Detection method and application of traditional Chinese medicine composition

Abstract

The invention discloses a detection method and application of a traditional Chinese medicine composition, and relates to the technical field of fingerprint spectrums of traditional Chinese medicinal materials. The method comprises the following steps: detecting by using a high performance liquid chromatography, and the conditions of the high performance liquid chromatography are as follows: a chromatographic column is a reverse phase silica gel bonded phase chromatographic column, and a mobile phase is an acid-containing aqueous solution; the phase B is an organic solvent; the traditional Chinese medicine composition is prepared from traditional Chinese medicine raw materials, and the traditional Chinese medicine raw materials comprise any one or a mixture of multiple of mint, fructus aurantii, pericarpium citri reticulatae, radix paeoniae alba, folium mori, semen armeniacae amarae, platycodon grandiflorum, radix asteris and liquorice. According to the method, the fingerprint spectrums of the product and the intermediate can be established through the same method, and the whole-process quality monitoring is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine fingerprint spectrum, and in particular to a detection method of a traditional Chinese medicine composition and application thereof. Background Art

[0002] Qingxuan Cough Relief Granules is a Chinese patent medicine that has been marketed and sold by Suzhong Pharmaceutical for many years. It is a compound preparation composed of nine ingredients: mulberry leaves, mint, bitter almonds (fried), platycodon, white peony root, bitter orange peel, tangerine peel, aster, and licorice. Its main effects are dispelling wind and heat, promoting lung function and relieving cough. It is generally used for cough caused by exogenous wind-heat in children. The main symptoms include cough, sputum, fever or nasal congestion, runny nose, slight aversion to wind and cold, red or sore throat, but its main functions and indications are dispelling wind and heat, promoting lung function and relieving cough as mentioned just now.

[0003] Traditional Chinese medicine fingerprint refers to the chromatogram or spectrum of a certain type or types of components that are common and characteristic in a certain type of traditional Chinese medicine or its raw materials and preparations. At present, when the effective ingredients of most traditional Chinese medicines are not clearly identified, the establishment of fingerprints is of great significance for effectively controlling the quality of traditional Chinese medicines, their raw materials and preparations.

[0004] For example, the document "High-performance liquid fingerprint of Xuanfei Zhike mixture" discloses a method for establishing the fingerprint of Xuanfei Zhike mixture using high-performance liquid chromatography, including the following steps: Test solution: accurately measure 5mL of Xuanfei Zhike mixture, place it in a 25mL volumetric bottle, add an appropriate amount of 50% methanol, shake well, ultrasonicate for 30min, cool, add 50% methanol to the scale, and filter through a 0.45μm microporous filter membrane. Establish the high-performance liquid fingerprint of Xuanfei Zhike mixture to provide a basis for the quality control and evaluation of Xuanfei Zhike mixture. Methods: Agilent, ZORBAX SB-C18 (4.6×250mm, 5μm) chromatographic column was used, and acetonitrile-0.1% formic acid water was used as the mobile phase for gradient elution at a detection wavelength of 254nm, with a flow rate of 1.0mL / min. Injection volume: 5μL; column temperature: 30℃.

[0005] When establishing fingerprints, because the drugs they target are different, that is, the specific active ingredient composition and content are different, the chromatographic conditions, mass spectrometry conditions, etc. cannot be universal, and there are great differences when applied to different drugs. At present, there is a lack of fingerprints and establishment methods for Qingxuan Anti-cough Granules and its intermediates, so as to better control the drug quality of Qingxuan Anti-cough Granules and ensure its clinical effectiveness and safety. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a detection method and a fingerprint establishment method for a Chinese medicine composition, and to improve the research on the drug quality control standard of the Chinese medicine composition.

[0007] To achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows: In one aspect, the present invention provides a method for detecting a Chinese medicine composition, characterized in that high performance liquid chromatography is used for detection, and the conditions of the high performance liquid chromatography are: Chromatographic column: reverse phase silica gel bonded phase chromatographic column, Mobile phase: Phase A, acid-containing aqueous solution; Phase B, organic solvent; The Chinese medicine composition is prepared from Chinese medicinal raw materials, which include any one or more mixtures of mint, fructus aurantii, tangerine peel, white peony root, mulberry leaf, bitter almond, platycodon, aster and licorice. Preferably, the Chinese medicinal raw materials include fructus aurantii and white peony root, and more preferably include mint, fructus aurantii, tangerine peel, white peony root, mulberry leaf, bitter almond, platycodon, aster and licorice.

[0008] Preferably, the reverse phase silica gel bonded phase chromatographic column is a C18 or C8 chromatographic column, preferably a C18 chromatographic column, more preferably a ZORBAX SB-C18 chromatographic column or a WondaSil C18 chromatographic column, further preferably an Agilent, ZORBAX SB-C18 chromatographic column or a Shimadzu WondaSil C18 chromatographic column.

[0009] Preferably, in the mobile phase, the acid is at least one of phosphoric acid, hydrochloric acid, sulfuric acid, and citric acid, more preferably phosphoric acid; the organic solvent is at least one of acetonitrile, ethanol, methanol, propanol, n-butanol, isobutanol, tetrahydrofuran, and DMF, more preferably acetonitrile.

[0010] More preferably, the mobile phase is: phase A, 0.05-0.15% phosphoric acid-water; phase B, acetonitrile; further preferably: phase A, 0.08-0.12% phosphoric acid-water; phase B, acetonitrile; further preferably, phase A, 0.1% phosphoric acid-water; phase B, acetonitrile.

[0011] Preferably, the flow rate of the HPLC is 0.4-1.0 mL / min, more preferably, the flow rate is 0.6-1.0 mL / min, and more preferably, it is 1.0 mL / min.

[0012] Preferably, the injection volume of the HPLC is 10-20 μL, and more preferably, the injection volume is 20 μL.

[0013] Preferably, the column temperature of the HPLC is 25-40°C, more preferably, the column temperature is 25-35°C, and more preferably, it is 35°C.

[0014] Preferably, the detection wavelength of the HPLC is 190-280 nm, and further preferably, the detection wavelength is 220 nm.

[0015] Preferably, the elution mode of the high performance liquid chromatography is gradient elution; further preferably, the gradient elution conditions are: Table 1.

[0016] More preferably: Table 2.

[0017] .

[0018] As a preferred embodiment of the present invention, the chromatographic conditions are as follows: Chromatographic column: Agilent, ZORBAX SB-C18; Mobile phase: Phase A, 0.1% phosphoric acid-water; Phase B, acetonitrile; Flow rate: 1.0 mL / min, Injection volume: 20 μL, Column temperature: 35°C, Detection wavelength: 220nm, Gradient elution: Table 3.

[0019] .

[0020] Preferably, the method comprises preparing the Chinese medicine composition into a test solution for testing; preferably, the method for preparing the test solution comprises: taking the test sample of the drug, extracting with a solvent, separating the solid and the liquid, and the obtained liquid is the test solution; further preferably, the method for preparing the test solution also satisfies any one or more of the following conditions: a. The solvent is an alcohol aqueous solution or an alcohol solvent; preferably, the alcohol is a C1~C6 alcohol, preferably any one or a combination of methanol, ethanol, propanol and butanol, more preferably methanol; preferably, the volume ratio of the alcohol solvent in the alcohol aqueous solution is 1%~90%, preferably 2%~60%, more preferably 5%~40%; b. In g / mL, the ratio of the test sample to the solvent is 1:30 to 100, preferably 1:40 to 80, and more preferably 50 to 80; c. The extraction method includes ultrasonic extraction or heating reflux extraction; preferably, when ultrasonic extraction is used, the extraction temperature is 15~40°C; d. The extraction time is 15 min or more, preferably 15 min to 2 h, and more preferably 30 to 60 min; e. The solid-liquid separation method is centrifugation and / or filtration; preferably, the centrifugation conditions are: 10000-15000r / min at 15-25°C for 5-30min; further preferably, the filtration is through a 0.4-0.5μm filter membrane.

[0021] The Chinese medicine composition is a mixture of peppermint extract, fructus aurantii extract, tangerine peel extract, white peony root extract, mulberry leaf extract, bitter almond extract, platycodon grandiflorum extract, aster root extract and licorice extract; further, the Chinese medicine composition is prepared from the following Chinese medicinal materials in parts by weight: 60-120 parts of peppermint, 60-120 parts of fructus aurantii, 100-150 parts of tangerine peel, 100-150 parts of white peony root, 150-220 parts of mulberry leaf, 50-150 parts of bitter almond, 80-160 parts of platycodon grandiflorum, 80-160 parts of aster root and 60-120 parts of licorice. 0 parts; most preferably, the raw materials of the Chinese medicine composition include 90 parts of mint, 90 parts of Citrus aurantium, 120 parts of dried orange peel, 120 parts of white peony root, 180 parts of mulberry leaf, 90 parts of bitter almond, 120 parts of Platycodon grandiflorum, 120 parts of Aster and 90 parts of Licorice in parts by weight; further, the Chinese medicine composition contains pharmaceutically acceptable excipients; further, the Chinese medicine composition is Qingxuan Anti-cough Granules or its intermediate product; further, the detection method is used for the detection of gallic acid, paeoniflorin, naringin, naringin, hesperidin and neohesperidin in the Chinese medicine composition. Further preferably, the intermediate product is the dry extract of Qingxuan Anti-cough Granules.

[0022] As a specific example of the present invention: take 0.0152g of Qingxuan Antitussive Granules, add 900μL of water to dissolve, add 200μL of chromatographic methanol to mix, centrifuge at 12000r / min at 20℃ for 15min, take the supernatant, and obtain the test solution of Qingxuan Antitussive Granules.

[0023] As a specific example of the present invention: the dry extract of the intermediate product of Qingxuan Antitussive Granules is dissolved in double distilled water to prepare a 30 mg / mL solution, and filtered through a 0.45 μm filter membrane to obtain a test solution of the intermediate product.

[0024] Preferably, the raw materials of the Qingxuan cough granules include 60-120 parts of mint, 60-120 parts of Citrus aurantium, 100-150 parts of dried orange peel, 100-150 parts of white peony root, 150-220 parts of mulberry leaf, 50-150 parts of bitter almond, 80-160 parts of Platycodon grandiflorum, 80-160 parts of Aster tataricus, and 60-120 parts of licorice. Most preferably, the raw materials of the Qingxuan cough granules include 90 parts of mint, 90 parts of Citrus aurantium, 120 parts of dried orange peel, 120 parts of white peony root, 180 parts of mulberry leaf, 90 parts of bitter almond, 120 parts of Platycodon grandiflorum, 120 parts of Aster tataricus, and 90 parts of licorice.

[0025] On the other hand, the present invention provides a method for establishing a fingerprint of a traditional Chinese medicine composition, comprising the steps of the aforementioned detection method.

[0026] Preferably, the characteristic spectrum includes 6 common peaks, namely: 2-gallic acid; 14-peonidin; 18-naringin; 19-naringin; 20-hesperidin; 21-neohesperidin. Further, the peak corresponding to gallic acid is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±5% of the specified value. The specified value of the relative retention time is: 1.00 (S), 4.38, 5.93, 6.25, 6.51, 6.83; further, 23 characteristic peaks are included.

[0027] Finally, the present invention provides the above-mentioned fingerprint establishment method or the application of the fingerprint in the qualitative and quantitative quality control of Qingxuan cough granules and their intermediates.

[0028] The detection method and establishment method of the present invention are used in the quality control of Qingxuan Anti-cough Granules and their intermediate products. The Qingxuan Anti-cough Granules can be prepared by a variety of methods, all of which have fingerprints that meet the requirements. One preparation method is: extract volatile oil from peppermint, tangerine peel, and fructus aurantii, and decoct the distilled water and the residue with white peony root, mulberry leaf, bitter almond, platycodon, aster and licorice twice in water, each time for 1 hour, combine the decoction, filter, and concentrate the filtrate into a thick paste to obtain; The Qingxuan cough-relieving granules can also be prepared by the following method: extracting volatile oil from peppermint, tangerine peel and fructus aurantii, and encapsulating the volatile oil with beta-cyclodextrin; boiling the distilled water and the drug residue with white peony root, mulberry leaf, bitter almond, platycodon, aster and licorice twice with water, each time for 1 hour, combining the decoctions, filtering, concentrating the filtrate into a thick paste, drying under reduced pressure and low temperature into a dry paste, crushing into fine powder, adding appropriate amounts of sucrose powder and dextrin, adding the above-mentioned inclusion compound, mixing evenly, and making granules to obtain the obtained granules; The Qingxuan cough-relieving granules can also be prepared by the following method: steam distilling mint, tangerine peel and fructus aurantii in weight proportion to extract volatile oil A1 for later use; combining the aqueous solution and the residues of the distilled mint, tangerine peel and fructus aurantii with mulberry leaves, bitter almonds, platycodon, white peony root, aster and licorice, decocting with water, combining the decoction, filtering, concentrating the filtrate, and drying to obtain a dry paste B for later use; mixing the volatile oil A1 and the dry paste B with pharmaceutically acceptable excipients to obtain; The Qingxuan cough-relieving granules can also be prepared by the following method: extracting peppermint, fructus aurantii, and tangerine peel with water to obtain volatile oil; extracting white peony root, mulberry leaf, stir-fried bitter almond, platycodon grandiflorum, aster, and licorice with the residues after water extraction, and concentrating under reduced pressure to obtain an extract; mixing the extract with sucrose and dextrin, granulating, sieving and granulating to obtain selected granules and fine powder passing through a sieve; mixing β-cyclodextrin and the fine powder, and then adding the volatile oil obtained in the above step and stirring again, and sieving the material to obtain an inclusion compound; mixing the inclusion compound and the selected granules to obtain; The Qingxuan cough-relieving granules can also be prepared by the following method: steam distilling peppermint, fructus aurantii, and tangerine peel to obtain volatile oil; soaking white peony root in water, and continuing to extract with mulberry leaves, fried bitter almonds, platycodon, aster, licorice and the distilled drug residues, separating the solid and liquid, concentrating the filtrate, drying, and crushing to obtain the granules.

[0029] The beneficial effects of the present invention are: The present invention provides a detection method and a fingerprint establishment method for a traditional Chinese medicine composition, improves the research on the drug quality control standard of Qingxuan Zhike Granules, and through the same method, the whole process monitoring of the process and quality from Qingxuan Zhike Granule intermediates to Qingxuan Zhike Granule products can be realized, which is convenient for confirming basic substances, evaluating production processes and quality, and providing standard indications for subsequent process improvements and perfections. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the HPLC chromatogram of Qingxuan Anti-cough Granules; Figure 2 This is the HPLC chromatogram of the repeatability experiment of the six components of Qingxuan Anti-cough Granules; Figure 3 This is the HPLC chromatogram of the stability experiment of Qingxuan cough granules; Figure 4 Comparison of HPLC chromatograms of 10 batches of Qingxuan cough granules; Figure 5 This is the HPLC chromatogram of Qingxuan Anti-Ke Granules; Figure 6 The liquid chromatograms of Qingxuan cough-relieving granules at wavelengths of 190nm, 220nm, 254nm, and 280nm; Figure 7 HPLC chromatograms of Qingxuan Zhike granules under different mobile phase conditions; Figure 8 HPLC chromatograms of Qingxuan Zhike granules under different column temperature conditions; Fig. 9 HPLC chromatograms of Qingxuan Zhike granules under different flow rate conditions; Fig.10 This is the HPLC chromatogram of the precision experiment of 6 chromatographic peaks in the intermediate dry extract; Fig.11 This is the HPLC chromatogram of the repeatability test of the six components in the intermediate dry extract; Fig.12 This is the HPLC chromatogram of the retention time stability experiment of the Qingxuan cough-relieving granules intermediate. DETAILED DESCRIPTION

[0031] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is only an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the invention of the present invention based on the disclosed content, and they should also fall within the scope of the present application.

[0032] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0033] The dosage form of the Chinese medicine composition of the present invention is suitable for various commonly used dosage forms, including but not limited to pills, powders, granules, sprays, tablets, capsules, etc., and any Chinese medicine composition with the same or similar medicinal ingredients can be applied to the method of the present invention. The following embodiment of the present invention is described by taking granules (Qingxuan Anti-cough Granules) as an example, but is not limited thereto.

[0034] The times mentioned in the present invention are relative to the weight of the materials to be processed, for example, "108 kg of mint, 108 kg of Citrus aurantium, and 144 kg of dried tangerine peel are added with 3 times the amount of water", wherein the 3 times weight of water is 3 times the total weight of "mint, Citrus aurantium, and dried tangerine peel"; The term "1 times amount of β-cyclodextrin in the amount of volatile oil and 4 times amount of volatile oil obtained in step (3)" refers to 1 times the mass of the volatile oil, and 4 times the amount refers to 1 times the weight of the volatile oil.

[0035] The relative amount of the residue, where the mass of the residue is calculated based on the mass of the Chinese medicinal raw materials before extraction, such as "144 kg of white peony root is soaked in water for 4 hours, combined with 216 kg of mulberry leaves, 108 kg of fried bitter almonds, 144 kg of platycodon, 144 kg of aster, and 108 kg of liquorice, and the residue after volatile oil extraction in step (1) is soaked in 10 times the amount of water for 30 minutes" The mass of the "dregs" is the total mass of "mint, fructus aurantii, and tangerine peel" before distillation.

[0036] The amount of water used for soaking in the present invention is enough to soak 2-5 cm of the medicinal material surface.

[0037] In the step (3) of the present invention, the "granules after granulation" are the sieve-down materials that pass through the 14-mesh sieve and the sieve-up materials that do not pass through the 60-mesh sieve, and the "fine powder passing through the sieve" are the sieve-down materials that pass through the 60-mesh sieve.

[0038] Unless otherwise specified, all percentages in the present invention are by volume.

[0039] In the present invention, the Qingxuan cough-relieving granules can be directly obtained from the market, or can be prepared according to any one of the following methods A, B or C: A method: (1) Weigh the raw materials of mulberry leaf, mint, bitter almond, platycodon, white peony root, fructus aurantii, tangerine peel, aster and liquorice and set aside; (2) steam distilling the peppermint, dried orange peel and fructus aurantii in the weight ratio to extract volatile oil A1 for later use; (3) The aqueous solution and residues of mint, tangerine peel and fructus aurantii distilled from the distillation are combined with mulberry leaves, bitter almonds, platycodon grandiflorum, white peony root, aster root and liquorice, and the mixture is decocted with water. The decoctions are combined, filtered, and the filtrate is concentrated and dried to obtain a dry paste B for later use. (4) The volatile oil A1 and the dry paste B are mixed with pharmaceutically acceptable excipients.

[0040] Method B: (1) Extract mint, fructus aurantii, and tangerine peel with water to obtain volatile oil; (2) extracting white peony root, mulberry leaf, fried bitter almond, platycodon grandiflorum, aster root, liquorice and the residue extracted from step (1) with water, and concentrating under reduced pressure to obtain an extract; (3) The extract is mixed with sucrose and dextrin, granulated, and sieved to obtain the selected granules and the fine powder that passes through the sieve; (4) mixing β-cyclodextrin and the fine powder obtained in step (3) and stirring, then adding the volatile oil obtained in step (1) and stirring again, and sieving the material to obtain an inclusion compound; (5) The inclusion compound obtained in step (4) and the selected particles obtained in step (3) are mixed to obtain.

[0041] C method: (1) Steam distill mint, fructus aurantii, and tangerine peel to obtain volatile oil; (2) soaking white peony root in water, and continuing to extract with mulberry leaf, fried bitter almond, platycodon, aster, liquorice and the residue after distillation in step 1), separating the solid and liquid, concentrating and drying the filtrate, and crushing to obtain a dry extract (i.e., the intermediate product of the present invention); (3) Mixing the dry extract with sucrose and dextrin, granulating, screening and granulating to obtain the selected granules and the fine powder passing through the sieve; (4) mixing β-cyclodextrin and the fine powder obtained in step (3) and stirring, adding the volatile oil obtained in step (1) and stirring again, and sieving the discharged material to obtain an inclusion compound; (5) The inclusion compound obtained in step (4) and the selected particles obtained in step (3) are mixed to obtain.

[0042] The following is a description of the preparation method of the Qingxuan Antitussive Granules and their intermediate dry extract used in the following examples.

[0043] A specific preparation method of Qingxuan cough granules is as follows: (1) 108 kg of mint, 108 kg of Citrus aurantium, and 144 kg of dried tangerine peel are added with 3 times the amount of water, and the volatile oil mass is 2.26 kg; (2) 144 kg of white peony root slices are soaked in water for 4 hours, and then combined with 216 kg of mulberry leaves, 108 kg of fried bitter almonds, 144 kg of platycodon, 144 kg of aster, and 108 kg of licorice. The residue after the volatile oil extraction in step 1) is soaked in 10 times the amount of water for 30 minutes, and decocted twice, each time for 1 hour, and the decoction is decocted in a can. The residue was filtered through an 80-mesh sieve, and the filtrate released was the first extract. The filtered residue was then added with 8 times the amount of water and operated in the same manner. The filtrate obtained was the second extract. The two extracts were combined and concentrated to a relative density of 1.03-1.09 (measured at 50°C). They were then sieved through a 300-mesh sieve while hot. The extract was further concentrated under reduced pressure to a relative density of 1.15-1.20 (measured at 50°C). The extract was then filtered through a 300-mesh sieve while hot to obtain an extract. The extract was dried using a vacuum dryer to obtain 360 kg of dry extract. (3) Sucrose was crushed through an 80-mesh sieve. Sucrose powder, dextrin and extract powder are sucked into a wet mixing granulator in appropriate amounts, and 95% ethanol is added after mixing. After stirring evenly, 55% ethanol is added and stirred; the soft material is granulated by shaking through a 20-mesh sieve; the wet granules are sucked into a boiling dryer, and when the material temperature reaches 75°C, the drying is continued for 15 minutes, the heating is stopped, and the temperature is lowered. When the material temperature reaches below 50°C, the material is discharged; the dried granules are selected, and the selection sieve mesh number is 14 mesh / 60 mesh, and the selected granules and the fine powder passing through the sieve are obtained; (4) 1 times the amount of volatile oil, β-cyclodextrin and The fine powder obtained in step (3) with a volume of 4 times the amount of volatile oil is added to a high-efficiency mixing granulator and stirred for 4 minutes. The volatile oil obtained in step (1) is poured into the granulator and stirred for 2 minutes. The material is sieved through a 14-mesh sieve to obtain an inclusion compound. (5) The inclusion compound obtained in step (4) and the selected granules obtained in step (3) are aspirated into a 3000L total mixing barrel three times, with each aspirating volume being 1 / 3 of the inclusion compound and 1 / 3 of the granules. After the aspiration is completed, the speed of the fixed hopper mixer is set to 8 rpm and mixed for 20 minutes to obtain Qingxuan cough-relieving granules (referred to as batch S1).

[0044] Another specific preparation method of Qingxuan cough granules is as follows: (1) add 3 times the amount of water to 90 kg of mint, 90 kg of Citrus aurantium, and 120 kg of dried tangerine peel, and distill to extract volatile oil with a mass of 1.88 kg; (2) soak 120 kg of white peony root slices in water for 4 hours, combine with 180 kg of mulberry leaves, 90 kg of stir-fried bitter almonds, 120 kg of Platycodon grandiflorum, 120 kg of Aster tataricus, and 90 kg of licorice; soak the residue after the volatile oil extraction in step (1) in 10 times the amount of water for 30 minutes, decoct twice, each time for 1 hour, filter the decoction through an 80-mesh sieve at the bottom of the tank, and concentrate to a relative density of 1.10-1.15, then pass through a 300-mesh sieve while hot, and continue to reduce pressure and concentrate to a relative density of 1.30 -1.35, release the fluid extract, filter it with a 300-mesh sieve while hot, and obtain 350 kg of extract; (3) 567.4 kg of sucrose was crushed with an 80-mesh grinder and added to a whole-grain wet mixing granulator with 283.7 kg of dextrin. The stirring blade speed was set to 100 rpm and the chopping speed was set to 1000 rpm. Mix for 300 seconds, add 6 kg of ethanol, stir for 100 seconds, weigh 50% of the extract and add it. When the current reaches 29 A after mixing for 30-80 seconds, stop mixing. Set the inlet temperature to 100 °C and the material temperature to 80 °C for drying. When the material temperature reaches 80 °C, continue drying for 10 minutes, stop heating and cool down, and discharge the material when the material temperature reaches below 50 °C. (4) Add the extract powder to a wet mixing granulator, set the stirring blade speed to 100 rpm, the chopping speed to 1000 rpm, mix for 300 seconds, add 21 kg of ethanol, stir for 100 seconds, weigh 50% of the extract and add, mix for 40 seconds, chop for 20 seconds, stop mixing when the current reaches 73A, discharge, granulate the soft material through 20 mesh, and dry the wet granules through boiling, set the inlet air temperature to 90°C and the material temperature to 75°C. When the material temperature reaches 75°C, continue drying for 10 minutes, stop heating and cool down, and discharge when the material temperature reaches below 50°C. Screen the granules through 14 mesh / 60 mesh, and granulate the granules that do not pass 14 mesh through 12 mesh. Then select the particles to obtain selected particles and fine powder that passes through the sieve; (5) add 1 times the amount of β-cyclodextrin of the volatile oil and 6 times the amount of the fine powder obtained in step 4) into the high-efficiency mixing granulator in sequence, set the stirring frequency to 50 Hz, set the flying knife to high speed, stir for 4 minutes, pour in the volatile oil obtained in step 1), stir for 2 minutes, and pass the discharged material through a 14-mesh sieve to obtain an inclusion compound; (6) The inclusion compound obtained in step 5) and the selected particles obtained in step 4) are aspirated into a 3000L total mixing barrel three times, with each aspirating amount being 1 / 3 of the inclusion compound and 1 / 3 of the particles. After aspiration, set the fixed hopper mixer speed to 8 rpm and mix for 20 minutes to obtain Qingxuan cough-relieving granules.

[0045] Another specific preparation method of Qingxuan cough granules is as follows: Prepared with reference to the current Chinese Pharmacopoeia method (2020 edition): [Prescription] 180g mulberry leaves, 95g mint, 90g fried bitter almonds, 120g platycodon, 120g white peony root, 90g aurantium, 125g tangerine peel, 120g aster, 95g licorice. The above nine flavors, mint, tangerine peel, and aurantium extract volatile oil, the volatile oil is encapsulated with beta-cyclodextrin. The distilled water and the residue are decocted twice with the remaining six flavors such as mulberry leaves, each for 1 hour, the decoction is combined, filtered, the filtrate is concentrated into a thick paste, and the dry paste is dried at low temperature under reduced pressure to about 250g (intermediate product), crushed into fine powder, added with sucrose powder and dextrin in appropriate amounts, added with the above inclusions, mixed, made into granules, dried, made into 1000g, and obtained (recorded as S1 batch).

[0046] Another specific preparation method of Qingxuan cough granules is as follows: Prepare according to the current Chinese Pharmacopoeia method: [Prescription] 180g mulberry leaves, 90g mint, 90g fried bitter almonds, 120g platycodon, 120g white peony root, 90g fructus aurantii, 120g tangerine peel, 120g aster, 90g licorice. The above nine flavors, mint, tangerine peel, fructus aurantii extract volatile oil, volatile oil with beta-cyclodextrin inclusion. The distilled water and the residues and the remaining six flavors such as mulberry leaves are decocted twice with water, each for 1 hour, the decoction is combined, filtered, the filtrate is concentrated into a thick paste, and the dry paste is dried at low temperature under reduced pressure to about 250g, crushed into fine powder, added with sucrose powder and dextrin in appropriate amounts, added with the above inclusion, mixed, made into granules, dried, made into 1000g, that is (recorded as S2-1 batch).

[0047] According to the same raw material feeding amount and experimental conditions as batch S2-1, another 9 batches of Qingxuan cough granules were prepared and recorded as batches S2-2 to S2-10 for future use. The above Chinese medicinal materials were purchased from Anhui Shengantang Pharmaceutical Co., Ltd.

[0048] Example 1 Establishment of fingerprint of Qingxuan cough-relieving granules 1.1 Methods (1) Preparation of test solution Take 0.0152 g of Qingxuan cough granules from batches S-1 and S2-1~S2-10 respectively, add 900 μL of water to dissolve, add 200 μL of chromatographic methanol to mix, centrifuge at 12000 r / min at 20°C for 15 min, take the supernatant for later use, and refrigerate at 4°C.

[0049] (2) High performance liquid chromatography was used to establish the fingerprint. The chromatographic conditions were: Chromatographic column: Agilent, ZORBAX SB-C 18(4.6×250mm, 5μm); mobile phase: phase A, 0.1% phosphoric acid-water; phase C, acetonitrile; flow rate: 1.0mL / min. Injection volume: 10μL; column temperature: 35℃; detection wavelength: 220nm.

[0050] The liquid phase gradient is: Table 4.

[0051] The liquid fingerprint spectrum of the Qingxuan cough-relieving granules of batch S1 obtained under the above liquid phase conditions is as follows: Figure 1 shown.

[0052] 1.2 Effect Verification In order to investigate the reliability of the analytical method, this experiment used Qingxuan Zhike Granules sample and selected Figure 1 The RSD values ​​of retention time and peak area of ​​characteristic peaks 2, 14, 18, 19, 20 and 21 were calculated respectively, and the repeatability and stability of the experimental method were investigated accordingly. The results showed that the precision, repeatability and stability all met the requirements of fingerprint determination.

[0053] (1) Repeatability experiment Take the same Qingxuan Zhike Granule (Batch S1) test solution (concentration 30mg / mL), inject 6 times, 20μL each time, test according to the best chromatographic conditions, record the retention time and peak area of ​​peaks 2, 14, 18, 19, 20, and 21 (automatically integrate at the lowest peak height of 20), and calculate the relative standard deviation RSD of the retention time and peak area of ​​each peak. The RSD of the retention time of each peak is between 0.05% and 0.32%, and the RSD of the peak area is between 0.15% and 0.53%, both less than 3%, indicating that this method has good repeatability and meets the detection requirements of the fingerprint spectrum. See Figure 2 and the table below.

[0054] Table 5.

[0055] Table 6.

[0056] (2) Stability test Take the same Qingxuan Zhike Granule (Batch S1) test solution (concentration 30 mg / mL), inject 6 times at 0h, 2h, 4h, 8h, 12h, and 24h, 20μL each time, detect according to the best chromatographic conditions, record the retention time and peak area of ​​each chromatographic peak (automatic integration at the minimum peak height of 20), and calculate the retention time and peak area RSD of peaks 2, 14, 18, 19, 20, and 21. The RSD of the retention time of each peak is 0.20%~0.83%, and the RSD of the peak area is 0.30%~2.45%, both less than 3%, indicating that the test solution is stable within 24h. See Figure 3 and the table below.

[0057] Table 7.

[0058] Table 8.

[0059] (3) Similarity analysis Liquid phase analysis was performed on 10 batches of Qingxuan Anti-cough Granules (batches S2-1 to S2-10). The sample concentration was 30 mg / mL, and 20 μL was loaded each time. According to the chromatographic conditions in Table 4, the components were basically the same, among which the peaks with stronger absorbance were 2, 14, 18, 19, 20, and 21 (such as Figure 4 as shown).

[0060] The HPLC fingerprints of 10 batches of Qingxuan Zhike Granules were imported into the fingerprint data of 10 batches of Qingxuan Zhike Granules using the software "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A Edition" issued by the Chinese Pharmacopoeia Committee. The S2-1 batch was set as the reference spectrum, the "time window width" was selected as 0.10min, and multiple characteristic peaks were selected for multi-point correction and automatic matching. The median method was used to generate the reference spectrum ( Figure 5 ).

[0061] (4) Calibration of common peaks and characteristic peaks The HPLC fingerprints of Qingxuan Antitussive Granules from batches S2-1 to S2-10 were matched in the above manner, with a minimum peak height of 10. After removing solvent peaks, there were 23-28 chromatographic peaks in different batches. Since this Chinese patent medicine is complex, the baseline is slightly raised and contains many dense chromatographic peaks that are not separated by the baseline. Therefore, 23 peaks contained in each batch were selected as characteristic peaks, among which peaks 2, 14, 18, 19, 20, and 21 had higher contents in the chromatograms of the 10 batches of samples and better separation, so these 6 peaks were calibrated as common peaks.

[0062] (5) Retention time, peak area and percentage of common peaks Under the established chromatographic conditions, the characteristic peaks of the Qingxuan cough-relieving granules sample were well separated and the retention time was relatively stable. At the same time, the six common peaks all appeared stably. The retention time, peak area and percentage of the common peaks were calculated, and the peak area percentage range statistics of the 23 characteristic peaks were established.

[0063] The evaluation of the common peak retention time of Qingxuan cough granules S2-1~S2-10 batches is as follows: Table 9.

[0064] The peak areas of the common peaks of 10 batches of Qingxuan cough granules are evaluated as follows: Table 10.

[0065] The peak area percentage (%) of the 10 batches of Qingxuan cough granules is evaluated as follows: Table 11.

[0066] The peak area percentage (%) of the characteristic peaks of 10 batches of Qingxuan cough granules is evaluated as follows: Table 12.

[0067] (6) Evaluation of similarity of fingerprint spectrum of Qingxuan cough granules HPLC fingerprints of 10 batches of Qingxuan Zhike granules (see Figure 4 ) to calculate the similarity, and the results are as follows.

[0068] Table 13.

[0069] Example 2 The difference from Example 1 is that the detection wavelengths are 190nm, 254nm, and 280nm respectively, and the rest are the same. Figure 6 (Compared with the detection wavelength of 220nm), the results show that the characteristic peaks of Qingxuan Antitussive Granules can also be separated at the detection wavelengths of 190nm, 254nm, and 280nm, but the peaks are more complete and the characteristic peaks are obvious at the detection wavelength of 220nm.

[0070] Example 3 The difference from Example 1 is that acetonitrile-0.1% TFA water and acetonitrile-0.1% formic acid water were used as mobile phases for gradient elution, and the rest were the same. Figure 7As shown in the figure (compared with acetonitrile-0.1% phosphoric acid water), the results show that the mobile phase of the present invention can effectively separate the characteristic peaks, but acetonitrile-0.1% phosphoric acid water has a better separation effect, the most stable baseline, and the best method reproducibility.

[0071] Example 4 The difference from Example 1 is that the experiments were conducted at 25°C, 30°C and 40°C column temperatures, and the rest were the same. Figure 8 As shown (compared with 35°C), the results show that the separation of the main chromatographic peaks is best at 35°C.

[0072] Example 5 Different from Example 1, the experiments were conducted at flow rates of 0.4 mL / min, 0.6 mL / min, and 0.8 mL / min, respectively. Fig. 9 As shown (compared with 1.0 mL / min), the experimental results show that the separation of the main chromatographic peaks is best when the flow rate is 1.0 mL / min.

[0073] Example 6 Establishment of fingerprint of dry extract of intermediate product of Qingxuan cough granules 2.1 Methods (1) Take the intermediate product dry extract of Qingxuan Antitussive Granules batch S1, dissolve it in double distilled water, and prepare a 30 mg / mL solution. Pass it through a 0.45 μm filter membrane to obtain the test solution of the intermediate product.

[0074] (2) High performance liquid chromatography was used to establish the fingerprint. The chromatographic conditions were: Chromatographic column: Agilent, ZORBAX SB-C18 (4.6×250 mm, 5 μm); Mobile phase: Phase A, 0.1% phosphoric acid-water; Phase B, acetonitrile; Flow rate: 1.0 mL / min, Injection volume: 20 μL, Column temperature: 35°C, Detection wavelength: 220nm, Gradient elution: Table 14.

[0075] 2.2 Effect Verification In order to investigate the reliability of the analytical method, this experiment used the intermediate product dry extract sample of batch S1, double distilled water as the solvent, prepared a 30 mg / mL solution, mixed it, passed it through a 0.45 μm filter membrane, and tested it according to the chromatographic conditions in 1.1. Figure 1The RSD (%) values ​​of retention time and peak area of ​​characteristic peaks No. 2, 14, 18, 19, 20 and 21 were calculated respectively, and the precision, repeatability and stability of the experimental method were investigated accordingly. The results showed that the precision, repeatability and stability all met the requirements of fingerprint determination.

[0076] (1) Precision experiment Take the same dry extract sample of Qingxuan Zhike Granules to make a test solution with a concentration of 30 mg / mL, inject 6 times continuously, 20 μL each time, record the retention time and peak area of ​​peaks 2, 14, 18, 19, 20, and 21 respectively, and calculate the relative standard deviation RSD of the retention time and peak area of ​​each peak. The RSD of the retention time of each peak is 0.12% to 0.17%, and the RSD of the peak area is 0.14% to 0.43%, both less than 3%, indicating that the instrument has good precision. See the chromatogram for details. Fig.10 , the calculation results are as follows.

[0077] Table 15.

[0078] Table 16.

[0079] (2) Repeatability experiment Take the same dry extract sample of Qingxuan Zhike Granules to make 6 test solutions with a concentration of 30 mg / mL, inject 20 μL of each solution, and detect according to the chromatographic conditions in 1.1. Record the retention time and peak area of ​​peaks 2, 14, 18, 19, 20, and 21, and calculate the relative standard deviation RSD of the retention time and peak area of ​​each peak. The RSD of the retention time of each peak is 0.22%~0.34%, and the RSD of the peak area is 0.60%~1.32%, both less than 3%, indicating that this method has good repeatability and meets the detection requirements of the fingerprint spectrum. See the chromatogram for details. Fig.11 , the calculation results are as follows.

[0080] Table 17.

[0081] Table 18.

[0082] (3) Stability test Take the same sample of Qingxuan Zhike Granule dry extract to make a test solution with a concentration of 30 mg / mL, inject 6 times at 0h, 2h, 4h, 8h, 12h, and 24h, 20μL each time, and detect according to the chromatographic conditions of 1.1, record the retention time and peak area of ​​each chromatographic peak, and calculate the RSD (%) of the retention time and peak area of ​​each peak. The RSD of the retention time of each peak is 0.36% to 0.75%, and the RSD of the peak area is 0.72% to 2.67%, both less than 3%, indicating that the test solution is stable within 24h. The chromatogram is shown in 12, and the calculation results are as follows.

[0083] Table 19.

[0084] Table 20.

[0085] According to the above methodological validation results, a fingerprint spectrum detection method for the dry extract of Qingxuan Zhike Granules intermediate product was established to provide a basis for the quality control method of Qingxuan Zhike Granules intermediate product.

[0086] The precision, repeatability and stability of the fingerprints of the Qingxuan cough-relieving granules and the intermediate product dry extract prepared by various methods in the present invention all meet the requirements of fingerprint spectrum determination.

[0087] In summary, the present invention analyzed the components of different batches of Qingxuan Antitussive Granules prepared by different methods, and found that their main components were basically the same. The similarities between Qingxuan Antitussive Granules samples S1-S10 and the control fingerprint spectrum R were all greater than 0.90, that is, the batches of Qingxuan Antitussive Granules were similar, the quality stability was good, and it met the requirements of the fingerprint spectrum. The whole process of process and quality monitoring of Qingxuan Antitussive Granules from intermediates to finished products can be achieved, which is convenient for confirming the basic substances, evaluating the production process and quality, and providing standard indicators for subsequent process improvement and perfection.

[0088] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for detecting a Chinese medicine composition, characterized in that: Detection was performed using high performance liquid chromatography, the conditions of which were: Chromatographic column: reverse phase silica gel bonded phase chromatographic column, Mobile phase: Phase A, acid-containing aqueous solution; Phase B, organic solvent; The Chinese medicine composition is prepared from Chinese medicinal raw materials, which include any one or more mixtures of mint, fructus aurantii, tangerine peel, white peony root, mulberry leaf, bitter almond, platycodon, aster and licorice. Preferably, the Chinese medicinal raw materials include fructus aurantii and white peony root, and more preferably include mint, fructus aurantii, tangerine peel, white peony root, mulberry leaf, bitter almond, platycodon, aster and licorice.

2. The method according to claim 1, characterized in that The reversed-phase silica gel bonded phase chromatographic column is a C18 or C8 chromatographic column, preferably a C18 chromatographic column, more preferably a ZORBAX SB-C18 chromatographic column or a WondaSil C18 chromatographic column, further preferably an Agilent, ZORBAX SB-C18 chromatographic column or a Shimadzu WondaSil C18 chromatographic column.

3. The detection method according to claim 1, characterized in that: In the mobile phase, the acid is at least one of phosphoric acid, hydrochloric acid, sulfuric acid, and citric acid, and phosphoric acid is further preferred; the organic solvent is at least one of acetonitrile, ethanol, methanol, propanol, n-butanol, isobutanol, tetrahydrofuran, and DMF, and acetonitrile is further preferred; further preferably, the mobile phase is: phase A, 0.05-0.15% phosphoric acid-water; phase B, acetonitrile; further preferably: phase A, 0.08-0.12% phosphoric acid-water; phase B, acetonitrile; further preferably, phase A, 0.1% phosphoric acid-water; phase B, acetonitrile; The flow rate of the HPLC is 0.4-1.0 mL / min, more preferably, the flow rate is 0.6-1.0 mL / min, more preferably 1.0 mL / min; The injection volume of the HPLC is 10-20 μL, and more preferably, the injection volume is 20 μL; The column temperature of the HPLC is 25-40°C, more preferably, the column temperature is 25-35°C, more preferably 35°C; The detection wavelength of the HPLC is 190-280 nm, and more preferably, the detection wavelength is 220 nm.

4. The detection method according to claim 1, characterized in that: The elution mode of the high performance liquid chromatography is gradient elution; preferably, the gradient elution conditions are: More preferably: 。 5. The detection method according to any one of claims 1 to 4, characterized in that: The chromatographic conditions are as follows: Chromatographic column: Agilent, ZORBAX SB-C18; Mobile phase: Phase A, 0.1% phosphoric acid-water; Phase B, acetonitrile; Flow rate: 1.0 mL / min, Injection volume: 20 μL, Column temperature: 35°C, Detection wavelength: 220nm, Gradient elution: 。 6. The detection method according to claim 1, characterized in that: The method comprises preparing the Chinese medicine composition into a test solution for testing; preferably, the method for preparing the test solution comprises: taking the test sample of the drug, extracting it with a solvent, separating the solid from the liquid, and the obtained liquid is the test solution; further preferably, the method for preparing the test solution also satisfies any one or more of the following conditions: a. The solvent is an alcohol aqueous solution or an alcohol solvent; preferably, the alcohol is a C1~C6 alcohol, preferably any one or a combination of methanol, ethanol, propanol and butanol, more preferably methanol; preferably, the volume ratio of the alcohol solvent in the alcohol aqueous solution is 1%~90%, preferably 2%~60%, more preferably 5%~40%; b. In g / mL, the ratio of the test sample to the solvent is 1:30 to 100, preferably 1:40 to 80, and more preferably 50 to 80; c. The extraction method includes ultrasonic extraction or heating reflux extraction; preferably, when ultrasonic extraction is used, the extraction temperature is 15~40°C; d. The extraction time is 15 min or more, preferably 15 min to 2 h, and more preferably 30 to 60 min; e. The solid-liquid separation method is centrifugation and / or filtration; preferably, the centrifugation conditions are: 10000-15000r / min at 15-25°C for 5-30min; further preferably, the filtration is through a 0.4-0.5μm filter membrane.

7. The detection method according to claim 1, characterized in that: The Chinese medicine composition is a mixture of peppermint extract, fructus aurantii extract, tangerine peel extract, white peony root extract, mulberry leaf extract, bitter almond extract, platycodon grandiflorum extract, aster root extract and licorice extract; further, the Chinese medicine composition is prepared from the following Chinese medicinal materials in parts by weight: 60-120 parts of peppermint, 60-120 parts of fructus aurantii, 100-150 parts of tangerine peel, 100-150 parts of white peony root, 150-220 parts of mulberry leaf, 50-150 parts of bitter almond, 80-160 parts of platycodon grandiflorum, 80-160 parts of aster root and 60-120 parts of licorice. 0 parts; most preferably, the raw materials of the Chinese medicine composition include, by weight, 90 parts of mint, 90 parts of fructus aurantii, 120 parts of dried tangerine peel, 120 parts of white peony root, 180 parts of mulberry leaf, 90 parts of bitter almond, 120 parts of platycodon, 120 parts of aster and 90 parts of liquorice; further, the Chinese medicine composition contains pharmaceutically acceptable excipients; further, the Chinese medicine composition is Qingxuan cough granules or its intermediates; further, the detection method is for the detection of gallic acid, paeoniflorin, naringin, naringin, hesperidin and neohesperidin in the Chinese medicine composition.

8. A method for establishing a characteristic spectrum of a traditional Chinese medicine composition, characterized in that: The method comprises the steps of any one of claims 1 to 7.

9. The establishment method according to claim 8, characterized in that: The characteristic spectrum includes 6 common peaks, namely: 2-gallic acid; 14-peonidin; 18-naringin; 19-naringin; 20-hesperidin; 21-neohesperidin; further, the peak corresponding to gallic acid is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±5% of the specified value, and the specified value of the relative retention time is: 1.00 (S), 4.38, 5.93, 6.25, 6.51, 6.83; further, it includes 23 characteristic peaks.

10. Application of the detection method according to any one of claims 1 to 7, or the establishment method according to claim 8 or 9 in the quality control of Qingxuan Zhike granules and their intermediates; Preferably, the Qingxuan cough-relieving granules are prepared by the following method: extracting volatile oil from peppermint, tangerine peel and fructus aurantii, decocting the distilled water and the residue with white peony root, mulberry leaf, bitter almond, platycodon, aster and licorice twice, each time for 1 hour, combining the decoctions, filtering, and concentrating the filtrate into a thick paste; Alternatively: extract volatile oil from mint, dried orange peel and fructus aurantii, and include the volatile oil with beta-cyclodextrin; decoct the distilled water and the residue with white peony root, mulberry leaf, bitter almond, platycodon, aster and liquorice twice with water, each time for 1 hour, combine the decoction, filter, concentrate the filtrate into a thick paste, dry it at low temperature under reduced pressure into a dry paste, grind it into fine powder, add appropriate amount of sucrose powder and dextrin, add the above inclusion material, mix well, and make granules to obtain the product; Alternatively: steam distill mint, dried orange peel and fructus aurantii in weight proportion to extract volatile oil A1, which is set aside; the water solution and residues after distillation of mint, dried orange peel and fructus aurantii are combined with mulberry leaves, bitter almonds, platycodon, white peony root, aster and liquorice, boiled with water, the decoctions are combined, filtered, the filtrate is concentrated and dried to obtain dry paste B, which is set aside; the volatile oil A1 and dry paste B are mixed with pharmaceutically acceptable excipients to obtain; Alternatively: extract peppermint, fructus aurantii, and tangerine peel with water to obtain volatile oil; continue to extract white peony root, mulberry leaf, stir-fried bitter almond, platycodon grandiflorum, aster, and licorice with the residue after water extraction, and concentrate under reduced pressure to obtain an extract; mix the extract with sucrose and dextrin, granulate, and sieve to obtain granules after granulation and fine powder after sieve; mix β-cyclodextrin and fine powder, stir, add the volatile oil obtained in the above step, stir again, and sieve the material to obtain inclusion compound; mix the inclusion compound and the granules after granulation to obtain; Alternatively: steam distill mint, fructus aurantii and tangerine peel to obtain volatile oil; soak white peony root in water, and continue to extract with mulberry leaf, fried bitter almond, platycodon, aster, licorice and the distilled residue, separate the solid and liquid, concentrate the filtrate, dry it, and grind it to obtain the product.

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