A fingerprint spectrum construction method for saponin components in a traditional Chinese medicine composition containing astragalus and its application

Abstract

The present invention discloses a method for determining the content of a Chinese medicine composition containing astragalus, and the method comprises the following steps: taking a test solution and a reference solution of the Chinese medicine composition for detection, and the chromatographic conditions of the detection are: using a chromatographic column whose filler is octadecylsilane bonded silica gel, the mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, and the mobile phase B is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution; according to the detection result, the content information of the Chinese medicine composition is obtained; wherein, the Chinese medicine composition comprises astragalus, rhizoma anemarrhenae, platycodon grandiflorum, radix bupleuri and rhizoma cimicifugae, and the reference substance is rhizoma anemarrhenae saponin BⅡ, platycodon grandiflorum saponin D, astragaloside Ⅳ and saikosaponin a. The present invention comprehensively and systematically analyzes the saponin chemical components of the Chinese medicine composition containing astragalus, and provides a theoretical basis for the in-depth study of quality control and the material basis of drug efficacy.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for constructing a fingerprint spectrum of saponin components in a traditional Chinese medicine composition containing astragalus and an application thereof. Background Art

[0002] According to the Traditional Chinese Medicine Law of the People's Republic of China, ancient classic prescriptions refer to "prescriptions recorded in ancient Chinese medicine classics that are still widely used, have definite efficacy, and have obvious characteristics and advantages". Ancient classic prescriptions have a long and rich history of human use in my country and are still used today. They are the summary of the essence of clinical practice of doctors of all dynasties, carrying the profound accumulation of the splendid civilization of Chinese medicine for thousands of years. They are the result of thousands of years of tempering of Chinese medicine theory, the summary of clinical experience of all dynasties, and the most essential part of the great treasure house of Chinese medicine. In-depth research and development of classic Chinese medicine prescriptions is a golden key to tap into the treasure house of traditional Chinese medicine.

[0003] As the most commonly used dosage form of traditional Chinese medicine clinical medicine, decoctions of classic Chinese medicine prescriptions have the advantages of reasonable formulation, rapid onset, significant efficacy, and easy absorption, and are deeply trusted by the majority of patients. However, due to the shortcomings of easy mold and deterioration during preparation, carrying, temporary decoction, and long-term storage, the bitter taste and large amount of decoction, and the difficulty in standardization, which seriously affects its clinical efficacy, it cannot adapt to the living requirements of modern people. In order to maintain the advantages of decoctions and overcome the various shortcomings of decoctions, the "Technical Guiding Principles for Pharmaceutical Research of Traditional Chinese Medicine Compound Preparations Managed by the Catalog of Ancient Classic Prescriptions (Trial)" issued by the Drug Evaluation Center of the State Food and Drug Administration on August 31, 2021 clearly pointed out that the quality of traditional Chinese medicine compound preparations managed by the catalog of ancient classic prescriptions should be basically consistent with the quality of the classic prescription benchmark samples. The benchmark sample represents the overall intrinsic quality of the preparation. Except for the molding process, the benchmark sample and the other quality control indicators of the preparation are basically consistent. Therefore, the benchmark sample is a physical comparison of the intrinsic quality of the preparation and a reference for the optimization of large-scale production processes and the formulation of its quality standards. Benchmark samples are the benchmark for classic prescriptions and even all Chinese medicine research and development, and are the reference materials to ensure the safety and effectiveness of drugs. The production process route formulation, parameter optimization and quality standard formulation of classic prescription compound preparations should be based on the benchmark samples of classic prescriptions. Benchmark samples are the link between clinic, enterprise and scientific research, and are the benchmark for inheriting, applying and promoting traditional Chinese medicine.

[0004] Fingerprint is based on the understanding of the overall effect of Chinese medicine substance groups. It uses spectral and chromatographic techniques to obtain the spectrum or chromatogram of Chinese medicine chemical components. It is a feasible model for identifying the authenticity of Chinese medicine, evaluating quality consistency and product stability. It has the characteristics of large amount of information, strong characteristics, integrity and fuzziness. Chinese medicine fingerprint can comprehensively reflect the relative relationship of chemical components contained in medicinal materials, embody the complexity and correlation of Chinese medicine components, and adapt to the traditional theory of Chinese medicine. It can truly effectively characterize, comprehensively evaluate and comprehensively control the intrinsic quality of Chinese medicine. It is especially suitable for quality control of Chinese medicinal materials and Chinese medicine products when the effective ingredients are not completely clear or do not need to be completely clear. In addition to being used to investigate the origin, harvesting season, harvesting part, processing, storage time and other factors of Chinese medicinal materials, so as to provide a basis for identifying the authenticity and quality of raw medicinal materials before production, Chinese medicine fingerprint can also be used for quality control of Chinese medicine production process: tracking the changes of certain chemical components in preparations, monitoring the consistency and stability of quality between raw medicinal materials and finished products, and between batches of finished products. Compared with the quality analysis method of determining the content of index components, fingerprint spectrum can more comprehensively reflect the types and quantities of chemical components of traditional Chinese medicine. At the current situation where the effective ingredients of traditional Chinese medicine compound preparations have not been fully elucidated, it can realize the comprehensive evaluation of the intrinsic quality of traditional Chinese medicine and the effective control of its overall substance. It is one of the effective means of quality control of traditional Chinese medicine and its preparations.

[0005] At present, there is no literature report on a comprehensive analysis and fingerprint study of the saponin chemical components of the traditional Chinese medicine composition containing Astragalus claimed in the present invention. In the prior art, only a single component in the traditional Chinese medicine compound is analyzed for quality, and there is no comprehensive and systematic quality control method to reflect the quality status of the main reference sample components in the traditional Chinese medicine compound and the finished product of the present invention, and it is impossible to effectively control its production process and product quality, and it is impossible to better guarantee its clinical efficacy. Therefore, it is necessary to adopt a fingerprint quality control method that can comprehensively control the overall quality of the traditional Chinese medicine compound of the present invention to control its key quality. Summary of the invention

[0006] Based on this, the present invention provides a method for determining the content of a traditional Chinese medicine composition containing Astragalus. The method comprises the following steps: taking a test solution and a reference solution of the traditional Chinese medicine composition for detection, and the chromatographic conditions of the detection are: using a chromatographic column whose filler is octadecylsilane bonded silica gel, a mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, a mobile phase B is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, and the gradient elution program is: 0-20 min, 90% B→77% B; 20-55 min, 77% B→75% B; 55-70 min, 75% B→68% B; 70-90 min, 68% B→65% B; 90-97 min, 65% B→55% B; 97-107 min, 55% B→40% B; 107-109 min, 40% B; 109-110 min, 40% B→90% B; 110-120 min, 90% B, flow rate is 0.1-1.0 ml / min, column temperature is 10-40°C, injection volume is 1-20 μl; according to the test results, the content information of the traditional Chinese medicine composition is obtained; wherein, the traditional Chinese medicine composition includes astragalus, anemarrhena, platycodon, bupleurum and cimicifuga, and the reference substance is anemarrhena saponin BⅡ, platycodon saponin D, astragaloside I and bupleurosaponin a.

[0007] In the present invention, when time, flow rate, temperature, wavelength, injection volume, concentration, power, frequency, ratio, voltage, volume, rotation speed, vacuum degree, number of times, mass, or other values ​​or parameters are expressed as a range, a preferred range, or a range limited by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of “10-40” is disclosed, the described range should be interpreted as including the range of “10-40”, “10-35”, “10-30”, “10-25”, “10-20”, “10-15”, “15-40”, “15-35”, “15-30”, “15-25”, “15-20”, “20-40”, “20-35”, “20-30”, “20-25”, “25-40”, “25-35”, “25-30”, “30-40”, “30-35”, “35-40”, etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within the range.

[0008] Furthermore, the information is the content of the Chinese medicine composition calculated according to the external standard method based on the corresponding peak areas in the recorded chromatogram of the test solution of the Chinese medicine composition and the chromatogram of the reference solution.

[0009] Furthermore, the 120 min in the gradient elution program was replaced by 130 min.

[0010] Furthermore, the detector for the detection is an electrospray detector.

[0011] Furthermore, the standard curve of the platycodon saponin D is y=0.0878x^0.8503, R 2 =0.9994.

[0012] Furthermore, the standard curve of astragaloside IV is y=0.1044x^0.8480, R 2 =0.9993.

[0013] Furthermore, the standard curve of saikosaponin a is y=0.1176x^0.8230, R 2 =0.9991.

[0014] Furthermore, the preparation method of the test solution of the Chinese medicine composition includes: weighing an appropriate amount of Chinese medicine composition powder containing astragalus, concentrating under reduced pressure, adding a first solvent containing an alkaline test solution, weighing the weight, cooling after heating to reflux, weighing the weight again, using the first solvent containing the alkaline test solution to make up for the lost weight, shaking, filtering, taking a first subsequent filtrate, evaporating to dryness, obtaining a residue, dissolving the obtained residue with a first solvent, shaking, filtering, taking a second subsequent filtrate, and obtaining the test solution of the Chinese medicine composition.

[0015] Furthermore, the alkaline test solution is an ammonia test solution.

[0016] Furthermore, the first solvent is alcohol, such as methanol.

[0017] Furthermore, the volume percentage concentration of the ammonia test solution is 1% to 10%, preferably 2% to 6%, for example about 4%.

[0018] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 4%" includes ±5% of 4%, or from 3.8% to 4.2%.

[0019] Furthermore, the volume percentage concentration of the methanol is 10% to 100%, preferably 70% to 90%, for example about 80%.

[0020] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 80%" includes ±5% of 80%, or from 76% to 84%.

[0021] Furthermore, the mass / volume (mg / ml) ratio of the powder of the Chinese medicinal composition containing Astragalus to the first solvent containing the alkaline test solution is 10-50, for example, about 20.

[0022] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0023] Furthermore, the heating reflux time is 30 to 120 minutes, for example, about 60 minutes.

[0024] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0025] Furthermore, the volume ratio of the first subsequent filtrate to the first solvent containing the alkaline test solution is 0.4 to 0.6, such as about 0.5.

[0026] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0027] Furthermore, the volume ratio of the second subsequent filtrate to the first subsequent filtrate is 0.1 to 0.4, for example, about 0.2.

[0028] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.2" includes ±5% of 0.2, or from 0.19 to 0.21.

[0029] Furthermore, the preparation method of the reference solution includes: weighing appropriate amounts of timosaponin BⅡ, platycodon saponin D, astragaloside I and bupleurosaponin a reference substances, adding methanol to prepare the reference solution with a concentration of timosaponin BⅡ, platycodon saponin D, astragaloside I and bupleurosaponin a of 0.01 to 200 μg / ml respectively.

[0030] Further, the concentration of Anemarrhena saponin BⅡ in the reference solution is about 100 μg / ml. Further, the concentration of Platycodon saponin D in the reference solution is about 100 μg / ml. Further, the concentration of Astragaloside IV in the reference solution is about 100 μg / ml. Further, the concentration of Bupleurum saponin A in the reference solution is about 100 μg / ml.

[0031] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105.

[0032] Furthermore, the flow rate of the HPLC detection is 0.5-1.0 ml / min, for example, about 0.8 ml / min.

[0033] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.8" includes ±5% of 0.8, or from 0.76 to 0.84.

[0034] Furthermore, the column temperature is 20-50°C, for example, about 35°C.

[0035] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 35" includes ±5% of 35, or from 33.25 to 36.75.

[0036] Furthermore, the injection volume is 1-20 μl, for example, about 10 μl.

[0037] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0038] Furthermore, the theoretical plate number of the chromatographic peak corresponding to astragaloside IV is not less than 5000.

[0039] Furthermore, the chromatographic column is a YMC-Pack ODS-A chromatographic column.

[0040] Furthermore, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0041] Furthermore, the mobile phase A is acetonitrile.

[0042] Furthermore, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.

[0043] Further, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid in different concentrations.

[0044] Furthermore, the acid aqueous solution is a 0.01% to 1% acid aqueous solution.

[0045] Furthermore, the acid aqueous solution is a 0.01% to 1% acetic acid aqueous solution.

[0046] Furthermore, the acid aqueous solution is about 0.2% acetic acid aqueous solution.

[0047] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.2%" includes ±5% of 0.2%, or from 0.19% to 0.21%.

[0048] Furthermore, the buffered saline solution is an acetate solution and / or an acetate solution.

[0049] Furthermore, the pH value of the buffered saline solution is not greater than 7.0.

[0050] Furthermore, the preparation method of the powder of the Chinese medicine composition containing astragalus comprises: (a) weighing appropriate amounts of astragalus, platycodon, bupleurum and cimicifuga, adding a first water for a first soaking, decocting over high heat until boiling, decocting over low heat for a period of time, adding rhizoma anemarrhenae for a second soaking, decocting over high heat until boiling, decocting over low heat for a period of time, and obtaining a decoction; (b) filtering the decoction to obtain a first filtrate and a filter residue, adding a second water to the filter residue, decocting over high heat until boiling, decocting over low heat for a period of time, filtering to obtain a second filtrate, combining the first filtrate and the second filtrate to obtain a combined filtrate, decompressing and concentrating the combined filtrate to an extract with a relative density of 1.10 to 1.20 at about 20° C., pre-freezing, drying in a freeze dryer, crushing, sieving, and mixing to obtain the powder of the Chinese medicine composition containing astragalus.

[0051] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0052] Furthermore, the ratio of the sum of the mass of the astragalus, platycodon, bupleurum and cimicifuga to the mass / volume (mg / ml) of the first water is 50-90, for example, about 74.62.

[0053] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 74.62" includes ±5% of 74.62, or from 70.889 to 78.351.

[0054] Furthermore, the first soaking time is 10 to 100 minutes, for example, about 60 minutes.

[0055] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0056] Furthermore, the high-fire decoction is high-fire decoction with a cover.

[0057] Furthermore, the voltage of the high fire is 150-300V, for example, about 220V.

[0058] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 220" includes ±5% of 220, or from 209 to 231.

[0059] Furthermore, the voltage of the slow fire is 100-250V, for example, about 175V.

[0060] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 175" includes ±5% of 175, or from 166.25 to 183.75.

[0061] Furthermore, the first slow-cooking time is 30 to 70 minutes, for example, about 55 minutes.

[0062] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 55" includes ±5% of 55, or from 52.25 to 57.75.

[0063] Furthermore, the second slow-cooking time is 1 to 10 minutes, for example, about 5 minutes.

[0064] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 5" includes ±5% of 5, or from 4.75 to 5.25.

[0065] Furthermore, the third slow-cooking time is 30 to 70 minutes, for example, about 50 minutes.

[0066] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5.

[0067] Furthermore, the second soaking time is 5 to 20 minutes, for example, about 10 minutes.

[0068] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0069] Furthermore, the filtration is a single-layer filtration of 100-mesh nylon filter cloth.

[0070] Furthermore, the volume of the second water is 300-500 ml, for example, about 400 ml.

[0071] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 400" includes ±5% of 400, or from 380 to 420.

[0072] Furthermore, the mass / volume (mg / ml) ratio of the Chinese medicinal composition comprising Astragalus to the second water is 100-150, for example, about 121.25.

[0073] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 121.25" includes ±5% of 121.25, or from 115.1875 to 127.3125.

[0074] Further, the relative density of the combined filtrate at about 20° C. is 1.010 to 1.025.

[0075] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0076] Furthermore, the instrument for the reduced pressure concentration is a rotary evaporator.

[0077] Furthermore, the rotation speed of the rotary evaporator is 50-150 r / min, for example, about 100 r / min.

[0078] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105.

[0079] Furthermore, the temperature of the reduced pressure concentration is not higher than 65°C.

[0080] Furthermore, the vacuum degree of the reduced pressure concentration is -0.08 to -0.099 MPa.

[0081] Furthermore, before the pre-freezing, the extract is placed in a stainless steel plate so that the layer thickness is 9 to 11 mm.

[0082] Furthermore, the pre-freezing degree is such that there is no wet core in the extract.

[0083] Furthermore, the cold trap temperature of the freeze-drying is ≤-50°C.

[0084] Furthermore, the vacuum degree of the freeze-drying is ≤100Pa.

[0085] Furthermore, the freeze-drying degree is such that the moisture content is no higher than 8%.

[0086] Furthermore, the dry paste rate of the powder of the traditional Chinese medicine composition containing astragalus is 27.4% to 33.5%.

[0087] Furthermore, the mass ratios of the astragalus, the rhizoma anemarrhenae, the platycodon, the bupleurum and the cimicifuga are (5-7):(0.1-2):(0.5-2.5):(2-4):(0.5-2.5).

[0088] Furthermore, the mass ratios of the astragalus, the rhizoma anemarrhenae, the platycodon, the bupleurum and the cimicifuga are (5.5-6.5):(0.5-1.5):(1-2):(2.5-3.5):(1-2).

[0089] Furthermore, the mass ratio of the Astragalus, the Anemarrhena, the Platycodon and the Cimicifuga is about 6: about 1: about 1.5: about 3: about 1.5.

[0090] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3; "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 1.5" includes ±5% of 1.5, or from 1.425 to 1.575; "about 3" includes ±5% of 3, or from 2.85 to 3.15.

[0091] Furthermore, the mass range of the astragalus is 10 to 30 g, for example, about 22.38 g.

[0092] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 22.38" includes ±5% of 22.38, or from 21.261 to 23.499.

[0093] Furthermore, the mass range of the Anemarrhena Rhizoma is 1 to 10 g, for example, about 3.73 g.

[0094] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3.73" includes ±5% of 3.73, or from 3.5435 to 3.9165.

[0095] Furthermore, the mass range of the Platycodon grandiflorum is 1 to 10 g, for example, about 5.60 g.

[0096] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5.60" includes ±5% of 5.60, or from 5.32 to 5.88.

[0097] Furthermore, the mass range of the Bupleurum is 10 to 20 g, for example, about 11.19 g.

[0098] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 11.19" includes ±5% of 11.19, or from 10.6305 to 11.7495.

[0099] Furthermore, the mass of the Cimicifuga heracleifolia is in the range of 1 to 10 g, for example, about 5.60 g.

[0100] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5.60" includes ±5% of 5.60, or from 5.32 to 5.88.

[0101] According to another aspect of the present invention, a method for constructing a fingerprint of a traditional Chinese medicine composition containing Astragalus is provided, the method comprising the following steps:

[0102] Preparation of a Chinese medicine composition test solution: weigh an appropriate amount of Chinese medicine composition powder containing astragalus, concentrate under reduced pressure, add a first solvent containing an alkaline test solution, weigh the weight, heat under reflux and then cool, weigh again, use the first solvent containing an alkaline test solution to make up for the lost weight, shake well, filter, take a first subsequent filtrate, evaporate to dryness, obtain a residue, dissolve the obtained residue with a first solvent, shake well, filter, take a second subsequent filtrate, and obtain the Chinese medicine composition test solution; wherein the Chinese medicine composition comprises astragalus, rhizoma anemarrhenae, platycodon grandiflorum, radix bupleuri and rhizoma cimicifuga;

[0103] Preparation of reference solution: weigh appropriate amounts of timosaponin BⅡ, platycodon saponin D, astragaloside I and saikosaponin a reference substances, add methanol to prepare reference solution with concentrations of timosaponin BⅡ, platycodon saponin D, astragaloside I and saikosaponin a of 0.01 to 200 μg / ml respectively;

[0104] Obtaining a fingerprint of the Chinese medicine composition according to the results of high performance liquid chromatography detection of the test solution and the reference solution of the Chinese medicine composition;

[0105] The chromatographic conditions of the high performance liquid phase detection are as follows: a chromatographic column whose filler is octadecylsilane bonded silica gel is used, a mobile phase A is selected from one or more of acetonitrile, methanol and tetrahydrofuran, a mobile phase B is an acid aqueous solution, an alkali aqueous solution and / or a buffered saline solution, a gradient elution program is as follows: 0-20 min, 90% B→77% B; 20-55 min, 77% B→75% B; 55-70 min, 75% B→68% B; 70-90 min, 68% B→65% B; 90-97 min, 65% B→55% B; 97-107 min, 55% B→40% B; 107-109 min, 40% B; 109-110 min, 40% B→90% B; 110-120 min, 90% B, a flow rate of 0.1-1.0 ml / min, a column temperature of 10-40° C., and an injection volume of 1-20 μl.

[0106] Furthermore, the preparation method of the powder of the Chinese medicine composition containing astragalus comprises: (a) weighing appropriate amounts of astragalus, platycodon, bupleurum and cimicifuga, adding a first water for a first soaking, decocting over high heat until boiling, decocting over low heat for a period of time, adding rhizoma anemarrhenae for a second soaking, decocting over high heat until boiling, decocting over low heat for a period of time, and obtaining a decoction; (b) filtering the decoction to obtain a first filtrate and a filter residue, adding a second water to the filter residue, decocting over high heat until boiling, decocting over low heat for a period of time, filtering to obtain a second filtrate, combining the first filtrate and the second filtrate to obtain a combined filtrate, decompressing and concentrating the combined filtrate to an extract with a relative density of 1.10 to 1.20 at about 20° C., pre-freezing, drying in a freeze dryer, crushing, sieving, and mixing to obtain the powder of the Chinese medicine composition containing astragalus.

[0107] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0108] Furthermore, the ratio of the sum of the mass of the astragalus, platycodon, bupleurum and cimicifuga to the mass / volume (mg / ml) of the first water is 50-90, for example, about 74.62.

[0109] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 74.62" includes ±5% of 74.62, or from 70.889 to 78.351.

[0110] Furthermore, the first soaking time is 10 to 100 minutes, for example, about 60 minutes.

[0111] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0112] Furthermore, the high-fire decoction is high-fire decoction with a cover.

[0113] Furthermore, the voltage of the high fire is 150-300V, for example, about 220V.

[0114] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 220" includes ±5% of 220, or from 209 to 231.

[0115] Furthermore, the voltage of the slow fire is 100-250V, for example, about 175V.

[0116] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 175" includes ±5% of 175, or from 166.25 to 183.75.

[0117] Furthermore, the first slow-cooking time is 30 to 70 minutes, for example, about 55 minutes.

[0118] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 55" includes ±5% of 55, or from 52.25 to 57.75.

[0119] Furthermore, the second slow-cooking time is 1 to 10 minutes, for example, about 5 minutes.

[0120] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 5" includes ±5% of 5, or from 4.75 to 5.25.

[0121] Furthermore, the third slow-cooking time is 30 to 70 minutes, for example, about 50 minutes.

[0122] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5.

[0123] Furthermore, the second soaking time is 5 to 20 minutes, for example, about 10 minutes.

[0124] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0125] Furthermore, the filtration is a single-layer filtration of 100-mesh nylon filter cloth.

[0126] Furthermore, the volume of the second water is 300-500 ml, for example, about 400 ml.

[0127] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 400" includes ±5% of 400, or from 380 to 420.

[0128] Furthermore, the mass / volume (mg / ml) ratio of the Chinese medicinal composition comprising Astragalus to the second water is 100-150, for example, about 121.25.

[0129] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 121.25" includes ±5% of 121.25, or from 115.1875 to 127.3125.

[0130] Further, the relative density of the combined filtrate at about 20° C. is 1.010 to 1.025.

[0131] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0132] Furthermore, the instrument for the reduced pressure concentration is a rotary evaporator.

[0133] Furthermore, the rotation speed of the rotary evaporator is 50-150 r / min, for example, about 100 r / min.

[0134] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105.

[0135] Furthermore, the temperature of the reduced pressure concentration is not higher than 65°C.

[0136] Furthermore, the vacuum degree of the reduced pressure concentration is -0.08 to -0.099 MPa.

[0137] Furthermore, before the pre-freezing, the extract is placed in a stainless steel plate so that the layer thickness is 9 to 11 mm.

[0138] Furthermore, the pre-freezing degree is such that there is no wet core in the extract.

[0139] Furthermore, the cold trap temperature of the freeze-drying is ≤-50°C.

[0140] Furthermore, the vacuum degree of the freeze-drying is ≤100Pa.

[0141] Furthermore, the freeze-drying degree is such that the moisture content is no higher than 8%.

[0142] Furthermore, the dry paste rate of the powder of the traditional Chinese medicine composition containing astragalus is 27.4% to 33.5%.

[0143] Furthermore, the mass ratios of the astragalus, the rhizoma anemarrhenae, the platycodon, the bupleurum and the cimicifuga are (5-7):(0.1-2):(0.5-2.5):(2-4):(0.5-2.5).

[0144] Furthermore, the mass ratios of the astragalus, the rhizoma anemarrhenae, the platycodon, the bupleurum and the cimicifuga are (5.5-6.5):(0.5-1.5):(1-2):(2.5-3.5):(1-2).

[0145] Furthermore, the mass ratio of the Astragalus, the Anemarrhena, the Platycodon and the Cimicifuga is about 6: about 1: about 1.5: about 3: about 1.5.

[0146] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3; "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 1.5" includes ±5% of 1.5, or from 1.425 to 1.575; "about 3" includes ±5% of 3, or from 2.85 to 3.15.

[0147] Furthermore, the mass range of the astragalus is 10 to 30 g, for example, about 22.38 g.

[0148] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 22.38" includes ±5% of 22.38, or from 21.261 to 23.499.

[0149] Furthermore, the mass range of the Anemarrhena Rhizoma is 1 to 10 g, for example, about 3.73 g.

[0150] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3.73" includes ±5% of 3.73, or from 3.5435 to 3.9165.

[0151] Furthermore, the mass range of the Platycodon grandiflorum is 1 to 10 g, for example, about 5.60 g.

[0152] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5.60" includes ±5% of 5.60, or from 5.32 to 5.88.

[0153] Furthermore, the mass range of the Bupleurum is 10 to 20 g, for example, about 11.19 g.

[0154] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 11.19" includes ±5% of 11.19, or from 10.6305 to 11.7495.

[0155] Furthermore, the mass of the Cimicifuga heracleifolia is in the range of 1 to 10 g, for example, about 5.60 g.

[0156] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5.60" includes ±5% of 5.60, or from 5.32 to 5.88.

[0157] Furthermore, in the preparation of the test solution of the traditional Chinese medicine composition, the alkaline test solution is an ammonia test solution.

[0158] Furthermore, the first solvent is alcohol, such as methanol.

[0159] Furthermore, the volume percentage concentration of the ammonia test solution is 1% to 10%, preferably 2% to 6%, for example about 4%.

[0160] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 4%" includes ±5% of 4%, or from 3.8% to 4.2%.

[0161] Furthermore, the volume percentage concentration of the methanol is 10% to 100%, preferably 70% to 90%, for example about 80%.

[0162] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 80%" includes ±5% of 80%, or from 76% to 84%.

[0163] Furthermore, the mass / volume (mg / ml) ratio of the powder of the Chinese medicinal composition containing Astragalus to the first solvent containing the alkaline test solution is 10-50, for example, about 20.

[0164] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0165] Furthermore, the heating reflux time is 30 to 120 minutes, for example, about 60 minutes.

[0166] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0167] Furthermore, the volume ratio of the first subsequent filtrate to the first solvent containing the alkaline test solution is 0.4 to 0.6, such as about 0.5.

[0168] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0169] Furthermore, the volume ratio of the second subsequent filtrate to the first subsequent filtrate is 0.1 to 0.4, for example, about 0.2.

[0170] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.2" includes ±5% of 0.2, or from 0.19 to 0.21.

[0171] Further, the concentration of Anemarrhena saponin BⅡ in the reference solution is about 100 μg / ml. Further, the concentration of Platycodon saponin D in the reference solution is about 100 μg / ml. Further, the concentration of Astragaloside IV in the reference solution is about 100 μg / ml. Further, the concentration of Bupleurum saponin A in the reference solution is about 100 μg / ml.

[0172] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105.

[0173] Furthermore, the 120 min in the gradient elution program was replaced by 130 min.

[0174] Furthermore, the detector for the detection is an electrospray detector.

[0175] Furthermore, the flow rate of the HPLC detection is 0.5-1.0 ml / min, for example, about 0.8 ml / min.

[0176] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.8" includes ±5% of 0.8, or from 0.76 to 0.84.

[0177] Furthermore, the column temperature is 20-50°C, for example, about 35°C.

[0178] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 35" includes ±5% of 35, or from 33.25 to 36.75.

[0179] Furthermore, the injection volume is 1-20 μl, for example, about 10 μl.

[0180] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0181] Furthermore, the theoretical plate number of the chromatographic peak corresponding to astragaloside IV is not less than 5000.

[0182] Furthermore, the chromatographic column is a YMC-Pack ODS-A chromatographic column.

[0183] Furthermore, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0184] Furthermore, the fingerprint spectrum includes peaks 1-16, among which peak 12 is astragaloside I as a reference peak, and the average retention times of peaks 1-13 correspond to about 12.9 min, about 20.1 min, about 26.1 min, about 39.5 min, about 40.6 min, about 61.1 min, about 62.1 min, about 63.1 min, about 64.8 min, about 66.5 min, about 76.4 min, about 90.1 min, about 95.5 min, about 101.1 min, about 101.5 min and about 110.9 min, respectively.

[0185] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 12.9" includes ±5% of 12.9, or from 12.255 to 13.545; "about 20.1" includes ±5% of 20.1, or from 19.095 to 21.105; "about 26.1" includes ±5% of 26.1, or from 24.795 to 27.405; "about 39.5" includes ±5% of 39.5, or from 37.525 to 41.475 ; "about 40.6" includes ±5% of 40.6, or from 38.57 to 42.63; "about 61.1" includes ±5% of 61.1, or from 58.045 to 64.155; "about 62.1" includes ±5% of 62.1, or from 58.995 to 65.205; "about 63.1" includes ±5% of 63.1, or from 59.945 to 66.255; "about 6 4.8" includes ±5% of 64.8, or from 61.56 to 68.04; "about 66.5" includes ±5% of 66.5, or from 63.175 to 69.825; "about 76.4" includes ±5% of 76.4, or from 72.58 to 80.22; "about 90.1" includes ±5% of 90.1, or from 85.595 to 94.605; "about 95.5" includes ±5% of 95.5, or from 90.725 to 100.275; "about 101.1" includes ±5% of 101.1, or from 96.045 to 106.155; "about 101.5" includes ±5% of 101.5, or from 96.425 to 106.575; "about 110.9" includes ±5% of 110.9, or from 105.355 to 116.445.

[0186] Furthermore, the fingerprint spectrum includes peaks 1-16, wherein peak 12 is astragaloside I as a reference peak, and the relative retention time average values ​​of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, peak 13, peak 14, peak 15 and peak 16 are approximately 0.14, approximately 0.22, approximately 0.29, approximately 0.44, approximately 0.45, approximately 0.68, approximately 0.69, approximately 0.70, approximately 0.72, approximately 0.74, approximately 0.85, approximately 1.06, approximately 1.12, approximately 1.13 and approximately 1.23, respectively.

[0187] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 0.14" includes ±5% of 0.14, or from 0.133 to 0.147; "about 0.22" includes ±5% of 0.22, or from 0.209 to 0.231; "about 0.29" includes ±5% of 0.29, or from 0.2755 to 0.3045; "about 0.44" includes ±5% of 0.44, or from 0.418 to 0.462; "about 0.45" includes ±5% of 0.45, or from 0.4275 to 0.4725; "about 0.68" includes ±5% of 0.68, or from 0.646 to 0.714; "about 0.69" includes ±5% of 0.69, or from 0.6555 to 0.7245; "about 0.70" includes ±5% of 0.70 ±5%, or from 0.665 to 0.735; "about 0.72" includes ±5% of 0.72, or from 0.684 to 0.756; "about 0.74" includes ±5% of 0.74, or from 0.703 to 0.777; "about 0.85" includes ±5% of 0.85, or from 0.8075 to 0.8925; "about 1.06" includes ±5% of 1.06, or from 1.007 to 1.113; "about 1.12" includes ±5% of 1.12, or from 1.064 to 1.176; "about 1.13" includes ±5% of 1.13, or from 1.0735 to 1.1865; "about 1.23" includes ±5% of 1.23, or from 1.1685 to 1.2915.

[0188] Furthermore, the mobile phase A is acetonitrile.

[0189] Furthermore, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.

[0190] Further, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid in different concentrations.

[0191] Furthermore, the acid aqueous solution is a 0.01% to 1% acid aqueous solution.

[0192] Furthermore, the acid aqueous solution is a 0.01% to 1% acetic acid aqueous solution.

[0193] Furthermore, the acid aqueous solution is about 0.2% acetic acid aqueous solution.

[0194] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.2%" includes ±5% of 0.2%, or from 0.19% to 0.21%.

[0195] Furthermore, the buffered saline solution is an acetate solution and / or an acetate solution.

[0196] Furthermore, the pH value of the buffered saline solution is not greater than 7.0.

[0197] Furthermore, when the flow rate was 0.8 ml / min and the column temperature was 35°C, peak No. 4 was anemarrhena saponin BⅡ, peak No. 7 was platycodon saponin D, peak No. 12 was astragaloside IV, and peak No. 14 was bupleurosaponin a.

[0198] Furthermore, when the flow rate is 0.8 ml / min and the column temperature is 35°C, peak 2 and peak 12 are from Astragalus membranaceus medicinal material, peak 1, peak 3, peak 4, peak 5 and peak 11 are from Anemarrhena asphodeloides medicinal material, peak 6, peak 7, peak 8, peak 9 and peak 10 are from Platycodon grandiflorum medicinal material, peak 14 and peak 15 are from Bupleurum chinense medicinal material, and peak 13 and peak 16 are from Anemarrhena asphodeloides medicinal material and Platycodon grandiflorum medicinal material.

[0199] According to another aspect of the present invention, a quality control method for a traditional Chinese medicine composition containing Astragalus is provided, the quality control method comprising the following steps: (1) establishing a standard fingerprint of a reference sample of the traditional Chinese medicine composition according to the fingerprint construction method; (2) taking a test sample solution of the traditional Chinese medicine composition and testing it according to the chromatographic conditions in the fingerprint construction method to obtain a fingerprint of a test sample of the traditional Chinese medicine composition; and (3) comparing the fingerprint of the test sample of the traditional Chinese medicine composition obtained in step (2) with the standard fingerprint of the reference sample of the traditional Chinese medicine composition obtained in step (1); a product that meets the requirements is a qualified product, and a product that does not meet the requirements is an unqualified product.

[0200] Furthermore, the compliance requirements include one or more of the following: (1) the fingerprint of the Chinese medicine composition sample to be tested presents 16 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard fingerprint of the Chinese medicine composition benchmark sample; (2) taking the astragaloside peak as the S peak, the relative retention time of each characteristic chromatographic peak in the fingerprint of the Chinese medicine composition sample to be tested and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard fingerprint of the Chinese medicine composition benchmark sample; and (3) according to the Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity between the fingerprint of the Chinese medicine composition sample to be tested and the standard fingerprint of the Chinese medicine composition benchmark sample shall not be less than 0.90 after similarity calculation.

[0201] According to another aspect of the present invention, there is provided a use of the above-mentioned determination method, the above-mentioned construction method or the above-mentioned quality control method in quality detection and / or quality evaluation and / or quality control of a traditional Chinese medicine composition containing Astragalus.

[0202] Beneficial effects of the present invention:

[0203] In short, the present invention provides a fingerprint spectrum determination and quality control method for a traditional Chinese medicine compound reference sample containing astragalus. The method confirms 13 common characteristic peaks, has simple conditions and short analysis time, solves the problems of difficulty in separating fingerprint characteristic peaks and interference from impurity peaks, ensures the chemical composition stability and safety of use of the reference sample, provides an important reference basis for the quality control of subsequent preparations, ensures the stability of product quality, guarantees the efficacy of the traditional Chinese medicine compound, and allows the traditional Chinese medicine compound to better serve human life and health.

[0204] Specifically, compared with the prior art, the present invention has the following beneficial effects:

[0205] (1) Establishing the fingerprint of the reference sample of the Chinese medicine compound of the present invention overcomes the defect that the determination of the content of a single component is difficult to reflect the overall content, and can control the intrinsic quality of the reference sample of the Chinese medicine compound of the present invention from a holistic and macroscopic perspective, thereby ensuring the efficacy of the drug, utilizing the main means of modern drug research, realizing the inheritance of classics, and enabling classic prescriptions to be subject to more formal quality control.

[0206] (2) The chemical components in the reference sample of the traditional Chinese medicine compound of the present invention are complex, and it is difficult to separate its characteristic peaks. In the process of establishing the fingerprint spectrum, the present invention adopts a gradient elution method to solve the problem of difficulty in separating the fingerprint characteristic peaks and interference of impurity peaks.

[0207] (3) In the process of establishing the fingerprint spectrum of the traditional Chinese medicine compound reference sample of the present invention, 16 common characteristic peaks were confirmed, and their relative retention time, relative peak area and similarity were studied, thereby ensuring the chemical composition stability and safety of the reference sample, and providing an important reference basis and quality reference for the quality control of subsequent compound preparations.

[0208] (4) The fingerprint spectra of each effective ingredient in the traditional Chinese medicine compound reference sample of the present invention are regarded as a whole, and the order and relationship of each characteristic peak are emphasized. This not only avoids the one-sidedness of judging the overall quality of the traditional Chinese medicine compound reference sample of the present invention due to only measuring one or two chemical components, but also reduces the possibility of artificial processing to meet the quality standards, thereby providing a new method and means for completely and accurately evaluating the quality of the traditional Chinese medicine compound reference sample of the present invention.

[0209] (5) The method of the present invention has good stability, high precision, good reproducibility, is convenient and easy to master. BRIEF DESCRIPTION OF THE DRAWINGS

[0210] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0211] Figure 1 This is the chromatogram of the test solution of the traditional Chinese medicine compound benchmark sample containing Astragalus.

[0212] Figure 2 The figure is the chromatogram of mixed reference solution. DETAILED DESCRIPTION

[0213] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0214] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0215] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0216] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0217] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0218] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0219] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0220] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0221] Example

[0222] Instruments and reagents

[0223] Instruments and equipment: Chromatograph: High performance liquid chromatograph: Vanquish VC-Corona Veo; Chromatographic system includes charged aerosol detector F, split sampler C, quaternary pump CN, column compartment C, Chormeleon 7 Chromatography management system; Electronic balance: Sartorius Scientific Instrument (Beijing) Co., Ltd. (BSA224S), Sartorius Scientific Instrument (Beijing) Co., Ltd. (BSA124S), Sartorius Scientific Instrument (Beijing) Co., Ltd. (BSA224S-CW), Shimadzu Enterprise Management (China) Co., Ltd. (AUW120D), Mettler Toledo (MS105DU), Changzhou Lucky Electronic Equipment Co., Ltd. (XY1000-2C); Ultrasonic instrument: KQ-500DB CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; Nitrogen generator: Beijing Antai Ruike Technology Co., Ltd. (ATEN-H10); Digital display constant temperature water bath: Beijing Yongguangming Medical Equipment Co., Ltd. (DZKW-S-6); Shanghai Lichen Bangxi Instrument Technology Co., Ltd. (HH-6 / HH-4); Chromatographic column: YMC-Pack ODS-A, 250*4.6mm, 5μm, SN:111EB20233; YMC-Pack ODS-A, 250*4.6mm, 5μm, SN:109IB20208; Reagents: acetonitrile, methanol, and glacial acetic acid were of chromatographic grade, water was distilled water (Watsons drinking water), and ammonia (ammonium hydroxide) was of analytical grade.

[0224] Test drugs: Anemarrhena saponin BⅡ (batch number: 111839-202107, mass fraction is 95.4%, China Food and Drug Inspection Institute); Platycodon saponin D (batch number: 111851-202209, mass fraction is 97.3%, China Food and Drug Inspection Institute); Astragaloside I (batch number: 110781-202219, mass fraction is 96.2%, China Food and Drug Inspection Institute); Bupleurum saponin a (batch number: 110777-201912, mass fraction is 94.8%, China Food and Drug Inspection Institute); Chinese medicine compound reference sample containing Astragalus (batch number: SXKL220926-G), Chinese medicine compound reference sample containing Astragalus (batch number: SXKL20230605-G), negative sample lacking Astragalus (batch number: S XKL2023051707-G), lack of Anemarrhena negative (batch number: SXKL2023051706-G), lack of Platycodon negative (batch number: SXKL2023060503-L), lack of Bupleurum negative (batch number: SXKL2023051708-G), lack of Cimicifuga negative (batch number: SXKL2023051710-G), single Astragalus negative ( Batch number: SXKL2023050801-G), single Anemarrhena asphodeloides is negative (Batch number: SXKL2023050802-G), single Platycodon grandiflorum is negative (Batch number: SXKL2023050804-G), single Bupleurum is negative (Batch number: SXKL2023060501-L), single Cimicifuga foetida is negative (Batch number: SXKL2023060502-L).

[0225] Description of the preparation method of Chinese medicine composition powder containing astragalus

[0226] [Prescription dosage] Astragalus 22.38g, Anemarrhena 3.73g, Platycodon 5.60g, Bupleurum 11.19g, Cimicifuga 5.60g.

[0227] [Preparation method] Take a daily dose of Astragalus, Bupleurum, Cimicifuga, and Platycodon, put them in a 1.8L multi-functional ceramic electric cooker, add 500ml of water, soak for 60 minutes, cover, heat with high heat (220V), boil, then simmer for 55 minutes with low heat (175V), add Anemarrhena asphodeloides, soak for 10 minutes, heat with high heat (220V), boil, then simmer for 5 minutes with low heat (175V), filter with a single layer of 100-mesh nylon filter cloth, and set aside the filtrate; add 400ml of water to the filter residue, heat with high heat (220V), boil, then simmer for 40 minutes with low heat (175V), filter with a single layer of 100-mesh nylon filter cloth, combine the filtrates, and the relative density is 1.010-1.025 (20℃). The filtrate is concentrated under reduced pressure using a rotary evaporator, the concentration temperature is not higher than 65°C, the vacuum degree is -0.08 to -0.099Mpa, the rotation speed is 100 rpm, and it is concentrated to a relative density of 1.10 to 1.20 (20°C). The concentrated solution is placed in a stainless steel plate, the thickness of the plate is 9 to 11mm, pre-frozen until there is no moisture inside, and dried in a freeze dryer, the drying cold trap temperature is ≤-50°C, the vacuum degree is ≤100Pa, and freeze-dried until the moisture content does not exceed 8.0%, taken out, crushed, and mixed to obtain the test sample powder.

[0228] Example 1 Fingerprint Study

[0229] 1. Establishment of chromatographic conditions

[0230] 1.1. Investigation of chromatographic elution gradient

[0231] The chromatographic elution conditions of the fingerprint spectrum of the traditional Chinese medicine compound benchmark sample containing Astragalus (batch number: SXKL220926-G) were explored.

[0232] Table 1 Elution gradient 1

[0233]

[0234] Table 2 Elution gradient 2

[0235]

[0236]

[0237] Table 3 Elution gradient 3

[0238]

[0239] Table 4 Elution gradient 4

[0240]

[0241] The results showed that compared with other elution gradients, the chromatographic peaks of Anemarrhenasaponin BⅡ, Platycodonosaponin D, Astragaloside I, Bupleurosaponin a, etc. under the elution gradient 4 were better separated and the peak shapes were better, while under the other elution gradients 1, 2, and 3, there was an overlapping phenomenon of chromatographic peaks and the peak shape was poor.

[0242] 1.2. Selection of different acid types

[0243] The effects of different types of acid (0.2% formic acid, 0.2% acetic acid) on the system suitability parameters (peak shape, separation, symmetry factor, total peak area, etc.) of each chromatographic peak of the fingerprint spectrum of the traditional Chinese medicine compound reference sample containing Astragalus were investigated, and appropriate chromatographic parameters were selected based on the results.

[0244] The results show that: under different acid conditions, when formic acid is used, t R =34.203 chromatographic peaks co-eluted with impurity peaks. When acetic acid was used, the peaks in the chromatogram of the test sample had better peak shapes and better separation, so acetic acid was determined to be used as the acid type for the characteristic spectrum method of the traditional Chinese medicine compound containing astragalus.

[0245] 1.3. Selection of different acid concentrations

[0246] The effects of different acetic acid concentrations (0.1%, 0.2%, 0.3%) on the system suitability parameters (peak shape, separation, symmetry factor, total peak area, etc.) of each chromatographic peak of the fingerprint spectrum of the traditional Chinese medicine compound reference sample containing Astragalus were investigated, and appropriate chromatographic parameters were selected based on the results.

[0247] The results showed that when the acetic acid concentration was 0.1%, the peak tR=85.037 co-eluted with the impurity peaks; when the acetic acid concentration was 0.2% and 0.3%, the peak shapes of the chromatographic peaks in the test sample chromatogram were better and the separation was better. Considering that the higher the acid concentration, the more severe the damage to the chromatographic column, 0.2% was determined as the acetic acid concentration for the characteristic spectrum method of the traditional Chinese medicine compound benchmark sample containing astragalus.

[0248] 1.4. Selection of different flow rates

[0249] Elution was performed according to the above-mentioned mobile phase gradient, and the effects of different flow rates (0.8 ml / min, 1.0 ml / min, 1.2 ml / min) on the system suitability parameters of each chromatographic peak of the fingerprint spectrum of the traditional Chinese medicine compound reference sample containing Astragalus (peak shape, separation, symmetry factor, total peak area, etc.) were investigated, and appropriate chromatographic parameters were selected based on the results.

[0250] The results showed that when the flow rate was 0.8-1.2 ml / min, the timosaponin BII (tR=37.300) and the miscellaneous peaks co-eluted at a flow rate of 0.8 ml / min; platycodon saponin D (tR=50.482) and the miscellaneous peaks co-eluted at a flow rate of 1.2 ml / min; and when the flow rate was 1.0 ml / min, the peaks of each chromatographic peak had better peak shapes and better separation. Therefore, 1.0 ml / min was selected as the characteristic chromatographic method flow rate for the benchmark sample of the traditional Chinese medicine compound containing astragalus.

[0251] 1.5. Selection of different column temperatures

[0252] Elution was performed according to the above-mentioned mobile phase gradient, and the effects of different column temperatures (25°C, 30°C, 35°C) on the system suitability parameters of each chromatographic peak of the fingerprint spectrum of the traditional Chinese medicine compound reference sample containing Astragalus (peak shape, separation, symmetry factor, total peak area, etc.) were investigated, and appropriate chromatographic parameters were selected based on the results.

[0253] The results showed that when the column temperature was set at 25°C, the peaks tR=23.837min and tR=33.888min co-eluted with the impurity peaks; when the column temperature was set at 35°C, the peak tR=34.423min co-eluted with the impurity peaks; when the column temperature was set at 30°C, the peak shapes of the chromatographic peaks in the sample chromatogram were better and the separation was better; therefore, 30°C was determined as the column temperature for the characteristic spectrum method of the benchmark sample of the traditional Chinese medicine compound containing Astragalus.

[0254] 1.6. Optimization of chromatographic conditions

[0255] The fingerprint method of the traditional Chinese medicine compound benchmark sample containing Astragalus was re-optimized. Combined with the previous single factor investigation results, the column temperature was changed to 35°C and the flow rate was changed to 0.8ml / min to achieve better separation of the chromatographic peaks of Platycodon saponin D and Astragaloside I. The influence of the system suitability parameters of each chromatographic peak before and after optimization (peak shape, resolution, symmetry factor, total peak area, etc.) was compared, and appropriate chromatographic parameters were selected according to the results.

[0256] The results show that: comparing the characteristic spectrum chromatograms of the traditional Chinese medicine compound containing Astragalus before and after optimization, the peak shapes of the chromatographic peaks are obviously better and the separation is better after optimization. Therefore, this chromatographic condition is determined as the characteristic spectrum method of the benchmark sample of the traditional Chinese medicine compound containing Astragalus.

[0257] 1.7. Determination of chromatographic conditions

[0258] The chromatographic conditions and system suitability test used octadecyl bonded silica gel as the filler (column length was 250 mm, inner diameter was 4.6 mm, particle size was 5 μm); acetonitrile was used as mobile phase A, 0.2% acetic acid was used as mobile phase B, and the gradient elution was performed according to Table 4; the flow rate was 0.8 ml per minute; the column temperature was 35° C.; and detection was performed using an electrospray detector.

[0259] 2. Investigation of test sample processing methods

[0260] 2.1. Investigation of extraction methods

[0261] The two extraction methods of heating reflux and ultrasonic extraction were compared, and the appropriate extraction method was determined based on the total peak area, symmetry factor and separation degree of each characteristic peak in the characteristic spectrum.

[0262] Take about 1.0g of the powder of this product, accurately weigh it, put it in a stoppered conical bottle, accurately add 50ml of 80% methanol containing 4% concentrated ammonia, seal it, weigh it, reflux extraction and ultrasonic treatment (300W, 40kHz) for 60min respectively, let it cool, weigh it again, make up the lost weight with 80% methanol containing 4% concentrated ammonia, shake it well, filter it, accurately measure 25ml of the filtrate, evaporate it to dryness, add 80% methanol to dissolve the residue, transfer it to a 5ml volumetric bottle, add 80% methanol to dilute to the scale, shake it well, filter it, and take the filtrate. According to the above chromatographic conditions, the effects of different extraction methods on the system suitability parameters of each characteristic peak of the characteristic spectrum of the traditional Chinese medicine compound benchmark sample containing astragalus (peak shape, separation, symmetry factor, etc.) are investigated, and appropriate chromatographic parameters are selected according to the results.

[0263] The results showed that there was no significant difference in the theoretical total peak area of ​​the characteristic spectrum of the traditional Chinese medicine compound benchmark sample containing Astragalus using the two extraction methods of ultrasonic extraction and reflux extraction. Considering that the content and characteristics of this method are the same method, reflux extraction was selected as the extraction method for the preparation of the characteristic spectrum of the traditional Chinese medicine compound benchmark sample containing Astragalus.

[0264] 2.2. Investigation of extraction solvent

[0265] The types of extraction solvents were investigated for the preparation of characteristic spectrum test samples of traditional Chinese medicine compound benchmark samples containing Astragalus. Methanol containing 4% concentrated ammonia, anhydrous ethanol containing 4% concentrated ammonia, 80% methanol containing 4% concentrated ammonia, 80% ethanol containing 4% concentrated ammonia, 50% methanol containing 4% concentrated ammonia, and 50% ethanol containing 4% concentrated ammonia were investigated as extraction solvents. The appropriate extraction solvent was determined based on the total peak area, symmetry factor, and separation of each characteristic peak in the characteristic spectrum.

[0266] Take about 1.0g of the powder (lyophilized powder) of this product, accurately weigh it, put it in a stoppered conical flask, accurately add 4% methanol containing concentrated ammonia, 4% anhydrous ethanol containing concentrated ammonia, 80% methanol containing 4% concentrated ammonia, 80% ethanol containing 4% concentrated ammonia, 50% methanol containing 4% concentrated ammonia, 50% ethanol containing 4% concentrated ammonia, 50% ethanol containing 4% concentrated ammonia, extract for 60 minutes according to the above conditions, take it out, cool it, weigh it, make up the lost weight with the above solvent, shake it well, filter it, accurately measure 25ml of the filtrate, evaporate it to dryness, add the corresponding solvent (excluding 4% concentrated ammonia) to dissolve the residue, transfer it to a 5ml volumetric flask, add the corresponding solvent (excluding 4% concentrated ammonia) to dilute to the scale, shake it well, filter it, and take the filtrate. Investigate the influence of different types of extraction solvents on the system suitability parameters of each characteristic peak of the characteristic spectrum of the traditional Chinese medicine compound benchmark sample containing astragalus (peak shape, resolution, symmetry factor, etc.), and select appropriate chromatographic parameters according to the results.

[0267] The results showed that different solvents were used to extract the characteristic spectrum of the traditional Chinese medicine compound benchmark sample containing Astragalus. Among them, 80% methanol containing 4% concentrated ammonia was used for extraction. The theoretical total peak area of ​​the characteristic spectrum was the highest after conversion. Therefore, 80% methanol containing 4% concentrated ammonia was selected as the extraction solvent for preparing the characteristic spectrum of the traditional Chinese medicine compound benchmark sample containing Astragalus.

[0268] 2.3. Investigation of extraction time

[0269] The extraction time of the characteristic spectrum of the traditional Chinese medicine compound containing Astragalus was investigated.

[0270] Take about 1.0g of the powder (lyophilized powder) of this product, weigh it accurately, put it in stoppered conical flasks, accurately add 50ml of the determined solvent, weigh the weight, extract it according to the determined method for 30, 60, 90, and 120 minutes, take it out, cool it, weigh it, make up the lost weight with the above solvent, shake it well, filter it, accurately measure 25ml of the filtrate, evaporate it to dryness, add the corresponding solvent (excluding 4% concentrated ammonia) to dissolve the residue, transfer it to a 5ml volumetric flask, add the corresponding solvent (excluding 4% concentrated ammonia) to dilute to the scale, shake it well, filter it, and take the filtrate to obtain it.

[0271] According to the above chromatographic conditions, the effects of different extraction times on the system suitability parameters of each characteristic peak of the characteristic spectrum of the traditional Chinese medicine compound reference sample containing Astragalus (peak shape, separation, symmetry factor, etc.) were investigated, and appropriate chromatographic parameters were selected based on the results.

[0272] Through the above data analysis, when the extraction time is 30 to 120 minutes, there is no significant difference in the extraction of characteristic spectra of the benchmark samples of the traditional Chinese medicine compound containing Astragalus. Considering the time cost and energy efficiency, 60 minutes is selected as the extraction time of the characteristic spectra of the benchmark samples of the traditional Chinese medicine compound containing Astragalus.

[0273] 2.4. Sampling volume investigation

[0274] The purpose of this study was to investigate whether the characteristic peaks in the Chinese herbal compound benchmark samples containing Astragalus were completely extracted under different sampling volumes.

[0275] Take about 0.5, 1.0, and 2.0 g of the powder (lyophilized powder) of this product, accurately weigh them, and place them in stoppered conical flasks and volumetric flasks, respectively. Accurately add 50 ml of the determined solvent, weigh the weight, and extract according to the determined method for 60 minutes. Take it out, cool, weigh it, make up the lost weight with the above solvent, shake it well, filter it, accurately measure 25 ml of the filtrate, evaporate it to dryness, add the corresponding solvent (excluding 4% concentrated ammonia) to dissolve the residue, transfer it to a 5 ml volumetric flask, add the corresponding solvent (excluding 4% concentrated ammonia) to dilute to the scale, shake it well, filter it, and take the filtrate to obtain it.

[0276] According to the above chromatographic conditions, the influence of different sampling amounts on the system suitability parameters of each characteristic peak of the characteristic spectrum of the traditional Chinese medicine compound reference sample containing Astragalus (peak shape, separation, symmetry factor, etc.) was investigated, and appropriate chromatographic parameters were selected according to the results.

[0277] The results show that when the sampling amount is 1.0g and 2.0g, there is no significant difference in the theoretical total area of ​​the characteristic spectrum of the Chinese herbal compound benchmark sample containing Astragalus, while when the sampling amount is 0.5g, the theoretical total area of ​​the characteristic spectrum of the Chinese herbal compound benchmark sample containing Astragalus is higher, mainly due to the nonlinearity of the peak area-sampling amount of the electrospray detector at low concentrations. Considering that the sampling amount is 1.0g, the overall response of the characteristic spectrum is consistent with that of the reference solution. Therefore, 1.0g is selected as the sampling amount for the preparation of the test sample of the characteristic spectrum of the Chinese herbal compound benchmark sample containing Astragalus.

[0278] 3. Determine system suitability conditions and solution preparation methods

[0279] 3.1 Chromatographic conditions and system suitability conditions

[0280] The chromatographic conditions and system suitability test used octadecyl bonded silica gel as the filler (column length was 250 mm, inner diameter was 4.6 mm, particle size was 5 μm); acetonitrile was used as mobile phase A, 0.2% acetic acid was used as mobile phase B, and the gradient elution was performed according to Table 4; the flow rate was 0.8 ml per minute; the column temperature was 35° C.; and detection was performed using an electrospray detector.

[0281] 3.2 Solution Preparation Method

[0282] Preparation of reference solution: Take appropriate amount of timosaponin BⅡ, platycodon saponin D, astragaloside I and bupleurosaponin a, weigh accurately, add 80% methanol to make a mixed solution containing 0.1 mg of timosaponin BⅡ, platycodon saponin D, astragaloside I and bupleurosaponin a per 1 ml, shake well and use as reference solution.

[0283] Preparation of test solution: Take 1.0 g of the reference sample (lyophilized powder), accurately weigh it, place it in a round-bottom flask, concentrate it to dryness under reduced pressure, accurately add 50 ml of 80% methanol solution containing 4% concentrated ammonia test solution (take 4 ml of concentrated ammonia test solution, add 80% methanol to 100 ml, shake well), stopper it, weigh the weight, heat it under reflux for 1 hour, cool it, weigh it again, make up the lost weight with 80% methanol solution containing 4% concentrated ammonia test solution, shake well, filter, accurately transfer 25 ml of the filtrate, evaporate to dryness, dissolve the residue with 80% methanol, transfer it to a 5 ml volumetric flask, add 80% methanol to the scale, shake well, filter, and take the filtrate.

[0284] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, measure, and record the chromatogram.

[0285] 4. Characteristic peak identification and selection

[0286] Through the above series of experimental optimization, under the chromatographic conditions of the optimal characteristic spectrum, the test sample presented a total of 16 obvious peaks. After positioning and identifying each peak using the reference substance, peak 4 was identified as timasaponin BⅡ, peak 7 as platycodon saponin D, peak 12 as astragaloside I, and peak 14 as bupleurosaponin a. Each chromatographic peak had a good peak shape, good symmetry, and moderate peak elution time, so peak 12 (astragaloside I) was taken as the S peak.

[0287] 5. Methodological Validation

[0288] The quality standard research samples were prepared according to the process determined by process validation, and the acceptable standard range was determined by referring to the 9101 Analytical Method Validation Guidelines of Part IV of the 2020 Edition of the Chinese Pharmacopoeia. The validation items and contents are as follows:

[0289] 5.1. Exclusivity Examination

[0290] 5.1.1. Exclusivity

[0291] According to the test sample preparation method, prepare blank solution, reference sample solution, negative test solution lacking Astragalus, negative test solution alone Astragalus, negative test solution lacking Platycodon, negative test solution alone Platycodon, negative test solution lacking Bupleurum, negative test solution alone Bupleurum, negative test solution lacking Cimicifuga, negative test solution alone Cimicifuga, and mixed reference solution, use "3. Determine system suitability conditions and solution preparation method" for sample injection and analysis, and record the chromatogram. Among them, the chromatogram of the reference sample solution is as follows: Figure 1 As shown, the chromatogram of the mixed reference solution is as follows Figure 2 shown.

[0292] The results showed that peak 4 was anemarrhena saponin BⅡ, peak 7 was platycodon saponin D, peak 12 was astragaloside IV, and peak 14 was bupleurosaponin a.

[0293] In the negative test sample lacking Astragalus, the peak with the corresponding retention time to the astragaloside IV reference substance was not detected, and there was no interference with the negative control; in the negative test sample lacking Anemarrhena, the peak with the corresponding retention time to the Anemarrhena saponin BⅡ reference substance was not detected, and there was no interference with the negative control; in the negative test sample lacking Platycodon grandiflorum, the peak with the corresponding retention time to Platycodon saponin D was not detected, and there was no interference with the negative control; in the negative test sample lacking Bupleurum, the peak with the corresponding retention time to the Bupleurosaponin a reference substance was not detected, and there was no interference with the negative control.

[0294] Peak 2 and Peak 12 are exclusive components of Astragalus, Peak 1, Peak 3, Peak 4, Peak 5 and Peak 11 are exclusive components of Anemarrhena, Peak 6, Peak 7, Peak 8, Peak 9 and Peak 10 are exclusive components of Platycodon, Peak 14 and Peak 15 are exclusive components of Bupleurum, Peak 13 and Peak 16 are common peaks of Anemarrhena and Platycodon.

[0295] There was no significant difference between the characteristic spectrum of the negative test sample lacking Cimicifuga and the characteristic spectrum of the benchmark sample solution. In the characteristic spectrum of the single Cimicifuga test sample, although chromatographic peaks were detected, the response values ​​were all low. This may be due to the low concentration, resulting in no chromatographic peak from this medicinal flavor being found in the characteristic spectrum detection of this benchmark sample.

[0296] 5.1.2. Holism

[0297] Take the test sample solution and perform HPLC analysis according to the determined chromatographic conditions by increasing the elution time by twice, and record the chromatogram. Within the running time of the chromatographic conditions, the peaks are completely eluted, and the chromatographic peaks of Anemarrhenasaponin BⅡ, Platycodonosaponin D, Astragaloside I, and Bupleurosaponin a have good peak shapes and better separation, so the overall performance of this method is good.

[0298] 5.2 Precision test

[0299] 5.2.1. Instrument precision test

[0300] Take the same sample solution and inject it 6 times continuously according to the above conditions to determine the relative retention time and relative peak area of ​​16 peaks.

[0301] The results showed that when the same test solution was injected six times in a row, the RSD values ​​of the relative retention times of the characteristic peaks in the characteristic spectra were all less than 2.0%, indicating that the method had good instrument precision.

[0302] 5.2.2 Intermediate precision study

[0303] The same batch of reference samples were operated independently by different experimenters, processed according to the test sample preparation method, and measured with different instruments. The relative retention time and relative peak area of ​​16 peaks were measured.

[0304] The results showed that six test solutions were prepared independently by different personnel, and the RSD values ​​of the relative retention times of the 16 characteristic peaks measured were all less than 1.0%, indicating that the method had good precision among different personnel.

[0305] 5.2.3 Repeatability Study

[0306] Take 6 portions of 1.0g of the same batch of test sample, weigh them accurately, prepare them according to the preparation method of the test sample solution, and perform the measurement under the above chromatographic conditions. Determine and calculate the relative retention time and relative peak area of ​​16 peaks.

[0307] The results showed that when 6 samples were prepared in parallel, the RSD values ​​of the relative retention times of the 16 characteristic peaks measured were all less than 1.0%, so the method had good repeatability.

[0308] 5.3 Durability test

[0309] 5.3.1 Solution stability

[0310] Take the same sample solution and inject it at 0, 4, 11, 18, 24, 30, 38, 50, and 56 hours after preparation, and determine and calculate the relative retention time and relative peak area of ​​16 peaks.

[0311] The results showed that when the same sample solution was injected within 56 hours, the RSD of the relative retention times of the 16 characteristic peaks measured were all less than 2.0%, indicating that the sample solution could remain stable within 56 hours.

[0312] 5.3.2 Durability test at different flow rates

[0313] Take 1.0 g of the test sample, weigh it accurately, and process it according to the test sample preparation method. Tests were carried out at different flow rates of 0.7 ml / min, 0.8 ml / min, and 0.9 ml / min, and the relative retention time and relative peak area of ​​16 peaks were determined and calculated.

[0314] The results show that different flow rates have a great influence on the retention time and the elution of each characteristic peak. The relative retention time RSD value is large, but still within ±10% of the specified value. When the flow rate is reduced, the elution time of each characteristic peak is prolonged, peak 13 and the impurity peak are eluted together, and the separation is poor. When the flow rate is 0.9ml / min, the symmetry of the saikosaponin peak is poor, and the elution time of peak 15 is advanced, which may be due to the interference of impurity peaks. In order to reproduce the spectrum, the recommended flow rate is 0.8ml / min.

[0315] 5.3.3. Durability test at different column temperatures

[0316] Take 1.0 g of the test sample, weigh it accurately, and process it according to the test sample preparation method. Test it at different column temperatures of 33°C, 35°C, and 37°C, and determine and calculate the relative retention time and relative peak area of ​​16 peaks.

[0317] The results showed that due to the high column temperature, this method had a strong selectivity for column temperature. When the column temperature decreased, the symmetry of the characteristic peaks of Anemarrhenasaponin BⅡ, Astragaloside I, and Bupleurosaponin a became worse. When the column temperature changed, split peaks appeared in Peak 5. It is recommended to fix the column temperature at 35℃.

[0318] 5.3.4. Durability of different chromatographic columns

[0319] Take 1.0 g of the test sample, weigh it accurately, process it according to the test sample preparation method, change to a chromatographic column with a different SN number (SN: 111EB20233) from the same manufacturer, and conduct the test. Determine and calculate the relative retention time and relative peak area of ​​16 peaks.

[0320] The results show that the RSD values ​​of the relative retention times of the 16 characteristic peaks measured for chromatographic columns with different SN numbers from the same manufacturer are all less than 2.0%, and this method has good durability for this type of chromatographic column.

[0321] 5.3.5 Durability test at different acid concentrations

[0322] Take 1.0 g of the test sample, weigh it accurately, and process it according to the test sample preparation method. Tests were carried out at different acid concentrations of 0.18% acetic acid, 0.2% acetic acid, and 0.22% acetic acid, and the relative retention times and relative peak areas of 16 peaks were determined and calculated.

[0323] The results show that under different acid concentrations, the RSD values ​​of the relative retention times of the 16 characteristic peaks measured are all less than 2.0%, and the method has good durability for different acid concentrations.

[0324] 6. Determination of multiple batches of samples

[0325] From the comprehensive analysis of the above experimental contents, it can be known that according to this method, the characteristic peaks (16 characteristic peaks) of the characteristic spectrum of saponin components of the traditional Chinese medicine compound benchmark sample containing Astragalus were determined, and a method for determining the characteristic spectrum of saponin components of the traditional Chinese medicine compound benchmark sample containing Astragalus was established. 30 batches of traditional Chinese medicine compound benchmark samples containing Astragalus were determined and analyzed. The established method is simple to prepare the test solution, and the chromatographic conditions are easy to achieve. The results of the analytical method verification show that the method is simple to operate, the results are accurate, and the reproducibility is good.

[0326] Example 2 Content Determination Study

[0327] 1. Establishment of chromatographic conditions

[0328] 1.1. Investigation of chromatographic elution gradient

[0329] Same as the first embodiment.

[0330] 1.2. Investigation of different types of acids

[0331] Elution was performed according to the above-mentioned mobile phase gradient, and the effects of different types of acids commonly found in the literature (acetic acid, formic acid) on the system suitability parameters of the peaks of Anemarrhenaposide BⅡ, Platycodonosaponin D, Astragaloside I, and Bupleurosaponin a in the content determination of the traditional Chinese medicine compound benchmark sample containing Astragalus were investigated (peak shape, separation, symmetry factor, content determination results, etc.), and appropriate chromatographic parameters were selected based on the results.

[0332] The results showed that when different types of acids were used to determine the content of the traditional Chinese medicine compound reference sample containing Astragalus, there was no significant difference in the content of platycoside D, astragaloside IV, and saikosaponin a. However, when formic acid was used, timosaponin BII, platycoside D, and astragaloside IV were significantly interfered by the miscellaneous peaks; when acetic acid was used, timosaponin BII, platycoside D, astragaloside IV, and saikosaponin a were better separated from the chromatographic peaks, and the peak shape was better. Therefore, acetic acid is recommended as the acid type for the determination of the content of the traditional Chinese medicine compound reference sample containing Astragalus.

[0333] 1.3. Investigation of different acid concentrations

[0334] Elution was performed according to the above-mentioned mobile phase conditions, and the effects of different acetic acid concentrations (0.1%, 0.2%, 0.3%) on the content determination of timosaponin BⅡ, platycodon saponin D, astragaloside I, and bupleurosaponin a in the traditional Chinese medicine compound benchmark sample containing astragalus were investigated (peak shape, separation, symmetry factor, content determination results, etc.), and appropriate chromatographic parameters were selected based on the results.

[0335] The results showed that when different acid concentrations were used to determine the content of the traditional Chinese medicine compound benchmark sample containing Astragalus, there was no significant difference in the content of Anemarrhenaposide BⅡ, Platycodon saponin D, Astragaloside Ⅳ, and Bupleurosaponin a. However, when 0.1% acetic acid was used, the separation of Astragaloside Ⅳ and Bupleurosaponin a was poor; when 0.3% acetic acid was used, the separation of Anemarrhenaposide BⅡ peaks was poor; and when 0.2% acetic acid was used, the separation of each chromatographic peak was good and the peak shape was better. Therefore, 0.2% was determined as the acid concentration for the determination method of the traditional Chinese medicine compound benchmark sample containing Astragalus.

[0336] 1.4. Investigation of different flow rates

[0337] Elution was carried out according to the above-mentioned mobile phase conditions, and the effects of different flow rates (0.8ml / min, 1.0ml / min, 1.2ml / min) on the system suitability parameters (peak shape, separation, symmetry factor, content determination results, etc.) of the peak of Anemarrhena saponin BⅡ, Platycodon saponin D, Astragaloside I, and Bupleurum saponin a in the content determination of the traditional Chinese medicine compound benchmark sample containing Astragalus were investigated, and appropriate chromatographic parameters were selected based on the results.

[0338] The results showed that when different flow rates were used to determine the content of the traditional Chinese medicine compound benchmark sample containing Astragalus, the contents of Anemarrhenasaponin BⅡ, Platycodonoside D, Astragaloside IV, and Bupleurosaponin a were quite different. When the flow rate was 0.8 ml / min, Anemarrhenasaponin BII and the impurity peaks co-eluted; when the flow rate was 1.2 ml / min, Platycodonoside D and the impurity peaks co-eluted; and when the flow rate was 1.0 ml / min, the peaks of each chromatographic peak had better peak shapes and better separation. Therefore, 1.0 ml / min was selected as the flow rate for the determination of the content of the traditional Chinese medicine compound benchmark sample containing Astragalus.

[0339] 1.5. Investigation of different column temperatures

[0340] Elution was performed according to the above-mentioned mobile phase gradient, and the effects of different column temperatures (20°C, 25°C, 30°C) on the system suitability parameters (peak shape, separation, symmetry factor, content determination results, etc.) of the peak of timosaponin BⅡ, platycodonosaponin D, astragaloside I, and bupleurosaponin a in the content determination of the traditional Chinese medicine compound benchmark sample containing astragalus were investigated, and appropriate chromatographic parameters were selected based on the results.

[0341] The results showed that when different column temperatures were used to determine the content of the traditional Chinese medicine compound benchmark sample containing Astragalus, the contents of Anemarrhenasaponin BⅡ, Platycodon saponin D, Astragaloside IV, and Bupleurosaponin a were quite different. When the column temperature was 25℃, the chromatographic peaks of Anemarrhenasaponin BII and Platycodon saponin D co-eluted with the impurity peaks; when the column temperature was 35℃, Anemarrhenasaponin BII co-eluted with the impurity peaks; therefore, 30℃ was used as the column temperature for the determination method of the traditional Chinese medicine compound benchmark sample containing Astragalus.

[0342] 1.6. Optimization of chromatographic conditions

[0343] The content determination method of the traditional Chinese medicine compound benchmark sample containing Astragalus was re-optimized. Combined with the previous single factor investigation results, the column temperature was changed to 35°C and the flow rate was changed to 0.8ml / min to achieve better separation of the chromatographic peaks of Platycodon saponin D and Astragaloside I. The influence of the system suitability parameters of each chromatographic peak before and after optimization (peak shape, resolution, symmetry factor, total peak area, etc.) was compared, and appropriate chromatographic parameters were selected according to the results.

[0344] The results showed that after using the re-optimized method, the chromatographic peaks of Anemarrhena saponin BII, Platycodon saponin D, Astragaloside IV, and Bupleurosaponin a had better peak shapes and better separation than before optimization. Therefore, the re-optimized method was selected as the content determination method for the benchmark sample of the traditional Chinese medicine compound containing Astragalus.

[0345] 1.7. Determination of chromatographic conditions

[0346] The chromatographic conditions and system suitability test used octadecyl bonded silica gel as the filler (column length was 250 mm, inner diameter was 4.6 mm, particle size was 5 μm); acetonitrile was used as mobile phase A, 0.2% acetic acid was used as mobile phase B, and the gradient elution was performed according to Table 4; the column temperature was 30°C; the flow rate was 1.0 ml per minute; and detection was performed using an electrospray detector.

[0347] 2. Investigation of test sample processing methods

[0348] 2.1. Investigation of extraction methods

[0349] The two extraction methods of heating reflux and ultrasonic extraction were compared, and the appropriate extraction method was determined according to the contents of timosaponin BⅡ, platycodonosaponin D, astragaloside IV and bupleurosaponin a.

[0350] Take about 1.0g of the powder of this product, accurately weigh it, put it in a stoppered conical bottle, accurately add 50ml of 80% methanol containing 4% concentrated ammonia, seal it, weigh it, reflux extraction and ultrasonic treatment (300W, 40KHz) for 60min respectively, cool it, weigh it again, make up the lost weight with 80% methanol containing 4% concentrated ammonia, shake it well, filter it, accurately measure 25ml of the filtrate, evaporate it to dryness, add 80% methanol to dissolve the residue, transfer it to a 5ml volumetric bottle, add 80% methanol to dilute to the scale, shake it well, filter it, and take the filtrate. According to the above chromatographic conditions, the effects of different extraction methods on the system suitability parameters (peak shape, resolution, symmetry factor, etc.) of the content determination of the traditional Chinese medicine compound benchmark sample containing astragalus were investigated, and appropriate chromatographic parameters were selected according to the results.

[0351] The results showed that the content of the traditional Chinese medicine compound benchmark sample containing Astragalus was determined by ultrasonic extraction and reflux extraction. The content of Anemarrhena saponin BII and Platycodon saponin D was higher in ultrasonic extraction than in reflux extraction; the content of Astragaloside IV was higher in reflux extraction than in ultrasonic extraction; and there was no significant difference in the content of Bupleurosaponin a between the two extraction methods. Considering that Astragalus is the main drug in this prescription, reflux extraction was selected as the extraction method for the preparation of the test sample for the content determination of the traditional Chinese medicine compound benchmark sample containing Astragalus.

[0352] 2.2. Investigation of extraction solvent

[0353] The types of extraction solvents were investigated in the preparation of test samples for content determination of the traditional Chinese medicine compound benchmark samples containing astragalus, including timosaponin BⅡ, platycodon saponin D, astragaloside Ⅳ, and bupleurosaponin a. The extraction solvents were respectively investigated using methanol containing 4% concentrated ammonia, anhydrous ethanol containing 4% concentrated ammonia, 80% methanol containing 4% concentrated ammonia, 80% ethanol containing 4% concentrated ammonia, 50% methanol containing 4% concentrated ammonia, and 50% ethanol containing 4% concentrated ammonia.

[0354] Take about 1.0g of the powder (lyophilized powder) of this product, accurately weigh it, put it in a stoppered conical flask, and accurately add 50ml of methanol containing 4% concentrated ammonia, anhydrous ethanol containing 4% concentrated ammonia, 80% methanol containing 4% concentrated ammonia, 80% ethanol containing 4% concentrated ammonia, 50% methanol containing 4% concentrated ammonia, and 50% ethanol containing 4% concentrated ammonia, respectively. Extract for 60 minutes according to the above-mentioned conditions, take out, cool, weigh, make up the lost weight with the above-mentioned solvent, shake well, filter, accurately measure 25ml of the filtrate, evaporate to dryness, add the corresponding solvent (excluding 4% concentrated ammonia) to dissolve the residue, transfer to a 5ml volumetric flask, add the corresponding solvent (excluding 4% concentrated ammonia) to dilute to the scale, shake well, filter, and take the filtrate. The effects of different types of extraction solvents on the system suitability parameters (peak shape, separation, symmetry factor, content determination results, etc.) of the peaks of timosaponin BⅡ, platycodon saponin D, astragaloside Ⅳ, and bupleurosaponin a in the traditional Chinese medicine compound benchmark sample containing astragalus were investigated, and appropriate chromatographic parameters were selected based on the results.

[0355] The results showed that when different extraction solvents were used to determine the content of the traditional Chinese medicine compound benchmark sample containing Astragalus, the contents of Anemarrhenasaponin BⅡ, Platycodonosaponin D, Astragaloside I and Bupleurosaponin A were higher than those of the other extraction solvents when 80% methanol containing 4% concentrated ammonia was used. Therefore, 80% methanol containing 4% concentrated ammonia was selected as the extraction solvent for the preparation of the test sample for the determination of the content of the traditional Chinese medicine compound benchmark sample containing Astragalus.

[0356] 2.3. Investigation of extraction time

[0357] The extraction time was investigated in the preparation of test samples for content determination of the traditional Chinese medicine compound benchmark samples containing astragalus, including timosaponin BⅡ, platycodonosaponin D, astragaloside Ⅳ and saikosaponin a.

[0358] Take about 1.0g of the powder (lyophilized powder) of this product, weigh it accurately, and place it in a stoppered conical flask and a measuring flask respectively. Accurately add 50ml of the determined solvent, weigh the weight, and extract it according to the determined extraction method for 30, 60, 90, and 120 minutes. Take it out, cool, weigh it, make up the lost weight with the above solvent, shake it well, filter it, accurately measure 25ml of the filtrate, evaporate it to dryness, add the corresponding solvent (excluding 4% concentrated ammonia) to dissolve the residue, transfer it to a 5ml measuring flask, add the corresponding solvent (excluding 4% concentrated ammonia) to dilute to the scale, shake it well, filter it, and take the filtrate to obtain it.

[0359] According to the above chromatographic conditions, the effects of different extraction times on the system suitability parameters (peak shape, separation, symmetry factor, content determination results, etc.) of the peaks of Anemarrhena saponin BⅡ, Platycodon saponin D, and Astragaloside IV in the content determination of the traditional Chinese medicine compound benchmark sample containing Astragalus were investigated, and appropriate chromatographic parameters were selected based on the results.

[0360] The results showed that the contents of timosaponin BⅡ, platycodon saponin D, astragaloside Ⅳ and bupleurosaponin a in the benchmark Chinese herbal compound containing astragalus could be extracted completely within an extraction time of 30 to 120 minutes, with no significant difference. Considering the time cost and energy consumption, 60 minutes was selected as the extraction time for the test sample for content determination of the benchmark Chinese herbal compound containing astragalus.

[0361] 2.4. Investigation of sampling quantity

[0362] The aim of this study was to investigate whether the contents of timosaponin BⅡ, platycodonosaponin D, astragaloside I and bupleurosaponin a in the benchmark samples of traditional Chinese medicine compound containing astragalus were completely extracted under different sampling amounts.

[0363] Take about 0.5, 1.0, and 2.0 g of the powder (lyophilized powder) of this product respectively, weigh them accurately, place them in stoppered conical flasks and volumetric flasks respectively, accurately add 50 ml of the determined solvent, weigh the weight, extract according to the determined method for 60 minutes, take out, cool, weigh, make up the lost weight with the above solvent, shake well, filter, accurately measure 25 ml of the filtrate, evaporate to dryness, add the corresponding solvent (excluding 4% concentrated ammonia) to dissolve the residue, transfer to a 5 ml volumetric flask, add the corresponding solvent (excluding 4% concentrated ammonia) to dilute to the scale, shake well, filter, and take the filtrate.

[0364] The effects of different sampling volumes on the system suitability parameters (peak shape, separation, symmetry factor, content determination results, etc.) of the peaks of timosaponin BⅡ, platycodon saponin D, astragaloside Ⅳ, and bupleurosaponin a in the traditional Chinese medicine compound benchmark samples containing astragalus were investigated, and appropriate chromatographic parameters were selected based on the results.

[0365] The results showed that when the sampling volume was 0.5g, the peak area of ​​the test sample was not proportional to the actual dilution factor (half the sampling volume), resulting in a higher content; while there was no significant difference in platycodon saponin D, astragaloside IV, and saikosaponin a when the sampling volume was 1.0g and 2.0g, and the extraction of timosaponin BII was incomplete when the sampling volume was 2.0g. Therefore, 1.0g was selected as the sample preparation sample for the benchmark sample content determination of the traditional Chinese medicine compound containing astragalus.

[0366] 3. Selection of content determination indicators

[0367] After an overall study of the saponin components in the benchmark samples, it was found that the content of Anemarrhena saponin BII in the test solution was unstable and there was a certain proportion of conversion. Considering that most of the flavonoid components in the corresponding natural plants have good antipyretic and anti-inflammatory pharmacological activities, the saponin content determination indicators of the Chinese herbal compound benchmark samples containing Astragalus were selected as Platycodon saponin D, Astragaloside I, and Bupleurum saponin a.

[0368] 4. Determine system suitability conditions and solution preparation methods

[0369] 4.1 Chromatographic conditions and system suitability conditions

[0370] The chromatographic conditions and system suitability test used octadecyl bonded silica gel as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); acetonitrile as mobile phase A, 0.2% acetic acid as mobile phase B, and gradient elution according to Table 4; column temperature: 35°C; flow rate: 0.8 ml per minute; detection by electrospray ionization detector; the theoretical plate number calculated based on the astragaloside peak should be no less than 5000.

[0371] 4.2. Preparation of test solution

[0372] Preparation of reference solution Take appropriate amount of timosaponin BⅡ, platycodon saponin D, astragaloside I, and saikosaponin a reference substance, add 80% methanol to make a mixed solution containing 0.1 mg of timosaponin BⅡ, platycodon saponin D, astragaloside I, and saikosaponin a per 1 ml.

[0373] Preparation of test solution: Take 1.0 g of the reference sample (lyophilized powder), accurately weigh it, place it in a round-bottom flask, concentrate it to dryness under reduced pressure, accurately add 50 ml of 80% methanol solution containing 4% concentrated ammonia test solution (take 4 ml of concentrated ammonia test solution, add 80% methanol to 100 ml, shake well), stopper it, weigh the weight, heat it under reflux for 1 hour, cool it, weigh it again, make up the lost weight with 80% methanol solution containing 4% concentrated ammonia test solution, shake well, filter, accurately transfer 25 ml of the filtrate, evaporate to dryness, dissolve the residue with 80% methanol, transfer it to a 5 ml volumetric flask, add 80% methanol to the scale, shake well, filter, and take the filtrate.

[0374] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, measure, and record the chromatogram.

[0375] 5. Methodological Validation

[0376] The quality standard research samples were prepared according to the process determined by process validation, and the acceptable standard range was determined by referring to the 9101 Analytical Method Validation Guidelines of Part IV of the 2020 Edition of the Chinese Pharmacopoeia. The validation items and contents are as follows:

[0377] 5.1. Exclusivity Examination

[0378] 5.1.1. Exclusivity

[0379] Take the test solution and blank control solution, perform HPLC analysis according to the determined chromatographic conditions, and record the chromatogram.

[0380] The results showed that there was no interference in the negative control experiment, and the chromatographic peaks of platycoside D, astragaloside I and saikosaponin a had good peak shapes and better separation, so the method had good specificity.

[0381] 5.1.2. Holism

[0382] Take the test sample solution, increase the elution time three times according to the determined chromatographic conditions, and perform HPLC analysis, and record the chromatogram.

[0383] The results showed that within the chromatographic operating time, the peaks were completely eluted, and the chromatographic peaks of platycoside D, astragaloside I and saikosaponin a had good peak shapes and were better separated, so the method had good overall performance.

[0384] 5.2 Detection Limit and Quantification Limit

[0385] 5.2.1 Detection Limit

[0386] The detection limit was determined in accordance with the guidance for validation of analytical methods in the quality standards of Part IV of the 2020 edition of the Chinese Pharmacopoeia. The signal measured from the test sample of known concentration was compared with the signal measured from the blank sample, and the signal-to-noise ratio was 3:1.

[0387] I.1 Detection limit of platycodon saponin D

[0388] After diluting the content determination standard by 22.2 times, the signal-to-noise ratio was 3.0, therefore, the minimum amount of platycodon saponin D that could be detected was 0.04685 μg.

[0389] I.2 Detection limit of astragaloside IV

[0390] After diluting the assay control substance 33.3 times, the signal-to-noise ratio was 3.1, so the minimum amount of astragaloside IV that could be detected was 0.03003 μg.

[0391] I.3 Detection limit of saikosaponin a

[0392] After diluting the assay control substance 100 times, the signal-to-noise ratio was 3.2, therefore, the minimum amount of saikosaponin a that could be detected was 0.01016 μg.

[0393] 5.2.2 Limit of Quantitation

[0394] The determination was carried out in accordance with the quantitative limit in the guidance principles for validation of analytical methods in the general quality standards of Part IV of the 2020 edition of the Chinese Pharmacopoeia. The signal measured from the test sample of known concentration was compared with the signal measured from the blank sample, and the signal-to-noise ratio was 10:1.

[0395] II.1 Limit of Quantification of Platycodon saponin D

[0396] After diluting the content determination standard by 13.33 times, the signal-to-noise ratio was 10.2, so the minimum amount of platycodon saponin D that could be quantified was 0.07808 μg.

[0397] II.2 Limit of quantification of astragaloside IV

[0398] After diluting the assay standard 20 times, the signal-to-noise ratio was 10.5, so the minimum amount of astragaloside IV that could be quantified was 0.05005 μg.

[0399] II.3 Limit of Quantification of saikosaponin a

[0400] After diluting the assay control substance 80 times, the signal-to-noise ratio was 10.7, so the minimum amount of saikosaponin a that could be quantified was 0.0127 μg.

[0401] 5.3 Linear Relationship Investigation

[0402] 5.3.1. Platycodon saponin D linearity

[0403] Investigation of the linear relationship at different concentrations: Take an appropriate amount of Platycodon saponin D reference substance, accurately weigh it, add 80% methanol to make solutions containing 9.10μg, 18.20μg, 36.41μg, 54.61μg, 91.02μg, 109.22μg, and 182.04μg per 1ml, accurately pipette 10μl of the above 7 different concentrations of Platycodon saponin D reference substance solutions and inject them into the liquid chromatograph to determine the peak area, and draw a standard curve with the concentration of Platycodon saponin D reference substance solution as the horizontal axis and the peak area (A) as the vertical axis.

[0404] Regression equation: y = 0.0878x^0.8503, R 2 =0.9994. The results showed that the linear relationship was good within the range of 9.10 μg / ml to 182.04 μg / ml of platycodon saponin D concentration.

[0405] 5.3.2. Astragaloside I Linearity

[0406] Investigation of the linear relationship at different concentrations: Take an appropriate amount of astragaloside IV reference substance, accurately weigh it, add methanol to make solutions containing 9.04μg, 18.08μg, 36.16μg, 54.23μg, 90.39μg, 108.47μg, and 180.78μg per 1ml. Accurately pipette 10μl of the above 7 different concentrations of astragaloside IV reference substance solutions and inject them into liquid chromatograph to determine the peak area. Draw a standard curve with the concentration of astragaloside IV reference substance solution as the horizontal axis and the peak area (A) as the vertical axis.

[0407] Regression equation: y = 0.1044x^0.8480, R 2 =0.9993. The results showed that the linear relationship was good within the range of astragaloside IV concentration from 9.04 μg / ml to 180.78 μg / ml.

[0408] 5.3.3. Linearity of saikosaponin a

[0409] Investigation of the linear relationship at different concentrations: Take an appropriate amount of saikosaponin a reference substance, accurately weigh it, add methanol to make solutions containing 9.04μg, 18.08μg, 36.16μg, 54.23μg, 90.39μg, 108.47μg, and 180.78μg per 1ml, accurately pipette 10μl of the above 7 different concentrations of saikosaponin a reference substance solutions and inject them into the liquid chromatograph to determine the peak area. Draw a standard curve with the concentration of saikosaponin a reference substance solution as the horizontal axis and the peak area (A) as the vertical axis.

[0410] Regression equation: y = 0.1176x^0.8230, R 2 =0.9991. The results showed that the linear relationship was good within the range of saikosaponin a concentration from 9.09 μg / ml to 181.80 μg / ml.

[0411] 5.4 Precision test

[0412] 5.4.1. Instrument precision test

[0413] Take the same sample solution and inject it 6 times continuously according to the above conditions to determine the peak areas of timosaponin BⅡ, platycodon saponin D, astragaloside I and bupleurosaponin a.

[0414] The test solution was sampled six times continuously. The RSD value of the retention time of platycoside D was 0.1%, and the RSD value of the peak area was 1.4%. The RSD value of the retention time of astragaloside I was 0.1%, and the RSD value of the peak area was 1.2%. The RSD value of the retention time of saikosaponin a was 0.1%, and the RSD value of the peak area was 0.9%, indicating that the instrument had good precision.

[0415] 5.4.2 Intermediate precision study

[0416] Six portions of 1.0 g of the same batch of test samples (batch number: SXKL20230605-G) were independently operated by another experimenter, processed according to the test sample preparation method, and measured using the same instrument.

[0417] The results showed that among the 12 test samples prepared by different personnel, the RSD value of platycoside D was 1.5%, the RSD value of astragaloside Ⅳ was 1.6%, and the RSD value of saikosaponin a was 1.4%. The separation effect of platycoside D, astragaloside Ⅳ and saikosaponin a was good. The results showed that this method had good precision among different operators.

[0418] 5.4.3 Repeatability Study

[0419] Take 6 portions of 1.0 g of the same batch of test sample (batch number: SXKL20230605-G), accurately weigh them, prepare them according to the preparation method of the test sample solution, and perform the measurement under the above-mentioned chromatographic conditions.

[0420] The results showed that the method had good repeatability. The RSD values ​​of platycodon saponin D, astragaloside IV, and saikosaponin a were 1.3%, 1.4%, and 1.7%, respectively.

[0421] 5.5. Accuracy test (sample recovery test)

[0422] 5.5.1. Platycodon saponin D sample recovery experiment

[0423] Weigh about 1.0g of 9 samples of the same batch number (batch number: SXKL20230605-G) accurately, place them in stoppered conical bottles, add 50ml of 80% methanol solution containing 4% concentrated ammonia test solution, weigh, reflux for 1 hour, cool, weigh again, make up the lost weight with 80% methanol solution containing 4% concentrated ammonia test solution, shake well, filter, accurately transfer 25ml of the filtrate, evaporate to dryness, dissolve the residue with 80% methanol, transfer to a 10ml volumetric flask, add 80% methanol to the scale, shake well, accurately transfer 5ml to a 10ml volumetric flask, accurately add 1ml, 2ml, and 3ml of the mixed reference solution (containing 0.2180mg / ml of platycodon saponin D) according to three different concentrations of high, medium and low, add 80% methanol to the scale, shake well, filter, and take the filtrate. Determine the content. Calculate the recovery rate and RSD.

[0424] The results showed that the recovery rate of platycodon saponin D ranged from 95.5% to 102.3%, with an RSD of 2.4%, which met the requirements of the 9101 analytical method validation guidelines of the fourth general chapter of the 2020 edition of the "Chinese Pharmacopoeia" (recovery rate was 85-110%, RSD was ≤4.0%). The results showed that the method had good accuracy.

[0425] 5.5.2. Astragaloside IV recovery experiment

[0426] Weigh about 1.0g of 9 samples of the same batch number (batch number: SXKL20230605-G) accurately, place them in stoppered conical bottles, add 50ml of 80% methanol solution containing 4% concentrated ammonia test solution, weigh, reflux for 1 hour, cool, weigh again, make up the lost weight with 80% methanol solution containing 4% concentrated ammonia test solution, shake well, filter, accurately transfer 25ml of the filtrate, evaporate to dryness, dissolve the residue with 80% methanol, transfer to a 10ml volumetric flask, add 80% methanol to the scale, shake well, accurately transfer 5ml to a 10ml volumetric flask, accurately add 1ml, 2ml, and 3ml of the mixed reference solution (containing 0.1855mg / ml of astragaloside IV) according to three different concentrations of high, medium and low, add 80% methanol to the scale, shake well, filter, and take the filtrate. Determine the content. Calculate the recovery rate and RSD.

[0427] 5.5.3. Recovery experiment of saikosaponin a

[0428] Weigh about 1.0g of 9 samples of the same batch number (batch number: SXKL20230605-G) accurately, place them in stoppered conical bottles, add 50ml of 80% methanol solution containing 4% concentrated ammonia test solution, weigh, reflux for 1 hour, cool, weigh again, make up the lost weight with 80% methanol solution containing 4% concentrated ammonia test solution, shake well, filter, accurately transfer 25ml of the filtrate, evaporate to dryness, dissolve the residue with 80% methanol, transfer to a 10ml volumetric flask, add 80% methanol to the scale, shake well, accurately transfer 5ml to a 10ml volumetric flask, accurately add 1ml, 2ml, and 3ml of the mixed reference solution (containing 0.09878mg / ml of saikosaponin a) according to three different concentrations of high, medium and low, add 80% methanol to the scale, shake well, filter, and take the filtrate. Determine the content. Calculate the recovery rate and RSD.

[0429] The results showed that the recovery rate of saikosaponin a ranged from 104.2% to 109.9%, with an RSD of 2.1%, which met the requirements of the 9101 analytical method validation guidelines of the 2020 edition of the Chinese Pharmacopoeia (recovery rate was 85-110%, RSD was ≤4.0%). The results showed that the method had good accuracy.

[0430] 5.6 Durability test

[0431] 5.6.1. Solution stability

[0432] Take the same sample solution and inject it at 0, 4, 11, 18, 24, 30, 38, 50, and 56 hours after preparation, and determine the peak area to investigate the stability of the solution.

[0433] From the results, it can be seen that when the same test solution was injected within 56 hours and the peak areas of platycodon saponin D, astragaloside I and saikosaponin a were determined, the retention time RSDs were all less than 1.0%, and the peak area RSDs were less than 2.0%, indicating that the test solution was basically stable within 56 hours and could meet the determination needs.

[0434] 5.6.2 Durability test at different flow rates

[0435] Take 1.0 g of the test sample (batch number: SXKL20230605-G), weigh it accurately, and process it according to the test sample preparation method. Tests were carried out at different flow rates of 0.7 ml / min, 0.8 ml / min, and 0.9 ml / min to determine the contents of platycodon saponin D, astragaloside I, and bupleurosaponin a.

[0436] The results showed that this method had great selectivity for different flow rates, with large differences in platycodon saponin D and bupleurosaponin a, with RSD values ​​greater than 4.0%. It had a good separation effect on the chromatographic peak of astragaloside IV. Considering the characteristic spectrum, a flow rate of 0.8 ml / min was selected.

[0437] 5.6.3. Durability test at different column temperatures

[0438] Take 1.0 g of the test sample (batch number: SXKL20230605-G), weigh it accurately, and process it according to the test sample preparation method. Tests were carried out at different column temperatures of 33°C, 35°C, and 37°C to determine the contents of platycodon saponin D, astragaloside I, and bupleurosaponin a.

[0439] The results showed that different column temperatures had a greater impact on platycoside D and astragaloside IV. When the column temperature was 37°C, peak 12 (astragaloside IV) had a slight tailing and a large content difference. Bupleurosaponin a had a small difference at different column temperatures. Considering the characteristic spectrum, a column temperature of 35°C was selected for detection.

[0440] 5.6.4. Durability of different chromatographic columns

[0441] Take 1.0 g of the test sample (batch number: SXKL20230605-G), accurately weigh it, process it according to the test sample preparation method, replace it with a chromatographic column with a different SN number (SN: 111EB20233) from the same manufacturer, and carry out the test to determine the contents of platycodon saponin D, astragaloside I, and saikosaponin a.

[0442] The results showed that different SN numbers of chromatographic columns from the same manufacturer had little effect on platycodon saponin D, astragaloside I and saikosaponin a, and the method had good durability for this type of chromatographic column.

[0443] 5.6.5 Durability test at different acid concentrations

[0444] Take 1.0 g of the test sample (batch number: SXKL20230605-G), weigh it accurately, and process it according to the test sample preparation method. Tests were carried out under different acid concentrations of 0.18% acetic acid, 0.2% acetic acid, and 0.22% acetic acid to determine the contents of platycoside D, astragaloside I, and saikosaponin a.

[0445] The results showed that under different acid concentrations, the effects on platycodon saponin D, astragaloside I and saikosaponin a were small. Therefore, this method has good durability to different acid concentrations.

[0446] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for determining the content of a Chinese medicine composition containing Astragalus, characterized in that: The determination method comprises the following steps: Take the test solution and the reference solution of the Chinese medicine composition for testing. The chromatographic conditions of the detection are: using a YMC-Pack ODS-A chromatographic column, the mobile phase A is acetonitrile, the mobile phase B is about 0.2% acetic acid aqueous solution, the gradient elution program is: 0-20min, 90% B→77% B; 20-55min, 77% B→75% B; 55-70min, 75% B→68% B; 70-90min, 68% B→65% B; 90-97min, 65% B→55% B; 97-107min, 55% B→40% B; 107-109min, 40% B; 109-110min, 40% B→90% B; 110-120min, 90% B, the flow rate is 0.1-1.0ml / min, the column temperature is 10-40°C, and the injection volume is 1-20μl; According to the test results, obtaining the content information of the Chinese medicine composition; The Chinese medicine composition comprises astragalus, rhizoma anemarrhenae, platycodon grandiflorum, bupleurum and cimicifuga, and the reference substances are rhizoma anemarrhenae saponin BⅡ, platycodon grandiflorum saponin D, astragaloside IV and bupleurosaponin a; The preparation method of the Chinese medicine composition test solution comprises: weighing an appropriate amount of Chinese medicine composition powder containing astragalus, concentrating under reduced pressure, adding about 80% methanol containing about 4% ammonia test solution, weighing the weight, cooling after heating under reflux, weighing the weight again, using about 80% methanol containing about 4% ammonia test solution to make up for the lost weight, shaking, filtering, taking a first subsequent filtrate, evaporating to dryness, obtaining a residue, dissolving the obtained residue with about 80% methanol, shaking, filtering, taking a second subsequent filtrate, and obtaining the Chinese medicine composition test solution; Wherein, the detector for the detection is an electrospray detector; The specifications of the chromatographic column are as follows: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm.

2. The measuring method according to claim 1, characterized in that The information is calculated according to the external standard method based on the corresponding peak areas in the recorded chromatogram of the test solution of the traditional Chinese medicine composition and the chromatogram of the reference solution.

3. The measuring method according to claim 1, characterized in that The 120 min in the gradient elution program was replaced by 130 min.

4. The measuring method according to claim 1, characterized in that The standard curve of Platycodon saponin D is y=0.0878x^0.8503, R 2 =0.9994.

5. The measuring method according to claim 1, characterized in that The standard curve of astragaloside IV is y=0.1044x^0.8480, R 2 =0.9993.

6. The measuring method according to claim 1, characterized in that The standard curve of saikosaponin a is y=0.1176x^0.8230, R 2 =0.9991.

7. The measuring method according to claim 1, characterized in that The mass / volume ratio of the Chinese medicinal composition powder containing astragalus to the about 80% methanol containing about 4% ammonia test solution is 10-50, and the unit is mg / ml.

8. The measuring method according to claim 7, characterized in that The mass / volume ratio of the Chinese medicinal composition powder containing Astragalus to the about 80% methanol containing about 4% ammonia test solution is about 20, in units of mg / ml.

9. The measuring method according to claim 1, characterized in that The heating reflux time is 30 to 120 minutes.

10. The measuring method according to claim 9, characterized in that The heating reflux time is about 60 min.

11. The measuring method according to claim 1, characterized in that The volume ratio of the first subsequent filtrate to the test solution containing about 4% ammonia and about 80% methanol is 0.4-0.

6.

12. The measuring method according to claim 11, characterized in that The volume ratio of the first subsequent filtrate to the test solution containing about 4% ammonia and about 80% methanol is about 0.

5.

13. The measuring method according to claim 1, characterized in that The volume ratio of the second subsequent filtrate to the first subsequent filtrate is 0.1 to 0.

4.

14. The measuring method according to claim 13, characterized in that The volume ratio of the second subsequent filtrate to the first subsequent filtrate is about 0.

2.

15. The measuring method according to claim 1, characterized in that The preparation method of the reference solution comprises: weighing appropriate amounts of timosaponin BⅡ, platycodon saponin D, astragaloside I and bupleurosaponin a reference substances, and adding methanol to prepare the reference solution with the concentrations of timosaponin BⅡ, platycodon saponin D, astragaloside I and bupleurosaponin a being 0.01 to 200 μg / ml respectively.

16. The measuring method according to claim 1, characterized in that The concentration of Anemarrhena saponin BⅡ in the reference solution is about 100 μg / ml.

17. The measuring method according to claim 1, characterized in that The concentration of platycodon saponin D in the reference solution is about 100 μg / ml.

18. The measuring method according to claim 1, characterized in that: The concentration of astragaloside IV in the reference solution is about 100 μg / ml.

19. The measuring method according to claim 1, characterized in that: The concentration of saikosaponin a in the reference solution is about 100 μg / ml.

20. The measuring method according to claim 1, characterized in that The flow rate of the detection is 0.5-1.0 ml / min.

21. The measuring method according to claim 20, characterized in that The flow rate of the assay was approximately 0.8 ml / min.

22. The determination method according to claim 1, characterized in that The column temperature was about 35°C.

23. The determination method according to claim 1, characterized in that The injection volume was about 10 μl.

24. The determination method according to claim 1, characterized in that The theoretical plate number of the chromatographic peak corresponding to the astragaloside IV is not less than 5000.

25. The determination method according to claim 1, characterized in that The preparation method of the powder of the Chinese medicine composition containing astragalus comprises: (a) weighing appropriate amounts of astragalus, platycodon, bupleurum and cimicifuga, adding first water for first soaking, decocting with high heat until boiling, decocting with low heat for a period of time, adding rhizoma anemarrhenae for second soaking, decocting with high heat until boiling, decocting with low heat for a period of time, and obtaining a decoction; (b) filtering the decoction to obtain a first filtrate and a filter residue, adding second water to the filter residue, decocting with high heat until boiling, decocting with low heat for a period of time, filtering to obtain a second filtrate, combining the first filtrate and the second filtrate to obtain a combined filtrate, decompressing and concentrating the combined filtrate to an extract with a relative density of 1.10 to 1.20 at about 20° C., pre-freezing, drying in a freeze dryer, crushing, sieving, and mixing to obtain the powder of the Chinese medicine composition containing astragalus.

26. The determination method according to claim 25, characterized in that The ratio of the sum of the mass of the astragalus, platycodon, bupleurum and cimicifuga to the mass / volume of the first water is 50-90, in units of mg / ml.

27. The assay method according to claim 26, characterized in that The ratio of the sum of the mass of the astragalus, platycodon, bupleurum and cimicifuga to the mass / volume of the first water is about 74.62, in mg / ml.

28. The determination method according to claim 25, characterized in that The first soaking time is 10 to 100 minutes.

29. The determination method according to claim 28, characterized in that The first soaking time is about 60 minutes.

30. The determination method according to claim 25, characterized in that The high heat decoction is high heat decoction with a cover.

31. The assay method according to claim 25, characterized in that The voltage of the high fire is 150-300V.

32. The assay method according to claim 31, characterized in that The voltage of the high fire is about 220V.

33. The assay method according to claim 25, characterized in that The voltage of the slow fire is 100-250V.

34. The assay method according to claim 33, characterized in that The voltage of the slow fire is about 175V.

35. The assay method according to claim 25, characterized in that The first slow-fire decocting time is 30 to 70 minutes.

36. The assay method according to claim 35, characterized in that The first slow-cooking time is about 55 minutes.

37. The assay method according to claim 25, characterized in that The second slow-fire decocting time is 1 to 10 minutes.

38. The determination method according to claim 37, characterized in that The second slow-cooking time is about 5 minutes.

39. The assay method according to claim 25, characterized in that The third slow-fire decocting time is 30 to 70 minutes.

40. The assay method according to claim 39, characterized in that The third slow-fire decocting time is about 50 minutes.

41. The assay method according to claim 25, characterized in that The second soaking time is 5 to 20 minutes.

42. The assay method according to claim 41, characterized in that The second soaking time is about 10 minutes.

43. The assay method according to claim 25, characterized in that The filtration is a single-layer filtration using 100-mesh nylon filter cloth.

44. The assay method according to claim 25, characterized in that The volume of the second water is 300-500 ml.

45. The assay method according to claim 44, characterized in that The volume of the second water is about 400 ml.

46. ​​The assay method according to claim 25, characterized in that The mass / volume ratio of the traditional Chinese medicine composition containing astragalus to the second water is 100-150, in units of mg / ml.

47. The assay method according to claim 46, characterized in that The mass / volume ratio of the traditional Chinese medicine composition containing astragalus to the second water is about 121.25, in mg / ml.

48. The assay method according to claim 25, characterized in that The relative density of the combined filtrate at about 20°C is 1.010 to 1.

025.

49. The assay method according to claim 25, characterized in that The instrument for the reduced pressure concentration is a rotary evaporator.

50. The assay method according to claim 49, characterized in that The rotating speed of the rotary evaporator is 50-150 r / min.

51. The assay method according to claim 50, characterized in that The rotation speed of the rotary evaporator is about 100 r / min.

52. The assay method according to claim 25, characterized in that The temperature of the reduced pressure concentration is not higher than 65°C.

53. The assay method according to claim 25, characterized in that The vacuum degree of the reduced pressure concentration is -0.08 to -0.099 MPa.

54. The assay method according to claim 25, characterized in that Before the pre-freezing, the extract is placed in a stainless steel plate so that the thickness of the plate is 9 to 11 mm.

55. The assay method according to claim 25, characterized in that The degree of pre-freezing is that there is no wet core in the extract.

56. The assay method according to claim 25, characterized in that The cold trap temperature of the freeze drying is ≤-50°C.

57. The assay method according to claim 25, characterized in that The vacuum degree of the freeze drying is ≤100Pa.

58. The assay method according to claim 25, characterized in that The freeze-drying degree is that the moisture content is no higher than 8%.

59. The assay method according to claim 25, characterized in that The dry extract rate of the powder of the traditional Chinese medicine composition containing astragalus is 27.4% to 33.5%.

60. The assay method according to claim 25, characterized in that The mass ratios of the astragalus, the rhizoma anemarrhenae, the platycodon, the radix bupleuri and the rhizoma cimicifugae are (5-7):(0.1-2):(0.5-2.5):(2-4):(0.5-2.5).

61. The assay method according to claim 60, characterized in that The mass ratio of the astragalus, the rhizoma anemarrhenae, the platycodon, the radix bupleuri and the rhizoma cimicifugae is (5.5-6.5):(0.5-1.5):(1-2):(2.5-3.5):(1-2).

62. The assay method according to claim 61, characterized in that The mass ratio of the astragalus, the rhizoma anemarrhenae, the platycodon and the cimicifuga is about 6: about 1: about 1.5: about 3: about 1.

5.

63. The assay method according to claim 25, characterized in that The mass range of the astragalus is 10 to 30 g.

64. The assay method according to claim 63, characterized in that The mass of the Astragalus membranaceus was about 22.38 g.

65. The assay method according to claim 25, characterized in that The mass range of the Anemarrhena Rhizoma is 1 to 10 g.

66. The assay method according to claim 65, characterized in that The mass of the Anemarrhena Rhizoma is about 3.73 g.

67. The assay method according to claim 25, characterized in that The mass range of the Platycodon grandiflorum is 1 to 10 g.

68. The assay method according to claim 67, characterized in that The mass of Platycodon grandiflorum was about 5.60 g.

69. The assay method according to claim 25, characterized in that The mass range of the Bupleurum is 10 to 20 g.

70. The assay method according to claim 69, characterized in that The mass of the Bupleurum Root is about 11.19 g.

71. The assay method according to claim 25, characterized in that The mass range of the Cimicifuga heracleifolia is 1 to 10 g.

72. The assay method according to claim 71, characterized in that The mass of the Cimicifuga was about 5.60 g.

73. A method for constructing a fingerprint of a traditional Chinese medicine composition containing Astragalus, characterized in that: The construction method comprises the following steps: preparing a test solution of a Chinese medicine composition: weighing an appropriate amount of powder of a Chinese medicine composition containing astragalus, concentrating under reduced pressure, adding about 80% methanol containing about 4% ammonia test solution, weighing the weight, cooling after heating under reflux, weighing the weight again, using about 80% methanol containing about 4% ammonia test solution to make up for the lost weight, shaking well, filtering, taking a first subsequent filtrate, evaporating to dryness, obtaining a residue, dissolving the obtained residue with about 80% methanol, shaking well, filtering, taking a second subsequent filtrate, and obtaining the test solution of the Chinese medicine composition; wherein the Chinese medicine composition comprises astragalus, rhizoma anemarrhenae, platycodon grandiflorum, radix bupleuri and rhizoma cimicifuga; Preparation of reference solution: weigh appropriate amounts of timosaponin BⅡ, platycodon saponin D, astragaloside I and saikosaponin a reference substances, add methanol to prepare reference solution with concentrations of timosaponin BⅡ, platycodon saponin D, astragaloside I and saikosaponin a of 0.01 to 200 μg / ml respectively; Obtaining a fingerprint of the Chinese medicine composition according to the results of high performance liquid chromatography detection of the Chinese medicine composition test solution and the reference solution; The chromatographic conditions of the high performance liquid phase detection are as follows: using a YMC-Pack ODS-A chromatographic column, the mobile phase A is acetonitrile, the mobile phase B is about 0.2% acetic acid aqueous solution, the gradient elution program is: 0-20 min, 90% B→77% B; 20-55 min, 77% B→75% B; 55-70 min, 75% B→68% B; 70-90 min, 68% B→65% B; 90-97 min, 65% B→55% B; 97-107 min, 55% B→40% B; 107-109 min, 40% B; 109-110 min, 40% B→90% B; 110-120 min, 90% B, the flow rate is 0.1-1.0 ml / min, the column temperature is 10-40° C., and the injection volume is 1-20 μl; The fingerprint spectrum includes peaks 1-16, wherein peak 12 is astragaloside I as a reference peak, and the relative retention time averages of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15 and 16 are about 0.14, about 0.22, about 0.29, about 0.44, about 0.45, about 0.68, about 0.69, about 0.70, about 0.72, about 0.74, about 0.85, about 1.06, about 1.12, about 1.13 and about 1.23, respectively; Wherein, the detector for the detection is an electrospray detector; The specifications of the chromatographic column are as follows: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm.

74. The construction method according to claim 73, characterized in that: The preparation method of the powder of the Chinese medicine composition containing astragalus comprises: (a) weighing appropriate amounts of astragalus, platycodon, bupleurum and cimicifuga, adding first water for first soaking, decocting with high heat until boiling, decocting with low heat for a period of time, adding rhizoma anemarrhenae for second soaking, decocting with high heat until boiling, decocting with low heat for a period of time, and obtaining a decoction; (b) filtering the decoction to obtain a first filtrate and a filter residue, adding second water to the filter residue, decocting with high heat until boiling, decocting with low heat for a period of time, filtering to obtain a second filtrate, combining the first filtrate and the second filtrate to obtain a combined filtrate, decompressing and concentrating the combined filtrate to an extract with a relative density of 1.10 to 1.20 at about 20° C., pre-freezing, drying in a freeze dryer, crushing, sieving, and mixing to obtain the powder of the Chinese medicine composition containing astragalus.

75. The construction method according to claim 74, characterized in that: The ratio of the sum of the mass of the astragalus, platycodon, bupleurum and cimicifuga to the mass / volume of the first water is 50-90, in units of mg / ml.

76. The construction method according to claim 75, characterized in that The ratio of the sum of the mass of the astragalus, platycodon, bupleurum and cimicifuga to the mass / volume of the first water is about 74.62, in mg / ml.

77. The construction method according to claim 74, characterized in that: The first soaking time is 10 to 100 minutes.

78. The construction method according to claim 77, characterized in that: The first soaking time is about 60 minutes.

79. The construction method according to claim 74, characterized in that: The high heat decoction is high heat decoction with a cover.

80. The construction method according to claim 74, characterized in that: The voltage of the high fire is 150-300V.

81. The construction method according to claim 80, characterized in that: The voltage of the high fire is about 220V.

82. The construction method according to claim 74, characterized in that: The voltage of the slow fire is 100-250V.

83. The construction method according to claim 82, characterized in that: The voltage of the slow fire is about 175V.

84. The construction method according to claim 74, characterized in that: The first slow-fire decocting time is 30 to 70 minutes.

85. The construction method according to claim 84, characterized in that: The first slow-cooking time is about 55 minutes.

86. The construction method according to claim 74, characterized in that: The second slow-fire decocting time is 1 to 10 minutes.

87. The construction method according to claim 86, characterized in that The second slow-cooking time is about 5 minutes.

88. The construction method according to claim 74, characterized in that: The third slow-fire decocting time is 30 to 70 minutes.

89. The construction method according to claim 88, characterized in that The third slow-fire decocting time is about 50 minutes.

90. The construction method according to claim 74, characterized in that: The second soaking time is 5 to 20 minutes.

91. The construction method according to claim 90, characterized in that: The second soaking time is about 10 minutes.

92. The construction method according to claim 74, characterized in that: The filtration is a single-layer filtration using 100-mesh nylon filter cloth.

93. The construction method according to claim 74, characterized in that: The volume of the second water is 300-500 ml.

94. The construction method according to claim 93, characterized in that: The volume of the second water is about 400 ml.

95. The construction method according to claim 74, characterized in that: The mass / volume ratio of the traditional Chinese medicine composition containing astragalus to the second water is 100-150, in units of mg / ml.

96. The construction method according to claim 95, characterized in that The mass / volume ratio of the traditional Chinese medicine composition containing astragalus to the second water is about 121.25, in mg / ml.

97. The construction method according to claim 74, characterized in that: The relative density of the combined filtrate at about 20°C is 1.010 to 1.

025.

98. The construction method according to claim 74, characterized in that: The instrument for the reduced pressure concentration is a rotary evaporator.

99. The construction method according to claim 98, characterized in that The rotating speed of the rotary evaporator is 50-150 r / min.

100. The construction method according to claim 99, characterized in that: The rotation speed of the rotary evaporator is about 100 r / min.

101. The construction method according to claim 74, characterized in that: The temperature of the reduced pressure concentration is not higher than 65°C.

102. The construction method according to claim 74, characterized in that: The vacuum degree of the reduced pressure concentration is -0.08 to -0.099 MPa.

103. The construction method according to claim 74, characterized in that: Before the pre-freezing, the extract is placed in a stainless steel plate so that the thickness of the plate is 9 to 11 mm.

104. The construction method according to claim 74, characterized in that: The degree of pre-freezing is that there is no wet core in the extract.

105. The construction method according to claim 74, characterized in that: The cold trap temperature of the freeze drying is ≤-50°C.

106. The construction method according to claim 74, characterized in that: The vacuum degree of the freeze drying is ≤100Pa.

107. The construction method according to claim 74, characterized in that: The freeze-drying degree is that the moisture content is no higher than 8%.

108. The construction method according to claim 74, characterized in that: The dry extract rate of the powder of the traditional Chinese medicine composition containing astragalus is 27.4% to 33.5%.

109. The construction method according to claim 74, characterized in that: The mass ratios of the astragalus, the rhizoma anemarrhenae, the platycodon, the radix bupleuri and the rhizoma cimicifugae are (5-7):(0.1-2):(0.5-2.5):(2-4):(0.5-2.5).

110. The construction method according to claim 109, characterized in that: The mass ratio of the astragalus, the rhizoma anemarrhenae, the platycodon, the radix bupleuri and the rhizoma cimicifugae is (5.5-6.5):(0.5-1.5):(1-2):(2.5-3.5):(1-2).

111. The construction method according to claim 110, characterized in that: The mass ratio of the astragalus, the rhizoma anemarrhenae, the platycodon and the cimicifuga is about 6: about 1: about 1.5: about 3: about 1.

5.

112. The construction method according to claim 74, characterized in that: The mass range of the astragalus is 10 to 30 g.

113. The construction method according to claim 112, characterized in that: The mass of the Astragalus membranaceus was about 22.38 g.

114. The construction method according to claim 74, characterized in that: The mass range of the Anemarrhena Rhizoma is 1 to 10 g.

115. The construction method according to claim 114, characterized in that: The mass of the Anemarrhena Rhizoma is about 3.73 g.

116. The construction method according to claim 74, characterized in that: The mass range of the Platycodon grandiflorum is 1 to 10 g.

117. The construction method according to claim 116, characterized in that: The mass of Platycodon grandiflorum was about 5.60 g.

118. The construction method according to claim 74, characterized in that: The mass range of the Bupleurum is 10 to 20 g.

119. The construction method according to claim 118, characterized in that: The mass of the Bupleurum Root is about 11.19 g.

120. The construction method according to claim 74, characterized in that: The mass range of the Cimicifuga heracleifolia is 1 to 10 g.

121. The construction method according to claim 120, characterized in that: The mass of the Cimicifuga was about 5.60 g.

122. The construction method according to claim 73, characterized in that: The mass / volume ratio of the Chinese medicinal composition powder containing astragalus to the about 80% methanol containing about 4% ammonia test solution is 10-50, and the unit is mg / ml.

123. The construction method according to claim 122, characterized in that: The mass / volume ratio of the Chinese medicinal composition powder containing Astragalus to the about 80% methanol containing about 4% ammonia test solution is about 20, in units of mg / ml.

124. The construction method according to claim 73, characterized in that: The heating reflux time is 30 to 120 minutes.

125. The construction method according to claim 124, characterized in that: The heating reflux time is about 60 min.

126. The construction method according to claim 73, characterized in that: The volume ratio of the first subsequent filtrate to the test solution containing about 4% ammonia and about 80% methanol is 0.4 to 0.

6.

127. The construction method according to claim 126, characterized in that: The volume ratio of the first subsequent filtrate to the test solution containing about 4% ammonia and about 80% methanol is about 0.

5.

128. The construction method according to claim 73, characterized in that: The volume ratio of the second subsequent filtrate to the first subsequent filtrate is 0.1 to 0.

4.

129. The construction method according to claim 128, characterized in that The volume ratio of the second subsequent filtrate to the first subsequent filtrate is about 0.

2.

130. The construction method according to claim 73, characterized in that: The concentration of Anemarrhena saponin BⅡ in the reference solution is about 100 μg / ml.

131. The construction method according to claim 73, characterized in that: The concentration of platycodon saponin D in the reference solution is about 100 μg / ml.

132. The construction method according to claim 73, characterized in that: The concentration of astragaloside IV in the reference solution is about 100 μg / ml.

133. The construction method according to claim 73, characterized in that: The concentration of saikosaponin a in the reference solution is about 100 μg / ml.

134. The construction method according to claim 73, characterized in that: The 120 min in the gradient elution program was replaced by 130 min.

135. The construction method according to claim 73, characterized in that: The flow rate of the high performance liquid phase detection is 0.5-1.0 ml / min.

136. The construction method according to claim 135, characterized in that: The flow rate of the HPLC detection is about 0.8 ml / min.

137. The construction method according to claim 73, characterized in that: The column temperature was about 35°C.

138. The construction method according to claim 73, characterized in that: The injection volume was about 10 μl.

139. The construction method according to claim 73, characterized in that: The theoretical plate number of the chromatographic peak corresponding to the astragaloside IV is not less than 5000.

140. The construction method according to claim 73, characterized in that: The fingerprint spectrum includes peaks 1-16, wherein peak 12 is astragaloside I as a reference peak, and the average retention times of peaks 1-16 correspond to about 12.9 min, about 20.1 min, about 26.1 min, about 39.5 min, about 40.6 min, about 61.1 min, about 62.1 min, about 63.1 min, about 64.8 min, about 66.5 min, about 76.4 min, about 90.1 min, about 95.5 min, about 101.1 min, about 101.5 min and about 110.9 min, respectively.

141. The construction method according to claim 73, characterized in that: When the flow rate is 0.8 ml / min and the column temperature is 35° C., peak No. 4 is anemarrhena saponin BⅡ, peak No. 7 is platycodon saponin D, peak No. 12 is astragaloside IV, and peak No. 14 is bupleurosaponin a.

142. The construction method according to claim 73, characterized in that: When the flow rate is 0.8 ml / min and the column temperature is 35°C, peak 2 and peak 12 are from Astragalus membranaceus medicinal material, peak 1, peak 3, peak 4, peak 5 and peak 11 are from Anemarrhena asphodeloides medicinal material, peak 6, peak 7, peak 8, peak 9 and peak 10 are from Platycodon grandiflorum medicinal material, peak 14 and peak 15 are from Bupleurum chinense medicinal material, and peak 13 and peak 16 are from Anemarrhena asphodeloides medicinal material and Platycodon grandiflorum medicinal material.

143. A quality control method for a Chinese medicine composition containing Astragalus, characterized in that: The quality control method comprises the following steps: (1) Establishing a standard fingerprint of a reference sample of a Chinese medicine composition according to the fingerprint construction method described in any one of claims 73 to 142; (2) taking a sample solution of the Chinese medicine composition, and testing it according to the chromatographic conditions in the fingerprint construction method according to any one of claims 73 to 142 to obtain a fingerprint of the Chinese medicine composition sample to be tested; as well as (3) Compare the fingerprint of the Chinese medicine composition sample obtained in step (2) with the standard fingerprint of the Chinese medicine composition reference sample obtained in step (1). If it meets the requirements, it is a qualified product, and if it does not meet the requirements, it is an unqualified product.

144. The quality control method according to claim 143, characterized in that: The compliance requirements include one or more of the following: (1) The fingerprint of the Chinese medicine composition sample to be tested presents 16 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard fingerprint of the Chinese medicine composition reference sample; (2) With the astragaloside peak as the S peak, the relative retention time of each characteristic chromatographic peak in the fingerprint of the Chinese medicine composition sample to be tested and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard fingerprint of the Chinese medicine composition reference sample; as well as (3) According to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the similarity between the fingerprint of the Chinese medicine composition sample to be tested and the standard fingerprint of the Chinese medicine composition reference sample is calculated, and the similarity shall not be less than 0.

90.

145. Use of the assay method according to any one of claims 1 to 72, the construction method according to any one of claims 73 to 142, or the quality control method according to claim 143 or 144 in quality detection and / or quality evaluation and / or quality control of a Chinese medicine composition comprising Astragalus.

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