A quality detection method for Acanthopanax acanthopanax formula granules

Abstract

The present invention provides a method for detecting the quality of acanthopanax cortex formula granules, including determining the properties, dry extract yield, thin layer identification, extract, characteristic spectrum and chlorogenic acid content of the acanthopanax cortex formula granules, and limiting the content standard of the acanthopanax cortex formula granules to 3.0-20.0 mg of chlorogenic acid per 1 g, wherein the thin layer identification is identified by thin layer chromatography; the extract is determined by hot immersion; the characteristic spectrum and chlorogenic acid content are both determined by liquid chromatography. The method for detecting the quality of the acanthopanax cortex formula granules of the present invention evaluates the quality of the acanthopanax cortex formula granules through systematic detection, which can comprehensively reflect the intrinsic quality of the formula granules and better control the quality of the acanthopanax cortex formula granules.

Description

Technical Field

[0001] The invention relates to the technical field of quality control of traditional Chinese medicines, and in particular to a quality detection method for a cortex acanthopanacis formula granules. Background Art

[0002] Modern drugs need to have three characteristics: stability, uniformity, safety and effectiveness. For Chinese patent medicines, multiple means are needed to detect them to ensure the reliability and stability of the test results. Formula granules are increasingly chosen by the public because they are easy to carry, convenient to take and have reliable efficacy. Formula granules are made from single medicinal materials through water extraction, concentration, drying, granulation and other processes. Compared with traditional medicinal materials, the morphological characteristics of formula granules have changed significantly, and the authenticity and quality of the products cannot be observed with the naked eye. Therefore, it is necessary to establish a quality evaluation system for Chinese medicine formula granules to comprehensively reflect the intrinsic quality of formula granules. Acanthopanax bark is the dried root bark of Acanthopanar gracilistμlus W.W.Smith, a plant of the Araliaceae family. The roots are excavated in summer and autumn, washed, peeled, and dried. It has the effects of removing rheumatism, nourishing the liver and kidneys, strengthening tendons and bones, and promoting diuresis and reducing swelling. At present, there is no systematic quality inspection method for the formula granules of Acanthopanax Cortex. It is not comprehensive to use only the existing inspection methods to inspect the formula granules of Acanthopanax Cortex. It cannot reflect the overall intrinsic quality of the formula granules of Acanthopanax Cortex and cannot meet the quality control requirements of the formula granules of traditional Chinese medicine. Therefore, it is necessary to establish a quality inspection method for the formula granules of Acanthopanax Cortex for the quality control of medicinal materials. Summary of the invention

[0003] The purpose of the present invention is to solve the deficiencies of the prior art and provide a quality detection method for the Acanthopanax Cortex formula granules which can comprehensively reflect the intrinsic quality of the formula granules and better control the quality of the Acanthopanax Cortex formula granules.

[0004] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0005] A method for detecting the quality of Acanthopanax acanthopanax formula granules, comprising the following detection methods:

[0006] Through the properties of the Acanthopanax Cortex Formula Granules, dry extract yield, thin layer identification, characteristic spectrum, extract and chlorogenic acid content determination, the content standard of the formula granules is limited to 3.0-20.0 mg of chlorogenic acid per 1g, among which, thin layer identification is carried out by thin layer chromatography; characteristic spectrum and chlorogenic acid content are determined by liquid chromatography; extract is determined by hot leaching method;

[0007] The characteristic spectrum is determined by liquid chromatography, including: performing liquid chromatography analysis, using a solution made from a cortex acanthopanacis reference medicinal material as a reference medicinal material reference solution b, using a solution made from a chlorogenic acid reference substance as a reference substance reference solution b, using a solution made from a cortex acanthopanacis formula granule sample as a test solution b, accurately aspirating the reference medicinal material reference solution b, the reference substance reference solution b and the test solution b, respectively, injecting them into a liquid chromatograph, and determining, to obtain; wherein the chromatographic conditions used are: chromatographic column: octadecylsilane bonded silica gel as filler, Shimadzu Shim-pack GIST C18-AQ (4.6mm×250mm, 5μm); mobile phase: acetonitrile as mobile phase A, 0.2% formic acid solution as mobile phase B, and gradient elution according to the provisions of Table a;

[0008] Table a Gradient elution program

[0009] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～25 0→15 100→85 25～45 15→20 85→80 45～85 20→48 80→52 85～88 48→0 52→100 88～98 0 100

[0010] Flow rate: 1.0 mL / min; column temperature: 20°C; injection volume: 10 μL; detection wavelength: 280 nm.

[0011] In one embodiment, the preparation method of the Acanthopanax Cortex formula granules is as follows: take 5600g of Acanthopanax Cortex decoction pieces, add water to boil, filter, concentrate the filtrate into a clear paste, add appropriate amount of auxiliary materials, dry or dry and crush, then add appropriate amount of auxiliary materials, mix well, granulate, and make 1000g.

[0012] In one embodiment, the thin layer chromatography method comprises the following steps: (1) preparing a test solution a: taking 2.0 g of a sample of acanthopanax cortex granules, adding 30 mL of water, ultrasonically treating for 30 min, extracting twice with dichloromethane shaking, 20 mL each time, combining the dichloromethane solutions, evaporating to dryness, adding 1 mL of methanol to the residue to dissolve it, and preparing a test solution a; (2) preparing a control medicinal material solution a: taking 1.0 g of acanthopanax cortex control medicinal material, adding 50 mL of water, boiling for 30 minutes, filtering, concentrating the filtrate to about 30 mL, cooling, and purifying with dichloromethane. Extract twice with 20 ml of dichloromethane each time, combine the dichloromethane solution, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, and prepare the reference medicinal material solution a; (3) perform thin layer chromatography analysis: the thin layer chromatography conditions are as follows: thin layer plate: silica gel G thin layer plate; sample volume: 2 uL of test solution a, 3 uL of reference medicinal material solution a; developing agent: petroleum ether (60-90°C)-ethyl acetate-formic acid (10:3:0.1) as the developing agent; color developer: 10% sulfuric acid ethanol test solution, heated at 105°C, and inspected under ultraviolet light at 365 nm.

[0013] In one embodiment, the hot dipping method uses ethanol as solvent, and the hot dipping method under the alcohol-soluble extract determination method is used to determine the extract range.

[0014] In one embodiment, the characteristic spectrum is determined by liquid chromatography, and the following steps are included: (1) preparing a reference medicinal material reference solution b: taking 0.2 g of Acanthopanax cortex reference medicinal material, adding 50 mL of 50% methanol, ultrasonically treating for 20 min, cooling, filtering, and taking a filtrate as a reference medicinal material reference solution b; (2) preparing a reference substance reference solution b: taking an appropriate amount of chlorogenic acid reference substance, accurately weighing, adding methanol to dissolve, and preparing a reference substance reference solution b with a concentration of 20 ug / mL; (3) preparing a test solution b: taking an appropriate amount of Acanthopanax cortex formula granules, grinding, taking about 0.2 g, placing in a stoppered conical flask, adding 50 mL of 50% methanol, weighing, ultrasonicating for 30 min, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking well, filtering, and taking a filtrate as a test solution b.

[0015] In one embodiment, the determination of the chlorogenic acid content by liquid chromatography includes: performing liquid chromatography analysis, using a solution made from a chlorogenic acid reference substance as a reference solution c, and using a solution made from a sample of acanthopanax cortex formula granules as a test solution c, accurately aspirating the reference solution c and the test solution c, respectively, injecting them into a liquid chromatograph, and determining to obtain; wherein, the chromatographic conditions used are: chromatographic column: octadecylsilane bonded silica gel as a filler (4.6 mm×250 mm, 5 μm); mobile phase: acetonitrile as mobile phase A, 0.2% formic acid solution as mobile phase B, and gradient elution as specified; flow rate: 1.0 mL / min; column temperature: 20°C; injection volume: 10 μL; detection wavelength: 325 nm.

[0016] In one embodiment, the determination of chlorogenic acid content by liquid chromatography also includes the following steps: (1) preparing a reference solution: taking an appropriate amount of chlorogenic acid reference substance, accurately weighing it, adding methanol to prepare a solution containing chlorogenic acid at a concentration of 20ug / ml, as the reference solution c; (2) preparing a test solution: taking an appropriate amount of Acanthopanax Cortex granules, grinding them, taking about 0.2g, accurately weighing it, placing it in a stoppered conical flask, accurately adding 50ml of 50% methanol, sealing it, weighing it, ultrasonicating it for 30min, cooling it, weighing it again, making up the lost weight with 50% methanol, shaking it well, filtering it, and taking the filtrate as the test solution c.

[0017] Compared with the prior art, the beneficial effect of the quality detection method of the scutellaria baicalensis formula granules provided by the present invention is: by studying the properties, dry extract yield, thin layer identification, extract, characteristic spectrum and chlorogenic acid content of the scutellaria baicalensis formula granules, through multi-faceted measurements, the quality of the scutellaria baicalensis formula granules is evaluated, a solid foundation is laid for the stability of product quality, a feasible quality standard for the scutellaria baicalensis formula granules can be established, and the quality of the scutellaria baicalensis formula granules can be effectively controlled. In addition, by using the chromatographic conditions of the present application for liquid phase analysis, a better and clearer characteristic spectrum can be obtained, and the method has good stability, high precision and good reproducibility. The scutellaria baicalensis decoction pieces are decocted to obtain the scutellaria baicalensis decoction pieces granules, and the average chlorogenic acid content is 7.84 mg / g. According to the "Technical Requirements for Quality Control and Standardization of Chinese Medicine Formula Granules", the chlorogenic acid content range is proposed to be 3.0 mg / g to 20.0 mg / g. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the TLC spectrum of thin layer identification of three batches of production samples of the formula granules of Acanthopanax Acanthopanax decoction pieces according to thin layer chromatography in one embodiment of the present invention.

[0019] Figure 2 The HPLC characteristic spectra of different extraction methods in the investigation of the characteristic spectrum determination extraction method of one embodiment of the present invention are shown; wherein S1 is the characteristic spectrum of the test solution extracted by ultrasonic extraction; and S2 is the characteristic spectrum of the test solution extracted by reflux extraction.

[0020] Figure 3 The HPLC characteristic spectra of different extraction times in the characteristic spectrum determination extraction time investigation of one embodiment of the present invention; wherein S1 is the characteristic spectrum of the test solution extracted by ultrasonic extraction for 40 minutes; S2 is the characteristic spectrum of the test solution extracted by ultrasonic extraction for 30 minutes; and S3 is the characteristic spectrum of the test solution extracted by ultrasonic extraction for 20 minutes.

[0021] Figure 4 The HPLC characteristic spectra of different extraction solvents in the characteristic spectrum determination extraction solvent investigation of one embodiment of the present invention; wherein S1 is the characteristic spectrum of the test solution extracted with 50% ethanol; S2 is the characteristic spectrum of the test solution extracted with 50% methanol; and S3 is the characteristic spectrum of the test solution extracted with 10% methanol.

[0022] Figure 5 HPLC characteristic spectra of different sample amounts in the characteristic spectrum determination sample amount investigation of one embodiment of the present invention; wherein S1 is the characteristic spectrum of the test solution with a sample amount of 0.3g; S2 is the characteristic spectrum of the test solution with a sample amount of 0.2g; S3 is the characteristic spectrum of the test solution with a sample amount of 0.1g

[0023] Figure 6It is the HPLC characteristic spectrum of different solutions in the investigation of the specificity of the characteristic spectrum determination of one embodiment of the present invention; wherein, S1 is the characteristic spectrum of the maltodextrin blank control solution, S2 is the characteristic spectrum of the Acanthopanax Cortex test solution, S3 is the characteristic spectrum of the Acanthopanax Cortex reference medicinal material solution, and S4 is the characteristic spectrum of the chlorogenic acid reference solution.

[0024] Figure 7 This is a common peak overlay spectrum of a repeatability test in a characteristic spectrum determination according to an embodiment of the present invention.

[0025] Figure 8 This is a common peak overlay spectrum of the precision test in the characteristic spectrum determination of one embodiment of the present invention.

[0026] Fig. 9 This is an overlay spectrum of common peaks in a stability test in the characteristic spectrum determination according to one embodiment of the present invention.

[0027] Fig.10 This is a characteristic spectrum of an embodiment of the present invention, in which different column temperatures (±2°C) are examined and superimposed on each other; wherein S1 is a chromatographic column with a column temperature of 18°C, S2 is a chromatographic column with a column temperature of 20°C, and S3 is a chromatographic column with a column temperature of 22°C.

[0028] Fig.11 This is a characteristic spectrum of an embodiment of the present invention, in which different column temperatures (±5°C) are measured and superimposed on the characteristic spectrum; wherein S1 is a chromatographic column with a column temperature of 15°C, S2 is a chromatographic column with a column temperature of 20°C, and S3 is a chromatographic column with a column temperature of 25°C.

[0029] Fig.12 This is a characteristic spectrum of a common peak superposition characteristic spectrum of a characteristic spectrum determination flow rate test (±0.02 ml / min) in one embodiment of the present invention; wherein S1 is a chromatographic column with a flow rate of 0.98 ml / min, S2 is a chromatographic column with a flow rate of 1.0 ml / min, and S3 is a chromatographic column with a flow rate of 1.02 ml / min.

[0030] Fig.13 This is a characteristic spectrum of a common peak superposition characteristic spectrum of a characteristic spectrum determination flow rate test (±0.2ml / min) of an embodiment of the present invention; wherein S1 is a chromatographic column with a flow rate of 0.8ml / min, S2 is a chromatographic column with a flow rate of 1.0ml / min, and S3 is a chromatographic column with a flow rate of 1.2ml / min.

[0031] Fig.14 This is an overlay of characteristic spectra of three batches of finished products in one embodiment of the present invention; among them, the finished product number of S1 is ZS220601, the finished product number of S2 is ZS220602, and the finished product number of S3 is ZS220603.

[0032] Fig.15It is an overlay graph of the Acanthopanax Cortex finished product, chlorogenic acid reference substance, and Acanthopanax Cortex reference medicinal material in one embodiment of the present invention, wherein S1 is a characteristic graph of the fitting of three Acanthopanax Cortex finished product solutions, S2 is a characteristic graph of the Acanthopanax Cortex reference medicinal material solution, and S3 is a characteristic graph of the chlorogenic acid reference substance solution.

[0033] Fig.16 This is a fitting diagram of the characteristic spectra of the Acanthopanax Cortex finished product solution and the Acanthopanax Cortex control medicinal material solution in one embodiment of the present invention.

[0034] Fig.17 This is a maltodextrin blank comparison diagram in the content determination specificity investigation of an embodiment of the present invention; wherein, S1 is maltodextrin, S2 is a chlorogenic acid reference substance, and S3 is a Acanthopanax cortex test substance. DETAILED DESCRIPTION

[0035] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0036] The present invention provides a quality detection method for a cortex acanthopanacis formula granules, comprising the following detection method, by determining the properties, dry extract yield, thin layer identification, characteristic spectrum, extract and chlorogenic acid content of the cortex acanthopanacis formula granules, the content standard of the formula granules is limited to 3.0-20.0 mg of chlorogenic acid per 1 g, wherein the thin layer identification is performed by thin layer chromatography; the characteristic spectrum and chlorogenic acid content are both determined by liquid chromatography; and the extract is determined by hot leaching.

[0037] In this embodiment:

[0038] Preparation of Acanthopanax gracilistylus formula granules: Formula granules made according to the "Technical Requirements" of the source and medicinal parts of Acanthopanax gracilistylus Chinese medicinal materials in the 2020 edition of the Chinese Pharmacopoeia, including the family name, Chinese name, Latin name and medicinal parts of the medicinal plant, that is, this product is a formula granule made by processing the root bark of Acanthopanax gracilistylus WWSmith, a plant of the Araliaceae family, and processing it according to the main quality indicators of the formula granules. The specific preparation method is: take 5600g of Acanthopanax gracilistylus slices, add water to decoct, filter, concentrate the filtrate into a clear paste (the dry extract yield is 9.0% to 15.9%), add an appropriate amount of auxiliary materials, dry and crush, add an appropriate amount of auxiliary materials, mix well, granulate, and make 1000g, that is, each 1g of formula granules is equivalent to 5.6g of slices. The dry extract yield is determined in the range of 70% to 130% based on the yield of 24 batches of standard decoctions, and the allowable range of the dry extract yield of Acanthopanax acanthopanax decoction piece formula granules is proposed to be 9.0% to 15.9%.

[0039] 1. Characteristic investigation

[0040] Properties: Take one sample each from three batches of large-scale production of Acanthopanax Cortex Granules, observe and record the sample's morphology, color and odor, and combine the color and odor of the standard soup sample. This product is a granule, yellow-brown to brown-brown in color; slight odor, bitter and slightly pungent in taste.

[0041] 2. Thin layer identification

[0042] With reference to the published thin-layer identification method of the quality standard of Acanthopanax Cortex in Hunan Province, the thin-layer identification method of this product was established by using Acanthopanax Cortex as a control medicinal material. After methodological investigation and 24 batches of sample tests, the chromatographic spots of the test product were clear and the negative control sample had no interference. Therefore, this method was included in the [Identification] item of the quality standard of Acanthopanax Cortex Granules. Specifically, the thin-layer identification method of Acanthopanax Cortex Granules is:

[0043] Preparation of test solution: Take 2.0g of the product, grind it into powder, add 30ml of water, ultrasonically treat it for 30 minutes, cool it, and extract it twice with dichloromethane by shaking, 20ml each time, combine the dichloromethane liquid, evaporate it to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the dichloromethane test solution.

[0044] Reference medicinal material solution: Take another 1.0g of Acanthopanax cortex as reference medicinal material, add 50ml of water, boil for 30 minutes, filter, concentrate the filtrate to about 30ml, cool, shake and extract with dichloromethane twice, and follow the above operation to prepare the reference medicinal material solution.

[0045] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 2 μl of the test sample solution and 3 μl of the control drug solution were respectively spotted on the same silica gel G thin layer plate, and petroleum ether (60-90°C)-ethyl acetate-formic acid (10:3:0.1) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% ethanolic sulfuric acid test solution, heated at 105°C until the spots were clearly colored, and inspected under ultraviolet light at 365nm.

[0046] According to the above detection method, take three batches of production sample test solution, negative sample (maltodextrin) solution and control medicinal material solution, spot and perform thin layer identification. Figure 1 As shown, the results show that in the chromatograms of the three batches of large-scale production samples, fluorescent spots of the same color appear at the corresponding positions of the chromatograms of the control medicinal materials.

[0047] 3. Feature Spectrum

[0048] 3.1 Characteristic spectrum determination method

[0049] 3.1.1 Chromatographic conditions and system suitability experiments Octadecylsilane bonded silica gel was used as the filler (Shimadzu Shim-pack GIST C18-AQ (4.6 mm × 250 mm, 5 μm)); acetonitrile was used as the mobile phase A, and 0.2% formic acid solution was used as the mobile phase B. Gradient elution was performed according to the provisions in Table 1; the flow rate was 1.0 ml / min; the column temperature was 20°C; the detection wavelength was 280 nm, and the injection volume was 10 μl. The number of theoretical plates calculated based on the chlorogenic acid peak should be no less than 5000.

[0050] Table 1 - Gradient elution program

[0051] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～25 0→15 100→85 25～45 15→20 85→80 45～85 20→48 80→52 85～88 48→0 52→100 88～98 0 100

[0052] 3.1.2 Preparation of reference solution

[0053] Take 0.2 g of Acanthopanax Cortex as a control medicinal material, add 50 ml of 50% methanol, weigh the weight, ultrasonicate (power 300 W, frequency 40 kHz) for 20 minutes, cool, filter, and take the filtrate as the control medicinal material reference solution.

[0054] Take an appropriate amount of chlorogenic acid reference substance, weigh it accurately, and add methanol to prepare a solution containing 20 μg of chlorogenic acid per 1 ml, which serves as the reference substance solution.

[0055] 3.1.3 Preparation of test solution

[0056] Take an appropriate amount of this product, grind it into powder, take about 0.2g, put it in a stoppered conical flask, add 50mL of 50% methanol, weigh it, ultrasonicate (power 300W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0057] 3.1.4 Determination method

[0058] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and measure to obtain the result.

[0059] 3.2 Methodological investigation of characteristic spectrum determination

[0060] 3.2.1 Investigation of extraction methods

[0061] Prepare the test sample solution using different extraction methods and perform the test according to the method in “3.1” above. Figure 2 As shown in Table 2, the results show that the number of main peaks in the chromatograms of the test sample solutions extracted by different extraction methods is consistent, and the peak shapes are not significantly different. However, the ultrasonic extraction method is simpler, so ultrasound is selected as the sample extraction method.

[0062] Table 2 - Comparison of extraction methods

[0063]

[0064] 3.2.2 Extraction time investigation

[0065] Prepare the test solution at different times and perform the test according to the method under "3.1" above. Figure 3 As shown in Table 3, the results show that the number of main peaks in the chromatogram of the test solution at different extraction times is consistent, and the peak shape difference is not obvious. In order to shorten the detection time, the extraction time of the sample is determined to be 20 minutes.

[0066] Table 3 - Comparison of extraction times

[0067]

[0068] 3.2.3 Investigation of extraction solvent

[0069] Prepare the test sample solution with different extraction solvents and perform the determination according to the method under "3.1" above. Figure 4 As shown in Table 4, the results show that the number of main peaks in the chromatograms of the test sample solutions with different extraction solvents is consistent, and the peak shapes are not significantly different, but the extraction effect of 50% methanol is slightly better than that of 10% methanol and 50% ethanol, so 50% methanol is selected as the extraction solvent.

[0070] Table 4 - Comparison of extraction solvents

[0071]

[0072] 3.2.4 Investigation of sampling volume

[0073] Prepare the test sample solution with different sampling volumes and perform the determination according to the method under "3.1" above. Figure 5 As shown in Table 5, the results show that the number of main peaks in the chromatogram of the test solution with different sampling amounts is consistent, and the peak shape difference is small, but the extraction effect is better when the sampling amount is 0.2g, so the sampling amount of the test solution preparation is selected as 0.2g.

[0074] Table 5 - Comparison of sampling volumes

[0075]

[0076] In summary, the method for preparing the test solution is determined as follows: take an appropriate amount of fine powder of the product, take about 0.2 g, place it in a stoppered conical flask, add 50 mL of 50% methanol, weigh the weight, ultrasonic (power 300 W, frequency 40 kHz) for 20 minutes, cool, weigh again, make up the lost weight with 50% methanol, filter, and take the filtrate to obtain.

[0077] 3.3 Verification of characteristic spectrum analysis method

[0078] 3.3.1 Specificity inspection

[0079] Take maltodextrin, about 0.2g, and prepare maltodextrin blank control solution according to the method under "3.1" above, and take Acanthopanax Cortex test solution, maltodextrin blank control solution, Acanthopanax Cortex control medicinal material solution and chlorogenic acid reference solution for determination.

[0080] The results showed that the main characteristic peaks in the chromatogram of the test sample were well separated, and the maltodextrin blank had no interference. The method had good specificity. Figure 6 .

[0081] 3.3.2 Repeatability test

[0082] Take about 0.2g of the sample of Acanthopanax Cortex Granules (batch number: 211103), a total of 6 portions, and measure them according to the method under "3.1". The results showed that the peak shape and number of peaks in the characteristic spectrum of the test sample were basically consistent. There are 5 common peaks in the characteristic spectrum. Taking the chlorogenic acid peak as the reference peak S, the relative retention time and relative peak area of ​​peak 1, peak 3, peak 4, peak 5 and peak S are calculated, and the RSD value is calculated. The calculation results show that the relative peak area RSD value and the relative retention time RSD value are both less than 3.0%, which are within the qualified range. The experiment showed that this method has good repeatability, see for details. Figure 7 and Tables 6-7.

[0083] Table 6-Relative retention time of characteristic spectra of repeatability test

[0084] Peak S1 S2 S3 S4 S5 S6 RSD(%) 1 0.793 0.792 0.794 0.797 0.797 0.797 0.26 2(S) 1 1 1 1 1 1 0.00 3 1.27 1.271 1.275 1.277 1.277 1.277 0.23 4 1.904 1.908 1.897 1.888 1.886 1.885 0.47 5 2.504 2.507 2.481 2.46 2.457 2.455 0.87

[0085] Table 7-Relative peak area of ​​characteristic spectrum of repeatability test

[0086] Peak S1 S2 S3 S4 S5 S6 RSD(%) 1 0.274 0.281 0.274 0.273 0.273 0.269 1.34 2(S) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 3 1.058 1.054 1.048 1.040 1.039 1.036 0.79 4 0.463 0.451 0.488 0.475 0.481 0.484 2.75 5 0.732 0.733 0.728 0.719 0.717 0.715 1.01

[0087] 3.3.3 Precision test

[0088] Take about 0.2g of the sample of Acanthopanax Cortex Granules (Batch No.: 211103), and perform 6 consecutive injections for determination according to the test method under "3.1". The test results show that the peak shape and number of peaks in the characteristic spectrum of the test sample are basically consistent. There are 5 common peaks in the characteristic spectrum. Taking the chlorogenic acid peak as the reference peak S, the relative retention time and relative peak area of ​​peak 1, peak 3, peak 4, peak 5 and peak S are calculated, and the RSD value is calculated. The calculation results show that the relative peak area RSD value and the relative retention time RSD value are both less than 3.0%, which are within the qualified range. The experiment shows that this method has good precision, see for details. Figure 8 and Tables 8-9.

[0089] Table 8 - Relative retention time of HPLC characteristic spectrum of precision test

[0090] Peak S1 S2 S3 S4 S5 S6 RSD(%) 1 0.805 0.804 0.804 0.804 0.803 0.802 0.12 2(S) 1 1 1 1 1 1 0.00 3 1.278 1.278 1.279 1.278 1.278 1.277 0.05 4 1.887 1.887 1.887 1.889 1.892 1.893 0.13 5 2.456 2.456 2.456 2.458 2.464 2.468 0.19

[0091] Table 9 - Relative peak area of ​​HPLC characteristic spectrum of precision test

[0092] Peak S1 S2 S3 S4 S5 S6 RSD(%) 1 0.2959 0.2948 0.2942 0.2931 0.2944 0.2896 0.68 2(S) 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 3 0.9705 0.9858 0.9883 0.9773 0.9871 0.9838 0.64 4 0.5013 0.4848 0.5080 0.4863 0.4767 0.4819 2.26 5 0.7019 0.7028 0.7039 0.7019 0.7031 0.7052 0.17

[0093] 3.3.4 Stability test

[0094] Take about 0.2g of the sample of Acanthopanax Cortex Granules (batch number: 211103) and test it at 0h, 2h, 4h, 8h, 12h and 24h according to the test method under "3.1". The results show that the peak shape and peak number of the characteristic spectrum of the test sample are basically the same, see Fig. 9 There are 5 common peaks in the characteristic spectrum. Taking the chlorogenic acid peak as the reference peak S, the relative retention time and relative peak area of ​​peak 1, peak 3, peak 4, peak 5 and peak S are calculated, and the RSD value is calculated. The calculation results show that the relative peak area RSD value and the relative retention time RSD value are both less than 3.0%, which is within the qualified range. See Table 10-11 for details, indicating that the test solution is relatively stable within 24 hours.

[0095] Table 10 - Relative retention time of characteristic spectrum of stability test

[0096] Peak 0h 2h 4h 8h 12h 24h RSD(%) 1 0.79 0.792 0.792 0.792 0.79 0.789 0.15 2(S) 1 1 1 1 1 1 0.00 3 1.314 1.314 1.314 1.312 1.314 1.314 0.06 4 1.822 1.818 1.814 1.816 1.821 1.832 0.32 5 2.347 2.342 2.336 2.34 2.347 2.364 0.38

[0097] Table 11 - Relative peak area of ​​characteristic spectrum of stability test

[0098] Peak 0h 2h 4h 8h 12h 24h RSD(%) 1 0.2775 0.2800 0.2788 0.2797 0.2832 0.2834 0.78 2(S) 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 3 0.9682 0.9850 1.0340 1.0111 0.9877 0.9920 2.11 4 0.4947 0.5058 0.5057 0.5082 0.5043 0.4869 1.52 5 0.7281 0.7385 0.7393 0.7430 0.7395 0.7407 0.64

[0099] 3.4 Durability test

[0100] 3.4.1 Investigation of different column temperatures (±2°C)

[0101] Take about 0.2g of the sample of Acanthopanax Cortex Granules (batch number: 211103) and perform the test according to the test method in item "3.1" at column temperatures of 18℃, 20℃, and 22℃. Fig.10 , the measurement results show that the peak shape and number of peaks of the characteristic spectrum of the test sample are basically the same. There are 5 common peaks in the characteristic spectrum. Taking the chlorogenic acid peak as the reference peak S, the relative retention time and relative peak area of ​​peak 1, peak 3, peak 4, peak 5 and peak S are calculated, and the RSD value is calculated. The calculation results show that the relative retention time RSD% value and the relative peak area RSD% value are both less than 3.0%, which is within the qualified range. See Table 12-13 for details. The experiment shows that different column temperature changes have little effect on the characteristic spectrum determination, that is, different column temperatures have good durability.

[0102] Table 12- Relative retention time of characteristic spectrum of column temperature test (±2℃)

[0103] Peak 18℃ 20℃ 22℃ RSD(%) 1 0.802 0.804 0.803 0.12 2(S) 1 1 1 0.00 3 1.28 1.278 1.271 0.37 4 1.891 1.887 1.89 0.11 5 2.461 2.457 2.471 0.29

[0104] Table 13- Relative peak area of ​​characteristic spectrum of column temperature test (±2℃)

[0105] Peak 18℃ 20℃ 22℃ RSD(%) 1 0.293 0.284 0.301 2.96 2(S) 1.000 1.000 1.000 0.00 3 1.045 0.991 1.027 2.73 4 0.509 0.520 0.506 1.48 5 0.698 0.707 0.717 1.30

[0106] 3.4.2 Investigation of different column temperatures (±5°C)

[0107] Take about 0.2g of the sample of Acanthopanax Cortex Granules (batch number: 211103) and perform the test according to the test method in item "3.1" at column temperatures of 15℃, 20℃, and 25℃. Fig.11 , the measurement results show that the peak shape and number of peaks of the characteristic spectrum of the test sample are basically the same. There are 5 common peaks in the characteristic spectrum. Taking the chlorogenic acid peak as the reference peak S, the relative retention time and relative peak area of ​​peak 1, peak 3, peak 4, peak 5 and peak S are calculated, and the RSD value is calculated. The calculation results show that the relative retention time RSD% value is less than 3.0%, and the relative peak area RSD% value is greater than 3.0%, see Table 14-15 for details. The experiment shows that different column temperature changes have a greater impact on the characteristic spectrum determination, that is, the column temperature is set to 20°C.

[0108] Table 14- Relative retention time of characteristic spectrum of column temperature test (±5℃)

[0109] Peak 15℃ 20℃ 25℃ RSD(%) 1 0.805 0.804 0.795 0.69 2(S) 1.000 1.000 1.000 0.00 3 1.287 1.278 1.26 1.08 4 1.888 1.887 1.906 0.56 5 2.448 2.457 2.52 1.59

[0110] Table 15- Relative peak area of ​​characteristic spectrum of column temperature test (±5℃)

[0111]

[0112]

[0113] 3.4.3 Investigation of different flow rates (±0.02ml / min)

[0114] Take about 0.2g of the sample of Acanthopanax Cortex Granules (batch number: 211103) and perform the test according to the test method in item "3.1" at flow rates of 0.98ml / min, 1.0ml / min, and 1.02ml / min. Fig.12 , the measurement results show that the peak shape and number of peaks of the characteristic spectrum of the test sample are basically the same. There are 5 common peaks in the characteristic spectrum. Taking the chlorogenic acid peak as the reference peak S, the relative retention time and relative peak area of ​​peak 1, peak 3, peak 4, peak 5 and peak S are calculated, and the RSD value is calculated. The calculation results show that the relative retention time RSD% value and the relative peak area RSD% value are both less than 3.0%, which is within the qualified range. See Table 16-17 for details. The experiment shows that slight changes in flow rate have little effect on the determination of the characteristic spectrum, that is, the durability of different flow rates is better.

[0115] Table 16 - Relative retention time of characteristic spectrum of flow rate test (±0.02ml / min)

[0116] Peak 0.98ml / min 1.0ml / min 1.02ml / min RSD(%) 1 0.802 0.803 0.800 0.19 2(S) 1.000 1.000 1.000 0.00 3 1.273 1.277 1.274 0.16 4 1.896 1.890 1.900 0.27 5 2.479 2.463 2.489 0.53

[0117] Table 17-Relative peak area of ​​characteristic spectrum of flow rate test (±0.02ml / min)

[0118] Peak 0.98ml / min 1.0ml / min 1.02ml / min RSD(%) 1 0.287 0.284 0.290 0.98 2(S) 1.000 1.000 1.000 0.00 3 1.012 0.979 0.962 2.58 4 0.458 0.449 0.435 2.63 5 0.717 0.716 0.688 2.31

[0119] 3.4.4 Investigation of different flow rates (±0.2ml / min)

[0120] Take about 0.2g of the sample of Acanthopanax Cortex Granules (batch number: 211103) and perform the test according to the test method in item "3.1" at flow rates of 0.8ml / min, 1.0ml / min, and 1.2ml / min. Fig.13 , the measurement results show that the peak shape and number of peaks of the characteristic spectrum of the test sample are basically the same. There are 5 common peaks in the characteristic spectrum. Taking the chlorogenic acid peak as the reference peak S, the relative retention time and relative peak area of ​​peak 1, peak 3, peak 4, peak 5 and peak S are calculated, and the RSD value is calculated. The calculation results show that the relative retention time RSD% value and the relative peak area RSD% value are both greater than 3.0%, see Table 18-19 for details. The experiment shows that slight changes in flow rate have a greater impact on the determination of the characteristic spectrum, that is, the flow rate is set to 1.0ml / min.

[0121] Table 18 - Relative retention time of characteristic spectrum of flow rate test (±0.2ml / min)

[0122]

[0123]

[0124] Table 19-Relative peak area of ​​characteristic spectrum of flow rate test (±0.2ml / min)

[0125] Peak 0.8ml / min 1.0ml / min 1.2ml / min RSD(%) 1 0.247 0.284 0.274 7.20 2(S) 1.000 1.000 1.000 0.00 3 1.105 0.979 1.127 7.46 4 0.529 0.449 0.477 8.31 5 0.760 0.716 0.703 4.14

[0126] 3.5 Summary

[0127] The characteristic spectrum method was tested for specificity, precision, repeatability, stability and durability, and the results met the requirements, indicating that the established method can be well used for the characteristic spectrum determination of Acanthopanax Cortex Granules.

[0128] 3.6 Characterization and analysis of characteristic spectra of three batches of finished products

[0129] 3.6.1 Characteristic spectra of three batches of finished products

[0130] According to the proposed characteristic spectrum analysis method, the characteristic spectrum of three batches of finished products was determined. The results showed that there were 5 common peaks in the characteristic chromatogram of the formula granules and the Chinese herbal medicine pieces used in their preparation, and the retention time should correspond to the 5 characteristic peaks in the chromatogram of the reference medicinal material reference. The peak corresponding to the chlorogenic acid reference substance is peak 2. The common peak characteristic spectrum is shown in detail. Figure 14-16 .

[0131] 3.6.2 Evaluation of characteristic chromatograms of three batches of formulated granules

[0132] According to the proposed characteristic spectrum analysis method, the characteristic spectra of three batches of finished products were measured. The results showed that there were 5 common peaks in the characteristic spectrum. The chlorogenic acid peak was used as the reference peak S, and the relative retention time and range of peak 1, peak 3, peak 4, peak 5 and peak S were calculated, as shown in Table 20.

[0133] Table 20-3 Relative retention time of common peaks in batches of finished products

[0134] Peak S1 S2 S3 Mean Relative retention time range ±10% 1 0.798 0.796 0.796 0.80 0.717-0.876 2(S) 1.000 1.000 1.000 1.00 / 3 1.281 1.278 1.275 1.28 1.150-1.406 4 1.901 1.909 1.909 1.91 1.716-2.097 5 2.490 2.504 2.506 2.50 2.250-2.750-

[0135] In summary, the characteristic spectrum determination method of the formula granules established by high performance liquid chromatography was investigated for stability in accordance with the "Guidelines for Validation of Analytical Methods (General Rule 9101)" of Part IV of the 2020 edition of the Chinese Pharmacopoeia, and it met the requirements. According to the characteristic spectrum analysis method proposed above, the characteristic spectra of three batches of finished products were determined. The results were analyzed, and five common characteristic peaks were calibrated. The chlorogenic acid peak was used as the reference peak S, and the relative retention times of peaks 1, 3, 4, 5 and S were calculated. The average relative retention time of the peaks of the three batches of samples was proposed as the specified values: 0.80 (peak 1), 1.28 (peak 3), 1.91 (peak 4), and 2.50 (peak 5). Considering the errors of multiple factors such as experimental operation, instruments, and reagents, the allowable range of the relative retention time is proposed to be ±10%.

[0136] 4 Extracts

[0137] 4.1 Test methods

[0138] The extraction solvent for Chinese herbal formula granules is pharmaceutical water. According to the "Technical Requirements", ethanol is used as the solvent and the determination is carried out according to the hot infusion method under the alcohol-soluble extract determination method (General Rules 2201 of the 2020 Edition of the Chinese Pharmacopoeia).

[0139] 4.2 Investigation of extractables

[0140] About 2 g of samples from three batches of large-scale production were taken and measured according to the law. The results are shown in Table 21.

[0141] Table 21 - Extraction results

[0142]

[0143] 4.3- Drafting of Extractables Range

[0144] Proposed plan 1:

[0145] The extraction limit is formulated based on the specifications of this product, the extraction rate and extraction limit of the standard decoction, and the extraction test results of three batches of large-scale production samples.

[0146] The specification of this product is that each 1g of formula granules is equivalent to 5.6g of medicinal pieces. According to the lower limit of the measured range of the extract yield of the standard decoction of this product (10.4%~13.1%), each 1g of granules contains no less than 5.6g×10.4% (i.e. 0.582g); on this basis, according to the proposed extract limit (20.2%) of the standard decoction of this product, it can be calculated that the extract in each 1g of granules is no less than 0.582g×20.2% (i.e. 0.118g), or in other words, the extract in this product (granules) is no less than 11.8% (0.118×100%), rounded up, that is, no less than 12.0%.

[0147] The measured extract values ​​of three batches of large-scale production samples of this product were 13.78%, 17.28% and 17.90% respectively, all of which were in line with the above-calculated particle extract limit (12.0%). Therefore, it is proposed that the alcohol-soluble extract of this product shall not be less than 12.0%.

[0148] Proposed plan 2:

[0149] The content of particles was converted from 24 batches of standard decoction extracts of Acanthopanax acanthopanax, and the summary is shown in Table 22:

[0150] Table 22-24 Summary of extracts converted from batches of Wujiapibiao decoction into granules

[0151]

[0152] manual

[0153]

[0154] Combined with the paste yield, according to the proposed product specification "each 1g of formula granules is equivalent to 5.6g of decoction pieces", the extracts of 24 batches of standard decoction samples are converted into granular extracts, with an average extract of 19.84%, and the measured value converted into granular extracts ranges from 16.78% to 23.37%, with an SD of 2.01; calculated at 70% to 130% of the mean, the allowable range of the extract is 13.89% to 25.79%. Calculated at mean -3SD to mean +3SD, the allowable range of the extract is 13.80% to 25.88%. For this reason, the proposed allowable range of the extract is: the alcohol-soluble extract of this product shall not be less than 13.8%.

[0155] After reviewing and comparing the above two approaches to extractables limits, and combining the content data of three batches of large-scale production samples, the final extractables limit range was proposed to be no less than 13.8%.

[0156] 5. Content determination

[0157] 5.1 Basis for selecting the components to be tested

[0158] Acanthopanax gracilistylus WWSmith is a plant of the Araliaceae family. It is mainly produced in Shaanxi, Hubei, Sichuan, Guizhou and other places. It contains polysaccharides, isofraxidin, chlorogenic acid, sesamin, β-sitosterol, stearic acid and other ingredients. Through literature review and research, chlorogenic acid is one of the main active ingredients of Acanthopanax gracilistylus WWSmith, and its content is relatively high in Acanthopanax gracilistylus. Acanthopanax gracilistylus WWSmith has the effects of dispelling wind and dampness, nourishing the liver and kidney, strengthening tendons and bones, promoting diuresis and reducing swelling. It is often used for rheumatic arthritis, weakness of tendons and bones, delayed walking in children, physical weakness, and edema. Modern pharmacological studies have shown that Acanthopanax gracilistylus WWSmith has anti-inflammatory, analgesic, sedative, anti-tumor and anti-ulcer effects. Therefore, establishing the content index of chlorogenic acid is more valuable for controlling the quality of Acanthopanax gracilistylus decoction.

[0159] 5.2 Chlorogenic acid content determination method

[0160] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0161] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler (4.6 mm×250 mm, 5 μm); acetonitrile-0.2% formic acid (15:85) was used as the mobile phase; the flow rate was 1.0 ml per minute; the column temperature was 20°C; and the detection wavelength was 325 nm.

[0162] Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, weigh it accurately, and add methanol to make a solution containing 20 μg per 1 ml.

[0163] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.2g, accurately weigh it, put it in a stoppered conical flask, accurately add 50ml of 50% methanol, stopper it, weigh it, ultrasonicate it for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0164] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0165] 5.3 Study on the methodological approach for determination of chlorogenic acid content

[0166] 5.3.1 Investigation of extraction methods

[0167] Referring to the method under "5.2" above, samples were extracted in different ways to prepare test solutions, and the test was carried out according to the law. The results showed that ultrasonic extraction was simpler, so ultrasonic extraction was selected to extract samples, see Table 23 for details.

[0168] Table 23 - Comparison of different extraction methods

[0169]

[0170] 5.3.2 Investigation of extraction time

[0171] Referring to the method under "5.2" above, samples were extracted at different times to prepare test solutions, and the test was performed according to the law. The results showed that ultrasonic extraction for 20 minutes can basically extract the sample completely, so the ultrasonic extraction time of the sample was determined to be 20 minutes, see Table 24 for details.

[0172] Table 24- Comparison of different extraction times

[0173]

[0174] 5.3.3 Investigation of extraction solvent

[0175] Refer to the method under "5.2" above, extract the samples with different solvents to prepare the test solution, and measure it according to the law. The results show that 50% methanol is the best solvent for extraction, so 50% methanol is selected as the solvent for extracting the samples, see Table 25 for details.

[0176] Table 25 - Comparison of different extraction solvents

[0177]

[0178] 5.3.4 Sampling volume investigation

[0179] Referring to the method under "5.2" above, the test solution was prepared with different sampling amounts and the test was carried out according to the law. The results showed that the extraction effect when the sampling amount was 0.2g was better than the other two groups, so 0.2g was selected as the sampling amount for preparing the test solution, see Table 26 for details.

[0180] Table 26 - Comparison of different sampling volumes

[0181]

[0182] In summary, the main parameters of the preparation method of the test solution of Acanthopanax acanthopanax were determined as follows: take an appropriate amount of fine powder of the product, take about 0.2 g, accurately weigh it, put it in a stoppered conical flask, accurately add 50 mL of 50% methanol, seal it, weigh it, ultrasonicate for 20 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0183] 5.4 Validation of analytical method for content determination

[0184] 5.4.1 Specificity inspection

[0185] Take about 0.2g of maltodextrin and prepare a maltodextrin blank solution according to the method in "5.2" above, and take the Acanthopanax Cortex test solution, maltodextrin blank solution and chlorogenic acid reference solution for determination. Fig.17 ,The test showed that the maltodextrin blank had no interference and the method had good specificity.

[0186] 5.4.2 Repeatability test About 0.2 g of the finished granule samples from the same batch (batch number: 211103) were taken, a total of 6 portions, and the test method under the above "5.2" was used for determination. The average chlorogenic acid content in the samples was 4.9321 mg / g, and the RSD value was 1.41%. The test showed that the method had good repeatability, see Table 27 for details.

[0187] Table 27 - Repeatability test

[0188]

[0189] 5.4.3 Precision test

[0190] Take about 0.2 g of the finished granule sample from the same batch (batch number: 211103), inject 6 times continuously according to the method under "5.2" above, measure the peak area, and calculate the RSD value of the chlorogenic acid content in the sample to be 0.67%. The test shows that the instrument has good precision, see Table 28 for details.

[0191] Table 28 - Precision test

[0192]

[0193]

[0194] 5.4.4 Stability test

[0195] About 0.2 g of a batch of Acanthopanax Cortex granules (211103) were taken and sampled at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h according to the test method under the above "5.2". The content was determined and the RSD value of the chlorogenic acid content in the sample was calculated to be 1.11%. The test showed that the test solution was relatively stable within 24 hours. See Table 29 for details.

[0196] Table 29 - Stability Test

[0197]

[0198] 5.4.5 Sample recovery test

[0199] Accurately weigh 9 portions of about 0.1g, 0.1g, and 0.1g of three groups of samples, 3 portions in each group, add 4.6ml, 5.7ml, and 6.9ml of chlorogenic acid reference solution of known concentration (104μg / ml) to each, prepare the test solution according to the method under "5.2", and measure it. The average recovery of chlorogenic acid is calculated to be 107.3423%, and the RSD is 0.28%. See Table 30 for details.

[0200] Table 30 - Sample recovery test results

[0201]

[0202] 5.4.6 Durability test

[0203] 5.4.6.1 Investigation of different mobile phase ratios

[0204] Take a batch of samples of about 0.2g, and according to the test method under "5.2", inject acetonitrile-0.2% formic acid (15:85), (14:86), and (16:84) respectively for determination. The calculation results show that the RSD value of the determined content is 0.60%, which is less than 2.0%. The test shows that this method has good durability for small changes in the mobile phase ratio, see Table 31 for details.

[0205] Table 31 - Comparison of different mobile phase ratios

[0206]

[0207] 5.4.6.2 Wavelength Investigation

[0208] Take a batch of samples of about 0.2g, and measure them at wavelengths of 330nm, 325nm, and 300nm according to the test method under "5.2". The calculation results show that the RSD value of the measured content is 1.22%, which is less than 2.0%. The test shows that this method has good durability against slight changes in wavelength, see Table 32 for details.

[0209] Table 32- Comparison of different wavelength ratios

[0210]

[0211] 5.5 Summary

[0212] In summary, the entire analytical method was tested for specificity, peak purity, precision, repeatability, stability, sample recovery and durability, and the results met the requirements, indicating that the established method can be well used for the determination of chlorogenic acid.

[0213] 6. Proposed content limit range

[0214] (1) Proposed content range plan 1:

[0215] Based on the specifications of this product and the paste yield and content control range of the standard decoction, combined with the test results of three batches of large-scale production samples, the content limit range is proposed.

[0216] The specification of this product is that each 1g of granules is equivalent to 5.6g of medicinal pieces. According to the allowable range of the paste yield of the standard decoction of this product (9.0% to 15.9%), the amount of dry paste contained in each 1g of granules is 5.6g×9.0% to 5.6g×15.9%; on this basis, according to the allowable range of chlorogenic acid content proposed for the standard decoction of this product (4.5mg / g to 31.3mg / g), it can be calculated that the allowable range of chlorogenic acid content in each 1g of granules is 5.6g×9.0%×4.5mg / g to 5.6g×15.9%×31.3mg / g (i.e. 2.27mg to 27.87mg).

[0217] Considering that this lower limit content (2.27 mg / g) is too low, combined with the content of the lowest batch of standard decoction (Y220612PT, paste yield 11.9%, containing 4.8324 mg / g) in the 24 batches of standard decoctions of this product converted into granules (i.e. the content of chlorogenic acid in each 1g of granules is: 5.6g×11.9%×4.8324 mg / g=3.22 mg), the lower limit of the content of this product can be preliminarily proposed to be 3.0 mg / g. The contents of the three batches of large-scale production samples of this product were 3.25 mg / g, 7.15 mg / g, and 7.60 mg / g, respectively, all within the above-mentioned granule content range calculated or proposed based on the standard decoction.

[0218] In summary, the chlorogenic acid content of this product is proposed to range from 3.0 mg / g to 27.8 mg / g.

[0219] (2) Proposed content range plan 2:

[0220] The content of 24 batches of standard decoction of Acanthopanax acanthopanax was converted into the content of particles, which is summarized as follows:

[0221] Table 33-24 Summary of the content of standard decoction of Acanthopanax acanthopanax converted into granules

[0222]

[0223] Combined with the paste yield, according to the proposed product specification "each 1g of formula granules is equivalent to 5.6g of decoction pieces", the content of 24 batches of standard decoction samples was converted into granule content, the average content of chlorogenic acid was 7.84mg / g, and the measured content was converted into granule content ranging from 3.22mg / g to 19.97mg / g, with an SD of 4.02; calculated at 70% to 130% of the mean, the allowable range of chlorogenic acid content is 5.49mg / g to 10.19mg / g. Calculated at mean -1.5SD to mean +3SD, the allowable range of chlorogenic acid content is 1.82mg / g to 19.89mg / g. For this reason, the allowable range of chlorogenic acid content is proposed to be 1.8mg / g to 19.9mg / g.

[0224] The above two content range considerations were reviewed and compared comprehensively, and combined with the content data of three batches of large-scale production samples, the final chlorogenic acid content range was proposed to be 3.0 mg / g to 20.0 mg / g.

[0225] 7 Specifications are determined based on the amount of raw materials added in the preparation method and the amount produced through process research, that is, every 1g of formula granules is equivalent to 5.6g of medicinal pieces.

[0226] 8 Storage is formulated according to the basic storage requirements of the variety.

[0227] The quality detection method of the scutellaria baicalensis formula granules provided by the present invention is to study the properties, dry extract yield, thin layer identification, extract, characteristic spectrum and chlorogenic acid content of the scutellaria baicalensis formula granules, and to evaluate the quality of the scutellaria baicalensis formula granules through multi-faceted measurements, so as to lay a solid foundation for the stable quality of the product, establish a feasible quality standard for the scutellaria baicalensis formula granules, and achieve effective control of the quality of the scutellaria baicalensis formula granules. In addition, by using the chromatographic conditions of the present application for liquid phase analysis, a characteristic spectrum with better separation and clearer can be obtained, and the method has good stability, high precision and good reproducibility. The scutellaria baicalensis formula granules are prepared by decocting the scutellaria baicalensis decoction pieces, and the average chlorogenic acid content is 7.84 mg / g. According to the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules", the chlorogenic acid content range is proposed to be 3.0 mg / g to 20.0 mg / g.

[0228] Those skilled in the art should understand that the discussion of any of the above embodiments is only exemplary and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples; any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting the quality of Acanthopanax Cortex granules, characterized in that: The following detection methods are included: Through the properties of the Acanthopanax Cortex Formula Granules, dry extract yield, thin layer identification, characteristic spectrum, extract and chlorogenic acid content determination, the content standard of the formula granules is limited to 3.0-20.0 mg of chlorogenic acid per 1g, among which, thin layer identification is carried out by thin layer chromatography; characteristic spectrum and chlorogenic acid content are determined by liquid chromatography; extract is determined by hot leaching method; The characteristic spectrum is determined by liquid chromatography, including: performing liquid chromatography analysis, using a solution made from a cortex acanthopanacis reference medicinal material as a reference medicinal material reference solution b, using a solution made from a chlorogenic acid reference substance as a reference substance reference solution b, using a solution made from a cortex acanthopanacis formula granule sample as a test solution b, accurately aspirating the reference medicinal material reference solution b, the reference substance reference solution b and the test solution b, respectively, injecting them into a liquid chromatograph, and determining, to obtain; wherein, the chromatographic conditions used are: chromatographic column: octadecylsilane bonded silica gel as a filler, with a specification of 4.6 mm×250 mm, 5µm, Shimadzu Shim-pack GIST C18-AQ; mobile phase: acetonitrile as mobile phase A, 0.2% formic acid solution as mobile phase B, and gradient elution according to the provisions of Table a; Table a Gradient elution program Flow rate: 1.0 mL / min; column temperature: 20°C; injection volume: 10 μL; detection wavelength: 280 nm.

2. The quality detection method of Acanthopanax Cortex Granules according to claim 1, characterized in that: The preparation method of the Acanthopanax Cortex granules is as follows: take 5600g of Acanthopanax Cortex slices, add water to boil, filter, concentrate the filtrate into a clear paste, add appropriate amount of auxiliary materials, dry or dry and crush, then add appropriate amount of auxiliary materials, mix well, granulate, and make 1000g.

3. The quality detection method of Acanthopanax Cortex Granules according to claim 1, characterized in that: The thin layer chromatography method comprises the following steps: (1) Preparation of test solution a: Take 2.0 g of Acanthopanax Cortex Granules, add 30 mL of water, and ultrasonically treat for 30 min. Extract twice with dichloromethane, 20 mL each time, combine the dichloromethane solutions, evaporate to dryness, and dissolve the residue with 1 mL of methanol to prepare test solution a. (2) Preparation of control medicinal material solution a: Take 1.0 g of Acanthopanax cortex as a control medicinal material, add 50 ml of water, boil for 30 minutes, filter, concentrate the filtrate to about 30 ml, cool, and extract twice with dichloromethane by shaking, 20 ml each time, combine the dichloromethane solutions, evaporate to dryness, and add 1 ml of methanol to the residue to dissolve it, thereby preparing control medicinal material solution a; (3) Thin layer chromatography analysis: Thin layer chromatography conditions are as follows: Thin layer plate: silica gel G thin layer plate; sample volume: test solution a is 2uL, control drug solution a is 3uL; developing agent: petroleum ether-ethyl acetate-formic acid as the developing agent, the ratio of petroleum ether, ethyl acetate, formic acid is 10:3:0.1, and the boiling range of petroleum ether is 60~90℃; color developer: 10% sulfuric acid ethanol test solution, heated at 105℃, and inspected under ultraviolet light at 365nm.

4. The quality detection method of Acanthopanax Cortex Granules according to claim 1, characterized in that: The hot soaking method uses ethanol as solvent, and the hot soaking method under the alcohol-soluble extract determination method is used to determine the extract range.

5. The quality detection method of Acanthopanax Cortex Granules according to claim 1, characterized in that: The method of measuring the characteristic spectrum by liquid chromatography also includes the following steps: (1) Preparation of reference medicinal material solution b: Take 0.2 g of Acanthopanax cortex as a reference medicinal material, add 50 mL of 50% methanol, ultrasonically treat for 20 min, cool, filter, and take the filtrate as the reference medicinal material solution b; (2) Preparation of reference substance solution b: Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, and dissolve it in methanol to prepare a reference substance solution b with a concentration of 20 ug / mL; (3) Preparation of test solution b: Take an appropriate amount of Acanthopanax Cortex granules, grind them into powder, take about 0.2 g, place in a stoppered conical flask, add 50 mL of 50% methanol, weigh the weight, sonicate for 30 min, cool, weigh again, make up the loss with 50% methanol, shake well, filter, and take the filtrate as the test solution b.

6. The quality detection method of Acanthopanax Cortex Granules according to claim 1, characterized in that: The liquid chromatography method for determining the chlorogenic acid content includes: performing liquid chromatography analysis, using a solution made from a chlorogenic acid reference substance as a reference substance solution c, and using a solution made from a sample of acanthopanax cortex formula granules as a test solution c, accurately aspirating the reference substance solution c and the test solution c, respectively, injecting them into a liquid chromatograph, and determining to obtain the content; wherein, the chromatographic conditions used are: chromatographic column: octadecylsilane bonded silica gel as a filler, with a specification of 4.6mm×250mm, 5µm; mobile phase: acetonitrile as mobile phase A, and 0.2% formic acid solution as mobile phase B, and gradient elution is performed as specified; flow rate: 1.0mL / min; column temperature: 20°C; injection volume: 10μL; detection wavelength: 325nm.

7. The quality detection method of Acanthopanax Cortex Granules according to claim 6, characterized in that: The liquid chromatography method for determining the chlorogenic acid content also includes the following steps: (1) Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, weigh it accurately, and add methanol to prepare a solution containing chlorogenic acid at a concentration of 20 ug / ml, which is used as reference solution c; (2) Preparation of test solution: Take an appropriate amount of Acanthopanax Cortex granules, grind them into powder, take about 0.2 g, weigh accurately, place in a stoppered conical flask, accurately add 50 ml of 50% methanol, stopper, weigh accurately, ultrasonicate for 30 min, cool, weigh again, make up the loss with 50% methanol, shake well, filter, and take the filtrate as the test solution c.

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