Extract preparation and isolation of pure compounds
Dried and powdered P. chinense (10 kg) was extracted with MeOH at room temperature and concentrated to dryness in a rotary evaporator under reduced pressure at below 40 °C. The MeOH extract (224 g) was suspended in 2 L of distillated water and consecutively partitioned with equal volumes of ethyl acetate (EtOAc) and butanol (BuOH).
The EtOAc layer (95.2 g) was separated on a Sephadex LH-20 (130 g, 70–100 μm, Sigma-Aldrich; 3.0 cm × 70 cm) with MeOH eluent. The fractions that showed similar TLC patterns were combined to yield more homogenous samples, Frs. 1 to 9. Fr. 2 (13.6 g) which contains a main component at Rf 0.83 on TLC (developed with EtOAc:MeOH 20:1 and showing a positive reaction with ferric chloride stain) was separated on a silica gel column [146 g silica gel 60 Å (40–63 μm), 3.4 × 40 cm] and was eluted with a dichloromethane (DCM)-acetone stepwise gradient (100: 1 to 100% acetone), yielding additional 12 fractions, Frs. 21 to 212. Fr. 26 contained a main compound and was crystalized with acetone to give crystals of PC1. Fr. 3 (7.2 g) was applied to a SiO2 column [142 g silica gel 60 Å (40–63 μm), 3.4 × 40 cm, packed with H:EtOAc (5:5, v/v)] and was eluted with a n-Hexane (H): EtOAc stepwise gradient (5:5 to 1:9 and EtOAc: MeOH 20:1 v/v, 500 mL each step) to produce five fractions, Frs. 31 to 35. Fr. 33 was further chromatographed on a silica gel column [SiO2 60 Å (15–40 μm), 2.0 × 45 cm] using DCM:MeOH (9:1, v/v) to give Fr. 34, which contained crystals of compound PC3. Compound PC4 was isolated from Fr. 35 by chromatography using silica gel [63 g, silica gel 60 Å (40–63 μm), 2.0 × 50 cm] and eluting with a H:EtOAc stepwise gradient (7:3 to 1:9, v/v) and mixtures of EtOAc:MeOH (50:1, 30:1, 20:1 and 8:2, 7:3, v/v, 150 mL each step). Fr. 31 was also isolated using a silica gel column [SiO2 60 Å (15–40 μm), 2.0 × 45 cm] with a stepwise gradient elution of H:EtOAc (10:1 to 3:7; 10% gradient and 100% EtOAc), yielding compound PC7. A portion of Fr. 4 (1.23 g) was subjected to column chromatography using a silica gel column [37 g SiO2 60 Å (40–63 μm), 3.0 × 45 cm] with eluting solvents of EtOAc: acetone (10:1, v/v, 200 mL) and DCM:MeOH (20:1, v/v, 400 mL), yielding three fractions, Frs. 41 to 43. PC2 was crystallized from the third fraction, Fr. 43. Fr. 6 (20 g) was applied to a Sephadex LH-20 column [100 g, 70–100 μm, Sigma-Aldrich; 2.2 × 60 cm] and eluted with MeOH to give five fractions, Frs. 61–65. Fr. 63 was chromatographed using silica gel and mixtures of E:MeOH (30: and 25:1) to furnish PC8. Compound PC5 was purified from Fr. 5 (4 g) using a Sep-pak C18 reverse-phase column and elution with a water-MeOH mixture with a MeOH proportion increasing from 70:30 to 100% MeOH.
A portion of Fr.1 (9.8 g) was separated on a silica gel [100 g silica gel 60 Å (40–63 μm), 4.0 × 45 cm] column and eluted using mixtures of H:acetone (98:2; 500 mL) and DCM:MeOH (95:5; 400 mL) to give seven fractions, Frs. 11 to 17. Frs. 13 and 14, which exhibited similar TLC patterns, were pooled into Fr. 134 (2.6 g) and then chromatographed on a silica gel column [70 g silica gel 60 Å (40–63 μm), 3.0 × 45 cm] with a mixture of H:acetone (8:2), yielding compound PC6.
Structure determination and characterization of the isolated compounds
The chemical structures were determined by spectroscopic methods, including electrospray ionization (ESI)-mass spectroscopy (MS) and nuclear magnetic resonance (NMR) spectroscopy, and by comparison of their spectral data with values reported previously [
12,
16]. The ESI-MS spectra of the purified compounds were recorded by LC-MS (Agilent 1100 Series LC/MSD Trap XCT Plus, Agilent Technologies, Palo Alto, CA). The
1H and
13C NMR, COSY, HMQC and HMBC spectra were recorded in deuterated NMR solvents using a Bruker AMX-500 FT-NMR spectrometer (Bruker Analytische Messtechnik Gmbh, Rheinstetten, Germany).
PC1 (Quercetin): ESI-MS (positive) (m/z) 303 [M + H]+, 1H–NMR (500 MHz, DMSO-d
6
), δ (ppm): 6.183 (1H, d, J = 2.0 Hz, H-6), 6.403 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H, d, J = 8.5 Hz, H-5′), 7.535 (1H, dd, J = 2.5, 8.5 Hz, H-8′), 7.67 (1H, d, J = 2.0 Hz, H-2′); 13C–NMR (125 MHz, DMSO-d
6
), δ(ppm): 93.308 (C8), 98.138(C6), 102.976 (C10), 115.028(C2’), 116.2 (C5′), 119.938 (C6’), 121.917(C1′), 135.683 (C3), 145.016 (C3’), 146.774 (C2), 147.662 (C4’), 156.098 (C9), 160.682 (C5), 163.837 (C7), 175.8 (C4).
PC2 (Protocatechuic acid methyl ester): ESI-MS (negative) (m/z) 167.01 [M-H]−, 1H–NMR (500 MHz, acetone-d
6
), δ (ppm): 3.801 (3H, s, −OCH
3), 6.893 (1H, d, J = 8.5 Hz, H-5); 7.435 (1H, dd, J = 2.0, 8.5 Hz, H-2), 7.493 (1H, d, J = 2.0 Hz, H-6); 13C–NMR (125 MHz, acetone-d
6
), δ (ppm): 51.84 (−OCH3), 115.74 (C2), 117.14 (C5), 122.83 (C1), 123.26 (C6), 145.62 (C3), 150.77 (C4), 167.07 (COO).
PC3 (Gallic acid): ESI-MS (positive) (m/z) 339.0 [2 M–H]−, 193 [M + Na]+; 1H–NMR (500 MHz, CD3OD-d
4
), δ (ppm): 7.076 (2H, s, H-2,6); 13C–NMR (125 MHz, CD3OD -d
4
), δ (ppm): 110.32 (C2, 6), 122.02 (C1), 139.33 (C4), 146.38 (C3, 5), 170.49 (COOH).
PC4 (Quercitrin): The compound was identified as quercetin 3-O-rhamnoside (also known as quercitrin) by comparison with an authentic sample (Rf 0.54) on a TLC plate using an E:A:M (40: 1:1, v/v/v) developing solvent and detected using the appropriate stains: AS and ferric chloride.
PC5 (Ellagic acid): ESI-MS (positive) (m/z) 325[M + Na]+, ESI-MS (negative) m/z 301 [M-H]−, 1H–NMR (125 MHz, DMSO-d
6), δ (ppm): 2.08–4.5 (s, −OH); 7.483 (2H, s, H-5,5′); 13C–NMR (500 MHz, DMSO- d
6), δ (ppm): 107.470 (C1,1’), 110.200 (C5,5’), 112.072 (C6,6′), 136.274 (C2, 2′), 139.377 (C3, 3′), 147.920 (C4, 4′), 158.728 (C7, 7′).
PC6 (β-Sitosterol): 1H–NMR (500 MHz, CDC13-d), δ (ppm): 0.69 (3H, s, H-18), 0.803 (3H, d, J = 6.0 Hz, H-27), 0.826 (3H, d, J = 4.0 Hz, H-26), 0.845 (3H, t, J = 2.0 Hz, H-29), 0.925 (3H, d, J = 4.0 Hz, H-21), 1.008 (3H, s, H-19), 2.282–1.071 (28H, m), 2.305 (2H, m), 3.486 (1H, m, H-3), 5.347 (1H, brs, H-6); 13C–NMR (125 MHz, CDC13-d), δ (ppm): 11.989 (C29), 12.215 (C24), 18.918 (C28), 19.180 (C19), 19.523 (C27), 19.945 (C26), 21.222 (C11), 23.215 (C23), 26.251 (C21), 28.376 (C16), 29.313 (C25), 31.779 (C2), 32.049 (C7), 32.049 (C8), 34.095 (C20), 36.279 (C18), 36.642(C10), 37.401 (C1), 39.923 (C12), 42.423 (C4), 42.460 (C13), 45.985 (C22), 50.286 (C9), 56.212 (C17), 56.911 (C14), 56.911 (C15), 71.908(C3), 121.817 (C6), 140.908(C5).
PC7 (Methyl gallate): ESI-MS (positive) m/z 185 [M + H]+, 1H–NMR (500 MHz, CD3OD-d
6), δ (ppm): 3.825 (3H, s, −OCH
3); 7.067 (2H, s, H-2, 6); 13C–NMR (125 MHz, CD3OD-d
6), δ (ppm): 52.250 (−OCH3); 110.044 (C2, 6), 121.449 (C1), 139.703 (C4), 146.441 (C3, 5), 169.007 (COO).
PC8 (Caffeic acid): 1H–NMR (500 MHz, CD3OD-d
4
), δ (ppm): 6.24 (1H, d, J = 16 Hz, H-8), 6.80 (1H, d, J = 8.5 Hz, H-5), 6.94 (1H, dd, J = 10.5 Hz, J = 2.0 Hz, H-6), 7.06 (1H, d, J = 2.0 Hz, H-2), 7.55 (1H, d, J = 15.5 Hz, H-7); 13C–NMR (125 MHz, CD3OD-d
4
), δ (ppm): 115.104 (C2), 115.513 (C8), 116.488 (C5), 122.840 (C6), 127.803 (C1), 146.763 (C3), 147.038 (C7), 149.418 (C4), 171.022 (COOH).