Journal of Natural Products
Article
62.5; HRESITOFMS m/z 639.0952 [M + Na]+ (calcd for
C28H24O16Na, 639.0962).
the inclusion of a pyrogallol moiety in the galloyl unit was of
greater importance in determining radical scavenging ability
than the presence of a catechol moiety in the flavonol B-ring.
Isoquercetin 6″-O-gallate (14c): yellow powder; IR (film) νmax
1
3449, 1728, 1287, 1073 cm−1; H NMR (400 MHz, methanol-d4) δ
7.55 (2H, m, H-2′ and H-6′), 6.93 (2H, s, galloyl), 6.71 (1H, d, J = 8.7
Hz, H-5′), 6.34 (1H, s, H-8), 6.17 (1H, s, H-6), 5.21 (1H, d, J = 7.8
Hz, H-1″), 4.34 (1H, dd, J = 11.9 and 4.6 Hz, H-6″α), 4.23 (1H, d, J =
11.4 Hz, H-6″β), 3.53−3.45 (4H, m, H-2″, H-3″, H-4″, and H-5″);
13C NMR (100 MHz, methanol-d4) δ 179.4, 168.2, 65.9, 162.9, 159.3,
158.4, 149.7, 146.3 (2C), 145.8, 139.7, 135.3, 123.5, 123.0, 121.2,
177.2, 115.9, 110.1 (2C), 105.5, 104.2, 99.9, 94.8, 78.0, 75.9, 75.7, 71.4,
64.3; HRESITOFMS m/z 639.0948 [M + Na]+ (calcd for
C28H24O16Na, 639.0962).
EXPERIMENTAL SECTION
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General Experimental Procedures. All solvents and reagents
were purchased from the suppliers and used without further
purification. IR spectra were recorded on a JASCO FT/IR-460 Plus
spectrophotometer. MS spectra were obtained using a JEOL JMS-700/
GI spectrometer and the Waters UPLC-MS system (Aquity UPLC
XevoQTof). The purities of compounds were assessed as >95% using
1
analytical UPLC. H (400 MHz) and 13C (100 MHz) NMR spectra
DPPH Radical Scavenging Assay. A 10 μL amount of sample
solutions (0.1 mM in MeOH) and 190 μL of DPPH solution (78 μM
in distilled H2O/MeOH = 5/3) were added to 96-well plates, resulting
in final concentrations of 5 μM for the samples and 74 μM for DPPH.
The solutions were vigorously mixed and allowed to stand. Visible
absorption (λ = 545 nm) was measured after 15, 30, and 60 min using
a microplate reader (Emax precision microplate reader, Molecular
Devices Japan, Tokyo, Japan). Wells without the compounds were
considered as negative controls. At least three replicates were
performed for each compound and control.
were recorded on a JEOL ECX 400 spectrometer with tetramethylsi-
lane as an internal standard. Silica gel column chromatography (CC)
was performed on silica gel N-60 (40−50 μm). TLC spots on plates
precoated with silica gel 60 F254 were detected with a UV lamp (254
nm). Fractionations for all CCs were based on TLC analyses.
Synthetic Methods. Detailed synthetic conditions and spectro-
The physical data of isoquercetin O-gallates are shown here.
Isoquercetin 2″,3″-O-digallate (12b): yellow powder; IR (film)
νmax 3417, 1633, 1203, 1086 cm−1; 1H NMR (400 MHz, methanol-d4)
δ 7.56 (1H, d, J = 1.8 Hz, H-2′), 7.50 (1H, dd, J = 8.7 and 2.3 Hz, H-
6′), 7.00 (2H, s, galloyl), 6.97 (2H, s, galloyl), 6.81 (1H, d, J = 8.7 Hz,
H-5′), 6.34 (1H, s, H-8), 6.17 (1H, s, H-6), 5.87 (1H, d, J = 7.3 Hz, H-
1″), 5.39 (1H, t, J = 9.6 Hz, H-3″), 5.36 (1H, t, J = 7.8 Hz, H-2″), 3.83
(1H, dd, J = 12.4 and 1.8 Hz, H-6″α), 3.78 (1H, t, J = 9.2 Hz, H-4″),
3.68 (1H, dd, J = 12.4 and 5.5 Hz, H-6″β), 3.50−3.47 (1H, m, H-5″);
13C NMR (100 MHz, methanol-d4) δ 179.0, 167.8, 167.3, 165.7, 163.1,
158.3, 149.8, 146.3 (3C), 146.2 (2C), 146.0, 140.0, 139.9, 135.0, 123.3,
123.0, 121.1, 117.0, 116.1, 110.5 (2C), 110.4 (3C), 105.9, 100.4, 99.7,
94.5, 78.7, 77.0, 74.0, 69.6, 62.1; HRESITOFMS m/z 769.1238 [M +
H]+ (calcd for C35H29O20, 769.1252), 791.1065 [M + Na]+ (calcd for
C35H28O20Na, 791.1072).
Pretreatment of Samples with CuSO4·5H2O. A 50 μL amount
of sample solutions (0.2 mM in MeOH) and 50 μL of CuSO4·5H2O
solution (0.4 mM in MeOH) were mixed and incubated in Eppendorf
tubes for 1.5 h at room temperature.
13C NMR Analysis of a Mixture of Methyl Gallate and 3-O-
Methylquercetin (3c) with DPPH. Methyl gallate (0.015 mmol), 3-
O-methylquercetin (3c, 0.015 mmol), and DPPH (0.045 mmol) were
dissolved in 0.8 mL of DMSO-d6. After mixing for 30 min, the 13C
NMR spectrum of the mixture was recorded.
ASSOCIATED CONTENT
* Supporting Information
■
S
Isoquercetin 4″-O-gallate (12c): yellow powder; IR (film) νmax
The Supporting Information is available free of charge on the
1
3440, 1633, 1289, 1042 cm−1; H NMR (400 MHz, methanol-d4) δ
7.73 (1H, s, H-2′), 7.61 (1H, d, J = 7.3 Hz, H-6′), 7.07 (2H, s, galloyl),
6.84 (1H, d, J = 6.8 Hz, H-5′), 6.40 (1H, s, H-8), 6.21 (1H, s, H-6),
5.42 (1H, d, J = 7.3 Hz, H-1″), 5.01 (1H, t, J = 9.6 Hz, H-4″), 3.77
(1H, t, J = 9.2 Hz, H-3″), 3.63 (1H, t, J = 8.2 Hz, H-2″), 3.54−3.51
(2H, m, H-5″ and H-6″α), 3.44 (1H, dd, J = 12.4 and 5.5 Hz, H-6″β);
13C NMR (100 MHz, methanol-d4) δ 179.4, 167.7, 166.0, 163.1, 159.0,
158.5, 149.9, 146.5 (2C), 145.9, 140.0, 135.5, 123.2, 123.0, 121.1,
117.5, 116.0, 110.3 (2C), 105.7, 104.0, 99.9, 94.7, 76.6, 76.0, 75.9, 72.2,
62.2; HRESITOFMS m/z 617.1130 [M + H]+ (calcd for C28H25O16,
617.1143), 640.1027 [M + Na]+ (calcd for C28H24O16Na, 640.1040).
Isoquercetin 3″-O-gallate (12d): yellow powder; IR (film) νmax
The synthetic protocols and physical data of compounds
AUTHOR INFORMATION
Corresponding Author
■
Notes
The authors declare no competing financial interest.
1
3449, 1631, 1203, 1042 cm−1; H NMR (400 MHz, methanol-d4) δ
7.72 (1H, s, H-2′), 7.59 (1H, d, J = 7.4 Hz, H-6′), 7.15 (2H, s, galloyl),
6.86 (1H, d, J = 6.9 Hz, H-5′), 6.40 (1H, s, H-8), 6.21 (1H, s, H-6),
5.43 (1H, d, J = 7.8 Hz, H-1″), 5.19 (1H, t, J = 9.2 Hz, H-3″), 3.75
(2H, t, J = 5.0 Hz, H-2″ and H-6″α), 3.69−3.62 (2H, m, H-4″ and H-
6″β), 3.39−3.35 (1H, m, H-5″); 13C NMR (100 MHz, methanol-d4) δ
179.4, 168.2, 166.0, 163.0, 158.9, 158.4, 149.9, 146.4 (2C), 145.9,
139.7, 135.5, 123.2, 123.0, 121.7, 117.5, 116.1, 110.4 (2C), 105.7,
104.0, 99.9, 94.7, 79.2, 78.3, 74.2, 69.5, 62.2; HRESITOFMS m/z
617.1135 [M + H]+ (calcd for C28H25O16, 617.1143), 639.0958 [M +
Na]+ (calcd for C28H24O16Na, 639.0962).
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