A method for constructing HPLC characteristic spectrum of Radix Codonopsis pilosulae medicinal materials, decoction pieces, standard decoctions, and formula granules

Abstract

The present invention provides a method for constructing an HPLC characteristic spectrum of a Herba Lycopodii medicinal material, a decoction piece, a standard decoction, and a formula granule, comprising: A) dissolving the Herba Lycopodii raw material in a solvent to obtain a test liquid; B) determining the test liquid by high performance liquid chromatography to obtain an HPLC characteristic spectrum of the Herba Lycopodii raw material; the chromatographic conditions of the high performance liquid chromatography are: a chromatographic column is a C18 column; mobile phase A is methanol, and mobile phase B is a 0.1% phosphoric acid aqueous solution, and gradient elution. The present invention adopts high performance liquid chromatography, selects methanol-0.1% phosphoric acid as the mobile phase for gradient elution, and uses dihydromyricetin reference substance and myricetin as reference substances to establish an HPLC characteristic spectrum of Herba Lycopodii medicinal materials, decoction pieces, standard decoctions, and formula granules, with good repeatability and precision, and the method is stable and reliable, and the quality of Herba Lycopodii medicinal materials, decoction pieces, standard decoctions, and formula granules can be controlled.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for constructing an HPLC characteristic spectrum of scutellaria baicalensis medicinal materials, decoction pieces, standard decoctions and formula granules. Background Art

[0002] This product is the dried whole herb of Shuteria pampaniniana Hand.-Mazz., a plant of the Leguminosae family, and is processed into formula granules according to standard processing.

[0003] In order to ensure the uniformity and stability of the quality of the Radix Rhizoma Rhizoma Rhizoma granules, it is necessary to control the quality of the Radix Rhizoma ... Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing an HPLC characteristic spectrum of Caulis Scutellariae medicinal materials, decoction pieces, standard decoctions, and formula granules. The HPLC characteristic spectrum method of Caulis Scutellariae medicinal materials, decoction pieces, standard decoctions, and formula granules constructed by the present invention is stable and reliable, and can control the quality of Caulis Scutellariae medicinal materials, decoction pieces, standard decoctions, and formula granules.

[0005] The present invention provides a method for constructing an HPLC characteristic spectrum of a Herba Lycopodii Herba, a decoction piece, a standard decoction, and a formula granule, comprising:

[0006] A) dissolving the raw material of Caulis Sargentodoxae in a solvent to obtain a test solution;

[0007] B) using high performance liquid chromatography to measure the test solution to obtain a HPLC characteristic spectrum of the Caulis Sargentodoxae raw material;

[0008] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the elution is performed in a gradient manner.

[0009] The invention provides a method for constructing an HPLC characteristic spectrum of a Caulis rubra medicinal material, a decoction piece, a standard decoction and a formula granule. Firstly, a Caulis rubra raw material is taken and dissolved with a solvent to obtain a test solution.

[0010] The raw material of the radix rubrae is radix rubrae medicinal material, granules, decoction pieces or decoction.

[0011] The extraction method of the invention is ultrasonic extraction or heating reflux extraction; the ultrasonic power is 300W, the frequency is 40kHz; and the extraction time is 30 to 40 minutes.

[0012] According to the present invention, the ratio of the mass g of the Rhizoma Ceratitise raw material to the volume mL of the solvent is (0.2-0.5): (25-50), and more preferably (0.2-3): (25-30).

[0013] In a preferred embodiment of the present invention, the preparation of the sample of the granules or decoction of Rhizoma Caulis is specifically as follows:

[0014] Add 70% ethanol to dissolve the granules or decoction of Rhizoma Coptidis, sonicate, cool, shake well and filter.

[0015] The ratio of the mass g of the raw material of Rhizoma Ceratitise to the volume mL of the solvent is (0.2-0.5):(25-50), and more preferably (0.2-3):(25-30).

[0016] In some preferred embodiments of the present invention, the preparation of the herbal medicine and decoction pieces of Rhizoma Caulis is as follows:

[0017] The extract is obtained by heating the Herba Lycopodii with water, filtering, and ultrasonically dissolving the filtrate with ethanol. More preferably, the Herba Lycopodii is boiled with water, cooled, filtered, and the filtrate is evaporated to dryness, and 70% ethanol is precisely added and ultrasonically dissolved.

[0018] Among them, the ratio of the mass g of the Herba Lycopodiellae medicinal materials, the volume mL of water and the volume mL of ethanol is 0.5:50:25.

[0019] The ultrasonic power is preferably 300 W, and the frequency is preferably 40 kHz; the extraction time is preferably 30 to 40 min, and more preferably 30 min.

[0020] When the above solvent addition amount is used, the characteristic spectrum chromatographic peak area is moderate.

[0021] When the present invention adopts 70% ethanol as the extraction solvent, the value of "total peak area / sample weight" is relatively large, and the amount of information of the chromatographic peak is large. Therefore, 70% ethanol is selected as the extraction solvent.

[0022] When using ultrasound and reflux extraction, the "total peak area / sample weight" values ​​of different extraction methods are not much different. Considering the simplicity of experimental operation, ultrasound is selected as the extraction method.

[0023] When the extraction time was 30 min, the chromatogram peak shape and separation were better, and the extraction was more complete.

[0024] The raw material of the Herba Lycopodiellae is Herba Lycopodiellae decoction, Herba Lycopodiellae decoction pieces or decoction. The present invention does not limit it, and the above raw materials can all be subjected to quality control and qualitative detection by the method of the present invention.

[0025] It also includes the preparation of reference substance solutions and the preparation of reference medicinal material solutions;

[0026] Preparation of reference substance solution: dihydromyricetin and myricetin reference substance are taken respectively, dissolved in methanol to obtain reference substance solution; the concentration of dihydromyricetin in the reference substance solution is 100 μg / mL; the concentration of myricetin in the reference substance solution is 100 μg / mL;

[0027] Preparation of control medicinal material solution: Extract the control medicinal material of Rhizoma Caulis by heating with water, filter, add 70% ethanol to the filtrate and ultrasonically dissolve it to obtain the control medicinal material solution.

[0028] The reference substance solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference substance; and the components of the HPLC characteristic spectra of the Herba Lycopodii Herba, decoction pieces, standard decoctions, and formula granules are qualitatively measured based on the chromatogram of the reference substance.

[0029] The mobile phase A of the present invention is methanol, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the elution is performed by gradient elution.

[0030] The gradient elution of the present invention is preferably specifically:

[0031] 0-10min, phase A: 20%, phase B: 80%;

[0032] 10-20min, phase A: 20-25%, phase B: 80-75%;

[0033] 20-30min, phase A: 25-30%, phase B: 75-70%;

[0034] 30-40min, phase A: 30-43%, phase B: 70-57%;

[0035] 40-45min, phase A: 43-65%, phase B: 57-35%;

[0036] 45-50min, phase A: 65-80%, phase B: 35-20%;

[0037] 50-55min, phase A: 80-20%, phase B: 20-80%;

[0038] 55-60min, phase A: 20%, phase B: 80%.

[0039] The present invention has good baseline separation under the above elution gradient, good separation of each peak and stable baseline.

[0040] C18 column, specifications are 1.8μm, 2.1×100mm.

[0041] Chromatographic columns of different brands and different numbers were selected. The peak shapes and separation degrees of each chromatographic peak were good. The relative retention time RSD values ​​of each characteristic peak and S peak were in the range of 2.34% to 9.75%, and the relative peak area RSD values ​​were in the range of 4.16% to 55.27%. The relative retention time and relative peak area were quite different, indicating that the relative peak area of ​​each characteristic peak was greatly affected by different chromatographic columns.

[0042] Column temperature: 30℃.

[0043] When the column temperature is 30° C., the peak shape of the chromatogram is relatively symmetrical, the separation degree is better, and the peak output is relatively complete.

[0044] The mobile phase flow rate is preferably 0.3 ml / min.

[0045] The present invention found that the peak shapes of each chromatogram were better and more symmetrical at a flow rate of 0.3 ml / min, which was the most preferred solution.

[0046] The detection wavelength of the present invention is preferably 230 nm.

[0047] The inventors found that at 230 nm, the chromatographic information is rich, each component has good absorption, the response value is moderate, the chromatographic peak information volume is large, the peak separation is good, and the baseline is stable.

[0048] The injection volume of the present invention is 1 μL.

[0049] The beneficial effect of the present invention is that under one liquid chromatography condition, the substance group of Radix Rhizoma Cynoglossi medicinal materials, decoction pieces, standard decoctions, and formula granules is controlled by fingerprint spectrum, and the fingerprint spectrum is positioned by dihydromyricetin reference substance and myricetin; the cost of detection can be greatly reduced and qualitative detection can be achieved.

[0050] The similarity of HPLC characteristic spectra of Radix Rhizoma Cynoglossi, decoction pieces, standard decoction and formula granules was evaluated by using the similarity evaluation system of Chinese medicine chromatographic fingerprint, and the HPLC standard characteristic spectra of Radix Rhizoma Cynoglossi, decoction pieces, standard decoction and formula granules consisting of 8 characteristic peaks were obtained, among which Peak 1 (S): dihydromyricetin; Peak 4: taxifolin; Peak 6: myricetin.

[0051] In the standard characteristic spectrum of the scutellaria baicalensis formula granules, the peak corresponding to the dihydromyricetin reference peak is taken as the S1 peak, and the relative retention times of peaks 2, 3, 4, and 5 with the S1 peak are calculated; the peak corresponding to the myricetin reference peak is taken as the S2 peak, and the relative retention times of peaks 7 and 8 with the S2 peak are calculated; the relative retention times should be within the prescribed range of ±10%, and the prescribed values ​​are: 1.22 (peak 2), 1.72 (peak 3), 2.72 (peak 4), 4.04 (peak 5), 1.14 (peak 7), and 1.19 (peak 8);

[0052] In the quasi-characteristic spectrum of the medicinal materials and decoction pieces of Rhizoma Coptidis, the peak corresponding to the dihydromyricetin reference peak is the S1 peak, and the relative retention times of peaks 2, 3, 4, and 5 with respect to the S1 peak are calculated, and their relative retention times should be within the prescribed range of ±10%, and the prescribed values ​​are 1.21 (peak 2), 1.73 (peak 3), 2.62 (peak 4), and 3.63 (peak 5); the peak corresponding to the myricetin reference peak is the S2 peak, and the relative retention times of peaks 7 and 8 with respect to the S2 peak are calculated, and should be within the prescribed range of ±10%, and the prescribed values ​​are 1.12 (peak 7) and 1.14 (peak 8).

[0053] In the standard characteristic spectrum of the standard decoction of Rhizoma Coptidis, the peak corresponding to the dihydromyricetin reference peak is the S1 peak, and the relative retention times of peaks 2, 3, 4, and 5 with the S1 peak are calculated, and their relative retention times should be within the prescribed range of ±10%, and the prescribed values ​​are 1.24 (peak 2), 1.73 (peak 3), 2.67 (peak 4), and 3.73 (peak 5); the peak corresponding to the myricetin reference peak is the S2 peak, and the relative retention times of peaks 7 and 8 with the S2 peak are calculated, and should be within the prescribed range of ±10%, and the prescribed values ​​are 1.12 (peak 7) and 1.15 (peak 8).

[0054] Quality judgment standard: Take samples of Radix Rhizoma Cynoglossi medicinal materials, decoction pieces, standard decoctions, and formula granules, and operate according to the same method as above to obtain the characteristic spectra of Radix Rhizoma Cynoglossi medicinal materials, decoction pieces, standard decoctions, and formula granules. Use the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 edition) of the National Pharmacopoeia Committee to analyze the standard characteristic spectra and sample characteristic spectra of Radix Rhizoma Cynoglossi medicinal materials, decoction pieces, standard decoctions, and formula granules, and the similarity is greater than 0.90.

[0055] The method provided by the present invention can effectively monitor the quality of different batches of Radix Salviae Miltiorrhizae medicinal materials, decoction pieces, standard decoctions, and formula granules to ensure stable quality. The method has the characteristics of high precision and good reproducibility, and is conducive to comprehensive monitoring of product quality.

[0056] The characteristic spectra of the Herba Lycopodiellae Radix, herbal slices, standard decoctions and formula granules established by the present invention take dihydromyricetin and myricetin as references, focus on the order of each characteristic peak and the correlation with the medicinal materials and intermediate products, can comprehensively evaluate the overall quality features of the products, and the method is scientific and reliable.

[0057] The newly developed characteristic spectrum method of the present invention can detect components with greater polarity in Radix Salviae Miltiorrhizae and its standard decoction. The test sample preparation method is simple and easy to operate, and relatively more characteristic peaks can be identified. Accurate and reliable characteristic spectrum detection can be performed on Radix Salviae Miltiorrhizae and its preparations. The authenticity, quality consistency and stability of Radix Salviae Miltiorrhizae and its preparations can be effectively detected and controlled. This provides a basis for effectively controlling and more comprehensively evaluating the quality of Radix Salviae Miltiorrhizae standard decoction. The uniformity and stability of the quality of Radix Salviae Miltiorrhizae and its standard decoction are ensured.

[0058] The present invention is applicable to the detection method of high-performance liquid phase characteristic spectrum of Radix Rhizoma Cynogaster medicinal materials, decoction pieces, standard decoctions and formula granules, and can comprehensively control the characteristic components in Radix Rhizoma Cynogaster medicinal materials, decoction pieces, standard decoctions and formula granules to ensure the overall stability of the quality of Radix Rhizoma Cynogaster medicinal materials, decoction pieces, standard decoctions and formula granules. The method is simple to operate, has high precision, good stability, good repeatability and high accuracy.

[0059] The present invention provides a method for constructing an HPLC characteristic spectrum of a Caulis dasyphylla medicinal material, a decoction piece, a standard decoction, and a formula granule, comprising: A) dissolving the Caulis dasyphylla raw material with a solvent to obtain a test solution; B) determining the test solution with a high performance liquid chromatography method to obtain an HPLC characteristic spectrum of the Caulis dasyphylla raw material; the chromatographic conditions of the high performance liquid chromatography method are: a chromatographic column is a C18 column; the mobile phase A is methanol, and the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the gradient elution is performed. The present invention adopts high performance liquid chromatography, selects methanol-0.1% phosphoric acid as the mobile phase for gradient elution, and uses dihydromyricetin reference substance and myricetin as reference substances to establish an HPLC characteristic spectrum of Caulis dasyphylla medicinal material, a decoction piece, a standard decoction, and a formula granule, which has good repeatability and precision, and the method is stable and reliable, and the quality of the Caulis dasyphylla medicinal material, a decoction piece, a standard decoction, and a formula granule can be controlled. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1A This is the reference characteristic spectrum of Herba Lycopodii Herba Granules;

[0061] Figure 1B This is the reference characteristic spectrum of the herbal medicine Rhizoma Caulis;

[0062] Figure 2A This is the reference characteristic spectrum of Herba Crotonii Herba Slices;

[0063] Figure 2B This is the reference characteristic spectrum of the standard decoction of Rhizoma Caulis;

[0064] Figure 3A Spectrum of dihydromyricetin - formula granules;

[0065] Figure 3B Myricetin spectrum - formula granules;

[0066] Figure 3CTaxifolin spectrum - formula granules;

[0067] Figure 4 Column temperature durability -28°C;

[0068] Figure 5 Column temperature durability -30°C;

[0069] Figure 6 Column temperature durability -32°C;

[0070] Figure 7 Flow rate durability - 0.28ml / min;

[0071] Figure 8 Flow rate durability - 0.30ml / min;

[0072] Fig. 9 Flow rate durability - 0.32ml / min;

[0073] Fig.10 Extraction solvent investigation - 30% methanol;

[0074] Fig.11 Extraction solvent investigation - 50% methanol;

[0075] Fig.12 Extraction solvent investigation - 70% methanol;

[0076] Fig.13 Extraction solvent investigation - methanol;

[0077] Fig.14 Extraction solvent investigation - 30% ethanol;

[0078] Fig.15 Extraction solvent investigation - 50% ethanol;

[0079] Fig.16 Extraction solvent investigation - 70% ethanol;

[0080] Fig.17 Extraction solvent investigation - ethanol;

[0081] Fig.18 Extraction solvent investigation - water;

[0082] Fig.19 Investigation of extraction methods - ultrasound;

[0083] Fig. 20 Investigation of extraction method - reflux;

[0084] Fig.21 Extraction time investigation - 15min;

[0085] Fig. 22 Extraction time investigation - 30min;

[0086] Fig.23 Extraction time investigation - 45min;

[0087] Fig.24 Solvent volume investigation-25ml;

[0088] Fig.25 Examination of solvent volume-50ml;

[0089] Fig.26 Solvent volume inspection-100ml;

[0090] Fig. 27 Chromatographic peak identification of Rhizoma Cynanchum var.

[0091] Fig.28 Intermediate precision inspection-repeatability;

[0092] Fig.29 Intermediate precision inspection-intermediate precision;

[0093] Fig.30 Column durability - Column 1;

[0094] Fig.31 Column durability - Column 2;

[0095] Fig.32 Column Durability - Column 3;

[0096] Fig.33 Verification diagram of characteristic spectrum of Picaohongteng formula granules;

[0097] Fig.34 Verification of the medicinal material of Rhizoma Cynoglossi;

[0098] Fig.35 Verification of Herba Lycopodii Scutellariae Decoction Pieces;

[0099] Fig.36 Verification of the Herba Lycopodii Tengbiao Decoction;

[0100] Fig.37 is the chromatogram of the particle-methanol-0.1% phosphoric acid of Comparative Example 1;

[0101] Fig.38 Comparative Example 1 Gradient Condition 2. Chromatogram of Particles-Methanol-0.1% Phosphoric Acid Fig.39 Comparative Example 1 Gradient Condition 3. Chromatogram of Particles-Methanol-0.1% Phosphoric Acid. DETAILED DESCRIPTION

[0102] The present invention provides a method for constructing HPLC characteristic spectra of Radix Salviae Miltiorrhizae medicinal materials, decoction pieces, standard decoctions, and formula granules. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0103] To further illustrate the present invention, a method for constructing an HPLC characteristic spectrum of a Caulis Scutellariae Radix, a decoction piece, a standard decoction, and a formula granule provided by the present invention is described in detail below in conjunction with examples.

[0104] High performance liquid chromatograph: WATERS, Agilent high performance liquid chromatograph;

[0105] Electronic balance: ME204E / 02, XPE26 (Mettler-Toledo Instrument Co., Ltd.);

[0106] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0107] Ultrasonic cleaner: KQ600DB (300w, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0108] Chromatographic columns: chromatographic column 1, chromatographic column 2, chromatographic column 3;

[0109] Methanol (Tianjin Damao Chemical Reagent Co., Ltd.) was of analytical grade, ethanol (Tianjin Damao Chemical Reagent Co., Ltd.) was of analytical grade, methanol (BCR, USA) was of HPLC grade, water was ultrapure water (self-made in the laboratory), and phosphoric acid (Tianjin Komiou Chemical Reagent Co., Ltd.) was of HPLC grade;

[0110] Dihydromyricetin reference substance (Guangzhou Jiatu Technology Co., Ltd., batch number: 27200-12-0, content is 99.6%), myricetin reference substance (Guangzhou Jiatu Technology Co., Ltd., batch number: 529-44-2, content is 100%), taxapine reference substance (China Food and Drug Inspection Institute, batch number: 111816-201102, content is 98.9%), and rhizoma serrata reference medicinal materials (Sichuan Institute of Drug Inspection, batch number: No. SCZD026-202111);

[0111] Herba Lycopodii Herba Granules: CHT-01, CHT-02, CHT-03;

[0112] Grass red vine medicinal materials: CHT-YC-01, CHT-YC-02, CHT-YC-03, CHT-YC-04, CHT-YC-05CHT-YC-06, CHT-YC-07, CHT-YC-08, CHT-YC-09, CH T-YC-10, CHT-YC-11, CHT-YC-12, CHT-YC-13, CHT-YC-14, CHT-YC-15, CHT-YC-16, CHT-YC-17, CHT-YC-18, CHT-YC-19;

[0113] Caohongteng decoction pieces: CHT-YP-01, CHT-YP-02, CHT-YP-03, CHT-YP-04, CHT-YP-05CHT-YP-06, CHT-YP-07, CHT-YP-08, CHT-YP-09, CH T-YP-10, CHT-YP-11, CHT-YP-12, CHT-YP-13, CHT-YP-14, CHT-YP-15, CHT-YP-16, CHT-YP-17, CHT-YP-18, CHT-YP-19;

[0114] Caohongteng standard decoction: CHT-BT-01, CHT-BT-02, CHT-BT-03, CHT-BT-04, CHT-BT-05CHT-BT-06, CHT-BT-07, CHT-BT-08, CHT-BT-09, C HT-BT-10, CHT-BT-11, CHT-BT-12, CHT-BT-13, CHT-BT-14, CHT-BT-15, CHT-BT-16, CHT-BT-17, CHT-BT-18, CHT-BT-19.

[0115] Example 1

[0116] 1.1 Formula particles:

[0117] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel is used as filler (column length is 100 mm, inner diameter is 2.1 mm, particle size is 1.8 μm); methanol is used as mobile phase A, 0.1% phosphoric acid solution is used as mobile phase B, and gradient elution is performed according to the provisions in the following table; the flow rate is 0.3 ml per minute; the column temperature is 30°C; the detection wavelength is 230 nm. The theoretical plate number calculated based on the dihydromyricetin peak should not be less than 5000.

[0118]

[0119] Preparation of reference solution Take 0.5g of Radix Rhizoma Cynoglossi reference medicinal material, add 50ml of water, boil, keep it slightly boiling for 30 minutes, filter, evaporate the filtrate, add 25ml of 70% ethanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take appropriate amount of dihydromyricetin reference substance and myricetin reference substance, accurately weigh, add methanol to prepare 1ml of mixed solution containing 100μg each, as the reference substance reference solution.

[0120] 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 25ml of 70% ethanol, ultrasonic (power 300W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the solution.

[0121] The determination method is to accurately pipette 1 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0122] There should be 8 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the dihydromyricetin reference peak is the S1 peak, and the relative retention time of peaks 2, 3, 4, and 5 with the S1 peak is calculated; the peak corresponding to the myricetin reference peak is the S2 peak, and the relative retention time of peaks 7 and 8 with the S2 peak is calculated. The relative retention time should be within the specified ±10% range. The specified values ​​are: 1.22 (peak 2), 1.72 (peak 3), 2.72 (peak 4), 4.04 (peak 5), 1.14 (peak 7), 1.19 (peak 8).

[0123] The results are as follows Figure 1A As shown, Figure 1A This is the reference characteristic spectrum of the Herba Lycopodii serrata formula granules, wherein Peak 1 (S1): dihydromyricetin; Peak 4: taxolanolide; Peak 6 (S2): myricetin.

[0124] 2.2 Medicinal Materials:

[0125] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length was 100 mm, inner diameter was 2.1 mm, particle size was 1.8 μm), methanol was used as mobile phase A, 0.1% phosphoric acid water was used as mobile phase B, gradient elution was performed according to the provisions in the following table; flow rate was 0.3 ml per minute; column temperature was 30°C; detection wavelength was 230 nm. The theoretical plate number calculated based on the dihydromyricetin peak should not be less than 5000.

[0126]

[0127]

[0128] Preparation of reference solution Take 0.5g of Herba Lycopodiellae Radix and add 50ml of water, boil, keep it slightly boiling for 30 minutes, cool, filter, evaporate the filtrate, add 25ml of 70% ethanol to the residue, ultrasonically treat (power 300W, frequency 40kHz) for 30min, take it out, cool, filter, and take the filtrate as the reference solution of the reference medicinal material. Take appropriate amount of dihydromyricetin reference substance and myricetin reference substance, accurately weigh them, and add methanol to prepare reference solution containing 100μg of dihydromyricetin reference substance, 100μg of myricetin reference substance, and 100μg of taxifolin per 1ml.

[0129] Preparation of test solution: Take an appropriate amount of the product powder (passed through No. 3 sieve), take about 0.5g, accurately weigh, place in a stoppered conical flask, add 50ml of water, boil, keep boiling for 30 minutes, cool, filter, evaporate the filtrate to dryness, accurately add 25ml of 70% ethanol, weigh the weight, ultrasonic (power 300W, frequency 40kHz) for 30min, take out, cool, shake well, filter, and take the filtrate.

[0130] Determination method: Accurately pipette 1 μl of reference solution and test solution, inject into liquid chromatograph, and determine.

[0131] There should be 8 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. The retention time of peak 1, peak 4, and peak 6 should correspond to the retention time of the chromatogram peaks of the dihydromyricetin reference substance and the myricetin reference substance. The peak corresponding to the dihydromyricetin reference peak is the S1 peak. The relative retention time of peaks 2, 3, 4, and 5 relative to the S1 peak should be within the specified ±10%, and the specified values ​​are 1.21 (peak 2), 1.73 (peak 3), 2.62 (peak 4), and 3.63 (peak 5); the peak corresponding to the myricetin reference peak is the S2 peak. The relative retention time of peaks 7 and 8 relative to the S2 peak should be within the specified ±10%, and the specified values ​​are 1.12 (peak 7) and 1.14 (peak 8).

[0132] The results are as follows Figure 1B As shown, Figure 1B This is a reference characteristic spectrum of the medicinal material Rhizoma Caulis Scutellariae, where Peak 1 (S1): dihydromyricetin; Peak 4: taxafoetida; Peak 6 (S2): myricetin.

[0133] 1.3 Pieces:

[0134] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length was 100 mm, inner diameter was 2.1 mm, particle size was 1.8 μm), methanol was used as mobile phase A, 0.1% phosphoric acid water was used as mobile phase B, gradient elution was performed according to the provisions in the following table; flow rate was 0.3 ml per minute; column temperature was 30°C; detection wavelength was 230 nm. The theoretical plate number calculated based on the dihydromyricetin peak should not be less than 5000.

[0135]

[0136]

[0137] Preparation of reference solution Take 0.5g of Herba Lycopodiellae Radix as reference medicinal material, add 50ml of water, boil, keep it slightly boiling for 30 minutes, cool, filter, evaporate the filtrate, add 25ml of 70% ethanol to the residue, ultrasonically treat (power 300W, frequency 40kHz) for 30min, take it out, cool, filter, and take the filtrate as the reference solution of reference medicinal material. Separately take appropriate amount of dihydromyricetin reference substance, myricetin reference substance, and cypermethrin reference substance, accurately weigh them, and add methanol to prepare reference substance solutions containing 100μg of dihydromyricetin reference substance, 100μg of myricetin reference substance, and 100μg of cypermethrin per 1ml.

[0138] Preparation of test solution Take an appropriate amount of the product powder (passed through No. 3 sieve), take about 0.5g, accurately weigh, place in a stoppered conical flask, add 50ml of water, boil, keep boiling for 30 minutes, cool, filter, evaporate the filtrate to dryness, accurately add 25ml of 70% ethanol, weigh, ultrasonic (power 300W, frequency 40kHz) for 30min, take out, cool, weigh again, make up the lost weight with 70% ethanol, shake well, filter, and take the filtrate.

[0139] Determination method: Accurately pipette 1 μl of reference solution and test solution, inject into liquid chromatograph, and determine.

[0140] There should be 8 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material. The retention time of peak 1, peak 4, and peak 6 should correspond to the retention time of the chromatogram peaks of the dihydromyricetin reference substance and the myricetin reference substance. The peak corresponding to the dihydromyricetin reference peak is the S1 peak. The relative retention time of peaks 2, 3, 4, and 5 relative to the S1 peak should be within the specified ±10%, and the specified values ​​are 1.21 (peak 2), 1.73 (peak 3), 2.62 (peak 4), and 3.63 (peak 5); the peak corresponding to the myricetin reference peak is the S2 peak. The relative retention time of peaks 7 and 8 relative to the S2 peak should be within the specified ±10%, and the specified values ​​are 1.12 (peak 7) and 1.14 (peak 8).

[0141] The results are as follows Figure 2A As shown, Figure 2A This is a reference characteristic spectrum of the Herba Lycopodii Scutellariae decoction pieces, wherein Peak 1 (S1): dihydromyricetin; Peak 4: taxafoetida; Peak 6 (S2): myricetin.

[0142] 1.4 Standard decoction:

[0143] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length was 100 mm, inner diameter was 2.1 mm, particle size was 1.8 μm), methanol was used as mobile phase A, 0.1% phosphoric acid water was used as mobile phase B, gradient elution was performed according to the provisions in the following table; flow rate was 0.3 ml per minute; column temperature was 30°C; detection wavelength was 230 nm. The theoretical plate number calculated based on the dihydromyricetin peak should not be less than 5000.

[0144]

[0145]

[0146] Preparation of reference solution Take 0.5g of Herba Lycopodiellae Radix and add 50ml of water, boil, keep it slightly boiling for 30 minutes, filter, evaporate the filtrate, add 25ml of 70% ethanol to the residue, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take appropriate amount of dihydromyricetin reference substance and myricetin reference substance, add methanol to prepare solutions containing 100μg of dihydromyricetin, 100μg of myricetin, and 100μg of taxifolin per 1ml, respectively, as the reference solution of the reference substance.

[0147] Preparation of test solution: Take an appropriate amount of the product, about 0.2 g, add 25 ml of 70% ethanol, weigh the weight, and treat with ultrasound (power 300 W, frequency 40 kHz) for 30 minutes. Take out, cool, shake well, filter, and take the filtrate as the test solution.

[0148] The determination method is to accurately pipette 100 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine them.

[0149] There should be 8 characteristic peaks in the chromatogram of the test sample, and the retention times of the 8 characteristic peaks in the chromatogram of the control medicinal material reference substance should correspond. The peak corresponding to the dihydromyricetin reference substance peak is the S1 peak. The relative retention times of peaks 2, 3, 4, and 5 are calculated to be within the prescribed range of ±10%, and the prescribed values ​​are 1.24 (peak 2), 1.73 (peak 3), 2.67 (peak 4), and 3.73 (peak 5). The peak corresponding to the myricetin reference substance peak is the S2 peak. The relative retention times of peaks 7 and 8 are calculated to be within the prescribed range of ±10%, and the prescribed values ​​are 1.12 (peak 7) and 1.15 (peak 8).

[0150] The results are as follows Figure 2B As shown, Figure 2B This is the reference characteristic spectrum of the standard decoction of Rhizoma Coptidis, wherein Peak 1 (S1): dihydromyricetin; Peak 4: taxolanate; Peak 6 (S2): myricetin.

[0151] Example 2 Exploration of the conditions of the formulation particles

[0152] 2.2.2 Spectral scanning

[0153] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning on the dihydromyricetin, myricetin, and taxifolin test solutions, such as Figures 3A to 3C As shown. Among them, Figure 3A Spectrum of dihydromyricetin - formula granules; Figure 3B Myricetin spectrum - formula granules; Figure 3C Taxol spectrum - formula granules.

[0154] 2.2.3 Column temperature investigation

[0155] Based on the experimental conditions proposed above, the column temperatures of 28°C, 30°C, and 32°C were investigated. Figure 4-6 shown.

[0156] Table 1 Column temperature investigation results of characteristic spectrum of Herba Lycopodii granules (relative retention time)

[0157]

[0158]

[0159] Table 2 Column temperature investigation results of characteristic spectrum of Radix Codonopsis pilosulae formula granules (relative peak area)

[0160]

[0161] Figure 4 Column temperature durability -28℃, Figure 5 Column temperature durability -30°C; Figure 6 Column temperature durability -32℃; the results of column temperature investigation showed that when the column temperature was 30℃, the chromatogram peaks were relatively symmetrical and the separation was good, so 30℃ was finally determined as the column temperature for the characteristic spectrum method of the herbaceous vine formula granules.

[0162] 2.2.4 Flow rate investigation

[0163] Based on the experimental conditions proposed above, the flow rates of 0.28mL / min, 0.30mL / min, and 0.32mL / min were investigated. Figure 7-9 shown.

[0164] Table 3 Flow rate investigation results of characteristic spectrum of granules of Herba Lycopodii (relative retention time)

[0165]

[0166] Table 4 Flow rate investigation results of characteristic spectrum of Radix Codonopsis pilosulae formula granules (relative peak area)

[0167]

[0168] Figure 7 Flow rate durability - 0.28ml / min, Figure 8 Flow rate durability - 0.30ml / min, Fig. 9 Flow rate durability - 0.32ml / min. The results show that when the flow rate is 0.3ml / min, the chromatogram peak shape is better and the separation is moderate. Therefore, the flow rate is determined to be 0.3ml / min.

[0169] 2.2.5 Extraction solvent investigation

[0170] Take an appropriate amount of this product (CHT-01), grind it into powder, take about 0.2g, put it in a stoppered conical flask, add 25ml of methanol, 30% methanol, 50% methanol, 70% ethanol, methanol, 30% ethanol, 50% ethanol, 70% ethanol, ethanol, water, ultrasonic treatment (power 300W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain. As shown in Table 5, Figure 10-18 shown.

[0171] Table 5 Characteristic spectrum of the granules of Rhizoma Coptidis formula and the results of the investigation of the solvent for extracting

[0172]

[0173] Fig.10 Extraction solvent investigation - 30% methanol; Fig.11 Extraction solvent investigation - 50% methanol; Fig.12 Extraction solvent investigation - 70% methanol; Fig.13Extraction solvent investigation - methanol; Fig.14 Extraction solvent investigation - 30% ethanol; Fig.15 Extraction solvent investigation - 50% ethanol; Fig.16 Extraction solvent investigation - 70% ethanol; Fig.17 Extraction solvent investigation - ethanol; Fig.18 Extraction solvent investigation - water.

[0174] The results showed that when 70% ethanol was used as the extraction solvent, the value of "total peak area / sample weight" was larger and the chromatographic peak information was larger. Therefore, 70% ethanol was selected as the extraction solvent.

[0175] 2.2.6 Investigation of extraction methods

[0176] Take an appropriate amount of this product (CHT-01), grind it into powder, take about 0.2g, put it in a stoppered conical bottle, add 25ml of 70% ethanol, and examine the extraction method of the test product when it is refluxed and ultrasonic respectively. The extraction time is 30 minutes, cool, shake well, filter, and take the filtrate. As shown in Table 6, Figure 19-20 shown.

[0177] Table 6 Results of investigation on the characteristic spectrum extraction method of Herba Lycopodii serrata formula granules

[0178]

[0179] Fig.19 Investigation of extraction methods - ultrasound; Fig. 20 The results showed that the "total peak area / sample weight" values ​​of different extraction methods were similar when using ultrasound and reflux extraction. Considering the simplicity of experimental operation, ultrasound was selected as the extraction method.

[0180] 2.2.7 Extraction time investigation

[0181] Take an appropriate amount of this product (CHT-01), grind it into powder, take about 0.2g, put it in a stoppered conical bottle, add 25ml of 70% ethanol, and inspect the ultrasonic extraction time of the test product for 15 minutes, 30 minutes, and 45 minutes respectively, cool it, shake it well, filter it, and take the filtrate. As shown in Table 7, Figure 21-23 .

[0182] Table 7 Results of the investigation on the extraction time of characteristic spectrum of Radix Codonopsis pilosulae formula granules

[0183]

[0184] Fig.21 Extraction time investigation - 15min; Fig. 22 Extraction time investigation - 30min; Fig.23The extraction time was 45 minutes. The results showed that different extraction times had little effect on the extraction of each characteristic peak. In order to ensure complete extraction, the extraction time was selected as 30 minutes.

[0185] 2.2.8 Investigation of solvent addition amount

[0186] Take an appropriate amount of this product (CHT-01), grind it into powder, take about 0.2g, put it in a stoppered conical bottle, add 70% ethanol 25ml, 50ml, 100ml respectively, stopper it, weigh it, ultrasonically treat it (power 300w, frequency 40kHz) for 30 minutes, shake it well, filter it, and take the filtrate to obtain it. As shown in Table 8, Figure 24-26 shown.

[0187] Table 8 Characteristic spectrum of the granules of the herba scutellariae formula and the results of the investigation of the solvent content

[0188]

[0189] Fig.24 Solvent volume investigation-25ml; Fig.25 Examination of solvent volume-50ml; Fig.26 Examination of solvent volume - 100ml.

[0190] The results show that when the amount of solvent added is 25 ml, the peak area of ​​the characteristic spectrum is moderate. Therefore, the amount of solvent added for the test sample is determined to be 25 ml.

[0191] 2.2.9 Determine the preparation method of the test product

[0192] Take an appropriate amount of this product, grind it into powder, take about 0.2g, put it in a stoppered conical flask, add 25ml of 70% ethanol, ultrasonically treat (power 300w, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0193] Example 3

[0194] 3.1 Methodological investigation

[0195] 3.1.1 Chromatographic peak identification

[0196] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Rhizoma Cynanchum wilfordii formula granules.

[0197] Preparation of reference solution: Take appropriate amount of dihydromyricetin reference substance and myricetin reference substance, weigh accurately, add methanol to prepare 1 ml of mixed solution containing 100 μg of each, as reference substance solution.

[0198] Preparation of control medicinal material solution: Take 0.5g of Herba Rhizoma ...

[0199] The characteristic spectrum peaks of the Herba Lycopodii granules were located. Fig. 27 shown. Fig. 27 Chromatographic peak identification of Rhizoma Coptidis formula granules.

[0200] 3.1.2 Precision test

[0201] Take the test solution of Herba Lycopodiellae Radix Granules (Batch No.: CHT-01), inject 6 times in succession according to the proposed experimental method, 1 μL each time, and calculate the retention time and peak area of ​​each characteristic peak. See Table 9-10.

[0202] Table 9 Precision investigation results of characteristic spectrum of Radix Codonopsis pilosulae formula granules (relative retention time)

[0203]

[0204] Table 10 Precision investigation results of characteristic spectrum of Radix Codonopsis pilosulae formula granules (relative peak area)

[0205]

[0206] The results showed that the same sample solution was injected 6 times continuously, with peak 1 as the reference peak S1 and peak 7 as the reference peak S2. The relative retention time RSD values ​​of each characteristic peak and the S peak were in the range of 0.01% to 0.12%, and the relative peak area RSD values ​​were in the range of 0.13% to 0.85%, indicating that the instrument had good precision.

[0207] 3.1.3 Repeatability study

[0208] Weigh 6 portions of Radix Rhizoma Cynoglossi formula granules accurately, and prepare and measure them according to the proposed experimental method. See Table 11-12.

[0209] Table 11 Results of repeatability study of characteristic spectra of Radix Codonopsis pilosulae formula granules (relative retention time)

[0210]

[0211] Table 12 Results of repeatability study of characteristic spectrum of Radix Codonopsis pilosulae formula granules (relative peak area)

[0212]

[0213] The results showed that the same batch of samples were measured six times, with peak 1 as the reference peak S1 and peak 7 as the reference peak S2. The relative retention time RSD values ​​of each characteristic peak and the S peak were in the range of 0.01% to 0.09%, and the relative peak area RSD values ​​were in the range of 0.17% to 1.56%. This method had good repeatability.

[0214] 3.1.4 Intermediate precision study

[0215] 3.1.4.1 Investigation with different instruments

[0216] Different analysts operated on different instruments at different times, accurately weighed 6 portions of Radix Rhizoma Cynoglossi formula granules, and prepared and measured them according to the proposed experimental method. See Table 13-14, Figure 28-29 .

[0217] Table 13 Intermediate precision investigation results of Radix Codonopsis pilosulae formula granules (relative retention time)

[0218]

[0219]

[0220] Table 14 Intermediate precision investigation results of Radix Codonopsis pilosulae formula granules (relative peak area)

[0221]

[0222] Fig.28 Intermediate precision inspection-repeatability; Fig.29 Intermediate precision investigation - intermediate precision. The results showed that the same batch of samples was measured 6 times by different analysts on different instruments at different times, with peak 1 as the reference peak S1 and peak 7 as the reference peak S2. The relative retention time RSD values ​​of each characteristic peak and the S peak were in the range of 0.01% to 0.06%, and the relative peak area RSD values ​​were in the range of 0.16% to 2.20%. The RSD values ​​of the relative retention time and the 6 data of the repeatability test were in the range of 0.29% to 2.42%, and the RSD values ​​of the relative peak area and the 6 data of the repeatability test were in the range of 1.17% to 8.32%, indicating that the intermediate precision of the relative peak area of ​​each characteristic peak was greatly affected by different instruments.

[0223] 3.1.5 Durability test

[0224] 3.1.5.1 Column durability investigation

[0225] Based on the above proposed experimental conditions, different chromatographic columns were investigated. See Table 15-16. Figure 30-31 .

[0226] Table 15 Characteristic spectra of Radix Codonopsis pilosulae formula granules Results of investigation of different brands of chromatographic columns (relative retention time)

[0227]

[0228] Table 16 Characteristic spectrum of Radix Codonopsis pilosulae formula granules Results of investigation of different brands of chromatographic columns (relative peak area)

[0229]

[0230] Fig.30 Column durability - Column 1 (Waters ACQUITY UPLC HSS T3, specification: 2.1*100mm, 1.8μm); Fig.31 Column durability - Column 2 (YMC-Triart C18, specification: 2.1*100mm, 1.8μm); Fig.32 Column durability - Column 3 (Agilent ZORBAX SB C18, specifications: 2.1*100mm, 1.8μm).

[0231] The results showed that when different brands of chromatographic columns with different numbers were used, the peak shapes and separation degrees of each chromatographic peak were good, the relative retention time RSD values ​​of each characteristic peak and S peak were in the range of 2.34% to 9.75%, and the relative peak area RSD values ​​were in the range of 4.16% to 55.27%. The relative retention time and relative peak area were quite different, indicating that the relative peak area of ​​each characteristic peak was greatly affected by different chromatographic columns.

[0232] 3.1.5.2 Stability study

[0233] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 2h, 4h, 8h, 12h and 24h respectively. See Table 17-18.

[0234] Table 17 Results of stability study of characteristic spectrum of Radix Codonopsis pilosulae formula granules (relative retention time)

[0235]

[0236] Table 18 Results of stability study of characteristic spectrum of Radix Codonopsis pilosulae formula granules (relative peak area)

[0237]

[0238] The results showed that the same test solution was analyzed within 24 hours, with peak 1 as the reference peak S1 and peak 7 as the reference peak S2. The relative retention time RSD values ​​of each characteristic peak and the S peak were in the range of 0.06% to 0.031%, and the relative peak area RSD values ​​were in the range of 0.49% to 1.78%. The relative peak area was not specified in the standard formulation, and the relative retention time of the test solution was stable within 24 hours.

[0239] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 9 characteristic peaks were included in the subsequent investigation.

[0240] 3.1.6 Determination of characteristic peaks and establishment of reference spectrum

[0241] 3.1.6.1 Establishment of relative retention time limit values

[0242] The summary of the methodological investigation items and verification results is shown in Table 19.

[0243] Table 19 Summary of RSD% results of each item of the methodology Standard - Retention time - Relative retention time

[0244]

[0245]

[0246] Therefore, the relative retention time of each peak is temporarily set at ±10%.

[0247] Peak 6 was deleted due to its small peak area after comprehensive consideration.

[0248] Example 4 Verification results of three batches of Radix Rhizoma Cynoglossi formula granules

[0249] The proposed method was used to measure the characteristic spectra of three batches of samples of this product, and the relative retention time and relative peak area were calculated. Fig.33 , as shown in Table 20 and Table 21. Fig.33 Verification chart of characteristic spectrum of Picaohongteng formula granules.

[0250] Table 20 Relative retention time of three batches of Radix Rhizoma Cynoglossi formula granules

[0251]

[0252] Table 21 Relative peak areas of three batches of Radix Codonopsis pilosulae formula granules

[0253]

[0254] According to the principle of stable relative retention time and the fact that each batch of samples can be detected and the peaks are relatively high, a total of 8 peaks with good repeatability were selected as characteristic peaks. The test sample chromatogram should show 8 characteristic peaks, and the retention time should correspond to the 8 characteristic peaks in the chromatogram of the reference medicinal material reference. The retention time of peak 1, peak 4, and peak 6 should correspond to the retention time of the chromatogram of the reference dihydromyricetin, myricetin, and taxifolin reference. The peak corresponding to the dihydromyricetin reference peak is the S1 peak. The relative retention time of peaks 2, 3, 4 and 5 relative to the S1 peak should be within the specified ±10%, and the specified values ​​are 1.22 (peak 2), 1.72 (peak 3), 2.72 (peak 4) and 4.04 (peak 5). The peak corresponding to the myricetin reference peak is the S2 peak. The relative retention time of peaks 7 and 8 relative to the S2 peak should be within the specified ±10%, and the specified values ​​are 1.14 (peak 7) and 1.19 (peak 8). Establishment of the limits of relative retention time specifications.

[0255] The summary of the methodology investigation items and verification results is shown in Table 22:

[0256] Table 22 Summary of RSD% results of each item of the methodology Standard - Retention time - Relative retention time

[0257]

[0258] Therefore, the relative retention time of each peak is temporarily set at ±10%.

[0259] Example 5 19 batches of Radix Rhizoma Cynoglossi medicinal materials verification results

[0260] The characteristic spectra of 19 batches of samples of this product were measured by the method proposed in Example 1, and the relative retention time and relative peak area were calculated. Fig.34 , as shown in Table 23 and Table 24.

[0261] Table 23 Relative retention time of characteristic spectra of Radix Codonopsis pilosulae

[0262]

[0263]

[0264] Table 24 Relative peak areas of characteristic spectrum of Radix Codonopsis pilosulae

[0265]

[0266] Based on the principle that the relative retention time is stable and each batch of samples can be detected and the peak is relatively high, a total of 8 peaks with good repeatability were selected as characteristic peaks. The results showed that the relative peak area RSD of the characteristic peaks of 19 batches of Radix Salviae Miltiorrhizae medicinal materials was too different, so it was not included in the main text of the quality standard. The relative retention time RSD of the 8 characteristic peaks of 19 batches of Radix Salviae Miltiorrhizae medicinal materials was less than 2.0%. Final regulations: There should be 8 characteristic peaks in the chromatogram of the test sample, and the retention times of the 8 characteristic peaks in the chromatogram of the control medicinal material reference substance should correspond to each other. The 8 peaks should be consistent with the retention times of the corresponding reference substance peaks. Peak 1 is taken as the S1 peak, and the relative retention times of peaks 2, 3, 4, and 5 are calculated to be within the prescribed range of ±10%, with the prescribed values ​​being 1.21 (peak 2), 1.73 (peak 3), 2.62 (peak 4), and 3.63 (peak 5). Peak 6 is taken as the S2 peak, and the relative retention times of peaks 7 and 8 are calculated to be within the prescribed range of ±10%, with the prescribed values ​​being 1.12 (peak 7) and 1.14 (peak 8).

[0267] Fig.34 Verification of the medicinal material of Rhizoma Coptidis.

[0268] Example 6 Verification results of 19 batches of Radix Rhizoma Cynoglossi

[0269] The characteristic spectra of 19 batches of samples of this product were measured by the method proposed in Example 1, and the relative retention time and relative peak area were calculated. Fig.35 , as shown in Table 25 and Table 26.

[0270] Table 25 Relative retention time of characteristic peaks of Herba Lycopodiellae Herba Slices

[0271]

[0272] Table 26 Relative peak areas of characteristic peaks of Herba Lycopodii Serratae

[0273]

[0274] According to the principle that the relative retention time is stable and each batch of samples can be detected and the peak is relatively high, a total of 8 peaks with good repeatability were selected as characteristic peaks. The results showed that the relative peak area RSD of the characteristic peaks of 19 batches of Herba Lycopodii Herba slices was too different, so it was not included in the main text of the quality standard. The relative retention time RSD of the 8 characteristic peaks of 19 batches of Herba Lycopodii Herba slices was less than 2.0%. Final regulations: There should be 8 characteristic peaks in the chromatogram of the test sample, and the retention times of the 8 characteristic peaks in the chromatogram of the control medicinal material reference substance should correspond to each other. The 8 peaks should be consistent with the retention times of the corresponding reference substance peaks. Peak 1 is taken as the S1 peak, and the relative retention times of peaks 2, 3, 4, and 5 are calculated to be within the prescribed range of ±10%, with the prescribed values ​​being 1.21 (peak 2), 1.73 (peak 3), 2.62 (peak 4), and 3.63 (peak 5). Peak 6 is taken as the S2 peak, and the relative retention times of peaks 7 and 8 are calculated to be within the prescribed range of ±10%, with the prescribed values ​​being 1.12 (peak 7) and 1.14 (peak 8).

[0275] Fig.35 Verification of Herba Crotonii Herba Scutellariae Decoction pieces.

[0276] Example 7 Verification results of 19 batches of Radix Rhizoma Rhizoma Caulis

[0277] The characteristic spectra of 19 batches of samples of this product were measured by the method proposed in Example 1, and the relative retention time and relative peak area were calculated. Fig.36 , as shown in Table 27 and Table 28.

[0278] Table 27 Relative retention time of 19 batches of Radix Rhizoma Cynoglossi standard decoction

[0279]

[0280]

[0281] Table 28 Relative peak areas of 19 batches of standard decoction of Rhizoma Caulis Scutellariae

[0282]

[0283] According to the principle of stable relative retention time and the fact that each batch of samples can be detected and the peaks are relatively high, a total of 8 peaks with good repeatability were selected as characteristic peaks. The peak corresponding to the dihydromyricetin reference peak is the S1 peak, and the relative retention time of peaks 2, 3, 4, and 5 relative to the S1 peak should be within the specified ±10%, and the specified values ​​are 1.24 (peak 2), 1.73 (peak 3), 2.67 (peak 4), and 3.73 (peak 5); the peak corresponding to the myricetin reference peak is the S2 peak, and the relative retention time of peaks 7 and 8 relative to the S2 peak should be within the specified ±10%, and the specified values ​​are 1.12 (peak 7) and 1.15 (peak 8). Fig.36 Verification of Caohongtengbiao Decoction.

[0284] Comparison of other gradient conditions in Comparative Example 1, except for the mobile phase and gradient, the rest are the same as those in Example 1 of the present invention.

[0285] Gradient conditions 1 Phase A: methanol, Phase B: 0.1% phosphoric acid

[0286] 0-12min, phase A: 18%, phase B: 82%;

[0287] 12-20min, phase A: 18-25%, phase B: 82-75%;

[0288] 20-31min, phase A: 25-29%, phase B: 75-71%;

[0289] 31-40min, phase A: 29-45%, phase B: 71-55%;

[0290] 40-48 min, phase A: 45-68%, phase B: 55-32%;

[0291] 48-52 min, phase A: 68-80%, phase B: 32-20%;

[0292] 52-55min, phase A: 80-18%, phase B: 20-82%;

[0293] 55-60min, phase A: 18%, phase B: 82%.

[0294] The results are as follows Fig.37 As shown, Fig.37 The chromatogram of the gradient condition 1 of comparative example 1: particles-methanol-0.1% phosphoric acid; Fig.37 It can be seen that the baseline of the chromatographic peak fluctuates greatly and some chromatographic peaks have peak wrapping phenomenon.

[0295] Gradient conditions 2 Phase A: methanol, Phase B: 0.1% phosphoric acid

[0296] 0-12min, phase A: 16%, phase B: 84%;

[0297] 12-20 min, phase A: 16-22%, phase B: 84-78%;

[0298] 20-31min, phase A: 22%, phase B: 78%;

[0299] 31-40min, phase A: 22-38%, phase B: 78-62%;

[0300] 40-48 min, phase A: 38-60%, phase B: 62-40%;

[0301] 48-52 min, phase A: 60-80%, phase B: 40-20%;

[0302] 52-55min, phase A: 80-16%, phase B: 20-84%;

[0303] 55-60min, phase A: 16%, phase B: 84%.

[0304] The results are as follows Fig.38 As shown, Fig.38 The chromatogram of the gradient condition 2 of comparative example 1: particles-methanol-0.1% phosphoric acid; Fig.38 It can be seen that the separation of some chromatographic peaks is not ideal and there is peak wrapping phenomenon.

[0305] Gradient conditions 3 Phase A: methanol, Phase B: 0.1% phosphoric acid

[0306] 0-5min, phase A: 2%, phase B: 98%;

[0307] 5-25min, phase A: 2-15%, phase B: 98-85%;

[0308] 25-35min, phase A: 15-20%, phase B: 85-80%;

[0309] 35-40 min, phase A: 20-22%, phase B: 80-78%

[0310] 40-50min, phase A: 22-25%, phase B: 78-75%;

[0311] 50-60min, phase A: 25%, phase B: 75%

[0312] The results are as follows Fig.39 As shown, Fig.39 The chromatogram of the gradient condition 3 of comparative example 1: particles-methanol-0.1% phosphoric acid; Fig.39 It can be seen that there are chromatographic peaks that overlap, and the peak missing phenomenon is due to the missing of some chromatographic peaks, so the chromatographic peaks are not marked.

[0313] According to the above chromatogram peak separation, peak number, and baseline drift degree, it can be seen that, compared with the above comparative example, the chromatogram of the present invention has richer peak information, a stable baseline, and a moderate peak response value.

[0314] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing HPLC characteristic spectrum of Radix Codonopsis pilosulae medicinal materials, decoction pieces, standard decoctions, and formula granules, comprising: A) extracting the raw material of Caulis Sargentodoxae with a solvent to obtain a test solution; the solvent is 70% ethanol; The extraction method is ultrasonic extraction or heating reflux extraction; B) using high performance liquid chromatography to measure the test solution to obtain a HPLC characteristic spectrum of the Caulis Sargentodoxae raw material; The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is a 0.1% phosphoric acid aqueous solution, and the elution is performed in a gradient manner; The gradient elution is specifically: 0-10min, phase A: 20%, phase B: 80%; 10-20min, phase A: 20-25%, phase B: 80-75%; 20-30min, phase A: 25-30%, phase B: 75-70%; 30-40min, phase A: 30-43%, phase B: 70-57%; 40-45min, phase A: 43-65%, phase B: 57-35%; 45-50min, phase A: 65-80%, phase B: 35-20%; 50-55min, phase A: 80-20%, phase B: 20-80%; 55-60min, phase A: 20%, phase B: 80%.

2. The method according to claim 1, characterized in that It also includes the preparation of reference substance solutions and the preparation of reference medicinal material solutions; Preparation of reference substance solution: dihydromyricetin and myricetin reference substance are taken respectively, dissolved in methanol to obtain reference substance solution; the concentration of dihydromyricetin in the reference substance solution is 100 μg / mL; the concentration of myricetin in the reference substance solution is 100 μg / mL; Preparation of control medicinal material solution: using the control medicinal material of Rhizoma Caulis to extract by heating with water, filtering, adding methanol to the filtrate for ultrasonic dissolution, and obtaining the control medicinal material solution; The reference substance solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference substance; and the components of the HPLC characteristic spectra of the Herba Lycopodii Herba, decoction pieces, standard decoctions, and formula granules are qualitatively measured based on the chromatogram of the reference substance.

3. The method according to claim 1, characterized in that The chromatographic column is a C18 column with specifications of 1.8 μm and 2.1×100 mm; the column temperature is 30° C.

4. The method according to claim 3, characterized in that The flow rate of the mobile phase was 0.3 mL / min; the detection wavelength was 230 nm; and the injection volume was 1 μL.

5. The method according to claim 1, characterized in that: The similarity of HPLC characteristic spectra of Radix Rhizoma Cynoglossi, decoction pieces, standard decoction and formula granules was evaluated by using the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, and the HPLC standard characteristic spectra of Radix Rhizoma Cynoglossi, decoction pieces, standard decoction and formula granules consisting of 8 characteristic peaks were obtained, among which Peak S1: dihydromyricetin; Peak 4: taxylin; Peak 6: myricetin.

6. The method according to claim 5, characterized in that In the standard characteristic spectrum of the scutellaria baicalensis formula granules, the peak corresponding to the dihydromyricetin reference peak is taken as the S1 peak, and the relative retention times of peaks 2, 3, 4, and 5 with the S1 peak are calculated; the peak corresponding to the myricetin reference peak is taken as the S2 peak, and the relative retention times of peaks 7 and 8 with the S2 peak are calculated; the relative retention times should be within the prescribed range of ±10%, and the prescribed values ​​are: 1.22 (peak 2), 1.72 (peak 3), 2.72 (peak 4), 4.04 (peak 5), 1.14 (peak 7), and 1.19 (peak 8); In the standard characteristic spectrum of the Herba Lycopodiellae and the decoction pieces, the peak corresponding to the dihydromyricetin reference peak is the S1 peak, and the relative retention time of peaks 2, 3, 4, and 5 with the S1 peak is calculated, and the relative retention time should be within the prescribed ±10%, and the prescribed values ​​are 1.21 (peak 2), 1.73 (peak 3), 2.62 (peak 4), and 3.63 (peak 5); the peak corresponding to the myricetin reference peak is the S2 peak, and the relative retention time of peaks 7 and 8 with the S2 peak is calculated, and the prescribed values ​​are 1.12 (peak 7) and 1.14 (peak 8); In the standard characteristic spectrum of the standard decoction of Rhizoma Coptidis, the peak corresponding to the dihydromyricetin reference peak is the S1 peak, and the relative retention times of peaks 2, 3, 4, and 5 with respect to the S1 peak are calculated, and their relative retention times should be within the prescribed range of ±10%, and the prescribed values ​​are 1.24 (peak 2), 1.73 (peak 3), 2.67 (peak 4), and 3.73 (peak 5); the peak corresponding to the myricetin reference peak is the S2 peak, and the relative retention times of peaks 7 and 8 with respect to the S2 peak are calculated, and should be within the prescribed range of ±10%, and the prescribed values ​​are 1.12 (peak 7) and 1.15 (peak 8).

7. The method according to claim 1, characterized in that Step A): the ultrasonic power is 300W and the frequency is 40kHz; the extraction time is 30 to 40 minutes.

8. The method according to claim 1, characterized in that: In step A), the ratio of the mass g of the Rhizoma Crotonissima raw material to the volume mL of the solvent is (0.2-0.5): (25-50).

9. The method according to claim 1, characterized in that: Step A): the raw material of the Herba Lycopodiellae is Herba Lycopodiellae medicinal materials, granules, decoction pieces or decoctions.

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