A construction method of HPLC characteristic chromatogram of a North Radix Astragali medicinal material decoction piece, a standard decoction and a formula granule

Abstract

This invention provides a method for constructing HPLC characteristic chromatograms of processed medicinal slices, standard decoctions, and formulated granules of *Sophora flavescens*, comprising: A) dissolving and extracting the test sample raw material using a solvent to obtain a test solution; B) determining the test solution using high-performance liquid chromatography (HPLC) to obtain HPLC characteristic chromatograms of processed medicinal slices, standard decoctions, and formulated granules of *Sophora flavescens*; the HPLC chromatographic conditions are as follows: a C18 column; mobile phase A is acetonitrile solution, mobile phase B is 0.05% trifluoroacetic acid aqueous solution, and gradient elution is performed; specifically, the gradient elution is as follows: 0–15 min, phase A: 5%–11%, phase B: 95%–89%; 15–40 min, phase A: 11%–18%, phase B: 89%–82%; 40–60 min, phase A: 18%–21%, phase B: 82%–79%. This invention employs high-performance liquid chromatography (HPLC) with acetonitrile-0.05% trifluoroacetic acid solution as the mobile phase for gradient elution. Using magnoflorine, isocoryne, styraxine, and styraxine as references, a method for establishing characteristic HPLC chromatograms of processed slices, standard decoctions, and formulated granules of *Sophora flavescens* was developed. This provides more scientific technical means for controlling the medicinal quality of processed slices, standard decoctions, and formulated granules of *Sophora flavescens*.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a method for constructing HPLC characteristic chromatograms of processed slices, standard decoctions, and formulated granules of Sophora flavescens. Background Technology

[0002] Northern Sophora Root is the medicinal material obtained by removing impurities, washing, soaking, slicing into thick pieces, and drying. The standard decoction of Northern Sophora Root is a freeze-dried powder made from the medicinal material according to a fixed preparation process; Northern Sophora Root granules are granules made from the processed Northern Sophora Root medicinal material according to the main quality indicators of the standard decoction. This process ensures the uniformity and stability of the quality of the Northern Sophora Root medicinal material, the standard decoction, and the granules.

[0003] Therefore, it is essential to establish a new characteristic spectral method to control its quality and to clearly distinguish it from Sophora flavescens and its preparations. 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 HPLC characteristic chromatograms of processed slices, standard decoctions, and formulated granules of Sophora flavescens. The method of the present invention is accurate and reliable.

[0005] This invention provides a method for constructing HPLC characteristic chromatograms of processed slices, standard decoctions, and formulated granules of *Radix Sophorae Flavescentis*, comprising:

[0006] A) Dissolve and extract the raw material of the test sample using a solvent to obtain the test solution;

[0007] B) The test solution was determined by high performance liquid chromatography to obtain the HPLC characteristic chromatograms of the prepared slices of Sophora flavescens, standard decoction, and formula granules.

[0008] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile solution, mobile phase B is 0.05% trifluoroacetic acid aqueous solution, and gradient elution is used.

[0009] The gradient elution specifically refers to:

[0010] 0–15 min, Phase A: 5%–11%, Phase B: 95–89%;

[0011] 15–40 min, Phase A: 11%–18%, Phase B: 89%–82%;

[0012] 40–60 min, Phase A: 18%–21%, Phase B: 82%–79%.

[0013] The raw materials for the test sample described in this invention are one or more of the following: Sophora flavescens medicinal material, Sophora flavescens medicinal slices, Sophora flavescens standard decoction, or Sophora flavescens formula granules.

[0014] A method for constructing HPLC characteristic chromatograms of Sophora flavescens medicinal materials, processed slices, standard decoctions, and formulated granules is provided. First, the raw materials of the test sample are dissolved and extracted with a solvent to obtain the test solution.

[0015] Specifically, the sample raw material is dissolved in a solvent, extracted, cooled, shaken, and filtered to obtain the final product.

[0016] The extraction method described in this invention is either reflux extraction or ultrasonic extraction; preferably, ultrasonic extraction. The ultrasonic power is 600W, the frequency is 40kHz, and the ultrasonic time is 30min.

[0017] The ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent in this invention is 0.1–0.2:25.

[0018] In some embodiments, the ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.2:25.

[0019] Specifically, the test solution is prepared by taking the medicinal material, slices, standard decoction, and formula granules of Sophora flavescens, weighing them accurately, placing them in a stoppered conical flask, adding methanol accurately, sealing the flask tightly, weighing it, sonicating it for 30 minutes, removing it, cooling it, shaking it well, filtering it, and collecting the filtrate.

[0020] All the raw materials mentioned above can be subjected to quality control using the methods of this invention.

[0021] The present invention also includes the preparation of a reference solution: magnoflorine, isocoryne, clopyralid, and clopyralid are dissolved in methanol to obtain a reference solution;

[0022] The reference solution was analyzed by high performance liquid chromatography to obtain the chromatogram of the reference; and the components of the prepared slices of Sophora flavescens, standard decoction, and formula granules were qualitatively determined based on the chromatogram of the reference.

[0023] The preferred concentrations of the reference solution in this invention are: 100 μg / mL of magnoflorine, 100 μg / mL of isocoryne, 20 μg / mL of piperine, and 20 μg / mL of piperine.

[0024] The test solution was analyzed by high performance liquid chromatography to obtain the HPLC characteristic chromatograms of the prepared slices, standard decoction, and formula granules of Sophora flavescens.

[0025] The chromatographic conditions for the high-performance liquid chromatography (HPLC) were as follows: the chromatographic column was a C18 column; the chromatographic column was a C18 column. 18 The specifications are 250×4.6mm 5μm; this invention does not limit the specific chromatographic column model, any C18 chromatographic column that meets the above specifications is acceptable.

[0026] The column temperature of this invention is 20–30°C; preferably 25°C. At the above column temperature, the chromatographic peaks of this invention are symmetrical, the resolution is good, and the peaks are eluted completely.

[0027] In this invention, mobile phase A is an acetonitrile solution, and mobile phase B is a 0.05% trifluoroacetic acid aqueous solution, with gradient elution.

[0028] Specifically, the gradient elution is as follows:

[0029] 0–15 min, Phase A: 5%–11%, Phase B: 95–89%;

[0030] 15–40 min, Phase A: 11%–18%, Phase B: 89%–82%;

[0031] 40–60 min, Phase A: 18%–21%, Phase B: 82%–79%.

[0032] The theoretical plate number, calculated based on magnolia alkaloids, should be no less than 5000.

[0033] The present invention exhibits good baseline separation, good peak separation, and stable baseline under the above-mentioned elution gradient.

[0034] The flow rate of the mobile phase described in this invention is 0.6 mL / min.

[0035] The present invention found that the chromatographic peaks were well separated and the resolution was moderate at the above flow rates, which is the optimal solution.

[0036] The injection volume is 5–10 μL.

[0037] The detection wavelength is 250 nm. The inventors have discovered that at 250 nm, the chromatographic peak information content is greater, the chromatographic baseline is more stable, and the peak areas are larger.

[0038] This invention is the first to establish a characteristic chromatographic detection method using ultra-high performance liquid chromatography (UHPLC) to distinguish between processed slices, standard decoctions, and formulated granules of Sophora flavescens. This method is beneficial for comprehensively evaluating the scientific validity and rationality of the related processes of Sophora flavescens and its processed extracts and preparations, and can better control the intrinsic quality of Sophora flavescens and its processed extracts and preparations, thus ensuring the clinical efficacy of Sophora flavescens and its processed extracts and preparations.

[0039] This invention uses a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine to evaluate the similarity of HPLC characteristic chromatograms of processed slices, standard decoctions, and formulated granules of Sophora flavescens. The resulting standard HPLC characteristic chromatogram consists of seven characteristic peaks, including peak 3 (S) magnoflorine; peak 5: isocotylein; peak 6: puerarin; and peak 7: puerarin.

[0040] In the characteristic chromatograms of the prepared slices, standard decoctions, and formula granules of Sophora flavescens, the relative retention time of each characteristic peak and the S peak is calculated with magnoflorine as the reference peak. The relative retention time is within ±10% of the specified value, which is 0.35 (peak 1), 0.60 (peak 2), and 1.12 (peak 4).

[0041] The inventors have creatively identified the characteristic peak of isocoryne alkaloid for the first time.

[0042] Quality judgment criteria: Take the extracts and preparations of Sophora flavescens and its processed products, and operate according to the above method to obtain the above characteristic chromatograms. Use the 2012 version of the "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine" of the National Pharmacopoeia Commission to analyze the characteristic chromatograms of chicken gizzard membrane and its processed products and their preparations and the sample characteristic chromatograms. The similarity is greater than 0.90.

[0043] This invention provides a method for constructing HPLC characteristic chromatograms of processed medicinal materials, standard decoctions, and formulated granules of Sophora flavescens, comprising: A) dissolving and extracting the raw material of the test sample using a solvent to obtain the test solution; B) determining the test solution using high performance liquid chromatography to obtain the HPLC characteristic chromatograms of processed medicinal materials, standard decoctions, and formulated granules of Sophora flavescens.

[0044] The chromatographic conditions for the high-performance liquid chromatography (HPLC) method are as follows: a C18 column; mobile phase A is acetonitrile solution, mobile phase B is 0.05% trifluoroacetic acid aqueous solution, and gradient elution is performed. Specifically, the gradient elution is as follows: 0–15 min, phase A: 5%–11%, phase B: 95%–89%; 15–40 min, phase A: 11%–18%, phase B: 89%–82%; 40–60 min, phase A: 18%–21%, phase B: 82%–79%. This invention employs HPLC with acetonitrile-0.05% trifluoroacetic acid solution as the mobile phase for gradient elution. Using magnoflorine, isocoryne, styraxine, and styraxine as references, a method for establishing HPLC characteristic chromatograms of *Radix Sophorae Flavescentis* (Beidougensis) medicinal slices, standard decoctions, and formulation granules was established. This provides more scientific technical means for controlling the medicinal quality of *Radix Sophorae Flavescentis* medicinal slices, standard decoctions, and formulation granules. Attached Figure Description

[0045] Figure 1 Extraction methods were examined;

[0046] Figure 2 Investigation of extraction solvents;

[0047] Figure 3 Investigation of solvent addition amount;

[0048] Figure 4 Extraction time consideration;

[0049] Figure 5 Chromatographic peak identification;

[0050] Figure 6 Spectrum of Magnoliaine - Reference Standard;

[0051] Figure 7 Spectrum of Magnolia alkaloids - Sophora flavescens root medicinal material;

[0052] Figure 8 Spectrum of isocyanidin for alkalization - reference standard;

[0053] Figure 9 Spectrum of isocyanidin - Sophora flavescens root medicinal material;

[0054] Figure 10 Spectrum of bat gazoline - reference standard;

[0055] Figure 11 Spectrum of bat gesulphine - Sophora flavescens root medicinal material;

[0056] Figure 12 Spectrum of puerarin - Reference Standard;

[0057] Figure 13 Spectrum of basidiosine from Sophora flavescens root.

[0058] Figure 14 Exploration using different instruments;

[0059] Figure 15 Investigations using different chromatographic columns;

[0060] Figure 16 Characteristic atlas of Sophora flavescens medicinal materials;

[0061] Figure 17 Characteristic atlas of Sophora flavescens medicinal materials;

[0062] Figure 18 Compare with the characteristic maps;

[0063] Figure 19 Characteristic spectrum of medicinal slices;

[0064] Figure 20 Characteristic spectrum of medicinal slices;

[0065] Figure 21 Compare with the atlas;

[0066] Figure 22 Chromatographic peak identification;

[0067] Figure 23 Spectrum of Magnoliaine - Reference Standard;

[0068] Figure 24 Magnolia alkaloid spectrum - standard decoction;

[0069] Figure 25Spectrum of isocyanidin for alkalization - reference standard;

[0070] Figure 26 Spectral diagram of isocyanidin for alkalization - standard decoction;

[0071] Figure 27 Spectrum of bat gazoline - reference standard;

[0072] Figure 28 Spectral diagram of bat gesulphine - standard decoction;

[0073] Figure 29 Spectrum of puerarin - Reference Standard;

[0074] Figure 30 Spectral diagram of puerarin in standard decoction;

[0075] Figure 31 Exploration using different instruments;

[0076] Figure 32 Investigations using different chromatographic columns;

[0077] Figure 33 Characteristic spectrum of standard decoction of Sophora flavescens;

[0078] Figure 34 Characteristic spectrum of standard decoction of Sophora flavescens;

[0079] Figure 35 Characteristic spectrum of standard decoction of Sophora flavescens;

[0080] Figure 36 Mobile phase selection;

[0081] Figure 37 Chromatograms of different wavelengths of granulated formula of Sophora flavescens;

[0082] Figure 38 Column temperature investigation;

[0083] Figure 39 Flow velocity study;

[0084] Figure 40 Delayed investigation;

[0085] Figure 41 Extraction methods were examined;

[0086] Figure 42 Investigation of extraction solvents;

[0087] Figure 43 Investigation of solvent addition amount;

[0088] Figure 44 Extraction time consideration;

[0089] Figure 45 Chromatographic peak identification;

[0090] Figure 46 Spectrum of Magnoliaine - Reference Standard;

[0091] Figure 47 Magnolia flower alkaloid spectrum - Sophora flavescens formula granules;

[0092] Figure 48 Spectrum of isocyanidin for alkalization - reference standard;

[0093] Figure 49 Spectrum of isocyanidin-based alkaloids - Sophora flavescens formula granules;

[0094] Figure 50 Spectrum of bat gazoline - reference standard;

[0095] Figure 51 Spectrum of bat gesulphine - Sophora flavescens formula granules;

[0096] Figure 52 Spectrum of puerarin - Reference Standard;

[0097] Figure 53 Spectrum of Ficus pumila alkaloids - Sophora flavescens formula granules;

[0098] Figure 54 Exploration using different instruments;

[0099] Figure 55 Investigations using different chromatographic columns;

[0100] Figure 56 Characteristic spectrum of Sophora flavescens formula granules (BDG-KL01);

[0101] Figure 57 Characteristic spectrum of Sophora flavescens formula granules (BDG-KL02);

[0102] Figure 58 Characteristic spectrum of Sophora flavescens formula granules (BDG-KL03);

[0103] Figure 59 Characteristic spectrum of granules formulated with Sophora flavescens;

[0104] Figure 60 This is a chromatogram of the peaks from Comparative Example 1;

[0105] Figure 61 This is a spectral overlay of Comparative Example 1;

[0106] Figure 62 This is a chromatogram of the peaks from Comparative Example 2;

[0107] Figure 63 This is a spectral overlay of Comparative Example 2. Detailed Implementation

[0108] This invention provides a method for constructing HPLC characteristic chromatograms of processed slices, standard decoctions, and formulated granules of *Radix Sophorae Flavescentis*. Those skilled in the art can refer to this method and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0109] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for constructing HPLC characteristic spectra of *Radix Sophorae Flavescentis* (a type of medicinal herb), standard decoction, and formulated granules provided by the present invention.

[0110] High performance liquid chromatograph: Agilent 1260 liquid chromatograph;

[0111] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0112] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0113] Ultrasonic cleaner: KQ600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0114] Chromatographic columns: Column 1 (YMC-Triart C18, 4.6*250mm, 5μm), Column 2 ( C18 (4.6*250mm, 5μm), column 3 (ZORBAX SB-Aq, 4.6*250mm, 5μm);

[0115] Acetonitrile and trifluoroacetic acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0116] Bat puerarin (China National Institutes for Food and Drug Control, batch number: 111867-202205, purity: 97.2%)

[0117] Bat guazoline (PCL (Chengdu Maisike Pharmaceutical Technology Co., Ltd.), batch number: D2308002, purity: 99.61%)

[0118] Magnolia alkaloid (OST, batch number: U40-1301095-03, purity: 99.9%)

[0119] Isocoridine (Chengdu Pusi Biotechnology Co., Ltd., batch number: PS011328, purity: 99.95%)

[0120] Reference material of Sophora flavescens (batch number: 120977-201905, China National Institutes for Food and Drug Control)

[0121] Batch numbers of northern bean root medicinal materials: BDG-YC-01, BDG-YC-02, BDG-YC-03, BDG-YC-04, BDG-YC-05, BDG-YC-06, BDG-YC-07, BDG-YC-08, BDG-YC-09, B DG-YC-10, BDG-YC-11, BDG-YC-12, BDG-YC-13, BDG-YC-14, BDG-YC-15, BDG-YC-16, BDG-YC-17, BDG-YC-18, BDG-YC-19.

[0122] Beidougen slices (BDG-YP-01, BDG-YP-02, BDG-YP-03, BDG-YP-04, BDG-YP-05, BDG-YP-06, BDG-YP-07, BDG-YP-08, BDG-YP-09, BDG-YP- 10. BDG-YP-11, BDG-YP-12, BDG-YP-13, BDG-YP-14, BDG-YP-15, BDG-YP-16, BDG-YP-17, BDG-YP-18, BDG-YP-19, BDG-YP-20).

[0123] Beanroot standard decoction (BDG-BT-01, BDG-BT-02, BDG-BT-03, BDG-BT-04, BDG-BT-05, BDG-BT-06, BDG-BT-07, BDG-BT-08, BDG-BT-09, B DG-BT-10, BDG-BT-11, BDG-BT-12, BDG-BT-13, BDG-BT-14, BDG-BT-15, BDG-BT-16, BDG-BT-17, BDG-BT-18, BDG-BT-19).

[0124] Northern Bean Root Formula Granules (Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers BDG-KL01, BDG-KL02, BDG-KL03)

[0125] Example 1: HPLC Characteristic Chromatography of Sophora flavescens Root

[0126] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A; and 0.05% trifluoroacetic acid as mobile phase B, with gradient elution as specified in the table below; column temperature 25℃; flow rate 0.6 ml / min; detection wavelength 250 nm; and theoretical plate number calculated based on the magnoflorine peak should be no less than 5000.

[0127]

[0128] Preparation of the reference solution: Take 0.2g of *Sophora tonkinensis* root reference material, place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, remove, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add methanol to prepare a solution containing 100μg of magnoflorine per ml, as the reference solution.

[0129] Preparation of the test solution: Take about 0.2g of powdered Sophora flavescens (passed through a No. 3 sieve), place it in a stoppered conical flask, add 15ml of 50% methanol, seal tightly, sonicate for 30 minutes, remove, cool and shake well, filter, and collect the filtrate to obtain the test solution.

[0130] The determination method involves precisely pipetting 5 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0131] The chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 3, 5, 6, and 7 should correspond to the retention times of the reference peaks. The peak corresponding to the magnoflorine reference peak is the S peak. Calculate the relative retention times of the remaining characteristic peaks and the S peak. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.60 (peak 2), and 1.12 (peak 4).

[0132] 2.2.1.2 Selection of Wavelength, Column Temperature, and Flow Rate

[0133] The chromatographic conditions and system adaptability of *Sophora flavescens* medicinal materials are consistent with the characteristic chromatographic methods of *Sophora flavescens* standard decoction and *Sophora flavescens* formula granules.

[0134] Using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), with acetonitrile as mobile phase A and 0.05% trifluoroacetic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the column temperature was 25℃; and the detection wavelength was 250 nm. The theoretical plate number, calculated based on the magnoflorine peak, should be no less than 5000.

[0135]

[0136] 2.2.1.3 Investigation into the preparation of the test solution

[0137] 2.2.1.3.1 Examination of Sample Preparation Methods

[0138] Take 0.2g of *Sophora flavescens* root powder (passed through a No. 3 sieve) (batch number: BDG-YC-01), place it in a stoppered conical flask, add 15ml of 50% methanol, seal tightly, reflux and sonicate for 30 minutes respectively, cool, shake well, filter, and collect the filtrate; Separately, take 0.2g of *Sophora flavescens* root powder (passed through a No. 3 sieve, batch number: BDG-YC-01), place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, shake well, filter, evaporate the filtrate to dryness, add 15ml of 50% methanol to the residue, sonicate for 30 minutes, shake well, filter, and collect the filtrate; see [link to product description]. Figure 1 . Figure 1 Extraction methods were examined.

[0139] The results showed that there was little difference in the effectiveness of reflux, ultrasonication, and ultrasonic extraction after decoction of the test sample. The ultrasonic method was fast and simple, so ultrasonic extraction was selected as the extraction method for the test sample.

[0140] 2.2.1.3.2 Investigation of Extraction Solvents

[0141] Take 0.2g of *Radix Sophorae Flavescentis* powder (batch number: BDG-YC-01), place it in a stoppered conical flask, and add water, 30% methanol, 50% methanol, 70% methanol, and 15ml of methanol respectively. Seal the flask tightly, sonicate for 30 minutes, cool, shake well, filter, and collect the filtrate. See [link to flask description]. Figure 2 . Figure 2 Investigation of extraction solvents.

[0142] The results showed that the peak shapes of the characteristic peaks did not differ much under different extraction solvents. Methanol was used as the extraction solvent because the sample was easy to filter. Methanol was tentatively chosen as the extraction solvent.

[0143] 2.2.1.3.3 Investigation of Solvent Addition Amount

[0144] Take 0.2g of *Radix Sophorae Flavescentis* powder (batch number: BDG-YC-01), place it in a stoppered conical flask, and add 15ml, 25ml, and 50ml of methanol respectively for testing. Seal tightly, sonicate for 30 minutes, cool, shake well, filter, and collect the filtrate. See [link to flask description]. Figure 3 . Figure 3 Investigation of the amount of solvent added.

[0145] The results showed that when the extraction solvent volume was 25 ml, the peak shapes and resolutions of each chromatographic peak were good, and the peak sizes were suitable; therefore, 25 ml of solvent was chosen. Furthermore, the peak elution times of the chromatographic peaks in the sequence investigated with 15 ml of extraction solvent differed from those in the sequence investigated with the solvent volume. This difference may be due to the volatilization of the 0.05% trifluoroacetic acid in the mobile phase. Therefore, during the experiment, care should be taken to ensure that the mobile phase preparation time is not too long and that the mobile phase is properly sealed, with a shelf life of 48 hours. The influence of the mobile phase on the relative retention time will be investigated subsequently.

[0146] 2.2.1.3.4 Examination of extraction time

[0147] Take 0.2g of *Radix Sophorae Flavescentis* powder (batch number: BDG-YC-01), place it in a stoppered conical flask, add 25ml of methanol, seal tightly, and sonicate for 15 minutes, 30 minutes, and 45 minutes respectively. Cool, shake well, filter, and collect the filtrate. See [link to flask]. Figure 4 . Figure 4 Extraction time was examined.

[0148] The results showed that extraction was complete at 30 minutes. Therefore, the extraction time was determined to be 30 minutes.

[0149] In summary, the preparation method of the test solution of the characteristic spectrum of Sophora flavescens medicinal material is determined as follows: Take 0.2g of the powder (passed through a No. 3 sieve), place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0150] 2.2.1.4 Methodological Examination

[0151] 2.2.1.4.1 Chromatographic Peak Identification

[0152] Preparation of the test solution: Prepare the test solution of Sophora flavescens root according to the experimental conditions proposed above.

[0153] Preparation of reference solutions: Take 0.2g of *Sophora tonkinensis* root reference material, place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, remove, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take appropriate amounts of magnoflorine, isocoryne, guaiacine, and guaiacine reference standards, accurately weigh them, and add methanol to prepare solutions containing 100μg of magnoflorine, 100μg of isocoryne, 20μg of guaiacine, and 20μg of guaiacine per ml, as reference solutions.

[0154] Preparation of negative control solution: Prepare negative control solution of Sophora flavescens root according to the experimental conditions proposed above.

[0155] The characteristic spectral peaks of *Radix Sophorae Flavescentis* were located. (See...) Figure 5-13 . Figure 5 Chromatographic peak identification; Figure 6 Spectrum of Magnoliaine - Reference Standard; Figure 7 Spectrum of Magnolia alkaloids - Sophora flavescens root medicinal material; Figure 8 Spectrum of isocyanidin for alkalization - reference standard; Figure 9 Spectrum of isocyanidin - Sophora flavescens root medicinal material; Figure 10 Spectrum of bat gazoline - reference standard; Figure 11 Spectrum of bat gesulphine - Sophora flavescens root medicinal material; Figure 12 Spectrum of puerarin - Reference Standard; Figure 13 Spectrum of basidiosine from Sophora flavescens root.

[0156] The results showed that peak 3 (S) was magnoflorine, peak 5 was isocoryne, peak 6 was guarcinine, and peak 7 was guarcinine. The following methodological investigation investigated these seven characteristic peaks in the sample.

[0157] 2.2.1.4.2 Precision Test

[0158] Take the test solution of Sophora flavescens root (BDG-YC-01) and inject it 6 times consecutively according to the proposed experimental method. Calculate the relative retention time of each characteristic peak. See Table 1.

[0159] Table 1 Precision test - retention time

[0160]

[0161] The results showed that the relative retention time (RSD) of each characteristic peak of the sample was 0.06%–0.14%, indicating that the instrument had good precision.

[0162] 2.2.1.4.3 Repeatability Test

[0163] Six portions of *Sophora flavescens* root powder (BDG-YC-01) were accurately weighed and prepared and measured according to the proposed experimental method. See Table 2.

[0164] Table 2 Repeatability Tests - Relative Retention Time

[0165]

[0166] The results showed that the relative retention time RSD of the six samples was 0.00%–0.28%, indicating that the method had good reproducibility.

[0167] 2.2.1.4.4 Intermediate Precision Examination

[0168] 2.2.1.4.4.1 Investigation with different instruments

[0169] Based on the above-planned experimental conditions, *Sophora flavescens* root (batch number: BDG-YC-01) was weighed and test solutions were prepared. The solutions were then analyzed using an Agilent 1260, Thermo Fisher Vanquish, and Waters e2695 high-performance liquid chromatograph. See Table 3. Figure 14 . Figure 14 Different instruments were used for the investigation.

[0170] Table 3 Instrument Durability Test - Relative Retention Time

[0171]

[0172] The results showed that when the test samples were tested using the above three instruments, the RSD of the relative retention time of each characteristic peak was 0.00% to 1.67%, indicating that the instruments had good durability.

[0173] 2.2.1.4.4.2 Investigations by different personnel and at different times

[0174] Based on the experimental conditions outlined above, different personnel (A and B) precisely weighed one sample of *Radix Sophorae Flavescentis* (BDG-YC-01) at different times (T1 and T2) to prepare test samples for determination. See Table 4.

[0175] Table 4. Personnel and Time Assessment - Relative Retention Time

[0176]

[0177]

[0178] The results showed that when different personnel measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.00% to 0.39%, indicating good method stability.

[0179] 2.2.1.4.3 Column robustness test

[0180] Based on the above-planned experimental conditions, chromatographic columns 1 (YMC-Triart C18, 4.6*250mm, 5μm) and 2 (…) were used respectively. The analysis was conducted using a C18 column (4.6*250mm, 5μm) and a ZORBAX SB-Aq column (4.6*250mm, 5μm). The results are shown in Table 5. Figure 15 . Figure 15 Investigation of different chromatographic columns.

[0181] Table 5. Column robustness study - relative retention time

[0182]

[0183] The results showed that when the samples were detected using the above three chromatographic columns, the RSD of the relative retention time of the characteristic peaks ranged from 1.36% to 9.37%, indicating that the columns had good robustness.

[0184] 2.2.1.4.4 Stability Assessment

[0185] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 3h, 6h, 9h, 12h, 18h, and 24h. See Table 6.

[0186] Table 6 Stability Study - Retention Time

[0187]

[0188] The results showed that the RSD of the retention time of the characteristic peak was between 0.47% and 0.69%, and the sample solution was stable within 24 hours.

[0189] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The above seven characteristic peaks will be included in subsequent investigations.

[0190] 2.2.1.5 Determination of characteristic peaks and establishment of reference spectra

[0191] Using this method, characteristic chromatographic analysis was performed on 20 batches of *Radix Sophorae Flavescentis* medicinal samples, and the relative retention times were calculated. (See attached image.) Figure 16-17 Table 7. Figure 16 Characteristic spectrum of *Radix Sophorae Flavescentis* (Beidougen) medicinal material: Peak 3 (S) magnoflorine; Peak 5: isocotylein; Peak 6: bacitracin; Peak 7: bacitracin (from bottom to top, S1-S10 are: BDG-YC-01, BDG-YC-02, BDG-YC-03, BDG-YC-04, BDG-YC-05, BDG-YC-06, BDG-YC-07, BDG-YC-08, BDG-YC-09, BDG-YC-10); Figure 17 Characteristic spectrum of *Radix Sophorae Flavescentis* (Beidougen) medicinal material: Peak 3 (S) magnoflorine; Peak 5: isocotylein; Peak 6: bacitracin; Peak 7: bacitracin (from bottom to top, S1-S10 are: BDG-YC-11, BDG-YC-12, BDG-YC-13, BDG-YC-14, BDG-YC-15, BDG-YC-16, BDG-YC-17, BDG-YC-18, BDG-YC-19, BDG-YC-20).

[0192] Table 7. Relative retention times of medicinal material characteristic atlases

[0193]

[0194]

[0195] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peak values, seven robust peaks were selected as characteristic peaks. Based on the methodological investigation results and validation results from 20 batches of medicinal slices, the theoretical plate number, calculated using the magnoflorine peak, is tentatively set at no less than 5000.

[0196] The final specification stipulates that the chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 3, 5, 6, and 7 should correspond to the retention times of the reference peaks. The peak corresponding to the magnoflorine reference peak is designated as the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.60 (peak 2), and 1.12 (peak 4).

[0197] Twenty batches of *Radix Sophorae Flavescentis* were synthesized using the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version), and a reference chromatogram of characteristic spectra of *Radix Sophorae Flavescentis* was established. (See...) Figure 18 . Figure 18 Compare with characteristic spectra; Peak 3 (S): Magnolia alkaloid; Peak 5: Isocoridine; Peak 6: Heterocorline; Peak 7: Heterocorline.

[0198] In summary, the feature map method is as follows:

[0199] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A; and 0.05% trifluoroacetic acid as mobile phase B, with gradient elution as specified in the table below; column temperature 25℃; flow rate 0.6 ml / min; detection wavelength 250 nm; and theoretical plate number calculated based on the magnoflorine peak should be no less than 5000.

[0200]

[0201] Preparation of the reference solution: Take 0.2g of *Sophora tonkinensis* root reference material, place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, remove, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add methanol to prepare a solution containing 100μg of magnoflorine per ml, as the reference solution.

[0202] Preparation of the test solution: Take about 0.2g of powdered Sophora flavescens (passed through a No. 3 sieve), place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, remove, cool and shake well, filter, and collect the filtrate to obtain the test solution.

[0203] The determination method involves precisely pipetting 5 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0204] The chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 3, 5, 6, and 7 should correspond to the retention times of the reference peaks. The peak corresponding to the magnoflorine reference peak is the S peak. Calculate the relative retention times of the remaining characteristic peaks and the S peak. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.60 (peak 2), and 1.12 (peak 4).

[0205] Example 2: HPLC Characteristic Chromatography of Sophora flavescens Slices

[0206] 2.2.2.2 Methods for determining characteristic spectra

[0207] Based on the methodology of the characterization of Sophora flavescens medicinal materials, the following method for characterization analysis of Sophora flavescens processed medicinal slices is determined:

[0208] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A; and 0.05% trifluoroacetic acid as mobile phase B, with gradient elution as specified in the table below; column temperature 25℃; flow rate 0.6 ml / min; detection wavelength 250 nm; and theoretical plate number calculated based on the magnoflorine peak should be no less than 5000.

[0209]

[0210]

[0211] Preparation of the reference solution: Take 0.2g of *Sophora tonkinensis* root reference material, place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, remove, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add methanol to prepare a solution containing 100μg of magnoflorine per ml, as the reference solution.

[0212] Preparation of the test solution: Take about 0.2g of powdered Sophora flavescens (passed through a No. 3 sieve), place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, remove, cool and shake well, filter, and collect the filtrate to obtain the test solution.

[0213] The determination method involves precisely pipetting 5 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0214] 2.2.2.3 Verification of the characteristic chromatogram of Sophora flavescens slices

[0215] The results of testing 20 batches of *Radix Sophorae Flavescentis* (Bei Dou Gen) slices are shown below. Figures 19-20 Table 8. Figure 19 Characteristic chromatogram of the processed medicinal slices: Peak 3 (S) magnoflorine; Peak 5: isocotylein; Peak 6: guarazine; Peak 7: guarazine; Figure 20 Characteristic chromatogram of the processed medicinal slices: Peak 3 (S) magnoflorine; Peak 5: isocotylein; Peak 6: guarsulinine; Peak 7: guarsulinine.

[0216] Table 8. Relative retention time of characteristic chromatograms of medicinal slices

[0217]

[0218] Based on the principles of stable relative retention times, detectability across all batches of samples, and relatively high peak values, seven peaks with good repeatability were selected as characteristic peaks. The results showed that the relative retention time RSD of the seven characteristic peaks from all 20 batches of *Toona sinensis* root slices was less than 1.0%.

[0219] The final specification stipulates that the chromatogram of the test sample should show 7 characteristic peaks, which should correspond to the retention times of the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 3, 5, 6, and 7 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the magnoflorine reference is designated as the S peak. The relative retention times of each characteristic peak and the S peak should be calculated and should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.60 (peak 2), and 1.12 (peak 4).

[0220] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize characteristic chromatograms of 20 batches of *Radix Sophorae Flavescentis* (Bei Dou Gen) slices, and a reference chromatogram for the characteristic chromatograms of *Radix Sophorae Flavescentis* slices was established. See [link / reference]. Figure 21 . Figure 21 Compare with the spectrum. Peak 3 (S): Magnolia alkaloid; Peak 5: Isocorynine; Peak 6: Piperine; Peak 7: Piperine.

[0221] In summary, the feature map method is as follows:

[0222] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A; and 0.05% trifluoroacetic acid as mobile phase B, with gradient elution as specified in the table below; column temperature 25℃; flow rate 0.6 ml / min; detection wavelength 250 nm; and theoretical plate number calculated based on the magnoflorine peak should be no less than 5000.

[0223]

[0224] Preparation of the reference solution: Take 0.2g of *Sophora tonkinensis* root reference material, place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, remove, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add methanol to prepare a solution containing 100μg of magnoflorine per ml, as the reference solution.

[0225] Preparation of the test solution: Take about 0.2g of powdered Sophora flavescens (passed through a No. 3 sieve), place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, remove, cool and shake well, filter, and collect the filtrate to obtain the test solution.

[0226] The determination method involves precisely pipetting 5 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0227] The final specification stipulates that the chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 3, 5, 6, and 7 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the magnoflorine reference is designated as the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.60 (peak 2), and 1.12 (peak 4). Peak 3 (S): magnoflorine; Peak 5: isocoryne; Peak 6: piracetamine; Peak 7: piracetamine.

[0228] Example 3: HPLC characteristic chromatogram of the standard decoction of Sophora flavescens

[0229] 2.2.3.2 Chromatographic conditions and system suitability test

[0230] The standard decoction and the finished granules have the same basic material composition, and the characteristic chromatographic conditions, system adaptability, and granule characteristic chromatographic methods are consistent.

[0231] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A; 0.05% trifluoroacetic acid was used as mobile phase B, and gradient elution was performed according to the specifications in the table below; column temperature was 25℃; flow rate was 0.6 ml / min; detection wavelength was 250 nm; and the theoretical plate number, calculated based on the magnoflorine peak, should not be less than 5000.

[0232]

[0233] Preparation of the reference solution: Take 0.2g of *Sophora tonkinensis* root reference material, place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, remove, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add methanol to prepare a solution containing 100μg of magnoflorine per ml, as the reference solution.

[0234] 2.2.3.2 Preparation of the test solution

[0235] The standard decoction and the finished granules have the same basic material composition, and the preparation method of the test solution is consistent with that of the finished granules.

[0236] The test solution is prepared as follows: Take 0.1g of this product, place it in a stoppered conical flask, add 25ml of methanol, stopper tightly, sonicate for 30 minutes, remove, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0237] 2.2.3.4 Methodological Examination

[0238] 2.2.2.4.1 Chromatographic Peak Identification

[0239] Preparation of the test solution: Prepare the test solution of the standard decoction of Sophora flavescens according to the experimental conditions proposed above.

[0240] Preparation of reference solutions: Take 0.2g of *Sophora tonkinensis* root reference material, place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, remove, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take appropriate amounts of magnoflorine, isocoryne, guaiacine, and guaiacine reference standards, accurately weigh them, and add methanol to prepare solutions containing 100μg of magnoflorine, 100μg of isocoryne, 20μg of guaiacine, and 20μg of guaiacine per ml, as reference solutions.

[0241] Preparation of negative control solution: Prepare negative control solution of standard decoction for *Sophora bean* root deficiency according to the experimental conditions proposed above.

[0242] The characteristic peaks of the standard decoction of *Radix Sophorae Flavescentis* were located. (See...) Figure 22-30 . Figure 22 Chromatographic peak identification; Figure 23 Spectrum of Magnoliaine - Reference Standard; Figure 24 Magnolia alkaloid spectrum - standard decoction; Figure 25 Spectrum of isocyanidin for alkalization - reference standard;

[0243] Figure 26 Spectral diagram of isocyanidin for alkalization - standard decoction; Figure 27 Spectrum of bat gazoline - reference standard; Figure 28Spectral diagram of bat gesulphine - standard decoction; Figure 29 Spectrum of puerarin - Reference Standard; Figure 30 Spectrum of Fibroside - Standard Decoction.

[0244] The results showed that peak 3 (S) was magnoflorine, peak 5 was isocoryne, peak 6 was guarcinine, and peak 7 was guarcinine. The following methodological investigation investigated these seven characteristic peaks in the sample.

[0245] 2.2.2.4.2 Precision Test

[0246] Take the standard decoction of Sophora flavescens (batch number: BDG-BT-01) as the test sample solution, and inject it 6 times consecutively according to the proposed experimental method, 1 μl each time, and calculate the retention time of each characteristic peak. See Table 9.

[0247] Table 9 Precision Examination - Retention Time

[0248]

[0249] The results showed that the retention time RSD of each characteristic peak was 0.10% to 0.26%, indicating that the instrument has good precision.

[0250] 2.2.2.4.3 Repeatability Test

[0251] Six portions of the standard decoction of Sophora flavescens (batch number: BDG-BT-01) were accurately weighed and prepared and measured according to the proposed experimental method. See Table 10.

[0252] Table 10 Repeatability Tests - Relative Retention Time

[0253]

[0254] The results showed that the relative retention time RSD of the six samples was 0.10%–0.37%, indicating that the method had good reproducibility.

[0255] 2.2.2.4.4 Intermediate Precision Examination

[0256] 2.2.2.4.4.1 Investigation with different instruments

[0257] Based on the above-established experimental conditions, standard decoction of *Radix Sophorae Flavescentis* (batch number: BDG-BT-01) was weighed and used to prepare test solutions. These solutions were then analyzed using Agilent 1260, Thermo Fisher Vanquish, and Waters e2695 high-performance liquid chromatographs, respectively, and the relative retention times of each characteristic peak were calculated. See Table 11. Figure 31 . Figure 31 Different instruments were used for the investigation.

[0258] Table 11 Instrument Durability Test - Relative Retention Time

[0259]

[0260] The results showed that when the test sample was tested using the above three instruments, the RSD of the relative retention time of each characteristic peak was 0.00% to 1.67%, indicating that the instruments had good durability.

[0261] 2.2.2.4.4..2 Investigations by different personnel and at different times

[0262] Based on the experimental conditions outlined above, different personnel (A and B) precisely weighed one portion of the standard decoction of *Radix Sophorae Flavescentis* (batch number: BDG-BT-01) at different times (T1 and T2) to prepare test samples for determination. See Table 12.

[0263] Table 12 Personnel and Time Assessment - Relative Retention Time

[0264]

[0265] The results showed that when different personnel measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.00% to 0.39%, indicating good method stability.

[0266] 2.2.2.4.5 Column robustness test

[0267] Based on the above-planned experimental conditions, chromatographic columns 1 (YMC-Triart C18, 4.6*250mm, 5μm) and 2 (…) were used respectively. The analysis was conducted using a C18 column (4.6*250mm, 5μm) and a ZORBAX SB-Aq column (4.6*250mm, 5μm), and the relative retention times of each characteristic peak were calculated. The results are shown in Table 13. Figure 32 . Figure 32 Investigation of different chromatographic columns.

[0268] Table 13 Column robustness study - relative retention time

[0269]

[0270] The results showed that the RSD of the relative retention time of the characteristic peaks when using the three chromatographic columns ranged from 1.35% to 9.43%, indicating that the columns had good robustness.

[0271] 2.2.2.4.6 Stability Assessment

[0272] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 3h, 6h, 9h, 12h, 18h, and 24h. See Table 14.

[0273] Table 14 Stability Study - Retention Time

[0274]

[0275] The results showed that the RSD of the retention time of the characteristic peak was between 0.44% and 0.68%, and the sample solution was stable within 24 hours.

[0276] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The aforementioned seven characteristic peaks will be included in subsequent investigations.

[0277] 2.2.3.5 Determination of characteristic peaks and establishment of reference spectra

[0278] 2.2.3.5.1 Verification results of 20 batches of standard decoction of Sophora flavescens

[0279] Using this method, characteristic spectral analysis was performed on 20 batches of samples, and the relative retention times were calculated. See [link / reference]. Figures 33-34 Table 15. Figure 33 Characteristic spectrum of standard decoction of Sophora flavescens: Peak 3 (S): magnoflorine; Peak 5: isocotylein; Peak 6: bacitracin; Peak 7: bacitracin (from bottom to top, S1-S10 are: BDG-BT-01, BDG-BT-02, BDG-BT-03, BDG-BT-04, BDG-BT-05, BDG-BT-06, BDG-BT-07, BDG-BT-08, BDG-BT-09, BDG-BT-10); Figure 34 Characteristic spectrum of standard decoction of Sophora flavescens: Peak 3 (S) magnoflorine; Peak 5: isocotylein; Peak 6: bacitracin; Peak 7: bacitracin (from bottom to top S1-S10 are: BDG-BT-11, BDG-BT-12, BDG-BT-13, BDG-BT-14, BDG-BT-15, BDG-BT-16, BDG-BT-17, BDG-BT-18, BDG-BT-19, BDG-BT-20).

[0280] Table 15 Relative retention time of standard decoction of Sophora flavescens

[0281]

[0282]

[0283] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peak values, seven robust peaks were selected as characteristic peaks. Based on the methodological investigation results and validation results from 20 batches of medicinal slices, the theoretical plate number, calculated using the magnoflorine peak, is tentatively set at no less than 5000.

[0284] The final specification stipulates that the chromatogram of the test sample should show 7 characteristic peaks, which should correspond to the retention times of the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 3, 5, 6, and 7 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the magnoflorine reference is designated as the S peak. The relative retention times of each characteristic peak and the S peak should be calculated and should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.60 (peak 2), and 1.12 (peak 4).

[0285] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize chromatograms of 20 batches of *Radix Sophorae Flavescentis* standard decoction, and a reference chromatogram of the characteristic chromatograms of *Radix Sophorae Flavescentis* standard decoction was established. See [link to relevant documentation]. Figure 35 . Figure 35 Compare with the characteristic chromatograms. Peak 3 (S): Magnolia alkaloid; Peak 5: Isocorynine; Peak 6: Piperine; Peak 7: Piperine.

[0286] In summary, the feature map method is as follows:

[0287] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A; and 0.05% trifluoroacetic acid as mobile phase B, with gradient elution as specified in the table below; column temperature 25℃; flow rate 0.6 ml / min; detection wavelength 250 nm; and theoretical plate number calculated based on the magnoflorine peak should be no less than 5000.

[0288]

[0289] Preparation of the reference solution: Take 0.2g of *Sophora tonkinensis* root reference material, place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, remove, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add methanol to prepare a solution containing 100μg of magnoflorine per ml, as the reference solution.

[0290] Preparation of the test solution: Take 0.1g of the standard decoction of Sophora flavescens, weigh it accurately, place it in a stoppered conical flask, add 25ml of methanol accurately, stopper tightly, weigh it, sonicate for 30 minutes, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and the test solution is obtained.

[0291] The determination method involves precisely pipetting 5 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0292] The chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 3, 5, 6, and 7 should correspond to the retention times of their respective reference peaks. The peak corresponding to the magnoflorine reference is designated as the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.60 (peak 2), and 1.12 (peak 4).

[0293] Example 4: HPLC Characteristic Chromatography of Sophora flavescens Formula Granules

[0294] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A; and 0.05% trifluoroacetic acid as mobile phase B, with gradient elution as specified in the table below; column temperature 20 °C; flow rate 0.6 ml / min; detection wavelength 250 nm; and theoretical plate number calculated based on the magnoflorine peak should be no less than 5000.

[0295]

[0296] Preparation of the reference solution: Take 0.2g of *Sophora tonkinensis* root reference material, place it in a stoppered conical flask, add 10ml of 50% methanol, seal tightly, sonicate for 30 minutes, remove, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add methanol to prepare a solution containing 100μg of magnoflorine per ml, as the reference solution.

[0297] Preparation of the test solution: Take an appropriate amount of granulated Sophora flavescens formula, grind it into a fine powder, take 0.1g, place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, take it out, cool it, shake it well, filter it, and take the filtrate to obtain the test solution.

[0298] The determination method involves precisely pipetting 5 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0299] 2.2.4.2 Selection of mobile phase

[0300] Based on the experimental conditions outlined above, the separation performance of four different mobile phases was investigated: acetonitrile-0.05% trifluoroacetic acid, acetonitrile-1% formic acid, acetonitrile-0.01% trifluoroacetic acid, and acetonitrile-0.05% triethylamine. (See attached diagram.) Figure 36 . Figure 36 Mobile phase selection; Note: The chromatogram of acetonitrile-0.05% triethylamine is the chromatogram from the official standard method of Sichuan Province for the quality standard of Beidougen formula granules.

[0301] The results showed that the gradient elution with acetonitrile-0.05% trifluoroacetic acid solution produced more chromatographic peaks and achieved better resolution. Therefore, gradient elution with acetonitrile-0.05% trifluoroacetic acid solution was chosen as the mobile phase for the determination of the characteristic chromatogram of the *Betu kumquat* root decoction.

[0302] 2.2.4.3 Wavelength Selection

[0303] Based on the above-specified experimental conditions, a diode array detector was used to perform a full-band scan of the test solution, and chromatograms of the test solution at wavelengths of 250 nm, 270 nm, 290 nm, 310 nm, and 330 nm were extracted. (See...) Figure 37 . Figure 37 Chromatograms of different wavelengths of Sophora flavescens formula granules

[0304] The results showed that the chromatographic peak information was greater and the chromatographic peak distribution was more uniform when the detection wavelength was 250 nm, so the detection wavelength was determined to be 250 nm.

[0305] 2.2.4.4 Column Temperature Investigation

[0306] Based on the above-specified experimental conditions, the results were investigated at column temperatures of 20℃, 25℃, and 30℃. (See...) Figure 38 See Tables 16 and 17. Figure 38 Column temperature investigation.

[0307] Table 16 Column Temperature Study - Retention Time

[0308]

[0309] Table 17 Column Temperature Study - Relative Retention Time

[0310]

[0311] The results showed that the chromatographic peaks could be well separated at column temperatures of 20-30℃, and the relative retention time RSD was 0.52% to 2.16. Therefore, column temperatures of 20-30℃ are acceptable, and a column temperature of 25℃ will be used for subsequent investigation.

[0312] 2.2.4.5 Flow velocity investigation

[0313] Based on the above-established experimental conditions, the flow rates of 0.5 ml / min, 0.6 ml / min, 0.7 ml / min, and 0.8 ml / min were investigated. (See...) Figure 39 Tables 18-19. Figure 39 Flow velocity study.

[0314] Table 18 Flow velocity study - retention time

[0315]

[0316]

[0317] Table 19 Flow velocity study - relative retention time

[0318]

[0319] The results showed that a flow rate of 0.6 ml / min resulted in good peak shape and moderate resolution. Therefore, the flow rate was determined to be 0.6 ml / min.

[0320] 2.2.4..6 Delayedness Examination

[0321] Based on the above-specified experimental conditions, a delay test was conducted. The results are shown below. Figure 40 . Figure 40 Delayed investigation.

[0322] The results showed that the sample had virtually no chromatographic peaks after 60 minutes, so the sample detection time was set at 60 minutes.

[0323] In summary, the chromatographic conditions and system suitability test for the characteristic chromatogram of the *Radix Sophorae Flavescentis* formula granules were determined as follows: Octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A; 0.05% trifluoroacetic acid was used as mobile phase B, with gradient elution as specified in the table below; column temperature was 25℃; flow rate was 0.6 ml / min; detection wavelength was 250 nm; and the theoretical plate number, calculated based on the magnolia alkaloid peak, should not be less than 5000.

[0324]

[0325] 2.2.4.7 Investigation into the preparation of the test solution

[0326] 2.2.4.7.1 Examination of Extraction Methods

[0327] Take an appropriate amount of *Radix Sophorae Flavescentis* granules (batch number: BDG-KL01), grind them finely, take 0.1g, place them in a stoppered conical flask, add 10ml of 50% methanol, seal tightly, reflux and sonicate for 30 minutes respectively, cool, shake well, filter, and collect the filtrate to obtain the final product. (See...) Figure 41 . Figure 41 Extraction methods were examined.

[0328] The results showed that there was little difference in the effectiveness of reflux and ultrasonic extraction of the test sample, and the ultrasonic method was fast and simple. Therefore, ultrasonic extraction was selected as the extraction method for the test sample.

[0329] 2.2.4.7.2 Investigation of Extraction Solvents

[0330] Take an appropriate amount of *Radix Sophorae Flavescentis* granules (batch number: BDG-KL01), grind them finely, take 0.1g, place them in a stoppered conical flask, add 10ml each of methanol, 30% methanol, 50% methanol, 70% methanol, and water, seal tightly, sonicate for 30 minutes, cool, shake well, filter, and collect the filtrate. See [link to flask description]. Figure 42 . Figure 42 Investigation of extraction solvents.

[0331] The results showed that the peak shapes of the characteristic peaks did not differ much under different extraction solvents. Methanol was used as the extraction solvent because the sample was easy to filter. Methanol was tentatively chosen as the extraction solvent.

[0332] 2.2.4.7.3 Investigation of Solvent Addition Amount

[0333] Take an appropriate amount of *Radix Sophorae Flavescentis* granules (batch number: BDG-KL01), grind them finely, and take 0.1g. Place the granules in a stoppered conical flask, and add 10ml, 25ml, and 50ml of methanol respectively for testing. Seal tightly, sonicate for 30 minutes, cool, shake well, filter, and collect the filtrate. See [link to flask]. Figure 43 . Figure 43 Investigation of the amount of solvent added.

[0334] The results showed that when the extraction solvent volume was 25 ml, the peak shapes and resolutions of each chromatographic peak were good, and the peak sizes were suitable; therefore, 25 ml of solvent was chosen. Furthermore, the peak elution times of the chromatographic peaks in the sequence investigated with 10 ml of extraction solvent differed from those in the sequence investigated with the solvent volume. This difference may be due to the volatilization of the 0.05% trifluoroacetic acid in the mobile phase. Therefore, during the experiment, care should be taken to ensure that the mobile phase preparation time is not too long and that the mobile phase is properly sealed, with a shelf life of 48 hours. The influence of the mobile phase on the relative retention time will be investigated subsequently.

[0335] 2.2.4.7.4 Examination of extraction time

[0336] Take an appropriate amount of *Radix Sophorae Flavescentis* granules (batch number: BDG-KL01), grind them finely, take 0.1g, place them in a stoppered conical flask, add 25ml of methanol, seal tightly, and sonicate for 15 minutes, 30 minutes, and 45 minutes respectively. Cool, shake well, filter, and collect the filtrate to obtain the final product. (See...) Figure 44 . Figure 44 Extraction time was examined.

[0337] The results showed that extraction was complete at a time of 30 minutes. Therefore, the extraction time was determined to be 30 minutes.

[0338] In summary, the preparation method of the test solution for the characteristic chromatogram of Beidougen decoction is determined as follows: Take an appropriate amount of this product, grind it into a fine powder, take 0.1g, place it in a stoppered conical flask, add 25ml of methanol, sonicate for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0339] 2.2.4.8 Methodological Examination

[0340] 2.2.4.8.1 Chromatographic Peak Identification

[0341] Preparation of the test solution: Prepare the test solution of the Sophora flavescens formula granules according to the experimental conditions proposed above.

[0342] Preparation of reference solutions: Take 0.2g of *Sophora tonkinensis* root reference material, place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, remove, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take appropriate amounts of magnoflorine, isocoryne, guaiacine, and guaiacine reference standards, accurately weigh them, and add methanol to prepare solutions containing 100μg of magnoflorine, 100μg of isocoryne, 20μg of guaiacine, and 20μg of guaiacine per ml, as reference solutions.

[0343] Preparation of negative control solution: Prepare negative control solution of *Sophora bean* formula granules according to the experimental conditions proposed above.

[0344] The characteristic peaks of the *Radix Sophorae Flavescentis* formula granules were located. (See...) Figures 45-53 . Figure 45 Chromatographic peak identification; Figure 46 Spectrum of Magnoliaine - Reference Standard; Figure 47 Magnolia flower alkaloid spectrum - Sophora flavescens formula granules; Figure 48 Spectrum of isocyanidin for alkalization - reference standard; Figure 49 Spectrum of isocyanidin-based alkaloids - Sophora flavescens formula granules; Figure 50 Spectrum of bat gazoline - reference standard;

[0345] Figure 51 Spectrum of bat gesulphine - Sophora flavescens formula granules; Figure 52 Spectrum of puerarin - Reference Standard; Figure 53 Spectrum of Ficus pumila alkaloids - Sophora flavescens formula granules.

[0346] The results showed that peak 3 (S) was magnoflorine, peak 5 was isocoryne, peak 6 was guarcinine, and peak 7 was guarcinine. The following methodological investigation investigated these seven characteristic peaks in the sample.

[0347] 2.2.4.8.2 Precision Test

[0348] Take the test solution of *Radix Sophorae Flavescentis* (batch number: BDG-KL01), inject it 6 times consecutively according to the proposed experimental method, 1 μl each time, and calculate the retention time of each characteristic peak. See Table 20.

[0349] Table 20 Precision Examination - Retention Time

[0350]

[0351] The results showed that the relative retention time (RSD) of each characteristic peak of the sample ranged from 0.06% to 0.18%, indicating that the method had good injection precision.

[0352] 2.2.4.8.3 Repeatability Test

[0353] Six portions of the *Beidougen* (Northern Bean Root) formula granules (batch number: BDG-KL01) were accurately weighed and prepared and measured according to the proposed experimental method. See Table 21.

[0354] Table 21 Repeatability Tests - Relative Retention Time

[0355]

[0356] The results showed that the relative retention time (RSD) of the six samples ranged from 0.00% to 0.28%, indicating that the method had good reproducibility.

[0357] 2.2.4.8.4 Investigation with different instruments

[0358] Based on the above-established experimental conditions, granules of the *Radix Sophorae Flavescentis* formula (batch number: 22070001) were weighed and used to prepare test solutions. These solutions were then analyzed using an Agilent 1260, Thermo Fisher Vanquish, and Waters e2695 high-performance liquid chromatograph. The relative retention times of each characteristic peak were calculated. (See...) Figure 54 Table 22. Figure 54 Different instruments were used for the investigation.

[0359] Table 22 Instrument Durability Assessment - Relative Retention Time

[0360]

[0361]

[0362] The results showed that when the test samples were detected using the above three instruments, the RSD of the relative retention time of each characteristic peak was 0.05% to 1.05%, indicating that the method has good instrument precision.

[0363] 2.2.3.4.4.1 Investigations by different personnel and at different times

[0364] Based on the experimental conditions outlined above, different personnel (A and B) precisely weighed one portion of the *Beidougen* formula granules (batch number: BDG-KL01) at different times (T1 and T2) to prepare test samples for determination. See Table 23.

[0365] Table 23 Personnel and Time Assessment - Relative Retention Time

[0366]

[0367] The results showed that when different personnel measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.00% to 0.39%, indicating that the method had good intermediate precision.

[0368] 2.2.4.8.4 Column robustness test

[0369] Based on the above-planned experimental conditions, chromatographic columns 1 (YMC-Triart C18, 4.6*250mm, 5μm) and 2 (…) were used respectively. The analysis was conducted using a C18 column (4.6*250mm, 5μm) and a ZORBAX SB-Aq column (4.6*250mm, 5μm), and the relative retention times of each characteristic peak were calculated. Results are shown below. Figure 55 Table 24. Figure 55 Investigation of different chromatographic columns.

[0370] Table 24 Column Robustness Study - Relative Retention Time

[0371]

[0372] The results showed that when the samples were detected using the above three chromatographic columns, the RSD of the relative retention time of the characteristic peaks ranged from 1.44% to 9.49%, indicating that the columns of this method have good robustness.

[0373] 2.2.4.8.5 Stability Assessment

[0374] Based on the experimental conditions outlined above, the same test solution was measured at 0h, 3h, 6h, 9h, 12h, 18h, and 24h. The retention times of each characteristic peak were calculated. See Table 25.

[0375] Table 25 Stability Study - Retention Time

[0376]

[0377] The results showed that the RSD of the retention time of the characteristic peak was between 0.45% and 0.60%, indicating that the sample solution was stable within 24 hours.

[0378] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The aforementioned seven characteristic peaks will be included in subsequent investigations.

[0379] 2.2.4.9 Determination of characteristic peaks and establishment of reference spectra

[0380] 2.2.4.9.1 Validation results of three batches of Sophora flavescens formula granules

[0381] Using this method, characteristic spectral analysis was performed on three batches of samples, and the relative retention times were calculated. See [link / reference]. Figures 56-57 Table 26. Figure 56 Characteristic spectrum of Sophora flavescens formula granules (BDG-KL01): Peak 3 (S): Magnolia alkaloid; Peak 5: Isocodinine; Peak 6: Heteropogoninine; Peak 7: Heteropogoninine; Figure 57 Characteristic spectrum of Beidougen formula granules (BDG-KL02): Peak 3 (S) magnoflorine; Peak 5: isocotylein; Peak 6: guarsulinine; Peak 7: guarsulinine.

[0382] Figure 58 Characteristic spectrum of *Radix Sophorae Flavescentis* (BDG-KL03) formula granules: Peak 3 (S): Magnolia alkaloid; Peak 5: Isocoridine; Peak 6: *Gynosteminium oleraceum*; Peak 7: *Gynosteminium oleraceum*.

[0383] Table 26 Relative Retention Time of Sophora flavescens Formula Granules

[0384]

[0385] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peak values, a total of 7 peaks were selected as characteristic peaks. Based on the methodological investigation results and the particle validation results of 3 batches, the theoretical plate number, calculated using the magnoflorine peak, is tentatively set to be no less than 5000.

[0386] 2.2.4.9.1 Establishment of Limits for Relative Retention Time

[0387] To better reflect the transfer of quantity values, the relative retention times of medicinal materials, processed medicinal pieces, standard decoctions, and formula granules are consistent. The average relative retention times of 20 batches of medicinal materials, 20 batches of processed medicinal pieces, 20 batches of standard decoctions, and 3 batches of formula granules are calculated as the final specified relative retention times. The results are shown in Table 27.

[0388] Table 27 Average Relative Retention Time

[0389]

[0390] The relative retention time (RSD) of the average values ​​of 20 batches of medicinal materials, 20 batches of processed medicinal slices, 20 batches of standard decoctions, and 3 batches of formula granules was less than 1.0%.

[0391] The final specification stipulates that the chromatogram of the test sample should show 7 characteristic peaks, which should correspond to the retention times of the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 3, 5, 6, and 7 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the magnoflorine reference is designated as the S peak. The relative retention times of each characteristic peak and the S peak should be calculated and should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.60 (peak 2), and 1.12 (peak 4).

[0392] Three batches of *Beidougen* (Radix Sophorae Flavescentis) formula granules were synthesized using the 2012 version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, and a reference chromatogram of the characteristic chromatograms of *Beidougen* formula granules was established. (See...) Figure 59 . Figure 59 According to the characteristic spectrum, peak 3 (S) is magnoflorine; peak 5 is isocotinine; peak 6 is guarsulinine; peak 7 is guarsulinine.

[0393] In summary, the feature map method is as follows:

[0394] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A; and 0.05% trifluoroacetic acid as mobile phase B, with gradient elution as specified in the table below; column temperature 25℃; flow rate 0.6 ml / min; detection wavelength 250 nm; and theoretical plate number calculated based on the magnoflorine peak should be no less than 5000.

[0395]

[0396] Preparation of the reference solution: Take 0.2g of *Sophora tonkinensis* root reference material, place it in a stoppered conical flask, add 25ml of methanol, seal tightly, sonicate for 30 minutes, remove, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add methanol to prepare a solution containing 100μg of magnoflorine per ml, as the reference solution.

[0397] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take 0.1 g, weigh it accurately, place it in a stoppered conical flask, add 25 ml of methanol accurately, stopper tightly, weigh it, sonicate for 30 minutes, take it out, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0398] The determination method involves precisely pipetting 5 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0399] The chromatogram of the test sample should show 7 characteristic peaks, which should correspond to the retention times of the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 3, 5, 6, and 7 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the magnoflorine reference is designated as the S peak. The relative retention times of each characteristic peak and the S peak should be calculated and should be within ±10% of the specified values. The specified values ​​are: 0.35 (peak 1), 0.60 (peak 2), and 1.12 (peak 4).

[0400] Comparative Example 1

[0401] The pretreatment is the same as in Example 1, except that...

[0402] Chromatographic conditions: Waters T3C18 column (4.6 mm × 250 mm, 5 μm), with octadecylsilane-bonded silica gel as the stationary phase; mobile phase: acetonitrile (A) - 0.05% triethylamine (B), gradient elution, program as shown below.

[0403] Table 1; Detection wavelength: 290 nm; Injection volume: 10 μL.

[0404]

[0405] The results are as follows Figure 60 and Figure 61 As shown, where Figure 60 This is a chromatogram of the peaks from Comparative Example 1. Figure 61 As shown in the spectral overlay of Comparative Example 1, it can be seen that the chromatographic peaks in the sample of the present invention do not match the peaks of the reference standard isocoryne, and the contrast spectra are inconsistent. Therefore, the method of Comparative Example 1 cannot detect isocoryne.

[0406] Comparative Example 2

[0407] Chromatographic conditions and system suitability were assessed using a WatersXBridge C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile (A) - 0.05% triethylamine (B), gradient elution (0–5 min, 5% A; 5–15 min, 5%–15% A; 15–25 min, 15%–20% A; 25–45 min, 20%–60% A; 45–50 min, 60% A); flow rate: 1 mL / min; column temperature: 30 °C; detection wavelength: 290 nm; injection volume: 10 μL. The theoretical plate number, calculated based on piracetamine, should be no less than 5,000.

[0408] The results are as follows Figure 62 and Figure 63 As shown, where Figure 62 This is a chromatogram of the peaks from Comparative Example 2. Figure 63As shown in the spectral overlay of Comparative Example 2, it can be seen that the chromatographic peaks in the sample of this invention do not match the peaks of the reference standard isocoryne, and the contrast spectra are inconsistent. Therefore, the method of Comparative Example 2 cannot detect isocoryne.

[0409] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing HPLC characteristic chromatograms of processed slices, standard decoctions, and formulated granules of *Radix Sophorae Flavescentis*, comprising: A) The test sample raw material is dissolved and extracted using a solvent to obtain the test solution; the solvent is methanol; B) The test solution was determined by high performance liquid chromatography to obtain the HPLC characteristic chromatograms of the prepared slices of Sophora flavescens, standard decoction, and formula granules. The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile solution, mobile phase B is 0.05% trifluoroacetic acid aqueous solution, and gradient elution is used. The gradient elution specifically refers to: 0–15 min, Phase A: 5%–11%, Phase B: 95–89%; 15–40 min, Phase A: 11%–18%, Phase B: 89%–82%; 40–60 min, Phase A: 18%–21%, Phase B: 82%–79%.

2. The method according to claim 1, characterized in that, It also includes the preparation of reference solutions: magnoflorine, isocoryne, clodexine, and clodexine are dissolved in methanol to obtain reference solutions; The reference solution was analyzed by high performance liquid chromatography to obtain the chromatogram of the reference; and the components of the prepared slices of Sophora flavescens, standard decoction, and formula granules were qualitatively determined based on the chromatogram of the reference.

3. The method according to claim 2, characterized in that, The specific concentrations of the reference solutions are as follows: magnoflorine 100 μg / mL, isocoryne 100 μg / mL, piperine 20 μg / mL, and piperine 20 μg / mL.

4. The method according to claim 1, characterized in that, The chromatographic column is C. 18 250×4.6mm 5μm; column temperature 20~30℃; theoretical plate number calculated based on magnolia alkaloids should not be less than 5000.

5. The method according to claim 4, characterized in that, The flow rate of the mobile phase is 0.6 mL / min; the injection volume is 5~10 μL.

6. The method according to claim 4, characterized in that, The detection wavelength is 250nm.

7. The method according to claim 4, characterized in that, The extraction in step A) is ultrasonic extraction; the ultrasonic power is 600W, the frequency is 40kHz, and the ultrasonic time is 30min.

8. The method according to claim 1, characterized in that, The ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.1~0.2:25; The raw materials for the test sample are one or more of the following: Sophora flavescens medicinal material, Sophora flavescens slices, Sophora flavescens standard decoction, or Sophora flavescens formula granules.

9. The method according to claim 1, characterized in that, The similarity of HPLC characteristic chromatograms of processed slices, standard decoctions, and formulated granules of *Radix Sophorae Flavescentis* was evaluated using a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. A standard HPLC characteristic chromatogram consisting of seven characteristic peaks was obtained, including peak 3: magnoflorine; peak 5: isocotinine; peak 6: puerarin; and peak 7: puerarin. In the characteristic chromatograms of the prepared slices, standard decoctions, and formula granules of Sophora flavescens, the relative retention time of each characteristic peak and the S peak is calculated with magnoflorine as the reference peak. The relative retention time is within ±10% of the specified value, which is 0.35 (peak 1), 0.60 (peak 2), and 1.12 (peak 4).

10. A method for identifying characteristic chromatograms of processed slices, standard decoctions, and formulated granules of *Radix Sophorae Flavescentis*, characterized in that... The detection is performed using the method described in any one of claims 1 to 9, and the detection results are analyzed.

Images