methanol-d4) of
castalagin (A) and dehydrocastalagin (B).
water), then adjusted to pH 6.0 with sodium hydroxide (1 mol/L),
freeze-dried, and, finally, isolated by means of preparative RP-HPLC.
Castalin and vescalin (yield: ∼20% each) were obtained as white
amorphous powders in a purity of more than 98% as measured by
LC-MS.
1H, J ) 7 Hz, H-C(3)], 5.21 [t, 1H, J ) 7 Hz, H-C(4)], 5.33 [d, 1H,
J ) 1 Hz, H-C(2)], 5.64 [d, 1H, J ) 7.3 Hz, H-C(5)], 6.59 [s, 1H,
H-C(2′, V)], 6.77 [s, 1H, H-C(2′, IV)], 6.83 [s, 1H, H-C(2′, III)].
13C NMR (100 MHz; CD3OD): δ 64.5 [C(6)], 68.5 [C(4)], 69.7 [C(3)],
70.6 [C(5)], 77.7 [C(2)], 106.7 [C(2′, V)], 107.0 [C(2′, IV)], 108.0 [C(2′,
III)], 111.5 [C(2′, I)], 112.1 [C(6′, I)], 113.4 [C(6′, II)], 113.9 [C(6′,
III)], 114.3 [C(6′, V)], 115.2 [C(6′, IV)], 115.4 [C(2′, II)], 120.5 [C(1′,
I)], 123.0 [C(1′, III)], 123.4 [C(1′, IV)], 125.4 [C(1′, V)], 126.3 [C(1′,
II)], 134.4 [C(4′, II)], 135.8 [C(4′, V)], 136.4 [C(4′, III)], 136.7 [C(4′,
IV)], 137.7 [C(4′, I)], 143.3-145.7 [10C, C(3′, I-V), C(5′, I-V)], 162.6
[C(7′, I)], 164.4 [C(7′, II)], 165.9 [C(7′, IV)], 166.5 [C(7′, III)], 168.9
[C(7′, V)], 190.1 [C(1)].
Castalin, 6 (Figure 1). UV/vis (water) λmax ) 229 nm. LC/TOF-
MS: C27H20O18. LC/MS (ESI-): m/z 315 (100, [M - 2H]2-, 631 (87,
[M - H]-). 1H NMR (400 MHz; D2O): δ 3.80 [dd, 1H, J ) 3; 12 Hz,
H-C(6)], 3.88 [d, 1H, J ) 3; 12 Hz, H-C(6)], 3.96 [t, 1H, J ) 7 Hz,
H-C(4)], 4.88 [d, 1H, J ) 7 Hz, H-C(3)], 5.02 [q, 1H, J ) 3, 7 Hz,
H-C(5)], 5.11 [d, 1H, J ) 3.0 Hz, H-C(2)], 5.55 [s, 1H, H-C(1)],
6.78 [s, 1H, H-C(2′, III)]. 13C NMR (100 MHz; D2O): δ 60.9 [C(6)],
65.8 [C(1)], 67.5 [C(4)], 68.7 [C(3)], 72.3 [C(2)], 74.5 [C(5)], 108.6
[C(2′, III)], 112.0 [C(6′, I)], 113.0 [C(6′, III)], 113.2 [C(6′, II)], 115.5
[C(2′, II)], 116.5 [C(2′, I)], 120.7 [C(1′, I)], 124.8 [C(1′, III)], 126.7
[C(1′, II)], 134.3 [C(4′, II)], 135.7 [C(4′, III)], 137.9 [C(4′, I)], 143.3
[C(5′, III)], 143.6 [C(5′, II)], 143.7 [C(5′, I)], 145.0 [C(3′, III)], 145.0
[C(3′, I)], 145.6 [C(3′, II)], 166.5 [C(7′, I)], 166.9 [C(6′, II)], 168.4
[C(7′, III)].
DeoxyVescalagin, 10 (Figure 4). UV/vis (water) λmax ) 229 nm.
LC/TOF-MS: C41H26O25. LC/MS (ESI-): m/z 458 (100, [M - 2H]2-
,
1
917 (67, [M - H]-). H NMR (400 MHz; D2O): δ 2.98 [d, 1H, J )
15.0 Hz, H-C(1)], 3.40 [d, 1H, J ) 15.0 Hz, H-C(1)], 4.16 [d, 1H,
J ) 12.5 Hz, H-C(6)], 4.89 [d, 1H, J ) 12.9 Hz, H-C(6)], 4.79 [d,
1H, J ) 7 Hz, H-C(3)], 5.11 [t, 1H, J ) 7 Hz, H-C(4)], 5.41 [d, 1H,
J ) 1 Hz, H-C(2)], 5.54 [d, 1H, J ) 7.3 Hz, H-C(5)], 6.70 [s, 1H,
H-C(2′, V)], 6.77 [s, 1H, H-C(2′, IV)], 6.93 [s, 1H, H-C(2′, III)].
13C NMR (100 MHz; D2O): δ 28.2 [C(1)], 65.5 [C(6)], 68.8 [C(4)],
70.4 [C(3)], 71.0 [C(5)], 72.0 [C(2)], 107.1 [C(2′, V)], 108.8 [C(2′,
IV)], 109.1 [C(2′, III)], 111.3 [C(2′, I)], 112.1 [C(6′, I)], 113.4 [C(6′,
II)], 113.5 [C(6′, V)], 113.7 [C(6′, III)], 115.2 [C(6′, IV)], 115.4 [C(2′,
II)], 120.5 [C(1′, I)], 123.0 [C(1′, III)], 123.4 [C(1′, IV)], 125.4 [C(1′,
V)], 126.3 [C(1′, II)], 134.4 [C(4′, II)], 135.0 [C(4′, V)], 136.4 [C(4′,
III)], 136.6 [C(4′, IV)], 137.6 [C(4′, I)], 143.3-146.7 [10C, C(3′, I-V),
C(5′, I-V)], 165.7 [C(7′, II)], 166.3 [C(7′, I)], 167.2 [C(7′, IV)], 167.6
[C(7′, III)], 170.2 [C(7′, V)].
Vescalin, 7 (Figure 1). UV/vis (water) λmax ) 229 nm. LC/TOF-
MS: C27H20O18. LC/MS (ESI-): m/z 315 (100, [M - 2H]2-, 631 (75,
1
[M - H]-). H NMR (400 MHz; D2O): δ 3.82 [dd, 1H, J ) 3; 12.5
Hz, H-C(6)], 3.88 [d, 1H, J ) 3; 12.5 Hz, H-C(6)], 3.95 [t, 1H, J )
7 Hz, H-C(4)], 4.46 [d, 1H, J ) 7 Hz, H-C(3)], 4.78 [s, 1H, H-C(1)],
5.04 [q, 1H, J ) 3; 7.0 Hz, H-C(5)], 5.32 [s, 1H, H-C(2)], 6.79 [s,
1H, H-C(2′, III)]. 13C NMR (100 MHz; D2O): δ 60.8 [C(6)], 64.0
[C(1)], 67.6 [C(4)], 70.7 [C(3)], 74.2 [C(5)], 76.0 [C(2)], 108.5 [C(2′,
III)], 112.3 [C(6′, I)], 113.0 [C(6′, III)], 113.9 [C(6′, II)], 115.2 [C(2′,
II)], 116.8 [C(2′, I)], 123.5 [C(1′, I)], 124.8 [C(1′, III)], 126.8 [C(1′,
II)], 134.4 [C(4′, II)], 135.7 [C(4′, III)], 137.8 [C(4′, I)], 143.6 [C(5′,
III)], 143.6 [C(5′, II)], 143.8 [C(5′, I)], 144.2 [C(3′, I)], 145.0 [C(3′,
III)], 147.4 [C(3′, II)], 166.7 [C(7′, I)], 166.8 [C(7′, II)], 168.3 [C(7′,
III)].
Thermal Treatment of Ellagitannins. The purified ellagitannins
1-7 (50 mg each) were separately dry-heated in a lab oven for 15, 30,
60, 90, 120, and 240 min at 180 °C. After cooling, the reaction mixtures
were taken up in water (5 mL), and an aliquot (200 µL) was analyzed
by means of RP-HPLC/DAD (Figure 2). The thermally treated samples
of castalagin and vescalagin were then separated by means of
preparative RP-HPLC and, after purification by rechromatography, the
main reaction products dehydrocastalagin (Figure 3B) and deoxyves-
calagin, respectively, were analyzed by means of LC/TOF-MS, LC-
MS/MS, and 1D/2D-NMR experiments. The reaction products generated
upon thermal treatment of castalin, vescalin, roburin A, roburin D, and
33-carboxy-33-deoxyvescalagin, respectively, were analyzed by means
of LC/TOF-MS and LC-MS/MS.
Dehydrocastalin, 11 (Figure 4). UV/vis (water) λmax ) 229 nm. LC/
TOF-MS: C27H18O18. LC/MS (ESI-): m/z 314 (100, [M - 2H]2-, 629
(87, [M - H]-).
DeoxyVescalin, 12 (Figure 4). UV/vis (water) λmax ) 229 nm. LC/
TOF-MS: C27H20O17. LC/MS (ESI-): m/z 307 (100, [M - 2H]2-, 615
(66, [M - H]-).
Dehydroroburin D, 13 (Figure 4). UV/vis (water) λmax ) 229 nm.
LC/TOF-MS: C82H48O51. LC/MS (ESI-): m/z 615 (100, [M - 3H]3-
,
923 (82, [M - 2H]2-).
Deoxyroburin A, 14 (Figure 4). UV/vis (water) λmax ) 229 nm. LC/
TOF-MS: C82H50O50. LC/MS (ESI-): m/z 610 (100, [M - 3H]3-, 916
(75, [M - 2H]2-).
Sensory Analyses. Twelve assessors (five male, seven female), who
had given informed consent to participate in the sensory tests of the
present investigation and had no history of known taste disorders, were
trained in sensory experiments at regular intervals for at least 2 years
as described earlier (9, 14-16) and were, therefore, familiar with the
techniques applied. Sensory analyses were performed in a sensory panel
room at 22-25 °C in three independent sessions. Prior to sensory
analysis, the fractions or compounds isolated were suspended in water,
and, after removing the volatiles in high vacuum (<5 mPa), were freeze-
Dehydrocastalagin, 9 (Figure 4). UV/vis (water) λmax ) 229 nm.
LC/TOF-MS: C41H24O26. LC/MS (ESI-): m/z 465 (100, [M - 2H]2-
,
931 (80, [M - H]-). 1H NMR (400 MHz; CD3OD): δ 4.02 [d, 1H, J
) 12.5 Hz, H-C(6)], 5.06 [d, 1H, J ) 12.9 Hz, H-C(6)], 4.81 [d,