CN112843124A - Preparation method and quality standard of salvia miltiorrhiza formula granules - Google Patents

Preparation method and quality standard of salvia miltiorrhiza formula granules Download PDF

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CN112843124A
CN112843124A CN201911237724.7A CN201911237724A CN112843124A CN 112843124 A CN112843124 A CN 112843124A CN 201911237724 A CN201911237724 A CN 201911237724A CN 112843124 A CN112843124 A CN 112843124A
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solution
fingerprint
water
precisely
weighing
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上官同强
韩风雨
刘爽
李海茹
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Inner Mongolia Traditional Chinese Medicine Innovation Center Co ltd
Inner Mongolia Tianqi Zhongmeng Pharmaceutical Co ltd
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Inner Mongolia Traditional Chinese Medicine Innovation Center Co ltd
Inner Mongolia Tianqi Zhongmeng Pharmaceutical Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/537—Salvia (sage)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/047—Standards external

Abstract

The invention discloses a preparation method and a quality standard of a salvia miltiorrhiza formula particle, the formula takes salvia miltiorrhiza decoction pieces as raw materials, the salvia miltiorrhiza formula particle is prepared through the processes of water extraction, drying and the like, a thin layer identification method, a fingerprint spectrum and content indexes of the salvia miltiorrhiza formula particle are determined through tests, the quality standard of the salvia miltiorrhiza formula particle is formulated, and data support is provided for ensuring the quality of the salvia miltiorrhiza formula particle to achieve stability, controllability, high efficiency and safety.

Description

Preparation method and quality standard of salvia miltiorrhiza formula granules
Technical Field
The invention relates to a preparation method and a quality standard of a traditional Chinese medicine formula particle, in particular to a preparation method and a quality standard of a salvia miltiorrhiza formula particle.
Background
The Salvia miltiorrhiza is dry root and rhizome of Salvia miltiorrhiza (Salvia millirrhiza Bge) belonging to Salvia of Labiatae, is mainly produced in Shaanxi, Anhui, Shanxi, Hebei, inner Mongolia and other places, has the effects of activating blood circulation, removing blood stasis, relieving pain, cooling blood, eliminating carbuncle, clearing away heart fire, relieving restlessness and the like, is usually used for treating angina pectoris, coronary heart disease, thoracico-abdominal pain, amenorrhea, dysmenorrhea and other diseases, and has a long history of being taken as a medicine. The main chemical components of salvia can be divided into two types, one is a fat-soluble tanshinone compound, mainly comprising ketone II A, tanshinone I, cryptotanshinone and the like; the second is water soluble salvianolic acid component, mainly including salvianolic acid B, protocatechuic aldehyde, tanshinol, etc. Tanshinone IIA has anti-tumor, antioxidant, anticoagulant and antithrombotic effects, and is one of the main effective components for treating coronary heart disease; the salvianolic acid B and tanshinol have the activities of resisting myocardial ischemia, anoxia and the like, and have very wide clinical application.
The traditional Chinese medicine formula particle is a novel formula medicine with unified specification, unified dosage and unified standard, which is prepared by taking traditional Chinese medicines as raw materials through the processes of extraction, concentration, granulation and the like, and is a particle prepared from single traditional Chinese medicine decoction pieces and used for a traditional Chinese medicine clinical formula. Compared with the traditional Chinese medicine, the traditional Chinese medicine formula granular preparation has the advantages of convenience in taking, easiness in carrying, unified quality and specification, convenience in storage and storage, convenience and rapidness in clinical use combination, flexibility and changeability and the like. However, the quality standard of the traditional Chinese medicine formula granule is not perfect at present and has no unified national standard.
The invention aims to provide a preparation method and a quality standard of a salvia miltiorrhiza formula particle, and provides a certain data support for the industrial production of the salvia miltiorrhiza formula particle by determining the quality standard and a detection method thereof.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a preparation method and a quality standard of a salvia miltiorrhiza formula particle, and the invention aims to be realized by the following technical scheme:
the preparation method comprises decocting Saviae Miltiorrhizae radix decoction pieces 2000g in water twice, filtering, concentrating the filtrate under reduced pressure, drying, adding appropriate amount of adjuvant, mixing, granulating to 1000g, and packaging.
The quality standard is as follows:
1. the characteristics are as follows: the product is a brown yellow to yellowish brown granule; light smell, slightly bitter and astringent taste.
2. And (3) identification:
grinding the product, collecting powder 0.3g, adding 50% ethanol 5ml, ultrasonic treating for 30 min, centrifuging, and collecting supernatant as test solution. Adding water 25ml into Saviae Miltiorrhizae radix control 1g, heating and refluxing for 60 min, filtering, evaporating filtrate, and dissolving residue with 50% ethanol 1ml to obtain control solution. Adding 50% ethanol into salvianolic acid B control to obtain 1ml solution containing 1mg as control solution. Testing by thin layer chromatography (0502 of the four parts of the design reside in the Chinese pharmacopoeia 2015), sucking 5 μ l of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-toluene-ethyl acetate-methanol-formic acid (6: 4: 8: 1: 4) as developing agent, taking out, air drying, spraying with 5% vanillin sulfuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution and the reference solution.
3. Examination of
Meets the regulations of granules (0104 in the four ministerial general rules of 2015, China pharmacopoeia).
4. Extract of plant
The product is ground into fine powder, about 2g is weighed precisely, 100ml of ethanol is added precisely, and the content of the product is not less than 30.0% as measured by hot dipping method under alcohol-soluble extract measuring method (2201 in the fourth pharmacopoeia 2015).
5. Finger print
The measurement is carried out by high performance liquid chromatography (0512 in the four-department general regulation of the 2015 edition in China pharmacopoeia).
Chromatographic conditions and system applicability test with octadecylsilane chemically bonded silica as filler (column length 25cm, inner diameter 4.6mm, particle size 5 μm); acetonitrile is taken as a mobile phase A, 0.05 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate was 1.0ml per minute; the detection wavelength is 286 nm; the column temperature was 30 ℃. The number of theoretical plates should not be less than 6000 calculated according to the salvianolic acid B peak.
Figure RE-GSB0000186436410000021
Preparing fingerprint reference solution by collecting Saviae Miltiorrhizae radix reference medicinal material 0.5g, placing in conical flask with plug, adding water 20ml precisely, sealing, weighing, heating and refluxing for 1 hr, standing to room temperature, adding water to compensate the lost weight, shaking, filtering, and collecting the filtrate.
The preparation method of the test solution comprises grinding the above materials, collecting powder 1.0g, precisely weighing, placing in a conical flask with a plug, precisely adding water 20ml, sealing, weighing, ultrasonic treating (power 250W, frequency 45kHz) for 30 min, cooling, weighing again, supplementing the weight loss with water, shaking, and filtering.
The determination method comprises precisely sucking 10 μ l of the fingerprint reference solution and sample solution respectively, injecting into liquid chromatograph, and determining.
According to the similarity evaluation system of the chromatographic fingerprint of the traditional Chinese medicine, the similarity of the sample fingerprint and the reference fingerprint is calculated, and the similarity of chromatographic peaks after 5 minutes is not less than 0.90.
6. Determination of content
The measurement was carried out by high performance liquid chromatography (0512 in the four-department general regulation of the 2015 edition, China pharmacopoeia).
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-0.1% phosphoric acid solution (22: 78) is used as a mobile phase; the flow rate was 1.2ml per minute; the detection wavelength was 286 nm. The number of theoretical plates should not be less than 6000 calculated according to the salvianolic acid B peak.
Preparation of control solution an appropriate amount of salvianolic acid B control is precisely weighed, and mixed solution of methanol and water (8: 2) is added to make into solution containing 0.10mg per 1 ml.
Preparing a test solution, grinding the test solution, taking 0.5g of powder, precisely weighing, placing the powder in a conical flask with a plug, precisely adding 50ml of methanol-water (8: 2) mixed solution, sealing the plug, weighing, carrying out ultrasonic treatment (power of 150W and frequency of 45kHz) for 30 minutes, cooling, weighing again, complementing the loss weight by using the methanol-water (8: 2) mixed solution, shaking up, filtering, precisely weighing 5ml of subsequent filtrate, placing the subsequent filtrate in a 10ml measuring flask, adding the methanol-water (8: 2) mixed solution to scale, and shaking up to obtain the test solution.
The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.
The product contains salvianolic acid B (C) 1g per dried product36H30O16) Not less than 20.0 mg.
8. Property, flavor and meridian tropism
Bitter and slightly cold. It enters heart and liver meridians.
9. Specification of
1g of the formula particle is equivalent to 3.0g of decoction pieces.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention defines the preparation process of the salvia miltiorrhiza formula granules, establishes quality standards such as salvia miltiorrhiza thin-layer chromatography identification, content determination indexes and the like, and can effectively control the quality of the salvia miltiorrhiza formula granules.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
Test example 1
Thin layer identification research of salvia miltiorrhiza formula granules
Referring to the method under the item of salvia miltiorrhiza [ identification ] in the first edition of 'Chinese pharmacopoeia' 2015 edition and the salvia miltiorrhiza identification method in the report of literature data, because tanshinone is not easy to dissolve in water, the content of tanshinone in the salvia miltiorrhiza formula granule preparation is too low, the quality of the salvia miltiorrhiza formula granule cannot be effectively reflected by controlling the content of tanshinone, the water solubility of total salvianolic acids is good, and a thin-layer chromatography is adopted: three batches of samples are identified by taking a salvianolic acid B reference substance and a salvia miltiorrhiza reference medicinal material as references, the result of the thin-layer chromatography is clear, the method is feasible, and the text is to be included as the identification method of the product. The concrete description is as follows: :
1. reagent and medicinal material
The salvia miltiorrhiza contrast medicinal material: 120923-201615, purchased from China institute for testing foodstuff and drug;
salvianolic acid B control: the batch number is 111562-201716, purchased from China institute for testing and testing food and drug;
and (3) testing the sample: batch numbers 171800901, 171800902, 171800903
Thin-layer plate: silica gel G plate.
All reagents are analytically pure.
2. Preparation of solutions
(1) Preparation of a test solution: grinding the product, collecting powder 0.3g, adding 50% ethanol 5ml, ultrasonic treating for 30 min, centrifuging, and collecting supernatant as test solution.
(2) Preparation of reference drug solution: collecting Saviae Miltiorrhizae radix reference material 1g, adding water 25ml, heating and refluxing for 60 min, filtering, evaporating filtrate, and dissolving residue with 50% ethanol 1ml to obtain reference material solution.
(3) Preparation of control solutions: adding ethanol into salvianolic acid B control to obtain 1ml solution containing 1mg as control solution.
3. Chromatographic conditions
Thin-layer plate: silica gel G thin layer plate.
Sample amount of spotting: mu.l each.
Developing agent: chloroform-toluene-ethyl acetate-methanol-formic acid (6: 4: 8: 1: 4).
Color development and inspection: spraying 5% vanillin-sulfuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Inspecting with the eye.
As a result: spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution and the reference solution.
4. Optimization of authentication methods
4.1 investigation of control drug solution preparation protocol
Because the salvia miltiorrhiza formula particles are extracted by water, in order to ensure that the reference medicinal material corresponds to the extraction method of the test sample, the research compares the reference medicinal material by different extraction methods, and the specific steps are as follows:
the method comprises the following steps: collecting Saviae Miltiorrhizae radix reference material 1g, adding water 25ml, heating and refluxing for 60 min, filtering, evaporating filtrate to dryness, adding ethanol 5ml into residue, ultrasonic treating for 15 min, centrifuging, concentrating supernatant to 1ml, and filtering to obtain the final product.
The method 2 comprises the following steps: collecting Saviae Miltiorrhizae radix reference material 1g, adding water 25ml, heating and refluxing for 60 min, filtering, evaporating filtrate to dryness, and dissolving residue with 50% ethanol 1 ml.
The method 3 comprises the following steps: collecting Saviae Miltiorrhizae radix control 1g, adding ethanol 5ml, ultrasonic treating for 15 min, centrifuging, concentrating supernatant to 1ml, and filtering.
Sucking 5 μ l of each of the 3 reference medicinal materials and salvianolic acid B reference solution, respectively dropping on the same silica gel G thin layer plate, spreading with chloroform-toluene-ethyl acetate-methanol-formic acid (6: 4: 8: 1: 4) as developing agent, taking out, air drying, spraying 5% vanillin-sulfuric acid ethanol test solution, and heating at 105 deg.C until the spots are clearly developed. The result shows that the main spot of the chromatogram of the method 2 is clearest, and the spot with the same color is displayed on the position corresponding to the chromatogram of the reference substance. Selecting the extraction method as a reference medicinal material solution preparation method, and determining the identification method as text.
Test example 2
Fingerprint detection of salvia miltiorrhiza formula granules
Refer to the method under the item of total salvianolic acid extract [ finger print ] in the edition of "Chinese pharmacopoeia" 2015.
1. Apparatus and materials
A Shimadzu LC-20A high performance liquid chromatograph system, japan; agilent1260 Infinity II hplc system, usa; XS-205DU electronic balance (1/10 ten thousand, Metler, Switzerland); a double-frequency digital display constant temperature ultrasonic cleaner (KQ-500 GVDV); an electric heating constant temperature water bath (DK-98-II); a chromatographic column: Shimadzu-GL InertSustain C18, the particle size is 5 μm, the inner diameter is 4.6mm, and the length is 250 mm; thermo SCIENTIFIC C18 with a particle size of 5 μm, an inner diameter of 4.6mm and a length of 250 mm;
the salvia miltiorrhiza contrast medicinal material: 120923-201615, purchased from China institute for testing foodstuff and drug;
protocatechuic aldehyde control: the batch number is 110810-201608, purchased from China food and drug testing research institute;
rosmarinic acid control: the batch number is 111871-;
levorotatory alkannin reference substance: the batch number is 110769 and 200506, purchased from China institute for testing and testing food and drug;
salvianolic acid B control: the batch number is 111562-201716, purchased from China institute for testing and testing food and drug;
acetonitrile is chromatographically pure (Fisher Chemicals); the water is the Wahaha purified water; the rest reagents are analytically pure.
2. Chromatographic conditions and system applicability
Octadecylsilane chemically bonded silica was used as a filler (column length 25cm, inner diameter 4.6mm, particle diameter 5 μm); acetonitrile is taken as a mobile phase A, 0.05 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate was 1.0ml per minute; the detection wavelength is 286 nm; the column temperature was 30 ℃. The number of theoretical plates should not be less than 6000 calculated according to the salvianolic acid B peak.
Figure RE-GSB0000186436410000051
3. Preparation of the solution
(1) Preparation of fingerprint reference solution
The salvia miltiorrhiza formula particle is obtained by carrying out water extraction, concentration and drying on salvia miltiorrhiza decoction pieces, and a reference solution obtained by carrying out water extraction on salvia miltiorrhiza reference medicinal materials can reflect the components in the salvia miltiorrhiza formula particle most, and is representative. And the salvia miltiorrhiza reference medicinal material is easy to obtain, and is obtained by selecting a reference substance as a salvia miltiorrhiza fingerprint and refluxing with water.
(2) Preparation of test solution
In order to embody the contained components in the fingerprint spectrum as much as possible, water is selected as a solvent according to the property that the salvia miltiorrhiza formula particles are extracted by water and dissolved in the water, and ultrasonic extraction is carried out.
4. Selection of instrumentation
Experiments were conducted on a Shimadzu LC-20A high performance liquid chromatograph system in japan and an Agilent1260 high performance liquid chromatograph system in the united states, respectively, using the same column, with the same column temperature, mobile phase, and detection wavelength. The result shows that the two types of instruments can obtain better analysis effect, but the former has better peak shape and separation degree, and a Japanese Shimadzu LC-20A high performance liquid chromatograph system is selected.
5. Selection of chromatography columns
Shimadzu-GL InertSustain C18 with the particle size of 5 μm, the inner diameter of 4.6mm and the length of 250mm and Thermo SCIENTIFIC C18 with the particle size of 5 μm, the inner diameter of 4.6mm and the length of 250mm are respectively selected, and the same flow is used for separation and measurement relative to the same reference substance solution, so that Thermo SCIENTIFIC C18 with the particle size of 5 μm, the inner diameter of 4.6mm and the length of 250mm has the best separation effect on the chromatographic column.
6. Selection of detection wavelength
And selecting an ultraviolet detector to obtain an HPLC fingerprint. Collecting chromatogram-spectrum data by DAD, comparing chromatograms with different wavelengths, referring to wavelength under fingerprint spectrum of total salvianolic acid extract of first part of 2015 of Chinese pharmacopoeia, selecting wavelength of 286nm
7. Establishment of fingerprint
7.1. Establishment of common patterns
Taking 10 parts of radix Salviae Miltiorrhizae and reference materials from three production areas, and preparing into 1-10 parts of medicinal solution according to the preparation method of fingerprint reference solution. Injecting into a liquid chromatograph according to the chromatographic condition of the fingerprint spectrum. And recording the chromatogram. And (3) establishing a common mode by taking the 10 batches of samples, introducing the 10 batches of sample chromatograms into a research edition (2004A edition) of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, taking the reference medicinal material fingerprint as a reference chromatogram, selecting a time window width of 0.1min and a production method of the reference chromatogram as a median, and automatically matching and establishing the fingerprint common mode.
7.2 establishment of reference fingerprint
The similarity of samples of each batch is calculated by adopting a system evaluation software analysis and inspection mode of a national pharmacopoeia committee 'Chinese medicine chromatogram fingerprint similarity evaluation system 2004 version A' and taking a fingerprint common mode as a contrast, and the result is shown in a table 13. The similarity can represent the overall similarity of the components in the types and the relative amounts of the components among different production places. The analysis result shows that the similarity value of all the medicinal materials is more than 0.9. Wherein the fingerprint similarity of the reference medicinal material is 0.979. Considering the stability and the cheapness of the reference fingerprint, the reference medicinal material fingerprint is adopted as the reference fingerprint.
7.3. Selection and identification of common characteristic peaks
The peak emergence time in 10 batches of salvia miltiorrhiza medicinal material fingerprint spectra is basically consistent, 8 peaks with larger peak areas are common characteristic chromatographic peaks, and the selection principle of the characteristic peaks is as follows: the separation degree of the main characteristic peak and the adjacent peak reaches more than 1.5, other characteristic peaks also reach certain separation, the distance from the peak tip to the peak valley is at least more than two thirds of the peak height, and if the distance is not reached, the main characteristic peak and the adjacent peak can be combined into one peak for calculation. According to the fingerprint condition of Saviae Miltiorrhizae radix, the mixed solution of protocatechuic aldehyde, rosmarinic acid, alkannin and salvianolic acid B as reference is injected for analysis, and compared with corresponding chromatographic peak in the reference fingerprint to determine the peak position of protocatechuic aldehyde, rosmarinic acid, alkannin and salvianolic acid B in the reference fingerprint.
8. Method for verifying fingerprint standard
8.1. Precision survey
Taking the same fingerprint reference substance solution, continuously injecting samples for 6 times according to the fingerprint chromatogram conditions, introducing the chromatograms for 6 times into the system evaluation software 'Chinese medicine chromatogram fingerprint similarity evaluation system 2004A edition', calculating the similarity, wherein the results are all larger than 0.99, and the precision of the instrument is good.
8.2. Repeatability survey
Taking the same sample, preparing 6 sample solutions, injecting into a liquid chromatograph according to the fingerprint chromatogram conditions, respectively, introducing the 6 chromatograms into the evaluation software of the Chinese medicine chromatogram fingerprint similarity evaluation system 2004A edition, calculating the similarity, and obtaining the result of more than 0.98. The method is good in repeatability.
8.3. Stability survey
Taking a fingerprint reference solution, determining according to fingerprint chromatogram conditions for 0, 4, 8, 12 and 24 hours after preparation, respectively, introducing the chromatogram into system evaluation software of 'Chinese medicine chromatogram fingerprint similarity evaluation system 2004 version A', calculating the similarity, wherein the results are all larger than 0.99. Indicating that the solution stability is good.
8.4. Durability examination
Taking the same batch of samples and reference medicinal materials, respectively carrying out 6 times of separation and measurement on the samples by using an Agilent1260 Infinity II high performance liquid chromatograph and a SHIMADZU LC-20A high performance liquid chromatograph according to the selected chromatographic conditions, wherein the separation degree of the results can meet the requirements, and the fingerprint spectrums obtained by the two devices are respectively led into system evaluation software of a traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system 2004A edition, and the similarity between the fingerprint spectrums and the reference spectrum is calculated. The results were all greater than 0.99. Indicating that the solution stability is good. RSD% of 12 similarity is not more than 1%, and the chromatographic condition has wide durability.
9. Study of similarity limits
Taking 10 batches of the salvia miltiorrhiza formula granules, and preparing the test solution 1-10 according to the preparation method of the test solution. Taking Saviae Miltiorrhizae radix reference medicinal material, preparing fingerprint reference solution according to the preparation method of fingerprint reference solution, taking sample solution and fingerprint reference solution, injecting into liquid chromatograph, and recording chromatogram. And (3) calculating the similarity of the test sample fingerprint and the reference fingerprint according to the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprints (see table 1). The result shows that the similarity of the fingerprint of the salvia miltiorrhiza formula particle and the comparison fingerprint is more than 0.88. In order to ensure the quality of the salvia miltiorrhiza formula granules, the similarity of fingerprint spectrums of the salvia miltiorrhiza formula granules is not less than 0.9, and the salvia miltiorrhiza formula granules are listed in the text.
TABLE 1 fingerprint similarity of Salvia miltiorrhiza formula granules
Figure RE-GSB0000186436410000081
Test example 3
Content determination of red sage root formula granule
1. Selection of index component
The total salvianolic acid and tanshinone are main components of the salvia miltiorrhiza, as the tanshinone is not easy to dissolve in water, the content of the tanshinone in the salvia miltiorrhiza formula particle preparation is too low, the quality of the salvia miltiorrhiza formula particle cannot be effectively reflected by controlling the content of the tanshinone, the water solubility of the total salvianolic acid is better, the total salvianolic acid is drawn as an index for measuring the content of the salvia miltiorrhiza formula particle, and the content is measured by adopting a high performance liquid chromatography method and taking salvianolic acid B as an index, and the content is listed in the text.
2. Instrument and reagent
A Japanese Shimadzu LC-20A high performance liquid chromatograph system comprises an SIL-20AC automatic sample injector, a DGU-20A degassing unit, an LC-20AT solution conveying unit, a CTO-20A column incubator, an SPD-20A ultraviolet detector, a Labsolutions LC workbench Ver.5 Multi LC-PDA (Chinese edition) data Workstation, a CBM-20A networked system controller, an XS-205DU electronic balance (1/10 ten thousand, Switzerland Miller), an ultraviolet visible spectrophotometer (UV-2600), a dual-frequency digital display constant temperature ultrasonic cleaner (KQ-500GVDV), an electric heating constant temperature water bath (DK-98-II), an electronic balance (TP-A500, Fuzhou Huazhi scientific instruments Co., Ltd.), an MZ-204 type electronic balance (1/ten thousand, Switzerland Miller Co.), and an electric heating constant temperature drying oven (WGLL-125 BZ).
Salvianolic acid B control: the batch number is 111562-201716, purchased from China institute for testing and testing food and drug;
acetonitrile is chromatographically pure (Fisher Chemicals), water is Wahaha purified water, and the remaining reagents are analytically pure.
3. Selection of detection wavelength
Taking a proper amount of salvianolic acid B reference substance, adding a methanol-water (8: 2) mixed solution for dissolving, and performing spectrum scanning (190 nm-400 nm) by an ultraviolet visible spectrophotometer (UV-2600), wherein the salvianolic acid B reference substance has maximum absorption at the wavelength of 288nm as seen from a spectrogram. The 286nm is selected as the detection wavelength by combining literature data and Chinese pharmacopoeia medicinal material standards.
4. Chromatographic conditions
Referring to the content determination method of salvianolic acid B under the item of Salvia miltiorrhiza [ content determination ] in the first edition of Chinese pharmacopoeia 2015, the chromatographic conditions are as follows: acetonitrile-0.1% phosphoric acid solution (22: 78) as mobile phase, flow rate: 1.2ml/min, using a C18 column (4.6X 250nm, 5 μm) as an analytical column.
5. Preparation of control solutions
Weighing appropriate amount of salvianolic acid B as reference, and adding methanol-water (8: 2) mixed solution to obtain 0.10mg solution per 1 ml.
6. Preparation of test solution
Taking a proper amount of the product, grinding, taking 0.5g of powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of methanol-water (8: 2) mixed solution, sealing the plug, weighing, carrying out ultrasonic treatment (power of 150W and frequency of 45kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with the methanol-water (8: 2) mixed solution, shaking uniformly, filtering, precisely weighing 5ml of subsequent filtrate, placing in a 10ml measuring flask, adding the methanol-water (8: 2) mixed solution to scale, and shaking uniformly to obtain the product.
7. Preparation of negative control solution
Taking 0.3g of blank auxiliary materials, and preparing a negative control solution by the same method as the preparation method of the test solution.
8. System applicability
Precisely sucking 10 μ l of the reference solution, injecting into a liquid chromatograph under selected chromatographic conditions, measuring for 6 times, and recording chromatogram, the results are shown in Table 2.
TABLE 2 System suitability test results
Figure RE-GSB0000186436410000091
As can be seen from the table, the RSD value of the peak area of the control was 0.10%, the RSD value of the retention time was 0.42%, the number of theoretical plates was 10641 at the lowest, and the system applicability was good.
9. Specificity
Precisely absorbing 10 mul of each of the blank solvent, the reference solution, the sample solution and the negative reference solution, injecting the mixture into a liquid chromatograph according to the selected chromatographic conditions, and recording the chromatogram, wherein the result is shown in Table 3.
TABLE 3 results of the specificity test
Figure RE-GSB0000186436410000101
As can be seen from the table, the blank solution and the negative sample solution have no interference on the detection of the salvianolic acid B, the separation degree between the main peak and the adjacent peak in the chromatogram of the test solution is more than 1.5, and the specificity is good.
10. Repeatability test
The method comprises the steps of sampling, preparing samples according to the preparation methods of the test solution and the reference solution, preparing 6 parts of the test solution in parallel, injecting the sample into a liquid chromatograph according to selected chromatographic conditions, recording a chromatogram, calculating the content by an external standard method, and obtaining the result shown in table 4, wherein the average value of the content is 35.18mg/g, and the repeatability is good.
TABLE 4 results of the repeatability tests
Figure RE-GSB0000186436410000102
11. Accuracy test
Sampling, adding reference solution into salvianolic acid B at equal ratio, preparing sample solution containing salvianolic acid B50%, 100%, and 150% in total 9 parts, injecting into liquid chromatograph according to selected chromatographic conditions, recording chromatogram, and calculating recovery rate, wherein the results are shown in Table 5.
TABLE 5 accuracy test results
Figure RE-GSB0000186436410000103
Figure RE-GSB0000186436410000111
The test shows that: the recovery rate of each concentration is between 95% and 105%, and the relative standard deviation RSD (%) is not more than 2.0%.
12. Sample assay
The content of three batches of the red sage root formula particles is measured according to the text chromatographic conditions, and the result is shown in table 6.
TABLE 6 Salvianolic acid B content determination results of the Salvia miltiorrhiza Bunge granules
Figure RE-GSB0000186436410000112
According to the measurement result and the actual situation, the product is prepared according to the dry product, and each 1g of the product contains salvianolic acid B (C)36H30O16) Not less than 20.0 mg.
Experimental example 1
A preparation method of a Salvia miltiorrhiza formula granule comprises the following steps:
the preparation method comprises decocting Saviae Miltiorrhizae radix decoction pieces 2000g in water twice, filtering, concentrating the filtrate under reduced pressure, drying, adding appropriate amount of adjuvant, mixing, granulating to 1000g, and packaging.
The quality standard of the salvia miltiorrhiza formula granules is as follows:
the quality standard is as follows:
1. the characteristics are as follows: the product is a brown yellow to yellowish brown granule; light smell, slightly bitter and astringent taste.
2. And (3) identification:
grinding the product, collecting powder 0.3g, adding 50% ethanol 5ml, ultrasonic treating for 30 min, centrifuging, and collecting supernatant as test solution. Adding water 25ml into Saviae Miltiorrhizae radix control 1g, heating and refluxing for 60 min, filtering, evaporating filtrate, and dissolving residue with 50% ethanol 1ml to obtain control solution. Adding 50% ethanol into salvianolic acid B control to obtain 1ml solution containing 1mg as control solution. Testing by thin layer chromatography (0502 of the four parts of the design reside in the Chinese pharmacopoeia 2015), sucking 5 μ l of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-toluene-ethyl acetate-methanol-formic acid (6: 4: 8: 1: 4) as developing agent, taking out, air drying, spraying with 5% vanillin sulfuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution and the reference solution.
3. Examination of
Meets the regulations of granules (0104 in the four ministerial general rules of 2015, China pharmacopoeia).
4. Extract of plant
The product is ground into fine powder, about 2g of the powder is precisely weighed, 100ml of ethanol is precisely added, and the content is 31.6% by hot dipping method according to alcohol-soluble extract determination method (2201 in the four departments of the pharmacopoeia of China 2015).
5. Finger print
The measurement is carried out by high performance liquid chromatography (0512 in the four-department general regulation of the 2015 edition in China pharmacopoeia).
Chromatographic conditions and system applicability test with octadecylsilane chemically bonded silica as filler (column length 25cm, inner diameter 4.6mm, particle size 5 μm); acetonitrile is taken as a mobile phase A, 0.05 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate was 1.0ml per minute; the detection wavelength is 286 nm; the column temperature was 30 ℃. The number of theoretical plates should not be less than 6000 calculated according to the salvianolic acid B peak.
Figure RE-GSB0000186436410000121
Preparing fingerprint reference solution by collecting Saviae Miltiorrhizae radix reference medicinal material 0.5g, placing in conical flask with plug, adding water 20ml precisely, sealing, weighing, heating and refluxing for 1 hr, standing to room temperature, adding water to compensate the lost weight, shaking, filtering, and collecting the filtrate.
The preparation method of the test solution comprises grinding the above materials, collecting powder 1.0g, precisely weighing, placing in a conical flask with a plug, precisely adding water 20ml, sealing, weighing, ultrasonic treating (power 250W, frequency 45kHz) for 30 min, cooling, weighing again, supplementing the weight loss with water, shaking, and filtering.
The determination method comprises precisely sucking 10 μ l of the fingerprint reference solution and sample solution respectively, injecting into liquid chromatograph, and determining.
According to the similarity evaluation system of the chromatographic fingerprint of the traditional Chinese medicine, the similarity of the fingerprint of the test sample and the fingerprint of the reference fingerprint is calculated, and the similarity of the chromatographic peak after 5 minutes is 0.94.
6. Determination of content
The measurement was carried out by high performance liquid chromatography (0512 in the four-department general regulation of the 2015 edition, China pharmacopoeia).
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-0.1% phosphoric acid solution (22: 78) is used as a mobile phase; the flow rate was 1.2ml per minute; the detection wavelength was 286 nm. The number of theoretical plates should not be less than 6000 calculated according to the salvianolic acid B peak.
Preparation of control solution an appropriate amount of salvianolic acid B control is precisely weighed, and mixed solution of methanol and water (8: 2) is added to make into solution containing 0.10mg per 1 ml.
Preparing a test solution, grinding the test solution, taking 0.5g of powder, precisely weighing, placing the powder in a conical flask with a plug, precisely adding 50ml of methanol-water (8: 2) mixed solution, sealing the plug, weighing, carrying out ultrasonic treatment (power of 150W and frequency of 45kHz) for 30 minutes, cooling, weighing again, complementing the loss weight by using the methanol-water (8: 2) mixed solution, shaking up, filtering, precisely weighing 5ml of subsequent filtrate, placing the subsequent filtrate in a 10ml measuring flask, adding the methanol-water (8: 2) mixed solution to scale, and shaking up to obtain the test solution.
The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.
The product contains salvianolic acid B (C) 1g per dried product36H30O16) Not less than 22.6 mg.
8. Specification of
1g of the formula particle is equivalent to 3.0g of decoction pieces.

Claims (1)

1. A preparation method and quality standard of a salvia miltiorrhiza formula particle comprise the following steps: decocting Saviae Miltiorrhizae radix decoction pieces 2000g in water twice, filtering, concentrating the filtrate under reduced pressure, drying, adding appropriate amount of adjuvant, mixing, granulating to 1000g, and packaging to obtain granule, wherein each 1g granule is equivalent to decoction piece 3.0 g;
the quality standard of the formula particle comprises character indexes, thin-layer chromatography identification indexes, inspection, extract indexes, fingerprint spectrums and content measurement, wherein the indexes are as follows:
(1) the characteristics are as follows: the product is a brown yellow to yellowish brown granule; light smell, slightly bitter and astringent taste;
(2) and (3) identification:
grinding the product, collecting powder 0.3g, adding 50% ethanol 5ml, ultrasonic treating for 30 min, centrifuging, and collecting supernatant as sample solution; adding water 25ml into Saviae Miltiorrhizae radix control 1g, heating and refluxing for 60 min, filtering, evaporating filtrate to dryness, and dissolving residue with 50% ethanol 1ml to obtain control solution; adding 50% ethanol into salvianolic acid B reference to obtain 1ml solution containing 1mg as reference solution; performing thin layer chromatography (0502 of the four parts of the book of the Chinese pharmacopoeia 2015), sucking 5 μ l of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-toluene-ethyl acetate-methanol-formic acid (6: 4: 8: 1: 4) as developing agent, taking out, air drying, spraying 5% vanillin sulfuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution;
(3) examination of
Meets the related regulations under the item of the granules (0104 in the four ministry of communications in 2015 pharmacopoeia of China;
(4) extract of plant
Grinding the product, precisely weighing about 2g, adding ethanol 100ml, and measuring by hot dipping method under alcohol soluble extract measuring method (2201 in the four parts of the pharmacopoeia 2015) to obtain extract of no less than 30.0%;
(5) finger print
Measuring by high performance liquid chromatography (0512 in the four-department general regulation of 2015 pharmacopoeia);
chromatographic conditions and system applicability test with octadecylsilane chemically bonded silica as filler (column length 25cm, inner diameter 4.6mm, particle size 5 μm); acetonitrile is taken as a mobile phase A, 0.05 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate was 1.0ml per minute; the detection wavelength is 286 nm; the column temperature is 30 ℃, and the number of theoretical plates is not less than 6000 according to the peak of salvianolic acid B;
Figure FSA0000196709830000011
preparing fingerprint reference solution by adding Saviae Miltiorrhizae radix reference medicinal material 0.5g into conical flask with plug, adding water 20ml precisely, sealing, weighing, heating and refluxing for 1 hr, standing to room temperature, adding water to compensate the lost weight, shaking, filtering, and collecting the filtrate;
grinding the test solution, collecting powder 1.0g, precisely weighing, placing in a conical flask with a plug, precisely adding water 20ml, sealing, weighing, ultrasonically treating (power 250W, frequency 45kHz) for 30 min, cooling, weighing again, supplementing the weight loss with water, shaking, and filtering;
the determination method comprises precisely sucking 10 μ l of fingerprint reference solution and sample solution respectively, injecting into liquid chromatograph, and determining;
according to the similarity evaluation system of the chromatographic fingerprint of the traditional Chinese medicine, the similarity of the sample fingerprint and the reference fingerprint is calculated, and the similarity of chromatographic peaks after 5 minutes is not less than 0.90;
(6) determination of content
Measuring by high performance liquid chromatography (0512 in the four ministry of communications in 2015 pharmacopoeia of China);
octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-0.1% phosphoric acid solution (22: 78) is used as a mobile phase; the flow rate was 1.2ml per minute; the detection wavelength is 286nm, and the number of theoretical plates is not less than 6000 according to the peak calculation of salvianolic acid B.
Preparing reference solution by precisely weighing appropriate amount of salvianolic acid B reference, and adding methanol-water (8: 2) mixed solution to obtain 0.10mg solution per 1 ml;
preparing a test solution, grinding the test solution, taking 0.5g of powder, precisely weighing, placing the powder in a conical flask with a plug, precisely adding 50ml of methanol-water (8: 2) mixed solution, sealing the plug, weighing, carrying out ultrasonic treatment (power of 150W and frequency of 45kHz) for 30 minutes, cooling, weighing again, complementing the loss weight by using the methanol-water (8: 2) mixed solution, shaking up, filtering, precisely weighing 5ml of subsequent filtrate, placing the subsequent filtrate in a 10ml measuring flask, adding the methanol-water (8: 2) mixed solution to scale, and shaking up to obtain the test solution;
the determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, and determining;
the product contains salvianolic acid B (C) 1g per dried product36H30O16) Not less than 20.0 mg.
CN201911237724.7A 2019-11-27 2019-11-27 Preparation method and quality standard of salvia miltiorrhiza formula granules Pending CN112843124A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115969900A (en) * 2022-12-23 2023-04-18 贵州威利德制药有限公司 Preparation and detection method of salviae miltiorrhizae ointment medicine

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115969900A (en) * 2022-12-23 2023-04-18 贵州威利德制药有限公司 Preparation and detection method of salviae miltiorrhizae ointment medicine

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