Quality control method for ramulus cinnamomi decoction with pueraria lobata

Abstract

A quality control method for a Ramulus cinnamomi decoction with Pueraria lobata, which method is applied to quality control over the Ramulus cinnamomi decoction with Pueraria lobata by using puerarin, paeoniflorin and liquiritin as detection markers. The quality control method provided by the present invention can more effectively and comprehensively detect and monitor the quality of the Ramulus cinnamomi decoction with Pueraria lobata, and can better distinguish Ramulus cinnamomi decoctions with Pueraria lobata having different qualities, thereby avoiding the quality of the Ramulus cinnamomi decoctions with Pueraria lobata being uneven. In addition, the quality control method provides a scientific quality control and evaluation system for standardized production in the pharmaceutical industry.

Description

Quality control method of Guizhiga Gegen decoction TECHNICAL FIELD

[0001] The present application belongs to the quality control field of traditional Chinese medicine preparations, and particularly relates to a quality control method of Guizhiga Gegen decoction. BACKGROUND

[0002] Guizhiga Gegen decoction is recorded in Zhang Zhongjing's Treatise on Febrile Diseases of Han Dynasty, and has the effects of relieving the exterior, promoting perspiration, and ascending and diffusing the fluid. It is used for treating the symptoms of the sun syndrome, such as stiff neck and back, reverse sweating, and aversion to wind. Modern Chinese medicine is mainly used for treating wind-cold type of common cold and the symptoms caused by it, such as fever, sweating, headache, and body pain. In addition, it is also used for treating nerve root type of cervical spondylosis, Parkinson's disease, and skin rash.

[0003] The preparation raw materials of Guizhiga Gegen decoction contain six medicinal materials, and the components are complex. The effective components for playing a therapeutic role are often not a single component, but two or more components that play a synergistic effect. For example, it is recorded in many literatures that puerarin is an isoflavone compound extracted from pueraria, which has a wide range of pharmacological effects, such as anti-inflammatory, antioxidant, and anticancer effects, and is suitable for treating coronary heart disease, angina pectoris, myocardial infarction, retinal arteriovenous obstruction, and sudden deafness. Soyasaponin and daidzein are isoflavone components, which are widely distributed in commonly used traditional Chinese medicines such as pueraria and licorice, and have antioxidant activity, cardiovascular protection, anticancer effect, anti-inflammatory effect, anti-ulcer effect, and estrogen and other pharmacological effects. Paeoniflorin and paeonol are the main active components of paeony, which have the effects of protecting the cardiovascular and cerebrovascular system, protecting nerve cells, anti-inflammatory, anti-depression, immune regulation, and analgesia. Licorice glycoside is one of the main components of licorice extract, which is a flavonoid compound, and has the effects of anti-depression, antioxidant, anti-inflammatory, anti-virus, and anti-tumor. The quality detection method of the single medicinal material in the Guizhiga Gegen decoction recorded in the Pharmacopoeia mainly takes puerarin as the main quality control object, which is not enough to comprehensively evaluate the overall therapeutic effect. Because the effective components for playing a therapeutic role are often not a single component, but two or more components that play a synergistic effect, the quality detection or monitoring of a single component cannot reflect the overall therapeutic effect.

[0004] The classical compound of Guizhiga Gegen decoction has important clinical value and historical records. Combined with the research on the wide pharmacological functions of the active components of the medicine by modern science and the comprehensive quality control of the multiple components with pharmacological activities of Guizhiga Gegen decoction established by the present application, it has important significance for the application of the traditional Chinese medicine compound in the pharmaceutical industry and clinic.

[0005] Guizhi Jia Gegen Tang, as a classic Chinese medicine formula, has important clinical value. Comprehensive quality testing and monitoring of it is essential in the pharmaceutical industry. However, there is currently no comprehensive quality testing and monitoring method for Guizhi Jia Gegen Tang, and it is not possible to effectively distinguish Guizhi Jia Gegen Tang with inconsistent quality.

[0006] Because traditional Chinese medicine and its compound preparations are complex multi-component systems, their quality should be evaluated using appropriate testing methods that provide rich identification information. However, current methods such as microscopic identification, physicochemical identification, and content determination are insufficient to comprehensively assess the quality of Guizhi Jia Gegen Tang (Cinnamon Twig and Kudzu Root Decoction). Therefore, there is an urgent need for a method that can comprehensively and accurately determine the quality of Guizhi Jia Gegen Tang. Summary of the Invention

[0007] Technical problem solved: This invention provides a quality control method for Guizhi Jia Gegen Tang (Cinnamon Twig and Kudzu Root Decoction). In addition to detecting puerarin, it adds the determination and quality control of the content of paeoniflorin, paeoniflorin lactone, daidzein, daidzein and glycyrrhizin, which can more effectively and comprehensively detect and monitor the quality of Guizhi Jia Gegen Tang.

[0008] Technical solution: Application of puerarin, paeoniflorin and glycyrrhizin as detection markers in the quality control of Guizhi Jia Gegen Tang.

[0009] Optimized, the above markers also include at least one of paeoniflorin, daidzein, and daidzeinogen.

[0010] The specific application methods are as follows: a. Preparation of control solutions: Prepare reference solutions of puerarin, paeoniflorin, paeoniflorin, glycyrrhizin, daidzein, and daidzein according to the required markers; b. Preparation of test samples: Prepare the Guizhi Jia Gegen Tang preparation to a concentration equivalent to 0.2–1.0 g / mL of the original medicinal materials. -1 The solution was diluted with methanol to a concentration of 0.1 g·mL⁻¹. -1 a. Ultrasonic centrifugation, collection of supernatant, and filtration through a microporous membrane to obtain the test solution; c. Detection: Chromatographic detection, with the following characteristic peaks as the quality control targets for the formulation, namely paeoniflorin, puerarin, paeoniflorin, daidzein, glycyrrhizin, and daidzeinogen; d. Result analysis: If the content of all indicator components of the test sample meets the proposed limits, it represents that its quality is qualified; if any component does not meet the proposed limits, the quality is unqualified.

[0011] The above-mentioned control solutions are prepared as follows: accurately weigh puerarin, paeoniflorin, paeoniflorin, glycyrrhizin, daidzein, and daidzein reference standards, add 60% methanol to each and mix well to prepare puerarin reference solution, paeoniflorin reference solution, paeoniflorin reference solution, glycyrrhizin reference solution, daidzein reference solution, and daidzein reference solution, with a concentration of 0.01-1 mg / mL.

[0012] The preparation method of the above-mentioned test sample is: weighing 0.5-50 g of Guizhiga Gegen Decoction preparation, and preparing a solution with a concentration equivalent to 0.2-1.0 g·mL -1 of the original medicinal material; if the preparation is a decoction, it is concentrated at 70°C under reduced pressure to a solution with a concentration equivalent to 1.0 g·mL -1 of the original medicinal material; diluted with 60% methanol to a concentration of 0.1 g·mL -1 , ultrasonic for 10 min, centrifuged at 12000 rpm for 10 min, take the supernatant, filter through 0.22 μm microporous filter membrane as test sample solution.

[0013] The above-mentioned chromatographic detection conditions are: the chromatographic column is Agela venusil MP-C18 column; the mobile phase: A water containing 0.1% (v / v) formic acid; B acetonitrile; gradient elution: 0-25 min, 5%-20% B; 25-50 min, 20%-63% B; 50-55 min, 63%-80% B; 55-58 min, 80%-5% B; 58-60 min, 5% B; equilibrate the chromatographic column for 10 min; the flow rate is 1.0 mL·min -1 ; the detection wavelength is 230 nm and 254 nm; the injection volume is 10 μL; the column temperature is 30°C.

[0014] The limit of the above-mentioned result analysis is: puerarin ≥ 4 mg / g, albiflorin ≥ 0.5 mg / g, paeonol ≥ 3.5 mg / g, glycyrrhizin ≥ 0.5 mg / g, daidzein ≥ 0.02 mg / g, daidzin ≥ 0.8 mg / g.

[0015] Beneficial effects: The quality control method provided by the present application has the characteristics of convenience, high accuracy, good repeatability and high stability. Compared with the traditional quality control means, the content determination and quality control of paeonol, albiflorin, daidzein, daidzin and glycyrrhizin are increased on the basis of detecting puerarin, and the fingerprint method is also increased to evaluate the accuracy and stability of the preparation preparation. In the research data of the present application, more than 30% of the preparation batches determined to be qualified by the traditional quality control method have poor stability between batches, and the contents of the other five components except puerarin are uneven, indicating that the traditional quality control method cannot comprehensively control and evaluate Guizhiga Gegen Decoction. The quality control method provided by the present application can more effectively and comprehensively detect and monitor the quality of Guizhiga Gegen Decoction, can better distinguish Guizhiga Gegen Decoction with different qualities, avoid the uneven quality of Guizhiga Gegen Decoction, and also provide a scientific quality control and evaluation system for standardized production of pharmaceutical industry. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1: Schematic diagram of the quality control method of Guizhiga Gegen Decoction.

[0017] Figure 2: Fingerprint characteristic peaks of Guizhiga Gegen Decoction and chromatogram of control substance, S1 is decoction, S2 and S3 are granules, S4 is paeonol glycoside control substance.

[0018] Figure 3: Fingerprint characteristic peaks of Guizhiga Gegen Decoction and chromatogram of control substance, S1 is decoction, S2 and S3 are granules, S4 is paeonol glycoside control substance.

[0019] Figure 4: Fingerprint characteristic peaks of Guizhiga Gegen Decoction and chromatogram of control substance, S1 is decoction, S2 and S3 are granules, S4 is paeonol glycoside control substance.

[0020] Figure 5: Fingerprint characteristic peaks of Guizhiga Gegen Decoction and chromatogram of control substance, S1 is decoction, S2 and S3 are granules, S4 is paeonol glycoside control substance.

[0021] Figure 6: Fingerprint characteristic peaks of Guizhiga Gegen Decoction and chromatogram of control substance, S1 is decoction, S2 and S3 are granules, S4 is paeonol glycoside control substance.

[0022] Figure 7: Fingerprint characteristic peaks of Guizhiga Gegen Decoction and chromatogram of control substance, S1 is decoction, S2 and S3 are granules, S4 is paeonol glycoside control substance.

[0023] Figure 8: Identification of fingerprint characteristic peaks of Guizhiga Gegen Decoction, S1 is decoction, S2 and S3 are granules

[0024] Figure 9: Accuracy of fingerprint, 1 is decoction, 2, 3, 4 are granules.

[0025] Figure 10: Precision and repeatability of fingerprint, S1-S6 are 6 injections of the same sample.

[0026] Figure 11: Stability of preparation, S1-S6 are injections of the same sample at 0, 2, 8, 12, 16 and 24h respectively.

[0027] Figure 12: Generation of control fingerprint using fingerprints of multiple batches of samples, S1-S9 are 9 batches of samples, R is the generated control fingerprint.

[0028] Figure 13: Chromatogram of control substance.

[0029] Figure 14: Chromatogram of content determination of Guizhiga Gegen Decoction, S1 is decoction, S2-S9 are granules.

[0030] Figure 15: Chromatogram of content determination of Guizhiga Gegen Decoction, S10 is decoction, S11-S20 are granules.

[0031] Figure 16: Chromatogram of content determination of Guizhiga Gegen Decoction (total of 12 samples), A is decoction, B, C, D are granules. Detailed Implementation

[0032] Unless otherwise stated, the technical and scientific terms used in this invention have the meanings commonly understood by a person skilled in the art to which this invention pertains.

[0033] The technical solution of the present invention will be further described in detail below through embodiments and in conjunction with the accompanying drawings. Unless otherwise stated, the methods and materials of the embodiments described below are all conventional products that can be purchased from the market. Those skilled in the art will understand that the methods and materials described below are merely exemplary and should not be considered as limiting the scope of the present invention.

[0034] The Guizhi Jia Gegen Tang formula involved in this invention is prepared from six medicinal materials, including kudzu root, white peony root, cinnamon or cassia twig, fresh ginger or dried ginger, licorice root, and jujube. The quality control method for Guizhi Jia Gegen Tang provided by this invention is applicable to the preparation of clinically acceptable Guizhi Jia Gegen Tang products using conventional processes. The applicable dosage form can be selected from granules, powders, tablets, capsules, pills, oral liquids, and injections.

[0035] Example 1: Preparation method of Guizhi Jia Gegen Tang decoction

[0036] The traditional Chinese medicine formula Guizhi Jia Gegen Tang involved in this invention is prepared from six medicinal materials, including kudzu root, white peony root, cinnamon or cassia twig, fresh ginger or dried ginger, licorice root, and jujube. The preparation method of Guizhi Jia Gegen Tang is as follows (but not limited to the preparation method in the examples): 20g of kudzu root, 10g of cinnamon, 15g of white peony root, 3.3g of dried ginger, 20g of jujube, and 10g of prepared licorice root are decocted with 10 times the amount of water for 30 minutes. After filtration, the residue is decocted with 10 times the amount of water for 30 minutes. The two decoctions are combined and filtered through a 0.22μm filter. The filtrate is concentrated under reduced pressure at 60-70℃ to a concentration of 1g / mL of the original medicinal materials to obtain Guizhi Jia Gegen Tang.

[0037] Example 2: Preparation method of Guizhi Jia Gegen Tang granules

[0038] The traditional Chinese medicine formula of Guizhi Jia Gegen Tang involved in this invention is prepared from six medicinal materials, including kudzu root, white peony root, cinnamon or cassia twig, fresh ginger or dried ginger, licorice root, and jujube. The preparation method of Guizhi Jia Gegen Tang granules is as follows (but not limited to the preparation method in the examples):

[0039] 1. Decoction: Take 20g of kudzu root, 20g of jujube, 15g of stir-fried white peony root, 10g of cinnamon, 3.3g of dried ginger, and 10g of prepared licorice root. Add 10 times the amount of water and decoct for 30 minutes. Pour out the decoction while it is still hot, filter the residue, add 10 times the amount of water and decoct for another 30 minutes. Combine the two decoctions and filter through a 0.22μm filter.

[0040] 2. Concentration of the filtrate: The filtrate was concentrated under reduced pressure (-0.06 to -0.10 MPa, ≤70°C) to a thick paste with a relative density of 1.20 to 1.25 (60 ± 2°C), maltodextrin (3.75% of the medicinal material amount) was added, and stirred uniformly to obtain a thick paste.

[0041] 3. Drying and pulverization: The thick paste was dried under reduced pressure (-0.06 to -0.10 MPa, ≤70°C) to dryness, and pulverized through an 80-mesh sieve to obtain dry extract powder.

[0042] 4. Dry mixing: 22.21 g of dry extract powder, 12.85 g of isomalt, and 3 g of lactose were placed in a wet granulator and mixed for 5 minutes to obtain a mixture.

[0043] 5. Granulation, drying, and sizing: 0.036 g of sucralose was dissolved in 95% ethanol and added to the mixture, 30-mesh granulation was performed, 45 ± 5°C drying (moisture control below 5%), and 30-mesh sizing was performed to obtain dry granules.

[0044] 6. Total mixing: 0.9 g of red date powder essence was mixed with an equal amount of dry granules in three increments, and then added to the remaining dry granules in a mixer. After the color was uniform, the mixture was discharged to obtain a total mixture, and 42 g of granules were obtained.

[0045] Example 3: Identification of puerarin and paeoniflorin in Guizhiga Gegen Decoction

[0046] Thin layer chromatography was used to identify puerarin and paeoniflorin in Guizhiga Gegen Decoction to determine whether the preparation contained the target components, thereby confirming that the prepared preparation was the correct preparation.

[0047] 1. Thin layer chromatography identification of puerarin

[0048] Referring to the thin layer chromatography test in Chinese Pharmacopoeia General 0502, the test solution and the control solution were prepared:

[0049] (1) Preparation of the test solution: 3 g of Guizhiga Gegen Decoction was added to 25 mL of methanol, ultrasonically treated for 30 minutes, filtered through a 0.22 μm filter, and the filtrate was evaporated to dryness. 5 mL of methanol was added to dissolve the residue, which was used as the test solution.

[0050] (2) Preparation of the control medicinal material solution: 3 g of Puerariae Radix control medicinal material was added to 25 mL of methanol, ultrasonically treated for 30 minutes, filtered through a 0.22 μm filter, and the filtrate was evaporated to dryness. 5 mL of methanol was added to dissolve the residue, which was used as the control medicinal material solution.

[0051] (3) Preparation of the control solution: Puerarin control was dissolved in methanol to prepare a solution containing 1 mg per 1 mL, which was used as the control solution.

[0052] After the preparation of the solution, 2 μL of each of the above three solutions was taken and spotted on the same silica gel G thin layer plate, developed with chloroform-methanol-water (28:10:1), taken out, air dried, and examined under UV light (365 nm).

[0053] Experimental results: In the test sample chromatogram, fluorescent spots of the same color appeared at positions corresponding to the chromatogram of the control medicinal material and the chromatogram of the control.

[0054] 2. Identification of paeoniflorin by thin layer chromatography

[0055] Referring to the test of thin layer chromatography in Chinese Pharmacopoeia General 0502, the test sample solution and the control solution were prepared first:

[0056] (1) Preparation of the test sample solution: 3 g of Guizhiga Gegen decoction preparation was taken, 25 mL of ethanol was added, and ultrasonic treatment was performed for 30 minutes. After filtration through a 0.22 μm filter, the filtrate was evaporated to dryness. The residue was dissolved in 10 mL of water, and then extracted with ethyl acetate twice, 20 mL each time. The ethyl acetate liquid was discarded, and the water layer was extracted with water-saturated n-butanol twice, 20 mL each time. The n-butanol liquid was combined and evaporated to dryness. The residue was dissolved in 2 mL of methanol to serve as the test sample solution.

[0057] (2) Preparation of the control solution: 1.5 g of the control Bai-sha medicinal material was taken, 25 mL of ethanol was added, and ultrasonic treatment was performed for 30 minutes. After filtration, the filtrate was evaporated to dryness. The residue was dissolved in 2 mL of methanol to serve as the control medicinal material solution.

[0058] (3) Preparation of the control solution: Paeoniflorin control was taken, and a solution containing 1 mg per 1 mL was prepared with methanol to serve as the control solution.

[0059] After the preparation of the solution, 15 μL of the test sample solution, 5 μL of the control medicinal material solution, and 5 μL of the control solution were taken and spotted on the same silica gel G thin layer plate. The lower layer solution of chloroform-methanol-water (13:7:2) that was placed below 10°C was used as the developing agent. After development, the sample was taken out, air dried, sprayed with 5% vanillin sulfuric acid solution, heated at 105°C until the spots developed clearly, and examined under daylight.

[0060] Experimental results: In the test sample chromatogram, spots of the same color appeared at positions corresponding to the chromatogram of the control medicinal material and the chromatogram of the control.

[0061] Example 4: Establishment of the fingerprint method of Guizhiga Gegen decoction

[0062] Guizhiga Gegen decoction contains a variety of components, and there is currently no fingerprint method for Guizhiga Gegen decoction. An effective fingerprint method needs to effectively separate the chromatographic peaks corresponding to different components.

[0063] 1. Materials

[0064] Medicinal materials: Puerariae radix, Jujubae fructus, Fried Paeoniae Radix, Cinnamomi Ramulus, Zingiberis rhizoma, Glycyrrhizae Radix et Rhizoma Praeparata cum Melle;

[0065] Reference substance: Puerarin, Albiflorin, Paeoniflorin, Liquiritin, Daidzein, Soybean Glycoside;

[0066] 2. Instruments

[0067] Agilent 1100 high performance liquid chromatograph (USA, DAD detector), rotary evaporator (RE-52A, Shanghai Sai Technology Co., Ltd.).

[0068] 3. Methods and results

[0069] 3.1 Preparation of test solution

[0070] Accurately weigh 50 g of Guizhifulang decoction preparation, add water to prepare a solution with a concentration equivalent to 1.0 g·mL -1 of the original medicinal material. If the preparation is a decoction, concentrate at 70°C under reduced pressure to a solution with a concentration equivalent to 1.0 g·mL -1 of the original medicinal material). Dilute with 60% methanol to a concentration of 0.1 g·mL -1 (equivalent to 0.1 g of crude drug per milliliter), ultrasonic for 10 min, centrifuge at 12000 rpm for 10 min, take the supernatant, filter through a microporous filter membrane (0.22 μm), and use as the test solution.

[0071] 3.2 Preparation of control solution

[0072] Accurately weigh Puerarin, Albiflorin, Paeoniflorin, Liquiritin, Daidzein, and Soybean Glycoside reference substances, respectively, add 60% methanol to prepare Puerarin reference solution, Albiflorin reference solution, Paeoniflorin reference solution, Liquiritin reference solution, Daidzein reference solution, and Soybean Glycoside reference solution, all with a concentration of 0.5 mg / mL.

[0073] 3.3 Chromatographic conditions

[0074] The chromatographic column is Agela venusil MP-C18 column (4.6 mm x 250 mm, i.d. 5 μm); mobile phase: A water (0.1% formic acid), B acetonitrile; gradient elution: 0-25 min, 5%-20% B; 25-50 min, 20%-63% B; 50-55 min, 63%-80% B; 55-58 min, 80%-5% B; 58-60 min, 5% B; equilibrate the chromatographic column for 10 min; flow rate 1.0 mL·min -1 ; detection wavelength 230 nm and 254 nm; injection volume 10 μL; column temperature 30 degrees.

[0075] 3.4 Identification of fingerprint characteristic peaks

[0076] The Guizhiga Gegen decoction and granules samples were prepared according to Examples 1 and 2, and were detected by fingerprint. By comparing the absorption spectrum and the retention time of the control, 6 characteristic peaks were mainly investigated and identified as the quality control objects of the group prescription preparation. The order of the chromatographic peak retention time was as shown in Figures 2-8, and was as follows: paeonol glucoside (18.120 min), puerarin (18.540 min), paeonol (20.020 min), daidzin (22.780 min), glycyrrhizin (26.459 min), and daidzein (34.473 min). The decoction and granules of different dosage forms all contained the above 6 characteristic peaks.

[0077] 3.5 Accuracy investigation of the fingerprint

[0078] The Guizhiga Gegen decoction and granules samples (1 batch of decoction and 3 batches of granules, a total of 4 batches of samples) were prepared according to Examples 1 and 2, and were detected by fingerprint. Whether the 6 characteristic peaks as the quality control of the preparation were contained was investigated, as shown in Figure 9. The decoction and granule samples all contained the 6 characteristic peaks, which were paeonol glucoside, puerarin, paeonol, daidzin, glycyrrhizin, and daidzein, respectively. This indicated that the accuracy of the fingerprint was good.

[0079] 3.6 Precision and repeatability investigation of the fingerprint

[0080] The Guizhiga Gegen decoction granules were prepared according to Example 2, and were detected by fingerprint. The same sample was continuously injected 6 times, and the retention time and peak area of the chromatographic peak were recorded. The results are shown in Figure 10 and Table 1. The similarity of the fingerprint (wavelength 254 nm) of the same sample injected 6 times was not less than 0.980, which indicated that the precision and repeatability were good.

[0081] Table 1: Fingerprint similarity results of Guizhiga Gegen decoction, S1-S6 are 6 injections of the same sample

[0082] 3.7 Stability investigation of the preparation

[0083] The Guizhiga Gegen decoction granule sample was prepared according to Example 2, and was detected by fingerprint. The same sample was continuously determined 6 times at 0, 2, 8, 12, 16, and 24 h, respectively, and the relative retention time and relative peak area of the common peaks were investigated. The results are shown in Figure 11 and Table 2. The similarity of the sample fingerprint (wavelength 254 nm) was not less than 0.975, which indicated that the stability of the sample within 24 h was good.

[0084] Table 2: Fingerprint similarity of Guizhiga Gegen decoction, S1-S6 are the injections of the same sample at 0, 2, 8, 12, 16, and 24 h, respectively

[0085] 3.8 Generation of Comparison Fingerprints

[0086] Multiple batches of Guizhi Jia Gegen Tang decoction and granules were prepared according to Examples 1 and 2, and fingerprint spectroscopy was performed. A control fingerprint spectrum was generated using the fingerprint spectra of multiple batches of Guizhi Jia Gegen Tang (as shown in Figure 12), where S1-S9 represent nine batches of samples, and R represents the generated control fingerprint spectrum. The control fingerprint spectrum can be used as a reference to monitor and evaluate the repeatability and stability of different batches of Guizhi Jia Gegen Tang preparations.

[0087] 3.9 Formulation of fingerprint spectrum quality control standards

[0088] The quality control method for the fingerprint spectrum of Guizhi Jia Gegen Tang mainly examines the similarity of the fingerprint spectrum and six characteristic peaks (including puerarin, paeoniflorin, paeoniflorin, glycyrrhizin, daidzein, and daidzein). If the similarity between batches of the fingerprint spectrum of Guizhi Jia Gegen Tang is not less than 0.9, and each batch contains the characteristic peaks of the six indicator components, it indicates good batch repeatability and stability.

[0089] Experimental conclusion: The fingerprinting method of Guizhi Jia Gegen Tang (Cinnamon Twig and Kudzu Root Decoction) has a good separation effect on each indicator component. Quality control was performed on the six characteristic peaks in the fingerprint spectrum. At the same time, the stability of different batches of Guizhi Jia Gegen Tang was analyzed and evaluated by fingerprint spectrum similarity. The method has wide applicability, high accuracy, good stability, and is convenient and fast.

[0090] Example 5: Content determination and limit of Guizhi Jia Gegen Tang (Cinnamon Twig and Kudzu Root Decoction)

[0091] 1. Materials

[0092] Medicinal herbs: kudzu root, jujube, stir-fried white peony root, cinnamon, dried ginger, and prepared licorice root;

[0093] Reference standards: puerarin, paeoniflorin, paeoniflorin, glycyrrhizin, daidzein, daidzein;

[0094] 2. Instruments

[0095] Agilent 1100 high-performance liquid chromatograph (USA, DAD detector), rotary evaporator (Xiniu Technology, RE-52A).

[0096] 3. Methods and Results

[0097] 3.1 Preparation of the test solution

[0098] Accurately weigh 50g of the Guizhi Jia Gegen Tang preparation and dissolve it in water to a concentration equivalent to 1.0g / mL of the original medicinal material. -1 The solution. If the preparation is a decoction, it is concentrated under reduced pressure at 70℃ to a concentration equivalent to 1.0 g / mL of the original medicinal material.-1 Dissolve 0.1 g of the reference substance in 100 mL of 60% methanol (equivalent to 0.1 g of crude drug per mL) and ultrasonicate for 10 min. Centrifuge at 12000 rpm for 10 min. Take the supernatant and filter through a microporous filter (0.22 μm) to obtain the test solution. -1 Dissolve 0.1 g of the reference substance in 100 mL of 60% methanol (equivalent to 0.1 g of crude drug per mL) and ultrasonicate for 10 min. Centrifuge at 12000 rpm for 10 min. Take the supernatant and filter through a microporous filter (0.22 μm) to obtain the test solution.

[0099] 3.2 Preparation of the control solution

[0100] Accurately weigh the puerarin, albiflorin, paeoniflorin, glycyrrhizin, daidzin, and daidzin reference substance, respectively, and mix with 60% methanol to prepare the puerarin reference substance solution, albiflorin reference substance solution, paeoniflorin reference substance solution, glycyrrhizin reference substance solution, daidzin reference substance solution, and daidzin reference substance solution, each with a concentration of 0.5 mg / mL.

[0101] 3.3 Chromatographic conditions

[0102] The chromatographic column is an Agela venusil MP-C18 column (4.6 mm x 250 mm, i.d. 5 μm); the mobile phase is A water (0.1% formic acid) and B acetonitrile; gradient elution: 0-25 min, 5%-20% B; 25-50 min, 20%-63% B; 50-55 min, 63%-80% B; 55-58 min, 80%-5% B; 58-60 min, 5% B; equilibrate the chromatographic column for 10 min; flow rate: 1.0 mL / min; detection wavelength: 230 nm and 254 nm; injection volume: 10 μL; column temperature: 30 degrees. -1

[0103] 3.4 Content determination of Guizhiga Gegen Decoction

[0104] Guizhiga Gegen Decoction and Guizhiga Gegen Decoction granules (9 samples in total) were prepared according to Examples 1 and 2, and content determination was performed. The liquid chromatogram of the reference substance is shown in Figure 13, the content determination chromatogram of Guizhiga Gegen Decoction is shown in Figure 14, and the content determination results of Guizhiga Gegen Decoction preparations are shown in Table 3.

[0105] Through content determination of the 6 indicative components of Guizhiga Gegen Decoction, it was found that the 6 indicative components of the granules of batches S3 and S9 were not lower than the content of the traditional decoction (S1), the 6 indicative components of the granules of batches S6 and S8 were higher than or lower than the content of the traditional decoction (S1). The 6 indicative components of the granules of the remaining batches S2, S4, S5, and S7 were generally lower than the content of the traditional decoction (S1). The results showed that the quality of the granules of different batches was uneven.

[0106] Table 3: Content determination results of Guizhiga Gegen Decoction preparations​

[0107] In order to obtain more data (data is the basis for the content of Guizhiga Gegen Decoction limit), according to the preparation of Guizhiga Gegen Decoction and Guizhiga Gegen Decoction granules (a total of 11 samples) according to examples 1 and 2 again, the content determination, the content determination chromatogram of Guizhiga Gegen Decoction is shown in figure 15, and the content determination results of Guizhiga Gegen Decoction preparation are shown in table 4.

[0108] Through the content determination of 6 indicative components of Guizhiga Gegen Decoction, it can be distinguished that the content of 6 indicative components of granules of S11, S13, S15-S18 and S20 batches is not less than that of traditional decoction (S10), and the content of 6 indicative components of granules of S14 and S19 batches is higher than that of traditional decoction (S10) or lower than that of traditional decoction (S10). The content of 6 indicative components of granules of the rest batch S12 is generally lower than that of traditional decoction (S10). The results show that the quality of granules of different batches is uneven.

[0109] On the basis of the same preparation process, the possible reasons for the uneven quality of Guizhiga Gegen Decoction are as follows:

[0110] 1) medicinal material: different origin, different quality, different batch, different content of effective components.

[0111] 2) production equipment and facilities: different types and scales of equipment and facilities, different efficiency and output results.

[0112] 3) operator: different proficiency of operation personnel in operation process.

[0113] 4) process parameter control: different control of equipment operation, temperature, humidity, speed, time and other detailed parameters in preparation process.

[0114] 5) environment: different preparation environment, different results.

[0115] Table 4: content determination results of Guizhiga Gegen Decoction preparation

[0116] 5. Content limit of Guizhiga Gegen Decoction

[0117] In order to avoid the uneven quality of Guizhiga Gegen Decoction and effectively control and evaluate the quality of different batches (or different preparation processes), the content of 6 indicative components in Guizhiga Gegen Decoction preparation is limited according to the data of different batches from different sources, as shown in table 5. If the content of 6 indicative components of Guizhiga Gegen Decoction meets the limit, it represents that the quality is qualified, and if any component does not meet the limit, the quality is unqualified.

[0118] By limiting the content of Guizhiga Gegen Decoction, it is helpful to screen the manufacturers and production processes that meet the requirements.

[0119] Table 5: Limitation of Guizhiga Gegen Decoction content

[0120] Example 6: Comprehensive quality control and evaluation of Guizhiga Gegen Decoction by the quality control method established in the application

[0121] A, B, C, D Different manufacturers prepare different batches of Guizhiga Gegen Decoction or Guizhiga Gegen Decoction granules according to Examples 1 and 2, and conduct comprehensive quality control: including thin layer chromatography identification, fingerprint detection, and determination of 6 indicative components. The fingerprint results are shown in Figure 16, and the content determination results are shown in Table 6. Through the comprehensive quality control and analysis of Guizhiga Gegen Decoction by the quality control method established in the application, the different batches and different manufacturers of Guizhiga Gegen Decoction are comprehensively evaluated, and the results are shown in Table 7.

[0122] The comprehensive quality control analysis results show that the quality of Guizhiga Gegen Decoction from C and D manufacturers is uneven, and the process stability is unqualified; the quality of Guizhiga Gegen Decoction from A and B manufacturers meets the proposed quality requirements.

[0123] If the traditional quality control methods are used: including thin layer chromatography identification and determination of puerarin content, it is difficult to distinguish the uneven process stability and preparation quality of C and D manufacturers.

[0124] Through the comprehensive quality control analysis of the application, the advantages and disadvantages of Guizhiga Gegen Decoction can be more efficiently distinguished, which is helpful for screening and evaluating the preparation processes of different manufacturers and different batches.

[0125] Table 6: Content determination results of Guizhiga Gegen Decoction preparation

[0126] Table 7: Comprehensive quality control and evaluation results of Guizhiga Gegen Decoction

[0127] Experimental conclusion: The Guizhiga Gegen Decoction quality control method established in the application can comprehensively and systematically control and evaluate Guizhiga Gegen Decoction, and has the characteristics of wide applicability, high accuracy, good repeatability and high stability, which can more effectively and comprehensively screen the unqualified preparation production processes and distinguish the uneven quality of preparations. By comprehensively and systematically detecting the quality and batch stability of Guizhiga Gegen Decoction, the process development and standardized production of the traditional Chinese medicine formula in the pharmaceutical industry can be promoted, and the uneven quality of Guizhiga Gegen Decoction can be avoided, which is more conducive to ensuring and improving the clinical efficacy.

[0128] The embodiments of the present application are not limited to the above-described examples, and various changes and improvements can be made in form and details by those skilled in the art without departing from the spirit and scope of the present application, and these are considered to fall within the scope of the present application.

Claims

1. Application of puerarin, paeoniflorin and glycyrrhizin as detection markers in the quality control of Guizhi Jia Gegen Tang.

2. The application according to claim 1, characterized in that, It also includes at least one of paeoniflorin, daidzein, and daidzeinogen.

3. The application according to claim 1, characterized in that, The steps are as follows: a. Preparation of control solutions: Prepare control solutions of puerarin, paeoniflorin, paeoniflorin, glycyrrhizin, daidzein, and daidzein according to the required markers; b. Preparation of the test sample: Prepare the Guizhi Jia Gegen Tang (Cinnamon Twig and Kudzu Root Decoction) preparation to a concentration equivalent to 0.2–1.0 g / mL of the original medicinal materials. -1 The solution was diluted with methanol to a concentration of 0.1 g·mL⁻¹. -1 The sample was centrifuged by ultrasonication, and the supernatant was filtered through a microporous membrane to obtain the test solution. c. Detection: The following characteristic peaks were used as the quality control targets for the formulation by chromatographic detection: paeoniflorin, puerarin, paeoniflorin, daidzein, glycyrrhizin and daidzeinogen. d. Results analysis: If the content of all the indicator components of the test sample meets the proposed limits, it means that its quality is qualified; if any component does not meet the proposed limits, the quality is unqualified.

4. The application according to claim 2, characterized in that, The preparation method of the control solution is as follows: accurately weigh puerarin, paeoniflorin, paeoniflorin, glycyrrhizin, daidzein, and daidzein reference standards, add 60% methanol to each and mix well to prepare puerarin reference solution, paeoniflorin reference solution, paeoniflorin reference solution, glycyrrhizin reference solution, daidzein reference solution, and daidzein reference solution, all with a concentration of 0.01-1 mg / mL.

5. The application according to claim 2, characterized in that, The preparation method of the test sample is as follows: weigh 0.5-50g of Guizhi Jia Gegen Tang preparation, add water to prepare a concentration equivalent to 0.2-1.0g·mL of the original medicinal material. -1 The solution; if the preparation is a decoction, it is concentrated under reduced pressure at 70℃ to a concentration equivalent to 1.0 g / mL of the original medicinal material. -1 The solution was diluted with 60% methanol to a concentration of 0.1 g / mL. -1 Sonicate for 10 min, centrifuge at 12000 rpm for 10 min, take the supernatant, filter through a 0.22 μm microporous membrane, and use it as the test solution.

6. The application according to claim 2, characterized in that, The chromatographic detection conditions were as follows: Column: Agela Venusil MP-C18 column; Mobile phase: A. Water containing 0.1% (v / v) formic acid; B. Acetonitrile; Gradient elution: 0–25 min, 5%–20% B; 25–50 min, 20%–63% B; 50–55 min, 63%–80% B; 55–58 min, 80%–5% B; 58–60 min, 5% B; Column equilibration 10 min; Flow rate: 1.0 mL / min -1 The detection wavelengths were 230 nm and 254 nm; the injection volume was 10 μL; and the column temperature was 30 °C.

7. The application according to claim 2, characterized in that, The proposed limits for the results analysis are as follows: puerarin ≥ 4 mg / g, paeoniflorin ≥ 0.5 mg / g, paeoniflorin ≥ 3.5 mg / g, glycyrrhizin ≥ 0.5 mg / g, daidzein ≥ 0.02 mg / g, and daidzein ≥ 0.8 mg / g.

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