Quality control method of traditional Chinese medicine composition freeze-dried powder

Abstract

The application discloses a quality control method of a traditional Chinese medicine composition freeze-dried powder, adopts a combination of a reference index component content, a fingerprint, an extract rate and a thin layer chromatography to evaluate a material reference of Daxiefei Tang, comprehensively detects the material reference of Daxiefei Tang through multiple technologies and multiple ways, and formulates a quality standard of the material reference of Daxiefei Tang, so as to provide a scientific basis for preparation development of Daxiefei Tang and provide a train of thought for development and research of similar prescriptions.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of quality detection of traditional Chinese medicine preparation, and particularly relates to a quality control method of a freeze-dried powder of a traditional Chinese medicine composition. BACKGROUND

[0002] The Daxiefei Decoction is from the Aiding Traveling Decoction for Zangfu Medication Method, and is composed of six medicines, including Platycladi Seed (blackened and pounded into mud), Radix et Rhizoma Rhei, Radix Paeoniae Alba, Radix Glycyrrhizae (charred), Radix Scrophulariae and Rhizoma Zingiberis Recens, and is used for treating sputum and phlegm in the chest, dyspnea, constipation, edema, fullness and desire for gas relief. The Platycladi Seed and Radix et Rhizoma Rhei are used for purging lung and relieving asthma, removing water and reducing edema, and are the monarch drugs; the Radix Scrophulariae is used for clearing heat and drying dampness and purging fire to assist the Radix et Rhizoma Rhei in clearing and discharging the evil heat in the bowels, and the Rhizoma Zingiberis Recens is used for warming the lung and resolving the phlegm and evil in the lung to assist the Platycladi Seed, and is the minister drugs; the Radix Paeoniae Alba is used for astringing and stopping sweating, and is the auxiliary drug; the Radix Glycyrrhizae is used for relieving urgency and pain, regulating all medicines, assisting the Radix Paeoniae Alba, and is the minister drug. The current researches on the Daxiefei Decoction are mainly in the clinical and application aspects, and the modern pharmaceutical researches show that the Daxiefei Decoction can be used for treating chronic obstructive pulmonary disease (COPD) and preventing and treating pneumonia, and has significant curative effect, safe use and good development prospect.

[0003] The current reports on the Daxiefei Decoction are mainly concentrated in the clinical application and curative effect evaluation aspects, and no literature report is found in the quality control aspect. The lack of quality control method cannot effectively control the stability of the quality of the therapeutic preparation, and thus affects the production and quality guarantee of the product. SUMMARY

[0004] The purpose of the present application is to provide a quality control method of a freeze-dried powder of a traditional Chinese medicine composition, that is, to comprehensively detect the quality of the material basis of the Daxiefei Decoction, establish the quality standard of the material basis of the Daxiefei Decoction, and provide a scientific basis for the development of the Daxiefei Decoction.

[0005] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0006] A quality control method of a freeze-dried powder of a traditional Chinese medicine composition, the traditional Chinese medicine composition comprising Platycladi Seed, Radix et Rhizoma Rhei, Radix Paeoniae Alba, Radix Glycyrrhizae, Radix Scrophulariae and Rhizoma Zingiberis Recens, and a content determination method of a reference index component in the traditional Chinese medicine composition is established.

[0007] S1, preparing a water decoction of the traditional Chinese medicine composition, freeze-drying to obtain a freeze-dried powder, and taking the freeze-dried powder as a material basis;

[0008] S2, preparing a test sample solution from the material basis obtained in S1;

[0009] S3, preparing a mixed control solution composed of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentio-bioside, paeoniflorin, glycyrrhizin, baicalin, aloe-emodin, 6-shogaol, rhein, chrysophanol and physcion;

[0010] S4, respectively, prepare negative sample solution containing no Platycladus orientalis, negative sample solution containing no Rheum officinale, negative sample solution containing no Radix Paeoniae Alba, negative sample solution containing no Radix Glycyrrhizae Preparata, negative sample solution containing no Scutellaria baicalensis and negative sample solution containing no Zingiber officinale;

[0011] S5, respectively, precisely take the test sample solution, mixed control sample solution and negative sample solution, and determine the content of the reference index component by high performance liquid chromatography, and the chromatographic conditions are as follows:

[0012] The chromatographic column is TC-C18 chromatographic column, the detection wavelength is 254 nm, the column temperature is 30℃, the flow rate is 1.0 mL / min, the injection amount is 10 μl, and the mobile phase is A acetonitrile-methanol-B 0.1% formic acid aqueous solution, the volume ratio of acetonitrile-methanol is 4:6, and the gradient elution program is as follows: 0-15 min, A is 5%→12%, B is 88%→95%; 15-40 min, A is 12%→20%, B is 80%→88%; 40-90 min, A is 20%→35%, B is 65%→80%; 90-110 min, A is 35%, B is 65%; 110-120 min, A is 35%→40%, B is 60%→65%; 120-160 min, A is 40%→72%, B is 28%→60%; 160-175 min, A is 72%→95%, B is 5%→28%; 175-180 min, A is 95%→100%, B is 0%→5%; 180-200 min, A is 100%, B is 0%;

[0013] The liquid phase data is obtained by determination, and the content of the reference index component in the traditional Chinese medicine composition is calculated.

[0014] Based on the above determination method, the content of each reference index component in the freeze-dried powder of the traditional Chinese medicine composition is further limited and required: each 1 g of the substance reference contains not less than 0.033 mg of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside; each 1 g of the substance reference contains not less than 2.329 mg of paeoniflorin; each 1 g of the substance reference contains not less than 0.100 mg of glycyrrhizin; each 1 g of the substance reference contains not less than 5.130 mg of baicalin; each 1 g of the substance reference contains not less than 0.009 mg of aloe-emodin; each 1 g of the substance reference contains not less than 0.200 mg of 6-shogaol; each 1 g of the substance reference contains not less than 0.029 mg of rhein; each 1 g of the substance reference contains not less than 0.013 mg of chrysophanol; and each 1 g of the substance reference contains not less than 0.006 mg of emodin methyl ether.

[0015] To further realize the present application, according to the original Fang recorded in the method of Daxie Fei decoction is "Tinglizi (black, grinding mud), Dahuang, Shaoyao (each 3 two), Gancao (zhizuo), Huangqi, Ganjiang (each 1 two), the six flavors, with water five liters, cook to take two liters, warm and divide again, two times a day", according to the literature research can know that the Eastern Han Dynasty 1 two is equal to 15.4 g now, 1 liter is equal to 200 mL, the preparation of water decoction of the reproduced traditional Chinese medicine composition: taking fried Tinglizi 46.2 g, Dahuang 46.2 g, Baishao 46.2 g, Zhigancao 15.4 g, Huangqi 15.4 g and Ganjiang 15.4 g, add pure water 1000 mL, soak for 1 h, then boil with strong fire and turn to small fire for 1 h to 400 ml of medicinal liquid, the obtained medicinal liquid is the water decoction of traditional Chinese medicine composition, combined with modern technology, the water decoction of traditional Chinese medicine composition is filtered, the filtrate is concentrated under reduced pressure to 185 mL, the water decoction of traditional Chinese medicine composition is concentrated liquid, the water decoction of traditional Chinese medicine composition concentrated liquid is placed in-80℃ refrigerator for 24 h, and then freeze-dried for 48 h, and the obtained freeze-dried powder is the material reference.

[0016] Due to the influence of the preparation process stability and the origin and batch of the decoction pieces, it is an important link in quality control, in order to further realize the present application, the extract yield (powder yield) of the material reference needs to be controlled in 15.48 %-22.40 %.

[0017] The set extract yield fluctuates within ±30 % of its average value, which ensures the stability of the preparation process.

[0018] The inventor found that the reference index component in the material reference of the present application, quercetin-3-O-beta-D-glucose-7-O-beta-D-gentisic disaccharide, only peaks when 70 % methanol is used as the solvent, and because quercetin-3-O-beta-D-glucose-7-O-beta-D-gentisic disaccharide is the index component of the monarch drug Tinglizi, 70 % methanol is determined as the sample solvent, in order to further realize the present application, the method for preparing the test solution of the material reference is designed: accurately take 0.1000 g of the material reference, add 70 % methanol to a 1 ml volumetric flask, ultrasonic for 30 min at a frequency of 50 Hz, supplement the lost weight with 70 % methanol, cool, pass through a 0.45 μm microporous filter membrane, and take the filtrate, which is obtained.

[0019] The RSD of the specificity, precision, repeatability and stability test results of the content determination method of the nine benchmark index components, quercetin-3-O-beta-D-glucose-7-O-beta-D-gentisides, paeoniflorin, glycyrrhizin, baicalin, aloe emodin, 6-gingerol, rhein, chrysophanol and emodin methyl ether, is less than 3.0%, indicating that the above test results are good; the quality concentration of the benchmark index components has good linear relationship within the respective range; the RSD of the average sample recovery rate is less than 3.0%, indicating that the method has good accuracy.

[0020] The HPLC fingerprint is established, the attribution of the characteristic peak is carried out, and the similarity evaluation is carried out, that is, by analyzing the liquid phase data, the similarity of the test product fingerprint and the control product fingerprint is calculated, and the similarity is limited to not less than 0.7 as the basis for quality control.

[0021] In order to further control the quality of the traditional Chinese medicine composition, the contents of total polysaccharide, total flavone and total polyphenol in the material reference are limited, that is, the contents of total polysaccharide, total flavone and total polyphenol in the material reference are not less than 16.89%, 28.40% and 0.49% respectively.

[0022] The content determination method of total polysaccharide, total flavone and total polyphenol in the material reference is also established, including the total polysaccharide content determination method:

[0023] S1, the test product solution is prepared from the material reference, and a glucose control solution is prepared;

[0024] S2, the ultraviolet spectrophotometry is adopted, phenol solution and concentrated sulfuric acid are used as color developing agents, the detection wavelength is 490nm, and then determination, calculation and obtaining are carried out;

[0025] The total flavone content determination method is:

[0026] S1, the test product solution is prepared from the material reference, and a rutin control solution is prepared;

[0027] S2, the ultraviolet spectrophotometry is adopted, aluminum nitrate solution and sodium hydroxide solution are used as color developing agents, the detection wavelength is 500nm, and then determination, calculation and obtaining are carried out;

[0028] The total polyphenol content determination method is:

[0029] S1, the test product solution is prepared from the material reference, and a gallic acid control solution is prepared;

[0030] S2, the ultraviolet spectrophotometry is adopted, Folin phenol solution and sodium carbonate solution are used as color developing agents, the detection wavelength is 760nm, and then determination, calculation and obtaining are carried out.

[0031] The invention also qualitatively identifies rhubarb, licorice, radix scrophulariae and dried ginger in the material reference.

[0032] In particular, the radix scrophulariae in the material reference is qualitatively identified by thin layer chromatography, including the following steps:

[0033] S1, taking the material reference to prepare a test solution;

[0034] S2, preparing a radix scrophulariae control solution, a baicalin control solution, a baicalein control solution and a negative sample solution without radix scrophulariae;

[0035] S3, taking the solutions obtained in S1 and S2 and spotting them on the same silica gel G thin layer plate, using ethyl acetate-methyl ketone-formic acid-water-methanol with a volume ratio of 5:3:0.6:1:0.4 as the developing agent, taking out after development, drying, spraying with 5% ferric trichloride ethanol test solution and observing under daylight.

[0036] The beneficial effects of the present invention compared with the prior art are:

[0037] The present invention adopts an evaluation mode combining reference index component content, fingerprint, extract yield and thin layer chromatography, and comprehensively detects the quality of the freeze-dried powder of the traditional Chinese medicine composition in the present invention, i.e. the material reference, through multiple technologies and multiple ways, formulates the quality standard of the material reference, so as to provide a scientific basis for the preparation development of the Daliufei Decoction and provide ideas for the development and research of similar prescriptions. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 It is the fingerprint superimposition graph in the experimental example 4 of the present invention, wherein S1-S15 are the HPLC fingerprints of 15 batches of material references, and R is a control graph;

[0039] Figure 2 It is the HPLC chromatogram of the test solution, the mixed control solution and each single decoction piece solution of the material reference S1 in the present invention in the experimental example 4 of the present invention, wherein 3 is quercetin-3-O-beta-D-glucose-7-O-beta-D-gentisic disaccharide; 4 is paeoniflorin; 6 is glycyrrhizin; 8 is baicalin; 12 is aloe emodin; 13 is 6-shogaol; 14 is rhein; 16 is chrysophanol; and 17 is emodin methyl ether;

[0040] Figure 3 It is the HPLC chromatogram of the test solution, the mixed control solution and each negative sample solution of the material reference S1 in the present invention in the experimental example 4 of the present invention, wherein 3 is quercetin-3-O-beta-D-glucose-7-O-beta-D-gentisic disaccharide; 4 is paeoniflorin; 6 is glycyrrhizin; 8 is baicalin; 12 is aloe emodin; 13 is 6-shogaol; 14 is rhein; 16 is chrysophanol; and 17 is emodin methyl ether;

[0041] Figure 4 Standard curve of total polysaccharide in Experimental Example 5 of the present application;

[0042] Figure 5 Standard curve of total flavonoids in Experimental Example 5 of the present application;

[0043] Figure 6 Standard curve of total polyphenols in Experimental Example 5 of the present application;

[0044] Figure 7 Thin layer identification chart in Experimental Example 6 of the present application, wherein: A is the thin layer identification chart of Rhubarb, 1-3 are test sample solutions, 4 is the Rhubarb medicinal material control solution, 5 is the emodin control solution, 6 is the rhein control solution, and 7 is the negative sample solution; B is the thin layer identification chart of Licorice, 1-3 are test sample solutions, 4 is the Licorice medicinal material control solution, 5 is the glycyrrhizic acid control solution, and 6 is the negative sample solution; C is the thin layer identification chart of Scutellaria, 1-3 are test sample solutions, 4 is the Scutellaria medicinal material control solution, 5 is the baicalin control solution, 6 is the baicalein control solution, and 7 is the negative sample solution; D is the thin layer identification chart of Dried Ginger, 1-3 are test sample solutions, 4 is the Dried Ginger medicinal material control solution, 5 is the gingerol control solution, and 6 is the negative sample solution. DETAILED DESCRIPTION

[0045] The quality control method of the freeze-dried powder of the traditional Chinese medicine composition provided by the present application is further described below in combination with experimental examples and the accompanying drawings.

[0046] Experimental Example 1: Preparation of the freeze-dried powder of the water decoction of the traditional Chinese medicine composition as a material reference:

[0047] I. Instruments and reagents

[0048] 1. Instruments 1260 II type high performance liquid chromatograph (Agilent, USA); N1210B type rotary evaporator (Shanghai Ailang Instrument Co., Ltd.); ultra-low temperature refrigerator (Matsushita Electric Co., Ltd., Japan); FDU-2110 type freeze dryer (Shanghai Ailang Instrument Co., Ltd.); ABJ80-4M type electronic analytical balance (one ten-thousandth); ABT100-5M type electronic analytical balance (one hundred-thousandth) (Germany); DT1028H ultrasonic cleaner; DHG-9070AE electric heating air drying oven (Guangdong Taishan Jun Science and Technology Instrument Co., Ltd.).

[0049] 2. Reagents

[0050] (1) Reference standards: Quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside (batch number ZC2306001), glycyrrhizin (batch number ZC06001), baicalin (batch number ZC2307001), 6-gingerol (batch number ZC2308001), Lanzhou Zhichun Biotechnology Co., Ltd., purity ≥98.0%; rhein (batch number 19010521), rutin (batch number 1609225), gallic acid (batch number 18102509), D-anhydrous glucose (batch number 22070596), Lanzhou Plante Biotechnology Co., Ltd., purity ≥98.0%. %; emodin (batch number 110756-200110), emodin methyl ether (batch number 110758-2000509), aloe-emodin (batch number 110795-200504), chrysophanol (batch number 110796-200513), paeoniflorin (batch number 110736-200630), China National Institutes for Food and Drug Control, purity ≥98.0%.

[0051] (2) Reagents: Petroleum ether (batch number: 20231101), anhydrous ethanol (batch number: 20221005), ethyl formate (batch number: 20230201), and glacial acetic acid (batch number: 20230504) were all analytical grade, Tianjin Damao Chemical Reagent Factory; ferric chloride (batch number: 20210301) and n-butanol (batch number: 20200301) were all analytical grade, Yantai Shuangshuang Chemical Co., Ltd.; ethyl acetate (batch number: 20180103) was analytical grade, Lianlong Bohua Tianjin Pharmaceutical Chemical Co., Ltd.; vanillin (batch number: 20201113) was from Tianjin Kemio Chemical Reagent Co., Ltd.; formic acid (batch number: 20200825) was chromatographic grade, Tianjin Damao Chemical Reagent Factory; methanol (batch number: 1143407112) and acetonitrile (batch number: JA100930) were both chromatographic grade, Merck GmbH, Germany.

[0052] (3) Medicinal materials: Each batch of stir-fried Lepidium apetalum, rhubarb, white peony root, dried ginger, prepared licorice root, and Scutellaria baicalensis slices was provided by the enterprise. Specific information is shown in Table 1. They were identified as *Dendrobium nobile*, a plant belonging to the Brassicaceae family. Descurainia sophia (L.) Webb.ex Prantl. The dried, mature seeds of Rheum palmatum, a plant in the Polygonaceae family. Rheum palmatum L The dried root of peony, a plant of the Ranunculaceae family. Paeonia lactiflora Pall. dried root of ginger, a plant of the ginger family. Zingiber officinale Rosc. dried rhizome of licorice, a legume. Glycyrrhiza uralensis Fisch. The dried rhizome of Scutellaria baicalensis, a plant of the Lamiaceae family. Scutellaria baicalensis Georgi The dried roots all meet the requirements of Part I of the 2020 edition of the Chinese Pharmacopoeia.

[0053]

[0054] II. Preparation of water decoction and freeze-dried powder of traditional Chinese medicine composition

[0055] According to the random number table method, 6 kinds of decoction pieces were randomly combined and sorted, and 15 batches of water decoction of traditional Chinese medicine composition corresponding to the batch of decoction pieces (as shown in Table 2). The preparation method of the traditional Chinese medicine composition recorded in the original prescription, namely Daxiefei Decoction, is “Tinglizi (blackened, pounded into mud), Dahuang, Shaoyao (each 3 liang), Gancao (fried), Huangqi, Ganjiang (each 1 liang), the above six kinds, with water 5 liters, cook to take 2 liters, warm and divide again, take twice a day, twice a day”. According to literature research, it is known that 1 liang in the Eastern Han Dynasty is equal to 15.4 g now, and 1 liter is equal to 200 mL, after conversion, 46.2 g of fried Tinglizi, 46.2 g of Dahuang, 46.2 g of Shaoyao, 15.4 g of Gancao, 15.4 g of Huangqi and 15.4 g of Ganjiang are taken, and 1000 mL of pure water is added and soaked for 1 h, then boiled with strong fire and then converted to small fire for decoction for 1 h to get 400 ml of medicinal liquid. The obtained medicinal liquid is the water decoction of traditional Chinese medicine composition, and the filtrate after filtration is concentrated under reduced pressure to 185 mL, which is the concentrated liquid of water decoction of traditional Chinese medicine composition. After being placed in a-80℃ refrigerator for 24 h, freeze-drying for 48 h, the freeze-dried powder of water decoction of traditional Chinese medicine composition obtained is used as the material reference.

[0056]

[0057] Experimental example 2, determination of the contents of 9 reference index components in the material reference:

[0058] I. Determination method:

[0059] S1, the water decoction of traditional Chinese medicine composition was prepared by the method of experimental example 1, freeze-dried to obtain freeze-dried powder, which was used as the material reference;

[0060] S2, the material reference was prepared to obtain the test solution: 0.1000 g of the material reference obtained in S1 was accurately weighed, 70% methanol was added to a 1 ml volumetric flask, ultrasonic was carried out for 30 min at a frequency of 50 Hz, 70% methanol was added to make up the weight loss, cooled, filtered through a 0.45 μm microporous filter membrane, and the filtrate was obtained, which was the test solution;

[0061] S3, preparation of mixed control solution:

[0062] (1) Respectively, precisely take quercetin-3-O-β-D-glucose-7-O-β-D-gentisides, paeoniflorin, glycyrrhizin, baicalin, aloe emodin, 6-gingerol, rhein, chrysophanol and rheochrysidin control substance, mix, methanol ultrasonic dissolution, make the mixed control stock solution with the mass concentration of 0.112, 0.432, 0.134, 0.324, 0.092, 0.30, 0.21, 0.092, 0.144 mg / ml respectively;

[0063] (2) Using step-by-step dilution method, precisely take 1 ml of mixed control stock solution, add methanol to dilute into 2 times, 4 times, 6 times, 8 times, 10 times of mixed control solution with different concentrations;

[0064] S4, respectively, prepare negative sample solution without Platycladus orientalis, negative sample solution without Rheum officinale, negative sample solution without Radix Paeoniae Alba, negative sample solution without Radix Glycyrrhizae Praeparata Cum Melle, negative sample solution without Scutellaria baicalensis and negative sample solution without Zingiber officinale:

[0065] (1) Preparation of negative sample solution without Platycladus orientalis

[0066] Take 46.2 g of Rheum officinale, 46.2 g of Radix Paeoniae Alba, 15.4 g of Radix Glycyrrhizae Praeparata Cum Melle, 15.4 g of Scutellaria baicalensis and 15.4 g of Zingiber officinale, add 1000 ml of pure water, soak for 1 h, then boil with strong fire, then change to small fire and decoct for 1 h to get 400 ml of medicinal liquid, get the water decoction of traditional Chinese medicine composition without Platycladus orientalis, filter the filtrate and concentrate under reduced pressure to 185 ml, get the water decoction of traditional Chinese medicine composition without Platycladus orientalis, put the obtained water decoction of traditional Chinese medicine composition in-80℃ refrigerator for 24 h, freeze dry for 48 h, get the freeze-dried powder as negative sample without Platycladus orientalis, precisely take 0.1000 g of negative sample, add 70% methanol to constant volume to 1 ml volumetric flask, ultrasonic for 30 min at frequency of 50 Hz, supplement the weight loss with 70% methanol, cool, pass through 0.45 μm microporous filter membrane, take the filtrate, get the negative sample solution without Platycladus orientalis;

[0067] (2) Preparation of negative sample solution without Rheum officinale

[0068] Take fried Platycladi seed 46.2 g, white peony root 46.2 g, honeyed licorice 15.4 g, scutellaria 15.4 g and dried ginger 15.4 g, add pure water 1000 mL, soak for 1 h, then use strong fire to boil and turn to small fire to decoct for 1 h to 400 ml of medicinal liquid, get the water decoction of traditional Chinese medicine composition without rhubarb, filter the filtrate, reduce pressure and concentrate to 185 mL, get the water decoction of traditional Chinese medicine composition without rhubarb concentrate, put the water decoction of traditional Chinese medicine composition concentrate obtained in-80℃ refrigerator for 24 h, freeze dry for 48 h, the freeze-dried powder obtained is the negative sample without rhubarb, accurately weigh 0.1000 g of the negative sample, add 70% methanol to constant volume to 1 ml volumetric flask, ultrasonic for 30 min at frequency 50 Hz, add 70% methanol to make up the weight loss, cool, pass through 0.45 μm microporous filter membrane, take the filtrate, get the negative sample solution without rhubarb;

[0069] (3) Preparation of negative sample solution without white peony root

[0070] Take fried Platycladi seed 46.2 g, rhubarb 46.2 g, honeyed licorice 15.4 g, scutellaria 15.4 g and dried ginger 15.4 g, add pure water 1000 mL, soak for 1 h, then use strong fire to boil and turn to small fire to decoct for 1 h to 400 ml of medicinal liquid, get the water decoction of traditional Chinese medicine composition without rhubarb, filter the filtrate, reduce pressure and concentrate to 185 mL, get the water decoction of traditional Chinese medicine composition without rhubarb concentrate, put the water decoction of traditional Chinese medicine composition concentrate obtained in-80℃ refrigerator for 24 h, freeze dry for 48 h, the freeze-dried powder obtained is the negative sample without rhubarb, accurately weigh 0.1000 g of the negative sample, add 70% methanol to constant volume to 1 ml volumetric flask, ultrasonic for 30 min at frequency 50 Hz, add 70% methanol to make up the weight loss, cool, pass through 0.45 μm microporous filter membrane, take the filtrate, get the negative sample solution without rhubarb;

[0071] (4) Preparation of negative sample solution without honeyed licorice

[0072] Take fried Platycladi seed 46.2 g, 46.2 g of large yellow, 46.2 g of white peony root, 15.4 g of Scutellaria and 15.4 g of dried ginger, soak in 1000 mL of pure water for 1 h, then boil with strong fire and turn to small fire for 1 h to 400 ml of medicinal liquid, get the water decoction of traditional Chinese medicine composition without fried licorice, filter the filtrate and concentrate under reduced pressure to 185 mL, get the water decoction of traditional Chinese medicine composition without fried licorice concentrate, put the water decoction of traditional Chinese medicine composition concentrate obtained in-80℃ refrigerator for 24 h, freeze dry for 48 h, the freeze-dried powder obtained is the negative sample without fried licorice, accurately weigh 0.1000 g of negative sample, add 70% methanol to 1 ml volumetric flask, ultrasonic for 30 min at frequency of 50 Hz, add 70% methanol to make up the weight loss, cool, filter through 0.45 μm microporous filter membrane, take the filtrate, get the negative sample solution without fried licorice;

[0073] (5) preparation of negative sample solution without Scutellaria

[0074] Take fried Platycladi seed 46.2 g, 46.2 g of large yellow, 46.2 g of white peony root, 15.4 g of Scutellaria and 15.4 g of dried ginger, soak in 1000 mL of pure water for 1 h, then boil with strong fire and turn to small fire for 1 h to 400 ml of medicinal liquid, get the water decoction of traditional Chinese medicine composition without fried licorice, filter the filtrate and concentrate under reduced pressure to 185 mL, get the water decoction of traditional Chinese medicine composition without fried licorice concentrate, put the water decoction of traditional Chinese medicine composition concentrate obtained in-80℃ refrigerator for 24 h, freeze dry for 48 h, the freeze-dried powder obtained is the negative sample without fried licorice, accurately weigh 0.1000 g of negative sample, add 70% methanol to 1 ml volumetric flask, ultrasonic for 30 min at frequency of 50 Hz, add 70% methanol to make up the weight loss, cool, filter through 0.45 μm microporous filter membrane, take the filtrate, get the negative sample solution without fried licorice;

[0075] (6) preparation of negative sample solution without dried ginger

[0076] Take fried Platycladi Seed 46.2 g, Radix et Rhizoma Rhei 46.2 g, Radix Paeoniae Alba 46.2 g, Radix Glycyrrhizae 15.4 g and Radix Scutellariae 15.4 g, add pure water 1000 mL, soak for 1 h, then use strong fire to boil and turn to small fire to decoct 1 h to 400 ml of medicinal liquid, get the water decoction of traditional Chinese medicine composition without Zingiberis Rhizoma Recens, filter the filtrate, reduce pressure and concentrate to 185 mL, get the water decoction concentrate of traditional Chinese medicine composition without Zingiberis Rhizoma Recens, put the water decoction concentrate of traditional Chinese medicine composition obtained in the-80℃ refrigerator for 24 h, freeze dry for 48 h, get the freeze-dried powder of negative sample without Zingiberis Rhizoma Recens, accurately weigh 0.1000 g of the negative sample, add 70% methanol to constant volume to 1 ml volumetric flask, ultrasonic for 30 min at 50 Hz, add 70% methanol to make up the weight loss, cool, filter through 0.45 μm microporous filter membrane, take the filtrate, get the negative sample solution without Zingiberis Rhizoma Recens;

[0077] S5, accurately weigh the test sample solution, mixed control sample solution and negative sample solution respectively, determine the content of the reference index component by high performance liquid chromatography, and the chromatographic conditions are as follows:

[0078] The chromatographic column is TC-C18 chromatographic column, the detection wavelength is 254 nm, the column temperature is 30℃, the flow rate is 1.0 mL / min, the injection volume is 10 μl, and the mobile phase is A acetonitrile-methanol-B 0.1% formic acid aqueous solution, the volume ratio of acetonitrile-methanol is 4:6, and the gradient elution program is as follows: 0-15 min, A is 5%→12%, B is 88%→95%; 15-40 min, A is 12%→20%, B is 80%→88%; 40-90 min, A is 20%→35%, B is 65%→80%; 90-110 min, A is 35%, B is 65%; 110-120 min, A is 35%→40%, B is 60%→65%; 120-160 min, A is 40%→72%, B is 28%→60%; 160-175 min, A is 72%→95%, B is 5%→28%; 175-180 min, A is 95%→100%, B is 0%→5%; 180-200 min, A is 100%, B is 0%;

[0079] Determine the liquid phase data, calculate, and get the content of the reference index component in the reference substance.

[0080] II. Methodology investigation:

[0081] 1. Specificity experiment, accurately weigh the test sample solution, mixed control sample solution and negative sample solution respectively, inject and analyze under the chromatographic conditions of S5 in "I. Content determination method", and the results of the negative sample solution are as shown in Figure 3 There is no interference at the peak time of the determined component, indicating that the method has good specificity.

[0082] 2. Linearity Examination: The mixed control stock solution prepared in S3(1) of "I. Content Determination Method" and five mixed control solutions of different concentrations prepared in S3(2) were injected and determined under the chromatographic conditions of S5 in "I. Content Determination Method". The abscissa is... （X） The mass concentrations of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, paeoniflorin, glycyrrhizin, baicalin, aloe-emodin, 6-gingerol, rhein, chrysophanol, and emodin methyl ether are represented on the ordinate. （Y） Linear regression was performed, and the regression equations are shown in Table 3. It can be seen that each component has a good linear relationship within its respective range.

[0083]

[0084] 3. Precision Test: Take one portion of the test solution and inject it six times consecutively under the chromatographic conditions specified in S5 of Section I, "Content Determination Method". The RSD values ​​of the peak areas of quercetin-3-O-β-D-glucan-7-O-β-D-gentioside, paeoniflorin, glycyrrhizin, baicalin, aloe-emodin, 6-gingerol, rhein, chrysophanol, and rhein methyl ether were 1.26%, 0.46%, 1.09%, 0.60%, 1.53%, 0.37%, 0.19%, 0.36%, and 0.56%, respectively. The RSD values ​​of the above components were all less than 2.0%, indicating that the instrument precision was good.

[0085] 4. Repeatability Test: Six aliquots of the same batch of test solution were injected separately under the chromatographic conditions specified in S5 of Section I, "Determination of Content". The RSDs of the mass fractions of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, paeoniflorin, glycyrrhizin, baicalin, aloe-rhubarb, 6-gingerol, rhein, chrysophanol, and emodin methyl ether were 1.64%, 1.07%, 1.10%, 1.16%, 1.38%, 1.74%, 1.07%, 0.91%, and 1.30%, respectively. The RSD values ​​of the above components were all less than 2.0%, indicating that the method has good repeatability.

[0086] 5、Stability investigation Take 1 portion of the test solution, under the chromatographic conditions of S5 in "I. Assay method", sample at 0, 4, 10, 17, 24 h, the RSD of peak area of quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, paeoniflorin, glycyrrhizin, baicalin, aloe-emodin, 6-shogaol, rhein, chrysophanol and physcion are 2.11%, 2.66%, 1.05%, 0.26%, 1.73%, 1.69%, 2.50%, 0.97%, 1.40% respectively, the RSD of the above components are all less than 3.0%, indicating that the sample has good stability.

[0087] 6、Recovery rate investigation Take 6 portions of 0.050 g of the substance reference with the content of each reference index component determined, place in a 1 ml volumetric flask, add mixed reference solution according to 50%, 100%, 150% of the reference index component respectively, then prepare test solution according to the method of S2 in "I. Assay method", prepare 3 portions of each mass concentration. Under the chromatographic conditions of S5 in "I. Assay method", sample and determine, then calculate the recovery rate of quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside, paeoniflorin, glycyrrhizin, baicalin, aloe-emodin, 6-shogaol, rhein, chrysophanol and physcion, the average recovery rate of the 9 components are 94.97%, 95.83, 95.54%, 96.09%, 92.01%, 95.23%, 97.17%, 94.06%, 96.80% respectively, the RSD are 1.68%, 2.70%, 1.90%, 2.18%, 2.65%, 2.59%, 2.71%, 2.71%, 1.73% respectively, the average recovery rate of each reference index component is 92%-98%, the RSD are all less than 3.0%, indicating that the method has good accuracy.

[0088] III. Determination of the content limit of the 9 reference index components in the substance reference

[0089] The minimum value of each group was determined as the content limit by determining the content of 9 reference index components in 15 batches of substance bases, i.e. each 1 g of substance reference contains not less than 0.033 mg of quercetin-3-O-beta-D-glucopyranoside-7-O-beta-D-gentiobioside; each 1 g of substance reference contains not less than 2.329 mg of paeoniflorin; each 1 g of substance reference contains not less than 0.100 mg of glycyrrhizin; each 1 g of substance reference contains not less than 5.130 mg of baicalin; each 1 g of substance reference contains not less than 0.009 mg of aloe-emodin; each 1 g of substance reference contains not less than 0.200 mg of 6-gingerol; each 1 g of substance reference contains not less than 0.029 mg of rhein; each 1 g of substance reference contains not less than 0.013 mg of chrysophanol; and each 1 g of substance reference contains not less than 0.006 mg of physcion, as shown in Table 4 and the continuation of Table 4.

[0090]

[0091]

[0092] Experimental Example 3, Reference Extracting Rate Detection:

[0093] The substance reference dry extract mass was obtained by proportionally converting the water decoction concentrate of the traditional Chinese medicine composition obtained in Experimental Example 1 and the substance reference, and the actual extracting rate of the substance reference was calculated. The dry extract mass of each single decoction piece solution and the negative sample solution was obtained by the same method, and the single decoction piece extracting rate was determined. The theoretical extracting rate of the substance reference was calculated according to the formula: theoretical extracting rate = ∑single extracting rate x single decoction piece mass / total amount of the whole prescription.

[0094]

[0095] As shown in Table 5, the average value of the actual extracting rate of the 15 batches of substance references was 18.11%, and the range of ±30% was 15.48%-22.40%. The actual extracting rate fluctuated within the range of ±30% of the average value, indicating that the preparation process of the substance reference was relatively stable, and the origin and batch of the decoction pieces had less influence.

[0096] Experimental Example 4, Fingerprint Study of Substance Reference:

[0097] I. Chromatographic conditions, preparation of test solution and preparation of mixed control solution: see Experimental Example 2.

[0098] II. Precision test: see Experimental Example 2.

[0099] III. Reproducibility investigation: see Experimental Example 2.

[0100] III. Stability investigation: see Experimental Example 2.

[0101] Four, the establishment of fingerprint and similarity evaluation

[0102] The sample solution of 15 batches of material reference was prepared by the method in experimental example 2, and was determined by injection under the chromatographic conditions in experimental example 2. The chromatograms of each batch were recorded, and the software of "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2004.A version)" was used for analysis (reference spectrum: S6; time window width: 0.1 min; median method; similarity calculation: multi-point correction and full spectrum peak matching). The fingerprint of the sample solution of 15 batches of material reference was calculated by the software to generate the control fingerprint. The results showed that the similarity of the sample solution (S1-S15) of 15 batches of material reference with the control fingerprint (R) was 0.789, 0.808, 0.854, 0.877, 0.815, 0.822, 0.937, 0.944, 0.967, 0.883, 0.903, 0.889, 0.939, 0.925, 0.933, respectively, all greater than 0.7, indicating that the overall similarity of 15 batches of material reference was good, and the fingerprint overlay of the 15 batches of material reference was shown in Figure 1 .

[0103] Five, identification and attribution of chromatographic peaks

[0104] The water decoction of the traditional Chinese medicine composition with serial number S1 in Table 2 was prepared into material reference by the method of experimental example 1. The sample solution of the material reference of S1 was prepared by the method of experimental example 2, and was compared with the fingerprint of each single herb decoction piece solution, each negative sample solution and the mixed control solution prepared in experimental example 3. It was found that the sample solution of the material reference of S1 had 17 common peaks at 254 nm wavelength, as shown in Figures 2-3 , in which peak 3 (quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside) and 5 belonged to the decoction piece of Semen Platycladi, peak 1 and 4 (paeoniflorin) belonged to the decoction piece of Radix Paeoniae Alba, peak 2, 6 (glycyrrhizin) and 15 belonged to the decoction piece of Glycyrrhiza, peak 8 (baicalin), 9, 10 and 11 belonged to the decoction piece of Radix Scutellariae, peak 13 (6-gingerol) belonged to the decoction piece of Rhizoma Zingiberis Recens, and peak 7, 12 (aloe emodin), 14 (rhein), 16 (chrysophanol) and 17 (emodin methyl ether) belonged to the decoction piece of Radix et Rhizoma Rhei. It can be seen that the main components of the material reference prepared by the process in experimental example 1 were completely transferred from the decoction piece to the material reference, and the attribution relationship was relatively clear.

[0105] Experimental example 5, determination of the contents of total polysaccharides, total flavonoids and total polyphenols in material reference:

[0106] I. Determination of total polysaccharides:

[0107] Accurately weigh 0.01091 g of glucose reference substance into a 25 ml volumetric flask, dissolve and dilute to the mark with purified water, shake well to obtain a glucose reference substance solution with a concentration of 0.4364 mg / ml. Accurately pipette 0.8, 1.0, 1.2, 1.4, 1.6 ml of the above glucose reference substance solution into 10 ml volumetric flasks, add purified water to the mark, shake well. Accurately pipette 2 ml of the above solution into a stoppered measuring cylinder, respectively add 1.0 ml of 4% phenol solution, shake well, quickly add 7 ml of concentrated sulfuric acid, shake well, incubate in a 40°C water bath for 30 min, take out, place in an ice water bath for 5 min, take out, use the corresponding reagent as a blank, measure the absorbance at a wavelength of 490 nm by ultraviolet-visible spectrophotometry, and the total polysaccharide standard curve is shown in Figure 4 .

[0108] Preparation of test sample solution: accurately weigh 0.1 g of 15 batches of material reference substance into a 25 ml volumetric flask, dilute to the mark with purified water, shake well, then take 5 ml into a 25 ml volumetric flask, shake well, then take 1 ml into a 10 ml volumetric flask, accurately pipette 2 ml of the sample solution into a stoppered measuring cylinder, respectively add 1.0 ml of 4% phenol solution, shake well, quickly add 7 ml of concentrated sulfuric acid, shake well, incubate in a 40°C water bath for 30 min, take out, place in an ice water bath for 5 min, take out, use the corresponding reagent as a blank, measure the absorbance at a wavelength of 490 nm by ultraviolet-visible spectrophotometry, and the content is measured according to the standard curve, as shown in Table 6, the content of total polysaccharide in the 15 batches of material reference substance is 16.89-27.77%.

[0109] II. Total flavone determination:

[0110] Accurately weigh 0.00507 g of rutin reference substance into a 25 ml volumetric flask, add 70% alcohol to the mark, shake well to obtain a rutin reference substance solution with a concentration of 0.2028 mg / ml. Accurately pipette 2.5 ml, 3.5 ml, 4.5 ml, 5.5 ml, 6.5 ml of the rutin reference substance solution into 25 ml volumetric flasks, respectively add 0.4 ml of 5% sodium nitrite solution, shake well and stand for 6 min, add 0.4 ml of 10% aluminum nitrate solution, stand for 6 min, add 4 ml of 4% sodium hydroxide solution, then add 70% ethanol to the mark, stand for 15 min, use 70% ethanol as a blank, measure the absorbance at a wavelength of 500 nm, and the standard curve is shown in Figure 5 .

[0111] Preparation of test solution: 0.1 g of lyophilized powder of each batch of the traditional Chinese medicine composition of the present application was precisely weighed into a 25 ml volumetric flask, and 70% ethanol was added and ultrasonically treated for 40 min. The weight was supplemented, and then 5 ml of the solution was taken into a 25 ml volumetric flask, and 70% ethanol was added to the calibration mark to obtain the test solution. 3 ml of the sample solution was precisely taken into a cylinder with a stopper, 0.4 ml of 5% sodium nitrite solution was added, and then shaken and placed for 6 min. 0.4 ml of 10% aluminum nitrate solution was added, and then placed for 6 min. 4 ml of 4% sodium hydroxide solution was added, and 70% ethanol was used as a blank. The absorbance was measured at a wavelength of 500 nm, and the total flavone content in the sample was obtained by substituting the standard curve equation. The total flavone content in the 15 batches of substance standards was 28.40-189.87%, as shown in Table 6.

[0112] III. Total polyphenol determination

[0113] 0.00251 g of gallic acid reference substance was precisely weighed into a 25 ml volumetric flask, and purified water was added to the calibration mark to obtain a gallic acid reference solution with a concentration of 0.1004 mg / ml. 1.5, 2.0, 2.5, 3.0, and 3.5 ml of the above reference solution was precisely taken into a 25 ml brown volumetric flask, and 1.5 ml of Folin phenol reagent was added in turn. After 8 min of reaction, 1.5 ml of 10% sodium carbonate solution was added, and purified water was added to the calibration mark. After standing at 25°C for 60 min (avoiding light), the corresponding reagent was used as a blank, and the absorbance was measured at a wavelength of 760 nm to prepare a standard curve, as shown in Figure 6 , and the regression equation was obtained.

[0114] Preparation of test solution: 0.1 g of lyophilized powder of each batch of the traditional Chinese medicine composition of the present application was precisely weighed into a 25 ml volumetric flask, and purified water was added to the calibration mark to obtain a gallic acid reference solution with a concentration of 0.1004 mg / ml. 1.5, 2.0, 2.5, 3.0, and 3.5 ml of the above reference solution was precisely taken into a 25 ml brown volumetric flask, and 1.5 ml of Folin phenol reagent was added in turn. After 8 min of reaction, 1.5 ml of 10% sodium carbonate solution was added, and purified water was added to the calibration mark. After standing at 25°C for 60 min (avoiding light), the corresponding reagent was used as a blank, and the absorbance was measured at a wavelength of 760 nm to prepare a standard curve, as shown in

[0115]

[0116] IV. Determination of the total polysaccharide, total flavone, and total polyphenol content limits in the substance standards

[0117] The minimum value in each group is determined as the content limit by determining the total polysaccharide, total flavonoid and total polyphenol content in 15 batches of substance reference, i.e. the content of total polysaccharide should not be less than 16.89%, the content of total flavonoid should not be less than 28.40%, and the content of total polyphenol should not be less than 0.49%.

[0118] Experimental Example 6, Qualitative Identification of Rhubarb, Licorice, Scutellaria and Dried Ginger in Substance Reference:

[0119] I. Thin Layer Chromatography Identification of Rhubarb

[0120] Substance reference was prepared by water decoction of the traditional Chinese medicine compositions with serial numbers S1, S2 and S3 in Table 2 according to the method of Experimental Example 1. 0.0782 g, 0.0713 g and 0.0695 g of the substance reference were precisely weighed, respectively. According to the thin layer chromatography method of rhubarb in Chinese Pharmacopoeia (Volume I, 2020 edition), the test solution of the substance reference, the rhubarb medicinal material reference solution, the aloe-emodin reference solution, the rhein acid reference solution, the chrysophanol reference solution, the physcion reference solution and the negative sample solution without rhubarb were obtained. The thin layer chromatography identification of rhubarb was carried out. The test solution of the substance reference showed the same orange yellow fluorescent spots at the corresponding position of the rhubarb medicinal material reference solution, and the negative sample solution without rhubarb showed no interference. See A of Figure 7

[0121] II. Thin Layer Chromatography Identification of Licorice

[0122] Substance reference was prepared by water decoction of the traditional Chinese medicine compositions with serial numbers S1, S2 and S3 in Table 2 according to the method of Experimental Example 1. 2.3172 g, 2.1291 g and 2.0780 g of the substance reference were precisely weighed, respectively. According to the thin layer chromatography method of licorice in Chinese Pharmacopoeia (Volume I, 2020 edition), the test solution of the substance reference, the licorice medicinal material reference solution, the glycyrrhizin reference solution and the negative sample solution without licorice were obtained. The thin layer chromatography identification of licorice was carried out. The test solution of the substance reference showed the same orange yellow fluorescent spots at the corresponding position of the licorice medicinal material reference solution, and the negative sample solution without licorice showed no interference. See B of Figure 7

[0123] III. Thin Layer Chromatography Identification of Scutellaria

[0124] ​​Take the water decoction of the traditional Chinese medicine composition with serial number S1, S2 and S3 in Table 2 respectively, and prepare them into substance reference according to the method of Experimental Example 1. Precisely take 2.0009 g, 2.1302 g and 2.0778 g of the substance reference respectively to prepare the test solution. According to the method of TLC of Scutellaria in Chinese Pharmacopoeia (2020 edition), the substance reference test solution, the Scutellaria medicinal material control solution, the baicalin control solution, the baicalein control solution and the negative sample solution without Scutellaria are obtained.

[0125] According to the TLC test (General test 0502), the above solutions are spotted on the same silica gel G thin layer plate, and developed with ethyl acetate-butanone-formic acid-water-methanol (5:3:0.6:1:0.4 by volume), taken out, dried, sprayed with 5% ferric trichloride ethanol solution, and observed under daylight. The results show that the same colored spots appear in the substance reference test solution at the position corresponding to the Scutellaria medicinal material control solution, and there is no interference in the negative sample solution without Scutellaria. See Figure 7 C.

[0126] IV. TLC identification of Zingiberis

[0127] Take the water decoction of the traditional Chinese medicine composition with serial number S1, S2 and S3 in Table 2 respectively, and prepare them into substance reference according to the method of Experimental Example 1. Precisely take 2.0009 g, 2.1302 g and 2.0778 g of the substance reference respectively to prepare the test solution. According to the method of TLC of Scutellaria in Chinese Pharmacopoeia (2020 edition), the substance reference test solution, the Scutellaria medicinal material control solution, the baicalin control solution, the baicalein control solution and the negative sample solution without Scutellaria are obtained. Figure 7 D.

Claims

1. A method for quality control of a freeze-dried powder of a traditional Chinese medicine composition, the traditional Chinese medicine composition comprising Semen Platycladi, Radix et Rhizoma Rhei, Radix Paeoniae Alba, Radix Glycyrrhizae Preparata, Radix Scrophulariae and Rhizoma Zingiberis Recens, characterized in that: A content determination method of the reference index component in the traditional Chinese medicine composition is established; S1, 46.2g of fried Platycladi Seed, 46.2g of Radix et Rhizoma Rhei, 46.2g of Radix Paeoniae Alba, 15.4g of Radix Glycyrrhizae Praeparata Cum Melle, 15.4g of Radix Scutellariae and 15.4g of Rhizoma Zingiberis Recens are taken, soaked in 1000ml of pure water for 1h, then boiled with strong fire and transferred to small fire for decocting for 1h to obtain 400ml of medicinal liquid, the obtained medicinal liquid is traditional Chinese medicine composition water decoction, filtered, and the filtrate is concentrated under reduced pressure to 185ml to obtain traditional Chinese medicine composition water decoction concentrate, the obtained traditional Chinese medicine composition water decoction concentrate is placed in a-80℃ refrigerator for 24h, and then freeze-dried for 48h to obtain freeze-dried powder, which is the material reference; S2, the material reference obtained in S1 is used to prepare a test sample solution; S3, a mixed control solution composed of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside, paeoniflorin, glycyrrhizin, baicalin, aloe-emodin, 6-shogaol, rhein, chrysophanol and physcion is prepared; S4, negative sample solutions without Platycladi Seed, negative sample solutions without Radix et Rhizoma Rhei, negative sample solutions without Radix Paeoniae Alba, negative sample solutions without Radix Glycyrrhizae Praeparata Cum Melle, negative sample solutions without Radix Scutellariae and negative sample solutions without Rhizoma Zingiberis Recens are prepared respectively in the traditional Chinese medicine composition; S5, the test sample solution, the mixed control solution and the negative sample solution are precisely weighed respectively, and the content of the reference index component is determined by high performance liquid chromatography, and the chromatographic conditions are as follows: The chromatographic column is TC-C18, the detection wavelength is 254nm, the column temperature is 30℃, the flow rate is 1.0ml / min, the injection amount is 10ul, the mobile phase is A acetonitrile-methanol-B 0.1% formic acid aqueous solution, the volume ratio of acetonitrile-methanol is 4:6, and the gradient elution program is as follows: 0-15min, A is 5%→12%, B is 88%→95%; 15-40min, A is 12%→20%, B is 80%→88%; 40-90min, A is 20%→35%, B is 65%→80%; 90-110min, A is 35%, B is 65%; 110-120min, A is 35%→40%, B is 60%→65%; 120-160min, A is 40%→72%, B is 28%→60%; 160-175min, A is 72%→95%, B is 5%→28%; 175-180min, A is 95%→100%, B is 0%→5%; 180-200min, A is 100%, B is 0%; The liquid phase data is obtained by determination, and the content of the reference index component in the traditional Chinese medicine composition is calculated; The content of each reference index component in the material reference is as follows: Each 1g of the material reference contains not less than 0.033mg of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside; Each 1g of the material reference contains not less than 2.329mg of paeoniflorin; Each 1g of the material reference contains not less than 0.100mg of glycyrrhizin; Each 1g of the material reference contains not less than 5.130mg of baicalin; The aloe-emodin content in each 1g of the substance reference shall not be less than 0.009mg; The 6-gingerol content in each 1g of the substance reference shall not be less than 0.200mg; The rhein content in each 1g of the substance reference shall not be less than 0.029mg; The chrysophanol content in each 1g of the substance reference shall not be less than 0.013mg; The physcion content in each 1g of the substance reference shall not be less than 0.006mg; The extract rate of the substance reference is 15.48%-22.40%; The method for preparing the test sample solution in S2 is as follows: 0.1000g of the substance reference is accurately weighed, added with 70% methanol to a 1ml volumetric flask, ultrasonically treated for 30min at a frequency of 50Hz, supplemented with 70% methanol to make up the weight loss, cooled, filtered through a 0.45μm microporous filter membrane, and the filtrate is taken to obtain the test sample solution. 2.The quality control method of the traditional Chinese medicine composition lyophilized powder according to claim 1, characterized in that: The liquid phase data obtained in S5 is analyzed, the control fingerprint spectrum is calculated by software from the test sample solution fingerprint spectrum, and the similarity of each test sample solution fingerprint spectrum to the control fingerprint spectrum is calculated, which shall not be less than 0.

7. 3.The quality control method of the traditional Chinese medicine composition lyophilized powder according to claim 1, characterized in that: The quality control method further comprises determination of the total polysaccharide, total flavone and total polyphenol contents in the substance reference, the total polysaccharide content in the substance reference shall not be less than 16.89%, the total flavone content shall not be less than 28.40%, and the total polyphenol content shall not be less than 0.49%.

4. The quality control method of the traditional Chinese medicine composition freeze-dried powder according to claim 3, characterized in that: The total polysaccharide content determination method is as follows: S1, the substance reference is taken to prepare a test sample solution, and a glucose control sample solution is prepared; S2, the ultraviolet spectrophotometry is adopted, phenol solution and concentrated sulfuric acid are used as the chromogenic agent, the detection wavelength is 490nm, and the determination and calculation are performed to obtain the result; The total flavone content determination method is as follows: S1, the substance reference is taken to prepare a test sample solution, and a rutin control sample solution is prepared; S2, the ultraviolet spectrophotometry is adopted, aluminum nitrate solution and sodium hydroxide solution are used as the chromogenic agent, the detection wavelength is 500nm, and the determination and calculation are performed to obtain the result; The total polyphenol content determination method is as follows: S1, the substance reference is taken to prepare a test sample solution, and a gallic acid control sample solution is prepared; S2, the ultraviolet spectrophotometry is adopted, Folin-phenol solution and sodium carbonate solution are used as the chromogenic agent, the detection wavelength is 760nm, and the determination and calculation are performed to obtain the result.

5. The quality control method of the traditional Chinese medicine composition lyophilized powder according to claim 4, characterized in that: The quality control method further comprises qualitative identification of rhubarb, licorice, scutellaria and zingiber in the traditional Chinese medicine composition freeze-dried powder. 6.The quality control method of the traditional Chinese medicine composition lyophilized powder according to claim 5, characterized in that: The qualitative identification of scutellaria adopts the thin layer chromatography, comprising the following steps: S1, the substance reference is taken to prepare a test sample solution; S2, a scutellaria medicinal material control sample solution, a baicalin control sample solution, a baicalein control sample solution and a negative sample solution without scutellaria are prepared; S3, the solutions obtained in S1 and S2 are taken and spotted on the same silica gel G thin layer plate, ethyl acetate-ketone-formic acid-water-methanol with a volume ratio of 5:3:0.6:1:0.4 is used as the developing agent, after development, the thin layer plate is taken out, dried, sprayed with 5% ferric trichloride ethanol reagent, and observed under sunlight.

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