CN101077852A - Preparation for chemical component of Shenmai injection and application of the same in curing cardio-cerebrovascular disease - Google Patents
Preparation for chemical component of Shenmai injection and application of the same in curing cardio-cerebrovascular disease Download PDFInfo
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Abstract
The present invention discloses process of extracting and separating compounds I-VIII from ginseng-ophiopogon injection. These compounds have functions of inhibiting myocardial cell apoptosis, promoting the release of CO from vascular endothelial cell, suppressing oxidation caused by AAPH, etc; and this show that these compounds are main active components for the ginseng-ophiopogon injection to treat cardiac and cerebral vascular diseases. The extracting and separating process includes the steps of: chromatographic separation in macroporous resin column, MCI columm, silica gel column, glucosan gel column and reverse silica gel column; efficient liquid phase separation; sublimation; crystallization; etc. The compound structure identification is completed through infrared method, 1D and 2D nuclear magnetic resonance, mass spectrum and other spectroscopic method.
Description
Technical field
The present invention relates to a kind of preparation method and the application aspect the treatment cardiovascular disorder of active chemical components of the traditional Chinese medicine that is applied to treat cardiovascular and cerebrovascular diseases.
Background technology
Shenmai injection (claims " ginseng wheat aqueous injection " again; hereinafter to be referred as " ginseng wheat pin ") be the development side of ancient prescription " SHENGMAI SAN "; by red ginseng and the tuber of dwarf lilyturf two kinds of Chinese medicinal materialss extract through modern crafts and process; the shock, coronary heart disease, viral myocarditis, chronic cardiopulmonary disease, the granulocytopenia that are used for the treatment of type of deficiency of both QI and YIN; for national Chinese medicine protection kind, be basic first-aid medicine.The clinical application in year surplus the ginseng wheat pin existing 20, but activeconstituents wherein is unclear always, the quality standard of present ginseng wheat pin is decided to be three ginsenoside Rb1s, Rg1 and Re of genseng (red ginseng) lining, but studies show that the pharmacological model not significantly effect of these three compounds to existing all treating cardiac and cerebral vascular diseases.Ginseng wheat pin on producing be by make respectively the medicinal material red ginseng and the tuber of dwarf lilyturf red ginseng stoste and the tuber of dwarf lilyturf stoste; after sterilizing, mix at 1: 1; we to ginseng wheat pin, red ginseng stoste and the tuber of dwarf lilyturf stoste carried out the protection apoptosis of cardiac muscle, suppressed the oxidation of AAPH and increased pharmacologically actives test such as the nitric oxide production release of vascular endothelial cell; the result shows that red ginseng stoste works hardly, and the tuber of dwarf lilyturf, stoste was close with the main activity index of ginseng wheat pin.
Summary of the invention
The objective of the invention is by to the contained chemical ingredients of the stoste tuber of dwarf lilyturf of ginseng wheat pin and the research of pharmacologically active thereof, determine wherein to be used for the treatment of the effective constituent of cardiovascular and cerebrovascular diseases, the secondary development that this research can be used to join the quality control index of wheat pin product and join the wheat pin improves the result of treatment of cardiovascular and cerebrovascular diseases.
The present invention mainly will join the wheat pin the tuber of dwarf lilyturf stoste through macroporous resin adsorption; with water with contain the aqueous ethanolic solution wash-out; again through chromatographic separation such as silicagel column, reverse silicagel column, sephadex columns; high performance liquid phase separates; distillation and method such as crystallization obtain this 9 compounds, and these 9 compounds have effects such as protecting apoptosis of cardiac muscle, anti-oxidant and increase vascular endothelial cell nitrogen protoxide release individually or simultaneously.
Embodiment
1. the preparation method of compound
1.1 the preparation method of Compound I-VIII:
I.) 10 kilograms the Chinese medicine Zhejiang medicinal material tuber of dwarf lilyturf is prepared into the stoste tuber of dwarf lilyturf according to the production technique of Shenmai injection, behind 40 ℃ of concentrating under reduced pressure, separate with macroporous resin column chromatography, with the water-alcohol elutriant wash-out of gradient, obtain water, 10%, 20%, 40%, 60%, the 80% and 95% alcohol wash-out part of the stoste tuber of dwarf lilyturf.
Ii.) the 95% alcohol wash-out part that obtains among the i is separated with silicagel column, is the developping agent wash-out with sherwood oil, chloroform, chloroform-acetone (1: 0 → 1: 1), chloroform-methanol (50: 1 → 1: 1); Chloroform-acetone (20: 1) wash-out partly continues to separate with silica gel column chromatography, can obtain compound VI with petroleum ether-ethyl acetate (5: 1) as the developping agent purifying; Chloroform-acetone (5: 1) wash-out part is further separated with silica gel column chromatography, with petroleum ether-ethyl acetate (2: 1) as developping agent, can obtain compound V and compound (VIIa+VIIb) enriching section, use dextrane gel Sephadex LH-20 column purification more respectively, launch to obtain respectively V and (VIIa+VIIb) with chloroform, the latter is a pair of epimer, high performance liquid phase (PreStar system with the preparation type, merckODS packed column 1.2 * 25cm, acetonitrile-water 30%-60%) prepares VIIa and VIIb; Chloroform-methanol (50: 1) wash-out partly uses silica gel column chromatography (developping agent is chloroform-methanol 25: 1) to be further purified can obtain the Compound I enriching section, can get pure I through distillation again; 25: 1 wash-out parts of chloroform-methanol wash-out position can obtain IV with being further purified with silica gel column chromatography (chloroform-methanol 15: 1).
Iii.) 80% alcohol wash-out part silicagel column among the i separates, and is the developping agent wash-out with chloroform-methanol (50: 1 → 1: 1); 10: 1 wash-out parts of chloroform-methanol wash-out position recycle silicon plastic column chromatography (chloroform-methanol-water 5: 1: 0.1) is further purified and can obtains Compound I I enriching section, use RP-18 reversed-phase column purifying again, can obtain II as distributing solution to wash with ethanol (or methyl alcohol)-water (2: 1).
Iv.) the 40% alcohol wash-out part that obtains among the i is separated with silicagel column, with chloroform (or ethyl acetate and methylene dichloride)-methyl alcohol (or ethanol) gradient elution.5: 1 wash-out parts of chloroform-methanol are further used RP-18 reversed-phase column purifying, can obtain compound III and VIII with methanol-water (4: 1) respectively as distribution solution.
1.2 physicochemical data
Compound I (2-baras camphor): white crystals (methyl alcohol), fusing point: 208 ℃, optically-active [α]
D 20-38 ° (c=5, EtOH).Molecular formula: C
10H
18O, molecule is heavy: 154; ESI-MS:m/z=155 ([M+H]
+).
1H NMR (CDCl
3, 300MHz): δ 0.81 (6H, s, Me-8 and Me-9), 0.83 (3H, s, Me-10), 0.95 (1H, dd, J=13.3,3.6Hz, Hax-3), 1.58 (1H, t, J=4.6Hz, H-4), 3.94 (1H, ddd, J=10.0,3.7,2.0Hz, H-2);
13C NMR (pyridine-d
5): δ 13.35 (C-10), 18.48 (C-8), 19.46 (C-9), 26.79 (C-6), 28.03 (C-5), 36.84 (C-3), 44.82 (C-4), 47.12 (C-7), 49.23 (C-1), 83.16 (C-2).
Compound I I (2-baras camphor glucoside): white, needle-shaped crystals (methyl alcohol), fusing point: 125-127 ℃, optically-active [α]
D 20-8.6 (c=0.3, MeOH).Molecular formula: C
16H
28O
6, molecule is heavy: 316; ESI-MS:m/z=317 ([M+H]
+).
1H NMR (pyridine-d
5): δ 0.75 (6H, s, Me-8 and Me-9), 1.05 (3H, s, Me-10), 4.51 (dd, J=11.4,2.2Hz, H-2), 4.89 (1H, d, J=7.8Hz, H-1 ');
13C NMR (pyridine-d
5): δ 14.3 (C-10), 18.9 (C-8), 19.9 (C-9), 27.2 (C-6), 28.6 (C-5), 38.1 (C-3), 45.5 (C-4), 48.8 (C-7), 50.0 (C-1), 86.2 (C-2), 106.2 (C-1 '), 75.6 (C-2 '), (78.7 C-3 '), (71.9 C-4 '), 78.2 (C-5 '), 63.1 (C-6 ').(pharmaceutical journal (Yakugaku Zasshi) 1983,103 (11): 1133)
Compound III (2-baras camphor-O-celery sugar (1-6) glucoside): white, needle-shaped crystals (methyl alcohol), fusing point: 183-185 ℃, optically-active [α]
D 23-53.4 (c=0.44, MeOH), molecular formula: C
21H
36O
10, molecule is heavy: 448; IR υ
KBr MaxCm
-1: 3400,2982,1054;
1H NMR (400MHz, pyridine-d
5): 5.86 (d, J=2.3Hz), 4.85 (d, J=7.7Hz, H-1 '), 4.77 (d, J=2.3Hz, H-1 "); 4.73 (dd, J=11.2,1.8, H-2); 4.59 (d, J=9.4, Ha-5 "), 4.37 (d, J=9.4, Hb-5 "), 1.04 (s, 10-Me) 0.81 (s; 8-Me), 0.74 (s, 9-Me).
13C NMR (pyridine-d
5): δ 86.14 (C-2), 49.89 (C-1), 47.57 (C-7), 45.33 (C-4), 38.08 (C-3), 28.54 (C-5), 27.08 (C-6), 19.81 (C-8), 18.89 (C-9), 14.13 (C-10), (106.16 C-1 '), 78.57 (C-3 '), 77.83 (C-5 '), (75.42 C-2 '), (71.84 C-4 '), 68.76 (C-6 '), 111.06 (C-1 "); 80.54 (C-3 "), 77.17 (C-2 "), 75.01 (C-4 "), 65.63 (C-5 "). (pharmaceutical journal (Yakugaku Zasshi); 1983,103 (11): 1133)
Compound IV (2,5-camphane glycol): white crystals (methyl alcohol), fusing point 255-257, optically-active [α]
D 25-16.12 (c=0.46, MeOH).Molecular formula: C
10H
18O
2, molecule is heavy: 170.251; ESI-MS m/z:193 ([M+Na]
+).
1H NMR (pyridine-d
5): δ 0.92 (3H, s, H-9 or H-10), 1.21 (3H, s, H-9 or H-10), 1.42 (3H, s, H-1), 4.36 (1H, br.s, H-3), 5.21 (1H, br.s, H-7);
13C NMR (pyridine-d
5): δ 13.2 (C-1), 50.4 (C-2), 74.5 (C-3), 34.9 (C-4), 53.1 (C-5), 40.1 (C-6), 82.5 (C-7), 47.3 (C-8), 20.0 (C-9), 21.2 (C-10).(Phytochemistry,1983,22(3):774)
Compound V (methyl dihydro tuber of dwarf lilyturf homoisoflavone B): white powder, molecular formula: C
19H
20O
6, molecule is heavy: 344; EI-MSm/z (%): 344[M]
+(80), 326[M-H
2O]
+, 220 (25), 208 (84), 207 (62), 137 (100), 121 (5), 84 (40), 77,56.
1H NMR (400MHz, CDCl
3) δ 4.25 (1H, dd, J=12.0,7.0Hz, H-2), 4.42 (1H, dd, J=12.0,3.0Hz, H-2), and 3.30-3.40 (1H, m, H-3), 13.10 (5-OH), 2.30 (3H, s, 6-Me), 2.39 (3H, s, 8-Me), 2.95 (1H, dd, J=10.0,10.0Hz, H-9), 3.75 (1H, d, J=10.0Hz, H-9), 7.10 (1H, d, J=8.2Hz, H-2 '), 6.58 (1H, dd, J=8.2,2.4Hz, H-3 '), (6.78 1H, d, J=2.4Hz, H-5 '), 3.80 (3H, s, 7 '-OMe);
13C NMR (100MHz) δ: 69.9 (C-2, t), 45.4 (C-3, d), 198.4 (C-4, s), 158.4 (C-5, s), 102.5 (C-6, s), 160.2 (C-7, s), 104.5 (C-8, s), 27.6 (C-9, t), 157.7 (C-10, s), 102.3 (C-11, s), 8.8 (6-Me), 8.5 (8-Me), 118.2 (C-1 ', s), 132.1 (C-2 ', d), 104.5 (C-3 ', d), 158.1 (C-4 ', s), 103.3 (C-5 ', d), 164.1 (C-6 ', s), 55.0 (5 '-OMe). (Chem Pharm Bull, 1980,28 (5): 1477)
Compound VI (hydroxymethyl dihydro tuber of dwarf lilyturf homoisoflavone B): fusing point: 159-160 ℃ of white, needle-shaped crystals (acetone), molecular formula: C
19H
20O
5, molecular weight: 328; EI-MS (m/z): 329 (M
+, 36), 310 (MH
2O), 207 (4), 135 (16), 121 (100).
1H NMR (400MHz, CDCl
3) δ 4.09 (1H, dd, J=12.0,7.0Hz, H-2), 4.31 (1H, dd, J=12.0,3.0Hz, H-2), 2.72-3.08 (1H, m, H-3), 12.34 (5-OH), 2.03 (3H, s, 6-Me), 2.08 (3H, s, 8-Me), 2.66 (1H, dd, J=10.0,10.0Hz, H-9), 3.20 (1H, d, J=10.0Hz, H-9), 7.15 (2H, d, J=9.0Hz, H-2 ' andH-6 '), 6.87 (2H, d, J=9.0Hz, H-3 ' and H-5 '), 3.80 (3H, s, 7 '-OMe);
13C NMR (100MHz, CDCl
3) δ: 68.6 (C-2, t), 46.6 (C-3, d), 198.3 (C-4, s), 159.3 (C-5, s), 102.6 (C-6, s), 160.4 (C-7, s), 102.0 (C-8, s), 31.8 (C-9, t), 157.5 (C-10, s), 101.3 (C-11, s), 8.0 (6-Me), 7.6 (8-Me), 129.8 (C-1 ', s), 129.8 (C-2 ', d), 113.8 (C-3 ', d), 158.1 (C-4 ', d), 113.8 (C-5 ', d), 129.8 (C-6 ', d), 55.0 (5 '-OMe).
Compound VI Ia (2 beta-hydroxy Methylophiopogonanone A B): yellow amorphous powder, molecular formula: C
19H
18O
7, molecular weight: 358; EIMS (%, m/z): 358[M]
+(56), 340[M-H
2O]
+(16), 329 (20), 181 (35), 135 (100);
1HNMR (400MHz, CDCl
3) δ δ 12.22 (1H, s, 5-OH), 6.70-6.65 (3H, overlapped signal, H-2 ', H-3 ' and H-6 '), 5.94 (2H, s, H-7 '), 5.56 (1H, br.s, H-2), 3.41 (1H, dd, J=14.1,4.7Hz, H-9a), 3.11 (1H, m, H-3), 2.71 (1H, dd, J=14.1,10.7Hz, H-9b), 2.03 (6H, s, 6-Me and 8-Me);
13C NMR (100MHz, DEPT) δ: 96.3 (C-2, t), 51.4 (C-3, d), 196.4 (C-4, s), 159.1 (C-5, s), 103.5 (C-6, s), 161.0 (C-7, s), 102.9 (C-8, s), 30.1 (C-9, t), 153.4 (C-10, s), 102.4 (C-11, s), 7.7 (6-Me), 7.2 (8-Me), 131.4 (C-1 ', s), 109.6 (C-2 ', d), 108.6 (C-3 ', d), 146.6 (C-4 '), 108.7 (C-5 ', s), 122.6 (C-6 ', d), 101.3, (7 '-C, t).
Compound VI Ib (2 Alpha-hydroxy Methylophiopogonanone A B): yellow amorphous powder, molecular formula: C
19H
18O
7, molecular weight: 358; EIMS (m/z): 358[M]
+(56), 340[M-H
2O]
+(16), 329 (20), 135 (100), 121 (12);
1H-NMR (400MHz, CDCl
3) δ 12.22 (1H, s, 5-OH), 6.70-6.65 (3H, overlapped signal, H-2 ', H-3 ' andH-6 '), 5.94 (2H, s, H-7 '), 5.50 (1H, br.s, H-2), 3.41 (1H, dd, J=14.1,4.7Hz, H-9a), 3.11 (1H, m, H-3), 2.71 (1H, dd, J=14.1,10.7Hz, H-9b), 2.03 (6H, s, 6-Me and 8-Me);
13C NMR (100MHz, DEPT) δ: 95.4 (C-2, t), 52.9 (C-3, d), 197.5 (C-4, s), 159.1 (C-5, s), 103.8, (C-6, s), 161.5 (C-7, s), 102.9 (C-8, s), 34.6 (C-9, t), 153.8 (C-10, s), 102.0 (C-11, s), 7.9 (6-Me), 7.2 (8-Me), 132.3 (C-1 ', s), 109.9 (C-2 ', d), 108.6 (C-3 ', d), 148.0 (C-4 '), 108.6 (C-5 ', s), 122.2 (C-6 ', d), 101.3 (7 '-C, t).
Compound VIII (river Ophiopogonin A, Ophiopojaponin A): white needle (chloroform-methanol, 1: 1), fusing point: 230-232 ℃, optically-active [α]
D 21-45.7 ° (c=0.35, MeOH); ESI-MS (m/z): 913 ([M+H]
+).
1H NMR (400MHz, C
5D
5N, Jin Hz, δ): δ 0.66 (3H, d, J=5.4Hz, Me-27), 0.95 (3H, s, H-18), 1.07 (3H, s, H-19), 1.22 (3H, d, J=7.1Hz, H-21), 1.78 (3H, d, J=6.6Hz, RhaH-6), 1.99 (3H, s, CH
3CO), 4.01 (1H, t, J=6.9Hz, Xyl-H-2), 4.17 (1H, t, overlapped signal, Glc-H-2), 4.17 (1H, overlapped signal, Glc-H-3), 4.17 (1H, overlapped signal, XylH-3), 4.20 (1H, overlapped signals, RhaH-5), 4.25 (1H, m, Rha-H-4), 4.72 (1H, m, Rha-H-3), 4.90 (1H, d, J=7.2Hz, Glc-H-1), 5.02 (1H, d, J=7.7Hz, Xyl-H-1), 5.27 (1H, m, H-6), 6.05 (1H, d, J=4.9Hz, Rha-H-2), 6.13 (1H, br.s Rha-H-1).
13C-NMR (125MHz, C
5D
5N, δ): part: 9.6 (C-21) of glucoside unit, 17.2 (C-18), 17.3 (C-27), 19.5 (C-19), 21.0 (C-11), 28.9 (C-24), 30.2 (C-2), 30.5 (C-25), 31.7 (C-23), 32.1 (C-7), 32.1 (C-12), 32.4 (C-8), 32.5 (C-15), 37.2 (C-10), 37.6 (C-1), 39.1 (C-4), 44.9 (C-20), 45.2 (C-13), 50.4 (C-9), 53.1 (C-14), 66.8 (C-26), 78.4 (C-3), 90.2 (C-16), 90.2 (C-17), 110.0 (C-22), 121.9 (C-6), 140.9 (C-5); Sugar moieties: 100.1 (C-1a), 77.1 (C-2a), 81.5 (C-3a), 70.8 (C-4a), 78.5 (C-5a), 61.8 (C-6a), 98.8 (C-1b), 74.0 (C-2b), 70.5 (C-3b), 74.2 (C-4b), 69.4 (C-5b), 18.6 (C-6b), 21.0 (CH
3CO), 177.6 (CH
3CO), 105.8 (C-1c), 75.0 (C-2c), 77.3 (C-3c), 70.8 (C-4c), 67.4 (C-5c). (Botany Gazette, 2001,43 (1): 97)
2. biological activity test
2.1 the test of compound protection apoptosis of cardiac muscle
2.1.1 test philosophy: in people myocardial cell, add the sample solution of different concns, cell death inducing under the ischemia/reperfusion condition, contrast does not add the blank of sample, and the assessment sample is to the provide protection of apoptosis of cardiac muscle.
2.1.2 experimental technique: 6 orifice plates and slide glass with 90% ethanol disinfection, are placed 7.5 * 10 in each hole
4Former generation individual layer myocardial cell (1m1) and 2ml DMEM (Dulbecco ' s Modified Eagle Medium) nutrient solution, grew 1.5 days, remove nutrient solution, add 2ml I/R Media (causing the medium of cell ischemic) and sample solution (20 or 50 μ l, corresponding 50 μ g/ml or 250 μ g/ml), remove nutrient solution after 60 minutes, add normal 2ml nutrient solution (colourless DMEM) and sample solution again, remove liquid after 30 minutes, wash 3 times with 1ml PBS, add 1ml organic solvent (methyl alcohol-acetone, 1: 1) fixed cell, 6 orifice plates are placed refrigerator-20 ℃ 10 minutes, taking-up adds 1ml methyl alcohol makes Premeabilisation of cells, put into refrigerator-20 again ℃ 10 minutes, take out with 1ml PBS washing 3 times, shift out wave carrier piece, blot liquid with blotting paper, to there be the wave carrier piece of cell to be fixed on the good wave carrier piece of mark with glue, add a PBS and prevent that cell from killing to the wave carrier piece, wave carrier piece is placed airtight Glass Containers to be made fully wetting in 10 minutes, take out, blot liquid, evenly add 200 μ l Hoechst staining agents in slide glass, slide glass was placed shady place 10 minutes, rinse out remaining staining agent with PBS then, blot with blotting paper, add an anti-decolourant,, place refrigerator in 4 ℃ of storages with the slide encapsulation.Use fluorescent microscope (excitation wavelength 352nm, wavelength of transmitted light 461nm) observation of cell apoptosis situation then.
2.1.3 experimental result
As shown in table 1, Compound I and II can obviously suppress the apoptosis of cardiac muscle that I/R causes, and have concentration to rely on effect.Compound III, IV, V, VIII can suppress the apoptosis that caused by ischemia/reperfusion at 100 μ g/ml.
The provide protection of the apoptosis of cardiac muscle that the chemical ingredients of table 1, ginseng wheat pin causes ischemia/reperfusion
Sample | Concentration (μ g/ml) | The mean apoptotic cell count | Standard deviation | t-test(vs I/R) | t-test(vs Ctrl) | Protection ratio |
Ctrl(DMSO) I/R(DMSO) I II Ctrl I/R III IV Ctrl I/R V VIII | 10 50 100 10 50 100 10 50 100 10 50 100 50 250 50 250 | 4.18 11.76 7.01 6.31 6.59 7.64 5.90 5.04 3.32 10.18 7.02 9.79 6.74 10.90 10.10 8.50 1.8 11.7 8.8 8.8 9.8 7.14 | 0.03 0.03 0.02 0.03 0.03 0.03 0.02 0.02 0.02 0.04 0.04 0.03 0.04 0.04 0.06 0.04 | 5.29669E-08 7.56089E-08 2.78667E-07 4.73911E-06 1.63507E-10 9.92895E-13 0.001874212 0.683212131 0.000778161 0.520209684 0.952033172 0.116025474 | 2.19E-12 8.09E-11 | 40.39% 46.34% 43.96% 35.03% 49.83% 57.14% 31.04% 3.83% 33.79% -7.07% 0.79% 16.50% 0.00% 24.79% 24.79% 16.24% 38.97% |
Sample number n=6, Ctrl=(blank) contrast; I/R: model control group.
2.2. detection compound activates the activity of endothelial cell nitric oxide synthetic enzyme (NOS)
2.2.1 test philosophy: NOS is the functional enzyme that quantitative catalysis generates nitrogen protoxide (NO), and the NO of generation has functions such as vasodilator, inhibition vascular smooth muscle cell proliferation, and in vivo, NO is with nitrate radical (NO
3 -), nitrite anions (NO
2 -) and the form of nitrosothiols (R-S-N=O) exist, nitrogen protoxide analyser (Nitric Oxide Analyzer) can quantitatively be reduced into above-mentioned NO material the NO of gas phase with vanadium trichloride-hydrochloric acid 90 ℃ the time, the amount of chemoluminescence (NO+ ozone) reaction detection NO by a gas phase can detect the activity of NOS total in the cell.
2.2.2 testing method: the human endothelial cell of buying (ECV304) is positioned over 24 orifice plates, add nutrient solution (500 μ L DMEMmedium+10%FBS+ microbiotic) overnight incubation, exhaust nutrient solution, other adds the DMEM that 500 μ L do not add FBS, every hole adds the compound sample (positive control or blank) of different concns, cultivated 4 hours, then in 4 ℃ of storages of refrigerator, with NO analyser (NOA
TM280) detect the NOS content of each sample.
2.2.3 test result
To promote the positive contrast of NO release rate (100%) of the strongest reactive site M2 of NO (100 μ g/m1) tuber of dwarf lilyturf, the effect that compound VI and VIII have significant promotion NO to discharge can reach about 40% the NO burst size of M2 (100 μ g/ml) when 0.01 μ g/ml concentration; The effect that Compound I and II also have stronger promotion NO to discharge.
The chemical ingredients of table 2, ginseng wheat pin promotes the effect that NO discharges to vascular endothelial cell
Sample | Concentration (μ g/ml) | The NO release rate |
M2 I(MDP-2A) II(MDP-2A) VIII(CMD-b2) VI(CMD-6-2) | 100 25 50 100 25 50 0.01 0.1 1 0.01 0.1 1 | 100.000 46.102 66.532 92.468 55.089 54.927 40.350 43.982 45.542 38.755 42.571 51.597 |
2.3 the inhibition of oxidation effect that compound causes AAPH
2.3.1 test philosophy: 2,2-azo two (2-amidine propane) dihydrochloride (AAPH) can (Dihydrorhodamine 123 with the dihydro rhodamine 123, dihydrochloride salt, DHR) (rhodamine 123 quantitatively to be oxidized to the rhodamine 123 that can send green fluorescence, Rh), the amount of Rh can detect with fluorescence microplate reader.
2.3.2 testing method: the sample solution or the positive control that on disinfectant 96 orifice plates, add 30 μ l, 500 μ M DHR, 15 μ l different concns, 45 μ l methyl alcohol, the 50mM AAPH of the 60 μ l that will newly dispose again is in every hole (except that blank), placement VersaMax fluorescence microplate reader operation 90min (set and detect wavelength 500nm).
2.3.3 experimental result:
As shown in the table, compound IV is the most obvious to the inhibition effect of AAPH inductive oxygenizement, and has concentration to rely on effect; Compound I and II also have stronger antioxidant effect.
The inhibition effect of the oxygenizement that the chemical ingredients of table 3, ginseng wheat pin causes AAPH
Test sample book | Sample number | Average inhibiting rate (%) | Standard deviation (%) | The T-check |
AAPH I(MDP-2) 1μg 10μg 100μg III(MDP-4) 1μg 10μg 100μg II(MDP-8) 1μg 10μg 100μg | 6 6 6 6 5 5 6 6 6 6 | 0.00 16.51 25.16 27.81 11.92 17.45 54.88 24.89 26.09 21.05 | 0.36 1.67 2.29 4.13 1.45 9.23 4.03 2.71 4.93 4.05 | - 6.1E-05 4.3E-05 0.00053 0.0004 0.06583 1.7E-05 0.00011 0.00157 0.00149 |
Trolox | 3 | 83.06 | 1.27 | 4E-05 |
Description of drawings
Fig. 1 Compound I (MDP-2A), II (MDP-2A), VI (CMD-6-2) and VIII (CMD-b2) promote the effect (annotate: MDP-2B is the Compound I that contains small amount of impurities) that NO discharges to vascular endothelial cell
The inhibition effect of the oxygenizement that Fig. 2 Compound I (MDP-2), II (MDP-8) and III (MDP-4) cause AAPH
Claims (4)
1. preparation method who separates the compound (structure and chemical name are as follows) obtain from Shenmai injection is characterized in that it may further comprise the steps:
A) 10 kilograms the Chinese medicine Zhejiang medicinal material tuber of dwarf lilyturf is prepared into the stoste tuber of dwarf lilyturf according to the production technique of Shenmai injection, behind 40 ℃ of concentrating under reduced pressure, separate with macroporous resin column chromatography, with the water-alcohol elutriant wash-out of gradient, obtain water, 10%, 20%, 40%, 60%, the 80% and 95% alcohol wash-out part of the stoste tuber of dwarf lilyturf.
B) the 95% alcohol wash-out part that obtains among a is separated with silicagel column, is the developping agent wash-out with sherwood oil, chloroform, chloroform-acetone (1: 0 → 1: 1), chloroform-methanol (50: 1 → 1: 1); Chloroform-acetone (20: 1) wash-out partly continues to separate with silica gel column chromatography, can obtain compound VI with petroleum ether-ethyl acetate (5: 1) as the developping agent purifying; Chloroform-acetone (5: 1) wash-out part is further separated with silica gel column chromatography, with petroleum ether-ethyl acetate (2: 1) as developping agent, can obtain compound V and compound (VIIa+VIIb) enriching section, use dextrane gel Sephadex LH-20 column purification more respectively, launch to obtain respectively V and (VIIa+VIIb) with chloroform, the latter is a pair of epimer, high performance liquid phase (PreStar system with the preparation type, merck ODS packed column 1.2 * 25cm, acetonitrile-water 30%-60%) prepares VIIa and VIIb; Chloroform-methanol (50: 1) wash-out partly uses silica gel column chromatography (developping agent is chloroform-methanol 25: 1) to be further purified can obtain the Compound I enriching section, can get pure I through distillation again; 25: 1 wash-out parts of chloroform-methanol wash-out position can obtain IV with being further purified with silica gel column chromatography (chloroform-methanol 15: 1).
C) 80% alcohol wash-out part silicagel column among a separates, and is the developping agent wash-out with chloroform-methanol (50: 1 → 1: 1); 10: 1 wash-out parts of chloroform-methanol wash-out position recycle silicon plastic column chromatography (chloroform-methanol-water 5: 1: 0.1) is further purified and can obtains Compound I I enriching section, use RP-18 reversed-phase column purifying again, can obtain II as distributing solution to wash with ethanol (or methyl alcohol)-water (2: 1).
D) the 40% alcohol wash-out part that obtains among a is separated with silicagel column, with chloroform (or ethyl acetate and methylene dichloride)-methyl alcohol (or ethanol) gradient elution.5: 1 wash-out parts of chloroform-methanol are further used RP-18 reversed-phase column purifying, can obtain compound III and VIII with methanol-water (4: 1) respectively as distribution solution.
2. a right characterizes as requiring 1 described compound to be used for the Shenmai injection quality control.
3. a right is used for the treatment of the purposes of cardiovascular and cerebrovascular diseases as requiring 1 described compound in preparation.
4. a right is as requiring preparation method and the application in being used for the treatment of the medicine of cardiovascular and cerebrovascular diseases thereof of 1 described new compound VIIa and VIIb.
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CN 200710122813 CN101077852A (en) | 2007-07-05 | 2007-07-05 | Preparation for chemical component of Shenmai injection and application of the same in curing cardio-cerebrovascular disease |
CNB2007101874224A CN100453538C (en) | 2007-07-05 | 2007-11-23 | Preparation for chemical composition of Shenmai injection and application in treating cardiovascular and cerebrovascular diseases thereof |
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CN 200710122813 CN101077852A (en) | 2007-07-05 | 2007-07-05 | Preparation for chemical component of Shenmai injection and application of the same in curing cardio-cerebrovascular disease |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102127042A (en) * | 2011-01-26 | 2011-07-20 | 浙江大学 | Homoisoflavonoid compounds and preparation method and use thereof |
CN102233094A (en) * | 2010-04-23 | 2011-11-09 | 正大青春宝药业有限公司 | Active component determination method for Shenmai injection |
CN103728332A (en) * | 2014-01-24 | 2014-04-16 | 西南民族大学 | Finished shenmai injection liquid quality identification method |
CN105004830A (en) * | 2015-07-21 | 2015-10-28 | 成都中医药大学 | HPLC-ELSD method for simultaneously determining five saponin components in ophiopogon japonicus |
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2007
- 2007-07-05 CN CN 200710122813 patent/CN101077852A/en active Pending
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102233094A (en) * | 2010-04-23 | 2011-11-09 | 正大青春宝药业有限公司 | Active component determination method for Shenmai injection |
CN102233094B (en) * | 2010-04-23 | 2012-09-26 | 正大青春宝药业有限公司 | Active component determination method for Shenmai injection |
CN102127042A (en) * | 2011-01-26 | 2011-07-20 | 浙江大学 | Homoisoflavonoid compounds and preparation method and use thereof |
CN102127042B (en) * | 2011-01-26 | 2013-04-03 | 浙江大学 | Homoisoflavonoid compounds and preparation method and use thereof |
CN103728332A (en) * | 2014-01-24 | 2014-04-16 | 西南民族大学 | Finished shenmai injection liquid quality identification method |
CN105004830A (en) * | 2015-07-21 | 2015-10-28 | 成都中医药大学 | HPLC-ELSD method for simultaneously determining five saponin components in ophiopogon japonicus |
CN105004830B (en) * | 2015-07-21 | 2016-06-08 | 成都中医药大学 | Measure the HPLC-ELSD method of 5 kinds of saponin constituents in Radix Ophiopogonis simultaneously |
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