A quality control method of a traditional Chinese medicine composition preparation

Abstract

The application relates to the technical field of analysis, and particularly discloses a quality control method for a traditional Chinese medicine composition preparation, which is prepared from raw medicinal materials such as Astragalus membranaceus, Gentiana macrophylla, Stephania tetrandra, Polygonum cuspidatum, Radix Paeoniae Alba, Angelica sinensis, Myrrha and Ramulus Cinnamomi; the quality control method comprises identification methods, limited quantity inspection methods and content determination methods. The quality control method has good specificity, good repeatability, good stability, good durability, high precision and high accuracy, and can effectively control the product quality of the traditional Chinese medicine composition preparation.

Description

Technical Field

[0001] This invention relates to the field of analytical technology, and in particular to a method for quality control of traditional Chinese medicine composition preparations. Background Technology

[0002] This traditional Chinese medicine composition consists of Astragalus membranaceus, Gentiana macrophylla, Stephania tetrandra, Polygonum cuspidatum, Paeonia lactiflora, Angelica sinensis, Myrrh (processed with vinegar), and Cinnamomum cassia. These herbs work together, combining warming and cooling properties, and invigorating and tonifying the body. They integrate the functions of dispelling wind, dispersing cold, eliminating dampness, clearing heat, tonifying qi, promoting blood circulation, resolving phlegm, and unblocking the meridians, aiming to "dispel pathogenic factors and relieve pain, tonify qi and unblock the meridians." This composition has the effects of dispelling wind, dispersing cold, eliminating dampness, tonifying qi, resolving blood stasis, and unblocking the meridians. It is mainly used to treat rheumatoid arthritis with wind-cold-dampness syndrome, qi deficiency and blood stasis, and phlegm and blood stasis obstruction.

[0003] With the vigorous development of traditional Chinese medicine, the types of Chinese medicinal materials used in clinical practice are increasing day by day. The determination and identification of the content of Chinese medicinal components has become an important task. Efficiently identifying the quality of Chinese medicinal materials and accurately determining the content of components can ensure the quality and efficacy of Chinese medicinal materials and improve the safety of medication.

[0004] Although there are reports that thin-layer chromatography and high-performance liquid chromatography can be used to determine the active ingredients of herbs such as Astragalus membranaceus, Polygonum cuspidatum, Paeonia lactiflora, and Angelica sinensis, there are currently no documents or reports disclosing methods for the identification and content determination of multiple components in this traditional Chinese medicine composition preparation.

[0005] This drug composition contains dozens or even hundreds of bioactive ingredients. For a marketed drug, how to comprehensively control the quality of this traditional Chinese medicine composition preparation and ensure the safety of people's medication is an urgent problem that needs to be solved. Summary of the Invention

[0006] The purpose of this invention is to provide a quality control method for traditional Chinese medicine composition preparations, including identification methods, limit testing methods, and content determination methods, which can effectively, quickly, and comprehensively control the quality of the traditional Chinese medicine composition preparations.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] A quality control method for a traditional Chinese medicine composition preparation, wherein the raw materials of the traditional Chinese medicine composition preparation include the following components in parts by weight: Astragalus membranaceus 120-190 parts, Gentiana macrophylla 120-190 parts, Stephania tetrandra 45-80 parts, Aconitum carmichaelii 45-80 parts, Polygonum cuspidatum 110-180 parts, Spatholobus suberectus 110-180 parts, Clematis chinensis 110-180 parts, Sinapis alba 20-40 parts, Paeonia lactiflora 45-80 parts, Rehmannia glutinosa 120-190 parts, Angelica sinensis 45-80 parts, Commiphora myrrha (processed with vinegar) 40-65 parts, Cyperus rotundus 40-65 parts, Cinnamomum cassia 45-80 parts, and Achyranthes bidentata 45-80 parts.

[0009] The quality control methods for the traditional Chinese medicine composition preparations include identification methods, limit testing methods, and content determination methods.

[0010] The identification method includes at least one of the following identification methods (1) to (5):

[0011] (1) Thin-layer chromatography with Polygonum cuspidatum as reference material and petroleum ether at 30-60℃-ethyl formate-formic acid upper layer solution with a volume ratio of 15:5:1 as the developing solvent.

[0012] (2) Thin-layer chromatography with tetrandrine and tebufenozide as reference standards and chloroform-acetone-methanol-ammonia water as the developing solvent in a volume ratio of 12:2:2:0.1;

[0013] (3) Thin-layer chromatography with cinnamaldehyde as reference standard and petroleum ether at 60-90℃-ethyl acetate at a volume ratio of 17:3 as the developing solvent;

[0014] (4) Thin-layer chromatography with myrrh as reference material and cyclohexane-ethyl ether-glacial acetic acid in a volume ratio of 4:1:0.1 as the developing solvent;

[0015] (5) High performance liquid chromatography with paeoniflorin as reference standard, octadecylsilane-bonded silica gel as stationary phase, and acetonitrile-0.1% phosphoric acid solution (14:86) as mobile phase;

[0016] The limit test method is the aconitine limit test method, which uses aconitine as the reference standard and thin-layer chromatography with cyclohexane-ethyl acetate-ammonia water at a volume ratio of 10:10:0.1 as the developing solvent.

[0017] The content determination methods include methods for determining the content of Astragalus membranaceus and Gentiana macrophylla, and the contents of astragaloside A and gentiopicroside are determined by high performance liquid chromatography.

[0018] The characteristic is that the identification in item (1) is a thin-layer chromatographic identification of Polygonum cuspidatum, and the method includes the following steps:

[0019] Take a traditional Chinese medicine composition preparation and use methanol extract as the test solution; take Polygonum cuspidatum reference material and use methanol extract as the reference material solution. Apply the test solution and the reference material solution separately to the same silica gel G thin layer plate. Use 15:5:1 petroleum ether 30-60℃-ethyl formate-formic acid upper layer solution as the developing solvent. Develop, remove, air dry, and examine under 365nm ultraviolet light. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0020] The identification method described in item (2) is a thin-layer chromatography identification of Stephania tetrandra, which includes the following steps:

[0021] Take the test solution under item (1), evaporate to dryness, dissolve the residue in 1% hydrochloric acid, adjust the pH value with sodium hydroxide solution, extract with chloroform by shaking, evaporate to dryness, dissolve the residue in methanol, and use it as the test solution; separately take the tetrandrine reference standard and the tetrandrine reference standard, add methanol to prepare a mixed solution, and use it as the reference solution. Take the test solution and the reference solution and spot them on the same silica gel G thin layer plate, use 12:2:2:0.1 chloroform-acetone-methanol-ammonia water as the developing solvent, spray with dilute bismuth potassium iodide test solution, and spot the same color in the test chromatogram at the corresponding position as in the reference chromatogram;

[0022] The identification method described in item (3) is a thin-layer chromatographic identification of cinnamon twigs, which includes the following steps:

[0023] Take a traditional Chinese medicine composition preparation and use water to extract the volatile oil as the test solution; take cinnamaldehyde reference standard, add ethanol to prepare a solution as the reference solution, and apply the test solution and the reference solution separately to the same silica gel G thin layer plate. Use 17:3 petroleum ether 60-90℃-ethyl acetate as the developing solvent and spray with dinitrophenylhydrazine ethanol test solution. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard.

[0024] The identification method described in item (4) is a thin-layer chromatography identification of myrrh, which includes the following steps:

[0025] Take myrrh reference material, add water to extract volatile oil as reference material solution, take the reference material solution and the test solution under (3) and spot them on the same silica gel G thin layer plate respectively, use 4:1:0.1 cyclohexane-ethyl ether-glacial acetic acid as the developing solvent, spray with sulfuric acid ethanol solution, heat until the spots are clearly visible, and in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference material;

[0026] The identification method described in item (5) is a thin-layer chromatography identification of white peony root, which includes the following steps:

[0027] Take the traditional Chinese medicine composition preparation, extract it with methanol, and use the filtrate as the test solution; separately take paeoniflorin reference standard, add methanol to prepare a solution as the reference solution, inject the test solution and the reference solution into the liquid chromatograph, use octadecylsilane bonded silica gel as the stationary phase, use acetonitrile-0.1% phosphoric acid solution (14:86) as the mobile phase, and use the detection wavelength at 230 nm. The chromatogram of the test sample should show a chromatographic peak with the retention time corresponding to that of the reference standard.

[0028] Furthermore, the method for testing the limit of aconitine includes the following steps:

[0029] Take a traditional Chinese medicine composition preparation, add ammonia test solution and let stand, then add ether, let stand, evaporate the filtrate to dryness, dissolve the residue in anhydrous ethanol to obtain the test solution; separately take aconitine reference standard, add anhydrous ethanol to prepare a solution to obtain the reference solution. Accurately pipette the test solution and the reference solution and spot them separately on the same silica gel G thin-layer plate, using cyclohexane-ethyl acetate-ammonia water as the developing solvent, and spray with dilute potassium bismuth iodide test solution. In the chromatogram of the test sample, the spot appearing at the corresponding position as in the chromatogram of the reference standard should be smaller than the spot of the reference standard, or no spot should appear.

[0030] Furthermore, the method for determining the content of Astragalus membranaceus includes the following steps:

[0031] HPLC detection: C18 column, mobile phase of methanol:water (v / v) at a ratio of 76–80:20–24, flow rate of 0.8–1.2 ml / min, evaporative light scattering detector, gas flow rate of 2.5–3.5 L / min, drift tube temperature of 75–85 °C;

[0032] Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, add the mobile phase to prepare a solution, and the solution is obtained.

[0033] Preparation of the test solution: Take the Chinese herbal medicine composition preparation, accurately weigh it, add methanol, shake well, filter, accurately measure the filtrate, evaporate to dryness, dissolve the residue in water, extract with chloroform by shaking, discard the chloroform layer, extract the aqueous layer with water-saturated n-butanol by shaking to obtain n-butanol solution, wash, evaporate the n-butanol layer to dryness, dissolve the residue in the mobile phase to obtain the test solution;

[0034] Assay: Accurately pipette the reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0035] Furthermore, the method for determining the content of Gentiana macrophylla includes the following steps:

[0036] HPLC detection: The chromatographic column was C18, the mobile phase was acetonitrile-water with a volume ratio of 10-12:88-90, the detection wavelength was 250-260 nm, and the flow rate was 0.8-1.2 ml / min;

[0037] Preparation of reference solution: Take an appropriate amount of gentiopicroside reference standard, accurately weigh it, add the mobile phase to prepare a solution, and the solution is obtained.

[0038] Preparation of the test solution: Take the traditional Chinese medicine composition preparation, weigh it accurately, add 70% methanol accurately, sonicate, filter, and take the filtrate to obtain the test solution;

[0039] Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

[0040] Furthermore, the dosage form of the above-mentioned traditional Chinese medicine composition is capsule, tablet, pill, oral liquid, granule or powder.

[0041] Furthermore, the active ingredient in the traditional Chinese medicine composition is prepared by the following steps:

[0042] A. Grind the Stephania tetrandra into a fine powder and set aside.

[0043] B. Take cinnamon twigs, vinegar, myrrh, and aromatics, and add 5-8 times the amount of water. Extract the volatile oil continuously by steam distillation for 5-10 hours. Collect the volatile oil, filter the distilled aqueous solution, collect it in a separate container, and set it aside for later use.

[0044] C. Take Gentiana macrophylla, Spatholobus suberectus, Angelica sinensis, Mustard seed, Paeonia lactiflora, and Polygonum cuspidatum, add 8-12 times the amount of 50-80% ethanol, reflux extract 1-3 times, 1 hour each time, filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear extract with a relative density of 1.15±0.05 for later use.

[0045] D. Take Astragalus membranaceus, Aconitum carmichaelii, Clematis chinensis, Rehmannia glutinosa, and Achyranthes bidentata, add 8-12 times the amount of water, decoct 2-5 times, 1.5 hours each time, filter the decoction and combine it with the aqueous solution after distillation of volatile oil, concentrate under reduced pressure to a clear extract with a relative density of 1.15±0.05, and set aside.

[0046] E. Combine the extracts from steps C and D, mix well, dry, and pulverize to obtain a mixed extract powder;

[0047] The fine powder obtained in step A, the volatile oil obtained in step B, and the clear extract powder obtained in step E together constitute the active components of the traditional Chinese medicine composition preparation.

[0048] Furthermore, the preparation process of the capsules is as follows:

[0049] A. Grind the Stephania tetrandra into a fine powder and set aside.

[0050] B. Take cinnamon twigs, vinegar, myrrh, and spice, add 8 times the amount of water, and continuously extract the volatile oil by steam distillation for 10 hours. Collect the volatile oil, filter the distilled aqueous solution, collect it in a separate container, and set it aside for later use.

[0051] C. Take Gentiana macrophylla, Spatholobus suberectus, Angelica sinensis, Sinapis alba, Paeonia lactiflora, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, 1 hour each time. Filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear extract with a relative density of 1.15±0.05 for later use.

[0052] D. Take Astragalus membranaceus, Aconitum carmichaelii, Clematis chinensis, Rehmannia glutinosa, and Achyranthes bidentata, add 8 times the amount of water, decoct 4 times, 1.5 hours each time, filter the decoction, combine it with the aqueous solution after distillation of volatile oil, and concentrate under reduced pressure to a clear extract with a relative density of 1.15±0.05 for later use.

[0053] E. Combine the clear extracts obtained in steps C and D, mix them evenly, dry them, and pulverize them to obtain a mixed clear extract powder. Add the volatile oil obtained in step B, add the fine powder from step A, granulate, shape the granules, and fill them into capsules to obtain the final product.

[0054] Furthermore, the tablet preparation process is as follows:

[0055] A. Grind the Stephania tetrandra into a fine powder and set aside.

[0056] B. Take cinnamon twigs, vinegar, myrrh, and spice, add 6 times the amount of water, and continuously extract the volatile oil by steam distillation for 8 hours. Collect the volatile oil, filter the distilled aqueous solution, collect it separately, and set it aside.

[0057] C. Take Gentiana macrophylla, Spatholobus suberectus, Angelica sinensis, Sinapis alba, Paeonia lactiflora, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, 1 hour each time. Filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear extract with a relative density of 1.15±0.05 for later use.

[0058] D. Take Astragalus membranaceus, Aconitum carmichaelii, Clematis chinensis, Rehmannia glutinosa, and Achyranthes bidentata, add 11 times the amount of water, decoct 3 times, 1.5 hours each time, filter the decoction, combine it with the aqueous solution after distillation of volatile oil, and concentrate under reduced pressure to a clear extract with a relative density of 1.15±0.05 for later use.

[0059] E. Combine the clear pastes obtained in steps C and D, mix them thoroughly, dry them, and pulverize them to obtain a mixed clear paste powder.

[0060] F. Take the volatile oil obtained in step B and incorporate it into the mixture for later use;

[0061] Granulate the fine powder obtained in step A and the clear paste powder obtained in step E, granulate them, add the inclusion complex obtained in step F, and compress them into tablets using conventional methods.

[0062] The inclusion process in step F above is as follows: Take 6-10 times the amount of cyclodextrin as volatile oil, add 6-10 times the amount of water as cyclodextrin, grind, then add an equal volume mixture of volatile oil and 90-95% ethanol, continue grinding until uniform, and then dry to obtain the final product.

[0063] Furthermore, the raw materials of the traditional Chinese medicine composition preparation of the present invention include the following components in parts by weight: Astragalus membranaceus 120 parts, Gentiana macrophylla 190 parts, Stephania tetrandra 45 parts, Aconitum carmichaelii 80 parts, Polygonum cuspidatum 110 parts, Spatholobus suberectus 180 parts, Clematis chinensis 110 parts, Sinapis alba 40 parts, Paeonia lactiflora 45 parts, Rehmannia glutinosa 190 parts, Angelica sinensis 45 parts, Myrrh (processed with vinegar) 65 parts, Cyperus rotundus 40 parts, Cinnamomum cassia 80 parts, and Achyranthes bidentata 45 parts.

[0064] Furthermore, the raw materials of the traditional Chinese medicine composition preparation of the present invention include the following components in parts by weight: 150 parts of Astragalus membranaceus, 160 parts of Gentiana macrophylla, 60 parts of Stephania tetrandra, 65 parts of Aconitum carmichaelii, 120 parts of Polygonum cuspidatum, 130 parts of Spatholobus suberectus, 135 parts of Clematis chinensis, 35 parts of Sinapis alba, 50 parts of Paeonia lactiflora, 160 parts of Rehmannia glutinosa, 60 parts of Angelica sinensis, 55 parts of Myrrh (processed with vinegar), 45 parts of Cyperus rotundus, 60 parts of Cinnamomum cassia, and 60 parts of Achyranthes bidentata.

[0065] Furthermore, the raw materials of the traditional Chinese medicine composition preparation of the present invention include the following components in parts by weight: Astragalus membranaceus 146 parts, Gentiana macrophylla 139 parts, Stephania tetrandra 58 parts, Aconitum carmichaelii 75 parts, Polygonum cuspidatum 120 parts, Spatholobus suberectus 125 parts, Clematis chinensis 135 parts, Mustard seed 28 parts, Paeonia lactiflora 60 parts, Rehmannia glutinosa 150 parts, Angelica sinensis 70 parts, Myrrh (processed with vinegar) 60 parts, Cyperus rotundus 60 parts, Cinnamomum cassia 70 parts, and Achyranthes bidentata 70 parts.

[0066] Furthermore, the raw materials of the traditional Chinese medicine composition preparation of the present invention include the following components in parts by weight: Astragalus membranaceus 156 parts, Gentiana macrophylla 156 parts, Stephania tetrandra 63 parts, Aconitum carmichaelii 63 parts, Polygonum cuspidatum 146 parts, Spatholobus suberectus 146 parts, Clematis chinensis 146 parts, Mustard seed 31 parts, Paeonia lactiflora 63 parts, Rehmannia glutinosa 156 parts, Angelica sinensis 63 parts, Myrrh (processed with vinegar) 52 parts, Cyperus rotundus 52 parts, Cinnamomum cassia 63 parts, and Achyranthes bidentata 63 parts.

[0067] Compared to existing technologies, the quality control method provided by this invention uses paeoniflorin, tetrandrine, fangchinorline, cinnamaldehyde, Polygonum cuspidatum, Angelica sinensis, and vinegar-processed myrrh as reference standards or reference medicinal materials, and employs a specific developing solvent. Thin-layer chromatography (TLC) can effectively and rapidly identify the corresponding active ingredients in the herbal composition preparations of this invention, producing clear spots with good separation and repeatability. Simultaneously, this invention also establishes a TLC method for the limit test of aconitine, enabling rapid and effective control of aconitine in the preparation. Furthermore, this invention establishes a high-performance liquid chromatography (HPLC) method for the determination of astragaloside A and gentianoside in the herbal composition preparation, exhibiting good specificity, repeatability, stability, robustness, precision, and accuracy. This method, when used in conjunction with TLC, effectively and rapidly controls the quality of the herbal composition preparation.

[0068] During the research process for quality standards, researchers examined various methods for identifying and determining the content of components, including but not limited to:

[0069] In the qualitative identification of Paeonia lactiflora, different methods for preparing test solution were investigated. The results showed that the optimal method was to use methanol for ultrasonic treatment, evaporate to dryness, dissolve in water, remove impurities with chloroform, extract the aqueous layer with water-saturated n-butanol, evaporate the n-butanol solution to dryness, dissolve the residue in methanol, and elute with methanol on a neutral alumina column to prepare the test solution. Paeoniflorin was used as a reference standard, and chloroform-methanol (4:1) was used as the developing solvent. The solution was developed by spraying with 5% vanillin-sulfuric acid solution and heating at 105℃ until the spots were clearly visible. In the chromatogram of the test sample, spots of the same color as those in the reference standard chromatogram appeared at the corresponding positions, and the spots were round, clear, and without interference from negative samples. However, during the subsequent implementation of the quality standards, poor reproducibility was found. The preparation method of the test sample solution was then optimized. After extraction with water-saturated n-butanol in the original method, washing with water saturated with n-butanol resulted in unsatisfactory thin-layer chromatography (TLC) results. Subsequently, liquid chromatography (LC) identification was used. The test sample chromatogram showed a chromatographic peak with a retention time corresponding to that of the reference standard, indicating good resolution. The robustness of different chromatographic columns, detection wavelengths, mobile phase flow rates, mobile phase ratios, column temperatures, and phosphoric acid concentrations was also investigated. The test sample chromatogram consistently showed chromatographic peaks with retention times consistent with the reference standard, and the specificity test was negative with no interference. Validation with three batches of samples demonstrated good reproducibility.

[0070] Thin-layer chromatography (TLC) identification studies were previously conducted on Angelica sinensis. The test solution from the Cinnamomum cassia identification section was used as the test sample for Angelica sinensis identification. 0.5g of Angelica sinensis reference material was added to 20ml of ether, sonicated for 10 minutes, filtered, and the ether evaporated. The residue was dissolved in 1ml of ethanol to obtain the reference material solution. These solutions were spotted onto the same silica gel G TLC plate, using n-hexane-ethyl acetate (4:1) as the developing solvent, and examined under UV light (365nm). Under these conditions, good experimental results were obtained. However, during standard implementation, poor reproducibility and resolution were found. Further tests were conducted using cyclohexane-ethyl acetate (4:1), petroleum ether (60℃~90℃)-ethyl acetate (8.5:1.5), and cyclohexane-ethyl acetate-glacial acetic acid (7:2:1) as developing solvents, and examined under UV light (365nm). The resolution remained poor, and the TLC results showed no improvement. Further studies were conducted on the preparation methods of the test samples. The solutions were prepared by sonication with ether, filtration, evaporation of the filtrate to dryness, and dissolution of the residue as test sample solution 1; sonication with ether, washing of the filtrate with 0.5% sodium hydroxide, evaporation of the ether solution to dryness, and dissolution of the residue as test sample solution 2; and sonication with hot water, followed by cooling and centrifugation, extraction of the supernatant with ethyl acetate, evaporation of the ethyl acetate layer to dryness, and dissolution of the residue as test sample solution 3. None of these three methods achieved satisfactory thin-layer chromatography (TLC) results, and a mature TLC identification method could not be established. Therefore, TLC identification of Angelica sinensis was not possible.

[0071] Thin-layer chromatography (TLC) identification of *Sinapis alba* seeds was previously studied. Methanol was extracted using ultrasound, and the extract was concentrated to serve as the test solution. Sinoprosinolate thiocyanate was used as a reference, and ethyl acetate-acetone-formic acid-water (5:3:1:0.5) was used as the developing solvent. Dilute potassium bismuth iodide reagent was sprayed onto the sample. The spots in the test sample chromatogram were not very clear. The sample was then examined under ultraviolet light (365 nm). In the test sample chromatogram, fluorescent spots of the same color as those in the reference chromatogram appeared at the corresponding positions, but the spots were dispersed. The sample was developed using acetone-methanol-formic acid-water (12:1:0.5:0.5), removed, dried, and examined under ultraviolet light (254 nm). In the chromatogram of the test sample, the background color was dark, and the spots were scattered and not round. Further studies were conducted on the identification of mustard seeds: using a mustard seed reference solution as a control, methanol ultrasonic extraction was performed, with ethyl acetate-acetone-formic acid-water (5:3:1:0.5) as the developing solvent and examined under UV light (365 nm). Similarly, with acetone-methanol-formic acid-water (12:1:0.5:0.5) as the developing solvent and examined under UV light (254 nm), the spots were not clear enough. Despite repeated changes to the developing and colorimetric solvents, a mature thin-layer chromatography identification method could not be established, therefore, thin-layer chromatography identification of mustard seeds was not possible.

[0072] Thin-layer chromatography (TLC) was used to identify *Spatholobus suberectus* (a type of vine). Methanol was extracted ultrasonically, the extract was evaporated to dryness, dissolved in water, and then extracted again with chloroform and ethyl acetate by shaking. The extract was purified using a silica gel column to prepare the test solution. Aromatin was used as a reference, and chloroform-methanol (30:1) was used as the developing solvent. The solution was then removed, dried, and examined under ultraviolet light (254 nm). The chromatogram of the test solution showed a dark background color, unclear spots, and interference from negative ions. Further investigation was conducted on the identification of *Spatholobus suberectus* (chicken blood vine): using a reference solution of *Spatholobus suberectus* as a control, the test sample was extracted with 80% acetone using ultrasound, with chloroform-methanol (20:1) as the developing solvent, and examined under ultraviolet light (254nm, 366nm). After fuming with ammonia, the sample was examined under ultraviolet light (366nm). The results showed that the spots were not clear enough, and the negative sample caused interference. Subsequently, methanol reflux extraction was performed, followed by elution with 70% ethanol on a D101 macroporous adsorption resin column, and the elution was discarded. Elution was performed with 95% ethanol, and the eluent was evaporated to dryness and dissolved in methanol to prepare the test solution. The reference herb, *Spatholobus suberectus*, was decocted in water and extracted with water-saturated n-butanol by shaking. The upper layer of a solution of toluene-ethyl formate-formic acid-water (20:10:1:1) was used as the developing solvent for the test. The sample was examined under ultraviolet light (254 nm, 366 nm) and after being fumigated in ammonia, it was examined under ultraviolet light (366 nm). The results showed that the spots were still not clear enough, and there was interference from negative samples. Therefore, a mature identification method could not be determined.

[0073] Thin-layer chromatography (TLC) identification studies were previously conducted on Clematis chinensis. The method involved ultrasonic extraction with methanol, removal of impurities with chloroform, followed by extraction with water-saturated n-butanol, evaporation of the n-butanol to dryness, dissolution of the residue in methanol to obtain the test solution, and use Clematis chinensis reference material as a reference. A chloroform-methanol-water (13:7:2) solution was used as the developing solvent, and the mixture was sprayed with 10% sulfuric acid in ethanol and heated to 105°C for color development. In the chromatogram of the test sample, the background color was dark and the spots were unclear. Subsequent purification using a neutral alumina column did not yield clear spots. An alternative method was then employed: ultrasonic extraction with ethanol, hydrolysis with hydrochloric acid, and extraction with petroleum ether (60–90°C). Oleanolic acid reference standard was used as a reference, and toluene-ethyl acetate-formic acid (20:3:0.2) was used as the developing solvent. The mixture was sprayed with 10% sulfuric acid in ethanol and heated to 105°C until the spots were clearly visible. The results showed negative interference, therefore a mature method was not established. Further research was conducted on the identification of Clematis chinensis: using a Clematis chinensis reference solution as a control, ethanol reflux extraction was performed, with cyclohexane-chloroform-ethyl acetate (4:1:1) as the developing solvent. The results showed interference from negative samples. Next, ethanol reflux extraction and concentration were performed, followed by reflux with hydrochloric acid, water addition, and shaking extraction with petroleum ether (60-90℃). Toluene-ethyl acetate-formic acid (20:3:0.2) was used as the developing solvent, and the results showed interference from negative samples. Therefore, a mature detection method was not established.

[0074] Thin-layer chromatography identification of Achyranthes bidentata was also studied. Using Achyranthes bidentata reference material solution as a control, methanol ultrasonic extraction, chloroform extraction to remove impurities, followed by water-saturated n-butanol extraction and neutral alumina column purification were performed. The lower layer of chloroform-methanol-water (13:7:2) was used as the developing solvent. The results showed that the background color of the sample was dark and the spots were not clear. Subsequently, methanol ultrasonic extraction, ethyl acetate extraction, and neutral alumina column purification were performed. The developing solvents were chloroform-methanol (10:1.5) and chloroform-ethyl acetate-methanol (10:3:1), respectively. The results did not yield clear thin-layer chromatograms. Further research was conducted on the identification of Achyranthes bidentata: using a reference solution of Achyranthes bidentata as a control, the test sample was extracted with methanol by ultrasonication, water-saturated n-butanol by shaking, washed with ammonia solution, and developed with toluene-chloroform-acetone (8:4:1). The results showed that the negative sample had interference. Next, methanol was extracted by ultrasonication, ether by shaking and evaporation, dissolved in chloroform, and petroleum ether (60-90℃)-ethyl acetate (3:2) as the developing solvent. The results showed that the background color of the test sample in the chromatogram was dark and the spots were not clear. Then, chloroform was extracted by ultrasonication, toluene-methanol (9:1) as the developing solvent, and water was extracted by ultrasonication and n-butanol by shaking. The lower layer of chloroform-formic acid-water (6:4:1) solution was used as the developing solvent. The sample was examined under ultraviolet light (365nm) and sprayed with 10% sulfuric acid ethanol solution and heated at 105℃ for color development. The results showed that the negative samples all had interference, so protection was not performed.

[0075] Thin-layer chromatography (TLC) was previously used to identify Rehmannia glutinosa. Using catalpol as a reference standard, the sample solution was extracted with methanol via ultrasonic extraction, purified with chloroform, and then further purified by shaking with water-saturated n-butanol. The upper and lower layers of chloroform-methanol-water (14:6:1), n-butanol-glacial acetic acid-water (4:1:5), and chloroform-methanol-water (13:7:2) solutions were used as developing solvents, respectively. Color development was performed by spraying with anisaldehyde solution and 10% sulfuric acid in ethanol solution. Clear TLC chromatograms were not obtained in any of these cases and therefore were not included in the main text.

[0076] Thin-layer chromatography (TLC) was used to identify Cyperus rotundus in traditional Chinese medicine. The test solution for Cinnamomum cassia was used, with α-cyperone as a reference. The developing solvent was toluene-ethyl acetate-glacial acetic acid (92:5:5), and the sample was examined under ultraviolet light (254 nm). Dinitrophenylhydrazine was then sprayed for color development. The test sample chromatogram showed spots of the same color at the corresponding positions as the reference chromatogram, but the resolution was poor. After adjusting the developing solvent toluene-ethyl acetate-glacial acetic acid (95:5:5), the spot resolution in the test sample chromatogram remained unsatisfactory and was therefore not included in the main text.

[0077] It is evident that complex traditional Chinese medicine formulas contain a variety of medicinal materials, each with complex and diverse components. Their interactions can interfere with and affect the test results. Therefore, when formulating quality control standards, it is not possible to obtain a stable and mature control method simply by following the identification methods of single medicinal materials. Technicians need to conduct repeated experiments and optimizations to achieve a feasible quality control method that can be used as a standard. Attached Figure Description

[0078] Figure 1 This is a thin-layer chromatogram of Polygonum cuspidatum for thin-layer identification in this invention;

[0079] Figure 2 This is a thin-layer chromatogram of the thin-layer identification of Stephania tetrandra in this invention;

[0080] Figure 3 This is a thin-layer chromatogram of the thin-layer identification of cinnamon twig in this invention;

[0081] Figure 4 This is a thin-layer chromatogram of myrrh thin-layer identification in this invention;

[0082] Figure 5 This is the chromatogram of thin-layer chromatography for the identification of white peony root in this invention;

[0083] Figure 6 This is a thin-layer chromatogram of the aconitine limit test in this invention;

[0084] Figure 7 This is a thin-layer chromatogram for investigating the detection limit of aconitine in this invention;

[0085] Figure 8 This is the specific chromatogram of Astragalus membranaceus in the specificity test of this invention;

[0086] Figure 9 This is the chromatogram of astragaloside A reference standard in this invention;

[0087] Figure 10 This is the standard curve of astragaloside A in the linearity study of this invention;

[0088] Figure 11 This is the spectrum of gentiopicrin in this invention;

[0089] Figure 12 This is the specific chromatogram of Gentiana macrophylla in the specificity test of this invention;

[0090] Figure 13 This is the chromatogram of gentiopicrin reference standard in this invention;

[0091] Figure 14 This is the standard curve of gentiopicroside in the linearity study of this invention. Detailed Implementation

[0092] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0093] Identification:

[0094] This invention establishes a method for identifying Polygonum cuspidatum, Stephania tetrandra, Cinnamomum cassia, Angelica sinensis, Myrrh (processed with vinegar), and Paeonia lactiflora using thin-layer chromatography. The tablets prepared according to Example 1 were used for identification and content determination. The results of the thin-layer chromatography identification of each herb in the formula are as follows:

[0095] 1. Materials

[0096] Traditional Chinese medicine composition preparations: tablets prepared according to Example 1, Shijiazhuang Yiling Pharmaceutical Co., Ltd., batch numbers 080701, 081201, 081202, 081203.

[0097] 2. Identification test

[0098] Thin-layer chromatographic identification of Polygonum cuspidatum: The methanol ultrasonic extract was used as the test solution, and the methanol extract of Polygonum cuspidatum reference material was used as a reference. The upper layer of petroleum ether (30–60℃)-ethyl formate-formic acid (15:5:1) was used as the developing solvent. The sample was examined under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color as those in the reference material were observed at the corresponding positions. The spots were clear, the separation was good, and there was no negative interference. Reproducibility was good after verification with three batches of samples (TLC chromatograms are shown in [reference image]). Figure 1 Among them, sample 1 was negative for Polygonum cuspidatum; sample 2 was the control material; and samples 3-5 were the test samples.

[0099] Thin-layer chromatographic identification of Stephania tetrandra: Extraction with methanol was performed, the extract was evaporated to dryness, the residue was dissolved in 1% hydrochloric acid, and the pH was adjusted with sodium hydroxide solution. Extraction with chloroform was then performed by shaking, and the residue was evaporated to dryness. The residue was dissolved in methanol as the test solution. A mixed standard of tetrandrine and tebufenozide was used as a reference. The developing solvents were chloroform-acetone-methanol (6:1:1), chloroform-acetone-methanol (6:1:1) saturated with ammonia, and chloroform-acetone-methanol-ammonia (12:2:2:0.1). Modified bismuth potassium iodide or dilute bismuth potassium iodide solution was sprayed. Results showed that chloroform-acetone-methanol-ammonia (12:2:2:0.1) as the developing solvent, sprayed with dilute bismuth potassium iodide solution, produced clear spots with good separation and no negative interference. Reproducibility was good after verification with three batches of samples (TLC chromatograms are shown in [link to TLC]). Figure 2 Among them, sample 1 is a negative result of Stephania tetrandra; sample 2 is a control; and samples 3-5 are test samples.

[0100] Thin-layer chromatographic identification of cinnamon twig: Water was used as the solvent, and the volatile oil was extracted. The collected volatile oil was used as the test solution. Cinnamaldehyde was used as a reference standard. Petroleum ether (60℃~90℃)-ethyl acetate (17:3) was used as the developing solvent, and dinitrophenylhydrazine ethanol solution was sprayed on. The results showed clear and round chromatographic spots with no negative interference. After verification with three batches of samples, the reproducibility was good (TLC chromatograms are shown in...). Figure 3 Among them, sample 1 is a negative test sample of cinnamon twig; sample 2 is a control sample; and samples 3-5 are test samples.

[0101] Thin-layer chromatography (TLC) identification of myrrh: The test solution for cinnamon twig identification was used, with myrrh as a reference. Cyclohexane-ethyl ether (4:1) and cyclohexane-ethyl ether-glacial acetic acid (4:1:0.1) were tested as the developing solvent. The results were obtained using cyclohexane-ethyl ether-glacial acetic acid (4:1:0.1) as the developing solvent, sprayed with 10% sulfuric acid ethanol solution, and heated to 105℃ for color development. The chromatographic spots were clear, with good separation and no interference from negative samples. Reproducibility was good after verification with three batches of samples (TLC chromatograms are shown in [link to TLC diagram]). Figure 4 Among them, sample 1 is myrrh negative; sample 2 is control material; and samples 3-5 are test samples.

[0102] Identification of Paeonia lactiflora: Remove the coating, grind finely, add 30 ml of methanol, sonicate for 30 minutes, filter, and use the filtrate as the test solution. Separately, prepare a paeoniflorin reference standard solution by adding methanol to a concentration of 0.1 mg / ml, as the reference solution. Perform high-performance liquid chromatography (HPLC) according to the Chinese Pharmacopoeia 2020 General Chapter 0512. Inject 5 μl each of the reference solution and the test solution into the HPLC system. Use octadecylsilane-bonded silica gel as the stationary phase; acetonitrile-0.1% phosphoric acid solution (14:86) as the mobile phase; flow rate: 1.0 ml / min; detection wavelength: 230 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should not be less than 2000. The chromatogram of the test sample should show a peak with a retention time corresponding to that of the reference standard. (See chromatogram for...) Figure 5 ).

[0103] Both Astragalus membranaceus and Gentiana macrophylla have established corresponding content determination methods, therefore thin-layer chromatography identification studies will no longer be conducted.

[0104] The identification method used in this invention can accurately and effectively identify the components of various medicinal materials in the traditional Chinese medicine composition. Due to the complexity of the components in large compound traditional Chinese medicines, not all medicinal materials can be significantly identified. Through numerous repeated experiments, the identification method for various medicinal materials was finally determined. Its specificity is extremely high, and it has achieved very accurate results. This effectively controls the quality of the traditional Chinese medicine composition product, improves the safety and reliability of the medicine, and ensures the safety of people's medication.

[0105] Aconitine Limit Test:

[0106] Take a traditional Chinese medicine composition preparation, add ammonia test solution and ether, let stand, evaporate the filtrate to dryness, dissolve the residue in anhydrous ethanol to obtain the test solution; separately take aconitine reference standard, add anhydrous ethanol to prepare a solution to obtain the reference solution. Accurately pipette the test solution and the reference solution and spot them separately on the same silica gel G thin-layer plate, use cyclohexane-ethyl acetate-ammonia water as the developing solvent, develop, remove, air dry, and spray with dilute bismuth potassium iodide test solution. In the chromatogram of the test sample, the spot appearing at the corresponding position as in the chromatogram of the reference standard should be smaller than the spot of the reference standard, or no spot should appear.

[0107] Three batches of this product (batch numbers: 081201, 081202, and 081203) were tested according to the aconitine limit test method. The results showed that no spots appeared at the corresponding positions in the chromatograms of the three batches of samples as in the chromatograms of the reference standard. (TLC chromatograms are shown in...) Figure 6 Among them, sample 1 is the sample from batch 081201; sample 2 is the reference standard; sample 3 is the sample from batch 081202; and sample 4 is the sample from batch 081203.

[0108] Aconitine Detection Limit Determination: Accurately weigh 10.02 mg of aconitine reference standard, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with anhydrous ethanol, shake well. Accurately measure 5 ml of this solution, place it in a 10 ml volumetric flask, dilute to the mark with anhydrous ethanol, shake well, and prepare a solution containing 0.501 mg per ml. Accurately pipette 1, 2, 5, 10, and 15 μl of the aconitine reference standard solution and spot them separately on the same silica gel G thin-layer plate. Use cyclohexane-ethyl acetate-ammonia (10:10:0.1) as the developing solvent, develop, remove, air dry, and spray with dilute bismuth potassium iodide reagent. Results: No spots were observed in the chromatogram of the reference standard with a sample volume of 1 μl, while spots appeared in the chromatograms of the reference standard with sample volumes of 2, 5, 10, and 15 μl. Therefore, the lowest detectable limit of aconitine using this method is 1.002 μg (TLC chromatogram shown in [reference needed]). Figure 7 (Among them, sample 1 is 1 μl; 2 is 2 μl; 3 is 5 μl; 4 is 10 μl; and 5 is 15 μl).

[0109] Content determination:

[0110] Astragaloside A content determination

[0111] Astragalus is the principal ingredient in the formula. In order to effectively control the quality of the finished product, astragaloside A was selected as the content determination index. A method for determining the content of astragaloside A in the finished product was established by high performance liquid chromatography.

[0112] 1. Experimental materials, instruments, and reagents:

[0113] Astragaloside A reference standard: purchased from the National Institutes for Food and Drug Control, batch number: 110781-200613, content calculated as 96.2%, for content determination.

[0114] Traditional Chinese medicine composition preparations: tablets prepared according to Example 1 of the present invention, provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd., batch numbers: 080701, 081201, 081202, 081203;

[0115] Agilent 1100 Series High Performance Liquid Chromatography (HPLC) with ELSD Evaporative Light Scattering Detector;

[0116] Methanol was of chromatographic grade, water was self-prepared redistilled water, and all other reagents and reagents were of analytical grade.

[0117] 2. Mobile phase selection:

[0118] The results showed that with methanol-water as the mobile phase, astragaloside A and the adjacent chromatographic peaks could achieve baseline separation, and the peak shapes were symmetrical.

[0119] 3. Chromatographic conditions:

[0120] Column: octadecylsilane-bonded silica gel is used as the packing material;

[0121] Mobile phase: methanol-water (78:22);

[0122] Flow rate: 1.0 ml / min;

[0123] Gas flow rate: 3.0 L / min;

[0124] Drift tube temperature: 80℃;

[0125] Column temperature: 25℃;

[0126] Theoretical plate number: calculated based on the astragaloside A peak, should be no less than 4000.

[0127] 4. Preparation of reference solution:

[0128] Accurately weigh 17.66 mg of astragaloside A reference standard, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well, accurately measure 3 ml, place it in a 10 ml volumetric flask, dilute to the mark with mobile phase, shake well, and prepare a reference standard solution containing 105.96 μg per ml.

[0129] 5. Preparation of the test solution:

[0130] This product was prepared by ultrasonic extraction with methanol, followed by impurity removal with chloroform and shaking extraction with water-saturated n-butanol. Under the chromatographic conditions used, astragaloside A and its adjacent chromatographic peaks achieved baseline separation, meeting the requirements for quantitative analysis. To ensure the accuracy of the content determination results, we conducted an experimental investigation on the preparation method of the test solution.

[0131] (1) Effect of chloroform impurity removal on astragaloside A content:

[0132] The sample was extracted with methanol using ultrasound, evaporated to dryness, and the residue was dissolved in water. Then, it was extracted with chloroform by shaking. The chloroform extract was evaporated to dryness, and the residue was dissolved in the mobile phase. The content was then determined. The results showed that astragaloside A was not detected in the chloroform extract, indicating that chloroform removal does not affect the content of astragaloside A.

[0133] (2) Effect of the number of water-saturated n-butanol extractions on the content of astragaloside A

[0134] The aqueous layer after purification with chloroform was extracted with water-saturated n-butanol by shaking. The first three extracts, the fourth extract, and the fifth and sixth extracts were collected. Each n-butanol extract was washed successively with 1% sodium hydroxide solution and water-saturated n-butanol solution. The n-butanol solution was evaporated to dryness, and the residue was dissolved in the mobile phase and the content was determined. The results showed that astragaloside A was not detected in the fifth and sixth n-butanol extracts. Therefore, four extractions with water-saturated n-butanol by shaking are recommended.

[0135] (3) Effects of alkaline washing solution and water washing solution on the content of astragaloside A

[0136] The alkaline and water washing solutions from the n-butanol extraction were combined, and the mixture was extracted twice with water-saturated n-butanol. The n-butanol solutions were collected, evaporated to dryness, and the residue was dissolved in the mobile phase before the content was determined. Astragaloside A was not detected; therefore, the alkaline and water washing solutions do not affect the content of astragaloside A and can be discarded.

[0137] (4) Effect of alkaline washing solution concentration on astragaloside A content

[0138] Take the same batch of samples (080701), wash the n-butanol shaking extract with ammonia test solution, 0.5% sodium hydroxide and 1% sodium hydroxide respectively, prepare the test solution, and determine the content. The results are shown in Table 1.

[0139] Table 1. Effect of different alkaline washing solution concentrations on content determination

[0140]

[0141] The results showed that different alkaline washing solutions did not significantly affect the determination of astragaloside A content. However, washing with ammonia solution did not easily result in separation, while washing with 0.5% and 1% sodium hydroxide solutions was relatively easy to separate into layers. Therefore, washing with 0.5% sodium hydroxide solution was preferred.

[0142] (5) Examination of ultrasound time

[0143] Take samples from the same batch (batch number 080701), and sonicate them (power 250W, frequency 40KHz) for 20, 30 and 40 minutes respectively to prepare test solutions and determine the content. The experimental results are shown in Table 2.

[0144] Table 2 Results of Ultrasonic Time-Based Study

[0145]

[0146] The results showed that there was no significant difference in the astragaloside A content between ultrasonic extraction for 30 minutes and 40 minutes, while the content was lower after ultrasonic extraction for 20 minutes. Therefore, the ultrasonic extraction time of 30 minutes was selected.

[0147] (6) Investigation of the extraction solvent

[0148] Samples from the same batch (batch number 080701) were extracted using 50% methanol, 75% methanol and methanol as extraction solvents, respectively, by ultrasonic extraction. Test solutions were prepared and the content was determined. The experimental results are shown in Table 3.

[0149] Table 3 Results of solvent extraction investigation

[0150]

[0151] The results showed that the content of astragaloside A was higher when methanol or 75% methanol was used as the extraction solvent, but the 75% methanol extract was not easy to filter. Therefore, methanol was chosen as the extraction solvent.

[0152] (7) Investigation on the amount of extraction solvent used

[0153] Take samples from the same batch (batch number 080701), add 30, 50, and 100 ml of methanol respectively for ultrasonic extraction, prepare test solutions, and determine the content. The experimental results are shown in Table 4.

[0154] Table 4 Results of the investigation on the amount of extraction solvent

[0155]

[0156] The results showed that there was little difference in the content determination results with different amounts of extraction solvent. Considering the large sample size, in order to ensure complete extraction of astragaloside A, the solvent volume was selected as 50 ml.

[0157] In summary, the test solution was prepared as follows: Take 20 tablets of this product, remove the coating, weigh accurately, grind finely, take 4g, weigh accurately, add 50ml of methanol accurately, weigh, sonicate (power 250W, frequency 40KHz) for 30 minutes, weigh again, replenish the lost weight with methanol, shake well, filter, accurately measure 25ml of the filtrate, evaporate to dryness, add 20ml of water to dissolve the residue, extract with chloroform 3 times by shaking, 15ml each time, discard the chloroform solution, extract the aqueous layer with water-saturated n-butanol 4 times by shaking, 15ml each time, combine the n-butanol solutions, wash twice with 0.5% sodium hydroxide solution, 10ml each time, then wash twice with n-butanol-saturated water, 10ml each time, evaporate the n-butanol solution to dryness, add mobile phase to dissolve the residue and transfer to a 5ml volumetric flask, dilute to the mark with mobile phase, shake well, filter, and the product is obtained.

[0158] 6. Specificity assessment:

[0159] Weigh out all medicinal ingredients except Astragalus membranaceus according to the prescription ratio, prepare negative samples according to the preparation process, and process them according to the test sample solution treatment method to obtain negative sample solutions. Take the reference solution, negative sample solution, and test sample solution separately, and perform detection according to the test specimen. The results show that no corresponding peak appears in the negative sample at the corresponding retention time of astragaloside A, indicating that the assay method is specific (chromatograms of the reference, negative sample, and test sample are shown in [reference]). Figure 8 ).

[0160] 7. Purity test of reference standard:

[0161] The purity of astragaloside A reference standard (batch number: 110781-200613, National Institutes for Food and Drug Control) was determined by area normalization method, and the results showed that the purity of astragaloside A reference standard was 100% (see...). Figure 9 ).

[0162] 8. Examination of linear relationships:

[0163] A standard solution of astragaloside A with a concentration of 105.96 μg / ml was precisely pipetted into the HPLC system at doses of 2, 5, 10, 20, 30, and 50 μl, respectively. The peak areas were measured, and a standard curve was plotted with the natural logarithm of the injection volume on the x-axis and the natural logarithm of the peak area integral on the y-axis. The experimental data are shown in Table 5.

[0164] Table 5. Experimental data for examining linear relationships.

[0165]

[0166] The results showed that astragaloside A exhibited good linearity within the injection range of 0.21192–5.2980 μg, with a regression equation of ln(y) = 1.9189 ln(x) + 6.5176 and a correlation coefficient r = 9999 (see standard curve). Figure 10 ).

[0167] 9. Precision test:

[0168] Accurately pipette the astragaloside A reference solution and the test sample (batch number: 080701) solution with a concentration of 105.96 μg / ml, and inject them five times consecutively. Measure the peak area. The results are shown in Table 6.

[0169] Table 6 Precision Experiment Results

[0170]

[0171] The results above show that the RSD of the peak area of ​​the reference sample is 1.21%, and the RSD of the peak area of ​​the test sample is 1.56%, indicating that the instrument has good precision.

[0172] 10. Stability test:

[0173] Accurately pipette the astragaloside A reference solution and the test sample (batch number: 080701) solution with a concentration of 105.96 μg / ml, and inject them at regular intervals to examine the stability over 24 hours. The results are shown in Table 7.

[0174] Table 7 Stability test results

[0175]

[0176] The experimental results show that the RSD of the peak area of ​​the astragaloside A reference solution was 2.77% and the RSD of the peak area of ​​the test solution was 2.20% within 24 hours, indicating that both the reference solution and the test solution were relatively stable within 24 hours.

[0177] 11. Repeatability Experiments:

[0178] Take samples from the same batch (batch number: 080701), set three sampling sizes (low, medium, and high), and make three replicates for each sampling size. Prepare the test solution according to the test solution preparation method, and then determine the results. The experimental results are shown in Table 8.

[0179] Table 8 Results of Repeatability Experiments

[0180]

[0181] The experimental results showed that the average content of astragaloside A in the test samples with low, medium and high sampling amounts was 0.3190 mg / g; the RSD was 2.42%, indicating that the determination method had good repeatability.

[0182] 12. Recovery rate experiment:

[0183] A spiking recovery test was conducted. Nine samples from the same batch (batch number: 080701) were divided into three groups. 50 ml of methanol solution containing high, medium, and low concentrations of astragaloside A reference standard was added to each group, and the samples were treated according to the sample solution treatment method. The recovery rates were then determined and calculated. The experimental results are shown in Table 9.

[0184] Table 9. Experimental Results of Recovery Rate

[0185]

[0186]

[0187] The results of the spiking recovery experiment showed that the average recovery rate of astragaloside A was 98.41%, and the RSD was 1.96%, indicating that the method has good accuracy.

[0188] 13. Durability test (sample batch number: 080701)

[0189] (1) Investigation of different drift tube temperatures

[0190] Take the same sample solution and investigate the effect of different drift tube temperatures on the content determination results. The experimental results are shown in Table 10.

[0191] Table 10 Results of Temperature Content Measurement in Different Drift Tubes

[0192]

[0193] The experimental results show that changes in the temperature of the drift tube within a small range have little impact on the determination of astragaloside A content.

[0194] (2) Investigation of different mobile phase ratios

[0195] Take the same sample solution and investigate the effect of different mobile phase ratios on the content determination results. The experimental results are shown in Table 11.

[0196] Table 11 Results of determination of different mobile phase ratios

[0197]

[0198] The experimental results show that changes in the mobile phase ratio within a small range have little impact on the content determination results.

[0199] (3) Investigation of different gas flow velocities

[0200] Take the same sample solution and investigate the effect of different gas flow rates on the content determination results. The experimental results are shown in Table 12.

[0201] Table 12 Results of gas flow rate content determination at different gas velocities

[0202]

[0203] The experimental results show that changes in gas flow rate within a small range have little impact on the determination of astragaloside A content.

[0204] (4) Investigation of different chromatographic columns

[0205] Take the same sample solution and investigate the effect of different chromatographic columns on the content determination results. The experimental results are shown in Table 13.

[0206] Table 13 Results of content determination at different chromatographic columns

[0207]

[0208] The experimental results show that different chromatographic columns have little effect on the determination of astragaloside A content.

[0209] (5) Investigation of different flow velocities of the mobile phase

[0210] Take the same sample solution and investigate the effect of different flow rates of the mobile phase on the content determination results. The experimental results are shown in Table 14.

[0211] Table 14 Results of determination of mobile phase content at different flow velocities

[0212]

[0213] The experimental results show that changes in the mobile phase velocity within a small range have little impact on the determination of astragaloside A content.

[0214] (6) Investigation of different column temperatures

[0215] Take the same sample solution and investigate the effect of different column temperatures on the content determination results. The experimental results are shown in Table 15.

[0216] Table 15 Results of content determination at different column temperatures

[0217]

[0218] The experimental results show that changes in column temperature within a small range have little impact on the determination of astragaloside A content.

[0219] 14. Content Limits:

[0220] Using the content determination method provided by this invention, the content of astragaloside A in three batches of samples was determined, and the content of astragaloside A in the astragalus slices used in the production samples was determined. The astragaloside A transfer rate was calculated, and the results are shown in Table 16.

[0221] Table 16 Results of Content Determination

[0222]

[0223] The results show that the average transfer rate of astragaloside A in the three batches of samples was 51.63%. According to the regulations under the Astragalus standard in the 2005 edition of the Chinese Pharmacopoeia (same as the 2010 edition), the content of astragaloside A in Astragalus membranaceus should not be less than 0.04%. Each tablet of this product contains 0.156g of Astragalus membranaceus. Therefore, the calculated content of astragaloside A in each tablet is: 0.156 × 0.04% × 51.63% × 1000 ≈ 0.032 (mg). Thus, the limit for Astragalus membranaceus content is set at not less than 0.032mg per tablet, calculated as astragaloside A. Based on the average content, an upper limit is set, meaning that the content of Astragaloside A (C41H68O14) per tablet should be between 0.032mg and 0.177mg.

[0224] (II) Determination of Gentianoside Content

[0225] Gentiana macrophylla is also the principal ingredient in the formula. In order to effectively control the quality of the finished product, gentiopicroside was selected as the content determination index. A method for determining the content of gentiopicroside in the finished product was established by high performance liquid chromatography.

[0226] The research process is as follows:

[0227] 1. Experimental materials, instruments, and reagents:

[0228] Gentianoside reference standard: purchased from the National Institutes for Food and Drug Control, batch number: 110770-200611, for content determination.

[0229] Traditional Chinese medicine composition preparations: tablets prepared according to Example 1 of the present invention, provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd., batch numbers: 080701, 081201, 081202, 081203;

[0230] Agilent 1100 Series High Performance Liquid Chromatograph with VWD UV Detector;

[0231] Acetonitrile was of chromatographic grade, and water was homemade redistilled water;

[0232] All other reagents and reagents were of analytical grade.

[0233] 2. Selection of detection wavelength:

[0234] Ultraviolet spectroscopy was performed on gentiopicrin. The results showed that gentiopicrin has a maximum absorption at 254 nm. Therefore, 254 nm was chosen as the detection wavelength (see spectrum). Figure 11 ).

[0235] 3. Mobile phase selection:

[0236] The mobile phases used for the determination of gentiopicrin content are mostly methanol-water and acetonitrile-water systems. Experiments have shown that using methanol-water system as mobile phase results in higher column pressure and poor peak shape of gentiopicrin. However, using acetonitrile-water (11:89) as mobile phase results in better peak shape of gentiopicrin and baseline separation from adjacent chromatographic peaks.

[0237] 4. Chromatographic conditions:

[0238] Column: octadecylsilane-bonded silica gel is used as the packing material;

[0239] Mobile phase: Acetonitrile-water (11:89);

[0240] Flow rate: 1.0 ml / min;

[0241] Detection wavelength: 254nm;

[0242] Column temperature: 25℃;

[0243] Theoretical plate number: calculated based on the gentiopicroside peak should be no less than 3000.

[0244] 5. Preparation of reference solution:

[0245] Accurately weigh 19.83 mg of gentiopicroside reference standard, place it in a 50 ml volumetric flask, dissolve it in methanol, dilute to the mark, and shake well. Accurately measure 2 ml of the solution, place it in a 10 ml volumetric flask, dilute to the mark with the mobile phase, and shake well to prepare a reference standard solution containing 79.32 μg per ml.

[0246] 6. Preparation of the test solution:

[0247] This product was extracted with methanol using ultrasound, and the filtrate was used as the test solution. Under the chromatographic conditions employed, gentiopicrin and its adjacent peaks achieved baseline separation, meeting the requirements for quantitative analysis. To ensure the accuracy of the content determination results, we conducted an experimental investigation into the preparation method of the test solution.

[0248] (1) Investigation of the extraction solvent

[0249] Samples from the same batch (batch number: 080701) were extracted using water, 50% methanol, 70% methanol and methanol as extraction solvents, respectively, by ultrasonic extraction. Test solutions were prepared and the content was determined. The experimental results are shown in Table 17.

[0250] Table 17 Results of investigation using different extraction solvents

[0251]

[0252] The results showed that the content determination results were slightly lower when methanol was used as the extraction solvent; there was no significant difference in the content determination results when water, 50% methanol and 70% methanol were used as the extraction solvents, but the extract was not easy to filter when water or 50% methanol was used as the solvent, so 70% methanol was chosen as the extraction solvent.

[0253] (2) Investigation on the amount of extraction solvent

[0254] Take samples from the same batch (batch number: 080701), add 25, 50 and 100 ml of 70% methanol respectively for ultrasonic extraction, prepare test solutions, and determine the content. The experimental results are shown in Table 18.

[0255] Table 18 Results of the investigation on the amount of extraction solvent

[0256]

[0257] The results showed that there was no significant difference in the determination of gentiopicrin content with different extraction solvent volumes, therefore, the extraction solvent volume of 25 ml was selected.

[0258] (3) Examination of ultrasound time

[0259] Take the same batch of samples (batch number: 080701), use 70% methanol as solvent, and sonicate (power 250W, frequency 40KHz) for 20 minutes, 30 minutes and 40 minutes respectively. Prepare and determine the content. The experimental results are shown in Table 19.

[0260] Table 19 Results of Ultrasonic Time-Based Study

[0261]

[0262] The results showed that there was no significant difference in the determination of gentiopicrin content with different ultrasound times, so the ultrasound time of 20 minutes was selected.

[0263] In summary, the test solution was prepared as follows: Take 20 tablets of this product, remove the coating, weigh accurately, grind finely, take 0.4g, weigh accurately, add 25ml of 70% methanol accurately, weigh, sonicate (power 250W, frequency 40KHz) for 20 minutes, remove, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and the solution is obtained.

[0264] 7. Specificity assessment:

[0265] Weigh out all medicinal ingredients except Gentiana macrophylla according to the prescription ratio, prepare negative samples according to the preparation process, and process them according to the test sample solution treatment method to obtain negative sample solutions. Take the reference solution, negative sample solution, and test sample solution separately, and detect them according to the assay conditions. The results show that no corresponding peak appears in the negative sample at the corresponding retention time of gentiopicroside, indicating that the method is specific (chromatograms of the reference, negative sample, and test sample are shown in [reference]). Figure 12 ).

[0266] 8. Purity test of reference standard:

[0267] The purity of gentiopicroside reference standard (batch number: 110770-200611, National Institutes for Food and Drug Control) was determined by area normalization method to be 98.51% (see [link to relevant documentation]). Figure 13 ).

[0268] 9. Examination of linear relationships:

[0269] Accurately weigh 19.83 mg of gentiopicroside reference standard into a 50 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well to prepare a gentiopicroside reference standard solution with a concentration of 396.6 μg / ml. Accurately pipette 1, 1, 2, 3, and 5 ml of each solution into 25, 10, 10, 10, and 10 ml volumetric flasks, respectively, dilute to the mark with 70% methanol, and shake well to prepare gentiopicroside reference standard solutions with concentrations of 15.864, 39.66, 79.32, 118.98, and 198.3 μg / ml, respectively. Accurately pipette 10 μl of each of these reference standard solutions into a high-performance liquid chromatograph (HPLC), and measure the peak area. Plot a standard curve with the injection volume as the x-axis and the peak area integral as the y-axis. The experimental data are shown in Table 20.

[0270] Table 20 Experimental data for examining linear relationships

[0271]

[0272] The results showed that gentiopicrin exhibited a good linear relationship within the injection range of 0.15864–3.966 μg, with a regression equation of y = 996.62x + 35.346 and a correlation coefficient r = 0.9998 (see standard curve). Figure 14 ).

[0273] 10. Precision Experiment:

[0274] Accurately pipette the gentiopicrin reference solution and the test sample (batch number: 080701) solution with a concentration of 79.32 μg / ml, and inject them five times consecutively. Measure the peak area. The results are shown in Table 21.

[0275] Table 21 Precision Experiment Results

[0276]

[0277] The experimental results above show that the peak area RSD of gentiopicrin reference standard is 0.29%, and the peak area RSD of test sample is 0.93%, indicating that the instrument has good precision.

[0278] 11. Stability test:

[0279] The stability of the gentiopicroside reference solution with a concentration of 79.32 μg / ml and the test sample (batch number: 080701) solution was investigated by precisely injecting them at regular intervals over 24 hours. The results are shown in Table 22.

[0280] Table 22 Stability Experiment Results

[0281]

[0282]

[0283] The stability test results show that the peak area RSD of gentiopicroside reference was 0.39% and that of the test sample was 0.17% within 24 hours, indicating that both the reference and test solutions were relatively stable within 24 hours.

[0284] 12. Repeatability Experiments:

[0285] Take samples from the same batch (batch number: 080701), set three sampling sizes (low, medium, and high), and make three replicates for each sampling size. Prepare the test solution according to the test solution preparation method, and then determine the results. The experimental results are shown in Table 23.

[0286] Table 23 Results of Repeatability Experiments

[0287]

[0288]

[0289] The experimental results showed that the average content of gentiopicroside in the three different concentrations of test samples (low, medium, and high) was 6.4681 mg / g, with an RSD of 1.00%, indicating that the determination method had good repeatability.

[0290] 13. Recovery rate experiment:

[0291] A spiking recovery test was conducted. Nine samples from the same batch (batch number: 080701) were divided into three groups. 25 ml of 70% methanol solution of high, medium, and low concentrations of gentiopicrin reference standard were added to each group, and the solutions were treated according to the sample preparation method. The recovery rates were then determined and calculated. The experimental results are shown in Table 24.

[0292] Table 24 Results of Recovery Rate Experiment

[0293]

[0294]

[0295] The recovery results of the spiking experiment showed that the average recovery rate of gentiopicroside was 98.05%, and the RSD was 2.15%, indicating that the method is accurate and reliable.

[0296] 14. Durability test (sample batch number: 080701)

[0297] (1) Investigation of different flow velocities of the mobile phase

[0298] Take the same sample solution and investigate the effect of different flow rates of the mobile phase on the content determination results. The results are shown in Table 25.

[0299] Table 25 Results of determination of mobile phase content at different flow velocities

[0300]

[0301] The experimental results show that changes in the mobile phase flow rate within a small range have little impact on the determination of gentiopicrin content.

[0302] (2) Investigation of different detection wavelengths

[0303] Take the same sample solution and investigate the effect of different detection wavelengths on the content determination results. The experimental results are shown in Table 26.

[0304] Table 26 Results of content determination at different detection wavelengths

[0305]

[0306] The experimental results show that changes in the detection wavelength within a small range have little impact on the determination of gentiopicrin content.

[0307] (3) Investigation of different mobile phase ratios

[0308] Take the same sample solution and investigate the effect of different mobile phase ratios on the content determination results. The experimental results are shown in Table 27.

[0309] Table 27 Results of determination of different mobile phase ratios

[0310]

[0311] The experimental results show that changes in the mobile phase ratio within a small range have little impact on the determination of gentiopicrin content.

[0312] (4) Investigation of different chromatographic columns

[0313] Take the same sample solution and investigate the effect of different chromatographic columns on the content determination results. The experimental results are shown in Table 28.

[0314] Table 28 Results of content determination at different chromatographic columns

[0315]

[0316] The experimental results show that different chromatographic columns have little effect on the determination of gentiopicrin content.

[0317] (5) Investigation at different column temperatures

[0318] Take the same sample solution and investigate the effect of different column temperatures on the content determination results. The experimental results are shown in Table 29.

[0319] Table 29 Results of content determination at different column temperatures

[0320]

[0321] The experimental results show that changes in column temperature within a small range have little impact on the determination of gentiopicrin content.

[0322] 15. Content Limits:

[0323] The content of gentiopicroside in three batches of samples was determined using the content determination method provided by this invention, and the content of Gentiana macrophylla used in the production of the samples was also determined. The gentiopicroside transfer rate was calculated, and the results are shown in Table 30.

[0324] Table 30 Results of Gentianoside Content Determination

[0325]

[0326] The results showed that the average transfer rate of gentiopicroside in the three batches of samples was 60.99%. According to the Chinese Pharmacopoeia 2005 edition, Part I, under the standard for Gentiana macrophylla, the content of gentiopicroside in Gentiana macrophylla should not be less than 2.0%. Each tablet of this product contains 0.156g of Gentiana macrophylla. Therefore, the lower limit of gentiopicroside content per tablet is calculated as: 0.156 × 2.0% × 60.99% × 1000 ≈ 1.90 (mg). Subsequent content determination of seven batches in large-scale production yielded an average content of 6.88 mg / tablet. A 70% lower limit is 2.06 mg / tablet, and a 70% upper limit is 11.70 mg / tablet. Therefore, the content limit for Gentiana macrophylla is set at gentiopicroside (C0.05) per tablet. 16 H 20 The total amount of O9 should be between 2.06 mg and 11.70 mg.

[0327] Example 1:

[0328] Methods for identification, limit testing and content determination of traditional Chinese medicine composition tablets according to the present invention

[0329] The raw material formula is as follows: Astragalus membranaceus 156g, Gentiana macrophylla 156g, Stephania tetrandra 63g, Aconitum carmichaelii 63g, Polygonum cuspidatum 146g, Spatholobus suberectus 146g, Clematis chinensis 146g, Sinapis alba 31g, Paeonia lactiflora 63g, Rehmannia glutinosa 156g, Angelica sinensis 63g, Myrrh (processed with vinegar) 52g, Cyperus rotundus 52g, Cinnamomum cassia 63g, Achyranthes bidentata 63g.

[0330] Preparation method:

[0331] A. Grind the Stephania tetrandra into a fine powder and set aside.

[0332] B. Take cinnamon twigs, vinegar, myrrh, and spice, add 6 times the amount of water, and continuously extract the volatile oil by steam distillation for 8 hours. Collect the volatile oil, filter the distilled aqueous solution, collect it in a separate container, and set it aside for later use.

[0333] C. Take Gentiana macrophylla, Spatholobus suberectus, Angelica sinensis, Sinapis alba, Paeonia lactiflora, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, 1 hour each time. Filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear extract with a relative density of 1.15±0.05 for later use.

[0334] D. Take Astragalus membranaceus, Aconitum carmichaelii, Clematis chinensis, Rehmannia glutinosa, and Achyranthes bidentata, add 11 times the amount of water, decoct 3 times, 1.5 hours each time, filter the decoction, concentrate under reduced pressure to a clear extract with a relative density of 1.10±0.05, then combine it with the aqueous solution after distillation of volatile oil, concentrate under reduced pressure to a clear extract with a relative density of 1.15±0.05, and set aside.

[0335] E. Combine the clear pastes obtained in steps C and D, mix them thoroughly, dry them, and pulverize them to obtain a mixed clear paste powder.

[0336] F. Take the volatile oil obtained in step B and incorporate it into the mixture for later use;

[0337] G. Granulate the fine powder obtained in step A and the clear paste powder obtained in step E, granulate them, add the inclusion complex obtained in step F, and compress them into 1000 tablets using conventional methods.

[0338] Identification:

[0339] For the thin-layer chromatographic identification of Polygonum cuspidatum, take the sample, remove the coating, grind finely, add methanol, sonicate, filter, and use the filtrate as the test solution. Separately take Polygonum cuspidatum reference material, add methanol, sonicate, and use the supernatant as the reference material solution. Apply 3-5 μl of the test solution and 2 μl of the reference material solution separately to the same silica gel G thin-layer plate. Use the upper layer of petroleum ether (30-60℃)-ethyl formate-formic acid (15:5:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0340] For the thin-layer chromatographic identification of Stephania tetrandra, take the test solution from the TLC identification of Polygonum cuspidatum, evaporate to dryness, dissolve the residue in 20 ml of 1% hydrochloric acid, adjust the pH to 9-10 with 6 mol / L sodium hydroxide solution, and extract three times with 20 ml of chloroform each time. Combine the chloroform extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution. Separately, prepare a mixed solution containing 1 mg of each of tetrandrine and fangchinorline reference standards in methanol as the reference solution. Apply 5-10 μl of the test solution and 5 μl of the reference solution separately to the same silica gel G TLC plate. Develop the plate using chloroform-acetone-methanol-ammonia (12:2:2:0.1) as the developing solvent, remove the plate, air dry, and spray with dilute potassium bismuth iodide reagent. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standards.

[0341] For the thin-layer chromatographic identification of cinnamon twig, take the contents of the product and extract the volatile oil using the cyclohexane solution of the volatile oil as the test solution; separately prepare a reference solution by dissolving cinnamaldehyde in ethanol. Apply 5–10 μl of the test solution and 2 μl of the reference solution separately to the same silica gel G thin-layer plate. Develop the plate using petroleum ether (60–90℃)-ethyl acetate (17:3) as the developing solvent. Remove the plate, air dry, and spray with dinitrophenylhydrazine ethanol reagent. The chromatogram of the test sample should show spots of the same color at the corresponding positions as the chromatogram of the reference sample.

[0342] For the thin-layer chromatography identification of myrrh, take the myrrh reference material, extract the volatile oil with water, and use this as the reference material solution. Apply 5–10 μl of the test solution and 2 μl of the reference material solution to the same silica gel G thin-layer plate. Develop the plate using cyclohexane-ether-glacial acetic acid (4:1:0.1) as the developing solvent. Remove the plate, air dry, spray with 10% sulfuric acid in ethanol, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0343] For the identification of white peony root, remove the coating, grind finely, add 30 ml of methanol, sonicate for 30 minutes, filter, and use the filtrate as the test solution. Separately, prepare a paeoniflorin reference standard solution by adding methanol to a concentration of 0.1 mg / ml, as the reference solution. Perform high-performance liquid chromatography (HPLC) according to the Chinese Pharmacopoeia 2020 General Chapter 0512. Inject 5 μl each of the reference solution and the test solution into the HPLC system. Use octadecylsilane-bonded silica gel as the stationary phase; acetonitrile-0.1% phosphoric acid solution (14:86) as the mobile phase; flow rate: 1.0 ml / min; detection wavelength: 230 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should not be less than 2000. The test sample chromatogram should show a peak with a retention time corresponding to that of the reference standard chromatogram.

[0344] Limited quantity inspection:

[0345] Aconitine Limit Test: Take the contents of this preparation, add ammonia solution and ether, let stand, evaporate the filtrate to dryness, dissolve the residue in anhydrous ethanol to obtain the test solution; separately take aconitine reference standard, add anhydrous ethanol to prepare a solution to obtain the reference solution. Accurately pipette the test solution and the reference solution, and spot them separately on the same silica gel G thin-layer plate. Develop with cyclohexane-ethyl acetate-ammonia water as the developing solvent, remove, air dry, and spray with dilute bismuth potassium iodide test solution. In the chromatogram of the test sample, the spot appearing at the corresponding position as in the chromatogram of the reference standard should be smaller than the spot of the reference standard, or no spot should appear.

[0346] Content determination:

[0347] Astragalus content determination:

[0348] HPLC detection: The chromatographic column was C18 (Phnomenex Gemini C18 4.6×250mm), the mobile phase was methanol-water with a volume ratio of 78:22, the flow rate was 1.0 ml / min, the gas flow rate was 3.0 L / min, the drift tube temperature was 80℃, and the column temperature was 25℃.

[0349] Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, add the mobile phase to prepare a solution, and the solution is obtained.

[0350] Preparation of the test solution: Weigh the contents of this product accurately, add methanol, shake well, filter, accurately measure the filtrate, evaporate to dryness, dissolve the residue in water, extract with chloroform by shaking, discard the chloroform layer, extract the aqueous layer with water-saturated n-butanol by shaking to obtain n-butanol solution, wash, evaporate the n-butanol layer to dryness, dissolve the residue in the mobile phase to obtain the test solution;

[0351] Assay: Accurately pipette the reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0352] Determination of Gentiana macrophylla content:

[0353] HPLC detection: The chromatographic column was C18 (Phenomenex Gemini C18 250×4.6mm), the mobile phase was acetonitrile-water with a volume ratio of 11:89, the detection wavelength was 254nm, the column temperature was 25℃, and the flow rate was 1.0ml / min.

[0354] Preparation of reference solution: Take an appropriate amount of gentiopicroside reference standard, accurately weigh it, add the mobile phase to prepare a solution, and the solution is obtained.

[0355] Preparation of the test solution: Weigh the contents of the traditional Chinese medicine composition preparation accurately, add 70% methanol accurately, shake well, filter, and take the filtrate to obtain the test solution;

[0356] Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

[0357] Example 2:

[0358] Methods for identification, limit testing and content determination of traditional Chinese medicine composition capsules according to the present invention

[0359] The raw material formula is as follows: Astragalus membranaceus 146g, Gentiana macrophylla 139g, Stephania tetrandra 58g, Aconitum carmichaelii 75g, Polygonum cuspidatum 120g, Spatholobus suberectus 125g, Clematis chinensis 135g, Sinapis alba 28g, Paeonia lactiflora 60g, Rehmannia glutinosa 150g, Angelica sinensis 70g, Myrrh (processed with vinegar) 60g, Cyperus rotundus 60g, Cinnamomum cassia 70g, Achyranthes bidentata 70g.

[0360] Preparation method:

[0361] A. Grind the Stephania tetrandra into a fine powder and set aside.

[0362] B. Take cinnamon twigs, vinegar, myrrh, and spice, add 8 times the amount of water, and continuously extract the volatile oil by steam distillation for 10 hours. Collect the volatile oil, filter the distilled aqueous solution, collect it in a separate container, and set it aside for later use.

[0363] C. Take Gentiana macrophylla, Spatholobus suberectus, Angelica sinensis, Sinapis alba, Paeonia lactiflora, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, 1 hour each time. Filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear extract with a relative density of 1.15±0.05 for later use.

[0364] D. Take Astragalus membranaceus, Aconitum carmichaelii, Clematis chinensis, Rehmannia glutinosa, and Achyranthes bidentata, add 8 times the amount of water, decoct 4 times, 1.5 hours each time, filter the decoction, concentrate under reduced pressure to a clear extract with a relative density of 1.10±0.05, then combine it with the aqueous solution after distillation of volatile oil, concentrate under reduced pressure to a clear extract with a relative density of 1.15±0.05, and set aside.

[0365] E. Combine the clear pastes obtained in steps C and D, mix them evenly, dry them, and pulverize them to obtain a mixed clear paste powder. Add the volatile oil obtained in step B, add the fine powder from step A, granulate, shape the granules, and fill them into 1000 capsules to obtain the final product.

[0366] Identification:

[0367] For the thin-layer chromatographic identification of Polygonum cuspidatum, take the sample, remove the coating, grind finely, add methanol, sonicate, filter, and use the filtrate as the test solution. Separately take Polygonum cuspidatum reference material, add methanol, sonicate, and use the supernatant as the reference material solution. Apply 3-5 μl of the test solution and 2 μl of the reference material solution separately to the same silica gel G thin-layer plate. Use the upper layer of petroleum ether (30-60℃)-ethyl formate-formic acid (15:5:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0368] For the thin-layer chromatographic identification of Stephania tetrandra, take the test solution from the TLC identification of Polygonum cuspidatum, evaporate to dryness, dissolve the residue in 20 ml of 1% hydrochloric acid, adjust the pH to 9-10 with 6 mol / L sodium hydroxide solution, and extract three times with 20 ml of chloroform each time. Combine the chloroform extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution. Separately, prepare a mixed solution containing 1 mg of each of tetrandrine and fangchinorline reference standards in methanol as the reference solution. Apply 5-10 μl of the test solution and 5 μl of the reference solution separately to the same silica gel G TLC plate. Develop the plate using chloroform-acetone-methanol-ammonia (12:2:2:0.1) as the developing solvent, remove the plate, air dry, and spray with dilute potassium bismuth iodide reagent. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standards.

[0369] For the thin-layer chromatographic identification of cinnamon twig, take the contents of the product and extract the volatile oil using the cyclohexane solution of the volatile oil as the test solution; separately prepare a reference solution by dissolving cinnamaldehyde in ethanol. Apply 5–10 μl of the test solution and 2 μl of the reference solution separately to the same silica gel G thin-layer plate. Develop the plate using petroleum ether (60–90℃)-ethyl acetate (17:3) as the developing solvent. Remove the plate, air dry, and spray with dinitrophenylhydrazine ethanol reagent. The chromatogram of the test sample should show spots of the same color at the corresponding positions as the chromatogram of the reference sample.

[0370] For the thin-layer chromatography identification of myrrh, take the myrrh reference material, extract the volatile oil with water, and use this as the reference material solution. Apply 5–10 μl of the test solution and 2 μl of the reference material solution to the same silica gel G thin-layer plate. Develop the plate using cyclohexane-ether-glacial acetic acid (4:1:0.1) as the developing solvent. Remove the plate, air dry, spray with 10% sulfuric acid in ethanol, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0371] For the identification of white peony root, remove the capsule shell, grind finely, add 30 ml of methanol, sonicate for 30 minutes, filter, and use the filtrate as the test solution. Separately, prepare a paeoniflorin reference standard solution by adding methanol to a concentration of 0.1 mg / ml, as the reference solution. Perform high-performance liquid chromatography (HPLC) according to the Chinese Pharmacopoeia 2020 General Chapter 0512. Inject 5 μl each of the reference solution and the test solution into the HPLC system. Use octadecylsilane-bonded silica gel as the stationary phase; acetonitrile-0.1% phosphoric acid solution (14:86) as the mobile phase; flow rate: 1.0 ml / min; detection wavelength: 230 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should not be less than 2000. The test sample chromatogram should show a peak with a retention time corresponding to that of the reference standard chromatogram.

[0372] Limited quantity inspection:

[0373] Aconitine Limit Test: Take the contents of this preparation, add ammonia solution and ether, let stand, evaporate the filtrate to dryness, dissolve the residue in anhydrous ethanol to obtain the test solution; separately take aconitine reference standard, add anhydrous ethanol to prepare a solution to obtain the reference solution. Accurately pipette the test solution and the reference solution, and spot them separately on the same silica gel G thin-layer plate. Develop with cyclohexane-ethyl acetate-ammonia water as the developing solvent, remove, air dry, and spray with dilute bismuth potassium iodide test solution. In the chromatogram of the test sample, the spot appearing at the corresponding position as in the chromatogram of the reference standard should be smaller than the spot of the reference standard, or no spot should appear.

[0374] Content determination:

[0375] Astragalus content determination:

[0376] HPLC detection: The chromatographic column was C18 (Agilent ZORBAX SB C18 4.6×150mm), the mobile phase was methanol-water with a volume ratio of 76:24, the flow rate was 1.2 ml / min, the gas flow rate was 2.5 L / min, the drift tube temperature was 85℃, and the column temperature was 10℃.

[0377] Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, add the mobile phase to prepare a solution, and the solution is obtained.

[0378] Preparation of the test solution: Weigh the contents of this product accurately, add methanol, shake well, filter, accurately measure the filtrate, evaporate to dryness, dissolve the residue in water, extract with chloroform by shaking, discard the chloroform layer, extract the aqueous layer with water-saturated n-butanol by shaking to obtain n-butanol solution, wash, evaporate the n-butanol layer to dryness, dissolve the residue in the mobile phase to obtain the test solution;

[0379] Assay: Accurately pipette the reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0380] Determination of Gentiana macrophylla content:

[0381] HPLC detection: The chromatographic column was C18 (Agilent ZORBAX SB C18 150×4.6mm), the mobile phase was acetonitrile-water with a volume ratio of 12:88, the detection wavelength was 250nm, the column temperature was 30℃, and the flow rate was 0.8ml / min;

[0382] Preparation of reference solution: Take an appropriate amount of gentiopicroside reference standard, accurately weigh it, add the mobile phase to prepare a solution, and the solution is obtained.

[0383] Preparation of the test solution: Weigh the contents of the traditional Chinese medicine composition preparation accurately, add 70% methanol accurately, shake well, filter, and take the filtrate to obtain the test solution;

[0384] Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

[0385] Example 3:

[0386] The present invention provides a method for the identification, limit testing, and content determination of traditional Chinese medicine composition granules.

[0387] The raw material formula is as follows: Astragalus membranaceus 150g, Gentiana macrophylla 160g, Stephania tetrandra 60g, Aconitum carmichaelii 65g, Polygonum cuspidatum 120g, Spatholobus suberectus 130g, Clematis chinensis 135g, Sinapis alba 35g, Paeonia lactiflora 50g, Rehmannia glutinosa 160g, Angelica sinensis 60g, Myrrh (processed with vinegar) 55g, Cyperus rotundus 45g, Cinnamomum cassia 60g, Achyranthes bidentata 60g.

[0388] Preparation method:

[0389] A. Grind the Stephania tetrandra into a fine powder and set aside.

[0390] B. Take cinnamon twigs, vinegar, myrrh, and spice, add 8 times the amount of water, and continuously extract the volatile oil by steam distillation for 10 hours. Collect the volatile oil, filter the distilled aqueous solution, collect it in a separate container, and set it aside for later use.

[0391] C. Take Gentiana macrophylla, Spatholobus suberectus, Angelica sinensis, Sinapis alba, Paeonia lactiflora, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, 1 hour each time. Filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear extract with a relative density of 1.15±0.05 for later use.

[0392] D. Take Astragalus membranaceus, Aconitum carmichaelii, Clematis chinensis, Rehmannia glutinosa, and Achyranthes bidentata, add 8 times the amount of water, decoct 4 times, 1.5 hours each time, filter the decoction, concentrate under reduced pressure to a clear extract with a relative density of 1.10±0.05, then combine it with the aqueous solution after distillation of volatile oil, concentrate under reduced pressure to a clear extract with a relative density of 1.15±0.05, and set aside.

[0393] E. Combine the clear pastes obtained in steps C and D, mix them evenly, dry them, and pulverize them to obtain a mixed clear paste powder. Add the volatile oil obtained in step B and the fine powder obtained in step A, granulate, and granulate to obtain 1000g of granules.

[0394] Identification:

[0395] For the thin-layer chromatographic identification of Polygonum cuspidatum, take the sample, remove the coating, grind finely, add methanol, sonicate, filter, and use the filtrate as the test solution. Separately take Polygonum cuspidatum reference material, add methanol, sonicate, and use the supernatant as the reference material solution. Apply 3-5 μl of the test solution and 2 μl of the reference material solution separately to the same silica gel G thin-layer plate. Use the upper layer of petroleum ether (30-60℃)-ethyl formate-formic acid (15:5:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0396] For the thin-layer chromatographic identification of Stephania tetrandra, take the test solution from the TLC identification of Polygonum cuspidatum, evaporate to dryness, dissolve the residue in 20 ml of 1% hydrochloric acid, adjust the pH to 9-10 with 6 mol / L sodium hydroxide solution, and extract three times with 20 ml of chloroform each time. Combine the chloroform extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution. Separately, prepare a mixed solution containing 1 mg of each of tetrandrine and fangchinorline reference standards in methanol as the reference solution. Apply 5-10 μl of the test solution and 5 μl of the reference solution separately to the same silica gel G TLC plate. Develop the plate using chloroform-acetone-methanol-ammonia (12:2:2:0.1) as the developing solvent, remove the plate, air dry, and spray with dilute potassium bismuth iodide reagent. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standards.

[0397] For the thin-layer chromatographic identification of cinnamon twig, take the contents of the product and extract the volatile oil using the cyclohexane solution of the volatile oil as the test solution; separately prepare a reference solution by dissolving cinnamaldehyde in ethanol. Apply 5–10 μl of the test solution and 2 μl of the reference solution separately to the same silica gel G thin-layer plate. Develop the plate using petroleum ether (60–90℃)-ethyl acetate (17:3) as the developing solvent. Remove the plate, air dry, and spray with dinitrophenylhydrazine ethanol reagent. The chromatogram of the test sample should show spots of the same color at the corresponding positions as the chromatogram of the reference sample.

[0398] For the thin-layer chromatography identification of myrrh, take the myrrh reference material, extract the volatile oil with water, and use this as the reference material solution. Apply 5–10 μl of the test solution and 2 μl of the reference material solution to the same silica gel G thin-layer plate. Develop the plate using cyclohexane-ether-glacial acetic acid (4:1:0.1) as the developing solvent. Remove the plate, air dry, spray with 10% sulfuric acid in ethanol, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0399] For the identification of white peony root, take an appropriate amount of the product, add 30 ml of methanol, sonicate for 30 minutes, filter, and use the filtrate as the test solution. Separately, take paeoniflorin reference standard, add methanol to prepare a solution containing 0.1 mg per ml, and use this as the reference solution. Perform the test according to high performance liquid chromatography (General Chapter 0512 of the Chinese Pharmacopoeia 2020 edition). Inject 5 μl each of the reference solution and the test solution into the liquid chromatograph. Use octadecylsilane-bonded silica gel as the stationary phase; acetonitrile-0.1% phosphoric acid solution (14:86) as the mobile phase; flow rate: 1.0 ml / min; detection wavelength: 230 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should not be less than 2000. The chromatogram of the test sample should show a peak with a retention time corresponding to that of the reference standard.

[0400] Limited quantity inspection:

[0401] Aconitine Limit Test: Take the contents of this preparation, add ammonia solution and ether, let stand, evaporate the filtrate to dryness, dissolve the residue in anhydrous ethanol to obtain the test solution; separately take aconitine reference standard, add anhydrous ethanol to prepare a solution to obtain the reference solution. Accurately pipette the test solution and the reference solution, and spot them separately on the same silica gel G thin-layer plate. Develop with cyclohexane-ethyl acetate-ammonia water as the developing solvent, remove, air dry, and spray with dilute bismuth potassium iodide test solution. In the chromatogram of the test sample, the spot appearing at the corresponding position as in the chromatogram of the reference standard should be smaller than the spot of the reference standard, or no spot should appear.

[0402] Content determination:

[0403] Astragalus content determination:

[0404] HPLC detection: The chromatographic column was C18 (Waters SunFire C18 4.6×150mm), the mobile phase was methanol-water with a volume ratio of 80:20, the flow rate was 0.8ml / min, the gas flow rate was 3.5L / min, the drift tube temperature was 75℃, and the column temperature was 40℃.

[0405] Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, add the mobile phase to prepare a solution, and the solution is obtained.

[0406] Preparation of the test solution: Weigh the contents of this product accurately, add methanol, shake well, filter, accurately measure the filtrate, evaporate to dryness, dissolve the residue in water, extract with chloroform by shaking, discard the chloroform layer, extract the aqueous layer with water-saturated n-butanol by shaking to obtain n-butanol solution, wash, evaporate the n-butanol layer to dryness, dissolve the residue in the mobile phase to obtain the test solution;

[0407] Assay: Accurately pipette the reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0408] Determination of Gentiana macrophylla content:

[0409] HPLC detection: The chromatographic column was C18 (Waters SunFire C18 150×4.6mm), the mobile phase was acetonitrile-water with a volume ratio of 10:90, the detection wavelength was 260nm, the column temperature was 20℃, and the flow rate was 1.2ml / min;

[0410] Preparation of reference solution: Take an appropriate amount of gentiopicroside reference standard, accurately weigh it, add the mobile phase to prepare a solution, and the solution is obtained.

[0411] Preparation of the test solution: Weigh the contents of the traditional Chinese medicine composition preparation accurately, add 70% methanol accurately, shake well, filter, and take the filtrate to obtain the test solution;

[0412] Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

[0413] Example 4:

[0414] Methods for identification, limit testing and content determination of traditional Chinese medicine composition pills according to the present invention

[0415] The raw material formula is as follows: Astragalus membranaceus 120g, Gentiana macrophylla 190g, Aconitum carmichaelii 45g, Aconitum carmichaelii 80g, Polygonum cuspidatum 110g, Spatholobus suberectus 180g, Clematis chinensis 110g, Sinapis alba 40g, Paeonia lactiflora 45g, Rehmannia glutinosa 190g, Angelica sinensis 45g, Myrrh (processed with vinegar) 65g, Cyperus rotundus 40g, Cinnamomum cassia 80g, Achyranthes bidentata 45g.

[0416] Preparation method:

[0417] A. Grind the Stephania tetrandra into a fine powder and set aside.

[0418] B. Take cinnamon twigs, vinegar, myrrh, and spice, add 8 times the amount of water, and continuously extract the volatile oil by steam distillation for 10 hours. Collect the volatile oil, filter the distilled aqueous solution, collect it in a separate container, and set it aside for later use.

[0419] C. Take Gentiana macrophylla, Spatholobus suberectus, Angelica sinensis, Sinapis alba, Paeonia lactiflora, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, 1 hour each time. Filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear extract with a relative density of 1.15±0.05 for later use.

[0420] D. Take Astragalus membranaceus, Aconitum carmichaelii, Clematis chinensis, Rehmannia glutinosa, and Achyranthes bidentata, add 8 times the amount of water, decoct 4 times, 1.5 hours each time, filter the decoction, concentrate under reduced pressure to a clear extract with a relative density of 1.10±0.05, then combine it with the aqueous solution after distillation of volatile oil, concentrate under reduced pressure to a clear extract with a relative density of 1.15±0.05, and set aside.

[0421] E. Combine the clear pastes obtained in steps C and D, mix them evenly, dry them, and pulverize them to obtain a mixed clear paste powder. Add the fine powder obtained in step A and the volatile oil obtained in step B, and make it into pills according to conventional methods.

[0422] The identification, limit test and content determination methods are the same as in Example 2.

[0423] Example 5:

[0424] The present invention provides a method for the identification, limit testing, and content determination of traditional Chinese medicine composition granules.

[0425] Astragalus membranaceus 190g, Gentiana macrophylla 120g, Stephania tetrandra 80g, Aconitum carmichaelii 45g, Polygonum cuspidatum 180g, Spatholobus suberectus 110g, Clematis chinensis 180g, Mustard seed 20g, Paeonia lactiflora 80g, Rehmannia glutinosa 120g, Angelica sinensis 80g, Myrrh (processed with vinegar) 40g, Cyperus rotundus 65g, Cinnamomum cassia 45g, Achyranthes bidentata 80g.

[0426] Preparation method:

[0427] A. Grind the Stephania tetrandra into a fine powder and set aside.

[0428] B. Take cinnamon twigs, vinegar, myrrh, and spice, add 8 times the amount of water, and continuously extract the volatile oil by steam distillation for 10 hours. Collect the volatile oil, filter the distilled aqueous solution, collect it in a separate container, and set it aside for later use.

[0429] C. Take Gentiana macrophylla, Spatholobus suberectus, Angelica sinensis, Sinapis alba, Paeonia lactiflora, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, 1 hour each time. Filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear extract with a relative density of 1.15±0.05 for later use.

[0430] D. Take Astragalus membranaceus, Aconitum carmichaelii, Clematis chinensis, Rehmannia glutinosa, and Achyranthes bidentata, add 8 times the amount of water, decoct 4 times, 1.5 hours each time, filter the decoction, concentrate under reduced pressure to a clear extract with a relative density of 1.10±0.05, then combine it with the aqueous solution after distillation of volatile oil, concentrate under reduced pressure to a clear extract with a relative density of 1.15±0.05, and set aside.

[0431] E. Combine the clear pastes obtained in steps C and D, mix them evenly, dry them, and pulverize them to obtain a mixed clear paste powder. Add the volatile oil obtained in step B, add the fine powder from step A, granulate, and granulate to obtain granules.

[0432] The identification, limit test and content determination methods are the same as in Example 3.

[0433] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for quality control of a traditional Chinese medicine composition preparation, characterized in that, The raw materials of the traditional Chinese medicine composition preparation include the following components in parts by weight: Astragalus membranaceus 120-190 parts, Gentiana macrophylla 120-190 parts, Stephania tetrandra 45-80 parts, Aconitum carmichaelii 45-80 parts, Polygonum cuspidatum 110-180 parts, Spatholobus suberectus 110-180 parts, Clematis chinensis 110-180 parts, Mustard seed 20-40 parts, Paeonia lactiflora 45-80 parts, Rehmannia glutinosa 120-190 parts, Angelica sinensis 45-80 parts, Myrrh (processed with vinegar) 40-65 parts, Cyperus rotundus 40-65 parts, Cinnamomum cassia 45-80 parts, and Achyranthes bidentata 45-80 parts. The quality control methods for the traditional Chinese medicine composition preparations include identification methods, limit testing methods, and content determination methods. The identification method includes at least one of the following identification methods (1) to (5): (1) Thin-layer chromatography with Polygonum cuspidatum as reference material and petroleum ether at 30-60℃-ethyl formate-formic acid upper layer solution with a volume ratio of 15:5:1 as the developing solvent. (2) Thin-layer chromatography with tetrandrine and tebufenozide as reference standards and chloroform-acetone-methanol-ammonia water as the developing solvent in a volume ratio of 12:2:2:0.1; (3) Thin-layer chromatography with cinnamaldehyde as reference standard and petroleum ether at 60-90℃-ethyl acetate at a volume ratio of 17:3 as the developing solvent; (4) Thin-layer chromatography with myrrh as reference material and cyclohexane-ethyl ether-glacial acetic acid in a volume ratio of 4:1:0.1 as the developing solvent; (5) Using paeoniflorin as a reference standard and octadecylsilane-bonded silica gel as a filler; High performance liquid chromatography using acetonitrile-0.1% phosphoric acid solution (14:86) as the mobile phase; The limit test method is the aconitine limit test method, which uses aconitine as the reference standard and thin-layer chromatography with cyclohexane-ethyl acetate-ammonia water at a volume ratio of 10:10:0.1 as the developing solvent. The content determination methods include methods for determining the content of Astragalus membranaceus and Gentiana macrophylla, and the contents of astragaloside A and gentiopicroside are determined by high performance liquid chromatography.

2. The quality control method as described in claim 1, comprising an identification method, a limit testing method, and a content determination method, wherein the identification method comprises at least one of the following identification methods (1) to (5): Its features are, The identification method for Polygonum cuspidatum described in item (1) is a thin-layer chromatographic identification, which includes the following steps: Take a traditional Chinese medicine composition preparation and use methanol extract as the test solution; take Polygonum cuspidatum reference material and use methanol extract as the reference material solution. Apply the test solution and the reference material solution separately to the same silica gel G thin layer plate. Use 15:5:1 petroleum ether 30-60℃-ethyl formate-formic acid upper layer solution as the developing solvent. Develop, remove, air dry, and examine under 365nm ultraviolet light. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference material. The identification method described in item (2) is a thin-layer chromatography identification of Stephania tetrandra, which includes the following steps: Take the test solution under item (1), evaporate to dryness, dissolve the residue in 1% hydrochloric acid, adjust the pH value with sodium hydroxide solution, extract with chloroform by shaking, evaporate to dryness, dissolve the residue in methanol, and use it as the test solution; separately take the tetrandrine reference standard and the tetrandrine reference standard, add methanol to prepare a mixed solution, and use it as the reference solution. Take the test solution and the reference solution and spot them on the same silica gel G thin layer plate, use 12:2:2:0.1 chloroform-acetone-methanol-ammonia water as the developing solvent, spray with dilute bismuth potassium iodide test solution, and spot the same color in the test chromatogram at the corresponding position as in the reference chromatogram; The identification method described in item (3) is a thin-layer chromatographic identification of cinnamon twigs, which includes the following steps: Take a traditional Chinese medicine composition preparation and use water to extract the volatile oil as the test solution; take cinnamaldehyde reference standard, add ethanol to prepare a solution as the reference solution, and apply the test solution and the reference solution separately to the same silica gel G thin layer plate. Use 17:3 petroleum ether 60-90℃-ethyl acetate as the developing solvent and spray with dinitrophenylhydrazine ethanol test solution. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard. The identification method described in item (4) is a thin-layer chromatography identification of myrrh, which includes the following steps: Take myrrh reference material, add water to extract volatile oil as reference material solution, take the reference material solution and the test solution under (3) and spot them on the same silica gel G thin layer plate respectively, use 4:1:0.1 cyclohexane-ethyl ether-glacial acetic acid as the developing solvent, spray with sulfuric acid ethanol solution, heat until the spots are clearly visible, and in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference material; The identification method described in item (5) is a thin-layer chromatography identification of white peony root, which includes the following steps: Take the traditional Chinese medicine composition preparation, extract it with methanol, and use the filtrate as the test solution; separately take paeoniflorin reference standard, add methanol to prepare a solution as the reference solution, inject the test solution and the reference solution into the liquid chromatograph, use octadecylsilane bonded silica gel as the stationary phase, use acetonitrile-0.1% phosphoric acid solution (14:86) as the mobile phase, and use the detection wavelength at 230 nm. The chromatogram of the test sample should show a chromatographic peak with the retention time corresponding to that of the reference standard.

3. The quality control method as described in claim 1, characterized in that, The method for testing the limit of aconitine includes the following steps: Take a traditional Chinese medicine composition preparation, add ammonia test solution and let stand, then add ether, let stand, evaporate the filtrate to dryness, dissolve the residue in anhydrous ethanol to obtain the test solution; separately take aconitine reference standard, add anhydrous ethanol to prepare a solution to obtain the reference solution. Accurately pipette the test solution and the reference solution and spot them separately on the same silica gel G thin-layer plate, using cyclohexane-ethyl acetate-ammonia water as the developing solvent, and spray with dilute potassium bismuth iodide test solution. In the chromatogram of the test sample, the spot appearing at the corresponding position as in the chromatogram of the reference standard should be smaller than the spot of the reference standard, or no spot should appear.

4. The quality control method as described in claim 1, characterized in that, The method for determining the content of Astragalus membranaceus includes the following steps: HPLC detection: C18 column, mobile phase of methanol:water (v / v) at a ratio of 76–80:20–24, flow rate of 0.8–1.2 ml / min, evaporative light scattering detector, gas flow rate of 2.5–3.5 L / min, drift tube temperature of 75–85 °C; Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, add the mobile phase to prepare a solution, and the solution is obtained. Preparation of the test solution: Take the Chinese herbal medicine composition preparation, accurately weigh it, add methanol, shake well, filter, accurately measure the filtrate, evaporate to dryness, dissolve the residue in water, extract with chloroform by shaking, discard the chloroform layer, extract the aqueous layer with water-saturated n-butanol by shaking to obtain n-butanol solution, wash, evaporate the n-butanol layer to dryness, dissolve the residue in the mobile phase to obtain the test solution; Assay: Accurately pipette the reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

5. The quality control method as described in claim 1, characterized in that, The method for determining the content of Gentiana macrophylla includes the following steps: HPLC detection: The chromatographic column was C18, the mobile phase was acetonitrile-water with a volume ratio of 10-12:88-90, the detection wavelength was 250-260 nm, and the flow rate was 0.8-1.2 ml / min; Preparation of reference solution: Take an appropriate amount of gentiopicroside reference standard, accurately weigh it, add the mobile phase to prepare a solution, and the solution is obtained. Preparation of the test solution: Take the traditional Chinese medicine composition preparation, weigh it accurately, add 70% methanol accurately, sonicate, filter, and take the filtrate to obtain the test solution; Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

6. The quality control method according to any one of claims 1 to 5, characterized in that, The dosage form of the traditional Chinese medicine composition is capsule, tablet, pill, oral liquid, granule or powder.

7. The quality control method according to any one of claims 1 to 5, characterized in that, The active components of the traditional Chinese medicine composition are prepared by the following steps: A. Grind the Stephania tetrandra into a fine powder and set aside. B. Take cinnamon twigs, vinegar, myrrh, and aromatics, and add 5-8 times the amount of water. Extract the volatile oil continuously by steam distillation for 5-10 hours. Collect the volatile oil, filter the distilled aqueous solution, collect it in a separate container, and set it aside for later use. C. Take Gentiana macrophylla, Spatholobus suberectus, Angelica sinensis, Mustard seed, Paeonia lactiflora, and Polygonum cuspidatum, add 8-12 times the amount of 50-80% ethanol, reflux extract 1-3 times, 1 hour each time, filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear extract with a relative density of 1.15±0.05 for later use. D. Take Astragalus membranaceus, Aconitum carmichaelii, Clematis chinensis, Rehmannia glutinosa, and Achyranthes bidentata, add 8-12 times the amount of water, decoct 2-5 times, 1.5 hours each time, filter the decoction and combine it with the aqueous solution after distillation of volatile oil, concentrate under reduced pressure to a clear extract with a relative density of 1.15±0.05, and set aside. E. Combine the extracts from steps C and D, mix well, dry, and pulverize to obtain a mixed extract powder; The fine powder obtained in step A, the volatile oil obtained in step B, and the clear extract powder obtained in step E together constitute the active components of the traditional Chinese medicine composition preparation.

8. The quality control method as described in claim 6, characterized in that, The preparation process of the capsule of the traditional Chinese medicine composition is as follows: A. Grind the Stephania tetrandra into a fine powder and set aside. B. Take cinnamon twigs, vinegar, myrrh, and spice, add 8 times the amount of water, and continuously extract the volatile oil by steam distillation for 10 hours. Collect the volatile oil, filter the distilled aqueous solution, collect it in a separate container, and set it aside for later use. C. Take Gentiana macrophylla, Spatholobus suberectus, Angelica sinensis, Sinapis alba, Paeonia lactiflora, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, 1 hour each time. Filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear extract with a relative density of 1.15±0.05 for later use. D. Take Astragalus membranaceus, Aconitum carmichaelii, Clematis chinensis, Rehmannia glutinosa, and Achyranthes bidentata, add 8 times the amount of water, decoct 4 times, 1.5 hours each time, filter the decoction, combine it with the aqueous solution after distillation of volatile oil, and concentrate under reduced pressure to a clear extract with a relative density of 1.15±0.05 for later use. E. Combine the clear extracts obtained in steps C and D, mix them evenly, dry them, and pulverize them to obtain a mixed clear extract powder. Add the volatile oil obtained in step B, add the fine powder from step A, granulate, shape the granules, and fill them into capsules to obtain the final product.

9. The quality control method as described in claim 6, characterized in that, The preparation process of the traditional Chinese medicine composition tablets is as follows: A. Grind the Stephania tetrandra into a fine powder and set aside. B. Take cinnamon twigs, vinegar, myrrh, and spice, add 6 times the amount of water, and continuously extract the volatile oil by steam distillation for 8 hours. Collect the volatile oil, filter the distilled aqueous solution, collect it separately, and set it aside. C. Take Gentiana macrophylla, Spatholobus suberectus, Angelica sinensis, Sinapis alba, Paeonia lactiflora, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, 1 hour each time. Filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear extract with a relative density of 1.15±0.05 for later use. D. Take Astragalus membranaceus, Aconitum carmichaelii, Clematis chinensis, Rehmannia glutinosa, and Achyranthes bidentata, add 11 times the amount of water, decoct 3 times, 1.5 hours each time, filter the decoction, combine it with the aqueous solution after distillation of volatile oil, and concentrate under reduced pressure to a clear extract with a relative density of 1.15±0.05 for later use. E. Combine the clear pastes obtained in steps C and D, mix them thoroughly, dry them, and pulverize them to obtain a mixed clear paste powder. F. Take the volatile oil obtained in step B and incorporate it into the mixture for later use; Granulate the fine powder obtained in step A and the clear paste powder obtained in step E, granulate them, add the inclusion complex obtained in step F, and compress them into tablets using conventional methods.

10. The quality control method as described in claim 9, characterized in that, The inclusion process in step F of the preparation of the traditional Chinese medicine composition tablet is as follows: Take 6-10 times the amount of cyclodextrin as volatile oil, add 6-10 times the amount of water as cyclodextrin, grind, then add an equal volume mixture of volatile oil and 90-95% ethanol, continue grinding until uniform, and then dry to obtain the final product.

11. The quality control method as described in claim 1, characterized in that, The raw materials of the traditional Chinese medicine composition preparation include the following components in parts by weight: Astragalus membranaceus 120 parts, Gentiana macrophylla 190 parts, Stephania tetrandra 45 parts, Aconitum carmichaelii 80 parts, Polygonum cuspidatum 110 parts, Spatholobus suberectus 180 parts, Clematis chinensis 110 parts, Mustard seed 40 parts, Paeonia lactiflora 45 parts, Rehmannia glutinosa 190 parts, Angelica sinensis 45 parts, Myrrh (processed with vinegar) 65 parts, Cyperus rotundus 40 parts, Cinnamomum cassia 80 parts, and Achyranthes bidentata 45 parts; or Astragalus membranaceus 150 parts, Gentiana macrophylla 160 parts, Stephania tetrandra 60 parts, Aconitum carmichaelii 65 parts, Polygonum cuspidatum 120 parts, Spatholobus suberectus 130 parts, Clematis chinensis 135 parts, Mustard seed 35 parts, Paeonia lactiflora 50 parts, Rehmannia glutinosa 160 parts, Angelica sinensis 60 parts, Myrrh (processed with vinegar) 55 parts, Cyperus rotundus 45 parts, Cinnamomum cassia 45 parts, and 60 parts of Astragalus membranaceus and 60 parts of Achyranthes bidentata; or 146 parts of Astragalus membranaceus, 139 parts of Gentiana macrophylla, 58 parts of Stephania tetrandra, 75 parts of processed Aconitum carmichaelii, 120 parts of Polygonum cuspidatum, 125 parts of Spatholobus suberectus, 135 parts of Clematis chinensis, 28 parts of Sinapis alba, 60 parts of Paeonia lactiflora, 150 parts of Rehmannia glutinosa, 70 parts of Angelica sinensis, 60 parts of vinegar-processed Myrrh, 60 parts of Cyperus rotundus, 70 parts of Cinnamomum cassia and 70 parts of Achyranthes bidentata; or 156 parts of Astragalus membranaceus, 156 parts of Gentiana macrophylla, 63 parts of Stephania tetrandra, 63 parts of processed Aconitum carmichaelii, 146 parts of Polygonum cuspidatum, 146 parts of Spatholobus suberectus, 146 parts of Clematis chinensis, 31 parts of Sinapis alba, 63 parts of Paeonia lactiflora, 156 parts of Rehmannia glutinosa, 63 parts of Angelica sinensis, 52 parts of vinegar-processed Myrrh, 52 parts of Cyperus rotundus, 63 parts of Cinnamomum cassia and 63 parts of Achyranthes bidentata.

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