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F. Tatsuzawa et al. / Phytochemistry 70 (2009) 672–674
analysis. Each fraction was transferred to a Diaion HP-20 column,
on which pigments were adsorbed. Anthocyanin pigments were
eluted with 5% HOAc–MeOH (5:95, v/v) followed by addition of ex-
cess Et2O and then dried. The purified pigments 1 (ca. 5 mg) and 2
(ca. 1 mg) were obtained as dried dark-violet powders.
glutaric acid: 2.53 (d, J = 4.3 Hz, H-2a or 4a), 2.53 (d, J = 4.3 Hz,
H-2b or 4b), 2.62 (d, J = 14.7 Hz, H-4a or 2a), 2.68 (d, J = 14.7 Hz,
H-4b or 2b), 1.25 (s, –CH3). 13C NMR (125.78 MHz, DCl–
CD3OD = 1:9): d for malvidin: 163.4 (C-2), 148.0 (C-3), 137.5 (C-
4), 156.3 (C-5), 103.8 (C-6), 170.9 (C-7), 98.3 (C-8), 158.1 (C-9),
113.6 (C-10), 119.7 (C-10), 110.8 (C-20), 149.8 (C-30), 145.6 (C-40),
149.8 (C-50), 110.8 (C-60), 57.4 (–CH3). For glucose: 103.8 (C-1),
75.0 (C-2), 77.9 (C-3), 71.4 (C-4), 76.0 (C-5), 64.5 (C-6). For 3-hy-
droxy-3-methyl-glutaric acid: 172.3 (CO), 46.1 (C-2 or 4), 70.7
(C-3), 46.1 (C-4 or 2), 173.3 (COOH), 27.8 (–CH3).
4.4. Chemical and spectroscopic analyses of purified anthocyanins
Acid hydrolyses of pigments 1 (ca. 1 mg) and 2 (ca. 1 mg) were
carried out using 2 N HCl (2 mL) at 100 °C for 2 h, to provide mal-
vidin, glucose and 3-hydroxy-3-methylglutaric acid from pigment
1, and malvidin, glucose and malonic acid from pigment 2. More-
over, alkaline hydrolyses of pigments 1 (ca. 1 mg) and 2 (ca.
1 mg) were carried out with 2 N NaOH solution (1 mL) under N2
gas at ambient temperature for 15 min to give the same deacylated
anthocyanin, whose structure was identified to be malvidin 3-glu-
coside in comparison with authentic sample obtained from H. syr-
iacus (Kim et al., 1989).
4.4.2. Malvidin 3-(600-malonyl)-glucoside (pigment 2)
UV–Vis (in 0.1% HCl–MeOH), kmax 539, 275 nm, E440/Emax (%) 24,
+AlCl3 0 shift; TLC (Rf x100) BAW 33, BuHCl 24, 1% HCl 5, AHW 19;
HPLC (Rt (min)) 27.6 min.
References
The authentic sample of 3-hydroxy-3-methylglutaric acid is
commercially available (Wako Pure Chemical Industries, Ltd.).
TLC, Yellow coloration with BCG (Bromocresol green (0.1 g) were
dissolved in 100 mL of ethanol. To this solution, 2 N NaOH was
added until the color of the solution become blue.), BAW 0.75,
15% HOAc 0.82, HPLC, Rt (min) 5.6 min. Malonic acid: TLC, Yellow
coloration with BCG, BAW 0.63, 15% HOAc 0.78, HPLC, Rt (min)
3.2 min. Glucose: TLC, Brown coloration with AHP (o-Phthalic acid
(1.66 g) and 0.91 mL of aniline were dissolved in 100 mL of n-BuOH
saturated with H2O.), BAW 0.21, EAA 0.33, ETN 0.83. Malvidin: TLC,
mauve coloration in visible light, Forestal 0.56, HPLC Rt (min)
32.3 min. Malvidin 3-glucoside: UV–Vis (in 0.1% HCl–MeOH), kmax
538, 280 nm, E440/Emax (%) 23, +AlCl3 0 shift; TLC (Rf x100) BAW 27,
BuHCl 14, 1% HCl 4, AHW 14; HPLC (Rt (min)) 22.0 min.
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4.4.1. Malvidin 3-[6-(3-hydroxy-3-methylglutaroyl)-glucoside]
(pigment 1)
UV–Vis (in 0.1% HCl–MeOH), kmax 540, 274 nm, E440/Emax (%) 22,
+AlCl3 0 shift; TLC (Rf x100) BAW 52, BuHCl 41, 1% HCl 10, AHW 36;
HPLC (Rt (min)) 28.8 min; FAB-MS m/z 637 [M]+ (calc. for
C29H33O16), HR-FAB MS (calc. 637.1769, found: 637.1780); 1H
NMR (500 MHz, DCl–CD3OD = 1:9): d for malvidin: 8.96 (s, H-4),
6.70 (br d, J = 1.9 Hz, H-6), 7.01 (br d, J = 1.9 Hz, H-8), 7.96 (2H, s,
H-20, 60), 4.00 (s, 2 x-CH3). For glucose: 5.37 (d, J = 7.6 Hz, H-1),
3.64 (t, J = 8.6 Hz, H-2), 3.5 (t, J = 8.9 Hz, H-3), 3.41 (t, J = 8.9 Hz,
H-4), 3.79 (ddd, J = 2.1, 7.2, 8.9 Hz, H-5), 4.21 (dd, J = 7.2, 12.2 Hz,
H-6a), 4.46 (dd, J = 2.1, 12.2 Hz, H-6b). For 3-hydroxy-3-methyl-
Ueno, N., Takemura, E., Hayashi, K., 1969. Additional data for the paper
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