A quality detection method for reference samples of Banxia Xiexin granules

Abstract

The present invention provides a quality detection method for a Banxia Xiexin granule reference sample, comprising constructing a characteristic spectrum of the Banxia Xiexin granule reference sample and determining the contents of individual components of baicalin, glycyrrhizic acid, and berberine, wherein the content standards are limited to 37.83-70.26 mg / g for baicalin, 3.91-7.27 mg / g for glycyrrhizic acid, and 1.86-3.46 mg / g for berberine, and liquid chromatography is used for constructing the characteristic spectrum and determining the contents of individual components of baicalin, glycyrrhizic acid, and berberine; and the chromatographic conditions adopted are as follows: a chromatographic column using octadecylsilane bonded silica gel as a filler, methanol as a mobile phase A, and a 0.1% by volume formic acid solution as a mobile phase B, and gradient elution is performed as specified. The chromatographic conditions adopted in the present invention can obtain a characteristic spectrum with better separation and clearer peak tips, and can more accurately reflect and control the quality of the Banxia Xiexin granule reference sample.

Description

Technical Field

[0001] The present invention relates to the technical field of quality control of Chinese patent medicine compound preparations, and in particular to a quality detection method for a reference sample of Banxia Xiexin granules. Background Art

[0002] Modern drugs must possess three key characteristics: stability, uniformity, safety, and efficacy. For compound preparations of traditional Chinese medicines, multiple testing methods are required to ensure the reliability and stability of test results. Banxia Xiexin Decoction, originating from the Treatise on Febrile Diseases in Zhang Zhongjing's Treatise on Febrile and Miscellaneous Diseases in the Eastern Han Dynasty, is a classic decoction widely used in clinical practice. The original recipe states: "Fullness without pain is considered a lump in the abdomen. Bupleurum is inappropriate for this condition; Banxia Xiexin Decoction is appropriate. Half a liter of washed Banxia; 3 liang each of Scutellaria baicalensis, dried ginger, ginseng, and roasted Licorice root; 1 liang of Coptis chinensis; 12 split jujubes. Add these seven ingredients to 1 dou of water and boil until 6 liters are obtained. Remove the residue and boil again until 3 liters are obtained. Take 1 liter warm three times a day." The main ingredient is Banxia, ​​which dissipates lumps and eliminates lump in the abdomen. The auxiliary ingredients are dried ginger, which warms the middle and dispels cold, while Coptis chinensis and Scutellaria baicalensis have bitter and cold properties that dissipate heat. Ginseng and jujube are supplementary ingredients to replenish deficiency and invigorate Qi. Licorice root strengthens the spleen and harmonizes the middle, which will naturally dissipate the lump and resolve all symptoms. Banxia Xiexin Decoction mainly contains flavonoids, alkaloids, saponins, and glycoside compounds. The compound preparation of Chinese patent medicine is composed of multiple Chinese medicinal materials. The multiple components work synergistically and have multiple targets. It is made by using modern scientific technology and imitating the traditional Chinese medicine decoction decoction method. It is refined through extraction, concentration, drying and other processes. It has the advantages of no need for decoction, easy to take, fast absorption, accurate dosage, safe and clean, and easy to carry. Compared with traditional medicinal raw materials, the morphological characteristics of Banxia Xiexin Granules have changed significantly. The authenticity and quality of the product cannot be observed with the naked eye. The existing technology does not have a detection method that can accurately reflect the overall intrinsic quality of Banxia Xiexin Granules, and it cannot meet the quality control requirements of Chinese patent medicine compound preparations. Therefore, it is very necessary to establish a Banxia Xiexin Granule benchmark sample quality detection method for quality control that can reflect the overall basic material situation of the classic prescription. Summary of the Invention

[0003] The purpose of the present invention is to solve the deficiencies of the prior art and provide a method for detecting the quality of a reference sample of Banxia Xiexin Granules that accurately reflects the intrinsic quality of a compound Chinese patent medicine preparation and better controls the quality of the compound Chinese patent medicine preparation.

[0004] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0005] The present invention provides a method for detecting the quality of a reference sample of Banxia Xiexin granules, comprising:

[0006] Characteristic spectrum construction and content determination of baicalin, glycyrrhizic acid, and berberine were performed on the reference sample of Banxia Xiexin Granule. The content standard of the reference sample of Banxia Xiexin Granule was limited to baicalin content of 37.83-70.26 mg / g, glycyrrhizic acid content of 3.91-7.27 mg / g, and berberine content of 1.86-3.46 mg / g. Characteristic spectrum construction and content determination of baicalin, glycyrrhizic acid, and berberine were all performed by liquid chromatography.

[0007] The characteristic spectrum of the Banxia Xiexin Granule reference sample determined by liquid chromatography includes: using a solution made from a berberine hydrochloride reference substance as a reference solution a1, a solution made from a baicalin reference substance as a reference solution a2, a solution made from a 6-gingerol reference substance as a reference solution a3, a solution made from an ammonium glycyrrhizate reference substance as a reference solution a4, and a solution made from the Banxia Xiexin Granule reference sample as a test solution a, accurately aspirating the reference solution a1, the reference solution a2, the reference solution a3, the reference solution a4, and the test solution a, respectively, and injecting them into a liquid chromatograph for determination, thereby obtaining the result; wherein the chromatographic conditions used are: a chromatographic column: octadecylsilane bonded silica gel as a filler, methanol as a mobile phase A, 0.1% by volume formic acid solution as a mobile phase B, and gradient elution according to the provisions in Table a;

[0008] Table a Gradient elution program

[0009] Time (min) Methanol (%) 0.1% formic acid water (%) 0 5 95 5 27 73 25 32 68 45 40 60 60 45 55 82 66 34 89 76 24 105 85 15

[0010] Flow rate: 0.8-1.2 ml / min; column temperature: 28-32°C; injection volume: 4-6 μl; detection wavelength for glycyrrhizic acid: 250-258 nm; detection wavelength for berberine hydrochloride, baicalin, and 6-gingerol: 275-285 nm.

[0011] In one embodiment, the method further comprises identifying the Banxia Xiexin granule reference sample by thin layer chromatography, wherein the thin layer chromatography method comprises the following steps:

[0012] (1) Take 3 g of Banxia Xiexin Granules reference sample powder, add 30 ml of methanol, ultrasonically treat for 15 min, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 ml of methanol to prepare the test solution b1; take 1 g of licorice control medicinal material, add 50 ml of water, decoct for 30 min, cool, filter, add double the amount of anhydrous ethanol to the filtrate, shake well, centrifuge, take the supernatant, evaporate to dryness, and dissolve the residue in 2 ml of methanol to prepare the control medicinal material solution b1; perform thin layer chromatography test, take 2 μl of the test solution b1 and 10 μl of the control medicinal material solution b1, respectively, spot them on the same silica gel G thin layer plate, use toluene-ethyl formate-formic acid solution with a volume ratio of 15:8:1.5 as the developing solvent, develop, take out, dry, and inspect under a 365 nm ultraviolet lamp;

[0013] (2) Take 4 g of the powder of the reference sample of Banxia Xiexin Granules, add 30 ml of ether, heat and reflux for 15 min, cool, filter, evaporate the filtrate, and dissolve the residue in 0.5 ml of ethyl acetate to prepare the test solution b2; take 1 g of the reference medicinal material of dried ginger, add 30 ml of ether, heat and reflux for 15 min, cool, filter, evaporate the filtrate, and dissolve the residue in 0.5 ml of ethyl acetate to prepare the control medicinal material solution b2; perform the thin layer chromatography test, aspirate 5 μl of the test solution b2 and 1 μl of the control medicinal material solution b2, and spot them on the same silica gel G thin layer plate, use a toluene-ethyl acetate solution with a volume ratio of 2:1 as the developing solvent, develop, remove, dry, spray with 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clearly colored, and inspect under sunlight;

[0014] (3) Take 0.5 g of the powder of the reference sample of Banxia Xiexin Granules, add 25 ml of methanol, ultrasonically treat for 30 minutes, filter, and take the filtrate as the test solution b3; take 0.25 g of the reference medicinal material of Coptis chinensis, add 25 ml of methanol, ultrasonically treat for 30 minutes, filter, and take the filtrate as the reference medicinal material solution b3; perform the thin layer chromatography test, take 2 μl of the test solution b3 and the reference medicinal material solution b3, respectively, and spot them on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine solution with a volume ratio of 3:3.5:1:1.5:0.5:1 as the developing solvent, place it in a developing cylinder pre-saturated with ammonia vapor, develop, take out, dry, and inspect under a 365 nm ultraviolet lamp;

[0015] (4) Take 2g of Banxia Xiexin Granules reference sample powder, add 50ml of ethanol, heat and reflux for 1 hour, let cool, filter, evaporate the filtrate to dryness, add 20ml of water to the residue, heat to dissolve, let cool, adjust the pH value to 1-2 with hydrochloric acid, shake and extract with 30ml of ethyl acetate, separate the ethyl acetate layer, evaporate to dryness, add 1ml of ethanol to dissolve the residue, and use it as the test solution b4; take 1g of Huangqin reference medicinal material, add 50ml of ethanol, heat and reflux for 1 hour, let cool, filter, evaporate the filtrate to dryness, add 20ml of water to the residue, add Heat to dissolve, cool, adjust the pH to 1-2 with hydrochloric acid, and extract by shaking with 30 ml of ethyl acetate. Separate the ethyl acetate layer and evaporate to dryness. Dissolve the residue in 1 ml of ethanol to prepare control medicinal solution B4. Perform thin-layer chromatography (TLC) testing by spotting 2 μl each of test solution B4 and control medicinal solution B4 onto the same polyamide film. Develop with a 4:1:10 by volume ethyl acetate-methanol-acetic acid solution. Remove the film, air-dry, spray with 2% ferric chloride solution, and examine under sunlight.

[0016] (5) Take 2g of Banxia Xiexin Granules reference sample powder, add 30ml of water, stir evenly, ultrasonically treat for 20min, centrifuge, take the supernatant, extract twice with chloroform, 20ml / time, discard the chloroform solution, extract the aqueous layer with water-saturated n-butanol twice, 30ml / time, combine the n-butanol solution, and then wash twice with n-butanol-saturated ammonia test solution, 60ml / time, take the n-butanol solution, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution b5; take 1g of ginseng control medicinal material, add 30ml of water, stir evenly, ultrasonically treat for 20min, centrifuge, take the supernatant, extract twice with chloroform, 20ml / time, discard the chloroform solution, and use the aqueous layer with Extract twice with water-saturated n-butanol, 30 ml each time, combine the n-butanol solutions, then wash twice with n-butanol-saturated ammonia test solution, 60 ml each time, take the n-butanol solution, evaporate to dryness, add 1 ml of methanol to dissolve the residue, as the control medicinal material solution b5; conduct thin layer chromatography test, aspirate 5 μl of test solution b5 and 2 μl of control medicinal material solution b5, respectively spot them on the same silica gel G thin layer plate, use chloroform-ethyl acetate-methanol-glacial acetic acid-water solution with a volume ratio of 10:4:5:2:2 at below 10°C as the lower layer solution as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under sunlight.

[0017] In one embodiment, the characteristic spectrum of the reference sample of Banxia Xiexin Granules determined by liquid chromatography also includes:

[0018] (1) Preparation of reference solutions: Take appropriate amounts of berberine hydrochloride reference substance, baicalin reference substance, 6-gingerol reference substance, and ammonium glycyrrhizate reference substance, accurately weigh them, and add methanol to prepare reference solution a1 containing 0.20 mg of berberine hydrochloride per 1 ml, reference solution a2 containing 0.50 mg of baicalin per 1 ml, reference solution a3 containing 0.40 mg of 6-gingerol per 1 ml, and reference solution a4 containing 0.25 mg of glycyrrhizic acid per 1 ml;

[0019] (2) Preparation of test solution a: Take 0.5 g of the reference sample of Banxia Xiexin Granules, accurately weigh it, place it in a stoppered conical flask, accurately add 10 ml of 50% ethanol, weigh it, reflux it for 30 min, let it cool, weigh it again, make up the weight loss with 50% ethanol, shake it well, filter it, and take the filtrate as the test solution a.

[0020] In one embodiment, the chromatogram of the test sample determined by liquid chromatography presents 11 characteristic peaks, of which 4 peaks correspond to the 4 reference peaks of the reference sample chromatogram, and peak 5 corresponding to the baicalin reference peak is used as the reference peak S. The relative retention times of peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, peak 10, and peak 11 in the chromatogram of the test sample and the reference peak S are calculated, and the specified values ​​are: peak 1 relative retention time 0.54, peak 2 relative retention time 0.60, peak 3 relative retention time 0.72, peak 4 relative retention time 0.75, peak 6 relative retention time 1.10, peak 7 relative retention time 1.14, peak 8 relative retention time 1.15, peak 9 relative retention time 1.19, peak 10 relative retention time 1.34, and peak 11 relative retention time 1.48. The relative retention times of the detected characteristic peaks should be within ±10% of the specified values.

[0021] In one embodiment, the liquid chromatography method for determining the content of baicalin, glycyrrhizic acid, and berberine comprises: performing liquid chromatography analysis, using a solution prepared from a berberine hydrochloride reference substance as a reference solution c1, a solution prepared from a baicalin reference substance as a reference solution c2, a solution prepared from an ammonium glycyrrhizate reference substance as a reference solution c3, and a solution prepared from a reference sample of Banxia Xiexin Granules as a test solution c, respectively accurately aspirating the reference solution c1, the reference solution c2, the reference solution c3, and the test solution c. The sample solution c was respectively injected into a liquid chromatograph and determined to obtain; wherein, the chromatographic conditions adopted were as follows: a chromatographic column: octadecylsilane bonded silica gel as a filler, methanol as a mobile phase A, 0.1% by volume formic acid solution as a mobile phase B, gradient elution according to the provisions in Table a, flow rate: 0.8-1.2 ml / min; column temperature: 28-32°C; injection volume: 4-6 μl; glycyrrhizic acid detection wavelength: 250-258 nm, berberine hydrochloride and baicalin detection wavelength: 275-285 nm.

[0022] In one embodiment, the liquid chromatography method for determining the content of baicalin, glycyrrhizic acid, and berberine as a single component also includes:

[0023] (1) Preparation of reference solution: Take appropriate amounts of berberine hydrochloride reference, baicalin reference, and ammonium glycyrrhizate reference, accurately weigh them, and add methanol to prepare reference solution C1 containing 0.20 mg of berberine hydrochloride per 1 ml, reference solution C2 containing 0.50 mg of baicalin per 1 ml, and reference solution C3 containing 0.25 mg of glycyrrhizic acid per 1 ml;

[0024] (2) Preparation of test solution: Take 0.5 g of the powder of the reference sample of Banxia Xiexin Granules, accurately weigh it, place it in a stoppered conical flask, accurately add 10 ml of 50% ethanol, weigh it, reflux it for 30 min, let it cool, weigh it again, make up the weight loss with 50% ethanol, shake it well, filter it, and take the filtrate as the test solution c.

[0025] In one embodiment, the method further comprises determining the content of 6-gingerol in the Banxia Xiexin Granules reference sample by liquid chromatography, and limiting the 6-gingerol content standard of the Banxia Xiexin Granules reference sample to 0.26-0.49 mg / g.

[0026] In one embodiment, the liquid chromatography method for determining the content of a single component of 6-gingerol includes: performing liquid chromatography analysis, using a solution prepared from a 6-gingerol reference substance as the reference solution c4, accurately aspirating the reference solution c4 and the test solution c, respectively, injecting them into the liquid chromatograph, and determining the content; wherein the chromatographic conditions used are: a chromatographic column: octadecylsilane bonded silica gel as a filler, methanol as a mobile phase A, 0.1% by volume formic acid solution as a mobile phase B, gradient elution according to the provisions in Table a, flow rate: 0.8-1.2 ml / min; column temperature: 28-32° C.; injection volume: 4-6 μl; detection wavelength: 275-285 nm;

[0027] Take an appropriate amount of 6-gingerol reference substance, accurately weigh it, and add methanol to prepare a reference substance solution C4 containing 0.40 mg of 6-gingerol per 1 ml;

[0028] Take 0.5 g of Banxia Xiexin Granules reference sample powder, accurately weigh it, place it in a stoppered conical flask, accurately add 10 ml of 50% ethanol, weigh it, reflux it for 30 minutes, cool it, weigh it again, make up the weight loss with 50% ethanol, shake it well, filter it, and take the filtrate as the test solution C.

[0029] In one embodiment, the properties, dry paste yield, and extract of the Banxia Xiexin Granule benchmark sample are also tested. The extract is tested by the hot soak method using ethanol as the solvent, and the extract range is determined by the hot soak method under the alcohol-soluble extract determination method.

[0030] In one embodiment, the chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0031] Compared with the prior art, the beneficial effect of the quality detection method of the Banxia Xiexin Granules reference sample provided by the present invention is: by constructing a characteristic spectrum of the Banxia Xiexin Granules reference sample and determining the content of single components of baicalin, glycyrrhizic acid, and berberine, the quality of the Banxia Xiexin Granules reference sample is evaluated, laying a solid foundation for the stable quality of the product, and being able to establish a feasible quality standard for the Banxia Xiexin Granules reference sample, and to achieve effective control of the quality of the Banxia Xiexin Granules reference sample. Moreover, by using the chromatographic conditions of the present application for liquid phase analysis, a characteristic spectrum with better separation and clearer peak tips can be obtained, which can more accurately reflect the quality of the Banxia Xiexin Granules reference sample. The method has good stability, and a quality standard for the Banxia Xiexin Granules reference sample with a baicalin content of 37.83-70.26 mg / g, a glycyrrhizic acid content of 3.91-7.27 mg / g, and a berberine content of 1.86-3.46 mg / g has been proposed. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the TLC spectrum of licorice in the reference sample of Banxia Xiexin Granules in one embodiment of the present invention; among them, group 1 is 1 μl of licorice control medicinal material solution, and groups 2 to 16 are 1 μl of test solution of 15 batches of reference samples.

[0033] Figure 2 This is the TLC spectrum of dried ginger in the reference sample of Banxia Xiexin Granules in one embodiment of the present invention; among them, group 1 is 1 μl of dried ginger control medicinal material solution, and groups 2 to 16 are 5 μl of test solution of 15 batches of reference samples.

[0034] Figure 3 This is the TLC spectrum of Coptis chinensis in the reference sample of Banxia Xiexin Granules in one embodiment of the present invention; among them, Group 1 is 2 μl of Coptis chinensis control medicinal material solution, and Groups 2 to 16 are 2 μl of test solution of 15 batches of reference samples.

[0035] Figure 4This is the TLC spectrum of Scutellaria baicalensis in the reference sample of Banxia Xiexin Granules in one embodiment of the present invention; wherein, in Figure (a), Group 1 is 2 μl of Scutellaria baicalensis control medicinal material solution, and Groups 2 to 6 are 2 μl of 5 batches of test solution of the reference sample; in Figure (b), Group 1 is 2 μl of Scutellaria baicalensis control medicinal material solution, and Groups 2 to 6 are 2 μl of 5 additional batches of test solution of the reference sample; in Figure (c), Group 1 is 2 μl of Scutellaria baicalensis control medicinal material solution, and Groups 2 to 6 are 2 μl of 5 additional batches of test solution of the reference sample.

[0036] Figure 5 This is the TLC spectrum of ginseng in the reference sample of Banxia Xiexin Granules in one embodiment of the present invention; among them, group 1 is 2 μl of ginseng control medicinal material solution, and groups 2 to 16 are 5 μl of test solution of 15 batches of reference samples.

[0037] Figure 6 This is an overlay of characteristic spectra of 15 batches of reference samples at a detection wavelength of 254 nm in the characteristic spectrum determination of one embodiment of the present invention.

[0038] Figure 7 This is an overlay of characteristic spectra of 15 batches of reference samples at a detection wavelength of 280 nm in the characteristic spectrum determination of one embodiment of the present invention.

[0039] Figure 8 This is a control characteristic spectrum of a reference solution at a detection wavelength of 280 nm in the characteristic spectrum determination of one embodiment of the present invention.

[0040] Figure 9 This is a control characteristic spectrum of the reference solution at a detection wavelength of 254 nm in the characteristic spectrum determination of one embodiment of the present invention; Figure 9 As shown, there are 11 common peaks, including peak 4: berberine hydrochloride; peak 5: baicalin; peak 10: 6-gingerol; peak 11: glycyrrhizic acid. DETAILED DESCRIPTION

[0041] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0042] 1. Process research on the reference sample of Banxia Xiexin Granules

[0043] 1.1 Prescription composition and dosage of Banxia Xiexin Decoction: 34.50g of dried pinellia, 41.40g of scutellaria, 41.40g of dried ginger, 41.40g of ginseng, 41.40g of stir-fried licorice, 13.80g of coptis, and 36.00g of jujube.

[0044] Dosage and administration: Add the above seven ingredients to 2000ml of water, boil and extract 1200ml, remove the residue, and then concentrate to 600ml. Take 200ml warm, 3 times a day.

[0045] 1.2 Pretreatment of prescription drug taste

[0046] The ingredients of this prescription are Pinellia ternata, Scutellaria baicalensis, dried ginger, ginseng, stir-fried licorice, Coptis chinensis, and jujube.

[0047] Clean Pinellia: Take clean Pinellia, separate the large and small ones, and soak them in 8% alum solution until there is no dry core left and a slight numbness on the tongue. Remove, wash, cut into thick slices, and dry. For every 100kg of clean Pinellia, use 12.5kg of alum for boiling and 20kg for soaking.

[0048] Scutellaria baicalensis: Remove impurities, steam for half an hour, take out, cut into thin slices, and dry (be careful to avoid direct sunlight).

[0049] Dried ginger: remove impurities, soak briefly, wash, moisten thoroughly, cut into thick slices or blocks, and dry.

[0050] Ginseng: moisten thoroughly, slice thinly, and dry

[0051] Stir-fry licorice: remove impurities, wash, soak, cut into thick slices, dry and sieve the medicine; take the licorice slices and stir-fry until slightly yellow, then take out and let cool.

[0052] Coptis chinensis: remove impurities, soak thoroughly, cut into thin slices, and dry.

[0053] Jujube: Remove impurities, wash, and dry in the sun. Crack or remove the core before use.

[0054] 1.3 Decoction process of the reference sample of Banxia Xiexin Granules

[0055] Prescription: Pinellia 34.5g, Scutellaria baicalensis 41.4g, Dried ginger 41.4g, Ginseng 41.4g, Fried licorice 41.4g, Coptis chinensis 13.8g, Jujube 36.0g.

[0056] The preparation method of the benchmark sample is as follows: according to the prescription ratio, 34.50g of Pinellia ternata, 41.40g of Scutellaria baicalensis, 41.40g of dried ginger, 41.40g of ginseng, 41.40g of stir-fried licorice, 13.80g of Coptis chinensis, and 36.00g of jujube were weighed, totaling 249.90g, placed in a ceramic jar, added with 2000ml of water, and soaked for 30min. First, heated to boiling with high heat (800w), and then decocted with low heat (500w) for 55min. The decoction was about 1200ml. The solid-liquid separation was carried out while hot (filtered with a 150-mesh pharmacopoeia sieve), and the decoction was concentrated under reduced pressure at 60℃ to 600ml. The concentrate was freeze-dried in a freeze dryer and freeze-dried for 48h. The freeze-dried powder was collected, weighed, and the yield was calculated. The freeze-dried powder was packaged with a pharmaceutical composite film and sealed for storage, thus obtaining 15 batches of benchmark samples.

[0057] Properties: This product is a light yellow to brown powder with a slight fragrance and a sweet taste followed by a bitter aftertaste.

[0058] 1.4 Investigation of the paste yield of benchmark samples

[0059] Decoction was performed according to the process in 1.3 above and the paste yield of the benchmark sample was calculated. The relevant information is shown in Table 1.

[0060] Table 1: Test data of paste yield of 15 batches of benchmark samples

[0061]

[0062] Method for determining the dry extract yield: Accurately measure 25 ml of a known volume of prepared medicinal solution and place it in an evaporating dish at a constant weight. Evaporate to dryness in a waterbath, dry at 105°C for 3 hours, cool in a desiccator for 30 minutes, quickly weigh, and continue drying in a 105°C oven until constant weight is reached. Calculate: Extract yield = (weight of dried extract and evaporating dish - weight of evaporating dish) × (volume of filtrate at constant volume / volume of aspirated filtrate) / weight of the medicinal piece × 100%.

[0063] The average yield of 15 batches of Banxia Xiexin Granule benchmark samples was 23.49%, with a standard deviation of 1.17 and a relative standard deviation (RSD) of 4.98%. Based on relevant technical guidelines, the yield range was established as 90% to 110% of the mean, resulting in a yield range of 21.1% to 25.8%. These results indicate that the yield of all 15 benchmark samples fell within this range, providing a reference for quality control studies of Banxia Xiexin Granules. Taking all these factors into consideration, the yield range was established as 21.1% to 25.8%.

[0064] 2. Study on the quality of the reference sample of Banxia Xiexin Granule

[0065] 2.1 Test according to thin layer chromatography (General Chapter 0502 of the Chinese Pharmacopoeia 2020 Edition)

[0066] Test (1): Take 3g of the powder of this product, add 30ml of methanol, ultrasonically treat for 15min, filter, evaporate the filtrate to dryness, add 2ml of methanol to dissolve the residue, and use it as the test solution; take 1g of licorice control medicinal material, add 50ml of water, decoct for 30min, let cool, filter, add double the amount of anhydrous ethanol to the filtrate, shake well, centrifuge, take the supernatant, evaporate to dryness, add 2ml of methanol to dissolve the residue, and use it as the control medicinal material solution; conduct the thin layer chromatography test, take 2μl of the test solution and 10μl of the control medicinal material solution, respectively, and spot them on the same silica gel G thin layer plate, use toluene-ethyl formate-formic acid solution with a volume ratio of 15:8:1.5 as the developing agent, develop, take out, dry, and inspect under a 365nm ultraviolet lamp. Figure 1 As shown, in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material.

[0067] Test (2): Take 4g of the powder of this product, add 30ml of ether, heat and reflux for 15min, cool, filter, evaporate the filtrate, add 0.5ml of ethyl acetate to the residue to dissolve it, and use it as the test solution; take 1g of the control medicinal material of dried ginger, and prepare the control medicinal material solution in the same way; perform the thin layer chromatography test, take 5μl of the test solution and 1μl of the control medicinal material solution, and spot them on the same silica gel G thin layer plate, use a toluene-ethyl acetate solution with a volume ratio of 2:1 as the developing agent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, heat at 105℃ until the spots are clearly colored, and inspect under sunlight. Figure 2 As shown, in the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material.

[0068] Test (3): Take 0.5g of the powder of this product, add 25ml of methanol, ultrasonically treat for 30 minutes, filter, and take the filtrate as the test solution; take 0.25g of the reference medicinal material Coptis chinensis, and prepare the reference medicinal material solution in the same way; perform the thin layer chromatography test, take 2μl of the test solution and the reference medicinal material solution, and spot them on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine solution with a volume ratio of 3:3.5:1:1.5:0.5:1 as the developing agent, place it in a developing cylinder pre-saturated with ammonia vapor, develop, take out, dry, and inspect under a 365nm ultraviolet lamp. Figure 3 As shown, in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material.

[0069] Test (4): Take 2g of the powder of this product, add 50ml of ethanol, heat and reflux for 1 hour, cool, filter, evaporate the filtrate to dryness, add 20ml of water to the residue, heat to dissolve, cool, adjust the pH value to 1-2 with hydrochloric acid, extract with 30ml of ethyl acetate by shaking, separate the ethyl acetate layer, evaporate to dryness, add 1ml of ethanol to the residue to dissolve, and use it as the test solution; take 1g of Scutellaria baicalensis as a control medicinal material, and prepare the control medicinal material solution in the same way; perform the thin layer chromatography test, take 2μl of the test solution and the control medicinal material solution, respectively, and spot them on the same polyamide film, use ethyl acetate-methanol-acetic acid solution with a volume ratio of 4:1:10 as the developing agent, develop, take out, dry, spray with 2% ferric chloride solution, and inspect under sunlight. Figure 4 As shown, in the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material.

[0070] Test (5): Take 2g of the powder of this product, add 30ml of water, stir evenly, ultrasonicate for 20min, centrifuge, take the supernatant, extract twice with chloroform (shake gently), 20ml / time, discard the chloroform solution, extract the water layer with water-saturated n-butanol twice, 30ml / time, combine the n-butanol solution, and then wash twice with n-butanol-saturated ammonia test solution, 60ml / time, take the n-butanol solution, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution; take another ginseng Prepare a control medicinal material solution in the same manner as for 1g of medicinal material; perform the thin layer chromatography test, taking 5μl of the test solution and 2μl of the control medicinal material solution, respectively, and spot them on the same silica gel G thin layer plate, using the lower layer solution of chloroform-ethyl acetate-methanol-glacial acetic acid-water solution with a volume ratio of 10:4:5:2:2 placed below 10℃ as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and inspect under sunlight. Figure 5 As shown, in the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material.

[0071] 2.2 Leaching matter

[0072] According to the hot infiltration method under the alcohol-soluble extract determination method (General Chapter 2201 of the 2020 edition of the Chinese Pharmacopoeia), ethanol was used as the solvent. The average extract determination results of 15 batches of benchmark samples were 41.37%, and the extract range of the mean ± 30% was: 28.96~53.78%, see Table 2 for details.

[0073] Table 2: Extractables from 15 batches of benchmark samples

[0074]

[0075] 2.3 Characteristic spectrum test method of the reference sample of Banxia Xiexin Granules

[0076] Banxia Xiexin Decoction primarily contains flavonoids, alkaloids, saponins, and glycosides. Traditional Chinese medicine compound preparations are composed of multiple Chinese medicinal materials, with multiple components acting synergistically and targeting multiple targets. Characteristic chromatograms combined with multi-index component content determination can reflect the overall basic substance content of classic prescriptions. Using a constructed HPLC characteristic chromatogram method for Banxia Xiexin Decoction, the mass fractions of four components—6-gingerol, baicalin, glycyrrhizic acid, and berberine hydrochloride—were simultaneously determined. This method provides a basis for the development and production of reference samples and granule preparations for the classic Banxia Xiexin Decoction.

[0077] The characteristic spectrum was determined by high performance liquid chromatography (General Chapter 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0078] 2.3.1 Determination of chromatographic conditions

[0079] (1) Selection of organic phase: Two organic phases, methanol and acetonitrile, were investigated in the experiment. When the organic phase in the mobile phase was methanol, the separation was better, so methanol was selected as the organic phase.

[0080] (2) Investigation of aqueous phase: The experiment investigated three different aqueous phases: 0.1% formic acid water, 0.1% acetic acid water and 0.1% phosphoric acid water. The results showed that the peak shape of 0.1% acetic acid water was poor, and the fingerprint spectra of 0.1% formic acid water and 0.1% phosphoric acid water were slightly different. Considering that phosphoric acid is an inorganic acid and has poor solubility with organic acids, 0.1% formic acid water was selected as the aqueous phase.

[0081] (3) Selection of extraction solvent: The experiment investigated five extraction solvents, including methanol, 50% methanol, pure water, 50% ethanol and ethanol. It was found that the extraction efficiency of methanol and ethanol was low and the chromatographic peaks were fewer. When the solvent was 50% methanol, the peak area was larger, so 50% ethanol was selected as the extraction solvent.

[0082] (4) Selection of extraction method: The experiment investigated two extraction methods: reflux and ultrasonic. The results showed that the peak areas of different extraction methods at a retention time of 36 min were 38287525 and 25550772 respectively. Reflux extraction efficiency was better, and the peak shape of reflux was better than that of ultrasonic. Therefore, reflux was selected as the extraction method.

[0083] (5) Selection of extraction time: The experiment examined reflux times of 30 min, 60 min, and 120 min. The results showed that the extraction time had little effect on the peak shape, separation, and peak area, so 30 min was finally selected as the extraction time.

[0084] (6) Optimization of separation conditions

[0085] Chromatographic column: InertSustain C18 (5μm, 4.6×250mm); mobile phase: methanol-0.1% formic acid water; volume flow rate: 1.0ml / min; column temperature, 30°C; wavelength: 254nm (glycyrrhizic acid), 280nm (berberine hydrochloride, baicalin, 6-gingerol); injection volume, 5μl, optimized chromatographic gradient conditions, the results showed that under the separation gradient 5, the separation degree of the index component peak was better, so gradient 5 was selected as the separation gradient.

[0086] Table 3: Gradient 1

[0087] Time (min) Methanol (%) 0.1% formic acid water (%) 0 5 95 5 27 73 15 34 66 40 45 55 70 66 34 77 76 24 95 85 15 110 5 95

[0088] Table 4: Gradient 2

[0089] Time (min) Methanol (%) 0.1% formic acid water (%) 0 5 95 10 35 65 40 45 55 50 51 49 65 60 40 75 66 34 82 76 24 85 78 22 90 85 15

[0090] Table 5: Gradient 3

[0091]

[0092]

[0093] Table 6: Gradient 4

[0094] Time (min) Methanol (%) 0.1% formic acid water (%) 0 5 95 5 27 73 50 40 60 65 45 55 87 66 34 94 76 24 110 85 15

[0095] Table 7: Gradient 5

[0096] Time (min) Methanol (%) 0.1% formic acid water (%) 0 5 95 5 27 73 25 32 68 45 40 60 60 45 55 82 66 34 89 76 24 105 85 15

[0097] In summary, the optimal chromatographic conditions are:

[0098] Chromatographic column: InertSustain C18 (5 μm, 4.6×250 mm); mobile phase: methanol-0.1% formic acid water; volume flow rate:

[0099] 1.0 ml / min; column temperature, 30°C; wavelength: 254 nm (glycyrrhizic acid), 280 nm (berberine hydrochloride, baicalin, 6-gingerol); injection volume, 5 μl; gradient, see Table 7.

[0100] Preparation of test solution: Take 0.5 g of Banxia Xiexin Granules standard sample powder, accurately weigh it, place it in a stoppered conical flask, add 10 ml of 50% ethanol, weigh it, reflux it for 30 minutes, let it cool, make up the weight loss with 50% ethanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0101] Preparation of reference solution: Accurately weigh appropriate amounts of berberine hydrochloride reference, baicalin reference, 6-gingerol reference, and ammonium glycyrrhizate reference. Add methanol to each solution to produce a solution containing 0.20 mg of berberine hydrochloride, 0.50 mg of baicalin, 0.40 mg of 6-gingerol, and 0.25 mg of ammonium glycyrrhizate per 1 ml.

[0102] 2.3.2 Methodological Investigation

[0103] (1) Repeatability About 0.5 g of the reference sample was weighed accurately and prepared in parallel for 6 times. The samples were injected separately and the chromatograms were recorded and integrated. Peak 5 (baicalin) was used as the reference peak to calculate the relative retention time and relative peak area. The results are shown in Tables 8 and 9. The RSD of the relative retention time and relative peak area was less than 5%. The experiment showed that the repeatability of this method was good.

[0104] Table 8: Repeatability data of the reference sample of Banxia Xiexin Granule (relative retention time)

[0105]

[0106] Table 9: Repeatability data of the benchmark sample of Banxia Xiexin Granule (relative peak area)

[0107]

[0108] (2) Precision: About 0.5 g of the reference sample was weighed, accurately weighed, processed, and injected six times continuously. The chromatogram was recorded. Peak 5 (baicalin) was used as the reference peak to calculate the relative retention time and relative peak area. The results are shown in Tables 10 to 11. The RSD values ​​of the relative retention time and relative peak area were less than 5%. The test showed that the precision of this method was good.

[0109] Table 10: Precision data of the reference sample of Banxia Xiexin Granule (relative retention time)

[0110]

[0111]

[0112] Table 11: Precision data of the reference sample of Banxia Xiexin Granule (relative peak area)

[0113]

[0114] (3) Stability About 0.5 g of the reference sample was weighed accurately and processed. The sample was injected at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, and 24 h, respectively. The chromatogram was recorded and integrated. Peak 5 (baicalin) was used as the reference peak to calculate the relative retention time and relative peak area. The results are shown in Tables 12 to 13. The RSD of the relative retention time and relative peak area was less than 10%. The test showed that the test solution had good stability within 24 h.

[0115] Table 12: Stability data of the reference sample of Banxia Xiexin Granules (relative retention time)

[0116]

[0117] Table 13: Stability data of the reference sample of Banxia Xiexin Granules (relative peak area)

[0118]

[0119]

[0120] (4) Durability: Take the reference sample of Banxia Xiexin Granules and prepare the test solution. Determine according to the method, record the chromatogram and integrate it. Take Peak 5 (baicalin) as the reference peak, calculate the relative retention time and relative peak area. The relative retention time and relative peak area RSD are less than 10%. The test shows that the test solution has good durability at different flow rates. The results are shown in Tables 14 to 17. The relative retention time and relative peak area RSD at different column temperatures are greater than 10%. It is recommended to control the column temperature at 30°C.

[0121] Table 14: Different flow rate data (relative retention time) of the Banxia Xiexin granule benchmark sample

[0122]

[0123] Table 15: Data of different flow rates of the Banxia Xiexin granule benchmark sample (relative peak area)

[0124]

[0125]

[0126] Table 16: Data of different column temperatures for the reference sample of Banxia Xiexin Granules (relative retention time)

[0127]

[0128] Table 17: Data of different column temperatures for the reference sample of Banxia Xiexin Granules (relative peak area)

[0129]

[0130] (5) Research on the identification of characteristic spectral peaks of reference samples

[0131] Confirmation of reference substance: Use reference substance to identify each chromatographic peak in the characteristic spectrum. The preparation of reference substance solution refers to the preparation of reference substance solution. Inject samples separately according to the optimal chromatographic conditions and identify them with reference substance.

[0132] Summary: The repeatability, precision, stability and durability of the characteristic spectrum method were tested, and the results met the requirements, indicating that the established method can be well used for the characteristic spectrum determination of the Banxia Xiexin Granules benchmark sample.

[0133] 2.3.3 Determination and evaluation of characteristic spectra of reference samples

[0134] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler, methanol as mobile phase A, and 0.1% formic acid solution as mobile phase B. Gradient elution was performed according to the specifications in Table 18. The flow rate was 1.0 ml / min, the column temperature was 30°C, and the detection wavelengths were 254 nm (glycyrrhizic acid) and 280 nm (berberine hydrochloride, baicalin, and 6-gingerol). The number of theoretical plates calculated for baicalin should be no less than 1500.

[0135] Table 18: Gradient elution program

[0136] Time (min) Mobile phase A (%) Mobile phase B (%) 0 5 95 5 27 73 25 32 68 45 40 60 60 45 55 82 66 34 89 76 24 105 85 15

[0137] Reference solution preparation

[0138] Accurately weigh appropriate amounts of berberine hydrochloride reference substance, baicalin reference substance, 6-gingerol reference substance, and ammonium glycyrrhizate reference substance. Add methanol to each of these solutions to prepare a solution containing 0.20 mg of berberine hydrochloride, 0.50 mg of baicalin, 0.40 mg of 6-gingerol, and 0.25 mg of glycyrrhizic acid per 1 ml.

[0139] Preparation of test solution

[0140] Take about 0.5g of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 10ml of 50% ethanol, weigh it, reflux it for 30 minutes, let it cool, weigh it again, make up the weight loss with 50% ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0141] Determination method: Accurately pipette 5 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0142] Determination of characteristic spectra of 15 batches of reference samples: 15 batches of reference samples were tested according to the above test method. The results are shown in Figure 6-9 . The test sample chromatogram should show 11 characteristic peaks, of which 4 peaks should correspond to the retention time of the corresponding reference peak. The peak corresponding to the baicalin reference peak is the S peak. See Tables 19 to 20 for details, calculate the relative retention time of each characteristic peak and the S peak, and the relative retention time should be within the range of ±10% of the specified value. The specified value is 0.54 (peak 1), 0.60 (peak 2), 0.72 (peak 3), 0.75 (peak 4), 1.10 (peak 6), 1.14 (peak 7), 1.15 (peak 8), 1.19 (peak 9), 1.34 (peak 10), 1.48 (peak 11).

[0143] Table 19: Relative retention time of Banxia Xiexin granule benchmark samples

[0144]

[0145]

[0146] Table 20: Relative peak areas of the reference samples of Banxia Xiexin Granules

[0147]

[0148] 2.4 Content determination

[0149] Determined by high performance liquid chromatography (General Chapter 0512 of the Chinese Pharmacopoeia 2020 edition).

[0150] (1) Chromatographic conditions

[0151] Chromatographic column: InertSustain C18 (5 μm, 4.6 × 250 mm); mobile phase: methanol-0.1% formic acid in water; flow rate: 1.0 ml / min; column temperature: 30°C; wavelength: 254 nm (glycyrrhizic acid), 280 nm (berberine hydrochloride, baicalin, 6-gingerol); injection volume: 5 μl; gradient: see Table 21:

[0152] Table 21:

[0153] Time (min) Methanol (%) 0.1% formic acid water (%) 0 5 95 5 27 73 25 32 68 45 40 60 60 45 55 82 66 34 89 76 24 105 85 15

[0154] Preparation of test solution

[0155] Take 0.5 g of the powder of the reference sample of Banxia Xiexin Granules, accurately weigh it, place it in a stoppered conical flask, add 10 ml of 50% ethanol, weigh it, reflux it for 30 minutes, let it cool, make up the weight loss with 50% ethanol, shake it well, filter it, and take the filtrate to obtain the product.

[0156] Preparation of reference solution

[0157] Accurately weigh appropriate amounts of berberine hydrochloride reference substance, baicalin reference substance, 6-gingerol reference substance, and ammonium glycyrrhizate reference substance. Add methanol to each of these solutions to prepare a solution containing 0.20 mg of berberine hydrochloride, 0.50 mg of baicalin, 0.40 mg of 6-gingerol, and 0.25 mg of glycyrrhizic acid per 1 ml.

[0158] (2) Linear range

[0159] Accurately weigh appropriate amounts of berberine hydrochloride standard, baicalin standard, 6-gingerol standard, and ammonium glycyrrhizate standard. Place in a volumetric flask and add methanol to volume to serve as the standard stock solutions. Dilute and measure the chromatograms in sequence. Record and integrate the chromatograms, using berberine hydrochloride (280 nm), baicalin (280 nm), 6-gingerol (280 nm), and glycyrrhizic acid (254 nm) as reference peaks, and calculate the peak area. Construct a standard curve using concentration as the abscissa and peak area as the ordinate. The results are shown in Table 22.

[0160] Table 22: Linearity and Range

[0161] Reference substances Standard curve Range (mg / ml) <![CDATA[R 2 ]]> Berberine hydrochloride y=15838230.0886x-95888.4714 0.0217～0.6519 0.9999 Baicalin y=23526845.0796x-1662569.0516 0.3476～3.4764 0.9998 6-gingerol y=2439141.2122x+13683.7450 0.0085～0.4233 0.9999 Glycyrrhizic acid y=4244819.6657x-28431.9923 0.0240～0.7210 0.9999

[0162] (3) Spike recovery

[0163] Weigh 6 portions of the same sample with determined content, accurately weigh them, add appropriate amount of reference substance to each of them, treat them according to the preparation method of the test solution, and determine them according to the law. The results are shown in Table 23. The test shows that this method has a good sample recovery rate.

[0164] Table 23: Spike recovery test data

[0165]

[0166]

[0167] (4) Sample determination Fifteen batches of Banxia Xiexin Granules were used to determine the contents of berberine, baicalin, 6-gingerol, and glycyrrhizic acid. The results are shown in Table 24. The average berberine content was 2.66 mg / g, with a mean ± 30% range of 1.86 to 3.46 mg / g. The measured content range was 2.28 to 3.29 mg / g. The average baicalin content was 54.04 mg / g, with a mean ± 30% range of 37.83 to 70.26 mg / g. The measured content range was 41.08 to 65.84 mg / g. The average 6-gingerol content was 0.37 mg / g, with a mean ± 30% range of 0.26 to 0.49 mg / g. The measured content range is 0.15-0.53 mg / g, the average glycyrrhizic acid content is 5.59 mg / g, the mean ± 30% content range is: 3.91-7.27 mg / g. The measured content range is 4.34-7.05 mg / g.

[0168] Table 24: Content of index components in 15 batches of Banxia Xiexin Granule benchmark samples

[0169]

[0170]

[0171] The experimental results indicate that a high-performance liquid chromatography (HPLC) method for detecting the characteristic spectrum of this product has been established. Methodological validation has demonstrated excellent repeatability, precision, and stability. This method was also used to determine the characteristic spectrum peaks (11 characteristic peaks) in a reference sample of Banxia Xiexin Granules. The characteristic spectrum of 15 batches of samples was analyzed and evaluated using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2004 Edition)" to determine the reference sample characteristic spectrum. The test sample chromatogram should exhibit 11 characteristic peaks, four of which should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the baicalin reference peak is the S peak. Calculate the relative retention time of each characteristic peak and S peak. The relative retention time should be within ±10% of the specified value, 0.54 (peak 1), 0.60 (peak 2), 0.72 (peak 3), 0.75 (peak 4), 1.10 (peak 6), 1.14 (peak 7), 1.15 (peak 8), 1.19 (peak 9), 1.34 (peak 10), 1.48 (peak 11).

[0172] The average berberine content of 15 batches of benchmark samples was 2.66 mg / g, with a range of ±30% of the mean of 1.86 to 3.46 mg / g. The actual content range was 2.28 to 3.29 mg / g, all within the range of ±30% of the mean, meeting the standard requirements.

[0173] The average baicalin content of 15 batches of benchmark samples was 54.04 mg / g, with a range of ±30% of the mean of 37.83 to 70.26 mg / g. The actual content range was 41.08 to 65.84 mg / g, all within the range of ±30% of the mean, meeting the standard requirements.

[0174] The average 6-gingerol content of 15 batches of benchmark samples was 0.37 mg / g, with a mean ± 30% range of 0.26 to 0.49 mg / g. The actual content range was 0.15 to 0.53 mg / g.

[0175] The average glycyrrhizic acid content of 15 batches of benchmark samples was 5.59 mg / g, with a range of ±30% of the mean of 3.91 to 7.27 mg / g. The actual content range was 4.34 to 7.05 mg / g, all within the range of ±30% of the mean, meeting the standard requirements.

[0176] The final benchmark sample content was determined to be 1.86-3.46 mg / g for berberine, 37.83-70.26 mg / g for baicalin, 0.26-0.49 mg / g for 6-gingerol, and 3.91-7.27 mg / g for glycyrrhizic acid. Because 6-gingerol is relatively discrete and falls outside the ±30% range of the mean, it is not included in the quality standard.

[0177] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples; any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting the quality of a reference sample of Banxia Xiexin granules, characterized in that: include: Characteristic profiles were constructed for the reference sample of Banxia Xiexin Granules, and the contents of baicalin, glycyrrhizic acid, and berberine were determined. The content standards of the reference sample of Banxia Xiexin Granules were limited to baicalin content of 37.83-70.26 mg / g, glycyrrhizic acid content of 3.91-7.27 mg / g, and berberine content of 1.86-3.46 mg / g. Liquid chromatography was used for the construction of characteristic profiles and the determination of the contents of baicalin, glycyrrhizic acid, and berberine. The characteristic spectrum of the Banxia Xiexin Granule reference sample determined by liquid chromatography includes: using a solution made from a berberine hydrochloride reference substance as a reference solution a1, a solution made from a baicalin reference substance as a reference solution a2, a solution made from a 6-gingerol reference substance as a reference solution a3, a solution made from an ammonium glycyrrhizate reference substance as a reference solution a4, and a solution made from the Banxia Xiexin Granule reference sample as a test solution a, accurately aspirating the reference solution a1, the reference solution a2, the reference solution a3, the reference solution a4, and the test solution a, respectively, and injecting them into a liquid chromatograph for determination, thereby obtaining the result; wherein, the chromatographic conditions used are: a chromatographic column: octadecylsilane bonded silica gel as a filler, methanol as a mobile phase A, 0.1% by volume formic acid solution as a mobile phase B, and gradient elution according to the provisions in Table a; Table a Gradient elution program Flow rate: 0.8-1.2 ml / min; column temperature: 28-32°C; injection volume: 4-6 μl; detection wavelength for glycyrrhizic acid: 250-258 nm; detection wavelength for berberine hydrochloride, baicalin, and 6-gingerol: 275-285 nm; The method also includes identifying the Banxia Xiexin granule reference sample using thin layer chromatography, wherein the thin layer chromatography method includes the following steps: (1) Take 3 g of Banxia Xiexin Granules reference sample powder, add 30 ml of methanol, ultrasonically treat for 15 min, filter, evaporate the filtrate to dryness, add 2 ml of methanol to dissolve the residue, and use it as the test solution b1; take 1 g of licorice control medicinal material, add 50 ml of water, decoct for 30 min, let cool, filter, add double the amount of anhydrous ethanol to the filtrate, shake well, centrifuge, take the supernatant, evaporate to dryness, add 2 ml of methanol to dissolve the residue, and use it as the control medicinal material solution b1; perform the thin layer chromatography test, take 2 μl of the test solution b1 and 10 μl of the control medicinal material solution b1, respectively, and spot them on the same silica gel G thin layer plate, use toluene-ethyl formate-formic acid solution with a volume ratio of 15:8:1.5 as the developing solvent, develop, take out, dry, and inspect under a 365 nm ultraviolet lamp; (2) Take 4 g of the powder of the reference sample of Banxia Xiexin Granules, add 30 ml of ether, heat and reflux for 15 min, cool, filter, evaporate the filtrate, and dissolve the residue in 0.5 ml of ethyl acetate to prepare the test solution b2; take 1 g of the reference medicinal material of dried ginger, add 30 ml of ether, heat and reflux for 15 min, cool, filter, evaporate the filtrate, and dissolve the residue in 0.5 ml of ethyl acetate to prepare the control medicinal material solution b2; perform the thin layer chromatography test, take 5 μl of the test solution b2 and 1 μl of the control medicinal material solution b2, and spot them on the same silica gel G thin layer plate respectively, use toluene-ethyl acetate solution with a volume ratio of 2:1 as the developing solvent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, heat at 105 °C until the spots are clearly colored, and inspect under sunlight; (3) Take 0.5 g of the reference sample powder of Banxia Xiexin Granules, add 25 ml of methanol, ultrasonically treat for 30 minutes, filter, and take the filtrate as the test solution b3; take 0.25 g of the reference medicinal material of Coptis chinensis, add 25 ml of methanol, ultrasonically treat for 30 minutes, filter, and take the filtrate as the reference medicinal material solution b3; perform the thin layer chromatography test, take 2 μl of the test solution b3 and the reference medicinal material solution b3, respectively, and spot them on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine solution with a volume ratio of 3:3.5:1:1.5:0.5:1 as the developing solvent, place it in a developing cylinder pre-saturated with ammonia vapor, develop, take out, dry, and inspect under a 365 nm ultraviolet lamp; (4) Take 2 g of Banxia Xiexin Granules reference sample powder, add 50 ml of ethanol, heat and reflux for 1 hour, let cool, filter, evaporate the filtrate to dryness, add 20 ml of water to the residue, heat to dissolve, let cool, adjust the pH value to 1-2 with hydrochloric acid, shake and extract with 30 ml of ethyl acetate, separate the ethyl acetate layer, evaporate to dryness, add 1 ml of ethanol to dissolve the residue, and use it as the test solution b4; take 1 g of Huangqin reference medicinal material, add 50 ml of ethanol, heat and reflux for 1 hour, let cool, filter, evaporate the filtrate to dryness, add 20 ml of water to the residue, and use it as the test solution b4. ml, heat to dissolve, cool, adjust the pH to 1-2 with hydrochloric acid, and extract by shaking with 30 ml of ethyl acetate. Separate the ethyl acetate layer, evaporate to dryness, and dissolve the residue in 1 ml of ethanol to serve as control medicinal solution B4. Perform thin-layer chromatography (TLC) tests by spotting 2 μl each of test solution B4 and control medicinal solution B4 onto the same polyamide film. Develop with a 4:1:10, volume ratio of ethyl acetate-methanol-acetic acid solution. Remove, air-dry, spray with 2% ferric chloride solution, and examine under sunlight. (5) Take 2 g of Banxia Xiexin Granules reference sample powder, add 30 ml of water, stir evenly, ultrasonically treat for 20 min, centrifuge, take the supernatant, extract twice with chloroform, 20 ml / time, discard the chloroform solution, extract the aqueous layer with water-saturated n-butanol twice, 30 ml / time, combine the n-butanol solution, wash twice with n-butanol-saturated ammonia test solution, 60 ml / time, take the n-butanol solution, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution b5; take 1 g of ginseng control medicinal material, add 30 ml of water, stir evenly, ultrasonically treat for 20 min, centrifuge, take the supernatant, extract twice with chloroform, 20 ml / time, discard the chloroform solution, extract the aqueous layer with water-saturated n-butanol twice, 30 ml / time, combine the n-butanol solution, wash twice with n-butanol-saturated ammonia test solution, 60 ml / time, ml / time, take the n-butanol solution, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the control medicinal material solution b5; carry out the thin layer chromatography test, take 5 μl of the test solution b5 and 2 μl of the control medicinal material solution b5, respectively, and spot them on the same silica gel G thin layer plate, use the lower layer solution of chloroform-ethyl acetate-methanol-glacial acetic acid-water solution with a volume ratio of 10:4:5:2:2 placed below 10℃ as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and inspect under sunlight.

2. The Banxia Xiexin Granule reference sample quality detection method according to claim 1, wherein: The characteristic spectrum of the reference sample of Banxia Xiexin Granules determined by liquid chromatography also includes: (1) Preparation of reference solutions: Take appropriate amounts of berberine hydrochloride reference substance, baicalin reference substance, 6-gingerol reference substance, and ammonium glycyrrhizate reference substance, accurately weigh them, and add methanol to prepare reference solution a1 containing 0.20 mg of berberine hydrochloride per ml, reference solution a2 containing 0.50 mg of baicalin per ml, reference solution a3 containing 0.40 mg of 6-gingerol per ml, and reference solution a4 containing 0.25 mg of glycyrrhizic acid per ml; (2) Preparation of test solution a: Take 0.5 g of the reference sample of Banxia Xiexin Granules, accurately weigh it, place it in a stoppered conical flask, accurately add 10 ml of 50% ethanol, weigh it, reflux it for 30 minutes, let it cool, weigh it again, make up the weight loss with 50% ethanol, shake it well, filter it, and take the filtrate as the test solution a.

3. The Banxia Xiexin Granule reference sample quality detection method according to claim 1 or 2, wherein: The chromatogram of the test sample determined by liquid chromatography showed 11 characteristic peaks, of which 4 peaks corresponded to the 4 reference peaks of the reference sample chromatogram. Peak 5 corresponding to the baicalin reference peak was used as the reference peak S. The relative retention times of peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, peak 10, and peak 11 in the chromatogram of the test sample and the reference peak S were calculated. The specified values ​​were: peak 1 relative retention time 0.54, peak 2 relative retention time 0.60, peak 3 relative retention time 0.72, peak 4 relative retention time 0.75, peak 6 relative retention time 1.10, peak 7 relative retention time 1.14, peak 8 relative retention time 1.15, peak 9 relative retention time 1.19, peak 10 relative retention time 1.34, and peak 11 relative retention time 1.

48. The relative retention times of the detected characteristic peaks should be within ±10% of the specified values.

4. The Banxia Xiexin Granule reference sample quality detection method according to claim 1 or 2, wherein: The liquid chromatography method for determining the content of a single component of baicalin, glycyrrhizic acid, and berberine includes: performing liquid chromatography analysis, using a solution prepared from a berberine hydrochloride reference substance as a reference solution c1, a solution prepared from a baicalin reference substance as a reference solution c2, a solution prepared from an ammonium glycyrrhizate reference substance as a reference solution c3, and a solution prepared from a reference sample of Banxia Xiexin Granules as a test solution c, accurately aspirating the reference solution c1, the reference solution c2, the reference solution c3, and the test solution c, respectively, and injecting them into the liquid chromatograph for determination, to obtain the results; wherein, the chromatographic conditions used are: a chromatographic column: octadecylsilane bonded silica gel as a filler, methanol as a mobile phase A, and a volume fraction of 0.1 % formic acid solution as mobile phase B, gradient elution according to the provisions in Table a, flow rate: 0.8-1.2 ml / min; column temperature: 28-32°C; injection volume: 4-6 μl; glycyrrhizic acid detection wavelength: 250-258 nm, berberine hydrochloride and baicalin detection wavelength: 275-285 nm.

5. The Banxia Xiexin Granule reference sample quality detection method according to claim 4, characterized in that: The content of baicalin, glycyrrhizic acid and berberine as single components determined by liquid chromatography also includes: (1) Preparation of reference solution: Take appropriate amount of berberine hydrochloride reference, baicalin reference, and ammonium glycyrrhizate reference, weigh accurately, and add methanol to prepare reference solution C1 containing 0.20 mg of berberine hydrochloride per ml, reference solution C2 containing 0.50 mg of baicalin per ml, and reference solution C3 containing 0.25 mg of glycyrrhizic acid per ml; (2) Preparation of test solution: Take 0.5 g of the powder of the reference sample of Banxia Xiexin Granules, accurately weigh it, place it in a stoppered conical flask, accurately add 10 ml of 50% ethanol, weigh it, reflux it for 30 min, let it cool, weigh it again, make up the weight loss with 50% ethanol, shake it well, filter it, and take the filtrate as the test solution C.

6. The Banxia Xiexin Granule reference sample quality detection method according to claim 4, wherein: It also includes the use of liquid chromatography to determine the content of 6-gingerol in the Banxia Xiexin Granules benchmark sample, and limiting the 6-gingerol content standard of the Banxia Xiexin Granules benchmark sample to 0.26~0.49 mg / g.

7. The Banxia Xiexin Granule reference sample quality detection method according to claim 6, wherein: The liquid chromatography method for determining the content of a single component of 6-gingerol includes: performing liquid chromatography analysis, using a solution prepared from a 6-gingerol reference substance as the reference solution c4, accurately aspirating the reference solution c4 and the test solution c, respectively, injecting them into the liquid chromatograph, and determining the content; wherein the chromatographic conditions used are: a chromatographic column: octadecylsilane bonded silica gel as a filler, methanol as mobile phase A, 0.1% by volume formic acid solution as mobile phase B, gradient elution according to the provisions in Table a, flow rate: 0.8-1.2 ml / min; column temperature: 28-32° C.; injection volume: 4-6 μl; detection wavelength: 275-285 nm; Take an appropriate amount of 6-gingerol reference substance, accurately weigh it, and add methanol to prepare reference substance solution C4 containing 0.40 mg of 6-gingerol per 1 ml; Take 0.5 g of Banxia Xiexin Granules reference sample powder, accurately weigh it, place it in a stoppered conical flask, accurately add 10 ml of 50% ethanol, weigh it, reflux it for 30 minutes, let it cool, weigh it again, make up the weight loss with 50% ethanol, shake it well, filter it, and take the filtrate as the test solution C.

8. The Banxia Xiexin Granule reference sample quality detection method according to claim 1 or 2, wherein: It also includes the properties, dry paste yield, and extract testing of the Banxia Xiexin Granules benchmark sample. The extract testing is carried out by the hot infusion method using ethanol as the solvent, and the extract range is determined by the hot infusion method under the alcohol-soluble extract determination method.

9. The Banxia Xiexin Granule reference sample quality detection method according to claim 1 or 2, wherein: The chromatographic column has a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

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