CN1899400B - Detection method for injection containing ginseng and aconite root in raw material - Google Patents

Detection method for injection containing ginseng and aconite root in raw material Download PDF

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CN1899400B
CN1899400B CN200610103234A CN200610103234A CN1899400B CN 1899400 B CN1899400 B CN 1899400B CN 200610103234 A CN200610103234 A CN 200610103234A CN 200610103234 A CN200610103234 A CN 200610103234A CN 1899400 B CN1899400 B CN 1899400B
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solution
water
preparation
ginsenoside
promptly
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CN1899400A (en
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孙明珍
艾劼
刘路
瞿冰
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Shenzhen Zifu Pharmaceutical Co., Ltd.
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SHENZHEN ZIFU PHARMACEUTICAL CO Ltd
XUANHONG MEDICINE TECHNOLOGY Co Ltd TIANJIN
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Abstract

The present invention relates to a kind of medicine preparation with ginseng component and aconite root component and its preparation process and quality control method. Of the medicine preparation, each unit contains total ginsenoside in 1-90 mg, total ginseng polysaccharide in 2-282 mg, and total aconite root alkaloid in 0.02-22 mg. The medicine preparation may have other synergistic medicine component added. Its preparation process includes extracting the total ginsenoside, the total ginseng polysaccharide and the total aconite root alkaloid; adding supplementary material; and forming different preparation forms.

Description

The detection method of the injection that a kind of bulk drug is made up of genseng and monkshood
Technical field
The present invention belongs to field of pharmaceutical preparations, specifically, relates to the pharmaceutical preparation that contains genseng and monkshood in a kind of raw material and preparation method thereof and method of quality control.
Background technology
Ancient prescription " shenfu decoction ", its side is made up of genseng, monkshood two flavor medicines.According to traditional Chinese medical theory, this side is the side of recuperating depleted yang, supplementing QI to prevent collapse, clinically is mainly used in fainting that yang-energy takes off cruelly and takes off disease.
Contain saponins, polysaccharide composition in the genseng; Containing the alkaloids composition in the monkshood, mainly is aconite alkaloids; Ester alkaloid is a kind of, poisonous in the aconite alkaloids.
There is ginseng aconite injection liquid to sell in the market, the disclosed patent that has multinomial relevant ginseng aconite injection liquid, ginseng aconite injection to apply in China simultaneously with freeze-dried powder.But it does not all have in the preparation finished product to keep polysaccharide composition in the genseng as a kind of main effective constituent, number is the publication of CN96117458.7, CN03135691.5, CN03135701.6, CN02149368.5, CN200510059785.0, CN200410013725.0, CN200510047701.1 as China's application (patent).
Polysaccharide composition in the genseng has various active, can bring into play antineoplastic action from number of ways as the polysaccharide composition in the genseng; Polysaccharide composition in the genseng all has obvious facilitation to the specific immunity and the nospecific immunity of body; And the polysaccharide composition in the genseng has multiple physiologically actives such as hypoglycemic activity.Polysaccharide composition in the genseng can improve the resistibility of shock patient and its recovery is had facilitation.
Further development quality is stable, controlled, the effective constituent kind keeps the attached pharmaceutical preparation of ginseng more complete, that active constituent content is higher, more complete reservation and develop the drug effect of traditional ancient prescription, can select for clinical use provides how better medication, also be the needs that the better development traditional Chinese medicine is learned.
Summary of the invention
The inventor finds in experimentation unexpectedly: for the pharmaceutical preparation of genseng and monkshood, the pharmacological action that has kept the pharmaceutical preparation of the polysaccharide composition in the genseng in the preparation finished product obviously is better than not keeping the pharmaceutical preparation of the polysaccharide composition in the genseng.
The inventor explores through a large amount of research on the basis of aforementioned discovery, in conjunction with novel unique technique, has developed the pharmaceutical preparation that contains genseng and monkshood in a kind of raw material.Contain the saponin component of genseng, the polysaccharide composition of genseng, the alkaloids composition of monkshood in this pharmaceutical preparation.
An object of the present invention is to provide the pharmaceutical preparation that contains genseng and monkshood in a kind of raw material.
At traditional ancient prescription, on the basis of proved recipe, utilize traditional Chinese medicine theory, theoretical and the modern medicine theory of modern traditional Chinese medicine, by at ancient prescription, increase and decrease flavour of a drug on the proved recipe, change the dose proportioning, the no incompatibility of increase, can play synergistic Chinese medicine, natural drug, Chinese medical extract, the natural drug extract, effective ingredient in Chinese, the natural drug active component, Chinese medicine active compound monomer, natural drug active compound monomer, medicines such as chemicals, to ancient prescription, proved recipe is further furtherd investigate and secondary development, be the exploitation better efficacy, security is better, a shortcut of the new drug that the research and development cost is lower.
Contain genseng, monkshood in the raw material in the pharmaceutical preparation of the present invention, simultaneously in this pharmaceutical preparation, can not add or add one or more no incompatibility, can play medicines such as synergistic Chinese medicine, natural drug, Chinese medical extract, natural drug extract, effective ingredient in Chinese, natural drug active component, Chinese medicine active compound monomer, natural drug active compound monomer, chemicals, as Radix Glycyrrhizae, the Radix Astragali, licorice, Astragalus Root P.E, naloxone hydrochloride, Dopamine hydrochloride etc.
Genseng in this pharmaceutical preparation and monkshood are wild product or artificial cultivation product, are genunie medicinal materials or non-genunie medicinal materials.Genseng as described can be genseng or mountain ginseng or garden ginsent or sylvan life ginseng etc.
Described genseng and monkshood are concocted processed product that forms or the processed product that forms according to the modernism process of preparing Chinese medicine for its bright medicinal material or according to classic method simultaneously.Genseng as described can be genseng or sun-dried mountain ginseng or sun-dried ginseng or red ginseng etc., and described monkshood can be black in sheet or RADIX ACONITILATERALIS PREPARATA or salted aconite root etc.
The pharmaceutical preparation that contains genseng and monkshood in a kind of raw material of the present invention can be any pharmaceutically useful formulation, and these formulations comprise oral solid formulation, oral semisolid preparation, oral liquid, injection.
Described oral solid formulation can be tablet, hard capsule, soft capsule, pill, pill, solid dispersion, powder, granule, fine granule, micropill preparation, microcapsules, microspheres agent and other pharmaceutically acceptable oral solid formulation.Described oral liquid can be oral liquid and other pharmaceutically acceptable oral liquid.Described injection can be parenteral solution, freeze-drying preparation for injection, sterile packaged preparation for injection, glucose injection, sodium chloride injection and other pharmaceutically acceptable injection.
Pharmaceutical preparation of the present invention, preferably tablet, hard capsule, soft capsule, solid dispersion, oral liquid, parenteral solution, freeze-drying preparation for injection, sterile packaged preparation for injection, glucose injection, sodium chloride injection.More preferably tablet, hard capsule, solid dispersion, oral liquid, parenteral solution, freeze-drying preparation for injection.More preferably tablet, hard capsule, oral liquid, parenteral solution, freeze-drying preparation for injection.
Pharmaceutical preparation of the present invention exists with unit dosage form, and described unit dosage form is meant single-dose preparations, as every of injection, and every bottle, every of oral formulations, every etc.
Pharmaceutical preparation of the present invention, genseng in the raw material: the ratio of monkshood is 1-10: 1-10, preferred 1-4: 1-8, more preferably 1-2: 1-4, most preferably 1: 2.
Pharmaceutical preparation of the present invention in the preparation of per unit dosage, contains genseng 0.1g-25g by raw material, monkshood 0.2g-50g; Preferably contain genseng 0.15g-20g, monkshood 0.3g-40g; More preferably contain genseng 0.2g-14g, monkshood 0.4g-28g; More preferably contain genseng 0.2g-11g, monkshood 0.4g-22g.
Pharmaceutical preparation of the present invention in the preparation of per unit dosage, contains general ginsenoside 1mg～90mg, genseng total polysaccharides 2mg～282mg, Radix Aconiti Lateralis Preparata total alkaloids 0.02mg～22mg.Preferably contain general ginsenoside 2mg～60mg, genseng total polysaccharides 4mg～188mg, Radix Aconiti Lateralis Preparata total alkaloids 0.05mg～15mg.More preferably contain general ginsenoside 2.5mg～45mg, genseng total polysaccharides 6mg～141mg, Radix Aconiti Lateralis Preparata total alkaloids 0.06mg～11mg.
Concrete can be: in the preparation of per unit dosage, contain general ginsenoside with ginsenoside Rb 1(C 54H 92O 23) meter, be 8mg～15mg; The genseng total polysaccharides is 25mg～47mg with glucose meter; Radix Aconiti Lateralis Preparata total alkaloids 0.2mg～3.6mg.
Or in the preparation of per unit dosage, contain general ginsenoside with ginsenoside Rb 1(C 54H 92O 23) meter, be 16mg～30mg; The genseng total polysaccharides is 50mg～94mg with glucose meter; Radix Aconiti Lateralis Preparata total alkaloids 0.4mg～7.2mg.
Or in the preparation of per unit dosage, contain general ginsenoside with ginsenoside Rb 1(C 54H 92O 23) meter, be 40mg～75mg; The genseng total polysaccharides is 125mg～235mg with glucose meter; Radix Aconiti Lateralis Preparata total alkaloids 1mg～18mg.
Or in the preparation of per unit dosage, contain general ginsenoside with ginsenoside Rb 1(C 54H 92O 23) meter, be 2.5mg～5mg; The genseng total polysaccharides is 8mg～16mg with glucose meter; Radix Aconiti Lateralis Preparata total alkaloids 0.06mg～1.2mg.
Ester alkaloid in the monkshood is a kind of, poisonous in the aconite alkaloids.Pharmaceutical preparation of the present invention in the preparation of per unit dosage, contains aconite alkaloids with aconitine (C 24H 47NO 11) meter, must not be higher than 10.0mg, preferably must not be higher than 5.0mg, more preferably must not be higher than 3.0mg, more preferably must not be higher than 2.0mg, more preferably must not be higher than 1.0mg.
The amount average molecular weight is 30～36 * 10 in the pharmaceutical preparation of the present invention 4, 8～12 * 10 4, 3.5～7.5 * 10 4The polysaccharide composition of genseng in one or more, wherein measuring average molecular weight is 33.0445 * 10 4, or 9.8878 * 10 4, or 5.4087 * 10 4The content of polysaccharide composition of genseng higher.
Pharmaceutical preparation of the present invention also contains where necessary or uses medicine acceptable auxiliary (or carrier), described auxiliary material is pharmaceutically acceptable preparation assistant.
Preferably contain the saponin component of the genseng of 0.05%～90% (percentage by weight), the polysaccharide composition of genseng and the alkaloids composition of monkshood in the pharmaceutical preparation of the present invention, and the pharmaceutically available auxiliary material of 99.95%～0% (percentage by weight).The content of the alkaloids composition of the polysaccharide composition of the saponin component of genseng, genseng and monkshood more preferably 0.08%～80% in described preparation, and more preferably 0.1%～75%, more preferably 0.15%～70%.
For oral solid formulation and oral semisolid preparation, described auxiliary material is selected from thinning agent, wetting agent, bonding agent, disintegrant, glidant, antisticking agent, lubricant, color and correctives thereof, solid dispersions carrier material, antioxidant, surfactant, stabilizing agent, PH correctives and other pharmaceutically acceptable oral solid formulation, oral semisolid preparation with one or more the material in the auxiliary material, and every kind of assistant can not select or select for use one or more the material in this kind assistant.
Described thinning agent is one or more the material that is selected from starch, amylum pregelatinisatum, Icing Sugar, dextrin, lactose, microcrystalline cellulose, sweet mellow wine, sorbierite, calcium sulphate, lime carbonate and other the pharmaceutically acceptable thinning agent;
Described wetting agent is one or more the material that is selected from ethanol, water and other the pharmaceutically acceptable wetting agent;
Described bonding agent is one or more the material that is selected from Hydroxypropyl methylcellulose, ethyl cellulose, sodium carboxymethyl cellulose, methylcellulose, L-HPC, starch slurry, polyvidone, gelatin, polyglycol, 50% to 70% sucrose solution, sodium alginate soln and other the pharmaceutically acceptable bonding agent;
Described disintegrant is one or more the material that is selected from sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, Ac-Di-Sol, dried starch, polyvinylpolypyrrolidone, gas-producing disintegrant and other the pharmaceutically acceptable disintegrant;
Described glidant, antisticking agent, lubricant are one or more the materials that is selected from talcum powder, superfine silica gel powder, dolomol, polyethylene glycols, sldium lauryl sulfate, magnesium laurylsulfate, hydrogenated vegetable oil and other pharmaceutically acceptable glidant, antisticking agent, the lubricant;
Described color and correctives thereof are one or more the materials that is selected from medicinal pigment, food coloring, essence and other pharmaceutically acceptable color and the correctives thereof;
Described solid dispersions carrier material is to be selected from polyethylene glycols, cellulose derivative, organic acid, surfactant-based, the polyvidone class, carbohydrate and alcohols, cellulose family, the polyacrylic resin class, cupreol, cholesterol, cholesterol ester stearic acid, tripalmitin, rilanit special, castor oil wax, beeswax, the material of one or more in Brazil wax and other pharmaceutically acceptable solid dispersions carrier material, every class solid dispersions carrier material can not select or select for use one or more the material in such solid dispersions carrier material;
Described pH regulator agent can be at least a pharmaceutically acceptable material that is used to regulate the pH value, and the material of described adjusting pH value is one or more the material that is selected from the pharmaceutically acceptable material that is used for regulating the pH value of alkali compounds, buffer system, acid and other.
For oral liquid, described auxiliary material is selected from one or more the material in solvent, solubilizer, cosolvent, cosolvent, antiseptic, flavouring, colorant, pH regulator agent, antioxidant, complexing of metal ion agent and other the pharmaceutically acceptable liquid preparation additives, and every kind of assistant can not select or select for use one or more the material in this kind assistant.
Described solvent is to be selected from water, certain density ethanol, glycerine, propylene glycol and other pharmaceutically acceptable liquid preparation with one or more the material in the solvent;
Described antiseptic is to be selected from parabens, sorbic acid and salt thereof, benzoic acid and salt thereof such as methyl p-hydroxybenzoate, ethyl ester, propyl ester, butyl ester, peppermint oil and other pharmaceutically acceptable liquid preparation with one or more the material in the antiseptic;
Described flavouring is to be selected from sucrose, simple syrup, rob, sorbierite, glycerine, sweet mellow wine, saccharin sodium, Herba Menthae Haplocalycis volatile oil and other pharmaceutically acceptable liquid preparation with one or more the material in the flavouring.
For injection, described auxiliary material is selected from pharmaceutically acceptable solvent for injection, solubilizer, bacteriostatic agent, antioxidant, stabilizing agent, complexing agent, analgestic, isotonic regulator, buffering agent, pH regulator agent, and one or more the material in the pharmaceutically acceptable medicine with other subsidiary function, every kind of assistant can not select or select for use one or more the material in this kind assistant.
Described solvent for injection is one or more the material that is selected from water for injection, certain density ethanol, glycerine, propylene glycol, phenmethylol and other the pharmaceutically acceptable solvent for injection;
Described solubilizer is to be selected from polysorbate 20, polyoxyethylene sorbitan monoleate, polysorbate 40, polysorbate 60 and other pharmaceutically acceptable injection with one or more the material in the solubilizer;
Described bacteriostatic agent is to be selected from hydroxypropyl butyl ester, hydroxypropyl methyl esters, phenmethylol, phenol, anesin and other pharmaceutically acceptable injection with one or more the material in the bacteriostatic agent;
Described antioxidant is to be selected from sodium pyrosulfite, sodium bisulfite, sodium sulphite, sodium thiosulfate and other pharmaceutically acceptable injection with one or more the material in the antioxidant;
Described complexing agent is one or more the material that is selected from Calcium Disodium Versenate, cyclohexanediamine four sodium acetates, N-hydroxyl diethylamine three acetic acid, diethyl triamine six acetic acid and other the pharmaceutically acceptable complexing agent.
Described pH regulator agent is one or more the material that is selected from certain density sodium hydroxide solution, certain density hydrochloric acid solution, phosphoric acid, acetic acid-sodium acetate buffer solution, acetate buffer solution, phosphate buffered solution, citrate buffer solution, citric acid-disodium hydrogen phosphate buffer solution and other the pharmaceutically acceptable pH regulator agent.
Wherein said freeze-drying preparation for injection can also not add or add one or more the acceptable an amount of excipient of medicine in preparation.Described excipient is selected from one or more the material in sweet mellow wine, sorbierite, glycocoll, lactose, sodium chloride, glucose and other the pharmaceutically acceptable excipient.The preferred sweet mellow wine of described excipient, sorbierite.
Pharmaceutical preparation of the present invention is determined usage and dosage according to patient's concrete condition in use, but uses every day 1～4 time, uses the pharmaceutical preparation of 1～20 unit dose at every turn, as 1～20 of each use or.
Another purpose of the present invention provides the preparation method who contains the pharmaceutical preparation of genseng and monkshood in a kind of raw material.
The preparation method of pharmaceutical preparation of the present invention is as follows:
Prescription: genseng, monkshood, a synergistic medicine (add or do not add), auxiliary material (add or do not add)
Preparation method's summary: extract the saponin component of the genseng in genseng and the monkshood, the polysaccharide composition of genseng, the alkaloids composition of monkshood, add or do not added synergistic medicine, add or do not add auxiliary material, make the oral solid formulation that contains genseng and monkshood in a kind of raw material, oral semisolid preparation, oral liquid, injection.
The preparation method
One, the extraction of the alkaloids composition of the polysaccharide composition of the saponin component of the genseng in genseng and the monkshood, genseng, monkshood:
(1) processing of genseng: genseng adopts certain density alcohol extract (extracting method adopts reflux extraction or ultrasonic extraction or percolation or infusion process or continuous extraction or decompression extraction method), the gained extract filters, filtrate recycling ethanol is to there not being the alcohol flavor, adjustment liquor strength extremely every 1ml is equivalent to contain genseng crude drug 0.5～4.5g, use water saturated extracting n-butyl alcohol, merge normal butyl alcohol liquid, reclaim normal butyl alcohol to there not being the normal butyl alcohol flavor, change water-soluble, filter, get the saponin component intermediate of genseng, standby;
(2) processing of the genseng dregs of a decoction: the dregs of a decoction elder generation water of genseng after by certain density alcohol extract extracts (extracting method adopts decocting method or reflux extraction or ultrasonic extraction or percolation or infusion process or continuous extraction or decompression extraction method), the gained extract filters, adjusting liquor strength is 0.2～2.4g crude drug/ml, temperature is 20～70 ℃, adds 85～95% ethanol alcohol precipitation and makes and contain the alcohol amount and reach 70～90%, standing over night, filter, precipitation is dry, add the suitable quantity of water dissolving after, centrifugal, it is (or not centrifugal to merge supernatant, filter the back and directly adjust liquor strength), adjusting the supernatant liquor strength is 0.7～6g crude drug/ml, adds 85～95% ethanol and makes and contain alcohol and measure and reach 60～80%, standing over night, filter, precipitation is dry, add the suitable quantity of water dissolving after, centrifugal, get supernatant (or not centrifugal, directly get filtrate after the filtration)
Previous step gained supernatant or filtrate are the polysaccharide composition intermediate of genseng, and is standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid;
Previous step gained supernatant adopts the ultra filtration membrane ultrafiltration of molecular cut off 100K～900K, and ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid, injection;
Or with of the ultra filtration membrane ultrafiltration of previous step gained supernatant elder generation with molecular cut off 100K～900K, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 1K～10K, promptly get the polysaccharide composition intermediate of genseng, standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid, injection;
(3) processing of monkshood: monkshood medicinal material elder generation water extracts (extracting method adopts decocting method or reflux extraction or ultrasonic extraction or percolation or infusion process or continuous extraction or decompression extraction method), the gained extract filters, the relative density of adjusting soup is 1.0～1.25,20～65 ℃ of temperature, ethanol alcohol precipitation with 85～95% makes ethanol content reach 60～80%, standing over night, filter, filtrate recycling ethanol is to there not being the alcohol flavor, the relative density of adjusting soup is that the ethanol of adding 85～95% in 1.05～1.45 o'clock makes pure content reach 75～90%, standing over night filters, and filtrate recycling ethanol is to there not being the alcohol flavor, filter, promptly get the monkshood intermediate, standby, can be used for oral solid formulation, oral semisolid preparation, the preparation of oral liquid;
Filtrate being heated to boiled, and keep little and boiled 10～60 minutes, cooling, refrigeration filters, and promptly gets the monkshood intermediate, and is standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid, injection;
Or precipitation solution reclaims ethanol to there not being the alcohol flavor for the first time, adjustment soup extremely every 1ml is equivalent to contain genseng crude drug 0.7～2.5g, the dichloromethane extraction of 0.5～3 times of amount of each adding 2～9 times, combined dichloromethane liquid reclaims methylene chloride to the greatest extent, changes water-soluble, filter, promptly get the monkshood intermediate, standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid, injection;
Two, play the processing of synergistic medicine:
(4) play the processing of synergistic medicine: playing a synergistic medicine is Chinese medicine or natural drug, extracts its active component or effective constituent monomer, and (make with extra care) is water-soluble, regulates pH value to 5.5～8.5, and filtration promptly gets intermediate, and is standby; Playing synergistic medicine is chemicals, and water-soluble, the pH value transfers to 5.5～8.5, filters, and promptly gets intermediate, standby; (remarks: can be not water-soluble when being used to prepare oral solid formulation, oral semisolid preparation)
Three, the preparation of each pharmaceutical preparation finished product:
(5) intermediate of merging " (1) ", " (2) ", " (3) " gained, mixing;
(6) intermediate with " (1) ", " (2) ", " (3) ", " (4) " gained merges, mixing, gained soup filter, and are condensed into clearly cream and [or " (5) " gained soup are filtered, be condensed into cream clearly, the intermediate not water-soluble with " (4) " mixes], add an amount of oral solid formulation, oral semisolid preparation auxiliary materials and mixing, the system softwood, granulate, oven dry, compressing tablet or incapsulate is made the sheet or the capsule that contain genseng and monkshood in a kind of raw material.
(7) " (5) " gained soup being adjusted volume with purified water or water for injection is 0.1～2.0g crude drug/ml to being equivalent to liquor strength, regulates pH value to 5.5～8.5, is heated to and boils, keep little and boiled 20～60 minutes, and cooling, refrigeration is spent the night; Filter that [or filtrate adds " (the 4) " intermediate of gained and an amount of oral liquid with auxiliary material or do not add auxiliary material, or filtrate adds an amount of oral liquid with auxiliary material or do not add auxiliary material; Mixing, (filtration)], regulate pH value to 5.5～8.5, add purified water or water for injection and adjust volume to full dose, survey the pH value, filter, according to dosage divide to be filled in the oral liquid packing container, seal, the conventional method sterilization promptly gets the oral liquid that contains genseng and monkshood in a kind of raw material.
(8) " (5) " gained soup being adjusted volume with water for injection is 0.1～2.0g crude drug/ml to being equivalent to liquor strength, regulates pH value to 5.5～8.5, is heated to and boils, keep little and boiled 20～60 minutes, and cooling, refrigeration is spent the night; Filter that [or filtrate adds " (the 4) " intermediate of gained and an amount of injection with auxiliary material or do not add auxiliary material, or filtrate adds an amount of injection with auxiliary material or do not add auxiliary material; Mixing, (filtration)], (regulating pH value to 5.5～8.5), adding 0.02～0.5% activated charcoal is heated to and boils, and keeps little and boils 10～60 minutes, filters, regulate pH value to 5.5～8.5, add the injection water and adjust volume, survey the pH value, smart filter (crossing the sintered glass filter in 0.22～0.50 μ m miillpore filter or similar aperture) to full dose, according to dosage divide and be filled to the infusion pump dress with in the container, seal, the conventional method sterilization promptly gets the parenteral solution that contains genseng and monkshood in a kind of raw material.(9) " (5) " gained soup being adjusted volume with water for injection is 0.3～2.0g crude drug/ml to being equivalent to liquor strength, regulates pH value to 5.5～8.5, is heated to and boils, keep little and boiled 20～60 minutes, and cooling, refrigeration is spent the night; Filter [or filtrate add " (the 4) " intermediate of gained and an amount of injection with auxiliary material, freeze-drying preparation for injection with auxiliary material or do not add auxiliary material, or an amount of injection of filtrate adding with auxiliary material, freeze-drying preparation for injection with auxiliary material or do not add auxiliary material; Mixing, (filtration)], regulate pH value to 5.5～8.5, adding 0.02～0.5% activated charcoal is heated to and boils, keeping little boiled 10～60 minutes, filter, regulate pH value to 5.5～8.5, add the injection water and adjust volume, survey the pH value, smart filter (crossing the sintered glass filter in 0.22～0.50 μ m miillpore filter or similar aperture) to full dose, aseptic subpackagedly contain 1～8ml soup to every control injection vial, freeze drying, is rolled lid at gland, promptly gets the freeze-drying preparation for injection that contains genseng and monkshood in a kind of raw material.
(10) freeze drying process is as follows:
The a pre-freeze stage: common pre-freeze, pre-freeze temperature-60 ℃～-40 ℃, about 2.5～6 hours of pre-freeze time; Or select pre-freeze repeatedly, pre-freeze temperature-60 ℃～-40 ℃, the temperature of rising again-15 ℃～-7 ℃, about 3～9 hours of pre-freeze time.
The b lyophilization stage: when the temperature of condenser reduce to-60 ℃～below-45 ℃, open vacuum pump, the temperature of shelf is arranged on-30 ℃～-15 ℃, goods slowly heat up, and observe the distillation interface, all walk only the end of lyophilization stage to free water.
C is drying stage again: shelf temperature slowly rises to-5 ℃～30 ℃, judges dry again terminal point, 3～12 hours drying times again with the vacuum tightness descent method.
Preferred manufacturing procedure
Pharmaceutical preparation preferred manufacturing procedure of the present invention is:
One, the extraction of the alkaloids composition of the polysaccharide composition of the saponin component of the genseng in genseng and the monkshood, genseng, monkshood:
(1) processing of genseng: genseng adds 3～9 times of amounts respectively (for the first time because be dried medicinal material, add 1～3 times of amount) 60～80% alcohol reflux or ultrasonic Extraction 1～4 time, each 0.5～3 hour, merge extract, filter, filtrate recycling ethanol is to there not being the alcohol flavor, adjustment liquor strength extremely every 1ml is equivalent to contain genseng crude drug 0.75～3.0g, the water saturated extracting n-butyl alcohol of 0.7～3 times of amount of each adding 2～9 times merges normal butyl alcohol liquid, reclaims normal butyl alcohol to there being the normal butyl alcohol flavor, change water-soluble, filter, get the saponin component intermediate of genseng, standby;
(2) processing of the genseng dregs of a decoction: the genseng dregs of a decoction after the alcohol extracting add 6～13.5 times of water gagings respectively (for the first time because be dried medicinal material, add 1～5 times of amount) decoct to extract or refluxing extraction 2～6 times, each 0.5～4 hour, merge extract, filter, adjusting liquor strength is 0.4～1.6g crude drug/ml, temperature is 20～60 ℃, adds 90～95% ethanol alcohol precipitation and makes and contain the alcohol amount and reach 75～85%, standing over night, filter, precipitation is dry, add the suitable quantity of water dissolving after, centrifugal, it is (or not centrifugal to merge supernatant, filter the back and directly adjust liquor strength, be used for oral solid formulation, oral semisolid preparation, the preparation of oral liquid), adjusting the supernatant liquor strength is 1.0～4.0g crude drug/ml, adding 90～95% ethanol makes and contains the alcohol amount and reach 65～75%, standing over night filters, and precipitation is dry, after adding the suitable quantity of water dissolving, centrifugal, get supernatant (or not centrifugal, directly get filtrate after the filtration, be used for oral solid formulation, oral semisolid preparation, the preparation of oral liquid)
Previous step gained supernatant or filtrate are the polysaccharide composition intermediate of genseng, and is standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid;
Previous step gained supernatant adopts the ultra filtration membrane ultrafiltration of molecular cut off 100K～700K, and ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid, injection;
Or with of the ultra filtration membrane ultrafiltration of previous step gained supernatant elder generation with molecular cut off 100K～700K, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 1K～8K, promptly get the polysaccharide composition intermediate of genseng, standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid, injection;
(3) processing of monkshood: the monkshood medicinal material adds 4～12 times of water gagings (for the first time because be dried medicinal material, add 1～4 times of amount), decoct extraction or refluxing extraction 2～6 times, each 0.5～3 hour, merge extract, filter, the relative density of adjusting soup is 1.01～1.21,20～60 ℃ of temperature, ethanol alcohol precipitation with 90～95% makes ethanol content reach 65～75%, standing over night filters, and filtrate recycling ethanol is to there not being the alcohol flavor, the relative density of adjusting soup is that the ethanol of adding 90～95% in 1.15～1.40 o'clock makes pure content reach 80～90%, standing over night filters, and filtrate recycling ethanol is to there not being the alcohol flavor, filter, promptly get the monkshood intermediate, standby, can be used for oral solid formulation, oral semisolid preparation, the preparation of oral liquid;
Filtrate being heated to boiled, and keep little and boiled 15～55 minutes, cooling, refrigeration filters, and promptly gets the monkshood intermediate, and is standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid, injection;
Or precipitation solution reclaims ethanol to there not being the alcohol flavor for the first time, adjustment soup extremely every 1ml is equivalent to contain genseng crude drug 0.8～2.0g, the dichloromethane extraction of 0.8～2 times of amount of each adding 3～7 times, combined dichloromethane liquid reclaims methylene chloride to the greatest extent, changes water-soluble, filter, promptly get the monkshood intermediate, standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid, injection;
Two, play the processing of synergistic medicine:
(4) play the processing of synergistic medicine: playing a synergistic medicine is Chinese medicine or natural drug, extracts its active component or effective constituent monomer, and (make with extra care) is water-soluble, regulates pH value to 6～8, and filtration promptly gets intermediate, and is standby; Playing synergistic medicine is chemicals, and water-soluble, the pH value transfers to 6～8, filters, and promptly gets intermediate, standby; (remarks: can be not water-soluble when being used to prepare oral solid formulation, oral semisolid preparation)
Three, the preparation of each pharmaceutical preparation finished product:
(5) intermediate of merging " (1) ", " (2) ", " (3) " gained, mixing;
(6) intermediate with " (1) ", " (2) ", " (3) ", " (4) " gained merges, mixing, gained soup filter, and are condensed into clearly cream and [or " (5) " gained soup are filtered, be condensed into cream clearly, the intermediate not water-soluble with " (4) " mixes], add an amount of oral solid formulation, oral semisolid preparation auxiliary materials and mixing, the system softwood, granulate, oven dry, compressing tablet or incapsulate is made the sheet or the capsule that contain genseng and monkshood in a kind of raw material.
(7) " (5) " gained soup being adjusted volume with purified water or water for injection is 0.1～1.5g crude drug/ml to being equivalent to liquor strength, regulates pH value to 6.0～8.0, is heated to and boils, keep little and boiled 20～60 minutes, and cooling, refrigeration is spent the night; Filter that [or filtrate adds " (the 4) " intermediate of gained and an amount of oral liquid with auxiliary material or do not add auxiliary material, or filtrate adds an amount of oral liquid with auxiliary material or do not add auxiliary material; Mixing, (filtration)], regulate pH value to 6.0～8.0, add purified water or water for injection and adjust volume to full dose, survey the pH value, filter, according to dosage divide to be filled in the oral liquid packing container, seal, the conventional method sterilization promptly gets the oral liquid that contains genseng and monkshood in a kind of raw material.
(8) " (5) " gained soup being adjusted volume with water for injection is 0.1～1.5g crude drug/ml to being equivalent to liquor strength, regulates pH value to 6.0～8.0, is heated to and boils, keep little and boiled 20～60 minutes, and cooling, refrigeration is spent the night; Filter that [or filtrate adds " (the 4) " intermediate of gained and an amount of injection with auxiliary material or do not add auxiliary material, or filtrate adds an amount of injection with auxiliary material or do not add auxiliary material; Mixing, (filtration)], (regulating pH value to 6.0～8.0), adding 0.02～0.3% activated charcoal is heated to and boils, and keeps little and boils 10～40 minutes, filters, regulate pH value to 6.0～8.0, add the injection water and adjust volume, survey the pH value, smart filter (crossing the sintered glass filter in 0.22～0.45 μ m miillpore filter or similar aperture) to full dose, according to dosage divide and be filled to the infusion pump dress with in the container, seal, the conventional method sterilization promptly gets the parenteral solution that contains genseng and monkshood in a kind of raw material.
(9) " (5) " gained soup being adjusted volume with water for injection is 0.5～2.0g crude drug/ml to being equivalent to liquor strength, regulates pH value to 6.0～8.0, is heated to and boils, keep little and boiled 20～60 minutes, and cooling, refrigeration is spent the night; Filter [or filtrate add " (the 4) " intermediate of gained and an amount of injection with auxiliary material, freeze-drying preparation for injection with auxiliary material or do not add auxiliary material, or an amount of injection of filtrate adding with auxiliary material, freeze-drying preparation for injection with auxiliary material or do not add auxiliary material; Mixing, (filtration)], regulate pH value to 6.0～8.0, adding 0.02～0.3% activated charcoal is heated to and boils, keeping little boiled 10～40 minutes, filter, regulate pH value to 6.0～8.0, add the injection water and adjust volume, survey the pH value, smart filter (crossing the sintered glass filter in 0.22～0.45 μ m miillpore filter or similar aperture) to full dose, aseptic subpackagedly contain 1～6ml soup to every control injection vial, freeze drying, is rolled lid at gland, promptly gets the freeze-drying preparation for injection that contains genseng and monkshood in a kind of raw material.
(10) freeze drying process is as follows:
The a pre-freeze stage: common pre-freeze, pre-freeze temperature-60 ℃～-45 ℃, about 2.5～6 hours of pre-freeze time; Or select pre-freeze repeatedly, pre-freeze temperature-60 ℃～-45 ℃, the temperature of rising again-13 ℃～-8 ℃, about 3～9 hours of pre-freeze time.
The b lyophilization stage: when the temperature of condenser reduce to-60 ℃～below-45 ℃, open vacuum pump, the temperature of shelf is arranged on-26 ℃～-15 ℃, goods slowly heat up, and observe the distillation interface, all walk only the end of lyophilization stage to free water.
C is drying stage again: shelf temperature slowly rises to 0 ℃～25 ℃, judges dry again terminal point, 3～12 hours drying times again with the vacuum tightness descent method.
The method of " extraction of the polysaccharide composition of the saponin component of the genseng in genseng and the monkshood, genseng, the alkaloids composition of monkshood " among the preparation method of pharmaceutical preparation of the present invention can be more preferably:
(1) processing of genseng: genseng adds 5～7 times of amounts respectively (for the first time because be dried medicinal material, add 1～2 times of amount) 65～75% alcohol reflux 2～3 times, each 1.5～2.5 hours, merge extract, filter, filtrate recycling ethanol is to there not being the alcohol flavor, adjustment liquor strength extremely every 1ml is equivalent to contain genseng crude drug 1.0～2.0g, the water saturated extracting n-butyl alcohol of 0.8～1.2 times of amount of each adding 5～7 times merges normal butyl alcohol liquid, reclaims normal butyl alcohol to there being the normal butyl alcohol flavor, change water-soluble, filter, get the saponin component intermediate of genseng, standby;
(2) processing of the genseng dregs of a decoction: the genseng dregs of a decoction after the alcohol extracting add 8～10 times of water gagings respectively (for the first time because be dried medicinal material, add 2～4 times of amounts) decoct to extract or refluxing extraction 2～4 times, each 1.5～2.5 hours, merge extract, filter, adjusting liquor strength is 0.6～1.0g crude drug/ml, temperature is 20～45 ℃, adds 95% ethanol alcohol precipitation and makes and contain the alcohol amount and reach 75～85%, standing over night, filter, precipitation is dry, add the suitable quantity of water dissolving after, centrifugal, it is (or not centrifugal to merge supernatant, filter the back and directly adjust liquor strength, be used for oral solid formulation, oral semisolid preparation, the preparation of oral liquid), adjusting the supernatant liquor strength is 1.5～2.5g crude drug/ml, adding 95% ethanol makes and contains the alcohol amount and reach 65～75%, standing over night filters, and precipitation is dry, after adding the suitable quantity of water dissolving, centrifugal, get supernatant (or not centrifugal, directly get filtrate after the filtration, be used for oral solid formulation, oral semisolid preparation, the preparation of oral liquid)
Previous step gained supernatant or filtrate are the polysaccharide composition intermediate of genseng, and is standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid;
Previous step gained supernatant adopts the ultra filtration membrane ultrafiltration of molecular cut off 100K～600K, and ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid, injection;
Or with of the ultra filtration membrane ultrafiltration of previous step gained supernatant elder generation with molecular cut off 100K～600K, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 1K～8K, promptly get the polysaccharide composition intermediate of genseng, standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid, injection;
(3) processing of monkshood: the monkshood medicinal material adds 7～9 times of water gagings (for the first time because be dried medicinal material, add 1～3 times of amount), decoct extraction or refluxing extraction 2～4 times, each 0.5～1.5 hour, merge extract, filter, the relative density of adjusting soup is 1.05～1.16,40～55 ℃ of temperature, ethanol alcohol precipitation with 95% makes ethanol content reach 65～75%, standing over night filters, and filtrate recycling ethanol is to there not being the alcohol flavor, the relative density of adjusting soup is that the ethanol of adding 95% in 1.21～1.35 o'clock makes pure content reach 80～90%, standing over night filters, and filtrate recycling ethanol is to there not being the alcohol flavor, filter, promptly get the monkshood intermediate, standby, can be used for oral solid formulation, oral semisolid preparation, the preparation of oral liquid;
Filtrate being heated to boiled, and keep little and boiled 30～45 minutes, cooling, refrigeration filters, and promptly gets the monkshood intermediate, and is standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid, injection;
Or precipitation solution reclaims ethanol to there not being the alcohol flavor for the first time, adjustment soup extremely every 1ml is equivalent to contain genseng crude drug 0.8～1.2g, the dichloromethane extraction that each adding 0.8-1.2 doubly measures 4～6 times, combined dichloromethane liquid reclaims methylene chloride to the greatest extent, changes water-soluble, filter, promptly get the monkshood intermediate, standby, can be used for the preparation of oral solid formulation, oral semisolid preparation, oral liquid, injection.
A further object of the present invention provides the method for quality control that contains the pharmaceutical preparation of genseng and monkshood in a kind of raw material.Make the stable and controllable for quality of pharmaceutical preparation of the present invention.
The present invention preferably provides the method for quality control that contains the injection of genseng and monkshood in a kind of raw material, comprises following discriminating, inspection, assay, finger-print.
Described discriminating, inspection, assay, finger-print and single concrete grammar wherein can be selected for use respectively or combination in any is used, and are used for the quality control of parenteral solution that a kind of raw material contains genseng and monkshood, freeze-drying preparation for injection, sterile packaged preparation for injection.
Described discriminating, inspection, assay, finger-print comprise genseng, ginsenoside Rg 1, ginsenoside Re's discriminating, the discriminating of monkshood, the limit examine of aconite alkaloids, ginsenoside Rg 1With the assay of polysaccharide composition in the assay of Re, Determination of Total Saponin Content in Panax Ginseng, the genseng, the finger-print of the ginsenoside in ginseng crude drug's finger-print, monkshood medicinal materials fingerprint, the preparation and alkaloidal finger-print.
The method of quality control of parenteral solution, freeze-drying preparation for injection, sterile packaged preparation for injection that contains genseng and monkshood in described a kind of raw material is as follows:
[1] differentiates
[1.1] genseng, ginsenoside Rg 1, the ginsenoside Re discriminating:
Get the injection content 1.0g for preparing, add water 30ml dissolving, add chloroform 30～50ml, jolting, water intaking liquid evaporate to dryness, residue adds water 2ml makes dissolving, added water saturated normal butyl alcohol 8～12ml sonicated 25～40 minutes, get normal butyl alcohol liquid and put in the separating funnel, add the ammonia solution of 2～4 times of amounts, washing, washing lotion discards, get normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Other gets ginseng crude drug 1g (crossing 60 mesh sieves), adds ethanol 20～40ml, and reflux 20～40 minutes filters, and filtrate evaporate to dryness, residue add water 30ml makes dissolving, filters, and filtrate is made ginseng crude drug's solution with method.Get the ginsenoside Rg more respectively 1, ginsenoside Re's reference substance is an amount of, add methyl alcohol and make the solution that every 1ml contains 2mg respectively, in contrast product solution.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B), draw each 2.5～5 μ l of above-mentioned four kinds of solution, put respectively on same silica gel g thin-layer plate, (14～16: 38～42: 21～23: 9～11) lower floor's solution of placing below 10 ℃ is developping agent with chloroform-ethyl acetate-methanol-water, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃.In the test sample chromatogram, with the corresponding position of ginseng crude drug's chromatogram on, show the spot of same color; With the corresponding position of reference substance chromatogram on, show the spot of same color.
[1.2] discriminating of monkshood:
Get the injection content 3.5g for preparing, add water 30ml dissolving, regulate pH value to 9～11 with ammonia solution, the jolting that adds diethyl ether is extracted 2～4 times, each 40～50ml, and ether liquid low temperature evaporate to dryness, residue adds absolute ethyl alcohol 2ml makes dissolving, as need testing solution.Other gets monkshood medicinal material coarse powder 20g, puts in the tool plug conical flask the 130～170ml that adds diethyl ether, jolting 9～12 minutes, add ammonia solution 9～11ml, jolting 25～35 minutes was placed 1～2 hour, divided and got the ether layer, volatilize, residue adds absolute ethyl alcohol 2ml makes dissolving, as monkshood medicinal material solution.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B), draw each 12～30 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate (thick 500 μ m), chromatography cylinder is earlier saturated with ammoniacal liquor, with sherwood oil-ether-acetone (4.8～5.2: 2.8～3.2: 2.8～3.2) be developping agent, launch, take out, dry, spray is with bismuth potassium iodide test solution, and it is clear to be heated to spot colour developing.In the test sample chromatogram, with the corresponding position of monkshood medicinal material chromatogram on, show the spot of same color.
[2] check
[2.1] limit examine of aconite alkaloids:
The preparation of [2.1.1] reference substance solution is learnt from else's experience the phosphorus pentoxide drying under reduced pressure to the aconitine reference substance 5mg of constant weight, accurately claims surely, puts in the 50ml measuring bottle, adds the 0.01mol/L hydrochloric acid solution and makes dissolving, and be diluted to scale, shakes up, promptly.
The preparation precision of [2.1.2] typical curve is measured above-mentioned reference substance solution 0.2～0.3ml, 0.4～0.6ml, 0.65～0.85ml, 0.9～1.1ml, 1.4～1.6ml, 1.65～1.85ml, 1.9～2.1ml, put in the separating funnel respectively, add 0.01mol/L hydrochloric acid solution 1.85～1.65ml respectively, 1.6～1.4ml, 1.35～1.15ml, 1.1～0.9ml, 0.6～0.4ml, 0.35～0.15ml, 0.00ml, accurate respectively adding acetic acid-sodium-acetate buffer (is got 0.2mol/L acetum 250ml, with 0.2mol/L sodium acetate solution adjust pH to 3.1) 8～12ml, bromcresol green liquid (is got bromcresol green 50mg, add 0.05mol/L sodium hydroxide solution 1.6ml grinding and make dissolving, add water to 100ml, extract 3 times with the chloroform jolting, each 30ml, discard chloroform solution) 1.5～2.5ml, chloroform 8～12ml, jolting 2～6 minutes, leave standstill, divide and get chloroform solution, retinue is blank, measure absorbance at 415nm wavelength place according to spectrophotometric degree method (appendix VA of Chinese Pharmacopoeia version in 2005). with concentration is horizontal ordinate, absorbance is an ordinate, the drawing standard curve.
The injection content 0.35g for preparing is got in the preparation of [2.1.3] need testing solution, and accurate the title decides, and adds water 10ml dissolving, is transferred in the separating funnel, adds ammonia solution and regulates pH value to 10～11, extracts combined chloroform liquid, evaporate to dryness 3～5 times with the chloroform jolting of equivalent.Residue adds 0.01mol/L hydrochloric acid solution gradation dissolving, and changes in the 10ml measuring bottle, is diluted to scale and shakes up, promptly.
[2.1.4] determination method precision is measured above-mentioned need testing solution 2ml, puts respectively in the separating funnel, measures absorbance according to " preparation of typical curve " item down from " each precision adds acetic acid-sodium-acetate buffer 10ml " in accordance with the law, calculates, promptly.
Every of the injection that [2.1.5] prepares contains aconite alkaloids with aconitine (C 24H 47NO 11) meter, must not be higher than 1.0mg.
[3] assay
[3.1] ginsenoside Rg 1Assay with Re:
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).
[3.1.1] chromatographic condition is filling agent with the octadecylsilane chemically bonded silica, acetonitrile-0.1% phosphoric acid solution (17～23: 75～85) be moving phase, detect wavelength 203nm.Number of theoretical plate calculates by the ginsenoside Re peak should be not less than 5000.
The preparation of [3.1.2] reference substance solution ginsenoside Rg of phosphorus pentoxide drying under reduced pressure that learn from else's experience to constant weight 1And ginsenoside Re's reference substance is an amount of, accurately claims surely, adds methyl alcohol and makes every 1ml respectively and contain the ginsenoside Rg 10.3mg, the solution of ginsenoside Re 0.2mg, promptly.
The injection content 0.35g for preparing under the content uniformity item is got in the preparation of [3.1.3] need testing solution, accurate claims surely, puts in the 5ml measuring bottle, is dissolved in water and is diluted to scale, shakes up, promptly.
Accurate respectively reference substance solution and each 10～30 μ l of need testing solution of drawing of [3.1.4] determination method inject liquid chromatograph, measure, promptly.
Every of the injection that [3.1.5] prepares contains the ginsenoside Rg 1, the ginsenoside Re the content sum should be 0.2～2.0mg.
[3.2] Determination of Total Saponin Content in Panax Ginseng:
The preparation of [3.2.1] reference substance solution ginsenoside Rb of phosphorus pentoxide drying under reduced pressure that learn from else's experience to constant weight 1Reference substance is an amount of, accurate claims surely, adds methyl alcohol and makes every 1ml and contain ginsenoside Rb 10.3mg solution, promptly.
The preparation precision of [3.2.2] typical curve is measured above-mentioned ginsenoside Rb 1Reference substance solution 0.05～0.15ml, 0.15～0.25ml, 0.25～0.35ml, 0.35～0.45ml, 0.45～0.55ml, 0.55～0.65ml, 0.65～0.75ml puts respectively in the tool plug test tube, fling to solvent, each adds 5% vanillic aldehyde-glacial acetic acid solution (fresh preparation) 0.15～0.25ml and perchloric acid 0.6～1.0ml, heats 12～18min in 55～65 ℃ of water-baths, takes out, cooling rapidly, add glacial acetic acid 3～7ml, shake up, simultaneously with reagent corresponding as blank, measure absorbance in 556nm wavelength place according to spectrophotometric method (appendix VA of Chinese Pharmacopoeia version in 2005), with the absorbance is ordinate, and sampling amount is a horizontal ordinate, the drawing standard curve.
The injection content 0.35g for preparing under the content uniformity item is got in the preparation of [3.2.3] sample solution, the accurate title, decide, adding distil water 10ml dissolving is put in the separating funnel, extracts 3～5 times with the chloroform jolting, each 8～12ml, discard chloroform solution, water liquid extracts 3～5 times with water saturated normal butyl alcohol jolting again, each 8～12ml, merge normal butyl alcohol liquid, use the saturated water washing of normal butyl alcohol 2～3 times again, each 8～12ml discards water liquid, normal butyl alcohol liquid is put evaporate to dryness in the water-bath, residue adds dissolve with methanol, is transferred in the 10ml measuring bottle, and is diluted to scale, shake up, promptly.
[3.2.4] determination method precision is measured sample solution 0.1ml and is put in the tool plug test tube, measures absorbance from " flinging to solvent " down according to " typical curve preparation " in accordance with the law, calculating, promptly.
Every of the injection that [3.2.5] prepares contains general ginsenoside with ginsenoside Rb 1(C 54H 92O 23) meter, should be 2.0～20.0mg.
[3.3] assay of polysaccharide composition in the genseng
The preparation of [3.3.1] reference substance solution phosphorus pentoxide drying under reduced pressure of learning from else's experience is an amount of to the glucose reference substance of constant weight, accurately claims surely, and adding distil water is made the solution that every 1ml contains glucose 0.2mg, promptly.
The preparation precision of [3.3.2] typical curve is measured glucose reference substance solution 0.05～0.15ml, 0.15～0.25ml, 0.25～0.35ml, 0.35～0.45ml, 0.45～0.55ml, 0.55～0.65ml, 0.65～0.75ml, 0.75～0.85ml puts respectively in the tool plug test tube, add water respectively and make into 1.0ml, each accurate freshly prepared 0.2% anthrone sulfuric acid (sulfuric acid concentration is 80%) solution 6～10ml that adds shakes up, and heats 8～12 minutes in 100 ℃ of water-baths, rapidly after the cooling, retinue is blank, measures absorbance at 620nm wavelength place according to spectrophotometric method (appendix VA of Chinese Pharmacopoeia version in 2005), is horizontal ordinate with concentration, absorbance is an ordinate, the drawing standard curve.
The injection content 3.5g for preparing under the content uniformity item is got in the preparation of [3.3.3] sample solution, accurate claim fixed, add water 25～35ml dissolving after, adding ethanol makes and contains the alcohol amount and reach 75～85%, centrifugal, add dissolved in distilled water after precipitation volatilizes ethanol, and be settled in the 100ml measuring bottle.Get above-mentioned solution 1～3ml and put in the 25ml measuring bottle, thin up shakes up to scale, promptly.
[3.3.4] determination method precision is measured 0.2ml in tool plug test tube, measures absorbance from " adding distil water is to 1.0ml respectively " down by " preparation of typical curve " item in accordance with the law, calculates, promptly.
Every of the injection that [3.3.5] prepares contains polysaccharide composition in the genseng with glucose meter, should be 4～60mg.
[4] finger-print
With reference to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D), measure in conjunction with the requirement of finger-print
[4.1] ginseng crude drug's finger-print
[4.1.1] medicinal material title and source
Genseng in this genseng medicinal materials fingerprint is selected red ginseng for use, and red ginseng is the dry root and rhizome of cultivation product (practise and claim " garden ginsent ") after steaming of Araliaceae genseng Panaxginseng C.A.Mey..
The mensuration of [4.1.2] ginseng crude drug finger-print
[4.1.2.1] chromatographic condition chromatographic column: ODS HYPERSIL C 18(post of Φ 4.6mm * 250mm), packing material size 5 μ m; Moving phase: acetonitrile and water are moving phase, and gradient condition sees the following form
Gradient condition
Column temperature: 25～35 ℃; Analysis time: 60～120min; Flow velocity: 0.6～1.0ml/min; 100～120 ℃ of ELSD drift tubes, flow rate of carrier gas 2.0～4.0ml/min.Number of theoretical plate is with ginsenoside Rb 1The peak meter should be not less than 5000.
The preparation of [4.1.2.2] reference substance solution ginsenoside Rb of phosphorus pentoxide drying under reduced pressure that learn from else's experience to constant weight 1Reference substance 2mg, the accurate title, decide, and puts in the 5ml measuring bottle, adds dissolve with methanol and be settled to scale, shakes up, promptly.
Red ginseng medicinal powder (crossing 20 mesh sieves) 2g is got in the preparation of [4.1.2.3] need testing solution, and accurate the title decides, and adds 70% alcohol reflux 2 times, and each 0.8～1.5 hour, add 6～8 times of amounts for the first time, add 5～7 times of amounts for the second time; The extract filtration, filtrate recycling ethanol concentrates, and extracts 4～6 times with the water saturated normal butyl alcohol jolting of equivalent, merges normal butyl alcohol liquid, and washs with the ammonia solution of equivalent; Cleansing solution discards, and normal butyl alcohol liquid evaporate to dryness, residue are dissolved in water and are transferred in the 10ml measuring bottle, are settled to scale, shake up, promptly.
Accurate respectively reference substance solution and each 5～20 μ l of need testing solution of drawing of [4.1.2.4] determination method inject liquid chromatograph, measure, promptly.
[4.1.2.5] result is with ginsenoside Rb 1The peak is set at the object of reference peak, and with the logarithm of its peak area as 1.000, according to ginsenoside Rb 1The retention time at peak is calculated, calibrate 10 total peaks altogether, its relative retention time is respectively: 0.056 ± 10%, 0.102 ± 10%, 0.372 ± 10%, 0.632 ± 10%, 0.931 ± 10%, 1.000,1.040 ± 10%, 1.084 ± 10%, 1.181 ± 10%, 1.662 ± 10%; And the regulation relative retention time is that the relative peak area at 0.632 ± 10%, 1.040 ± 10%, 1.084 ± 10%, 1.181 ± 10% peak is 0.682～1.266,0.346～0.644,0.262～0.486,0.103～0.191 than (peak area logarithm ratio).
[4.2] monkshood medicinal materials fingerprint
The title and the source of [4.2.1] monkshood medicinal material
Monkshood is the processed goods of the sub-root of ranunculaceae plant rhizome of Chinese monkshood Aconitum carmichaeli Debx..Monkshood in this monkshood medicinal materials fingerprint is selected for use black in sheet.
The mensuration of [4.2.2] monkshood medicinal materials fingerprint
[4.2.2.1] chromatographic condition: chromatographic column: ZORBAX SB C 18(Φ 4.6mm * 150mm), packing material size 5 μ m; Moving phase: methyl alcohol and 0.1% phosphoric acid-0.04% triethylamine-aqueous solution are moving phase, and gradient condition sees the following form
Gradient condition
Figure G2006101032344D00171
Column temperature: 25～35 ℃; Detect wavelength: 235nm; Analysis time: 60～120min; Flow velocity: 0.7～1.3ml/min; Number of theoretical plate calculates with the mesaconine peak should be not less than 2000.
The preparation of [4.2.2.2] reference substance solution is learnt from else's experience the phosphorus pentoxide drying under reduced pressure to the mesaconine reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml measuring bottle, adds methyl alcohol and is diluted to scale, shakes up, promptly.
The preparation monkshood medicinal material coarse powder 10g of [4.2.2.3] need testing solution spends the night with ammonia solution 3～5ml, ether 40～60ml cold soaking, filters; The dregs of a decoction add diethyl ether: chloroform (2.8～3.2: 0.8～1.2) mixed solution 40～60ml, sonicated 25～40min, filter, the dregs of a decoction wash 3～4 times with mixed solution, each 14～16ml, and washing lotion and filtrate merge, the low temperature evaporate to dryness, residue adds methyl alcohol 5ml dissolving, with the filter membrane filtration of 0.45 μ m, can measure.
Accurate respectively inner mark solution 15～25 μ l and need testing solution 30～50 μ l of drawing of [4.2.2.4] determination method inject liquid chromatograph, measure, promptly.
[4.2.2.5] result is set at the object of reference peak with the mesaconine peak, retention time according to the mesaconine peak is calculated, calibrate 8 total peaks altogether, its relative retention time is respectively: 0.146 ± 10%, 0.254 ± 10%, 0.531 ± 10%, 0.630 ± 10%, 0.727 ± 10%, 0.833 ± 10%, 1.000,1.114 ± 10%; And with the stable and the biggest relative retention time of peak area be the peak area at 0.531 ± 10% peak as 1.000, and the regulation relative retention time is that the relative peak area ratio at 0.727 ± 10%, 1.114 ± 10% peak is 0.182～0.272,0.373～0.552.
[4.3] ginsenoside finger-print
[4.3.1] chromatographic condition chromatographic column: ODS HYPERSIL C 18(post of Φ 4.6mm * 250mm), packing material size 5 μ m; Acetonitrile and water are moving phase, and gradient condition sees the following form:
Gradient condition
Figure G2006101032344D00172
Column temperature: 25～35 ℃; Analysis time: 60～120min; Flow velocity: 0.6～1.0ml/min; ELSD parameter: 100～120 ℃ of drift tubes, flow rate of carrier gas 2.0～4.0ml/min.Number of theoretical plate is with ginsenoside Rb 1The peak calculates should be not less than 5000.
The preparation of [4.3.2] reference substance solution ginsenoside Rb of phosphorus pentoxide drying under reduced pressure that learn from else's experience to constant weight 1Reference substance 2mg, the accurate title, decide, and puts in the 5ml measuring bottle, adds dissolve with methanol and be settled to scale, shakes up, promptly.
The injection content 0.35g for preparing is got in the preparation of [4.3.3] need testing solution, the accurate title, decide, be dissolved in water to 8～12ml, extract 4～6 times, merge normal butyl alcohol liquid with the water-saturated n-butanol jolting of equivalent, ammonia solution with equivalent washs 1～2 time, divide and to get normal butyl alcohol liquid, reclaim normal butyl alcohol, residue is with dissolved in distilled water and be settled in the 5ml measuring bottle, shake up, promptly.
Accurate respectively reference substance solution and each 5～15 μ l of need testing solution of drawing of [4.3.4] determination method inject liquid chromatograph, measure, promptly.
[4.3.5] result is with ginsenoside Rb 1The peak is set at the object of reference peak, and with the logarithm of its peak area as 1.000, according to ginsenoside Rb 1The retention time at peak is calculated, calibrate 10 total peaks altogether, its relative retention time is respectively: 0.441 ± 10%, 0.632 ± 10%, 0.942 ± 10%, 1.000,1.042 ± 10%, 1.062 ± 10%, 1.089 ± 10%, 1.188 ± 10%, 1.701 ± 10%, 1.737 ± 10%; And the regulation relative retention time is that the relative peak area at 0.441 ± 10%, 0.632 ± 10%, 1.042 ± 10%, 1.089 ± 10% peak is respectively 0.688～1.148,0.673～1.121,0.692～1.154,0.688～1.146 than (peak area logarithm ratio).
[4.4] fingerprint of alkaloid
[4.4.1] chromatographic condition chromatographic column: ZORBAX SB C 18(post of Φ 4.6mm * 150mm), packing material size 5 μ m; Methyl alcohol and 0.1% phosphoric acid-0.04% triethylamine aqueous solution are moving phase, and gradient condition sees the following form:
Gradient condition
25～35 ℃ of column temperatures; Detect wavelength: 235nm; Analysis time: 60～120min; Number of theoretical plate calculates with the mesaconine peak should be not less than 2000.
The preparation of [4.4.2] inner mark solution is learnt from else's experience the phosphorus pentoxide drying under reduced pressure to the mesaconine reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml measuring bottle, adds 1% phosphoric acid solution 3ml, is diluted to scale with methyl alcohol, shakes up, promptly.
The preparation precision of [4.4.3] need testing solution is measured the injection content 1.0g for preparing, be dissolved in water to 8～12ml, put in the separating funnel, add ammonia solution adjust pH to 9～11, with the E-C (2.8～3.2: mixed liquor jolting extraction 0.8～1.2) 4～6 times of equivalent, combining extraction liquid, low temperature volatilizes, and residue adds methyl alcohol 1ml makes dissolving, adds inner mark solution 40～60 μ l, filter, as need testing solution.
Accurate respectively inner mark solution 15～25 μ l and need testing solution 30～50 μ l of drawing of [4.4.4] determination method inject liquid chromatograph, measure, promptly.
[4.4.5] result is set at the mesaconine peak at the object of reference peak of relative retention time, retention time according to the mesaconine peak is calculated, calibrate 5 total peaks altogether, its relative retention time is respectively: 0.259 ± 10%, 0.342 ± 10%, 0.532 ± 10%, 0.724 ± 10%, 1.000 ± 10%; And with the stable and the biggest relative retention time of peak area be the peak area at 0.532 ± 10% peak as 1.000, and the regulation relative retention time is that the relative peak area ratio at 0.724 ± 10% peak is 0.455～0.683.
Contain parenteral solution, the freeze-drying preparation for injection of genseng and monkshood, the method for quality control of sterile packaged preparation for injection in described a kind of raw material, its each concrete steps are as follows:
[1] differentiates
[1.1] genseng, ginsenoside Rg 1, the ginsenoside Re discriminating:
Get the injection content 1.0g for preparing, add water 30ml dissolving, add chloroform 40ml, jolting, water intaking liquid evaporate to dryness, residue adds water 2ml makes dissolving, added water saturated normal butyl alcohol 10ml sonicated 30 minutes, get normal butyl alcohol liquid and put in the separating funnel, add the ammonia solution of 3 times of amounts, washing, washing lotion discards, get normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Other gets ginseng crude drug 1g (crossing 60 mesh sieves), adds ethanol 30ml, and reflux 30 minutes filters, and filtrate evaporate to dryness, residue add water 30ml makes dissolving, filters, and filtrate is made ginseng crude drug's solution with method.Get the ginsenoside Rg more respectively 1, ginsenoside Re's reference substance is an amount of, add methyl alcohol and make the solution that every 1ml contains 2mg respectively, in contrast product solution.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B), draw each 2.5～5 μ l of above-mentioned four kinds of solution, put respectively on same silica gel g thin-layer plate, (15: 40: 22: 10) lower floor's solution of placing below 10 ℃ was developping agent with chloroform-ethyl acetate-methanol-water, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃.In the test sample chromatogram, with the corresponding position of ginseng crude drug's chromatogram on, show the spot of same color; With the corresponding position of reference substance chromatogram on, show the spot of same color.
[1.2] discriminating of monkshood:
Get the injection content 3.5g for preparing, add water 30ml dissolving, regulate pH value to 10 with ammonia solution, the jolting that adds diethyl ether is extracted 3 times, each 45ml, ether liquid low temperature evaporate to dryness, residue adds absolute ethyl alcohol 2ml makes dissolving, as need testing solution. and other gets monkshood medicinal material coarse powder 20g, puts in the tool plug conical flask, 150ml adds diethyl ether, jolting 10 minutes adds ammonia solution 10ml, jolting 30 minutes, placed 1～2 hour, divide and get the ether layer, volatilize, residue adds absolute ethyl alcohol 2ml makes dissolving, as monkshood medicinal material solution. according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B) test, draw each 12～30 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate (thick 500 μ m), chromatography cylinder is earlier saturated with ammoniacal liquor, with sherwood oil-ether-acetone (5: 3: 3) is developping agent, launch, take out, dry, spray is with bismuth potassium iodide test solution, and it is clear to be heated to the spot colour developing.In the test sample chromatogram, with the corresponding position of monkshood medicinal material chromatogram on, show the spot of same color.
[2] check
[2.1] limit examine of aconite alkaloids:
The preparation of [2.1.1] reference substance solution is learnt from else's experience the phosphorus pentoxide drying under reduced pressure to the aconitine reference substance 5mg of constant weight, accurately claims surely, puts in the 50ml measuring bottle, adds the 0.01mol/L hydrochloric acid solution and makes dissolving, and be diluted to scale, shakes up, promptly.
The preparation precision of [2.1.2] typical curve is measured above-mentioned reference substance solution 0.25ml, 0.50ml, 0.75ml, 1.00ml, 1.50ml, 1.75ml, 2.00ml, put in the separating funnel respectively, add 0.01mol/L hydrochloric acid solution 1.75ml respectively, 1.50ml, 1.25ml, 1.00ml, 0.50ml, 0.25ml, 0.00ml, accurate respectively adding acetic acid-sodium-acetate buffer (is got 0.2mol/L acetum 250ml, with 0.2mol/L sodium acetate solution adjust pH to 3.1) 10ml, bromcresol green liquid (is got bromcresol green 50mg, add 0.05mol/L sodium hydroxide solution 1.6ml grinding and make dissolving, add water to 100ml, extract 3 times with the chloroform jolting, each 30ml, discard chloroform solution) 2ml, chloroform 10ml, jolting 3 minutes is left standstill, and divides and gets chloroform solution, retinue is blank, measures absorbance according to spectrophotometric degree method (appendix VA of Chinese Pharmacopoeia version in 2005) 415nm wavelength place.With concentration is horizontal ordinate, and absorbance is an ordinate, the drawing standard curve.
The injection content 0.35g for preparing is got in the preparation of [2.1.3] need testing solution, and accurate the title decides, and adds water 10ml dissolving, is transferred in the separating funnel, adds ammonia solution and regulates pH value to 10～11, extracts combined chloroform liquid, evaporate to dryness 4 times with the chloroform jolting of equivalent.Residue adds 0.01mol/L hydrochloric acid solution gradation dissolving, and changes in the 10ml measuring bottle, is diluted to scale and shakes up, promptly.
[2.1.4] determination method precision is measured above-mentioned need testing solution 2ml, puts respectively in the separating funnel, measures absorbance according to " preparation of typical curve " item down from " each precision adds acetic acid-sodium-acetate buffer 10ml " in accordance with the law, calculates, promptly.
Every of the injection that [2.1.5] prepares contains aconite alkaloids with aconitine (C 24H 47NO 11) meter, must not be higher than 1.0mg.
[3] assay
[3.1] ginsenoside Rg 1Assay with Re:
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).
[3.1.1] chromatographic condition and system suitability test are filling agent with the octadecylsilane chemically bonded silica, and acetonitrile-0.1% phosphoric acid solution (20: 80) is a moving phase, detects wavelength 203nm.Number of theoretical plate calculates by the ginsenoside Re peak should be not less than 5000.
The preparation of [3.1.2] reference substance solution ginsenoside Rg of phosphorus pentoxide drying under reduced pressure that learn from else's experience to constant weight 1And ginsenoside Re's reference substance is an amount of, accurately claims surely, adds methyl alcohol and makes every 1ml respectively and contain the ginsenoside Rg 10.3mg, the solution of ginsenoside Re 0.2mg, promptly.
The injection content 0.35g for preparing under the content uniformity item is got in the preparation of [3.1.3] need testing solution, accurate claims surely, puts in the 5ml measuring bottle, is dissolved in water and is diluted to scale, shakes up, promptly.
Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of [3.1.4] determination method inject liquid chromatograph, measure, promptly.
Every of the injection that [3.1.5] prepares contains the ginsenoside Rg 1, the ginsenoside Re the content sum should be 0.8～1.4mg.
[3.2] Determination of Total Saponin Content in Panax Ginseng:
The preparation of [3.2.1] reference substance solution ginsenoside Rb of phosphorus pentoxide drying under reduced pressure that learn from else's experience to constant weight 1Reference substance is an amount of, accurate claims surely, adds methyl alcohol and makes every 1ml and contain ginsenoside Rb 10.3mg solution, promptly.
The preparation precision of [3.2.2] typical curve is measured above-mentioned ginsenoside Rb 1Reference substance solution 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml 0.7ml puts respectively in the tool plug test tube, fling to solvent, each adds 5% vanillic aldehyde-glacial acetic acid solution (fresh preparation) 0.2ml and perchloric acid 0.8ml, heats 15min in 60 ℃ of water-baths, takes out, cooling rapidly, add glacial acetic acid 5ml, shake up, simultaneously with reagent corresponding as blank, measure absorbance in 556nm wavelength place according to spectrophotometric method (appendix VA of Chinese Pharmacopoeia version in 2005), with the absorbance is ordinate, and sampling amount is a horizontal ordinate, the drawing standard curve.
The injection content 0.35g for preparing under the content uniformity item is got in the preparation of [3.2.3] sample solution, the accurate title, decide, adding distil water 10ml dissolving is put in the separating funnel, extracts 4 times with the chloroform jolting, each 10ml, discard chloroform solution, water liquid extracts 4 times with water saturated normal butyl alcohol jolting again, each 10ml, merge normal butyl alcohol liquid, use the saturated water washing of normal butyl alcohol 2 times again, each 10ml discards water liquid, normal butyl alcohol liquid is put evaporate to dryness in the water-bath, residue adds dissolve with methanol, is transferred in the 10ml measuring bottle, and is diluted to scale, shake up, promptly.
[3.2.4] determination method precision is measured sample solution 0.1ml and is put in the tool plug test tube, measures absorbance from " flinging to solvent " down according to " typical curve preparation " in accordance with the law, calculating, promptly.
Every of the injection that [3.2.5] prepares contains general ginsenoside with ginsenoside Rb 1(C 54H 92O 23) meter, should be 8.0～15.0mg.
[3.3] assay of polysaccharide composition in the genseng
The preparation of [3.3.1] reference substance solution phosphorus pentoxide drying under reduced pressure of learning from else's experience is an amount of to the glucose reference substance of constant weight, accurately claims surely, and adding distil water is made the solution that every 1ml contains glucose 0.2mg, promptly.
The preparation precision of [3.3.2] typical curve is measured glucose reference substance solution 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml, 0.7ml 0.8ml puts respectively in the tool plug test tube, add water respectively and make into 1.0ml, each accurate freshly prepared 0.2% anthrone sulfuric acid (sulfuric acid concentration is 80%) solution 8ml that adds shakes up, and heating is 10 minutes in 100 ℃ of water-baths, rapidly after the cooling, retinue is blank, measures absorbance at 620nm wavelength place according to spectrophotometric method (appendix VA of Chinese Pharmacopoeia version in 2005), is horizontal ordinate with concentration, absorbance is an ordinate, the drawing standard curve.
The injection content 3.5g for preparing under the content uniformity item is got in the preparation of [3.3.3] sample solution, accurate claim fixed, add water 30ml dissolving after, adding ethanol makes and contains the alcohol amount and reach 80%, centrifugal, add dissolved in distilled water after precipitation volatilizes ethanol, and be settled in the 100ml measuring bottle.Get above-mentioned solution 2ml and put in the 25ml measuring bottle, thin up shakes up to scale, promptly.
[3.3.4] determination method precision is measured 0.2ml in tool plug test tube, measures absorbance from " adding distil water is to 1.0ml respectively " down by " preparation of typical curve " item in accordance with the law, calculates, promptly.
Every of the injection that [3.3.5] prepares contains polysaccharide composition in the genseng with glucose meter, should be 27～47mg.
[4] finger-print
With reference to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D), measure in conjunction with the requirement of finger-print
[4.1] ginseng crude drug's finger-print
[4.1.1] medicinal material title and source
Genseng in this genseng medicinal materials fingerprint is selected red ginseng for use, and red ginseng is the dry root and rhizome of cultivation product (practise and claim " garden ginsent ") after steaming of Araliaceae genseng PanaxginsengC.A.Mey..
The mensuration of [4.1.2] ginseng crude drug finger-print
[4.1.2.1] chromatographic condition chromatographic column: ODS HYPERSIL C 18(post of Φ 4.6mm * 250mm), packing material size 5 μ m; Moving phase: acetonitrile and water are moving phase, and gradient condition sees the following form
Gradient condition
Figure G2006101032344D00221
Column temperature: 30 ℃; Analysis time: 60min; Flow velocity: 0.8ml/min; 110 ℃ of ELSD drift tubes, flow rate of carrier gas 3.0ml/min.Number of theoretical plate is with ginsenoside Rb 1The peak meter should be not less than 5000.
The preparation of [4.1.2.2] reference substance solution: the phosphorus pentoxide drying under reduced pressure of learning from else's experience is to ginsenoside Rb1's reference substance 2mg of constant weight, accurately claims surely, puts in the 5ml measuring bottle, adds dissolve with methanol and is settled to scale, shakes up, promptly.
Red ginseng medicinal powder (crossing 20 mesh sieves) 2g is got in the preparation of [4.1.2.3] need testing solution, and accurate the title decides, and adds 70% alcohol reflux twice, and each 1 hour, add 7 times of amounts for the first time, add 6 times of amounts for the second time; The extract filtration, filtrate recycling ethanol concentrates, and extracts 5 times with the water saturated normal butyl alcohol jolting of equivalent, merges normal butyl alcohol liquid, and washs with the ammonia solution of equivalent; Cleansing solution discards, and normal butyl alcohol liquid evaporate to dryness, residue are dissolved in water and are transferred in the 10ml measuring bottle, are settled to scale, shake up, promptly.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of [4.1.2.4] determination method inject liquid chromatograph, measure, promptly.
[4.1.2.5] result is with ginsenoside Rb 1The peak is set at the object of reference peak, and with the logarithm of its peak area as 1.000, according to ginsenoside Rb 1The retention time at peak is calculated, calibrate 10 total peaks altogether, its relative retention time is respectively: 0.056 ± 10%, 0.102 ± 10%, 0.372 ± 10%, 0.632 ± 10%, 0.931 ± 10%, 1.000,1.040 ± 10%, 1.084 ± 10%, 1.181 ± 10%, 1.662 ± 10%; And the regulation relative retention time is that the relative peak area at 0.632 ± 10%, 1.040 ± 10%, 1.084 ± 10%, 1.181 ± 10% peak is 0.682～1.266,0.346～0.644,0.262～0.486,0.103～0.191 than (peak area logarithm ratio).
[4.2] monkshood medicinal materials fingerprint
The title and the source of [4.2.1] monkshood medicinal material
Monkshood is the processed goods of the sub-root of ranunculaceae plant rhizome of Chinese monkshood Aconitum carmichaeli Debx..Monkshood in this monkshood medicinal materials fingerprint is selected for use black in sheet.
The mensuration of [4.2.2] monkshood medicinal materials fingerprint
[4.2.2.1] chromatographic condition: chromatographic column: ZORBAX SBC 18(post of Φ 4.6mm * 150mm), packing material size 5 μ m; Moving phase: methyl alcohol and 0.1% phosphoric acid-0.04% triethylamine-aqueous solution are moving phase, and gradient condition sees the following form
Gradient condition
Figure G2006101032344D00231
Column temperature: 30 ℃; Detect wavelength: 235nm; Analysis time: 60min; Flow velocity: 1.0ml/min; Number of theoretical plate calculates with the mesaconine peak should be not less than 2000.
The preparation of [4.2.2.2] reference substance solution is learnt from else's experience the phosphorus pentoxide drying under reduced pressure to the mesaconine reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml measuring bottle, adds methyl alcohol and is diluted to scale, shakes up, promptly.
The preparation monkshood medicinal material coarse powder 10g of [4.2.2.3] need testing solution spends the night with ammonia solution 4ml, ether 50ml cold soaking, filters; The dregs of a decoction add diethyl ether: chloroform (3: 1) mixed solution 50ml, and sonicated 30min filters, and the dregs of a decoction wash 3～4 times with mixed solution, each 15ml, washing lotion and filtrate merge, the low temperature evaporate to dryness, residue adds methyl alcohol 5ml dissolving, with the filter membrane filtration of 0.45 μ m, can measure.
Accurate respectively inner mark solution 20 μ l and the need testing solution 40 μ l of drawing of [4.2.2.4] determination method inject liquid chromatograph, measure, promptly.
[4.2.2.5] result is set at the object of reference peak with the mesaconine peak, retention time according to the mesaconine peak is calculated, calibrate 8 total peaks altogether, its relative retention time is respectively: 0.146 ± 10%, 0.254 ± 10%, 0.531 ± 10%, 0.630 ± 10%, 0.727 ± 10%, 0.833 ± 10%, 1.000,1.114 ± 10%; And with the stable and the biggest relative retention time of peak area be the peak area at 0.531 ± 10% peak as 1.000, and the regulation relative retention time is that the relative peak area ratio at 0.727 ± 10%, 1.114 ± 10% peak is 0.182～0.272,0.373～0.552.
[4.3] ginsenoside finger-print
[4.3.1] chromatographic condition chromatographic column: ODS HYPERSIL C 18(post of Φ 4.6mm * 250mm), packing material size 5 μ m; Acetonitrile and water are moving phase, and gradient condition sees the following form:
Gradient condition
Column temperature: 30 ℃; Analysis time: 60min; Flow velocity: 0.8ml/min; ELSD parameter: 110 ℃ of drift tubes, flow rate of carrier gas 3.0ml/min.Number of theoretical plate is with ginsenoside Rb 1The peak calculates should be not less than 5000.
The preparation of [4.3.2] reference substance solution ginsenoside Rb of phosphorus pentoxide drying under reduced pressure that learn from else's experience to constant weight 1Reference substance 2mg, the accurate title, decide, and puts in the 5ml measuring bottle, adds dissolve with methanol and be settled to scale, shakes up, promptly.
The injection content 0.35g for preparing is got in the preparation of [4.3.3] need testing solution, the accurate title, decide, be dissolved in water to 10ml, extract 5 times, merge normal butyl alcohol liquid with the water-saturated n-butanol jolting of equivalent, ammonia solution with equivalent washs 1 time, divide and to get normal butyl alcohol liquid, reclaim normal butyl alcohol, residue is with dissolved in distilled water and be settled in the 5ml measuring bottle, shake up, promptly.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of [4.3.4] determination method inject liquid chromatograph, measure, promptly.
[4.3.5] result is with ginsenoside Rb 1The peak is set at the object of reference peak, and with the logarithm of its peak area as 1.000, according to ginsenoside Rb 1The retention time at peak is calculated, calibrate 10 total peaks altogether, its relative retention time is respectively: 0.441 ± 10%, 0.632 ± 10%, 0.942 ± 10%, 1.000,1.042 ± 10%, 1.062 ± 10%, 1.089 ± 10%, 1.188 ± 10%, 1.701 ± 10%, 1.737 ± 10%; And the regulation relative retention time is that the relative peak area at 0.441 ± 10%, 0.632 ± 10%, 1.042 ± 10%, 1.089 ± 10% peak is respectively 0.688～1.148,0.673～1.121,0.692～1.154,0.688～1.146 than (peak area logarithm ratio).
[4.4] fingerprint of alkaloid
[4.4.1] chromatographic condition chromatographic column: ZORBAX SB C 18(post of Φ 4.6mm * 150mm), packing material size 5 μ m; Methyl alcohol and 0.1% phosphoric acid-0.04% triethylamine aqueous solution are moving phase, and gradient condition sees the following form:
Gradient condition
Figure G2006101032344D00251
30 ℃ of column temperatures; Detect wavelength: 235nm; Analysis time: 60min; Number of theoretical plate calculates with the mesaconine peak should be not less than 2000.
The preparation of [4.4.2] inner mark solution is learnt from else's experience the phosphorus pentoxide drying under reduced pressure to the mesaconine reference substance 5mg of constant weight, accurately claims surely, puts in the 25ml measuring bottle, adds 1% phosphoric acid solution 3ml, is diluted to scale with methyl alcohol, shakes up, promptly.
The preparation precision of [4.4.3] need testing solution is measured the injection content 1.0g for preparing, be dissolved in water to 10ml, put in the separating funnel, add ammonia solution adjust pH to 10, use the mixed liquor jolting of the E-C (3: 1) of equivalent to extract 5 times, combining extraction liquid, low temperature volatilizes, and residue adds methyl alcohol 1ml makes dissolving, adds inner mark solution 50 μ l, filter, as need testing solution.
Accurate respectively inner mark solution 20 μ l and the need testing solution 40 μ l of drawing of [4.4.4] determination method inject liquid chromatograph, measure, promptly.
[4.4.5] result is set at the mesaconine peak at the object of reference peak of relative retention time, retention time according to the mesaconine peak is calculated, calibrate 5 total peaks altogether, its relative retention time is respectively: 0.259 ± 10%, 0.342 ± 10%, 0.532 ± 10%, 0.724 ± 10%, 1.000 ± 10%; And with the stable and the biggest relative retention time of peak area be the peak area at 0.532 ± 10% peak as 1.000, and the regulation relative retention time is that the relative peak area ratio at 0.724 ± 10% peak is 0.455～0.683.
Pharmaceutical preparation of the present invention, kept the polysaccharide composition in the genseng, can add synergistic medicine, more comprehensively keep and developed the good drug effect of traditional ancient prescription " shenfu decoction ", adopted advanced method of quality control simultaneously, as fingerprint pattern technology, make the stable and controllable for quality of pharmaceutical preparation of the present invention, change the relatively poor shortcoming of quality controllability of Chinese medicine preparation.Can bring into play better clinical effect, select for clinical use provides how better medication.
Oral solid formulation of the present invention carries taking convenience, significantly reduced injection volume, changed the traditional Chinese medicine amount big, carry the shortcoming of taking inconvenience; The existing commercially available ginseng aconite injection liquid of the kind analogy of the effective constituent of parenteral solution of the present invention is many, can more comprehensively keep and develop the curative effect of ancient prescription, and onset is rapid; Freeze-drying preparation for injection of the present invention is on the basis of all advantages of parenteral solution, and the storage time is longer, and character is more stable, and onset is rapid.The inventor is by a large amount of quadrature screening experiments simultaneously, and preferred suitable preparation method can adopt hyperfiltration technique in the present invention, removes big molecular impurity, also can remove small molecular weight impurity again, better guarantees stability of formulation.
The preparation method of pharmaceutical preparation of the present invention is not loaded down with trivial details simultaneously, can control cost preferably.The preparation nature of making is stable, and is quality controllable, Orally-administrable or drug administration by injection.Can be used for treating fainting that yang-energy takes off cruelly and take off disease (infectivity, losing blood property, hypovolemic shock etc.); Also can be used for due to the deficiency of yang (deficiency of vital energy) palpitation with fear, palpitation, breath with cough, have a stomach-ache, have loose bowels, rheumatism etc., and other former " shenfu decoction " disease of controlling.Can select for clinical use provides how better medication.
The main pharmacodynamics experiment of pharmaceutical preparation of the present invention shows that pharmaceutical preparation of the present invention has good pharmacological action.
From the experiment of the main pharmacodynamics of pharmaceutical preparation of the present invention as can be seen, " the injection ginseng attached (freeze-drying) " that the present invention makes, the main pharmacodynamics experiment shows under Isodose, (Sanjiu Pharmaceutical Industry Co., Ltd., Ya'an City produces with the ginseng aconite injection liquid of selling in the market for it, lot number: 041103) compare, the time-to-live of the anti-anoxic of injection ginseng attached (freeze-drying) high dose group prolongation mouse normal pressure is longer significantly, and the effect that increases coronary flow, inhibition CK, LDH release is stronger.Proved beneficial effect of the present invention.
Embodiment
Below will the invention will be further described by embodiment, these descriptions are not that content of the present invention is done further qualification.One skilled in the art will understand that the equal replacement that technical characterictic of the present invention is done, or corresponding the improvement, still belong within protection scope of the present invention.
Embodiment 1
Prescription:
Figure G2006101032344D00261
The preparation method:
(1) genseng adds 6 times of amounts (because be dried medicinal material, adding 1 times of amount for the first time), 70% alcohol reflux 2 times, each 2 hours respectively, filter, filtrate recycling ethanol is not to there being the alcohol flavor, and soup is concentrated into every 1ml and is equivalent to contain genseng crude drug 1.45～1.55g, and the water-saturated n-butanol that at every turn adds equivalent extracts 6 times, merge normal butyl alcohol liquid, reclaim normal butyl alcohol to there not being the normal butyl alcohol flavor, change water-soluble, filter, get the ginsenoside intermediate, standby;
(2) the red ginseng dregs of a decoction after the alcohol extracting add 9 times of water gagings respectively (for the first time because be dried medicinal material, add 3 times of amounts) decoct to extract or refluxing extraction 3 times, each 2 hours, filter, being concentrated into liquor strength is 0.75～0.85g crude drug/ml, temperature is 38～42 ℃, adds 95% ethanol alcohol precipitation and makes and contain the alcohol amount and reach 80%, standing over night, filter, precipitation is dry, add the suitable quantity of water dissolving after, centrifugal, merge supernatant, adjusting the supernatant liquor strength is 1.95～2.05g crude drug/ml, adds 95% ethanol and makes and contain the alcohol amount and reach 70%, standing over night, filter, precipitation is dry, add the suitable quantity of water dissolving after, centrifugal, it is (or not centrifugal to get supernatant, directly get filtrate after the filtration), previous step gained supernatant or filtrate are the polysaccharide composition intermediate of genseng, and is standby;
Or the ultra filtration membrane ultrafiltration of previous step gained supernatant employing molecular cut off 100K, ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby;
Or the ultra filtration membrane ultrafiltration of previous step gained supernatant employing molecular cut off 200K, ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby;
Or the ultra filtration membrane ultrafiltration of previous step gained supernatant employing molecular cut off 500K, ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby;
Or the ultra filtration membrane ultrafiltration of molecular cut off 200K of previous step gained supernatant elder generation, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 5K, promptly get the polysaccharide composition intermediate of genseng, standby;
Or the ultra filtration membrane ultrafiltration of molecular cut off 500K of previous step gained supernatant elder generation, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 2K, promptly get the polysaccharide composition intermediate of genseng, standby;
Or the ultra filtration membrane ultrafiltration of molecular cut off 500K of previous step gained supernatant elder generation, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 5K, promptly get the polysaccharide composition intermediate of genseng, standby;
(3) the monkshood medicinal material adds 8 times of water gagings (for the first time because be dried medicinal material, add 2 times of amounts), decoct extraction or refluxing extraction 3 times, each 1 hour, filter, be concentrated into relative density of medicine liquid 1.09～1.13,47～53 ℃ of temperature, ethanol alcohol precipitation with 95% makes ethanol content reach 70%, and standing over night filters, filtrate recycling ethanol is to there not being the alcohol flavor, the relative density of adjusting soup is that the ethanol of adding 95% in 1.27～1.31 o'clock makes pure content reach 85%, and standing over night filters, filtrate recycling ethanol is to there not being the alcohol flavor, filter, promptly get the monkshood intermediate, standby;
Filtrate being heated to boiled, and keep little and boiled 38～45 minutes, cooling, refrigeration filters, and promptly gets the monkshood intermediate, and is standby;
Or for the first time precipitation solution reclaims ethanol to there not being the alcohol flavor, adjusts soup and is equivalent to contain genseng crude drug 0.95～1.05g to every 1ml, adds the dichloromethane extraction 5 times of 1.0～1.1 times of amounts at every turn, combined dichloromethane liquid reclaims methylene chloride to the greatest extent, changes water-soluble, filter, promptly get the monkshood intermediate, standby;
(4) intermediate with " (1) ", " (2) ", " (3) " gained merges, and mixing filters, and is condensed into cream clearly;
(5) will cross 60～80 mesh sieves except that the auxiliary material 95% ethanol, the dolomol, mixing with " (4) " gains mixing, adds 95% an amount of ethanol, make softwood, pushed 20～24 mesh sieves and granulate, 40 ℃ to 65 ℃ dry 1h to 4h cross the whole grain of 20 mesh sieves, add dolomol, mixing
(6) with above-mentioned particle compressing tablet, promptly get the sheet that contains genseng and monkshood in a kind of raw material;
(7), promptly get the tablet that contains genseng and monkshood in the raw material with " (6) " gained sheet bag film-coating or sugar coating;
(8) " (5) " gained is particles filled to capsule, with the polishing of gained capsule, promptly get the capsule that contains genseng and monkshood in a kind of raw material then.
Embodiment 2
Prescription:
Figure G2006101032344D00281
Figure G2006101032344D00282
Figure G2006101032344D00283
Figure G2006101032344D00284
Figure G2006101032344D00285
The preparation method:
Remarks: the preparation method of similar preparation prescription is identical in the present embodiment.
(1) genseng adds 6 times of amounts (because be dried medicinal material, adding 1 times of amount for the first time), 70% alcohol reflux 2 times, each 2 hours respectively, filter, filtrate recycling ethanol is not to there being the alcohol flavor, and soup is concentrated into every 1ml and is equivalent to contain genseng crude drug 1.45～1.55g, and the water-saturated n-butanol that at every turn adds equivalent extracts 6 times, merge normal butyl alcohol liquid, reclaim normal butyl alcohol to there not being the normal butyl alcohol flavor, change water-soluble, filter, get the ginsenoside intermediate, standby;
(2) the red ginseng dregs of a decoction after the alcohol extracting add 9 times of water gagings respectively (for the first time because be dried medicinal material, add 3 times of amounts) decoct to extract or refluxing extraction 3 times, each 2 hours, filter, being concentrated into liquor strength is 0.75～0.85g crude drug/ml, temperature is 38～42 ℃, adds 95% ethanol alcohol precipitation and makes and contain the alcohol amount and reach 80%, standing over night, filter, precipitation is dry, add the suitable quantity of water dissolving after, centrifugal, merge supernatant, adjusting the supernatant liquor strength is 1.95～2.05g crude drug/ml, adds 95% ethanol and makes and contain the alcohol amount and reach 70%, standing over night, filter, precipitation is dry, add the suitable quantity of water dissolving after, centrifugal, it is (or not centrifugal to get supernatant, directly get filtrate after the filtration, be used for the preparation of oral liquid)
Previous step gained supernatant or filtrate are the polysaccharide composition intermediate of genseng, and is standby, is used for the preparation of oral liquid;
Or the ultra filtration membrane ultrafiltration of previous step gained supernatant employing molecular cut off 100K, ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby, is used for the preparation of oral liquid, injection;
Or the ultra filtration membrane ultrafiltration of previous step gained supernatant employing molecular cut off 200K, ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby, is used for the preparation of oral liquid, injection;
Or the ultra filtration membrane ultrafiltration of previous step gained supernatant employing molecular cut off 500K, ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby, is used for the preparation of oral liquid, injection;
Or previous step gained supernatant is used the ultra filtration membrane ultrafiltration of molecular cut off 200K earlier, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 5K, promptly get the polysaccharide composition intermediate of genseng, standby, be used for the preparation of oral liquid, injection;
Or previous step gained supernatant is used the ultra filtration membrane ultrafiltration of molecular cut off 500K earlier, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 2K, promptly get the polysaccharide composition intermediate of genseng, standby, be used for the preparation of oral liquid, injection;
Or previous step gained supernatant is used the ultra filtration membrane ultrafiltration of molecular cut off 500K earlier, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 5K, promptly get the polysaccharide composition intermediate of genseng, standby, be used for the preparation of oral liquid, injection;
(3) the monkshood medicinal material adds 8 times of water gagings (because be dried medicinal material, adding 2 times of amounts for the first time), decocts extraction or refluxing extraction 3 times, each 1 hour, filter, be concentrated into relative density of medicine liquid 1.09～1.13,47～53 ℃ of temperature, the ethanol alcohol precipitation with 95% makes ethanol content reach 70%, standing over night, filter, filtrate recycling ethanol does not extremely have the alcohol flavor, and the relative density of adjusting soup is that the ethanol of adding 95% in 1.27～1.31 o'clock makes pure content reach 85%, standing over night, filter, filtrate recycling ethanol filters to there not being the alcohol flavor, and filtrate being heated to boiled, keeping little boiled 38～45 minutes, cooling, refrigeration filters, promptly get the monkshood intermediate, standby;
Or for the first time precipitation solution reclaims ethanol to there not being the alcohol flavor, adjusts soup and is equivalent to contain genseng crude drug 0.95～1.05g to every 1ml, adds the dichloromethane extraction 5 times of 1.0～1.1 times of amounts at every turn, combined dichloromethane liquid reclaims methylene chloride to the greatest extent, changes water-soluble, filter, promptly get the monkshood intermediate, standby;
(4) intermediate with " (1) ", " (2) ", " (3) " gained merges, and mixing adds purified water or water for injection to full dose, regulates pH value to 7.0, is heated to and boils, and keep little and boiled 38～42 minutes, cooling, refrigeration is spent the night; Filter, filtrate is regulated pH value to 7.0, adds purified water or water for injection and adjusts volume to full dose, surveys the pH value, filters, and according to dosage divides to be filled in the oral liquid packing container, seals, and the conventional method sterilization promptly gets the oral liquid that contains genseng and monkshood in a kind of raw material.
(5) intermediate with " (1) ", " (2) ", " (3) " gained merges, and mixing adds to the full amount of water for injection, and regulates pH value to 7.0, is heated to and boils, and keep little and boiled 40～45 minutes, cooling, refrigeration is spent the night; Filter, (regulating pH value to 7.0) adds 0.1% activated charcoal and is heated to and boils, keeping little boiled 20～25 minutes, filter, regulate pH value to 7.0, add the injection water and adjust volume to full dose, survey the pH value, cross 0.45 μ m miillpore filter, according to dosage divide to be filled to the infusion pump dress, seal with in the container, the conventional method sterilization promptly gets the parenteral solution that contains genseng and monkshood in a kind of raw material.
(6) intermediate with " (1) ", " (2) ", " (3) " gained merges, and mixing adds to the full amount of water for injection, and regulates pH value to 7.0, is heated to and boils, and keep little and boiled 40～45 minutes, cooling, refrigeration is spent the night; Filter, add sweet mellow wine/or sorbierite in the filtrate, stir evenly, regulate pH value to 7.0, add 0.1% activated charcoal and be heated to and boil, keep little and boiled 20～25 minutes, filter, regulate pH value to 7.0, add the injection water and adjust volume to full dose, survey the pH value, cross 0.45 μ m miillpore filter, aseptic subpackaged to control injection vial, freeze drying, gland, roll lid, promptly get the freeze-drying preparation for injection that contains genseng and monkshood in a kind of raw material.
(7) freeze drying process is as follows:
The a pre-freeze stage: pre-freeze temperature-56 ℃～-45 ℃, about 2.5～6 hours of pre-freeze time;
The b lyophilization stage: when the temperature of condenser reduce to-56 ℃～below-45 ℃, open vacuum pump, the temperature of shelf is arranged on-21 ℃～-19 ℃, goods slowly heat up, and observe the distillation interface, all walk only the end of lyophilization stage to free water.
C is drying stage again: shelf temperature slowly rises to 0 ℃～20 ℃, judges dry again terminal point, 4～11 hours drying times again with the vacuum tightness descent method.
Embodiment 3
Prescription:
Figure G2006101032344D00301
The preparation method:
(1) genseng adds 6 times of amounts (because be dried medicinal material, adding 1 times of amount for the first time), 70% alcohol reflux 2 times, each 2 hours respectively, filter, filtrate recycling ethanol is not to there being the alcohol flavor, and soup is concentrated into every 1ml and is equivalent to contain genseng crude drug 1.45～1.55g, and the water-saturated n-butanol that at every turn adds equivalent extracts 6 times, merge normal butyl alcohol liquid, reclaim normal butyl alcohol to there not being the normal butyl alcohol flavor, change water-soluble, filter, get the ginsenoside intermediate, standby;
(2) the red ginseng dregs of a decoction after the alcohol extracting add 9 times of water gagings (for the first time because be dried medicinal material, add 3 times of amounts) respectively and decoct and extract or refluxing extraction 3 times each 2 hours, filter, being concentrated into liquor strength is 0.75～0.85g crude drug/ml, and temperature is 37～43 ℃, adds 95% ethanol alcohol precipitation and makes and contain the alcohol amount and reach 80%, standing over night, filter, precipitation is dry, add the suitable quantity of water dissolving after, centrifugal, merge supernatant, adjusting the supernatant liquor strength is 1.95～2.05g crude drug/ml, adds 95% ethanol and makes and contain the alcohol amount and reach 70%, standing over night, filter, precipitation is dry, add the suitable quantity of water dissolving after, centrifugal, get supernatant, supernatant earlier with the ultra filtration membrane ultrafiltration of molecular cut off 500K, is removed small-molecular weight impurity with the gained ultrafiltrate with the ultra filtration membrane of molecular cut off 5K then, promptly get the polysaccharide composition intermediate of genseng, standby;
(3) the monkshood medicinal material adds 8 times of water gagings (because be dried medicinal material, adding 2 times of amounts for the first time), decocts extraction or refluxing extraction 3 times, each 1 hour, filter, be concentrated into relative density of medicine liquid 1.09～1.13,47～53 ℃ of temperature, the ethanol alcohol precipitation with 95% makes ethanol content reach 70%, standing over night, filter, filtrate recycling ethanol does not extremely have the alcohol flavor, and the relative density of adjusting soup is that the ethanol of adding 95% in 1.27～1.31 o'clock makes pure content reach 85%, standing over night, filter, filtrate recycling ethanol filters to there not being the alcohol flavor, and filtrate being heated to boiled, keeping little boiled 38～45 minutes, cooling, refrigeration filters, promptly get the monkshood intermediate, standby;
(4) intermediate with " (1) ", " (2) ", " (3) " gained merges, and mixing adds to the full amount of water for injection, and regulates pH value to 7.0, is heated to and boils, and keep little and boiled 40～45 minutes, cooling, refrigeration is spent the night; Filter, add sweet mellow wine in the filtrate, stir evenly, regulate pH value to 7.0, add 0.1% activated charcoal and be heated to and boil, keep little and boiled 20～25 minutes, filter, regulate pH value to 7.0, add the injection water and adjust volume to full dose, survey the pH value, cross 0.45 μ m miillpore filter, aseptic subpackaged to control injection vial, freeze drying, gland, roll lid, promptly get the freeze-drying preparation for injection that contains genseng and monkshood in a kind of raw material.(remarks: regulate the pH value and use the 1mol/L sodium hydroxide solution, or use the 1mol/L hydrochloric acid solution.)
(5) freeze drying process is as follows:
The a pre-freeze stage: select pre-freeze repeatedly, pre-freeze temperature-56 ℃～-45 ℃, the temperature of rising again-11 ℃～-10 ℃, about 4～7 hours of pre-freeze time.
The b lyophilization stage: when the temperature of condenser reduce to-56 ℃～below-45 ℃, open vacuum pump, the temperature of shelf is arranged on-21 ℃～-19 ℃, goods slowly heat up, and observe the distillation interface, all walk only the end of lyophilization stage to free water.
C is drying stage again: shelf temperature slowly rises to 0 ℃～20 ℃, judges dry again terminal point, 4～11 hours drying times again with the vacuum tightness descent method.
Embodiment 4
Prescription:
The preparation method:
(1) genseng adds 6 times of amounts (because be dried medicinal material, adding 1 times of amount for the first time), 70% alcohol reflux 2 times, each 2 hours respectively, filter, filtrate recycling ethanol is not to there being the alcohol flavor, and soup is concentrated into every 1ml and is equivalent to contain genseng crude drug 1.45～1.55g, and the water-saturated n-butanol that at every turn adds equivalent extracts 6 times, merge normal butyl alcohol liquid, reclaim normal butyl alcohol to there not being the normal butyl alcohol flavor, change water-soluble, filter, get the ginsenoside intermediate, standby;
(2) the red ginseng dregs of a decoction after the alcohol extracting add 9 times of water gagings (for the first time because be dried medicinal material, add 3 times of amounts) respectively and decoct and extract or refluxing extraction 3 times each 2 hours, filter, being concentrated into liquor strength is 0.75～0.85g crude drug/ml, and temperature is 37～43 ℃, adds 95% ethanol alcohol precipitation and makes and contain the alcohol amount and reach 80%, standing over night, filter, precipitation is dry, add the suitable quantity of water dissolving after, centrifugal, merge supernatant, adjusting the supernatant liquor strength is 1.95～2.05g crude drug/ml, adds 95% ethanol and makes and contain the alcohol amount and reach 70%, standing over night, filter, precipitation is dry, add the suitable quantity of water dissolving after, centrifugal, get supernatant, supernatant earlier with the ultra filtration membrane ultrafiltration of molecular cut off 500K, is removed small-molecular weight impurity with the gained ultrafiltrate with the ultra filtration membrane of molecular cut off 5K then, promptly get the polysaccharide composition intermediate of genseng, standby;
(3) the monkshood medicinal material adds 8 times of water gagings (for the first time because be dried medicinal material, add 2 times of amounts), decoct extraction or refluxing extraction 3 times, each 1 hour, filter, be concentrated into relative density of medicine liquid 1.09～1.13,47～53 ℃ of temperature, the ethanol alcohol precipitation with 95% makes ethanol content reach 70%, standing over night, filter, filtrate recycling ethanol is not to there being the alcohol flavor, adjusts soup and is equivalent to contain genseng crude drug 0.95～1.05g to every 1ml, adds the dichloromethane extraction 5 times of 1.0～1.1 times of amounts at every turn, combined dichloromethane liquid, reclaim methylene chloride to the greatest extent, change water-soluble, filter, promptly get the monkshood intermediate, standby;
(4) intermediate with " (1) ", " (2) ", " (3) " gained merges, and mixing adds to the full amount of water for injection, and regulates pH value to 7.0, is heated to and boils, and keep little and boiled 40～45 minutes, cooling, refrigeration is spent the night; Filter, add sweet mellow wine/or sorbierite in the filtrate, stir evenly, regulate pH value to 7.0, add 0.1% activated charcoal and be heated to and boil, keep little and boiled 20～25 minutes, filter, regulate pH value to 7.0, add the injection water and adjust volume to full dose, survey the pH value, cross 0.45 μ m miillpore filter, aseptic subpackaged to control injection vial, freeze drying, gland, roll lid, promptly get the freeze-drying preparation for injection that contains genseng and monkshood in a kind of raw material.(remarks: regulate the pH value and use the 1mol/L sodium hydroxide solution, or use the 1mol/L hydrochloric acid solution.)
(5) freeze drying process is as follows:
The a pre-freeze stage: select pre-freeze repeatedly, pre-freeze temperature-56 ℃～-45 ℃, the temperature of rising again-11 ℃～-10 ℃, about 4～7 hours of pre-freeze time.
The b lyophilization stage: when the temperature of condenser reduce to-56 ℃～below-45 ℃, open vacuum pump, the temperature of shelf is arranged on-21 ℃～-19 ℃, goods slowly heat up, and observe the distillation interface, all walk only the end of lyophilization stage to free water.
C is drying stage again: shelf temperature slowly rises to 0 ℃～20 ℃, judges dry again terminal point, 4～11 hours drying times again with the vacuum tightness descent method.
Embodiment 5
Prescription:
Figure G2006101032344D00331
The preparation method:
(1) genseng adds 6 times of amounts (because be dried medicinal material, adding 1 times of amount for the first time), 70% alcohol reflux 2 times, each 2 hours respectively, filter, filtrate recycling ethanol is not to there being the alcohol flavor, and soup is concentrated into every 1ml and is equivalent to contain genseng crude drug 1.45～1.55g, and the water-saturated n-butanol that at every turn adds equivalent extracts 6 times, merge normal butyl alcohol liquid, reclaim normal butyl alcohol to there not being the normal butyl alcohol flavor, change water-soluble, filter, get the ginsenoside intermediate, standby;
(2) the red ginseng dregs of a decoction after the alcohol extracting add 9 times of water gagings (for the first time because be dried medicinal material, add 3 times of amounts) respectively and decoct and extract or refluxing extraction 3 times each 2 hours, filter, being concentrated into liquor strength is 0.75～0.85g crude drug/ml, and temperature is 37～43 ℃, adding 95% ethanol alcohol precipitation makes and contains the alcohol amount and reach 80%, standing over night filters, and precipitation is dry, after adding the suitable quantity of water dissolving, centrifugal, merge supernatant, adjusting the supernatant liquor strength is 1.95～2.05g crude drug/ml, adding 95% ethanol makes and contains the alcohol amount and reach 70%, standing over night filters, and precipitation is dry, after adding the suitable quantity of water dissolving, centrifugal, get supernatant
Or the ultra filtration membrane ultrafiltration of previous step gained supernatant employing molecular cut off 100K, ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby;
Or the ultra filtration membrane ultrafiltration of previous step gained supernatant employing molecular cut off 200K, ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby;
Or the ultra filtration membrane ultrafiltration of previous step gained supernatant employing molecular cut off 500K, ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby;
Or the ultra filtration membrane ultrafiltration of molecular cut off 200K of previous step gained supernatant elder generation, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 5K, promptly get the polysaccharide composition intermediate of genseng, standby;
Or the ultra filtration membrane ultrafiltration of molecular cut off 200K of previous step gained supernatant elder generation, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 2K, promptly get the polysaccharide composition intermediate of genseng, standby;
Or the molecular cut off 500K ultra filtration membrane ultrafiltration of previous step gained supernatant ripple elder generation, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 2K, promptly get the polysaccharide composition intermediate of genseng, standby;
(3) the monkshood medicinal material adds 8 times of water gagings (because be dried medicinal material, adding 2 times of amounts for the first time), decocts extraction or refluxing extraction 3 times, each 1 hour, filter, be concentrated into relative density of medicine liquid 1.09～1.13,47～53 ℃ of temperature, the ethanol alcohol precipitation with 95% makes ethanol content reach 70%, standing over night, filter, filtrate recycling ethanol does not extremely have the alcohol flavor, and the relative density of adjusting soup is that the ethanol of adding 95% in 1.27～1.31 o'clock makes pure content reach 85%, standing over night, filter, filtrate recycling ethanol filters to there not being the alcohol flavor, and filtrate being heated to boiled, keeping little boiled 35～45 minutes, cooling, refrigeration filters, promptly get the monkshood intermediate, standby;
Or for the first time precipitation solution reclaims ethanol to there not being the alcohol flavor, adjusts soup and is equivalent to contain genseng crude drug 0.95～1.05g to every 1ml, adds the dichloromethane extraction 5 times of 1.0～1.1 times of amounts at every turn, combined dichloromethane liquid reclaims methylene chloride to the greatest extent, changes water-soluble, filter, promptly get the monkshood intermediate, standby;
(4) intermediate with " (1) ", " (2) ", " (3) " gained merges, and mixing adds to the full amount of water for injection, and regulates pH value to 7.0, is heated to and boils, and keep little and boiled 40～45 minutes, cooling, refrigeration is spent the night; Filter, add sweet mellow wine in the filtrate, stir evenly, regulate pH value to 7.0, add 0.1% activated charcoal and be heated to and boil, keep little and boiled 20～25 minutes, filter, regulate pH value to 7.0, add the injection water and adjust volume to full dose, survey the pH value, cross 0.45 μ m miillpore filter, aseptic subpackaged to control injection vial, freeze drying, gland, roll lid, promptly get the freeze-drying preparation for injection that contains genseng and monkshood in a kind of raw material.
(5) freeze drying process is as follows:
The a pre-freeze stage: select pre-freeze repeatedly, pre-freeze temperature-56 ℃～-45 ℃, the temperature of rising again-11 ℃～-10 ℃, about 4～7 hours of pre-freeze time.
The b lyophilization stage: when the temperature of condenser reduce to-56 ℃～below-45 ℃, open vacuum pump, the temperature of shelf is arranged on-21 ℃～-19 ℃, goods slowly heat up, and observe the distillation interface, all walk only the end of lyophilization stage to free water.
C is drying stage again: shelf temperature slowly rises to 0 ℃～20 ℃, judges dry again terminal point, 4～11 hours drying times again with the vacuum tightness descent method.
Embodiment 6
Prescription:
Figure G2006101032344D00351
The preparation method:
(1) genseng adds 6 times of amounts (because be dried medicinal material, adding 1 times of amount for the first time), 70% alcohol reflux 2 times, each 2 hours respectively, filter, filtrate recycling ethanol is not to there being the alcohol flavor, and soup is concentrated into every 1ml and is equivalent to contain genseng crude drug 1.45～1.55g, and the water-saturated n-butanol that at every turn adds equivalent extracts 6 times, merge normal butyl alcohol liquid, reclaim normal butyl alcohol to there not being the normal butyl alcohol flavor, change water-soluble, filter, get the ginsenoside intermediate, standby;
(2) the red ginseng dregs of a decoction after the alcohol extracting add 9 times of water gagings (for the first time because be dried medicinal material, add 3 times of amounts) respectively and decoct and extract or refluxing extraction 3 times each 2 hours, filter, being concentrated into liquor strength is 0.75～0.85g crude drug/ml, and temperature is 37～43 ℃, adding 95% ethanol alcohol precipitation makes and contains the alcohol amount and reach 80%, standing over night filters, and precipitation is dry, after adding the suitable quantity of water dissolving, centrifugal, merge supernatant, adjusting the supernatant liquor strength is 1.95～2.05g crude drug/ml, adding 95% ethanol makes and contains the alcohol amount and state 70%, standing over night filters, and precipitation is dry, after adding the suitable quantity of water dissolving, centrifugal, get supernatant
Previous step gained supernatant adopts the ultra filtration membrane ultrafiltration of molecular cut off 100K, and ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby;
Or the ultra filtration membrane ultrafiltration of previous step gained supernatant employing molecular cut off 200K, ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby;
Or the ultra filtration membrane ultrafiltration of previous step gained supernatant employing molecular cut off 500K, ultrafiltrate is the polysaccharide composition intermediate of genseng, and is standby;
Or the ultra filtration membrane ultrafiltration of molecular cut off 200K of previous step gained supernatant elder generation, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 5K, promptly get the polysaccharide composition intermediate of genseng, standby;
Or the ultra filtration membrane ultrafiltration of molecular cut off 200K of previous step gained supernatant elder generation, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 2K, promptly get the polysaccharide composition intermediate of genseng, standby;
Or the ultra filtration membrane ultrafiltration of molecular cut off 500K of previous step gained supernatant elder generation, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 2K, promptly get the polysaccharide composition intermediate of genseng, standby;
Or the ultra filtration membrane ultrafiltration of molecular cut off 500K of previous step gained supernatant elder generation, then the gained ultrafiltrate is removed small-molecular weight impurity with the ultra filtration membrane of molecular cut off 5K, promptly get the polysaccharide composition intermediate of genseng, standby;
(3) the monkshood medicinal material adds 8 times of water gagings (because be dried medicinal material, adding 2 times of amounts for the first time), decocts extraction or refluxing extraction 3 times, each 1 hour, filter, be concentrated into relative density of medicine liquid 1.09～1.13,47～53 ℃ of temperature, the ethanol alcohol precipitation with 95% makes ethanol content reach 70%, standing over night, filter, filtrate recycling ethanol does not extremely have the alcohol flavor, and the relative density of adjusting soup is that the ethanol of adding 95% in 1.27～1.31 o'clock makes pure content reach 85%, standing over night, filter, filtrate recycling ethanol filters to there not being the alcohol flavor, and filtrate being heated to boiled, keeping little boiled 38～45 minutes, cooling, refrigeration filters, promptly get the monkshood intermediate, standby;
Or for the first time precipitation solution reclaims ethanol to there not being the alcohol flavor, adjusts soup and is equivalent to contain genseng crude drug 0.95～1.05g to every 1ml, adds the dichloromethane extraction 5 times of 1.0～1.1 times of amounts at every turn, combined dichloromethane liquid reclaims methylene chloride to the greatest extent, changes water-soluble, filter, promptly get the monkshood intermediate, standby;
(4) intermediate with " (1) ", " (2) ", " (3) " gained merges, and mixing adds to the full amount of water for injection, and regulates pH value to 7.0, is heated to and boils, and keep little and boiled 40～45 minutes, cooling, refrigeration is spent the night; Filter, add in the filtrate naloxone hydrochloride/or Dopamine hydrochloride (naloxone hydrochloride/or Dopamine hydrochloride be dissolved in the low amounts of water, adjust about pH value of water solution to 7, and filter), sweet mellow wine, stir evenly, regulate about pH value to 7.0, add 0.1% activated charcoal and be heated to and boil, keeping little boiled 20～25 minutes, filter, regulate about pH value to 7.0, add the injection water and adjust volume to full dose, survey the pH value, cross 0.45 μ m miillpore filter, aseptic subpackaged to control injection vial, freeze drying, gland, roll lid, promptly get the freeze-drying preparation for injection that contains genseng and monkshood in a kind of raw material.
(5) freeze drying process is as follows:
The a pre-freeze stage: pre-freeze temperature-56 ℃～-45 ℃, about 2.5～6 hours of pre-freeze time; Or select pre-freeze repeatedly, pre-freeze temperature-56 ℃～-45 ℃, the temperature of rising again-11 ℃～-10 ℃, about 4～7 hours of pre-freeze time.
The b lyophilization stage: when the temperature of condenser reduce to-56 ℃～below-45 ℃, open vacuum pump, the temperature of shelf is arranged on-21 ℃～-19 ℃, goods slowly heat up, and observe the distillation interface, all walk only the end of lyophilization stage to free water.
C is drying stage again: shelf temperature slowly rises to 0 ℃～20 ℃, judges dry again terminal point, 4～11 hours drying times again with the vacuum tightness descent method.
Embodiment 7
It below is the Pharmacodynamic test of active extract of pharmaceutical preparation of the present invention.
1 experiment purpose and content
Clinical application according to injection ginseng attached (freeze-drying), on animal, carry out the main pharmacodynamics checking, mainly carry out following experiment: injection ginseng attached (freeze-drying) high, medium and low dosage group is to the influence of anti-anoxic time-to-live of mouse normal pressure, hypophysis is held the influence of Acute Myocardial Ischemia in Rats due to the foline (Pit), influence to acute rat blood stasis model, influence to the isolated rat heart ischemical reperfusion injury, to the influence of miocardial infarction due to the anesthetized dog coronary ligation, to the hemodynamic influence of anesthetized dog.
2 experiment materials
2.1 be subjected to the reagent thing: injection ginseng attached (freeze-drying) is provided lot number by Xuanhong Medicine Technology Co., Ltd., Tianjin: 040710, and adopt the embodiment of the invention 3 used prescriptions and preparation method to make.Face with preceding 0.9% sodium chloride injection and be mixed with needed concentration with certain volume.
2.2 medicine and reagent:
Ginseng aconite injection liquid, Sanjiu Pharmaceutical Industry Co., Ltd., Ya'an City, lot number: 041103 (being the used medicine of the attached former medicine group of the ginseng of indication in this experiment);
Danshen injections, Zhengda Qingchunbao Pharmaceutical Co., Ltd, lot number: 0405202;
Nitroglycerin injection, Beijing Yimin Pharmaceutical Co., Ltd., lot number: 040217;
Heparin sodium injection, the biological thousand red pharmaceutical Co. Ltds in Changzhou, lot number: 040615;
Sodium citrate, the sincere chemical reagent in Shanghai company limited, lot number: 040128;
Adrenalin hydrochloride, Tianjin gold credit amino acid company limited, lot number: 0403031;
Yellow Jackets, Beijing chemical reagents corporation, lot number: 020402;
Pituitrin, Shanghai No.1 Bio-Chemical Pharmacetical Industry Co., Ltd, lot number: 040501;
Urethane, Shanghai chemical reagents corporation of China Drug Co., lot number 20021104;
Lactic dehydrogenase (LDH-L) kit, the medical company limited of sysmex Jinan Sysmex, lot number: ZG4001;
Creatine kinase (CK) kit, the medical company limited of sysmex Jinan Sysmex, lot number: ZG4004;
Sodium chloride, AR level, the sincere chemical reagent in Shanghai company limited, lot number: 050705;
Potassium chloride, AR level, Shanghai Ling Feng chemical reagent company limited, lot number: 021031;
Lime chloride, AR level, Shanghai Ling Feng chemical reagent company limited, lot number: 030619;
Sodium bicarbonate, AR level, last marine rainbow photoinitiator chemical factory, lot number: 030604;
Glucose, the AR level, last Nereid analyses Chemical Industry Science Co., Ltd, lot number: 030823;
Activated partial prothrombin time (APTT) is measured kit, Beijing Steellex Scientific Instrument Company's lot number: ST20201-20;
Fibrinogen (FIB) is measured kit, Beijing Steellex Scientific Instrument Company's lot number: ST20401-16;
Prothrombin time (PT) is measured kit, Beijing Steellex Scientific Instrument Company's lot number: ST10101-22;
Rockwell liquid preparation: NaCl 9.0g/L, KCl 0.42g/L, CaCl 20.24g/L, NaHCO 30.2g/L, Glu 1.0g/L;
Formaldehyde: Nanjing chemical reagent factory, lot number: 20040721.
2.3 experimental apparatus
RM-6000 eight road physiograph and annexes, Japanese NIHON KOHDEN company;
The DH140 Spirophore, Zhejiang Medical university medical instrument trial (demonstration) plant; DHG-9053A type Constant Temp. Oven, the medical thermostatic equipment in Shanghai factory; The last ware electronic balance of FA2104, Shanghai balance equipment factory;
JNA type precision torsion balance, Shanghai Second Balance Factory; Electrocardiograph, Japanese NIHON KOHDEN company; Vitalab 200 type automatic clinical chemistry analyzers, Dutch vital scientific company;
BT01-100 type constant flow pump, Baoding LanGe constant flow pump Co., Ltd; Perfusion device: self-designed L angendorff perfusion device;
LMS-2B type two road physiographs, Chengdu Instruement Factory; The WC/09-05 calibration cell, Chinese Chongqing Yinhe Experimental Equipment Co., Ltd..
2.4 animal
Kunming mouse, body weight 18～22g; The SD rat, body weight 180-220g; Rabbit, body weight 2.0～2.4kg, male and female half and half are supplied with by Shanghai Si Laike animal used as test responsibility company limited.Conformity certification: SCXK (Shanghai) 2003-0003.
The hybrid dog, body weight 7-10kg, the male and female dual-purpose by the hybrid dog of Nanjing University of Traditional Chinese Medicine's animal center purchase from suburbs, is raised and train at this center before the experiment, after quarantine and expelling parasite, is used for this experiment.
3 experimental techniques and result
Data are represented (X ± S) with mean ± standard deviation.All measurement datas adopt the Student-t check, and enumeration data adopts rank test.
Before rate of change (incidence) %=(after the X administration-X administration before)/X administration * 100%
Be observed reading after the administration after the X administration, be observed reading before the administration before the X administration.
3.1 influence to the anti-anoxic of mouse normal pressure
3.1.1 experimental technique: get 120 of Kunming mouses, male and female half and half, be divided into 6 groups at random, promptly control group (giving isometric physiological saline), positive drug group (danshen injections 20.0g crude drug/kg), the attached former medicine group of ginseng (and 3.2g crude drug/kg), injection ginseng attached (freeze-drying) high, medium and low (3.2,1.6,0.8g crude drug/kg).Each group is all by the tail intravenously administrable, and the administration volume is 20ml/kg.Behind the intravenously administrable 15min, mouse is placed the airtight wide-necked bottle of 125ml (built-in soda lime 20g).Stopping with the mouse mouth breathing is the dead mouse sign, the time-to-live of record mouse, surpass 60min in 60min.
3.1.2 experimental result:
Experimental result sees Table 1.
Table 1 injection ginseng attached (freeze-drying) is to the protective effect of the anti-anoxic of mouse normal pressure (X ± SD)
Compare with control group: * P＜0.05, * * P＜0.01.
Compare with injection ginseng attached (freeze-drying) high dose group: ▲: P＜0.05.
The result shows, injection ginseng attached (freeze-drying) is high, middle dosage group and join attached former medicine group and all can prolong the time-to-live of the anti-anoxic of mouse normal pressure, with remarkable (P＜0.01 of control group comparing difference, P＜0.05, and join attached (freeze-drying) high dose group mouse time-to-live and significantly be longer than former medicine group (P＜0.05) P＜0.05).
3.2 influence to rat heart muscle ischemic due to the pituitrin (Pit)
3.2.1 experimental technique: get 56 of healthy SD rats, male and female half and half, body weight 180～220g, be divided into 7 groups at random, that is: normal group, model group (above two groups give isometric physiological saline), positive drug group (monobel 0.5mg/kg), injection ginseng attached (freeze-drying) basic, normal, high (0.4,0.8,3 dosage groups of 1.6g crude drug/kg), the attached former medicine group of ginseng (1.6g crude drug/kg).Water 12h is can't help in the rat fasting before the experiment, and dorsal position is got in lumbar injection urethane 1g/kg anesthesia, and it is subcutaneous that needle electrode carefully thrusts four limbs, measures the II lead electrocardiogram, and 15min is stablized in the operation back, and the record normal ECG.Each treated animal is respectively through the tail vein injection administration, and behind the 15min, except that the normal control group, each group is no more than 5s inject time respectively through sublingual vein injection of pituitrin 1.5u/kg.The cardiogram of 15s, 30s, 1min, 2min, 3min, 5min, 7min, 10min, 15min behind the immediate record injection of pituitrin.
Observation index mainly is decided to be the variation of T wave height, changes in heart rate.It is baseline that the T wave height is measured with the PR section, and every time point is surveyed 3 continuous wave modes, gets its mean value.Record also calculates the changing value (no matter raise or reduce, get the absolute value of variation) of T wave height.For the index that dynamic observes of sequential relationships such as variation of T wave height and heart rate, on each time point, with t check between the changing value work group after the medication.
20.3.2.2 experimental result:
Experimental result sees Table 2, table 3.
(1) the attached influence to rats with myocardial ischemia ECG T wave displacement due to the pituitrin of injection ginseng sees Table 2.
The influence of rats with myocardial ischemia ECG T wave due to the table 2 pair pituitrin (n=8)
Figure G2006101032344D00402
Compare * P＜0.05, * * P＜0.01 with model group; Compare * P＜0.05 with the attached high dose group of ginseng
Behind the rats in normal control group intravenous injection pituitrin, electrocardio takes place obviously to change, and the first phase, (1～30s) tangible T wave height occurs alarmmed; The second phase (after the 30s), present the low flat or inversion of tangible T ripple.
Injection ginseng attached (freeze-drying) high, medium and low dosage and former medicine group can significantly be resisted the electrocardio T ripple displacement (P＜0.01) that pituitrin causes at the 15s time point, join attached (freeze-drying) height, middle dosage and former medicine group and can significantly resist the electrocardio T ripple displacement (P＜0.05) that pituitrin causes at the 30s time point, and there is certain dose-effect relationship between the high, medium and low dosage, joins attached high dose group and compare in 15s, 30s, 1min, 5min also significant difference (P＜0.05) with former medicine.
(2) the attached influence to rats with myocardial ischemia heart rate due to the pituitrin of injection ginseng the results are shown in Table 3.
The influence of rats with myocardial ischemia heart rate due to the table 3 pair pituitrin (n=8)
Compare * P＜0.05, * * P＜0.01 with model group;
Behind the rats in normal control group intravenous injection pituitrin, the rat heart rate is obviously slowed down, not recover yet to 15min.Injection ginseng attached (freeze-drying) high dose can be alleviated the decreased heart rate due to the pituitrin when 10min, join attached former medicine and when 2min, can alleviate decreased heart rate due to the pituitrin, with model group than difference remarkable (P＜0.05), compare with the attached former medicine of ginseng the using of alleviating due to the pituitrin of decreased heart rate and there is no significant difference (P＞0.05) but join attached (freeze-drying) high dose.
3.2.3 conclusion:
Injection ginseng attached (freeze-drying) high, medium and low dosage and former medicine can obviously resist between the variation of the cardiogram T section that pituitrin causes and the high, medium and low dosage and have certain dose-effect relationship, join attached (freeze-drying) high dose and former medicine and can alleviate decreased heart rate due to the pituitrin at some time point, prompting injection ginseng attached (freeze-drying) has the effect of myocardial ischemia due to certain antagonism pituitrin.
3.3 to the hemorheological influence of stasis syndrome rat model
3.3.1 experimental implementation method:
Get 56 of SD male rats, body weight 350～450g, be divided into 7 groups at random, it is the normal control group, model group (above two groups give isometric physiological saline), positive drug group (10g crude drug/kg danshen injections), injection ginseng attached (freeze-drying) is low, in, high (0.4,0.8,1.6g 3 dosage groups of crude drug/kg), join attached former medicine group (1.6g crude drug/kg), the administration volume is 5ml/kg, vein successive administration 3 days, after the 2nd administration, except that the normal control group, each treated animal hypodermic injection adrenalin hydrochloride parenteral solution (Adr) 0.8ml/kg totally 2 times, interval 4h, before injecting Adr for the second time, place frozen water to soak 5min rat, cause the Blood stasis model.Fasting, the 15min administration is 1 time before operation, with 10% chloral hydrate anesthesia (300mg/kg, ip), face upward the position and fix the bloodletting of arteria carotis intubate, sodium citrate with 3.8% or liquaemin normal saline solution (500ul/ml) detect hemorheological every index by anti-freezing in 1: 9.
3.3.2 experiment detects index
(1) to the influence of rat platelet aggregation rate
The blood of the sodium citrate anti-freezing with 3.8% is with the centrifugal 10min of 800r/min, get platelet rich plasma (PRP), remainder is centrifugal with 3000r/min, get platelet poor plasma (PPP), utilize LG-PABER type platelet aggregation and clotting factor analyser record MA and calculate inhibiting rate by following formula.Assemble inhibiting rate (%)
=[(model group MA-administration group MA)/model group MA] * 100%
(2) blood viscosity detects:
1. whole blood viscosity: adopt LG-R-80 series blood viscosity instrument to measure the blood of anticoagulant heparin.
2. plasma viscosity: after the whole blood test finished, the residue blood sample was centrifugal with 3000r/min, carries out the test of plasma viscosity.
3. erythrocyte deformability, erythrocyte aggregation ability detect: the blood of anticoagulant heparin is adopted LG-R-190 type red blood cell deformation/ability of aggregation analyzer.
(3) detection of blood coagulation system:
The blood of the sodium citrate anti-freezing with 3.8% is with the centrifugal 10min of 3000r/min, separate platelet poor plasma, adopt LG-PABER type platelet aggregation and clotting factor analysis-e/or determining activated partial prothrombin time (APTT), prothrombin time (PT), plasma fibrinogen (FIB).
3.3.3 experimental result
Experimental result sees Table 4～table 7.
(1) injection ginseng attached (freeze-drying) the results are shown in Table 4 to the influence of Blood stasis rat model platelet aggregation rate.
Table 4 injection ginseng attached (freeze-drying) is to the influence of Blood stasis rat model platelet aggregation rate (X ± s)
Compare with model group: * * P＜0.01, * P＜0.05.
(2) injection ginseng attached (freeze-drying) the results are shown in Table 5 to the influence of Blood stasis rat model red blood cell deformation and ability of aggregation.
Table 5 injection ginseng attached (freeze-drying) is to the influence of Blood stasis rat model red blood cell deformation and ability of aggregation (X ± s)
Compare with model group: * P＜0.05; * P＜0.01.
(3) injection ginseng attached (freeze-drying) the results are shown in Table 6 to the influence of Blood stasis rat model APTT, PT, FIB.
Table 6 injection ginseng attached (freeze-drying) is to the influence of Blood stasis rat model APTT, PT, FIB (n=8, X ± s)
Compare with model group: * P＜0.05.
(4) injection ginseng attached (freeze-drying) the results are shown in Table 7 to the influence of Blood stasis rat model blood viscosity.
Table 7 injection ginseng attached (freeze-drying) is to the influence of Blood stasis rat model blood viscosity (n=8, X ± s)
Figure G2006101032344D00442
Compare with model group: * P＜0.05; * P＜0.01
Result: join attached (freeze-drying) high dose and can suppress platelet aggregation, reduction erythrocyte aggregation ability, the whole blood viscosity that reduces (low cutting) and plasma viscosity, prolong blood plasma PT, reduce plasma fibrin (FIB) content, with model group comparing difference remarkable (P＜0.05, P＜0.01).Middle dosage also can suppress platelet aggregation, reduction whole blood viscosity (low cutting) and plasma viscosity, with model group comparing difference remarkable (P＜0.05).Join attached former medicine and can suppress platelet aggregation, reduction erythrocyte aggregation ability, the whole blood viscosity that reduces (low cutting) and plasma viscosity, prolong blood plasma PT, reduce plasma fibrin (FIB) content, with model group comparing difference remarkable (P＜0.05); But joining attached former medicine compares and there is no significant difference (P＞0.05) with ginseng attached (freeze-drying) high dose.
3.4 influence to the isolated rat heart ischemical reperfusion injury
3.4.1 experimental implementation:
(1) grouping and dosage
Get 56 of SD rats, body weight 200g～300g is divided into 7 groups at random, and 8 every group, male and female half and half.I.e. (1) normal control group: contain oxygen Rockwell liquid; (2) model group: nitrification Rockwell liquid; (3) positive drug group: nitrification danshen injections, 1.3mg/ml; (4) injection ginseng attached (freeze-drying) high dose group: the nitrification ginseng is attached, 0.31mg/ml; (5) dosage group in the injection ginseng attached (freeze-drying): nitrification is joined attached 0.16mg/ml; (6) injection ginseng attached (freeze-drying) low dose group: the nitrification ginseng is attached, 0.08mg/ml; (7) the attached former medicine group of ginseng: the former ginseng aconite injection liquid of nitrification, 0.31mg/ml.Used medicine is all prepared with Rockwell liquid.
Rat tail vein injecting heparin sodium (1000U/kg) hits dizzyly behind the 15min, open chest, heart is taken out rapidly, move in the double dish of 4 ℃ of Rockwell liquid of contain, wash out heart blood as far as possible, under liquid level, rapidly sustainer is connected on the sleeve pipe of perfusion, and pricks fixing with toe-in.Carry out perfusion to contain oxygen Rockwell liquid immediately.Perfusion pressure 70cm water column, 38 ± 0.5 ℃ of perfusion temperature, flow velocity 11.5 ± 0.5ml/min.Tangle apex and link to each other with frog heart clip, be connected in two road physiographs and trace changes in heart rate with transducer.Recovering beat of heart and heart rate are stablized 30min after showing normally, experimentize again, arrhythmia cordis person occurs during this period and discard.
(2) method:
Adopt the modeling of anoxic reperfusion injury method:
Model group: with after containing oxygen Rockwell liquid perfusion 30min and making heart working stable, regulate three-way cock, use in advance that nitrification Rockwell liquid pours into 40min as anoxic instead, recover to contain oxygen Rockwell liquid perfusion 40min again to perfusion pipe one side of nitrification Rockwell liquid in advance.Cause anoxic reperfusion injury model.
Positive drug group and each administration group: method is the same substantially, only pours into 40min adding different pharmaceutical when nitrification Rockwell liquid is done the anoxic perfusion in advance.
Normal control group: to contain oxygen Rockwell liquid perfusion 110min.
Coronary flow and heart rate when record normally and again pours into back the 20th, 40min, the spill-out of lactic dehydrogenase (LDH), creatine kinase (CK) is calculated rate of change in the mensuration each point perfusate.Experiment with 10% formaldehyde fixed heart, is done the pathology histological examination after finishing.
Rate of descent %=(before stopping irritating-after pouring into again)/before stopping irritating * 100%
Escalating rate %=(perfusion back-before stopping irritating) again/before stopping irritating * 100%
3.4.2 statistical procedures adopts the Student-t check.
3.4.3 the results are shown in Table 8～table 11.
(1) injection ginseng attached (freeze-drying) the results are shown in Table 8 to the influence of anoxic reperfusion injury isolated rat heart heart rate.
Table 8 injection ginseng attached (freeze-drying) is to influence (X ± S) (n=8) of anoxic reperfusion injury isolated rat heart heart rate
Figure G2006101032344D00451
Compare * P＜0.05 with model group; * P＜0.01.
Experimental result: each group equal indifference of heart rate before stopping irritating, when pouring into back 20min and 40min again, injection ginseng attached (freeze-drying) high, medium and low dosage group heart rate all is lower than model group, and rate of descent and model group are relatively, statistical significance (P＜0.01, P＜0.05) is arranged.Injection ginseng attached (freeze-drying) high dose group heart rate rate of descent is compared no difference of science of statistics (P＞0.05) with join attached former medicine with concentration.
(2) injection ginseng attached (freeze-drying) the results are shown in Table 9 to the influence of anoxic reperfusion injury isolated rat heart coronary artery perfusion flow.
Table 9 injection ginseng attached (freeze-drying) is to influence (X ± S) (n=8) of anoxic reperfusion injury isolated rat heart coronary artery perfusion flow
Compare * P＜0.05 with model group; * P＜0.01.
Experimental result: each group is perfusion flow and the relatively more equal indifference of normal group before stopping irritating, when pouring into back 20min and 40min again, injection ginseng attached (freeze-drying) height, middle dosage group perfusion flow are apparently higher than model group, and the low and model group of its rate of descent has significant difference (P＜0.01).Join attached (freeze-drying) low dose group perfusion flow rate of descent and all be lower than model group, but not statistically significant (P＞0.05).Join attached (freeze-drying) three dosage groups and in influence, present certain dose-effect relationship the coronary artery perfusion flow.Join attached (freeze-drying) high dose group coronary artery perfusion flow rate of descent and be lower than the attached former medicine group of ginseng, but no difference of science of statistics (P＞0.05).
(3) injection ginseng attached (freeze-drying) the results are shown in Table 10 to the influence of anoxic reperfusion injury isolated rat heart CK.
Experimental result: each group is CK value and the relatively more equal indifference of normal group before stopping irritating, when pouring into back 20min and 40min again, injection ginseng attached (freeze-drying) high, medium and low dosage group CK value significantly is lower than model group, its escalating rate and model group are relatively, be lower than model group, significant difference (P＜0.01) is arranged.Join attached (freeze-drying) three dosage groups and can suppress all that CK overflows due to the myocardial cell injury, present certain dose-effect relationship.Join attached (freeze-drying) high dose group CK value escalating rate and be lower than with concentration and join attached former medicine group, but no difference of science of statistics (P＞0.05).
Table 10 injection ginseng attached (freeze-drying) is to influence (X ± S) (n=8) of anoxic reperfusion injury isolated rat heart CK
Figure G2006101032344D00471
Compare * P＜0.05 with model group; * P＜0.01.
(4) injection ginseng attached (freeze-drying) the results are shown in Table 11 to the influence of anoxic reperfusion injury isolated rat heart LDH.
Table 11 injection ginseng attached (freeze-drying) is to influence (X ± S) (n=8) of anoxic reperfusion injury isolated rat heart LDH
Figure G2006101032344D00472
Compare * P＜0.05 with model group; * P＜0.01.High dose group and control group comparison ▲ P＜0.05.
Experimental result: each group is LDH value and the relatively more equal indifference of normal group before stopping irritating, when pouring into back 20min and 40min again, injection ginseng attached (freeze-drying) height, middle dosage group LDH value all is lower than model group, its escalating rate and model group are relatively, statistical significance (P＜0.01 is arranged, P＜0.05). join attached (freeze-drying) low dose group when pouring into back 20min again, the LDH escalating rate is lower than model group (P＜0.05), when pouring into back 40min again, escalating rate also is lower than model group, but not statistically significant (P＞0.05). join attached (freeze-drying) three dosage groups and can suppress all that LDH overflows due to the myocardial cell injury, present certain dose-effect relationship. join attached (freeze-drying) high dose group LDH value and all be lower than with concentration and join attached former medicine group, and certain difference (P＜0.05) is arranged, high dose group LDH escalating rate is lower than with concentration joins attached former medicine group, but no difference of science of statistics (P＞0.05).
3.4.4 conclusion:
Injection ginseng attached (freeze-drying) height, middle dosage group are when pouring into back 20min and 40min again, and heart rate, perfusion flow rate of descent and CK, LDH escalating rate all are lower than model group (P＜0.01, P＜0.05).Prompting ginseng attached (freeze-drying) is high, middle dosage group can be improved the heart rate decline that the anoxic reperfusion injury is caused, and increases coronary flow simultaneously, suppresses CK, LDH and discharges.The effect that wherein joining attached (freeze-drying) high dose group increases coronary flow, inhibition CK, LDH release demonstrates the trend of joining attached former medicine group with concentration that is better than.
Injection ginseng attached (freeze-drying) low dose group is when pouring into back 20min and 40min again, and heart rate rate of descent, CK escalating rate all have certain difference (P＜0.05, P＜0.01) with model group, and when pouring into back 20min again, the LDH escalating rate is lower than model group (P＜0.05).Prompting injection ginseng attached (freeze-drying) low dose group can be improved heart rate decline, inhibition CK, the LDH release that ischemia-reperfusion causes.
Anoxic reperfusion injury isolated rat heart is the result show; injection ginseng attached (freeze-drying) is mainly by improving the protective effect of bringing into play heart of overflowing of heart rate decline that the anoxic reperfusion injury caused, coronary artery dilator, inhibition myocardium enzyme; have certain coronary artery dilator simultaneously, increase coronary flow; improve the effect that heart rate that the anoxic reperfusion injury caused descends; three dosage groups are to coronary artery perfusion flow and CK; in the influence of LDH, all present certain dose-effect relationship.
Isolated rat heart ischemical reperfusion injury tissue pathology checking report shows: injection ginseng attached (freeze-drying) has the certain protection effect to damage due to the isolated rat myocardial ischemia-reperfusion.
3.5 influence to miocardial infarction due to the anesthetized dog coronary ligation
3.5.1 experimental technique
Get 35 of domesticated dogs, body weight is 7-10kg, is divided into 7 groups at random, every group of 5 animals, male and female dual-purpose: (1) sham operated rats: give isometric(al) physiological saline.(2) model group: give isometric(al) physiological saline.(3) positive drug danshen injections control group: 2.0g crude drug/kg.(4) injection ginseng attached (freeze-drying) low dose group: 0.2g crude drug/kg.(5) dosage group in the injection ginseng attached (freeze-drying): 0.4g crude drug/kg.(6) injection ginseng attached (freeze-drying) high dose group: 0.8g crude drug/kg.(7) the attached former medicine control group of ginseng: 0.4g crude drug/kg.The used medicine of each treated animal is dissolved in 0.9% sodium chloride injection, and the administration volume is 1ml/kg.
After domesticated dog is anaesthetized with 3% yellow Jackets (30mg/kg) by the forelimb small saphenous vein, back of the body position is fixed on the operating table, cutting off the hair of neck, chest and left hind inboard. it is subcutaneous that needle electrode is inserted the dog four limbs, recording ecg (ECG). separate tracheae and insert trachea cannula. separate left side femoral artery, femoral vein, separate femoral vein and insert venous cannula, slowly constant speed input physiological saline (about 1ml/min); Separate femoral artery insertion arterial cannulation (being full of the heparin-saline of 500u/ml in the pipe) and connect pressure transducer (TP-400T), measure SAP (SAP) through amplifying (AP-641G), auterial diastole is pressed (DAP), mean arterial pressure (MAP), calibration sensitivity is that the line connection board that 13.33kPa (100mmHg)/cm. connects registering instrument directly triggers heart rate numeration instrument (AT-601G) recorded heart rate (HR) by angiosthenia. in the left side the 4th, five intercostals are opened chest, expose heart, and meet the breathing apparatus. open pericardium, be sewn in the wall of the chest, make the pocket cradle. expose right auricle of heart, quiet notes heparin 5-10mg/kg makes heparinize, clamp right auricle of heart with auricular clamp, make a pocket sealing, suture is through in a bit of proofed sleeve, with little steel wire line is colluded into proofed sleeve, cut an osculum in pocket mouth center, insert coronary sinus cannula (internal diameter 4-5mm rapidly, the mouth of pipe is the oblique angle, nearly mouth of pipe place is that circle is expanded, the oblique angle face avoids the mouth of pipe and Dou Bi to be close to, the portion of expanding is for making coronary sinus blood unlikely excessive), with pocket suture tension in the small rubber hose, withstand intubate by small rubber hose, to avoid hemorrhage, rapidly intubate is accurately inserted coronary sinus, ligation right auricle of heart place pocket suture, fixing intubate. the far-end of this intubate connects a three-way pipe to be on the waiting list blood. with 1/3 place down in No. 0 silk thread ligation ramus descendens anterior arteriae coronariae sinistrae, a sham operated rats not ligation of threading. 12 the mapping point seams in position are put the epicardial electrogram electrode near selecting infarct, and selecting a control point away from infarct, carry out mapping with hand-held insulated metal point-like electrode by the mapping dot sequency, through sensor (JB-642G), amplifier (AB-621G), with eight road physiographs record epicardial electrogram (EECG) (calibration: 1mm=1mv). ligation 15min is after femoral vein constant flow pump constant speed administration (1ml/min), directly write down SAP (SAP), diastolic pressure (DAP), mean arterial pressure (MAP), heart rate (HR), and trace EECG and angiosthenia (BP) curve. before the record ligation, after ligation 15min and the administration 5,15,30,60,90, the EECG of 120min, SAP, DAP, MAP, HR. add up ST field offset ∑-ST of each mapping point EECG, and the ST section raise 〉=above each group of N-ST. of 2mv is respectively at before the administration and behind the administration 60min, left ventricle is got blood and is surveyed arterial blood oxygen, coronary sinus is got blood and is surveyed coronary sinus blood oxygen, both oxygen difference reflecting myocardium oxygen consumptions; Each group is respectively at before the ligation, femoral vein is got blood behind ligation 15min, the administration 60min, with kit measurement serum CK, LDH value. after experiment finishes, take out heart immediately, behind physiological saline flush away blood, take by weighing heavy whole-heartedly and ventricular weight, and the ventricle crosscut become 5, place 37 ℃ of 1%TTC solution 15min that dyes, cut off the non-infarct that each myocardium sheet is colored, undyed infarct cardiac muscle is weighed, obtains infarction size heavily respectively divided by heavy or ventricle whole-heartedly and account for heavy whole-heartedly or account for the heavy percent of ventricle.
Myocardial consumption of oxygen (kpa/min)=MAP*HR
3.5.2 experimental result
(1) injection ginseng attached (freeze-drying) is to the miocardial infarction BP (influence of SAP, DAP, MAP, HR and myocardial consumption of oxygen due to the anesthetized dog coronary ligation
Experimental result shows: injection ginseng attached (freeze-drying) height, in the dosage group can reduce DAP, MAP, HR and the myocardial consumption of oxygen of miocardial infarction anesthetized dog due to the coronary ligation, with model group notable difference (P＜0.05 is arranged relatively, P＜0.01), the dosage group relatively has significant difference (P＜0.05) with the attached former medicine group of ginseng and wherein.
(2) injection ginseng attached (freeze-drying) is to anesthetized dog arteriovenous blood oxygen partial pressure (Po 2), arteriovenous blood partial pressure of carbon dioxide (Pco 2) influence
The result shows: injection ginseng attached (freeze-drying) is high, middle dosage group and model group relatively, the arteriovenous oxygen difference that can significantly suppress due to the coronary ligation increases (P＜0.05), and wherein the dosage group with join some point of attached former medicine group comparison significant difference (P＜0.01) arranged.
(3) injection ginseng attached (freeze-drying) is to the influence of miocardial infarction ∑-ST due to the anesthetized dog coronary ligation
The result shows: injection ginseng attached (freeze-drying) high, medium and low dosage group can reduce miocardial infarction anesthetized dog ∑-ST due to the coronary ligation, part-time point and remarkable (P＜0.05 of model group comparing difference, P＜0.01), show that injection ginseng attached (freeze-drying) can obviously alleviate degree of myocardial ischemia, and wherein the dosage group there is significant difference (P＜0.01) with some point of the attached former medicine group of ginseng comparison.
(4) injection ginseng attached (freeze-drying) is to the influence of miocardial infarction N-ST due to the anesthetized dog coronary ligation
The result shows: injection ginseng attached (freeze-drying) high, medium and low dosage group can reduce miocardial infarction anesthetized dog N-ST due to the coronary ligation, part-time point and remarkable (P＜0.05 of model group comparing difference, P＜0.01), and wherein the dosage group relatively has significant difference (P＜0.01) with some point of the attached former medicine group of ginseng.
(5) injection ginseng attached (freeze-drying) is to the serum CK of miocardial infarction due to the anesthetized dog coronary ligation, the influence of LDH.
Experimental result shows: injection ginseng attached (freeze-drying) high, medium and low dosage group, can reduce the release of CK, LDH in the serum after the miocardial infarction due to the anesthetized dog coronary ligation, with model group comparing difference significantly (P＜0.01).
(6) injection ginseng attached (freeze-drying) is to the influence of miocardial infarction infarction size due to the anesthetized dog coronary ligation
Experimental result shows: injection ginseng attached (freeze-drying) is high, middle dosage group can reduce miocardial infarction infarction size due to the anesthetized dog coronary ligation, with remarkable (P＜0.05 of model group comparing difference, P＜0.01), and wherein the dosage group relatively has significant difference (P＜0.01) with the attached former medicine group of ginseng.
3.5.3 conclusion:
The miocardial infarction experimental result shows due to the anesthetized dog coronary ligation: injection ginseng attached (freeze-drying) height, in the dosage group can reduce DAP, MAP, HR and the myocardial consumption of oxygen of miocardial infarction anesthetized dog due to the coronary ligation, arteriovenous oxygen difference due to the inhibition coronary ligation increases, part-time point and model group comparing difference remarkable (P＜0.05, P＜0.01); High, medium and low dosage group can reduce miocardial infarction anesthetized dog ∑-ST due to the coronary ligation, and N-ST, part-time point and model group comparing difference be (P＜0.05, P＜0.01) significantly; High, medium and low dosage group can reduce due to the anesthetized dog coronary ligation after the miocardial infarction release of LDH and CK in the serum, and is with model group comparing difference significantly (P＜0.05), remarkable with former medicine group of last index and middle dosage group comparing difference.Show that injection ginseng attached (freeze-drying) can obviously alleviate degree of myocardial ischemia.
3.6 to the hemodynamic influence of anesthetized dog [5-6]
3.6.1 experimental technique:
Get 30 of domesticated dogs, the male and female dual-purpose is divided into 6 groups at random, every group of 5 animals, i.e. (1) normal control group: give isometric(al) physiological saline; (2) positive drug injection of danshen group: 2.0g crude drug/kg.(3) injection ginseng attached (freeze-drying) low dose group: 0.2g crude drug/kg.(4) dosage group in the injection ginseng attached (freeze-drying): 0.4g crude drug/kg.(5) injection ginseng attached (freeze-drying) high dose group: 0.8g crude drug/kg.(6) the attached former medicine group of ginseng: 0.4g crude drug/kg, the used medicine of each treated animal is dissolved in 0.9% sodium chloride injection, the administration volume is 1ml/kg.
With the subcutaneous cephalic vein anesthesia of 3% yellow Jackets 30mg/kg forelimb, back of the body position is fixed on the operating table, and the inboard unhairing of neck, chest and left hind is standby.Separate tracheae and insert trachea cannula; Separate femoral vein and insert venous cannula, slowly constant speed input physiological saline (about 1ml/min); Separate femoral artery and insert arterial cannulation (being full of the heparin-saline of 500u/ml in the pipe), to measure arterial pressure.Under the artificial respiration, open chest, cut off pericardium in the 4th intercostal, be sewn in the wall of the chest, separate root of ascending aorta and ramus descendens anterior arteriae coronariae sinistrae, place the electromagnetic blood flowmeter probe (12mm of suitable internal diameter, 2mm), be connected in and measure cardiac output (CO) and coronary flow (CBF) on the electromagnetic blood flowmeter.Left ventricular cannulation (being full of heparin-saline in the pipe) in left ventricle apex wound inserts left ventricle, is measured left indoor pressure (LVSP), left chamber diastasis pressure (LVEDP), the maximum climbing speed (LVdp/dtmax) of intraventricular pressure; It is subcutaneous that needle electrode is inserted the dog four limbs, recording ecg (ECG).After waiting to stablize, with constant flow pump through femoral vein administration (1ml/min), directly write down SAP (SAP), diastolic pressure (DAP), mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), coronary flow (CBF), and on eight road physiographs, trace ECG, left indoor pressure (LVSP), LVdp/dtmax, LVEDP and angiosthenia (BP) curve.Measure and calculate the BP (SAP of each time period anesthetized dog of administration front and back, DAP, MAP), HR, CO, CBF, LVSP, LVEDP, + dp/dtmax (myocardial contraction parameter),-dp/dtmax (myocardial relaxation parameter), t～dp/dtmax (left chamber begins to be contracted to left indoor pressure climbing speed time to peak), ejection time, SV (stroke output), CI (cardiac index), SI (SI), LVWI (stroke work index), TTI (total oxygen consumption index), TPVR (total peripheral vascular resistance).
Computing formula:
CI (cardiac index)=CO/ body surface area; SV (stroke output)=CO*1000/HR;
SI (SI)=CI*1000/HR; TTI (total oxygen consumption index)=MAP*HR* ejection time;
TPVR (total peripheral vascular resistance)=MAP/CO;
LVWI (stroke work index)=CI*1.025* (SAP-0.667) * 13.6*0.001
3.6.2 experimental result
(1) injection ginseng attached (freeze-drying) is to the influence of anesthetized dog SAP, DAP, MAP, HR
The result shows: the DAP that injection ginseng attached (freeze-drying) is high, middle dosage group can reduce anesthetized dog, reducing heart rate is with normal control group comparing difference remarkable (P＜0.05, P＜0.01).
(2) injection ginseng attached (freeze-drying) is to the influence of anesthetized dog CO, CBF, SV, CI, SI
The result shows: injection ginseng attached (freeze-drying) is high, middle dosage group can increase anesthetized dog coronary flow (CBF), SI (SI), stroke output (SV), with normal control group comparing difference remarkable (P＜0.05, P＜0.01); High, middle dosage group can obviously increase anesthetized dog cardiac output (CO), cardiac index (CI), (with normal control group P＜0.01 relatively), and wherein dosage group and the attached former medicine group comparing difference of ginseng significantly (P＜0.05).
(3) to the influence of anesthetized dog ejection time, LVSP, LVEDP
The result shows: injection ginseng attached (freeze-drying) three each dosage groups all do not make significant difference to anesthetized dog ejection time, LVSP, LVEDP.
(4) to anesthetized dog+dp/dtmax ,-influence of dp/dtmax, t-dp/dtmax
The result shows: injection ginseng attached (freeze-drying) three dosage groups to anesthetized dog+dp/dtmax ,-dp/dtmax and t-dp/dtmax do not make significant difference (with the normal control group relatively, P＞0.05).
(5) to the influence of anesthetized dog LVWI, TTI, TPVR
The result shows: injection ginseng attached (freeze-drying) is high, in two dosage groups can reduce anesthetized dog total peripheral resistance (TPVR), total oxygen utilization (TTI), compare with the normal control group, significant difference (P＜0.05, P＜0.01), and wherein dosage group and the attached former medicine group comparing difference of ginseng significantly (P＜0.05).
3.6.3 experiment conclusion:
The experiment of anesthetized dog haemodynamics shows: the DAP that injection ginseng attached (freeze-drying) is high, middle dosage group can reduce anesthetized dog, reducing heart rate, reduce anesthetized dog total peripheral resistance (TPVR), total oxygen utilization (TTI), increase anesthetized dog cardiac output (CO), cardiac index (CI), with normal control group comparing difference remarkable (P＜0.05, P＜0.01).High, middle dosage group can increase anesthetized dog coronary flow (CBF), SI (SI), stroke output (SV), with normal control group comparing difference remarkable (P＜0.05, P＜0.01) and remarkable with former medicine group of last index and middle dosage group comparing difference.
More than two experimental results show: injection ginseng attached (freeze-drying) has the improvement effect to the myocardial ischemia and the heart function of anesthetized dog anterior descending coronary caused by ligature, on the basis that does not reduce the heart blood supply function, the alleviate myocardial ischemia degree.
Learn experimental result as can be known by this main pharmacodynamics, " injection ginseng attached (freeze-drying) " stable in properties that the present invention makes, the main pharmacodynamics experiment shows under Isodose, it is compared with the ginseng aconite injection liquid of selling in the market, the time-to-live of the anti-anoxic of high dose group prolongation mouse normal pressure is longer significantly, and it is stronger that high dose group increases the effect of coronary flow, inhibition CK, LDH release.Proved beneficial effect of the present invention.
Embodiment 8
Get and write out a prescription 12 among the embodiment 2, prescription 13 employed raw materials and the parenteral solution made by corresponding preparation method (having kept the polysaccharide composition in the genseng of 5K～500K molecular weight), detect (sample volume is converted accordingly by the weight of the corresponding crude drug of weight of preparation finished product content among embodiment 3, the embodiment 4) by the method in " containing parenteral solution, the freeze-drying preparation for injection of genseng and monkshood, the method for quality control of sterile packaged preparation for injection in a kind of raw material " in " summary of the invention ", meet the regulation in this method of quality control.
Embodiment 9
Get embodiment 3, embodiment 4 employed raw materials and gained preparation, detect by the method in " containing parenteral solution, the freeze-drying preparation for injection of genseng and monkshood, the method for quality control of sterile packaged preparation for injection in a kind of raw material " in " summary of the invention ", meet the regulation in this method of quality control.

Claims (9)

1. a bulk drug is made up of genseng and monkshood and its ratio is 1-10: the detection method of the injection of 1-10, it is characterized in that described detection method comprises finger print method, and it comprises:
(1) ginseng crude drug's finger-print
(1.1) medicinal material title and source
Genseng in this genseng medicinal materials fingerprint is selected red ginseng for use, and red ginseng is the dry root and rhizome of cultivation product after steaming of Araliaceae genseng Panax ginseng C.A.Mey.;
(1.2) mensuration of ginseng crude drug's finger-print
(1.2.1) chromatographic condition
Chromatographic column: ODS HYPERSIL C 18Post, Φ 4.6mm * 250mm, packing material size 5 μ m; Moving phase: acetonitrile and water are moving phase,
Gradient condition:
Time: 0min; Acetonitrile: 10%-20%; Water: 90%-80%;
Time: 20min; Acetonitrile: 25%-35%; Water: 75%-65%;
Time: 55min; Acetonitrile: 45%-55%; Water: 55%-45%;
Time: 60min; Acetonitrile: 10%-20%; Water: 90%-80%;
Column temperature: 25～35 ℃; Analysis time: 60～120min; Flow velocity: 0.6～1.0ml/min; 100～120 ℃ of ELSD drift tubes, flow rate of carrier gas 2.0～4.01/min; Number of theoretical plate is with ginsenoside Rb 1The peak meter should be not less than 5000;
(1.2.2) preparation of reference substance solution
The ginsenoside Rb of phosphorus pentoxide drying under reduced pressure to constant weight learns from else's experience 1Reference substance 2mg, the accurate title, decide, and puts in the 5ml measuring bottle, adds dissolve with methanol and be settled to scale, shakes up, promptly;
(1.2.3) preparation of need testing solution
Got the red ginseng medicinal powder 2g of 20 mesh sieves, the accurate title, decide, and adds 70% alcohol reflux 2 times, and each 0.8～1.5 hour, add 6～8 times of amounts for the first time, add 5～7 times of amounts for the second time; The extract filtration, filtrate recycling ethanol concentrates, and extracts 4～6 times with the water saturated normal butyl alcohol jolting of equivalent, merges normal butyl alcohol liquid, and washs with the ammonia solution of equivalent; Cleansing solution discards, and normal butyl alcohol liquid evaporate to dryness, residue are dissolved in water and are transferred in the 10ml measuring bottle, are settled to scale, shake up, promptly;
(1.2.4) determination method
Accurate respectively reference substance solution and each 5～20 μ l of need testing solution of drawing inject liquid chromatograph, measure, promptly;
(1.2.5) result
With ginsenoside Rb 1The peak is set at the object of reference peak, and with the logarithm of its peak area as 1.000, according to ginsenoside Rb 1The retention time at peak is calculated, calibrate 10 total peaks altogether, its relative retention time is respectively: 0.056 ± 10%, 0.102 ± 10%, 0.372 ± 10%, 0.632 ± 10%, 0.931 ± 10%, 1.000,1.040 ± 10%, 1.084 ± 10%, 1.181 ± 10%, 1.662 ± 10%; And the regulation relative retention time is the relative peak area ratio at 0.632 ± 10%, 1.040 ± 10%, 1.084 ± 10%, 1.181 ± 10% peak, and promptly the peak area logarithm ratio is 0.682～1.266,0.346～0.644,0.262～0.486,0.103～0.191;
(2) monkshood medicinal materials fingerprint
(2.1) title of monkshood medicinal material and source
Monkshood is the processed goods of the sub-root of ranunculaceae plant rhizome of Chinese monkshood Aconitum carmichaeli Debx.; Monkshood in this monkshood medicinal materials fingerprint is selected for use black in sheet;
(2.2) mensuration of monkshood medicinal materials fingerprint
(2.2.1) chromatographic condition
Chromatographic column: ZORBAX SB C 18Post, Φ 4.6mm * 150mm, packing material size 5 μ m; Moving phase: methyl alcohol and 0.1% phosphoric acid-0.04% triethylamine-aqueous solution are moving phase,
Gradient condition:
Time: 0min; Methyl alcohol: 25%-35%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 75%-65%;
Time: 10min; Methyl alcohol: 25%-35%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 75%-65%;
Time: 45min; Methyl alcohol: 37%-47%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 63%-53%;
Time: 60min; Methyl alcohol: 37%-47%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 63%-53%;
Column temperature: 25～35 ℃; Detect wavelength: 235nm; Analysis time: 60～120min; Flow velocity: 0.7～1.3ml/min; Number of theoretical plate calculates with the mesaconine peak should be not less than 2000;
(2.2.2) preparation of reference substance solution
Learn from else's experience the phosphorus pentoxide drying under reduced pressure to the mesaconine reference substance 5mg of constant weight, accurately claim surely, put in the 25ml measuring bottle, add methyl alcohol and be diluted to scale, shake up, promptly;
(2.2.3) preparation of need testing solution
Monkshood medicinal material coarse powder 10g spends the night with ammonia solution 3～5ml, ether 40～60ml cold soaking, filters; The dregs of a decoction add 2.8～3.2: 0.8～1.2 ether: chloroform mixed solution 40～60ml, sonicated 25～40min, filter, the dregs of a decoction wash 3～4 times with mixed solution, each 14～16ml, and washing lotion and filtrate merge, the low temperature evaporate to dryness, residue adds methyl alcohol 5ml dissolving, with the filter membrane filtration of 0.45 μ m, can measure;
(2.2.4) determination method
Accurate respectively inner mark solution 15～25 μ l and need testing solution 30～50 μ l of drawing inject liquid chromatograph, measure, promptly;
(2.2.5) result
The mesaconine peak is set at the object of reference peak, retention time according to the mesaconine peak is calculated, calibrate 8 total peaks altogether, its relative retention time is respectively: 0.146 ± 10%, 0.254 ± 10%, 0.531 ± 10%, 0.630 ± 10%, 0.727 ± 10%, 0.833 ± 10%, 1.000,1.114 ± 10%; And with the stable and the biggest relative retention time of peak area be the peak area at 0.531 ± 10% peak as 1.000, and the regulation relative retention time is that the relative peak area ratio at 0.727 ± 10%, 1.114 ± 10% peak is 0.182～0.272,0.373～0.552;
(3) ginsenoside finger-print
(3.1) chromatographic condition
Chromatographic column: ODS HYPERSIL C 18Post, Φ 4.6mm * 250mm, packing material size 5 μ m; Acetonitrile and water are moving phase,
Gradient condition:
Time: 0min; Acetonitrile: 10%-20%; Water: 90%-80%;
Time: 20min; Acetonitrile: 25%-35%; Water: 75%-65%;
Time: 55min; Acetonitrile: 45%-55%; Water: 55%-45%;
Time: 60min; Acetonitrile: 10%-20%; Water: 90%-80%;
Column temperature: 25～35 ℃; Analysis time: 60～120min; Flow velocity: 0.6～1.0ml/min; ELSD
Parameter: 100～120 ℃ of drift tubes, flow rate of carrier gas 2.0～4.01/min; Number of theoretical plate is with ginsenoside Rb 1The peak calculates should be not less than 5000;
(3.2) preparation of reference substance solution
The ginsenoside Rb of phosphorus pentoxide drying under reduced pressure to constant weight learns from else's experience 1Reference substance 2mg, the accurate title, decide, and puts in the 5ml measuring bottle, adds dissolve with methanol and be settled to scale, shakes up, promptly;
(3.3) preparation of need testing solution
Get the injection content 0.35g for preparing, the accurate title, decide, be dissolved in water to 8～12ml, extract 4～6 times, merge normal butyl alcohol liquid with the water-saturated n-butanol jolting of equivalent, ammonia solution with equivalent washs 1～2 time, divide and to get normal butyl alcohol liquid, reclaim normal butyl alcohol, residue is with dissolved in distilled water and be settled in the 5ml measuring bottle, shake up, promptly;
(3.4) determination method
Accurate respectively reference substance solution and each 5～15 μ l of need testing solution of drawing inject liquid chromatograph, measure, promptly;
(3.5) result
With ginsenoside Rb 1The peak is set at the object of reference peak, and with the logarithm of its peak area as 1.000, according to ginsenoside Rb 1The retention time at peak is calculated, calibrate 10 total peaks altogether, its relative retention time is respectively: 0.441 ± 10%, 0.632 ± 10%, 0.942 ± 10%, 1.000,1.042 ± 10%, 1.062 ± 10%, 1.089 ± 10%, 1.188 ± 10%, 1.701 ± 10%, 1.737 ± 10%; And the regulation relative retention time is the relative peak area ratio at 0.441 ± 10%, 0.632 ± 10%, 1.042 ± 10%, 1.089 ± 10% peak, and promptly the peak area logarithm ratio is respectively 0.688～1.148,0.673～1.121,0.692～1.154,0.688～1.146;
(4) fingerprint of alkaloid
(4.1) chromatographic condition
Chromatographic column: ZORBAX SB C 18Post, Φ 4.6mm * 150mm, packing material size 5 μ m; Methyl alcohol and 0.1% phosphoric acid-0.04% triethylamine aqueous solution are moving phase,
Gradient condition:
Time: 0min; Methyl alcohol: 25%-35%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 75%-65%;
Time: 10min; Methyl alcohol: 25%-35%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 75%-65%;
Time: 45min; Methyl alcohol: 37%-47%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 63%-53%;
Time: 60min; Methyl alcohol: 37%-47%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 63%-53%;
25～35 ℃ of column temperatures; Detect wavelength: 235nm; Analysis time: 60～120min; Number of theoretical plate calculates with the mesaconine peak should be not less than 2000;
(4.2) preparation of inner mark solution
Learn from else's experience the phosphorus pentoxide drying under reduced pressure to the mesaconine reference substance 5mg of constant weight, accurately claim surely, put in the 25ml measuring bottle, add 1% phosphoric acid solution 3ml, be diluted to scale, shake up, promptly with methyl alcohol;
(4.3) preparation of need testing solution
Precision is measured the injection content 1.0g for preparing, be dissolved in water to 8～12ml, put in the separating funnel, add ammonia solution adjust pH to 9～11, extract 4～6 times with the mixed liquor jolting of 2.8～3.2: 0.8～1.2 E-C of equivalent, combining extraction liquid, low temperature volatilizes, and residue adds methyl alcohol 1ml makes dissolving, adds inner mark solution 40～60 μ l, filter, as need testing solution;
(4.4) determination method
Accurate respectively inner mark solution 15～25 μ l and need testing solution 30～50 μ l of drawing inject liquid chromatograph, measure, promptly;
(4.5) result
The mesaconine peak is set at the object of reference peak of relative retention time, retention time according to the mesaconine peak is calculated, calibrate 5 total peaks altogether, its relative retention time is respectively: 0.259 ± 10%, 0.342 ± 10%, 0.532 ± 10%, 0.724 ± 10%, 1.000 ± 10%; And with the stable and the biggest relative retention time of peak area be the peak area at 0.532 ± 10% peak as 1.000, and the regulation relative retention time is that the relative peak area at 0.724 ± 10% peak is than being 0.455-0.683.
2. detection method according to claim 1 is characterized in that described finger print method comprises:
(1) ginseng crude drug's finger-print
(1.1) medicinal material title and source
Genseng in this genseng medicinal materials fingerprint is selected red ginseng for use, and red ginseng is the dry root and rhizome of cultivation product after steaming of Araliaceae genseng Panax ginseng C.A.Mey.;
(1.2) mensuration of ginseng crude drug's finger-print
(1.2.1) chromatographic condition
Chromatographic column: ODS HYPERSIL C 18Post, Φ 4.6mm * 250mm, packing material size 5 μ m; Moving phase: acetonitrile and water are moving phase,
Gradient condition:
Time: 0min; Acetonitrile: 15%; Water: 85%;
Time: 20min; Acetonitrile: 30%; Water: 70%;
Time: 55min; Acetonitrile: 50%; Water: 50%;
Time: 60min; Acetonitrile: 15%; Water: 85%;
Column temperature: 30 ℃; Analysis time: 60min; Flow velocity: 0.8ml/min; 110 ℃ of ELSD drift tubes, flow rate of carrier gas 3.01/min; Number of theoretical plate is with ginsenoside Rb 1The peak meter should be not less than 5000;
(1.2.2) preparation of reference substance solution
The ginsenoside Rb of phosphorus pentoxide drying under reduced pressure to constant weight learns from else's experience 1Reference substance 2mg, the accurate title, decide, and puts in the 5ml measuring bottle, adds dissolve with methanol and be settled to scale, shakes up, promptly;
(1.2.3) preparation of need testing solution
Got the red ginseng medicinal powder 2g of 20 mesh sieves, the accurate title, decide, and adds 70% alcohol reflux twice, and each 1 hour, add 7 times of amounts for the first time, add 6 times of amounts for the second time; The extract filtration, filtrate recycling ethanol concentrates, and extracts 5 times with the water saturated normal butyl alcohol jolting of equivalent, merges normal butyl alcohol liquid, and washs with the ammonia solution of equivalent; Cleansing solution discards, and normal butyl alcohol liquid evaporate to dryness, residue are dissolved in water and are transferred in the 10ml measuring bottle, are settled to scale, shake up, promptly;
(1.2.4) determination method
Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject liquid chromatograph, measure, promptly;
(1.2.5) result
With ginsenoside Rb 1The peak is set at the object of reference peak, and with the logarithm of its peak area as 1.000, according to ginsenoside Rb 1The retention time at peak is calculated, calibrate 10 total peaks altogether, its relative retention time is respectively: 0.056 ± 10%, 0.102 ± 10%, 0.372 ± 10%, 0.632 ± 10%, 0.931 ± 10%, 1.000,1.040 ± 10%, 1.084 ± 10%, 1.181 ± 10%, 1.662 ± 10%; And the regulation relative retention time is the relative peak area ratio at 0.632 ± 10%, 1.040 ± 10%, 1.084 ± 10%, 1.181 ± 10% peak, and promptly the peak area logarithm ratio is 0.682～1.266,0.346～0.644,0.262～0.486,0.103～0.191;
(2) monkshood medicinal materials fingerprint
(2.1) title of monkshood medicinal material and source
Monkshood is the processed goods of the sub-root of ranunculaceae plant rhizome of Chinese monkshood Aconitum carmichaeli Debx.; Monkshood in this monkshood medicinal materials fingerprint is selected for use black in sheet;
(2.2) mensuration of monkshood medicinal materials fingerprint
(2.2.1) chromatographic condition
Chromatographic column: ZORBAX SB C18 post, Φ 4.6mm * 150mm, packing material size 5 μ m; Moving phase: methyl alcohol and 0.1% phosphoric acid-0.04% triethylamine-aqueous solution are moving phase,
Gradient condition:
Time: 0min; Methyl alcohol: 30%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 70%;
Time: 10min; Methyl alcohol: 30%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 70%;
Time: 45min; Methyl alcohol: 42%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 58%;
Time: 60min; Methyl alcohol: 42%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 58%;
Column temperature: 30 ℃; Detect wavelength: 235nm; Analysis time: 60min; Flow velocity: 1.0ml/min; Number of theoretical plate calculates with the mesaconine peak should be not less than 2000;
(2.2.2) preparation of reference substance solution
Learn from else's experience the phosphorus pentoxide drying under reduced pressure to the mesaconine reference substance 5mg of constant weight, accurately claim surely, put in the 25ml measuring bottle, add methyl alcohol and be diluted to scale, shake up, promptly;
(2.2.3) preparation of need testing solution
Monkshood medicinal material coarse powder 10g spends the night with ammonia solution 4ml, ether 50ml cold soaking, filters; The dregs of a decoction add 3: 1 ether: chloroform mixed solution 50ml, and sonicated 30min filters, and the dregs of a decoction wash 3～4 times with mixed solution, each 15ml, washing lotion and filtrate merge, the low temperature evaporate to dryness, residue adds methyl alcohol 5ml dissolving, with the filter membrane filtration of 0.45 μ m, can measure;
(2.2.4) determination method
Accurate respectively inner mark solution 20 μ l and the need testing solution 40 μ l of drawing inject liquid chromatograph, measure, promptly;
(2.2.5) result
The mesaconine peak is set at the object of reference peak, retention time according to the mesaconine peak is calculated, calibrate 8 total peaks altogether, its relative retention time is respectively: 0.146 ± 10%, 0.254 ± 10%, 0.531 ± 10%, 0.630 ± 10%, 0.727 ± 10%, 0.833 ± 10%, 1.000,1.114 ± 10%; And with the stable and the biggest relative retention time of peak area be the peak area at 0.531 ± 10% peak as 1.000, and the regulation relative retention time is that the relative peak area ratio at 0.727 ± 10%, 1.114 ± 10% peak is 0.182～0.272,0.373～0.552;
(3) ginsenoside finger-print
(3.1) chromatographic condition
Chromatographic column: ODS HYPERSIL C 18Post, Φ 4.6mm * 250mm, packing material size 5 μ m; Acetonitrile and water are moving phase,
Gradient condition:
Time: 0min; Acetonitrile: 15%; Water: 85%;
Time: 20min; Acetonitrile: 30%; Water: 70%;
Time: 55min; Acetonitrile: 50%; Water: 50%;
Time: 60min; Acetonitrile: 15%; Water: 85%;
Column temperature: 30 ℃; Analysis time: 60min; Flow velocity: 0.8ml/min; ELSD parameter: 110 ℃ of drift tubes, flow rate of carrier gas 3.0l/min; Number of theoretical plate is with ginsenoside Rb 1The peak calculates should be not less than 5000;
(3.2) preparation of reference substance solution
The ginsenoside Rb of phosphorus pentoxide drying under reduced pressure to constant weight learns from else's experience 1Reference substance 2mg, the accurate title, decide, and puts in the 5ml measuring bottle, adds dissolve with methanol and be settled to scale, shakes up, promptly;
(3.3) preparation of need testing solution
Get the injection content 0.35g for preparing, the accurate title, decide, be dissolved in water to 10ml, extract 5 times, merge normal butyl alcohol liquid with the water-saturated n-butanol jolting of equivalent, ammonia solution with equivalent washs 1 time, divide and to get normal butyl alcohol liquid, reclaim normal butyl alcohol, residue is with dissolved in distilled water and be settled in the 5ml measuring bottle, shake up, promptly;
(3.4) determination method
Accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing inject liquid chromatograph, measure, promptly;
(3.5) result
With ginsenoside Rb 1The peak is set at the object of reference peak, and with the logarithm of its peak area as 1.000, according to ginsenoside Rb 1The retention time at peak is calculated, calibrate 10 total peaks altogether, its relative retention time is respectively: 0.441 ± 10%, 0.632 ± 10%, 0.942 ± 10%, 1.000,1.042 ± 10%, 1.062 ± 10%, 1.089 ± 10%, 1.188 ± 10%, 1.701 ± 10%, 1.737 ± 10%; And the regulation relative retention time is the relative peak area ratio at 0.441 ± 10%, 0.632 ± 10%, 1.042 ± 10%, 1.089 ± 10% peak, and promptly the peak area logarithm ratio is respectively 0.688～1.148,0.673～1.121,0.692～1.154,0.688～1.146;
(4) fingerprint of alkaloid
(4.1) chromatographic condition
Chromatographic column: ZORBAX SB C 18Post, Φ 4.6mm * 150mm, packing material size 5 μ m; Methyl alcohol and 0.1% phosphoric acid-0.04% triethylamine aqueous solution are moving phase,
Gradient condition:
Time: 0min; Methyl alcohol: 30%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 70%;
Time: 10min; Methyl alcohol: 30%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 70%;
Time: 45min; Methyl alcohol: 42%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 58%;
Time: 60min; Methyl alcohol: 42%; 0.1% phosphoric acid-0.04% triethylamine-aqueous solution: 58%;
30 ℃ of column temperatures; Detect wavelength: 235nm; Analysis time: 60min; Number of theoretical plate calculates with the mesaconine peak should be not less than 2000;
(4.2) preparation of inner mark solution
Learn from else's experience the phosphorus pentoxide drying under reduced pressure to the mesaconine reference substance 5mg of constant weight, accurately claim surely, put in the 25ml measuring bottle, add 1% phosphoric acid solution 3ml, be diluted to scale, shake up, promptly with methyl alcohol;
(4.3) preparation of need testing solution
Precision is measured the injection content 1.0g for preparing, be dissolved in water to 10ml, put in the separating funnel, add ammonia solution adjust pH to 10, use the mixed liquor jolting of 3: 1 E-C of equivalent to extract 5 times, combining extraction liquid, low temperature volatilizes, and residue adds methyl alcohol 1ml makes dissolving, adds inner mark solution 50 μ l, filter, as need testing solution;
(4.4) determination method
Accurate respectively inner mark solution 20 μ l and the need testing solution 40 μ l of drawing inject liquid chromatograph, measure, promptly;
(4.5) result
The mesaconine peak is set at the object of reference peak of relative retention time, retention time according to the mesaconine peak is calculated, calibrate 5 total peaks altogether, its relative retention time is respectively: 0.259 ± 10%, 0.342 ± 10%, 0.532 ± 10%, 0.724 ± 10%, 1.000 ± 10%; And with the stable and the biggest relative retention time of peak area be the peak area at 0.532 ± 10% peak as 1.000, and the regulation relative retention time is that the relative peak area at 0.724 ± 10% peak is than being 0.455-0.683.
3. detection method as claimed in claim 1, it also comprises discriminating:
(1) genseng, ginsenoside Rg 1, the ginsenoside Re discriminating:
Get the injection content 1.0g for preparing, add water 30ml dissolving, add chloroform 30～50ml, jolting, water intaking liquid evaporate to dryness, residue adds water 2ml makes dissolving, added water saturated normal butyl alcohol 8～12ml sonicated 25～40 minutes, get normal butyl alcohol liquid and put in the separating funnel, add the ammonia solution of 2～4 times of amounts, washing, washing lotion discards, get normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other got the ginseng crude drug 1g of 60 mesh sieves, added ethanol 20～40ml, and reflux 20～40 minutes filters, and filtrate evaporate to dryness, residue add water 30ml makes dissolving, filters, and filtrate is made ginseng crude drug's solution with method; Get the ginsenoside Rg more respectively 1, ginsenoside Re's reference substance is an amount of, add methyl alcohol and make the solution that every 1ml contains 2mg respectively, in contrast product solution; Thin-layered chromatography test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B, draw each 2.5～5 μ l of above-mentioned four kinds of solution, put respectively on same silica gel g thin-layer plate, with 14～16: 38～42: 21～23: lower floor's solution that chloroform-ethyl acetate of 9～11-methanol-water is placed below 10 ℃ is developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of ginseng crude drug's chromatogram on, show the spot of same color; With the corresponding position of reference substance chromatogram on, show the spot of same color;
(2) discriminating of monkshood:
Get the injection content 3.5g for preparing, add water 30ml dissolving, regulate pH value to 9～11 with ammonia solution, the jolting that adds diethyl ether is extracted 2～4 times, each 40～50ml, and ether liquid low temperature evaporate to dryness, residue adds absolute ethyl alcohol 2ml makes dissolving, as need testing solution; Other gets monkshood medicinal material coarse powder 20g, puts in the tool plug conical flask the 130～170ml that adds diethyl ether, jolting 9～12 minutes, add ammonia solution 9～11ml, jolting 25～35 minutes was placed 1～2 hour, divided and got the ether layer, volatilize, residue adds absolute ethyl alcohol 2ml makes dissolving, as monkshood medicinal material solution; Thin-layered chromatography test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B, draw each 12～30 μ l of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate of thick 500 μ m, chromatography cylinder is earlier saturated with ammoniacal liquor, and with 4.8～5.2: 2.8～3.2: sherwood oil-ether of 2.8～3.2-acetone is developping agent, launches, take out, dry, spray is with bismuth potassium iodide test solution, and it is clear to be heated to spot colour developing; In the test sample chromatogram, with the corresponding position of monkshood medicinal material chromatogram on, show the spot of same color.
4. detection method as claimed in claim 3 is characterized in that described discriminating comprises:
(1) genseng, ginsenoside Rg 1, the ginsenoside Re discriminating:
Get the injection content 1.0g for preparing, add water 30ml dissolving, add chloroform 40ml, jolting, water intaking liquid evaporate to dryness, residue adds water 2ml makes dissolving, added water saturated normal butyl alcohol 10ml sonicated 30 minutes, get normal butyl alcohol liquid and put in the separating funnel, add the ammonia solution of 3 times of amounts, washing, washing lotion discards, get normal butyl alcohol liquid evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other got the ginseng crude drug 1g of 60 mesh sieves, added ethanol 30ml, and reflux 30 minutes filters, and filtrate evaporate to dryness, residue add water 30ml makes dissolving, filters, and filtrate is made ginseng crude drug's solution with method; Get the ginsenoside Rg more respectively 1, ginsenoside Re's reference substance is an amount of, add methyl alcohol and make the solution that every 1ml contains 2mg respectively, in contrast product solution; Thin-layered chromatography test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B, draw each 2.5～5 μ l of above-mentioned four kinds of solution, put respectively on same silica gel g thin-layer plate, with 15: 40: 22: lower floor's solution that chloroform-ethyl acetate of 10-methanol-water is placed below 10 ℃ was developping agent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing at 105 ℃; In the test sample chromatogram, with the corresponding position of ginseng crude drug's chromatogram on, show the spot of same color; With the corresponding position of reference substance chromatogram on, show the spot of same color;
(2) discriminating of monkshood:
Get the injection content 3.5g for preparing, add water 30ml dissolving, regulate pH value to 10 with ammonia solution, the jolting that adds diethyl ether is extracted 3 times, each 45ml, and ether liquid low temperature evaporate to dryness, residue adds absolute ethyl alcohol 2ml makes dissolving, as need testing solution; Other gets monkshood medicinal material coarse powder 20g, puts in the tool plug conical flask, and the 150ml that adds diethyl ether, jolting 10 minutes adds ammonia solution 10ml, and jolting 30 minutes was placed 1～2 hour, divided and got the ether layer, volatilized, and residue adds absolute ethyl alcohol 2ml makes dissolving, as monkshood medicinal material solution; Thin-layered chromatography test according to an appendix VI of Chinese Pharmacopoeia version in 2005 B, draw each 12～30 μ l of above-mentioned two kinds of solution, put respectively on the same silica gel g thin-layer plate of thick 500 μ m, chromatography cylinder is earlier saturated with ammoniacal liquor, is developping agent with sherwood oil-ether of 5: 3: 3-acetone, launches, take out, dry, spray is with bismuth potassium iodide test solution, and it is clear to be heated to spot colour developing; In the test sample chromatogram, with the corresponding position of monkshood medicinal material chromatogram on, show the spot of same color.
5. detection method as claimed in claim 1, it also comprises inspection:
(1) limit examine of aconite alkaloids:
(1.1) preparation of reference substance solution
Learn from else's experience the phosphorus pentoxide drying under reduced pressure to the aconitine reference substance 5mg of constant weight, accurately claim surely, put in the 50ml measuring bottle, add the 0.01mol/L hydrochloric acid solution and make dissolving, and be diluted to scale, shake up, promptly;
(1.2) preparation of typical curve
Precision is measured above-mentioned reference substance solution 0.2～0.3ml, 0.4～0.6ml, 0.65～0.85ml, 0.9～1.1ml, 1.4～1.6ml, 1.65～1.85ml, 1.9～2.1ml, put in the separating funnel respectively, add 0.01mol/L hydrochloric acid solution 1.85～1.65ml respectively, 1.6～1.4ml, 1.35～1.15ml, 1.1～0.9ml, 0.6～0.4ml, 0.35～0.15ml, 0.00ml, accurate respectively acetic acid-sodium-acetate buffer 8～12ml that adds, bromcresol green liquid 1.5～2.5ml, chloroform 8～12ml, jolting 2～6 minutes, leave standstill, divide and get chloroform solution, retinue is blank, measures absorbance at 415nm wavelength place according to the spectrophotometric method of an appendix VA of Chinese Pharmacopoeia version in 2005; With concentration is horizontal ordinate, and absorbance is an ordinate, the drawing standard curve; Wherein said acetic acid-sodium-acetate buffer is by getting 0.2mol/L acetum 250ml, make with 0.2mol/L sodium acetate solution adjust pH to 3.1, described bromcresol green liquid is by getting bromcresol green 50mg, add 0.05mol/L sodium hydroxide solution 1.6ml grinding and make dissolving, add water to 100ml, extract 3 times with the chloroform jolting, each 30ml discards chloroform solution and makes;
(1.3) preparation of need testing solution
Get the injection content 0.35g for preparing, the accurate title, decide, and adds water 10ml dissolving, is transferred in the separating funnel, adds ammonia solution and regulate pH value to 10～11, extracts combined chloroform liquid, evaporate to dryness 3～5 times with the chloroform jolting of equivalent; Residue adds 0.01mol/L hydrochloric acid solution gradation dissolving, and changes in the 10ml measuring bottle, is diluted to scale and shakes up, promptly;
(1.4) determination method
Precision is measured above-mentioned need testing solution 2ml, puts respectively in the separating funnel, down from " accurate respectively acetic acid-sodium-acetate buffer 8～12ml that adds ", measures absorbance according to " preparation of typical curve " item in accordance with the law, calculating, promptly;
(1.5) every of the injection for preparing contains aconite alkaloids with aconitine, i.e. C 24H 47NO 11Meter must not be higher than 1.0mg.
6. detection method as claimed in claim 5 is characterized in that described inspection comprises:
(1) limit examine of aconite alkaloids:
(1.1) preparation of reference substance solution
Learn from else's experience the phosphorus pentoxide drying under reduced pressure to the aconitine reference substance 5mg of constant weight, accurately claim surely, put in the 50ml measuring bottle, add the 0.01mol/L hydrochloric acid solution and make dissolving, and be diluted to scale, shake up, promptly;
(1.2) preparation of typical curve
Precision is measured above-mentioned reference substance solution 0.25ml, 0.50ml, 0.75ml, 1.00ml, 1.50ml, 1.75ml, 2.00ml, put in the separating funnel respectively, add 0.01mol/L hydrochloric acid solution 1.75ml, 1.50ml, 1.25ml, 1.00ml, 0.50ml, 0.25ml, 0.00ml respectively, the accurate respectively acetic acid-sodium-acetate buffer 10ml that adds, bromcresol green liquid 2ml, chloroform 10ml, jolting 3 minutes, leave standstill, divide and get chloroform solution, retinue is blank, measures absorbance at 415nm wavelength place according to spectrophotometric method; With concentration is horizontal ordinate, and absorbance is an ordinate, the drawing standard curve; Wherein said acetic acid-sodium-acetate buffer is by getting 0.2mol/L acetum 250ml, make with 0.2mol/L sodium acetate solution adjust pH to 3.1, described bromcresol green liquid is by getting bromcresol green 50mg, add 0.05mol/L sodium hydroxide solution 1.6ml grinding and make dissolving, add water to 100ml, extract 3 times with the chloroform jolting, each 30ml discards chloroform solution and makes;
(1.3) preparation of need testing solution
Get the injection content 0.35g for preparing, the accurate title, decide, and adds water 10ml dissolving, is transferred in the separating funnel, adds ammonia solution and regulate pH value to 10～11, extracts combined chloroform liquid, evaporate to dryness 4 times with the chloroform jolting of equivalent; Residue adds 0.01mol/L hydrochloric acid solution gradation dissolving, and changes in the 10ml measuring bottle, is diluted to scale and shakes up, promptly;
(1.4) determination method
Precision is measured above-mentioned need testing solution 2ml, puts respectively in the separating funnel, down from " the accurate respectively acetic acid-sodium-acetate buffer 10ml that adds ", measures absorbance according to " preparation of typical curve " item in accordance with the law, calculating, promptly;
(1.5) every of the injection for preparing contains aconite alkaloids in aconitine, must not be higher than 1.0mg.
7. detection method as claimed in claim 1, it also comprises assay:
(1) ginsenoside Rg 1Assay with Re:
High performance liquid chromatography according to an appendix VI of Chinese Pharmacopoeia version in 2005 D is measured;
(1.1) chromatographic condition
With the octadecylsilane chemically bonded silica is filling agent, and 17～23: 75～85 acetonitrile-0.1% phosphoric acid solution is a moving phase, detects wavelength 203nm; Number of theoretical plate calculates by the ginsenoside Re peak should be not less than 5000;
(1.2) preparation of reference substance solution
The ginsenoside Rg of phosphorus pentoxide drying under reduced pressure to constant weight learns from else's experience 1And ginsenoside Re's reference substance is an amount of, accurately claims surely, adds methyl alcohol and makes every 1ml respectively and contain the ginsenoside Rg 10.3mg, the solution of ginsenoside Re 0.2mg, promptly;
(1.3) preparation of need testing solution
Get the injection content 0.35g for preparing under the content uniformity item, accurate claim surely, put in the 5ml measuring bottle, be dissolved in water and be diluted to scale, shake up, promptly;
(1.4) determination method
Accurate respectively reference substance solution and each 10～30 μ l of need testing solution of drawing inject liquid chromatograph, measure, promptly;
(1.5) every of the injection for preparing contains the ginsenoside Rg 1, the ginsenoside Re the content sum should be 0.2～2.0mg;
(2) Determination of Total Saponin Content in Panax Ginseng:
(2.1) preparation of reference substance solution
The ginsenoside Rb of phosphorus pentoxide drying under reduced pressure to constant weight learns from else's experience 1Reference substance is an amount of, accurate claims surely, adds methyl alcohol and makes every 1ml and contain ginsenoside Rb 10.3mg solution, promptly;
(2.2) preparation of typical curve
Precision is measured above-mentioned ginsenoside Rb 1Reference substance solution 0.05～0.15ml, 0.15～0.25ml, 0.25～0.35ml, 0.35～0.45ml, 0.45～0.55ml, 0.55～0.65ml, 0.65～0.75ml puts respectively in the tool plug test tube, fling to solvent, each adds freshly prepared 5% vanillic aldehyde-glacial acetic acid solution 0.15～0.25ml and perchloric acid 0.6～1.0ml, heats 12～18min in 55～65 ℃ of water-baths, takes out, cooling rapidly, add glacial acetic acid 3～7ml, shake up, simultaneously with reagent corresponding as blank, spectrophotometric method according to an appendix VA of Chinese Pharmacopoeia version in 2005 is measured absorbance in 556nm wavelength place, with the absorbance is ordinate, and sampling amount is a horizontal ordinate, the drawing standard curve;
(2.3) preparation of sample solution
Get the injection content 0.35g for preparing under the content uniformity item, the accurate title, decide, adding distil water 10ml dissolving is put in the separating funnel, extracts 3～5 times with the chloroform jolting, each 8～12ml, discard chloroform solution, water liquid extracts 3～5 times with water saturated normal butyl alcohol jolting again, each 8～12ml, merge normal butyl alcohol liquid, use the saturated water washing of normal butyl alcohol 2～3 times again, each 8～12ml discards water liquid, normal butyl alcohol liquid is put evaporate to dryness in the water-bath, residue adds dissolve with methanol, is transferred in the 10ml measuring bottle, and is diluted to scale, shake up, promptly;
(2.4) determination method
Precision is measured sample solution 0.1ml and is put in the tool plug test tube, measures absorbance from " flinging to solvent " down according to " typical curve preparation " in accordance with the law, calculating, promptly;
(2.5) every of the injection for preparing contains general ginsenoside with ginsenoside Rb 1Meter should be 2.0～20.0mg;
(3) assay of polysaccharide composition in the genseng
(3.1) preparation of reference substance solution
The phosphorus pentoxide drying under reduced pressure of learning from else's experience is an amount of to the glucose reference substance of constant weight, and accurate the title decides, and adding distil water is made the solution that every 1ml contains glucose 0.2mg, promptly;
(3.2) preparation of typical curve
Precision is measured glucose reference substance solution 0.05～0.15ml, 0.15～0.25ml, 0.25～0.35ml, 0.35～0.45ml, 0.45～0.55ml, 0.55～0.65ml, 0.65～0.75ml, 0.75～0.85ml puts respectively in the tool plug test tube, adding distil water is to 1.0ml respectively, the anthrone sulfuric acid solution 6～10ml of each accurate adding freshly prepared 0.2%, wherein sulfuric acid concentration is 80%, shakes up the back and heats 8～12 minutes in 100 ℃ of water-baths, rapidly after the cooling, retinue is blank, measures absorbance at 620nm wavelength place according to spectrophotometric method, is horizontal ordinate with concentration, absorbance is an ordinate, the drawing standard curve;
(3.3) preparation of sample solution
Get the injection content 3.5g for preparing under the content uniformity item, accurate claim fixed, add water 25～35ml dissolving after, add ethanol and make and contain the alcohol amount and reach 75～85%, centrifugal, add dissolved in distilled water after precipitation volatilizes ethanol, and be settled in the 100ml measuring bottle; Get above-mentioned solution 1～3ml and put in the 25ml measuring bottle, thin up shakes up to scale, promptly;
(3.4) determination method
Precision is measured 0.2ml in tool plug test tube, measures absorbance from " adding distil water is to 1.0ml respectively " down by " preparation of typical curve " item in accordance with the law, calculates, promptly;
(3.5) every of the injection for preparing contains polysaccharide composition in the genseng with glucose meter, should be 4～60mg.
8. detection method as claimed in claim 7 is characterized in that described assay comprises:
(1) ginsenoside Rg 1Assay with Re:
According to high effective liquid chromatography for measuring;
(1.1) chromatographic condition
With the octadecylsilane chemically bonded silica is filling agent, and 20: 80 acetonitrile-0.1% phosphoric acid solution is a moving phase, detects wavelength 203nm; Number of theoretical plate calculates by the ginsenoside Re peak should be not less than 5000;
(1.2) preparation of reference substance solution
The ginsenoside Rg of phosphorus pentoxide drying under reduced pressure to constant weight learns from else's experience 1And ginsenoside Re's reference substance is an amount of, accurately claims surely, adds methyl alcohol and makes every 1ml respectively and contain the ginsenoside Rg 10.3mg, the solution of ginsenoside Re 0.2mg, promptly;
(1.3) preparation of need testing solution
Get the injection content 0.35g for preparing under the content uniformity item, accurate claim surely, put in the 5ml measuring bottle, be dissolved in water and be diluted to scale, shake up, promptly;
(1.4) determination method
Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject liquid chromatograph, measure, promptly;
(1.5) every of the injection for preparing contains the ginsenoside Rg 1, the ginsenoside Re the content sum should be 0.8～1.4mg;
(2) Determination of Total Saponin Content in Panax Ginseng:
(2.1) preparation of reference substance solution
The ginsenoside Rb of phosphorus pentoxide drying under reduced pressure to constant weight learns from else's experience 1Reference substance is an amount of, accurate claims surely, adds methyl alcohol and makes every 1ml and contain ginsenoside Rb 10.3mg solution, promptly;
(2.2) preparation of typical curve
Precision is measured above-mentioned ginsenoside Rb 1Reference substance solution 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml, 0.7ml puts respectively in the tool plug test tube, fling to solvent, each adds freshly prepared 5% vanillic aldehyde-glacial acetic acid solution 0.2ml and perchloric acid 0.8ml, heats 15min in 60 ℃ of water-baths, take out, cooling adds glacial acetic acid 5ml rapidly, shake up, the while as blank, in 556nm wavelength place is measured absorbance according to spectrophotometric method with reagent corresponding, with the absorbance is ordinate, and sampling amount is a horizontal ordinate, the drawing standard curve;
(2.3) preparation of sample solution
Get the injection content 0.35g for preparing under the content uniformity item, the accurate title, decide, and adding distil water 10ml dissolving is put in the separating funnel, extract 4 times with the chloroform jolting, each 10ml discards chloroform solution, water liquid extracts 4 times with water saturated normal butyl alcohol jolting again, and each 10ml merges normal butyl alcohol liquid, use the saturated water washing of normal butyl alcohol 2 times again, each 10ml discards water liquid, normal butyl alcohol liquid is put evaporate to dryness in the water-bath, and residue adds dissolve with methanol, is transferred in the 10ml measuring bottle, and be diluted to scale, shake up, promptly;
(2.4) determination method
Precision is measured sample solution 0.1ml and is put in the tool plug test tube, measures absorbance from " flinging to solvent " down according to " typical curve preparation " in accordance with the law, calculating, promptly;
(2.5) every of the injection for preparing contains general ginsenoside with ginsenoside Rb 1, i.e. C 54H 92O 23Meter should be 8.0～15.0mg;
(3) assay of polysaccharide composition in the genseng
(3.1) preparation of reference substance solution
The phosphorus pentoxide drying under reduced pressure of learning from else's experience is an amount of to the glucose reference substance of constant weight, and accurate the title decides, and adding distil water is made the solution that every 1ml contains glucose 0.2mg, promptly;
(3.2) preparation of typical curve
Precision is measured glucose reference substance solution 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml, 0.7ml 0.8ml puts respectively in the tool plug test tube, adding distil water is to 1.0ml respectively, the freshly prepared 0.2% anthrone sulfuric acid solution 8ml of each accurate adding, wherein sulfuric acid concentration is 80%, shakes up the back and heats 10 minutes in 100 ℃ of water-baths, rapidly after the cooling, retinue is blank, measures absorbance at 620nm wavelength place according to the spectrophotometric method of an appendix VA of Chinese Pharmacopoeia version in 2005, is horizontal ordinate with concentration, absorbance is an ordinate, the drawing standard curve;
(3.3) preparation of sample solution
Get the injection content 3.5g for preparing under the content uniformity item, accurate claim fixed, add water 30ml dissolving after, add ethanol and make and contain the alcohol amount and reach 80%, centrifugal, add dissolved in distilled water after precipitation volatilizes ethanol, and be settled in the 100ml measuring bottle; Get above-mentioned solution 2ml and put in the 25ml measuring bottle, thin up shakes up to scale, promptly;
(3.4) determination method
Precision is measured 0.2ml in tool plug test tube, measures absorbance from " adding distil water is to 1.0ml respectively " down by " preparation of typical curve " item in accordance with the law, calculates, promptly;
(3.5) every of the injection for preparing contains polysaccharide composition in the genseng with glucose meter, should be 27～47mg.
9. detection method as claimed in claim 1 is characterized in that described injection is parenteral solution, freeze-drying preparation for injection or sterile packaged preparation for injection.
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