2.5. Extraction and Isolation of Flavonoid Glycosides

The fresh seeds of N. lappaceum (1.2 kg) were extracted with 70% EtOH at room temperature, and the filtered extract was concentrated in vacuo to yield 93.1 g of residue. The dried crude extract was sequentially partitioned between the n-hexane (10.3 g), EA (9.8 g), n-BuOH (25.8) and water (46.7 g). A precipitate was obtained from the ethyl acetate-soluble fraction, and this precipitate was purified by semipreparative HPLC to obtain compound 9 (50.0 mg). The n-BuOH-soluble fraction was initially subjected to Sephadex LH-20 column chromatography eluting with 100% MeOH to yield subfractions Bu1 (14.0 g), Bu2 (6.0 g) and Bu3 (5.8 g). Obtained fraction Bu1 was purified on an Optima Pak C18 column eluting with 20% MeOH/H₂O containing 0.1% formic acid by semipreparative HPLC to obtain compounds 5 (15.0 mg), 8 (18.1 mg), and 7 (8.0 mg). The obtained fraction Bu2 was purified by semipreparative HPLC using an Optima Pak C18 column with 35% MeOH/H₂O to yield subfraction Bu2.1 (15 mg) and compounds 1 (6.0 mg), 2 (15.0 mg), and 3 (4.0 mg). Subtraction Bu2.1 was further purified by semipreparative HPLC eluting with 20% MeOH/H₂O containing 0.1% formic acid on a YMC-Triart phenyl column to obtain compound 4 (4.2 mg). Compound 6 (4.8 mg) was isolated from subfraction Bu3 by semipreparative HPLC using an Optima Pak C18 column eluting with 45% MeOH/H₂O containing 0.1% formic acid.

Kaempferol 3-O-β-d-galactopyranosyl-7-O-α-l-rhamnopyranoside (1): Yellow, amorphous powder; [α]D20 −40.4 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 200 (4.76), 265 (4.40), 350 (4.17) nm; IR ν_max 3390, 1648, 1488, 1355, 1268, 1205, 1060, 993, 891 cm⁻¹; ¹H and ¹³C NMR data, see Figures S1–S4, Tables S1 and S2 in Supplementary Materials; HRESIMS m/z 593.1505 [M − H]^─ (calcd for C₂₇H₂₉O₁₅, 593.1506).

Kaempferol 3-O-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside (2): Yellow, amorphous powder; [α]D20 −134.8 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 200 (4.29), 265 (4.04), 350 (3.78) nm; IR ν_max 3389, 2925, 1661, 1495, 1310, 1208, 1078, 811 cm⁻¹; ¹H and ¹³C NMR data, see Figures S5–S8, Tables S1 and S2 in Supplementary Materials; HRESIMS m/z 593.1489 [M − H]^─ (calcd for C₂₇H₂₉O₁₅, 593.1506).

Kaempferol-3-O-α-l-arabinopyranosyl-7-O-α-l-rhamnopyranoside (3): Yellow, amorphous powder; [α]D20 −51.6 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 200 (4.19), 265 (3.87), 350 (3.65) nm; IR ν_max 3325, 2938, 1685, 1174, 1108, 1054, 810 cm⁻¹; ¹H and ¹³C NMR data, see Figures S9–S12, Tables S1 and S2 in Supplementary Materials; HRESIMS m/z 563.1376 [M − H] ^─ (calcd for C₂₆H₂₇O₁₄, 563.1401).

Kaempferol 3-O-rutinoside (4): Yellow, amorphous powder; [α]D20 −81.8 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 200 (4.32), 265 (3.93), 350 (3.73) nm; IR ν_max 3385, 2920, 1657, 1508, 1362, 1181, 1024, 840 cm⁻¹; ¹H and ¹³C NMR data, see Figures S13–S16, Tables S1 and S2 in Supplementary Materials; HRESIMS m/z 593.1525 [M − H]^─ (calcd for C₂₇H₂₉O₁₅, 593.1506).

Ternatumoside X (5): Yellow, gummy solid; [α]D20 −154.4 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 200 (4.83), 225 (4.45), 265 (4.47), 350 (4.57) nm; IR ν_max 3393, 2937, 1657, 1603, 1514, 1348, 1173, 1032, 832 cm⁻¹; ¹H and ¹³C NMR data, see Figures S17–S20, Tables S1 and S2 in Supplementary Materials; HRESIMS m/z 1047.2950 [M − H]^─ (calcd for C₄₈H₅₅O₂₆, 1047.2982).

Astragalin (6): Yellow, amorphous powder; [α]D20 −112.7 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 200 (4.24), 265 (3.78), 370 (3.66) nm; IR ν_max 3393, 1657, 1608, 1508, 1362, 1181, 1024, 828 cm⁻¹; ¹H and ¹³C NMR data, see Figures S21–S24, Tables S1 and S2 in Supplementary Materials; HRESIMS m/z 447.0919 [M − H]^─ (calcd for C₂₁H₁₉O₁₁, 447.0927).

Compound 7 (7): Pale-yellow, gummy solid; [α]D20 −83.3 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 200 (4.20), 265 (3.79), 370 (3.75) nm; IR ν_max 3373, 1655, 1605, 1514, 1353, 1175, 1024, 828 cm⁻¹; ¹H and ¹³C NMR data, see Figures S25–S31, Table S3 in Supplementary Materials; HRESIMS m/z 885.2432 [M − H]^─ (calcd for C₄₂H₄₅O₂₁, 885.2453).

5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-7-[(α-l-rhamnopyranosyl)oxy]-4H-chromen-3-yl [6-O-[(2E)-3-(4-hydroxyphenyl)prop-2-enoyl]-β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→4)]-[6-O-[(2E)-3-(4-hydroxyphenyl)prop-2-enoyl]-β-d-glucopyranosyl-(1→3)]-α-l-rhamnopyranoside (8): Yellow, gummy solid; [α]D20 −130.8 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 200 (4.13), 225 (3.94), 270 (3.79), 350 (3.80) nm; IR ν_max 3371, 1604, 1514, 1350, 1262, 1172, 1080, 832 cm⁻¹; ¹H and ¹³C NMR data, see Figures S32–S35, Tables S1 and S2 in Supplementary Materials; HRESIMS m/z 1355.3875 [M − H]^─ (calcd for C₆₃H₄₁O₃₃, 1355.3878).

Kaempferol 7-O-α-l-rhamnopyranoside (9): Pale-yellow, amorphous powder; [α]D20 −29.0 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 200 (4.38), 265 (3.95), 370 (3.84) nm; IR ν_max 3383, 2921, 1657, 1597, 1489, 1351, 1287, 1207, 1180, 1061, 1011, 824 cm⁻¹; ¹H and ¹³C NMR data, see Figures S36–S39, Tables S1 and S2 in Supplementary Materials; HRESIMS m/z 431.0976 [M − H]^─ (calcd for C₂₁H₁₉O₁₀, 431.0978).