Quality control method of traditional Chinese medicine composition

Abstract

The invention provides a quality control method of a traditional Chinese medicine composition, which comprises the following steps: establishing a spinosin content determination method of jujube and cypress nerve-soothing granules: A) extracting the jujube and cypress nerve-soothing granules by adopting a solvent to obtain a to-be-determined solution; b) taking a spinosin standard substance, and dissolving the spinosin standard substance with a solvent to obtain a reference substance reference substance solution; c) respectively obtaining chromatograms of the liquid to be detected in the step A) and the reference substance reference substance in the step B) under the same detection conditions by adopting a high performance liquid chromatography, and D) according to the reference substance concentration, the peak area of the reference substance reference substance solution in the chromatogram and the peak area of the component corresponding to the reference substance reference substance solution in the chromatogram, based on the detection conditions in the step C), calculating the content of the chemical component in the jujube and cypress nerve-soothing particle by an external standard method. The method for determining the content of the main component spinosin, established by the invention, is good in precision, stability and repeatability.

Description

[0001] This application claims the priority of a Chinese patent application filed with the China Patent Office on June 30, 2023, with application number 202310805062.9 and invention name “A Quality Control Method for Zaobai Anshen Granules”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The invention relates to the technical field of drug analysis, and in particular to a quality control method for a traditional Chinese medicine composition. Background Art

[0003] The prescription of Zaobai Anshen Granules is composed of nine Chinese medicines: stir-fried spiny jujube seeds, salvia miltiorrhiza, cypress seeds, Polygonum multiflorum, vinegar Schisandra chinensis, coptis chinensis, pearl powder, jujube flowers, and Hypericum perforatum. Among them, stir-fried spiny jujube seeds are the main medicine, salvia miltiorrhiza, cypress seeds, Polygonum multiflorum, vinegar Schisandra chinensis are the ministerial medicines, coptis chinensis and pearl powder are adjuvants, and jujube flowers and Hypericum perforatum are the guiding medicines. The combination of these medicines has the effects of nourishing blood and calming the soul, clearing away heat and relieving restlessness. The prescription of Zaobai Anshen Granules has been used in the clinic for many years in the form of decoction, with a definite therapeutic effect and high development and application value. However, there is no research report on the preparation process and quality research method of Zaobai Anshen Granules.

[0004] Therefore, it is very necessary to establish its quality standards to comprehensively evaluate the quality of the preparation. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a quality control method for a Chinese medicine composition. The quality control method provided by the present invention can comprehensively control the quality of the product, and the method has good stability, precision and repeatability.

[0006] The terms "include", "comprising" and "having" are used interchangeably herein and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions herein also provides a "consisting of..." solution.

[0007] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0008] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0009] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the following processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0010] The present invention provides a quality control method for a traditional Chinese medicine composition, comprising: establishing a method for determining the content of spinosin in Zaobai Anshen granules:

[0011] A) extracting Zaobai Anshen granules with a solvent to obtain a test solution;

[0012] B) taking a spinosin standard and dissolving it in a solvent to obtain a reference substance solution;

[0013] C) using high performance liquid chromatography to obtain chromatograms of the test solution in step A) and the reference substance in step B) under the same detection conditions, and the specific chromatographic parameters are as follows:

[0014] The chromatographic column was a C18 column, the detection wavelength was 335 nm; the mobile phase was methanol-0.1% phosphoric acid;

[0015] The elution procedure is:

[0016] 0-5min, phase A 20%-30%, phase B 80%-70%;

[0017] 5-30min: 30%-40% of phase A, 70%-60% of phase B;

[0018] 30-35min: Phase A 40%-100%, Phase B 60%-0%;

[0019] 35-40min: Phase A 100%, Phase B 0%;

[0020] 40-45min: Phase A 100%-20%, Phase B 0%-80%;

[0021] D) calculating the content of chemical components in Zaobai Anshen Granules by external standard method according to the concentration of the reference substance, the peak area of ​​the reference substance solution in the chromatogram and the peak area of ​​the components in Zaobai Anshen Granules corresponding to the reference substance solution in the chromatogram, and based on the detection conditions in step C).

[0022] The quality control method of Zaobai Anshen granules provided by the present invention comprises establishing a method for determining the content of spinosin in Zaobai Anshen granules;

[0023] Also includes any one or more of the following determination methods:

[0024] i) HPLC determination of berberine hydrochloride content in Zaobai Anshen granules;

[0025] ii) Using thin layer chromatography to identify the stir-fried spiny jujube seeds in Zaobai Anshen granules;

[0026] iii) Identification of Coptis chinensis by thin layer chromatography;

[0027] iv) identification of Salvia miltiorrhiza using thin layer chromatography;

[0028] v) identifying Schisandra chinensis var. vinegar by thin layer chromatography;

[0029] vi) Identification of Polygonum multiflorum by thin layer chromatography.

[0030] The present invention finds corresponding characteristic spots from a series of thin layer chromatographic identifications of medicinal materials and preparations, which provides a basis for tracing the characteristic components in the preparations back to the medicinal materials, and confirms a certain correlation between the medicinal materials and the preparations through content determination.

[0031] The method for determining the contents of the two main components, spinosum and berberine hydrochloride, established in the present invention has good precision, stability and repeatability.

[0032] The thin layer identification method for five medicinal materials established by the present invention has good durability.

[0033] The present invention can ensure the safety, effectiveness and stability of clinical medication, and also provides a detailed and reliable research basis for subsequent preparation process research, quality standard establishment and stability investigation of Zaobai Anshen granule preparations.

[0034] On the one hand, the method for determining the content of spinosin in Zaobai Anshen Granules of the present invention is as follows:

[0035] Firstly, Zaobai Anshen Granules are extracted with a solvent to obtain a test solution.

[0036] In some preferred embodiments of the present invention, the solvent is 70% methanol.

[0037] The inventors found that 70% methanol had the highest extraction rate, and too many impurities extracted by methanol caused the main peak of spinotoxin to be wrapped with other peaks, resulting in a high content of spinotoxin, so 70% methanol was selected as the extraction solvent.

[0038] In some preferred embodiments of the present invention, the extraction is ultrasonic extraction; the time of the ultrasonic extraction is 10 to 20 minutes.

[0039] According to the present invention, the ratio of the mass g of the Zaobai Anshen granules to the volume mL of the solvent is 1:15-45; more preferably 1:25.

[0040] Take the spinosum standard product, dissolve it in a solvent, and obtain a reference substance solution.

[0041] The spinosin of the present invention has good linearity within the range of 0.01362-0.2180 mg / mL.

[0042] The chromatograms of the test solution in step A) and the reference substance in step B) were obtained under the same detection conditions by high performance liquid chromatography. The specific chromatographic parameters are as follows:

[0043] In some preferred embodiments of the present invention, the mobile phase flow rate is 1 ml / min, the chromatographic column temperature is 25° C., and the injection volume is 10 μL;

[0044] In some preferred embodiments of the present invention, the chromatographic column is a C18 chromatographic column;

[0045] In some preferred embodiments of the present invention, the chromatogram in the Phenomenex C18 chromatographic column has a better separation effect than other chromatographic index components, so it is used as the chromatographic column.

[0046] The detection wavelength of the present invention is 335nm. According to the previous experimental research, the maximum absorption wavelength of spinoside is 335nm. Under this wavelength condition, the response of spinoside is high and stable, so the selected detection wavelength is 335nm.

[0047] The mobile phase of the present invention is methanol-0.1% phosphoric acid;

[0048] In some preferred embodiments of the present invention, the elution procedure is:

[0049] 0-5min, phase A 20%-30%, phase B 80%-70%;

[0050] 5-30min: 30%-40% of phase A, 70%-60% of phase B;

[0051] 30-35min: Phase A 40%-100%, Phase B 60%-0%;

[0052] 35-40min: Phase A 100%, Phase B 0%;

[0053] 40-45min: Phase A 100%-20%, Phase B 0%-80%;

[0054] According to the concentration of the reference substance, the peak area of ​​the reference substance solution in the chromatogram and the peak area of ​​the components in Zaobai Anshen Granules corresponding to the reference substance solution in the chromatogram, and based on the detection conditions in step C), the content of the chemical components in Zaobai Anshen Granules is calculated by the external standard method.

[0055] On the one hand, the HPLC determination of the berberine hydrochloride content in Zaobai Anshen granules is specifically:

[0056] a) extracting Zaobai Anshen granules with a solvent to obtain a test solution;

[0057] b) taking a berberine hydrochloride standard product and dissolving it in a solvent to obtain a reference substance solution;

[0058] c) using high performance liquid chromatography to obtain chromatograms of the test solution in step a) and the reference substance in step b) under the same detection conditions, and performing qualitative and quantitative analysis on the components of the test solution based on the chromatograms of the reference substance;

[0059] The specific chromatographic parameters are as follows:

[0060] The chromatographic column was a C18 column with a detection wavelength of 345 nm; the mobile phase was acetonitrile-0.05 mol / L potassium dihydrogen phosphate solution (50:50), with 0.4 g of sodium dodecyl sulfate added to every 100 ml, and the pH value was adjusted to 4.0 with phosphoric acid; isocratic elution, mobile phase flow rate of 1.0 ml / min; the column temperature was 30°C, and the injection volume was 10 μL.

[0061] The HPLC method provided by the present invention for determining the content of berberine hydrochloride in Zaobai Anshen granules comprises first extracting Zaobai Anshen granules with a solvent to obtain a test solution.

[0062] According to the present invention, the step a) is specifically:

[0063] Zaobai Anshen Granules are ultrasonically extracted with a mixed solution of methanol and hydrochloric acid, and the ultrasonic extraction time is 10 to 30 minutes, more preferably 20 minutes;

[0064] The inventors found that ultrasonic extraction has a high efficiency, and for ease of operation, ultrasonic extraction was selected as the extraction method.

[0065] In some preferred embodiments of the present invention, the volume ratio of methanol to hydrochloric acid is 100:1;

[0066] The inventors found that the extraction rate was higher when methanol-hydrochloric acid (100:1, V / V) solution was used as the extraction solvent, so this was selected as the extraction solvent.

[0067] According to the present invention, the ratio of the mass g of the Zaobai Anshen granules to the volume mL of the solvent is 0.1:25-100; more preferably 0.1:50.

[0068] Take a berberine hydrochloride standard product, dissolve it in a solvent, and obtain a reference substance solution;

[0069] The berberine hydrochloride has good linearity in the range of 0.005377 to 0.2151 mg / mL.

[0070] Using high performance liquid chromatography, under the same detection conditions, respectively obtain the chromatograms of the test solution in step a) and the reference substance in step b), and perform qualitative and quantitative analysis on the components of the test solution according to the chromatogram of the reference substance;

[0071] The specific chromatographic parameters are as follows:

[0072] In some preferred embodiments of the present invention, the chromatographic column is a C18 chromatographic column, and the detection wavelength is 345 nm;

[0073] The maximum absorption wavelength of berberine hydrochloride is 345nm. Under this wavelength condition, the response of berberine hydrochloride is high and stable, so the selected detection wavelength is 345nm.

[0074] In some preferred embodiments of the present invention, the mobile phase is: acetonitrile-0.05 mol / L potassium dihydrogen phosphate solution (50:50), 0.4 g of sodium dodecyl sulfate is added to every 100 ml, and the pH value is adjusted to 4.0 with phosphoric acid; isocratic elution, the mobile phase flow rate is 1.0 ml / min;

[0075] The present invention found that the chromatogram in the mobile phase elution condition 3 has a better separation effect than other chromatogram index components, so this is used as the elution condition.

[0076] In some preferred embodiments of the present invention, the temperature of the chromatographic column is 30° C. and the injection volume is 10 μL.

[0077] On the one hand, the method of using thin layer chromatography to identify the stir-fried Chinese date seeds in Zaobai Anshen granules is specifically as follows:

[0078] a) Extracting Zaobai Anshen Granules, stir-fried spinach seed negative test sample and spinach seed control medicinal material with solvent to obtain test sample solution, stir-fried spinach seed negative test sample and spinach seed control medicinal material solution respectively;

[0079] Add methanol to dissolve jujuboside A reference substance and jujuboside B reference substance to obtain jujuboside A reference substance solution and jujuboside B reference substance solution;

[0080] b) performing thin layer chromatography on the test solution, the stir-fried Ziziphus jujuba seed negative test sample, the Ziziphus jujuba seed control medicinal material solution, the Ziziphus jujuba seed saponin A reference substance, and the Ziziphus jujuba seed saponin B reference substance solution, the thin layer plate is a silica gel G thin layer plate; the developing solvent is n-butanol-glacial acetic acid-water, and the volume ratio of n-butanol-glacial acetic acid-water is 4:1:5;

[0081] c) Inspection: If the test sample, the Chinese jujube seed reference material, the Chinese jujube seed saponin A reference material, and the Chinese jujube seed saponin B reference material have spots of the same color at the same Rf value, and the Chinese jujube seed negative test sample has no spots, then it is stir-fried Chinese jujube seed.

[0082] The invention provides a method for identifying stir-fried spinach seeds in Zaobai Anshen granules by using thin layer chromatography. Zaobai Anshen granules, stir-fried spinach seeds negative test sample and spinach seeds control medicinal material are extracted by solvent to obtain a test sample solution, a stir-fried spinach seeds negative test sample and a spinach seeds control medicinal material solution, respectively.

[0083] The above extraction is specifically as follows: Zaobai Anshen Granules are added with ether and heated under reflux for extraction for 1 hour, methanol is added after evaporation, and the mixture is heated under reflux for extraction for 30 minutes, filtered, dissolved in water, and extracted by shaking with water-saturated n-butanol for 2 to 3 times, the n-butanol solution is combined, washed twice with ammonia test solution, the ammonia solution is discarded, the n-butanol solution is evaporated to dryness, and the residue is dissolved in methanol to serve as the test solution;

[0084] In some preferred embodiments of the present invention, the mass volume ratio of Zaobai Anshen Granules, ether, methanol and water is: 5g:30mL:30mL:20mL.

[0085] Take the negative prescription of stir-fried spiny jujube seeds and process it in the same way to obtain the negative test sample of stir-fried spiny jujube seeds;

[0086] Take another reference medicinal material of Ziziphus jujuba seeds and prepare a reference medicinal material solution in the same manner;

[0087] Take jujuboside A reference substance and jujuboside B reference substance, add methanol to make a mixed solution containing 1 mg of each per 1 ml, as the reference substance solution.

[0088] According to the thin layer chromatography test, the developing solvent is the upper layer solution of n-butanol-glacial acetic acid-water (4:1:5) placed below 10°C, 5-10 μl of each of the above solutions is taken, spotted on a silica gel G plate, developed, taken out, dried, sprayed with 5% vanillin sulfuric acid solution, and blown with hot air until the spots are clearly colored, and examined immediately;

[0089] If the test sample, the Chinese jujube seed control medicinal material, the Chinese jujube seed saponin A control substance, and the Chinese jujube seed saponin B control substance have spots of the same color at the same Rf value, and the Chinese jujube seed negative test sample has no spots, then the quality is qualified.

[0090] On the one hand, the identification of Rhizoma Coptidis by thin layer chromatography is specifically as follows:

[0091] a) extracting Zaobai Anshen granules, Coptis chinensis negative test sample and Coptis chinensis control medicinal material with a solvent to obtain a test sample solution, a Coptis chinensis negative test sample and a Coptis chinensis control medicinal material solution, respectively;

[0092] Dissolve the berberine hydrochloride reference substance in methanol to obtain a berberine hydrochloride reference substance solution;

[0093] b) performing thin layer chromatography on the test solution, the negative test product of Coptis chinensis, the reference medicinal material solution of Coptis chinensis, and the reference solution of berberine hydrochloride, wherein the thin layer plate is a silica gel G thin layer plate; the developing solvent is cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine, and the volume ratio of the cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine is 3:3.5:1:1.5:0.5:1;

[0094] c) Inspection: If the test sample, the Rhizoma Coptidis reference material, and the berberine hydrochloride reference material have fluorescent spots of the same color at the same Rf value, and the Rhizoma Coptidis negative test sample has no spots, then it is Rhizoma Coptidis;

[0095] The inspection was performed under ultraviolet light; the wavelength of the ultraviolet light was 365 nm; and the sample spotting volume was 1 μL.

[0096] The invention firstly extracts Zaobai Anshen granules with a solvent to obtain a test sample solution.

[0097] The above extraction is specifically as follows: extract Zaobai Anshen granules with methanol and perform ultrasonic treatment for 20 to 30 minutes;

[0098] The mass volume ratio of Zaobai Anshen Granules and methanol is 0.5g:10mL.

[0099] Take the negative prescription of Coptis chinensis and process it in the same way to obtain the negative test sample of Coptis chinensis;

[0100] Take another reference herbal medicine of Coptis chinensis and prepare a reference herbal medicine solution in the same way;

[0101] Then take berberine hydrochloride reference substance, add methanol to make a solution containing 0.5 mg per 1 ml, as the reference substance solution;

[0102] According to the thin layer chromatography test, the developing solvent is cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine (3:3.5:1:1.5:0.5:1) and the upper layer solution is placed below 10°C. 1 μl of each of the above solutions is taken and spotted on a silica gel G plate, which is placed in a developing cylinder pre-saturated with concentrated ammonia test solution for 20 minutes, developed, taken out, dried, and examined under an ultraviolet lamp (365nm);

[0103] If the test sample has fluorescent spots of the same color at the same Rf value as the chromatogram of the Rhizoma Coptidis reference material and the chromatogram of the berberine hydrochloride reference substance, and the Rhizoma Coptidis negative test sample has no spots, then the quality is qualified.

[0104] On the one hand, the identification of Salvia miltiorrhiza by thin layer chromatography is specifically as follows:

[0105] a) extracting Zaobai Anshen granules, Danshen negative test sample and Danshen control medicinal material with a solvent to obtain a test sample solution, a Danshen negative test sample and a Danshen control medicinal material solution, respectively;

[0106] Dissolve the Tanshinone ⅡA reference substance in ethyl acetate to obtain a Tanshinone ⅡA reference substance solution;

[0107] b) performing thin layer chromatography on the test solution, the negative test sample of Danshen, the control medicinal material solution of Danshen and the reference solution of Tanshinone ⅡA, the thin layer plate is a silica gel G thin layer plate; the developing solvent is toluene-ethyl acetate, and the volume ratio of toluene-ethyl acetate is 19:1;

[0108] c) Inspection: If the test sample, the control drug of Danshen, and Tanshinone ⅡA have spots of the same color at the same Rf value, and the negative test sample of Danshen has no spots, then it is Danshen;

[0109] The inspection was performed under daylight; the spotting volume was 4 to 10 μL.

[0110] The method for identifying Danshen miltiorrhiza by thin layer chromatography provided by the present invention first extracts Zaobai Anshen granules by solvent to obtain a test solution;

[0111] Take Zaobai Anshen Granules of this product, add ethyl acetate, ultrasonically treat for 20 minutes, filter, evaporate the filtrate, add ethyl acetate to the residue to dissolve it, and use it as the test solution.

[0112] The mass volume ratio of the Zaobai Anshen granules and ethyl acetate of the present invention is 1g:25mL.

[0113] Take the negative prescription of Danshen, and process it in the same way to obtain the negative test sample of Danshen; take another 1g of Danshen reference medicinal material, and prepare the reference medicinal material solution in the same way; then take the Tanshinone ⅡA reference substance, add ethyl acetate to prepare a solution containing 1mg per 1ml, as the reference substance solution;

[0114] According to the thin layer chromatography test, the developing solvent is toluene-ethyl acetate (19:1), take 4 μl of the above-mentioned Tanshinone ⅡA reference solution and Danshen reference medicinal material solution, and 5-10 μl of the test solution, spot them on the silica gel G plate, develop, take out, dry, and examine under sunlight;

[0115] If the test sample has spots of the same color at the same Rf value as the chromatogram of the Danshen reference medicinal material and the chromatogram of the Tanshinone ⅡA reference substance, and the negative Danshen test sample has no spots, the quality is qualified;

[0116] On the one hand, the identification of Schisandra chinensis var. vinegar by thin layer chromatography is specifically as follows:

[0117] a) extracting Zaobai Anshen granules, vinegar Schisandra chinensis negative test sample and Schisandra chinensis control medicinal material with a solvent to obtain a test sample solution, vinegar Schisandra chinensis negative test sample and Schisandra chinensis control medicinal material solution, respectively;

[0118] Dissolve the Schisandrin A reference substance in petroleum ether to obtain a Schisandrin A reference substance solution;

[0119] b) The test solution, the vinegar Schisandra negative test sample, the vinegar Schisandra control medicinal material solution and the Schisandrae Chinensis A reference solution are subjected to thin layer chromatography, the thin layer plate is a silica gel GF254 thin layer plate; the developing solvent is petroleum ether-ethyl formate-formic acid, and the volume ratio of the petroleum ether-ethyl formate-formic acid is 15:5:1;

[0120] c) Inspection: If the test sample, Schisandra chinensis control medicinal material, and Schisandrin A have spots of the same color at the same Rf value, and the negative test sample of Schisandra chinensis vinegar has no spots, then it is Schisandra chinensis vinegar;

[0121] The inspection was performed under ultraviolet light; the wavelength of the ultraviolet light was 254 nm; and the sample spotting volume was 5 μL.

[0122] Take Zaobai Anshen granules, grind them into powder, add petroleum ether Ultrasonic treatment for 20 minutes, filtered, the filtrate evaporated to dryness, and the residue added petroleum ether Dissolve it as the test solution;

[0123] Among them, the mass volume ratio of Zaobai Anshen Granules and petroleum ether is 4g:25mL.

[0124] Take the negative prescription of Schisandra chinensis vinegar and process it in the same way to obtain the negative test sample of Schisandra chinensis vinegar;

[0125] Take another reference medicinal material of Schisandra chinensis and prepare a reference medicinal material solution in the same manner;

[0126] Take Schisandrin A reference substance and add petroleum ether Prepare a solution containing 1 mg per 1 ml as the reference solution;

[0127] According to the thin layer chromatography test, the developing solvent is petroleum ether -ethyl formate-formic acid (15:5:1) upper layer solution placed below 10°C, take 5 μl of each of the above solutions, spot on silica gel GF254 plate, develop, take out, dry, and inspect under ultraviolet light (254 nm);

[0128] If the chromatograms of the test sample, Schisandra chinensis reference medicinal material and reference sample have spots of the same color at the same Rf value, and the vinegar Schisandra chinensis negative test sample has no spots, then the quality is qualified.

[0129] On the one hand, the identification of Radix Polygoni Multiflori using thin layer chromatography specifically comprises:

[0130] a) extracting Zaobai Anshen granules, Radix Polygoni Multiflori negative test sample and Radix Polygoni Multiflori control medicinal material with solvent to obtain test sample solution, Radix Polygoni Multiflori negative test sample and Radix Polygoni Multiflori control medicinal material solution respectively;

[0131] The rheum officinale reference substance was dissolved in methanol to obtain a rheum officinale reference substance solution;

[0132] b) performing thin layer chromatography on the test solution, Radix Polygoni Multiflori negative test sample, Radix Polygoni Multiflori control medicinal material solution and rhein reference solution, the thin layer plate is a silica gel G thin layer plate; the developing solvent is petroleum ether-ethyl formate-formic acid, and the volume ratio of the petroleum ether-ethyl formate-formic acid is 15:5:1;

[0133] c) Inspection: If the test sample, Radix Polygoni Multiflori control medicinal material, and Rhein have fluorescent spots of the same color at the same Rf value, and the Radix Polygoni Multiflori negative test sample has no spots, then it is Radix Polygoni Multiflori;

[0134] The inspection was performed under ultraviolet light; the wavelength of the ultraviolet light was 365 nm; and the sample spotting volume was 5 μL.

[0135] Take 2g of Zaobai Anshen Granules, add 30ml of ether and 2ml of hydrochloric acid, and treat with ultrasound for 30 minutes. Filter, evaporate the filtrate, and dissolve the residue in 1ml of methanol to prepare the test solution.

[0136] Among them, the mass volume ratio of Zaobai Anshen Granules, ether and hydrochloric acid is 2g:30mL:2mL.

[0137] Take the Radix Polygoni Multiflori negative prescription and process it in the same way to obtain the Radix Polygoni Multiflori negative test sample;

[0138] Take another Radix Polygoni Multiflori as a control medicinal material, and prepare a control medicinal material solution in the same manner;

[0139] Take the rheum officinale reference substance and add methanol to make a solution containing 0.1 mg per 1 ml as the reference substance solution;

[0140] According to the thin layer chromatography test, the developing solvent is petroleum ether (60-90°C)-ethyl formate-formic acid (15:5:1) placed below 10°C, 5 μl of each of the above solutions is taken, spotted on a silica gel G plate, developed, taken out, dried, and examined under an ultraviolet lamp (365nm);

[0141] If the test sample and the reference drug chromatogram of Radix Polygoni Multiflori and the reference drug chromatogram have the same color fluorescent spots at the same Rf value, and the Radix Polygoni Multiflori negative test sample has no spots, the quality is qualified;

[0142] From a series of thin-layer chromatographic identifications of medicinal materials and preparations, corresponding characteristic spots were found, which provided a basis for tracing the characteristic components in the preparations back to the medicinal materials. Content determination confirmed that there was a certain correlation between the medicinal materials and preparations.

[0143] The thin layer identification method for five medicinal materials established by the present invention has good durability.

[0144] The present invention provides a quality control method for a Chinese medicine composition, comprising: establishing a method for determining the content of spinotoxin in Zaobai Anshen granules: A) extracting Zaobai Anshen granules with a solvent to obtain a test solution; B) taking a spinotoxin standard product and dissolving it with a solvent to obtain a reference reference product solution; C) using high performance liquid chromatography to obtain the chromatograms of the test solution in step A) and the reference reference product in step B) under the same detection conditions, respectively; D) according to the reference concentration, the peak area of ​​the reference reference product solution in the chromatogram, and the peak area of ​​the component corresponding to the reference reference product solution in Zaobai Anshen granules in the chromatogram, and based on the detection conditions in step C), the content of the chemical components in Zaobai Anshen granules is calculated by an external standard method. The content determination methods of the two main components, spinotoxin and berberine hydrochloride, established by the present invention have good precision, stability and repeatability. The present invention can ensure the safety, effectiveness and stability of clinical medication, and also provides a detailed and reliable research basis for the subsequent preparation process research, quality standard establishment and stability investigation of Zaobai Anshen granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0145] Figure 1 .TLC chart of stir-fried Chinese date seeds in Zaobai Anshen granules;

[0146] Figure 2 .TLC chart of Coptis chinensis in Zaobai Anshen granules;

[0147] Figure 3 .TLC chart of Salvia miltiorrhiza in Zaobai Anshen granules;

[0148] Figure 4 .TLC chart of Schisandrae Chinensis Fruit in Zaobai Anshen Granules;

[0149] Figure 5 .TLC chart of Polygonum multiflorum in Zaobai Anshen granules;

[0150] Figure 6 .AB correspond to the HPLC diagrams (acetonitrile system) of the screening of elution conditions for spinosin;

[0151] Figure 7 .AC correspond to the HPLC diagrams of the screening of elution conditions of spinosin;

[0152] Figure 8 .AD correspond to the HPLC charts of chromatographic columns screened by different manufacturers of spinosin;

[0153] Fig. 9 .Linear relationship diagram of spinosum;

[0154] Fig.10 .AC correspond to the HPLC diagrams of berberine hydrochloride elution condition screening;

[0155] Fig.11 .Linear relationship diagram of berberine hydrochloride. DETAILED DESCRIPTION

[0156] The present invention provides a quality control method for a Chinese medicine composition, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0157] In order to further illustrate the present invention, a quality control method for a traditional Chinese medicine composition provided by the present invention is described in detail below in conjunction with examples.

[0158] Example 1

[0159] 1. Study on the quality standard of Zaobai Anshen granules

[0160] 1.1 Qualitative study of Zaobai Anshen granules

[0161] 1.1.1 Thin layer chromatography study of Zaobai Anshen granules

[0162] (1) TLC Identification of Stir-fried Ziziphus jujuba Seeds

[0163] Take 5g of this product, grind it, add 30ml of ether, heat and reflux for 1 hour, filter, evaporate the solvent from the residue, add 30ml of methanol, heat and reflux for 30 minutes, filter, evaporate the filtrate, add 20ml of water to the residue to dissolve it, shake and extract it with water-saturated n-butanol 3 times, 25ml each time, combine the n-butanol solution, wash it with ammonia test solution 2 times, 20ml each time, discard the ammonia solution, evaporate the n-butanol solution, and add 2ml of methanol to the residue to dissolve it as the test solution; take the negative prescription of stir-fried spinach kernel, and treat it in the same way to obtain the negative test product of stir-fried spinach kernel; take 1g of spinach kernel reference medicinal material, and prepare the reference medicinal material solution in the same way. Then take the spinach kernel saponin A reference substance and the spinach kernel saponin B reference substance, add methanol to prepare a mixed solution containing 1mg of each in 1ml, as the reference solution.

[0164] According to the thin layer chromatography test, the developing solvent is the upper layer solution of n-butanol-glacial acetic acid-water (4:1:5) placed below 10°C, 5-10 μl of each of the above solutions is aspirated, spotted on a silica gel G plate, developed, taken out, dried, sprayed with 5% vanillin sulfuric acid solution, and blown with hot air until the spots are clearly colored and examined immediately.

[0165] The three batches of test samples, the reference medicinal materials of sour jujube seeds, the reference materials of sour jujube seeds saponin A and sour jujube seeds saponin B had spots of the same color at the same Rf value, and the negative test sample of fried sour jujube seeds had no spots. Figure 1 (1. Jujube seed saponin A and Jujube seed saponin B mixed reference substance, 2. Jujube seed reference medicinal material, 3. Jujube seed negative test sample, 4. 1 to 3 batches of preparation test samples).

[0166] (2) Thin layer identification of Coptis chinensis

[0167] Take 0.5g of this product, grind it into powder, add 10ml of methanol, ultrasonically treat it for 30 minutes, filter it, and take the filtrate as the test solution; take the negative prescription of Coptis chinensis, treat it in the same way to get the negative test product of Coptis chinensis; take another 0.25g of Coptis chinensis reference medicinal material, and prepare the reference medicinal material solution in the same way; then take the berberine hydrochloride reference substance, add methanol to prepare a solution containing 0.5mg per 1ml, as the reference substance solution;

[0168] According to the thin layer chromatography test, the developing solvent is cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine (3:3.5:1:1.5:0.5:1) and the upper layer solution is placed below 10°C. 1 μl of each of the above solutions is taken and spotted on a silica gel G plate, which is placed in a developing cylinder pre-saturated with concentrated ammonia test solution for 20 minutes, developed, taken out, dried, and examined under an ultraviolet lamp (365nm);

[0169] The three batches of test samples had fluorescent spots of the same color at the same Rf value as the chromatograms of the reference medicinal material of Coptis chinensis and the chromatograms of the reference substance of berberine hydrochloride, while the negative test sample of Coptis chinensis had no spots. Figure 2 (1. Berberine hydrochloride reference substance, 2. Coptis chinensis reference medicinal material, 3. Coptis chinensis negative test substance, 4. 1 to 3 batches of preparation test substances).

[0170] (3) Thin layer identification of Salvia miltiorrhiza

[0171] Take 1g of this product, grind it into powder, add 25ml of ethyl acetate, ultrasonically treat it for 20 minutes, filter it, evaporate the filtrate, and dissolve the residue in 1ml of ethyl acetate to prepare the test solution; take the negative prescription of Danshen, treat it in the same way to obtain the negative test product of Danshen; take 1g of Danshen control medicinal material, prepare the control medicinal material solution in the same way; take Tanshinone ⅡA reference substance, add ethyl acetate to prepare a solution containing 1mg per 1ml, as the reference substance solution;

[0172] According to the thin layer chromatography test, the developing solvent is toluene-ethyl acetate (19:1), take 4 μl of the above-mentioned Tanshinone ⅡA reference solution and Danshen reference medicinal material solution, and 5-10 μl of the test solution, spot them on the silica gel G plate, develop, take out, dry, and examine under sunlight;

[0173] The three batches of test samples had spots of the same color at the same Rf value as the chromatograms of the Danshen reference medicinal material and the chromatograms of the Tanshinone ⅡA reference substance, while the negative Danshen test sample had no spots. Figure 3 (1. Tanshinone ⅡA reference substance 2. Danshen reference medicinal material, 3. Danshen negative test sample, 4. 1 to 3 batches of preparation test samples).

[0174] (4) Thin layer chromatography identification of Schisandra chinensis

[0175] Take 4g of this product, grind it into powder, add 25ml of petroleum ether (60-90℃), ultrasonically treat it for 20 minutes, filter it, evaporate the filtrate to dryness, add 0.5ml of petroleum ether (60-90℃) to dissolve the residue, and use it as the test solution; take the negative prescription of Schisandrae Chinensis Vinegar, treat it in the same way to obtain the negative test product of Schisandrae Chinensis Vinegar; take 1g of Schisandrae Chinensis reference medicinal material, and prepare the reference medicinal material solution in the same way; take the Schisandrae Chinensis A reference substance, add petroleum ether (60-90℃) to prepare a solution containing 1mg per 1ml, and use it as the reference substance solution;

[0176] According to the thin layer chromatography test, the developing solvent is petroleum ether (60-90°C)-ethyl formate-formic acid (15:5:1) placed below 10°C, 5 μl of each of the above solutions is taken, spotted on a silica gel GF254 plate, developed, taken out, dried, and examined under an ultraviolet lamp (254nm);

[0177] The three batches of test samples had spots of the same color at the same Rf value as the chromatograms of the Schisandra chinensis reference medicinal material and the chromatograms of the Schisandra chinensis A reference substance, but the vinegar Schisandra chinensis negative test sample had no spots. Figure 4 (1. Schisandrin A reference substance, 2. Schisandra control medicinal materials, 3. Schisandra negative test substances, 4. 1 to 3 batches of preparation test substances).

[0178] (5) TLC Identification of Polygonum multiflorum

[0179] Take 2g of this product, grind it into powder, add 30ml of ether and 2ml of hydrochloric acid, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, add 1ml of methanol to dissolve the residue, and use it as the test solution; take the negative prescription of Radix Polygoni Multiflori, treat it in the same way to obtain the negative test product of Radix Polygoni Multiflori; take 0.25g of Radix Polygoni Multiflori reference medicinal material, and prepare the reference medicinal material solution in the same way; take the rhein reference substance, add methanol to prepare a solution containing 0.1mg per 1ml, and use it as the reference substance solution;

[0180] According to the thin layer chromatography test, the developing solvent is petroleum ether (60-90°C)-ethyl formate-formic acid (15:5:1) placed below 10°C, 5 μl of each of the above solutions is taken, spotted on a silica gel G plate, developed, taken out, dried, and examined under an ultraviolet lamp (365nm);

[0181] The three batches of test samples had fluorescent spots of the same color at the same Rf value as the chromatograms of the Radix Polygoni Multiflori reference medicinal materials and the chromatograms of the Rhein reference substance, while the Radix Polygoni Multiflori negative test sample had no spots. Figure 5 (1. Rhein reference substance, 2. Radix Polygoni Multiflori reference medicinal material, 3. Radix Polygoni Multiflori negative test substance, 4. 1 to 3 batches of preparation test substances).

[0182] 1.2 Quantitative study of Zaobai Anshen granules

[0183] 1.2.1 Quantitative Methodological Study of Index Components

[0184] (1) Determination of the content of spinosin

[0185] ①Optimization of chromatographic conditions

[0186] a. Selection of detection wavelength

[0187] According to the preliminary experimental research, the maximum absorption wavelength of spinoside is 335nm. Under this wavelength condition, the response of spinoside is high and stable, so the selected detection wavelength is 335nm.

[0188] b. Investigation of mobile phase elution conditions

[0189] Agilent C18 column (250 mm × 4.6 mm), detection wavelength 335 nm, acetonitrile (A)-water (B), acetonitrile (A)-0.1% phosphoric acid (B) as mobile phase, eluted according to the ratios in Tables 1 to 2, and compared the chromatograms, see Figure 6 , (A to B in the figure correspond to the elution conditions in sequence). The experimental results show that the separation effect of the main peak of spinosin and other impurity peaks in the chromatogram of the test sample is poor when acetonitrile is used as the elution condition, so it is considered to change to methanol system elution.

[0190] Table 1. Mobile phase elution conditions 1

[0191]

[0192] Table 2. Mobile phase elution conditions 2

[0193]

[0194] c. Investigation of mobile phase elution gradient

[0195] Agilent C18 chromatographic column (250mm×4.6mm), detection wavelength of 335nm, methanol (A)-0.1% phosphoric acid (B) as mobile phase, eluted according to the ratios in Tables 3 to 5, and compared the chromatograms. Figure 7, (A to C in the figure correspond to the elution conditions in sequence). The experimental results show that the chromatogram in the mobile phase elution condition 5 has a better separation effect than other chromatogram index components, so this is used as the gradient elution condition.

[0196] Table 3. Mobile phase elution conditions 3

[0197]

[0198] Table 4. Mobile phase elution conditions 4

[0199]

[0200] Table 5. Mobile phase elution conditions 5

[0201]

[0202] b. Selection of chromatographic columns from different manufacturers

[0203] Agilent, Waters, Elite, Phenomenex C18 chromatographic columns (250mm×4.6mm), detection wavelength of 335nm, methanol (A)-0.1% phosphoric acid (B) as mobile phase, elution according to the ratio in Table 5, the influence of different manufacturers' chromatographic columns on the chromatogram was investigated, see Figure 8 , (A to D in the figure correspond to chromatographic columns of different manufacturers). The experimental results show that the main peak of spinoside in the Waters manufacturer's chromatographic column is wrapped by impurity peaks, and the main peak of spinoside in the Agilent and Elite manufacturer's chromatographic columns is easily interfered by impurity peaks before and after, while the chromatogram in the Phenomenex C18 column has a better separation effect than other chromatographic index components, so it is used as the chromatographic column.

[0204] c. Determination of liquid phase conditions

[0205] Phenomenex C18 column (250mm×4.6mm), detection wavelength of 335nm, methanol (A)-0.1% phosphoric acid (B) as mobile phase, elution according to the ratio in Table 5; flow rate of 1mL / min; column temperature of 25°C; injection volume of 10μL.

[0206] ②Specificity inspection

[0207] Weigh Zaobai Anshen Granules and obtain the test sample according to the test sample treatment item; weigh the stir-fried spiny jujube seed negative prescription and prepare the stir-fried spiny jujube seed negative test sample in the same way; take 10 μL of each of the above test samples and spinotin reference solution, and analyze by HPLC injection. The results show that the spiny jujube seed negative has no interference.

[0208] ③Linear relationship investigation

[0209] Take the spinosulin reference substance, add methanol to dilute, and obtain a spinosulin solution with a concentration of 0.8720 mg / mL. Use this as the mother solution, and use methanol to prepare standard solutions with concentrations of 0.01362, 0.02725, 0.05450, 0.1090, and 0.2180 mg / mL. Take 10 μL of the reference solution of the above five concentrations, inject it into the HPLC injection system, and use the spinosulin concentration-peak area as the coordinate axis to draw a standard curve Y=24454.8312X+14.7494, r=1.0000. Spinosulin has good linearity in the range of 0.01362-0.2180 mg / mL. The results are shown in Table 6. Fig. 9 .

[0210] Table 6. Spinosin linearity test

[0211]

[0212]

[0213] ③ Investigation of the pretreatment method of the test sample

[0214] a. Investigation of extraction methods

[0215] Take this product, grind it into powder, take about 1g, weigh it accurately, put it in a stoppered conical flask, parallel 2 parts, accurately add 70% methanol 25ml, weigh it, ultrasonically treat it for 30 minutes, heat it under reflux for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to get it. HPLC injection analysis was used to determine the content of spinosin, and the results are shown in Table 7. The results show that the extraction effects of the two extraction methods are equivalent. For the convenience of operation, the ultrasonic method is selected as the extraction method.

[0216] Table 7. Results of investigation on spinosum extraction method (n=2)

[0217]

[0218] b. Selection of extraction solvent

[0219] Take this product, grind it into powder, take about 1g, weigh it accurately, put it in a stoppered conical flask, parallel 7 parts, accurately add methanol, 70% methanol, 50% methanol, ethanol, 70% ethanol, 50% ethanol, 25ml of pure water, weigh it, ultrasonic treatment for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate. HPLC injection analysis was used to determine the content of spinosin, and the results are shown in Table 8. The results show that 70% methanol has the highest extraction rate. Too many impurities extracted by methanol cause the main peak of spinosin to be wrapped with other peaks, resulting in a high content, so 70% methanol was selected as the extraction solvent.

[0220] Table 8. Results of investigation on the solvent for extracting spinosad (n=7)

[0221]

[0222] c. Investigation of extraction time

[0223] Take this product, grind it into powder, take about 1g, weigh it accurately, put it in a stoppered conical flask, parallel 4 parts, accurately add 25ml of 70% methanol, weigh it, ultrasonically treat it for 10 minutes, 20 minutes, 30 minutes, and 40 minutes respectively, take it out, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to get it. HPLC injection analysis was used to determine the content of spinosin, and the results are shown in Table 9. The results show that the extraction effect is equivalent under different extraction times. In order to save time and ensure that the components are completely extracted, the ultrasonic time is selected to be a more moderate 20 minutes.

[0224] Table 9. Results of investigation on the extraction time of spinosadine (n=4)

[0225]

[0226] d. Investigation of the amount of extraction solvent

[0227] Take this product, grind it into powder, take about 1g, weigh it accurately, put it in a stoppered conical flask, parallel 4 parts, accurately add 70% methanol 15ml, 25ml, 35ml, 45ml, weigh it, ultrasonically treat it for 20 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate. HPLC injection analysis was used to determine the content of spinosin, and the results are shown in Table 10. The results show that the extraction effect is equivalent under different extraction volumes. In order to save solvent and ensure that the components are completely extracted, the extraction volume is selected as 25ml.

[0228] Table 10. Results of investigation on the amount of solvent used for extraction of spinosad (n=4)

[0229]

[0230] ④ Handling of test samples

[0231] Take this product, grind it into powder, take about 1g, weigh it accurately, put it in a stoppered conical flask, add 25ml of 70% methanol accurately, weigh it, treat it with ultrasound for 20 minutes, take it out, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the spinosulin test sample.

[0232] ⑤Precision experiment

[0233] 10 μL of each low, medium and high concentration reference solution was accurately drawn and measured according to the established HPLC analysis method. The sample was injected 6 times continuously. The results are shown in Table 11. The results show that the instrument has good precision.

[0234] Table 11. Spinosin precision test results (n=6)

[0235]

[0236] ⑥Repeat experiment

[0237] Take this product, prepare the test sample according to the test sample processing method, and perform 6 parallel tests according to the established HPLC analysis method. The results are shown in 12. The results show that the method has good repeatability, and the content of spinosin in Zaobai Anshen Granules is 1.05 mg / g.

[0238] Table 12. Spinosin repeatability test results (n=6)

[0239]

[0240] ⑦Stability test

[0241] The test sample was prepared according to the test sample treatment method, and the same test sample solution was placed at room temperature for 0, 2, 4, 8, 12, and 24 hours, respectively, and HPLC analysis was performed. The results are shown in Table 13. The results show that the sample has good stability within 24 hours.

[0242] Table 13. Spinosin stability test results

[0243]

[0244]

[0245] ⑧ Sample recovery experiment

[0246] Take 9 portions of Zaobai Anshen granules with known content, add 50%, 100%, and 150% of the reference substance of the spinotoxin content in the test sample, respectively, prepare the test sample according to the test sample processing method, process 3 portions in parallel, and perform HPLC analysis and determination. The results are shown in Table 14. The results show that the method has high recovery rate and good accuracy in determining the content of spinotoxin.

[0247] Table 14. Spinosin recovery test results (n = 9)

[0248]

[0249] ⑨Durability test

[0250] The tolerance level of the results was determined by making slight changes in the column temperature, flow rate, chromatographic column, etc. of the chromatographic conditions, and the results are shown in Table 15. The results show that the method has strict requirements for the determination conditions and has a narrow tolerance range for parameters such as flow rate, column temperature, and chromatographic column. In order to ensure the stability of the method, the chromatographic parameters need to be fixed.

[0251] Table 15. Spinosin durability test results

[0252]

[0253]

[0254] Note: “ / ” indicates that the separation does not meet the requirements and the data are not included in the calculation.

[0255] (2) Determination of berberine hydrochloride content

[0256] ①Optimization of chromatographic conditions

[0257] a. Selection of detection wavelength

[0258] According to preliminary experimental studies, the maximum absorption wavelength of berberine hydrochloride is 345nm. Under this wavelength condition, the response of berberine hydrochloride is high and stable, so the selected detection wavelength is 345nm.

[0259] b. Investigation of mobile phase elution conditions

[0260] Hanbang Lichrospher C18 chromatographic column (250mm×4.6mm), detection wavelength of 345nm, acetonitrile-0.033mol / L potassium dihydrogen phosphate solution (30:70, V / V) (adjust pH value to 3-4 with phosphoric acid) (condition 1), acetonitrile-0.05mol / L potassium dihydrogen phosphate solution (30:70, V / V) (add 0.4g sodium dodecyl sulfate per 100ml, and adjust pH to 4 with phosphoric acid) (condition 2), acetonitrile-0.05mol / L potassium dihydrogen phosphate solution (50:50, V / V) (add 0.4g sodium dodecyl sulfate per 100ml, and adjust pH to 4 with phosphoric acid) (condition 3) as mobile phase, isocratic elution, compare chromatograms, see Fig.10 , (A to C in the figure correspond to the elution conditions in sequence). The experimental results show that the separation degree between the main peak of berberine hydrochloride and the impurities in conditions 1 and 2 is poor, while the chromatogram in mobile phase elution condition 3 has a good separation effect compared with other chromatogram index components, so this is used as the elution condition.

[0261] c. Determination of liquid phase conditions

[0262] Hanbang Lichrospher C18 chromatographic column (250mm×4.6mm), detection wavelength of 345nm, acetonitrile-0.05mol / L potassium dihydrogen phosphate solution (50:50, V / V) (0.4g of sodium dodecyl sulfate was added to every 100ml, and the pH was adjusted to 4 with phosphoric acid) as the mobile phase; the flow rate was 1mL / min; the column temperature was 30℃; the injection volume was 10μL.

[0263] ②Specificity inspection

[0264] Weigh Zaobai Anshen Granules and prepare the test sample according to the test sample treatment item; weigh Coptis chinensis negative prescription and prepare Coptis chinensis negative test sample in the same way; take 10μL of each of the above test samples and berberine hydrochloride reference solution and analyze by HPLC injection. The results show that Coptis chinensis negative has no interference.

[0265] ③Linear relationship investigation

[0266] Take berberine hydrochloride reference substance, dilute with methanol, and prepare standard solutions with concentrations of 0.005377, 0.01075, 0.02689, 0.05377, 0.1075, and 0.2151 mg / mL, respectively. Take 10 μL of the reference solution of the above six concentrations, inject it into the HPLC injection system, and use berberine hydrochloride concentration-peak area as the coordinate axis to draw a standard curve Y=44547.9213X-3.8220, r=1.0000. Berberine hydrochloride has good linearity in the range of 0.005377-0.2151 mg / mL. The results are shown in Table 16. Fig.11 .

[0267] Table 16 Berberine hydrochloride linearity test

[0268]

[0269] ③ Investigation of the pretreatment method of the test sample

[0270] a. Investigation of extraction methods

[0271] Take this product, grind it into powder, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, parallel 2 parts, accurately add 50ml of methanol-hydrochloric acid (100:1, V / V) solution, weigh it, ultrasonically treat it and heat it under reflux for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with methanol-hydrochloric acid (100:1, V / V) solution, shake it well, filter it, and take the filtrate to get it. HPLC injection analysis was used to determine the content of berberine hydrochloride, and the results are shown in Table 17. The results show that the ultrasonic extraction efficiency is high, so the ultrasonic method is selected as the extraction method for the convenience of operation.

[0272] Table 17. Results of the investigation of berberine hydrochloride extraction method (n=2)

[0273]

[0274] b. Selection of extraction solvent

[0275] Take this product, grind it into powder, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, and add 8 parts in parallel, respectively, accurately add methanol, ethanol, 50% methanol, 50% ethanol, methanol-hydrochloric acid (100:1, V / V) solution, ethanol-hydrochloric acid (100:1, V / V) solution, 50% methanol-hydrochloric acid (100:1, V / V) solution, 50% ethanol-hydrochloric acid (100:1, V / V) solution 50ml, weigh it, ultrasonic treatment for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with the corresponding reagent, shake it well, filter it, and take the filtrate to get it. HPLC injection analysis was used to determine the content of berberine hydrochloride, and the results are shown in Table 18. The results show that the extraction rate is higher when methanol-hydrochloric acid (100:1, V / V) solution is used as an extractant, so this is selected as the extraction solvent.

[0276] Table 18. Results of investigation on berberine hydrochloride extraction solvents (n=8)

[0277]

[0278] c. Investigation of extraction time

[0279] Take this product, grind it into powder, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, parallel 4 parts, accurately add 50ml of methanol-hydrochloric acid (100:1, V / V) solution, weigh it, ultrasonically treat it for 10 minutes, 20 minutes, 30 minutes, and 40 minutes respectively, take it out, let it cool, weigh it again, make up the lost weight with methanol-hydrochloric acid (100:1, V / V) solution, shake it well, filter it, and take the filtrate to get it. HPLC injection analysis was used to determine the content of berberine hydrochloride, and the results are shown in Table 19. The results show that the extraction effect is equivalent under different extraction times. In order to save time and ensure that the components are completely extracted, the ultrasonic time is selected to be 20 minutes.

[0280] Table 19. Results of the investigation on the extraction time of berberine hydrochloride (n=4)

[0281]

[0282] d. Investigation of the amount of extraction solvent

[0283] Take this product, grind it into powder, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, and add 4 parts in parallel. Accurately add 25ml, 50ml, 75ml, and 100ml of methanol-hydrochloric acid (100:1, V / V) solution, weigh it, ultrasonicate it for 20 minutes, take it out, let it cool, weigh it again, make up the lost weight with methanol-hydrochloric acid (100:1, V / V) solution, shake it well, filter it, and take the filtrate. HPLC injection analysis was used to determine the content of berberine hydrochloride. The results are shown in Table 20. The results show that when the extraction solvent is 50ml, the berberine hydrochloride in the sample has been completely extracted, so the extraction volume is selected to be 50ml.

[0284] Table 20. Results of the investigation on the amount of solvent used for berberine hydrochloride extraction (n=4)

[0285]

[0286] ④ Handling of test samples

[0287] Take this product, grind it into powder, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, accurately add 50ml of methanol-hydrochloric acid (100:1, V / V) solution, weigh it, treat it with ultrasound for 20 minutes, let it cool, weigh it again, make up the lost weight with methanol-hydrochloric acid (100:1, V / V) solution, shake it well, filter it, and take the filtrate to obtain it.

[0288] ⑤Precision experiment

[0289] 10 μL of each low, medium and high concentration reference solution was accurately drawn and measured according to the established HPLC analysis method. The sample was injected 6 times continuously. The results are shown in Table 21. The results show that the instrument has good precision.

[0290] Table 21. Berberine hydrochloride precision test results (n = 6)

[0291]

[0292] ⑥Repeat experiment

[0293] Take this product, prepare the test sample according to the test sample processing method, and make 6 parallel copies. Determine according to the established HPLC analysis method. The results are shown in 22. The results show that the method has good repeatability, and the berberine hydrochloride content in Zaobai Anshen Granules is 22.88 mg / g.

[0294] Table 22. Berberine hydrochloride repeatability test results (n=6)

[0295]

[0296]

[0297] ⑦Stability test

[0298] The test sample was prepared according to the test sample treatment method, and the same test sample solution was placed at room temperature for 0, 2, 4, 8, 12, and 24 hours, respectively, and HPLC analysis was performed. The results are shown in Table 23. The results show that the sample has good stability within 24 hours.

[0299] Table 23. Berberine hydrochloride stability test results

[0300]

[0301] ⑧ Sample recovery experiment

[0302] Take 9 portions of Zaobai Anshen granules with known content, add 50%, 100%, and 150% of the berberine hydrochloride content in the test sample, respectively, prepare the test sample according to the test sample processing method, process 3 portions in parallel, and perform HPLC analysis and determination. The results are shown in Table 24. The results show that this method has a high recovery rate and good accuracy in determining the content of berberine hydrochloride.

[0303] Table 24. Berberine hydrochloride sample recovery test results (n = 9)

[0304]

[0305]

[0306] ⑨Durability test

[0307] The results of the determination of the tolerance level of the results without being affected by slight changes in the chromatographic conditions such as column temperature, flow rate, and chromatographic column are shown in Table 25. The results show that the determination conditions of this method have good durability.

[0308] Table 25. Berberine hydrochloride durability test results

[0309]

[0310] In summary, the present invention determines the qualitative and quantitative quality evaluation methods of Zaobai Anshen Granules, which can be used for the comprehensive quality control of the prescription.

[0311] It should be noted that the present invention has conducted a large number of research experiments on chromatographic conditions during long-term and extensive experimental research, but they cannot be described one by one here. The above only uses typical experiments as an illustration.

[0312] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for quality control of a Chinese medicine composition, characterized in that: include: Establishment of the method for determining the content of spinosin in Zaobai Anshen granules: A) extracting Zaobai Anshen granules with a solvent to obtain a test solution; B) taking a spinosin standard and dissolving it in a solvent to obtain a reference substance solution; C) using high performance liquid chromatography to obtain chromatograms of the test solution in step A) and the reference substance in step B) under the same detection conditions, and the specific chromatographic parameters are as follows: The chromatographic column was a C18 column, the detection wavelength was 335 nm; the mobile phase was methanol-0.1% phosphoric acid; The elution procedure is: 0-5min, phase A 20%-30%, phase B 80%-70%; 5-30min: 30%-40% of phase A, 70%-60% of phase B; 30-35min: Phase A 40%-100%, Phase B 60%-0%; 35-40min: Phase A 100%, Phase B 0%; 40-45min: Phase A 100%-20%, Phase B 0%-80%; D) calculating the content of chemical components in Zaobai Anshen Granules by external standard method according to the concentration of the reference substance, the peak area of ​​the reference substance solution in the chromatogram and the peak area of ​​the components in Zaobai Anshen Granules corresponding to the reference substance solution in the chromatogram, and based on the detection conditions in step C).

2. The method according to claim 1, characterized in that The mobile phase flow rate is 1 ml / min, the column temperature is 25°C, and the injection volume is 10 μL; the solvent in step A) is 70% methanol, and the extraction is ultrasonic extraction; the ultrasonic extraction time is 10 to 20 minutes; The spinosin has good linearity in the range of 0.01362 to 0.2180 mg / mL.

3. The method according to claim 1, characterized in that The quality control method also includes any one or more of the following determination methods: i) HPLC determination of berberine hydrochloride content in Zaobai Anshen granules; ii) Using thin layer chromatography to identify the stir-fried spiny jujube seeds in Zaobai Anshen granules; iii) Identification of Coptis chinensis by thin layer chromatography; iv) identification of Salvia miltiorrhiza using thin layer chromatography; v) identifying Schisandra chinensis var. vinegar by thin layer chromatography; vi) Identification of Polygonum multiflorum by thin layer chromatography.

4. The method according to claim 3, characterized in that The HPLC determination of the berberine hydrochloride content in Zaobai Anshen granules is specifically as follows: a) extracting Zaobai Anshen granules with a solvent to obtain a test solution; b) taking a berberine hydrochloride standard product and dissolving it in a solvent to obtain a reference substance solution; c) using high performance liquid chromatography to obtain chromatograms of the test solution in step a) and the reference substance in step b) under the same detection conditions, and performing qualitative and quantitative analysis on the components of the test solution based on the chromatograms of the reference substance; The specific chromatographic parameters are as follows: The chromatographic column was a C18 column with a detection wavelength of 345 nm; the mobile phase was acetonitrile-0.05 mol / L potassium dihydrogen phosphate solution (50:50), with 0.4 g of sodium dodecyl sulfate added to every 100 ml, and the pH value was adjusted to 4.0 with phosphoric acid; isocratic elution, mobile phase flow rate of 1.0 ml / min; the column temperature was 30°C, and the injection volume was 10 μL.

5. The method according to claim 4, characterized in that The step a) is specifically as follows: Zaobai Anshen granules are ultrasonically extracted with a mixed solution of methanol and hydrochloric acid, and the ultrasonic extraction time is 10 to 30 minutes; The berberine hydrochloride has good linearity in the range of 0.005377 to 0.2151 mg / mL.

6. The method according to claim 3, characterized in that: The method of using thin layer chromatography to identify the stir-fried spiny jujube seeds in Zaobai Anshen granules is specifically as follows: a) Extracting Zaobai Anshen Granules, stir-fried spinach seed negative test sample and spinach seed control medicinal material with solvent to obtain test sample solution, stir-fried spinach seed negative test sample and spinach seed control medicinal material solution respectively; Add methanol to dissolve jujuboside A reference substance and jujuboside B reference substance to obtain jujuboside A reference substance solution and jujuboside B reference substance solution; b) performing thin layer chromatography on the test solution, the stir-fried Ziziphus jujuba seed negative test sample, the Ziziphus jujuba seed control medicinal material solution, the Ziziphus jujuba seed saponin A reference substance, and the Ziziphus jujuba seed saponin B reference substance solution, the thin layer plate is a silica gel G thin layer plate; the developing solvent is n-butanol-glacial acetic acid-water, and the volume ratio of n-butanol-glacial acetic acid-water is 4:1:5; c) Inspection: If the test sample, the Chinese jujube seed reference material, the Chinese jujube seed saponin A reference material, and the Chinese jujube seed saponin B reference material have spots of the same color at the same Rf value, and the Chinese jujube seed negative test sample has no spots, then it is stir-fried Chinese jujube seed.

7. The method according to claim 3, characterized in that The identification of Rhizoma Coptidis by thin layer chromatography is specifically as follows: a) extracting Zaobai Anshen granules, Coptis chinensis negative test sample and Coptis chinensis control medicinal material with a solvent to obtain a test sample solution, a Coptis chinensis negative test sample and a Coptis chinensis control medicinal material solution, respectively; Dissolve the berberine hydrochloride reference substance in methanol to obtain a berberine hydrochloride reference substance solution; b) performing thin layer chromatography on the test solution, the negative test product of Coptis chinensis, the reference medicinal material solution of Coptis chinensis, and the reference solution of berberine hydrochloride, wherein the thin layer plate is a silica gel G thin layer plate; the developing solvent is cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine, and the volume ratio of the cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine is 3:3.5:1:1.5:0.5:1; c) Inspection: If the test sample, the Rhizoma Coptidis reference material, and the berberine hydrochloride reference material have fluorescent spots of the same color at the same Rf value, and the Rhizoma Coptidis negative test sample has no spots, then it is Rhizoma Coptidis; The inspection was performed under ultraviolet light; the wavelength of the ultraviolet light was 365 nm; and the sample spotting volume was 1 μL.

8. The method according to claim 3, characterized in that The method of using thin layer chromatography to identify Danshen is specifically as follows: a) extracting Zaobai Anshen granules, Danshen negative test sample and Danshen control medicinal material with a solvent to obtain a test sample solution, a Danshen negative test sample and a Danshen control medicinal material solution, respectively; Dissolve the Tanshinone ⅡA reference substance in ethyl acetate to obtain a Tanshinone ⅡA reference substance solution; b) performing thin layer chromatography on the test solution, the negative test sample of Danshen, the control medicinal material solution of Danshen and the reference solution of Tanshinone ⅡA, the thin layer plate is a silica gel G thin layer plate; the developing solvent is toluene-ethyl acetate, and the volume ratio of toluene-ethyl acetate is 19:1; c) Inspection: If the test sample, the control drug of Danshen, and Tanshinone ⅡA have spots of the same color at the same Rf value, and the negative test sample of Danshen has no spots, then it is Danshen; The inspection was performed under daylight; the spotting volume was 4 to 10 μL.

9. The method according to claim 3, characterized in that: The method of using thin layer chromatography to identify Schisandra chinensis vinegar is specifically as follows: a) extracting Zaobai Anshen granules, vinegar Schisandra chinensis negative test sample and Schisandra chinensis control medicinal material with a solvent to obtain a test sample solution, vinegar Schisandra chinensis negative test sample and Schisandra chinensis control medicinal material solution, respectively; Dissolve the Schisandrin A reference substance in petroleum ether to obtain a Schisandrin A reference substance solution; b) The test solution, the vinegar Schisandra negative test sample, the vinegar Schisandra control medicinal material solution and the Schisandrae Chinensis A reference solution are subjected to thin layer chromatography, the thin layer plate is a silica gel GF254 thin layer plate; the developing solvent is petroleum ether-ethyl formate-formic acid, and the volume ratio of the petroleum ether-ethyl formate-formic acid is 15:5:1; c) Inspection: If the test sample, Schisandra chinensis control medicinal material, and Schisandrin A have spots of the same color at the same Rf value, and the negative test sample of Schisandra chinensis vinegar has no spots, then it is Schisandra chinensis vinegar; The inspection was performed under ultraviolet light; the wavelength of the ultraviolet light was 254 nm; and the sample spotting volume was 5 μL.

10. The method according to claim 3, characterized in that: The method of using thin layer chromatography to identify Radix Polygoni Multiflori is specifically as follows: a) extracting Zaobai Anshen granules, Radix Polygoni Multiflori negative test sample and Radix Polygoni Multiflori control medicinal material with solvent to obtain test sample solution, Radix Polygoni Multiflori negative test sample and Radix Polygoni Multiflori control medicinal material solution respectively; Add methanol to dissolve the rheum-emodin reference substance to obtain a rheum-emodin reference substance solution; b) The test solution, Radix Polygoni Multiflori negative test sample, Radix Polygoni Multiflori control medicinal material solution and Rhein reference solution are subjected to thin layer chromatography, the thin layer plate is a silica gel G thin layer plate; the developing solvent is petroleum ether-ethyl formate-formic acid (the upper layer solution placed below 10° C.), and the volume ratio of petroleum ether-ethyl formate-formic acid is 15:5:1; c) Inspection: If the test sample, Radix Polygoni Multiflori control medicinal material, and Rhein have fluorescent spots of the same color at the same Rf value, and the Radix Polygoni Multiflori negative test sample has no spots, then it is Radix Polygoni Multiflori; The inspection was performed under ultraviolet light; the wavelength of the ultraviolet light was 365 nm; and the sample spotting volume was 5 μL.

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