CN103977163A - Pharmaceutical composition with myocardial ischemia resisting effect and preparation method and application thereof - Google Patents
Pharmaceutical composition with myocardial ischemia resisting effect and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a traditional Chinese medicine substance composition and a myocardial ischemia resisting effect thereof. The traditional Chinese medicine substance composition disclosed by the invention is obtained by extracting heart tonifying soup with ethanol and purifying with macroporous adsorption resin, and phenol and triterpenoid saponin ingredients in the heart tonifying soup are enriched; meanwhile, proved by experiments on rats with acute myocardial ischemia, the traditional Chinese medicine drug substance composition has an obvious myocardial ischemia resisting effect, and after prophylactic administration, the content of serum creatine kinase and lactate dehydrogenase in blood serum of model rats can be lowered.
Description
Technical field
The invention belongs to drug development research field, relate to a kind of Chinese medicine combinations of substances, particularly a kind of Chinese medicine combinations of substances with function of resisting myocardial ischemia, with the method that can prepare this combination simultaneously.
Background technology
Myocardial ischemia, refers to the minimizing due to heart blood perfusion, causes the oxygen supply of heart to reduce, and energy metabolism of myocardial is undesired, can not support a kind of pathological state of the normal work of heart.Narrow and small or the solidifying plug of arteria coronaria that coronary atherosclerosis causes is to cause main, the modal cause of disease of myocardial ischemia, and then causes myocardial ischemia-anoxemia, causes thus " coronary heart disease " that everybody often says, so coronary heart disease is myocardial ischemia " arch-criminal ".The medicine resisting myocardial ischemia clinically has a lot, and mechanism of action is also all different.Chinese medicine can be by improving myocardial cell metabolism, reduce myocardial oxygen consumption, regulate NO/ET, suppress calcium overload, resisting oxidation free radical, improve myocardial ischemia causes the approach such as apoptosis and Angiogensis and plays the effect resisting myocardial ischemia, Chinese medicine is emphasized Overall View and determination for the treatment of based on pathogenesis obtained through differentiation of symptoms and signs, present the model of action of the many target spots of multipath, each mechanism of action is a process connecting each other, there is not the impact of single factors, therefore there is exploratory meaning in conjunction with the feature of myocardial ischemia pathogenesis and Chinese medicine to finding more effective new Chinese medicine future.
The present invention is former as side taking classical master bushing soup, exploration discovery a kind of Chinese medicine combinations of substances with the use of resisting myocardial ischemia, and taking the type composition of drug effect association and index composition as comprehensive consideration, set up reliable and stable, be applicable to preparation technology and scientific comprehensive method of quality control when actual production, be conducive to the research and development of further original new drug.At present, have no the relevant report of purification medicaments for resisting myocardial ischemia combinations of substances from reinforcing the heart soup, also have no about said composition is at the correlational study report aspect treatment myocardial ischemia disease.
Summary of the invention
The object of the present invention is to provide a kind of Chinese medicine combinations of substances with function of resisting myocardial ischemia, its drug system is mainly made up of phenols, triterpene saponin, and its side was the compositions such as Radix Ginseng, Radix Polygalae, Poria, Pollen Typhae originally.
Another object of the present invention is to provide its preparation method and technique, and the 3rd object of the present invention is to provide its quality testing and control method thereof.The 4th object of the present invention is to provide its protective effect to rats with myocardial ischemia.The 5th object of the present invention is to provide the goods of this Chinese medicine composition of matter and the application at medicine, field of food thereof.
The object of the invention is to realize by following approach:
Chinese medicine combinations of substances of the present invention can and substitute kind by following 4 groups of medicinal plants, comprises its medicinal part and dis-medicinal part, and medical material and decoction pieces combination thereof are made, and its drug system is mainly made up of phenols, triterpene saponin.
Group 1: panax japonicus c.a.mey.var.major (burk.) c.y.wu et k.m.feng, Panax pseudoginseng Wall., Panax zingiberensis C. Y. Wu. Et Feng．, Radix Ginseng, Radix Notoginseng, Radix Panacis Quinquefolii, narrow leaf Panax pseudoginseng Wall., beautiful Panax pseudoginseng Wall., Rhizoma Panacis bipinnatifidi, panax japonicus are equal to Araliaceae, and processed product.
Group 2: the rogation flowers such as Radix Polygalae, ovum leaf Radix Polygalae, caudal lobe Radix Polygalae, kidney fruit Radix Polygalae, Herba Polygalae Japonicae, hinge grass, largeleaf milkwort herb, Radix Crotalariae szemoensis, Radix Polygalae fallacis, Siberia Radix Polygalae, and Huang Yeshu, Radix securidacae, tooth fruit grass be equal to milk wort, and processed product.
Group 3: Poria, Polyporus, Coriolous Dersicolor (Fr.) Quel, Grifola frondosa, Laetiporus sulphureus (Bull. Ex Fr.) Bond. Et Singer. are equal to Polyporaceae plant, and processed product.
Group 4: Herba Typhae, Typha angustifolia, typha angustifolia, common cattail, hair wax candle, black cutter candle, Typha angustifolia Herba Typhae, typha orientalis are equal to Typhaceae plant, and processed product.
Chinese medicine combinations of substances of the present invention, can be after above-mentioned raw materials medicine combination with ethanol or other alcohols, rare alcohol or other organic solvents or water extraction again by macroporous adsorbent resin or other chromatographic processes, as polyamide chromatography etc., or the purification such as solvent extraction obtains.
Chinese medicine combinations of substances of the present invention can be by the extract of above-mentioned raw materials medicine after further enriching and purifying or the macroporous resin prepared product of above-mentioned raw materials medicine mix.
Chinese medicine combinations of substances of the present invention can be through chemosynthesis or structural modification, and biosynthesis or biology the approach such as turn and obtain.
Chinese medicine combination of the present invention is preferably made up of following crude drug:
Radix Ginseng 100-300 weight portion Poria 100-300 weight portion
Radix Polygalae 100-200 weight portion Pollen Typhae 100-200 weight portion.
The preparation method of Chinese medicine combinations of substances of the present invention comprises the steps:
Step l: choose above-mentioned raw materials medicine;
Step 2: ethanol extraction;
Step 3: purification with macroreticular resin;
This Chinese medicine combinations of substances is made up of two parts, Part I be yellowish-brown to yellowish-brown powder, slightly delicate fragrance of gas, bitter in the mouth.Wherein total phenol content is 10%-90%, and 3,6 '-bis-mustard acyl content are 1%-10%; typhaneoside content is 0.5%-5%, and preferably total phenol content is 40%-80%, preferably 3; 6 '-bis-mustard acyl content are 2%-10%, and preferably typhaneoside content is 0.8%-5%.Therefore be called phenols drug substance because Part I phenols occupies chief component; Part II is yellowish-brown powder, and tool special aroma is lightly seasoned, is called terpenoid drug substance.Wherein ginsenoside Rb
1content is 2%-10%.
In above-mentioned steps 2, crude drug is doubly measured with 8-12,0-70% alcohol reflux 1-3 time extracts 1-2 hour at every turn.Merge extracted twice liquid concentrating under reduced pressure, to the clear paste that relative density is 1.15-1.20g/ml, add aqueous dispersion, making sample solution concentration is 0.01-0.1g/mL.
In above-mentioned steps 3, sample solution is by macroporous adsorbent resin, and resin blade diameter length ratio is l: 4-1: 10, sample solution concentration is 0.01-0.1g/mL, applied sample amount is counted 0.2g/ml-0.5g/ml with full side's raw medicinal herbs, and absorption flow velocity is 1-4BV/h, and water elution 1-3 times of resin volume carries out remove impurity, first with 0-8 times of resin volume of 40-70% ethanol elution, elution flow rate is 2-6BV/h, collects ethanol elution, reclaims solvent, drying under reduced pressure, obtains phenols drug substance; With 0-13 times of resin volume of 70-95% ethanol elution, elution flow rate is 2-6BV/h again, collects ethanol elution, reclaims solvent, and drying under reduced pressure, obtains terpenoid drug substance.
In above-mentioned steps 3, big pore adsorption resin is preferably AB-8, DM130, low pole or the non-polar macroporous resin such as HPD-100 type.
Chinese medicine combinations of substances of the present invention, is made up of 2 parts.The summation of aldehydes matter percentage composition reaches 50%-80%.In terpenoid drug substance, contain ginsenoside Rb
1deng triterpenoid saponins and other types composition, terpenoid percentage composition summation is 50%-80%.
Above gained two parts Chinese medicine material is carried out to compatible combination, add conventional adjuvant, preparation process is routinely made the acceptable any conventional dosage form of pharmaceutics, comprises capsule, tablet, granule, gel, slow releasing agent, oral liquid etc.
Chinese medicine combinations of substances of the present invention is obtained through ethanol extraction, purification with macroreticular resin by reinforcing the heart soup, enrichment phenols component and the ter penoids in reinforcing the heart soup, pass through Acute Myocardial Ischemia in Rats experimental verification simultaneously, there is the effect obviously resisting myocardial ischemia.After preventive administration, can reduce the content of serum creatine kinase and lactic acid dehydrogenase in rat model serum.
Experimental example 1 the present invention causes the therapeutical effect of Acute Myocardial Ischemia in Rats to sublingual vein injection pituitrin.
1 experiment material
1.1 equipment
UV-2000 ultraviolet spectrophotometer (Shanghai You Nike company),
KQ-500DE type ultrasonic cleaner (Kunshan ultrasonic instrument company limited),
QL-901 vortex mixed machine (its woods Bel instrument manufacturing company).
1.2 reagent and medicine
Injection pituitrin (Nanjing Xinbai Pharmaceutical Co),
FUFANG DANSHEN DIWAN (lot number: 131008; Tasly Pharmaceutical Group Co., Ltd.),
Creatine kinase (CK) test kit and lactic acid dehydrogenase (LDH) test kit all build up Bioengineering Research Institute's Chinese medicine composition of matter purchased from Nanjing: combined in paste-forming rate ratio by phenols drug substance and terpenoid drug substance.
2 experimental techniques
Dosage and the time of 2.1 groupings and administration
SD rat is divided into 6 groups at random by body weight, be respectively: Normal group, model control group, Composite Salvia Dropping Pill group, reinforcing the heart soup extract group, reinforcing the heart soup ethanol extract group (extracting by extraction process), reinforcing the heart soup enriched substance group (by preparation technology's preparation, and two positions being merged in proportion).Three groups of reinforcing the heart soup are all converted into rat dosage by crude drug amount 3g/kg gastric infusion, 10 every group, administration every day 1 time, successive administration 3 days.
The variation of Biochemical Index in 2.2 serum
After the administration of each administration group rat last, lumbar injection 10% chloral hydrate anesthesia, lies on the back and is fixed on operating-table, and every sublingual vein is to pituitrin 0.6U/mL.Abdominal aortic blood, separation of serum, the content of mensuration serum creatine kinase (CK), lactic acid dehydrogenase (LDH) are injected after 20min.
3 results
In each group rat blood serum, the measurement result of CK, LDH is in table 1.By CK, LDH content in the visible model control group rat blood serum of table 1 far away higher than each administration group and rats in normal control group (P < 0.01).Reinforcing the heart soup ethanol extract group and reinforcing the heart soup enriched substance group CK, LDH content are well below reinforcing the heart soup extract group (P < 0.01), but there was no significant difference between reinforcing the heart soup ethanol extract group and enriched substance group.
The variation of table 1 rats with myocardial ischemia serum creatine kinase (CK) and lactic acid dehydrogenase (LDH) content
Note: compare with model control group: * P < 0.01; Compare with reinforcing the heart soup extract group:
#p < 0.01,
▲p < 0.05
The preparation of the prepared product that contains the present invention's proposition can be according to method preparation well known in the art.Prepared product and one or more solids that the present invention can be proposed or liquid medicine excipient and/or adjuvant are combined, and make the suitable administration form or the dosage form that can be used as people's medicine or veterinary drug use.
Contain the preparation of prepared product thing that the present invention proposes, can unit dosage form administration, route of administration can be intestinal or non-intestinal, as oral, muscle, nasal cavity, oral mucosa, skin, transdermal, subcutaneous, Intradermal, peritoneum, rectum etc.Form of administration can be liquid dosage form, solid dosage forms, if liquid dosage form can be true solution dosage form, colloid solution dosage form, particulate formulations, emulsion dosage form, mixed suspension form.Liquid dosage form form can be syrup, medicated wine, injection solution, non-aqueous solution, suspension or emulsion etc.; Solid dosage forms is tablet, lozenge, capsule, drop pill, pill, granule, powder, cream, suppository, powder, unguentum etc. such as.
The preparation of the prepared product that contains the present invention's proposition, can be ordinary preparation, can be also slow releasing preparation, controlled release preparation, targeting preparation and various particulate delivery systems etc.
For unit form of administration is made to tablet, can be widely used various carrier well known in the art.Example about carrier comprises, excipient is as calcium carbonate, lactose, calcium phosphate, sodium phosphate; Diluent and absorbent are as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, carbamide, calcium carbonate, kaolin, microcrystalline Cellulose, aluminium silicate, glucosan, colloidal silica, arabic gum, gelatin, magnesium trisilicate, keratin etc.; Wetting agent and binding agent are as water, glycerol, Polyethylene Glycol, ethanol, propanol, starch slurry, dextrin, syrup, Mel, glucose solution, mucialga of arabic gummy, gelatine size, sodium carboxymethyl cellulose, lac, methylcellulose, potassium phosphate, polyvinylpyrrolidone etc.; Disintegrating agent is as dry starch, sodium alginate, agar powder, laminaran, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, dodecyl sodium sulfate, methylcellulose, ethyl cellulose etc.; Disintegrate inhibitor is as sucrose, glyceryl tristearate, cocoa ester, hydrogenated vegetable wet goods; Absorption enhancer is as quaternary ammonium salt, sodium lauryl sulphate etc.; Lubricant is as Pulvis Talci, triethylamine magnesium stearate, triethylamine stearic acid, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, Polyethylene Glycol etc.Tablet further can also be made to coated tablet, as sugar coated tablet, thin membrane coated tablet, ECT, or double-layer tablet and multilayer tablet etc.
For unit form of administration is made to pill, can be widely used various carrier well known in the art.Example about carrier is that for example diluent and absorbent, as glucose, lactose, starch, cocoa ester, hydrogenated vegetable oil, polyvinylpyrrolidone, Kaolin, Pulvis Talci etc.; Binding agent is as arabic gum, tragacanth gum, gelatin, ethanol, Mel, rice paste or batter etc.; Disintegrating agent is as agar powder, dry starch, sodium alginate, dodecyl sodium sulfate, methylcellulose, ethyl cellulose etc.
For unit form of administration is made to capsule, the prepared product that the present invention can be proposed mixes with above-mentioned various carriers, and the mixture obtaining is thus placed in to hard gelatin capsule or soft capsule.The prepared product that also the present invention can be proposed is made microcapsule, is suspended in aqueous medium and forms suspensoid, also can pack in hard capsule or make injection application.
For unit form of administration is made to oral liquid, as emulsion, solution, suspension, syrup etc., optional additives is as coloring agent, antiseptic, emulsifying agent, suspending agent, correctives (as Herba Menthae, Ilicis Purpureae wet goods), sweeting agent (as sucrose, lactose etc.) or other materials as required.
For unit form of administration being made to the moisture or non-water formulation of injection, as solution, suspension type solution, Emulsion, lyophilized injectable powder, can be containing acceptable carrier on a kind of and/or multiple pharmacodynamics, as diluent, wetting agent, emulsifying agent, lubricant, antiseptic, surfactant or dispersant, and conventional cosolvent, buffer agent, pH adjusting agent etc.The isooctadecanol of the optional plain boiled water of diluent, ethanol, Polyethylene Glycol, 1,3-PD, ethoxylation, the isooctadecanol of polyoxy, vegetable oil (as olive oil, Semen Maydis wet goods), gelatin, organic ester (as ethyl oleate, fatty acid ester etc.), polyoxyethylene sorbitol etc. for injectable.To ooze injection in order preparing etc., can also to add appropriate sodium chloride, glucose or glycerol.
Following embodiment all can realize the effect of above-mentioned experimental example.
Detailed description of the invention
Embodiment 1: the Chinese medicine combinations of substances that resists myocardial ischemia preparation technology
Radix Ginseng 300g Poria 300g
Radix Polygalae 200g Pollen Typhae 200g
Get according to the above ratio crude drug decoction pieces 1kg, measure 50% alcohol reflux 3 times with 6 times, each extraction 1.5 hours, merge extracted twice liquid (20L altogether) concentrating under reduced pressure, to the relative density clear paste that is 1.15-1.20g/ml, add aqueous dispersion, making sample solution concentration is 0.05g/mL (to roll over raw medicinal herbs amount), sample solution is adsorbed by DM130 type macroporous adsorbent resin, applied sample amount and resin volume ratio are 0.25g: 1mL, resin column blade diameter length ratio is 1: 8.7, and absorption flow velocity is 1bv/h; After absorption, with the remove impurity of 2 times of resinite hydrops eluting, washing flow velocity is 2bv/h; After washing remove impurity, with 4 times of resin volumes of 40% ethanol elution, elution flow rate is 2bv/h, collects 40% ethanol elution, reclaims solvent, with 3 times of resin volumes of 90% ethanol elution, elution flow rate is 2bv/h, collects 90% ethanol elution, reclaims solvent, vacuum drying, obtains reinforcing the heart soup phenols active substance and terpenoid active substance based on drug system.
Total phenol content assay method:
The preparation of reference substance solution: precision takes 3,6`-, bis-mustard acyl control sucrose product 2.15mg, with 70% methanol solution ultrasonic dissolution, is settled to 10mL graduation mark, shakes up, and obtaining concentration is 3 of 0.2150mg/mL, 6`-bis-mustard acyl control sucrose product solution;
Reagent: 0.6% liquor ferri trichloridi, 0.9% potassium ferricyanide solution, 0.3% sodium dodecyl sulfate solution, 50% methanol solution, 0.1mol/L hydrochloric acid solution;
Coloration method: precision pipettes sample solution and is placed in right amount the dark brown volumetric flask of 25ml, add 50% methanol to 5mL, add again 0.3% sodium dodecyl sulfate solution 2mL, shake up, add 0.6% ferric chloride-0.9% potassium ferricyanide new system mixed solution 1.0mL of 1: 1,5min is placed in dark place, add to 25mL graduation mark with 0.1mol/L hydrochloric acid, after shaking up, leave standstill 30min in dark place, and accompany and do blank, in order to measuring;
Assay method: sample after above-mentioned colour developing is measured in 770 ± 5nm wave-length coverage to absorbance, phenols drug substance is pressed dry product and calculated, and with 3,6`-, bis-mustard acyl sucrose meters, is no less than 40.0% containing total phenols.
Terpenoid content assaying method:
The preparation of reference substance solution: precision takes ginsenoside Rb
1reference substance 3.00mg, with 70% methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and obtaining concentration is ginsenoside Rb1's reference substance solution of 0.1200mg/mL;
Reagent: 5% vanillin-glacial acetic acid solution, perchloric acid solution, glacial acetic acid solution;
Coloration method: accurate absorption reinforcing the heart decoction extract sample liquid 1mL5 part, is placed in respectively cillin bottle, after boiling water bath evaporate to dryness, be cooled to room temperature, respectively add 5% vanillin-glacial acetic acid solution (developer) 0.2mL,, then add perchloric acid 0.8mL, shake up, in 60 DEG C of heating in water bath 15min, taking-up is put in cold water cooling, adds glacial acetic acid 5mL, blank is done in retinue, in order to measuring;
Assay method: sample after above-mentioned colour developing is measured in 560 ± 5nm wave-length coverage to absorbance, terpenoid drug substance is pressed dry product and calculated, contains terpenoid with ginsenoside Rb
1meter, is no less than 50.0%.
Index composition 3,6`-bis-mustard acyl sucrose, typhaneoside content assaying method simultaneously
The preparation of reference substance solution: precision takes 3,6 ' two mustard acyl control sucrose product 3.09mg, use methanol solution ultrasonic dissolution, be settled to 25mL graduation mark, shake up, the accurate 2mL that draws adds 3 times of methanol dilutions, shakes up, obtaining concentration is 3,6 '-bis-mustard acyl control sucrose product solution of 0.0412mg/mL; Precision takes typhaneoside reference substance 3.09mg, uses methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and the accurate 2mL that draws adds 18 times of methanol dilutions, shakes up, and obtaining concentration is the typhaneoside reference substance solution of 0.006867mg/mL;
Chromatographic condition: taking octadecylsilane chemically bonded silica as filler; Taking acetonitrile as mobile phase (A), taking 0.1% formic acid water as mobile phase (B), according to the form below, regulation is carried out gradient elution; Detection wavelength is 330nm; 30 DEG C of column temperatures, flow velocity is 1.0ml/min;
The preparation of standard curve: accurate absorption 3 respectively; 6`-bis-mustard acyl control sucrose product solution 3.0,6.0,9.0,12.0,15.0,18.0 μ L; typhaneoside reference substance solution 10,20,30,40,50,60,70 μ L injection liquid chromatographies; by above-mentioned chromatographic condition; measure 3,6`-, bis-mustard acyl sucrose, typhaneoside chromatographic peak peak area.Taking reference substance sample size as abscissa, taking chromatographic peak peak area as vertical coordinate drawing standard curve, and carry out respectively linear regression, obtain regression equation and be:
The preparation of need testing solution: precision takes reinforcing the heart decoction extract 60.0mg after 70% methanol ultrasonic dissolution,, shakes up by 70% methanol constant volume to 10mL volumetric flask, and 0.45 μ m filtering with microporous membrane, obtains reinforcing the heart decoction extract need testing solution.
Index composition ginsenoside Rb
1content assaying method
The preparation of reference substance solution: precision takes ginsenoside Rb1's reference substance 3.10mg, with 70% methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and obtaining concentration is the ginsenoside Rb of 0.1240mg/mL
1reference substance solution;
Chromatographic condition: taking octadecylsilane chemically bonded silica as filler; Taking acetonitrile as mobile phase (A), taking 0.1% formic acid water as mobile phase (B), according to the form below, regulation is carried out gradient elution; Detection wavelength is 203nm; 30 DEG C of column temperatures, flow velocity is 1.0ml/min;
The preparation of standard curve: the accurate ginsenoside Rb that draws respectively
1reference substance solution 10,20,30,40,50,60,70 μ L injection liquid chromatographies, by above-mentioned chromatographic condition, measure ginsenoside Rb
1chromatographic peak peak area.Taking reference substance sample size as abscissa, taking chromatographic peak peak area as vertical coordinate drawing standard curve, and carry out respectively linear regression, obtain regression equation;
The preparation of need testing solution: precision takes reinforcing the heart decoction extract 60.0mg after 70% methanol ultrasonic dissolution,, shakes up by 70% methanol constant volume to 10mL volumetric flask, and 0.45 μ m filtering with microporous membrane, obtains reinforcing the heart decoction extract need testing solution;
Algoscopy: the accurate phenols need testing solution 20ul that draws, terpenoid need testing solution 20ul, injection liquid chromatography, measures, and by peak area substitution standard curve, obtains as calculated the amount of each index composition.
Phenols drug substance is pressed dry product and is calculated, and must not be less than 3.0% containing 3,6`-, bis-mustard acyl sucrose, must not be less than 0.8% containing typhaneoside.Terpenoid drug substance is pressed dry product and is calculated, containing ginsenoside Rb
1must not be less than 2.0%.
Embodiment 2:
Radix Ginseng 300g Poria 300g
Radix Polygalae 200g Pollen Typhae 200g
Get according to the above ratio crude drug decoction pieces 1kg, measure 50% alcohol reflux 3 times with 6 times, each extraction 1.5 hours, merge extracted twice liquid (20L altogether) concentrating under reduced pressure, to the relative density clear paste that is 1.15-1.20g/ml, add aqueous dispersion, making sample solution concentration is 0.05g/mL (to roll over raw medicinal herbs amount), sample solution is adsorbed by DM130 type macroporous adsorbent resin, applied sample amount and resin volume ratio are 0.25g: 1mL, resin column blade diameter length ratio is 1: 8.7, and absorption flow velocity is 1bv/h; After absorption, with the remove impurity of 2 times of resinite hydrops eluting, washing flow velocity is 2bv/h; After washing remove impurity, with 4 times of resin volumes of 40% ethanol elution, elution flow rate is 2bv/h, collects 40% ethanol elution, reclaims solvent, with 3 times of resin volumes of 90% ethanol elution, elution flow rate is 2bv/h, collects 90% ethanol elution, reclaims solvent, vacuum drying, obtains reinforcing the heart soup phenols active substance and terpenoid active substance based on drug system.
Total phenol content assay method:
The preparation of reference substance solution: precision takes 3,6`-, bis-mustard acyl control sucrose product 2.15mg, with 70% methanol solution ultrasonic dissolution, is settled to 10mL graduation mark, shakes up, and obtaining concentration is 3 of 0.2150mg/mL, 6`-bis-mustard acyl control sucrose product solution;
Reagent: 0.6% liquor ferri trichloridi, 0.9% potassium ferricyanide solution, 0.3% sodium dodecyl sulfate solution, 50% methanol solution, 0.1mol/L hydrochloric acid solution;
Coloration method: precision pipettes sample solution and is placed in right amount the dark brown volumetric flask of 25ml, add 50% methanol to 5mL, add again 0.3% sodium dodecyl sulfate solution 2mL, shake up, add 0.6% ferric chloride-0.9% potassium ferricyanide new system mixed solution 1.0mL of 1: 1,5min is placed in dark place, add to 25mL graduation mark with 0.1mol/L hydrochloric acid, after shaking up, leave standstill 30min in dark place, and accompany and do blank, in order to measuring;
Assay method: sample after above-mentioned colour developing is measured in 770 ± 5nm wave-length coverage to absorbance, phenols drug substance is pressed dry product and calculated, and with 3,6`-, bis-mustard acyl sucrose meters, is no less than 40.0% containing total phenols.
Terpenoid content assaying method:
The preparation of reference substance solution: precision takes ginsenoside Rb
1reference substance 3.00mg, with 70% methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and obtaining concentration is ginsenoside Rb1's reference substance solution of 0.1200mg/mL;
Reagent: 5% vanillin-glacial acetic acid solution, perchloric acid solution, glacial acetic acid solution;
Coloration method: accurate absorption reinforcing the heart decoction extract sample liquid 1mL5 part, is placed in respectively cillin bottle, after boiling water bath evaporate to dryness, be cooled to room temperature, respectively add 5% vanillin-glacial acetic acid solution (developer) 0.2mL,, then add perchloric acid 0.8mL, shake up, in 60 DEG C of heating in water bath 15min, taking-up is put in cold water cooling, adds glacial acetic acid 5mL, blank is done in retinue, in order to measuring;
Assay method: sample after above-mentioned colour developing is measured in 560 ± 5nm wave-length coverage to absorbance, terpenoid drug substance is pressed dry product and calculated, contains terpenoid with ginsenoside Rb
1meter, is no less than 50.0%.
Index composition 3,6`-bis-mustard acyl sucrose, typhaneoside content assaying method simultaneously
The preparation of reference substance solution: precision takes 3,6 ' two mustard acyl control sucrose product 3.09mg, use methanol solution ultrasonic dissolution, be settled to 25mL graduation mark, shake up, the accurate 2mL that draws adds 3 times of methanol dilutions, shakes up, obtaining concentration is 3,6 '-bis-mustard acyl control sucrose product solution of 0.0412mg/mL; Precision takes typhaneoside reference substance 3.09mg, uses methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and the accurate 2mL that draws adds 18 times of methanol dilutions, shakes up, and obtaining concentration is the typhaneoside reference substance solution of 0.006867mg/mL;
Chromatographic condition: taking octadecylsilane chemically bonded silica as filler; Taking acetonitrile as mobile phase (A), taking 0.1% formic acid water as mobile phase (B), according to the form below, regulation is carried out gradient elution; Detection wavelength is 330nm; 30 DEG C of column temperatures, flow velocity is 1.0ml/min;
The preparation of standard curve: accurate absorption 3 respectively; 6`-bis-mustard acyl control sucrose product solution 3.0,6.0,9.0,12.0,15.0,18.0 μ L; typhaneoside reference substance solution 10,20,30,40,50,60,70 μ L injection liquid chromatographies; by above-mentioned chromatographic condition; measure 3,6`-, bis-mustard acyl sucrose, typhaneoside chromatographic peak peak area.Taking reference substance sample size as abscissa, taking chromatographic peak peak area as vertical coordinate drawing standard curve, and carry out respectively linear regression, obtain regression equation and be:
The preparation of need testing solution: precision takes reinforcing the heart decoction extract 60.0mg after 70% methanol ultrasonic dissolution,, shakes up by 70% methanol constant volume to 10mL volumetric flask, and 0.45 μ m filtering with microporous membrane, obtains reinforcing the heart decoction extract need testing solution.
Index composition ginsenoside Rb
1content assaying method
The preparation of reference substance solution: precision takes ginsenoside Rb1's reference substance 3.10mg, with 70% methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and obtaining concentration is the ginsenoside Rb of 0.1240mg/mL
1reference substance solution;
Chromatographic condition: taking octadecylsilane chemically bonded silica as filler; Taking acetonitrile as mobile phase (A), taking 0.1% formic acid water as mobile phase (B), according to the form below, regulation is carried out gradient elution; Detection wavelength is 203nm; 30 DEG C of column temperatures, flow velocity is 1.0ml/min;
The preparation of standard curve: the accurate ginsenoside Rb that draws respectively
1reference substance solution 10,20,30,40,50,60,70 μ L injection liquid chromatographies, by above-mentioned chromatographic condition, measure ginsenoside Rb
1chromatographic peak peak area.Taking reference substance sample size as abscissa, taking chromatographic peak peak area as vertical coordinate drawing standard curve, and carry out respectively linear regression, obtain regression equation;
The preparation of need testing solution: precision takes reinforcing the heart decoction extract 60.0mg after 70% methanol ultrasonic dissolution,, shakes up by 70% methanol constant volume to 10mL volumetric flask, and 0.45 μ m filtering with microporous membrane, obtains reinforcing the heart decoction extract need testing solution;
Algoscopy: the accurate phenols need testing solution 20ul that draws, terpenoid need testing solution 20ul, injection liquid chromatography, measures, and by peak area substitution standard curve, obtains as calculated the amount of each index composition.
Phenols drug substance is pressed dry product and is calculated, and must not be less than 3.0% containing 3,6`-, bis-mustard acyl sucrose, must not be less than 0.8% containing typhaneoside.Terpenoid drug substance is pressed dry product and is calculated, containing ginsenoside Rb
1must not be less than 2.0%.
Embodiment 3:
Radix Ginseng 300g Poria 300g
Radix Polygalae 200g Pollen Typhae 200g
Get according to the above ratio crude drug decoction pieces 1kg, measure 50% alcohol reflux 3 times with 6 times, each extraction 1.5 hours, merge extracted twice liquid (20L altogether) concentrating under reduced pressure, to the relative density clear paste that is 1.15-1.20g/ml, add aqueous dispersion, making sample solution concentration is 0.05g/mL (to roll over raw medicinal herbs amount), sample solution is adsorbed by DM130 type macroporous adsorbent resin, applied sample amount and resin volume ratio are 0.25g: 1mL, resin column blade diameter length ratio is 1: 8.7, and absorption flow velocity is 1bv/h; After absorption, with the remove impurity of 2 times of resinite hydrops eluting, washing flow velocity is 2bv/h; After washing remove impurity, with 4 times of resin volumes of 40% ethanol elution, elution flow rate is 2bv/h, collects 40% ethanol elution, reclaims solvent, with 3 times of resin volumes of 90% ethanol elution, elution flow rate is 2bv/h, collects 90% ethanol elution, reclaims solvent, vacuum drying, obtains reinforcing the heart soup phenols active substance and terpenoid active substance based on drug system.
Total phenol content assay method:
The preparation of reference substance solution: precision takes 3,6`-, bis-mustard acyl control sucrose product 2.15mg, with 70% methanol solution ultrasonic dissolution, is settled to 10mL graduation mark, shakes up, and obtaining concentration is 3 of 0.2150mg/mL, 6`-bis-mustard acyl control sucrose product solution;
Reagent: 0.6% liquor ferri trichloridi, 0.9% potassium ferricyanide solution, 0.3% sodium dodecyl sulfate solution, 50% methanol solution, 0.1mol/L hydrochloric acid solution;
Coloration method: precision pipettes sample solution and is placed in right amount the dark brown volumetric flask of 25ml, add 50% methanol to 5mL, add again 0.3% sodium dodecyl sulfate solution 2mL, shake up, add 0.6% ferric chloride-0.9% potassium ferricyanide new system mixed solution 1.0mL of 1: 1,5min is placed in dark place, add to 25mL graduation mark with 0.1mol/L hydrochloric acid, after shaking up, leave standstill 30min in dark place, and accompany and do blank, in order to measuring;
Assay method: sample after above-mentioned colour developing is measured in 770 ± 5nm wave-length coverage to absorbance, phenols drug substance is pressed dry product and calculated, and with 3,6`-, bis-mustard acyl sucrose meters, is no less than 40.0% containing total phenols.
Terpenoid content assaying method:
The preparation of reference substance solution: precision takes ginsenoside Rb
1reference substance 3.00mg, with 70% methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and obtaining concentration is ginsenoside Rb1's reference substance solution of 0.1200mg/mL;
Reagent: 5% vanillin-glacial acetic acid solution, perchloric acid solution, glacial acetic acid solution;
Coloration method: accurate absorption reinforcing the heart decoction extract sample liquid 1mL5 part, is placed in respectively cillin bottle, after boiling water bath evaporate to dryness, be cooled to room temperature, respectively add 5% vanillin-glacial acetic acid solution (developer) 0.2mL,, then add perchloric acid 0.8mL, shake up, in 60 DEG C of heating in water bath 15min, taking-up is put in cold water cooling, adds glacial acetic acid 5mL, blank is done in retinue, in order to measuring;
Assay method: sample after above-mentioned colour developing is measured in 560 ± 5nm wave-length coverage to absorbance, terpenoid drug substance is pressed dry product and calculated, contains terpenoid with ginsenoside Rb
1meter, is no less than 50.0%.
Index composition 3,6`-bis-mustard acyl sucrose, typhaneoside content assaying method simultaneously
The preparation of reference substance solution: precision takes 3,6 ' two mustard acyl control sucrose product 3.09mg, use methanol solution ultrasonic dissolution, be settled to 25mL graduation mark, shake up, the accurate 2mL that draws adds 3 times of methanol dilutions, shakes up, obtaining concentration is 3,6 '-bis-mustard acyl control sucrose product solution of 0.0412mg/mL; Precision takes typhaneoside reference substance 3.09mg, uses methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and the accurate 2mL that draws adds 18 times of methanol dilutions, shakes up, and obtaining concentration is the typhaneoside reference substance solution of 0.006867mg/mL;
Chromatographic condition: taking octadecylsilane chemically bonded silica as filler; Taking acetonitrile as mobile phase (A), taking 0.1% formic acid water as mobile phase (B), according to the form below, regulation is carried out gradient elution; Detection wavelength is 330nm; 30 DEG C of column temperatures, flow velocity is 1.0ml/min;
The preparation of standard curve: accurate absorption 3 respectively; 6`-bis-mustard acyl control sucrose product solution 3.0,6.0,9.0,12.0,15.0,18.0 μ L; typhaneoside reference substance solution 10,20,30,40,50,60,70 μ L injection liquid chromatographies; by above-mentioned chromatographic condition; measure 3,6`-, bis-mustard acyl sucrose, typhaneoside chromatographic peak peak area.Taking reference substance sample size as abscissa, taking chromatographic peak peak area as vertical coordinate drawing standard curve, and carry out respectively linear regression, obtain regression equation and be:
The preparation of need testing solution: precision takes reinforcing the heart decoction extract 60.0mg after 70% methanol ultrasonic dissolution,, shakes up by 70% methanol constant volume to 10mL volumetric flask, and 0.45 μ m filtering with microporous membrane, obtains reinforcing the heart decoction extract need testing solution.
Index composition ginsenoside Rb
1content assaying method
The preparation of reference substance solution: precision takes ginsenoside Rb1's reference substance 3.10mg, with 70% methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and obtaining concentration is the ginsenoside Rb of 0.1240mg/mL
1reference substance solution;
Chromatographic condition: taking octadecylsilane chemically bonded silica as filler; Taking acetonitrile as mobile phase (A), taking 0.1% formic acid water as mobile phase (B), according to the form below, regulation is carried out gradient elution; Detection wavelength is 203nm; 30 DEG C of column temperatures, flow velocity is 1.0ml/min;
The preparation of standard curve: the accurate ginsenoside Rb that draws respectively
1reference substance solution 10,20,30,40,50,60,70 μ L injection liquid chromatographies, by above-mentioned chromatographic condition, measure ginsenoside Rb
1chromatographic peak peak area.Taking reference substance sample size as abscissa, taking chromatographic peak peak area as vertical coordinate drawing standard curve, and carry out respectively linear regression, obtain regression equation;
The preparation of need testing solution: precision takes reinforcing the heart decoction extract 60.0mg after 70% methanol ultrasonic dissolution,, shakes up by 70% methanol constant volume to 10mL volumetric flask, and 0.45 μ m filtering with microporous membrane, obtains reinforcing the heart decoction extract need testing solution;
Algoscopy: the accurate phenols need testing solution 20ul that draws, terpenoid need testing solution 20ul, injection liquid chromatography, measures, and by peak area substitution standard curve, obtains as calculated the amount of each index composition.
Phenols drug substance is pressed dry product and is calculated, and must not be less than 3.0% containing 3,6`-, bis-mustard acyl sucrose, must not be less than 0.8% containing typhaneoside.Terpenoid drug substance is pressed dry product and is calculated, containing ginsenoside Rb
1must not be less than 2.0%.
Embodiment 4:
Radix Ginseng 300g Poria 300g
Radix Polygalae 200g Pollen Typhae 200g
Get according to the above ratio crude drug decoction pieces 1kg, measure 50% alcohol reflux 3 times with 6 times, each extraction 1.5 hours, merge extracted twice liquid (20L altogether) concentrating under reduced pressure, to the relative density clear paste that is 1.15-1.20g/ml, add aqueous dispersion, making sample solution concentration is 0.05g/mL (to roll over raw medicinal herbs amount), sample solution is adsorbed by DM130 type macroporous adsorbent resin, applied sample amount and resin volume ratio are 0.25g: 1mL, resin column blade diameter length ratio is 1: 8.7, and absorption flow velocity is 1bv/h; After absorption, with the remove impurity of 2 times of resinite hydrops eluting, washing flow velocity is 2bv/h; After washing remove impurity, with 4 times of resin volumes of 40% ethanol elution, elution flow rate is 2bv/h, collects 40% ethanol elution, reclaims solvent, with 3 times of resin volumes of 90% ethanol elution, elution flow rate is 2bv/h, collects 90% ethanol elution, reclaims solvent, vacuum drying, obtains reinforcing the heart soup phenols active substance and terpenoid active substance based on drug system.
Total phenol content assay method:
The preparation of reference substance solution: precision takes 3,6`-, bis-mustard acyl control sucrose product 2.15mg, with 70% methanol solution ultrasonic dissolution, is settled to 10mL graduation mark, shakes up, and obtaining concentration is 3 of 0.2150mg/mL, 6`-bis-mustard acyl control sucrose product solution;
Reagent: 0.6% liquor ferri trichloridi, 0.9% potassium ferricyanide solution, 0.3% sodium dodecyl sulfate solution, 50% methanol solution, 0.1mol/L hydrochloric acid solution;
Coloration method: precision pipettes sample solution and is placed in right amount the dark brown volumetric flask of 25ml, add 50% methanol to 5mL, add again 0.3% sodium dodecyl sulfate solution 2mL, shake up, add 0.6% ferric chloride-0.9% potassium ferricyanide new system mixed solution 1.0mL of 1: 1,5min is placed in dark place, add to 25mL graduation mark with 0.1mol/L hydrochloric acid, after shaking up, leave standstill 30min in dark place, and accompany and do blank, in order to measuring;
Assay method: sample after above-mentioned colour developing is measured in 770 ± 5nm wave-length coverage to absorbance, phenols drug substance is pressed dry product and calculated, and with 3,6`-, bis-mustard acyl sucrose meters, is no less than 40.0% containing total phenols.
Terpenoid content assaying method:
The preparation of reference substance solution: precision takes ginsenoside Rb
1reference substance 3.00mg, with 70% methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and obtaining concentration is ginsenoside Rb1's reference substance solution of 0.1200mg/mL;
Reagent: 5% vanillin-glacial acetic acid solution, perchloric acid solution, glacial acetic acid solution;
Coloration method: accurate absorption reinforcing the heart decoction extract sample liquid 1mL5 part, is placed in respectively cillin bottle, after boiling water bath evaporate to dryness, be cooled to room temperature, respectively add 5% vanillin-glacial acetic acid solution (developer) 0.2mL,, then add perchloric acid 0.8mL, shake up, in 60 DEG C of heating in water bath 15min, taking-up is put in cold water cooling, adds glacial acetic acid 5mL, blank is done in retinue, in order to measuring;
Assay method: sample after above-mentioned colour developing is measured in 560 ± 5nm wave-length coverage to absorbance, terpenoid drug substance is pressed dry product and calculated, contains terpenoid with ginsenoside Rb
1meter, is no less than 50.0%.
Index composition 3,6`-bis-mustard acyl sucrose, typhaneoside content assaying method simultaneously
The preparation of reference substance solution: precision takes 3,6 ' two mustard acyl control sucrose product 3.09mg, use methanol solution ultrasonic dissolution, be settled to 25mL graduation mark, shake up, the accurate 2mL that draws adds 3 times of methanol dilutions, shakes up, obtaining concentration is 3,6 '-bis-mustard acyl control sucrose product solution of 0.0412mg/mL; Precision takes typhaneoside reference substance 3.09mg, uses methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and the accurate 2mL that draws adds 18 times of methanol dilutions, shakes up, and obtaining concentration is the typhaneoside reference substance solution of 0.006867mg/mL;
Chromatographic condition: taking octadecylsilane chemically bonded silica as filler; Taking acetonitrile as mobile phase (A), taking 0.1% formic acid water as mobile phase (B), according to the form below, regulation is carried out gradient elution; Detection wavelength is 330nm; 30 DEG C of column temperatures, flow velocity is 1.0ml/min;
The preparation of standard curve: accurate absorption 3 respectively; 6`-bis-mustard acyl control sucrose product solution 3.0,6.0,9.0,12.0,15.0,18.0 μ L; typhaneoside reference substance solution 10,20,30,40,50,60,70 μ L injection liquid chromatographies; by above-mentioned chromatographic condition; measure 3,6`-, bis-mustard acyl sucrose, typhaneoside chromatographic peak peak area.Taking reference substance sample size as abscissa, taking chromatographic peak peak area as vertical coordinate drawing standard curve, and carry out respectively linear regression, obtain regression equation and be:
The preparation of need testing solution: precision takes reinforcing the heart decoction extract 60.0mg after 70% methanol ultrasonic dissolution,, shakes up by 70% methanol constant volume to 10mL volumetric flask, and 0.45 μ m filtering with microporous membrane, obtains reinforcing the heart decoction extract need testing solution.
Index composition ginsenoside Rb
1content assaying method
The preparation of reference substance solution: precision takes ginsenoside Rb1's reference substance 3.10mg, with 70% methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and obtaining concentration is the ginsenoside Rb of 0.1240mg/mL
1reference substance solution;
Chromatographic condition: taking octadecylsilane chemically bonded silica as filler; Taking acetonitrile as mobile phase (A), taking 0.1% formic acid water as mobile phase (B), according to the form below, regulation is carried out gradient elution; Detection wavelength is 203nm; 30 DEG C of column temperatures, flow velocity is 1.0ml/min;
The preparation of standard curve: the accurate ginsenoside Rb that draws respectively
1reference substance solution 10,20,30,40,50,60,70 μ L injection liquid chromatographies, by above-mentioned chromatographic condition, measure ginsenoside Rb
1chromatographic peak peak area.Taking reference substance sample size as abscissa, taking chromatographic peak peak area as vertical coordinate drawing standard curve, and carry out respectively linear regression, obtain regression equation;
The preparation of need testing solution: precision takes reinforcing the heart decoction extract 60.0mg after 70% methanol ultrasonic dissolution,, shakes up by 70% methanol constant volume to 10mL volumetric flask, and 0.45 μ m filtering with microporous membrane, obtains reinforcing the heart decoction extract need testing solution;
Algoscopy: the accurate phenols need testing solution 20ul that draws, terpenoid need testing solution 20ul, injection liquid chromatography, measures, and by peak area substitution standard curve, obtains as calculated the amount of each index composition.
Phenols drug substance is pressed dry product and is calculated, and must not be less than 3.0% containing 3,6`-, bis-mustard acyl sucrose, must not be less than 0.8% containing typhaneoside.Terpenoid drug substance is pressed dry product and is calculated, containing ginsenoside Rb
1must not be less than 2.0%.
Embodiment 5: the Chinese medicine combinations of substances that resists myocardial ischemia preparation technology
Radix Ginseng 300g Poria 300g
Radix Polygalae 200g Pollen Typhae 200g
Get according to the above ratio crude drug decoction pieces 1kg, measure 50% alcohol reflux 3 times with 6 times, each extraction 1.5 hours, merge extracted twice liquid (20L altogether) concentrating under reduced pressure, to the relative density clear paste that is 1.15-1.20g/ml, add aqueous dispersion, making sample solution concentration is 0.05g/mL (to roll over raw medicinal herbs amount), sample solution is adsorbed by DM130 type macroporous adsorbent resin, applied sample amount and resin volume ratio are 0.25g: 1mL, resin column blade diameter length ratio is 1: 8.7, and absorption flow velocity is 1bv/h; After absorption, with the remove impurity of 2 times of resinite hydrops eluting, washing flow velocity is 2bv/h; After washing remove impurity, with 4 times of resin volumes of 40% ethanol elution, elution flow rate is 2bv/h, collects 40% ethanol elution, reclaims solvent, with 3 times of resin volumes of 90% ethanol elution, elution flow rate is 2bv/h, collects 90% ethanol elution, reclaims solvent, vacuum drying, obtains reinforcing the heart soup phenols active substance and terpenoid active substance based on drug system.
Total phenol content assay method:
The preparation of reference substance solution: precision takes 3,6`-, bis-mustard acyl control sucrose product 2.15mg, with 70% methanol solution ultrasonic dissolution, is settled to 10mL graduation mark, shakes up, and obtaining concentration is 3 of 0.2150mg/mL, 6`-bis-mustard acyl control sucrose product solution;
Reagent: 0.6% liquor ferri trichloridi, 0.9% potassium ferricyanide solution, 0.3% sodium dodecyl sulfate solution, 50% methanol solution, 0.1mol/L hydrochloric acid solution;
Coloration method: precision pipettes sample solution and is placed in right amount the dark brown volumetric flask of 25ml, add 50% methanol to 5mL, add again 0.3% sodium dodecyl sulfate solution 2mL, shake up, add 0.6% ferric chloride-0.9% potassium ferricyanide new system mixed solution 1.0mL of 1: 1,5min is placed in dark place, add to 25mL graduation mark with 0.1mol/L hydrochloric acid, after shaking up, leave standstill 30min in dark place, and accompany and do blank, in order to measuring;
Assay method: sample after above-mentioned colour developing is measured in 770 ± 5nm wave-length coverage to absorbance, phenols drug substance is pressed dry product and calculated, and with 3,6`-, bis-mustard acyl sucrose meters, is no less than 40.0% containing total phenols.
Terpenoid content assaying method:
The preparation of reference substance solution: precision takes ginsenoside Rb
1reference substance 3.00mg, with 70% methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and obtaining concentration is ginsenoside Rb1's reference substance solution of 0.1200mg/mL;
Reagent: 5% vanillin-glacial acetic acid solution, perchloric acid solution, glacial acetic acid solution;
Coloration method: accurate absorption reinforcing the heart decoction extract sample liquid 1mL5 part, is placed in respectively cillin bottle, after boiling water bath evaporate to dryness, be cooled to room temperature, respectively add 5% vanillin-glacial acetic acid solution (developer) 0.2mL,, then add perchloric acid 0.8mL, shake up, in 60 DEG C of heating in water bath 15min, taking-up is put in cold water cooling, adds glacial acetic acid 5mL, blank is done in retinue, in order to measuring;
Assay method: sample after above-mentioned colour developing is measured in 560 ± 5nm wave-length coverage to absorbance, terpenoid drug substance is pressed dry product and calculated, contains terpenoid with ginsenoside Rb
1meter, is no less than 50.0%.
Index composition 3,6`-bis-mustard acyl sucrose, typhaneoside content assaying method simultaneously
The preparation of reference substance solution: precision takes 3,6 ' two mustard acyl control sucrose product 3.09mg, use methanol solution ultrasonic dissolution, be settled to 25mL graduation mark, shake up, the accurate 2mL that draws adds 3 times of methanol dilutions, shakes up, obtaining concentration is 3,6 '-bis-mustard acyl control sucrose product solution of 0.0412mg/mL; Precision takes typhaneoside reference substance 3.09mg, uses methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and the accurate 2mL that draws adds 18 times of methanol dilutions, shakes up, and obtaining concentration is the typhaneoside reference substance solution of 0.006867mg/mL;
Chromatographic condition: taking octadecylsilane chemically bonded silica as filler; Taking acetonitrile as mobile phase (A), taking 0.1% formic acid water as mobile phase (B), according to the form below, regulation is carried out gradient elution; Detection wavelength is 330nm; 30 DEG C of column temperatures, flow velocity is 1.0ml/min;
The preparation of standard curve: accurate absorption 3 respectively; 6`-bis-mustard acyl control sucrose product solution 3.0,6.0,9.0,12.0,15.0,18.0 μ L; typhaneoside reference substance solution 10,20,30,40,50,60,70 μ L injection liquid chromatographies; by above-mentioned chromatographic condition; measure 3,6`-, bis-mustard acyl sucrose, typhaneoside chromatographic peak peak area.Taking reference substance sample size as abscissa, taking chromatographic peak peak area as vertical coordinate drawing standard curve, and carry out respectively linear regression, obtain regression equation and be:
The preparation of need testing solution: precision takes reinforcing the heart decoction extract 60.0mg after 70% methanol ultrasonic dissolution,, shakes up by 70% methanol constant volume to 10mL volumetric flask, and 0.45 μ m filtering with microporous membrane, obtains reinforcing the heart decoction extract need testing solution.
Index composition ginsenoside Rb
1content assaying method
The preparation of reference substance solution: precision takes ginsenoside Rb1's reference substance 3.10mg, with 70% methanol solution ultrasonic dissolution, is settled to 25mL graduation mark, shakes up, and obtaining concentration is the ginsenoside Rb of 0.1240mg/mL
1reference substance solution;
Chromatographic condition: taking octadecylsilane chemically bonded silica as filler; Taking acetonitrile as mobile phase (A), taking 0.1% formic acid water as mobile phase (B), according to the form below, regulation is carried out gradient elution; Detection wavelength is 203nm; 30 DEG C of column temperatures, flow velocity is 1.0ml/min;
The preparation of standard curve: the accurate ginsenoside Rb that draws respectively
1reference substance solution 10,20,30,40,50,60,70 μ L injection liquid chromatographies, by above-mentioned chromatographic condition, measure ginsenoside Rb
1chromatographic peak peak area.Taking reference substance sample size as abscissa, taking chromatographic peak peak area as vertical coordinate drawing standard curve, and carry out respectively linear regression, obtain regression equation;
The preparation of need testing solution: precision takes reinforcing the heart decoction extract 60.0mg after 70% methanol ultrasonic dissolution,, shakes up by 70% methanol constant volume to 10mL volumetric flask, and 0.45 μ m filtering with microporous membrane, obtains reinforcing the heart decoction extract need testing solution;
Algoscopy: the accurate phenols need testing solution 20ul that draws, terpenoid need testing solution 20ul, injection liquid chromatography, measures, and by peak area substitution standard curve, obtains as calculated the amount of each index composition.
Phenols drug substance is pressed dry product and is calculated, and must not be less than 3.0% containing 3,6`-, bis-mustard acyl sucrose, must not be less than 0.8% containing typhaneoside.Terpenoid drug substance is pressed dry product and is calculated, containing ginsenoside Rb
1must not be less than 2.0%.
Embodiment 6: the preparation of capsule
Get Chinese medicine combinations of substances 200g of the present invention, pulverize, cross 80 mesh sieves, 100g is mixed homogeneously with microcrystalline Cellulose, with 95% alcohol granulation, dry, with 20 mesh sieve granulate, fill capsule.
Embodiment 7: the preparation of tablet
Get Chinese medicine combinations of substances 50g of the present invention, pulverize, cross 80 mesh sieves, mix homogeneously with microcrystalline Cellulose 70g, carboxymethyl starch sodium 5g, granulate with 5%PVP, dry, with 20 mesh sieve granulate, add magnesium stearate 2g, tabletting.
Embodiment 8: the preparation of drop pill
Get Chinese medicine combinations of substances 60g of the present invention, pulverize, cross 80 mesh sieves, mix homogeneously, drop in the polyethylene glycol 6000 of 180g heating and melting, be stirred to dissolving, be transferred in reservoir, airtight and insulation at 80～90 DEG C, regulate pill dripping machine drop quantitative valve, splash into from top to bottom in the liquid paraffin of 10～15 DEG C, the drop pill of formation is drained and erasing liquor paraffin body, dry.
Embodiment 9: the preparation of oral liquid
Get Chinese medicine combinations of substances 70g of the present invention, pulverize, cross 80 mesh sieves, mix homogeneously, mix with Mel 1000g, sucrose 200g, sodium benzoate 10g and distilled water 2000ml, be heated to 85～90 DEG C, be stirred to dissolve, insulation 30min, filter, filtrate is diluted with water to 4000ml, stirs, embedding, sterilizing.
Embodiment 10: the preparation of injection
Get Chinese medicine combinations of substances 100g of the present invention, inject water and make in right amount to dissolve, 0.02% the active carbon that adds configuration amount stirs 5～10min, filters, filtrate is diluted to 10L left and right, adds sodium chloride and regulates osmotic pressure to ooze to waiting, and regulates pH7.5～8.0, ultrafiltration, embedding, 100 DEG C of sterilizing 30min.
Embodiment 11: the preparation of injectable powder
Get Chinese medicine combinations of substances 100g of the present invention, inject water and dilute sodium hydroxide and make in right amount to dissolve, 0.02% the active carbon that adds configuration amount stirs 5～10min, filter, filtrate is diluted to 1L, regulates pH6.5～7.8, ultrafiltration, spraying is dry, and dry powder is aseptic subpackaged.Every 100mg, injects before use water and makes in right amount to dissolve, with slowly intravenous drip after sodium chloride transfusion 250～500ml dilution.
Claims (12)
1. have function of resisting myocardial ischemia a Chinese medicine combinations of substances, it is characterized in that the preparation method of this Chinese medicine combinations of substances comprises the steps:
Step 1: choose crude drug;
Step 2: ethanol extraction;
Step 3: purification with macroreticular resin;
This Chinese medicine combinations of substances is made up of two parts, and Part I is phenols, and wherein phenols content is 10-50%, Part II terpenoid, and wherein terpenoid content is 10-20%.
2. Chinese medicine extract as claimed in claim 1, is characterized in that consisting of of step 1 Raw medicine:
Radix Ginseng 300g Poria 300g
Radix Polygalae 200g Pollen Typhae 200g.
3. Chinese medicine extract as claimed in claim 1 or 2, is characterized in that in step 2, by crude drug 10-80% alcohol reflux 2-4 time, extracts 0.5-2 hour at every turn.
4. Chinese medicine extract as claimed in claim 1 or 2, is characterized in that in step 3, and by extract obtained step 2 dispersing and dissolving that adds water, making concentration of aqueous solution is 0.02-0.1g/mL.
5. Chinese medicine extract as claimed in claim 1 or 2, it is characterized in that in step 3, aqueous dispersions is by low pole or nonpolar macroporous adsorption resin, resin blade diameter length ratio is 1: 4-1: 10, sample solution concentration is 0.01-0.1g/mL, applied sample amount is counted 0.2g/ml-0.5g/ml with full side's raw medicinal herbs, absorption flow velocity is 1-4BV/h, water elution 1-3 times of resin volume carries out remove impurity, and first with 0-8 times of resin volume of 40-70% ethanol elution, elution flow rate is 2-6BV/h, collect ethanol elution, reclaim solvent, drying under reduced pressure, obtains phenols drug substance; With 0-13 times of resin volume of 70-95% ethanol elution, elution flow rate is 2-6BV/h again, collects ethanol elution, reclaims solvent, and drying under reduced pressure, obtains terpenoid drug substance.
6. as described in right 1-5, Chinese medicine combinations of substances is characterized in that and substituting kind by following 4 groups of medicinal plants, comprises its medicinal part and dis-medicinal part, and medical material and decoction pieces combination thereof are made:
Group 1: panax japonicus c.a.mey.var.major (burk.) c.y.wu et k.m.feng, Panax pseudoginseng Wall., Panax zingiberensis C. Y. Wu. Et Feng．, Radix Ginseng, Radix Notoginseng, Radix Panacis Quinquefolii, narrow leaf Panax pseudoginseng Wall., beautiful Panax pseudoginseng Wall., Rhizoma Panacis bipinnatifidi, panax japonicus are equal to Araliaceae, and processed product.
Group 2: the rogation flowers such as Radix Polygalae, ovum leaf Radix Polygalae, caudal lobe Radix Polygalae, kidney fruit Radix Polygalae, Herba Polygalae Japonicae, hinge grass, largeleaf milkwort herb, Radix Crotalariae szemoensis, Radix Polygalae fallacis, Siberia Radix Polygalae, and Huang Yeshu, Radix securidacae, tooth fruit grass be equal to milk wort, and processed product.
Group 3: Poria, Polyporus, Coriolous Dersicolor (Fr.) Quel, Grifola frondosa, Laetiporus sulphureus (Bull. Ex Fr.) Bond. Et Singer. are equal to Polyporaceae plant, and processed product.
Group 4: Herba Typhae, Typha angustifolia, typha angustifolia, common cattail, hair wax candle, black cutter candle, Typha angustifolia Herba Typhae, typha orientalis are equal to Typhaceae plant, and processed product.
As described in right 1-5 Chinese medicine combinations of substances it is characterized in that can be after above-mentioned raw materials medicine combination with ethanol or other alcohols, rare alcohol or other organic solvents or water extraction again by macroporous adsorbent resin or other chromatographic processes, as polyamide chromatography etc., or the purification such as solvent extraction obtains.
As described in right 1-5 Chinese medicine combinations of substances it is characterized in that can be by the extract of crude drug described in right 6 after further enriching and purifying or the macroporous resin prepared product of above-mentioned raw materials medicine mix.
As described in right 1-5 Chinese medicine combinations of substances it is characterized in that can be through chemosynthesis or structural modification, biosynthesis or biology the approach such as turn and obtain.
10. Chinese medicine combinations of substances as described in claim 1-9, it is characterized in that adding various pharmaceutic adjuvant well known in the art, preparation process is routinely made the acceptable any conventional dosage form of pharmaceutics, also can be made into other transmission system drug-delivery preparations such as long-acting and slow releasing preparation, controlled release preparation, targeting preparation.Obtained medicament can be used as people's medicine, veterinary drug, plant medication use or uses.
11. as described in claim 1-9 Chinese medicine combinations of substances, it is characterized in that adding known food additive to make health food or the beverage with prevention and health care function as antiseptic, antioxidant agent, coloring agent, thickening agent and stabilizing agent, bulking agent, sweeting agent, acidity agent, brightening agent, spice etc.
12. Chinese medicine combinations of substances as described in as arbitrary in claim 1-9 have the application in medicaments for resisting myocardial ischemia in preparation.
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| CN112858567A (en) * | 2021-01-07 | 2021-05-28 | 成都大帝汉克生物科技有限公司 | Method for measuring and evaluating effective total acid content of acidifier and acidity regulator in feed |
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| CN102119986A (en) * | 2011-03-07 | 2011-07-13 | 北京中医药大学 | Traditional Chinese medicine extract with anti-dementia effect and preparation method thereof |
| CN103385903A (en) * | 2013-08-02 | 2013-11-13 | 石任兵 | Stevia rebaudiana phenol extract and application thereof in preparing lipid-lowering products |
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| CN102119986A (en) * | 2011-03-07 | 2011-07-13 | 北京中医药大学 | Traditional Chinese medicine extract with anti-dementia effect and preparation method thereof |
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| CN112858567A (en) * | 2021-01-07 | 2021-05-28 | 成都大帝汉克生物科技有限公司 | Method for measuring and evaluating effective total acid content of acidifier and acidity regulator in feed |
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