A detection method of a breviscapine formula granule

Abstract

This invention belongs to the field of drug detection technology, specifically relating to a detection method for *Erigeron breviscapus* (Dendrobium nobile) formula granules. These granules are made from *Erigeron breviscapus* medicinal slices through decoction, concentration, drying, and granulation. The detection method includes morphology, identification, inspection, extractives, characteristic chromatograms, and content determination. The identification method uses thin-layer chromatography; the characteristic chromatogram uses high-performance liquid chromatography, with the baicalin reference peak as the S peak, indicating 11 characteristic peaks; and the content determination measures the content of baicalin. The detection method established by this invention improves the detection requirements for *Erigeron breviscapus* medicinal materials, medicinal slices, standard decoctions, and formula granules, contributing to the establishment of a comprehensive quality control system for various *Erigeron breviscapus* products and helping to ensure the quality and clinical efficacy of *Erigeron breviscapus* formula granules.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a detection method for *Erigeron breviscapus* formula granules. Background Technology

[0002] Traditional Chinese medicine (TCM) granules are made from single-herb medicinal slices through one or more extraction and concentration processes, and are intended for use in TCM clinical prescriptions. They have the advantages of being stable and controllable, safe and efficient, requiring no decoction, easy to prepare, convenient to carry, and ready to take with a simple dissolving process.

[0003] *Erigeron breviscapus* (Vant.) Hand.-Mazz., also known as lampflower, lamp chrysanthemum, asarum grass, and in Guizhou, as a dried whole herb, is a plant belonging to the Asteraceae family. It is mainly produced in Guizhou, Guangxi, Yunnan, Sichuan, and Hunan provinces. Its properties are pungent, slightly bitter, and warm; it enters the heart and liver meridians. It has the effects of promoting blood circulation, relieving pain, dispelling wind and cold. It is used for stroke hemiplegia, chest pain, rheumatic pain, headache, toothache, etc.

[0004] Currently, the standards for *Erigeron breviscapus* medicinal materials only include the determination of baicalin content and its identification by liquid chromatography. With the increasing clinical use of formulated granules, there is an urgent need for more comprehensive, convenient, and controllable testing methods to control the quality of *Erigeron breviscapus* formulated granules. Summary of the Invention

[0005] This invention addresses the shortcomings of existing detection methods for *Erigeron breviscapus* formula granules by providing a quality detection method for *Erigeron breviscapus* formula granules, specifically:

[0006] The testing items for the *Dendrobium nobile* formula granules include properties, identification, inspection, extractives, characteristic chromatograms, and content determination.

[0007] In some embodiments, the Asarum sieboldii granules are light brown granules; they have a faint odor and a slightly bitter and astringent taste.

[0008] In some embodiments, the Asarum sieboldii granules are brownish-brown granules; they have a faint odor and a slightly bitter and astringent taste.

[0009] The identification method is as follows: Take 0.5g of *Erigeron breviscapus* granules, grind them finely, add 20ml of methanol, sonicate for 30 minutes, filter, recover the solvent from the filtrate to dryness, add 20ml of water to the residue, dissolve it with a little warmth, extract with ether 3 times, 20ml each time, discard the ether layer, adjust the pH of the aqueous layer to 2-3 with dilute hydrochloric acid, extract with ethyl acetate 3 times, 20ml each time, combine the ethyl acetate, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution; separately take 2.0g of *Erigeron breviscapus* reference material, add 60ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 20ml of methanol to the residue, and prepare the reference drug solution in the same way. Perform the thin-layer chromatography test (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Apply 5–10 μl of each of the above solutions as a strip, spotting them separately onto the same polyamide film. Develop with glacial acetic acid as the developing solvent. Remove the film, air-dry, spray with 5% ferric chloride ethanol solution, and examine under sunlight. In the chromatogram of the test sample, a main spot of the same color should appear at the corresponding position as in the chromatogram of the reference medicinal material.

[0010] The inspection method shall comply with all relevant provisions under the General Rules for Preparations of Chinese Pharmacopoeia, specifically for granules.

[0011] The method for determining the extractives is as follows: Take the granules of *Erigeron breviscapus* formula and determine them according to the hot extraction method under the "Determination of Alcohol-Soluble Extractives" section (General Chapter 2201 of the Chinese Pharmacopoeia 2020), using ethanol as the solvent.

[0012] The characteristic chromatogram detection method is as follows: high performance liquid chromatography (HPLC) according to the General Rules of the Chinese Pharmacopoeia, specifically including the following operations:

[0013] Chromatographic conditions and system suitability experiment: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A; and 0.1%-0.5% formic acid solution was used as mobile phase B for gradient elution. The flow rate was 0.2-1 ml per minute; the column temperature was 35-45℃; the detection wavelength was 300-350 nm; and the theoretical plate number, calculated based on the baicalin peak, should be no less than 2000-5000. The gradient elution conditions were as follows:

[0014] 0-5 min, mobile phase A 2%, mobile phase B 98%;

[0015] 5-8 min, mobile phase A 2→5%, mobile phase B 98→95%;

[0016] 8-24 min, mobile phase A 5→15%, mobile phase B 95→85%;

[0017] 24-55 min, mobile phase A 15→18%, mobile phase B 85→82%;

[0018] Preparation of the reference solution: Weigh 0.5-2g of *Erigeron breviscapus* reference material, add 20-50ml of water, heat under reflux for 30-60 minutes, filter, evaporate the filtrate to dryness, add 20-50ml of 25%-50% methanol to the residue, sonicate for 10-60 minutes, cool, filter, and take the filtrate as the reference solution for the reference material; Separately, take an appropriate amount of baicalin reference standard, add methanol to prepare a solution containing 0.2mg per ml, and use it as the reference solution for the reference standard.

[0019] Preparation of the test solution: Take an appropriate amount of Asarum sieboldii granules, grind them into a fine powder, weigh 0.1-1g, add 20-50ml of 25%-50% methanol, sonicate for 10-60min, cool, filter, and take the filtrate to obtain the test solution.

[0020] Determination method: Accurately pipette 2 μl of the reference solution and the test solution into the liquid chromatograph, and perform the determination according to the working conditions of high performance liquid chromatography. Record the chromatogram from 0 to 55 min.

[0021] The chromatogram of the test sample should show 11 characteristic peaks, and the retention times should correspond to the 11 characteristic peaks in the chromatogram of the reference medicinal material. Peak 5 should correspond to the retention time of the baicalin reference peak. Using the baicalin reference peak as peak S, calculate the relative retention times of each characteristic peak and peak S. The relative retention times should be within ±10% of the specified value. Specifically, the relative retention times for peak 1 are 0.43, peak 2 is 0.59, peak 3 is 0.63, and peak 4 is... The relative retention times of peaks are 0.68, 1.11, 1.16, 1.21, 1.24, 1.26, and 1.33 respectively; among them, peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 5(S) is baicalin, peak 6 is isochlorogenic acid B, peak 7 is 3,5-O-dicaffeoylquinic acid, and peak 11 is 4,5-di-O-caffeoylquinic acid.

[0022] Preferably, the mobile phase B in the characteristic spectrum detection is a 0.1% formic acid solution;

[0023] Preferably, the flow rate in the feature spectrum detection is 0.2 ml per minute;

[0024] Preferably, the column temperature in the feature spectrum detection is 40℃;

[0025] Preferably, the detection wavelength in the feature spectrum detection is 326 nm;

[0026] Preferably, the theoretical plate number in the characteristic spectrum detection, calculated based on the baicalin peak, should be no less than 3000;

[0027] Preferably, the chromatographic column used in the characteristic chromatogram detection is a Waters ACQUITY UPLC HSS T3, C18, 2.1 × 100 mm, 1.8 μm;

[0028] Preferably, the preparation of the reference solution in the characteristic spectrum detection is as follows: 0.5g of *Erigeron breviscapus* reference material is weighed, 50ml of water is added, the mixture is heated under reflux for 30 minutes, filtered, the filtrate is evaporated to dryness, the residue is added to 25ml of 50% methanol, sonicated for 30 minutes, cooled, filtered, and the filtrate is used as the reference solution for the reference material; separately, an appropriate amount of baicalin reference standard is accurately weighed, and methanol is added to prepare a solution containing 0.2mg per 1ml, which is used as the reference solution for the reference standard.

[0029] Preferably, the test solution for the characteristic spectrum detection is prepared by taking an appropriate amount of *Erigeron breviscapus* formula granules, grinding them finely, weighing 0.2g, adding 25ml of 50% methanol, sonicating for 30min, cooling, filtering, and taking the filtrate.

[0030] In some embodiments, the test solution is prepared by taking an appropriate amount of standard decoction of *Erigeron breviscapus*, weighing 0.2g, adding 25ml of 50% methanol, sonicating for 30min, cooling, filtering, and taking the filtrate.

[0031] In some embodiments, the test solution is prepared by taking an appropriate amount of Asarum sieboldii, crushing it, weighing 1.0g, adding 50ml of 50% methanol, sonicating for 30min, cooling, filtering, and taking the filtrate.

[0032] In some embodiments, the test solution is prepared by taking an appropriate amount of Asarum sieboldii slices, crushing them, weighing 0.5g, adding 50ml of 50% methanol, sonicating for 30min, cooling, filtering, and taking the filtrate.

[0033] The content determination method is as follows: high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0034] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol-0.1% phosphoric acid solution-tetrahydrofuran (13:74:13) as the mobile phase; and a detection wavelength of 335 nm. The theoretical plate number, calculated based on the baicalin peak, should be no less than 5000.

[0035] Preparation of the reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.2 mg per ml.

[0036] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 80% methanol, weigh it, sonicate it (power 500W, frequency 53KHz) for 30 minutes, cool it, weigh it again, make up the weight loss with 80% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0037] The assay involves precisely pipetting 5 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0038] The formula granules for *Dendrobium nobile* are made by taking 4000g of *Dendrobium nobile* slices, adding water to decoct, filtering, concentrating the filtrate, drying, pulverizing, adding appropriate excipients, mixing, granulating, and making 1000g.

[0039] In some embodiments, the granules of *Erigeron breviscapus* are prepared by taking 4000g of *Erigeron breviscapus* slices, adding water and decocting, filtering, concentrating the filtrate, vacuum drying, pulverizing, adding an appropriate amount of maltodextrin, mixing, granulating, and making 1000g.

[0040] In some embodiments, the granules of *Erigeron breviscapus* are prepared by taking 4000g of *Erigeron breviscapus* slices, adding water to decoct, filtering, concentrating the filtrate, vacuum drying, pulverizing, adding an appropriate amount of starch, mixing, granulating, and making 1000g.

[0041] Some embodiments involve preparing a standard decoction of *Erigeron breviscapus*, specifically by taking 4000g of *Erigeron breviscapus* slices, adding water and decocting, filtering, concentrating the filtrate under reduced pressure to obtain a clear extract, dispensing it into vials, and freeze-drying it (-40℃) until it is dry.

[0042] Beneficial effects:

[0043] 1. This invention is a detection method for the formulation granules of *Erigeron breviscapus*. This detection method has comprehensive detection items, is easy to operate, has high precision, strong specificity, and good reproducibility.

[0044] 2. Furthermore, this invention adds the detection of characteristic spectra to the conventional detection items, which can more comprehensively reflect the types and quantities of medicinal chemical components contained in the product. In particular, the characteristic spectra are detected by UPLC, which has the characteristics of short detection time, high precision, and good reproducibility. At the same time, the characteristic spectra also clearly identify a total of 11 characteristic peaks, of which peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 5(S) is baicalin, peak 6 is isochlorogenic acid B, peak 7 is 3,5-O-dicaffeoylquinic acid, and peak 11 is 4,5-di-O-caffeoylquinic acid, which makes the control of product quality more targeted.

[0045] 3. Through testing, the detection method of this invention is also applicable to medicinal materials, processed slices, and standard decoctions of *Erigeron breviscapus*, and has a wide range of applications. Attached Figure Description

[0046] Figure 1 Thin-layer chromatography for the identification of granules of *Erigeron breviscapus* formula

[0047] Figure 2 The characteristic chromatogram of the standard decoction of *Erigeron breviscapus* is shown below. Peak 1: neochlorogenic acid; Peak 2: chlorogenic acid; Peak 3: cryptochlorogenic acid; Peak 5(S): baicalin; Peak 6: isochlorogenic acid B; Peak 7: 3,5-O-dicaffeoylquinic acid; Peak 11: 4,5-di-O-caffeoylquinic acid.

[0048] Figure 3 The characteristic chromatogram of the *Erigeron breviscapus* formula granules is shown below. Peak 1: neochlorogenic acid; Peak 2: chlorogenic acid; Peak 3: cryptochlorogenic acid; Peak 5(S): baicalin; Peak 6: isochlorogenic acid B; Peak 7: 3,5-O-dicaffeoylquinic acid; Peak 11: 4,5-di-O-caffeoylquinic acid.

[0049] Figure 4 Characteristic spectrum and specificity spectrum of granules for the formula of *Erigeron breviscapus*

[0050] Figure 5 Determination of Baicalin Content in *Erigeron breviscapus* Formula Granules - Specificity Spectroscopy

[0051] Figure 6 Chromatograms and corresponding spectra of the reference standard and test sample in Comparative Example 1

[0052] Figure 7 Example 5: Chromatograms and corresponding spectra of the reference standard and test sample Detailed Implementation

[0053] To make the technical solutions and effects of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. The embodiments described below are some embodiments of the present invention, but not all embodiments. In conjunction with the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] Example 1: Establishment of a thin-layer chromatography method for identifying granules of *Erigeron breviscapus* formula.

[0055] This invention uses *Erigeron breviscapus* as a reference herb as a positive control, and the excipients used in the *Erigeron breviscapus* formula granules and methanol as negative controls, and uses glacial acetic acid as a developing solvent to investigate the thin-layer chromatography identification method.

[0056] 1.1 Solution Preparation

[0057] (1) Preparation of the test solution

[0058] Take 0.5g of the granulated formula of *Erigeron breviscapus*, grind it into a fine powder, add 20ml of methanol, sonicate for 30 minutes, filter, recover the solvent from the filtrate until dry, add 20ml of water to the residue, dissolve it with a little warmth, extract with ether 3 times, 20ml each time, discard the ether layer, adjust the pH of the aqueous layer to 2-3 with dilute hydrochloric acid, extract with ethyl acetate 3 times, 20ml each time, combine the ethyl acetate, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution.

[0059] (2) Preparation of control herbal solution

[0060] Take 2.0g of Asarum sieboldii (a type of medicinal herb), add 60ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, and prepare the reference herb solution from the residue starting from "add 20ml of methanol".

[0061] (3) Preparation of blank negative solution

[0062] A. Preparation of blank excipient solution: Take 0.5g of maltodextrin, the excipient used in the formula granules of *Erigeron breviscapus*, and prepare a negative excipient solution using the same method as the test solution.

[0063] B. Preparation of blank solvent solution: Methanol was used as the negative solvent solution.

[0064] 1.2 Chromatographic conditions

[0065] Carrier: Polyamide film

[0066] Developing solvent: glacial acetic acid

[0067] Inspection: Spray with 5% ferric chloride ethanol solution and inspect under sunlight.

[0068] 1.3 Methodological Examination

[0069] 1.3.1 Specificity: Prepare solutions of *Erigeron breviscapus* granules, negative-labeled solvent, negative-labeled excipient, and *Erigeron breviscapus* reference material according to the above solution preparation method. Apply 10 μl of each solution to the same polyamide film, develop under the above thin-layer chromatographic conditions, remove, air dry, spray with 5% ferric chloride ethanol solution, and examine under sunlight. The results show that the test sample chromatogram shows a main spot of the same color at the corresponding position as the reference material chromatogram. Negative-labeled solvent and negative-labeled excipient showed no interference. See [link to relevant documentation]. Figure 1 .

[0070] 1.3.2 Spotting volume: Take 2 μl, 3 μl, 5 μl, 15 μl and 20 μl of the test solution respectively, spot them on the same polyamide film, develop according to the above thin-layer chromatography conditions, remove, air dry, spray with 5% ferric chloride ethanol solution, and examine under sunlight. Based on the separation and clarity of the spots, determine the spotting volume of the test solution to be 5 to 10 μl.

[0071] 1.3.3 Durability:

[0072] 1.3.3.1 Testing under different temperatures and humidity: 10 μl each of the anion exchange solvent, anion exchange excipient, reference drug, and test sample solution were spotted onto the same polyamide film. Thin-layer chromatography was performed under the conditions described above: low temperature (18℃), room temperature (25℃), high temperature (40℃), low humidity (18%), normal humidity (49%), and high humidity (72%). The films were then removed, air-dried, sprayed with 5% ferric chloride ethanol solution, and examined under sunlight. Results: In the chromatogram of the test sample, a main spot of the same color appeared at the corresponding position as in the chromatogram of the reference drug. Negative-solvent and anion exchange excipient showed no interference. The film exhibited good durability under different temperatures and humidity.

[0073] 1.3.3.2 Examination of Thin-Layer Plates from Different Manufacturers: 10 μl each of the negative-labeled solvent, negative-labeled excipient, reference drug, and test sample solution were spotted onto polyamide films from different manufacturers (Zhejiang Sijia, Qingdao Bangkai, and Shandong Xiya). The films were developed under the aforementioned thin-layer chromatographic conditions, removed, dried, sprayed with 5% ferric chloride ethanol solution, and examined under sunlight. Results showed that using polyamide films from different manufacturers had no significant impact on thin-layer identification, indicating that this thin-layer identification method is robust.

[0074] 1.3.3.3 Durability Study by Different Personnel: Different testing personnel prepared test samples, negative samples, and control drug solutions using the same method at different times. 10 μl of each of the negative-solvent, negative-excipient, control drug, and test sample solutions were spotted onto the same polyamide film. The films were developed under the aforementioned thin-layer chromatography conditions, removed, dried, sprayed with 5% ferric chloride ethanol solution, and examined under sunlight. Results showed that the use of the same testing method by different personnel had no significant impact on the thin-layer identification, indicating that the thin-layer identification method has good durability.

[0075] The thin-layer chromatography identification method of this invention has been examined methodologically and has good specificity and robustness, and can be used for the thin-layer chromatography identification of granules of *Erigeron breviscapus* formula.

[0076] Example 2: Establishment of a method for detecting the characteristic chromatograms of the standard decoction of *Erigeron breviscapus*

[0077] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0078] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 0.2 mL / min; column temperature 40 °C; detection wavelength 326 nm. The theoretical plate number, calculated based on the baicalin peak, should be no less than 3000.

[0079]

[0080] Preparation of reference solution

[0081] (1) Preparation of reference solution

[0082] Take an appropriate amount of baicalin as a reference standard, add methanol to prepare a solution containing 0.2 mg per 1 ml, and use it as a reference solution.

[0083] (2) Preparation of reference solution of control medicinal materials

[0084] Take about 0.5g of Asarum sieboldii reference material, place it in a round-bottom flask, add 50ml of water, heat under reflux for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25ml of 50% methanol to the residue, transfer to a stoppered conical flask, sonicate for 30 minutes, cool, filter, and take the filtrate as the reference solution for the reference material.

[0085] Preparation of test solution

[0086] Take about 0.2g of the standard decoction of *Erigeron breviscapus*, add 25ml of 50% methanol, sonicate for 30 minutes, cool, filter, and collect the filtrate.

[0087] Determination method

[0088] Accurately pipette 2 μl of the reference solution and the test solution into the liquid chromatograph, measure, record and analyze the chromatograms.

[0089] Identification of characteristic peaks in the characteristic spectrum and selection of the reference peak (S peak)

[0090] Take appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, baicalin, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid reference standards, and add methanol to prepare standard peak reference solutions respectively.

[0091] Accurately pipette 2 μl each of the standard solution and the test solution for each peak, inject them into the liquid chromatograph, determine the concentrations, and record the chromatograms. The results are shown in the table below:

[0092]

[0093] By reviewing literature and comparing chromatographic retention times, peaks 1, 2, 3, 5, 6, 7, and 11 in the characteristic chromatogram were found to have retention times and absorption spectra consistent with the reference standards neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, baicalin, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid, respectively. Based on literature review, peak 1 was identified as neochlorogenic acid, peak 2 as chlorogenic acid, peak 3 as cryptochlorogenic acid, peak 5 as baicalin, peak 6 as isochlorogenic acid B, peak 7 as 3,5-O-dicaffeoylquinic acid, and peak 11 as 4,5-di-O-caffeoylquinic acid.

[0094] Baicalin can be obtained from the China National Institutes for Food and Drug Control, a legally authorized institution. This component is also a content assay indicator, exhibiting a high response value in the characteristic chromatogram. Therefore, baicalin was designated as the reference peak (S peak) to calculate the relative retention time of the characteristic peak.

[0095] Determination of characteristic peaks in characteristic spectrum

[0096] The relative retention times of the characteristic chromatograms were determined based on the research results. Analysis of the characteristic chromatograms of 21 batches of *Erigeron breviscapus* standard decoctions revealed 11 characteristic peaks in the test sample chromatogram, corresponding to the retention times of the 11 characteristic peaks in the reference chromatograms of the control medicinal materials. Peak 5 should correspond to the retention time of the baicalin reference peak. Using the baicalin reference peak as peak S, the relative retention times of each peak and peak S were calculated. These relative retention times should be within ±10% of the specified values, which are: 0.43 (peak 1), 0.59 (peak 2), 0.63 (peak 3), 0.68 (peak 4), 1.11 (peak 6), 1.16 (peak 7), 1.21 (peak 8), 1.24 (peak 9), 1.26 (peak 10), and 1.33 (peak 11). Since the relative peak areas differed significantly, no specific regulations were imposed on the relative peak areas. The characteristic chromatogram of the standard decoction of *Erigeron breviscapus* is shown below. Figure 2 .

[0097] Example 3: Establishment of a method for detecting the characteristic spectra of *Erigeron breviscapus* formula granules

[0098] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0099] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 0.2 mL / min; column temperature 40 °C; detection wavelength 326 nm. The theoretical plate number, calculated based on the baicalin peak, should be no less than 3000.

[0100]

[0101] Preparation of reference solution

[0102] (1) Preparation of reference solution

[0103] Take an appropriate amount of baicalin as a reference standard, add methanol to prepare a solution containing 0.2 mg per 1 ml, and use it as a reference solution.

[0104] (2) Preparation of reference solution of control medicinal materials

[0105] Take about 0.5g of Asarum sieboldii reference material, place it in a round-bottom flask, add 50ml of water, heat under reflux for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25ml of 50% methanol to the residue, transfer to a stoppered conical flask, sonicate for 30 minutes, cool, filter, and take the filtrate as the reference solution for the reference material.

[0106] Preparation of test solution

[0107] Take the granules of *Erigeron breviscapus* and grind them into a fine powder. Take about 0.2g of the powder, add 25ml of 50% methanol, sonicate for 30 minutes, cool, filter, and collect the filtrate.

[0108] Determination method

[0109] Accurately pipette 2 μl of the reference solution and the test solution into the liquid chromatograph, measure and record the chromatograms.

[0110] The chromatogram of the test sample showed 11 characteristic peaks, corresponding to the retention times of the 11 characteristic peaks in the chromatogram of the reference medicinal material. Peak 5 corresponds to the retention time of the baicalin reference peak. Using the baicalin reference peak as peak S, the relative retention times of each peak with peak S were calculated. These relative retention times should be within ±10% of the specified values, which are: 0.43 (peak 1), 0.59 (peak 2), 0.63 (peak 3), 0.68 (peak 4), 1.11 (peak 6), 1.16 (peak 7), 1.21 (peak 8), 1.24 (peak 9), 1.26 (peak 10), and 1.33 (peak 11). The characteristic chromatogram of the *Erigeron breviscapus* formula granules is shown below. Figure 3 .

[0111] Example 4: Methodological validation of the spectral characteristic detection method for *Erigeron breviscapus* formula granules

[0112] The practicality and accuracy of the feature spectrum detection method in Example 3 were verified by examining its specificity, linear range, precision, solution stability, and robustness.

[0113] In these examples, all original reagents and materials were commercially available. Experimental methods not specifically described were conventional methods and conditions well-known in the art, or as recommended by the instrument manufacturer. The reagents, reagents, and instruments used in these examples are detailed in the table below:

[0114]

[0115]

[0116] 4.1 Specificity test

[0117] Blank solvent test solution: Take 50% methanol solution.

[0118] Blank excipient test solution: Take an appropriate amount of maltodextrin, the excipient used in the formula granules of *Erigeron breviscapus*, and prepare it according to the method of "Preparation of test solution" in Example 3.

[0119] Preparation of reference solution: Prepared according to the method of "Preparation of reference solution" in Example 3.

[0120] Preparation of test solution: Prepared according to the method of "Preparation of test solution" in Example 3.

[0121] Each solution was injected separately for analysis. Results showed that the blank solvent and blank excipient had no interference with the detection, indicating good method specificity. See [link to documentation]. Figure 4 .

[0122] 4.2 Instrument precision test

[0123] The reference solution was prepared according to the method described in Example 3, "Preparation of Reference Solution," and injected once. The Asarum sieboldii granules were used to prepare the test solution according to the method described in Example 3, "Preparation of Test Solution," and injected six times. The relative retention time and relative peak area of ​​the remaining peaks were calculated using peak 5 as the reference peak. The results showed that the RSD of the relative retention time of each characteristic peak was 0.03%–0.10%, and the RSD of the relative peak area was 0.20%–1.40%, both less than 2.0%. The relative retention time of each peak was within the specified range, indicating good instrument precision.

[0124] 4.3 Sample stability

[0125] A reference solution was prepared according to the method described in Example 3, "Preparation of Reference Solution," and injected once at each time. A test solution was prepared using *Erigeron breviscapus* granules according to the method described in Example 3, "Preparation of Test Solution." Samples were injected and detected at 0h, 3h, 6h, and 12h. The relative retention time and relative peak area of ​​the remaining peaks were calculated using peak 5 as the reference peak. The results showed that the RSD of the relative retention time of each characteristic peak was 0.03%–0.14%, and the RSD of the relative peak area was 0.41%–1.63%, both less than 2%. The relative retention time of each peak was within the specified range, indicating that the test solution was stable within 12 hours.

[0126] 4.4 Repeatability Test

[0127] The reference solution was prepared according to the method described in Example 3, "Preparation of Reference Solution," and each sample was injected once. Six test solutions were prepared using the *Erigeron breviscapus* formula granules, following the method described in Example 3, "Preparation of Test Solution." The relative retention time and relative peak area of ​​the remaining peaks were calculated using peak 5 as the reference peak. The results are shown in the table below. The results show that the RSD of the relative retention time of each characteristic peak is 0.04%–0.12%, and the RSD of the relative peak area is 0.32%–2.48%, both less than 3.0%. The relative retention time of each peak is within the specified range, indicating that the method has good repeatability.

[0128] 4.5 Intermediate Precision

[0129] Samples were prepared by different inspectors at different times, using different instruments, according to the following method, and then tested using different instruments.

[0130] A 50% methanol solution was used as a blank solvent for the test solution. An appropriate amount of maltodextrin was used to prepare a blank excipient test solution according to the method described in Example 3, "Preparation of Test Solution". A reference solution was prepared according to the method described in Example 3, "Preparation of Reference Solution". Six test solutions were prepared using *Erigeron breviscapus* granules according to the method described in Example 3, and each solution was injected once. The relative retention time and relative peak area of ​​the remaining peaks were calculated using peak 5 as the reference peak. The results showed that the RSD of the relative retention time of each characteristic peak was 0.76%–0.95%, and the RSD of the relative peak area was 4.52%–18.89%, depending on the testing personnel and the instruments used. The relative retention time of each peak was less than 2%, indicating good intermediate precision of the method. However, the RSD of the relative peak area was greater than 2%, indicating that different instruments had a significant impact on the relative peak area of ​​the test sample. Therefore, the relative peak area was not used as a detection indicator when formulating the standard.

[0131] 4.6 Durability

[0132] 4.6.1 Investigation at different column temperatures

[0133] A reference solution was prepared according to the method described in Example 3, "Preparation of Reference Solution"; granules of *Erigeron breviscapus* were used to prepare a test solution according to the method described in Example 3, "Preparation of Test Solution". Samples were injected and detected at column temperatures of 35℃, 40℃, and 45℃, respectively. The relative retention time and relative peak area of ​​the remaining peaks were calculated using peak 5 as the reference peak. The results showed that the RSD of the relative retention time of each characteristic peak was 0.21%–2.58%, all less than 3%, and the relative retention times of each peak were within the specified range, indicating that the method has good column temperature robustness.

[0134] 4.6.2 Investigation of different flow velocities

[0135] A reference solution was prepared according to the method described in Example 3, "Preparation of Reference Solution"; granules of *Erigeron breviscapus* were used to prepare a test solution according to the method described in Example 3, "Preparation of Test Solution". Samples were injected and detected at a flow rate of 0.2 ± 0.02 ml / min. The relative retention time and relative peak area of ​​the remaining peaks were calculated using peak 5 as the reference peak. The results showed that the RSD of the relative retention time of each characteristic peak was 0.34%–1.63%, all less than 2%, and the relative retention times of each peak were within the specified range, indicating that the method has good flow rate robustness.

[0136] 4.6.3 Investigation of Chromatographic Columns of Different Brands

[0137] A reference solution was prepared according to the method described in Example 3, "Preparation of Reference Solution"; the *Erigeron breviscapus* formula granules were used, and a test solution was prepared according to the method described in Example 3, "Preparation of Test Solution". Aglient ZORBAX SB-Aq RRHD, Aglient ZORBAX SB, and Waters ACQUITY UPLC HSS T3 columns were used for injection and detection. The relative retention times and relative peak areas of the remaining peaks were calculated using peak 5 as the reference peak. The results showed that the relative retention times (RSD) of each characteristic peak ranged from 0.43% to 19.35%, indicating significant differences in relative retention times between different brands of columns. Considering all chromatographic peaks, the Waters ACQUITY UPLC HSS T3 column was consistently used for the determination of the *Erigeron breviscapus* formula granules.

[0138] 4.6.4 Investigation of chromatographic columns from different batches of the same brand

[0139] The reference solution was prepared according to the method described in Example 3, "Preparation of Reference Solution"; the *Erigeron breviscapus* granules were used to prepare the test solution according to the method described in Example 3, "Preparation of Test Solution". Different batches of Waters ACQUITY UPLC HSS T3 columns (serial number: 0270313563) and Waters ACQUITY UPLC HSS T3 column (serial number: 0243302951) were used for injection and detection. The relative retention times and relative peak areas of the remaining peaks were calculated using peak 5 as the reference peak. The results showed that the relative retention times of the characteristic peaks from different batches of columns differed by 0.01% to 0.03%, indicating that using different batches of the same brand of Waters ACQUITY UPLC HSS T3 columns had no significant impact on the detection results of the relative retention times of the characteristic peaks.

[0140] Example 5: Establishment of a method for determining the content of *Erigeron breviscapus* granules in a formula.

[0141] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0142] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; methanol-0.1% phosphoric acid solution-tetrahydrofuran (13:74:13) as the mobile phase; and a detection wavelength of 335 nm. The theoretical plate number, calculated based on the baicalin peak, should be no less than 5000.

[0143] Preparation of the reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.2 mg per ml.

[0144] Preparation of the test solution: Take an appropriate amount of *Dendrobium nobile* formula granules, grind them finely, take about 0.2g, weigh accurately, place in a stoppered conical flask, accurately add 25ml of 80% methanol, weigh, sonicate (power 500W, frequency 53KHz) for 30 minutes, cool, weigh again, replenish the lost weight with 80% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0145] For the assay, accurately pipette 5 μl each of the reference solution and the test solution and inject them into the liquid chromatograph. Record the chromatograms.

[0146] Example 6: Methodological Validation of the Method for Determining the Granular Content of *Erigeron breviscapus* Formula

[0147] 6.1 Exclusivity

[0148] Take the granules of *Erigeron breviscapus* and prepare a test solution according to the method described in Example 5, "Preparation of Test Solution"; take approximately 0.2 g of maltodextrin and prepare a blank excipient solution according to the same method; use 80% methanol as the blank solvent solution. The above solutions were analyzed under the chromatographic conditions described in Example 5. The results show that the baicalin peak in the chromatogram of the test solution was well separated from adjacent peaks, and the blank and negative results did not interfere with the detection, indicating that the specificity of this method meets the requirements. See... Figure 5 .

[0149] 6.2 Linearity and Range

[0150] Accurately weigh 13.180 mg of baicalin reference standard, place it in a 25 ml volumetric flask, and add methanol to prepare a reference standard stock solution containing 848.39 μg of baicalin per ml.

[0151] A precise amount of the above-mentioned stock solution was taken to prepare reference solutions with concentrations of 26.51 μg / ml, 53.02 μg / ml, 106.05 μg / ml, 212.10 μg / ml, and 424.20 μg / ml, respectively. The five different concentrations of reference solutions and the reference stock solution were then analyzed under the chromatographic conditions of Example 5. The peak areas of the chromatograms were recorded. A standard curve was plotted with peak area as the ordinate (Y) and reference concentration as the abscissa (X). The linear regression equation for baicalin was y = 17692x + 50005, with a correlation coefficient r = 0.9998 and SS = 184, indicating that the linear relationship between the injection concentration and peak area was good within the range of 26.51 μg / ml to 848.39 μg / ml.

[0152] 6.3 Stability Test

[0153] Take an appropriate amount of *Erigeron breviscapus* granules, grind them finely, take about 0.2g, weigh accurately, and prepare the test solution according to the test solution preparation method in Example 5. Inject the sample at 0, 3, 6, 9, and 12 hours according to the chromatographic conditions in Example 5, and record the peak area and RSD value of baicalin in the sample. The results show that for the same test solution, the RSD value of the peak area of ​​baicalin within 12 hours is 0.27%, which is less than 2%, indicating that the test solution has good stability within 12 hours.

[0154] 6.4 Intermediate Precision Examination

[0155] Different personnel used different instruments to prepare six test solutions. Approximately 0.2g of the *Erigeron breviscapus* formula granules were finely ground and accurately weighed. Following the test solution preparation method in Example 5, the solutions were injected separately. The baicalin content and RSD value in the samples were calculated. The same batch of samples was measured six times by different personnel at different times using different detection instruments. The average baicalin content was 21.04 mg / ml and 20.99 mg / ml, respectively, with an RSD value of 0.61%, indicating good repeatability and intermediate precision of the method.

[0156] 6.5 Accuracy Test

[0157] Accurately weigh 22.312 mg of baicalin reference standard, place it in a 250 ml volumetric flask, add 80% methanol to dissolve it, and prepare a solution containing 0.08166 mg of baicalin per ml.

[0158] Take an appropriate amount of *Erigeron breviscapus* granules (content 21.07 mg / g), grind them finely, take about 0.1 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of the above-mentioned reference stock solution, weigh it, sonicate for 30 minutes, remove it, cool it, weigh it again, replenish the lost weight with 80% methanol, shake well, filter, and collect the filtrate. Prepare 6 test solutions using the same method, and detect them according to the chromatographic conditions of Example 5. Calculate the content of baicalin in the test solutions, calculate the recovery rate, and the results show that the recovery rate of baicalin ranges from 100.08% to 100.94%, with an average recovery rate of 100.49% and an RSD of 0.37%, indicating that the analytical method has good accuracy.

[0159] 6.6 Durability Test

[0160] Take an appropriate amount of *Erigeron breviscapus* granules, grind them finely, take about 0.2g, accurately weigh it, and prepare the test solution according to the test solution preparation method in Example 5. The solution was then measured using different chromatographic columns (Waters XBridge C18 column (4.6×250mm, 5μm); GL InertSμstain C18 column (4.6×250mm, 5μm); Aglient Eclipse XDB-C18 column (4.6×250mm, 5μm)). The results showed good separation efficiency for each column, with baicalin content of 21.34 mg / ml, 21.51 mg / ml, and 21.07 mg / ml, and an RSD of 1.1%, indicating good robustness of the analytical method under different chromatographic columns.

[0161] The determination was performed using different column temperatures (25℃, 30℃ and 35℃), and the separation effect was good. The RSD value of baicalin content was 0.46%, indicating that the analytical method has good robustness within the column temperature range of ±5℃.

[0162] The granules of *Erigeron breviscapus* were analyzed using different flow rates (0.9 ml / min, 1.0 ml / min, and 1.1 ml / min). The results showed that the chromatographic peaks were well separated at different flow rates, and the RSD value of baicalin was 0.20%, indicating that the analytical method has good robustness within a flow rate range of ±0.1 ml / min.

[0163] Comparative Example 1

[0164] The difference between Comparative Example 1 and Example 5 lies in the mobile phase used in the chromatographic conditions. The chromatographic conditions for Comparative Example 1 are those specified in the method for determining the content of *Erigeron breviscapus* in the Chinese Pharmacopoeia.

[0165] Column: Aglient Eclipse XDB-C18

[0166] Mobile phase: methanol - 0.1% phosphoric acid solution (40:60)

[0167] The detection wavelength is 335nm;

[0168] Accurately pipette 10 μl each of the test solution and the reference solution into the liquid chromatograph, record the chromatograms, and see [see figure]. Figure 6

[0169] Example 5: The mobile phase was methanol - 0.1% phosphoric acid solution - tetrahydrofuran (13:74:13);

[0170] Accurately pipette 10 μl each of the test solution and the reference solution into the liquid chromatograph, record the chromatograms, and see [see figure]. Figure 7 .

[0171] As a result, by Figure 6 It can be seen that under these conditions, the separation effect of baicalin in the chromatogram of the test sample is not good, and the separation degree does not meet the requirements; however, after changing the mobile phase in Example 5, baicalin in the chromatogram of the test sample can achieve baseline separation, and the separation effect is good.

[0172] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A detection method of Gypenoside granules, wherein the Gypenoside granules are prepared by decocting, concentrating, drying, pulverizing and granulating Gypenoside decoction pieces; the detection method comprises characteristics, identification, examination, extract, characteristic spectrum and content determination, and is characterized in that, The characteristic map detection method is detected by high performance liquid chromatography, and the working conditions of the high performance liquid chromatography are as follows: the high performance liquid chromatograph is an ultra-high performance liquid chromatograph, the chromatographic column is Waters ACQUITY UPLC HSS T3, C18, 2.1*100 mm, 1.8 mu m, acetonitrile is used as mobile phase A, and 0.1%-0.5% formic acid solution is used as mobile phase B for gradient elution, the flow rate is 0.2-1 ml per minute, the column temperature is 35-45 DEG C, the detection wavelength is 300-350 nm, the theoretical plate number calculated according to the wild scutellarein peak should be not less than 2000-5000, and the gradient elution conditions are as follows: 0-5 min, mobile phase A 2%, mobile phase B 98%; 5-8 min, mobile phase A 2-5%, mobile phase B 98-95%; 8-24 min, mobile phase A 5-15%, mobile phase B 95-85%; 24-55 min, mobile phase A 15-18%, mobile phase B 85-82%; The detection method comprises the following operations: Preparation of reference solution: 0.5-2 g of the control medicinal material of Herba Erigerontis is weighed, 20-50 ml of water is added, and heating reflux is carried out for 30-60 minutes, then filtration is carried out, the filtrate is evaporated to dryness, 20-50 ml of 25%-50% methanol is added to the residue, ultrasonic treatment is carried out for 10-60 minutes, the solution is cooled, filtration is carried out, and the continuous filtrate is used as the reference solution of the control medicinal material; and the reference solution of the control sample is prepared by adding the scutellarein control sample to methanol to prepare a solution containing 0.2 mg per 1 ml; Preparation of test sample solution: the Herba Erigerontis formula granules are finely ground, 0.1-1 g is weighed, 20-50 ml of 25%-50% methanol is added, ultrasonic treatment is carried out for 10-60 minutes, the solution is cooled, filtration is carried out, and the continuous filtrate is obtained; Determination method: 1-4 mu l of the reference solution and the test sample solution is precisely taken respectively, injected into the liquid chromatograph, and determination is carried out according to the working conditions of the high performance liquid chromatography, and the chromatogram of 0-55 min is recorded. The test sample chromatogram should present 11 characteristic peaks, and the retention time of the 11 characteristic peaks in the chromatogram of the reference sample of the control medicinal material should correspond to that of the reference sample of the control medicinal material, wherein peak 5 should correspond to the retention time of the reference peak of wild scutellarein; the reference peak of wild scutellarein is taken as the S peak, and the relative retention time of each characteristic peak to the S peak is calculated, which should be within ±10% of the specified value; wherein the relative retention time of peak 1 is 0.43, the relative retention time of peak 2 is 0.59, the relative retention time of peak 3 is 0.63, the relative retention time of peak 4 is 0.68, the relative retention time of peak 6 is 1.11, the relative retention time of peak 7 is 1.16, the relative retention time of peak 8 is 1.21, the relative retention time of peak 9 is 1.24, the relative retention time of peak 10 is 1.26, and the relative retention time of peak 11 is 1.33; wherein peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 5 (S) is wild scutellarein, peak 6 is isochlorogenic acid B, peak 7 is 3, 5-O-dicaffeoylquinic acid, and peak 11 is 4, 5-di-O-caffeoylquinic acid.

2. The detection method according to claim 1, characterized in that, The working conditions of the high performance liquid chromatography are as follows: gradient elution is performed with acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, the flow rate is 0.2 ml per minute, the column temperature is 40°C, and the detection wavelength is 326 nm, and the theoretical plate number calculated according to the wild scutellarein peak should be not less than 3000; The preparation of the reference solution: 0.5 g of the control medicinal material of Herba eriotae is added to 50 ml of water, heated and refluxed for 30 minutes, filtered, and the filtrate is evaporated to dryness; 25 ml of 50% methanol is added to the residue, ultrasonic treatment is performed for 30 minutes, the solution is cooled and filtered, and the filtrate is taken as the reference solution of the control medicinal material; in addition, an appropriate amount of wild scutellarein reference substance is precisely weighed and dissolved in methanol to prepare a solution containing 0.2 mg per 1 ml, which is taken as the reference solution of the reference substance; The preparation of the test sample solution: an appropriate amount of the formula granules of Herba eriotae is finely ground, 0.2 g of the ground sample is weighed, 25 ml of 50% methanol is added, ultrasonic treatment is performed for 30 minutes, the solution is cooled and filtered, and the filtrate is obtained. Determination method: 2 μl of the reference solution and the test sample solution is precisely taken respectively, injected into the liquid chromatograph, and determined according to the working conditions of the high performance liquid chromatography, and the chromatogram from 0 to 55 minutes is recorded.

3. The assay of any one of claims 1 or 2, wherein, The identification method is as follows: 0.2-1 g of breviscapine dispensing granules is finely ground, 20-50 ml of methanol is added, and ultrasonic treatment is performed for 30-60 minutes. The filtrate is recovered to dryness, the residue is dissolved in 20 ml of water, and the solution is warmed and dissolved. The solution is extracted with ether three times, 20 ml each time, the ether layer is discarded, the water layer is adjusted to pH 2-3 with dilute hydrochloric acid, and extracted with ethyl acetate three times, 20 ml each time. The ethyl acetate is combined and evaporated to dryness. The residue is dissolved in 1 ml of methanol to obtain a test solution. Another 2.0 g of breviscapine reference material is added to 60 ml of water, boiled and decocted for 30 minutes, and filtered. The filtrate is evaporated to dryness, and the residue is dissolved in 20 ml of methanol to obtain a reference solution. The above solutions are each taken 5-10 μl, spotted in a strip shape on the same polyamide film, and developed with glacial acetic acid as the developing agent. After being taken out, dried, and sprayed with 5% ferric trichloride ethanol solution, the film is observed under daylight. In the test sample chromatogram, the same color main spot should appear at the position corresponding to the reference material chromatogram.

4. The assay of any one of claims 1 or 2, wherein, The content determination is to determine the content of wild scutellarein by high performance liquid chromatography. The detection conditions of the high performance liquid chromatography are as follows: octadecylsilane-bonded silica gel is used as the filler; methanol: 0.1%-0.5% phosphoric acid solution: tetrahydrofuran = 13:74:13 is used as the mobile phase; the detection wavelength is 335 nm, and the theoretical plate number calculated according to the wild scutellarein peak should not be less than 2000-8000.

5. The detection method according to claim 4, characterized in that, The content determination method comprises the following operations: Preparation of the reference solution: an appropriate amount of wild scutellarein reference substance is accurately weighed and dissolved in methanol to obtain a solution containing 0.2 mg per 1 ml. Preparation of the test solution: an appropriate amount of breviscapine dispensing granules is finely ground, about 0.2 g is accurately weighed, placed in a conical flask with a plug, 25 ml of 80% methanol is accurately added, the weight is determined, ultrasonic treatment is performed for 30 minutes, the weight is determined after cooling, the lost weight is made up with 80% methanol, shaken, filtered, and the filtrate is obtained. Determination method: 5 ul of the reference solution and the test solution are respectively taken and injected into the liquid chromatograph for determination, and the chromatogram is recorded. The content of wild scutellarein is calculated by the external standard method.

6. The assay of any one of claims 1-2, wherein, The leachate detection method is to take breviscapine dispensing granules, and determine the hot leaching method under the alcohol-soluble leachate determination method in the Chinese Pharmacopoeia with ethanol as the solvent.

7. The assay of any one of claims 1-2, wherein, The breviscapine dispensing granules are prepared by taking breviscapine decoction pieces, adding water, boiling and decocting, concentrating, drying, crushing, adding pharmaceutically acceptable excipients, mixing, granulating, and the like. 1 g of the dispensing granules is equivalent to 4.0 g of breviscapine decoction pieces.

8. Use of the detection method according to any one of claims 1 to 7, characterized in that, The detection method is applied to the quality detection of breviscapine medicinal materials, breviscapine decoction pieces, breviscapine standard decoction, and breviscapine dispensing granules.

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