A quality detection method for Elsholtzia scoparia formula granules

Abstract

The present invention provides a quality detection method for scutellaria formula granules, including determining the properties, dry extract yield, thin layer identification, extract, characteristic spectrum and rosmarinic acid content of scutellaria formula granules, and limiting the content standard of scutellaria formula granules to 4.0-23.2mg of rosmarinic acid per 1g, wherein the thin layer identification is identified by thin layer chromatography; the extract is determined by hot dipping; the characteristic spectrum and rosmarinic acid content are both determined by liquid chromatography. The quality detection method for scutellaria formula granules of the present invention evaluates the quality of scutellaria formula granules through systematic detection, and can comprehensively reflect the intrinsic quality of the formula granules and better control the quality of scutellaria 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 Elsholtzia scoparia formula granules. Background Art

[0002] Modern drugs need to have three characteristics: stability, uniformity, safety and effectiveness. For Chinese patent medicines, multiple means of testing are needed to ensure the reliability and stability of the test results. Formula granules are increasingly chosen by the public because of their advantages of being easy to carry, convenient to take and reliable in 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. Stone sedge is the dried aerial part of the Lamiaceae plant Stone sedge Moslachinensis Maxim., which can sweat and relieve the symptoms, eliminate dampness and harmonize the middle, promote diuresis and reduce swelling. It is mainly used to treat cold and cold drinks in summer, headache and fever, aversion to cold and no sweating, chest fullness and abdominal pain, vomiting and diarrhea, edema, and athlete's foot. At present, there is no systematic quality inspection method for the formula granules of Ligustrum lucidum. It is not comprehensive to use only the existing inspection methods to inspect the formula granules of Ligustrum lucidum. It cannot reflect the overall intrinsic quality of the formula granules of Ligustrum lucidum 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 Ligustrum lucidum 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 to provide a quality detection method for the Elsholtzia scoparia formula granules which can comprehensively reflect the intrinsic quality of the formula granules and better control the quality of the Elsholtzia scoparia formula granules.

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

[0005] A quality detection method for Elsholtzia schrenkiana formula granules, comprising the following detection method, through the properties, dry extract yield, thin layer identification, characteristic spectrum, extract and rosmarinic acid content determination of the Elsholtzia schrenkiana formula granules, the content standard of the formula granules is limited to 4.0-23.2 mg of rosmarinic acid per 1g, wherein the thin layer identification is performed by thin layer chromatography; the characteristic spectrum and rosmarinic acid content are both determined by liquid chromatography; the extract is determined by hot leaching;

[0006] The characteristic spectrum is determined by liquid chromatography, including: performing liquid chromatography analysis, using a solution made from a reference medicinal material of Elsholtzia as a reference medicinal material reference solution b, using a solution made from a reference substance of caffeic acid and a reference substance of rosmarinic acid as a reference substance reference solution b, using a solution made from a sample of Elsholtzia formula granules 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: a chromatographic column: using octadecylsilane bonded silica gel as a filler, Shimadzu Shim-packGIST C18-AQ (4.6 mm×250 mm, 5 μm); a mobile phase: using acetonitrile as a mobile phase A, using 0.05% phosphoric acid solution as a mobile phase B, and performing gradient elution according to the provisions of Table a;

[0007] Table a Gradient elution program

[0008] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～20 5→11 95→89 20～30 11→15 89→85 30～60 15→30 85→70 60～65 30→50 70→50 65～70 50 50 70～75 50→95 50→5 75～80 95→5 5→95

[0009] Flow rate: 1.0 mL / min; column temperature: 35°C; injection volume: 10 μL; detection wavelength: 330 nm.

[0010] In one embodiment, the preparation method of the Herba Cynoglossi formula granules is as follows: take 5000g of Herba Cynoglossi decoction pieces, add water and boil, collect an appropriate amount of volatile oil, encapsulate the volatile oil with β-cyclodextrin for later use, filter, concentrate the filtrate into a clear paste, add the volatile oil inclusion compound, dry, add an appropriate amount of auxiliary materials, mix well, granulate, and make 1000g.

[0011] In one embodiment, the thin layer chromatography method comprises the following steps: (1) preparing a test solution a: taking a sample of the formula granules of the Herba Cynoglossi, grinding it into powder, weighing 2.0 g, adding 30 ml of 70% ethanol, ultrasonically treating it for 30 minutes, cooling it, filtering it, evaporating the filtrate to dryness, adding 1 ml of ethanol 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 the Herba Cynoglossi control medicinal material, grinding it into powder, adding 30 ml of 70% ethanol, ultrasonically treating it for 30 minutes, cooling it, filtering it, evaporating the filtrate to dryness, adding 1 ml of ethanol to the residue to dissolve it, and preparing a Obtain reference medicinal material solution a; (3) Prepare reference substance solution a: Take rosmarinic acid reference substance, add methanol to make a solution of 0.3 mg / ml, and prepare reference substance solution a (4) Perform thin layer chromatography analysis: Thin layer chromatography conditions are as follows: thin layer plate: silica gel G thin layer plate; spot volume: 2uL each of test solution a, reference medicinal material solution a, and reference substance solution a; use n-hexane-ethyl acetate-isopropanol-formic acid (15:3:3.5:0.5) solution as the developing agent; color developer: 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clearly colored.

[0012] 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.

[0013] In one embodiment, the method of determining the characteristic spectrum by liquid chromatography further comprises the following steps:

[0014] (1) Preparation of reference medicinal material reference solution b: Take 0.2 g of Elsholtzia ciliata reference medicinal material, place it in a stoppered conical flask, add 15 ml of 70% ethanol, ultrasonically treat for 30 minutes, let cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution b; (2) Preparation of reference substance reference solution b: Take appropriate amounts of caffeic acid reference substance and rosmarinic acid reference substance, accurately weigh, add methanol to each of them to make a mixed solution containing 20 μg of caffeic acid and 100 μg of rosmarinic acid per 1 ml, respectively, as the reference substance reference solution b; (3) Preparation of test sample solution b: Take appropriate amount of Elsholtzia ciliata formula granules, grind them into powder, take about 0.2 g, place it in a stoppered conical flask, add 15 ml of 70% ethanol, ultrasonically treat for 30 minutes, let cool, shake well, filter, and take the filtrate as the test sample solution b.

[0015] In one embodiment, the determination of rosmarinic acid content by liquid chromatography includes: performing liquid chromatography analysis, using a solution made from a rosmarinic acid reference substance as a reference solution c, and using a solution made from a sample of a scutellaria formula granule 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 the content; wherein, the chromatographic conditions used are: a chromatographic column: octadecylsilane bonded silica gel as a filler (4.6mm×250mm, 5μm); acetonitrile-0.1% phosphoric acid (20:80) as a mobile phase, and performing gradient elution as specified; flow rate: 1.0mL / min; column temperature: 35°C; injection volume: 10μL; detection wavelength: 330nm.

[0016] In one embodiment, the determination of rosmarinic acid content by liquid chromatography also includes the following steps: (1) preparing a reference solution: taking an appropriate amount of rosmarinic acid reference substance, accurately weighing it, adding methanol to prepare a solution containing rosmarinic acid at a concentration of 100ug / ml, as the reference solution c; (2) preparing a test solution: taking an appropriate amount of Elsholtzia scoparia granules, grinding them, taking about 0.2g, accurately weighing it, placing it in a stoppered conical flask, accurately adding 15ml of 70% ethanol, sealing it, weighing it, ultrasonically treating it for 30 minutes, cooling it, weighing it again, making up the lost weight with 70% ethanol, shaking it well, filtering it, and taking the filtrate as the test solution c.

[0017] In one embodiment, it also includes using liquid chromatography to respectively determine the characteristic spectra of the Elsholtzia jiangxiensis reference medicinal material and the Elsholtzia jiangxiensis reference medicinal material of the source variety of the Elsholtzia jiangxiensis formula granules, specifically: using octadecylsilane bonded silica gel as a filler (4.6mm×250mm, 5μm), acetonitrile as a mobile phase A, and 0.05% phosphoric acid solution as a mobile phase B, gradient elution is performed according to the provisions of Table a, the flow rate is 1.0ml per minute, the column temperature is 35°C, and the detection wavelength is 330nm; preparing Elsholtzia jiangxiensis reference solution d: taking 0.5g of Elsholtzia jiangxiensis reference medicinal material, adding 15ml of 70% ethanol, ultrasonically treating for 30 minutes, cooling, shaking, filtering, and taking the filtrate to obtain; preparing Elsholtzia jiangxiensis reference solution d: taking 0.5g of Elsholtzia jiangxiensis reference medicinal material, adding 15ml of 70% ethanol, ultrasonically treating Prepare the reference substance solution d: take appropriate amount of caffeic acid and rosmarinic acid reference substances, accurately weigh them, add methanol to prepare mixed solutions containing 20μg caffeic acid and 100μg rosmarinic acid per 1ml, as the reference substance solution d; prepare the test solution d: take about 0.5g of Elsholtzia scoparia granules, accurately weigh them, put them in a stoppered conical flask, accurately add 15ml of 70% ethanol, seal it, weigh it, ultrasonically treat it for 30 minutes, cool it, weigh it again, make up the lost weight with 70% ethanol, shake it, filter it, and obtain the subsequent filtrate; accurately aspirate 10μl of Elsholtzia scoparia reference solution d, Elsholtzia jiangxiensis reference solution d, reference substance solution d, and test solution d, inject them into liquid chromatograph, and determine them.

[0018] Compared with the prior art, the quality detection method for the Elsholtzia schrenkiana formula granules provided by the present invention has the following beneficial effects: by studying the properties, dry extract yield, thin layer identification, extract, characteristic spectrum and rosmarinic acid content determination of the Elsholtzia schrenkiana formula granules, the quality of the Elsholtzia schrenkiana formula granules is evaluated through multi-faceted measurements, a solid foundation is laid for the stable quality of the product, a feasible quality standard for the Elsholtzia schrenkiana formula granules can be established, and effective control of the quality of the Elsholtzia schrenkiana formula granules can be achieved, and liquid phase analysis using the chromatographic conditions of the present application can obtain a characteristic spectrum with better separation and clearer clarity, and the obtained characteristic spectrum of the Elsholtzia schrenkiana formula granules can be well distinguished from the characteristic spectrum of the Elsholtzia jiangxiensis control medicinal material, which is beneficial to distinguishing the two basic sources of Elsholtzia schrenkiana and Elsholtzia jiangxiensis in production or clinical application, and the method has good stability, high precision and good reproducibility. The Shiquan decoction slices were boiled to prepare the Shiquan decoction slices formula granules, the average content of rosmarinic acid was 10.966 mg / g. According to the "Technical Requirements for Quality Control and Standardization of Chinese Medicine Formula Granules", the rosmarinic acid content was proposed to range from 4.0 mg / g to 23.2 mg / g, and the volatile oil content of the Shiquan formula granules was proposed to range from 0.3% to 1.6%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the TLC spectrum of three batches of production samples of the Herba Cyperi formula granules according to thin layer chromatography in one embodiment of the present invention.

[0020] 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 reflux; and S2 is the characteristic spectrum of the test solution extracted by ultrasonic extraction.

[0021] 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 90 minutes; S2 is the characteristic spectrum of the test solution extracted by ultrasonic extraction for 60 minutes; and S3 is the characteristic spectrum of the test solution extracted by ultrasonic extraction for 30 minutes.

[0022] Figure 4 The characteristic spectrum of the HPLC of different extraction solvents in the extraction solvent investigation of the characteristic spectrum determination of one embodiment of the present invention; wherein S1 is the characteristic spectrum of the test solution extracted with 70% ethanol; S2 is the characteristic spectrum of the test solution extracted with 70% methanol; and S3 is the characteristic spectrum of the test solution extracted with 30% ethanol.

[0023] 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

[0024] Figure 6 The characteristic spectrum of the HPLC is superimposed in the specificity test in the specificity investigation 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 coffee color and rosmarinic acid reference solution, S3 is the characteristic spectrum of the Ligusticum chuanxiong reference medicinal material solution, and S4 is the characteristic spectrum of the Ligusticum chuanxiong test solution.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] Fig.10This is a characteristic spectrum of a common peak superposition characteristic spectrum of a characteristic spectrum determination flow rate test (±0.02ml / min) of an embodiment of the present invention; wherein S1 is a chromatographic column with a flow rate of 1.02ml / min, S2 is a chromatographic column with a flow rate of 1.00ml / min, and S3 is a chromatographic column with a flow rate of 0.98ml / min.

[0029] Fig.11 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 1.2ml / 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 0.8ml / min.

[0030] Fig.12 The characteristic spectra of three batches of finished products in one embodiment of the present invention are superimposed with the HPLC characteristic spectra; among them, the finished product number of S1 is ZS220602, the finished product number of S2 is ZS220601, and the finished product number of S3 is 220601.

[0031] Fig.13 The HPLC characteristic spectrum is superimposed with the characteristic spectrum of three batches of Elsholtzia scoparia finished products, reference medicinal materials, and reference substance reference materials in one embodiment of the present invention.

[0032] Fig.14 The HPLC characteristic spectrum is fitted for the characteristic spectrum of the finished product of Elsholtzia ciliata and the reference medicinal material in one embodiment of the present invention; wherein, peak 2 (S1) corresponds to the caffeic acid reference substance, and peak 6 (S2) corresponds to the rosmarinic acid reference substance.

[0033] Fig.15 This is an overlay of thymol and carvacrol determined by gas phase analysis of 15 batches of standard decoctions in one embodiment of the present invention.

[0034] Fig.16 The HPLC characteristic spectrum is superimposed in the specificity test in the specificity investigation of content determination in one embodiment of the present invention; wherein S1 is the characteristic spectrum of the Elsholtzia test sample, S2 is the characteristic spectrum of the rosmarinic acid reference substance, and S3 is the characteristic spectrum of the maltodextrin blank reference substance.

[0035] Fig.17 The present invention is a curve diagram of different concentrations of rosmarinic acid reference substance in a linear range test in one embodiment of the present invention, wherein the amount of rosmarinic acid is the abscissa and the rosmarinic acid peak area is the ordinate.

[0036] Fig.18 This is a thin layer identification diagram of Elsholtzia schrenkiana and Elsholtzia jiangxiensis in one embodiment of the present invention.

[0037] Fig.19 HPLC characteristic spectra of the test sample, Elsholtzia scoparia reference medicinal material, and Elsholtzia jiangxiensis reference medicinal material in one embodiment of the present invention. DETAILED DESCRIPTION

[0038] 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.

[0039] The present embodiment provides a quality detection method for Elsholtzia scoparia formula granules, including the following detection method, by analyzing the properties, dry extract yield, thin layer identification, characteristic spectrum, extract and rosmarinic acid content determination of the Elsholtzia scoparia formula granules, the content standard of the formula granules is limited to 3.8-23.2 mg of rosmarinic acid per 1g, wherein the thin layer identification is carried out by thin layer chromatography; the characteristic spectrum and rosmarinic acid content determination are both determined by liquid chromatography; the extract is determined by hot immersion method.

[0040] In this embodiment:

[0041] Preparation of Ligusticum chuanxiong formula granules: Formula granules made according to the "technical requirements" of the source and medicinal parts of Ligusticum chuanxiong Chinese medicinal materials in the 2020 edition of the Chinese Pharmacopoeia, including the family name, Chinese name, Latin and medicinal parts of the medicinal plant, that is, this product is the root bark of Acanthopanax gracilistylus W.W. Smith, a plant of the Araliaceae family, which is processed and processed according to the main quality indicators of the formula granules. The specific preparation method is: take 5000g of Ligusticum chuanxiong slices, add water to decoct, collect an appropriate amount of volatile oil (enclosed in β-cyclodextrin for standby use), filter, concentrate the filtrate into a clear paste (the dry extract yield is 10.0% to 16.6%), add volatile oil inclusion compound, dry (or dry and crush), add an appropriate amount of auxiliary materials, mix well, granulate, and make 1000g. The dry extract yield was determined as 70% to 130% of the average yield of 21 batches of standard decoctions.

[0042] 1. Characteristic investigation

[0043] Properties: Take one sample each from three batches of large-scale production of Elsholtzia scabra granules, observe and record the morphology, color and smell of the samples, and combine the color and smell of the standard decoction samples. This product is a granule, with yellow-brown to brown particles; it has a fresh fragrance and a slightly spicy and cool taste.

[0044] 2. Thin layer identification

[0045] With reference to the published thin-layer identification method of Elsholtzia odorata quality standard of Hunan Province TCM formula granules, the thin-layer identification method of this product was established by using Elsholtzia odorata reference medicinal materials and rosmarinic acid reference substances as controls. After methodological investigation and 21 batches of sample tests, the chromatographic spots of the test sample were clear and the negative control samples had no interference. Therefore, this method was included in the [Identification] item of the text of the Elsholtzia odorata formula granules quality standard. Specifically, the thin-layer identification method of Elsholtzia odorata formula granules is:

[0046] Preparation of test solution: Take the product, grind it into powder, weigh about 2.0g, add 30ml of 70% ethanol, ultrasonically treat for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 1ml of ethanol to the residue to dissolve it, and use it as the test solution.

[0047] Preparation of control medicinal material solution: Take 1.0 g of Elsholtzia ciliata control medicinal material and prepare the control medicinal material solution in the same way.

[0048] Preparation of reference solution: Take another rosmarinic acid reference substance and add methanol to make a solution containing 0.3 mg per 1 ml as the reference solution.

[0049] 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 control medicinal material solution, reference substance solution and test solution prepared by the above method were taken and spotted on the same silica gel G thin layer plate, and n-hexane-ethyl acetate-isopropanol-formic acid (15:3:3.5:0.5) solution was used as the developing solvent. The plate was developed, taken out, dried, sprayed with 5% vanillin sulfuric acid solution, and heated at 105°C until the spots were clearly colored.

[0050] 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, spots of the same color appear at the corresponding positions in the chromatograms of the control medicinal materials.

[0051] 3. Feature Spectrum

[0052] 3.1 Characteristic spectrum determination method

[0053] 3.1.1 Chromatographic conditions and system suitability experiments Octadecylsilane bonded silica gel was used as the filler (Shimadzu Shim-pack GISTC18-AQ (4.6 mm × 250 mm, 5 μm)); acetonitrile was used as the mobile phase A, 0.05% phosphoric acid solution was used as the mobile phase B, and gradient elution was performed according to the provisions in Table 1; the flow rate was 1.0 ml / min; the column temperature was 35°C; the detection wavelength was 330 nm. The injection volume was 10 μl. The number of theoretical plates calculated based on the rosmarinic acid peak should not be less than 50,000.

[0054] Table 1 - Gradient elution program

[0055] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～20 5→11 95→89 20～30 11→15 89→85 30～60 15→30 85→70 60～65 30→50 70→50 65～70 50 50 70～75 50→95 50→5 75～80 95→5 5→95

[0056] 3.1.2 Preparation of reference solution

[0057] Take 0.2 g of Elsholtzia ciliata reference medicinal material, place it in a stoppered conical flask, add 15 ml of 70% ethanol, ultrasonically treat (power 300 W, frequency 50 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the control medicinal material.

[0058] Take appropriate amount of caffeic acid reference substance and rosmarinic acid reference substance, weigh them accurately, add methanol to prepare mixed solutions containing 20 μg of caffeic acid and 100 μg of rosmarinic acid per 1 ml, respectively, as reference substance solutions.

[0059] 3.1.3 Preparation of test solution

[0060] Take an appropriate amount of this product, grind it into powder, take about 0.2g, put it in a stoppered conical flask, add 15ml of 70% ethanol, ultrasonically treat (power 300W, frequency 50kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0061] 3.1.4 Determination method

[0062] Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0063] 3.2 Methodological investigation of characteristic spectrum determination

[0064] The sample pretreatment methods of the characteristic spectrum of the Herba Cynoglossi granules (batch number: 220601) were investigated, mainly focusing on the effects of the extraction solvent, extraction method, extraction time and extraction solvent dosage (or sampling volume) on the characteristic spectrum of the Herba Cynoglossi granules.

[0065] 3.2.1 Investigation of extraction methods

[0066] 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 of the chromatographic solution of the test sample with different extraction methods is consistent, and the peak shape difference is not obvious, but the extraction effect of ultrasonic method is better than that of reflux method, so ultrasonic extraction is selected as the sample extraction method.

[0067] Table 2 - Comparison of extraction methods

[0068]

[0069] 3.2.2 Extraction time investigation

[0070] 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 chromatograms of the test sample solutions with different extraction times is consistent, the peak shapes are not much different, and the extraction effects are similar. In order to shorten the detection time, the extraction time of the test sample is selected as 30 minutes.

[0071] Table 3 - Comparison of extraction times

[0072]

[0073] 3.2.3 Investigation of extraction solvent

[0074] 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 of the chromatograms of the test sample solutions extracted with different extraction solvents is consistent, the peak shapes are quite different at different extraction times, and the extraction effect of 70% ethanol is better than that of the other two groups. Therefore, 70% ethanol is selected as the extraction solvent for the test sample.

[0075] Table 4 - Comparison of extraction solvents

[0076]

[0077] 3.2.4 Investigation of sampling volume

[0078] 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 chromatograms of the test solution with different sampling amounts is consistent, the peak shapes are slightly different, and the extraction effects are similar. However, when the sampling amount is 0.2 g, its extraction effect is significantly better than the other two groups. Therefore, the sampling amount of the test solution prepared is 0.2 g.

[0079] Table 5 - Comparison of sampling volumes

[0080]

[0081] In summary, the main parameters of the test solution preparation method are determined as follows: take an appropriate amount of the product, grind it into powder, take about 0.2 g, put it in a stoppered conical flask, add 15 ml of 70% ethanol, ultrasonically treat (power 300 W, frequency 50 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate.

[0082] 3.3 Verification of characteristic spectrum analysis method

[0083] 3.3.1 Specificity inspection

[0084] Take maltodextrin, about 0.2g, and prepare maltodextrin blank control solution according to the method under "3.1" above, and take maltodextrin blank control solution, caffeic acid and rosmarinic acid reference solution, Elsholtzia ciliata reference medicinal material solution, and Elsholtzia ciliata test solution for determination.

[0085] 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 .

[0086] 3.3.2 Repeatability test

[0087] Take about 0.2g of the sample of Elsholtzia scoparia formula granules (batch number: 220601), a total of 6 portions, and measure it according to the method under "3.1", and inject 6 injections for measurement. The measurement results show that the peak shape and number of peaks in the characteristic spectrum of the test sample are basically consistent. There are 7 common peaks in the characteristic spectrum. Caffeic acid (peak 2) is used as the reference peak S1, and rosmarinic acid (peak 6) is used as the reference peak S2. The relative retention time and relative peak area of ​​peak 1, peak 3, peak 4 and peak S1 are calculated. The relative retention time and relative peak area of ​​peak 5, peak 7 and peak S2 are calculated, and the RSD value is calculated. The calculation results show that the relative retention time and relative peak area RSD% values ​​are within the qualified range. The experiment shows that this method has good reproducibility. For details, see Figure 7 and Tables 6-7.

[0088] Table 6-Relative retention time of characteristic spectra of repeatability test

[0089] Peak S1 S2 S3 S4 S5 S6 RSD(%) 1 0.951 0.951 0.951 0.951 0.951 0.951 0.01 2(S1) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 3 1.053 1.053 1.053 1.053 1.053 1.053 0.02 4 1.155 1.154 1.155 1.154 1.155 1.154 0.05 5 0.981 0.981 0.981 0.981 0.981 0.981 0.00 6(S2) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 7 1.369 1.369 1.369 1.370 1.368 1.368 0.05

[0090] Table 7-Relative peak area of ​​repeatability test characteristic spectrum

[0091] Peak S1 S2 S3 S4 S5 S6 RSD(%) 1 1.284 1.284 1.285 1.288 1.284 1.293 0.28 2(S1) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 3 0.324 0.324 0.324 0.325 0.324 0.325 0.19 4 0.878 0.878 0.876 0.881 0.877 0.879 0.21 5 0.303 0.303 0.302 0.302 0.303 0.302 0.08 6(S2) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 7 0.080 0.080 0.080 0.080 0.080 0.080 0.18

[0092] 3.3.3 Precision test

[0093] Take about 0.2g of the sample of Cyperus rotundus formula granules (batch number: 220601), and perform the measurement by continuous injection of 6 injections according to the test method under "3.1". Perform the measurement by continuous injection of 6 injections. The measurement results show that the peak shape and number of peaks in the characteristic spectrum of the test sample are basically consistent. There are 7 common peaks in the characteristic spectrum. Taking caffeic acid (peak 2) as the reference peak S1 and rosmarinic acid (peak 6) as the reference peak S2, calculate the relative retention time and relative peak area of ​​peak 1, peak 3, peak 4 and peak S1, calculate the relative retention time and relative peak area of ​​peak 5, peak 7 and peak S2, and calculate the RSD value. The calculation results show that the relative retention time RSD% value and the relative peak area RSD% value are both within the qualified range. The experiment shows that this method has good precision. For details, see Figure 8 and Tables 8-9.

[0094] Table 8 - Relative retention time of HPLC characteristic spectrum of precision test

[0095] Peak S1 S2 S3 S4 S5 S6 RSD(%) 1 0.954 0.954 0.954 0.954 0.954 0.954 0.01 2(S1) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 3 1.053 1.053 1.053 1.053 1.054 1.053 0.02 4 1.153 1.154 1.154 1.153 1.154 1.153 0.05 5 0.981 0.981 0.981 0.981 0.981 0.981 0.00 6(S2) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 7 1.369 1.368 1.368 1.367 1.367 1.367 0.07

[0096] Table 9 - Relative peak area of ​​HPLC characteristic spectrum of precision test

[0097] Peak S1 S2 S3 S4 S5 S6 RSD(%) 1 1.170 1.168 1.168 1.168 1.166 1.168 0.11 2(S1) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 3 0.454 0.454 0.455 0.454 0.454 0.454 0.08 4 0.686 0.685 0.687 0.687 0.685 0.687 0.12 5 0.179 0.179 0.179 0.178 0.178 0.179 0.10 6(S2) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 7 0.032 0.032 0.032 0.032 0.032 0.032 0.14

[0098] 3.3.4 Stability test

[0099] Take about 0.2g of the sample of Elsholtzia scoparia formula granules (batch number: 220601), and according to the test method under "3.1", sample injection and determination were performed at 0h, 2h, 4h, 8h, 12h, and 24h. The measurement results showed that the peak shape and number of peaks in the characteristic spectrum of the test sample were basically consistent. There are 7 common peaks in the characteristic spectrum. Taking caffeic acid (peak 2) as the reference peak S1, and rosmarinic acid (peak 6) as the reference peak S2, the relative retention time and relative peak area of ​​peak 1, peak 3, peak 4 and peak S1 were calculated, and the relative retention time and relative peak area of ​​peak 5, peak 7 and peak S2 were calculated, and the RSD value was calculated. The calculation results showed that the relative retention time RSD% value and the relative peak area RSD% value were both within the qualified range. The experiment showed that the test solution was relatively stable within 24 hours, see for details. Fig. 9 and Tables 10-11.

[0100] Table 10 - Relative retention time of characteristic spectrum of stability test

[0101] Peak S1 S2 S3 S4 S5 S6 RSD(%) 1 0.954 0.954 0.954 0.954 0.954 0.955 0.03 2(S1) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 3 1.053 1.053 1.053 1.054 1.053 1.053 0.01 4 1.153 1.154 1.154 1.154 1.153 1.154 0.04 5 0.981 0.981 0.981 0.981 0.981 0.981 0.00 6(S2) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 7 1.369 1.368 1.368 1.367 1.366 1.369 0.10

[0102] Table 11 - Relative peak area of ​​characteristic spectrum of stability test

[0103] Peak S1 S2 S3 S4 S5 S6 RSD(%) 1 1.170 1.168 1.168 1.166 1.167 1.164 0.17 2(S1) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 3 0.454 0.454 0.455 0.454 0.454 0.454 0.09 4 0.686 0.685 0.687 0.685 0.687 0.686 0.11 5 0.179 0.179 0.179 0.178 0.178 0.179 0.11 6(S2) 1.000 1.000 1.000 1.000 1.000 1.000 0.00 7 0.032 0.032 0.032 0.032 0.032 0.032 0.13

[0104] 3.4 Durability test

[0105] 3.4.1 Investigation of different flow rates (±0.02ml / min)

[0106] Take about 0.2g of Elsholtzia scoparia granules (batch number: 220601) and perform the test according to the test method in item "3.1" at flow rates of 0.98ml / min, 1.00ml / min, and 1.02ml / min. Fig.10As shown, 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 7 common peaks in the characteristic spectrum. Caffeic acid (peak 2) is used as the reference peak S1, and rosmarinic acid (peak 6) is used as the reference peak S2. The relative retention time and relative peak area of ​​peak 1, peak 3, peak 4 and S1 are calculated, and the relative retention time and relative peak area of ​​peak 5, peak 7 and S2 are calculated, and the RSD value is calculated. The calculation results show that the relative retention time and relative peak area RSD% values ​​are both within the qualified range. 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 good, see Table 12-13 for details.

[0107] Table 12 - Relative retention time of HPLC test characteristic spectrum at different flow rates (±0.02 ml / min)

[0108] Peak 0.98ml / min 1.00ml / min 1.02ml / min RSD% 1 0.954 0.954 0.954 0.02 2(S1) 1.000 1.000 1.000 0.00 3 1.051 1.053 1.056 0.24 4 1.148 1.153 1.159 0.48 5 0.981 0.981 0.981 0.02 6(S2) 1.000 1.000 1.000 0.00 7 1.366 1.365 1.366 0.05

[0109] Table 13 - Relative peak area of ​​HPLC characteristic spectrum in different flow rate tests (±0.02 ml / min)

[0110] Peak 0.98ml / min 1.00ml / min 1.02ml / min RSD% 1 1.171 1.143 1.162 1.23 2(S1) 1.000 1.000 1.000 0.00 3 0.463 0.451 0.464 1.51 4 0.705 0.669 0.679 2.71 5 0.178 0.178 0.179 0.35 6(S2) 1.000 1.000 1.000 0.00 7 0.033 0.032 0.032 0.85

[0111] 3.4.2 Investigation of different flow rates (±0.2ml / min)

[0112] Take about 0.2g of Elsholtzia scoparia formula granules (batch number: 220601) and perform the test according to the test method in item "3.1" at flow rates of 0.80ml / min, 1.00ml / min, and 1.20ml / min. Fig.11 As shown, 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 7 common peaks in the characteristic spectrum. Caffeic acid (peak 2) is used as the reference peak S1, and rosmarinic acid (peak 6) is used as the reference peak S2. The relative retention time and relative peak area of ​​peak 1, peak 3, peak 4 and S1 are calculated, and the relative retention time and relative peak area of ​​peak 5, peak 7 and S2 are calculated, and the RSD value is calculated. The calculation results show that the relative retention time and relative peak area RSD% values ​​are not within the qualified range, see Table 14-15 for details. The experiment shows that the flow rate change of ±0.2ml / min has a greater impact on the characteristic spectrum determination, that is, the specified flow rate is 1.0ml per minute.

[0113] Table 14 - Relative retention time of HPLC characteristic spectrum in different flow rates (±0.2ml / min)

[0114]

[0115]

[0116] Table 15 - Relative peak area of ​​HPLC characteristic spectrum of different flow rates (±0.2ml / min)

[0117] Peak 0.8ml / min 1.0ml / min 1.2ml / min RSD% 1 1.045 1.145 1.105 4.61 2(S1) 1.000 1.000 1.000 0.00 3 0.431 0.449 0.456 2.95 4 0.645 0.676 0.732 6.45 5 0.173 0.178 0.190 4.62 6(S2) 1.000 1.000 1.000 0.00 7 0.031 0.032 0.033 2.78

[0118] 3.5 Summary

[0119] The characteristic spectrum method was tested for specificity, repeatability, precision, stability, intermediate precision, reproducibility and durability, and the results met the requirements, indicating that the established method can be well used for the determination of the characteristic spectrum of the Elsholtzia lappa formula granules.

[0120] 3.6 Characterization and analysis of characteristic spectra of three batches of finished products

[0121] 3.6.1 Characteristic spectra of three batches of finished products

[0122] According to the proposed characteristic spectrum analysis method, the characteristic spectrum of three batches of finished products was determined and located by caffeic acid (concentration: 20.6μg / ml) and rosmarinic acid (concentration: 98.84μg / ml) reference substances. The results showed that there were 7 common peaks in the characteristic chromatograms of the three batches of finished products, and the retention times of the 7 characteristic peaks in the chromatograms of the reference medicinal material reference substances should correspond to each other. Among them, the peaks corresponding to the caffeic acid reference substance and the rosmarinic acid reference substance reference substances are peak 2 (S1) and peak 6 (S2), see for details. Figure 12-14 .

[0123] 3.6.2 Evaluation of characteristic chromatograms of three batches of formulated granules

[0124] According to the characteristic spectrum analysis method proposed above, the characteristic spectrum of three batches of finished products was determined. The results showed that there were 7 common peaks in the characteristic spectrum, with caffeic acid (peak 2) as the reference peak S1 and rosmarinic acid (peak 6) as the reference peak S2. The relative retention time and relative peak area of ​​peak 1, peak 2, peak 4 and S1 were calculated, and the relative retention time of peak 5, peak 7 and S2 was calculated. The relative retention time and range are shown in Table 16.

[0125] Table 16-Relative retention time of common peaks in three batches of finished products

[0126]

[0127]

[0128] In summary, the determination method of the characteristic spectrum of the formula granules established by high performance liquid chromatography was verified for repeatability, precision, stability, intermediate precision, reproducibility and durability 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 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 7 common characteristic peaks were calibrated. Caffeic acid (peak 2) was used as the reference peak S1, and rosmarinic acid (peak 6) was used as the reference peak S2. The relative retention times of peaks 1, 3, and 4 with S1 were calculated, and the relative retention times of peaks 5 and 7 with S2 were calculated. The average relative retention times of the peaks of the three batches of samples were proposed as the specified values: 0.95 (peak 1), 1.05 (peak 3), 1.15 (peak 4), 0.98 (peak 5), and 1.37 (peak 7). Taking into account the errors of multiple factors such as experimental operation, instruments, reagents, etc., the allowable range of relative retention time is proposed to be ±10%.

[0129] 4 Extracts

[0130] 4.1 Test methods

[0131] 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).

[0132] 4.2 Investigation of extractables

[0133] About 2 g of samples from three batches of large-scale production were taken and measured according to the method. The results are shown in Table 17.

[0134] Table 17 - Extraction results

[0135]

[0136] 4.3 Drafting of extractable range

[0137] Proposed plan 1:

[0138] 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.

[0139] The specification of this product is that each 1g of granules is equivalent to 5g of medicinal pieces. According to the lower limit of the allowable range of the extract yield of the standard decoction of this product (10.0%~16.6%), the amount of dry extract contained in each 1g of granules is not less than 5g×10.0% (i.e. 0.5g); on this basis, according to the extract limit proposed for the standard decoction of this product (26.0%), it can be calculated that the extract in each 1g of granules is not less than 0.5g×26.0% (i.e. 0.13g), or in other words, the extract in this product (granules) is not less than 13.0% (0.13×100%).

[0140] The extract values ​​of three batches of large-scale production samples of this product were 28.4%, 29.8%, and 28.3%, respectively, which were far higher than the particle extract limit (13%) calculated above, so it was considered to appropriately increase the extract limit of this product. To this end, according to 70% of the extract limit (26.0%) proposed for the standard decoction, that is, 26.0% × 0.7 = 18.2%, it was proposed that the alcohol-soluble extract of this product should not be less than 18.2%.

[0141] Proposed plan 2:

[0142] The content of particles was converted from 21 batches of standard decoction extracts of Herba Cynoglossi, and the summary is shown in Table 18:

[0143] Table 18-21 Summary of extracts from batches of Shixiangru Biao decoction converted into granules

[0144]

[0145] Combined with the paste yield, according to the proposed product specification "each 1g of formula granules is equivalent to 5g of decoction pieces", the extracts of 21 batches of standard decoction samples are converted into granular extracts, with an average extract of 24.21%, and the actual measured value converted into granular extracts ranges from 13.30% to 34.04%, with an SD of 6.86; calculated at 70% to 130% of the mean, the allowable range of extracts is 16.95% to 31.47%. Calculated at mean -1.5SD to mean +3SD, the allowable range of extracts is 13.92% to 44.78%. Considering that the 21 batches of samples of this product are quite different, the lowest value of 13.3% converted into granular extracts is used as the lower limit content. For this reason, the proposed allowable range of extracts is: the alcohol-soluble extract of this product shall not be less than 13.3%.

[0146] After reviewing and comparing the above two approaches to extractables limits, and combining them with the content data of three batches of large-scale production samples, the final extractables limit range was proposed to be no less than 18.2%.

[0147] 5. Content determination

[0148] 5.1 Basis for selecting the components to be tested

[0149] This product is the dried aerial part of the Lamiaceae plant Moslachinensis Maxim. Its medicinal materials are distributed in East China, Central and South China, etc. The whole root or aerial part is used as medicine. It is slightly warm and spicy. It can induce sweating and relieve the symptoms, eliminate dampness and harmonize the middle, and promote diuresis and reduce swelling. The chemical components contained are volatile oils, as well as flavonoids, inorganic elements, etc. It is used clinically to dispel wind and cold, and has the effect of inducing sweat and relieving the symptoms, but it is mostly used for fever, chills, headache, and anhidrosis caused by colds and wind-colds in summer. Pharmacological experiments have shown that the volatile oil in Moslachinensis has broad-spectrum antibacterial and bactericidal effects, antiviral effects, and antibacterial effects. According to the content determination item of Moslachinensis in the 2020 edition of the Chinese Pharmacopoeia, thymol and carvacrol are used as the content determination components.

[0150] The 15 batches of standard decoction of Herba Elsholtziae were tested by gas chromatography, and no volatile components were detected. The contents of thymol and carvacrol were Fig.15 . In order to establish a method for determining the content of Elsholtzia ciliata, it was found from the literature and chromatographic positioning that it contains caffeic acid and rosmarinic acid. The content of caffeic acid and rosmarinic acid is quite different. Rosmarinic acid has strong anti-inflammatory activity. At the same time, rosmarinic acid also has antibacterial, antiviral and anti-tumor activities. Considering the pharmacological effects of Elsholtzia ciliata, rosmarinic acid was selected as the measured component.

[0151] 5.2 Determination of rosmarinic acid content

[0152] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0153] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel as filler (4.6 mm × 250 mm, 5 μm); acetonitrile-0.1% phosphoric acid (20:80) as mobile phase; detection wavelength of 330 nm. The theoretical plate number calculated based on the rosmarinic acid peak should be no less than 10,000.

[0154] Preparation of reference solution: Take an appropriate amount of rosmarinic acid reference substance, weigh it accurately, and add methanol to make a solution containing 100 μg of rosmarinic acid per 1 ml.

[0155] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.2g, weigh it accurately, put it in a stoppered conical flask, accurately add 15ml of 70% ethanol, stopper it, weigh it, ultrasonically treat it (power 300W, frequency 50kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0156] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0157] 5.3 Investigation on the pretreatment of samples for determination of Elsholtzia content

[0158] The sampling amount (or the amount of solvent) for the determination of rosmarinic acid content in Elsholtzia scoparia granules (batch number: 220601) was investigated to determine the best method for preparing the test solution.

[0159] 5.3.1 Sampling volume investigation

[0160] 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 content of different sampling amounts was not much different, and 0.2g had a better extraction effect than 0.1g and 0.3g, so 0.2g was selected as the sampling amount for preparing the test solution, see Table 19 for details.

[0161] Table 19 - Comparison of different sampling volumes

[0162]

[0163]

[0164] In summary, the main parameters of the test solution preparation method are determined as follows: take an appropriate amount of fine powder of the product, take about 0.2 g, accurately weigh, place in a stoppered conical flask, accurately add 15 mL of 70% ethanol, seal, weigh, ultrasonically treat (power 250 W, frequency 50 kHz) for 30 minutes, cool, weigh again, make up the lost weight with 70% ethanol, shake well, filter, and take the filtrate.

[0165] 5.4 Validation of analytical method for content determination

[0166] 5.4.1 Specificity inspection

[0167] Take about 0.2g of maltodextrin and prepare a maltodextrin blank control solution according to the method under "5.2" above, and take the test solution of Elsholtzia scoparia granules, the maltodextrin blank control solution, the Elsholtzia scoparia control medicinal material solution and the rosmarinic acid reference solution for determination. Fig.16 As shown, the results show that the main characteristic peaks in the chromatogram of the test sample are well separated, the maltodextrin blank has no interference, and the method has good specificity.

[0168] 5.4.2 Linear relationship test

[0169] Take the rosmarinic acid reference solution with the concentrations of 1.3728μg / ml, 27.4557μg / ml, 54.9115μg / ml, 109.8230μg / ml, 219.6459μg / ml, and 439.2918μg / ml and perform the determination according to the chromatographic conditions under "5.2". Draw the standard curve with the amount of rosmarinic acid as the abscissa and the peak area of ​​rosmarinic acid as the ordinate, and perform linear regression.

[0170] The regression equation is: y = 3E + 06x – 12529R2 = 1

[0171] It can be seen that rosmarinic acid has a good linear relationship with its peak area in the range of 0.0137μg to 4.3929μg, see Fig.17 and Table 20.

[0172] Table 20 - Linearity test results of rosmarinic acid

[0173] serial number Injection volume μg Peak area Linear 1 0.0137 43649 Linear 2 0.2746 887030 Linear 3 0.5491 1813355 Linear 4 1.0982 3699690 Linear 5 2.1965 7354606 Linear 6 4.3929 14712788

[0174] 5.4.3 Sample recovery test

[0175] About 0.1 g of the sample of Elsholtzia scoparia formula granules (batch number: 220601) was taken, a total of 9 portions, divided into 3 groups, 3 portions in each group, 2.1 ml, 2.8 ml, and 3.2 ml of rosmarinic acid reference substance of known concentration (354.3372 μg / ml) were added to each, and the test solution was prepared according to the method under "5.2", and the test was carried out. The average recovery rate of rosmarinic acid was calculated to be 103.4830%, and the RSD was 1.36%. See Table 21 for details.

[0176] Table 21 - Sample recovery test results

[0177]

[0178] 5.4.4 Durability test

[0179] 5.4.4.1 Experiments with different mobile phase ratios

[0180] About 0.2g of the sample of Elsholtzia scoparia formula granules (batch number: 220601) was taken and tested according to the test method under item "5.2" with acetonitrile-0.1% phosphoric acid (19:81), (20:80), and (21:79). The calculation results showed that the RSD value of the measured content was 0.46%, which was less than 2.0%. The test showed that the method had good durability for small changes in the mobile phase ratio, as shown in Table 22.

[0181] Table 22 - Comparison of different mobile phase ratios

[0182]

[0183] 5.4.4.2 Tests at different wavelengths

[0184] About 0.2g of the sample of Elsholtzia scoparia formula granules (batch number: 220601) was taken and tested at wavelengths of 290nm, 329nm, and 330nm according to the test method under "5.2". The calculation results showed that the RSD value of the measured content was 0.10%, which was less than 2.0%. The test showed that the method had good durability against wavelength changes, as shown in Table 23.

[0185] Table 23- Comparison of different wavelength ratios

[0186]

[0187] 5.5 Summary

[0188] In summary, the results of the entire analytical method met the requirements after specificity, linearity investigation, sample recovery and durability tests, indicating that the established method can be well used for the determination of rosmarinic acid in the Elsholtzia scoparia granules.

[0189] 5.6 Assay of three batches of finished products

[0190] According to the above-mentioned content analysis method, the rosmarinic acid content of three batches of finished products was determined. The results are shown in Table 24.

[0191] Table 24 - Test results of three batches of finished products

[0192]

[0193] 5.7 Proposed content limit range

[0194] (1) Proposed content range plan 1:

[0195] 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.

[0196] The specification of this product is that each 1g of granules is equivalent to 5g of medicinal pieces. According to the allowable range of the extract yield of the standard decoction of this product (10.0% to 16.6%), the amount of dry extract contained in each 1g of granules is 5g×10.0% to 5g×16.6%; on this basis, according to the allowable range of rosmarinic acid content proposed for the standard decoction of this product (5.8mg / g to 28.0mg / g), it can be calculated that the allowable range of rosmarinic acid content in each 1g of granules is 5g×10.0%×5.8mg / g to 5g×16.6%×28.0mg / g (i.e. 2.90mg to 23.24mg). Considering that this lower limit content (2.90 mg / g) is too low, combined with the content of the lowest batch of standard decoction (Y220521PT, 10.6% paste yield, 7.2337 mg / g) in the 21 batches of standard decoctions of this product converted into granules (i.e., the content of rosmarinic acid in each 1g of granules is: 5g×10.6%×7.2337 mg / g=3.834 mg), the lower limit of the content of this product can be preliminarily proposed to be 4.0 mg / g. The contents of the three batches of large-scale production samples of this product were 9.43 mg / g, 9.67 mg / g, and 10.68 mg / g, respectively, all within the above-mentioned granule content range calculated or proposed based on the standard decoction.

[0197] In summary, the rosmarinic acid content of this product is proposed to range from 4.0 mg / g to 23.2 mg / g.

[0198] (2) Proposed content range plan 2:

[0199] The content of 21 batches of standard decoction of Herba Cynomorii was converted into the content of granules, which is summarized as follows:

[0200] Table 25-21 Summary of the content of standard decoction of Elsholtzia scoparia converted into granules

[0201]

[0202]

[0203] Combined with the paste yield, according to the proposed product specification "each 1g of formula granules is equivalent to 5g of decoction pieces", the content of 21 batches of standard decoction samples was converted into granule content, the average content of rosmarinic acid was 10.966mg / g, and the actual content was converted into granule content in the range of 3.834mg / g to 17.050mg / g, with an SD of 4.123; calculated at 70% to 130% of the mean, the allowable range of rosmarinic acid content was 7.68mg / g to 14.26mg / g. Calculated at mean -2SD to mean +3SD, the allowable range of rosmarinic acid content was 2.75mg / g to 23.23mg / g. Considering that the 21 batches of samples of this product are quite different, the lowest value of 3.83mg / g converted into granules is adopted as the lower limit content, and for this reason, the allowable range of rosmarinic acid content is proposed to be 3.8mg / g to 23.2mg / g.

[0204] 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 rosmarinic acid content range was proposed to be 4.0 mg / g to 23.2 mg / g.

[0205] 5.8 Volatile Oil

[0206] 5.8.1 Test methods

[0207] The relative density of the volatile oil of Elsholtzia ciliata is about 0.9, which is lower than 1.0, so it is determined according to Method A under the volatile oil determination method (General Rules 2204 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0208] 5.8.2 Determination of volatile oil content

[0209] 100g of each sample from three batches of large-scale production was taken and determined according to the law. The results of the volatile oil content of the three batches of samples are shown in Table 26.

[0210] Table 26 - Volatile oil content determination results

[0211]

[0212] 5.8.3 Proposed range of volatile oil content

[0213] The average volatile oil content of three batches of scale production samples is 0.5%. According to the specifications of this product (each 1g of formula granules is equivalent to 5g of decoction pieces), the allowable range of volatile oil in 21 batches of standard decoctions is 0.13% to 0.38% of decoction pieces, and the measured range of the paste yield of 21 batches of standard decoctions is 10.0% to 16.6%, and the range of auxiliary materials is 17% to 50%. The range of volatile oil content of the formula granules is calculated as follows: 0.13×(1-50%)×5=0.325%, 0.38×(1-17%)×5=1.58%, that is, the acceptable range of volatile oil content is 0.325% to 1.58%. Combined with the above three batches of scale production samples, comprehensive consideration is taken, so the volatile oil content of this product is proposed to be 0.3% to 1.6%. The volatile oil content of the pilot samples is within the proposed range and meets the requirements.

[0214] 6. Origin identification

[0215] There are two sources of this product, which are the dried aerial parts of Moslachinensis Maxim. or Moslachinensis'Jiangxiangru' of the Lamiaceae family. The sources of Moslachinensis Maxim. and Moslachinensis'Jiangxiangru' were identified by using properties, thin layer identification, characteristic spectrum and other methods.

[0216] 6.1 Properties

[0217] Elsholtzia: 30-50cm long, purple-red at base, yellow-green or light yellow at upper part, densely covered with white hairs all over, stem square-cylindrical, sub-circular at base, 1-2mm in diameter, obvious nodes, 4-7cm long internodes; brittle, easy to break, leaves opposite, mostly wrinkled or falling off, leaves flattened to be oblong or lanceolate, dark green or yellow-green, with 3-5 sparse shallow serrations on the edges, spike inflorescence terminal and axillary, bracts round-ovate or round-obovate, falling off or remaining; calyx persistent, bell-shaped, light purple-red or gray-green, 5-lobed at apex, densely covered with hairs, 4 nutlets, 0.7-1.1mm in diameter, nearly spherical, with reticular patterns, strong and fresh fragrance, slightly spicy and cool taste.

[0218] Elsholtzia jiangensis: 55-66cm long, yellow-green in surface, soft in texture, with 5-9 sparse shallow serrations on the edge, the fruit has a diameter of 0.9-1.4mm, and has sparse reticular patterns on the surface.

[0219] 6.2 Thin layer identification

[0220] Take the volatile oil under "5.8.2 Determination of volatile oil content" and add ether to make a solution containing 3μl per 1ml as the test solution.

[0221] Take thymol reference substance and carvacrol reference substance separately, add ether to make solutions containing 1 mg per 1 ml, as reference substance solutions.

[0222] According to the thin layer chromatography method (General Rule 0502), 5 μl of the test solution, thymol reference solution, and carvacrol reference solution were taken and spotted on the same silica gel G thin layer plate, and toluene was used as the developing solvent. The plate was developed with a development distance of more than 15 cm. The plate was taken out, dried, sprayed with 5% vanillin sulfuric acid solution, and heated at 105°C until the spots were clearly colored. Fig.18 As shown, in the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the reference sample.

[0223] 6.3 Feature Spectrum

[0224] Take the control medicinal materials of Elsholtzia schrenkiana and Elsholtzia jiangxiensis, and determine them according to the following high performance liquid chromatography method (General Rules 0512 of the 2020 Edition of the Chinese Pharmacopoeia).

[0225] Octadecylsilane bonded silica gel was used as the filler (4.6 mm × 250 mm, 5 μm); acetonitrile was used as the mobile phase A, 0.05% phosphoric acid solution was used as the mobile phase B, and gradient elution was performed as specified in the following table; the flow rate was 1.0 ml per minute; the column temperature was 35°C; the detection wavelength was 330 nm, and the number of theoretical plates calculated based on rosmarinic acid was not less than 50,000.

[0226] Preparation of Elsholtzia ciliata reference solution: Take 0.5 g of Elsholtzia ciliata reference medicinal material, add 15 ml of 70% ethanol, ultrasonically treat (power 250 W, frequency 50 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the Elsholtzia ciliata reference solution.

[0227] Preparation of Elsholtzia jiangru reference solution: Take 0.5 g of Elsholtzia jiangru reference medicinal material, add 15 ml of 70% ethanol, ultrasonically treat (power 250 W, frequency 50 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the Elsholtzia jiangru reference solution.

[0228] Preparation of reference substance solution: Take appropriate amount of caffeic acid and rosmarinic acid reference substances, weigh accurately, add methanol to prepare mixed solutions containing 20 μg of caffeic acid and 100 μg of rosmarinic acid per 1 ml, as reference substance solutions.

[0229] Preparation of test solution: Take about 0.5 g of the powder of the product, weigh it accurately, put it in a stoppered conical flask, add 15 ml of 70% ethanol accurately, stopper it, weigh it, ultrasonically treat it (power 300 W, frequency 50 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% ethanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0230] Determination method: Accurately pipette 10 μl of each of the Elsholtzia schrenkiana reference solution, Elsholtzia jiangxiensis reference solution, reference substance reference solution and test solution, inject into the liquid chromatograph, and determine. Fig.19 .from Fig.19 It can be seen that the characteristic peaks appearing on the HPLC characteristic spectrum of Lithospermum officinale are significantly more than those appearing on the HPLC characteristic spectrum of Elsholtzia jiangxiensis. The above chromatographic conditions are conducive to distinguishing Lithospermum officinale and Elsholtzia jiangxiensis as two different medicinal materials in production and clinical applications, making the quality detection of Lithospermum officinale formula granules more accurate.

[0231] 6.4 Comparison of the medicinal parts of Elsholtzia schrenkiana and Elsholtzia jiangxiensis

[0232] This study on the Elsholtzia formula granules referred to ancient literature and was based on the standards of the 2020 edition of the "Chinese Pharmacopoeia", and the medicinal part used was the dried above-ground part.

[0233] First, the quality of stems, branches and leaves (rosmarinic acid content) of the source variety of this product, Elsholtzia paniculata, was studied. The results are shown in Table 27.

[0234] Table 27- Comparison of rosmarinic acid content in different parts of Elsholtzia ciliata

[0235]

[0236] The content of the main active ingredient (rosmarinic acid) in different parts of Elsholtzia ciliata was determined. The results showed that the content of rosmarinic acid was highest in Elsholtzia ciliata leaves, followed by Elsholtzia ciliata branches, and least in Elsholtzia ciliata stems. Combined with the results of the source identification of this sample, it was confirmed that the medicinal parts of the Elsholtzia ciliata formula granules met the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0237] Then, the rosmarinic acid content in different parts of similar / different origin varieties of Elsholtzia elata was studied, and the results are shown in Table 28.

[0238] Table 28- Comparison of rosmarinic acid content in different parts of Elsholtzia jiangensis

[0239]

[0240] The content of the main active ingredient (rosmarinic acid) in different parts of Elsholtzia jiangxiensis was determined. The results showed that the content of rosmarinic acid was highest in Elsholtzia jiangxiensis leaves, followed by Elsholtzia jiangxiensis stems, and least in Elsholtzia jiangxiensis branches. In comparison, the content of rosmarinic acid in Elsholtzia jiangxiensis was higher than that in Elsholtzia jiangxiensis.

[0241] At the same time, three batches of Elsholtzia ciliata and Elsholtzia jiangxiensis were taken, and the stems, branches and leaves of Elsholtzia ciliata and Elsholtzia jiangxiensis were classified and weighed respectively, and the average percentage was calculated. The results are shown in Table 29. It can be seen that compared with the two, Elsholtzia ciliata has more leaves, while the stems of Elsholtzia jiangxiensis are thicker and the branches account for a larger proportion.

[0242] 39-Comparison table of stem, branch and leaf proportions of Elsholtzia schrenkiana and Elsholtzia jiangxiensis

[0243] Product Name Origin sampling Stem ratio Branch proportion Leaf ratio Elsholtzia Hunan 100g 6.4％ 47.1％ 46.5％ Elsholtzia jiangensis Hubei 100g 16.2％ 60.8％ 23.0％

[0244] By analyzing the data in Tables 27-29, it can be seen that under the same gram weight, the rosmarinic acid content in the medicinal material of Lithospermum officinale is higher than that in Lithospermum officinale.

[0245] 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 5g of medicinal pieces.

[0246] 8 Storage is formulated according to the basic storage requirements of the variety.

[0247] 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 Elsholtzia scoparia granules, characterized in that: The following detection methods are included: Through the properties, dry extract yield, thin layer identification, characteristic spectrum, extract and rosmarinic acid content determination of the formula granules of Elsholtzia lappa, the content standard of the formula granules is limited to 4.0-23.2 mg of rosmarinic acid per 1g, among which, thin layer identification is carried out by thin layer chromatography; characteristic spectrum and rosmarinic acid content determination are both carried out 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 reference medicinal material of Herba Cynoglossi as a reference medicinal material reference solution b, using a solution made from a reference substance of caffeic acid and a reference substance of rosmarinic acid as a reference substance reference solution b, using a solution made from a sample of Herba Cynoglossi formula granules 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: a chromatographic column: using octadecylsilane bonded silica gel as a filler, with a specification of 4.6 mm×250 mm, 5µm, Shimadzu Shim-pack GIST C18-AQ; a mobile phase: using acetonitrile as mobile phase A, using 0.05% phosphoric acid solution as mobile phase B, and performing gradient elution according to the provisions of Table a; Table a Gradient elution program Flow rate: 1.0 mL / min; column temperature: 35°C; injection volume: 10 μL; detection wavelength: 330 nm.

2. The quality detection method of Elsholtzia scoparia formula granules according to claim 1, characterized in that: The preparation method of the Elsholtzia scoparia formula granules is as follows: take 5000g of Elsholtzia scoparia slices, add water to boil, collect a proper amount of volatile oil, encapsulate the volatile oil with β-cyclodextrin for later use, filter, concentrate the filtrate into a clear paste, add the volatile oil inclusion compound, dry, add a proper amount of auxiliary materials, mix well, granulate, and make 1000g.

3. The quality detection method of Elsholtzia scoparia formula granules according to claim 1, characterized in that: The thin layer chromatography method comprises the following steps: (1) Preparation of test solution a: Take a sample of Elsholtzia scoparia granules, grind it into powder, weigh 2.0 g, add 30 ml of 70% ethanol, ultrasonically treat for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 1 ml of ethanol to dissolve the residue, and prepare test solution a; (2) Preparation of control medicinal material solution a: Take 1.0 g of Elsholtzia ciliata control medicinal material, grind it into powder, add 30 ml of 70% ethanol, ultrasonically treat it for 30 minutes, cool it, filter it, evaporate the filtrate to dryness, add 1 ml of ethanol to dissolve the residue, and prepare control medicinal material solution a; (3) Preparation of reference solution a: Take rosmarinic acid reference substance and add methanol to make a 0.3 mg / ml solution to prepare reference solution a. (4) Thin layer chromatography analysis: Thin layer chromatography conditions are as follows: Thin layer plate: silica gel G thin layer plate; spot volume: 2uL each of test solution a, reference drug solution a, and reference substance solution a; n-hexane-ethyl acetate-isopropanol-formic acid solution is used as the developing solvent, and the ratio of n-hexane, ethyl acetate, isopropanol, and formic acid is 15:3:3.5:0.5; color developer: 5% vanillin sulfuric acid solution, heated at 105°C until the spots are clearly colored.

4. The quality detection method of Elsholtzia scoparia formula 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 Elsholtzia scoparia formula 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 Elsholtzia ciliata reference medicinal material, place it in a stoppered conical flask, add 15 ml of 70% ethanol, ultrasonically treat for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material solution b; (2) Preparation of reference substance solution b: Take appropriate amount of caffeic acid reference substance and rosmarinic acid reference substance, weigh accurately, and add methanol to prepare a mixed solution containing 20 µg of caffeic acid and 100 µg of rosmarinic acid per 1 ml respectively, which is used as reference substance solution b; (3) Preparation of test solution b: Grind an appropriate amount of Elsholtzia scoparia granules into powder. Take about 0.2 g and place in a stoppered conical flask. Add 15 ml of 70% ethanol and ultrasonically treat for 30 min. Cool, shake well, filter, and take the filtrate as the test solution b.

6. The quality detection method of Elsholtzia scoparia formula granules according to claim 1, characterized in that: The liquid chromatography method for determining the rosmarinic acid content includes: performing liquid chromatography analysis, using a solution made from a rosmarinic acid reference substance as a reference substance solution c, using a solution made from a sample of the Elsholtzia scoparia formula granules as a test solution c, accurately aspirating the reference substance solution c and the test solution c, respectively, injecting them into the liquid chromatograph, and determining to obtain the content; wherein, the chromatographic conditions used are: a chromatographic column: octadecylsilane bonded silica gel as a filler, with a specification of 4.6mm×250mm, 5µm; acetonitrile-0.1% phosphoric acid as a mobile phase, with a ratio of acetonitrile to 0.1% phosphoric acid of 20:80, and gradient elution as specified; flow rate: 1.0mL / min; column temperature: 35°C; injection volume: 10μL; detection wavelength: 330nm.

7. The quality detection method of Elsholtzia scoparia formula granules according to claim 6, characterized in that: Adopting liquid chromatography to measure rosmarinic acid content also comprises the following steps: (1) Preparation of reference solution: Take an appropriate amount of rosmarinic acid reference substance, weigh it accurately, and add methanol to prepare a solution containing rosmarinic acid at a concentration of 100 ug / ml, which is used as reference solution C; (2) Preparation of test solution: Grind an appropriate amount of Elsholtzia scoparia granules, take about 0.2 g, weigh accurately, place in a stoppered conical flask, accurately add 15 ml of 70% ethanol, stopper, weigh, ultrasonically treat for 30 minutes, cool, weigh again, make up the loss with 70% ethanol, shake well, filter, and take the filtrate as the test solution c.

8. The method for detecting the quality of the Elsholtzia scoparia formula granules according to claim 1, characterized in that: It also includes using liquid chromatography to respectively determine the characteristic spectra of the Elsholtzia jiangxiensis reference medicinal material and the Elsholtzia schrenkiana reference medicinal material of the Elsholtzia schrenkiana formula granule base variety, specifically: Octadecylsilane bonded silica gel was used as a filler, with a specification of 4.6 mm × 250 mm, 5 µm, acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, gradient elution was performed according to the provisions of Table a, the flow rate was 1.0 ml per minute, the column temperature was 35 ° C, and the detection wavelength was 330 nm; Preparation of Elsholtzia ciliata reference solution d: Take 0.5 g of Elsholtzia ciliata reference medicinal material, add 15 ml of 70% ethanol, ultrasonically treat for 30 minutes, cool, shake well, filter, and take the filtrate to obtain; Preparation of Elsholtzia jiangruta reference solution d: Take 0.5 g of Elsholtzia jiangruta reference medicinal material, add 15 ml of 70% ethanol, ultrasonically treat for 30 minutes, cool, shake well, filter, and take the filtrate to obtain; Preparation of reference substance solution d: Take appropriate amount of caffeic acid and rosmarinic acid reference substances, weigh accurately, add methanol to prepare mixed solutions containing 20µg caffeic acid and 100µg rosmarinic acid per 1ml, as reference substance solution d; Prepare the test solution d: take about 0.5g of Elsholtzia scoparia granules, weigh accurately, put into a stoppered conical flask, add 15ml of 70% ethanol accurately, stopper tightly, weigh, ultrasonically treat for 30 minutes, cool, weigh again, make up the loss with 70% ethanol, shake well, filter, and take the filtrate to obtain; Accurately pipette 10µl of each of Elsholtzia ciliata reference solution d, Elsholtzia jiangxiensis reference solution d, reference substance solution d, and test solution d, inject into liquid chromatograph, and measure to obtain the result.

Images