CN1836717A

CN1836717A - Quality controlling method for pulse restoring injection

Info

Publication number: CN1836717A
Application number: CN 200510200759
Authority: CN
Inventors: 于文勇
Original assignee: Yunyanxichuang Medicinal Science And Technology Development Co Ltd Guiyang C
Current assignee: Beijing Liushenghe Medical Technology Co.,Ltd.
Priority date: 2004-12-13
Filing date: 2005-12-02
Publication date: 2006-09-27
Anticipated expiration: 2025-12-02
Also published as: CN100529753C

Abstract

The quality control method for Shengmai injection includes fingerprint test, identification, content measurement and other items. The present invention obtains ideal fingerprint of red ginseng or ginseng, ophiopogon root and schisandra in certain conditions. The quality control method is effective on the Chinese medicine injection prepared with red ginseng or ginseng, ophiopogon root and schisandra and possesses high precision and high stability.

Description

The method of quality control of pulse restoring injection

Technical field

The present invention is a kind of method of quality control of pulse restoring injection, belongs to the technical field of quality control, particularly a kind of method of quality control for traditional medicine Injectio.

Background technology

Pulse restoring injection is that raw material is made with Radix Ginseng, Radix Ophiopogonis, Fructus Schisandrae Chinensis, have supplementing QI and nourishing YIN, answer the effect that arteries and veins takes off admittedly, be used for QIYINLIANGXU, the cardiopalmus that deficient pulse is desired to take off, the deficiency of vital energy, extreme cold of the limbs and myocardial infarction, cardiogenic shock, septic shock also are used for the treatment of diseases such as angina pectoris, weakness of the spleen and stomach, acute viral myocarditis, malignant tumor, severe pneumonia of infants, viral pneumonia, viral hepatitis, optic atrophy, diabetes, cervical spondylosis, psoriasis, sudden deafness at present clinically; Be to change agent by traditional SHENGMAI SAN to form, it has overcome the slow shortcoming of conventional formulation onset, number of research projects has been done to it by many inventors and medicine enterprise, as: number of patent application is " 93110807.1 ", application, number of patent application that name is called " SHENGMAI ZHUSHEYE and preparation technology thereof " are called the application of " a kind of SHENGMAI ZHUSHEYE and preparation method thereof " for " 96117457.9 ", name; Applicant's Yu Wenyong had also once applied for to Patent Office of the People's Republic of China that name was called: " a kind of pharmaceutical composition for the treatment of cardiovascular and cerebrovascular disease and preparation method thereof ", number of patent application are 02153312.1 patent of invention, but pharmaceutical preparation must be on the basis that guarantees the constant product quality controllable safety, constantly more new development, in order better to control the quality of said preparation, guarantee the safety of medication, better instruct and produce, make technology controlling and process rationally strict more, make consumer's energy full appreciation product quality, need this injection method for quality of research control; At present, in the related drugs preparation of formulation of ' Sheng Mai ', generally only with ginsenoside Re, Rb ₁Be to detect index, but its quality of reactor product comprehensively at all not very reasonable with this quality that is used for controlling injection only; If control,, relatively be difficult to implement owing to do not have ready-made detection scheme, testing conditions or the like with other index.

Summary of the invention

The objective of the invention is to: a kind of method of quality control of pulse restoring injection is provided, and this method provides means, technical method of the index that detects, detection or the like to relevant production, testing agency; So that better control the quality of said preparation, guarantee the safety of medication, can better instruct production, make controlling of production process rationally strict more, make consumer's energy full appreciation product quality.

The present invention constitutes like this:

The method of quality control of pulse restoring injection comprises following all or part of content:

(1) finger printing of pulse restoring injection test, comprise based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics finger printing and based in the finger printing of Fructus Schisandrae Chinensis composition characteristics one or more;

(2) Radix Ginseng Rubra or ginseng crude drug, Radix Ophiopogonis medical material, schisandra chinensis medicinal material, ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re, ginsenoside Rf, ophiopogonin B, ophiopogonin D, ophiopogonin D ', the differential test method of all or part of composition in the Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B;

(3) ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re, ophiopogonin B, ophiopogonin D, ophiopogonin D ', the content test method of all or part of composition in the Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, schisandrin, schisantherin B, schisantherin A, deoxyschizandrin, schisandrin B, schisandrin C, total saponins, total lignans, total polysaccharides.

The method of quality control of described pulse restoring injection, this method comprises one or both in the following finger printing:

A, adopt liquid chromatography test Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics be main finger printing:

(1) preparation of need testing solution: it is an amount of to get Shengmai injection to be measured, adds water or methanol or dissolve with ethanol or dilution, shakes up, and filters, and gets subsequent filtrate as need testing solution;

(2) preparation of object of reference solution: get the main active reference substance in an amount of Radix Ginseng Rubra or Radix Ginseng and the Radix Ophiopogonis medical material, comprise the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re, ginsenoside Rf, ophiopogonin B, ophiopogonin D, Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, ophiopogonin D ' in a kind of, water or methanol or dissolve with ethanol, be settled to suitable concn, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler: mobile phase A is 0.01mol/L～2mol/L biphosphate sodium water solution or 0.01mol/L～2mol/L potassium dihydrogen phosphate aqueous solution or 0.01mol/L～2mol/L sodium hydrogen phosphate aqueous solution or water or 0.1%～5% glacial acetic acid solution or 0.1%～5% formic acid solution or 0.02%～5% phosphoric acid solution; B is 10%～100% acetonitrile solution or 10%～absolute methanol solution, and gradient elution, flow velocity be 0.5～2.0ml/min, detect wavelength is that one or several or evaporation photodetector in 190～410nm scope detects, and column temperature is in 20～60 ℃ of scopes;

(4) formulation of standard finger-print: with said method as formulate based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics the means of testing of standard finger-print; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～50;

(5) based on the described method in (1)～(3) as Radix Ginseng Rubra or Radix Ginseng in the injection to be measured and Radix Ophiopogonis composition characteristics the means of testing of finger printing, the finger printing of preparation testing sample;

(6) with the finger printing of injection to be measured and the contrast of above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:

I. calculate the similarity of the finger printing and the standard finger-print of injection to be measured, should be 0.80～1.00;

II. in the injection finger printing to be measured, non-total peak area must not surpass 10% of total peak area;

In the ratio that 30%～80% total peak area arranged in the injection finger printing III. to be measured and the standard finger-print odds ratio of each corresponding total peak area, its difference must not be greater than soil 50%;

B, employing liquid chromatography are tested the finger printing based on the Fructus Schisandrae Chinensis composition characteristics:

(2) preparation of object of reference solution: get the main active reference substance in an amount of schisandra chinensis medicinal material, comprise a kind of in schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, the schisantherin B, water or methanol or dissolve with ethanol, be settled to suitable concn, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is acetonitrile or methanol, Mobile phase B is water or 0.01%～3% phosphoric acid solution or 0.1%～5% glacial acetic acid solution or 0.1%～5% formic acid solution, gradient elution, flow velocity is that 0.5～2.0ml/min, detection wavelength are one or several in the 190-410nm scope, and column temperature is in 20～60 ℃ of scopes;

(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the standard finger-print of Fructus Schisandrae Chinensis composition characteristics; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～50;

(5) based on the means of testing of the described method in (1)～(3) as the finger printing of Fructus Schisandrae Chinensis composition characteristics in the injection to be measured, the finger printing of preparation testing sample;

(6) with the contrast of injection finger printing to be measured and above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:

In the ratio that 30%～80% total peak area arranged in the injection finger printing III. to be measured and the standard finger-print odds ratio of each corresponding total peak area, its difference must not be greater than soil 50%.

(ratio of total peak area is meant: as 1, calculate the ratio of each total fingerprint peaks area and object of reference peak area with the object of reference peak area)

A, adopt liquid chromatography for measuring based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics finger printing:

(1) preparation of need testing solution: it is an amount of that precision takes by weighing Shengmai injection to be measured, adds water and make the solution that every 1ml contains 50mg, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;

(2) preparation of object of reference solution: precision takes by weighing the ginsenoside Rg ₁In right amount, add methanol and make the solution that every 1ml contains 0.3mg, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is the 0.05mol/L potassium dihydrogen phosphate, and Mobile phase B is acetonitrile-water 80: 20, gradient elution, solvent ratios is from 0 minute to 5 minutes, the ratio of Mobile phase B is 25%, and from 5 minutes to 40 minutes, the ratio of Mobile phase B rose to 64% by 25%, from 40 minutes to 50 minutes, the ratio of Mobile phase B is 64%, and from 50 minutes to 65 minutes, the ratio of Mobile phase B rose to 80% by 64%, from 65 minutes to 75 minutes, the ratio of Mobile phase B reduced to 25% by 80%; Flow velocity is 1.0ml/min, and the detection wavelength is 203 ± 2nm, and column temperature is 40 ℃;

(4) formulation of standard finger-print: with said method as formulate based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics the means of testing of standard finger-print; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～30;

(6) with the contrast of pulse restoring injection finger printing to be measured and above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:

I. calculate the similarity of the finger printing and the standard finger-print of injection to be measured, should be 0.90～1.00;

II. in the injection finger printing to be measured, non-total peak area must not surpass 5% of total peak area;

In the ratio that 40%～60% total peak area arranged in the injection finger printing III. to be measured and the standard finger-print odds ratio of each corresponding total peak area, its difference must not be greater than soil 30%;

B, adopt the finger printing of liquid chromatography for measuring based on the Fructus Schisandrae Chinensis composition characteristics:

(2) preparation of object of reference solution: it is an amount of that precision takes by weighing the schisandrin reference substance, adds water and make the solution that every 1ml contains 0.1mg, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase is methanol-water, gradient elution, solvent ratios are from 0 minute to 6 minutes, and the ratio of methanol is 68%, from 6 minutes to 8 minutes, the ratio of methanol rises to 76% by 68%, and from 8 minutes to 10 minutes, the ratio of methanol was 76%, from 10 minutes to 15 minutes, the ratio of methanol reduces to 68% by 76%, and from 15 minutes to 60 minutes, the ratio of methanol was 68%; Flow velocity is 1.0ml/min, and the detection wavelength is 250 ± 2nm, and column temperature is 30 ℃;

(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the standard finger-print of Fructus Schisandrae Chinensis composition characteristics; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulating standard finger-print, is benchmark with the retention time of the object of reference chromatographic peak determined, calculates the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 3～30;

In the ratio that 40%～60% total peak area arranged in the injection finger printing III. to be measured and the standard finger-print odds ratio of each corresponding total peak area, its difference must not be greater than soil 30%.

The method of quality control of described pulse restoring injection, the discrimination method of described injection comprise following all or part of content:

One or more thin layer chromatography discrimination method in Fructus Schisandrae Chinensis, schisandrin, schisantherin B, Fructus Schisandrae Chinensis first element, schisandrin B, schisandrin C, schisantherin A, the schisantherin B in a, the injection:

It is an amount of to get Shengmai injection to be measured, adds chloroform or ether or ethyl acetate or normal hexane or 10%～dehydrated alcohol or 10%～absolute methanol and extracts, and filters, and filtrate volatilizes, and residue adds chloroform or ethyl acetate dissolving, as need testing solution; Other gets one or more preparation contrast solutions in Fructus Schisandrae Chinensis control medicinal material, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, Fructus Schisandrae Chinensis third element, schisantherin A, the schisantherin B; The preparation of Fructus Schisandrae Chinensis control medicinal material solution: it is an amount of to get the Fructus Schisandrae Chinensis control medicinal material, add chloroform or ether or ethyl acetate or normal hexane or 10%～dehydrated alcohol or 10%～absolute methanol and extract, filter, filtrate volatilizes, residue adds chloroform or ethyl acetate dissolving, medical material solution in contrast; The preparation of reference substance solution: get one or more reference substances in schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, the schisantherin B; Add chloroform or ethyl acetate respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned need testing solution and reference substance solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F ₂₅₄On the lamellae, with petroleum ether (30～60 ℃) or petroleum ether (60～90 ℃): Ethyl formate or ethyl acetate: formic acid or acetic acid=2～40: 0.2～15: 0.1～5 upper solution or benzene or toluene: ethyl acetate or Ethyl formate=1～30: 0.2～10 or normal hexane: benzene or toluene: Ethyl formate or ethyl acetate: formic acid or acetic acid=0.2～5: 0.5～10: 1～15: 0.1～5 is developing solvent, launch, take out, dry, putting uviol lamp 365nm or 254nm inspects or sprays with 2%～20% chromotropic acid-concentrated sulfuric acid solution or 2%～20% phosphomolybdic acid ethanol solution or anisaldehyde sulfuric acid solution, dry by the fire the clear or smoked colour developing of iodine at 80 ℃～160 ℃ to the speckle colour developing, in the test sample chromatograph, with control medicinal material chromatograph or the corresponding position of reference substance chromatograph on, should show the speckle of same color;

One or more liquid chromatograph discriminating in schisandrin, deoxyschizandrin, the schisandrin B in b, the injection:

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methanol or mobile phase or ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With schisandrin, deoxyschizandrin, the methanol of one or more reference substances or alcoholic solution are contrast in the schisandrin B, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile: water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% aqueous formic acid=5%～80%: 95%～20% is mobile phase, the detection wavelength is one or several in 190～410nm scope, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the main peak in the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;

The thin layer chromatography discrimination method of Radix Ophiopogonis in c, the injection:

It is an amount of to get Shengmai injection to be measured, adds chloroform-methanol mixed solution or n-butyl alcohol or chloroform or ethyl acetate or dichloromethane extraction, as need testing solution; Other gets control medicinal material Radix Ophiopogonis, adds chloroform-methanol mixed solution or ethyl acetate or n-butyl alcohol or chloroform or dichloromethane extraction, filters, and evaporate to dryness, residue add chloroform or dichloromethane dissolving, medical material solution in contrast; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F ₂₅₄On the lamellae, with benzene or toluene or chloroform or dichloromethane: methanol or ethanol: glacial acetic acid or formic acid or water=8～300: 0.5～100: 0.01～30 or ethyl acetate or Ethyl formate: pyridine: water=0.2～10: 0.1～5: 0.2～10 is developing solvent, launch, take out, dry, put and inspect under uviol lamp 365nm or the 254nm or spray with 1%～50% sulphuric acid or vanillin reagent or 1%～25% vanillin reagent or anisaldehyde reagent, it is clear to dry by the fire to the speckle colour developing at 80 ℃～160 ℃, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the fluorescence speckle of same color;

Ophiopogonin B in d, the injection, ophiopogonin D, ophiopogonin D ' in one or more thin layer chromatography discrimination method:

It is an amount of to get Shengmai injection to be measured, with behind the water dissolution with n-butyl alcohol or ether or ethyl acetate or chloroform or dichloromethane extraction, extract volatilizes, residue is with methanol or dissolve with ethanol, as need testing solution; Other get ophiopogonin B, ophiopogonin D, ophiopogonin D ' in one or more reference substances, add methanol or ethanol respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F ₂₅₄On the lamellae, with ethyl acetate or chloroform or dichloromethane: methanol or ethanol: water=3～50: 1～15: 0.1～5 is developing solvent, launch, take out, dry, spray is with 5%～50% sulphuric acid ethanol reagent, it is clear to dry by the fire to the speckle colour developing at 80 ℃～160 ℃, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, should show the speckle of same color;

Ophiopogonin B in e, the injection, ophiopogonin D, ophiopogonin D ' in one or more liquid chromatograph differentiate:

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methanol or mobile phase or ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With ophiopogonin B, ophiopogonin D, ophiopogonin D ' in the methanol of one or more reference substances or alcoholic solution be contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile: water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% aqueous formic acid=1%～99%: 99%～1% is mobile phase, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;

One or more thin layer chromatography discrimination method in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin in f, the injection:

It is an amount of to get Shengmai injection to be measured, add to regulate pH behind sulphuric acid or the hydrochloric acid hydrolysis to neutral, and evaporate to dryness, residue is with chloroform or dichloromethane or ethyl acetate or methanol or dissolve with ethanol, as need testing solution; Other gets one or more reference substances in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin, adds chloroform or dichloromethane or ethyl acetate or methanol or ethanol respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F ₂₅₄On the lamellae, with normal hexane or cyclohexane extraction or methanol or ethanol: ethyl acetate or chloroform or dichloromethane or Ethyl formate: water=0.2～15: 0.2～40: 0.1～5 is developing solvent, launch, take out, dry, spray is with 5%～50% sulphuric acid ethanol reagent, it is clear to dry by the fire to the speckle colour developing at 80 ℃～160 ℃, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, should show the speckle of same color;

One or more liquid chromatograph discriminating in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin in g, the injection:

It is an amount of to get Shengmai injection to be measured, put in the round-bottomed flask, after vulcanization acid or hydrochloric acid hydrolysis are dissolved in water, take out, put to room temperature, with chloroform or dichloromethane or n-butyl alcohol or ethyl acetate extraction, extracting solution volatilizes accent pH to neutral back, residue filters with microporous filter membrane after with methanol or dissolve with ethanol, gets subsequent filtrate as need testing solution; With Ruscus aculeatus L. sapogenin, diosgenin, the methanol of one or more reference substances or alcoholic solution are contrast in the ruscogenin, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile: water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% aqueous formic acid=5%～95%: 95%～5% is mobile phase, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;

Radix Ginseng Rubra or Radix Ginseng, ginsenoside Rg in h, the injection ₁, ginsenoside Rb ₁, one or more thin layer chromatography discrimination method among the ginsenoside Re, ginsenoside Rf:

It is an amount of to get Shengmai injection to be measured, with n-butyl alcohol or ethanol or methanol or ethyl acetate or chloroform or dichloromethane extraction, filters, and filtrate is as need testing solution; Other gets Radix Ginseng Rubra or Radix Ginseng control medicinal material, ginsenoside Rg ₁, ginsenoside Rb ₁, among the ginsenoside Re, ginsenoside Rf one or more, the preparation contrast solution; The preparation of Radix Ginseng Rubra or Radix Ginseng control medicinal material solution: get Radix Ginseng Rubra or the Radix Ginseng control medicinal material is an amount of, adding chloroform or methylene chloride reflux extracts, discard chloroform or dichloromethane solution, residue adds water-saturated n-butanol or ethyl acetate extraction, extracting solution adds ammonia solution, divides and gets upper strata, evaporate to dryness, residue is with methanol or dissolve with ethanol, medical material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁, one or more reference substances among the ginsenoside Re, ginsenoside Rf, add methanol or ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively in same silica gel g thin-layer plate or silica gel H lamellae or silica gel G F ₂₅₄On the lamellae, with chloroform or dichloromethane or normal hexane or cyclohexane extraction: ethyl acetate or Ethyl formate: methanol or ethanol or acetone: water=5～40: 10～100: 5～50: 0.2～30 at lower floor's solution or the n-butyl alcohol placed below 10 ℃: ethyl acetate or Ethyl formate: water=1～10: 0.2～2: 1～15 upper strata is developing solvent, launch, take out, dry, spray is with 5%～50% sulphuric acid ethanol reagent, it is clear to dry by the fire to the speckle colour developing at 80 ℃～160 ℃, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, should show the speckle of same color;

Ginsenoside Rg in i, the injection ₁, ginsenoside Rb ₁, one or more liquid chromatograph is differentiated among the ginsenoside Re:

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methanol or mobile phase or ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁The methanol of one or more reference substances or alcoholic solution are contrast among the ginsenoside Re, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile: water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% formic acid solution=5%～40%: 95%～60% is mobile phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

One or more thin layer chromatography discrimination method in Fructus Schisandrae Chinensis, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, the schisantherin B in a, the injection:

It is an amount of to get Shengmai injection to be measured, adds chloroform extraction, filters, and filtrate volatilizes, and residue adds the chloroform dissolving, as need testing solution; Other gets in Fructus Schisandrae Chinensis control medicinal material, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, the Fructus Schisandrae Chinensis ester second one or more, the preparation contrast solution; The preparation of Fructus Schisandrae Chinensis control medicinal material solution: it is an amount of to get the Fructus Schisandrae Chinensis control medicinal material, adds chloroform extraction, filters, and filtrate volatilizes, and residue adds chloroform dissolving, medical material solution in contrast; The preparation of reference substance solution: get one or more reference substances in schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, the schisantherin B, add chloroform respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put in same silica gel G F respectively ₂₅₄On the lamellae, with petroleum ether (30～60 ℃): Ethyl formate: the upper solution of formic acid=15: 5: 1 is developing solvent, launch, take out, dry, put under the uviol lamp 254nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color;

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with one or more reference substances in schisandrin, deoxyschizandrin, the schisandrin B is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, methanol: water=65%: 35% is mobile phase, the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;

It is an amount of to get Shengmai injection to be measured, adds 7: 3 mixed solutions of chloroform-methanol and extracts, and filters, and filtrate volatilizes, and residue adds the chloroform dissolving, as need testing solution; Other gets control medicinal material Radix Ophiopogonis, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put in same silica gel G F respectively ₂₅₄On the lamellae, with toluene: methanol: glacial acetic acid=80: 5: 0.1 is developing solvent, launches, and takes out, dry, put under the uviol lamp 254nm and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color, negative noiseless;

It is an amount of to get Shengmai injection to be measured, and with using n-butanol extraction behind the water dissolution, extract volatilizes, and the residue dissolve with methanol is as need testing solution; Other get ophiopogonin B, ophiopogonin D, ophiopogonin D ' in one or more reference substances, add methanol respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with ethyl acetate: methanol: water=15: 5: 1 is developing solvent, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With ophiopogonin B, ophiopogonin D, ophiopogonin D ' in the methanol solution of one or more reference substances be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;

It is an amount of to get Shengmai injection to be measured, adds 3% sulphuric acid hydrolysis 4 hours, takes out, and puts to room temperature, regulates pH to neutral, filters, and filtrate volatilizes, and residue dissolves with chloroform, as need testing solution; Other gets one or more reference substances in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin, adds chloroform respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with normal hexane: ethyl acetate: water=1: 1: 1 is developing solvent, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 90 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color;

It is an amount of to get Shengmai injection to be measured, puts in the round-bottomed flask, and refluxing 4 hours with 3% sulphuric acid in the back that is dissolved in water, takes out, put to room temperature, transfer pH to neutral, use chloroform extraction, extracting solution volatilizes, the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; By the methanol solution with one or more reference substances in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;

It is an amount of to get Shengmai injection to be measured, uses n-butanol extraction, filters, and filtrate is as need testing solution; Other gets Radix Ginseng Rubra or Radix Ginseng control medicinal material, ginsenoside Rg ₁, ginsenoside Rb ₁, among the ginsenoside Re, ginsenoside Rf one or more, preparation control medicinal material solution; The preparation of Radix Ginseng Rubra or Radix Ginseng control medicinal material solution: get Radix Ginseng Rubra or the Radix Ginseng control medicinal material is an amount of, add the chloroform reflux, extract,, discard chloroform liquid, residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁, one or more reference substances among the ginsenoside Re, ginsenoside Rf, add methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with chloroform: ethyl acetate: methanol: water=15: 40: 22: 10 is developing solvent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ are dried by the fire to speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color;

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, the methanol solution of one or more reference substances is contrast among the ginsenoside Re, adopts liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

The method of quality control of described pulse restoring injection, the method for testing of described injection content should comprise following all or part of content:

Ginsenoside Rg in a, the injection ₁, ginsenoside Rb ₁, one or more assay among the ginsenoside Re:

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methanol or mobile phase or ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁The methanol of one or more reference substances or alcoholic solution are contrast among the ginsenoside Re, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile: water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% formic acid solution=5%～40%: 95%～60% is mobile phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; Calculate with external standard method or standard curve method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

The per unit amount contains the ginsenoside Rg ₁Limit must not be less than 0.8mg;

The limit that the per unit amount contains the ginsenoside Re must not be less than 0.4mg;

The per unit amount contains ginsenoside Rb ₁Limit must not be less than 0.5mg;

(4) the per unit amount contains the ginsenoside Rg ₁, the ginsenoside Re the limit of summation must not be less than 1.7mg;

The assay of total saponins in b, the injection:

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, and adding distil water or methanol or ethanol make dissolving and fixed to scale in right amount, shake up, precision is measured in right amount, puts in the measuring bottle, water bath method takes out immediately, and precision adds 1%～50% vanillin-glacial acetic acid solution 0.1ml～10ml, perchloric acid 0.1ml～15ml shakes up, and heats 3～50 minutes in 30 ℃～80 ℃ water-baths, take out, with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale immediately, shake up, as need testing solution, with the ginsenoside Rg ₁Or ginsenoside Rb ₁Or ginsenoside Re or ginsenoside Rf or ophiopogonin B or ophiopogonin D or ophiopogonin D ' be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, wavelength place at 547 ± 10nm measures trap, calculate with external standard method or standard curve method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with the ginsenoside Rg ₁Or ginsenoside Rb ₁Or ginsenoside Re or ginsenoside Rf or ophiopogonin B or ophiopogonin D or ophiopogonin D ' meter, must not be less than 10mg;

Total polysaccharides assay in c, the injection:

It is an amount of that monosaccharide is got Shengmai injection to be measured, puts in the iodine flask, and pH is regulated to neutral with sodium hydroxide test solution in adding distil water dissolving back, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, add dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear, and titration results is proofreaied and correct with blank assay, promptly to blue.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg, calculates the content of monosaccharide thus;

It is an amount of that total sugar is got Shengmai injection to be measured, put in the iodine flask, adding distil water adds dilute sulfuric acid 25ml after making dissolving, reflux 1～4 hour, put cold, add 1～2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, and precision adds iodine liquid (0.1mol/L) 25ml, shake up, dropwise add sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, add dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear, and titration results is proofreaied and correct with blank assay, promptly to blue.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg, calculates sugar contents with this;

The content that deducts monosaccharide with sugar contents promptly gets the content of total polysaccharides;

Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous glucose, must not be less than 50mg;

Total lignans assay in d, the injection:

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, and it is fixed to scale to add water, behind salt Acid precipitation saponin component, with ethyl acetate or chloroform or dichloromethane extraction and and extracting solution, volatilize, residue is with methanol or dissolve with ethanol, as need testing solution; Methanol or alcoholic solution with schisandrin or schisantherin B are reference substance solution.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, wavelength place at 254 ± 10nm measures trap, calculate with external standard method or standard curve method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, the per unit amount contains total lignans in schisandrin or schisantherin B, must not be less than 1.5mg;

One or more assay in schisandrin, deoxyschizandrin, the schisandrin B in e, the injection:

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methanol or mobile phase or ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With schisandrin, deoxyschizandrin, the methanol of one or more reference substances or alcoholic solution are contrast in the schisandrin B, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile: water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% aqueous formic acid=5%～80%: 95%～20% is mobile phase, the detection wavelength is one or several in 190～410nm scope, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or standard curve method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

(1) the per unit amount limit that contains schisandrin must not be less than 0.2mg;

(2) the per unit amount limit that contains deoxyschizandrin must not be less than 0.025mg;

(3) the per unit amount limit that contains schisandrin B must not be less than 0.075mg;

(4) the per unit amount limit that contains the summation of schisandrin, deoxyschizandrin, schisandrin B must not be less than 0.3mg;

Ophiopogonin B in f, the injection, ophiopogonin D, ophiopogonin D ' in one or more assay:

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methanol or mobile phase or ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With ophiopogonin B, ophiopogonin D, ophiopogonin D ' in the methanol of one or more reference substances or alcoholic solution be contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile: water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% aqueous formic acid=1%～99%: 99%～1% is mobile phase, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or standard curve method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several.

(1) the per unit amount limit that contains ophiopogonin B must not be less than 0.05mg;

(2) the per unit amount limit that contains ophiopogonin D must not be less than 0.05mg;

(3) the per unit amount contain ophiopogonin D ' limit must not be less than 0.01mg;

(4) the per unit amount contain ophiopogonin B, ophiopogonin D, ophiopogonin D ' the limit of summation must not be less than 0.11mg;

One or more assay in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin in g, the injection:

It is an amount of to get Shengmai injection to be measured, put in the round-bottomed flask, after vulcanization acid or hydrochloric acid hydrolysis are dissolved in water, take out, put to room temperature, transfer pH to neutral back with chloroform or n-butyl alcohol or ethyl acetate extraction, extracting solution volatilizes, residue filters with microporous filter membrane with methanol or dissolve with ethanol, gets subsequent filtrate as need testing solution; With Ruscus aculeatus L. sapogenin, diosgenin, the methanol of one or more reference substances or alcoholic solution are contrast in the ruscogenin, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile: water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% aqueous formic acid=5%～95%: 95%～5% is mobile phase, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; Calculate with one point external standard method or standard curve method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

(1) the per unit amount limit that contains Ruscus aculeatus L. sapogenin must not be less than 0.01mg;

(2) the per unit amount limit that contains diosgenin must not be less than 0.01mg;

(3) the per unit amount limit that contains ruscogenin must not be less than 0.01mg;

(4) the per unit amount limit that contains the summation of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin must not be less than 0.03mg.

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, the methanol solution of one or more reference substances is contrast among the ginsenoside Re, adopts liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is 30 ℃; Calculate with one point external standard method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

(1) the per unit amount contains the ginsenoside Rg ₁Limit must not be less than 1.6mg;

(2) the per unit amount limit that contains the ginsenoside Re must not be less than 0.8mg

(3) the per unit amount contains ginsenoside Rb ₁Limit must not be less than 1.0mg;

(4) the per unit amount contains the ginsenoside Rg ₁, ginsenoside Rb ₁, the ginsenoside Re the limit of summation must not be less than 3.4mg;

The assay of total saponins in b, the injection:

It is an amount of to get Shengmai injection to be measured, puts in the 10ml measuring bottle, adds water to scale, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillin-glacial acetic acid solution 0.5ml, perchloric acid 2.0ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, immediately with ice-water bath cooling 2 minutes, fixed to scale with glacial acetic acid, shake up, as need testing solution; With the ginsenoside Rg ₁Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, measure trap, calculate with one point external standard method at the wavelength place of 547nm, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with the ginsenoside Rg ₁Meter must not be less than 20mg;

Total polysaccharides assay in c, the injection:

It is an amount of that monosaccharide is got Shengmai injection to be measured, puts in the iodine flask, and adding distil water makes dissolving, hydro-oxidation sodium test solution is to neutral, and accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, dropwise add sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, uses the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg, calculates the content of monosaccharide thus;

It is an amount of that total sugar is got Shengmai injection to be measured, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 4 hours is put coldly, adds 1～2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg, calculates sugar contents with this;

Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous glucose, must not be less than 100mg;

Total lignans assay in d, the injection:

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, and it is fixed to scale to add water, behind salt Acid precipitation saponin component, use ethyl acetate extraction and extracting solution also, water bath method, and the residue dissolve with methanol is as need testing solution; With the schisandrin is reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, wavelength place at 254nm measures trap, calculate with one point external standard method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, the per unit amount contains total lignans in schisandrin, must not be less than 3mg;

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with one or more reference substances in schisandrin, deoxyschizandrin, the schisandrin B is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-water is a mobile phase at 65%: 35%, the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

(1) the per unit amount limit that contains schisandrin must not be less than 0.4mg;

(2) the per unit amount limit that contains deoxyschizandrin must not be less than 0.05mg;

(3) the per unit amount limit that contains schisandrin B must not be less than 0.10mg;

(4) the per unit amount limit that contains the summation of schisandrin, deoxyschizandrin, schisandrin B must not be less than 0.55mg;

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With ophiopogonin B, ophiopogonin D, ophiopogonin D ' in the methanol solution of one or more reference substances be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90%: 10%, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

(1) the per unit amount limit that contains ophiopogonin B must not be less than 0.1mg;

(2) the per unit amount limit that contains ophiopogonin D must not be less than 0.1mg;

(3) the per unit amount contain ophiopogonin D ' limit must not be less than 0.02mg;

(4) the per unit amount contain ophiopogonin B, ophiopogonin D, ophiopogonin D ' the limit of summation must not be less than 0.22mg;

It is an amount of to get Shengmai injection to be measured, puts in the round-bottomed flask, is dissolved in water, and refluxes 4 hours with 3% sulphuric acid, takes out, put to room temperature, transfer pH, use chloroform extraction and extracting solution also to neutral, evaporate to dryness, the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with one or more reference substances in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

(1) the per unit amount limit that contains Ruscus aculeatus L. sapogenin must not be less than 0.02mg;

(2) the per unit amount limit that contains diosgenin must not be less than 0.02mg;

(3) the per unit amount limit that contains ruscogenin must not be less than 0.02mg;

(4) the per unit amount limit that contains the summation of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin must not be less than 0.06mg.

The method of quality control of described pulse restoring injection, all or part of total content of surveying composition of the saponins in the described injection, polysaccharide, lignanoids account for more than 25% of total solid of deducting adjuvant amount and water quantities in the preparation.

(total solid is meant: the weight of content deducts the weight after adjuvant amount and the water quantities)

Compared with prior art, the present invention's quality of the Chinese medicine injection products made with Radix Ginseng Rubra or Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae Chinensis of perfect control more.The composition more complicated of this Chinese medicine preparation, if only with wherein one, two kind of composition illustrate its inherent quality, has certain one-sidedness, more can't judge the index components of its drug effect.Therefore the applicant formulated based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics finger printing and control the quality of injection based on the finger printing of Fructus Schisandrae Chinensis composition characteristics comprehensively.But because contained complex chemical composition between each medical material in the pulse restoring injection causes interference to the formulation of finger printing, cause each several part finger printing feature instability, thus must control mobile phase isochromatic spectrum condition, just can obtain good finger printing.That is to say, the finger printing of formulation of ' Sheng Mai ' is not that the finger printing simple superposition of Radix Ginseng Rubra or Radix Ginseng, Radix Ophiopogonis and schisandra chinensis medicinal material or preparation is just getable, because three kinds of medical material ingredients interference effect each other in the prescription, cause the finger printing characteristic peak of Radix Ginseng Rubra in the formulation of ' Sheng Mai ' or Radix Ginseng and Radix Ophiopogonis part, Fructus Schisandrae Chinensis part to change, and have only the condition of the present invention of employing, just can obtain ideal finger printing.

Proof by experiment, method of quality control of the present invention is more effective to the quality control of the Chinese medicine injection products made with Radix Ginseng Rubra or Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae Chinensis, and method precision, stability are all higher.

Experimental example 1 based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics the preparation of finger printing

A, experimental apparatus, reagent and sample:

Reference substance: ginsenoside Rg ₁: Nat'l Pharmaceutical ﹠ Biological Products Control Institute

B, chromatographic condition and system suitability experiment:

1. the selection of chromatographic column:

In the research process, having selected conventional octadecylsilane chemically bonded silica for use is the liquid-phase chromatographic column of filler, tried out Zorbax, Inertsil ODS-3 respectively, Diamonsil ODS (is C18,4.6mm * 200mm, 5 μ m) chromatographic column of three kinds of trades mark, result show that the chromatographic column of three kinds of trades mark all can reach separating effect preferably, and wherein Diamonsil ODS chromatographic column separating effect is best, post is imitated the highest, can reach 5400 (calculating with the object of reference ginsenoside Rg1).So finally selecting DiamonsilODS chromatographic column (4.6mm * 200mm, 5 μ m) for use is the experimentation post.

2. the selection of mobile phase:

Investigated (1) methanol-water (20: 80) in the research process respectively, (2) acetonitrile-water (10: 90), (3) acetonitrile-water (gradient elution) (4) A is 50mmol.L ^-1KH ₂PO ₄Solution, B is that (the gradient elution volume proportion is from 0 minute to 5 minutes to acetonitrile-water (80: 20), the ratio of Mobile phase B is 25%, and from 5 minutes to 40 minutes, the ratio of Mobile phase B rose to 64% by 25%, from 40 minutes to 50 minutes, the ratio of Mobile phase B is 64%, and from 50 minutes to 65 minutes, the ratio of Mobile phase B rose to 80% by 64%, from 65 minutes to 75 minutes, the ratio of Mobile phase B reduced to 25% by 80%) four kinds of flow phase system.The result shows that peak shape is relatively poor under mobile phase (1) condition, and it is less to go out the peak in 1 hour, goes out the peak after still having many components to be trapped in; Under mobile phase (2) the mobile phase condition, peak shape is relatively poor, separates bad; (3) under the condition, the peak hangover is serious, and it is incomplete to go out the peak; Under mobile phase (4) condition, peak shape is better, goes out the peak fully and be evenly distributed, so finally selected.

3. detection wavelength determination:

Be 50mmol.L at A in the research ^-1KH ₂PO ₄Solution, B are under acetonitrile-water (80: 20) (gradient elution) the mobile phase condition, have investigated the chromatographic peak situation under different-waveband typical wavelengths 203,210,230,254 respectively, the result shows, chromatographic peak is more under 203nm, and peak shape is better, so finally select for use 203nm as detecting wavelength.

4. instrument, chromatographic column and integral parameter:

4.1 instrument parameter: selected liquid-phase chromatographic analysis field mainstream configuration and well behaved Agilent 1100 series of high efficiency chromatograph of liquid for use, the Chemstation chromatographic work station.Chromatographic column is Diamonsil ODS (4.6mm * 200mm, 5 μ m); 40 ℃ of column temperatures, flow velocity 1.0ml/min.

4.2 integral parameter: Slope Sensitivity:1, peak width:0.05, smallest peaks area are 5% of object of reference (S) peak-to-peak area, minimum peak height be the S peak-to-peak high 5%.So setting can be avoided some very calculating of the chromatographic peak of small size (unimodal area accounts for total peak area less than 0.5%), guarantees the dependency with object of reference simultaneously.

5. the preparation of need testing solution:

It is an amount of that precision takes by weighing this product, adds water and make the solution that every 1ml contains 50mg, promptly.

6. the preparation of object of reference solution:

The ginsenoside Rg ₁Be one of Radix Ginseng Rubra or Radix Ginseng main active, its integral area proportion in finger printing more greatly and more stable is taken into account the research of intermediate and medical material simultaneously, therefore selected ginsenoside Rg ₁As object of reference.

7. finger printing and technical parameter:

Formulate standard finger-print according to 10 batch samples, test sample finger printing and standard finger-print are compared, similarity is all between 0.90～1.00.

Experimental example 2 is based on the preparation of the finger printing of Fructus Schisandrae Chinensis composition characteristics

A, experimental apparatus, reagent and sample:

Reference substance: schisandrin: Nat'l Pharmaceutical ﹠ Biological Products Control Institute.

B, chromatographic condition and system suitability experiment:

1. the selection of chromatographic column:

In the research process, having selected conventional octadecylsilane chemically bonded silica for use is the liquid-phase chromatographic column of filler, tried out Zorbax, Inertsil ODS-3 respectively, Diamonsil ODS (is C18,250mm * 4.6mm, 5 μ m) chromatographic column of three kinds of trades mark, result show that the chromatographic column of three kinds of trades mark all can reach separating effect preferably, and wherein Diamonsil ODS chromatographic column separating effect is best, post is imitated the highest, can reach 5400 (calculating with object of reference Fructus Schisandrae Chinensis alcohol first).So finally selecting DiamonsilODS chromatographic column (250mm * 4.6mm, 5 μ m) for use is the experimentation post.

2. the selection of mobile phase:

Investigated (1) methanol-water (20: 80) in the research process respectively, (2) acetonitrile-water (10: 80), (3) (the gradient elution volume proportion is from 0 minute to 6 minutes to acetonitrile-water (gradient elution) (4) methanol-water, the ratio of methanol is 68%, from 6 minutes to 8 minutes, the ratio of methanol rises to 76% by 68%, from 8 minutes to 10 minutes, the ratio of methanol is 76%, from 10 minutes to 15 minutes, the ratio of methanol reduces to 68% by 76%, and from 15 minutes to 60 minutes, the ratio of methanol was 68%) four kinds of flow phase system.The result shows that peak shape is relatively poor under mobile phase (1) condition, and it is less to go out the peak in 1 hour, goes out the peak after still having many components to be trapped in; Under mobile phase (2) the mobile phase condition, peak shape is relatively poor, separates bad; (3) under the condition, the peak hangover is serious, and it is incomplete to go out the peak; Under mobile phase (4) condition, peak shape is better, goes out the peak fully and be evenly distributed, so finally selected.

3. detection wavelength determination:

In the research under methanol-water (gradient elution) mobile phase condition, investigated the chromatographic peak situation under different-waveband typical wavelengths 203,210,230,250,300nm respectively, the result shows that chromatographic peak is more under 250nm, peak shape is better, so finally select for use 250nm as detecting wavelength.

4. instrument, chromatographic column and integral parameter:

4.1 instrument parameter: selected liquid-phase chromatographic analysis field mainstream configuration and well behaved Agilent 1100 series of high efficiency chromatograph of liquid for use, the Chemstation chromatographic work station.Chromatographic column is Diamonsil ODS (4.6mm * 200mm, 5 μ m); 30 ℃ of column temperatures, flow velocity 1.0ml/min.

5. the preparation of need testing solution:

6. the preparation of object of reference solution:

Schisandrin is one of main active in the Fructus Schisandrae Chinensis, and its integral area proportion in finger printing more greatly and more stable is taken into account the research of intermediate and medical material simultaneously, and therefore selected schisandrin is as object of reference.

7. finger printing and technical parameter:

The thin layer chromatography discrimination method of Fructus Schisandrae Chinensis, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B in experimental example 3 pulse restoring injections:

Feature for outstanding Fructus Schisandrae Chinensis, selected Fructus Schisandrae Chinensis, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, Fructus Schisandrae Chinensis ester first, schisantherin B is as its feature speckle, but owing to exist more and schisandrin in the medical material, schisantherin B, Fructus Schisandrae Chinensis first element, schisandrin B, schisandrin C, schisantherin A, the schisantherin B structure is close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition to the Fructus Schisandrae Chinensis schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B launches:

Condition	Problem
Condition	Problem	Normal hexane-toluene-methanol-water (5: 5: 10: 4) silica gel H lamellae	Reference substance is expanded to the forward position
Cyclohexane extraction-benzene-ethanol-formic acid (5: 5: 5: 3) silica gel H lamellae	Reference substance does not separate, and feminine gender has interference		Reference substance is expanded to the forward position
		With petroleum ether (30～60 ℃)-Ethyl formate-methanol (10: 10: 3) silica gel g thin-layer plate	Reference substance is expanded to the forward position
With petroleum ether (60～90 ℃)-ethyl acetate-methanol (15: 5: 1) silica gel G F ₂₅₄Lamellae	Reference substance does not separate, and feminine gender has interference		Reference substance is expanded to the forward position
		Toluene-methanol (10-5) silica gel H lamellae	Reference substance does not separate, and feminine gender has interference
Benzene-methanol (15-5) silica gel g thin-layer plate	Reference substance does not separate, and feminine gender has interference	Toluene-methanol (10-5) silica gel H lamellae
Benzene-methanol (15-5) silica gel g thin-layer plate		Upper solution silica gel G F with petroleum ether (30～60 ℃)-Ethyl formate-formic acid (15: 5: 1) ₂₅₄Lamellae	It is clear to separate, and the moderate feminine gender of Rf value is noiseless

Through screening, determined with the silica gel g thin-layer plate to be immobile phase, upper solution with petroleum ether (30～60 ℃)-Ethyl formate-formic acid (15: 5: 1) is developing solvent, with this understanding, the Rf value of schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B is moderate, it is clear to separate with other speckle, negative noiseless.

The liquid chromatograph discrimination method of schisandrin, deoxyschizandrin, schisandrin B in experimental example 4 pulse restoring injections:

Feature for outstanding Fructus Schisandrae Chinensis, except the thin layer discrimination method, selected schisandrin, deoxyschizandrin, schisandrin B as its characteristic component, but because have in the medical material that more and schisandrin, deoxyschizandrin, schisandrin B structure are close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and with mobile phase schisandrin, deoxyschizandrin, schisandrin B are separated:

Condition	Problem
Condition	Problem	Methanol-0.02mol/L sodium dihydrogen phosphate (80: 20) octadecylsilane chemically bonded silica	Appearance time is too fast

Acetonitrile-0.02mol/L sodium dihydrogen phosphate (40: 60) octadecylsilane chemically bonded silica	Feminine gender has interference
	Feminine gender has interference	Methanol-0.05mol/L sodium hydrogen phosphate (70: 30) eight alkyl silane bonded silica gels	Feminine gender has interference
Acetonitrile-0.05mol/L sodium hydrogen phosphate (70: 30) octadecylsilane chemically bonded silica	Peak shape is slightly asymmetric		Feminine gender has interference
	Peak shape is slightly asymmetric	Acetonitrile-0.05mol/L potassium dihydrogen phosphate (70: 30) octadecylsilane chemically bonded silica	Peak shape is slightly asymmetric
Methanol-0.05mol/L potassium dihydrogen phosphate (50: 50) eight alkyl silane bonded silica gels	Feminine gender has interference		Peak shape is slightly asymmetric
	Feminine gender has interference	Methanol-water (65: 35) octadecylsilane chemically bonded silica	Retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless

Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, methanol-water (65: 35) be a mobile phase, and with this understanding, schisandrin, deoxyschizandrin, schisandrin B retention time are moderate, and the peak is capable sharp-pointed, symmetry, feminine gender is noiseless.

The thin layer chromatography discrimination method of Radix Ophiopogonis in experimental example 5 pulse restoring injections:

Feature for outstanding Radix Ophiopogonis, selected the Radix Ophiopogonis control medicinal material as its feature speckle, but because have that more structure is close in the medical material, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition to launching Radix Ophiopogonis:

Condition	Problem
Condition	Problem	Benzene-methanol-ethyl acetate (50: 10: 3) silica gel G F ₂₅₄Lamellae	Reference substance does not separate, and feminine gender has interference
Toluene-methanol-methyl acetate (50: 10: 3) silica gel H lamellae	Reference substance does not separate, and feminine gender has interference
		Chloroform-normal hexane-formic acid (50: 20: 5) silica gel g thin-layer plate	Reference substance is expanded to the forward position
Dichloromethane-cyclohexane extraction (5: 2) silica gel g thin-layer plate	Reference substance is expanded to the forward position		Reference substance is expanded to the forward position
	Reference substance is expanded to the forward position	Ethyl acetate-pyridine-acetone (5: 3: 5) silica gel H lamellae	Reference substance does not separate, and feminine gender has interference
Benzene-Ethyl formate-formic acid (60: 10: 2) silica gel g thin-layer plate	Reference substance does not separate, and feminine gender has interference
		Toluene-methanol-glacial acetic acid (80: 5: 0.1) silica gel G F ₂₅₄Lamellae	It is clear to separate, and the moderate feminine gender of Rf value is noiseless

Through screening, determined with silica gel G F ₂₅₄Lamellae is an immobile phase, is developing solvent with toluene-methanol-glacial acetic acid (80: 5: 0.1), and with this understanding, the Rf value of principal spot is moderate, and it is clear to separate with other speckle, and is negative noiseless.

Ophiopogonin B in experimental example 6 pulse restoring injections, ophiopogonin D, ophiopogonin D ' the thin layer chromatography discrimination method:

Feature for outstanding ophiopogonin, ophiopogonin B, ophiopogonin D, ophiopogonin D ' have been selected as its feature speckle, but because have in the medical material that more and ophiopogonin B, ophiopogonin D, ophiopogonin D ' structure are close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition to launching Radix Ophiopogonis:

Condition

Problem

Ethyl acetate-methanol-formic acid (10: 10: 0.5) silica gel g thin-layer plate	Reference substance is expanded to the forward position
	Reference substance is expanded to the forward position	Chloroform-ethanol-glacial acetic acid (10: 10: 0.5) silica gel H lamellae	Reference substance is expanded to the forward position
Ethyl acetate-methanol-acetone (10: 3: 0.2) silica gel g thin-layer plate	Reference substance does not separate, and feminine gender has interference		Reference substance is expanded to the forward position
		Methylene chloride-methanol-normal hexane (15: 10: 2) silica gel G F ₂₅₄Lamellae	Reference substance is expanded to the forward position
Ethyl acetate-methanol-butanone (20: 3: 1) silica gel G F ₂₅₄Lamellae	Reference substance does not separate, and feminine gender has interference		Reference substance is expanded to the forward position
		Ethyl acetate-methanol-cyclohexane extraction (20: 8: 1) silica gel g thin-layer plate	Reference substance does not separate, and feminine gender has interference
With ethyl acetate-methanol-water (15: 5: 1) silica gel g thin-layer plate	It is clear to separate, and the moderate feminine gender of Rf value is noiseless

Through screening, determined with the silica gel g thin-layer plate to be immobile phase, be developing solvent with ethyl acetate-methanol-water (15: 5: 1), with this understanding, the Rf value of ophiopogonin B, ophiopogonin D, ophiopogonin D ' speckle is moderate, and it is clear to separate with other speckle, and is negative noiseless.

Ophiopogonin B in experimental example 7 pulse restoring injections, ophiopogonin D, ophiopogonin D ' the liquid chromatograph discrimination method:

Feature for outstanding ophiopogonin, except the thin layer discrimination method, ophiopogonin B, ophiopogonin D, ophiopogonin D ' have been selected as its characteristic component, but because have in the medical material that more and ophiopogonin B, ophiopogonin D, ophiopogonin D ' structure are close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and mobile phase to ophiopogonin B, ophiopogonin D, ophiopogonin D ' separate:

Condition	Problem
Condition	Problem	Methanol-0.05mol/L sodium dihydrogen phosphate (99: 1) octadecylsilane chemically bonded silica	Appearance time is too fast
Acetonitrile-0.05mol/L sodium dihydrogen phosphate (95: 5) octadecylsilane chemically bonded silica	Appearance time is too fast		Appearance time is too fast
	Appearance time is too fast	Methanol-0.02mol/L sodium hydrogen phosphate (95: 5) eight alkyl silane bonded silica gels	Feminine gender has interference
Methanol-0.02mol/L potassium dihydrogen phosphate (95: 5) octadecylsilane chemically bonded silica	Feminine gender has interference		Feminine gender has interference
	Feminine gender has interference	Acetonitrile-0.02mol/L sodium hydrogen phosphate (95: 5) eight alkyl silane bonded silica gels	There is bifurcated at the peak,
Acetonitrile-oxolane-1% glacial acetic acid (80: 10: 10) octadecylsilane chemically bonded silica	Feminine gender has interference		There is bifurcated at the peak,
	Feminine gender has interference	Acetonitrile-water (90: 10) octadecylsilane chemically bonded silica	Retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless

Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, acetonitrile-water (90: 10) be a mobile phase, and with this understanding, ophiopogonin B, ophiopogonin D, ophiopogonin D ' retention time are moderate, and the peak is capable sharp-pointed, symmetry, feminine gender is noiseless.

The thin layer chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in experimental example 8 pulse restoring injections:

Feature for outstanding ophiopogonin unit, selected Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin as its feature speckle, but because have in the medical material that more and Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin structure are close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition to launching Radix Ophiopogonis:

Condition	Problem
Condition	Problem	Methanol-chloroform-glacial acetic acid (5: 3: 2) silica gel g thin-layer plate	Reference substance is expanded to the forward position
Normal hexane-ethyl acetate-glacial acetic acid (5: 3: 1) silica gel H lamellae	Reference substance is expanded to the forward position		Reference substance is expanded to the forward position
	Reference substance is expanded to the forward position	Normal hexane-Ethyl formate (3: 2) silica gel G F ₂₅₄Lamellae	Feminine gender has interference
Methanol-ethyl acetate-formic acid (2: 1: 1) silica gel g thin-layer plate	Feminine gender has interference		Feminine gender has interference
	Feminine gender has interference	Methanol-ethyl acetate-acetone (1: 1: 1) silica gel H lamellae	Feminine gender has interference
Methanol-Ethyl formate (2: 1.5) silica gel H lamellae	Feminine gender has interference		Feminine gender has interference
Methanol-Ethyl formate (2: 1.5) silica gel H lamellae	Feminine gender has interference	Normal hexane-ethyl acetate-water (1: 1: 1) silica gel g thin-layer plate	It is clear to separate, and the moderate feminine gender of Rf value is noiseless

Through screening, determined with the silica gel g thin-layer plate to be immobile phase, be developing solvent with normal hexane-ethyl acetate-water (1: 1: 1), with this understanding, the Rf value of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin is moderate, and it is clear to separate with other speckle, and is negative noiseless.

The liquid chromatograph discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in experimental example 9 pulse restoring injections

Feature for outstanding ophiopogonin unit, except the thin layer discrimination method, selected Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin as its characteristic component, but because have in the medical material that more and Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin structure are close, composition like the polar phase, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and with mobile phase Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin are separated:

Condition	Problem
Condition	Problem	Methanol-0.05mol/L sodium dihydrogen phosphate (99: 1) octadecylsilane chemically bonded silica	Appearance time is too fast
Acetonitrile-0.05mol/L sodium dihydrogen phosphate (95: 5) octadecylsilane chemically bonded silica	Appearance time is too fast		Appearance time is too fast
	Appearance time is too fast	Methanol-0.05mol/L sodium hydrogen phosphate (95: 5) eight alkyl silane bonded silica gels	Feminine gender has interference
Methanol-0.05mol/L sodium hydrogen phosphate (95: 5) octadecylsilane chemically bonded silica	Feminine gender has interference		Feminine gender has interference
	Feminine gender has interference	Acetonitrile-oxolane-1% glacial acetic acid aqueous solution (90: 5: 5) eight alkyl silane bonded silica gels	There is bifurcated at the peak
Acetonitrile-oxolane 0.5% phosphoric acid aqueous acid (90: 5: 5) octadecylsilane chemically bonded silica	Feminine gender has interference		There is bifurcated at the peak
	Feminine gender has interference	Acetonitrile-water (90: 10) octadecylsilane chemically bonded silica	Retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless

Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, acetonitrile-water (90: 10) is a mobile phase, with this understanding, wheat Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin retention time are moderate, and the peak is capable sharp-pointed, symmetry, negative noiseless.

Ginsenoside Rg in experimental example 10 pulse restoring injections ₁, ginsenoside Rb ₁, ginsenoside Re, ginsenoside Rf the thin layer chromatography discrimination method

For the feature of outstanding Radix Ginseng Rubra or Radix Ginseng, selected the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re, ginsenoside Rf be as its feature speckle, but owing to exist more and the ginsenoside Rg in the medical material ₁, ginsenoside Rb ₁, composition like close, the polar phase of ginsenoside Re, ginsenoside Rf's structure, usual terms is difficult to reach requirements for quality control, so we have screened following lamellae and unfolding condition to launching Radix Ophiopogonis:

Condition	Problem
Condition	Problem	Lower floor's solution silica gel g thin-layer plate that chloroform-normal hexane-water (20: 60: 10) is placed below 10 ℃	Reference substance is expanded to the forward position
Dichloromethane-Ethyl formate-cyclohexane extraction (10: 1: 5) silica gel H lamellae	Reference substance is expanded to the forward position		Reference substance is expanded to the forward position
	Reference substance is expanded to the forward position	Chloroform-butanone-methanol (15: 40: 22) silica gel g thin-layer plate	Reference substance does not separate, and feminine gender has interference
Lower floor's solution silica gel g thin-layer plate that chloroform-ethyl acetate-water (10: 40: 15) is placed below 10 ℃	Reference substance does not separate, and feminine gender has interference
		N-butyl alcohol-Ethyl formate-water (5: 1: 5) upper solution silica gel g thin-layer plate	Reference substance does not separate, and feminine gender has interference
Isopropyl alcohol-Ethyl formate-water (2: 1.5: 0.5) upper solution silica gel H lamellae	Reference substance does not separate, and feminine gender has interference
		Chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) lower floor's solution silica gel g thin-layer plate of placing below 10 ℃	It is clear to separate, and the moderate feminine gender of Rf value is noiseless

Through screening, determined with the silica gel g thin-layer plate to be immobile phase, with chloroform-ethyl acetate-methanol-water (15: 40: 22: 10) lower floor's solution of placing below 10 ℃ was developing solvent, with this understanding, the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re, ginsenoside Rf Rf value moderate, it is clear to separate with other speckle, negative noiseless.

Ginsenoside Rg in experimental example 11 pulse restoring injections ₁, ginsenoside Rb ₁, the ginsenoside Re the liquid chromatograph discrimination method

For the feature of outstanding Radix Ginseng Rubra or Radix Ginseng, except the thin layer discrimination method, selected the ginsenoside Rg ₁, ginsenoside Rb ₁, the ginsenoside Re is as its characteristic component, but owing to exist more and the ginsenoside Rg in the medical material ₁, ginsenoside Rb ₁, composition like close, the polar phase of ginsenoside Re's structure, usual terms is difficult to reach requirements for quality control, so we have screened following chromatographic column and mobile phase to the ginsenoside Rg ₁, ginsenoside Rb ₁, the ginsenoside Re separates:

Condition	Problem
Condition	Problem	Methanol-oxolane-water (85: 5: 10) octadecylsilane chemically bonded silica	Appearance time is too fast
Acetonitrile-oxolane water (90: 5: 5) octadecylsilane chemically bonded silica	Appearance time is too fast		Appearance time is too fast

Methanol-0.005mol/L sodium dihydrogen phosphate (80: 20) eight alkyl silane bonded silica gels	Feminine gender has interference
	Feminine gender has interference	Methanol-0.02mol/L sodium hydrogen phosphate (80: 20) octadecylsilane chemically bonded silica	Feminine gender has interference
Acetonitrile-0.005mol/L sodium dihydrogen phosphate ((90: 10) eight alkyl silane bonded silica gels	Feminine gender has interference		Feminine gender has interference
	Feminine gender has interference	Acetonitrile-0.05mol/L potassium dihydrogen phosphate ((85: 15) octadecylsilane chemically bonded silica	Feminine gender has interference
Acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 35 minutes, the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, from 55 minutes to 70 minutes, the ratio of acetonitrile is 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% octadecylsilane chemically bonded silica by 29%	Retention time is moderate, and the peak is capable sharp-pointed, and symmetry is negative noiseless		Feminine gender has interference

Through screening, determined with the octadecylsilane chemically bonded silica to be immobile phase, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rises to 40% by 29%, with this understanding, and the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re's retention time is moderate, the peak is capable sharp-pointed, symmetry is negative noiseless.

Ginsenoside Rg in experimental example 12 pulse restoring injections ₁, ginsenoside Rb ₁, the ginsenoside Re content assaying method

1. instrument and reagent (1) instrument: Agilent1100 high performance liquid chromatograph, chemstationsys work station.The TU-1800SPC ultraviolet spectrophotometer.

(2) reagent: ginsenoside Rg ₁, ginsenoside Rb ₁, the ginsenoside Re: Nat'l Pharmaceutical ﹠ Biological Products Control Institute;

2. detect the selection of wavelength: precision takes by weighing the ginsenoside Rg ₁, ginsenoside Rb ₁, the ginsenoside Re, split in the 10ml measuring bottle, add methanol dilution and make the solution that every 1ml contains 0.5mg, scan in the 200-400nm wave-length coverage.The ginsenoside Rg ₁, ginsenoside Rb ₁, the ginsenoside Re all has absorption maximum at the 203nm place, therefore selects the detection wavelength of 203nm as assay.

3. chromatographic condition: Dikma ODS (4.6mm * 250mm, 5um); Mobile phase: acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 35 minutes, the ratio of acetonitrile is 19%, and from 35 minutes to 55 minutes, the ratio of acetonitrile rose to 29% by 19%, from 55 minutes to 70 minutes, the ratio of acetonitrile is 29%, and from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; The detection wavelength is 203nm; Flow velocity: 1.0ml/min.

4. the preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁An amount of with the ginsenoside Re, accurate claim surely, add methanol and make 1ml and contain the ginsenoside Rg ₁0.30mg, ginsenoside Rb ₁0.30mg, the mixed solution of ginsenoside Re 0.2mg.

5. the preparation of need testing solution: get under the content uniformity item 5 bottles of this product, get content, mixing is therefrom got about 0.5g, accurately claims surely, puts in the 5ml measuring bottle, adds and flows mutual-assistance dissolving and be diluted to scale, shakes up, promptly.

With this understanding, negative sample ginsenoside Rg in the disturbed specimen not ₁, ginsenoside Rb ₁With ginsenoside Re's mensuration, and separating degree is good.

6. ginsenoside Rg ₁, ginsenoside Rb ₁Take by weighing the ginsenoside Rg with ginsenoside Re's linear relationship precision ₁10.24mg, ginsenoside Rb ₁10.09mg, ginsenoside Re 7.54mg, put altogether in the 10ml measuring bottle, it is fixed to scale to add mobile phase, shakes up, product solution (contains the ginsenoside Rg among every 1ml in contrast ₁1.024mg, ginsenoside Rb ₁1.009mg, contain ginsenoside Re 0.754mg), precision is measured 2.5ml, puts in the 10ml measuring bottle, adds mobile phase to scale, shakes up, in contrast the product dilute solution.(contain the ginsenoside Rg among every 1ml ₁0.256mg, contain ginsenoside Rb ₁0.25225mg, contain ginsenoside Re 0.1885mg) and accurate reference substance solution 3 μ l, 6 μ l, the 10 μ l of drawing; Reference substance dilute solution 2 μ l, 5 μ l, 10 μ l; Injecting chromatograph of liquid, is vertical coordinate with the peak area, the ginsenoside Rg ₁Be figure for abscissa, the drawing standard curve with ginsenoside Re's amount.

The ginsenoside Rg ₁Linear relationship

Numbering	The ginsenoside Rg ₁(μg)	Peak area
Numbering	The ginsenoside Rg ₁(μg)	Peak area	1	0.512	150.63
2	1.280	475.54	1	0.512	150.63
2	1.280	475.54	3	2.560	890.23
4	3.072	1058.85	3	2.560	890.23
4	3.072	1058.85	5	6.144	2107.02
6	10.24	3538.96	5	6.144	2107.02

Regression equation: Y=345.01X+1.20;

The coefficient of determination: γ=0.9996;

The ginsenoside Rg ₁Good in 0.512～10.240 μ g scope internal linear relation.

Ginsenoside Rb ₁Linear relationship

Numbering	Ginsenoside Rb ₁(μg)	Peak area
Numbering	Ginsenoside Rb ₁(μg)	Peak area	1	0.5045	142.08
2	1.2613	353.07	1	0.5045	142.08
2	1.2613	353.07	3	2.5225	715.48
4	3.0270	860.21	3	2.5225	715.48
4	3.0270	860.21	5	6.0540	1709.14
6	10.090	2855.26	5	6.0540	1709.14

Regression equation: Y=282.95X-0.44;

The coefficient of determination: γ=0.9999;

Ginsenoside Rb ₁Good in 0.5045～10.090 μ g scope internal linear relation.

Ginsenoside Re's linear relationship

Numbering	Ginsenoside Re (μ g)	Peak area
Numbering	Ginsenoside Re (μ g)	Peak area	1	0.3770	184.37
2	0.9425	466.91	1	0.3770	184.37
2	0.9425	466.91	3	1.8850	928.26
4	2.2620	1104.76	3	1.8850	928.26
4	2.2620	1104.76	5	4.5240	2216.15
6	7.5400	3698.24	5	4.5240	2216.15

Regression equation: Y=490.12X+1.11;

The coefficient of determination: γ=0.9999;

The ginsenoside Re is good in 0.3770～7.5400 μ g scope internal linear relation.

7. reference substance precision and stability test precision is drawn ginsenoside Rg under the standard curve item ₁, the ginsenoside Rg ₁(contain the ginsenoside Rg among every 1ml with ginsenoside Re's reference substance dilute solution ₁0.256mg, ginseng saponin Rb ₁0.25225mg, contain ginsenoside Re 0.1885mg) and 10 μ l, inject chromatograph of liquid, the record peak area is measured at 0,6,12,24,48 hour sample introduction.

The test of reference substance solution precision

Testing time (h)	0	6	12	24	48	On average	RSD(％)
Testing time (h)	0	6	12	24	48	On average	RSD(％)	The ginsenoside Rg ₁	884.26	893.71	899.04	892.35	895.89	893.05	0.62
Ginsenoside Rb ₁	726.23	722.57	724.92	725.86	722.78	724.47	0.24	The ginsenoside Rg ₁	884.26	893.71	899.04	892.35	895.89	893.05	0.62
Ginsenoside Rb ₁	726.23	722.57	724.92	725.86	722.78	724.47	0.24	The ginsenoside Re	921.63	922.44	925.36	917.03	930.58	923.41	0.54

The result shows, the ginsenoside Rg ₁, ginsenoside Rb ₁With ginsenoside Re's reference substance solution precision and having good stability.

8 need testing solution stability tests are got under the weight differential item 5 bottles of this product, get content, mixing is therefrom got about 0.5g, and accurate the title decides, put in the 5ml measuring bottle, add and flow mutual-assistance dissolving and decide to shake up the accurate 10 μ l that draw to scale, inject chromatograph of liquid, measure at 0,6,12,24,48 hour sample introduction respectively.

Need testing solution stability test result

Testing time (h)	0	6	12	24	48	Average	RSD(％)
Testing time (h)	0	6	12	24	48	Average	RSD(％)	The ginsenoside Rg ₁(mg/ bottle)	1.06	1.02	1.04	1.03	1.05	1.04	1.52

Ginsenoside Rb ₁(mg/ bottle)	0.99	1.00	0.97	1.01	0.98	0.99	1.60
Ginsenoside Rb ₁(mg/ bottle)	0.99	1.00	0.97	1.01	0.98	0.99	1.60	Ginsenoside Re's (mg/ bottle)	0.52	0.54	0.53	0.53	0.54	0.53	1.57

The result shows that need testing solution has good stability.

9. replica test is got under this product content uniformity item 5 bottles on powder pin, get content, porphyrize is therefrom got about 0.5g (totally 5 parts), the accurate title, decide, split in the 5ml measuring bottle, add the mutual-assistance dissolving and fixed of flowing, shake up to scale, the accurate 10 μ l that draw, inject chromatograph of liquid, calculate with one point external standard method, promptly.

Replica test

Test number	1	2	3	4	5	Average	RSD(％)
Test number	1	2	3	4	5	Average	RSD(％)	The ginsenoside Rg ₁(mg/ bottle)	1.08	1.11	1.07	1.10	1.09	1.09	1.45
Ginsenoside Rb ₁(mg/ bottle)	1.02	1.04	1.01	1.03	1.02	1.02	1.11	The ginsenoside Rg ₁(mg/ bottle)	1.08	1.11	1.07	1.10	1.09	1.09	1.45
Ginsenoside Rb ₁(mg/ bottle)	1.02	1.04	1.01	1.03	1.02	1.02	1.11	Ginsenoside Re's (mg/ bottle)	0.55	0.54	0.54	0.55	0.53	0.54	1.54

10. the application of sample absorption method is adopted in average recovery test, gets this product under the weight differential item, gets third tolerantly, and mixing is therefrom got about 250mg (totally 5 parts), and accurate the title decides, and splits in the 5ml measuring bottle; Precision takes by weighing the ginsenoside Rg ₁11.58mg, ginsenoside Rb ₁10.84mg ginsenoside Re 5.76mg puts in the 25ml measuring bottle altogether, add an amount of supersound process of mobile phase and make dissolving, take out, put to room temperature, add mobile phase to scale, shake up, precision is measured 1ml (totally 5 parts), split in the above-mentioned 5ml measuring bottle, add mobile phase, shake up to scale, the accurate 10 μ l that draw, inject chromatograph of liquid, calculate with one point external standard method, promptly.

Ginsenoside Rg in the pulse restoring injection ₁Content: 0.1812%;

Ginsenoside Rb in the pulse restoring injection ₁Content: 0.1695%;

Ginsenoside Re's content in the pulse restoring injection: 0.0897%;

The ginsenoside Rg ₁The average recovery test

Numbering	Test sample weighing (mg)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample weighing (mg)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1	246.93	0.4474	0.4632	0.8974	97.15
2	255.12	0.4623	0.4632	0.9173	98.23	1	246.93	0.4474	0.4632	0.8974	97.15
2	255.12	0.4623	0.4632	0.9173	98.23	3	253.08	0.4586	0.4632	0.9055	96.49

4	249.47	0.4520	0.4632	0.9097	98.81
4	249.47	0.4520	0.4632	0.9097	98.81	5	261.25	0.4734	0.4632	0.9270	97.94

The ginsenoside Rg ₁Average recovery rate=97.72%; RSD=0.93%

Ginsenoside Rb ₁The average recovery test

Numbering	Test sample weighing (mg)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample weighing (mg)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1	246.93	0.4185	0.4336	0.8446	98.25
2	255.12	0.4324	0.4336	0.8630	99.31	1	246.93	0.4185	0.4336	0.8446	98.25
2	255.12	0.4324	0.4336	0.8630	99.31	3	253.08	0.4290	0.4336	0.8541	98.04
4	249.47	0.4229	0.4336	0.8503	98.57	3	253.08	0.4290	0.4336	0.8541	98.04
4	249.47	0.4229	0.4336	0.8503	98.57	5	261.25	0.4428	0.4336	0.8601	96.23

Ginsenoside Rb ₁Average recovery rate=98.08%; RSD=1.16%

The test of ginsenoside Re's average recovery

Numbering	Test sample weighing (mg)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample weighing (mg)	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1	246.93	0.2215	0.2304	0.4455	97.21
2	255.12	0.2288	0.2304	0.4500	95.98	1	246.93	0.2215	0.2304	0.4455	97.21
2	255.12	0.2288	0.2304	0.4500	95.98	3	253.08	0.2270	0.2304	0.4473	95.62
4	249.47	0.2238	0.2304	0.4503	98.33	3	253.08	0.2270	0.2304	0.4473	95.62
4	249.47	0.2238	0.2304	0.4503	98.33	5	261.25	0.2343	0.2304	0.4590	97.51

Ginsenoside Re's average recovery rate=96.93%; RSD=1.15%

11. three batches of pilot scale sample sizes are measured

Get injection and give birth to three batches in arteries and veins sample, press the described method of text and handle.

Three batch sample assay results

Lot number	The ginsenoside Rg ₁(mg/ bottle)	Ginsenoside Rb ₁(mg/ bottle)	Ginsenoside Re's (mg/ bottle)
Lot number	The ginsenoside Rg ₁(mg/ bottle)	Ginsenoside Rb ₁(mg/ bottle)	Ginsenoside Re's (mg/ bottle)	1	1.12	0.97	0.55
2	1.08	1.03	0.52	1	1.12	0.97	0.55
2	1.08	1.03	0.52	3	1.14	1.06	0.58

From above test as can be known, adopt the method for the invention to measure ginsenoside Rg in the pulse restoring injection ₁, ginsenoside Rb ₁, the ginsenoside Re content precision, stability and repeatability good, response rate height, method is easy, data accurately, reliable.

Radix Ginseng total saponins content assaying method in experimental example 13 pulse restoring injections

Instrument, reagent (1) instrument: the general logical TU-1810SPC ultraviolet/visible spectrophotometer SARTORIUS BP211D electronic analytical balance of analysing

(2) reagent: vanillin analytical pure Tianjin recovery fine chemistry industry institute

Methanol chromatographically pure J.T.Baker

Glacial acetic acid analytical pure Shanghai reagent one factory

Chemical plant, prosperous source, perchloric acid analytical pure Tianjin

The 2 selection precisions that detect wavelength take by weighing the ginsenoside Rg ₁6.11mg, put in the 100ml measuring bottle, add an amount of methanol supersound process (power 250W, frequency 33KHz) makes dissolving, add methanol to scale, shake up, precision is measured 1.2ml, put in the 10ml tool plug test tube, the water bath method solvent takes out, the accurate 5% vanillin-glacial acetic acid solution 0.2ml that adds, perchloric acid 0.8ml, shake up, put in 60 ℃ of water-baths and heated 15 minutes, take out, in ice-water bath, cooled off 2 minutes immediately, the accurate glacial acetic acid 5ml that adds shakes up, and scans in 700～400nm wave-length coverage, the result shows, the ginsenoside Rg ₁At the 547nm place absorption maximum is arranged, blank noiseless, therefore selecting 547nm is the detection wavelength of Radix Ginseng total saponins in the spectrophotometry pulse restoring injection.

The investigation precision of 3 linear relationships takes by weighing the ginsenoside Rg ₁Reference substance 6.85mg puts in the 100ml measuring bottle, adds an amount of supersound process of methanol (power 250W, frequency 33KHZ) makes dissolving, take out, put to room temperature, add methanol to scale, shake up, precision measures 0.4,0.8,1.2,1.6,2.0ml, split in the 10ml tool plug test tube, the water bath method solvent takes out, the accurate 5% vanillin-glacial acetic acid solution 0.2ml that adds, perchloric acid 0.8ml, shake up, put in 60 ℃ of water-baths and heated 15 minutes, take out, in ice-water bath, cooled off 2 minutes immediately, the accurate glacial acetic acid 5ml that adds shakes up, and is blank with the retinue solvent, according to spectrophotography (appendix VA of Chinese Pharmacopoeia version in 2000), measure trap at the wavelength place of 547nm.With the trap is vertical coordinate, the ginsenoside Rg ₁Amount (μ g) be abscissa, the drawing standard curve.

Regression equation Y=0.0049X-0.0077; R=0.999

The ginsenoside Rg ₁Linear good in 27.4～137.0 μ g scopes.

The ginsenoside Rg ₁The standard curve determination data

Numbering	The ginsenoside Rg ₁(μg)	Trap
Numbering	The ginsenoside Rg ₁(μg)	Trap	1	27.4	0.121
2	54.8	0.261	1	27.4	0.121
2	54.8	0.261	3	82.2	0.404
4	109.6	0.534	3	82.2	0.404
4	109.6	0.534	5	137.0	0.656

4 precision test precision is measured the ginsenoside Rg ₁Reference substance solution (0.0685mg/ml) 1.2ml puts in the 10ml tool plug test tube water bath method solvent, take out accurate 5% vanillin-glacial acetic acid solution 0.2ml, the perchloric acid 0.8ml of adding, shake up, put in 60 ℃ of water-baths and heated 15 minutes, take out, cooled off 2 minutes in ice-water bath immediately, the accurate glacial acetic acid 5ml that adds shakes up, with the retinue solvent is blank, according to spectrophotography (Chinese Pharmacopoeia version-appendix VA of portion in 2000), measure trap, METHOD FOR CONTINUOUS DETERMINATION 5 times at the wavelength place of 547nm.

The precision experiment

Numbering	1	2	3	4	5	X is average	RSD％
Numbering	1	2	3	4	5	X is average	RSD％	Absorbance	0.429	0.426	0.426	0.425	0.424	0.426	0.44

5 stability tests

5.1 the stability test precision of reference substance solution is measured the ginsenoside Rg ₁Reference substance solution (0.0727mg/ml) 1.2ml, put in the 10ml tool plug test tube, the water bath method solvent, take out, the accurate 5% vanillin-glacial acetic acid solution 0.2ml that adds, perchloric acid 0.8ml shakes up, and puts in 60 ℃ of water-baths and heats 15 minutes, take out, cooled off 2 minutes in ice-water bath immediately, the accurate glacial acetic acid 5ml that adds shakes up, with the retinue solvent is blank, according to spectrophotography (appendix VA of Chinese Pharmacopoeia version in 2000), measure trap at the wavelength place of 547nm, in the time of 0,10,20,40,60 minute, measure respectively.

The reference substance solution stability experiment

Time (min)	0	10	20	40	60	Average	RSD％
Time (min)	0	10	20	40	60	Average	RSD％	Absorbance	0.453	0.453	0.450	0.451	0.449	0.451	0.40

5.2 the stability experiment of need testing solution is got under this product content uniformity item 5 bottles on powder pin, gets content, porphyrize, therefrom get about 100mg, the accurate title, decide, and puts in the 10ml measuring bottle, adds water and make dissolving and fixed to scale in right amount, shake up, precision is measured 0.2ml, puts in the 10ml tool plug test tube water bath method solvent, take out, the accurate 5% vanillin-glacial acetic acid solution 0.2ml that adds, perchloric acid 0.8ml shakes up, put in 60 ℃ of water-baths and heated 15 minutes, take out, in ice-water bath, cooled off 2 minutes immediately, the accurate glacial acetic acid 5ml that adds, shake up, with the retinue solvent is blank, according to spectrophotography (appendix VA of Chinese Pharmacopoeia version in 2000), measures trap at the wavelength place of 547nm, calculate with one point external standard method, respectively 0,10,20,40, measure in the time of 60 minutes.

The need testing solution stability experiment

Time (min)	0	10	20	40	60	Average	RSD％
Time (min)	0	10	20	40	60	Average	RSD％	Content (mg/ bottle)	22.18	21.74	22.03	21.59	22.55	22.02	1.71

6 replica tests are got under this product content uniformity item 5 bottles on powder pin, get content, porphyrize is therefrom got about 100mg (totally 5 parts), split in the 10ml measuring bottle, add water and make dissolving and fixed to scale in right amount, shake up, precision is measured 0.2ml, split in the 10ml tool plug test tube, the water bath method solvent takes out, the accurate 5% vanillin-glacial acetic acid solution 0.2ml that adds, perchloric acid 0.8ml, shake up, put in 60 ℃ of water-baths and heated 15 minutes, take out, in ice-water bath, cooled off 2 minutes immediately, the accurate glacial acetic acid 5ml that adds shakes up, and is blank with the retinue solvent, according to spectrophotography (appendix VA of Chinese Pharmacopoeia version in 2000), wavelength place at 547nm measures trap, calculates with one point external standard method, promptly.

Repeated experiment

Numbering	1	2	3	4	5	X is average	RSD％
Numbering	1	2	3	4	5	X is average	RSD％	Content (mg/ bottle)	22.05	21.77	21.62	22.49	22.38	22.06	1.70

7 recovery tests adopt the application of sample absorption method, get under this product content uniformity item 5 bottles on powder pin, get content, and porphyrize is therefrom got about 50mg (totally 5 parts), and accurate the title splits in the 10ml measuring bottle calmly; Other gets the ginsenoside Rg ₁The about 1.8mg of reference substance (totally 5 parts), the accurate title, decide, split in the above-mentioned 10ml measuring bottle, add water and make dissolving and fixed in right amount, shake up to scale, precision is measured 0.2ml, putting in the 10ml tool plug test tube, by operating under the text algoscopy item, is blank with the retinue solvent, measure trap in accordance with the law, read ginsenoside Rg the need testing solution from standard curve ₁Content, calculate with one point external standard method, promptly.

The content of total saponins in the pulse restoring injection: 3.666%

The experiment of Radix Ginseng total saponins average recovery

Numbering	Test sample weighing (mg)	Total saponins amount (mg) in the test sample	The ginsenoside Rg ₁Addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Test sample weighing (mg)	Total saponins amount (mg) in the test sample	The ginsenoside Rg ₁Addition (mg)	Measured value (mg)	The response rate (%)	1	50.24	1.842	1.77	3.581	98.25
2	47.81	1.753	1.92	3.628	97.66	1	50.24	1.842	1.77	3.581	98.25
2	47.81	1.753	1.92	3.628	97.66	3	55.23	2.025	2.13	4.140	99.32
4	50.94	1.867	2.04	3.876	98.47	3	55.23	2.025	2.13	4.140	99.32
4	50.94	1.867	2.04	3.876	98.47	5	51.67	1.894	1.86	3.697	96.93

Average recovery rate=99.53% RSD=1.88%

8 three batches of pilot scale sample sizes are measured

Get three batches in this product sample, press the described method of text and handle.

Three batch sample assay results

Lot number	Radix Ginseng total saponins (mg/ bottle)
Lot number	Radix Ginseng total saponins (mg/ bottle)	1	22.17
2	22.39	1	22.17
2	22.39	3	21.94

From above test as can be known, it is good to adopt the method for the invention to measure the content precision of Radix Ginseng total saponins in the pulse restoring injection, stability and repeatability, response rate height, and method is easy, and data are accurate, reliable.

The content assaying method of schisandrin, deoxyschizandrin, schisandrin B in experimental example 14 pulse restoring injections

1. instrument and reagent

(1) instrument: Agilent1100 high performance liquid chromatograph, chemstationsys work station; The TU-1800SPC ultraviolet spectrophotometer; The AE240 electronic balance.

(2) reagent: schisandrin, deoxyschizandrin, schisandrin B: Nat'l Pharmaceutical ﹠ Biological Products Control Institute;

2. it is an amount of that the selection precision that detects wavelength takes by weighing schisandrin, deoxyschizandrin, schisandrin B reference substance, add methanol respectively and be diluted to scale, shake up, get every 1ml and contain schisandrin 0.10mg respectively, deoxyschizandrin 0.10mg, the solution of schisandrin B 0.10mg scans in the 200-400nm wave-length coverage respectively.The result shows: schisandrin, deoxyschizandrin, schisandrin B all have absorption maximum at the 250nm place, therefore select the detection wavelength of 250nm as assay.

3. chromatographic condition: Dikma ODS (4.6 * 250mm, 5um); Mobile phase: methanol-water (65: 35) is a mobile phase; The detection wavelength is 250nm; Flow velocity: 1.0ml/min.

4. system suitability test

4.1 it is an amount of that the preparation precision of reference substance solution takes by weighing schisandrin, deoxyschizandrin, schisandrin B reference substance, adding methanol makes every 1ml and contains schisandrin 0.02mg, deoxyschizandrin 0.003mg, the reference substance mixed solution of schisandrin B 0.005mg, promptly.

4.2 the preparation of need testing solution is got SHENGMAI ZHUSHEYE as need testing solution.

4.3 the preparation of negative need testing solution takes by weighing the negative sample that does not contain Fructus Schisandrae Chinensis, according to the preparation method preparation of need testing solution, as negative need testing solution.

Accurate respectively reference substance solution, need testing solution, each 10 μ l of negative need testing solution of drawing inject chromatograph of liquid, measure the record chromatogram.As seen from the figure, schisandrin in the test sample, schisandrin B, deoxyschizandrin peak separate good, and it is clear to separate with close peak, and fully, schisandrin, deoxyschizandrin in the negative test sample, the schisandrin B peak is noiseless.

5. linear relationship is investigated precision and is taken by weighing schisandrin reference substance 10.27mg, deoxyschizandrin reference substance 10.03mg, schisandrin B reference substance 9.86mg, split in the 50ml measuring bottle, add methanol respectively and make dissolving and fixed to scale in right amount, shake up, precision is measured schisandrin reference substance solution 2ml, deoxyschizandrin reference substance solution 0.3ml, schisandrin B reference substance solution 0.6ml puts in the 10ml measuring bottle altogether, and it is fixed to scale to add methanol, shake up, the accurate 2 μ l that draw, 4 μ l, 6 μ l, 8 μ l, 10 μ l inject chromatograph of liquid, are vertical coordinate with the peak area, sample size (μ g) is figure for abscissa, the drawing standard curve.

The schisandrin linear relationship is investigated

Numbering	Schisandrin (μ g)	Peak area
Numbering	Schisandrin (μ g)	Peak area	1	0.08216	121.45

2	0.16432	238.71
2	0.16432	238.71	3	0.24648	362.58
4	0.32864	487.62	3	0.24648	362.58
4	0.32864	487.62	5	0.41080	601.91

Regression equation: Y=1472.5X-0.495;

The coefficient of determination: γ=0.9998;

Schisandrin is good in 0.08216～0.4108 μ g scope internal linear relation.

The deoxyschizandrin linear relationship is investigated

Numbering	Deoxyschizandrin (μ g)	Peak area
Numbering	Deoxyschizandrin (μ g)	Peak area	1	0.012036	121.36
2	0.024072	245.71	1	0.012036	121.36
2	0.024072	245.71	3	0.036108	371.96
4	0.048144	494.18	3	0.036108	371.96
4	0.048144	494.18	5	0.060180	610.42

Regression equation: Y=10191X+0.749;

The coefficient of determination: γ=0.9999;

Deoxyschizandrin is good in 0.012036～0.060180 μ g scope internal linear relation.

The schisandrin B linear relationship is investigated

Numbering	Schisandrin B (μ g)	Peak area
Numbering	Schisandrin B (μ g)	Peak area	1	0.023664	184.61
2	0.047328	381.09	1	0.023664	184.61
2	0.047328	381.09	3	0.070992	561.82
4	0.094656	745.96	3	0.070992	561.82
4	0.094656	745.96	5	0.118320	940.83

Regression equation: Y=7993.2X-0.331;

The coefficient of determination: γ=0.9999;

Schisandrin B is good in 0.023664～0.11832 μ g scope internal linear relation.

6. precision test precision is drawn reference substance mixed solution (every 1ml contains schisandrin 20.54 μ g respectively, deoxyschizandrin 3.009 μ g, schisandrin B 5.916 μ g) 10 μ l, inject chromatograph of liquid, the record peak area, METHOD FOR CONTINUOUS DETERMINATION 5 times, the precision of investigation reference substance solution.

The test of reference substance precision

Test number (TN)	1	2	3	4	5	Average	RSD(％)
Test number (TN)	1	2	3	4	5	Average	RSD(％)	Fructus Schisandrae Chinensis alcohol first	305.17	311.24	309.33	315.27	314.62	311.13	1.33
Deoxyschizandrin	315.47	320.09	322.18	318.45	312.22	317.68	1.23	Fructus Schisandrae Chinensis alcohol first	305.17	311.24	309.33	315.27	314.62	311.13	1.33
Deoxyschizandrin	315.47	320.09	322.18	318.45	312.22	317.68	1.23	Schisandrin B	474.90	466.33	470.15	477.36	480.42	474.83	1.19

The result shows that reference substance solution precision is good.

7. the accurate SHENGMAI ZHUSHEYE 10 μ l that draw of stability test inject chromatograph of liquid, at 0,6,12,24,48 hour sample introduction, calculate with one point external standard method, promptly respectively.

The reference substance solution stability test

Time (h)	0	6	12	24	48	Average	RSD(％)
Time (h)	0	6	12	24	48	Average	RSD(％)	Schisandrin (mg/ props up)	0.2514	0.2547	0.2533	0.2529	0.2568	0.2538	0.80
Deoxyschizandrin (mg/ props up)	0.0338	0.0331	0.0336	0.0341	0.0339	0.0337	1.13	Schisandrin (mg/ props up)	0.2514	0.2547	0.2533	0.2529	0.2568	0.2538	0.80
Deoxyschizandrin (mg/ props up)	0.0338	0.0331	0.0336	0.0341	0.0339	0.0337	1.13	Schisandrin B (mg/ props up)	0.0658	0.0642	0.0666	0.0675	0.0651	0.0658	1.95

The result shows that reference substance solution has good stability.

8. the accurate SHENGMAI ZHUSHEYE 10 μ l that draw of replica test inject chromatograph of liquid, calculate with one point external standard method, promptly.

Replica test

Numbering	1	2	3	4	5	Average	RSD(％)
Numbering	1	2	3	4	5	Average	RSD(％)	Schisandrin (mg/ props up)	0.2528	0.2503	0.2519	0.2542	0.2581	0.2535	1.17
Deoxyschizandrin (mg/ props up)	0.0342	0.0335	0.0351	0.0346	0.0347	0.0344	1.76	Schisandrin (mg/ props up)	0.2528	0.2503	0.2519	0.2542	0.2581	0.2535	1.17
Deoxyschizandrin (mg/ props up)	0.0342	0.0335	0.0351	0.0346	0.0347	0.0344	1.76	Schisandrin B (mg/ props up)	0.0662	0.0657	0.0682	0.0673	0.0665	0.0668	1.47

The result shows that repeatability is good.

9. the application of sample absorption method is adopted in the average recovery test, and precision is measured SHENGMAI ZHUSHEYE 5ml, splits in the 10ml measuring bottle; Precision is measured reference substance mixed solution (every 1ml contains schisandrin 0.1044mg respectively, deoxyschizandrin 0.01446mg, schisandrin B 0.02552mg) 1ml (totally 5 parts), split in the above-mentioned 10ml measuring bottle, add the about 6ml of methanol, jolting adds methanol to scale, shake up, filter, the accurate subsequent filtrate 10 μ l that draw inject chromatograph of liquid, calculate with one point external standard method, promptly.

Schisandrin content in the SHENGMAI ZHUSHEYE: 0.2535mg/ props up;

Deoxyschizandrin content in the SHENGMAI ZHUSHEYE: 0.0344mg/ props up;

Schisandrin B content in the SHENGMAI ZHUSHEYE: 0.0668mg/ props up.

The test of schisandrin average recovery

Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1	0.12675	0.1044	0.2293	98.25
2	0.12675	0.1044	0.2301	99.03	1	0.12675	0.1044	0.2293	98.25
2	0.12675	0.1044	0.2301	99.03	3	0.12675	0.1044	0.2306	99.47
4	0.12675	0.1044	0.2282	97.19	3	0.12675	0.1044	0.2306	99.47
4	0.12675	0.1044	0.2282	97.19	5	0.12675	0.1044	0.2307	99.56

Schisandrin average recovery rate=98.70%; RSD=1.00%.

The test of deoxyschizandrin average recovery

Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1	0.0172	0.01446	0.03115	96.48
2	0.0172	0.01446	0.03125	97.15	1	0.0172	0.01446	0.03115	96.48
2	0.0172	0.01446	0.03125	97.15	3	0.0172	0.01446	0.03152	99.01
4	0.0172	0.01446	0.03128	97.34	3	0.0172	0.01446	0.03152	99.01
4	0.0172	0.01446	0.03128	97.34	5	0.0172	0.01446	0.03141	98.25

Deoxyschizandrin average recovery rate=97.65%; RSD=1.01%.

The test of schisandrin B average recovery

Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1	0.0334	0.02552	0.05779	95.59
2	0.0334	0.02552	0.05802	96.18	1	0.0334	0.02552	0.05779	95.59
2	0.0334	0.02552	0.05802	96.18	3	0.0334	0.02552	0.05783	95.74
4	0.0334	0.02552	0.05849	98.32	3	0.0334	0.02552	0.05783	95.74
4	0.0334	0.02552	0.05849	98.32	5	0.0334	0.02552	0.05822	97.26

Schisandrin B average recovery rate=96.62%; RSD=1.19%.

10. three batches of pilot scale sample sizes are measured

Three batch sample assay results

Lot number	1	2	3
Lot number	1	2	3	Schisandrin (mg/ props up)	0.2541	0.2551	0.2536
Deoxyschizandrin (mg/ props up)	0.0351	0.0344	0.0352	Schisandrin (mg/ props up)	0.2541	0.2551	0.2536
Deoxyschizandrin (mg/ props up)	0.0351	0.0344	0.0352	Schisandrin B (mg/ props up)	0.0664	0.0672	0.0668

From above test as can be known, it is good to adopt the method for the invention to measure the content precision of schisandrin, deoxyschizandrin, schisandrin B in the SHENGMAI ZHUSHEYE, stability and repeatability, response rate height, and method is easy, and data are accurate, reliable.

The assay of total lignans in experimental example 15 pulse restoring injections

1 instrument and reagent

(1) key instrument:

Ultraviolet spectrophotometer TU-1810SPC Beijing Puxi General Instrument Co., Ltd

Electronic analytical balance BP211D SARTORIUS

Ultrasonic washing unit KQ250DB Kunshan Ultrasonic Instruments Co., Ltd.

(2) reagent:

Schisandrin Nat'l Pharmaceutical ﹠ Biological Products Control Institute

Ethanol analytical pure atropic is Fine Chemical Co., Ltd now

The schisandrin reference substance is got in 2 selections that detect wavelength, adds methanol and makes the solution that every 1ml contains 20 μ g, in contrast product solution.Measure SHENGMAI ZHUSHEYE 4ml, put in the 50ml measuring bottle, add an amount of supersound process of methanol solution (power 250W, frequency 33KHz) and make dissolving, take out, put, be diluted to scale, shake up, filter, get subsequent filtrate as need testing solution with methanol solution to room temperature.Draw the schisandrin reference substance solution, need testing solution, press the text total lignans and measure item method suggested down, in 200～400nm wave-length coverage, scan, the result shows, reference substance solution and need testing solution all have absorption maximum at 250nm, and solvent is noiseless, therefore select the detection wavelength of 250nm as total lignans content in the spectrophotometry SHENGMAI ZHUSHEYE.

Schisandrin reference substance 15mg is got in the preparation of 3 reference substance solution, puts in the 50ml measuring bottle, adds an amount of supersound process of methanol solution (power 250W, frequency 33KHz) makes dissolving, take out, put to room temperature, add methanol solution to scale, shake up, promptly get (containing schisandrin 0.3mg among every 1ml).

The preparation precision of 4 standard curves is measured reference substance solution (C=0.2908mg/ml) 0.1ml, 0.2ml, 0.4ml, 0.6ml 0.8ml, 1.0ml split in the 10ml measuring bottle, add methanol to scale, shaking up, is blank with the retinue solvent, according to spectrophotography (appendix VB of Chinese Pharmacopoeia version in 2000), measure trap at 250nm wavelength place, (μ g/ml) is abscissa with concentration, and trap is that vertical coordinate is figure, the drawing standard curve.

Total lignans standard curve determination result

Numbering	Schisandrin concentration (μ g/ml)	Trap
Numbering	Schisandrin concentration (μ g/ml)	Trap	1	2.908	0.118
2	5.816	0.245	1	2.908	0.118
2	5.816	0.245	3	11.632	0.491

4	17.448	0.732
4	17.448	0.732	5	23.264	0.971
6	29.080	1.217	5	23.264	0.971

Regression equation: Y=0.0418x+0.0003;

Correlation coefficient: γ=0.9999;

The result shows: schisandrin is good in 2.908～29.080 μ g/ml scope internal linear.

As calculated, the standard curve of schisandrin is crossed initial point, therefore selects for use one point external standard method to measure the content of total lignans in the pulse restoring injection.

The accurate schisandrin reference substance solution (concentration: 0.2908mg/ml) 0.6ml of drawing of 5 precision test, put in the 10ml measuring bottle, add methanol to scale, shake up, with the retinue solvent is blank, according to spectrophotography (appendix VB of Chinese Pharmacopoeia version in 2000), measure trap, replication 5 times at 250nm wavelength place.

The precision test

Numbering	1	2	3	4	5	Average	RSD(％)
Numbering	1	2	3	4	5	Average	RSD(％)	Trap	0.732	0.733	0.731	0.734	0.732	0.732	0.16

The result shows that reference substance solution precision is good.

6 stability tests

6.1 the accurate schisandrin reference substance solution (concentration: 0.2908mg/ml) 0.6ml of drawing of reference substance solution stability test, put in the 10ml measuring bottle, add methanol to scale, shake up, with the retinue solvent is blank, according to spectrophotography (appendix VB of Chinese Pharmacopoeia version in 2000), measure trap at 250nm wavelength place, respectively once at 0,2,4,8,12 hour replication.

The reference substance solution stability test

Time (h)	0	2	4	8	12	Average	RSD(％)
Time (h)	0	2	4	8	12	Average	RSD(％)	Trap	0.732	0.733	0.731	0.734	0.732	0.732	0.16

The result shows that reference substance solution has good stability.

6.2 need testing solution stability test precision is measured SHENGMAI ZHUSHEYE 4ml, puts in the 50ml measuring bottle, adds an amount of supersound process of methanol solution (power 250W, frequency 33KHz) makes dissolving, take out, put to room temperature, be diluted to scale with methanol solution, shaking up, filter, is blank with the retinue solvent, according to spectrophotography (appendix VB of Chinese Pharmacopoeia version in 2000), measure trap at 250nm wavelength place, calculate with one point external standard method, respectively once at 0,2,4,8,12 hour replication.

The need testing solution stability test

Time (h)	0	2	4	8	12	Average	RSD(％)
Time (h)	0	2	4	8	12	Average	RSD(％)	Content (mg/ props up)	2.03	2.05	2.02	2.04	2.03	2.03	0.56

The result shows that need testing solution is good at the 30min internal stability.

7 replica test precisions are measured SHENGMAI ZHUSHEYE 4ml (totally 5 parts), split in the 50ml measuring bottle, add an amount of supersound process of methanol solution (power 250W, frequency 33KHz) makes dissolving, take out, put to room temperature, be diluted to scale with methanol solution, shaking up, filter, is blank with the retinue solvent, according to spectrophotography (appendix VB of Chinese Pharmacopoeia version in 2000), measure trap at 250nm wavelength place, calculate with one point external standard method, promptly.

Replica test

Numbering	1	2	3	4	5	Average	RSD(％)
Numbering	1	2	3	4	5	Average	RSD(％)	Content (mg/ props up)	2.06	2.01	2.04	2.06	2.05	2.04	1.01

The result shows that repeatability is good.

The application of sample absorption method is adopted in the test of 8 average recoveries, and precision is measured SHENGMAI ZHUSHEYE 2ml (totally 5 parts), and accurate the title decides, split in the 50ml measuring bottle, precision is measured reference substance solution (C=0.2722mg/ml) 1.5ml, splits in the above-mentioned 50ml measuring bottle, add an amount of supersound process of methanol solution (power 250W, frequency 33KHz) and make dissolving, take out, put to room temperature, be diluted to scale, shake up with methanol solution, filter, wavelength place at 250nm measures trap, calculates with one point external standard method, promptly.

The content of total lignans is in the SHENGMAI ZHUSHEYE: 2.04mg/ props up.

The average recovery test

Numbering	Total lignans content (mg) in the test sample	Schisandrin addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Total lignans content (mg) in the test sample	Schisandrin addition (mg)	Measured value (mg)	The response rate (%)	1	0.408	0.4083	0.8060	97.47
2	0.408	0.4083	0.8087	98.13	1	0.408	0.4083	0.8060	97.47
2	0.408	0.4083	0.8087	98.13	3	0.408	0.4083	0.8024	96.60
4	0.408	0.4083	0.8125	99.08	3	0.408	0.4083	0.8024	96.60
4	0.408	0.4083	0.8125	99.08	5	0.408	0.4083	0.8092	98.26

Average recovery rate=97.91%, RSD=0.95%.

The assay of 9 samples is got this product three batch samples, according to the preparation of text need testing solution and the operation under the algoscopy item, working sample content.

The assay of total lignans

Lot number	Total lignans content (mg/ props up)
Lot number	Total lignans content (mg/ props up)	1	2.09

2	2.07
2	2.07	3	2.01

From above test as can be known, it is good to adopt the method for the invention to measure the content precision of total lignans in the pulse restoring injection, stability and repeatability, response rate height, and method is easy, and data are accurate, reliable.

The content assaying method of total polysaccharides in experimental example 16 pulse restoring injections

Monosaccharide is got under the content uniformity item 5 bottles of this product, gets content, mixing, therefrom get about 500mg, the accurate title, decide, and puts in the iodine flask, and adding distil water 60ml makes dissolving, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose (C of 9.008mg ₆H ₁₂O ₆), convert out the content of every bottle of monosaccharide thus.

Total sugar is got under the content uniformity item 5 bottles of this product, gets content, mixing, therefrom get about 500mg, the accurate title, decide, and puts in the iodine flask, adding distil water 20ml makes dissolving, adds dilute sulfuric acid 25ml, reflux 4 hours, put coldly, add 1～2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose (C of 9.008mg ₆H ₁₂O ₆), calculate sugar contents in every bottle in the sample with this.

The standard curve precision takes by weighing 105 ℃ of anhydrous glucose reference substance 50.24mg that are dried to constant weight, 101.87mg, 149.58mg, 201.36mg, 252.81mg, 300.79mg, split in the iodine flask, adding distil water 60ml makes dissolving, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose (C of 9.008mg ₆H ₁₂O ₆).Consuming volumetric solution volume (ml) with sample is abscissa, is that vertical coordinate is figure with the amount (mg) of anhydrous glucose, the drawing standard curve.

The anhydrous glucose standard curve

Numbering	The sample volumetric solution consumes volume (ml)	Anhydrous glucose (mg)
Numbering	The sample volumetric solution consumes volume (ml)	Anhydrous glucose (mg)	1	5.65	50.24
2	11.25	101.87	1	5.65	50.24
2	11.25	101.87	3	16.85	149.58

4	22.65	201.36
4	22.65	201.36	5	28.25	252.81
6	33.74	300.79	5	28.25	252.81

Regression equation: Y=8.9093x+0.3139;

Correlation coefficient: γ=0.9999;

The result shows: anhydrous glucose is good in 50.24～300.79 μ g scope internal linear.

As calculated, the standard curve of anhydrous glucose is crossed initial point, therefore selects for use one point external standard method to measure the content of total polysaccharides in the pulse restoring injection.

Repeated experiment is got this product under the content uniformity item, gets content, and mixing is therefrom got about 500mg (5 parts), presses operation under the text algoscopy item, measures, promptly.

Repeated experiment

Numbering	Weighing (mg)	Total sugar (mg/ bottle)	Monosaccharide (mg/ bottle)	Total polysaccharides (mg/ bottle)
Numbering	Weighing (mg)	Total sugar (mg/ bottle)	Monosaccharide (mg/ bottle)	Total polysaccharides (mg/ bottle)	1	470.54	172.65	85.64	87.01
2	502.03	177.84	89.21	88.63	1	470.54	172.65	85.64	87.01
2	502.03	177.84	89.21	88.63	3	512.39	171.29	86.37	84.92
4	502.47	176.01	88.59	87.42	3	512.39	171.29	86.37	84.92
4	502.47	176.01	88.59	87.42	5	508.61	174.39	86.87	87.52
Average	-	174.44	87.34	87.10	5	508.61	174.39	86.87	87.52
Average	-	174.44	87.34	87.10	RSD(％)	-	1.49	1.73	1.56

The application of sample absorption method is adopted in response rate experiment, gets this product under the content uniformity item, gets content, and mixing is therefrom got about 250mg (6 parts), splits in the iodine flask; Be taken at 105 ℃ of about 35mg of anhydrous glucose that are dried to constant weight, the accurate title, decide, split in the above-mentioned iodine flask, adding distil water 60ml makes dissolving, and hydro-oxidation sodium test solution is to neutral, and precision adds iodine liquid (0.1mol/L) 25ml, shake up, dropwise add sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, add dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear, and titration results is proofreaied and correct with blank assay, promptly to blue.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose (C of 9.008mg ₆H ₁₂O ₆).

Pulse restoring injection contents of monosaccharides: 87.34mg/ bottle.

The test of anhydrous glucose average recovery

Numbering

Test sample weighing (mg)

Monosaccharide amount (mg) in the test sample

Anhydrous glucose addition (mg)

Measured value (mg)

The response rate (%)

1	244.86	35.54	34.92	70.12	99.03
1	244.86	35.54	34.92	70.12	99.03	2	250.17	36.31	37.26	72.91	98.23
3	239.64	34.79	35.84	70.33	99.16	2	250.17	36.31	37.26	72.91	98.23
3	239.64	34.79	35.84	70.33	99.16	4	255.73	37.12	36.11	71.94	96.43
5	260.92	37.87	35.58	72.93	98.54	4	255.73	37.12	36.11	71.94	96.43
5	260.92	37.87	35.58	72.93	98.54	6	251.53	36.51	34.79	70.82	98.62

Average recovery rate=98.34%; RSD=1.01%.

The assay of sample is got in this product three batches of test agents, presses the preparation and the operation down of algoscopy item of text need testing solution.

Total polysaccharides assay result

Lot number	Total polysaccharides content (mg/ bottle)
Lot number	Total polysaccharides content (mg/ bottle)	1	84.93
2	90.22	1	84.93
2	90.22	3	87.65

From above test as can be known, it is good to adopt the method for the invention to measure the content precision of total polysaccharides in the pulse restoring injection, stability and repeatability, response rate height, and method is easy, and data are accurate, reliable.

Ophiopogonin B in experimental example 17 pulse restoring injections, ophiopogonin D, ophiopogonin D ' content assaying method

1. instrument and reagent

(1) instrument: SHIMADZU 2010Aht high performance liquid chromatograph; The TU-1810SPC ultraviolet spectrophotometer; SARTORIUS BP211D electronic analytical balance.

(2) reagent: ophiopogonin B, ophiopogonin D, ophiopogonin D '

2. it is an amount of that the selection precision that detects wavelength takes by weighing ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance, adds methanol respectively and make the solution that every 1ml contains 0.1mg, scans in the 200-400nm wave-length coverage respectively.The result shows: ophiopogonin B, ophiopogonin D, ophiopogonin D ' all absorption maximum is arranged at the 203nm place, therefore select the detection wavelength of 203nm as assay.

3. chromatographic condition: Dikma ODS (4.6 * 250mm, 5um); Mobile phase: acetonitrile-water (90: 10) is a mobile phase; The detection wavelength is 203nm; Flow velocity: 1.0ml/min.

4. system suitability test

4.1 it is an amount of that the preparation precision of reference substance solution takes by weighing ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance, adding methanol makes every 1ml and contains ophiopogonin B 0.007mg, ophiopogonin D 0.007mg, the reference substance mixed solution of ophiopogonin D ' 0.002mg, promptly.

4.3 the preparation of negative need testing solution takes by weighing the negative sample that does not contain Radix Ophiopogonis, according to the preparation method preparation of need testing solution, as negative need testing solution.

Accurate respectively reference substance solution, need testing solution, each 10 μ l of negative sample solution of drawing inject chromatograph of liquid, measure the record chromatogram.As seen from the figure, ophiopogonin B in the test sample, ophiopogonin D, ophiopogonin D ' peak separate good, and it is clear to separate with close peak, and fully, ophiopogonin B, ophiopogonin D in the negative test sample, ophiopogonin D ' peak is noiseless.

5. linear relationship is investigated precision and is taken by weighing ophiopogonin B reference substance 10.14mg, ophiopogonin D reference substance 10.06mg, ophiopogonin D ' reference substance 6.59mg, split in the 50ml measuring bottle, add methanol respectively and make dissolving and fixed to scale in right amount, shake up, precision is measured ophiopogonin B reference substance solution 0.7ml, ophiopogonin D reference substance solution 0.7ml, ophiopogonin D ' reference substance solution 0.4ml is put in the 10ml measuring bottle altogether, and it is fixed to scale to add methanol, shake up, the accurate 2 μ l that draw, 4 μ l, 6 μ l, 8 μ l, 10 μ l inject chromatograph of liquid, are vertical coordinate with the peak area, sample size (μ g) is figure for abscissa, the drawing standard curve.

The ophiopogonin B linear relationship is investigated

Numbering	Ophiopogonin B (μ g)	Peak area
Numbering	Ophiopogonin B (μ g)	Peak area	1	0.028392	21894
2	0.056784	43805	1	0.028392	21894
2	0.056784	43805	3	0.085176	66601
4	0.113568	88329	3	0.085176	66601
4	0.113568	88329	5	0.141960	109588

Regression equation: Y=774556.21X+69.80;

The coefficient of determination: γ=0.9999;

Ophiopogonin B is good in 0.028392～0.14196 μ g scope internal linear relation.

The ophiopogonin D linear relationship is investigated

Numbering	Ophiopogonin D (μ g)	Peak area
Numbering	Ophiopogonin D (μ g)	Peak area	1	0.028168	18411
2	0.056336	36824	1	0.028168	18411
2	0.056336	36824	3	0.084504	55119
4	0.112672	73802	3	0.084504	55119
4	0.112672	73802	5	0.140840	92108

Regression equation: Y=654544.16X-58.80;

The coefficient of determination: γ=0.9999;

Ophiopogonin D is good in 0.028168～0.14084 μ g scope internal linear relation.

Ophiopogonin D ' linear relationship is investigated

Numbering	Ophiopogonin D ' (μ g)	Peak area
Numbering	Ophiopogonin D ' (μ g)	Peak area	1	0.010544	18859
2	0.021088	37804	1	0.010544	18859
2	0.021088	37804	3	0.031632	56821
4	0.042176	75621	3	0.031632	56821
4	0.042176	75621	5	0.052720	94338

Regression equation: Y=1790354.70X+56.10;

The coefficient of determination: γ=0.9999;

Ophiopogonin D ' good in 0.010544～0.052720 μ g scope internal linear relation.

6. precision test precision is drawn reference substance mixed solution (every 1ml contains ophiopogonin B 8.112 μ g respectively, ophiopogonin D 8.048 μ g, ophiopogonin D ' 2.636 μ g) 10 μ l, inject chromatograph of liquid, the record peak area, METHOD FOR CONTINUOUS DETERMINATION 5 times, the precision of investigation reference substance solution.

The test of reference substance precision

Test number (TN)	1	2	3	4	5	Average	RSD(％)
Test number (TN)	1	2	3	4	5	Average	RSD(％)	Ophiopogonin B	63356	64187	62195	63314	62868	63184	1.16
Ophiopogonin D	51196	52137	53268	52571	53094	52453	1.59	Ophiopogonin B	63356	64187	62195	63314	62868	63184	1.16
Ophiopogonin D	51196	52137	53268	52571	53094	52453	1.59	Ophiopogonin D '	47615	45508	46309	47703	46925	46672	1.71

The result shows that reference substance solution precision is good.

7. the accurate SHENGMAI ZHUSHEYE 10 μ l that draw of stability test inject chromatograph of liquid, calculate with one point external standard method, that is, measure at 0,6,12,24,48 hour sample introduction respectively.

The reference substance solution stability test

Time (h)	0	6	12	24	48	Average	RSD(％)
Time (h)	0	6	12	24	48	Average	RSD(％)	Ophiopogonin B (mg/ props up)	0.0814	0.0822	0.0813	0.0807	0.0842	0.0820	1.66
Ophiopogonin D (mg/ props up)	0.0818	0.0805	0.0834	0.0816	0.0822	0.0819	1.28	Ophiopogonin B (mg/ props up)	0.0814	0.0822	0.0813	0.0807	0.0842	0.0820	1.66
Ophiopogonin D (mg/ props up)	0.0818	0.0805	0.0834	0.0816	0.0822	0.0819	1.28	Ophiopogonin D ' (mg/ props up)	0.0203	0.0205	0.0206	0.0201	0.0208	0.0205	1.32

The result shows that reference substance solution has good stability.

Replica test

Numbering	1	2	3	4	5	Average	RSD(％)
Numbering	1	2	3	4	5	Average	RSD(％)	Ophiopogonin B (mg/ props up)	0.0827	0.0834	0.0818	0.0825	0.0836	0.0828	0.88
Ophiopogonin D (mg/ props up)	0.0821	0.0831	0.0827	0.0826	0.0818	0.0825	0.62	Ophiopogonin B (mg/ props up)	0.0827	0.0834	0.0818	0.0825	0.0836	0.0828	0.88
Ophiopogonin D (mg/ props up)	0.0821	0.0831	0.0827	0.0826	0.0818	0.0825	0.62	Ophiopogonin D ' (mg/ props up)	0.0211	0.0207	0.0205	0.0214	0.0208	0.0209	1.69

The result shows that repeatability is good.

9. the application of sample absorption method is adopted in the average recovery test, and precision is measured SHENGMAI ZHUSHEYE 5ml, splits in the 10ml measuring bottle; Precision is measured the reference substance mixed solution, and (every 1ml contains ophiopogonin B 0.03728mg respectively, ophiopogonin D 0.03564mg, the 1ml (totally 5 parts) of ophiopogonin D ' 0.00928mg), split in the above-mentioned 10ml measuring bottle, add an amount of supersound process of methanol (power 250W, frequency 33KHz) 10 minutes, take out, put, add methanol to scale to room temperature, shake up, filter, accurate subsequent filtrate 10 μ 1 that draw inject chromatograph of liquid, calculate with one point external standard method, promptly.

Ophiopogonin B content in the SHENGMAI ZHUSHEYE: 0.0828mg/ props up;

Ophiopogonin D content in the SHENGMAI ZHUSHEYE: 0.0825mg/ props up;

Ophiopogonin D ' content in the SHENGMAI ZHUSHEYE: 0.0209mg/ props up.

The test of ophiopogonin B average recovery

Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1	0.0414	0.03728	0.07798	98.12
2	0.0414	0.03728	0.07832	99.04	1	0.0414	0.03728	0.07798	98.12
2	0.0414	0.03728	0.07832	99.04	3	0.0414	0.03728	0.07777	97.56
4	0.0414	0.03728	0.07809	98.41	3	0.0414	0.03728	0.07777	97.56
4	0.0414	0.03728	0.07809	98.41	5	0.0414	0.03728	0.07840	99.25

Ophiopogonin B average recovery rate=98.48%; RSD=0.70%.

The test of ophiopogonin D average recovery

Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1	0.04125	0.03564	0.07608	97.72
2	0.04125	0.03564	0.07623	98.15	1	0.04125	0.03564	0.07608	97.72
2	0.04125	0.03564	0.07623	98.15	3	0.04125	0.03564	0.07673	99.56
4	0.04125	0.03564	0.07631	98.37	3	0.04125	0.03564	0.07673	99.56

5

0.04125

0.03564

0.07654

99.03

Ophiopogonin D average recovery rate=98.57%; RSD=0.74%.

Ophiopogonin D ' average recovery test

Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1	0.01045	0.00928	0.01965	99.15
2	0.01045	0.00928	0.01966	99.26	1	0.01045	0.00928	0.01965	99.15
2	0.01045	0.00928	0.01966	99.26	3	0.01045	0.00928	0.01934	95.84
4	0.01045	0.00928	0.01945	97.03	3	0.01045	0.00928	0.01934	95.84
4	0.01045	0.00928	0.01945	97.03	5	0.01045	0.00928	0.01964	99.08

Ophiopogonin D ' average recovery rate=98.07%; RSD=1.58%.

10. three batches of pilot scale sample determinations

Three batch sample assay results

Lot number	1	2	3
Lot number	1	2	3	Ophiopogonin B (mg/ props up)	0.0834	0.0819	0.0839
Ophiopogonin D (mg/ props up)	0.0821	0.0816	0.0824	Ophiopogonin B (mg/ props up)	0.0834	0.0819	0.0839
Ophiopogonin D (mg/ props up)	0.0821	0.0816	0.0824	Ophiopogonin D ' (mg/ props up)	0.0232	0.0235	0.0243

From above test as can be known, adopt the method for the invention measure ophiopogonin B in the SHENGMAI ZHUSHEYE, ophiopogonin D, ophiopogonin D ' content precision, stability and repeatability good, response rate height, method is easy, data are accurately, reliably.

The content assaying method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in experimental example 18 pulse restoring injections

1. instrument and reagent

(2) reagent: Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin

2. it is an amount of that the selection precision that detects wavelength takes by weighing Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance, adds methanol respectively and make the solution that every 1ml contains 0.1mg, scans in the 200-400nm wave-length coverage respectively.The result shows: Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin all have absorption maximum at the 203nm place, therefore select the detection wavelength of 203nm as assay.

4. system suitability test

4.1 it is an amount of that the preparation precision of reference substance solution takes by weighing Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance, adding methanol makes every 1ml and contains Ruscus aculeatus L. sapogenin 0.002mg, diosgenin 0.002mg, the reference substance mixed solution of ruscogenin 0.002mg, promptly.

4.2 the preparation precision of need testing solution is measured SHENGMAI ZHUSHEYE 10ml, puts in the round-bottomed flask, adds 3% sulphuric acid 10ml and refluxes 4 hours, take out, put, transfer pH to neutral with 3% sodium hydroxide solution to room temperature, with chloroform extraction 3 times, each 15ml and and extracting solution, evaporate to dryness, residue makes dissolving in right amount with methanol and quantitatively is transferred in the 10ml measuring bottle, and it is fixed to scale to add methanol, shakes up, filter with microporous filter membrane, get subsequent filtrate promptly.

Accurate respectively reference substance solution, need testing solution, each 10 μ l of negative sample solution of drawing inject chromatograph of liquid, measure the record chromatogram.As seen from the figure, Ruscus aculeatus L. sapogenin in the test sample, diosgenin, ruscogenin peak separate good, and it is clear to separate with close peak, and fully, Ruscus aculeatus L. sapogenin, diosgenin in the negative test sample, the ruscogenin peak is noiseless.

5. linear relationship is investigated precision and is taken by weighing Ruscus aculeatus L. sapogenin reference substance 6.71mg, diosgenin reference substance 6.94mg, ruscogenin reference substance 6.63mg, put altogether in the 50ml measuring bottle, add methanol and make dissolving and fixed in right amount to scale, shake up, precision is measured 0.4ml, puts in the 10ml measuring bottle, and it is fixed to scale to add methanol, shake up, accurate 2 μ l, 4 μ l, 6 μ l, 8 μ l, the 10 μ l of drawing inject chromatograph of liquid, are vertical coordinate with the peak area, sample size (μ g) is figure for abscissa, the drawing standard curve.

The Ruscus aculeatus L. sapogenin linear relationship is investigated

Numbering	Ruscus aculeatus L. sapogenin (μ g)	Peak area
Numbering	Ruscus aculeatus L. sapogenin (μ g)	Peak area	1	0.010736	15729
2	0.021472	31526	1	0.010736	15729
2	0.021472	31526	3	0.032208	47338
4	0.042944	62952	3	0.032208	47338
4	0.042944	62952	5	0.053680	78903

Regression equation: Y=1469578.99X-42.60;

The coefficient of determination: γ=0.9999;

Ruscus aculeatus L. sapogenin is good in 0.010736～0.053680 μ g scope internal linear relation.

The diosgenin linear relationship is investigated

Numbering

Diosgenin (μ g)

Peak area

1	0.011104	16593
1	0.011104	16593	2	0.022208	33041
3	0.033312	49825	2	0.022208	33041
3	0.033312	49825	4	0.044416	66407
5	0.055520	82831	4	0.044416	66407

Regression equation: Y=1493533.86X-13.20;

The coefficient of determination: γ=0.9999;

Diosgenin is good in 0.011104～0.055520 μ g scope internal linear relation.

The ruscogenin linear relationship is investigated

Numbering	Ruscogenin (μ g)	Peak area
Numbering	Ruscogenin (μ g)	Peak area	1	0.010608	20521
2	0.021216	41137	1	0.010608	20521
2	0.021216	41137	3	0.031824	61459
4	0.042432	82003	3	0.031824	61459
4	0.042432	82003	5	0.053040	102711

Regression equation: Y=1934822.78X-7.60;

The coefficient of determination: γ=0.9999;

Ruscogenin is good in 0.010608～0.053040 μ g scope internal linear relation.

6. precision test precision is drawn reference substance mixed solution (every 1ml contains Ruscus aculeatus L. sapogenin 2.684 μ g respectively, diosgenin 2.776 μ g, ruscogenin 2.652 μ g) 10 μ l, inject chromatograph of liquid, the record peak area, METHOD FOR CONTINUOUS DETERMINATION 5 times, the precision of investigation reference substance solution.

The test of reference substance precision

Test number (TN)	1	2	3	4	5	Average	RSD(％)
Test number (TN)	1	2	3	4	5	Average	RSD(％)	Ruscus aculeatus L. sapogenin	39429	40107	41135	40863	40526	40412	1.66
Diosgenin	41137	40928	42056	40764	41963	41370	1.45	Ruscus aculeatus L. sapogenin	39429	40107	41135	40863	40526	40412	1.66
Diosgenin	41137	40928	42056	40764	41963	41370	1.45	Ruscogenin	50847	51093	50625	51147	52036	51150	1.05

The result shows that reference substance solution precision is good.

7. the stability test precision is measured SHENGMAI ZHUSHEYE 10ml, put in the round-bottomed flask, add 3% sulphuric acid 10ml and refluxed 4 hours, take out, put to room temperature, transfer pH to neutral with 3% sodium hydroxide solution, with chloroform extraction 3 times, each 15ml and and extracting solution, evaporate to dryness, residue makes dissolving in right amount with methanol and quantitatively is transferred in the 10ml measuring bottle, and it is fixed to scale to add methanol, shakes up, filter, the accurate 10 μ l that draw inject chromatograph of liquid, calculate with one point external standard method, that is, measure at 0,6,12,24,48 hour sample introduction respectively.

The reference substance solution stability test

Time (h)	0	6	12	24	48	Average	RSD(％)
Time (h)	0	6	12	24	48	Average	RSD(％)	Ruscus aculeatus L. sapogenin (mg/ props up)	0.0214	0.0205	0.0211	0.0208	0.0206	0.0209	1.77
Diosgenin (mg/ props up)	0.0203	0.0199	0.0205	0.0207	0.0201	0.0203	1.56	Ruscus aculeatus L. sapogenin (mg/ props up)	0.0214	0.0205	0.0211	0.0208	0.0206	0.0209	1.77
Diosgenin (mg/ props up)	0.0203	0.0199	0.0205	0.0207	0.0201	0.0203	1.56	Ruscogenin (mg/ props up)	0.0214	0.0217	0.0223	0.0221	0.0219	0.0219	1.60

The result shows that reference substance solution has good stability.

8. the replica test precision is measured SHENGMAI ZHUSHEYE 10ml (totally 5 parts), the accurate title, decide, and splits in the round-bottomed flask, adds 3% sulphuric acid 10ml and refluxed 4 hours, take out, put to room temperature, transfer pH to neutral, use chloroform extraction 3 times with 3% sodium hydroxide solution, each 15ml, with and extracting solution, evaporate to dryness, residue with methanol make in right amount the dissolving and quantitatively be transferred in the 10ml measuring bottle, it is fixed to scale to add methanol, shake up, filter, the accurate 10 μ l that draw, the accurate 10 μ l that draw, inject chromatograph of liquid, calculate with one point external standard method, promptly.

Replica test

Numbering	1	2	3	4	5	Average	RSD(％)
Numbering	1	2	3	4	5	Average	RSD(％)	Ruscus aculeatus L. sapogenin (mg/ props up)	0.0223	0.0221	0.0226	0.0218	0.0225	0.0223	1.44
Diosgenin (mg/ props up)	0.0214	0.0209	0.0211	0.0207	0.0204	0.0209	1.82	Ruscus aculeatus L. sapogenin (mg/ props up)	0.0223	0.0221	0.0226	0.0218	0.0225	0.0223	1.44
Diosgenin (mg/ props up)	0.0214	0.0209	0.0211	0.0207	0.0204	0.0209	1.82	Ruscogenin (mg/ props up)	0.0226	0.0227	0.0229	0.0224	0.0225	0.0226	0.85

The result shows that repeatability is good.

9. the application of sample absorption method is adopted in the average recovery test, and precision is measured SHENGMAI ZHUSHEYE 5ml (totally 5 parts), splits in the round-bottomed flask; Precision is measured the reference substance mixed solution, and (every 1ml contains Ruscus aculeatus L. sapogenin 0.00904mg respectively, diosgenin 0.00948mg, ruscogenin 0.00848mg) 1ml (totally 5 parts), split in the above-mentioned round-bottomed flask, adding 3% sulphuric acid 10ml respectively refluxed 4 hours, take out, put, transfer pH to neutral with 3% sodium hydroxide solution to room temperature, with chloroform extraction 3 times, 15ml and also extracting solution at every turn, evaporate to dryness, residue makes dissolving in right amount with methanol and quantitatively is transferred in the 10ml measuring bottle, add methanol and decide to shake up, filter to scale, the accurate subsequent filtrate 10 μ l that draw, inject chromatograph of liquid, calculate with one point external standard method, promptly.

Ruscus aculeatus L. sapogenin content in the SHENGMAI ZHUSHEYE: 0.0223mg/ props up;

Diosgenin content in the SHENGMAI ZHUSHEYE: 0.0209mg/ props up;

Ruscogenin content in the SHENGMAI ZHUSHEYE: 0.0226mg/ props up.

The test of Ruscus aculeatus L. sapogenin average recovery

Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1	0.01115	0.00904	0.01994	97.25
2	0.01115	0.00904	0.02007	98.63	1	0.01115	0.00904	0.01994	97.25
2	0.01115	0.00904	0.02007	98.63	3	0.01115	0.00904	0.02011	99.06
4	0.01115	0.00904	0.01987	96.42	3	0.01115	0.00904	0.02011	99.06
4	0.01115	0.00904	0.01987	96.42	5	0.01115	0.00904	0.01993	97.15

Ruscus aculeatus L. sapogenin average recovery rate=97.70%; RSD=1.13%.

The test of diosgenin average recovery

Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1	0.01045	0.00948	0.01975	98.12
2	0.01045	0.00948	0.01974	98.03	1	0.01045	0.00948	0.01975	98.12
2	0.01045	0.00948	0.01974	98.03	3	0.01045	0.00948	0.01986	99.27
4	0.01045	0.00948	0.01961	96.64	3	0.01045	0.00948	0.01986	99.27
4	0.01045	0.00948	0.01961	96.64	5	0.01045	0.00948	0.01984	99.05

Diosgenin average recovery rate=98.22%; RSD=1.06%.

The test of ruscogenin average recovery

Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)
Numbering	Pure product amount (mg) in the test sample	Pure product addition (mg)	Measured value (mg)	The response rate (%)	1	0.0113	0.00848	0.01970	99.05
2	0.0113	0.00848	0.01962	98.16	1	0.0113	0.00848	0.01970	99.05
2	0.0113	0.00848	0.01962	98.16	3	0.0113	0.00848	0.01964	98.34
4	0.0113	0.00848	0.01953	97.06	3	0.0113	0.00848	0.01964	98.34
4	0.0113	0.00848	0.01953	97.06	5	0.0113	0.00848	0.01948	96.47

Ruscogenin average recovery rate=97.82%; RSD=1.06%.

10. three batches of pilot scale sample determinations

Three batch sample assay results

Lot number	1	2	3
Lot number	1	2	3	Ruscus aculeatus L. sapogenin (mg/ props up)	0.0221	0.0246	0.0239
Diosgenin (mg/ props up)	0.0226	0.0218	0.0242	Ruscus aculeatus L. sapogenin (mg/ props up)	0.0221	0.0246	0.0239
Diosgenin (mg/ props up)	0.0226	0.0218	0.0242	Ruscogenin (mg/ props up)	0.0236	0.0229	0.0241

From above test as can be known, it is good to adopt the method for the invention to measure the content precision of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the SHENGMAI ZHUSHEYE, stability and repeatability, response rate height, and method is easy, and data are accurate, reliable.

The specific embodiment:

Embodiment 1: adopt liquid chromatography for measuring based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics finger printing:

(1) preparation of need testing solution: it is an amount of that precision takes by weighing freeze-dried powder to be measured, adds water and make the solution that every 1ml contains 50mg, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;

(4) formulation of standard finger-print: with said method as formulate based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics the means of testing of standard finger-print; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 11;

(5) based on the described method in (1)～(3) as Radix Ginseng Rubra or Radix Ginseng in the freeze-dried powder to be measured and Radix Ophiopogonis composition characteristics the means of testing of finger printing, the finger printing of preparation testing sample;

(6) with the contrast of freeze-dried powder finger printing to be measured and above-mentioned standard finger-print, in should meeting the following requirements partly or entirely:

I. calculate the similarity of the finger printing and the standard finger-print of freeze-dried powder to be measured, should be 0.90～1.00;

II. in the freeze-dried powder finger printing to be measured, non-total peak area is no more than 5% of total peak area;

III., the odds ratio that each total peak area in the ratio of 40%～60% total peak area and the standard finger-print arranged in the test sample finger printing, its difference is not more than ± 30%.

Embodiment 2: adopt the finger printing of liquid chromatography for measuring based on the Fructus Schisandrae Chinensis composition characteristics:

(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the standard finger-print of Fructus Schisandrae Chinensis composition characteristics; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 17

(5) based on the means of testing of the described method in (1)～(3) as the finger printing of freeze-dried powder Fructus Schisandrae Chinensis composition characteristics to be measured, the finger printing of preparation testing sample;

The odds ratio that each total peak area in the ratio of 40%～60% total peak area and the standard finger-print arranged in the freeze-dried powder finger printing III. to be measured, its difference is not more than ± 30%.

Embodiment 3: adopt liquid chromatography for measuring based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics finger printing:

(1) preparation of need testing solution: it is an amount of that precision takes by weighing freeze-dried powder to be measured, adds methanol and make the solution that every 1ml contains 50mg, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate as need testing solution;

(2) preparation of object of reference solution: precision takes by weighing ginsenoside Rb ₁In right amount, add ethanol and make the solution that every 1ml contains 0.3mg, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is the 0.05mol/L sodium dihydrogen phosphate, and Mobile phase B is acetonitrile-water 85: 15, gradient elution, solvent ratios is from 0 minute to 4 minutes, the ratio of Mobile phase B is 22%, and from 4 minutes to 40 minutes, the ratio of Mobile phase B rose to 60% by 22%, from 40 minutes to 50 minutes, the ratio of Mobile phase B is 60%, and from 50 minutes to 65 minutes, the ratio of Mobile phase B rose to 77% by 60%, from 65 minutes to 75 minutes, the ratio of Mobile phase B reduced to 22% by 77%; Flow velocity is 1.0ml/min, and the detection wavelength is 203 ± 2nm, and column temperature is 40 ℃;

(4) formulation of standard finger-print: with said method as formulate based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics the means of testing of standard finger-print; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 13;

Embodiment 4: adopt the finger printing of liquid chromatography for measuring based on the Fructus Schisandrae Chinensis composition characteristics:

(2) preparation of object of reference solution: it is an amount of that precision takes by weighing the schisantherin B reference substance, adds methanol and make the solution that every 1ml contains 0.1mg, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase is acetonitrile-0.1% phosphoric acid, gradient elution, solvent ratios are from 0 minute to 5 minutes, and the ratio of acetonitrile is 60%, from 5 minutes to 10 minutes, the ratio of acetonitrile rises to 70% by 60%, and from 10 minutes to 12 minutes, the ratio of acetonitrile was 70%, from 12 minutes to 20 minutes, the ratio of acetonitrile reduces to 62% by 70%, and from 12 minutes to 60 minutes, the ratio of acetonitrile was 62%; Flow velocity is 1.0ml/min, and the detection wavelength is 250 ± 2nm, and column temperature is 30 ℃;

(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the standard finger-print of Fructus Schisandrae Chinensis composition characteristics; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 20;

(5) based on the means of testing of the described method in (1)～(3) as the finger printing of Fructus Schisandrae Chinensis composition characteristics in the freeze-dried powder to be measured, the finger printing of preparation testing sample;

Embodiment 5: adopt liquid chromatography for measuring based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics finger printing:

(1) preparation of need testing solution: get injection as need testing solution;

(2) preparation of object of reference solution: it is an amount of that precision takes by weighing the ginsenoside Rf, adds ethanol and make the solution that every 1ml contains 0.3mg, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase A is 1% glacial acetic acid aqueous solution, Mobile phase B is methanol-water 95: 5, gradient elution, solvent ratios are from 0 minute to 10 minutes, and the ratio of Mobile phase B is 30%, from 10 minutes to 35 minutes, the ratio of Mobile phase B rises to 85% by 30%, and from 35 minutes to 55 minutes, the ratio of Mobile phase B was 85%, from 55 minutes to 70 minutes, the ratio of Mobile phase B reduced to 30% by 85%; Flow velocity is 1.0ml/min, and the detection wavelength is 203 ± 2nm, and column temperature is 40 ℃;

(4) formulation of standard finger-print: with said method as formulate based on Radix Ginseng Rubra or Radix Ginseng and Radix Ophiopogonis composition characteristics the means of testing of standard finger-print; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 15;

II. in the injection finger printing to be measured, non-total peak area is no more than 5% of total peak area;

The odds ratio that each total peak area in the ratio of 40%～60% total peak area and the standard finger-print arranged in the injection finger printing III. to be measured, its difference is not more than ± 30%.

Embodiment 6: adopt the finger printing of liquid chromatography for measuring based on the Fructus Schisandrae Chinensis composition characteristics:

(2) preparation of object of reference solution: it is an amount of that precision takes by weighing the deoxyschizandrin reference substance, adds ethanol and make the solution that every 1ml contains 0.1mg, as object of reference solution;

(3) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler; Mobile phase is acetonitrile-1% glacial acetic acid aqueous solution, gradient elution, solvent ratios is from 0 minute to 5 minutes, the ratio of acetonitrile is 60%, and from 5 minutes to 10 minutes, the ratio of acetonitrile rose to 70% by 60%, from 10 minutes to 15 minutes, the ratio of acetonitrile is 70%, and from 15 minutes to 60 minutes, the ratio of acetonitrile reduced to 54% by 70%; Flow velocity is 1.0ml/min, and the detection wavelength is 250 ± 2nm, and column temperature is 30 ℃;

(4) formulation of standard finger-print: with said method as the means of testing of formulating based on the standard finger-print of Fructus Schisandrae Chinensis composition characteristics; According to 10 batches or 10 batches of collection of illustrative plates that above test sample is measured, formulate standard finger-print, be benchmark with the retention time of the object of reference chromatographic peak determined, calculate the relative retention time of other total chromatographic peak, in the described standard finger-print, total peak has 14;

The odds ratio that each total peak area in the ratio of 40%～60% total peak area and the standard finger-print arranged in the injection finger printing III. to be measured, its difference is not more than 30%.

Embodiment 7: the thin layer chromatography discrimination method of Fructus Schisandrae Chinensis, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, adds chloroform extraction, filters, and filtrate volatilizes, and residue adds the chloroform dissolving, as need testing solution; The preparation of Fructus Schisandrae Chinensis control medicinal material solution: it is an amount of to get the Fructus Schisandrae Chinensis control medicinal material, adds chloroform extraction, filters, and filtrate volatilizes, and residue adds chloroform dissolving, medical material solution in contrast; The preparation of reference substance solution: get schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B reference substance, add chloroform respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put in same silica gel G F respectively ₂₅₄On the lamellae, with petroleum ether (30～60 ℃): Ethyl formate: the upper solution of formic acid=15: 5: 1 is developing solvent, launch, take out, dry, put under the uviol lamp 254nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color.

Embodiment 8: the liquid chromatograph of schisandrin, deoxyschizandrin, schisandrin B is differentiated in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-water is a mobile phase at 65%: 35%, the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 9: the thin layer chromatography discrimination method of Radix Ophiopogonis in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, adds 7: 3 mixed solutions of chloroform-methanol and extracts, and filters, and filtrate volatilizes, and residue adds the chloroform dissolving, as need testing solution; Other gets control medicinal material Radix Ophiopogonis, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put in same silica gel G F respectively ₂₅₄On the lamellae, with toluene: methanol: glacial acetic acid=80: 5: 0.1 is developing solvent, launches, and takes out, dry, put under the uviol lamp 254nm and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color, negative noiseless.

Embodiment 10: ophiopogonin B in the freeze-dried powder, ophiopogonin D, ophiopogonin D ' the thin layer chromatography discrimination method:

It is an amount of to get freeze-dried powder to be measured, and with using n-butanol extraction behind the water dissolution, extract volatilizes, and the residue dissolve with methanol is as need testing solution; Other gets ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance, adds methanol respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with ethyl acetate: methanol: water=15: 5: 1 is developing solvent, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

Embodiment 11: ophiopogonin B in the freeze-dried powder, ophiopogonin D, ophiopogonin D ' liquid chromatograph differentiate:

It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopts liquid chromatography, and the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, and the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 12: the thin layer chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, adds 3% sulphuric acid hydrolysis 4 hours, takes out, and puts to room temperature, regulates pH to neutral, filters, and filtrate volatilizes, and residue dissolves with chloroform, as need testing solution; Other gets Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance, adds chloroform respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with normal hexane: ethyl acetate: water=1: 1: 1 is developing solvent, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 90 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

Embodiment 13: the liquid chromatograph of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin is differentiated in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, puts in the round-bottomed flask, and refluxing 4 hours with 3% sulphuric acid in the back that is dissolved in water, takes out, put to room temperature, transfer pH to neutral, use chloroform extraction, extracting solution volatilizes, the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 14: Radix Ginseng Rubra or Radix Ginseng, ginsenoside Rg in the freeze-dried powder ₁, ginsenoside Rb ₁, ginsenoside Re, ginsenoside Rf the thin layer chromatography discrimination method:

It is an amount of to get freeze-dried powder to be measured, uses n-butanol extraction, filters, and filtrate is as need testing solution; The preparation of Radix Ginseng Rubra or Radix Ginseng control medicinal material solution: get Radix Ginseng Rubra or the Radix Ginseng control medicinal material is an amount of, add the chloroform reflux, extract,, discard chloroform liquid, residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re, ginsenoside Rf's reference substance, add methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with chloroform: ethyl acetate: methanol: water=15: 40: 22: 10 is developing solvent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ are dried by the fire to speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color.

Embodiment 15: ginsenoside Rg in the freeze-dried powder ₁, ginsenoside Rb ₁, the ginsenoside Re liquid chromatograph differentiate:

It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 16: the thin layer chromatography discrimination method of Fructus Schisandrae Chinensis, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, adds ethyl acetate extraction, filters, and filtrate volatilizes, and residue adds the ethyl acetate dissolving, as need testing solution; The preparation of Fructus Schisandrae Chinensis control medicinal material solution: it is an amount of to get the Fructus Schisandrae Chinensis control medicinal material, adds ethyl acetate extraction, filters, and filtrate volatilizes, and residue adds ethyl acetate dissolving, medical material solution in contrast; The preparation of reference substance solution: get schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, Fructus Schisandrae Chinensis ester second reference substance, add ethyl acetate respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put in same silica gel G F respectively ₂₅₄On the lamellae, with petroleum ether (60～90 ℃): ethyl acetate: the upper solution of formic acid=15: 5: 1 is developing solvent, launch, take out, dry, put under the uviol lamp 254nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color.

Embodiment 17: the liquid chromatograph of schisandrin, deoxyschizandrin, schisandrin B is differentiated in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with eight alkyl silane bonded silica gels, and acetonitrile-water is a mobile phase at 60%: 40%, the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 18: the thin layer chromatography discrimination method of Radix Ophiopogonis in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, adds ethyl acetate extraction, filters, and filtrate volatilizes, and residue adds the ethyl acetate dissolving, as need testing solution; Other gets control medicinal material Radix Ophiopogonis, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put in same silica gel G F respectively ₂₅₄On the lamellae, with benzene: methanol: formic acid=85: 5: 0.3 is developing solvent, launches, and takes out, and dries, put under the uviol lamp 254nm and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color, negative noiseless.

Embodiment 19: ophiopogonin B in the freeze-dried powder, ophiopogonin D, ophiopogonin D ' the thin layer chromatography discrimination method:

It is an amount of to get freeze-dried powder to be measured, and with using ethyl acetate extraction behind the water dissolution, extract volatilizes, and the residue dissolve with methanol is as need testing solution; Other gets ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance, adds methanol respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel H lamellae, with chloroform: methanol: water=25: 4: 1 is developing solvent, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

Embodiment 20: ophiopogonin B in the freeze-dried powder, ophiopogonin D, ophiopogonin D ' liquid chromatograph differentiate:

It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with eight alkyl silane bonded silica gels, and acetonitrile-1% glacial acetic acid is a mobile phase at 90: 10, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 21: the thin layer chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, adds 15% hydrochloric acid hydrolysis 4 hours, takes out, and puts to room temperature, regulates pH to neutral, filters, and filtrate volatilizes, and residue dissolves with dichloromethane, as need testing solution; Other gets Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance, adds chloroform respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel H lamellae, with cyclohexane extraction: Ethyl formate: water=1: 1: 0.5 is developing solvent, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 90 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

Embodiment 22: the liquid chromatograph of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin is differentiated in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, puts in the round-bottomed flask, and the back that is dissolved in water is with 15% hydrochloric acid reflux 4 hours, taking-up, put to room temperature, transfer pH to neutral, use dichloromethane extraction, extracting solution volatilizes, the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Alcoholic solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with eight alkyl silane bonded silica gels, and methanol-water is a mobile phase at 96: 4, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 23: Radix Ginseng Rubra or Radix Ginseng, ginsenoside Rg in the freeze-dried powder ₁, ginsenoside Rb ₁, ginsenoside Re, ginsenoside Rf the thin layer chromatography discrimination method:

It is an amount of to get freeze-dried powder to be measured, uses ethyl acetate extraction, filters, and filtrate is as need testing solution; The preparation of Radix Ginseng Rubra or Radix Ginseng control medicinal material solution: get Radix Ginseng Rubra or the Radix Ginseng control medicinal material is an amount of, the reflux, extract, that adds methylene chloride discards dichloromethane solution, residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re, ginsenoside Rf's reference substance, add ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel H lamellae, with normal hexane: ethyl acetate: acetone: water=20: 30: 15: 15 is developing solvent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ are dried by the fire to speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color.

Embodiment 24: ginsenoside Rg in the freeze-dried powder ₁, ginsenoside Rb ₁, the ginsenoside Re liquid chromatograph differentiate:

It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid chromatography, chromatographic column is a filler with eight alkyl silane bonded silica gels, acetonitrile-0.5% glacial acetic acid aqueous solution is a mobile phase, gradient elution, solvent ratios are from 0 minute to 30 minutes, and the ratio of acetonitrile is 20%, from 30 minutes to 50 minutes, the ratio of acetonitrile rises to 25% by 20%, and from 50 minutes to 70 minutes, the ratio of acetonitrile was 25%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 25%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 25: the thin layer chromatography discrimination method of Fructus Schisandrae Chinensis, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B in the injection:

It is an amount of to get injection to be measured, and the extraction that adds diethyl ether filters, and filtrate volatilizes, and residue adds the chloroform dissolving, as need testing solution; The preparation of Fructus Schisandrae Chinensis control medicinal material solution: it is an amount of to get the Fructus Schisandrae Chinensis control medicinal material, adds ethyl acetate extraction, filters, and filtrate volatilizes, and residue adds ethyl acetate dissolving, medical material solution in contrast; The preparation of reference substance solution: get schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, Fructus Schisandrae Chinensis ester second reference substance, add chloroform respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put in same silica gel G F respectively ₂₅₄On the lamellae, with petroleum ether (60～90 ℃): Ethyl formate: the upper solution of acetic acid=15: 5: 3 is developing solvent, launch, take out, dry, put under the uviol lamp 254nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color.

Embodiment 26: the liquid chromatograph of schisandrin, deoxyschizandrin, schisandrin B is differentiated in the injection:

It is an amount of to get injection to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Alcoholic solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with the dialkyl silane bonded silica gel, and acetonitrile-0.5% glacial acetic acid aqueous solution is a mobile phase at 57%: 43%, the detection wavelength is 250nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 27: the thin layer chromatography discrimination method of Radix Ophiopogonis in the injection:

It is an amount of to get injection to be measured, and the extraction that adds methylene chloride filters, and filtrate volatilizes, and residue adds the chloroform dissolving, as need testing solution; Other gets control medicinal material Radix Ophiopogonis, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put in same silica gel G F respectively ₂₅₄On the lamellae, with chloroform: ethanol: formic acid=70: 10: 2 is developing solvent, launches, and takes out, dry, put under the uviol lamp 254nm and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color, negative noiseless.

Embodiment 28: ophiopogonin B in the injection, ophiopogonin D, ophiopogonin D ' the thin layer chromatography discrimination method:

It is an amount of to get injection to be measured, uses dichloromethane extraction, and extract volatilizes, and the residue dissolve with methanol is as need testing solution; Other gets ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance, adds ethanol respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put in same silica gel G F respectively ₂₅₄On the lamellae, with dichloromethane: ethanol: water=20: 8: 2 is developing solvent, launches, and takes out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

Embodiment 29: ophiopogonin B in the injection, ophiopogonin D, ophiopogonin D ' liquid chromatograph differentiate:

It is an amount of to get injection to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Ethanol alcoholic solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with the dialkyl silane bonded silica gel, and acetonitrile-0.1% phosphate aqueous solution is a mobile phase at 87: 13, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 30: the thin layer chromatography discrimination method of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the injection:

It is an amount of to get injection to be measured, adds 5% sulphuric acid hydrolysis 3 hours, takes out, and puts to room temperature, regulates pH to neutral, filters, and filtrate volatilizes, and residue dissolves with ethyl acetate, as need testing solution; Other gets Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance, adds ethyl acetate respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put in same silica gel G F respectively ₂₅₄On the lamellae, with methanol: dichloromethane: water=2: 1: 0.2 is developing solvent, launches, and takes out, dry, spray is with 10% sulphuric acid ethanol reagent, 90 ℃ dry by the fire to speckle colour developing clear, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

Embodiment 31: the liquid chromatograph of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin is differentiated in the injection:

It is an amount of to get injection to be measured, puts in the round-bottomed flask, with 5% sulphuric acid hydrolysis 3 hours, takes out, and puts to room temperature, transfers pH to neutral, use ethyl acetate extraction, and extracting solution volatilizes, and the residue dissolve with ethanol with the microporous filter membrane filtration, is got subsequent filtrate as need testing solution; Alcoholic solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with the dialkyl silane bonded silica gel, and methanol-0.2% glacial acetic acid aqueous solution is a mobile phase at 95: 5, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 32: Radix Ginseng Rubra or Radix Ginseng, ginsenoside Rg in the injection ₁, ginsenoside Rb ₁, ginsenoside Re, ginsenoside Rf the thin layer chromatography discrimination method:

It is an amount of to get injection to be measured, uses ethyl acetate extraction, filters, and filtrate is as need testing solution; The preparation of Radix Ginseng Rubra or Radix Ginseng control medicinal material solution: get Radix Ginseng Rubra or the Radix Ginseng control medicinal material is an amount of, the reflux, extract, that adds methylene chloride discards dichloromethane solution, residue adds ethyl acetate extraction, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re, ginsenoside Rf's reference substance, add ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put in same silica gel G F respectively ₂₅₄On the lamellae, with n-butyl alcohol: Ethyl formate: water=20: 30: 15 upper solution be developing solvent, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ are dried by the fire to speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color.

Embodiment 33: ginsenoside Rg in the injection ₁, ginsenoside Rb ₁, the ginsenoside Re liquid chromatograph differentiate:

It is an amount of to get injection to be measured, puts in the measuring bottle, adds ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid chromatography, chromatographic column is a filler with the dialkyl silane bonded silica gel, acetonitrile-0.2% phosphate aqueous solution is a mobile phase, gradient elution, solvent ratios are from 0 minute to 20 minutes, and the ratio of acetonitrile is 15%, from 20 minutes to 45 minutes, the ratio of acetonitrile rises to 22% by 15%, and from 45 minutes to 60 minutes, the ratio of acetonitrile was 22%, from 60 minutes to 80 minutes, the ratio of acetonitrile rose to 35% by 22%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

Embodiment 34: ginsenoside Rg in the freeze-dried powder ₁, ginsenoside Rb ₁, the ginsenoside Re assay:

It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios are from 0 minute to 35 minutes, and the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is 30 ℃; Calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:

(2) the per unit amount limit that contains the ginsenoside Re must not be less than 0.8mg;

(4) the per unit amount contains the ginsenoside Rg ₁, ginsenoside Rb ₁, the ginsenoside Re the limit of summation must not be less than 3.4mg.

Embodiment 35: the assay of total saponins in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, puts in the 10ml measuring bottle, adds water to scale, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillin-glacial acetic acid solution 0.5ml, perchloric acid 2.0ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, immediately with ice-water bath cooling 2 minutes, fixed to scale with glacial acetic acid, shake up, as need testing solution; With the ginsenoside Rg ₁Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, measure trap, calculate with one point external standard method at the wavelength place of 547nm, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with the ginsenoside Rg ₁Meter must not be less than 20mg.

Embodiment 36: total polysaccharides assay in the freeze-dried powder:

It is an amount of that monosaccharide is got freeze-dried powder to be measured, puts in the iodine flask, and adding distil water makes dissolving, hydro-oxidation sodium test solution is to neutral, and accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, dropwise add sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, uses the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg, calculates the content of monosaccharide thus;

It is an amount of that total sugar is got freeze-dried powder to be measured, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 4 hours is put coldly, adds 1～2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg, calculates sugar contents with this;

Freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous glucose, must not be less than 100mg.

Embodiment 37: total lignans assay in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, and it is fixed to scale to add water, behind salt Acid precipitation saponin component, use ethyl acetate extraction and extracting solution also, water bath method, and the residue dissolve with methanol is as need testing solution; With the schisandrin is reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, wavelength place at 254nm measures trap, calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, the per unit amount contains total lignans in schisandrin, must not be less than 3mg.

Embodiment 38: the assay of schisandrin, deoxyschizandrin, schisandrin B in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-water is a mobile phase at 65%: 35%, the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:

(4) the per unit amount limit that contains the summation of schisandrin, deoxyschizandrin, schisandrin B must not be less than 0.55mg.

Embodiment 39: ophiopogonin B in the freeze-dried powder, ophiopogonin D, ophiopogonin D ' assay:

It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90%: 10%, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:

(4) the per unit amount contain ophiopogonin B, ophiopogonin D, ophiopogonin D ' the limit of summation must not be less than 0.22mg.

Embodiment 40: the assay of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, puts in the round-bottomed flask, is dissolved in water, and refluxes 4 hours with 3% sulphuric acid, takes out, put to room temperature, transfer pH, use chloroform extraction and extracting solution also to neutral, evaporate to dryness, the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water is a mobile phase at 90: 10, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:

Embodiment 41: ginsenoside Rg in the freeze-dried powder ₁, ginsenoside Rb ₁, the ginsenoside Re assay:

It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid chromatography, chromatographic column is a filler with eight alkyl silane bonded silica gels, acetonitrile-0.5% glacial acetic acid aqueous solution is a mobile phase, gradient elution, solvent ratios are from 0 minute to 30 minutes, and the ratio of acetonitrile is 20%, from 30 minutes to 50 minutes, the ratio of acetonitrile rises to 25% by 20%, and from 50 minutes to 70 minutes, the ratio of acetonitrile was 25%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 25%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; Calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:

(3) the per unit amount contains ginsenoside Rb ₁Limit must not be less than 1.0mg

Embodiment 42: the assay of total saponins in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, puts in the 10ml measuring bottle, adds methanol to scale, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillin-glacial acetic acid solution 0.5ml, perchloric acid 2.0ml shakes up, and heating is 30 minutes in 60 ℃ of water-baths, take out, immediately with ice-water bath cooling 2 minutes, fixed to scale with glacial acetic acid, shake up, as need testing solution; With ginsenoside Rb ₁Be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, measure trap, calculate with one point external standard method at the wavelength place of 547nm, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total saponins with ginsenoside Rb ₁Meter must not be less than 20mg.

Embodiment 43: total polysaccharides assay in the freeze-dried powder:

It is an amount of that total sugar is got freeze-dried powder to be measured, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 2 hours is put coldly, adds 1～2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg, calculates sugar contents with this;

Embodiment 44: total lignans assay in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, and it is fixed to scale to add water, behind salt Acid precipitation saponin component, use chloroform extraction and extracting solution also, water bath method, and the residue dissolve with methanol is as need testing solution; With the schisantherin B is reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, wavelength place at 254nm measures trap, calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, the per unit amount contains total lignans in schisantherin B, must not be less than 3mg.

Embodiment 45: the assay of schisandrin, deoxyschizandrin, schisandrin B in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with eight alkyl silane bonded silica gels, and acetonitrile-water is a mobile phase at 60%: 40%, the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:

Embodiment 46: ophiopogonin B in the freeze-dried powder, ophiopogonin D, ophiopogonin D ' assay:

It is an amount of to get freeze-dried powder to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with eight alkyl silane bonded silica gels, and acetonitrile-1% glacial acetic acid is a mobile phase at 90: 10, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:

Embodiment 47: the assay of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the freeze-dried powder:

It is an amount of to get freeze-dried powder to be measured, puts in the round-bottomed flask, and the back that is dissolved in water is with 15% hydrochloric acid reflux 4 hours, taking-up, put to room temperature, transfer pH to neutral, use dichloromethane extraction, extracting solution volatilizes, the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Alcoholic solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with eight alkyl silane bonded silica gels, and methanol-water is a mobile phase at 96: 4, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method or standard curve method, freeze-dried powder to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:

Embodiment 48: ginsenoside Rg in the injection ₁, ginsenoside Rb ₁, the ginsenoside Re assay:

It is an amount of to get injection to be measured, puts in the measuring bottle, adds ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg ₁, ginsenoside Rb ₁, ginsenoside Re's reference substance methanol solution be contrast, adopt liquid chromatography, chromatographic column is a filler with the dialkyl silane bonded silica gel, acetonitrile-0.2% phosphate aqueous solution is a mobile phase, gradient elution, solvent ratios are from 0 minute to 20 minutes, and the ratio of acetonitrile is 15%, from 20 minutes to 45 minutes, the ratio of acetonitrile rises to 22% by 15%, and from 45 minutes to 60 minutes, the ratio of acetonitrile was 22%, from 60 minutes to 80 minutes, the ratio of acetonitrile rose to 35% by 22%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; Calculate with one point external standard method, injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:

Embodiment 49: the assay of total saponins in the injection:

It is an amount of to get injection to be measured, puts in the 10ml measuring bottle, adds ethanol to scale, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method takes out immediately, and precision adds 10% vanillin-glacial acetic acid solution 0.3ml, perchloric acid 3.0ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, immediately with the ice-water bath cooling, fixed to scale with glacial acetic acid, shake up, as need testing solution; With ginsenoside Re is reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, wavelength place at 547nm measures trap, calculate with one point external standard method, injection to be measured is unit quantity to be equivalent to every day with output, the per unit amount contains total saponins in the ginsenoside Re, must not be less than 20mg.

Embodiment 50: total polysaccharides assay in the injection:

It is an amount of that monosaccharide is got injection to be measured, puts in the iodine flask, and hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, add dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear, and titration results is proofreaied and correct with blank assay, promptly to blue.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg), calculate the content of monosaccharide thus;

It is an amount of that total sugar is got injection to be measured, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 3 hours is put coldly, adds 1～2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, accurate iodine liquid (0.1mol/L) 25ml that adds shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly.The iodine liquid (0.1mol/L) of every 1ml is equivalent to the anhydrous glucose of 9.008mg, calculates sugar contents with this;

Injection to be measured is unit quantity to be equivalent to every day with output, and the per unit amount contains total polysaccharides in anhydrous glucose, must not be less than 100mg.

Embodiment 51: total lignans assay in the injection:

It is an amount of to get injection to be measured, puts in the measuring bottle, and it is fixed to scale to add water, behind salt Acid precipitation saponin component, use dichloromethane extraction and extracting solution also, water bath method, and the residue dissolve with methanol is as need testing solution; With the schisandrin is reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, wavelength place at 254nm measures trap, calculate with one point external standard method, injection to be measured is unit quantity to be equivalent to every day with output, the per unit amount contains total lignans in schisandrin, must not be less than 3mg.

Embodiment 52: the assay of schisandrin, deoxyschizandrin, schisandrin B in the injection:

It is an amount of to get injection to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Alcoholic solution with schisandrin, deoxyschizandrin, schisandrin B reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with the dialkyl silane bonded silica gel, and acetonitrile-0.5% glacial acetic acid aqueous solution is a mobile phase at 57%: 43%, the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:

(1) the per unit amount limit that contains schisandrin must not be less than 0.4mg

Embodiment 53: ophiopogonin B in the injection, ophiopogonin D, ophiopogonin D ' assay:

It is an amount of to get injection to be measured, puts in the measuring bottle, adds water to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Ethanol alcoholic solution with ophiopogonin B, ophiopogonin D, ophiopogonin D ' reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with the dialkyl silane bonded silica gel, and acetonitrile-0.1% phosphate aqueous solution 87:13 is a mobile phase, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:

Embodiment 54: the assay of Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin in the injection:

It is an amount of to get injection to be measured, puts in the round-bottomed flask, with 5% sulphuric acid hydrolysis 3 hours, takes out, and puts to room temperature, transfers pH to neutral, use ethyl acetate extraction, and extracting solution volatilizes, and the residue dissolve with ethanol with the microporous filter membrane filtration, is got subsequent filtrate as need testing solution; Alcoholic solution with Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin reference substance is contrast, adopt liquid chromatography, chromatographic column is a filler with the dialkyl silane bonded silica gel, and methanol-0.2% glacial acetic acid aqueous solution is a mobile phase at 95: 5, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate with one point external standard method, injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be following several:

Claims

1. the method for quality control of a pulse restoring injection, it is characterized in that: this method comprises following all or part of content:

(2) Radix Ginseng Rubra or ginseng crude drug, Radix Ophiopogonis medical material, schisandra chinensis medicinal material, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rf, ophiopogonin B, ophiopogonin D, ophiopogonin D ', the differential test method of all or part of composition in the Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, schisantherin B;

(3) ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ophiopogonin B, ophiopogonin D, ophiopogonin D ', the content test method of all or part of composition in the Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, schisandrin, schisantherin B, schisantherin A, deoxyschizandrin, schisandrin B, schisandrin C, total saponins, total lignans, total polysaccharides.

2. according to the method for quality control of the described pulse restoring injection of claim 1, it is characterized in that this method comprises one or both in the following finger printing:

(2) preparation of object of reference solution: get the main active reference substance in an amount of Radix Ginseng Rubra or Radix Ginseng and the Radix Ophiopogonis medical material, comprise ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rf, ophiopogonin B, ophiopogonin D, Ruscus aculeatus L. sapogenin, diosgenin, ruscogenin, ophiopogonin D ' in a kind of, water or methanol or dissolve with ethanol, be settled to suitable concn, as object of reference solution;

3. according to the method for quality control of the described pulse restoring injection of claim 2, it is characterized in that:

This method comprises one or both in the following finger printing:

(2) preparation of object of reference solution: it is an amount of that precision takes by weighing the ginsenoside Rg1, adds methanol and make the solution that every 1ml contains 0.3mg, as object of reference solution;

4. according to the method for quality control of the described pulse restoring injection of claim 1, it is characterized in that: the discrimination method of described injection comprises following all or part of content:

It is an amount of to get Shengmai injection to be measured, adds chloroform or ether or ethyl acetate or normal hexane or 10%～dehydrated alcohol or 10%～absolute methanol and extracts, and filters, and filtrate volatilizes, and residue adds chloroform or ethyl acetate dissolving, as need testing solution; Other gets one or more preparation contrast solutions in Fructus Schisandrae Chinensis control medicinal material, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, the schisantherin B; The preparation of Fructus Schisandrae Chinensis control medicinal material solution: it is an amount of to get the Fructus Schisandrae Chinensis control medicinal material, add chloroform or ether or ethyl acetate or normal hexane or 10%～dehydrated alcohol or 10%～absolute methanol and extract, filter, filtrate volatilizes, residue adds chloroform or ethyl acetate dissolving, medical material solution in contrast; The preparation of reference substance solution: get one or more reference substances in schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, the schisantherin B; Add chloroform or ethyl acetate respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned need testing solution and reference substance solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae, petroleum ether with 30～60 ℃ petroleum ether or 60～90 ℃: Ethyl formate or ethyl acetate: formic acid or acetic acid=2～40: 0.2～15: 0.1～5 upper solution or benzene or toluene: ethyl acetate or Ethyl formate=1～30: 0.2～10 or normal hexane: benzene or toluene: Ethyl formate or ethyl acetate: formic acid or acetic acid=0.2～5: 0.5～10: 1～15: 0.1～5 is developing solvent, launch, take out, dry, putting uviol lamp 365nm or 254nm inspects or sprays with 2%～20% chromotropic acid-concentrated sulfuric acid solution or 2%～20% phosphomolybdic acid ethanol solution or anisaldehyde sulfuric acid solution, dry by the fire the clear or smoked colour developing of iodine at 80 ℃～160 ℃ to the speckle colour developing, in the test sample chromatograph, with control medicinal material chromatograph or the corresponding position of reference substance chromatograph on, should show the speckle of same color;

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methanol or mobile phase or ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol or alcoholic solution with one or more reference substances in schisandrin, deoxyschizandrin, the schisandrin B are contrast, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile-water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% aqueous formic acid 5%～80%: 95%～20% are mobile phase, the detection wavelength is one or several in 190～410nm scope, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the main peak in the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;

It is an amount of to get Shengmai injection to be measured, adds chloroform-methanol mixed solution or n-butyl alcohol or chloroform or ethyl acetate or dichloromethane extraction, as need testing solution; Other gets control medicinal material Radix Ophiopogonis, adds chloroform-methanol mixed solution or ethyl acetate or n-butyl alcohol or chloroform or dichloromethane extraction, filters, and evaporate to dryness, residue add chloroform or dichloromethane dissolving, medical material solution in contrast; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae, with benzene or toluene or chloroform or dichloromethane: methanol or ethanol: glacial acetic acid or formic acid or water=8～300: 0.5～100: 0.01～30 or ethyl acetate or Ethyl formate: pyridine: water=0.2～10: 0.1～5: 0.2～10 is developing solvent, launch, take out, dry, put and inspect under uviol lamp 365nm or the 254nm or spray with 1%～50% sulphuric acid or vanillin reagent or 1%～25% vanillin reagent or anisaldehyde reagent, it is clear to dry by the fire to the speckle colour developing at 80 ℃～160 ℃, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, should show the fluorescence speckle of same color;

It is an amount of to get Shengmai injection to be measured, with behind the water dissolution with n-butyl alcohol or ether or ethyl acetate or chloroform or dichloromethane extraction, extract volatilizes, residue is with methanol or dissolve with ethanol, as need testing solution; Other get ophiopogonin B, ophiopogonin D, ophiopogonin D ' in one or more reference substances, add methanol or ethanol respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae, with ethyl acetate or chloroform or dichloromethane: methanol or ethanol: water=3～50: 1～15: 0.1～5 is developing solvent, launch, take out, dry, spray is with 5%～50% sulphuric acid ethanol reagent, it is clear to dry by the fire to the speckle colour developing at 80 ℃～160 ℃, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, should show the speckle of same color;

It is an amount of to get Shengmai injection to be measured, add to regulate pH behind sulphuric acid or the hydrochloric acid hydrolysis to neutral, and evaporate to dryness, residue is with chloroform or dichloromethane or ethyl acetate or methanol or dissolve with ethanol, as need testing solution; Other gets one or more reference substances in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin, adds chloroform or dichloromethane or ethyl acetate or methanol or ethanol respectively and makes the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae, with normal hexane or cyclohexane extraction or methanol or ethanol: ethyl acetate or chloroform or dichloromethane or Ethyl formate: water=0.2～15: 0.2～40: 0.1～5 is developing solvent, launch, take out, dry, spray is with 5%～50% sulphuric acid ethanol reagent, it is clear to dry by the fire to the speckle colour developing at 80 ℃～160 ℃, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, should show the speckle of same color;

One or more thin layer chromatography discrimination method among Radix Ginseng Rubra or Radix Ginseng, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, the ginsenoside Rf in h, the injection:

It is an amount of to get Shengmai injection to be measured, with n-butyl alcohol or ethanol or methanol or ethyl acetate or chloroform or dichloromethane extraction, filters, and filtrate is as need testing solution; Other gets among Radix Ginseng Rubra or Radix Ginseng control medicinal material, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, the ginsenoside Rf one or more, the preparation contrast solution; The preparation of Radix Ginseng Rubra or Radix Ginseng control medicinal material solution: get Radix Ginseng Rubra or the Radix Ginseng control medicinal material is an amount of, adding chloroform or methylene chloride reflux extracts, discard chloroform or dichloromethane solution, residue adds water-saturated n-butanol or ethyl acetate extraction, extracting solution adds ammonia solution, divides and gets upper strata, evaporate to dryness, residue is with methanol or dissolve with ethanol, medical material solution in contrast; The preparation of reference substance solution: get one or more reference substances among ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, the ginsenoside Rf, add methanol or ethanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 1～30 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate or silica gel H lamellae or silica GF254 lamellae, with chloroform or dichloromethane or normal hexane or cyclohexane extraction: ethyl acetate or Ethyl formate: methanol or ethanol or acetone: water=5～40: 10～100: 5～50: 0.2～30 at lower floor's solution or the n-butyl alcohol placed below 10 ℃: ethyl acetate or Ethyl formate: water=1～10: 0.2～2: 1～15 upper strata is developing solvent, launch, take out, dry, spray is with 5%～50% sulphuric acid ethanol reagent, it is clear to dry by the fire to the speckle colour developing at 80 ℃～160 ℃, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, should show the speckle of same color;

One or more liquid chromatograph discriminating among ginsenoside Rg1, ginsenoside Rb1, the ginsenoside Re in i, the injection:

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methanol or mobile phase or ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg1, the ginsenoside Rb1, the methanol of one or more reference substances or alcoholic solution are contrast among the ginsenoside Re, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile: water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% formic acid solution=5%～40%: 95%～60% is mobile phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; In the test sample chromatograph, answer tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

5. according to the method for quality control of the described pulse restoring injection of claim 4, it is characterized in that:

The discrimination method of described injection comprises following all or part of content:

It is an amount of to get Shengmai injection to be measured, adds chloroform extraction, filters, and filtrate volatilizes, and residue adds the chloroform dissolving, as need testing solution; Other gets in Fructus Schisandrae Chinensis control medicinal material, schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, the schisantherin B one or more, the preparation contrast solution; The preparation of Fructus Schisandrae Chinensis control medicinal material solution: it is an amount of to get the Fructus Schisandrae Chinensis control medicinal material, adds chloroform extraction, filters, and filtrate volatilizes, and residue adds chloroform dissolving, medical material solution in contrast; The preparation of reference substance solution: get one or more reference substances in schisandrin, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, schisantherin A, the schisantherin B, add chloroform respectively and make the solution that every 1ml contains 1mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 2 μ l of above-mentioned solution, put respectively on same silica GF254 lamellae, with 30～60 ℃ petroleum ether: Ethyl formate: the upper solution of formic acid=15: 5: 1 is developing solvent, launches, take out, dry, put under the uviol lamp 254nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color;

It is an amount of to get Shengmai injection to be measured, adds 7: 3 mixed solutions of chloroform-methanol and extracts, and filters, and filtrate volatilizes, and residue adds the chloroform dissolving, as need testing solution; Other gets control medicinal material Radix Ophiopogonis, shines medical material solution in pairs with legal system; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 10 μ l of above-mentioned solution, put respectively on same silica GF254 lamellae, with toluene: methanol: glacial acetic acid=80: 5: 0.1 is developing solvent, launch, take out, dry, put under the uviol lamp 254nm and inspect, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color, negative noiseless;

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With ophiopogonin B, ophiopogonin D, ophiopogonin D ' in the methanol solution of one or more reference substances be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile: water=90: 10 is mobile phase, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;

It is an amount of to get Shengmai injection to be measured, puts in the round-bottomed flask, and refluxing 4 hours with 3% sulphuric acid in the back that is dissolved in water, takes out, put to room temperature, transfer pH to neutral, use chloroform extraction, extracting solution volatilizes, the residue dissolve with methanol filters with microporous filter membrane, gets subsequent filtrate as need testing solution; By the methanol solution with one or more reference substances in Ruscus aculeatus L. sapogenin, diosgenin, the ruscogenin is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile: water=90: 10 is mobile phase, the detection wavelength is 203nm, 30 ℃ of column temperatures; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end;

It is an amount of to get Shengmai injection to be measured, uses n-butanol extraction, filters, and filtrate is as need testing solution; Other gets among Radix Ginseng Rubra or Radix Ginseng control medicinal material, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, the ginsenoside Rf one or more, preparation control medicinal material solution; The preparation of Radix Ginseng Rubra or Radix Ginseng control medicinal material solution: get Radix Ginseng Rubra or the Radix Ginseng control medicinal material is an amount of, add the chloroform reflux, extract,, discard chloroform liquid, residue adds water-saturated n-butanol and extracts, and extracting solution adds ammonia solution, divides and gets the upper strata, evaporate to dryness, residue dissolve with methanol, medical material solution in contrast; The preparation of reference substance solution: get one or more reference substances among ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re, the ginsenoside Rf, add methanol respectively and make the solution that every 1ml contains 2mg, in contrast product solution; Adopt the disclosed thin layer chromatography test of Chinese Pharmacopoeia appendix, draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with chloroform: ethyl acetate: methanol: water=15: 40: 22: 10 is developing solvent at lower floor's solution of placing below 10 ℃, launch, take out, dry, spray is with 10% sulphuric acid ethanol reagent, 105 ℃ are dried by the fire to speckle colour developing clearly, put respectively under daylight and the ultra-violet lamp 365nm and inspect, in the test sample chromatograph, with control medicinal material chromatograph and the corresponding position of reference substance chromatograph on, show the speckle of same color;

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with one or more reference substances among ginsenoside Rg1, ginsenoside Rb1, the ginsenoside Re is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 35 minutes, the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is at 30 ℃; In the test sample chromatograph, tool and the reference substance chromatograph consistent chromatographic peak of time of withing a hook at the end.

6. according to the method for quality control of the described pulse restoring injection of claim 1, it is characterized in that:

The method of testing of described injection content should comprise following all or part of content:

One or more assay among ginsenoside Rg1, ginsenoside Rb1, the ginsenoside Re in a, the injection:

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds water or methanol or mobile phase or ethanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With the ginsenoside Rg1, the ginsenoside Rb1, the methanol of one or more reference substances or alcoholic solution are contrast among the ginsenoside Re, adopt liquid chromatography, chromatographic column is a filler with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels or dialkyl silane bonded silica gel, methanol or acetonitrile: water or 0.05%～5% glacial acetic acid aqueous solution or 0.02%～5% phosphate aqueous solution or 0.05%～5% formic acid solution=5%～40%:95%～60% is a mobile phase, gradient elution, flow velocity is 0.5～2.0ml/min, one or several or the evaporation photodetector that detect wavelength and be in 190～410nm scope detect, and column temperature is in 20～60 ℃ of scopes; Calculate with external standard method or standard curve method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

(1) the unit quantity limit that contains the ginsenoside Rg1 must not be less than 0.8mg;

(2) the per unit amount limit that contains the ginsenoside Re must not be less than 0.4mg;

(3) the per unit amount limit that contains the ginsenoside Rb1 must not be less than 0.5mg

(4) the per unit amount limit that contains ginsenoside Rg1, ginsenoside Re's summation must not be less than 1.7mg;

The assay of total saponins in b, the injection:

It is an amount of to get Shengmai injection to be measured, put in the measuring bottle, adding distil water or methanol or ethanol make dissolving in right amount and decide and shakes up to scale, and precision is measured in right amount, put in the measuring bottle, water bath method takes out immediately, and precision adds 1%～50% vanillin-glacial acetic acid solution 0.1ml～10ml, perchloric acid 0.1ml～15ml, shake up, heated 3～50 minutes in 30 ℃～80 ℃ water-baths, take out, immediately with ice-water bath or flowing water cooling, precision adds glacial acetic acid to scale, shakes up, as need testing solution, with ginsenoside Rg1 or ginsenoside Rb1 or ginsenoside Re or ginsenoside Rf or ophiopogonin B or ophiopogonin D or ophiopogonin D ' be reference substance, get reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, wavelength place at 547 ± 10nm measures trap, calculate with external standard method or standard curve method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, the per unit amount contain total saponins in ginsenoside Rg1 or ginsenoside Rb1 or ginsenoside Re or ginsenoside Rf or ophiopogonin B or ophiopogonin D or ophiopogonin D ', must not be less than 10mg;

Total polysaccharides assay in c, the injection:

It is an amount of that monosaccharide is got Shengmai injection to be measured, puts in the iodine flask, and pH is regulated to neutral with sodium hydroxide test solution in adding distil water dissolving back, the accurate iodine liquid 25ml that adds 0.1mol/L shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, add dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear, and titration results is proofreaied and correct with blank assay, promptly to blue; The iodine liquid of every 1ml0.1mol/L is equivalent to the anhydrous glucose of 9.008mg, calculates the content of monosaccharide thus;

It is an amount of that total sugar is got Shengmai injection to be measured, put in the iodine flask, adding distil water adds dilute sulfuric acid 25ml after making dissolving, reflux 1～4 hour, put cold, add 1～2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, and precision adds 0.1mol/L iodine liquid 25ml, shake up, dropwise add sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, add dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear, and titration results is proofreaied and correct with blank assay, promptly to blue; The iodine liquid of every 1ml 0.1mol/L is equivalent to the anhydrous glucose of 9.008mg, calculates sugar contents with this;

Total lignans assay in d, the injection:

7. according to the method for quality control of the described pulse restoring injection of claim 6, it is characterized in that:

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; Methanol solution with one or more reference substances among ginsenoside Rg1, ginsenoside Rb1, the ginsenoside Re is contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, acetonitrile-water is a mobile phase, gradient elution, solvent ratios is from 0 minute to 35 minutes, the ratio of acetonitrile is 19%, from 35 minutes to 55 minutes, the ratio of acetonitrile rises to 29% by 19%, and from 55 minutes to 70 minutes, the ratio of acetonitrile was 29%, from 70 minutes to 100 minutes, the ratio of acetonitrile rose to 40% by 29%; Flow velocity is 1.0ml/min, detects wavelength 203nm, and column temperature is 30 ℃; Calculate with one point external standard method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

(1) the per unit amount limit that contains the ginsenoside Rg1 must not be less than 1.6mg;

(3) the per unit amount limit that contains the ginsenoside Rb1 must not be less than 1.0mg;

(4) the per unit amount limit that contains ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re's summation must not be less than 3.4mg;

The assay of total saponins in b, the injection:

It is an amount of to get Shengmai injection to be measured, puts in the 10ml measuring bottle, adds water to scale, shake up, precision is measured 0.6ml, to the 25ml measuring bottle, water bath method takes out immediately, and precision adds 5% vanillin-glacial acetic acid solution 0.5ml, perchloric acid 2.0ml shakes up, and heating is 15 minutes in 60 ℃ of water-baths, take out, immediately with ice-water bath cooling 2 minutes, fixed to scale with glacial acetic acid, shake up, as need testing solution; With ginsenoside Rg1 is reference substance, gets reference substance solution with legal system.With the retinue solvent is blank, adopt the disclosed spectrophotography of Chinese Pharmacopoeia appendix, wavelength place at 547nm measures trap, calculate with one point external standard method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, the per unit amount contains total saponins in the ginsenoside Rg1, must not be less than 20mg;

Total polysaccharides assay in c, the injection:

It is an amount of that monosaccharide is got Shengmai injection to be measured, puts in the iodine flask, and adding distil water makes dissolving, hydro-oxidation sodium test solution is to neutral, and the accurate iodine liquid 25ml that adds 0.1mol/L shakes up, dropwise add sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, uses the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly; Every 1ml 0.1mol/L iodine liquid be equivalent to the anhydrous glucose of 9.008mg, calculate the content of monosaccharide thus;

It is an amount of that total sugar is got Shengmai injection to be measured, puts in the iodine flask, and adding distil water makes dissolving, add dilute sulfuric acid 25ml, reflux 4 hours is put coldly, adds 1～2 of instructions phenolphthalein solution, hydro-oxidation sodium test solution is to neutral, the accurate iodine liquid 25ml that adds 0.1mol/L shakes up, and dropwise adds sodium hydroxide test solution 4ml, the violent jolting of limit edged, close plug, placed 10 minutes the dark place, adds dilute sulfuric acid 4ml, use the titration of 0.1mol/L sodium thiosulfate volumetric solution immediately, during to nearly terminal point, add starch indicator solution 2ml, continue titration and disappear to blue, and titration results proofreaied and correct with blank assay, promptly; The iodine liquid of every 1ml 0.1mol/L is equivalent to the anhydrous glucose of 9.008mg, calculates sugar contents with this;

Total lignans assay in d, the injection:

It is an amount of to get Shengmai injection to be measured, put in the measuring bottle, add methanol to scale, shake up, filter with microporous filter membrane, get continuous solution and be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and methanol-water 65%:35% is a mobile phase, the detection wavelength is 250nm, 30 ℃ of column temperatures; Calculate with one point external standard method, Shengmai injection to be measured is unit quantity to be equivalent to every day with output, and content limit should be with the next item down or several:

It is an amount of to get Shengmai injection to be measured, puts in the measuring bottle, adds methanol to scale, shakes up, and filters with microporous filter membrane, gets subsequent filtrate as need testing solution; With ophiopogonin B, ophiopogonin D, ophiopogonin D ' in the methanol solution of one or more reference substances be contrast, adopt liquid chromatography, the chromatographic column octadecylsilane chemically bonded silica is a filler, and acetonitrile-water 90%:10% is a mobile phase, the detection wavelength is 203nm, 30 ℃ of column temperatures; Calculate Shengmai injection to be measured with one point external standard method

8. according to the method for quality control of claim 6 or 7 described pulse restoring injections, it is characterized in that: all or part of total content of surveying composition of the saponins in the described injection, polysaccharide, lignanoids accounts for more than 25% of total solid of deducting adjuvant amount and water quantities in the preparation.