A Use of Alkaloid in Preparing Pharmaceutical Compositions for Preventing or Treating Pulmonary Fibrosis

Abstract

A new use of a compound as indicated in structural formula I in preparing medications for preventing and/or treating pulmonary fibrosis includes the compound having the structure as indicated in structural formula 1 that substantially reduces the inflammation of diseased lung tissue, lowers the concentration of fibrosis factors TGF-βI in diseased lung tissue, decreases the excessive deposition of collagen in diseased lung tissue, and has substantial prevention and treatment effectiveness against fibrosis.

Claims

1. A use of alkaloid having a structure as indicated in structural Formula I in preparing pharmaceutical compositions for one or both of preventing and treating pulmonary fibrosis: ##STR00013## wherein, R.sub.1 is selected from H, α or β OH, α or β COOH, α or β halogen substitution group, α or β alkoxy groups, α or β alkyl group, and α or β alpha-methyl-gamma-butyrolactone; R.sub.2 is selected from H, α or β OH, α or β COOH, α or β halogen substitution group, α or β alkoxy groups, and α or β alkyl group; R.sub.3 is selected from H, α or β OH, α or β COOH, α or β halogen substitution group, a or β alkoxy groups, and α or β alkyl group; or R.sub.2 and R.sub.3 are cyclized as the following structure by α or β configuration: ##STR00014## wherein, R.sub.8 is selected from α or β alkyl group; R.sub.4 is selected from α or β alkyl group; R.sub.5 is selected from α or β H; R.sub.6 is selected from α or β H, and α or β OH; and R.sub.7 is selected from α or β H.

2. A use of compounds having a structure as indicated in structural Formula Ia in preparing pharmaceutical compositions for one or both of preventing and treating pulmonary fibrosis according to claim 1, ##STR00015## wherein R.sub.1 is selected from H, α or β OH, α or β COOH, α or β halogen substitution group, α or β alkoxy groups, α or β alkyl group, and α or β alpha-methyl-gamma-butyrolactone; R.sub.2 is selected from α or β H; R.sub.3 is selected from α or β H; R.sub.4 is selected from α or β alkyl group; R.sub.5 is selected from α or β H; R.sub.6 is selected from α or β H and α or β OH; R.sub.7 is selected from α or β H; and R.sub.8 is selected from α or β alkyl group.

3. The use according to claim 2, wherein the compounds are alkaloids having the structure as indicated in structural Formula I or pharmaceutical derivatives thereof.

4. The use according to claim 3, wherein the pharmaceutical derivatives of the compounds are salts or esters of alkaloids having the structure as indicated in structural Formula I.

5. The use according to claim 1, wherein the pharmaceutical compositions include at least one active ingredient such as alkaloids having the structure as indicated in structural Formula I and a pharmaceutical carrier.

6. The use according to claim 5, wherein the pharmaceutical compositions include ingredients with the following mass percentages: 0.01%-99% alkaloids having the structure as indicated in structural Formula I and 0.01%-99% of the pharmaceutical carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIGS. 1A-1C show the effects of 8 alkaloids and pentoxyverine on pulmonary fibrosis mice induced by bleomycin.

[0052] Wherein, FIG. 1A: Content of Hydroxyproline; FIG. 1B: HE stained pathological sections; FIG. 1C: Masson stained pathological sections. Wherein, 1:sham operation group, 2:model group, 3:pentoxyverine, 4:pirfenidone, 5:compound I, 6:compound II, 7:compound III, 8:compound IV, 9:compound V, 10:compound VI, 11:compound VII, 12:compound VIII. Compared with sham operation group, #P<0.05, ##P<0.01; compared with model group, *P<0.05, **P<0.01.

[0053] FIG. 2 shows the effects of 3 alkaloids on lung index of pulmonary fibrosis mice induced by bleomycin. Wherein, A: prevention groups, B: treatment group; 1:sham operation group, 2:model group, 3:compound I low-dose (30 mg/kg) group, 4:compound I high-dose (60 mg/kg) group, 5:compound VII low-dose (30 mg/kg) group, 6:compound VII high-dose (60 mg/kg) group, 7:compound VIII low-dose (30 mg/kg) group, 8:compound VIII high-dose (60 mg/kg) group, 9:pirfenidone (300 mg/kg) group. Compared with sham operation group, #P<0.05, ##P<0.01; compared with model group, *P<0.05, **P<0.01.

[0054] FIG. 3 shows the effects of 3 alkaloids on pulmonary histopathological changes of pulmonary fibrosis mice induced by bleomycin (HE stained). Wherein, A: prevention groups, B: treatment group; 1:sham operation group, 2:model group, 3:compound I low-dose (30 mg/kg) group, 4:compound I high-dose (60 mg/kg) group, 5:compound VII low-dose (30 mg/kg) group, 6:compound VII high-dose (60 mg/kg) group, 7:compound VIII low-dose (30 mg/kg) group, 8:compound VIII high-dose (60 mg/kg) group, 9:pirfenidone (300 mg/kg) group.

[0055] FIG. 4 shows the effects of 3 alkaloids on lung tissue inflammation scores of pulmonary fibrosis mice induced by bleomycin (HE stained). Wherein, A: prevention groups, B: treatment group; 1:sham operation group, 2:model group, 3:compound I low-dose (30 mg/kg) group, 4:compound I high-dose (60 mg/kg) group, 5:compound VII low-dose (30 mg/kg) group, 6:compound VII high-dose (60 mg/kg) group, 7:compound VIII low-dose (30 mg/kg) group, 8:compound VIII high-dose (60 mg/kg) group, 9:pirfenidone (300 mg/kg) group. Compared with sham operation group, #P<0.05, ##P<0.01; compared with model group, *P<0.05, **P<0.01.

[0056] FIG. 5 shows the effects of 3 alkaloids on pulmonary histopathological changes of pulmonary fibrosis mice induced by bleomycin (Masson stained). Wherein, A: prevention groups, B: treatment group; 1:sham operation group, 2:model group, 3; compound I low-dose (30 mg/kg) group, 4:compound I high-dose (60 mg/kg) group, 5:compound VII low-dose (30 mg/kg) group, 6:compound VII high-dose (60 mg/kg) group, 7:compound VIII low-dose (30 mg/kg) group, 8:compound VIII high-dose (60 mg/kg) group, 9:pirfenidone (300 mg/kg) group.

[0057] FIG. 6 shows the effects of 3 alkaloids on collagen contents of lung tissues of pulmonary fibrosis mice induced by bleomycin (Average optical density value statistics). Wherein, A: prevention groups, B: treatment group; 1:sham operation group, 2:model group, 3:compound I low-dose (30 mg/kg) group, 4:compound I high-dose (60 mg/kg) group, 5:compound VII low-dose (30 mg/kg) group, 6:compound VII high-dose (60 mg/kg) group, 7:compound VIII low-dose (30 mg/kg) group, 8:compound VIII high-dose (60 mg/kg) group, 9:pirfenidone (300 mg/kg) group. Compared with sham operation group, #P<0.05, ##P<0.01; compared with model group, *P<0.05, **P<0.01.

[0058] FIG. 7 shows the effects of 3 alkaloids on content of Hydroxyproline (Hyp) in lung tissue of pulmonary fibrosis mice induced by bleomycin. Wherein, A: prevention groups, B: treatment group; 1:sham operation group, 2:model group, 3:compound I low-dose (30 mg/kg) group, 4:compound I high-dose (60 mg/kg) group, 5:compound VII low-dose (30 mg/kg) group, 6:compound VII high-dose (60 mg/kg) group, 7:compound VIII low-dose (30 mg/kg) group, 8:compound VIII high-dose (60 mg/kg) group, 9:pirfenidone (300 mg/kg) group. Compared with sham operation group, #P<0.05, ##P<0.01; compared with model group, *P<0.05, **P<0.01.

[0059] FIG. 8 shows the effects of 3 alkaloids on content of TGF-β1 in lung tissue of pulmonary fibrosis mice induced by bleomycin (ELISA). Wherein, A: prevention groups, B: treatment group; 1:sham operation group, 2:model group, 3:compound I low-dose (30 mg/kg) group, 4:compound I high-dose (60 mg/kg) group, 5:compound VII low-dose (30 mg/kg) group, 6:compound VII high-dose (60 mg/kg) group, 7:compound VIII low-dose (30 mg/kg) group, 8:compound VIII high-dose (60 mg/kg) group, 9:pirfenidone (300 mg/kg) group. Compared with sham operation group, #P<0.05, ##P<0.01; compared with model group, *P<0.05, **P<0.01.

DETAILED DESCRIPTION

Embodiment 1: Preparation and Structure Identification of Alkaloids

[0060] 1. Medicinal Material and Reagents

[0061] The medicinal material is the dried roots of Stemona tuberosa Lour, and is purchased from Anguo medicine market, Hebei. Ethanol, dichloromethane, methanol and other reagents are all analytical grade.

[0062] Extraction and Isolation

[0063] Stemona tuberosa Lour is extracted with 90% ethanol by infiltrating, until the detection results of TLC show that the condensing percolation solution has no bismuth potassium iodide reactivity. Adjust the pH of condensing percolation solution to 1-2 by adding 5% diluted hydrochloric acid, then filter the condensing percolation solution to obtain filtrate, then adjust the pH of the filtrate to 10 by adding concentrated ammonia, then extract the aqueous layer with chloroform to get the total alkaloids, then separate the total alkaloids by silica gel column chromatography. Wherein, dichloromethane-methanol (100:0˜1:5) is used in gradient elution. Based on results of TLC, the same effluent is blended and then separated by silica gel column chromatography repeatedly. Compound I and compound VII are obtained by partial isolation with eluent of dichloromethane-methanol (100:0), and the rest is isolated by preparative HPLC method with eluent of acetonitrile-water (42:58) to get compound II. Compound IV and compound III are obtained by partial isolation with eluent of dichloromethane-methanol (100:2), and the rest is isolated by preparative HPLC method with eluent of acetonitrile-water (23:77) to get compound V and compound VI. Compound VIII is obtained by partial isolation with eluent of dichloromethane-methanol (100:4). Purities of the above compounds are over 99% after HPLC analysis.

[0064] 3. Structure Identification

[0065] Compound I (Tuberostemonine): colorless raphide (methanol), improved bismuth potassium iodide reactivity is positive.

[0066] ESI-MS (m/z: 376[M+H]+). .sup.1HNMR (CDCl.sub.3, 300 MHz) δ:1.80 (1H, m, H-1), 2.18 (1H, m, H-2 α), 1.10 (1H, m, H-2β), 3.43 (1H, m, H-3), 3.47 (1H, m, H-5α), 2.67 (1H, m, H-5β), 1.57 (1H, m, H-8), 1.82 (1H, m, H-9), 3.07 (1H, dd, J=3.5, 4.0 Hz, H-9α), 1.55 (1H, m, H-10), 4.44 (1H, dd, J=3.0, 3.5 Hz, H-11), 2.00 (1H, m, H-12), 2.41 (1H, dq, J=6.5, 7.5 Hz, H-13), 1.28 (3H, d, J=7.0 Hz, H-15), 1.52 (1H, m, H-16), 0.96 (3H, t, J=7.5 Hz, H-17), 4.31 (1H, m, H-18), 2.38 (1H, ddd, J=5.5, 13.5, 15.5 Hz, H-19), 2.60 (1H, ddq, J=7.0, 5.5, 12.0 Hz, H-20), 1.26 (3H, d, J=7.0 Hz, H-22). .sup.13C NMR (CDCl.sub.3, 75 MHz) δ: 41.6 (C-1), 32.1 (C-2), 65.0 (C-3), 48.1 (C-5), 28.1 (C-6), 29.9 (C-7), 30.4 (C-8), 40.7 (C-9), 63.6 (C-9a), 45.0 (C-10), 80.3 (C-11), 47.3 (C-12), 40.9 (C-13), 179.2 (C-14), 14.7 (C-15), 24.3 (C-16), 11.2 (C-17), 81.4 (C-18), 34.6 (C-19), 34.8 (C-20), 179.4 (C-21), 14.9 (C-22).

##STR00005##

[0067] Compound II (Tuberostemonine A): colorless raphide (methanol), improved bismuth potassium iodide reactivity is positive, ESI-MS (m/z:376[M+H]+). .sup.1HNMR (CDCl.sub.3, 300 MHz) δ:1.81 (1H, m, H-1), 1.84 (1H, m, H-2), 1.60 (1H, m, H-2), 3.09 (1H, m, H-3), 2.98 (1H, m, H-5), 2.44 (1H, m, H-5), 1.65 (1H, m, H-8), 1.57 (1H, m, H-9), 2.63 (1H, dd, J=3.5, 4.0 Hz, H-9α), 1.42 (1H, m, H-10), 4.44 (1H, dd, J=3.0, 3.5 Hz, H-11), 2.01 (1H, m, H-12), 2.33 (1H, dq, J=6.5, 7.5 Hz, H-13), 1.26 (3H, d, J=7.0 Hz, H-15), 1.64 (1H, m, H-16), 0.91 (3H, t, J=7.5 Hz, H-17), 4.31 (1H, m, H-18), 2.37 (1H, ddd, J=5.5, 13.5, 15.5 Hz, H-19), 2.69 (1H, ddq, J=7.0, 5.5, 12.0 Hz, H-20), 1.30 (3H, d, J=7.0 Hz, H-22). .sup.13C NMR (CDCl.sub.3, 75 MHz) δ:41.4 (C-1), 32.6 (C-2), 65.1 (C-3), 53.3 (C-5), 26.0 (C-6), 29.9 (C-7), 29.9 (C-8), 40.6 (C-9), 68.3 (C-9a), 42.5 (C-10), 80.2 (C-11), 46.9 (C-12), 40.1 (C-13), 179.2 (C-14), 15.2 (C-15), 22.4 (C-16), 9.8 (C-17), 80.8 (C-18), 32.5 (C-19), 35.6 (C-20), 179.4 (C-21), 15.2 (C-22).

##STR00006##

[0068] Compound III (Tuberostemonine J): colorless raphide (methanol), improved bismuth potassium iodide reactivity is positive, ESI-MS (m/z:376[M+H]+). .sup.1HNMR (CDCl.sub.3, 300 MHz) δ:1.40˜2.10 (15H, H-1, 2, 6˜10, 12, 16, 19), 3.02 (2H, m, H-3, H-9a), 2.98 (1H, m, H-5), 2.74 (2H, H-5, H-13), 4.46 (1H, m, H-11), 1.18 (3H, d, J=7.5 Hz, H-15), 0.91 (3H, t, J=7.5 Hz, H-17), 4.39 (1H, m, H-18), 2.25 (1H, m, H-19), 2.50 (1H, m, H-20), 1.22 (3H, d, J=7.5 Hz, H-22). .sup.13C NMR (CDCl.sub.3, 75 MHz) δ:38.4 (C-1), 32.4 (C-2), 64.6 (C-3), 50.1 (C-5), 33.3 (C-6), 29.5 (C-7), 30.6 (C-8), 34.8 (C-9), 66.3 (C-9a), 34.5 (C-10), 80.3 (C-11), 41.1 (C-12), 45.8 (C-13), 179.3 (C-14), 11.6 (C-15), 25.4 (C-16), 12.9 (C-17), 81.2 (C-18), 34.3 (C-19), 45.1 (C-20), 179.2 (C-21), 14.8 (C-22).

##STR00007##

[0069] Compound IV (Tuberostemonine H): colorless raphide (methanol), improved bismuth potassium iodide reactivity is positive, ESI-MS (m/z:376[M+H]+). .sup.1HNMR (CDCl.sub.3, 300 MHz) δ:1.30˜2.00 (16H, H-1, 2, 6˜10, 12, 16, 19), 3.20 (1H, m, H-3), 2.84 (1H, m, H-5), 2.78 (1H, H-5), 4.57 (1H, m, H-11), 2.61 (1H, m, H-13), 1.18 (3H, d, J=7.2 Hz, H-15), 1.00 (3H, t, J=7.2 Hz, H-17), 4.37 (1H, m, H-18), 2.35 (1H, m, H-19), 2.45 (1H, m, H-20), 1.22 (3H, d, J=7.2 Hz, H-22). .sup.13C NMR (CDCl.sub.3, 75 MHz) δ:41.9 (C-1), 31.1 (C-2), 78.0 (C-3), 54.7 (C-5), 27.3 (C-6), 24.1 (C-7), 27.1 (C-8), 41.1 (C-9), 67.5 (C-9a), 35.3 (C-10), 80.7 (C-11), 44.1 (C-12), 47.2 (C-13), 179.4 (C-14), 11.6 (C-15), 21.2 (C-16), 11.9 (C-17), 79.2 (C-18), 33.4 (C-19), 44.8 (C-20), 179.1 (C-21), 15.0 (C-22).

##STR00008##

[0070] Compound V (Tuberostemonine N): colorless raphide (methanol) improved bismuth potassium iodide reactivity is positive, ESI-MS (m/z:376[M+H]+). .sup.1HNMR (CDCl.sub.3, 300 MHz) δ:1.75 (1H, m, H-1), 2.21 (1H, ddd, J=11.8, 6.3, 5.6 Hz, H-2α), 1.09 (1H, m, H-2β), 3.21 (1H, m, H-3), 3.31 (1H, dd, J=15.1, 6.3 Hz, H-5α), 2.79 (1H, m, H-5β), 1.33 (1H, m, H-6), 1.49 (1H, m, H-6), 1.19 (1H, m, H-7), 1.76 (1H, m, H-7), 1.60 (1H, m, H-8), 1.77 (1H, m, H-8), 2.01 (1H, d, J=12.0 Hz, H-9), 3.12 (1H, dd, J=11.9, 3.8 Hz, H-9a), 1.98 (1H, t, J=7.3 Hz, H-10), 4.20 (1H, dd, J=4.3, 1.8 Hz, H-11), 2.261 (1H, ddd, J=10.2, 7.3, 4.3 Hz, H-12), 2.81 (1H, dq, J=7.3, 7.3 Hz, H-13), 1.27 (3H, d, J=7.3 Hz, H-15), 1.42 (2H, m, H-16), 1.00 (3H, t, J=7.3 Hz, H-17), 4.19 (1H, m, H-18), 1.54 (1H, m, H-19α), 2.36 (1H, ddd, J=12.5, 8.4, 5.4 Hz, H-19β), 2.62 (1H, m, H-20), 1.27 (3H, d, J=7.1 Hz, H-22). .sup.13C NMR (CDCl.sub.3, 75 MHz) δ:35.2 (C-1), 33.7 (C-2), 65.2 (C-3), 50.7 (C-5), 26.9 (C-6), 29.8 (C-7), 32.8 (C-8), 40.1 (C-9), 64.2 (C-9a), 46.9 (C-10), 81.7 (C-11), 45.2 (C-12), 41.5 (C-13), 178.7 (C-14), 11.9 (C-15), 25.9 (C-16), 13.1 (C-17), 83.4 (C-18), 34.2 (C-19), 35.2 (C-20), 179.4 (C-21), 15.0 (C-22).

##STR00009##

[0071] Compound VI (Tuberostemonine K): colorless raphide (methanol), improved bismuth potassium iodide reactivity is positive, ESI-MS (m/z:376[M+H]+). .sup.1HNMR (CDCl.sub.3, 300 MHz) δ:1.79 (1H, m, H-1), 2.25 (1H, m, H-2α), 1.10 (1H, m, H-2β), 3.22 (1H, dd, J=9.0, 12.1 Hz, H-3), 2.81 (1H, dd, J=9.3, 15.1 Hz, H-5α), 3.41 (1H, dd, J=5.8, 9.3 Hz, H-5β), 1.28 (2H, m, H-6), 1.57 (2H, m, H-7), 1.06 (1H, m, H-8α), 1.88 (1H, m, H-8β), 1.89 (1H, m, H-9), 3.11 (1H, dd, J=3.9, 11.2 Hz, H-9a), 1.89 (1H, m, H-10), 4.20 (1H, d, J=2.0 Hz, H-11), 2.17 (1H, m, H-12), 2.88 (1H, dq, J=6.8, 7.4 Hz, H-13), 1.32 (3H, d, J=7.4 Hz, H-15), 1.2˜1.4 (2H, m, H-16), 0.87 (3H, t, J=7.4 Hz, H-17), 4.26 (1H, m, H-18), 1.49 (1H, ddd, J=12 Hz, H-19α), 2.21 (1H, m, H-19β), 2.64 (1H, dq, J=4.0, 6.8 Hz, H-20), 1.23 (3H, d, J=6.8 Hz, H-22). .sup.13C NMR (CDCl.sub.3, 75 MHz) δ:35.6 (C-1), 33.6 (C-2), 65.5 (C-3), 50.4 (C-5), 27.6 (C-6), 30.1 (C-7), 33.0 (C-8), 47.3 (C-9), 64.1 (C-9a), 41.0 (C-10), 81.6 (C-11), 45.1 (C-12), 41.6 (C-13), 178.9 (C-14), 12.1 (C-15), 26.1 (C-16), 13.1 (C-17), 83.6 (C-18), 34.2 (C-19), 35.3 (C-20), 179.5 (C-21), 15.2 (C-22).

##STR00010##

[0072] Compound VII (Neotuberostemonine): colorless raphide (methanol), mp:160.5-162° C., improved bismuth potassium iodide reactivity is positive, ESI-MS (m/z:376[M+H]+). .sup.1HNMR (CDCl.sub.3, 500 MHz) δ:1.75 (1H, m, H-1), 1.65 (2H, m, H-2), 3.33 (1H, dd, J=7.5, 14.0 Hz, H-3), 3.08 (1H, m, H-5α), 2.98 (1H, m, H-5β), 1.94 (1H, m, H-8), 1.84 (1H, m, H-9), 3.21 (1H, dd, J=3.5, 4.0 Hz, H-9α), 1.72 (1H, m, H-10), 4.52 (1H, dd, J=3.0, 3.5 Hz, H-11), 2.10 (1H, m, H-12), 2.88 (1H, dq, J=6.5, 7.5 Hz, H-13), 1.23 (3H, d, J=7.0 Hz, H-15), 1.37 (1H, m, H-16), 1.00 (1H, t, J=7.5 Hz, H-17), 4.43 (1H, m, H-18), 2.39 (1H, ddd, J=5.5, 13.5, 15.5 Hz, H-19), 2.61 (1H, ddq, J=7.0, 5.5, 12.0 Hz, H-20), 1.26 (3H, d, J=7.0 Hz, H-22). .sup.13C NMR (CDCl.sub.3, 125 MHz) δ:37.3 (C-1), 32.6 (C-2), 67.7 (C-3), 51.0 (C-5), 29.1 (C-6), 22.8 (C-7), 28.6 (C-8), 35.9 (C-9), 67.2 (C-9a), 34.8 (C-10), 80.4 (C-11), 41.5 (C-12), 42.6 (C-13), 178.8 (C-14), 10.2 (C-15), 21.1 (C-16), 11.1 (C-17), 78.4 (C-18), 34.5 (C-19), 34.9 (C-20), 178.6 (C-21), 14.8 (C-22).

##STR00011##

[0073] Compound VIII (Sessilifoline B): colorless column crystal (methanol), improved bismuth potassium iodide reactivity is positive, ESI-MS (m/z:278[M+H]+). .sup.1H-NMR (CDCl.sub.3, 500 MHz) δ:4.52 (1H, d, J=3.5 Hz, H-11), 3.29 (1H, m, H-3), 2.94 (1H, m, H-5), 2.86 (1H, m, H-13), 2.51 (2H, m, H-9α, H-3), 2.40 (1H, m, H-5), 2.30 (1H, m, H-12), 2.02 (1H, m, H-2), 1.93 (1H, m, H-9), 1.58 (1H, m, H-7), 1.21 (3H, d, J=7.5 Hz, 15-CH.sub.3), 0.99 (3H, d, J=7.5 Hz, 17-CH.sub.3). .sup.13C-NMR (CDCl.sub.3, 75 MHz) δ:37.3 (C-1), 29.9 (C-2), 55.9 (C-3), 55.8 (C-5), 28.1 (C-6), 21.0 (C-7), 28.0 (C-8), 34.0 (C-9), 71.1 (C-9a), 37.2 (C-10), 79.2 (C-11), 42.5 (C-12), 42.4 (C-13), 179.5 (C-14), 10.1 (C-15), 21.1 (C-16), 11.4 (C-17).

##STR00012##

Embodiment 2: Preparation of Pulmonary Fibrosis Animal Model

[0074] 1. Main Reagents and Experimental Animals

[0075] Bleomycin for experiment is purchased from Nippon Kayaku Co., Ltd., and the batch number is 730342.

[0076] C57BL/6 mice of SPF level for experiment (female, eight-week-old) are purchased from Comparative Medicine Center, Yangzhou University.

[0077] 2. Model Preparation

[0078] Pulmonary fibrosis mice induced by bleomycin are the internationally recognized animal models for screening anti pulmonary fibrosis drugs. Acute lung injury of models induced by bleomycin is characterized by inflammation at the early stage. Then fibrosis begins to appear after 10 days around and lots of collagen is accumulated in lung. The pathological changes have close similarities with that of idiopathic pulmonary fibrosis. Although pathogenesis and mechanism of idiopathic pulmonary fibrosis are still unclear, pulmonary fibrosis and idiopathic pulmonary fibrosis induced by bleomycin are same that pulmonary injury causes immunity and inflammation, and leads to pathological changes of pulmonary fibrosis. Therefore, idiopathic pulmonary fibrosis and other pulmonary fibrosis disease causing by a series of various etiological factors which lead to similar pathological changes can be represented by pulmonary fibrosis induced by bleomycin.

[0079] Preparation Method of Pulmonary Fibrosis Animal Model

[0080] Female C57BL/6 mice (eight-week-old) are established for models after 7 days, the time is set for the mice to adapt to the environment. After fasts for overnight, the mice are anaesthetized by 3% chloral hydrate and fixed. Sterilize and incise neck skin longitudinally as little injury as possible. Split fascia and muscle with tweezers longitudinally and expose the windpipe. Then, about 35 μl (3.5 mg/kg) bleomycin are injected into windpipe with microinjector. Put the mice upright rapidly and rotate the mice for 3˜5 minutes, so that bleomycin is able to enter into the pulmones uniformly. Observe the breathing of mice. Sterilize neck injury with 75% alcohol tampon and stitch the wound, then 1 drop or 2 drops of benzylpenicillin injection is/are dripped into the suture. Put the mice back into dry and clean cages, and feed them normally after waking up. Operations are done on operating table at about 60° C. Physiological saline using in injection is injected into windpipe of sham operation group.

Embodiment 3: Identification of Activity of 8 Alkaloids and Pentoxyverine Towards Anti Pulmonary Fibrosis in Mice

[0081] This embodiment aims at studying the activity of anti-pulmonary fibrosis in mice by alkaloids obtained from embodiment 1, so as to identity whether the alkaloids have effects on anti-pulmonary fibrosis or not. The alkaloids mentioned above are all active ingredients of traditional Chinese medicine stemonae in cough suppressing. In order to study whether cough medicines have effect on anti-pulmonary fibrosis or not, this embodiment has chosen pentoxyverine citrate tablets, which are anti-cough drugs used frequently in clinic and are positive contrast medicine used frequently in pharmacology experiments of cough suppressing, to screen the activity of pulmonary fibrosis synchronously.

[0082] Main Reagents and Experimental Animals

[0083] Compounds I˜VIII are obtained from embodiment 1, and their purities are all high than 98%. Pentoxyverine is purchased from Sinopharm Group Rong Sheng Pharmaceutical Co., Ltd., and the batch number is 13110221. Bleomycin for experiment is purchased from Nippon Kayaku Co., Ltd., and the batch number is 730342. Prifenidone (Positive drug) is purchased from Dalian Meilun Biological Technology Co., Ltd., and the purity is higher than 99%. Prifenidone, a new drug used in treating pulmonary fibrosis, is developed by American Marnac Inc. Development rights in Japan, Taiwan and Korea are given to Shionogi & Co., Ltd. The drug firstly come into the market in Japan in Oct. 17, 2008.

[0084] C57BL/6 mice of SPF level for experiment (female, eight-weeks-old) are purchased from Comparative Medicine Center, Yangzhou University.

[0085] 2. Experimental Method

[0086] Experimental mice are randomly divided into 12 groups: 10 mice in model group, 10 mice in pentoxyverine group, and 5 mice in any other group. Group 1 is sham operation group, group 2 is model group, group 3 is pentoxyverine group, group 4 is positive drug pirfenidone group, groups 5-12 are compounds I-VIII administration group respectively. Models of groups 2-12 are established by using bleomycin with the same method used in embodiment 2. From the first day after successfully establishing model, 30 mg/kg pentoxyverine and 300 mg/kg pirfenidone are given to group 3 and group 4 by means of intragastric administration respectively. 30 mg/kg compounds I-VIII are given to groups 5-12 also by means of intragastric administration respectively. Besides, same doses of solvent are given to sham operation group and model group until the 21th day. After the last intragastric administration, the mice are killed to collect the lung tissue, and the right lobule is used for determining the content of hydroxyproline, while the left lobule is used for preparing pathological sections.

[0087] 3. Experimental Results

[0088] As is shown in FIG. 1A, comparing with sham operation group, the content of hydroxyproline in lung tissue of mice in model group increases significantly, indicating that the collagen has increased obviously and the fibrosis changes are serious. The 8 stemona alkaloids can significantly decrease the increasing degrees of the content of hydroxyproline caused by establishing mouse model and the alkaloids are more effective than the positive drug pirfenidone. The content of Hyp shows no significant difference in pentoxyverine group and model group, however, the content of Hyp in pentoxyverine group is significantly higher than that in sham operation group. As is shown in FIG. 1A and FIG. 1B, 21 days later after successfully establishing model, structures of pulmonary lobules of sham operation group are normal and the alveolar wall are complete without inflammation and fibrosis pathological changes. Obvious inflammation injury and masses of tissue begin to appear in lung of mice in model group, then the normal lung tissue disappears, alveolar is blocked and the collagen accumulates obviously. The 8 stemona alkaloids can significantly alleviate the pulmonary inflammation of mice caused by bleomycin and decrease the degree of pulmonary injury. Compounds I, II, III, VII and VIII enable the mice pulmonary to recover to nearly normal structure. Pentoxyverine cannot decrease the degree of pulmonary injury and fibrosis, the tissue section of pentoxyverine group is similar with that in model group, inflammatory injury is serious, alveolar is blocked, and the collagen accumulates obviously. The results illustrate that alkaloids possessing mother nucleus structure as indicated in structural Formula I have the significant effect in inhibiting or decreasing pulmonary fibrosis of model mice induced by bleomycin, however, anti-cough drugs are not able to improve pulmonary fibrosis of model mice.

Embodiment 4: The Effect of Compounds I, VII and VIII in Preventing or Treating Pulmonary Fibrosis

[0089] Experimental Materials and Method

[0090] (1) Main Reagents and Experimental Animals

[0091] Compounds I, VII and VIII are obtained from embodiment 1, and purities are higher than 98%. Prifenidone (positive drug) is purchased from Dalian Meilun Biological Technology Co., Ltd., and the purity is higher than 99%.

[0092] Experimental animals are model mice which are prepared according to embodiment 2. The day when the model is established is counted as day 0.

[0093] (2) Experimental Method

[0094] Model animals are divided into prevention groups and treatment groups, then drugs are given to the two groups respectively. According to internationally methods of drug administration, drugs are given continuously to the prevention groups by means of intragastric administration from the second day after successfully establishing model to the 14th day, and drugs are given continuously to treatment groups by means of intragastric administration from the 8th day after successfully establishing model to the 21th day. Groups and dosage regimen are shown in tab.1.

TABLE-US-00001 TABLE 1 Groups and dosage regimen of pulmonary fibrosis mice after successfully establishing model Amount of NO. Group animals Dosage and dosage regimen Prevention 1 Sham 10 Sham operation group, same doses of solvent, from groups (A) the first day after establishing model to the 14th day 2 Model 10 Model group, same doses of solvent, from the first day after establishing model to the 14th day 3 I-30 10 Compound I, 30 mg/kg, from the first day after establishing model to the 14th day 4 I-60 10 Compound I, 60 mg/kg, from the first day after establishing model to the 14th day 5 VII-30 10 Compound VII, 30 mg/kg, from the first day after establishing model to the 14th day 6 VII-60 10 Compound VII, 60 mg/kg, from the first day after establishing model to the 14th day 7 VIII-30 10 Compound VIII, 30 mg/kg, from the first day after establishing model to the 14th day 8 VIII-60 10 Compound VIII, 60 mg/kg, from the first day after establishing model to the 14th day 9 Pir 10 Pirfenidone, 300 mg/kg, from the first day after establishing model to the 14th day Treatment 1 Sham 10 Sham operation group, same doses of solvent, from groups (B) the 8th day after establishing model to the 21th day 2 Model 10 Model group, same doses of solvent, from the 8th day after establishing model to the 21th day 3 I-30 10 Compound I, 30 mg/kg, from the 8th day after establishing model to the 21th day 4 I-60 10 Compound I, 60 mg/kg, from the 8th day after establishing model to the 21th day 5 VII-30 10 Compound VII, 30 mg/kg, from the 8th day after establishing model to the 21th day 6 VII-60 10 Compound VII, 60 mg/kg, from the 8th day after establishing model to the 21th day 7 VIII-30 10 Compound VIII, 30 mg/kg, from the 8th day after establishing model to the 21th day 8 VIII-60 10 Compound VIII, 60 mg/kg, from the 8th day after establishing model to the 21th day 9 Pir 10 Pirfenidone, 300 mg/kg, from the 8th day after establishing model to the 21th day

[0095] 2. Determination of Mice Mortality

[0096] From the 0th day (the day when the model is established) to 14th day, drugs are given continuously to prevention groups. From the 8th day after successfully establishing model to 21th day, drugs are given continuously to treatment groups. Statistical analysis of death of animals in each group is carried on every day and the survival rate of animals in each group is calculated. Results are shown in tab.2.

[0097] As is shown in tab. 2, comparing with sham operation group, the mortality of model mice induced by bleomycin within 14 days is 20%, and the mortality within 21 days is 33%. Comparing with model group, the 3 compounds have protective effect on model mice, wherein compounds I and VII of prevention groups are more effective than compound VIII, and groups with lower dose (30 mg/kg) have better effect. In treatment groups, the 3 compounds have a similar effect in decreasing the mortality of mice, wherein groups with lower dose are slightly more effective than groups with higher dose. The present experimental results show that groups with lower dose have better protective effect on model mice, whether in prevention groups or treatment groups, than groups with higher dose. In general, the 3 compounds have better effect in decreasing the mortality of mice than positive drug pirfenidone.

TABLE-US-00002 TABLE 2 Death of prevention group and treatment group I- I- VIII- VIII- Group Sham Model 30 60 VII-30 VII-60 30 60 Pir Prevention Amount of 10 10 10 10 10 10 10 10 10 groups animal Mortality 0 2 0 1 0 2 1 2 3 Survival rate (%) 100 80 100 90 100 80 90 80 70 Treatment Amount of 10 18 18 18 18 18 18 18 18 groups animal Mortality 0 6 2 3 2 4 3 4 6 Survival rate (%) 100 67 89 83 89 78 83 78 67

[0098] 3. Detection of the Mice Lung Index

[0099] After the last intragastric administration, the mice are killed to strip and weigh the lung, and the lung index is the lung weight divided by mouse weight (FIG. 2). As is shown in FIGS. 1A-1C, the lung index of model group is significantly higher than that of sham operation group. The groups with doses of 30 and 60 mg/kg of compounds I, VII and VIII can significantly decrease the lung index of mice, whether in prevention groups or treatment groups, wherein group with lower dose in prevention groups has a better effect. In general, the 3 compounds have better effect in decreasing the mortality of mice than the positive drug pirfenidone.

[0100] 4. HE Stained Pathological Evaluation and Inflammation Scores

[0101] After the last intragastric administration, the mice are anaesthetized and killed to collect the lung tissue. Immerse the left lobule in 10% formalin, and embed it by paraffin after being fixed, then cut it into slices to observe the pathological changes by HE staining. As is shown in FIG. 3, structures of lobules of sham operation group are normal and the alveolar walls are complete without inflammation and fibrosis pathological changes. Obvious inflammation injury and masses of tissue begin to appear in lung of mice in model group, then the normal lung tissue disappears and alveolar is blocked. Whether in prevention groups or treatment groups, compounds I, VII and VIII can significantly alleviate the pulmonary inflammation caused by bleomycin and decrease the pulmonary injury, and recover lung to normal structure. The groups with lower dose (30 mg/kg) of the 3 compounds show better effect than the groups with higher dose. The pulmonary inflammation disappears and alveolar structure is clear in groups with lower dose.

[0102] Inflammatory classification semi quantitative statistics analysis is made according to results of HE staining. Grade 0 represents normal tissue or minimal inflammation change. Grade 1 (+) represents mild to moderate inflammation change, and lung tissue without obvious damage. Grade 2 (++) represents moderate to severe inflammation injury, and alveolar septum is thickened to form masses of tissue, or lung tissue with damage caused by partial area inflammation. Grade 3 (+++) represents severe inflammation injury, and structure of restricted regions in lung tissue is severely damaged, thus resulting in obliteration. Inflammation scores of prevention groups and treatment groups are shown in FIG. 4. Comparing with sham operation group, there is significantly pulmonary inflammation appearing in model mice induced by bleomycin. The groups with doses of 30, 60 mg/kg of compounds I, VII and VIII can significantly decrease the pulmonary inflammation induced by bleomycin. The groups with lower dose show better effect than the groups with higher dose. In general, the 3 compounds have better effect in decreasing the pulmonary inflammation than the positive drug pirfenidone.

[0103] 5. Masson Stained Pathological Evaluation and Imaging Analysis

[0104] Masson staining is a method aiming at specific staining of fibrillar collagen. After the last intragastric administration, the mice are anaesthetized and killed to collect the lung tissue. Immerse the left lobule in 10% formalin, and embed it by paraffin after being fixed, then is cut it into slices to observe the collagen deposition by Masson staining. The results of Masson staining in prevention groups and treatment groups are shown in FIG. 5. Semi quantitative analysis is made by using Image-Pro Plus6.0. Integrated optical density (IOD) of collagen in each visual field is measured after Masson staining. 5 samples are studied in each group and 5 visual fields are picked out from each group. Statistics analysis is made by calculating mean value which is considered as relative amount of collagen in each group. The analysis results of prevention groups and treatment groups are shown in FIG. 6.

[0105] As is shown in FIGS. 5 and 6, there is no obvious Masson collagen deposition can be seen in mice pulmonary of sham operation group. Obvious Masson collagen deposition can be seen in mice pulmonary after giving bleomycin for 14 or 21 days, massive fibrosis tissues are formed, and fibrosis in 21th day is more severe than that in 14th day. The groups with doses of 30, 60 mg/kg of compounds I, VII and VIII can significantly decrease collagen deposition caused by bleomycin and the effect in decreasing pulmonary fibrosis is extremely obvious. Wherein, the groups with lower dose of the 3 compounds in prevention groups show better effect than the groups with higher dose, and the groups with higher dose of the 3 compounds in treatment groups show better effect than the groups with lower dose. In general, effects of compounds I and VII in decreasing the collagen deposition are slightly better than that of compound VIII, and the effects of 3 compounds are obviously more effective than that of the positive drug pirfenidone.

[0106] 6. Content Determination of Hydroxyproline

[0107] Hydroxyproline mainly exists in collagen protein, but there is only a very small amounts in elastin proteins and it cannot found in other proteins. Therefore the content of collagen protein can be indicated by measuring the content of hydroxyproline, so as to evaluate the degree of pulmonary fibrosis. Method of test kit (which is purchased from Nanjing Jiancheng Biological Technology Co., Ltd.) according to specification is used in determination of hydroxyproline. Results can be seen in FIG. 7.

[0108] As is shown in FIG. 7, comparing with sham operation group, the content of hydroxyproline in lung tissue of model mice significantly increases, indicating that there are serious fibrosis changes in lung tissue of model mice. Both the groups with lower dose (30 mg/kg) and the groups with higher dose (60 mg/kg) of compounds I, VII and VIII can effectively decrease the content of hydroxyproline in lung tissue of model mice whether in prevention groups or treatment groups. The effect of groups with lower dose is slightly better than the groups with higher dose, indicating that the 3 compounds can significantly improve the pulmonary fibrosis of model mice induced by bleomycin and decrease the collagen deposition.

[0109] 7. ELISA Content Determination of TGF-β1

[0110] TGF-β1 is a universally recognized strong pro-fibrotic cytokine. It can stimulate the cells to synthesize and secrete extracellular matrix as well as exchange the activity of matrix-degrading enzyme component and directly increase the deposition of ECM. The progress of pulmonary fibrosis can be slowed down by decreasing the content of TGF-β1 in lung tissue. Elisa test kit (which is purchased from Shanghai Excell Biological Technology Co., Ltd.) according to specification is used in the determination of TGF-β1 in mice lung tissue. Results can be seen in FIG. 8.

[0111] As is shown in FIG. 8, comparing with sham operation group, the content of TGF-β1 in mice lung tissue after giving bleomycin significantly increases. Comparing with model group, both groups with lower dose (30 mg/kg) and groups with higher dose (60 mg/kg) of compounds I, VII and VIII can effectively decrease the content of TGF-β1 in model mice whether in prevention groups or treatment groups, and significantly reduce the degree of pulmonary fibrosis. Wherein, groups with lower dose are more effective than groups with higher dose and effects of compound I and VIII are slightly better than that of compound VII.

[0112] Discussion

[0113] The present invention has shown that compounds possessing mother nucleus structure as indicated in structural Formula I, such as compounds I-VIII, have significantly effect for preventing and/or treating pulmonary fibrosis (embodiment 3). Surveys on activity of compounds I and VII (containing R1 side chain) together with VIII (no R1 side chain) have further shown that structures as indicated in structural Formula I have the obvious effect of anti-pulmonary fibrosis. Multi-index animal experiments have shown that the compounds are more effective than the positive contrast medicine pirfenidone which has just come into the market in Japan. The compounds in present invention can obviously decrease the mortality of pulmonary fibrosis mice induced by bleomycin, the lung index of model mice and the extent of pulmonary fibrosis of model mice, and the contents of Hyp and pro-fibrogenic factor TGF-β1 in lung tissue. As mentioned above, compounds having the structure as indicated in structural Formula I can relieve lung inflammation of mice induced by bleomycin and reduce the accumulation of lung collagen.

[0114] Results in present invention have provided a scientific basis for compounds having the structure as indicated in structural Formula I using in pharmaceutical compositions for preventing and/or treating pulmonary fibrosis.