Study on chemical constituents, biological activities of conifer species: Dacrycarpus imbricatus and fokienia hodginsii

5.88 (1H, d, J = 2.5 Hz, H-6); 4.59 (1H, d, J = 8 Hz, H-2); 4.01 (1H, ddd, J = 5.5, 5, 5.5 Hz, H-3); 2.86 (1H, dd, J = 5.5, 5.5 Hz, H-4a); 2.53 (1H, dd, J = 8, 8 Hz, H-4b). 13 C-NMR (125MHz, CD3OD), δ (ppm): 157.8 (C-5); 157.5 (C-7); 156.9 (C-9); 146.2 (C-4’); 146.21 (C-3’); 132.2 (C-1); 120.0 (C-6’); 116.1 (C-2’); 115.2 (C-5’); 100.8 (C-10); 96.3 (C-6); 95.5 (C-8); 82.8 (C-2); 68.8 (C-3); 28.46 (C-4). ● Compound 87 (ponasterone A) white powder, Rf = 0.3 (DCM:MeOH = 4.5:0.5), t o nc = 259-260 o C, HR-ESI-MS m/z = 487,3029 [M+Na] + (C27H44O6Na), m/z = 465.3209 [M+H] + (C27H45O6), molecular formula C27H44O6 1 H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 5.83 (1H, d, J = 2 Hz, H-7); 3.97 (1H, d, J = 2 Hz, H-3); 3.86 (1H, ddd, J = 4, 4.5, 3 Hz, H-2ax); 3.36 (1H, H-22); 3.17 (1H, m, H-9); 2.38 (1H, m, H-5); 2.14 (1H, m, H- 12ax); 2.02 (1H, H-17); 2.00 (1H, H-15α); 1.9 (1H, H-16α); 1.8 (1H, H- 12eq); 1.78 (1H, H-1eq); 1.78 (1H, H-11eq); 1.75 (1H, H-4eq); 1.75 (1H, H- 24a); 1.72 (1H, H-20); 1.60 (1H, H-23a); 1.65 (1H, H-11ax); 1.65 (1H, H- 4ax); 1.53 (1H, H-15β); 1.48 (1H, H-16β); 1.45 (1H, H-24b); 1.43 (1H, H- 1ax); 1.30 (1H, H-23b); 1.20 (3H, s, H-27); 0.98 (3H, s, H-26); 0.97 (3H, s, H-19); 0.94 (3H, H-21); 0.91 (3H, s, H-18). 13 C-NMR (125MHz, CD3OD), δ (ppm): 206.40 (C-6); 167.94 (C-8); 122.14 (C-7); 85.23 (C-14); 77.96 (C-20); 77.84 (C-22); 68.70 (C-2); 68.51 (C-3); 51.78 (C-5); 50.47 (C-17); 49.62 (C-13); 39.26 (C-10); 37.65 (C-23); 37.37 (C-1); 35.10 (C-9); 32.86 (C-4); 32.52 (C-12); 31.75 (C-15); 30.47 (C- 24); 29.21 (C-25); 24.40 (C-19); 23.40 (C-27); 22.75 (C-26); 21.50 (C-11); 21.50 (C-16); 20.99 (C-21); 18.32 (C-18). ● Compound 88 (20-hydroxyecdysone) colorless solid, Rf = 0.3 (DCM:MeOH = 4:1), [α] 25 D = 58 o (MeOH, c 0.1), t o nc = 244-246 o C HR-ESI-MS m/z = 503.2972 [M+Na] + (C27H44O7Na), m/z = 481.3151 [M+H] + (C27H45O7), molecular formula C27H44O7. 1 H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 5.83 (1H, J = 2 Hz, H- 7); 3.97 (1H, d, J = 1.5 Hz, H-3); 3.86 (1H, ddd, J = 3.5, 5, 3.5 Hz, H-2ax); 3.36 (1H, H-22); 3.17 (1H, t, J = 9Hz, H-9); 2.38 (1H, dd, J = 9.5; 5 Hz, H- 5); 2.16 (1H, m, H-12 ax); 2.02 (1H, H-17); 2.00 (1H, H-15α); 1.98 (1H, H- 16α); 1.8 (1H, H-12eq); 1.79 (1H, H-1eq); 1.78 (1H, H-11eq); 1.75 (1H, H- 4eq); 1.75 (1H, H-24a); 1.72 (1H, H-20); 1.67 (1H, H-23a); 1.65 (1H, H- 11ax); 1.65 (1H, H-4ax); 1.59 (1H, H-15β); 1.48 (1H, H-16β); 1.45 (1H, H- 24b); 1.43 (1H, H-1ax); 1.31 (1H, H-23b); 1.22 (3H, s, H-27); 1.22 (3H, s, H-26); 1.21 (3H, s, H-19); 0.98 (3H, H-21); 0.91 (3H, s, H-18). 7 13 C-NMR (125MHz, CD3OD), δ (ppm): 206.36 (C-6); 167.93 (C-8); 122.12 (C-7); 85.20 (C-14); 78.39 (C-20); 77.88 (C-22); 71.27 (C-25); 68.68 (C-2); 68.49 (C-3); 51.75 (C-5); 50.51 (C-17); 49.50 (C-13); 42.36 (C-24); 39.25 (C-10); 37.36 (C-1); 35.07 (C-9); 32.82 (C-4); 32.50 (C-12); 31.77 (C-15); 29.68 (C-26); 28.99 (C-27); 27.32 (C-23); 24.40 (C-19); 21.49 (C-16); 21.06 (C-21); 18.04 (C-18). ● Compound 84 (lambertic acid) white powder, Rf = 0.5 (n- hexane:EtOAc = 4:1), [α] 25 D = +58 o (MeOH, c 0.1), t o nc = 129-130 o C, molecular formula C20H28O3 1 H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 6.98 (1H, d, J = 8.3 Hz, H-12); 6.52 (1H, d, J = 8.5 Hz, H-11); 3.12-3.14 (1H, m, H-15); 2.94 (1H, dd, J = 14.1, 6.3 Hz, H-7α); 2.74-2.76 (1H, m, H-7β); 2.26-2.28 (1H, m, H-6α); 2.01(1H, dd, J = 13.8 Hz; H-1α); 1.86 (1H, d, J = 13.4 Hz; H- 3α); 1.71-1.73 (1H, m, H-2α); 1.67-1.69 (1H, m, H-1β); 1.62-1.64 (1H, m, H-2β); 1.42 (1H, d, J = 13.4 Hz; H-5); 1.38 (1H, d, J = 13.4 Hz; H-6β); 1.34 (3H, d, J = 7 Hz, H-16); 1.33 (3H, d, J = 7 Hz, H-17); 1.18 (3H, s, H- 20); 1.06 (3H, s, H-18); 1.00 (1H, dd, J = 13, 4.3 Hz; H-3β). 13 C-NMR (125MHz, CD3OD), δ (ppm): 181,64 (C-19); 153,38 (C- 12); 147,37 (C-9); 133,61 (C-8); 127,30 (C-14); 127,17 (C-13); 112,58 (C- 11); 54,41 (C-5); 44,87 (C-4); 40,91 (C-6); 39,45 (C-10); 38,83 (C-1); 32,49 (C-3); 29,29 (C-15); 27,73 (C-18); 23,69 (C-20); 23,16 (C-16); 23,12 (C-17); 22,63 (C-7); 21,20 (C-2). ● Compound 79 (cassipouryl hexadecanoat, new compound) colorless oil, Rf = 0.5 (n-hexane:EtOAc = 5:0.2), m/z: 555.5126 [M+Na] + (C36H68NaO2), molecular formula C36H68O2 . 1 H-NMR (500MHz, CDCl3), δ (ppm), J (Hz): 5.33 (1H, t, J = 7.0 Hz, H-14); 4.58 (2H, d, J = 7.0 Hz; H-15); 2.29 (2H, t, J = 7.5 Hz, H-2’); 2.00 (1H, m, H-12a); 1.69 (3H, br s, H-20); 1.60 (2H, m, H-11); 1.58-1.60 (2H, m, H-3’); 1,52 (1H, m, H-5); 1.38 (1H, m, H-6); 1.36 (2H, m, H-9); 1.30 (2H, m, H-8); 1.28 (2H, m, H-7); 1.25 (2H, m, H-3); 1.25 (2H, m, H-4); 1.25 (1H, m, H-10a); 1.25 (26H, m, H-4’- H-13’); 1.25 (2H, s, H-14’); 1.25 (2H, br s, H-15’); 1.14 (2H, m, H-2); 1.08 (1H, m, H-10b); 1.08 (1H, m, H- 12b); 0.87 (3H, t, J = 7 Hz; H-16’); 0.86 (3H, s, H-16); 0.85 (3H, s, H-17); 0.845 (3H, d, J = 7; H-18); 0.838 (3H, d, J = 7 Hz; H-19). 13 C-NMR (125MHz, CDCl3), δ (ppm): 173.93 (C-1’); 142.58 (C-13); 118.21 (C-14); 61.19 (C-15); 39.86 (C-12); 39.38 (C-2); 37.44 (C-10); 37.37 (C-4); 37.30 (C-8); 36.64 (C-1); 34.42 (C-2’); 32.80 (C-6); 32.68 (C-9); 31.93 (C-14’); 29.70-29.16 (C4’-C13’); 27.98 (C-5); 25.04 (C-11); 24.80 (C-7); 24.80 (C-3’); 24.47 (C-3); 22.69 (C-16); 22.62 (C-17); 22.71 (C-15’); 19.74 (C-18); 19.71 (C-19); 16.36 (C-20); 14.10 (C-16’). 8 ● Compound 85, (+)-spathulenol: colorless oil, Rf = 0.25 (n- hexane:EtOAc = 5:0.5), [α] 25 D = 5° (CHCl3, c 0.1) ESI-MS m/z: 221.3 [M+H] + , molecular formula C15H24O 1 H-NMR (500 MHz, CDCl3), δ (ppm), J (Hz): 4.69 (1H, s, H-14a); 4.66 (1H, s, H-14b); 2.42 (1H, dd, J = 13.0, 6.0 Hz, H-4a); 2.20 (1H, m, H- 6); 2.06 (1H, d, J = 13.0 Hz, H-4b); 1.77 (1H, m, H-8a); 1.55 (1H, m, H-8b); 1.32 (1H, d, J = 10.5 Hz, H-10); 1.28 (3H, s, H-15); 1.06 (3H, s, H-12); 1.03 (3H, s, H-13); 0.71 (1H, ddd, J = 11.0, 9.5, 6.0 Hz, H-2); 0.47 (1H, dd, J = 11.0, 9.5 Hz, H-1). 13 C-NMR (125MHz, CDCl3), δ (ppm): 153.46 (C-5); 106.26 (C-14); 80.99 (C-9); 54.35 (C-10); 53.41 (C-6); 41.75 (C-8); 38.87 (C-4); 29.92 (C-1); 28.66 (C-12); 27.50 (C-2); 26.72 (C-7); 26.08 (C-15); 24.79 (C-3); 20.27 (C-11); 16.34 (C-13). ● Compound cassipourol: colorless oil, Rf = 0.5 (n-hexane:DCM = 3:1.5), [α] 25 D = + 10.9° (CHCl3, c 0.1), molecular formula C20H38O 1 H-NMR (500MHz, CDCl3), δ (ppm), J (Hz): 5.41 (1H, qt, J = 1.5, 7.0 Hz, H-14); 4.15 (2H, d, J = 7.0 Hz; H-15); 1.99 (1H, m, H-12a); 1.67 (3H, br s, H-20); 1.52 (1H, m, H-5); 1.51 (2H, m, H-11); 1.40 (1H, m, H-9); 1.38 (1H, m, H-10a), 1.38 (1H, m, H-6); 1.34 (2H, m, H-8); 1.29 (1H, m, H-10b); 1.28 (1H, m, H-2α); 1.28 (2H, m, H-7); 1.26 (2H, m, H-3); 1.25 (2H, m, H-4); 1.09 (1H, m, H-12b); 1.04 (1H, m, H-2β); 0.86 (3H, s, H-16); 0.853 (3H, d, J = 7, H-18); 0.85 (3H, s, H-17); 0.846 (3H, d, J = 7 Hz, H-19). 13 C-NMR (125MHz, CDCl3), δ (ppm): 140.30 (C-13); 123.11(C-14); 59.43 (C-15); 39.87 (C-12); 39.38 (C-2); 37.44 (C-10); 37.37 (C-8); 37.37 (C- 4); 36.67 (C-1); 32.70 (C-9); 32.70 (C-6); 27.98 (C-5); 25.14 (C-11); 24.79 (C-3); 24.47 (C-7); 22.71 (C-16); 22.62 (C-17); 19.75 (C-18); 19.71 (C-19); 16.17 (C-20). ● DIH03 the mixture of trans-communic acid (compound 81) and (compound 82) cis–communic acid, Yellowish oil, molecular formula C20H30O2 1 H-NMR compound 81 (500MHz, CDCl3), δ (ppm), J (Hz): 6.32 (1H, dd, J = 17.0, 10.5 Hz, H-14); 5.41 (1H, t, J = 6.0 Hz, H-12); 5.04 (1H, d, J = 17.0 Hz, H-15a); 4.88 (1H, d, J = 10.5 Hz, H-15b); 4.84 (1H, s, H-17a); 4.47 (1H, s, H-17b); 1.75 (3H, s, H-16); 1.25 (3H, s, H-18); 0.65 (3H, s, H-20). 13 C-NMR compound 81 (125MHz, CDCl3), δ (ppm): 183.90 (C-19); 147.93 (C-8); 141.61 (C-12); 133.90 (C-14); 133.45 (C-13); 109.91 (C- 15); 107.66 (C-17); 56.43 (C-5); 56.29 (C-9); 44.21 (C-4); 40.37 (C-10); 39.27 (C-6); 38.49 (C-1); 37.93 (C-3); 29.04 (C-18); 25.84 (C-7); 23.31 (C-11); 19.93 (C-2); 12.83 (C-20); 11.84 (C-16). 9 1 H-NMR compound 82 (500MHz, CDCl3), δ (ppm), J (Hz): 6.78 (1H, dd, J = 17.5, 11.0 Hz, H-14); 5.31 (1H, t, J = 6.0 Hz, H-12); 5.18 (1H, d, J = 17.5 Hz, H-15a); 5.08 (1H, d, J = 11.0 Hz, H-15b); 4.84 (1H, s, H-17a); 4.49 (3H, s, H-17b); 1.77 (3H, s, H-16); 1.25 (3H, s, H-18); 0.65 (3H, s, H-20). 13 C-NMR compound 82 (125MHz, CDCl3), δ (ppm): 183.90 (C-19); 147.93 (C-8); 133.86 (C-14); 131.67 (C-12); 131.56 (C-13); 113.25 (C- 15); 107.80 (C-17); 56.70 (C-9); 56.43 (C-5); 44.21 (C-4); 40.43 (C-10); 39.27 (C-6); 38.52 (C-1); 37.93 (C-3); 29.04 (C-18); 25.84 (C-7); 22.28 (C-11); 19.93 (C-2); 19.72 (C-16); 12.83 (C-20). ● Compound 86 (β-sitosterol): colorless solid, Rf = 0.34 (n- hexane:DCM = 1:3). ● Compound 83 (pimaric acid): colorless solid, Rf = 0.5 (n- hexane:EtOAc = 4:1), [α] 20 D = + 87.3 o (CHCl3, c 0.4), t o nc = 217-219 o C ESI-MS m/z: 303.6 [M+H] + , molecular formula C20H30O2 1 H-NMR (500MHz, CDCl3), δ (ppm), J (Hz): 5.77 (1H, dd, J = 17.0, 10.5 Hz, H-15); 5.22 (1H, s, H-14); 4.90 (1H, dd, J = 17.0, 1.5 Hz, H- 16a); 4.88 (1H, dd, J = 10.5, 1.5 Hz, H-16b); 1.21 (3H, s, H-19); 1.04 (3H, s, H-17); 0.84 (3H, s, H-20). 13 C-NMR (125MHz, CDCl3), δ (ppm): 184.92 (C-18); 148.91 (C- 15); 136.63 (C-8); 129.13 (C-14); 110.16 (C-16); 50.58 (C-9); 48.84 (C-5); 47.28 (C-40); 38.30 (C-1); 37.74 (C-10); 37.40 (C-13); 37.05 (C-3); 35.48 (C-7); 34.46 (C-12); 26.04 (C-17); 24.91 (C-6); 18.57 (C-11); 18.16 (C-2); 16.78 (C-19); 15.22 (C-20). ● Compound 80 (12,13-dihydroxylabda-8(17),14-dien-19-oic acid): colorless oil, Rf = 0.2 (n-hexane:EtOAc = 2:1), molecular formula C20H32O4 1 H-NMR (500MHz, CDCl3), δ (ppm), J (Hz): 5.93 (1H, dd, J = 11, 11 Hz, H-14); 5.34 (1H, d, J = 17 Hz, H-15); 5.21 (1H, dd, J = 10.5, 10.5 Hz, H-15); 4.85 (1H, s, H-17a); 4.44 (1H, s, H-17b); 3.48 (1H, d, J = 10.5 Hz, H-12); 2.4 (1H, m, H-7); 2.18 (1H, d, J = 13 Hz, H-3); 2.03 (1H, m, H- 9); 1.97 (1H, m, H-7); 1.85 (1H, m, H-2α); 1.81-2.0 (2H, m, H-6); 1.79 (1H, m, H-1); 1.6 (1H, m, H-11); 1.53 (1H, m, H-2β); 1.4 (1H, m, H-5); 1.4 (1H, m, H-11); 1.33 (3H, s, H-16); 1.24 (3H, s, H-18); 1.15 (1H, m, H- 1); 1.07 (1H, m, H-3); 0.59 (3H, s, H-20). 13 C-NMR (125MHz, CDCl3), δ (ppm): 183.04 (C-19); 148.36 (C- 19); 140.88 (C-8); 114.57 (C-15); 106.71 (C-17); 75.98 (C-13); 75.84 (C- 12); 56.24 (C-5); 51.65 (C-9); 44.21 (C-4); 40.13 (C-10); 39.03 (C-1); 38.70 (C-7); 37.97 (C-3); 29.02 (C-16); 26.06 (C-6); 24.54 (C-18); 19.87 (C-2); 12.95 (C-20). 10 2.3.2. Fokienia hodginsii Figure 2.4. Schematic diagram showing steps for the isolation of pure compounds from the twig and leaves of Fokienia hodginsii ● Compound 81 (trans-communic acid) colorless solid, Rf = 0.5 (n- hexane: EtOAc = 4:1), [α] 25 D = +38 o (MeOH, c 0.1), t o nc = 130-132 o C, molecular formula C20H30O2 1 H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 6.34 (1H, dd, J = 17.5, 17 Hz, H-14); 5.40 (1H, t, J = 6.5 Hz, H-12); 5.04 (1H, d, J = 17.5 Hz, H- 15a); 4.88 (1H, d, J = 9.5 Hz; H-15b); 4.85 (1H, br s, H-17a); 4.47 (1H, br s, H-17b); 1.75 (3H, s, H-16); 1.22 (3H, s, H-18); 0.70 (3H, s, H-20). 13 C-NMR (125MHz, CD3OD), δ (ppm): 181.20 (C-19); 149.55 (C-8); 142.85 (C-14); 134.89 (C-12); 134.55 (C-13); 110.15 (C-15); 107.95 (C-17); 11 57.90 (C-9); 57.43 (C-5); 45.14 (C-4); 41.39 (C-10); 40.62 (C-1); 39.65 (C-3); 39.34 (C-7); 29.62 (C-18); 27.31 (C-6); 24.32 (C-11); 21.20 (C-2); 13.43 (C- 20); 12.02 (C-16). ● Compound 86 (β-sitosterol): colorless solid, Rf = 0.34 (n- hexane:DCM = 1:3). ● Compound 92 (totarolone): colorless solid, Rf = 0.5 (n- hexane:EtOAc = 4:1), [α] 25 D = +101.5 o (MeOH, c 0.1), t o nc = 188-189 o C, molecular formula C20H28O2. 1 H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 6.95 (1H, d, J = 8.5 Hz, H-11); 6.57 (1H, d, J = 8.5 Hz, H-12); 3.26 (1H, s, H-15); 1.34 (3H, d, J = 3.5 Hz, H-16); 1.35 (3H, d, J = 3.5 Hz, H-17); 1.28 (3H, s, H-18); 1.16 (3H, s, H-19); 1.14 (3H, s, H-20). 13 C-NMR (125MHz, CD3OD), δ (ppm): 220.56 (C-3); 155.12 (C- 13); 140.11 (C-9); 134.93 (C-8); 132.05 (C-14); 124.81 (C-11); 115.58 (C- 12); 51.52 (C-5); 48.32 (C-4); 39.62 (C-1); 38.39 (C-10); 35.79 (C-2); 30.14 (C-7); 28.85 (C-15); 27.30 (C-18); 25.07 (C-20); 21,68 (C-6); 21,47 (C-19); 20,58 (C-16); 20,51 (C-17). ● Compound 90 (15-nor-labda-8(17),12E-diene-14-carboxaldehyde- 19-oic acid): colorless oil, Rf = 0.5 (n-hexane:EtOAc = 4:1), molecular formula C19H28O3 1 H-NMR (500MHz, CD3OD): δ (ppm), J (Hz): 9.31 (1H, d, J = 3 Hz; H- 15); 6.56 (1H, ddd, J = 3, 3, 6 Hz; H-12); 4.86 (1H, s, H-17a); 4.44 (1H, s, H- 17b); 1.76 (3H, d, J = 1.1 Hz, H-16); 1.24 (3H, s, H-18); 0.71 (3H, s, H-20). 13 C-NMR (125MHz, CD3OD), δ (ppm): 197.07 (C-14); 181.13 (C-19); 158.69 (C-12); 149.57 (C-8); 140.01 (C-13); 108.03 (C-17); 57.25 (C-5); 57.15 (C-9); 45.15 (C-4); 41.38 (C-10); 40.55 (C-1); 39.42 (C-3); 39.26 (C-7); 29.54 (C-18); 27.24 (C-6); 25.73 (C-11); 21.14 (C-2); 13.37 (C-20); 9.29 (C-16). ● Compound 91 (13-oxo-15,16-dinor-labda-8(17),11E-diene-19-oic acid): colorless oil, Rf = 0.57 (n-hexane:EtOAc = 4:1), [α] 25 D = +20.5 o (MeOH, c 0.1), molecular formula C18H26O3 1 H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 7.01 (1H, dd, J = 10.5, 10 Hz, H-11); 6.10 (1H, d, J = 16 Hz, H-12); 4.85 (1H, H-17a); 4.45 (1H, s, H-17b); 2.6 (1H, dd, J = 10.5 Hz, H-9); 2.47 (1H, m, H-3α); 2.29 (3H, s, H- 16); 2.18 (1H, m, H-7α); 2.04 (1H, m, H-3β); 1.98 (2H, m, H-6); 1.89 (1H, m, H-2α); 1.48 (1H, m, H-1α); 1.45 (1H, m, H-2β); 1.33 (1H, m, H-5); 1.24 (3H, s, H-18); 1.09 (1H, m, H-1β); 1.07 (1H, m, H-7β); 0.86 (3H, s, H-20). 13 C-NMR (125MHz, CD3OD), δ (ppm): 200.88 (C-13); 180.12 (C- 19); 149.89 (C-8); 148.67 (C-11); 134.58 (C-12); 108.87 (C-17); 61.35 (C- 9); 56.38 (C-5); 45.08 (C-4); 42.13 (C-1); 40.95 (C-10); 39.31 (C-3); 38.24 12 (C-7); 29.42 (C-18); 27.11 (C-14); 26.33 (C-6); 20.86 (C-2); 14.14 (C-20). ● Compound 93 (3β-hydroxytotarol): colorless oil, Rf = 0.55 (n- hexane:EtOAc = 4:1), [α] 25 D = +29 o (MeOH, c 0.1), molecular formula C20H30O2 1 H-NMR (500MHz, CD3OD),δ (ppm), J (Hz): 6.92 (1H, d, J = 8.5 Hz, H-11); 6.53 (1H, d, J = 8.5 Hz, H-12); 4.1 (1H, d, J = 7 Hz, H-3); 3,24 (1H, dd, J = 5, 5 Hz, H-15); 2.97 (1H, dd, J = 6; 6 Hz, H-7α); 2.71 (1H, dd, J = 4, 7.5 Hz, H-7β); 2.30 (1H, m, H-1α); 2.03 (1H, s, H-6α); 19.6 (2H, m, H-2); 1.82 (1H, m, H-6β); 1.42 (1H, m, H-1β); 1.35 (3H, H-17); 1.32 (3H, H-16); 1.29 (1H, m, H-5); 1.17 (3H, s, H-18); 1.07 (3H, s, H-19); 0.88 (3H, s, H-20). 13 C-NMR (125MHz, CD3OD), δ (ppm): 155.32 (C-13); 142.29 (C-9); 134.89 (C-8); 132.07 (C-14); 123.78 (C-11); 115.26 (C-12); 79.58 (C-3); 50.93 (C-5); 39.87 (C-4); 39.27 (C-1); 38.56 (C-10); 30.29 (C-7); 28.96 (C-2); 28.76 (C-19); 25.61 (C-20); 20.56 (C-16&C-17); 20.43 (C-6); 16.08 (C-18). ● Compound 80 (12,13-dihydroxylabda-8(17),14-dien-19-oic acid) colorless solid, Rf = 0.5 (n-hexane:EtOAc = 4:1), molecular formula C20H32O4 ● Compound 94 [(9S)-drummondol]: colorless oil, Rf = 0.5 (n- hexane:EtOAc = 4:1), molecular formula C13H20O4 1 H-NMR (500MHz, CD3OD) δ (ppm), J (Hz): 6.19 (1H, dd, J = 16.5, 5.5 Hz, H-8); 6.05 (1H, d, J = 16.5 Hz, H-7); 4.40 (1H, dq, J = 5.2, 6.5 Hz, H-9); 3.93 (1H, dd, J = 8, 2.5 Hz, H-11α); 3.67 (1H, d, J = 8 Hz, H-11β); 2.68 (1H, dd, J = 18, 2.5 Hz; H-2α); 2.37 (1H, dd, J = 18, 2.5 Hz, H-2β); 2.80 (1H, d, J = 17.5 Hz, H-4α); 2.45 (1H, dd, J = 17.5, 2.5 Hz; H-4β); 1.30 (3H, d, J = 6.5 Hz, H-10); 1.20 (3H, s, H-13); 0.98 (3H, s, H-12). 13 C-NMR (125MHz, CD3OD), δ (ppm): 211.23 (C-3); 140.68 (C-8); 125.66 (C-7); 87.45 (C-5); 82.34 (C-6); 78.39 (C-11); 68.87 (C-9); 53.94 (C-4); 53.23 (C-2); 48.49 (C-1); 23.97 (C-10); 19.17 (C-13); 15.65 (C-12). ● Compound 95 (vomifoliol): colorless oil, Rf = 0.5 (n- hexane:EtOAc = 4:1), molecular formula C13H20O3 1 H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 5.90 (1H, d, J = 1 Hz, H-4); 5.83 (1H, d, J = 15.5 Hz, H-8); 5.79 (1H, d, J = 15.5 Hz, H-7); 4.34 (1H, dq, J = 6.5, 4 Hz, H-9); 2.54 (1H, d, J = 17 Hz, H-2α); 2.18 (1H, d, J = 17 Hz, H-2β); 1.95 (3H, d, J = 1 Hz, H-13); 1.26 (3H, d, J = 7 Hz, H-10); 1.06 (3H, s, H-12); 1.03 (3H, s, H-11). 13 C-NMR (125MHz, CD3OD), δ (ppm): 201.34 (C-3); 167.53 (C-5); 136.91 (C-8); 130.08 (C-7); 127.10 (C-4); 79.97 (C-6); 68.72 (C-9); 50.72 (C-2); 42.43 (C-1); 24.48 (C-11); 23.81 (C-10); 23.47 (C-12); 19.59 (C-13). 13 Chapter 3: RESULTS AND DISCUSSION 3.1. Determination the chemical structure of isolated compounds from Dacrycarpus imbricatus 13 Compounds have been isolated and determined the structure from Dacrycarpus imbricatus, including: 3 steroid 86, 87 and 88; 1 flavonoid 89; 1 sugar saccaroza (GB2), 1 secquiterpene 85; 7 diterpene, 3 compound diterpene labdane 80; 81 and 82, 2 compound diterpene abietane 83, 84 and 2 diterpene monocycle 78 and 79. ● Compound 78 and 79: Cassipourol and cassipouryl hexadecanoate (new compound) Compounds 78 and 79 were obtained as colorless semi-liquid compounds. The molecular formula of 78 was established as C20H38O from its HR-ESI-MS in combination with 13 C NMR, and DEPT spectroscopic data. The 13 C NMR and DEPT spectra indicated the presence of 20 14 carbons in this compound including 5xCH3, 9xCH2, 4xCH, 2xCq. These spectra also revealed the existence of the only one double bond (C 140.3, 123.1) in the structure suggesting compound 78 must be a monocyclic diterpene. The l H NMR showed signals of an olefinic proton as a quartet triplets at  5.41 (J = 1.5, 7.0 Hz); two oxymethylene protons as a doublet at H 4.15 (2H, J = 7.0 Hz). Remaining proton signals in the l H NMR spectrum were extremely overlapped, thus the chemical shift assignments were determined by HSQC experiment. Namely, three methyl singlets at H 1.67, 0.86, 0.85 correlated with the carbon signals at C 16.2, 22.6, 22.7, respectively in the HSQC spectra. In addtion, it could be assigned for two methyl doublets at H 0.846; 0.853/C19.7, 19.8; eight methylenes and three methines between  1.04 and  1.99 (Table 1). A comparison of the 1 H and 13 C NMR data of 78 with those of cassipourol indicated that they were identical [45] A comparison of the 1 H and 13 C NMR data of 79 with those of 78 indicated that they were almost identical except for the presence of signals for an aliphatic long-chain acid derivative. This group was determined through the apperance of one carbonyl ester (C 173.9), fourteen methylenes (between C 29.2 and 29.7 ppm) and one methyl group (C 14.1) beside the 20 carbons of cassipourol in the 13 C NMR spectrum of 79 (Table 3.1). Similarly, it could be seen not only the resonances of 78 but also the resonances of methyl triplet at H 0.87 (J = 7 Hz), strong broad singlet at H ca 1.25 and a triplet at H 2.29 (J = 7.5 Hz) in the 1 H NMR of 79. The singlet at  1.25 correlated to the signal of methylene carbon (at C ca. 29 ppm), while triplet at H 2.29 (J = 7.5 Hz) connected to a shifted downfield methylene at C 34.4 in the HSQC spectrum. The HR-ESI-MS exhibited molecular ion peak at m/z 555.5126 [M+Na] + (calcd for C36H68NaO2, 555.5117) establishing a molecular formula of C36H68O2 to compound 79 and the fragment at m/z 318.2965 (C20H39NaO required 318.2899) indicating the presence of a cassipouryl moiety. These data allowed to identify aliphatic chain as a hexadecanoic acid derivative. The esterification of this acid with cassipourol on C-15 was suggested by the chemical shift of C-15 at C 61.19 (shifted downfield,  +1.76 ppm due to este RCOO group). Furthermore, it was also confirmed by the key HMBC correlations H-14/C-12, C-15; H-15/C-14, C-13, C-1’ (C=O); H- 2’/C-1’, C-3’, C-4’ and H-3’/C-2’, C-4’. 15 (CH2)11CH3 O1' 2' 3' 4' O 1 3 5 6 7 9 19 11 13 14 2 4 8 10 17 18 16 HMBC (H/C ) and NOESY ( ) 15 H Figure 3.4. Selected HMBC and NOESY correlations of compounds 79 The configurations of the chiral centers at C-5, C-6 and three methyl groups at C-16, C-17, and C-18 of 79 were similar to those of 78 due to the same phyto origin. This finding was deduced from their almost identical 13 C NMR values and coupling constants. In addition, it was also supported by the correlations of Me-16 with H-6 and the Me-17, Me-18 with methylene group H2-7 in the NOESY spectrum. The trans (E stereochemistry) of the double bond C-13 and C-14 was determined by the correlation of Me-20 with oxymethylene protons H2-15 (no correlation between Me-20 with H-14). Consequently, the structure of 79 was elucidated as cassipouryl hexadecanoate, a new cassipourol dervative. Table 3.1.: 13 C- and 1 H NMR data for compounds 78 and 79 (125/500 MHz, CDCl3, ppm) a C 78 79 C H C H HMBC (HC) 1 36.7 - 36.6 - - 2 39.4 1.28 m, 1.04 m 39.4 1.14 m, 2H C-3; C-4; C-16; C-17 3 24.8 1,26 m 24.5 1.25 m C-2 4 37.4 1,25 m 37.4 d * 1.25 m C-5; C-18 5 28.0 1.52 m 28.0 1.52 m C-3; 6 32.7 1.38 m 32.8 1.38 m C-4; C-7; C-18 7 24.5 1.28 m 24.8 1.28 m C-6; C-8, 8 37.4 1.34 m 37.3 d * 1.30* m C-7; C-19 9 32.7 1.40 m 32.7 1.36 m C-8 10 37.4 1.38 m, 1.29 m 37.4 1.25 m, 1.08m C-8; C-9; C-19 11 25.1 1.51 m 25.0 1.60 m C-10; C-12 12 39.9 1.99 m, 1.09 m 39.9 2.00 m, 1.08 m C-20; C-11; C-10; C-14; C-13 13 140.3 - 142.6 - - 14 123.1 5.41 qt (1.5. 7.0) 118.2 5.33 t (7.0) C-20; C-12; C-15 (w) 15 59.4 4.15 d (7.0) 61.2 4.58 d (7.0) C-14; C-13; C-1’ (C=O); 16 22.7 b 0.86 s 22.7 e 0.86 s* C-1; C-2; C-17 17 22.6 b 0.85 s 22.6 e 0.85 s* C-1; C-2; C-16; 18 19.8 c 0.853b d (7) 19.7 f 0.845 d (7) C-5 16 19 19.7 c 0.846 d (7) 19.7 f 0.838 d (7) C-8; C-9 20 16.2 1.67 br s 16.4 1.69 br s C-12; C-14 C-1’ - - 173.9 - - 2’ - - 34.4 2.29 t (7.5) C-1’, C-3’, C-4’ 3’ - - 24.8 1.58-1.60 m C-2’, C-4’ 4’-13’ - - 29.7 - 29.2 1.25 m, 26 H * 14’ - - 31.9 1.25 s * 15’ - - 22.7 1.25* br s * 16’ - - 14.1 0.87 t (7) * a Assignments made on the basis of HSQC and HMBC spectroscopic data and in comparison with the literature values [45] b-h values having the same superscript in the respective columns may be interchanged. * Overlap signals (of C1’-C16’= hexadecanoate). Figure 3.5. HR-ESI-MS spectrum of compound 79 17 Figure 3.6. 1 H-NMR spectrum of compound 79 (CDCl3, 500MHz) Figure 3.7. 13 C-NMR spectrum of compound 79 (CDCl3, 125 MHz) 18 Figure 3.8. HSQC spectrum of compound 79 Figure 3.8. HSQC spectrum of compound 79 19 Figure 3.8. HSQC spectrum of compound 79 Figure 3.9. HMBC spectrum of compound 79 20 Figure 3.10. COSY spectrum of compound 79 Figure 3.11. NOESY spectrum of compound 79 3.2. Determination the structure of isolated compounds from Fokienia hodginsii 9 Compounds have been isolated and determined the structure from Fokienia hodginsii, including: 1 steroid 86; 6 diterpene, 2 compound 21 abietane diterpene 92, 93, 2 compound labdane diterpene 81, 80, 2 nor- labdane 90, 91; 2 megastigmen 94 and 95. ● Compound 92: Totarolone The 1 H NMR spectrum of 92 revealed two aromatic ortho-coupled protons at δH 6.95 (1H, d, J = 8.5 Hz, H-11); 6.58 (1H, d, J = 8.5 Hz, H- 12), three singlet methyl groups at δH 1.29 (3H, s, H-20); 1.17 (3H, s, H- 19); 1.14 (3H, s, H-18), an isopropyl group attached to a benzene ring by two doublet methyl signals at δH 1.34, 1.33 (each 3H, J = 6.5 Hz) and one methine proton at δH 3.27 (1H, br s, H-15), and aliphatic protons in the range from δH 1.73 to 3.03 ppm. Its 13 C-NMR and DEPT spectra showed signals of 20 carbon atoms including a ketone group, 5xCH3, 4xCH2, 4xCH, and 6xCq. The presence of an aromatic ring was confirmed by two methine signals at δC 124.81 (C-11), 115.58 (C-12); one quaternary carbon bonded to an oxygen atom at δC 155.12 (C-13) and three other quaternary carbons at δC 140.11 (C-9), 134.93 (C-8), 132.05 (C-14). The HMBC correlations observed between H-16, H-17 and C-14; H-11 and the C-13 indicated that the isopropyl group was at C-14, and the phenolic hydroxyl group at C-13. Thus, the structure of 92 was concluded to be 13-hydroxy- 8,11,13-totaratrien-3-one (3-oxototarol, 3-ketototarol). This compound showed the strong activity against some gram-positive and gram-negative bacteria as well as against some human pathogenic fungi [87-91]. Figure 3.58. 1 H-NMR spectrum of compound 92 (500MHz, CD3OD) 22 Figure 3.59. 13 C-NMR spectrum of compound 92 (125 MHz, CD3OD) 3.3. Biological activities of some isolated compounds Some of the compounds of Dacrycarpus imbricatus and Fokienia hodginsii: pimaric acid (83), lambetic acid (84), 12,13-dihydroxylabda-8(17),14- dien-19-oic acid (80), trans–communic acid (81), 15-nor-labda-8(17),12E- diene-14-carboxaldehyde-19-oic a