CN101375990B - Composition for treating cardiovascular disease and preparation thereof - Google Patents
Composition for treating cardiovascular disease and preparation thereof Download PDFInfo
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Abstract
The invention discloses a composition for treating cardiovascular diseases and a preparation method thereof. The composition comprises (by weight part) Radix Ginseng Rubra saponin 2-20, Ophiopogon japonicus saponin 0.2-3, and Ophiopogon japonicus flavone 0.02-3; wherein the Radix Ginseng Rubra saponin extract contains (by dry weight) ginsenoside Rg1 not less than 9.0%, ginsenoside Re not less than 3.0%, ginsenoside Rb1 not less than 12.0%, and Panax ginseng total saponins not less than 80%; the Ophiopogon japonicus saponin extract contains (by dry weight) ophiopogonin D not less than 6.0%, ophiopogonin D' not less than 6.0%, and Ophiopogon japonicus total saponins not less than 80%; and the Ophiopogon japonicus flavone extract contains (by dry weight) methylophiopogonone A and methylophiopogonone B in total not less than 80%. Animal experiments showed that the composition has good therapeutic action on myocardial ischemia and has better anti-shock effect.
Description
Technical field
The invention belongs to pharmaceutical field, relate to a kind of compositions of the chemical constituent that is used to treat cardiovascular disease and the method for preparing of injection preparation thereof.
Background technology
SHENMAI ZHUSHEYE is the Chinese medicine preparation of on ancient prescription Rhizoma Zingiberis Recens drink basis, succeeding in developing with modern science and technology, selects refining forming of Radix Ginseng Rubra, Radix Ophiopogonis for use, uses compound formulation for vein, has invigorating vital energy and reinforce the heart, the multiple arteries and veins that promotes the production of body fluid, blood circulation promoting and blood stasis dispelling, a function of regulating the flow of vital energy and having one's ideas straightened out.Wherein Radix Ginseng has the effect of similar cardiotonic glycoside, can strengthen myocardial contraction, coronary artery dilator, microcirculation improvement; Can promote humoral immunization Radix Ophiopogonis, regulates nucleic acid metabolism; Share and to play heart tonifying, expand blood vessel and regulate the effect of neuroendocrine function.Experimentation shows that its pharmacotoxicological effect mainly shows coronary artery dilator, increases coronary flow, increases myocardial contraction, reduces Peripheral resistance, reduces myocardial oxygen consumption, improves energy metabolism of myocardial, improves the body anti-anoxia ability, removes oxygen-derived free radicals, protects myocardium effect.Simultaneously to stablizing myocardial cell membrane, keep the rhythm of the heart and have good effect, and do not find obvious toxic-side effects, safe and effective.
The main effective ingredient of Radix Ginseng Rubra is the ginsenoside, and by the chemical constitution of its aglycon, the ginsenoside can be divided three classes: the panoxadiol is that saponin, panaxatriol are that saponin, oleanolic acid are the ginsenoside of aglycon.The ginsenoside can be divided into 3 types by the structure of aglycon; Oleanolic acid type such as ginsenoside Ro mainly contain antiinflammatory, antiplatelet release action.Protopanoxadiol type such as ginsenoside-Rb1 and-Rb2 show as central nerve inhibition, reduce intracellular Ca2+, antioxidation, removing free radical and improve effect such as myocardial ischemia reperfusion injury, and the protopanoxadiol saponins does not have hemolytic activity.Protopanaxatriol's saponins has hemolytic activity, and Protopanaxatriol's type saponin then shows as central nervous excitation like-Rg1, and nootropics promotes the effect such as synthetic of protein, DNA and RNA;-Re is the arrhythmia effective ingredient, can suppress morphine and bring out the effects such as drug resistance that mice produces;-Rg2 can suppress effects such as rabbit platelet release reaction.Radix Ophiopogonis, main component was ophiopogonin, flavone.Contain multiple steroidal saponin: the aglycon of Ophiopogonin A, B, C, D is Lu Sikao sapogenin, ophiopogonin B ', the aglycon of C ', D ' is diosgenin.Ophiopogonin strengthens cardiac contractile force, increases coronary flow; Can improve the tolerance of mice ischemic myocardium, improve the effect of myocardial cell nutritional blood flow amount hypoxia; Arrhythmia and Antishock function.Ophiopogonone has cardiotonic, improves the body anti-anoxia ability, removes oxygen-derived free radicals, protects myocardium effect.
SHENMAI ZHUSHEYE has been widely used in clinical treatment cardiovascular, respiratory system disease; There is the quality instability problem in existing injection containing ginseng extract preparation; This mainly be because just through extraction, simple refining back dosing embedding sterilization and getting, contain a large amount of impurity in the Chinese herbal medicine extract like the matter of mixing, pigment, antibacterial thermal source etc., can occur color and luster standing time for a long time to deepen, produce problems such as deposition, content reduction; And active ingredient is indeterminate in the preparation; The quality standard imperfection, each batch quality exists than big-difference, has influenced the stable and standardization production of determined curative effect of injection.
Summary of the invention
The object of the invention is to provide a kind of compositions that is used to treat cardiovascular disease;
The object of the invention also is to provide a kind of composite preparation that is used to treat cardiovascular disease;
The object of the invention also is to provide a kind of purification process that is used to treat the composition material medicine of cardiovascular disease;
The object of the invention also is to provide a kind of method of quality control that is used to treat the compositions of cardiovascular disease.
The present invention seeks to realize through following technical scheme:
Technical scheme 1:
A kind of compositions that is used to treat cardiovascular disease is to be processed by the crude drug of following weight ratio:
Red ginseng saponin 2-20 weight portion ophiopogonin 0.1-3 weight portion
Preferably: red ginseng saponin 5-10 weight portion ophiopogonin 0.5-1 weight portion
Preferably: red ginseng saponin 12-18 weight portion ophiopogonin 0.5-1 weight portion
Preferably: red ginseng saponin 12-18 weight portion ophiopogonin 1.5-2.8 weight portion
Wherein red ginseng saponin extract is pressed dry product calculating, contains the ginsenoside Rg
1Must not be less than 9.0%, the ginsenoside Re must not be less than 3.0%, ginsenoside Rb
1Must not be less than 12.0%, Radix Ginseng total saponins content must not be less than 80%;
Wherein radix ophiopogonis saponin extract is pressed dry product calculating, and contain ophiopogonin D and must not be less than 6.0%, ophiopogonin D ' must not be less than 6.0%, Radix Ophiopogonis total saponins content must not be less than 80%;
Technical scheme 2:
A kind of compositions that is used to treat cardiovascular disease according to the invention can be by being that crude drug by following weight ratio is processed:
Red ginseng saponin 2-20 weight portion ophiopogonin 0.2-3 weight portion
Ophiopogonone 0.02-3 weight portion
Preferably: red ginseng saponin 5-10 weight portion ophiopogonin 0.5-1 weight portion
Ophiopogonone 0.1-1 weight portion
Preferably: red ginseng saponin 12-18 weight portion ophiopogonin 0.5-1 weight portion
Ophiopogonone 2.2-3 weight portion
Preferably: red ginseng saponin 5-10 weight portion ophiopogonin 1.5-3 weight portion
Ophiopogonone 0.2-1 weight portion
Further be preferably: red ginseng saponin: ophiopogonin: ophiopogonone=10 weight portions: 1 weight portion: 0.1 weight portion
Further be preferably: red ginseng saponin: ophiopogonin: ophiopogonone=10 weight portions: 3 weight portions: 0.3 weight portion
Wherein red ginseng saponin extract is pressed dry product calculating, contains the ginsenoside Rg
1Must not be less than 9.0%, the ginsenoside Re must not be less than 3.0%, ginsenoside Rb
1Must not be less than 12.0%, Radix Ginseng total saponins content must not be less than 80%;
Wherein radix ophiopogonis saponin extract is pressed dry product calculating, and contain ophiopogonin D and must not be less than 6.0%, ophiopogonin D ' must not be less than 6.0%, Radix Ophiopogonis total saponins content must not be less than 80%;
Wherein the ophiopogonone extract calculate by dry product contain the methyl Methylophiopogonanone A, the B total content must not be less than 80%;
Technical scheme 3:
A kind of compositions that is used to treat cardiovascular disease according to the invention can be by being that crude drug by following weight ratio is processed:
Ophiopogonin 0.2-3 weight portion ophiopogonone 0.2-3 weight portion
Preferably: ophiopogonin 0.5-1 weight portion ophiopogonone 0.4-1 weight portion
Preferably: ophiopogonin 0.5-1 weight portion ophiopogonone 2.2-3 weight portion
Preferably: ophiopogonin 1.5-2.8 weight portion ophiopogonone 1.2-2 weight portion
Wherein radix ophiopogonis saponin extract is pressed dry product calculating; Contain ophiopogonin D and must not be less than 6.0%; Ophiopogonin D ' must not be less than 6.0% also contains other main six kinds of ophiopogonin constituents except that containing above-mentioned ophiopogonin D, D ' composition, Radix Ophiopogonis total saponins content must not be less than 80%;
Wherein the ophiopogonone extract calculate by dry product contain the methyl Methylophiopogonanone A, the B total content must not be less than 80%;
Above-mentioned three kinds of technical schemes are described to be used to treat the cardiovascular disease compositions, adds acceptable accessories/carrier, can be made into clinical acceptable various dosage forms, like capsule, granule, soft capsule etc.
The preferred dosage form of compositions that above-mentioned three kinds of technical schemes are described to be used to treat cardiovascular disease is an injection, can in prescription, add mannitol 80-200 weight portion water for injection 800-2000 parts by volume; Method for preparing is: get the recipe quantity crude drug and under aseptic condition, add in the 300-500 parts by volume water for injection, add 8-12%NaOH solution adjust pH to 7-8, add recipe quantity mannitol and make abundant dissolving; Add injection and be diluted with water to 1000 parts by volume; Add the 0.01-0.06% activated carbon adsorption and remove thermal source, filter packing with microporous filter membrane; Frozen drying 24-36 hour, under aseptic condition, encapsulate.Wherein said weight portion/parts by volume is corresponding with g/ml.
For example: wherein a kind of method for preparing of composition injection is:
Choose: red ginseng saponin 2-20 weight portion ophiopogonin 0.2-3 weight portion
Ophiopogonone 0.2-3 weight portion
Mannitol 80-200 weight portion water for injection 800-2000 parts by volume;
Get red ginseng saponin 2-20 weight portion ophiopogonin 0.2-3 weight portion ophiopogonone 0.2-3 weight portion and under aseptic condition, add in the 300-500 parts by volume water for injection, add 8-12%NaOH solution adjust pH, add recipe quantity mannitol and make abundant dissolving to 7-8; Add injection and be diluted with water to 1000ml; Add the 0.01-0.06% activated carbon adsorption and remove thermal source, filter packing with microporous filter membrane; Frozen drying 24-36 hour, under aseptic condition, encapsulate.
Red ginseng saponin of the present invention, ophiopogonin, ophiopogonone can be taken from commercially available, also can be through the method preparation of existing document record; Also can the following method for preparing red ginseng saponin, ophiopogonin, ophiopogonone provided by the present invention.
The invention provides a kind of preferred method for preparing red ginseng saponin, ophiopogonin, ophiopogonone:
Wherein the red ginseng saponin extract method for preparing is:
Extract: with Radix Ginseng Rubra with ethanol or a certain proportion of ethanol water (0: 100-100: 0) make solvent, adopt percolation to extract or the thermal cycle reflux, extract,, collect extracting solution to red ginseng saponin and extract fully;
Water precipitating: extracting solution is evaporated to 0.8-1.5 times of crude drug amount, puts coldly, and thin up is to 1-3 times of crude drug amount, cold preservation, filter Radix Ginseng Rubra water liquid;
Extraction: Radix Ginseng Rubra water liquid adds the natrium carbonicum calcinatum stirring and dissolving and transfers pH value 9-10, uses n-butanol extraction, and is abundant to the red ginseng saponin extraction;
Column chromatography absorption: collect butanol extraction liquid, reclaim n-butyl alcohol and evaporate to dryness, add water and fully dissolve, transfer pH value 9～10, macroporous adsorptive resins absorption on the medicinal liquid like the natrium carbonicum calcinatum stirring and dissolving;
The column chromatography eluting: first water is washed till eluent for neutral, and reuse 60-90% ethanol elution is complete to the red ginseng saponin eluting, collects ethanol elution;
Refining: merge ethanol elution, reclaim ethanol and be concentrated into 1 times of crude drug amount, add the decolouring of 0.01% active carbon stirring at normal temperature, evaporate to dryness gets dry extract; Pulverize, add anhydrous alcohol solution under the room temperature, filter, reclaim ethanol with the 0.45um microporous filter membrane; Evaporate to dryness gets dry extract, and pulverizes, and promptly gets;
Wherein the red ginseng saponin extract method for preparing is preferably:
With Radix Ginseng Rubra medical material 10 weight portions, make solvent with 60-80% ethanol, after the solvent impregnated 10-14 of 15 parts by volume hour, carry out the percolation extraction earlier, collect percolate to red ginseng saponin and extract fully.
Percolate is evaporated to about 10 parts by volume, put cold, thin up to 20 parts by volume, cold preservation 10-14 hour, filter Radix Ginseng Rubra water liquid.
Radix Ginseng Rubra water liquid adds natrium carbonicum calcinatum 0.2 weight portion stirring and dissolving (pH value 9～10), and with n-butanol extraction 8-12 time, first three time adds n-butyl alcohol 18-22 parts by volume at every turn; The follow-up n-butyl alcohol 8-12 parts by volume that at every turn adds is abundant to the red ginseng saponin extraction;
Collect butanol extraction liquid; Reclaim n-butyl alcohol and evaporate to dryness, add water 10 parts by volume and fully dissolve, add natrium carbonicum calcinatum 0.1 weight portion stirring and dissolving (pH value 9～10); D101 macroporous adsorptive resins on the medicinal liquid (amount of resin 10 weight portions) absorption, absorption flow velocity be the 1.5-3 column volume/hour.
With washing 4～6 column volumes, be neutral to eluent, reuse 65-80% ethanol elution, eluting 4-6 column volume, elution flow rate be the 1.5-3 column volume/hour, collection 65-80% ethanol elution;
Merge collected column chromatography ethanol elution, reclaim ethanol and be concentrated into the 9-12 parts by volume, add 0.01% solution amount active carbon stirring at normal temperature decolouring 10 minutes; Evaporate to dryness gets dry extract, and pulverizes, and adds anhydrous alcohol solution under the room temperature; Filter with the 0.45um microporous filter membrane, reclaim ethanol, evaporate to dryness gets dry extract; Pulverize, promptly get.
Radix ophiopogonis saponin extract method for preparing wherein:
Extract: will make solvent with ethanol or a certain proportion of ethanol water Radix Ophiopogonis, and adopt percolation to extract or the thermal cycle reflux, extract,, and collect extracting solution to ophiopogonin, ophiopogonone extraction fully;
Water precipitating: extracting solution is evaporated to 1 times of crude drug amount, puts coldly, and thin up is to 1.5-2.5 times of crude drug amount, cold preservation, filter Radix Ophiopogonis water liquid;
The flavone part: collecting precipitation fully dissolves with petroleum ether heating extraction to ophiopogonone, gets ophiopogonone deposition part, and Radix Ophiopogonis, water liquid was abundant that ophiopogonone extracts part with the extraction of petroleum ether extraction to ophiopogonone, and merging two parts promptly get the ophiopogonone bullion;
Extraction: Radix Ophiopogonis, water liquid added natrium carbonicum calcinatum stirring and dissolving accent pH value 9～10, used n-butanol extraction, and is abundant to the ophiopogonin extraction;
Column chromatography absorption: collect butanol extraction liquid, reclaim n-butyl alcohol and evaporate to dryness, add water and fully dissolve, add the natrium carbonicum calcinatum stirring and dissolving and transfer pH value 9～10, macroporous adsorptive resins absorption on the medicinal liquid;
The column chromatography eluting: first water is washed till eluent for neutral, and reuse 60-90% ethanol elution is complete to the ophiopogonin eluting, collects ethanol elution;
Refining: merge ethanol elution, reclaim ethanol and be concentrated into 1 times of crude drug amount, add the decolouring of 0.01% active carbon stirring at normal temperature, evaporate to dryness gets dry extract; Pulverize, add anhydrous alcohol solution under the room temperature, filter, reclaim ethanol with the 0.45um microporous filter membrane; Evaporate to dryness gets dry extract, and pulverizes, and promptly gets;
The radix ophiopogonis saponin extract method for preparing preferably be:
With medical material 10 weight portions Radix Ophiopogonis, section or pulverizing are coarse powder, make solvent with 65-75% ethanol, after the solvent impregnated 10-13 of 14-16 parts by volume hour, carry out the percolation extraction earlier, collect the extraction of percolate to ophiopogonin and flavone fully.
Percolate is evaporated to about 10 parts by volume, put cold, thin up to 20 parts by volume, cold preservation 20-28 hour, filter, respectively Radix Ophiopogonis water liquid and ophiopogonone deposition part.
Filtrating adds petroleum ether 10 parts by volume with petroleum ether extraction 10 times at every turn, merges petroleum ether extraction liquid, reclaim petroleum ether, and be concentrated into thick paste, ophiopogonone petroleum ether extraction part, with above-mentioned ophiopogonone deposition partly merge the ophiopogonone bullion, retain subsequent use.
Mother solution adds natrium carbonicum calcinatum 0.2 weight portion stirring and dissolving behind the Radix Ophiopogonis petroleum ether extraction, and pH value 9～10 adds n-butanol extraction 9-12 time, and first three time adds n-butyl alcohol 20 parts by volume at every turn; Follow-up n-butyl alcohol 10 parts by volume that at every turn add are abundant to the ophiopogonin extraction.
Merge butanol extraction liquid, be concentrated into 5 parts by volume, gradation adds low amounts of water washing n-butyl alcohol liquid, adds 1 parts by volume at every turn, washs 3-4 time, and reuse 1% natrium carbonicum calcinatum solution washing adds 1 parts by volume to colourless at every turn, washs 6-8 time; Washing adds 1 parts by volume to neutral at every turn, washs 6-8 time.Get n-butyl alcohol liquid, reclaim n-butyl alcohol and evaporate to dryness, add water 5 parts by volume and fully dissolve, D101 macroporous adsorptive resins on the medicinal liquid, the absorption of amount of resin 5 weight portions, the absorption flow velocity be 2 column volumes/hour.
Elder generation's water is washed till eluent for neutral, washes 4～6 column volumes, and reuse 65-80% ethanol elution, eluting 4-6 column volume, HPLC detect whether eluting is complete, elution flow rate be 2 column volumes/hour, collect 75% ethanol elution.
Merge collected column chromatography ethanol elution, reclaim ethanol and be concentrated into about 5 parts by volume, add 0.01% active carbon stirring at normal temperature decolouring 10 minutes, evaporate to dryness gets dry extract; Pulverize, add anhydrous alcohol solution under the room temperature, filter, reclaim ethanol with the 0.45um microporous filter membrane; Evaporate to dryness gets dry extract, and pulverizes, and promptly gets.
Ophiopogonone method for preparing extractive wherein:
Get above-mentioned ophiopogonone bullion and add the silica gel mixed sample that 2-4 doubly measures, be splined on silica gel column chromatography, add the remove impurity of petroleum ether eluting earlier; Reuse petroleum ether: ethyl acetate=95: 5-90: 10 eluting, it is abundant to be collected in Radix Ophiopogonis the flavone component eluting, merges main flavone eluting stream part; 40-60 ℃ of decompression and solvent recovery, a small amount of petroleum ether sample, 30-50 ℃ of vacuum drying; Pulverize dry powder, promptly get.
The ophiopogonone method for preparing extractive is preferably:
Get the silica gel mixed sample that the ophiopogonone bullion adds 3 times of amounts, evaporate to dryness grinds well; Be splined on silica gel column chromatography, add 4 times of column volume amounts of petroleum ether eluting earlier, reuse petroleum ether: ethyl acetate=95: 5-90: 10 eluting; Collect 1/4 column volume and be first-class part, the whole eluting of flavone component are abundant to the Radix Ophiopogonis, merge main flavone eluting stream part; 50 ℃ of decompression and solvent recoveries, a small amount of petroleum ether sample, 40 ℃ of vacuum dryings; Pulverize dry powder, promptly get.
Weight portion/parts by volume is corresponding with kg/L described in the said extracted thing preparation technology.
Pharmaceutical composition method of quality control of the present invention comprises one or both (this method of quality control is applicable to the various dosage forms of compositions) in the following assay:
1, red ginseng saponin detects: the high effective liquid chromatography for measuring ginsenoside Rg
1, Re, Rb
1Chromatographic condition and system suitability test: using octadecylsilane chemically bonded silica to be filler, is mobile phase A with the acetonitrile, is Mobile phase B with water, and gradient elution method is:
0～30 minute, mobile phase A 20 → 21 (%), Mobile phase B 80 → 79 (%);
30～31 minutes, mobile phase A 21 → 28 (%), Mobile phase B 79 → 72 (%);
31～50 minutes, mobile phase A 28 → 30 (%), Mobile phase B 72 → 70 (%);
50～60 minutes, mobile phase A 30 → 40 (%), Mobile phase B 70 → 60 (%);
60～80 minutes, mobile phase A 40 → 65 (%), Mobile phase B 60 → 35 (%);
80～80.1 minutes, mobile phase A 65 → 20 (%), Mobile phase B 35 → 80 (%);
80.1～90 minutes, mobile phase A 20 (%), Mobile phase B 80 (%);
Flow velocity: 1.0mL/min; Sample size: 20 μ L; Evaporative light scattering detector detects, 110 ℃ of drift tube temperatures, and gas flow rate 2.9L/min does not shunt; Column temperature: 25 ℃.
It is an amount of that the preparation precision of need testing solution takes by weighing red ginseng saponin, adds methanol and process the solution that every 1ml contains these article 4.0mg, promptly gets.
The preparation of reference substance solution: precision takes by weighing the ginsenoside Rg through drying under reduced pressure to constant weight
1, ginsenoside Re, ginsenoside Rb
1Reference substance is an amount of, adds methanol and processes the mixed solution that contains 0.64mg, 0.24mg, 0.80mg among every 1ml respectively, shakes up, and promptly gets.
Accurate respectively reference substance solution 10ul, 20ul and the need testing solution 20ul of drawing of algoscopy injects chromatograph of liquid, measures, and writes down 80 minutes collection of illustrative plates, calculates with external standard two-point method logarithmic equation, promptly gets.
These article are pressed dry product and are calculated, and contain the ginsenoside Rg
1(C
42H
72O
14) must not be less than 9.0%, ginsenoside Re (C
48H
82O
18) must not be less than 3.0%, ginsenoside Rb
1(C
54H
92O
23) must not be less than 12.0%.
2, ophiopogonin detects:
Chromatographic condition and system suitability test: using octadecylsilane chemically bonded silica to be filler, is mobile phase A with the acetonitrile, is Mobile phase B with water, and gradient elution method is:
0～25 minute, mobile phase A 100 (%), Mobile phase B 0 (%);
25～25.1 minutes, mobile phase A 100 → 0 (%), Mobile phase B 0 → 100 (%);
25.1～50 minutes, mobile phase A 0 (%), Mobile phase B 100 (%);
50～60 minutes, mobile phase A 0 → 100 (%), Mobile phase B 100 → 0 (%);
Flow velocity: 1.0mL/min; Sample size: 20 μ L; Evaporative light scattering detector detects, 95.2 ℃ of drift tube temperatures, and gas flow rate 2.5L/min does not shunt; Column temperature: 25 ℃.
The preparation of need testing solution: get radix ophiopogonis saponin extract and add methanol and be mixed with the solution that every 1ml contains 2.0mg, promptly get.
The preparation of reference substance solution: get ophiopogonin D, ophiopogonin D ' reference substance is an amount of, add methanol and process solution that every 1ml contains 0.3mg respectively as object of reference solution;
Algoscopy: accurate respectively reference substance solution 10ul, 20ul and the need testing solution 20ul of drawing, inject chromatograph of liquid, measure, write down 60 minutes collection of illustrative plates, calculate with external standard two-point method logarithmic equation, promptly get;
Radix ophiopogonis saponin extract contains by dry product that ophiopogonin D should be not less than 6.0%, ophiopogonin D ' should be not less than 6.0%.
3, methyl Methylophiopogonanone A, B assay:
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With 50-60: 4-8: 35-45 acetonitrile-methanol-0.2% glacial acetic acid aqueous solution is a mobile phase; UV-detector detects wavelength 296nm, and theoretical cam curve is calculated by methyl flavanone Radix Ophiopogonis A peak should be not less than 6000;
The preparation of reference substance solution: methyl Methylophiopogonanone A, B reference substance that precision takes by weighing through 55-65 ℃ of drying under reduced pressure to constant weight are an amount of, add methanol and process the solution that every 1ml contains methyl Methylophiopogonanone A, each 0.1mg of B;
The preparation of need testing solution: get flavone extract and add methanol and be mixed with the solution that every 1ml contains 0.25mg, promptly get
Algoscopy: respectively accurate reference substance solution and the need testing solution 20 μ l of drawing, inject chromatograph of liquid, measure, with external standard method calculating content, promptly get, flavone extract by dry product contain the methyl Methylophiopogonanone A, the B total content should be not less than 80%.
Three described compositionss of technical scheme of the present invention have good drug effect; Through further clearly reaching the percentage composition of effective ingredient in the control combination thing; Some unnecessary impurity have been removed; Thereby the crude extract preparation of on drug effect, comparing has better effect, and can avoid the generation of some untoward reaction.As in zoopery, showing myocardial ischemia there is good therapeutical effect and has better Antishock function.
The invention also discloses exquisite method Radix Ginseng Rubra Radix Ophiopogonis, Radix Ginseng Rubra contained active ingredient red ginseng saponin in Radix Ophiopogonis, ophiopogonin are all soluble in water, ethanol and certain density ethanol water, and ophiopogonone is dissolved in ethanol and high concentration ethanol aqueous solution, and is water insoluble.Prior art adopts water, alcohol heat reflux to extract more, and it is more that impurity is extracted in hot reflux, and high more pigment that extracts of temperature and oil-soluble impurities are high more; Pure water extracts and then extracts non-active ingredient such as a large amount of saccharides, protein, polysaccharide, pigment inorganic salt; Increased the difficulty of subsequent purification, technological operation is complicated, and cost is high; The extraction means that the present invention adopts percolation or warm macerating hot reflux to extract; Do to extract solvent with certain density ethanol water, saponin and flavones ingredient are extracted fully, guarantee to leach less the impurity component of volume again as far as possible.Radix Ginseng Rubra and ophiopogonin constituents water solublity are better; Can be dissolved in n-butyl alcohol again, in conjunction with the character of saponin component, extracting solution concentrates the first cold preservation water precipitation in back and removes water-insoluble impurity; To guarantee clarity; Be beneficial to subsequent purification and preparation, water precipitating liquid combines to handle with n-butanol extraction and two kinds of means of purification of absorption with macroporous adsorbent resin, has obtained very special effect.
The invention also discloses method of quality control, this method is the quality of control product very effectively, and injection can be used safely clinically.
The present invention improves its safety and quality controllability on the injection dosage form; Active chemical to wherein carries out deep analysis and evaluation; Adopt resin absorption, solvent extraction and crystallization purifying means process combined method; Make the pure degree of drug effect reach controlled composition and account for more than 80%, removed the related impurities of influence stability; Increase finger printing control product quality, guarantee that steady quality and the standardization between each batch produced.
Following experimental example and embodiment are used to further specify but are not limited to the present invention.
Test Example 1 Rhizoma Zingiberis Recens extract is to the influence of rat heart muscle ischemic injuries due to the Iso
1, process of the test:
Get 70 of healthy rats; 220-260g; Male and female half and half; Be divided into 7 groups immediately by body weight, be respectively blank group, model group, A group (red ginseng saponin 10mg/kg+ ophiopogonin 1mg/kg), B group (ophiopogonone 0.1mg/kg), C organizes (red ginseng saponin 10mg/kg+ ophiopogonin 1mg/kg+ ophiopogonone 0.1mg/kg), 10 every group.
The medicinal liquid 10ml/kg of the different components of each administration group intravenous injection; Isometric 5% glucose injection of model group and the intravenous injection of blank group, once a day, continuous 10 days; 30min is except that blank control group after the administration in the 10th day; All the other each components are lumbar injection Iso 8mg/kg respectively, and the blank group gives isometric normal saline, the electrocardiogram of 30min behind the record injection Iso; Measure T ripple and heart rate, calculate after the administration with administration before the mV number average value that raises of T ripple as the index of treating myocardial ischemia damage degree.
2, result of the test:
Table 1 Rhizoma Zingiberis Recens extract is to the protective effect
of rat heart muscle ischemic injuries due to the isoproterenol
P＜0.05, compare with model group * * P＜0.01
Visible by last table, the apparent in view rising of model group rat T wave-amplitude relatively has notable difference with normal group; A, C group rat T wave-amplitude obviously reduce, and with model group notable difference are arranged relatively; B group rat T wave-amplitude also reduces, but compares no significant difference with model group.A, B, C group rat heart rate raise to some extent, but compare no significant difference with model group.
Rising all has the reduction effect to the T ripple for A group, C group, and prompting has therapeutical effect to myocardial ischemia, and single is that the B group also has effect trend with ophiopogonone, but intensity is less; The C group is the most obvious to the rising effect trend of heart rate.The C group relatively has the addition synergism with folk prescription, and therefore according to this result of the test, red ginseng saponin 10mg/kg+ ophiopogonin 1mg/kg+ ophiopogonone 0.1mg/kg resisting myocardial ischemia effect is the most obvious.
Test Example 2 Rhizoma Zingiberis Recens extracts are to the influence test of dog cardiogenic shock
1, process of the test
Get 12 of healthy dogs; 12-15kg; Male and female half and half; Be divided into 6 groups immediately by body weight, be respectively model group, A group (red ginseng saponin 4mg/kg+ ophiopogonin 0.4mg/kg), B group (ophiopogonone 0.04mg/kg), C and organize (red ginseng saponin 4mg/kg+ ophiopogonin 0.4mg/kg+ ophiopogonone 0.04mg/kg), 8 every group.
Animal via pentobarbital sodium (30mg/kg) intravenous anesthesia, tracheal intubation connects respirator; External jugular vein intubate persistent instillation 5% glucose (30 droplets/minute); Separate the right lateral thigh vein, connect the constant speed infusion pump, in order to the input pentobarbital sodium; Separate common carotid artery, aortic cannulation is measured aortic blood pressure; Left side the 4th intercostal is opened breast, cuts off pericardium bag bed; Separate aorta, place the electromagnetic flowmeter probe, use the electromagnetic flowmeter determination cardiac output.Above-mentioned observation index is recorded in eight and leads on the physiograph.
Operation finishes, intravenous injection heparin 125 (U/kg), stablize 20 minutes after; Record aortic blood pressure and kinemic normal value as the basic value before the cardiogenic shock, are imported 2% pentobarbital sodium (0.2ml/kg/min) with the infusion pump constant speed then; With aortic blood pressure and cardiac output decline 40% index as cardiogenic shock; After this speed with 0.08ml/kg/min kept 10 minutes, and record each item index is as the basic value before the administration after the modeling; 1,5,15,30,60,90 and 120 minute aortic blood pressure and cardiac output behind the medicine of constant speed vein injection medicine, and mensuration then.
2, result of the test
Cardiac output changing value (behind the medicine-medicine before) as follows behind table 2 medicine:
Visible by last table, each time point A, B, C group dog cardiac output and model group more all have increase behind the medicine, and be the most obvious with the C group.
Mean arterial pressure changing value (behind the medicine-medicine before) as follows behind table 3 medicine:
Visible by last table, each time point is respectively organized the dog mean arterial pressure behind the medicine more all has in various degree with model group and increases, the most obvious with B group, C group, points out flavone in this model, to have Antishock function.
Following embodiment all can realize the described effect of above-mentioned experimental example
The specific embodiment
Embodiment 1:
Red ginseng saponin 100g ophiopogonin 10g mannitol 100g, water for injection 1000ml
Wherein red ginseng saponin extract is pressed dry product calculating, contains the ginsenoside Rg
1Must not be less than 10.0%, the ginsenoside Re must not be less than 3.6%, ginsenoside Rb
1Must not be less than 12.6%, Radix Ginseng total saponins content must not be less than 80%;
Wherein radix ophiopogonis saponin extract is pressed dry product calculating, and contain ophiopogonin D and must not be less than 8.0%, ophiopogonin D ' must not be less than 8.0%, Radix Ophiopogonis total saponins content must not be less than 80%;
Get the recipe quantity red ginseng saponin, ophiopogonin adds in the 300-500ml water for injection under aseptic condition, adds 10%NaOH solution adjust pH to 7-8, makes abundant dissolving; Add 0.02% activated carbon adsorption and remove thermal source; Add injection and be diluted with water to 1000ml, filter, be sub-packed in cillin bottle with the 0.22um microporous filter membrane; Every bottled amount 1ml, under aseptic condition, encapsulated in frozen drying 24-36 hour.
Embodiment 2:
Red ginseng saponin 10g ophiopogonin 1g ophiopogonone 0.8g, mannitol 100g, water for injection 1000ml
Get recipe quantity red ginseng saponin, ophiopogonin, ophiopogonone and under aseptic condition, add in the 300-500ml water for injection, add 10%NaOH solution adjust pH, make abundant dissolving to 7-8; Add 0.02% activated carbon adsorption and remove thermal source; Add injection and be diluted with water to 1000ml, filter, be sub-packed in cillin bottle with the 0.22um microporous filter membrane; Every bottled amount 1ml, under aseptic condition, encapsulated in frozen drying 24-36 hour.
Embodiment 3:
Ophiopogonin 1g ophiopogonone 0.8g, mannitol 100g, water for injection 1000ml
Get the recipe quantity ophiopogonin, ophiopogonone adds in the 300-500ml water for injection under aseptic condition, adds 10%NaOH solution adjust pH to 7-8, makes abundant dissolving; Add 0.02% activated carbon adsorption and remove thermal source; Add injection and be diluted with water to 1000ml, filter, be sub-packed in cillin bottle with the 0.22um microporous filter membrane; Every bottled amount 1ml, under aseptic condition, encapsulated in frozen drying 24-36 hour.
Embodiment 4:
Red ginseng saponin 10g ophiopogonin 1g ophiopogonone 0.8g, mannitol 100g, water for injection 1000ml
Get recipe quantity red ginseng saponin, ophiopogonin, ophiopogonone and under aseptic condition, add in the 300-500ml water for injection, add 10%NaOH solution adjust pH, make abundant dissolving to 7-8; Add 0.02% activated carbon adsorption and remove thermal source; Add injection and be diluted with water to 1000ml, filter, be sub-packed in cillin bottle with the 0.22um microporous filter membrane; Every bottled amount 1ml, under aseptic condition, encapsulated in frozen drying 24-36 hour.
Embodiment 5: injection
Red ginseng saponin 16g ophiopogonin 2g ophiopogonone 2g, mannitol 100g, water for injection 1000ml
Get recipe quantity red ginseng saponin, ophiopogonin, ophiopogonone and under aseptic condition, add in the 300-500ml water for injection, add 10%NaOH solution adjust pH, make abundant dissolving to 7-8; Add 0.02% activated carbon adsorption and remove thermal source; Add injection and be diluted with water to 1000ml, filter, be sub-packed in cillin bottle with the 0.22um microporous filter membrane; Every bottled amount 1ml, under aseptic condition, encapsulated in frozen drying 24-36 hour.
Embodiment 6: injection
Red ginseng saponin 13g ophiopogonin 1g ophiopogonone 0.8g, mannitol 100g, water for injection 1000ml
Get recipe quantity red ginseng saponin, ophiopogonin, ophiopogonone and under aseptic condition, add in the 300-500ml water for injection, add 10%NaOH solution adjust pH, make abundant dissolving to 7-8; Add 0.02% activated carbon adsorption and remove thermal source; Add injection and be diluted with water to 1000ml, filter, be sub-packed in cillin bottle with the 0.22um microporous filter membrane; Every bottled amount 1ml, under aseptic condition, encapsulated in frozen drying 24-36 hour.
Embodiment 7:
With Radix Ginseng Rubra medical material 10kg, cut into the thin slice (or pulverizing is coarse powder) of 2～3mm, make solvent with 75% ethanol, carry out percolation after solvent impregnated 12 hours with 15L earlier and extract, collect percolate to red ginseng saponin extraction fully (about 200-250L, HPLC detects).Percolate is evaporated to about 10L, puts coldly, and thin up is to 20L, cold preservation 12 hours, filter Radix Ginseng Rubra water liquid.Radix Ginseng Rubra water liquid adds natrium carbonicum calcinatum 200g stirring and dissolving (pH value 9～10), uses n-butanol extraction, and first three time adds n-butyl alcohol 20L at every turn; The follow-up n-butyl alcohol 10L that at every turn adds is to red ginseng saponin extraction abundant (HPLC detects, and need extract approximately 10 times).Collect butanol extraction liquid, reclaim n-butyl alcohol and evaporate to dryness, add water 10L and fully dissolve, add natrium carbonicum calcinatum 100g stirring and dissolving (pH value 9～10), D101 macroporous adsorptive resins (amount of resin 10kg) absorption on the medicinal liquid, the absorption flow velocity be 2 column volumes/hour.Elder generation's water is washed till eluent and is neutral (washing 4～6 column volumes approximately), reuse 75% ethanol elution, the about 4-6 column volume of eluting (HPLC detects whether eluting is complete), elution flow rate be 2 column volumes/hour, collect 75% ethanol elution.Merge 75% ethanol elution, reclaim ethanol and be concentrated into about 10L, add 0.01% active carbon stirring at normal temperature decolouring 10min, evaporate to dryness gets dry extract; Pulverize, add anhydrous alcohol solution under the room temperature, filter, reclaim ethanol with the 0.45um microporous filter membrane; Evaporate to dryness gets dry extract, and pulverizes, and promptly gets.
With medical material 10kg Radix Ophiopogonis, cut into the thin slice (or pulverizing is coarse powder) of 2～3mm, make solvent with 75% ethanol, carry out percolation after solvent impregnated 12 hours with 15L earlier and extract, collect percolate to ophiopogonin and flavone extraction fully (about 200-250L, HPLC detects).Percolate is evaporated to about 10L, puts coldly, and thin up is to 20L, and cold preservation 24 hours filters, respectively Radix Ophiopogonis water liquid and ophiopogonone deposition part.Filtrate and use petroleum ether extraction, add petroleum ether 10L (HPLC detects and whether extracts fully, need extract approximately 10 times) at every turn, merge petroleum ether extraction liquid, reclaim petroleum ether, and be concentrated into thick paste, get ophiopogonone extraction part.Mother solution adds natrium carbonicum calcinatum 200g stirring and dissolving (pH value 9～10) behind the Radix Ophiopogonis petroleum ether extraction, adds n-butanol extraction, and first three time adds n-butyl alcohol 20L at every turn; The follow-up n-butyl alcohol 10L that at every turn adds is to ophiopogonin extraction abundant (HPLC detects, and need extract approximately 10 times).Merge butanol extraction liquid, be concentrated into 5L, gradation adds low amounts of water washing n-butyl alcohol liquid (add 1L at every turn, wash about 3-4 time), and reuse 1% natrium carbonicum calcinatum solution washing (adds 1L at every turn, washs about 6-8 time to colourless; ), washing is to neutral (add 1L, wash about 6-8 time) at every turn.Get n-butyl alcohol liquid, reclaim n-butyl alcohol and evaporate to dryness, add water 5L and fully dissolve, D101 macroporous adsorptive resins (amount of resin 5kg) absorption on the medicinal liquid, the absorption flow velocity be 2 column volumes/hour.Elder generation's water is washed till eluent and is neutral (washing 4～6 column volumes approximately), reuse 75% ethanol elution, the about 4-6 column volume of eluting (HPLC detects whether eluting is complete), elution flow rate be 2 column volumes/hour, collect 75% ethanol elution.Merge 75% ethanol elution, reclaim ethanol and be concentrated into about 5L, add 0.01% active carbon stirring at normal temperature decolouring 10min, evaporate to dryness gets dry extract; Pulverize, add anhydrous alcohol solution under the room temperature, filter, reclaim ethanol with the 0.45um microporous filter membrane; Evaporate to dryness gets dry extract, and pulverizes, and promptly gets.
Get the silica gel mixed sample that ophiopogonone partly adds 3 times of amounts, evaporate to dryness grinds well; Be splined on silica gel column chromatography, add 4 times of column volume amounts of petroleum ether eluting earlier, reuse petroleum ether: ethyl acetate=95: 5-90: 10 eluting; Collect 1/4 column volume and be first-class part, the whole eluting of flavone component are abundant to the Radix Ophiopogonis, merge main flavone eluting stream part; 50 ℃ of decompression and solvent recoveries, a small amount of petroleum ether sample, 40 ℃ of vacuum dryings; Pulverize dry powder, promptly get.
Get red ginseng saponin 10g, ophiopogonin 1g, ophiopogonone 0.1g; Mannitol 100g adds in the 300-500ml water for injection under aseptic condition, adds 10%NaOH solution adjust pH to 7-8, makes abundant dissolving; Add 0.02% activated carbon adsorption and remove thermal source, add injection and be diluted with water to 1000ml, filter with the 0.22um microporous filter membrane; Be sub-packed in cillin bottle, every bottled amount 1ml, under aseptic condition, encapsulated in frozen drying 24-36 hour.
Embodiment 8: method of quality control (being suitable for the quality control of the said preparation raw material of the foregoing description 1-7 in this method)
1 red ginseng saponin detects: the high effective liquid chromatography for measuring ginsenoside Rg
1, Re, Rb
1
Chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filler, are mobile phase A with the acetonitrile, are Mobile phase B with water, and the regulation in the according to the form below is carried out gradient elution; Flow velocity: 1.0mL/min; Sample size: 20 μ L; Evaporative light scattering detector detects, 110 ℃ of drift tube temperatures, and gas flow rate 2.9L/min does not shunt; Column temperature: 25 ℃.
It is an amount of that the preparation precision of need testing solution takes by weighing formulation samples, adds methanol and process the solution that every 1ml contains red ginseng saponin 4.0mg, promptly gets.
The preparation precision of reference substance solution takes by weighing the ginsenoside Rg through drying under reduced pressure to constant weight
1, ginsenoside Re, ginsenoside Rb
1Reference substance is an amount of, adds methanol and processes the mixed solution that contains 0.64mg, 0.24mg, 0.80mg among every 1ml respectively, shakes up, and promptly gets.
Accurate respectively reference substance solution 10ul, 20ul and the need testing solution 20ul of drawing of algoscopy injects chromatograph of liquid, measures, and writes down 80 minutes collection of illustrative plates, calculates with external standard two-point method logarithmic equation, promptly gets.
Contain the ginsenoside Rg in these article Radix Ginseng Rubra saponin
1(C
42H
72O
14) must not be less than 10.0%, ginsenoside Re (C
48H
82O
18) must not be less than 3.6%, ginsenoside Rb
1(C
54H
92O
23) must not be less than 12.6%.
2 ophiopogonins detect: high effective liquid chromatography for measuring ophiopogonin D, ophiopogonin D '
Chromatographic condition and system suitability test are filler (first-selected COSMOSIL 5u-MS-II post) with the octadecylsilane chemically bonded silica, column temperature: 25 ℃; With acetonitrile-isopropyl alcohol-water (160: 40: 245) is mobile phase A, is Mobile phase B with acetonitrile-water (45: 55), and the regulation in the according to the form below is carried out gradient elution; Evaporative light scattering detection ((gas flow rate 2.5L/min does not shunt for preferred Alltech ELSD2000ES, 95.2 ℃ of drift tube temperatures); Column temperature: 25 ℃, theoretical cam curve is calculated by the ophiopogonin D peak should be not less than 6000.
It is an amount of that the preparation precision of need testing solution takes by weighing formulation samples, adds methanol and be mixed with the solution that every 1ml contains the 2.0mg radix ophiopogonis saponin extract, promptly gets.
Ophiopogonin D is got in the preparation of reference substance solution, ophiopogonin D ' reference substance is an amount of, adds methanol and processes solution that every 1ml contains 0.3mg respectively as object of reference solution.
Accurate respectively reference substance solution 10ul, 20ul and the need testing solution 20ul of drawing of algoscopy injects chromatograph of liquid, measures, and writes down 60 minutes collection of illustrative plates, calculates with external standard two-point method logarithmic equation, promptly gets.
Contain in these article ophiopogonin that ophiopogonin D should be not less than 6.0%, ophiopogonin D ' should be not less than 6.0%.
3, methyl Methylophiopogonanone A, B
[assay] methyl Methylophiopogonanone A, B are according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test: with the octadecylsilane chemically bonded silica is filler; With acetonitrile-methanol-0.2% glacial acetic acid aqueous solution (54: 6: 40) is mobile phase; UV-detector detects wavelength 296nm, and theoretical cam curve is calculated by methyl flavanone Radix Ophiopogonis A peak should be not less than 6000.
The preparation of reference substance solution: methyl Methylophiopogonanone A, B reference substance that precision takes by weighing through 60 ℃ of drying under reduced pressure to constant weights are an amount of, add methanol and process the solution that every 1ml contains methyl Methylophiopogonanone A, each 0.1mg of B.
The preparation of need testing solution: it is an amount of that precision takes by weighing formulation samples, adds methanol and be mixed with the solution that every 1ml contains the 0.25mg flavone extract, promptly gets
Algoscopy: accurate respectively reference substance solution and the need testing solution 20 μ l of drawing, inject chromatograph of liquid, measure, calculate content with external standard method, promptly get.
Contain the methyl Methylophiopogonanone A in these article flavone extract, the B total content should be not less than 80%.
Embodiment 9:
Red ginseng saponin 10g ophiopogonin 3g ophiopogonone 0.3g, mannitol 100g, water for injection 1000ml
Preparation technology:
Get recipe quantity red ginseng saponin, ophiopogonin, ophiopogonone and under aseptic condition, add in the 300-500ml water for injection, add 10%NaOH solution adjust pH, make abundant dissolving to 7-8; Add 0.02% activated carbon adsorption and remove thermal source; Add injection and be diluted with water to 1000ml, filter, be sub-packed in cillin bottle with the 0.22um microporous filter membrane; Every bottled amount 1ml, under aseptic condition, encapsulated in frozen drying 24-36 hour.
Method of quality control to raw material:
Red ginseng saponin detects: the high effective liquid chromatography for measuring ginsenoside Rg
1, Re, Rb
1
Chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filler, are mobile phase A with the acetonitrile, are Mobile phase B with water, and the regulation in the according to the form below is carried out gradient elution; Flow velocity: 1.0mL/min; Sample size: 20 μ L; Evaporative light scattering detector detects, 110 ℃ of drift tube temperatures, and gas flow rate 2.9L/min does not shunt; Column temperature: 25 ℃.
It is an amount of that the preparation precision of need testing solution takes by weighing formulation samples, adds methanol and process the solution that every 1ml contains red ginseng saponin 4.0mg, promptly gets.
The preparation precision of reference substance solution takes by weighing the ginsenoside Rg through drying under reduced pressure to constant weight
1, ginsenoside Re, ginsenoside Rb
1Reference substance is an amount of, adds methanol and processes the mixed solution that contains 0.64mg, 0.24mg, 0.80mg among every 1ml respectively, shakes up, and promptly gets.
Accurate respectively reference substance solution 10ul, 20ul and the need testing solution 20ul of drawing of algoscopy injects chromatograph of liquid, measures, and writes down 80 minutes collection of illustrative plates, calculates with external standard two-point method logarithmic equation, promptly gets.
These article Radix Ginseng Rubra saponin contains the ginsenoside Rg
1(C
42H
72O
14) must not be less than 10.0%, ginsenoside Re (C
48H
82O
18) must not be less than 3.6%, ginsenoside Rb
1(C
54H
92O
23) must not be less than 12.6%.
Embodiment 10:
Red ginseng saponin 10g ophiopogonin 3g ophiopogonone 0.3g, mannitol 100g, water for injection 1000ml
Preparation technology:
Get recipe quantity red ginseng saponin, ophiopogonin, ophiopogonone and under aseptic condition, add in the 300-500ml water for injection, add 10%NaOH solution adjust pH, make abundant dissolving to 7-8; Add 0.02% activated carbon adsorption and remove thermal source; Add injection and be diluted with water to 1000ml, filter, be sub-packed in cillin bottle with the 0.22um microporous filter membrane; Every bottled amount 1ml, under aseptic condition, encapsulated in frozen drying 24-36 hour.
Method of quality control to raw material:
1, red ginseng saponin detects: the high effective liquid chromatography for measuring ginsenoside Rg
1, Re, Rb
1
Chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filler, are mobile phase A with the acetonitrile, are Mobile phase B with water, and the regulation in the according to the form below is carried out gradient elution; Flow velocity: 1.0mL/min; Sample size: 20 μ L; Evaporative light scattering detector detects, 110 ℃ of drift tube temperatures, and gas flow rate 2.9L/min does not shunt; Column temperature: 25 ℃.
It is an amount of that the preparation precision of need testing solution takes by weighing these article, adds methanol and process the solution that every 1ml contains these article 4.0mg, promptly gets.
The preparation precision of reference substance solution takes by weighing the ginsenoside Rg through drying under reduced pressure to constant weight
1, ginsenoside Re, ginsenoside Rb
1Reference substance is an amount of, adds methanol and processes the mixed solution that contains 0.64mg, 0.24mg, 0.80mg among every 1ml respectively, shakes up, and promptly gets.
The preparation precision of need testing solution takes by weighing these article 30mg, puts in the 10ml volumetric flask, adds dissolve with methanol and is diluted to scale, shakes all, filters, and gets subsequent filtrate, promptly gets.
Accurate respectively reference substance solution 10ul, 20ul and the need testing solution 20ul of drawing of algoscopy injects chromatograph of liquid, measures, and writes down 80 minutes collection of illustrative plates, calculates with external standard two-point method logarithmic equation, promptly gets.
These article Radix Ginseng Rubra saponin contains the ginsenoside Rg
1(C
42H
72O
14) must not be less than 10.0%, ginsenoside Re (C
48H
82O
18) must not be less than 3.6%, ginsenoside Rb
1(C
54H
92O
23) must not be less than 12.6%.
2 ophiopogonins detect: high effective liquid chromatography for measuring ophiopogonin D, ophiopogonin D '
Chromatographic condition and system suitability test are filler (first-selected COSMOSIL 5u-MS-II post) with the octadecylsilane chemically bonded silica, column temperature: 25 ℃; With acetonitrile-isopropyl alcohol-water (160: 40: 245) is mobile phase A, is Mobile phase B with acetonitrile-water (45: 55), and the regulation in the according to the form below is carried out gradient elution; Evaporative light scattering detection ((gas flow rate 2.5L/min does not shunt for preferred Alltech ELSD2000ES, 95.2 ℃ of drift tube temperatures); Column temperature: 25 ℃, theoretical cam curve is calculated by the ophiopogonin D peak should be not less than 6000.
It is an amount of that the preparation precision of need testing solution takes by weighing formulation samples, adds methanol and be mixed with the solution that every 1ml contains the 2.0mg radix ophiopogonis saponin extract, promptly gets.
Ophiopogonin D is got in the preparation of reference substance solution, ophiopogonin D ' reference substance is an amount of, adds methanol and processes solution that every 1ml contains 0.3mg respectively as object of reference solution.
Accurate respectively reference substance solution 10ul, 20ul and the need testing solution 20ul of drawing of algoscopy injects chromatograph of liquid, measures, and writes down 60 minutes collection of illustrative plates, calculates with external standard two-point method logarithmic equation, promptly gets.
These article ophiopogonin contains that ophiopogonin D should be not less than 6.0%, ophiopogonin D ' should be not less than 6.0%.
Claims (8)
1. compositions that is used to treat cardiovascular disease is characterized in that the crude drug of this pharmaceutical composition consists of:
Red ginseng saponin 2-20 weight portion ophiopogonin 0.2-3 weight portion
Ophiopogonone 0.02-3 weight portion;
Wherein red ginseng saponin extract is pressed dry product calculating, contains the ginsenoside Rg
1Must not be less than 9.0%, the ginsenoside Re must not be less than 3.0%, ginsenoside Rb
1Must not be less than 12.0%, Radix Ginseng total saponins content must not be less than 80%, and the method for preparing of above-mentioned red ginseng saponin extract is:
Extract: Radix Ginseng Rubra is made solvent with ethanol, adopt percolation to extract or the thermal cycle reflux, extract,, collect extracting solution to red ginseng saponin and extract fully;
Water precipitating: extracting solution is evaporated to 0.8-1.5 times of crude drug amount, puts coldly, and thin up is to 1-3 times of crude drug amount, cold preservation, filter Radix Ginseng Rubra water liquid;
Extraction: Radix Ginseng Rubra water liquid adds natrium carbonicum calcinatum stirring and dissolving adjust pH 9-10, uses n-butanol extraction, and is abundant to the red ginseng saponin extraction;
Column chromatography absorption: collect butanol extraction liquid, reclaim n-butyl alcohol and evaporate to dryness, add water and fully dissolve, add natrium carbonicum calcinatum stirring and dissolving adjust pH 9～10, macroporous adsorptive resins absorption on the medicinal liquid;
The column chromatography eluting: first water is washed till eluent for neutral, and reuse 60-90% ethanol elution is complete to the red ginseng saponin eluting, collects ethanol elution;
Refining: merge ethanol elution, reclaim ethanol and be concentrated into 1 times of crude drug amount, add the decolouring of 0.01% active carbon stirring at normal temperature, evaporate to dryness gets dry extract; Pulverize, add anhydrous alcohol solution under the room temperature, filter, reclaim ethanol with the 0.45um microporous filter membrane; Evaporate to dryness gets dry extract, and pulverizes, and promptly gets red ginseng saponin extract;
Wherein radix ophiopogonis saponin extract is pressed dry product calculating, and contain ophiopogonin D and must not be less than 6.0%, ophiopogonin D ' must not be less than 6.0%, Radix Ophiopogonis total saponins content must not be less than 80%, and the method for preparing of above-mentioned radix ophiopogonis saponin extract is:
Extract: will make solvent with ethanol Radix Ophiopogonis, and adopt percolation to extract or the thermal cycle reflux, extract,, and collect extracting solution to ophiopogonin, ophiopogonone extraction fully;
Water precipitating: extracting solution is evaporated to 1 times of crude drug amount, puts coldly, and thin up is to 1.5-2.5 times of crude drug amount, cold preservation, filter Radix Ophiopogonis water liquid;
The flavone part: collecting precipitation fully dissolves with petroleum ether heating extraction to ophiopogonone, gets ophiopogonone deposition part, and Radix Ophiopogonis, water liquid was abundant that ophiopogonone extracts part with the extraction of petroleum ether extraction to ophiopogonone, and merging two parts promptly get the ophiopogonone bullion;
Extraction: Radix Ophiopogonis, water liquid added natrium carbonicum calcinatum stirring and dissolving adjust pH 9～10, used n-butanol extraction, and is abundant to the ophiopogonin extraction;
Column chromatography absorption: collect butanol extraction liquid, reclaim n-butyl alcohol and evaporate to dryness, add water and fully dissolve, add natrium carbonicum calcinatum stirring and dissolving adjust pH 9～10, macroporous adsorptive resins absorption on the medicinal liquid;
The column chromatography eluting: first water is washed till eluent for neutral, and reuse 60-90% ethanol elution is complete to the ophiopogonin eluting, collects ethanol elution;
Refining: merge ethanol elution, reclaim ethanol and be concentrated into 1 times of crude drug amount, add the decolouring of 0.01% active carbon stirring at normal temperature, evaporate to dryness gets dry extract; Pulverize, add anhydrous alcohol solution under the room temperature, filter, reclaim ethanol with the 0.45um microporous filter membrane; Evaporate to dryness gets dry extract, and pulverizes, and promptly gets radix ophiopogonis saponin extract;
Wherein the ophiopogonone extract calculate by dry product contain the methyl Methylophiopogonanone A, the B total content must not be less than 80%, above-mentioned ophiopogonone preparation method of extract is:
Extract: will make solvent with ethanol Radix Ophiopogonis, and adopt percolation to extract or the thermal cycle reflux, extract,, and collect extracting solution to ophiopogonin, ophiopogonone extraction fully;
Water precipitating: extracting solution is evaporated to 1 times of crude drug amount, puts coldly, and thin up is to 1.5-2.5 times of crude drug amount, cold preservation, filter Radix Ophiopogonis water liquid;
The flavone part: collecting precipitation fully dissolves with petroleum ether heating extraction to ophiopogonone, gets ophiopogonone deposition part, and Radix Ophiopogonis, water liquid was abundant that ophiopogonone extracts part with the extraction of petroleum ether extraction to ophiopogonone, and merging two parts promptly get the ophiopogonone bullion;
Get above-mentioned ophiopogonone bullion and add the silica gel mixed sample that 2-4 doubly measures, be splined on silica gel column chromatography, add the remove impurity of petroleum ether eluting earlier; Reuse petroleum ether: ethyl acetate=95: 5-90: 10 eluting, it is abundant to be collected in Radix Ophiopogonis the flavone component eluting, merges main flavone eluting stream part; 40-60 ℃ of decompression and solvent recovery, a small amount of petroleum ether sample, 30-50 ℃ of vacuum drying; Pulverize dry powder, promptly get the ophiopogonone extract.
2. compositions as claimed in claim 1 is characterized in that the crude drug of this pharmaceutical composition consists of:
Red ginseng saponin 5-10 weight portion ophiopogonin 0.5-1 weight portion
Ophiopogonone 0.1-1 weight portion.
3. compositions as claimed in claim 1 is characterized in that the crude drug of this pharmaceutical composition consists of:
Red ginseng saponin 12-18 weight portion ophiopogonin 0.5-1 weight portion
Ophiopogonone 2.2-3 weight portion.
4. compositions as claimed in claim 1 is characterized in that the crude drug of this pharmaceutical composition consists of:
Red ginseng saponin 5-10 weight portion ophiopogonin 1.5-3 weight portion
Ophiopogonone 0.2-1 weight portion.
5. compositions as claimed in claim 1 is characterized in that the crude drug of this pharmaceutical composition consists of: red ginseng saponin: ophiopogonin: ophiopogonone=10 weight portions: 1 weight portion: 0.1 weight portion.
6. compositions as claimed in claim 1 is characterized in that the crude drug of this pharmaceutical composition consists of:
Red ginseng saponin: ophiopogonin: ophiopogonone=10 weight portions: 3 weight portions: 0.3 weight portion.
7. compositions that is used to treat cardiovascular disease is characterized in that the crude drug of this pharmaceutical composition consists of:
Ophiopogonin 0.2-3 weight portion ophiopogonone 0.2-3 weight portion;
Wherein radix ophiopogonis saponin extract is pressed dry product calculating; Contain ophiopogonin D and must not be less than 6.0%; Ophiopogonin D ' must not be less than 6.0% also contains other main six kinds of ophiopogonin constituents except that containing above-mentioned ophiopogonin D, D ' composition, Radix Ophiopogonis total saponins content must not be less than 80%;
Wherein the ophiopogonone extract calculate by dry product contain the methyl Methylophiopogonanone A, the B total content must not be less than 80%; Wherein radix ophiopogonis saponin extract is pressed dry product calculating, and contain ophiopogonin D and must not be less than 6.0%, ophiopogonin D ' must not be less than 6.0%, Radix Ophiopogonis total saponins content must not be less than 80%, and the method for preparing of above-mentioned radix ophiopogonis saponin extract is:
Extract: will make solvent with ethanol Radix Ophiopogonis, and adopt percolation to extract or the thermal cycle reflux, extract,, and collect extracting solution to ophiopogonin, ophiopogonone extraction fully;
Water precipitating: extracting solution is evaporated to 1 times of crude drug amount, puts coldly, and thin up is to 1.5-2.5 times of crude drug amount, cold preservation, filter Radix Ophiopogonis water liquid;
The flavone part: collecting precipitation fully dissolves with petroleum ether heating extraction to ophiopogonone, gets ophiopogonone deposition part, and Radix Ophiopogonis, water liquid was abundant that ophiopogonone extracts part with the extraction of petroleum ether extraction to ophiopogonone, and merging two parts promptly get the ophiopogonone bullion;
Extraction: Radix Ophiopogonis, water liquid added natrium carbonicum calcinatum stirring and dissolving adjust pH 9～10, used n-butanol extraction, and is abundant to the ophiopogonin extraction;
Column chromatography absorption: collect butanol extraction liquid, reclaim n-butyl alcohol and evaporate to dryness, add water and fully dissolve, add natrium carbonicum calcinatum stirring and dissolving adjust pH 9～10, macroporous adsorptive resins absorption on the medicinal liquid;
The column chromatography eluting: first water is washed till eluent for neutral, and reuse 60-90% ethanol elution is complete to the ophiopogonin eluting, collects ethanol elution;
Refining: merge ethanol elution, reclaim ethanol and be concentrated into 1 times of crude drug amount, add the decolouring of 0.01% active carbon stirring at normal temperature, evaporate to dryness gets dry extract; Pulverize, add anhydrous alcohol solution under the room temperature, filter, reclaim ethanol with the 0.45um microporous filter membrane; Evaporate to dryness gets dry extract, and pulverizes, and promptly gets radix ophiopogonis saponin extract;
Wherein the ophiopogonone extract calculate by dry product contain the methyl Methylophiopogonanone A, the B total content must not be less than 80%, above-mentioned ophiopogonone preparation method of extract is:
Extract: will make solvent with ethanol Radix Ophiopogonis, and adopt percolation to extract or the thermal cycle reflux, extract,, and collect extracting solution to ophiopogonin, ophiopogonone extraction fully;
Water precipitating: extracting solution is evaporated to 1 times of crude drug amount, puts coldly, and thin up is to 1.5-2.5 times of crude drug amount, cold preservation, filter Radix Ophiopogonis water liquid;
The flavone part: collecting precipitation fully dissolves with petroleum ether heating extraction to ophiopogonone, gets ophiopogonone deposition part, and Radix Ophiopogonis, water liquid was abundant that ophiopogonone extracts part with the extraction of petroleum ether extraction to ophiopogonone, and merging two parts promptly get the ophiopogonone bullion;
Get above-mentioned ophiopogonone bullion and add the silica gel mixed sample that 2-4 doubly measures, be splined on silica gel column chromatography, add the remove impurity of petroleum ether eluting earlier; Reuse petroleum ether: ethyl acetate=95: 5-90: 10 eluting, it is abundant to be collected in Radix Ophiopogonis the flavone component eluting, merges main flavone eluting stream part; 40-60 ℃ of decompression and solvent recovery, a small amount of petroleum ether sample, 30-50 ℃ of vacuum drying; Pulverize dry powder, promptly get the ophiopogonone extract.
8. like the injection of any pharmaceutical composition among the claim 1-7, it is characterized in that the method for preparing of this injection is:
Add mannitol 80-200 weight portion water for injection 800-2000 parts by volume in the prescription; Method for preparing is: get the recipe quantity crude drug and under aseptic condition, add in the 300-500 parts by volume water for injection, add 8-12%NaOH solution adjust pH to 7-8, add recipe quantity mannitol and make abundant dissolving; Add injection and be diluted with water to 1000 parts by volume; Add the 0.01-0.06% activated carbon adsorption and remove thermal source, filter packing with microporous filter membrane; Frozen drying 24-36 hour, under aseptic condition, encapsulate.
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| CN101879274B (en) * | 2010-07-12 | 2012-10-17 | 哈尔滨吉尔生物科技有限公司 | Medicine composition containing red ginseng saponin extract and radix ophiopogonis saponin extract as well as preparation method and application thereof |
| CN102133333B (en) * | 2011-03-25 | 2013-06-12 | 浙江大学 | Quality control method for shenmai injection mass spectrum finger prints |
| CN102988399A (en) * | 2012-12-21 | 2013-03-27 | 周志欢 | Medicine composition and application and preparation thereof |
| CN103393708B (en) * | 2013-08-20 | 2015-06-03 | 大理药业股份有限公司 | Ginsenoside-ophiopogonin D composition for treating cardiovascular and cerebrovascular diseases |
| CN104623262B (en) * | 2013-11-12 | 2017-12-19 | 华润三九（雅安）药业有限公司 | A kind of Shenmai injection and preparation method thereof |
| 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 |
| CN107823420A (en) * | 2017-11-09 | 2018-03-23 | 华润三九（雅安）药业有限公司 | The preparation method and Shenmai injection of Shenmai injection |
| CN109709250B (en) * | 2018-12-28 | 2020-12-25 | 南京海昌中药集团有限公司 | Method for detecting fingerprint of ginseng and pilose antler wine |
| CN112294794B (en) * | 2019-08-01 | 2021-07-30 | 四川大学 | Application of methyl ophiopogonone A in preparation of medicine for preventing and/or treating non-alcoholic fatty liver disease and liver injury |
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| CN1927339A (en) * | 2005-09-08 | 2007-03-14 | 刘玉辉 | Complex traditional Chinese medicine preparation for treating myocardial ischemia and improving microcirculation and preparing method thereof |
| CN1943710A (en) * | 2005-10-03 | 2007-04-11 | 徐东铭 | The application of radix ophioponis in the medicines for treating myocarditis and medicines containing its extracts with active elements |
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| CN1927339A (en) * | 2005-09-08 | 2007-03-14 | 刘玉辉 | Complex traditional Chinese medicine preparation for treating myocardial ischemia and improving microcirculation and preparing method thereof |
| CN1943710A (en) * | 2005-10-03 | 2007-04-11 | 徐东铭 | The application of radix ophioponis in the medicines for treating myocarditis and medicines containing its extracts with active elements |
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