The study did not involve animal or human experiments; thus, no ethical approvals are required.
Materials
Rabbit primary chondrocytes (iCell Bioscience Inc., Shanghai, China); Fetal Bovine Serum (Gibco, 10099141); Trypsin 0.25% EDTA (Gibco, 25200114); DMEM High Glucose (HyClone, SH30022.01); Penicillin–Streptomycin Solution (HyClone, SV30010); DPBS (Gibco, C14190500BT); Recombinant Human IL-1β (Invitrogen, USA); Cell Counting Kit-8 (Keygen, Nanjing, China); Annexin V-FITC/PI Apoptosis Detection Kit (Keygen, Nanjing, China); Diacerein (Aladdin, Shanghai, China); dimethyl sulfoxide (Sigma-Aldrich, Germany); RNase-free water (Tiangen, Beijing, China); Revertra Ace (TOYOBO, Japan); Recombinant Rnasin Ribonuclcase Inhibitor (Promega, USA); Advanced SYBR Green Supermix (Bio-Rad, USA); Oligo (dT) 18 Primer (TaKara, Japan); TRI Reagent (MRC, USA); HPLC-grade MeOH (Fisher Scientific, NJ, USA). Analytical-grade solvents for extraction and chromatography (Beijing Beihua Fine Chemicals Company, Beijing, China); ODS-A C18 reversed-phase silica gel (50 μm, YMC).
Plant material
The seed shells of Paeonia suffruticosa were obtained from Heze, Shandong province, P. R. China and were identified by Dr. Chun-nian He. The voucher specimen (2012001) has been deposited in the Seed Resource Bank of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China.
Extraction and isolation
The dried seed shells (1.2 kg) were pulverized and extracted with 70% ethyl alcohol (EtOH,9 L) by soaking at room temperature for 24 h twice. The combined EtOH extract was concentrated under reduced pressure at 60 °C to afford a dark-brown residue (245 g). A portion of the EtOH extract of the seed shells (120 g) that was dissolved in MeOH–H2O (60: 40, v/v) was chromatographed through an ODS-A C18 reversed-phase silica gel column (5.0 cm i.d. × 50 cm) eluted with MeOH-H2O (20: 80 → 80: 20, v/v) gradient to afford fractions A-D. Fraction B (40 g) was rechromatographed through a YMC-pack ODS-A column (250 mm × 20 mm, 5 μm) on pre-HPLC (BUCHI Reveleris PREP) eluted with MeOH-H2O (40: 60, v/v) to obtain suffruticosol A (1, 2.0 g), suffruticosol B (2, 5.0 g), and suffruticosol C (3, 55 mg), respectively. Fraction C (20 g) was rechromatographed through a YMC-pack ODS-A column (250 mm × 20 mm, 5 μm) on pre-HPLC (BUCHI Reveleris PREP) eluted with MeOH-H2O (45: 55, v/v) to obtain trans-resveratrol (4800 mg), cis-ε-viniferin (5200 mg), and trans-ε-viniferin (6, 1.2 g), respectively. Fraction D (38 g) was rechromatographed through a YMC-pack ODS-A column (250 mm × 20 mm, 5 μm) on pre-HPLC (BUCHI Reveleris PREP) eluted with MeOH-H2O (52: 48, v/v) to obtain cis-suffruticosol D (7, 55 mg), cis-gnetin H (8, 45 mg), trans-suffruticosol D (9, 1.5 g), and trans-gnetin H (10, 5.2 g), respectively (flowchart was shown in Additional file 3).
Confirmation of the ten compound was made by comparison with the reference substance isolated previously by the author [9]. 1H- and 13C-NMR spectra were measured on a Bruker Avance DRX-500 spectrometer (1H at 500 MHz and 13C at 125 MHz) in MeOH-d4. Chemical shifts are given in δ values (ppm) relative to tetramethylsilane (TMS) as an internal standard (shown in Additional file 4). UV spectra were measured on a Shimadzu UV-2550 UV–visible spectrophotometer. ESI–MS spectra were recorded on an ABI 3200 Qtrap spectrometer.
Suffruticosol A (1)
UV λmax nm: 230, 285 (MeOH). ESI–MS m/z: 679 (M+ - H). 1H-NMR (500 MHz, CD3OD) δ (ppm): 7.10 (2H, d, J = 8.5 Hz, H-2″, 6″), 6.95 (2H, d, J = 8.5 Hz, H-2, 6), 6.69 (2H, d, J = 8.5 Hz, H-3″, 5″), 6.48 (2H, d, J = 8.5 Hz, H-2′, 6′), 6.36 (2H, d, J = 8.5 Hz, H-3, 5), 6.24 (1H, d, J = 2.5 Hz, H-12″), 6.19 (1H, d, J = 1.0 Hz, H-12′), 6.11 (2H, d, J = 8.5 Hz, H-3′, 5′), 6.05 (1H, t, J = 2.5 Hz, H-12), 5.98 (2H, d, J = 2.5 Hz, H-10, 14), 5.93 (1H, d, J = 2.5 Hz, H-14″), 5.68 (1H, d, J = 12.0 Hz, H-7″), 5.42 (1H, d, J = 3.5 Hz, H-7′), 4.74 (1H, s, H-8), 4.34 (1H, d, J = 12.0 Hz, H-8″), 3.93 (1H, m, H-8′), 3.68 (1H, d, J = 8.0 Hz, H-8). 13C-NMR (125 MHz, CD3OD) δ (ppm): 160.7 (C-11′), 159.8 (C-11, 13), 159.4 (C-4″), 157.2 (C-13″), 157.0 (C-4), 155.6 (C-13′), 155.4 (C-11″), 155.0 (C-4′), 148.9 (C-9), 145.1 (C-9′), 142.3 (C-9″), 136.0 (C-1), 134.4 (C-1′), 131.3 (C-1″), 131.2 (C-2′, 6′), 131.0 (C-2, 2″, 6, 6″), 127.4 (C-10″), 123.5 (C-14′), 117.7 (C-10′), 116.7 (C-3″, 5″), 115.9 (C-3, 5), 114.7 (C-3′, 5′), 107.3 (C-10, 14), 106.4 (C-14″), 102.4 (C-12″), 101.9 (C-12), 96.7 (C-12′), 92.0 (C-7″), 61.5 (C-7), 55.0 (C-8), 49.5 (C-8″), 49.1 (C-8′), 40.2 (C-7′).
Suffruticosol B (2)
UV λmax nm: 230, 284 (MeOH). ESI–MS m/z: 679 (M+ - H). 1H-NMR (500 MHz, CD3OD) δ (ppm): 7.58 (2H, d, J = 8.5 Hz, H-2″, 6″), 6.95 (2H, brd, H-2′, 6′), 6.91 (2H, d, J = 8.5 Hz, H-3″, 5″), 6.50 (2H, d, J = 8.5 Hz, H-3′, 5′), 6.28 (2H, d, J = 8.5 Hz, H-3, 5), 6.26 (2H, d, J = 8.5 Hz, H-2, 6), 6.22 (1H, d, J = 2.0 Hz, H-10, 14), 6.19 (1H, s, H-12′), 6.17 (2H, d, J = 2.0 Hz, H-12″), 6.16 (1H, t, J = 2.0 Hz, H-12), 5.94 (2H, d, J = 2.5 Hz, H-14″), 5.86 (1H, d, J = 11.5 Hz, H-7″), 5.08 (1H, d, J = 11.5 Hz, H-8″), 4.22 (1H, d, J = 11.5 Hz, H-7′), 4.11 (1H, m, H-8′), 4.09 (1H, s, H-8), 3.81 (1H, d, J = 6.0 Hz, H-7). 13C-NMR (125 MHz, CD3OD) δ (ppm): 160.7 (C-11′), 159.9 (C-11, 13), 159.6 (C-4″), 158.9 (C-13″), 157.7 (C-11″), 156.6 (C-4′), 156.5 (C-4), 156.2 (C-13′), 148.0 (C-9, 9′), 142.9 (C-9″), 136.0 (C-1), 134.3 (C-1′), 133.6 (C-2′, 6′), 131.4 (C-1″), 131.0 (C-2″, 6″), 129.9 (C-2, 6), 124.1 (C-14′), 123.4 (C-10″), 119.0 (C-10′), 117.0 (C-3″, 5″), 115.6 (C-3, 5), 115.2 (C-3′, 5′), 107.8 (C-10, 14), 105.4 (C-12″), 104.2 (C-14″), 101.9 (C-12), 96.7 (C-12′), 91.6 (C-7″), 63.6 (C-7), 57.4 (C-8), 49.3 (C-8″), 48.3 (C-8′), 47.0 (C-7′).
Suffruticosol C (3)
UV λmax nm: 230, 285 (MeOH). ESI–MS m/z: 679 (M+ - H). 1H-NMR (500 MHz, CD3OD) δ (ppm): 7.23 (2H, d, J = 8.4 Hz, H-2′, 6′), 7.12 (2H, d, J = 8.4 Hz, H-2″, 6″), 6.95 (2H, d, J = 9.0 Hz, H-2, 6), 6.72 (2H, d, J = 7.8 Hz, H-3′, 5′, 3″, 5″), 6.54 (2H, d, J = 8.4 Hz, H-3, 5), 6.41 (1H, d, J = 2.5 Hz, H-10″), 6.27 (1H, d, J = 2.5 Hz, H-12″), 6.03 (3H, s, H-7″, 12, 12′), 5.89 (2H, d, J = 2.5 Hz, H-10, 10′), 5.10 (1H, s, H-7), 4.29 (1H, d, J = 9.6 Hz, H-8′), 4.26 (1H, s, H-8″), 4.17 (1H, d, J = 11.4 Hz, H-8), 3.01 (1H, dd, J = 10.2, 11.4 Hz, H-7′).
Resveratrol (4)
UV λmax nm: 219,230, 285 (MeOH). ESI–MS m/z: 227 (M+ - H). 1H-NMR (500 MHz, CD3OD) δ (ppm): 7.04 (2H, d, J = 8.5 Hz, H-2, 6), 6.61 (2H, d, J = 8.5 H-3, 5), 6.36 (1H, d, J = 12.0 Hz, H-7), 6.26 (1H, d, J = 12.0 Hz, H-8), 6.18 (2H, d, J = 2.5 H-10, 14), 6.10 (1H, t, J = 2.5 Hz, H-12). 13C-NMR (125 MHz, CD3OD) δ (ppm): 159.8 (C-11, 13), 158.2 (C-4), 141.5 (C-9), 131.3 (C-7), 130.6 (C-2, 6), 130.3 (C-1), 129.6 (C-8), 116.4 (C-3, 5), 108.5 (C-10, 14), 102.9 (C-12).
cis-ε-Viniferin (5)
UV λmax nm: 230, 280 (MeOH). ESI–MS m/z: 453 (M+ - H). 1H-NMR (500 MHz, CD3OD) δ (ppm): 6.94 (2H, d, J = 8.4 Hz, H-2, 6), 6.93 (2H, d, J = 8.4 Hz, H-2′, 6′), 6.71 (2H, d, J = 8.4 Hz, H-3, 5), 6.59 (2H, d, J = 8.4 H-3′, 5′), 6.24 (1H, d, J = 2.4 H-14′), 6.22 (1H, d, J = 2.4 Hz, H-12′), 6.20 (1H, d, J = 12.0 H-7′), 6.09 (1H, t, J = 2.4 Hz, H-12), 6.03 (1H, d, J = 12.0 Hz, H-8′), 5.93 (2H, d, J = 2.4 Hz, H-10, 14), 5.18 (1H, d, J = 6.0 Hz, H-7), 3.78 (1H, d, J = 6.0 Hz, H-8). 13C-NMR (125 MHz, CD3OD) δ (ppm): 163.2 (C-11′), 160.1 (C-11, 13), 159.6 (C-13′), 159.0 (C-4′), 158.3 (C-4), 147.7 (C-9), 137.9 (C-9′), 134.3 (C-1), 131.8 (C-7′), 131.2 (C-2′, 6′), 130.6 (C-1′), 128.8 (C-2, 6), 127.2 (C-8′), 120.8 (C-10′), 116.4 (C-3′, 5′), 116.6 (C-3, 5), 109.4 (C-14′), 107.9 (C-10, 14), 102.3 (C-12), 97.2 (C-12′), 95.4 (C-7), 58.2 (C-8).
trans-ε-Viniferin (6)
UV λmax nm: 230, 330 (MeOH). ESI–MS m/z: 453 (M+ - H). 1H-NMR (500 MHz, CD3OD) δ (ppm): 7.14 (2H, d, J = 9.0 Hz, H-2, 6), 7.03 (2H, d, J = 9.0 Hz, H-2′, 6′), 6.81 (1H, d, J = 16.0 Hz, H-7′), 6.76 (2H, d, J = 9.0 Hz, H-3, 5), 6.65 (2H, d, J = 9.0 H-3′, 5′), 6.63 (1H, d, J = 2.0 H-14′), 6.56 (1H, d, J = 16.0 Hz, H-8′), 6.25 (1H, d, J = 2.0 Hz, H-12′), 6.18 (1H, t, J = 2.0 Hz, H-12), 6.16 (2H, d, J = 2.0 Hz, H-10, 14), 5.36 (1H, d, J = 6.5 Hz, H-7), 4.35 (1H, d, J = 6.5 Hz, H-8). 13C-NMR (125 MHz, CD3OD) δ (ppm): 163.2 (C-11′), 160.5 (C-11, 13), 160.2 (C-13′), 159.0 (C-4′), 158.9 (C-4), 147.9 (C-9), 137.4 (C-9′), 134.4 (C-1), 130.9 (C-7′), 130.8 (C-1′), 129.3 (C-2′, 6′), 128.7 (C-2, 6), 124.2 (C-8′), 120.6 (C-10′), 116.9 (C-3′, 5′), 116.8 (C-3, 5), 108.0 (C-10, 14), 104.9 (C-14′), 102.7 (C-12), 97.4 (C-12′), 95.3 (C-7), 58.8 (C-8).
cis-Suffruticosol D (7)
UV λmax nm: 203, 230 (sh), 282, 328 (MeOH). ESI–MS m/z: 679 (M+–H). 1H-NMR (500 MHz, CD3OD) δ (ppm): 7.17 (2H, d, J = 8.5 Hz, H-2, 6), 6.95 (2H, d, J = 8.5 Hz, H-2″, 6″), 6.78 (2H, d, J = 8.5 Hz, H-3, 5), 6.75 (2H, d, J = 8.5 Hz, H-2′, 6′), 6.55 (2H, d, J = 8.5 Hz, H-3″, 5″), 6.54 (2H, d, J = 8.5 Hz, H-3′, 5′), 6.42 (H, d, J = 12.0 Hz, H-7′), 6.46 (H, s, H-12′), 6.36 (H, d, J = 12.0 Hz, H-8′), 6.19 (H, t, J = 2.0 Hz, H-12), 6.17 (2H, d, J = 2.0 Hz, H-10,14), 5.94 (H, t, J = 2.0 Hz, H-12″), 5.82 (H, d, J = 8.0 Hz, H-7″), 5.78 (2H, d, J = 2.0 Hz, H-10″,14″), 5.43 (H, d, J = 6.0 Hz, H-7), 4.50 (H, d, J = 8.0 Hz, H-8″), 4.39 (H, d, J = 6.0 Hz, H-8). 13C-NMR (125 MHz, CD3OD) δ (ppm): 162.6 (C-11′13′), 159.9 (C-11, 13), 159.0 (C-11″,13″), 158.9 (C-4), 158.4 (C-4′), 157.9 (C-4″), 148.3 (C-9), 143.9 (C- 9″), 135.1 (C-7′), 134.7 (C-1), 133.9(C-9′), 131.1 (C-1′), 130.1 (C-1″), 129.8(C-2″,6″), 129.5 (C-2′, 6′), 128.7 (C-2, 6), 125.0(C-8′), 122.4(C-14′), 121.6 (C-10′), 116.7 (C-3, 5), 116.4(C-3″,5″), 115.8 (C-3′, 5′), 109.8(C-10″,14″), 107.9 (C-10, 14), 102.3 (C-12), 102.0 (C-12″), 95.7 (C-7), 92.3 (C-12′), 91.6 (C-7″), 59.3 (C-8), 55.3(C-8″).
cis-gnetin H (8)
UV λmax nm: 203, 230 (sh), 282 (MeOH). ESI–MS m/z: 679 (M+ –H). 1H-NMR (500 MHz, CD3OD) δ (ppm): 6.98 (4H, d, J = 8.5 Hz, H-2,6,2″6″), 6.77 (2H, d, J = 8.5 Hz, H-2′, 6′), 6.69(4H, d, J = 8.5 Hz, H-3,5,3″, 5″), 6.50 (2H, d, J = 8.5 Hz, H-3′, 5′), 6.35 (H, s, H-12′), 6.05 (2H, t, J = 2.0 Hz, H-12,12″), 5.95 (2H, d, J = 2.0 Hz, H-10,14), 5.92 (H, d, J = 12.0 Hz, H-7′), 5.78(2H, d, J = 2.0 Hz, H-10″,14″), 5.74 (H, d, J = 12.0 Hz, H-8′), 5.24 (2H, d, J = 5.0 Hz, H-7,7″), 3.86 (2H, d, J = 5.0 Hz, H-8,8″). 13C-NMR (125 MHz, CD3OD) δ(ppm): 163.6(C-11′,13′), 159.9(C-11,13,11″,13″), 158.8 (C-4,4″), 158.7 (C-4′), 148.2 (C-9,9″), 135.1 (C-9′), 134.1 (C-1, 1″), 132.3 (C-7′), 131.1 (C-1′), 129.5 (C-2′, 6′), 128.6 (C-2,6,2″,6″), 124.6 (C-8′), 121.2 (C-10′,14′), 116.8 (C-3, 5, 3″,5″), 116.6 (C-3′, 5′), 107.6 (C-10, 14,10″,14″), 102.4 (C-12, 12″), 95.6 (C-7, 7″), 92.3 (C-12′), 59.2 (C-8, 8″).
trans-Suffruticosol D (9)
UV λmax nm: 203, 230 (sh), 282 (MeOH). ESI–MS m/z: 679 (M+–H). 1H-NMR (500 MHz, CD3OD) δ (ppm): 7.19 (2H, d, J = 8.5 Hz, H- 2, 6), 6.98 (2H, d, J = 8.5 Hz, H-2″, 6″), 6.79 (2H, d, J = 8.5 Hz, H-3, 5), 6.75 (2H, d, J = 8.5 Hz, H-2′, 6′), 6.56 (2H, d, J = 8.5 Hz, H-3″, 5″), 6.54 (2H, d, J = 8.5 Hz, H-3′, 5′), 6.51 (H, d, J = 16.0 Hz, H-7′), 6.46 (H, s, H-12′), 6.43 (H, d, J = 16.0 Hz, H-8′), 6.19 (H, t, J = 2.0 Hz, H-12), 6.17 (2H, d, J = 2.0 Hz, H-10,14), 5.94 (H, t, J = 2.0 Hz, H-12″), 5.83 (H, d, J = 8.0 Hz, H-7″), 5.78 (2H, d, J = 2.0 Hz, H-10″,14″), 5.43 (H, d, J = 6.0 Hz, H-7), 4.52 (H, d, J = 8.0 Hz, H-8″), 4.39 (H, d, J = 6.0 Hz, H-8). 13C-NMR (125 MHz, CD3OD) δ (ppm):163.4 (C-13′), 162.8 (C-11′), 160.7 (C-11, 13), 159.5 (C-11″,13″), 159.1 (C-4), 158.9 (C-4′), 158.2 (C-4″), 148.1 (C-9), 144.1 (C- 9″), 134.9 (C-7′), 134.6 (C-1), 134.1 (C-9′), 131.1 (C-1′), 130.0 (C-1″), 129.8 (C-2″,6″), 129.1 (C-2′, 6′), 128.6 (C-2, 6), 122.9 (C-8′), 122.6 (C-14′), 121.7 (C-10′), 116.9 (C-3, 5), 116.8 (C-3″,5″), 115.8 (C-3′, 5′), 109.7 (C-10″,14″), 107.9 (C-10, 14), 102.7 (C-12), 102.3 (C-12″), 95.4 (C-7), 92.4 (C-12′), 91.9 (C-7″), 59.3 (C-8), 55.3 (C-8″).
trans-gnetin H (10)
UV λmax nm: 230, 325 (MeOH). ESI–MS m/z: 679 (M+ - H). 1H-NMR (500 MHz, CD3OD) δ (ppm): 7.18 [4H, dd, J = 2.0, 8.5 Hz, H-2(6), 2″ (6″)], 6.78 [4H, dd, J = 2.0, 8.5 Hz, H-3(5), 3″ (5″)], 6.69 (2H, d, J = 8.5 Hz, H-2′, 6′), 6.51 (2H, d, J = 8.5 Hz, H-3′, 5′), 6.42 (1H, s, J = 8.5 Hz, H-12), 6.38 (2H, s, H-7′, 8′), 6.14 [6H, s, H-10(10″), 12(12″), 14(14″)], 5.40 (2H, d, J = 5.5 Hz, H-7, 7″), 4.40 (2H, d, J = 5.5 Hz, H-8, 8″). 13C-NMR (125 MHz, CD3OD) δ (ppm):163.5 (C-11′, 13′), 160.6 [C-11(11″), 13(13″)], 159.0 (C-4, 4″), 158.8 (C-4′), 148.0 (C-9, 9″), 135.0 (C-9′), 134.7 (C-1″), 134.2 (C-1), 131.1 (C-1′), 129.2 (C-2′, 6′, 8′), 128.6 [C-2(2″), 6(6″)], 123.0 (C-7′), 120.8 (C-10′, 14′), 116.9 [C-3(3″), 5(5″)], 116.7 (C-3′, 5′), 107.8 [C-10(10″), 14(14″)], 102.7 (C-12, 12″), 95.3 (C-7, 7″), 92.0 (C-12′), 59.4 (C-8, 8″).
The purity of ten compounds were detected using the protocol previously described by He et al. [31]. Briefly, an Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with an online vacuum degasser, a quaternary pump, an autosampler, a thermostated column compartment, and a diode array detector was used. HPLC separation was performed by a YMC-pack ODS-A column (250 mm × 4.6 mm, 5 μm) using the mobile phase containing water (A) and methanol (B) in a gradient: 0–10 min, linear gradient from 35% to 52% B; 10–30 min, linear gradient from 52% to 60% B. The flow rate was at 1.0 mL/min. The column temperature was set at 25 °C. The injection volume was 5 μL, and the UV detection wavelength was set at 230 nm. The retention times of 1 to 10 were 9.23 min, 10.61 min, 11.18 min, 13.56 min, 15.39 min, 18.80 min, 20.31 min, 21.63 min, 25.68 min and 26.51 min, respectively. The purities of suffruticosol A, suffruticosol B, suffruticosol C, trans-resveratrol, cis-ε-viniferin, trans-ε-viniferin, cis-suffruticosol D, cis-gnetin H, trans-suffruticosol D, and trans-gnetin H were determined by HPLC using normalization of the peak area and were 98.2%, 96.1%, 98.6%, 98.6%, 95.8%, 98.1%, 95.5%, 94.5%, 86.7%, and 85.8%, respectively (shown in Additional file 5).
Finally, the compounds were resuspended in dimethyl sulfoxide (DMSO) to yield a concentration of 50 mM and were stored at 4 °C.