A characteristic spectrum construction method of Simiao Yongan Decoction and detection methods of various raw medicinal materials

Abstract

The application provides a characteristic map construction method of Simiao Yongan Decoction. The application also provides a quality detection method of honeysuckle medicinal materials, radix scrophulariae medicinal materials, angelica medicinal materials and licorice medicinal materials. The method is suitable for the identification control of honeysuckle, radix scrophulariae, angelica and licorice single medicinal materials, has the characteristics of comprehensive detection and simple operation, can effectively identify and control the whole process of quality transmission of medicinal materials, decoction pieces and reference samples, and ensures the uniformity and stability of honeysuckle, radix scrophulariae, angelica, licorice medicinal materials and Simiao Yongan Decoction reference sample quality.

Description

Technical Field

[0001] This invention relates to a method for constructing a characteristic spectrum of Simiao Yong'an Decoction and a method for detecting each raw material. Background Technology

[0002] Simiao Yong'an Decoction is a prescription selected by the State Administration of Traditional Chinese Medicine from the first batch of the "Catalogue of Famous Ancient Prescriptions." It originates from the "Osteoarthritis" section of Bao Xiang'ao's "New Compilation of Effective Prescriptions" from the Qing Dynasty and is included in planned textbooks for Traditional Chinese Medicine, making it widely used throughout the country. It consists of four herbs: honeysuckle, scrophularia, angelica, and licorice. It is used to clear heat and detoxify, invigorate blood and nourish blood, and unblock meridians and relieve pain. The research and development of this prescription is based on ancient principles with innovation. In Simiao Yong'an Decoction, honeysuckle and scrophularia are used as the chief herbs to clear heat and detoxify. The combination of these two herbs clears pathogenic heat in the qi level and resolves heat toxins in the blood level. Furthermore, scrophularia also has the effect of nourishing yin and dispersing stagnation. Angelica, with its warming and moistening properties, invigorates blood and removes blood stasis, unblocks blood vessels, and nourishes yin and blood to moisten the extremities. Licorice, used raw, serves three purposes: first, it assists honeysuckle in clearing heat and detoxifying; second, it combines with angelica and scrophularia to nourish yin and generate fluids; and third, it harmonizes the other herbs, acting as an adjuvant. Although it contains only four ingredients, their dosage is potent and specific, working together to clear heat and detoxify, invigorate blood circulation, and relieve pain. Currently, in clinical practice, Simiao Yong'an Decoction plays an important role in many aspects, including endocrine, cardiovascular, and dermatological diseases.

[0003] Currently, there is a lack of official quality standards for Simiao Yong'an Decoction. Quality control mainly relies on methods developed by individual research institutions. The main challenges in detection are: the active ingredients are not yet standardized; serum pharmacochemistry studies have identified approximately 20 precursor components entering the bloodstream, with flavonoids and triterpenoids being the most prevalent, but there is no consensus on which are the core indicators that "must be quantified." Different literature lists chlorogenic acid, luteolin, ferulic acid, harbazoside, glycyrrhizic acid, glycyrrhizin, isorhamnetin, and glycyrrhetinic acid as "potential Q-markers," leading to a lack of unified targets when companies or hospitals establish standards, resulting in poor reproducibility. The decoction process of the compound formula involves significant dynamic changes. When the four herbs are decocted together, phenolic acids such as chlorogenic acid and ferulic acid are easily oxidized and degraded by heat; harbazoside and harbazoside contained in Scrophularia can interconvert at high temperatures; and glycyrrhizic acid easily forms insoluble complexes with Angelica polysaccharides, resulting in content differences of 20-40% between different batches of the same herb. This places higher demands on the determination of sampling time points and decoction endpoints. Matrix interference is severe, making method development difficult. The volatile oil of Angelica sinensis, glycyrrhizin, and honeysuckle polyphenols exhibit overlapping peaks on HPLC. Chlorogenic acid and isochlorogenic acid C are isomers, and the resolution on conventional C18 columns is often <1.2. While LC-MS / MS can improve selectivity, the sample needs to be removed by solid-phase extraction to remove the high-content saponin matrix; otherwise, ion inhibition is significant, and the recovery rate fluctuates greatly. The supply and cost of reference standards are also problematic. Characteristic components such as angoloside C, harpagoside, and crude glycyrrhizin C lack national reference standards and must be isolated or custom-made, which is expensive (approximately 3000–6000 RMB / 20mg) and has poor stability (purity decreases by >5% after 6 months at -20℃), posing difficulties for routine content determination and stability studies.

[0004] There are relevant literature reports on the preparation process and quality testing of Simiao Yong'an Decoction, such as: Guo Zixian et al., Establishment of Standard Decoction Process for Simiao Yong'an Decoction, Chinese Patent Medicine, May 2021, which investigated the soaking time of medicinal ingredients, the amount of water added, and the decoction time. Single-factor experiments were conducted to investigate the filtration method of the decoction. The content and yield of harpagoside, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, ferulic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, angoloside C, harpagoside, cinnamic acid, glycyrrhizic acid, and extract were used as evaluation indicators. Wei Shuchang et al., Optimization of Extraction Process of Simiao Yong'an Decoction, Journal of Gansu University of Traditional Chinese Medicine, September 1999, which used orthogonal method and chlorogenic acid extraction amount as the indicator to optimize the extraction process of Simiao Yong'an Decoction.

[0005] Li Huihong, et al., Determination of chlorogenic acid and catalpol content in Jiawei Simiao Yong'an Decoction by reversed-phase high-performance liquid chromatography, Chinese Journal of Hospital Drug Evaluation and Analysis, Vol. 23, No. 1, 2023. A method for determining the content of chlorogenic acid and catalpol in Jiawei Simiao Yong'an Decoction extract by reversed-phase high-performance liquid chromatography was established.

[0006] Application No. 202110208860.4, Invention Title: A Preparation Process and Quality Control Method for Simiao Yong'an Decoction, provides a preparation process and quality control method for Simiao Yong'an Decoction. The quality control method for the preparation process includes: obtaining a standard decoction and a test sample; using an aqueous decoction of Simiao Yong'an Decoction as the standard decoction, and using a physical reference sample or compound preparation of Simiao Yong'an Decoction as the test sample. The chromatograms obtained by this method do not identify and control the individual herbs honeysuckle, scrophularia, angelica, and licorice, and the experimental conditions, such as detection wavelength and elution gradient, cannot achieve the identification and control of the herbal properties of honeysuckle, scrophularia, angelica, and licorice.

[0007] Application No. 202211617635.7, Invention Title: A Method for Constructing, Quality Controlling, and Preparing a Characteristic Chromatography of a Simiao Yong'an Decoction Reference Sample, Discloses a method for constructing a characteristic chromatogram of a Simiao Yong'an Decoction reference sample, a quality control method, and a preparation method. It employs two sets of detection methods to control the Simiao Yong'an Decoction reference sample, the detection process is complex, and it does not perform identification and control of the individual herbs honeysuckle, scrophularia, angelica, and licorice. Summary of the Invention

[0008] This invention provides a method for constructing the characteristic spectrum of Simiao Yong'an Decoction and a method for detecting each raw material.

[0009] This invention, through qualitative analysis of the characteristic spectrum of the Simiao Yong'an Decoction reference sample, can simultaneously identify and control the four medicinal ingredients: honeysuckle, scrophularia, angelica, and licorice. It is also applicable to the identification and control of each individual medicinal ingredient: honeysuckle, scrophularia, angelica, and licorice.

[0010] The present invention provides a standard sample of Simiao Yong'an Decoction with 13 characteristic peaks. Peaks 1, 2, 3, 6, 7, and 9 are derived from honeysuckle; peaks 8 and 11 from scrophularia; peaks 4, 10, and 13 from angelica; and peaks 5 and 12 from licorice. This method can effectively identify the four herbs. Honeysuckle has 7 characteristic peaks, scrophularia has 7 characteristic peaks, angelica has 7 characteristic peaks, and licorice has 8 characteristic peaks. This method is applicable to the identification and control of individual honeysuckle, scrophularia, angelica, and licorice herbs. It features comprehensive detection and simple operation, effectively controlling the quality transfer of herbs, processed herbs, and standard samples throughout the entire process, ensuring the uniformity and stability of the quality of honeysuckle, scrophularia, angelica, licorice, and the Simiao Yong'an Decoction standard sample. Attached Figure Description

[0011] Figure 1 Characteristic chromatogram of the Si Miao Yong An Tang reference sample (peak 1: neochlorogenic acid, peak 2: chlorogenic acid, peak 3: cryptochlorogenic acid, peak 4: ferulic acid, peak 5: glycyrrhizin, peak 7: 3,5-di-O-caffeoylquinic acid, peak 8: Angoloside C, peak 9: 4,5-di-O-caffeoylquinic acid, peak 10: ligusticoside I, peak 11: cinnamic acid, peak 12: glycyrrhizic acid, peak 13: ligustilide);

[0012] Figure 2 Chromatogram of honeysuckle (peak 1: neochlorogenic acid, peak 2: chlorogenic acid, peak 3: cryptochlorogenic acid, peak 6: 3,5-di-O-caffeoylquinic acid, peak 7: 4,5-di-O-caffeoylquinic acid);

[0013] Figure 3 Chromatographic diagram of Scrophularia ningpoensis (peak 5: Angoloside C, peak 6: harpagoside, peak 7: cinnamic acid);

[0014] Figure 4 Chromatogram of Angelica sinensis (peak 2: chlorogenic acid, peak 4: ferulic acid, peak 5: ligustilide I, peak 6: ligustilide H, peak 7: ligustilide);

[0015] Figure 5 Chromatogram of licorice root (peak 3: glycyrrhizin, peak 5: isoglycyrrhizin, peak 6: glycyrrhizin, peak 8: glycyrrhizic acid);

[0016] Figure 6 Different wavelengths;

[0017] Figure 7 Different mobile phases for the Si Miao Yong An Tang reference sample;

[0018] Figure 8 Investigation of different column temperatures on the reference sample of Simiao Yong'an Decoction;

[0019] Figure 9Investigation of different flow rates on the reference sample of Simiao Yong'an Decoction;

[0020] Figure 10 Delayed investigation;

[0021] Figure 11 Examining the extraction method;

[0022] Figure 12 Investigation of extraction solvents;

[0023] Figure 13 Extraction time consideration;

[0024] Figure 14 Investigation of sample weight / solvent addition amount;

[0025] Figure 15 Chromatographic peak identification;

[0026] Figure 16 Specificity assessment;

[0027] Figure 17 Exploration using different instruments;

[0028] Figure 18 Investigations using different chromatographic columns;

[0029] Figure 19 Chromatogram of Comparative Example 1;

[0030] Figure 20 Chromatogram of Comparative Example 2;

[0031] Figure 21 Chromatogram of Comparative Example 3. Detailed Implementation

[0032] Example 1: Characteristic spectrum of the Si Miao Yong An Tang reference sample of the present invention

[0033] 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, 0.4% phosphoric acid solution as mobile phase B, a flow rate of 0.7 mL / min, and a column temperature of 25 °C. Gradient elution was performed according to the specifications in the table below. The detection wavelength was 276 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 5000.

[0034]

[0035] Preparation of reference solutions: Take appropriate amounts of chlorogenic acid and 3,5-di-O-caffeoylquinic acid reference standards, accurately weigh them, and add 50% methanol to prepare solutions containing 20 μg per mL.

[0036] Preparation of the test solution: Weigh approximately 0.5 g of this product accurately, place it in an Erlenmeyer flask, add 50 mL of 50% methanol accurately, reflux for 30 minutes, cool, add 50% methanol to make up the weight, shake well, filter, and collect the filtrate to obtain the test solution.

[0037] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0038] The chromatogram of the test sample should show 13 characteristic peaks, two of which should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the chlorogenic acid reference peak is S1, and the peak corresponding to the 3,5-di-O-caffeoylquinic acid reference peak is S2. Calculate the relative retention times of each characteristic peak (peaks 1-3) with peak S1, and calculate the relative retention times of each characteristic peak (peaks 4-13) with peak S2. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.73 (peak 1), 1.08 (peak 3), 0.75 (peak 4), 0.80 (peak 5), 0.97 (peak 6), 1.02 (peak 8), 1.04 (peak 9), 1.06 (peak 10), 1.41 (peak 11), 2.01 (peak 12), 2.31 (peak 13). (See...) Figure 1 )

[0039] Example 2: Detection method of honeysuckle medicinal material according to the present invention

[0040] Chromatographic conditions and system suitability test are the same as those for the Simiao Yong'an Decoction reference sample [Chromatographic conditions and system suitability test].

[0041] Preparation of reference solutions: Take appropriate amounts of chlorogenic acid and 3,5-di-O-caffeoylquinic acid reference standards, accurately weigh them, and add 50% methanol to prepare solutions containing 20 μg per mL.

[0042] Preparation of the test solution: Weigh approximately 0.5 g of this product (passed through a No. 3 sieve) accurately, place it in a stoppered conical flask, add 50 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25 mL of 50% methanol, sonicate (power 600 W, frequency 40 kHz) for 15 minutes, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0043] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0044] The chromatogram of the test sample should show 7 characteristic peaks, two of which should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the chlorogenic acid reference peak is S1, and the peak corresponding to the 3,5-di-O-caffeoylquinic acid reference peak is S2. Calculate the relative retention times of each characteristic peak (peaks 1-3) with peak S1, and calculate the relative retention times of each characteristic peak (peaks 4-7) with peak S2. These relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.73 (peak 1), 1.08 (peak 3), 0.91 (peak 4), 0.97 (peak 5), and 1.04 (peak 7). (See...) Figure 2 )

[0045] Example 3: Detection method of Scrophularia ningpoensis in this invention

[0046] Chromatographic conditions and system suitability test are the same as those for the Simiao Yong'an Decoction reference sample [Chromatographic conditions and system suitability test].

[0047] Preparation of the reference solution: Take an appropriate amount of Angoloside C reference standard, accurately weigh it, and add 50% methanol to prepare a solution containing 20 μg per 1 mL.

[0048] Preparation of the test solution: Weigh approximately 0.5 g of this product (passed through a No. 3 sieve) accurately, place it in a stoppered conical flask, add 25 mL of 50% methanol, sonicate (power 600 W, frequency 40 kHz) for 15 minutes, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0049] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0050] The chromatogram of the test sample should show 7 characteristic peaks, of which the peak corresponding to the Angoloside C reference standard is S. Calculate the relative retention times of each characteristic peak (peaks 1-7) and peak S; the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.17 (peak 1), 0.89 (peak 2), 0.91 (peak 3), 0.95 (peak 4), 1.26 (peak 6), and 1.40 (peak 7). (See...) Figure 3 )

[0051] Example 4: Detection method of Angelica sinensis in this invention

[0052] Chromatographic conditions and system suitability test are the same as those for the Simiao Yong'an Decoction reference sample [Chromatographic conditions and system suitability test].

[0053] Preparation of the reference solution: Take an appropriate amount of ligustrazine lactone I reference standard, accurately weigh it, and add 50% methanol to prepare a solution containing 20 μg per 1 mL.

[0054] Preparation of the test solution: Weigh approximately 0.5 g of this product (passed through a No. 3 sieve) accurately, place it in a stoppered conical flask, add 25 mL of 50% methanol, sonicate (power 600 W, frequency 40 kHz) for 15 minutes, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0055] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0056] The chromatogram of the test sample should show 7 characteristic peaks, of which the peak corresponding to the reference standard ligustrazine I is S. Calculate the relative retention times of each characteristic peak (peaks 1-7) and peak S; the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.27 (peak 1), 0.30 (peak 2), 0.40 (peak 3), 0.72 (peak 4), 1.05 (peak 6), and 2.18 (peak 7). (See...) Figure 4 )

[0057] Example 5: Detection method of licorice medicinal materials according to the present invention

[0058] Chromatographic conditions and system suitability test are the same as those for the Simiao Yong'an Decoction reference sample [Chromatographic conditions and system suitability test].

[0059] Preparation of the reference solution: Take an appropriate amount of glycyrrhizin reference standard, accurately weigh it, and add 50% methanol to prepare a solution containing 20 μg per 1 mL.

[0060] Preparation of the test solution: Weigh approximately 0.5 g of this product (passed through a No. 3 sieve) accurately, place it in a stoppered conical flask, add 50 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25 mL of 50% methanol, sonicate (power 600 W, frequency 40 kHz) for 15 minutes, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0061] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0062] The chromatogram of the test sample should show 8 characteristic peaks, of which the peak corresponding to the glycyrrhizin reference standard is S. Calculate the relative retention times of each characteristic peak (peaks 1-8) and peak S; these relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.45 (peak 1), 0.49 (peak 2), 1.27 (peak 4), 1.37 (peak 5), 1.56 (peak 6), 2.09 (peak 7), and 2.51 (peak 8). (See...) Figure 5 )

[0063] Example 6: Screening Experiment of Feature Map Conditions of the Invention

[0064] 1. Experimental Instruments and Materials

[0065] High performance liquid chromatographs: Agilent high performance liquid chromatographs, Waters high performance liquid chromatographs, Shimadzu high performance liquid chromatographs;

[0066] Electronic balances: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instruments Ltd.);

[0067] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0068] Ultrasonic cleaner: KQ5200DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0069] Column: Agilent ZORBAX Extend-C18 250×4.6mm, 5μm;

[0070] Chromatographic column: Waters 250×4.6mm, 5μm;

[0071] Column: Agilent ZORBAX Eclipse XDB-C18 250×4.6mm, 5μm.

[0072] Methanol, acetonitrile, formic acid, and phosphoric acid were of chromatographic grade; water was ultrapure water; and all other reagents were of analytical grade.

[0073] Chlorogenic acid (China National Institutes for Food and Drug Control, batch number: 110753-202119, content calculated as 96.3%);

[0074] 3,5-O-dicaffeoylquinic acid (China National Institutes for Food and Drug Control, batch number: 111782-202309, content calculated as 95.0%);

[0075] 4,5-O-dicaffeoylquinic acid (China National Institutes for Food and Drug Control, batch number: 111894-202406, content calculated as 95.9%);

[0076] Harpagoside (batch number: 111730-202110, China National Institutes for Food and Drug Control, content calculated as 96.8%);

[0077] Ferulic acid (batch number: 110773-202316, China National Institutes for Food and Drug Control, content calculated as 99.3%);

[0078] Ligusticolone (batch number: 111737-202311, China National Institutes for Food and Drug Control);

[0079] Glycyrrhizin (batch number: 111610-202209, China National Institutes for Food and Drug Control, content calculated as 95.2%);

[0080] Ammonium glycyrrhizate (batch number: 110731-202122, China National Institutes for Food and Drug Control, content calculated as 94.4%);

[0081] Honeysuckle reference material (China National Institutes for Food and Drug Control, batch number: 121060-201608);

[0082] Scrophularia ningpoensis reference material (China National Institutes for Food and Drug Control, batch number: 121008-202110);

[0083] Angelica sinensis reference material (China National Institutes for Food and Drug Control, batch number: 120927-202118);

[0084] Licorice reference material (China National Institutes for Food and Drug Control, batch number: 120904-202021);

[0085] Simiao Yong'an Decoction Standard Sample: BT-01;

[0086] Honeysuckle Decoction: Single JYH-BT-01;

[0087] Scrophularia Decoction: Single XS-BT-01;

[0088] Dang Gui Biao Tang: Single DG-BT-01;

[0089] Licorice Decoction: Single GC-BT-01;

[0090] Missing Honeysuckle Decoction: Missing JYH-BT-01;

[0091] Missing Scrophularia Decoction: Missing XS-BT-01;

[0092] Missing Angelica Root Decoction: Missing DG-BT-01;

[0093] Deficiency of Licorice Decoction: Deficiency of GC-BT-01;

[0094] Honeysuckle medicinal material: JYH-YC-01;

[0095] Scrophularia ningpoensis herb: XS-YC-01;

[0096] Angelica sinensis medicinal material: DG-YC-01;

[0097] Licorice herb: GC-YC-01;

[0098] 2. Determination of detection wavelength

[0099] Based on the above-planned experimental conditions, a diode array detector was used to perform a full-band scan of the test solution, and chromatograms of the test solution were extracted at wavelengths of 210 nm, 230 nm, 250 nm, 276 nm, 290 nm, 310 nm, and 330 nm. For example... Figure 6 As shown.

[0100] The results showed that honeysuckle had many impurity peaks at wavelengths of 210, 230, and 250 nm, and the separation of characteristic peaks was poor. Scrophularia had few characteristic peaks at wavelengths of 310 and 330 nm. Angelica had no characteristic peaks at wavelengths of 210, 230, 310, and 330 nm for 50 minutes, and the separation of characteristic peaks at wavelength of 290 nm for 98 minutes was poor. Licorice had many impurity peaks at wavelengths of 210, 230, and 250 nm, and the separation of characteristic peaks was poor. The information of characteristic peaks at wavelengths of 290, 310, and 330 nm was also poor. In summary, honeysuckle, scrophularia, angelica, licorice, and the Simiao Yong'an Decoction reference sample all showed a large amount of chromatographic peak information and a more stable chromatographic baseline at a detection wavelength of 276 nm. Therefore, the detection wavelength was determined to be 276 nm.

[0101] 3 Investigation of different mobile phases

[0102] Based on the above-specified experimental conditions, the separation effects of nine different mobile phases were investigated: acetonitrile-0.4% phosphoric acid, methanol-0.4% phosphoric acid, acetonitrile-water, acetonitrile-0.4% glacial acetic acid, acetonitrile-0.4% formic acid, acetonitrile-0.5% phosphoric acid, acetonitrile-0.3% phosphoric acid, acetonitrile-0.2% phosphoric acid, and acetonitrile-0.1% phosphoric acid. Figure 7 As shown.

[0103] The results showed that the chromatogram baseline was more stable, with more chromatographic peaks and uniform distribution when using acetonitrile-0.4% phosphoric acid. Therefore, acetonitrile-0.4% phosphoric acid was selected as the mobile phase for the determination of the characteristic chromatogram of Simiao Yong'an Decoction reference sample.

[0104] 4. Column temperature investigation

[0105] Based on the above-planned experimental conditions, the results were investigated at column temperatures of 20℃, 25℃, and 30℃. Figure 8 As shown.

[0106] The results showed that at a column temperature of 30℃, peaks 8 and 9 were not separated; at a column temperature of 20℃, peak 9 was missing, and the positions of peaks 11 and 12 were different from those at column temperatures of 25℃ and 30℃; at a column temperature of 25℃, the separation of each chromatographic peak was good, so the column temperature was tentatively set at 25℃.

[0107] 5. Flow velocity investigation

[0108] Based on the above-established experimental conditions, flow rates of 0.8 mL / min, 0.7 mL / min, 0.6 mL / min, and 0.5 mL / min were investigated. Figure 9 As shown.

[0109] The results showed that peak 10 was missing at a flow rate of 0.8 mL / min; peaks 4 and 11 were missing at a flow rate of 0.6 mL / min; peak 4 was missing at a flow rate of 0.5 mL / min, peak 5 was severely affected by peak engulfment, and the positions of peaks 11 and 12 were opposite to those at other flow rates; at a flow rate of 0.7 mL / min, the resolution of each chromatographic peak was good, so the flow rate was tentatively set at 0.7 mL / min.

[0110] 6. Delayed testing

[0111] Based on the above-planned experimental conditions, the chromatogram acquisition time was extended to 220 minutes. For example... Figure 10 As shown.

[0112] The results showed that the sample had virtually no chromatographic peaks after 110 minutes, so the sample detection time was set at 110 minutes.

[0113] 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, 0.4% phosphoric acid solution as mobile phase B, a flow rate of 0.7 mL / min, and a column temperature of 25 °C. Gradient elution was performed according to the specifications in the table below. The detection wavelength was 276 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 5000.

[0114]

[0115]

[0116] 7. Examination of extraction methods

[0117] Take approximately 0.5g of this product (batch number: BT-01) as the reference sample for Simiao Yong'an Decoction. Accurately weigh the sample and place it in a stoppered conical flask. Add 50mL of 50% methanol and heat under reflux and sonicate (power 600W, frequency 40kHz) for 30 minutes respectively. Cool, shake well, filter, and collect the filtrate to obtain the sample.

[0118] To obtain the honeysuckle herb, take approximately 0.5g of this product (batch number: JYH-YC-01), accurately weigh it, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 50mL of 50% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the subsequent filtrate; or take approximately 0.5g of this product (batch number: JYH-YC-01), accurately weigh it, place it in a stoppered conical flask, add 50mL of 50% methanol, heat under reflux and sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the subsequent filtrate.

[0119] For the preparation of Scrophularia ningpoensis, take approximately 0.5g of this product (batch number: XS-YC-01), accurately weigh it, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 50mL of 50% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the subsequent filtrate; or take approximately 0.5g of this product (batch number: XS-YC-01), accurately weigh it, place it in a stoppered conical flask, add 50mL of 50% methanol, heat under reflux and sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the subsequent filtrate.

[0120] Take approximately 0.5g of Angelica sinensis (batch number: DG-YC-01), accurately weigh it, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 50mL of 50% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the subsequent filtrate to obtain the product; take approximately 0.5g of Angelica sinensis (batch number: DG-YC-01), accurately weigh it, place it in a stoppered conical flask, add 50mL of 50% methanol, heat under reflux and sonicate (power 600W, frequency 40kHz) for 30 minutes respectively, cool, shake well, filter, and collect the subsequent filtrate to obtain the product;

[0121] For the preparation of licorice root, take approximately 0.5g of this product (batch number: GC-YC-01), accurately weigh it, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 50mL of 50% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the subsequent filtrate; or take approximately 0.5g of this product (batch number: GC-YC-01), accurately weigh it, place it in a stoppered conical flask, add 50mL of 50% methanol, heat under reflux and sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the subsequent filtrate.

[0122] The results are as follows Figure 11 .

[0123] The results showed that the chromatographic peaks of the extraction methods for the Simiao Yong'an Decoction reference sample, Scrophularia ningpoensis, and Angelica sinensis were consistent, and ultrasonic extraction was simpler. Therefore, ultrasonic extraction was chosen for subsequent studies. Peak 5 of Lonicera japonica significantly increased after decoction, so decoction-ultrasound was chosen for subsequent studies. The baseline of Glycyrrhiza uralensis was stable with fewer impurities after 90-110 minutes of decoction, so decoction-ultrasound was determined as the extraction method.

[0124] 8. Investigation of extraction solvents

[0125] Take approximately 0.5g of the Simiao Yong'an Decoction reference sample (BT-01), accurately weigh it, place it in a stoppered conical flask, add 50mL of water, 30% methanol, 50% methanol, 70% methanol, methanol, and ethanol respectively, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the final product.

[0126] Take approximately 0.5g of honeysuckle (batch number: JYH-YC-01), accurately weigh it, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 50mL each of water, 30% methanol, 50% methanol, 70% methanol, methanol, and ethanol, and sonicate (600W power, 40kHz frequency) for 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the final product.

[0127] Take approximately 0.5g of this product (batch number: XS-YC-01) of Scrophularia ningpoensis, accurately weigh it, place it in a stoppered conical flask, add 50mL of water, 30% methanol, 50% methanol, 70% methanol, methanol, and ethanol respectively, and sonicate (power 600W, frequency 40kHz) for 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the product.

[0128] Take approximately 0.5g of Angelica sinensis (batch number: DG-YC-01), accurately weigh it, place it in a stoppered conical flask, add 50mL each of water, 30% methanol, 50% methanol, 70% methanol, methanol, and ethanol, and sonicate (600W power, 40kHz frequency) for 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the final product.

[0129] Take approximately 0.5g of this product (batch number: GC-YC-01) of licorice root, accurately weigh it, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 50mL each of water, 30% methanol, 50% methanol, 70% methanol, methanol, and ethanol, and sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product.

[0130] like Figure 12 As shown.

[0131] The results showed that the peak shapes of each characteristic peak were better when using 50% methanol, so 50% methanol was chosen for further research.

[0132] 9. Extraction time assessment

[0133] Take approximately 0.5g of the Simiao Yong'an Decoction reference sample (BT-01), accurately weigh it, place it in a stoppered conical flask, add 50mL of 50% methanol, and sonicate it (power 600W, frequency 40kHz) for 15, 30, and 45 minutes. Cool it, shake it well, filter it, and collect the filtrate to obtain the final product.

[0134] Take approximately 0.5g of honeysuckle (batch number: JYH-YC-01), accurately weigh it, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 50mL of 50% methanol, and sonicate (power 600W, frequency 40kHz) for 15, 30, and 45 minutes, cool, shake well, filter, and collect the filtrate to obtain the honeysuckle.

[0135] Take approximately 0.5g of this product (batch number: XS-YC-01) of Scrophularia ningpoensis, accurately weigh it, place it in a stoppered conical flask, add 50mL of 50% methanol, and sonicate it (power 600W, frequency 40kHz) for 15, 30 and 45 minutes respectively. Cool it, shake it well, filter it, and take the filtrate to obtain the product.

[0136] Take approximately 0.5g of Angelica sinensis (batch number: DG-YC-01), accurately weigh it, place it in a stoppered conical flask, add 50mL of 50% methanol, and sonicate it (power 600W, frequency 40kHz) for 15, 30, and 45 minutes respectively. Cool it, shake it well, filter it, and collect the filtrate to obtain the final product.

[0137] Take approximately 0.5g of this product (batch number: GC-YC-01) of licorice root, accurately weigh it, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 50mL of 50% methanol, and sonicate (power 600W, frequency 40kHz) for 15, 30, and 45 minutes, cool, shake well, filter, and collect the filtrate to obtain the product.

[0138] like Figure 13 As shown.

[0139] The results showed that the peak shapes of each characteristic peak were suitable after 15 minutes of extraction. Therefore, the extraction time for the test sample was determined to be 15 minutes.

[0140] 10. Sample weight / solvent addition amount investigation

[0141] Take approximately 0.2 and 0.5 g of the Simiao Yong'an Decoction reference sample (BT-01), accurately weigh them, place them in a stoppered conical flask, add 15, 25, and 50 mL of 50% methanol, sonicate (power 600W, frequency 40kHz) for 15 minutes, cool, shake well, filter, and collect the filtrate to obtain the final product.

[0142] Take approximately 0.2 or 0.5 g of honeysuckle (batch number: JYH-YC-01), accurately weigh it, place it in a stoppered conical flask, add 50 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 15, 25, and 50 mL of 50% methanol, and sonicate (600 W power, 40 kHz frequency) for 15 minutes, cool, shake well, filter, and collect the filtrate to obtain the honeysuckle extract.

[0143] Take approximately 0.2 and 0.5 g of this product (batch number: XS-YC-01), accurately weigh it, place it in a stoppered conical flask, add 15, 25, and 50 mL of 50% methanol, sonicate (power 600W, frequency 40kHz) for 15 minutes, cool, shake well, filter, and collect the filtrate to obtain the product.

[0144] Take approximately 0.2 and 0.5 g of Angelica sinensis (batch number: DG-YC-01), accurately weigh them, place them in a stoppered conical flask, add 15, 25, and 50 mL of 50% methanol, sonicate (600W power, 40kHz frequency) for 15 minutes, cool, shake well, filter, and collect the filtrate to obtain the final product.

[0145] Take approximately 0.2 or 0.5 g of this product (batch number: GC-YC-01) of licorice root, accurately weigh it, place it in a stoppered conical flask, add 50 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 15, 25, and 50 mL of 50% methanol, and sonicate (power 600 W, frequency 40 kHz) for 15 minutes, cool, shake well, filter, and collect the filtrate to obtain the product.

[0146] like Figure 14 As shown.

[0147] The results showed that the characteristic peaks of the Simiao Yong'an Decoction standard sample were appropriate at 0.2g and 25ml. Therefore, the sample weight was determined to be 0.2g and the solvent volume was determined to be 25ml. The characteristic peaks of the honeysuckle, scrophularia, angelica, and licorice herbs were appropriate at 0.5g and 25ml. Therefore, the sample weight was determined to be 0.5g and the solvent volume was determined to be 25ml.

[0148] 11. Determine the method for preparing the test sample.

[0149] Take 0.2g of this product as the reference sample for Simiao Yong'an Decoction. Accurately weigh it and place it in a stoppered conical flask. Add 25mL of 50% methanol and sonicate (600W power, 40kHz frequency) for 15 minutes. Cool, shake well, filter, and collect the filtrate to obtain the sample.

[0150] Take about 0.5g of honeysuckle (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25mL of 50% methanol, sonicate (power 600W, frequency 40kHz) for 15 minutes, cool, shake well, filter, and collect the filtrate to obtain the honeysuckle.

[0151] Take approximately 0.5g of the Scrophularia ningpoensis (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, add 25mL of 50% methanol, sonicate (600W power, 40kHz frequency) for 15 minutes, cool, shake well, filter, and collect the filtrate to obtain the product.

[0152] Take approximately 0.5g of Angelica sinensis (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, add 25mL of 50% methanol, sonicate (600W power, 40kHz frequency) for 15 minutes, cool, shake well, filter, and collect the filtrate to obtain the final product.

[0153] Take about 0.5g of this product (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25mL of 50% methanol, sonicate (power 600W, frequency 40kHz) for 15 minutes, cool, shake well, filter, and collect the filtrate to obtain the product.

[0154] 12. Methodological Examination

[0155] 12.1 Chromatographic peak identification and specificity investigation

[0156] Preparation of test solutions: Prepare test solutions of honeysuckle, scrophularia, angelica, licorice and the Simiao Yong'an Decoction reference sample according to the experimental conditions proposed above.

[0157] Preparation of reference solutions: Take appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, ferulic acid, glycyrrhizin, 3,5-O-dicaffeoylquinic acid, angoloside C, 4,5-O-dicaffeoylquinic acid, ligusticatin I, cinnamic acid, glycyrrhizic acid, and ligustilide reference standards, accurately weigh them, and add 50% methanol to prepare individual solutions containing 20 μg of the above reference standards per ml.

[0158] Preparation of reference solutions: Accurately weigh 0.5g of honeysuckle reference material, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25mL of 50% methanol, sonicate (600W power, 40kHz frequency) for 15 minutes, cool, shake well, filter, and collect the filtrate as the honeysuckle reference solution. Accurately weigh 0.5g of scrophularia reference material, place it in a stoppered conical flask, add 25mL of 50% methanol, sonicate (600W power, 40kHz frequency) for 15 minutes, cool, shake well, filter, and collect the filtrate as the scrophularia reference solution. Accurately weigh 0.5g of Angelica sinensis reference material and place it in a stoppered conical flask. Add 25mL of 50% methanol and sonicate (600W, 40kHz) for 15 minutes. Cool, shake well, filter, and collect the filtrate as the reference solution for Angelica sinensis. Accurately weigh 0.5g of Glycyrrhiza uralensis reference material and place it in a stoppered conical flask. Add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25mL of 50% methanol, sonicate (600W, 40kHz) for 15 minutes, cool, shake well, filter, and collect the filtrate as the reference solution for Glycyrrhiza uralensis.

[0159] Preparation of negative control solutions: According to the experimental conditions proposed above, prepare negative control solutions for single honeysuckle decoction, single scrophularia decoction, single angelica decoction, single licorice decoction, decoction lacking honeysuckle, decoction lacking scrophularia, decoction lacking angelica, and decoction lacking licorice.

[0160] The characteristic spectral peaks of the Si Miao Yong An Tang reference sample, honeysuckle, scrophularia, angelica, and licorice were located. For example... Figure 15 , Figure 16 As shown.

[0161] From the chromatographic peak identification diagram, the characteristic peaks of the Simiao Yong'an Decoction reference sample are as follows: Peak 1 is neochlorogenic acid, Peak 2 is chlorogenic acid, Peak 3 is cryptochlorogenic acid, Peak 4 is ferulic acid, Peak 5 is glycyrrhizin, Peak 7 is 3,5-di-O-caffeoylquinic acid, Peak 8 is angoloside C, Peak 9 is 4,5-di-O-caffeoylquinic acid, Peak 10 is ligusticoside I, Peak 11 is cinnamic acid, Peak 12 is glycyrrhizic acid, and Peak 13 is ligustilide.

[0162] From the chromatographic peak identification diagram, the characteristic peaks of honeysuckle are: peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 6 is 3,5-O-dicaffeoylquinic acid, and peak 7 is 4,5-O-dicaffeoylquinic acid.

[0163] From the chromatographic peak identification diagram, the characteristic peaks of Scrophularia ningpoensis are: peak 5 is Angoloside C, peak 6 is harpagoside, and peak 7 is cinnamic acid.

[0164] From the chromatographic peak identification diagram, the characteristic peaks of Angelica sinensis are: peak 2 is chlorogenic acid, peak 4 is ferulic acid, peak 5 is ligustilide I, peak 6 is ligustilide H, and peak 7 is ligustilide.

[0165] Based on the chromatographic peak identification, the characteristic peaks of licorice in the chromatogram are: peak 3 is glycyrrhizin, peak 5 is isoglycyrrhizin, peak 6 is glycyrrhizin, and peak 8 is glycyrrhizic acid.

[0166] From the specific graph, the characteristic peaks of the Simiao Yong'an Decoction reference sample, peaks 1, 2, 3, 6, 7, and 9 are derived from honeysuckle, peaks 8 and 11 from scrophularia, peaks 4, 10, and 13 from angelica, and peaks 5 and 12 from licorice. This method can effectively distinguish the honeysuckle, scrophularia, angelica, and licorice components in Simiao Yong'an Decoction.

[0167] 12.2 Precision Test

[0168] Take the following test solutions: Simiao Yong'an Decoction (batch number: BT-01), honeysuckle (batch number: JYH-YC-01), scrophularia (batch number: XS-YC-01), angelica (batch number: DG-YC-01), and licorice (batch number: GC-YC-01). Inject 10 μL of each solution six times consecutively according to the proposed experimental method. Calculate the retention time and peak area of ​​each characteristic peak. See Tables 1-12.

[0169] Table 1. Precision Study of the Si Miao Yong An Tang Reference Samples - Relative Retention Time

[0170]

[0171] Table 2 Precision Study of Simiao Yong'an Decoction Reference Samples - Relative Retention Time

[0172]

[0173] Table 3 Precision Study of Honeysuckle Medicinal Material - Relative Retention Time

[0174]

[0175]

[0176] Table 4. Precision Study of Scrophularia ningpoensis - Relative Retention Time

[0177]

[0178] Table 5 Precision Study of Angelica sinensis - Relative Retention Time

[0179]

[0180] Table 6 Precision Study of Licorice Herbs - Relative Retention Time

[0181]

[0182] Table 7 Precision Study of Simiao Yong'an Decoction Reference Samples - Relative Peak Area

[0183]

[0184]

[0185] Table 8 Precision Study of Simiao Yong'an Decoction Reference Samples - Relative Peak Area

[0186]

[0187] Table 9 Precision Study of Honeysuckle Medicinal Material - Relative Peak Area

[0188]

[0189] Table 10 Precision Study of Scrophularia ningpoensis - Relative Peak Area

[0190]

[0191]

[0192] Table 11 Precision Study of Angelica sinensis - Relative Peak Area

[0193]

[0194] Table 12 Precision Study of Licorice Herbs - Relative Peak Area

[0195]

[0196] The results show that the instrument has good precision.

[0197] 12.3 Repeatability Test

[0198] Take six portions of the following test solutions: Simiao Yong'an Decoction (batch number: BT-01), honeysuckle (batch number: JYH-YC-01), scrophularia (batch number: XS-YC-01), angelica (batch number: DG-YC-01), and licorice (batch number: GC-YC-01). Prepare and determine the solutions according to the proposed experimental method. See Tables 13-24.

[0199] Table 13 Repeatability Study of Simiao Yong'an Decoction Reference Samples - Relative Retention Time

[0200]

[0201]

[0202] Table 14 Repeatability Study of Simiao Yong'an Decoction Reference Samples - Relative Retention Time

[0203]

[0204] Table 15 Repeatability Study of Honeysuckle Medicinal Material - Relative Retention Time

[0205]

[0206] Table 16 Repeatability Study of Scrophularia ningpoensis - Relative Retention Time

[0207]

[0208]

[0209] Table 17 Repeatability Study of Angelica sinensis - Relative Retention Time

[0210]

[0211] Table 18 Repeatability Study of Licorice Herbs - Relative Retention Time

[0212]

[0213] Table 19 Repeatability Study of Simiao Yong'an Decoction Reference Samples - Relative Peak Area

[0214]

[0215] Table 20 Repeatability Study of Simiao Yong'an Decoction Reference Samples - Relative Peak Area

[0216]

[0217]

[0218] Table 21 Repeatability Study of Honeysuckle Medicinal Material - Relative Peak Area

[0219]

[0220] Table 22 Repeatability Study of Scrophularia ningpoensis - Relative Peak Area

[0221]

[0222] Table 23 Repeatability Study of Angelica sinensis - Relative Peak Area

[0223]

[0224]

[0225] Table 24 Repeatability Study of Licorice Herbs - Relative Peak Area

[0226]

[0227] The results show that the method has good repeatability.

[0228] 12.4 Intermediate Precision Examination

[0229] 12.4.1 Investigation with different instruments

[0230] Based on the above-planned experimental conditions, the following test solutions were taken: Simiao Yong'an Decoction (batch number: BT-01), honeysuckle (batch number: JYH-YC-01), scrophularia (batch number: XS-YC-01), angelica (batch number: DG-YC-01), and licorice (batch number: GC-YC-01). These solutions were then analyzed using Agilent, Waters, and Shimadzu high-performance liquid chromatographs, respectively. Figure 17 As shown in Tables 25-36.

[0231] Table 25 Instrument Durability Study of Simiao Yong'an Decoction Reference Samples - Relative Retention Time

[0232]

[0233] Table 26 Instrument Durability Study of Simiao Yong'an Decoction Reference Samples - Relative Retention Time

[0234]

[0235] Table 27 Instrument Durability Study of Honeysuckle Medicinal Material - Relative Retention Time

[0236]

[0237] Table 28 Instrument Durability Study of Scrophularia ningpoensis Medicinal Material - Relative Retention Time

[0238]

[0239] Table 29 Instrument Durability Study of Angelica sinensis - Relative Retention Time

[0240]

[0241] Table 30 Durability Study of Licorice Herbal Instruments - Relative Retention Time

[0242]

[0243] Table 31 Instrument durability test of Simiao Yong'an Decoction reference sample - relative peak area

[0244]

[0245] Table 32 Instrument Durability Study of Simiao Yong'an Decoction Reference Samples - Relative Peak Area

[0246]

[0247] Table 33 Instrument Durability Study of Honeysuckle Medicinal Material - Relative Peak Area

[0248]

[0249] Table 34 Instrument Durability Study of Scrophularia ningpoensis - Relative Peak Area

[0250]

[0251] Table 35 Instrument Durability Study of Angelica sinensis - Relative Peak Area

[0252]

[0253] Table 36 Instrument Durability Test of Licorice Herbs - Relative Peak Area

[0254]

[0255] 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 less than 5.0%.

[0256] 12.4.2 Investigations by different personnel and at different times

[0257] Based on the above-established experimental conditions, different personnel (A and B) collected test solutions of Simiao Yong'an Decoction (batch number: BT-01), honeysuckle (batch number: JYH-YC-01), scrophularia (batch number: XS-YC-01), angelica (batch number: DG-YC-01), and licorice (batch number: GC-YC-01) at different times (T1 and T2) for determination. The results are shown in Tables 37 and 48.

[0258] Table 37. Personnel and Time-Based Investigation of Simiao Yong'an Decoction Reference Samples - Relative Retention Time

[0259]

[0260] Table 38. Personnel and Time-Based Investigation of Simiao Yong'an Decoction Reference Samples - Relative Retention Time

[0261]

[0262] Table 39. Personnel and Time Survey of Honeysuckle Medicinal Materials - Relative Retention Time

[0263]

[0264] Table 40. Personnel and Time-Related Retention Time of Scrophularia ningpoensis Medicinal Materials

[0265]

[0266] Table 41. Personnel and Time Survey of Angelica sinensis Medicinal Materials - Relative Retention Time

[0267]

[0268] Table 42 Personnel and Time Survey of Licorice Herbs - Relative Retention Time

[0269]

[0270] Table 43. Personnel and Time-Based Investigation of Simiao Yong'an Decoction Reference Samples - Relative Peak Area

[0271]

[0272] Table 44. Personnel and Time-Based Investigation of the Si Miao Yong An Tang Reference Sample - Relative Peak Area

[0273]

[0274] Table 45. Personnel and Time-Based Investigation of Honeysuckle Medicinal Herbs - Relative Peak Area

[0275]

[0276]

[0277] Table 46. Personnel and Time Survey of Scrophularia ningpoensis Medicinal Materials - Relative Peak Area

[0278]

[0279] Table 47 Personnel and Time Survey of Angelica sinensis Medicinal Materials - Relative Peak Area

[0280]

[0281] Table 48 Personnel and Time Survey of Licorice Herbs - Relative Peak Area

[0282]

[0283] 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 less than 2.0%, indicating good method stability.

[0284] 13 Durability Test

[0285] 13.1 Column robustness test

[0286] Based on the above-planned experimental conditions, the following tests were conducted: Column 1: Agilent ZORBAX Extend-C18 250×4.6mm; Column 2: Agilent ZORBAX Eclipse XDB-C18 250×4.6mm, 5μm; Column 3: Waters... The sample size was 250 × 4.6 mm, with a diameter of 5 μm, and was investigated. For example... Figure 18 As shown in Tables 49-60.

[0287] Table 49. Column robustness study of Simiao Yong'an Decoction reference sample - relative retention time

[0288]

[0289] Table 50. Column robustness study of Simiao Yong'an Decoction reference sample - relative retention time

[0290]

[0291] Table 51. Chromatographic column durability study of honeysuckle medicinal material - relative retention time

[0292]

[0293] Table 52. Column durability study of Scrophularia ningpoensis - relative retention time

[0294]

[0295] Table 53. Column durability study of Angelica sinensis - relative retention time

[0296]

[0297]

[0298] Table 54. Column durability study of licorice medicinal materials - relative retention time

[0299]

[0300] Table 55. Column robustness test of Simiao Yong'an Decoction reference sample - relative peak area

[0301]

[0302] Table 56. Column robustness test of Simiao Yong'an Decoction reference sample - relative peak area

[0303]

[0304] Table 57. Chromatographic column durability study of honeysuckle medicinal material - relative peak area

[0305]

[0306] Table 58. Chromatographic column durability study of Scrophularia ningpoensis - relative peak area

[0307]

[0308] Table 59. Column durability study of Angelica sinensis - relative peak area

[0309]

[0310] Table 60. Column durability study of licorice medicinal materials - relative peak area

[0311]

[0312] The results showed that the characteristic peaks exhibited good durability on different chromatographic columns when the samples were detected using the three types of columns mentioned above.

[0313] 13.2 Stability Test

[0314] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h, respectively. As shown in Tables 61-72.

[0315] Table 61 Stability-Relative Retention Time of Simiao Yong'an Decoction Reference Samples

[0316]

[0317] Table 62 Stability and Relative Retention Time of Simiao Yong'an Decoction Reference Samples

[0318]

[0319] Table 63 Stability of Honeysuckle Medicinal Material - Relative Retention Time

[0320]

[0321] Table 64 Stability and Relative Retention Time of Scrophularia ningpoensis

[0322]

[0323] Table 65 Stability of Angelica sinensis - Relative Retention Time

[0324]

[0325]

[0326] Table 66 Stability of Licorice Herbs - Relative Retention Time

[0327]

[0328] Table 67 Stability of Simiao Yong'an Decoction Reference Samples - Relative Peak Area

[0329]

[0330] Table 68 Stability of Simiao Yong'an Decoction Reference Samples - Relative Peak Area

[0331]

[0332]

[0333] Table 69 Stability of Honeysuckle Medicinal Material - Relative Peak Area

[0334]

[0335] Table 70 Stability of Scrophularia ningpoensis - Relative Peak Area

[0336]

[0337] Table 71 Stability of Angelica sinensis - Relative Peak Area

[0338]

[0339] Table 72 Stability of Licorice Herbs - Relative Peak Area

[0340]

[0341]

[0342] The results showed that the RSD of the relative retention time of each characteristic peak was less than 2.5%, indicating that the sample solution was relatively stable within 24 hours.

[0343] In summary, all characteristic peaks are incorporated into the standard.

[0344] Comparative Example 1 refers to Bu Hongpeng, “Research on Preparation Process and Quality Standard of Simiao Yong'an Granules” (Bu Hongpeng. Research on Preparation Process and Quality Standard of Simiao Yong'an Granules [D]. Shandong University of Traditional Chinese Medicine, 2016.)

[0345] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm). Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in the table below. Detection wavelengths: 325 nm (chlorogenic acid, ferulic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid), 280 nm (harpagoside), 250 nm (glycyrrhizic acid); flow rate: 1.0 ml / min; column temperature: 40 ℃.

[0346]

[0347] For the baseline sample of Simiao Yong'an Decoction, the identification of honeysuckle, scrophularia, angelica, and licorice herbs is as follows: Figure 19 :

[0348] The method showed poor separation of the Si Miao Yong An Tang reference sample within 50-78 minutes, failing to effectively identify it; honeysuckle, scrophularia, and angelica showed few characteristic peak information and could not be used for the characteristic identification of single honeysuckle, scrophularia, and angelica; angelica and licorice showed a large amount of characteristic peak information within 80-95 minutes but were not identified and separated.

[0349] Comparative Example 2

[0350] Refer to Zhang Xing's "Study on the decoction process of Simiao Yong'an Decoction" (Zhang Xing, Lai Wenjing, Lin Xia, et al. Box-Behnken design-response surface methodology optimization of the classic formula Simiao Yong'an Decoction [J]. Chinese Traditional and Herbal Drugs, 2023, 54(10):3109-3119.)

[0351] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm). Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in the table below. Detection wavelength: 273 nm; flow rate: 1.0 ml / min; column temperature: 38 ℃.

[0352]

[0353]

[0354] Identification of the reference sample of Simiao Yong'an Decoction Figure 20 .

[0355] The method failed to separate the peak at 20 minutes and the separation was poor at 40-54 minutes, thus it could not effectively identify the Simiao Yong'an Decoction reference sample.

[0356] Comparative Example 3 references the "Quality Standard for Honeysuckle Formula Granules" (National Pharmacopoeia Commission).

[0357] 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, 0.4% phosphoric acid solution as mobile phase B, a flow rate of 1.0 mL / min, and a column temperature of 35 °C. Gradient elution was performed according to the specifications in the table below. The detection wavelength was 350 nm. The theoretical plate number, calculated based on the luteolin peak, should be no less than 2000.

[0358]

[0359] Identification of the reference sample of Simiao Yong'an Decoction Figure 21 .

[0360] This method fails to capture all the information of the Simiao Yong'an Decoction reference sample.

Claims

1. A method for constructing a feature map of Simiao Yong'an Decoction, characterized by comprising the following steps: a. Preparation of the test solution: The Simiao Yong'an Decoction was taken as a test sample, dissolved and extracted with 50% methanol aqueous solution to obtain the test sample solution; b. The test solution was analyzed by high performance liquid chromatography to obtain the characteristic chromatogram of Simiao Yong'an Decoction; The characteristic chromatographic conditions for the Simiao Yong'an Decoction are as follows: The chromatographic column was a C18 column; acetonitrile was used as mobile phase A, and 0.4% phosphoric acid solution was used as mobile phase B, with gradient elution. The elution conditions were as follows: The C18 column has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; the flow rate is 0.7 ml per minute; the column temperature is 25 °C; the detection wavelength is 276 nm; and the theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 5000. It also includes the preparation of the reference solution; the method for preparing the reference solution is as follows: Prepare a reference solution by adding methanol aqueous solution to the reference standard; the reference standard is chlorogenic acid and 3,5-di-O-caffeoylquinic acid. The characteristic spectrum contains 13 characteristic peaks. Peak S1 corresponds to the chlorogenic acid reference standard, and peak S2 corresponds to the 3,5-di-O-caffeoylquinic acid reference standard. The relative retention times of each characteristic peak (peaks 1-3) with peak S1 were calculated, and the relative retention times of each characteristic peak (peaks 4-13) with peak S2 were calculated. The relative retention times of each characteristic peak are as follows: peak 1: 0.73, peak 3: 1.08, peak 4: 0.75, peak 5: 0.80, peak 6: 0.97, peak 8: 1.02, peak 9: 1.04, peak 10: 1.06, peak 1 ...12: 0.73, peak 3: 1.04, peak 10: 0.06, peak 11: 0.73, peak 12: 0.73, peak 13: 0.74, peak 14: 0.75, peak 15: 0.80, peak 16: 0.97, peak 17: 0.97, peak 18: 0.97, peak 19: 0.97, peak 10: 0.97, peak 11: 0.97, peak 12: 0.97, peak 13: 0.97, peak 14: 0.97, peak 15: 0.97, peak 16: 0.97, peak 17: 0.97, peak 18: 0 1.41, Peak 12: 2.01, Peak 13: 2.31; relative retention time fluctuates within ±10%; The characteristic peaks are as follows: Peak 1: Neochlorogenic acid, Peak 2: Chlorogenic acid, Peak 3: Cryptochlorogenic acid, Peak 4: Ferulic acid, Peak 5: Glycyrrhizin, Peak 7: 3,5-di-O-caffeoylquinic acid, Peak 8: Angiologin C, Peak 9: 4,5-di-O-caffeoylquinic acid, Peak 10: Ligusticum lactone I, Peak 11: Cinnamic acid, Peak 12: Glycyrrhizic acid, Peak 13: Ligusticum lactone.

2. The method for constructing the feature map of Simiao Yong'an Decoction according to claim 1, characterized in that: The method for preparing the test solution in step a is as follows: Take the Simiao Yong'an Decoction as a test sample, add 50% methanol aqueous solution, reflux to extract, filter, and take the filtrate as the test sample solution.

3. A method for identifying genuine or qualified Simiao Yong'an Decoction, characterized in that: It includes the following steps: a. Weigh the sample to be tested; b. Detect the characteristic peaks according to the characteristic spectrum construction method of Simiao Yong'an Decoction according to any one of claims 1-2. If the 13 characteristic peaks are completely consistent, it is a genuine or qualified product.

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