p-Coumaroyl Anthocyanins from the Tuber Epidermis of a Colored Potato Solanum tuberosum L. cv Jayoung

Experimental

General Experimental Procedures. 1D and 2D NMR experiments were performed on a Bruker 400 MHz or Verian 500 MHz FT-NMR instrument with tetramethylsilane (TMS) or solvent residues as internal standard. Mass spectra were obtained using an Agilent 1200 series coupled to a 6120 Quadrupole LC/MS system. Silica gel (70-230 mesh and 230-400 mesh, Merck, Germany) was used for column chromatography (CC). Thin-layer chromatographic (TLC) analysis was performed on Kieselgel 60 F 254 plates (silica gel, 0.25 mm layer thickness, Merck, Germany); compounds were visualized by UV light (254 and 365 nm) and 20% (v/v) H2SO4 reagent (Aldrich). All solvents used for the chromatographic separations were distilled before use.

Plant Material. The purple colored potato cultivar, “Jayoung” (Solanum tuberosum L. cv Jayoung, Solanaceae) was supplied by Highland Agriculture Research Center (HARC), the Korean National Institute of Crop Science, and the Rural Development Administration (RDA), Republic of Korea, in September 2012. A voucher specimen (KHP-2012-SOTU1) was deposited in the Lab. of Natural Product Medicine, College of Pharmacy, Kyung Hee University. The epidermis (< 5 mm thickness) from the fresh tubers of “Jayoung” were cut into small pieces and were freeze-dried.

Extraction and Isolation. The freeze-dried samples (7 kg) were extracted with 30 L of 70% EtOH three times by maceration. The extracts were combined and concentrated in vacuo to give a 70% EtOH extract (642 g). The 70% EtOH extract (641 g) was suspended in distilled water (2 L) and then successively extracted with n-hexane (3 × 2 L), CH2Cl2 (3 × 2 L), EtOAc (3 × 2 L), and n-butanol (3 × 2 L) to give n-hexane- (66.2 g), CH2Cl2- (5.1 g), EtOAc- (2.3 g), n-butanol- (61.1 g), and water-soluble fractions (506.0 g). The CH2Cl2- soluble extract was chromatographed over silica gel (230-400 mesh, ϕ 4.2 × 41.5 cm) as stationary phase with a CH2Cl2-MeOH gradient (from 95:5 to 85:15 v/v; final stage, MeOH 100%) as mobile phase to afford 14 pooled fractions (M1-M14). The fraction M4 (108 mg) was further fractionated using a Sephadex column (ϕ 2.5 × 75 cm) with CH2Cl2-MeOH mixture (1:1 v/v), yielding compound 5 (5.0 mg). The fraction M9 (125.7 mg) was subjected to a Sephadex column (ϕ 3.6 × 73.5 cm) with CH2Cl2-MeOH mixture (1:1 v/v) to give compound 9 (9.0 mg). Compound 10 (135.1 mg) was purified from the fraction M12 (408 mg) using a reversed phase column chromatography (CC) (YMC gel, ϕ 2.8 × 28.5 cm) with a MeOH-H2O gradient (from 1:1 to 1:0 v/v) as mobile phase. A portion of the BuOH-soluble extract (20.2 g) was separated by Diaion HP20 CC (ϕ 5.0 × 59.0 cm), using gradient mixtures of a MeOH-H2O (from 0:1 to 1:0 v/v) as mobile phases, affording 20 fractions (1B1-1B20). Compounds 6 (58.0 mg) and 7 (110.8 mg) were purified from the fraction 1B5 (570 mg) using a flash CC system with Redi Sep-C18 (48 g, MeOH-H2O-formic acid = 15:85/1→30/70/1 v/v). Compound 8 (139.8 mg) was obtained from the fraction 1B9 (1.68 g) by using repeated silica gel CC. The fraction 1B12 (310 mg) was subjected to a Sephadex column (ϕ 3.2 × 37.8 cm) with MeOH to obtain compound 11 (38.8 mg). For the isolation of anthocyanins, a portion of the BuOH-soluble extract (10.26 g) was separated by Diaion HP20 CC (ϕ 4.4 × 48.8 cm), using gradient mixtures of a MeOH-H2O-TFA (from 0:100:0.1 to 100:0:0.1 v/v) as mobile phases, affording five fractions (2B1-2B5). The fraction 2B4 (2.29 g) was further fractionated using a Sephadex column (ϕ 3.5 × 55.0 cm) with MeOH with 0.1% TFA, yielding seven fractions 2B4-1-2B4-7. An anthocyanin-rich fraction (2B4-4, 245.2 mg) was separated by using preparative HPLC with a gradient of MeCN-H2O-TFA (15:85:0.1 to 35:65:0.1), resulting in the isolation of compound 1 (11.0 mg, violet powder) and a mixture of compounds 2-4 (28.0 mg, purple powder).

Petanin (1): Amorphous violet powder; UV (On-line HPLC-DAD) λmax nm: 532; 1H-NMR (0.1% CF3COOD in CD3OD, 400 MHz), see Table 1; 13C-NMR (0.1% CF3COOD in CD3OD, 100 MHz), aglycone: see Table 2, 3-O-β-Glu: δ 101.3 (C-1''), 73.3 (C-2''), 76.8 (C-3''), 69.9 (C-4''), 76.5 (C-5''), 65.9 (C-6''), 5-O-β-Glu: δ 101.4 (C-1'''), 73.4 (C-2'''), 76.2 (C-3'''), 69.6 (C-4'''), 77.3 (C-5'''), 60.8 (C-6'''), 6''-O-α-Rha: δ 100.7 (C-1''''), 70.7 (C-2''''), 68.9 (C-3''''), 73.9 (C-4''''), 66.4 (C-5''''), 16.5 (C-6''''), 4''''-E-p-Cou: δ 125.7 (C-1), 129.9 (C-2/C-6), 115.5 (C-3/C-5), 159.9 (C-4), 145.6 (C-7), 113.6 (C-8), 167.6 (C-9); APT-ES-MS (positive mode) m/z = 933 [M]+.

Peonanin (2): Amorphous purple powder; UV (On-line HPLC-DAD) λmax nm: 520; 1H-NMR (0.1% CF3COOD in CD3OD, 500 MHz), see Table 1; 13C-NMR (0.1% CF3COOD in CD3OD, 125 MHz), aglycone: see Table 2, 3-O-β-Glu: δ 101.4 (C-1''), 73.3 (C-2''), 76.8 (C-3''), 69.8 (C-4''), 76.5 (C-5''), 65.9 (C-6''), 5-O-β-Glu: δ 101.3 (C-1'''), 73.4 (C-2'''), 76.2 (C-3'''), 69.6 (C-4'''), 77.3 (C-5'''), 60.7 (C-6'''), 6''-O-α-Rha: δ 100.7 (C-1''''), 70.7 (C-2''''), 68.9 (C-3''''), 73.9 (C-4''), 66.4 (C-5''), 16.5 (C-6''), 4''''-E-p-Cou: δ 125.7 (C-1), 129.8 (C-2/C-6), 115.4 (C-3/C-5), 159.9 (C-4), 145.6 (C-7), 113.6 (C-8), 167.5 (C-9); APT-ES-MS (positive mode) m/z = 917 [M]+

Malvanin (3): 1H-NMR (0.1% CF3COOD in CD3OD, 500 MHz): δ 9.00 (1H, s, H-4), 7.98 (2H, s, H-2'/H-6'); 13C-NMR (0.1% CF3COOD in CD3OD, 125 MHz), aglycone: see Table 2; APT-ES-MS (positive mode) m/z = 947 [M]+.

Pelanin (4): 1H-NMR (0.1% CF3COOD in CD3OD, 500 MHz): δ 9.00 (1H, s, H-4), 8.59 (2H, d, J = 9.0 Hz, H-2'/H-6'), 7.06 (2H, d, J = 9.0 Hz, H-3'/H-5'); 13C-NMR (0.1% CF3COOD in CD3OD, 125 MHz), aglycone: see Table 2; APT-ES-MS (positive mode) m/z = 887 [M]+.