Structural elucidation
As a result, twenty compounds were obtained (Fig. 1). Structural elucidation of the isolated compounds was done based on HR-ESI-MS, NMR analysis, and compared with the data reported in literatures.
2,2',4,4',6,6',7,7'-octahydroxy-1,1'-biphenanthrene (1)
Brown powder; HR-ESI-MS (m/z): 483.1088 [M + H] +. 1H-NMR (600MHz, CD3OD), δH: 9.27 (1H, s, H-5,5′), 7.21 (1H, d, J = 9.2Hz, H-9,9′), 7.05 (1H, s, H-8,8′), 6.83 (1H, d, J = 9.2Hz, H-10,10′), 6.81 (1H, s, H-3,3′)
13C-NMR (151MHz, CD3OD), δC:158.1 (C-4,4′), 154.3 (C-2,2′), 146.2 (C-6,6′), 145.1 (C-7,7′), 136.0 (C-10a,10a′), 127.8 (C-8a,8a′), 127.7 (C-9,9′), 127.0 (C-4b,4b′), 123.2 (C-10,10′), 115.6 (C-4a,4a′), 114.1 (C-5,5′), 112.4 (C-8,8′), 110.2 (C-1,1′), 102.9 (C-3,3′). The data are consistent with those reported previously [6]. Compound 1 is isolated from the rhizome of Dioscorea bulbifera L. for the first time.
2,7-dihydroxy-4-methoxyphenanthrene (2)
C15H12O3, m/z 240.26; 1H-NMR (600MHz, CD3OD), δH: 9.33(1H, d, J = 9.3 Hz, H-5), 7.50 (1H, d, J = 8.8 Hz, H-9), 7.46 (1H, d, J = 8.8 Hz, H-10), 7.13 (1H, d, J = 2.8 Hz, H-8), 7.07 (1H, dd, J = 9.3, 2.8 Hz, H-6), 6.81(1H, d, J = 2.4 Hz, H-1), 6.75 (1H, d, J = 2.4 Hz, H-3), 4.08 (3H, s, 4-OCH3).
13C-NMR (151MHz, CD3OD), δC: 160.6 (C-4), 156.3 (C-2), 155.3 (C-7), 135.8 (C-8a), 134.7 (C-4b), 130.1 (C-6), 128.4 (C-9), 127.9 (C-10), 125.5 (C-10a), 117.2 (C-5), 116.0 (C-4a), 112.3 (C-8), 105.7 (C-1), 100.3 (C-3), 55.9 (4-OCH3). The data are consistent with those reported previously [7].
2,7-dihydroxy-3,4-dimethoxyphenanthrene (3)
C16H14O4, m/z 270.28; 1H-NMR (600MHz, CD3OD), δH: 9.26 (1H, d, J = 9.2 Hz, H-5), 7.42 (2H, d, J = 1.8 Hz, H-9, 10), 7.13 (1H, d, J = 2.8 Hz, H-8), 7.09 (1H, dd, J = 9.2, 2.8 Hz, H-6), 7.04 (1H, s, H-1), 3.99 (3H, s, 3-OCH3), 3.93 (3H, s, 4-OCH3).
13C-NMR (151MHz, CD3OD), δC: 155.9 (C-7), 152.6 (C-2), 150.1 (C-4), 143.2 (C-3), 134.9 (C-10a), 130.7 (C-8a), 129.0 (C-5), 127.7 (C-9), 127.2 (C-10), 124.6 (C-4b), 119.5 (C-4a), 117.5 (C-8), 112.5 (C-6), 110.1 (C-1), 61.4 (3-OCH3), 60.3 (4-OCH3). The data are consistent with those reported previously [8].
2,3,7-trihydroxy-4-methoxyphenanthrene (4)
C15H12O4, m/z 256.26; 1H-NMR (600MHz, CD3OD), δH: 9.25 (1H, d, J = 9.2 Hz, H-5), 7.45 (1H, d, J = 8.8 Hz, H-9), 7.37 (1H, d, J = 8.8 Hz, H-10), 7.14 (1H, d, J = 2.8 Hz, H-8), 7.10 (1H, dd, J = 9.2, 2.8 Hz, H-6), 7.04 (1H, s, H-1), 3.87 (3H, s, 4-OCH3).
13C-NMR (151MHz, CD3OD), δC: C: 155.7 (C-7), 146.4 (C-4), 145.8 (C-2), 140.6 (C-3), 135.1 (C-8a), 128.9 (C-5), 127.9 (C-10), 127.2 (C-10a), 125.3 (C-9), 124.2 (C-4b), 119.3 (C-4a), 117.2 (C-8), 112.2 (C-6), 109.6 (C-1), 59.7 (4-OCH3). The data are consistent with those reported previously [9].
3,7-dihydroxy-2,4-dimethoxyphenanthrene (5)
C16H14O4, m/z 270.28;1H-NMR (600MHz, CD3OD), δH: 9.29 (1H, d, J = 9.2 Hz, H-5), 7.57 (1H, d, J = 8.7 Hz, H-9), 7.43 (1H, d, J = 8.7 Hz, H-10), δH 7.18 (1H, s, H-1), 7.16 (1H, d, J = 2.7Hz, H-8), 7.11 (1H, dd, J = 9.2, 2.7 Hz, H-6), 4.02 (3H, s, 2-OCH3), 3.90 (3H, s, 4-OCH3).
13C-NMR (151MHz, CD3OD), δC: 156.0 (C-7), 148.9 (C-2), 145.7 (C-4), 141.1 (C-3), 135.3 (C-8a), 129.1 (C-9), 128.2 (C-10), 127.0 (C-10a), 125.5 (C-5), 124.2 (C-4a), 120.3 (C-4b), 117.3 (C-8), 112.2 (C-6), 106.1 (C-1), 69.8 (4-OCH3), 56.4 (2-OCH3). The data are consistent with those reported previously [8].
Hircinol (6)
C15H14O3, m/z 242.27; 1H-NMR (600MHz, CD3OD), δH: 7.06 (1H, t, J = 7.7 Hz, H-7), 6.82 (2H, dd, J = 7.7, 3.7 Hz, H-6, 8), 6.55 (1H, d, J = 2.4 Hz, H-1), 6.50 (1H, d, J = 2.4 Hz, H-3), 3.94 (3H, s, 4-OCH3), 2.65 (2H, d, J = 7.2 Hz, H-9), 2.61 (2H, d, J = 7.4 Hz, H-10).
13C-NMR (151MHz, CD3OD), δC: 159.0 (C-2), 156.8 (C-4), 154.4 (C-5), 144.6 (C-10a), 141.8 (C-8a), 128.3 (C-7), 122.0 (C-4b), 120.6 (C-8), 118.2 (C-6), 115.0 (C-4a), 110.1 (C-1), 100.2 (C-3), 57.5 (4-OCH3), 32.2 (C-10), 32.0 (C-9). The data are consistent with those reported previously [10].
7-hydroxy-2,3,4-trimethoxy-9,10-dihydrophenanthrene (7)
C17H18O4, m/z 286.33; 1H-NMR (600MHz, CD3OD), δH: 8.06 (1H, d, J = 9.3 Hz, H-5), 6.70 (1H, s, H-1), 6.67 (2H, d, J = 6.9 Hz, H-6, 8), 3.85 (3H, s, 3-OCH3), 3.86 (3H, s, 2-OCH3), 3.70 (3H, s, 4-OCH3), 2.68 (4H, q, J = 2.7Hz, H-9, 10).
13C-NMR (151MHz, CD3OD), δC: 157.0 (C-7), 152.7 (C-2), 152.7 (C-3), 142.6 (C-4), 140.8 (C-8a), 135.6 (C-10a), 129.5 (C-5), 125.5 (C-4b), 122.3 (C-4a), 115.3 (C-8), 114.2 (C-6), 108.8 (C-1), 61.4 (3-OCH3), 60.8 (4-OCH3), 56.4 (2-OCH3), 31.3 (C-9), 31.1 (C-10). The data are consistent with those reported previously [11].
2,3,5,7-tetrahydroxy-9,10-dihydrophenanthrene (8)
C14H12O4, m/z 244.25; 1H-NMR (600MHz, CD3OD), δH: 7.89 (1H, s, H-1), 6.60 (1H, s, H-4), 6.24 (1H, d, J = 2.4 Hz, H-6), 6.19 (1H, d, J = 2.4Hz, H-8), 2.60 (2H, m, H-9), 2.56 (2H, m, H-10).
13C-NMR (151MHz, CD3OD), δC:156.9 (C-5), 156.3 (C-7), 143.6(C-2), 143.6 (C-3), 141.9 (C-8a), 130.6 (C-10a), 126.7 (C-4a), 116.6 (C-8), 115.3 (C-4), 115.2 (C-4b), 107.6 (C-1), 102.6 (C-6), 32.2 (C-9), 30.3 (C-10). The data are consistent with those reported previously [12].
3,7-dimethoxy-3',4',5-trihydroxyflavone (9)
Yellow powder. C17H14O7, m/z 330.07; 1H-NMR(600MHz, CD3OD), δH: 7.62 (1H, d, J = 2.2 Hz, H-2'), 7.52 (1H, dd, J = 8.5, 2.2 Hz, H-6'), 6.90 (1H, d, J = 8.5 Hz, H-5'), 6.47 (1H, d, J = 2.1 Hz, H-8), 6.39 (1H, d, J = 2.1 Hz, H-6).
13C-NMR (151MHz, CD3OD), δC:176.1 (C-4), 166.0 (C-7), 162.4 (C-8a), 160.3 (C-5), 155.3 (C-2), 149.5 (C-4'), 146.4 (C-3'), 141.3 (C-3), 123.1 (C-1'), 121.9 (C-6'), 116.3 (C-2'), 116.2 (C-5'), 108.3 (C-4a), 97.5 (C-6), 96.2 (C-8), 60.2 (7-OCH3), 56.3 (3-OCH3). The data are consistent with those reported previously [13].
3,7-dimethoxy-5,4'-dihydroxyflavone (10)
Pale yellow powder. C17H14O6, m/z 314.08; 1H-NMR(600MHz, CD3OD), δH: 7.98 (2H, d, J = 8.9 Hz, H-2', 6'), 6.93 (2H, d, J = 8.9 Hz, H-3', 5'), 6.51 (1H, d, J = 2.1Hz, H-8), 6.41 (1H, d, J = 2.1 Hz, H-6).
13C-NMR (151MHz, CD3OD), δC: 176.1 (C-4), 165.0 (C-7), 162.5 (C-8a), 161.3 (C-4'), 160.2 (C-5), 155.5 (C-2), 141.3 (C-3), 131.1 (C-2',6'), 122.7 (C-1'), 116.4 (C-3',5'), 108.6 (C-4a), 97.2 (C-6), 96.1 (C-8), 60.2 (7-OCH3), 56.4 (3-OCH3). The data are consistent with those reported previously [9].
Kaempferol (11)
Yellow powder. C15H10O6, m/z 286.05; 1H-NMR (600MHz, CD3OD), δH: 8.10 (2H, d, J = 8.9 Hz, H-2', 6'), 6.92 (2H, d, J = 8.9 Hz, H-3', 5'), 6.41 (1H, d, J = 2.1 Hz, H-8), 6.19 (1H, d, J = 2.1 Hz, H-6).
13C NMR (151 MHz, MeOD), δC: 177.3 (C-4), 165.5 (C-7), 162.5 (C-5), 160.5 (C-4′), 158.2 (C-9), 148.0 (C-2), 137.1 (C-3), 130.6 (C-2',6'), 123.7(C-1'), 116.3 (C-3',5'), 104.5 (C-10), 99.2 (C-6), 94.4 (C-8).The data are consistent with those reported previously [14].
Myricetin (12)
Yellow acicular crystal. C15H10O8, m/z 318.04; 1H-NMR (600MHz, CD3OD), δH: 7.36 (2H, s, H-2', 6'), 6.38 (1H, d, J = 2.1 Hz, H-8), 6.19 (1H, d, J = 2.1Hz, H-6). The data are consistent with those reported previously [12].
Quercetin (13)
Yellow acicular crystal. C15H10O7, m/z 318.04; 1H-NMR (600MHz, CD3OD), δH: 7.74 (1H, d, J = 2.2 Hz, H-2'), 7.64 (1H, dd, J = 8.5, 2.2 Hz, H-6'), 6.89 (1H, d, J = 8.5 Hz, H-5'), 6.39 (1H, d, J = 2.1 Hz, H-8), 6.19 (1H, d, J = 2.1 Hz, H-6).
13C NMR (151 MHz, MeOD), δC:177.3 (C-4), 165.5 (C-7), 162.5 (C-5), 158.2 (C-9), 148.7 (C-2), 147.9 (C-4′), 146.2 (C-3′), 137.2 (C-3), 124.1 (C-1′), 121.6 (C-6′), 116.2 (C-5′), 115.9 (C-2′), 104.5 (C-10), 99.2 (C-6), 94.4 (C-8).The data are consistent with those reported previously [14].
Hyperoside (14)
Yellow acicular crystal. C21H20O12, m/z 464.10; 1H-NMR(600MHz, CD3OD), δH: 7.72 (1H, d, J = 2.2 Hz, H-2'), 7.60 (1H, dd, J = 8.5, 2.2 Hz, H-6'), 6.88 (1H, d, J = 8.5 Hz, H-5'), 6.41 (1H, d, J = 1.9 Hz, H-8), 6.22 (1H, d, J = 2.1 Hz, H-6), 5.27 (1H, d, J = 7.7 Hz, Glu-1'')。3.72 (1H, dd, J = 11.9, 2.4 Hz, Glu-6''), 3.58 (1H, dd, J = 11.9, 5.4 Hz, Glu-6''), 3.49 (1H, dd, J = 9.1, 7.6 Hz, Glu-2''), 3.43(1H, t, J = 9.0Hz, Glu-3''), 3.36 (1H, m, Glu-4''), 3.23 (1H, ddd, J = 9.7, 5.4, 2.4 Hz, Glu-5'')。
13C-NMR (151MHz, CD3OD), δC: 179.5 (C-4), 166.0 (C-7), 163.0 (C-5), 159.0 (C-2), 158.4 (C-8a), 149.8 (C-4'), 145.9 (C-3'), 135.6 (C-3), 123.1 (C-6'), 123.0 (C-1'), 117.5 (C-5'), 116.0 (C-2'), 105.6(C-4a), 104.2 (C-1''), 99.9 (C-6), 94.7 (C-8), 78.4 (C-5''), 78.1 (C-3''), 75.7 (C-2''), 71.2 (C-4''), 62.5 (C-6''). The data are consistent with those reported previously [14].
(+) Catechin (15)
Dark red powder. C15H14O6, m/z 290.08; 1H-NMR (600MHz, CD3OD), δH: 6.85 (1H, d, J = 2.0 Hz, H-2'), 6.77 (1H, d, J = 8.1 Hz, H-5'), 6.73 (1H, dd, J = 8.2, 2.0 Hz, H-6'), 5.94 (1H, d, J = 2.3 Hz, H-8), 5.87 (1H, d, J = 2.3 Hz, H-6), 4.58 (1H, d, J = 7.5 Hz, H-2), 3.99 (1H, td, J = 7.9, 5.5 Hz, H-3), 2.86 (1H, dd, J = 16.1, 5.5 Hz, H-4α), 2.52 (1H, dd, J = 16.1, 8.2 Hz, H-4β).
13C-NMR (151MHz, CD3OD), δC: 157.8 (C-8a), 157.5 (C-7), 156.9 (C-5), 146.2 (C-4'), 146.2 (C-3'), 132.2 (C-1'), 120.0(C-2'), 116.0(C-5'), 115.2 (C-6'), 100.8 (C-4a), 96.2 (C-6), 95.4 (C-8), 82.8 (C-2), 68.8 (C-3), 28.5 (C-4). The data are consistent with those reported previously [14].
Quercetin-3-O -β- D-glucopyranoside-(3'→O-3''')-quercetin-3''-O -β- D-galactopyranoside (16)
1H-NMR (600MHz, CD3OD), δH: 7.84 (1H, d, J = 2.2 Hz, H-2'''), 7.71 (1H, d, J = 2.1 Hz, H-2'), 7.58 (1H, d, J = 8.4 Hz, H-6', 6'''), 6.86 (1H, dd, J = 8.4, 3.2 Hz, H-5', 5'''), 6.38 (1H, dd, J = 4.1, 2.1 Hz, H-8, 8''), 6.19 (1H, d, J = 2.1 Hz, H-6, 6''), 5.25 (1H, d, J = 7.6 Hz, H-Glu-1''''), 5.16 (1H, d, J = 7.8 Hz, H-Gal-1'''''), 3.87 (1H, d, J = 3.4 Hz, H-Gal-4'''''), 3.82 (1H, dd, J = 9.7, 7.8 Hz, H-Gal-2'''''), 3.71 (1H, dd, J = 11.9, 2.3 Hz, H-Glu-6''''), 3.64 (1H, dd, J = 11.2, 6.0 Hz, H-Gal-6'''''), 3.57 (3H, td, J = 11.6, 5.8 Hz, H-Glu-1'''', Gal-3''''', 5'''''), 3.48 (2H, m, H-Glu-2'''', 5''''), 3.43 (1H, t, J = 9.0 Hz, Gal-6'''''), 3.35 (1H, t, J = 9.3 Hz, H-Glu-4''''), 3.23 (1H, dq, J = 7.3, 2.7 Hz, H-Glu-3'''').
13C-NMR (151MHz, CD3OD), δC: 179.5 (C-4''), 179.4 (C-4), 166.2 (C-7''), 166.1 (C-7), 163.0 (C-5, 5''), 158.9(C-2), 158.7 (C-2''), 158.4 (C-9, 9'') 149.93 (C-4'''), 149.84 (C-4'), 145.8 (C-3'''), 145.7(C-4'), 135.7 (C-3''), 135.6 (C-3), 123.1 (C-6'''), 123.06 (C-6'), 122.9 (C-1'''), 122.8 (C-1'), 117.7 (C-2'''), 117.5 (C-2'), 116.0 (C-5'''), 115.9 (C-5'), 105.6 (C-10''), 105.5 (C-10), 105.4(C-Gal-1'''''), 104.31 (C-Glu-1''''), 99.9 (C-6, 6''), 94.7 (C-8, 8''), 78.3 (C-5''''), 78.1 (C-3''''), 77.1 (C-5'''''), 75.7 (C-2''''), 75.0 (C-3'''''), 73.1 (C-2'''''), 71.2 (C-4''''), 70.0 (C-4'''''), 62.5 (C-6''''), 61.9 (C-6'''''). The data are consistent with those reported previously [15].
8-epidiosbulbin E acetate (17)
White powder.C21H24O7, m/z 388.15; 1H-NMR (600MHz, CD3OD), δH: 7.65 (1H, m, H-16), 7.50 (1H, t, J = 1.8 Hz, H-15), 6.56 (1H, dd, J = 1.8, 0.9 Hz, H-14), 5.56 (1H, dd, J = 12.7, 3.5 Hz, H-12), 5.09 (1H, q, J = 2.7 Hz, H-6), 4.88 (1H, t, J = 5.2 Hz, H-2), 2.70 (1H, dt, J = 15.0, 2.6 Hz, H-7a), 2.51 (1H, m, H-3a, 4, 10), 2.17 (3H, m, H-1a, 8, 11a), 2.04 (1H, ddd, J = 15.0, 6.2, 2.5 Hz, H-7b), 2.05 (1H, ddd, J = 15.0, 6.2, 2.5 Hz, H-5), 1.92(1H, m, H-11b), 1.90 (3H, s, 21-CH3),1.88(1H, m, H-3b), 1.59 (1H, ddd, J = 15.0, 6.2, 2.5 Hz, H-1b), 1.19 (3H, s, 19-CH3).
13C-NMR (151MHz, CD3OD), δC: 179.2 (C-20), 174.6 (C-18), 172.3 (C-17), 144.9 (C-15), 141.5 (C-16), 126.7 (C-13), 109.5 (C-14), 79.1 (C-12), 72.0 (C-2), 71.9 (C-6), 46.8 (C-8), 43.9 (C-4), 41.9 (C-5), 40.3 (C-9), 39.3 (C-10), 35.6 (C-3), 32.8 (C-11), 28.9 (C-1), 27.5 (C-7), 21.7 (C-21), 21.0 (C-19). The data are consistent with those reported previously [16].
8-epidiosbulbin E (18)
White powder. C19H22O6, m/z 346.14. 1H-NMR (600MHz, CDCl3),1H NMR (600 MHz, Chloroform-d). δH: 7.57 (t, J = 1.2 Hz, 1H, H-16), 7.40 (t, J = 1.8 Hz, 1H, H-15), 6.94 (dd, J = 1.9, 0.9 Hz, 1H, H-14), 5.30 (dd, J = 11.0, 5.4 Hz, 1H, H-12), 4.88 (t, J = 5.1 Hz, 1H, H-2), 4.71 (d, J = 5.7 Hz, 1H, H-6), 2.76 (d, J = 5.4 Hz, 1H, H-7a), 2.64–2.60 (m, 1H, H-10), 2.60–2.55 (m, 1H, H-4), 2.18–2.12 (m, 2H, H-3a, 8), 2.11–2.06 (m, 2H, H-11,1a), 2.01(1H, d, J = 15.0, 6.2, 2.5 Hz, H-5),1.98 (ddd, J = 12.0, 5.5, 1.1 Hz, 1H, H-7b), 1.93 (d, J = 11.9 Hz, 1H, H-11b), 1.80 (d, J = 11.7 Hz, 1H, H-3b), 1.66–1.59 (m, 1H, H-1b), 1.25 (s, 3H, 19-CH3). The data are consistent with those reported previously [17].
Batatasin Ⅲ (19)
White powder. C15H16O3, m/z 244.29.1H-NMR (600MHz, CD3OD), δH:7.06 (1H, t, J = 7.8 Hz, H-5'), 6.65 (1H, dt, J = 7.6, 1.2 Hz, H-6'), 6.62 (2H, t, J = 2.0 Hz, H-4', 2'), 6.24 (1H, t, J = 1.8 Hz, H-6), 6.22 (1H, t, J = 1.8 Hz, H-2), 6.18 (1H, t, J = 1.8 Hz, H-4), 3.70 (3H, s, 5-OCH3), 2.79 (2H, m, a), 2.76 (2H, m, a').
13C-NMR (151MHz, CD3OD), δC: 162.2 (C-5), 159.3 (C-3), 158.3 (C-3'), 145.4(C-1), 144.6 (C-1'), 130.2 (C-5'), 120.8 (C-6'), 116.3 (C-2'), 113.7 (C-4'), 109.0 (C-4), 106.5 (C-6), 99.9 (C-2), 55.5 (5-OCH3), 39.1 (C-a), 38.8 (C-a'). The data are consistent with those reported previously [14].
Protocatechuic acid (20)
White powder. C7H6O4, m/z 154.03; 1H-NMR (600MHz, CD3OD), δH: 7.43(2H, m, H-5, 6), 6.79 (1H, d, J = 2.1Hz, H-2)
13C-NMR (150MHz, CD3OD), δC: 170.3 (C-COOH), 151.6 (C-4), 146.1 (C-3), 124.0 (C-1), 123.2 (C-6), 117.8 (C-5), 115.8 (C-2). The data are consistent with those reported previously [18].
In the present study, twenty compounds, including eight phenanthrenes (1–8), eight flavonoids (9–16), two diterpenes (17, 18), one bibenzyl derivatives (19), and one acid (20) were isolated from the rhizome of Dioscorea bulbifera. Among them, compound 1 is first reported from the Dioscorea bulbifera and compound 16 first reported from the genus Dioscorea.
Cytotoxic activity against CT26 cell lines
Compound 1 and compound 16 against CT26 cell lines were evaluated at different concentrations (2.5, 5, 10, 20, 40, 80µg/ml) to calculate IC50 values. The results were shown in Table 1.
Table 1
Cytotoxic activity of the isolates of Dioscorea bulbifera against CT26 cell lines
Compound | IC50 (µM) |
1 | 83.23 |
16 | > 100 |