under vacuum (40°C) to an aqueous residue that was extracted with hexane and EtOAc to produce an EtOAc fraction (216 g)
that was separated in 20-g portions over Sephadex LH-20 (5 × 100 cm) with elution by Me CO–H O (100:0→0:100).
2
2
Subfractions of similar composition were combined and rechromatographed over Sephadex LH-20 under the same conditions
followed by separation over RP-SiO (2 × 30 cm, MeCN–H O eluent, 100:00:100) and by preparative HPLC [gradient mode
2
2
(
[
%B): 5–90 min, 5–30%] to isolate eight compounds, i.e., 1 (12 mg), 2 (18), amritoside (1-O-ellagoylgentiobiose, 22 mg, 3)
14], 1-O-ellagoylglucose (18 mg, 4) [7], punicalagins A/B (hexahydroxydiphenoylgallagyl-α/β-D-glucopyranose as the total
with α/β ratio 1:18, 9.7 g, 5/6) [15], granatin B (galloyl hexahydroxydiphenoyldehydrohexahydroxydiphenoyl-β-D-
glucopyranose, 107 mg, 7) [16], and ellagic acid (15.2 g, 8) [7].
–
Granatoside A (1). C H O . UV spectrum (ÌåÎÍ, λ , nm): 256, 357. HR-ESI-MS, m/z 909.640 ([M – H] ;
4
0
30 25
max
–
–
–
calcd 909.662). ESI-MS, m/z: 909 [M – H] , 463 [(M – H) – C H O – C H O ] , 341 [(M – H) – 2 × C H O ] , 301
1
4
5
8
6
10
5
14 5 8
–
–
[
(M – H) – C H O – 2 × C H O ] , 161 [(M – H) – 2 × C H O – C H O ] . Table 1 lists the PMR (500 MHz, 300 K,
14 5 8 6 10 5 14 5 8 6 10 5
1
3
Py-d , δ, ppm) and C NMR spectra (125 MHz, 300 K, Py-d , δ, ppm).
5
5
–
Granatoside B (2). C H O . UV spectrum (ÌåÎÍ, λ , nm): 254, 355. HR-ESI-MS, m/z: 747.503 ([M – H] ;
3
4
20 20
max
–
–
–
calcd 747.519). ESI-MS, m/z: 747 [M – H] , 463 [(M – H) – C H O ] , 301 [(M – H) – C H O – C H O ] , 161 [(M – H) –
1
4
5
8
14
5
8
6 10 5
–
13
2
× C H O ] . Table 1 lists the PMR (500 MHz, 300 K, Py-d , δ, ppm) and C NMR spectra (125 MHz, 300 K, Py-d , δ, ppm).
14 5 8 5 5
Acid Hydrolysis of 1 and 2. The compound (2 mg) was dissolved in trifluoroacetic acid (TFA, 5%, 5 mL) in Me CO
2
and heated at 100°C for 2 h. The hydrolysate was concentrated to dryness with MeOH in vacuo. The dry residue was
dissolved in EtOH (50%, 2 mL) and passed over RP-SiO (5 g) with elution successively by H O (40 mL, eluate I) and MeCN
2
2
(
40%, 60 mL, eluate II). A portion of eluate I was derivatized with 3-methyl-1-phenyl-2-pyrazolin-5-one [21] and analyzed by
analytical HPLC (conditions 1). A second portion of eluate I underwent reductive amination with L-tryptophan [22] followed
by analytical HPLC (conditions 2) to determine if the monosaccharides were the D- or L-form. Eluate II was analyzed by
1
3
C NMR spectroscopy and mass spectrometry. Hydrolysates of 1 and 2 contained ellagic acid (8) [7] and D-glucose.
Compounds 1 and 2 were methylated in K CO –DMF–MeI according to Moalin et al. [23] followed by GC-MS
2
3
analysis of the hydrolysate [24].
Anti-α-glucosidase activity was determined by a microplate spectrophotometric method as described earlier [25].
The positive control was ellagic acid (≥95%, No. E2250), and acarbose (≥95%, A8980, Sigma-Aldrich).
Analytical HPLC. Conditions 1: mobile phase CH COONH (100 mM, pH 4.5) (A) and MeCN (B); gradient mode
3
4
(
%B): 0–20 min, 20–26%, ν 150 μL/min; column temperature 35°C; UV detector at 250 nm. Retention times of monosaccharide
derivatives with 3-methyl-1-phenyl-2-pyrazolin-5-one (t , min) were glucose 12.50. Conditions 2: mobile phase NaH PO
4
R
2
(
10 mM) and Na B O (50 mM) (1:1, pH 9.6); isocratic mode; ν 200 μL/min; column temperature 35°C; UV detector
2 4 7
at 220 nm. Retention times of monosaccharide derivatives with L-tryptophan (t , min) were D-glucose 8.32
R-
and L-glucose 8.67.
ACKNOWLEDGMENT
The work was financially supported by the RFBR in the framework of Science Project No. 18-33-00414 and by the
Ministry of Science and Higher Education of the Russian Federation (Project No. AAAA-A17-117011810037-0).
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