Preparation method and application of a standard reference material of salvia miltiorrhiza formula granules

Abstract

The application provides a preparation method and application of a standard reference material of Danshen formula granules, and the method comprises the following steps: crushing Danshen medicinal materials into Danshen powder; mixing the Danshen powder to obtain Danshen powder for feeding; processing to obtain an extraction liquid, filtering and combining to obtain Danshen extraction liquid; obtaining Danshen permeate; loading the Danshen permeate onto a first preparation column, collecting an upflow eluate, and eluting to obtain a first eluate a; loading the upflow eluate and the first eluate onto a second preparation column, and eluting after elution to obtain an eluate b; combining the eluate a and the eluate b to obtain a preparation liquid of the standard reference material of Danshen formula granules; and obtaining the standard reference material of Danshen formula granules according to the preparation liquid of the standard reference material of Danshen formula granules. The standard reference material of Danshen formula granules can solve the problem of difficult positioning of characteristic peaks in a Danshen formula granule fingerprint, and can be applied to thin layer identification of Danshen formula granules and detection of the content of salvianolic acid B.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine quality control technology, and in particular to a method for preparing and applying a standard reference for Danshen formula granules. Background Technology

[0002] Traditional Chinese medicine (TCM) granules are granules made from single-herb TCM decoction pieces through water extraction, concentration, drying, granulation, and quantitative packaging. They offer advantages such as being easy to take without decoction, convenient to carry, and easy to dispense. As a promising area for the development of traditional Chinese medicine, the quality control of TCM granules has always been a key focus of the industry. Since the National Medical Products Administration, together with several other organizations, jointly issued the "Announcement on Ending the Pilot Program for TCM Granules," national drug standards for 265 varieties of granules have been released, further improving the quality of granules currently on the market. Currently, the quality control model for TCM granules mainly uses standard decoctions as a reference, with characteristic / fingerprint spectroscopy as the core, and uses reference standards or reference medicinal materials for quality control. In the actual application of national drug standards for TCM granules, problems still exist in areas such as thin-layer chromatography identification, characteristic / fingerprint spectroscopy, and content determination. These include the inability to accurately locate characteristic peaks in the characteristic / fingerprint spectroscopy detection item, which can easily lead to misinterpretations; the cumbersome and time-consuming extraction of reference medicinal materials in the standards; and the large variety, poor accessibility, and high price of reference materials, which have become key factors hindering the implementation of the standards.

[0003] The standard reference material for traditional Chinese medicine (TCM) formula granules is prepared based on the extraction of standard decoctions. It contains a variety of characteristic components and is mainly used as a national / provincial drug standard material for the detection of characteristics / fingerprint spectra, content determination, thin-layer chromatography identification, etc. of TCM formula granules. It is used to apply for the application of reference extracts, which focuses on solving common problems such as the difficulty in locating characteristic peaks, the high price of reference materials, and the poor applicability of reference medicinal materials. It significantly improves the quality control level and applicability of TCM formula granule standards, and can also be used for the quality evaluation of TCM formula granules.

[0004] Danshen is the dried root and rhizome of *Salvia miltiorrhiza* Bge., a plant in the Lamiaceae family. It possesses properties such as promoting blood circulation and removing blood stasis, regulating menstruation and relieving pain, clearing the mind and relieving irritability, and cooling the blood and reducing swelling. Danshen granules, as a supplement to danshen decoction pieces, have a wide range of clinical applications and possess high research value and broad application prospects.

[0005] In the application of the national standard for Danshen formula granules, it has been found that there are problems such as difficulty in locating characteristic peaks in the fingerprint spectrum, high price of reference standards, and poor applicability of reference medicinal materials, which seriously affect the implementation of the national standard and make it difficult to control product quality. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide a method for preparing and applying a standard reference for Danshen formula granules, so as to at least solve the shortcomings of the prior art.

[0007] In a first aspect, the present invention provides a method for preparing a standard reference material for Danshen (Salvia miltiorrhiza) formula granules, the method comprising:

[0008] Take the raw or processed Danshen medicinal material and crush it into coarse powder to obtain Danshen powder;

[0009] The danshen powder is mixed in a preset ratio to obtain the danshen powder for feeding.

[0010] The added Danshen powder is soaked in an extraction solvent and decocted, and then subjected to shaking, ultrasonication and / or multiple reflux extractions in sequence to obtain an extract. The extract obtained from each extraction is filtered to obtain a filtrate, and the filtrates are combined to obtain Danshen extract.

[0011] The pH value of the danshen extract was adjusted, and the danshen extract was clarified and filtered to obtain danshen permeate.

[0012] The first preparation column is acid-equilibrated, and the permeate of the danshen is loaded onto the first preparation column to collect the sample flow-through. The column is then rinsed with a first acidic aqueous solution to obtain a first eluent, and eluted with an ethanol aqueous solution to obtain eluent a.

[0013] The second preparation column is acid-equilibrated. The sample flow-through solution and the first eluent are combined and loaded onto the second preparation column. The column is then eluted with a second acid aqueous solution to obtain a second eluent. Finally, an ethanol aqueous solution is used to elute the column to obtain eluent b.

[0014] The eluent a and eluent b are combined to obtain the preparation solution of the standard reference material for Danshen formula granules;

[0015] The preparation solution of the standard reference material for Danshen formula granules was concentrated and dried sequentially to obtain Danshen formula granule extract;

[0016] Several extracts of the described Danshen formula granules were mixed to obtain a standard reference for Danshen formula granules.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: by adjusting the pH value of the danshen extract, and then clarifying and filtering to obtain the danshen permeate, and then preparing the eluent a through the first preparation column and the eluent b through the second preparation column, the target components can be accurately retained, which has strong specificity and can solve the problem of difficult localization of characteristic peaks under fingerprint spectrum. The eluent a and the eluent b are combined to obtain the danshen formula granule standard reference preparation solution, which is then concentrated and dried to obtain the danshen formula granule extract. The danshen formula granule extract is then mixed to ensure batch-to-batch consistency.

[0018] Furthermore, the particle size of the danshen powder is 10 mesh to 150 mesh;

[0019] The extraction solvent is water, and the weight of the extraction solvent is 5 to 15 times the weight of the danshen powder. The soaking time is 0.5 to 2 hours, the decoction temperature is 40°C to 100°C, the number of extractions is 1 to 5, and the extraction time is 0.5 to 3 hours.

[0020] Furthermore, the pH value of the danshen extract is adjusted by using one or at least two of hydrochloric acid, formic acid, acetic acid, hypochlorous acid, etc., the pH value of the danshen extract is 2 to 6, and the concentration of the danshen permeate is 0.1 mg / mL to 100 mg / mL.

[0021] The clarification and filtration of the danshen extract is performed using a filter membrane, which is an inorganic or organic membrane, and is one or more of the following: ceramic membrane, flat sheet membrane, spiral wound membrane, tubular membrane, or hollow fiber membrane. The pore size of the filter membrane is 50nm~0.1μm, 5kd~1000kd, or 0.1μm~75μm. The filter membrane has at least two layers, and the pore size of the two layers decreases sequentially from top to bottom.

[0022] Furthermore, the membrane flux of the filter membrane is 0.01 L / min to 10.0 L / min, the inlet pressure of the filter membrane is 0 MPa to 2.0 MPa, and the temperature of the filter membrane is 15℃ to 35℃.

[0023] Furthermore, after the step of clarifying and filtering the tanshinone extract, the method further includes:

[0024] The filter membrane is washed with a third acidic solution with a pH of 2 to 5;

[0025] The amount of the third acid water added during the washing and filtration of 100g to 10000g of the tanshinone extract is 1L to 150L.

[0026] Furthermore, the first and second preparation columns were equilibrated using 1 to 10 column volumes of an acidic aqueous solution with a pH of 2 to 6.

[0027] Both the first acid solution and the second acid solution are solutions with a pH of 2 to 6, which are 1 to 10 column volumes.

[0028] The permeate from the danshen was eluted with an aqueous ethanol solution with a volume concentration of 10% to 95% at a column volume of 1 to 10 times.

[0029] The sample loading capacity of both the first and second preparative columns is 50% to 80%. The first and second preparative columns are washed with 1 to 10 column volumes of a fourth acid-water equilibrium solution with a pH of 2 to 6. The washing volume is 1 to 10 column volumes, the washing flow rate is 10 to 10000 mL / min, and the elution is one or more of isocratic elution, linear gradient elution, or step gradient elution.

[0030] Furthermore, both the first acidic solution and the second acidic solution are one or more of hydrochloric acid solution, formic acid solution, and acetic acid solution, and the pH range of the first acidic solution and the second acidic solution is 2 to 6.

[0031] Furthermore, the concentration is one or more of atmospheric pressure evaporation, reduced pressure evaporation, thin film evaporation, and multi-effect evaporation, and the drying is one or more of reduced pressure drying, freeze drying, microwave drying, and spray drying.

[0032] Furthermore, the content of salvianolic acid B in the standard reference material of the danshen formula granules is ≥35%, and the content of characteristic components in the standard reference material of the danshen formula granules is ≥60%.

[0033] The chromatogram of the Danshen formula granule standard reference material obtained by thin-layer chromatography is located at the corresponding position of the chromatogram of Danshen reference medicinal material and the chromatogram of Danshen acid B reference standard, and shows spots of the same color.

[0034] The standard reference material for Danshen formula granules was obtained by high performance liquid chromatography (HPLC). Based on the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine, the similarity between the chromatographic fingerprint and the fingerprint of the national standard for Danshen formula granules was calculated using characteristic peaks. Except for peak B of salvianolic acid (No. 7), the similarity between the chromatographic fingerprint and the fingerprint of the national standard for Danshen formula granules was not less than 0.990, and the similarity between the chromatographic fingerprint and the first batch of standard reference materials for Danshen formula granules was not less than 0.990.

[0035] Secondly, the present invention also provides an application of a standard reference for Danshen formula granules. The standard reference for Danshen formula granules prepared according to the above-described preparation method is applied to the thin-layer identification, fingerprinting, and detection of salvianolic acid B content of Danshen formula granules. Attached Figure Description

[0036] Figure 1 This is a flowchart of the preparation method of the standard reference material for Danshen formula granules in the first embodiment of the present invention;

[0037] Figure 2 These are the liquid chromatograms of each sample in the first embodiment of the present invention;

[0038] Figure 3 TLC chromatogram of the standard reference material for Danshen formula granules;

[0039] Figure 4 The HPLC fingerprint overlay diagram obtained by high performance liquid chromatography for the three batches of reference granule standard materials in the first embodiment;

[0040] Figure 5 The thin-layer chromatograms were obtained by thin-layer chromatography using chemical reference standards, reference medicinal materials, and Danshen formula granules as reference materials.

[0041] Figure 6 This is an overlay of HPLC fingerprints obtained by high-performance liquid chromatography (HPLC) of commercially available formula granules using a standard reference material of Danshen (Salvia miltiorrhiza) formula granules as a reference.

[0042] The following detailed description, in conjunction with the accompanying drawings, will further illustrate the present invention. Detailed Implementation

[0043] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Several embodiments of the invention are illustrated in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0044] It should be noted that when a component is said to be "fixed to" another component, it can be directly on the other component or there may be an intervening component. When a component is said to be "connected to" another component, it can be directly connected to the other component or there may be an intervening component. The terms "vertical," "horizontal," "left," "right," and similar expressions used in this document are for illustrative purposes only.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0046] Example 1

[0047] Please see Figure 1 The diagram illustrates a method for preparing a standard reference material for Danshen (Salvia miltiorrhiza) formula granules according to the first embodiment of the present invention. The method includes steps S1 to S9:

[0048] S1, Take Danshen medicinal material or processed slices, and crush the Danshen medicinal material or processed slices into coarse powder to obtain Danshen powder;

[0049] In this embodiment, during specific implementation, three batches of Salvia miltiorrhiza slices were taken and crushed into powder. The particle size of the powder was 10 mesh to 150 mesh, resulting in three batches of Salvia miltiorrhiza powder. The information of the Salvia miltiorrhiza powder is shown in Table 1.

[0050] Table 1

[0051] 1 22011008-1 230509-1 15kg Shandong 2 22011008-2 230509-2 15kg Shanxi 3 22011008-3 230509-3 15kg Hebei

[0052] S2, the danshen powder is mixed in a preset ratio to obtain the danshen powder for feeding;

[0053] In practice, the danshen powder was mixed in the required proportions, as shown in Table 2. Three batches of danshen powder were obtained. The information of the danshen powder, which is the feed powder, is shown in Table 2.

[0054] Table 2

[0055]

[0056] S3, the added Danshen powder is soaked in the extraction solvent and decocted, and then subjected to shaking, ultrasonication and / or multiple reflux extractions in sequence to obtain an extract. The extract obtained from each extraction is filtered to obtain a filtrate, and the filtrates are combined to obtain Danshen extract.

[0057] It should be explained that, in this embodiment, the extraction solvent is water, and the weight of the extraction solvent is 12 times the weight of the danshen powder. In other optional embodiments, the weight of the water can be selected to be in the range of 5 to 15 times the weight of the danshen powder. The soaking time is 0.5 hours. In other optional embodiments, the soaking time can be selected to be in the range of 0.5 hours to 2 hours. The decoction temperature is 100°C. In other optional embodiments, the decoction temperature can be selected to be in the range of 40°C to 100°C. The number of extractions is 2. In other optional embodiments, the number of extractions can be selected to be in the range of 1 to 5. The extraction time is 1.5 hours. In other optional embodiments, the extraction time can be selected to be in the range of 0.5 hours to 3 hours.

[0058] In practice, 10 kg of danshen powder is put into an extraction tank, 120 L of pure water is added, soaked for 30 minutes, decocted for 30 minutes, filtered through a 200-mesh filter, and the filtrate is collected. The residue is added to 100 L of pure water, put into the extraction tank again, decocted for 30 minutes, filtered through a 200-mesh filter, and the filtrate is collected. The filtrates are combined to obtain danshen extract.

[0059] It is worth noting that after the above-mentioned specific implementation process, three batches of danshen powder were fed into the feed, resulting in three batches of danshen extract.

[0060] S4, adjust the pH value of the danshen extract and clarify and filter the danshen extract to obtain danshen permeate;

[0061] It should be explained that in this embodiment, the pH value of the tanshinone extract is adjusted by adding formic acid to the tanshinone extract and adjusting the pH value of the tanshinone extract to 3.1. Then, the extract with the adjusted pH value is passed through a 50nm ceramic membrane to obtain the tanshinone permeate.

[0062] It is worth noting that in other optional embodiments, the pH value of the tanshinone extract is adjusted by one or at least two of hydrochloric acid, formic acid, acetic acid, hypochlorous acid, etc., and the pH value of the tanshinone extract can be adjusted within the range of 2 to 6, and the concentration of the tanshinone permeate can be adjusted within the range of 0.1 mg / mL to 100 mg / mL.

[0063] Additionally, it should be explained that the filter membrane is either an inorganic or organic membrane, and is one or more of a ceramic membrane, flat sheet membrane, spiral wound membrane, tubular membrane, or hollow fiber membrane. The pore size of the filter membrane is 50 nm to 0.1 μm, 5 kd to 1000 kd, or 0.1 μm to 75 μm. The filter membrane has at least two layers, and the pore size of the two layers decreases sequentially from top to bottom. The membrane flux of the filter membrane is 0.01 L / min to 10.0 L / min, the inlet pressure of the filter membrane is 0 MPa to 2.0 MPa, and the temperature of the filter membrane is 15°C to 35°C.

[0064] After filtration, the filter membrane is washed with a third acidic solution with a pH of 2 to 5. The amount of the third acidic solution added for washing 100g to 10000g of Salvia miltiorrhiza extract is 1L to 150L.

[0065] S5, the first preparation column is acid-equilibrated, and the permeate of the danshen is loaded onto the first preparation column to collect the sample flow-through. The column is then rinsed with a first acidic aqueous solution to obtain a first elution solution, and eluted with an ethanol aqueous solution to obtain eluent a.

[0066] S6, the second preparation column is acid-equilibrated, the sample flow-through solution and the first eluent are combined and loaded onto the second preparation column, and then eluted with a second acid aqueous solution to obtain a second eluent, and then eluted with an ethanol aqueous solution to obtain eluent b;

[0067] It should be explained that the first and second preparative columns are equilibrated with 1 to 10 column volumes of an acidic aqueous solution with a pH of 2 to 6. The permeate of the tanshinone is eluted with 1 to 10 column volumes of a first acidic aqueous solution with a pH of 2 to 6. The permeate is eluted with 1 to 10 column volumes of an ethanolic aqueous solution with a volume concentration of 10% to 95%. The sample loading of the first preparative column is 50% to 80%. The chromatographic columns on the first and second preparative columns are washed with 1 to 10 column volumes of a fourth acidic aqueous equilibration solution with a pH of 2 to 6. The washing volume is 1 to 10 column volumes, the washing flow rate is 10 to 10000 mL / min, and the elution is one or more of isocratic elution, linear gradient elution, or step gradient elution.

[0068] It is worth noting that the first acid solution and the second acid solution are both made of one or more of hydrochloric acid solution, formic acid solution, and acetic acid solution, and the pH range of the first acid solution and the second acid solution is 2 to 6.

[0069] In practice, the first preparative column was packed with C18YE material with a particle size of 30 μm, an inner diameter of 300 mm, and a column length of 250 mm. The first preparative column was connected to a high-pressure liquid chromatography system with a designed flow rate of 4 L / min. After equilibration with 3 column volumes of formic acid aqueous solution at pH 3.1, the permeate from S4 was loaded onto preparative column 1, with a sample loading of approximately 65%. The permeate was collected, and the sample was first eluted with 3 column volumes of formic acid aqueous solution at pH 3.1. The eluent was collected, and then eluted with 3 column volumes of 70% ethanol aqueous solution. Collect eluent a; the second preparative column is packed with PS-NVP, with a particle size of 60μm, an inner diameter of 400mm, and a column length of 250mm. The second preparative column is connected to a high-pressure liquid chromatography system with a designed flow rate of 5L / min. After equilibration with 3 column volumes of acidic aqueous solution with pH 3.1, the sample flow-through liquid obtained in S5 is combined with the eluent and loaded onto preparative column 2, with a sample loading of approximately 60%. First, it is eluented with 3 column volumes of acidic aqueous solution with pH 3.1, and the eluent is collected. Then, it is eluted with 3 column volumes of 70% ethanol aqueous solution, and the eluent b is collected.

[0070] In other alternative embodiments, the filler type of the first preparation column and the second preparation column is one or two of macroporous resin and silica gel, wherein the macroporous resin includes one or at least two of PS NVP, WA30, etc.; and the silica gel includes one or at least two of C18YE, C18HCE, etc.

[0071] S7, combine the eluent a and eluent b to obtain the preparation solution of the standard reference material for Danshen formula granules;

[0072] In practice, eluent a and eluent b are combined to obtain the preparation solution of the standard reference material for Danshen formula granules.

[0073] S8, the preparation solution of the standard reference material of Danshen formula granules is concentrated and dried in sequence to obtain Danshen formula granule extract;

[0074] In practice, the preparation solution of the standard reference material for Danshen formula granules is subjected to rotary evaporation at 50°C to remove ethanol, followed by spray drying at an inlet air temperature of 170°C and an outlet air temperature of 120°C. The lyophilized powder is then collected, which is the extract of Danshen formula granules. Specific information about the extract of Danshen formula granules is shown in Table 3.

[0075] Table 3

[0076] 1 230703-01 0.988 5.66 2 230711-01 0.972 5.82 3 230717-01 0.978 5.73

[0077] It is worth noting that in other alternative embodiments, concentration is one or more of atmospheric pressure evaporation, reduced pressure evaporation, thin film evaporation, and multi-effect evaporation, and drying is one or more of reduced pressure drying, freeze drying, microwave drying, and spray drying.

[0078] S9, Mix several of the above-mentioned Danshen formula granule extracts to obtain Danshen formula granule standard reference material.

[0079] It should be explained that since there were 3 batches of Danshen powder, there were also 3 batches of Danshen formula granule extract. The 3 batches of Danshen formula granule extract were mixed to obtain 3 batches of Danshen formula granule standard reference materials. The specific information of the 3 batches of Danshen formula granule standard reference materials is shown in Table 4:

[0080] Table 4

[0081]

[0082] It is worth noting that the content of salvianolic acid B in the standard reference material for Danshen formula granules is ≥35%, and the content of characteristic components in the standard reference material for Danshen formula granules is ≥60%. The chromatogram for monitoring the entire process is shown below. Figure 2 As shown, Figure 2 The chromatograms, from top to bottom, are as follows: (A) chromatogram of the extract of *Salvia miltiorrhiza*, (B) chromatogram of the permeate of *Salvia miltiorrhiza*, (C) chromatogram of the preparation solution of the standard reference material of *Salvia miltiorrhiza* granules, (D) chromatogram of the extract of *Salvia miltiorrhiza* granules, and (E) chromatogram of the standard reference material of *Salvia miltiorrhiza* granules. Peak 1 is tanshinone, peak 2 is protocatechuic aldehyde, peak 3 is caffeic acid, peak 4 is salvianolic acid E, peak 5 is rosmarinic acid, peak 6 is shikonin, peak 7 is salvianolic acid B, and peak 8 is salvianolic acid L. The total titer values ​​were stable throughout the process, and all characteristic components were fully preserved.

[0083] In addition, in this embodiment, the mixing standard is as follows: the final Danshen formula granule standard reference material should have a Danshen acid B content ≥35% and a total characteristic component content ≥60%. The Danshen formula granule standard reference material is obtained by high performance liquid chromatography (HPLC). The similarity is calculated based on characteristic peaks using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system. Except for the Danshen acid B peak (peak number 7), the similarity with the national standard fingerprint of Danshen formula granules is not less than 0.990. Compared with the first batch of Danshen formula granule standard reference materials (in this embodiment, the first batch, i.e., batch number 1), the similarity is not less than 0.990. The content range of each component in this mixed standard is also the optimal data obtained through repeated experiments to maintain stability, consistency, and subsequent application.

[0084] It should be explained that the chromatogram of the Danshen formula granule standard reference material, detected by thin-layer chromatography, shows spots of the same color at corresponding positions as the chromatograms of the Danshen reference herb and the Danshen phenolic acid B reference standard. Through mass spectrometry analysis and reference standard localization, the Danshen formula granule standard reference material identified eight components, which are consistent with the eight characteristic peak components in the fingerprint spectrum of the national standard for Danshen formula granules. The Danshen formula granule standard reference material is a newly proposed concept, submitted as a national drug standard substance—a reference extract—for application in the national drug standard for Danshen formula granules, including thin-layer identification, fingerprinting, and content determination.

[0085] Example 2

[0086] The second embodiment of the present invention provides a method for identifying the standard reference material of Danshen formula granules. The Danshen formula granule standard reference material prepared in Example 1 is in the form of brown powder in all three batches.

[0087] The specific morphological information of the three batches of Danshen formula granules standard reference materials is shown in Table 5:

[0088] Table 5

[0089]

[0090]

[0091] In this embodiment, the standard reference material for Danshen formula granules was identified by thin-layer chromatography. Three batches of Danshen formula granules standard reference materials obtained in Example 1 were analyzed using thin-layer chromatography, and the detection results are as follows: Figure 3 As shown, Figure 3 In the chromatograms, 1-6 represent: 1 corresponding to the negative sample, 2 corresponding to the Danshen B reference standard, 3 corresponding to the Danshen reference material, 4 corresponding to the Danshen formula granule standard reference material with batch number 230720-01, 5 corresponding to the Danshen formula granule standard reference material with batch number 230720-02, and 6 corresponding to the Danshen formula granule standard reference material with batch number 230720-03. The results show that the chromatograms of the three batches of Danshen formula granule standard reference materials detected by thin-layer chromatography all show spots of the same color at the corresponding positions as the chromatograms of the Danshen reference material and the Danshen B reference standard.

[0092] In this embodiment, the fingerprint chromatograms of the Danshen formula granule standard reference materials were obtained by analyzing the three batches of Danshen formula granule standard reference materials obtained in Example 1 using mass spectrometry and reference standard localization. Eight components were identified in each batch, consistent with the eight characteristic peaks in the fingerprint chromatogram of the national standard for Danshen formula granules. The Danshen formula granule standard reference material with batch number 230720-01 was designated as the first batch of Danshen formula granule standard reference materials. The above three batches of Danshen formula granule standard reference materials were then analyzed using high-performance liquid chromatography (HPLC). The results showed that the chromatograms obtained by HPLC for the Danshen formula granule standard reference materials all exhibited eight characteristic peaks corresponding to the fingerprint chromatogram of the national standard for Danshen formula granules. According to the similarity evaluation system for chromatographic fingerprint chromatograms of traditional Chinese medicine, the similarity was calculated based on the characteristic peaks. Except for peak 7 (tanshinone B), the similarity results compared with the fingerprint chromatogram of the national standard for Danshen formula granules and with the first batch of Danshen formula granule standard reference materials are shown in Table 6. The overlay HPLC fingerprint chromatograms are shown in Table 6. Figure 4 As shown, Figure 4 From bottom to top, 1-3 correspond to the standard reference materials for Danshen formula granules with batch numbers 230720-01, 230720-02, and 230720-03, respectively. 4 corresponds to the fingerprint spectrum of the national standard for Danshen formula granules.

[0093] Table 6

[0094]

[0095] The chromatograms of the three batches of Danshen formula granules obtained in Example 1 were detected by high performance liquid chromatography. According to the similarity evaluation system of chromatographic fingerprint chromatograms of traditional Chinese medicine, the similarity was calculated based on the characteristic peaks. Except for peak B of salvianolic acid No. 7, the similarity with the fingerprint chromatogram of the national standard of Danshen formula granules was not less than 0.990. The similarity with the first batch of Danshen formula granules standard reference materials was not less than 0.990, which meets the requirements for mixing.

[0096] In this embodiment, the content of the standard reference material for Danshen formula granules was determined. The three batches of standard reference materials for Danshen formula granules obtained in Example 1 were determined by high performance liquid chromatography using reference standards. The content determination results are shown in Table 7, and the results meet the quality requirements.

[0097] Table 7

[0098]

[0099] In Example 1, the three batches of Danshen formula granule standard reference materials had a salvianolic acid B content ≥35% and a total characteristic component content ≥60%, meeting the mixing requirements. The RSD of salvianolic acid B content and the RSD of characteristic component content were both less than 2.00%. Therefore, the Danshen formula granule standard reference materials prepared using the preparation method of the present invention meet the mixing requirements and have good batch-to-batch consistency.

[0100] In this embodiment, the stability of the Danshen formula granule standard reference material was tested. Three batches of Danshen formula granule standard reference material prepared in Example 1 were taken, and the indicators included appearance and solubility were examined in accordance with the relevant provisions of the "Technical Requirements for the Development of National Drug Standard Materials" formulated by the National Pharmacopoeia Commission.

[0101] The test sample was placed in a suitable clean container with the opening open and placed at 60°C for 10 days. Samples were taken on the 5th and 10th days and tested according to the key stability test items.

[0102] The results showed that there were no significant changes in the properties of the test sample before and after the high-temperature test, and there were also no significant changes in the thin-layer chromatograms before and after the high-temperature test. The dissolution rate was determined by independent samples t-test using SPSS, and the calculated result was P>0.05, indicating that there was no significant difference.

[0103] Example 3

[0104] The third embodiment of the present invention provides an application of the standard reference material for Danshen formula granules prepared in Example 1. The standard reference material for Danshen formula granules is used for thin-layer identification, fingerprinting, and detection of Danshen phenolic acid B content of Danshen formula granules.

[0105] Commercially available Danshen formula granules were analyzed using thin-layer chromatography and high-performance liquid chromatography, with reference medicinal materials, chemical reference standards, and Danshen formula granule standard reference materials as reference substances.

[0106] Thin-layer chromatography detection begins with sample preparation:

[0107] Chemical reference solution: Take salvianolic acid B reference standard, add ethanol to prepare a solution containing 0.2 mg per 1 mL, and use it as the reference solution.

[0108] Reference herb solution: Take 0.5g of Salvia miltiorrhiza reference herb, add 20ml of water, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 10mL of methanol to the residue, sonicate for 30 minutes, shake well, and filter through a 0.22μm filter membrane to obtain the reference herb solution.

[0109] Danshen formula granule standard reference solution: Take about 30mg of Danshen formula granule standard reference, add 10mL of methanol, stopper tightly, shake well, and filter through a 0.22μm filter membrane to obtain Danshen formula granule standard reference solution.

[0110] Danshen formula granule test solution: Take 1g of Danshen formula granules, grind them finely, add 10ml of methanol, sonicate for 30 minutes, and filter through a 0.22μm filter membrane to obtain the Danshen formula granule test solution. It is worth noting that in this embodiment, 7 batches of Danshen formula granules were used and obtained through different channels, and then 7 batches of Danshen formula granule test solutions were prepared, namely Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, Sample 6 and Sample 7.

[0111] Thin-layer chromatography was performed, and the detection conditions were as follows:

[0112] Thin-layer plate: Silicone GF254 thin-layer plate

[0113] Spotting: 4 μL, in strip form;

[0114] Developing solvent: chloroform-toluene-ethyl acetate-methanol-formic acid, volume ratio 3:2:4:0.5:2;

[0115] Inspection: Examine under a 254nm ultraviolet light.

[0116] Test results as follows Figure 5 As shown, Figure 5 In the figure, 1-11 represent: 1 negative sample, 2 salvianolic acid B reference standard, 3 tanshinone reference medicinal material, 4 tanshinone formula granule standard reference material, and 5-11 correspond to 7 batches of tanshinone formula granule samples: sample 1, sample 2, sample 3, sample 4, sample 5, sample 6, and sample 7, respectively. The figure shows the thin-layer chromatogram under ultraviolet light at a wavelength of 254 nm. In the chromatogram, 1-11 represent: 1 negative sample, 2 salvianolic acid B reference standard, 3 tanshinone reference medicinal material, 4 tanshinone formula granule standard reference material, and 5-11 correspond to 7 batches of tanshinone formula granule samples: sample 1, sample 2, sample 3, sample 4, sample 5, sample 6, and sample 7, respectively.

[0117] Results Analysis: Figure 5 It was found that, using thin-layer chromatography under ultraviolet light (254 nm), negative samples showed no spots. Using the reference medicinal material and reference standard as controls, seven batches of commercially available Danshen formula granules showed spots of the same color at the corresponding positions as the reference standard and the reference medicinal material. Using the Danshen formula granule standard reference material as a control, all seven batches of commercially available formula granules showed spots of the same color at the corresponding positions as the Danshen formula granule standard reference material. The results were consistent with those obtained using the reference standard and reference medicinal material as controls. Therefore, using the Danshen formula granule standard reference material as a reference substance for thin-layer identification and detection of commercially available Danshen formula granules is feasible.

[0118] Fingerprint analysis begins with sample preparation:

[0119] Formula granule standard reference solution: Weigh approximately 10 mg of Danshen formula granule standard reference solution accurately, place it in a 50 ml volumetric flask, add 80% methanol aqueous solution to dilute to the mark, stopper tightly, shake well, sonicate for 30 minutes, and filter through a 0.22 μm filter membrane to obtain the formula granule standard reference solution.

[0120] Danshen Formula Granules Test Solution: Take an appropriate amount of Danshen formula granules, grind them finely, take about 0.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of 80% methanol aqueous solution, seal tightly, weigh, and sonicate (power 140W, frequency 42kHz) for 30 minutes. Remove, cool, and weigh again. Make up the lost weight with 80% methanol aqueous solution, shake well, filter through a 0.22μm filter membrane, accurately measure 5mL of the filtrate into a 10mL volumetric flask, dilute to the mark with 80% methanol aqueous solution, shake well, and the Danshen formula granules test solution is obtained. In this example, 7 batches of Danshen formula granules were used and obtained through different channels, and then 7 batches of Danshen formula granules test solutions were prepared, namely Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, Sample 6, and Sample 7.

[0121] High performance liquid chromatography (HPLC) detection, detection conditions are as follows:

[0122] The testing conditions are as follows:

[0123] Chromatography instrument: Waters Alliance e2695 high performance liquid chromatograph;

[0124] Detector: Waters PDA detector;

[0125] Column: Waters Xbridge C18 4.6mm × 250mm, 5μm;

[0126] Mobile phase: (A) Acetonitrile; (B) 0.05% phosphoric acid solution;

[0127] Gradient elution program: 0–15 min: 10 → 20% A; 15–40 min: 20 → 25% A; 40–50 min: 20 → 30% A.

[0128] Detection wavelength: 286 nm; flow rate: 1 mL / min; injection volume: 10 μl; column temperature: 30 ℃; run time: 50 min.

[0129] High-performance liquid chromatography fingerprint detection results:

[0130] Measure and record the chromatogram to obtain the result. Figure 6 The HPLC fingerprint overlay shown is as follows: Figure 6From bottom to top, 1-7 correspond to 7 batches of Danshen formula granule samples: Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, Sample 6, Sample 7. 8 corresponds to the standard reference material for Danshen formula granules, and 9 corresponds to the fingerprint spectrum of the national standard for Danshen formula granules. From bottom to top, 1-7 correspond to 7 batches of Danshen formula granules from different channels, namely Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, Sample 6, Sample 7. 8 corresponds to the standard reference material for Danshen formula granules, and 9 corresponds to the fingerprint spectrum of the national standard for Danshen formula granules.

[0131] According to the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity was calculated based on characteristic peaks. Except for peak B of tanshinone (No. 7), the similarity results compared with the standard reference material of Danshen formula granules and the fingerprint comparison of the national standard of Danshen formula granules are shown in Table 8:

[0132] Table 8

[0133]

[0134] Depend on Figure 6 As shown in Table 8, fingerprint analysis of the standard reference material of Danshen granules and seven batches of commercially available Danshen granules samples was performed using HPLC at a detection wavelength of 286 nm. The results showed that the eight characteristic peaks in the fingerprint spectrum of the standard reference material of Danshen granules could accurately identify the eight characteristic peaks in the fingerprint spectra of the seven batches of commercially available Danshen granules samples. Using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity was calculated based on the characteristic peaks. Except for peak B of salvianolic acid (No. 7), the similarity results were compared with those of the standard reference material of Danshen granules and the national standard reference fingerprint spectrum of Danshen granules. The similarity of both met the national drug standards, indicating that the judgment results of the standard reference material and the granule reference spectrum were consistent. Therefore, using the fingerprint spectrum of the standard reference material of Danshen granules to replace the national standard reference fingerprint spectrum of Danshen granules is feasible.

[0135] For content determination, the first step is sample preparation:

[0136] Chemical reference solution: Take an appropriate amount of salvianolic acid B reference standard, accurately weigh it, and add 80% methanol aqueous solution to prepare a solution containing 0.10 mg per ml.

[0137] Formula granule standard reference solution: Weigh approximately 10 mg of Danshen formula granule standard reference solution accurately, place it in a 50 ml volumetric flask, add 80% methanol aqueous solution to dilute to the mark, stopper tightly, shake well, sonicate for 30 minutes, and filter through a 0.22 μm filter membrane to obtain the solution.

[0138] Danshen Formula Granules Test Solution: Take an appropriate amount of Danshen formula granules, grind them finely, and accurately weigh approximately 0.2g. Place the weighed 0.2g in a stoppered conical flask, accurately add 50mL of 80% methanol aqueous solution, seal tightly, weigh, and sonicate (140W power, 42kHz frequency) for 30 minutes. Remove, cool, and weigh again. Replenish the lost weight with 80% methanol aqueous solution, shake well, filter through a 0.22μm filter membrane, accurately measure 5mL of the filtrate into a 10mL volumetric flask, dilute to the mark with 80% methanol aqueous solution, and shake well to obtain the Danshen formula granules test solution. Seven batches of Danshen formula granules samples came from different channels, namely Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, Sample 6, and Sample 7.

[0139] High performance liquid chromatography (HPLC) detection, detection conditions are as follows:

[0140] Chromatography instrument: Waters Alliance e2695 high performance liquid chromatograph;

[0141] Detector: Waters PDA detector;

[0142] Column: Waters Xbridge C18 4.6mm × 250mm, 5μm;

[0143] Mobile phase: (A) acetonitrile; (B) 0.1% phosphoric acid solution;

[0144] Gradient elution program: using acetonitrile-0.1% phosphoric acid solution (22:78) as the mobile phase, elute isocratically for 20 minutes.

[0145] Detection wavelength: 286 nm; flow rate: 1.2 mL / min; injection volume: 10 μl; column temperature: 30 ℃; run time: 20 min.

[0146] Results of content determination by high performance liquid chromatography:

[0147] The content was determined by high performance liquid chromatography using standard reference material and control substance of Danshen formula granules. The results are shown in Table 9.

[0148] Table 9

[0149]

[0150] As shown in Table 9, using HPLC at a detection wavelength of 286 nm, the content of salvianolic acid B in seven batches of commercially available Danshen formula granules was determined by using a standard reference and a control. The results showed that the content of salvianolic acid B in the Danshen formula granule samples measured by both methods met the national drug standard for Danshen formula granules, and the RSD of the content was less than 2.00%, indicating consistent results. Therefore, the use of the standard reference for Danshen formula granules for content determination in commercially available Danshen formula granules is feasible.

[0151] The standard for Danshen (Salvia miltiorrhiza) formula granules includes several testing items requiring reference substances, such as thin-layer chromatography (TLC), fingerprinting, and content determination. However, the reference substances for different control items are inconsistent. TLC uses Danshen reference material and the chemical reference standard salvianolic acid B as reference substances, but the method of using Danshen reference material is cumbersome. Fingerprinting lacks accompanying controls; if Danshen reference material is used, it cannot qualitatively identify characteristic components; if the chemical reference standard is used, multiple chemical reference standards are required, which are difficult to obtain and expensive. Content determination uses chemical reference standards, resulting in high costs. Using the standard reference material for Danshen formula granules as the reference material can simultaneously meet the requirements of multiple testing items for Danshen formula granules, identify all characteristic peaks, is easy to use, has a simple preparation process, low cost, and allows for qualitative and quantitative research on characteristic components. This forms a new overall quality control model based on the standard reference material for traditional Chinese medicine formula granules, achieving the goal of cost reduction and efficiency improvement, and has excellent application prospects.

[0152] The comparison between the standard reference material of the Danshen formula granules prepared in this invention and the chemical reference standard and reference medicinal material is shown in Table 10:

[0153] Table 10

[0154]

[0155] Note: High: +++; Medium: ++; Low: +

[0156] In summary, this invention presents a method for preparing and applying a standard reference for Danshen (Salvia miltiorrhiza) formula granules, which has the following beneficial effects:

[0157] 1) Compared with the difficulty in obtaining chemical reference standards and the high cost of preparation, the preparation process of the present invention is simple, stable, reproducible, and low in cost;

[0158] 2) Compared to the poor overall consistency of chemical reference standards, the quality of reference medicinal materials is affected by factors such as the place of origin and processing methods, resulting in poor batch-to-batch consistency. This invention uses self-developed materials and a multi-column combination method to accurately retain target components, which has strong specificity and integrity; the use of mixing technology ensures batch-to-batch consistency.

[0159] 3) Chemical reference standards can only be used for qualitative and quantitative analysis of single components. The use of reference medicinal materials is cumbersome and cannot be used for qualitative and quantitative analysis of special components. The standard reference material of Danshen formula granules prepared by this invention is easy to use and can be used for qualitative analysis of all components and quantitative analysis of multiple components. It can be applied to detection items such as identification, fingerprinting, and content determination.

[0160] 4) The preparation method of this invention is fast and efficient, easy to operate, and uses non-toxic and harmless reagents. It is low in cost and can be used for large-scale production, with good prospects for industrialization.

[0161] 5) The standard reference material for Danshen formula granules obtained by the preparation method in this invention is a new concept proposed for the first time. It is applied for as a national drug standard substance - control extract. It is used in the detection items such as thin-layer identification, fingerprint spectrum and content determination in the national drug standard for Danshen formula granules, forming a new overall quality control model based on the standard reference material for traditional Chinese medicine formula granules. It has the advantages of strong practicality, low preparation cost and simple operation.

[0162] Comparative Example

[0163] The difference from Example 1 is as follows:

[0164] In Example 1, a single-column preparation using WA30 packing material was used instead of two preparation columns (C18YE and PS-NVP). When the sample loading was greater than 20%, characteristic components were lost, and some characteristic components were not completely recovered. This shows that the efficiency of single-column preparation is significantly lower than that of dual-column preparation, and it is impossible to accurately retain characteristic components while maintaining high throughput efficiency.

[0165] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0166] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing a standard reference material for Danshen (Salvia miltiorrhiza) formula granules, characterized in that, The method includes: Take the raw or processed Danshen medicinal material and crush it into coarse powder to obtain Danshen powder; The danshen powder is mixed in a preset ratio to obtain the danshen powder for feeding. The danshen powder is soaked in an extraction solvent and decocted to obtain an extract. The extract obtained from each extraction is filtered to obtain a filtrate, and the filtrates are combined to obtain a danshen extract. The extraction solvent is water, and the weight of the extraction solvent is 5 to 15 times the weight of the danshen powder. The soaking time is 0.5 to 2 hours, the decoction temperature is 40℃ to 100℃, the number of extractions is 1 to 5, and the extraction time is 0.5 to 3 hours. The pH value of the danshen extract is adjusted and the danshen extract is clarified and filtered to obtain danshen permeate, wherein the pH value of the danshen extract is adjusted to 2~6. The first preparation column is acid-equilibrated, and the permeate of the danshen is loaded onto the first preparation column to collect the sample flow-through. The column is then rinsed with a first acid aqueous solution to obtain a first eluent. The eluent is then eluted with a 70% ethanol aqueous solution to obtain eluent a. The packing material of the first preparation column is C18YE. The second preparation column is acid-equilibrated. The sample flow-through solution and the first eluent are combined and loaded onto the second preparation column. The column is then eluted with a second acidic aqueous solution to obtain a second eluent. The eluent is then eluted with a 70% ethanol aqueous solution to obtain eluent b. The packing material of the second preparation column is PS-NVP. The first and second preparative columns were equilibrated using 1 to 10 column volumes of an acidic aqueous solution with a pH of 2 to 6. Both the first and second acidic solutions were 1 to 10 column volumes of a solution with a pH of 2 to 6. The sample loading capacity of both the first and second preparative columns was 50% to 80%. The elution volume was 1 to 10 column volumes, and the elution flow rate was 10 to 10000 mL / min. The eluent a and eluent b are combined to obtain the preparation solution of the standard reference material for Danshen formula granules; The preparation solution of the standard reference material for Danshen formula granules was concentrated and dried sequentially to obtain Danshen formula granule extract; Several extracts of the described Danshen formula granules were mixed to obtain a standard reference for Danshen formula granules.

2. The method for preparing the standard reference material of Danshen formula granules according to claim 1, characterized in that, The particle size of the danshen powder is 10 mesh to 150 mesh.

3. The method for preparing the standard reference material of Danshen formula granules according to claim 1, characterized in that, The pH value of the danshen extract is adjusted by using one or more of hydrochloric acid, formic acid, acetic acid, and hypochlorous acid, and the concentration of the danshen permeate is 0.1 mg / mL to 100 mg / mL. The clarification and filtration of the danshen extract is performed using a filter membrane, which is either an inorganic or organic membrane. The filter membrane has at least two layers, and the pore size of the two layers decreases sequentially from top to bottom.

4. The method for preparing the standard reference material of Danshen formula granules according to claim 3, characterized in that, The membrane flux of the filter membrane is 0.01 L / min to 10.0 L / min, the inlet pressure of the filter membrane is 0 MPa to 2.0 MPa, and the temperature of the filter membrane is 15℃ to 35℃.

5. The method for preparing the standard reference material of Danshen formula granules according to claim 3, characterized in that, After the step of clarifying and filtering the tanshinone extract, the method further includes: The filter membrane is washed with a third acidic solution with a pH of 2-5; The amount of the third acid water added during the washing and filtration of 100g to 10000g of the tanshinone extract is 1L to 150L.

6. The method for preparing the standard reference material of Danshen formula granules according to claim 1, characterized in that, Both the first acidic solution and the second acidic solution are one or more of hydrochloric acid solution, formic acid solution, and acetic acid solution, and the pH range of the first acidic solution and the second acidic solution is 2 to 6.

7. The method for preparing the standard reference material of Danshen formula granules according to claim 1, characterized in that, The concentration is achieved by atmospheric pressure evaporation or vacuum evaporation, and the drying is achieved by spray drying.

8. The method for preparing the standard reference material of Danshen formula granules according to claim 1, characterized in that, The Danshen formula granule standard reference material contains ≥35% salvianolic acid B, and the characteristic components in the Danshen formula granule standard reference material contain ≥60%; The chromatogram of the Danshen formula granule standard reference material obtained by thin-layer chromatography shows spots of the same color at corresponding positions as the chromatogram of Danshen reference medicinal material and the chromatogram of Danshen acid B reference standard. The standard reference material for Danshen formula granules was obtained by high performance liquid chromatography, and the similarity between the chromatographic fingerprint of the granules and the national standard reference fingerprint of Danshen formula granules was calculated based on the characteristic peaks using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system. Except for peak B of tanshinone (No. 7), the similarity between the chromatographic fingerprint of the granules and the national standard reference fingerprint of Danshen formula granules was not less than 0.

990.

9. The application of a standard reference material for Danshen (Salvia miltiorrhiza) formula granules, characterized in that, The standard reference material for Danshen formula granules prepared by the method according to any one of claims 1 to 8 is applied to the thin-layer identification and fingerprinting of Danshen formula granules.

Images