Method for identifying and detecting tan yu tong bi decoction

Abstract

The application provides a method for identifying and detecting Tan Yu Tong Bi Decoction, which comprises three modules of characteristic pattern construction of medicinal materials, optional thin layer chromatography identification and effective component content determination. The characteristic pattern construction adopts a UPLC analysis strategy of two-stage segmented extraction of 'diethyl ether-ethyl acetate', overcomes the interference of complex components, and can comprehensively characterize the overall chemical characteristics of the compound; the thin layer chromatography identification provides a specific identification method for Rhizoma Coptidis, Radix et Rhizoma Curcumae, Radix et Rhizoma Rhei and the like; the content determination adopts a high performance liquid chromatography method to detect effective components of chlorogenic acid, salvianolic acid B, glycyrrhizic acid and berberine hydrochloride. The application constructs a multi-dimensional and configurable quality control system, realizes comprehensive quality control of Tan Yu Tong Bi Decoction from qualitative analysis, overall characterization to quantitative analysis, has strong specificity, and the result is reliable.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis and quality control technology, specifically relating to a method for identifying and detecting the traditional Chinese medicine compound preparation "Tanyu Tongbi Fang". Background Technology

[0002] Phlegm-Stasis-Clearing Formula is a traditional Chinese medicine compound preparation used clinically to treat related diseases, with definite efficacy. "Phlegm-Stasis-Clearing Granules," made primarily from this formula, are commonly used traditional Chinese medicine preparations. In industrial production, the homogeneous powder obtained after extraction, concentration, and drying of medicinal materials is called an "intermediate" or dry extract powder, and its quality is fundamental to the efficacy and safety of the finished product.

[0003] Traditional Chinese medicine (TCM) compound formulas have extremely complex chemical compositions, typically containing compounds with vastly different structures and polarities, such as alkaloids, saponins, flavonoids, terpenes, and polysaccharides. This complexity presents a significant challenge to their quality control, as single analytical methods are often insufficient to comprehensively and accurately reflect their intrinsic quality.

[0004] Currently, conventional quality control methods for this type of compound preparation have the following main shortcomings:

[0005] I. Isolated Detection Methods and Fragmented Information: Single TLC methods are often used to identify a few medicinal ingredients or to determine the content of only one or two indicator components. TLC is rapid but provides limited information and is difficult to quantify; content determination is accurate but cannot reflect the overall chemical profile; while characteristic chromatographic techniques that can characterize the overall features often suffer from severe interference in complex matrices and difficulties in chromatographic conditions that accommodate components with a wide range of polarities, leading to the loss of key information or non-specific spectra. The conclusions of various methods are isolated from each other, making it difficult to form a comprehensive evaluation.

[0006] Second, there is a lack of systematic and configurable integrated solutions: Existing technologies rarely offer mature solutions that systematically integrate specific TLC identification, high-information-content spectroscopy, and multi-index content determination, and can be flexibly combined and applied according to different quality control objectives (such as rapid screening, in-depth identification, or full release). This results in either insufficient testing items or an inability to balance testing costs and efficiency when facing quality control needs in different scenarios.

[0007] Third, there is a lack of pretreatment and separation strategies for complex systems: For the Tanyu Tongbi formula, its components are widely distributed from strong polarity to weak polarity, resulting in severe interference from direct analysis. Existing methods lack targeted sample pretreatment strategies to effectively remove interference and enrich target components, and also lack innovative chromatographic analysis strategies to simultaneously obtain chemical information with high coverage and high resolution.

[0008] Therefore, developing a comprehensive identification and detection method for the phlegm-stasis-relieving and arthralgia-relieving formula that can integrate multiple analytical dimensions, be flexibly configured according to needs, and effectively overcome interference from complex matrices is of great significance for improving its quality control level and ensuring clinical efficacy and safety. Summary of the Invention

[0009] The purpose of this invention is to provide a comprehensive quality analysis method for the Phlegm-Stasis-Clearing and Pain-Relieving Formula, in order to solve the problem that the existing quality control methods are singular, isolated, and unable to comprehensively evaluate the quality of complex Chinese herbal compound formulas. The composition of the Phlegm-Stasis-Clearing and Pain-Relieving Formula is as follows: Artemisia capillaris 8-13 parts, Atractylodes lancea 8-12 parts, Alisma plantago-aquatica 8-12 parts, Curcuma longa 8-14 parts, Salvia miltiorrhiza 10-20 parts, Curcuma longa 8-14 parts, Panax notoginseng powder 1-5 parts, Corydalis yanhusuo 5-10 parts, Pinellia ternata 8-12 parts, Coptis chinensis 5-10 parts, Panax ginseng 4-10 parts, Citrus reticulata peel 5-12 parts, and Glycyrrhiza uralensis 4-10 parts.

[0010] This invention provides a comprehensive technical solution that systematically integrates characteristic spectrum construction, thin-layer chromatography identification, and multi-index content determination. Users can flexibly select and combine the above-mentioned technical modules according to actual quality control needs to form a configurable quality control strategy from rapid screening to comprehensive evaluation. By optimizing the sample pretreatment, chromatographic conditions, and detection parameters of each module, the specificity, reproducibility, and reliability of the method are ensured, achieving multi-dimensional and precise control over the quality of the Tanyu Tongbi formula.

[0011] In this invention, the colon ":" used when describing the developing solvent formulation indicates that the solvent components are mixed in a volume ratio. When no specific numerical value is specified after the colon, and only the solvent name is connected (e.g., A:B:C), it indicates a developing solvent system composed of the listed solvents, the specific volume ratio of which can be flexibly adjusted and selected. In this case, the colon indicates the mixing relationship of the components, rather than a fixed ratio.

[0012] In this invention, the term "petroleum ether at 60-90°C" refers to the boiling range specification of the solvent "petroleum ether" used, rather than the ambient temperature or operating temperature at which the process is carried out. Petroleum ether is not a single compound, but a mixture of low-boiling fractions of petroleum. "60-90°C" indicates the boiling point range of the solvent, that is, the distillation temperature of the main component is between 60°C and 90°C.

[0013] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0014] A method for identifying and detecting phlegm-stasis-relieving formulas, the method comprising constructing a feature map, which includes:

[0015] (1.1) Preparation of test solution V: Take the powder of the phlegm-stasis-relieving and pain-relieving formula, extract it with methanol and concentrate it, dissolve the residue in water, and perform two-stage liquid-liquid extraction with ether and ethyl acetate in sequence. Collect and combine the ethyl acetate extract fraction to obtain test solution V.

[0016] (1.2) Chromatographic analysis: The test solution V was injected into an ultra-high performance liquid chromatograph, and gradient elution was performed using 0.1% formic acid aqueous solution and acetonitrile as the mobile phase. Detection was performed at a wavelength of 280 nm.

[0017] The ultra-high performance liquid chromatograph uses a C18 column bonded with surface-charged hybrid silica particles. The column size is 2~3 mm × 80~120 mm, and the packing particle size is 1.5~2 μm.

[0018] According to the method provided by the present invention, preferably, the gradient elution procedure in step (1.2) is as follows:

[0019] 0~9 min: The volume percentage of acetonitrile increases from 5% to 15%;

[0020] 9-11 min: The volume percentage of acetonitrile remains at 15%;

[0021] 11-15 min: The volume percentage of acetonitrile increases from 15% to 20%;

[0022] 15~25 min: The volume percentage of acetonitrile is maintained at 20%;

[0023] 25-26 min: The volume percentage of acetonitrile increases from 20% to 21%;

[0024] 26-30 min: The volume percentage of acetonitrile remains at 21%.

[0025] The design principle of this invention lies in overcoming the technical challenge of comprehensively analyzing this compound under single chromatographic conditions due to its numerous compounds with vastly different structures and wide polarity ranges, such as polysaccharides, saponins, sesquiterpenes, lignin, flavonoids, and terpenes. This invention innovatively develops a "segmented extraction" analytical strategy. First, through a two-stage liquid-liquid extraction process using diethyl ether and ethyl acetate, the complex chemical components are segmented according to polarity, effectively removing strongly polar interfering substances such as polysaccharides and specifically enriching the target components. The selection of 280 nm as the uniform detection wavelength is based on extensive experimental findings, indicating that compounds with conjugated structures, such as flavonoids and salvianolic acids, contained in the compound exhibit characteristic UV absorption at this wavelength, enabling the acquisition of abundant and highly responsive chromatographic peaks. Ethyl acetate chromatographic information is chosen because this detection wavelength provides the highest number of retained peaks and the best resolution, thus comprehensively reflecting the overall chemical characteristics of the compound and solving the problem of insufficient chromatographic information coverage. This strategy overcomes the interference of complex components and can comprehensively characterize the overall chemical characteristics of the compound.

[0026] In some preferred embodiments of the present invention, the method may further include thin-layer chromatography identification of the medicinal material, which includes:

[0027] (2.1) Identification of Coptis chinensis: Take the powder of the phlegm-stasis-relieving and pain-relieving formula, add methanol and extract by ultrasonication, and use the filtrate as test solution I; use berberine hydrochloride reference solution as control; use silica gel G thin layer plate, develop with petroleum ether: acetone: methanol: ammonia water at 60~90℃ as developing solvent, and examine under ultraviolet light at 365nm.

[0028] (2.2) Identification of Curcuma longa: Take the powder of the phlegm-stasis-relieving and pain-relieving formula, add anhydrous ethanol for ultrasonic extraction, evaporate the filtrate to dryness, dissolve the residue in ethanol to prepare the test solution II; use Curcuma longa as the reference material and prepare the solution in the same way as the control; use silica gel G thin layer plate, develop with petroleum ether: chloroform at 60~90℃, spray with sulfuric acid ethanol solution, heat to develop color, and examine under sunlight;

[0029] (2.3) Identification of Corydalis rhizome in vinegar: Take the powder of the phlegm-stasis-relieving and pain-relieving formula, add methanol for ultrasonic extraction, evaporate the filtrate to dryness, dissolve the residue in water, alkalize it, extract it with ether, evaporate the ether solution to dryness, dissolve the residue in methanol to prepare test solution III; use the Corydalis rhizome standard solution as a reference; use alkaline silica gel G thin layer plate, develop it with petroleum ether: ethyl acetate: triethylamine at 60~90℃ as the developing solvent, and examine it under ultraviolet light at 365nm after iodine fuming.

[0030] In some preferred embodiments of the present invention, the method may further include the determination of the content of the active ingredient, which includes:

[0031] (3.1) Preparation of test solution IV: Take the powder of the phlegm-stasis-relieving and pain-relieving formula, add methanol aqueous solution for ultrasonic extraction, and filter;

[0032] (3.2) Chlorogenic acid determination: elution was performed using 0.2% phosphoric acid aqueous solution-methanol as the mobile phase, and the detection wavelength was 327 nm;

[0033] (3.3) Determination of salvianolic acid B and glycyrrhizic acid: Gradient elution was performed using 0.2% phosphoric acid aqueous solution-acetonitrile as the mobile phase, and the detection wavelengths were 286 nm for salvianolic acid B and 237 nm for glycyrrhizic acid.

[0034] (3.4) Berberine hydrochloride determination: elution was performed using phosphate buffer-methanol as the mobile phase, and the detection wavelength was 345 nm.

[0035] In some preferred embodiments of the present invention, the coverage of the thin-layer chromatography identification module can be further expanded, increasing the specificity for identifying licorice and / or ginseng, and providing a more comprehensive means of identifying the authenticity and dosage of medicinal materials. Specifically, the identification methods for licorice and ginseng are as follows:

[0036] (2.4) Identification of licorice: Take the powder of the phlegm-stasis-relieving and pain-relieving formula, defatt it with ether, extract it by reflux with methanol, evaporate the extract to dryness, dissolve the residue in water, extract with water-saturated n-butanol, evaporate the n-butanol solution to dryness, dissolve the residue in methanol to prepare the test solution; use glycyrrhizin as the reference standard, use dichloromethane:methanol:formic acid:water (e.g. 8:3:0.1:1) as the developing solvent, spray with sulfuric acid ethanol solution (e.g. 10%) and heat (e.g. 105℃) to develop color, and examine under ultraviolet light at 365nm;

[0037] (2.5) Identification of ginseng: Take the powder of the phlegm-stasis-relieving and pain-relieving formula, defatt it with chloroform, moisten the residue with water, add water-saturated n-butanol for ultrasonic extraction, purify the extract with ammonia solution, take the upper layer liquid and evaporate it to dryness, dissolve the residue in methanol to use as the test solution; use ginsenosides Rb1, Re, Rf, and Rg1 as reference standards, use ethyl acetate:acetone:water (e.g., 40:25:10) as the developing solvent, spray with sulfuric acid ethanol solution and heat to develop color, and examine under sunlight.

[0038] In the identification of Coptis chinensis in (2.1), the volume ratio of petroleum ether: acetone: methanol: ammonia in the developing solvent can be (2.5~3.5):(3~5):(1.5~2.5):(0.2~0.8); the concentration of the berberine hydrochloride reference solution can be 0.4~0.6 mg / ml.

[0039] In the identification of Curcuma longa in (2.2), the volume ratio of petroleum ether to chloroform in the developing solvent can be (88~80):(18~14); the concentration of the sulfuric acid ethanol solution can be 8%~12%.

[0040] In the identification of Corydalis rhizome in (2.3), the volume ratio of petroleum ether: ethyl acetate: triethylamine in the developing solvent can be (6~8):(1.5~2.5):(0.5~1.5); the alkaline silica gel G thin-layer plate can be prepared using 1% sodium hydroxide solution.

[0041] In the determination of the effective component content, the ratio of the mobile phase for isocratic elution in step (3.2) for the determination of chlorogenic acid can be: 0.2% phosphoric acid aqueous solution: methanol = (88~94):(12~6).

[0042] In the determination of the content of the active ingredient, the gradient elution procedure for the determination of salvianolic acid B and glycyrrhizic acid in step (3.3) is as follows:

[0043] 0~15 min: Acetonitrile volume percentage remains at 18%;

[0044] 15~20 min: The volume percentage of acetonitrile increases from 18% to 35%;

[0045] 20-30 min: The volume percentage of acetonitrile remains at 35%.

[0046] In the determination of the effective component content, in the mobile phase of the berberine hydrochloride determination in step (3.4), the phosphate buffer solution can be a 0.05 mol / L potassium dihydrogen phosphate solution with the pH adjusted to 3.0 by phosphoric acid, and its volume ratio with methanol can be 70:30.

[0047] In some specific embodiments of the present invention, in the identification of Coptis chinensis in (2.1), the sample amount of the phlegm-stasis-relieving and pain-relieving formula powder can be 0.2g~0.3g, for example 0.25g; the amount of methanol can be 20ml~30ml, for example 25ml; the ultrasonic treatment time can be 20min~40min, for example 30min. The volume ratio of petroleum ether:acetone:methanol:ammonia water at 60~90℃ in the developing solvent is 3:4:2:0.5; the concentration of the berberine hydrochloride reference solution is 0.4~0.6 mg / ml. The sample spotting volume is 0.5μl~2μl, for example 1μl.

[0048] According to the method provided by the present invention, in the identification of *Curcuma longa* in (2.2), the sample amount of the *Tanyu Tongbi Fang* powder can be 1.5g~2.5g, for example 2g; the amount of anhydrous ethanol can be 20ml~30ml, for example 25ml. The volume ratio of petroleum ether at 60~90℃ to chloroform in the developing solvent is (88~80):(18~14); the concentration of the sulfuric acid ethanol solution is 8%~12%, preferably 10%. The sample spotting volume can be 4μl~6μl, for example 5μl.

[0049] According to the method provided by the present invention, in the identification of Corydalis rhizome in vinegar (1.3), the sample amount of the phlegm-stasis-relieving and pain-relieving formula powder can be 0.8g~1.2g, for example 1g; the amount of methanol can be 40ml~60ml, for example 50ml; the concentrated ammonia solution used for alkalization is adjusted to alkaline; the volume of each extraction with ether can be 8ml~12ml, for example 10ml. The volume ratio of petroleum ether:ethyl acetate:triethylamine at 60~90℃ in the developing solvent is (6~8):(1.5~2.5):(0.5~1.5), preferably 7:2:1. The alkaline silica gel G thin-layer plate is prepared using 1% sodium hydroxide solution. The sample volume of the test solution can be 4μl~8μl, for example 6μl; the sample volume of the reference solution can be 1μl~3μl, for example 2μl.

[0050] According to the method provided by the present invention, in the determination of the content of the active ingredient, in step (2.1), in the preparation of the test solution IV for the determination of chlorogenic acid and berberine hydrochloride, the concentration of the methanol aqueous solution can be 45%~55%, for example 50%; in the preparation of the test solution IV for the determination of salvianolic acid B and glycyrrhizic acid, the concentration of the methanol aqueous solution can be 65%~75%, for example 70%.

[0051] The isocratic elution mobile phase ratio for chlorogenic acid determination in step (2.2) is: 0.2% phosphoric acid aqueous solution: methanol = (88~94):(12~6), preferably 93:7. A BEH C18 column (2.1 × 100 mm, 1.7 μm) can be used; the column temperature can be 25°C~35°C, for example 30°C; the flow rate can be 0.1~0.4 mL / min, for example 0.3 mL / min.

[0052] In step (2.4), the mobile phase for the determination of berberine hydrochloride is a phosphate buffer solution with a concentration of 0.05 mol / L potassium dihydrogen phosphate, and the pH is adjusted to 3.0 with phosphoric acid. The volume ratio of the buffer solution to methanol is 70:30.

[0053] According to the method provided by the present invention, in the determination of the content of the active ingredient, the gradient elution procedure for the determination of salvianolic acid B and glycyrrhizic acid in step (2.3) is as follows:

[0054] 0~15 min: Acetonitrile volume percentage remains at 18%;

[0055] 15~20 min: The volume percentage of acetonitrile increases from 18% to 35%;

[0056] 20-30 min: The volume percentage of acetonitrile remains at 35%.

[0057] According to the method provided by the present invention, in the construction of the characteristic spectrum, the ultra-high performance liquid chromatography detection in step (1.2) uses a surface-charged hybrid silica-bonded C18 column (e.g., Waters CORIECST3), with a column size of 2.1 × 100 mm and a packing particle size of 1.6 μm. The flow rate is preferably 0.3 mL / min; the column temperature is preferably 30 °C.

[0058] According to the method provided by the present invention, the thin-layer chromatography identification steps (2.1), (2.2), (2.3), (2.4), and (2.5) all include corresponding negative control experiments. That is, negative samples of the Tanyu Tongbi formula lacking the corresponding medicinal ingredients are taken respectively, and negative control solutions are prepared in the same way. During inspection, the negative control solutions should not show the same spots at the corresponding positions as the corresponding reference standard or reference medicinal material chromatogram, so as to ensure the specificity of the method.

[0059] This invention constructs a multi-dimensional quality control system with characteristic chromatograms as its core and flexible expansion capability. It achieves comprehensive quality control of the Tanyu Tongbi formula, from overall chemical characterization to qualitative identification of specific medicinal materials, and then to quantitative analysis of key components. The system is highly specific and yields reliable results. The method provided by this invention systematically integrates ultra-high performance liquid chromatography (UPLC) characteristic chromatogram construction, thin-layer chromatography (TLC) qualitative identification, and multi-index component content determination techniques. It is applicable to the quality control and evaluation of the raw materials, intermediates (such as extract powder), and preparations (such as granules) of this compound formula, and has the following beneficial effects:

[0060] By constructing a holistic characteristic spectrum based on a "segmented extraction analysis" strategy as the core method, and organically combining it with specific thin-layer chromatography identification and multi-index component content determination, a more comprehensive, complete, and configurable quality analysis method was established. This integrated quality control scheme exhibits high accuracy and specificity (as verified by negative controls), and reliable detection results. It achieves comprehensive characterization and precise control of the chemical basis of the Tanyu Tongbi formula, meeting the quality monitoring needs of modern Chinese medicine production. This is of great significance for ensuring the quality uniformity, safety, and clinical efficacy of this Chinese medicine compound preparation. Attached Figure Description

[0061] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0062] Figure 1The image shows the characteristic chromatograms of the phlegm-stasis-relieving formula constructed in Example 1. Image A is the control chromatogram of direct methanol extraction, and Image B is the characteristic chromatogram of ethyl acetate extraction. In the image, 1 is neochlorogenic acid, 2 is caffeic acid, 3 is chlorogenic acid, 4 is salicylic acid, 5 is salvianolic acid B, 6 isorhamnetin, 7 isoglycyrrhizin, 8 isochlorogenic acid A, 9 is salvianolic acid A, 10 is kaempferol rutin, 11 is quercetin glucoside, 12 is glycyrrhizin diglucoside, and 13 is hesperidin.

[0063] Figure 2 The chromatogram for thin-layer identification of licorice in Example 1 is shown below. In the figure: ① is glycyrrhizin reference standard, ② is the test sample of Tanyu Tongbi Formula, and ③ is the negative control sample lacking licorice.

[0064] Figure 3 The chromatogram for thin-layer identification of ginseng in Example 1 is shown below. In the figure: ① is a mixed reference standard of ginsenosides Rb1, Re, Rf and Rg1, ② is the test sample of the phlegm-stasis-relieving formula, and ③ is a negative control sample lacking ginseng.

[0065] Figure 4 The chromatogram for thin-layer identification of Coptis chinensis in Example 1 is shown in the figure. In the figure: ① is berberine hydrochloride reference standard, ② is the test sample of Tanyu Tongbi Formula, and ③ is the negative control sample lacking Coptis chinensis.

[0066] Figure 5 The chromatogram for thin-layer identification of Curcuma longa in Example 1 is shown in the figure. In the figure: ① Curcuma longa is the reference material, ② is the test sample of the Tanyu Tongbi Formula, and ③ is the negative control sample for Curcuma longa deficiency.

[0067] Figure 6 The chromatogram for thin-layer identification of vinegar-treated Corydalis rhizome in Example 1 is shown in the figure. In the figure: ① is the reference standard of corydalis ethylsine, ② is the test sample of the phlegm-stasis-relieving and pain-relieving formula, and ③ is the negative control sample of vinegar-treated Corydalis rhizome.

[0068] Figure 7 The above is an exemplary chromatogram for the determination of chlorogenic acid content in Example 1, wherein curve A is the chromatogram of chlorogenic acid reference standard and curve B is the chromatogram of the phlegm-stasis-relieving and pain-relieving formula test sample.

[0069] Figure 8 The following is an exemplary chromatogram for the determination of the contents of salvianolic acid B and glycyrrhizic acid in Example 1. Curve A is the chromatogram of ammonium glycyrrhizate reference standard, curve B is the chromatogram of salvianolic acid B reference standard, and curve C is the chromatogram of the test sample of Tanyu Tongbi Formula at 286nm. In this chromatogram, 1 represents salvianolic acid B and 2 represents glycyrrhizic acid.

[0070] Figure 9The following is an exemplary chromatogram for the determination of the contents of salvianolic acid B and glycyrrhizic acid in Example 1. Curve A is the chromatogram of ammonium glycyrrhizate reference standard, curve B is the chromatogram of salvianolic acid B reference standard, and curve C is the chromatogram of the test sample of Tanyu Tongbi Formula at 237 nm. In this chromatogram, 1 represents salvianolic acid B and 2 represents glycyrrhizic acid.

[0071] Figure 10 This is an exemplary chromatogram for the determination of berberine hydrochloride content in Example 1, wherein curve A is the chromatogram of berberine hydrochloride reference standard and curve B is the chromatogram of the test sample of Tanyu Tongbi Formula.

[0072] Figure 11 These are the characteristic spectra detected at 340 nm wavelength under different extraction conditions in Example 3.

[0073] Figure 12 These are characteristic spectra of samples extracted with diethyl ether and ethyl acetate at different detection wavelengths in Example 3.

[0074] Figure 13 The images shown are characteristic spectra obtained in Example 3 under the condition of a mobile phase system of 0.2% phosphoric acid-acetonitrile. Graph A is the characteristic spectrum obtained by gradient program 1, and graph B is the characteristic spectrum obtained by gradient program 2.

[0075] Figure 14 This is a comparison of the effects of different mobile phase systems (0.2% phosphoric acid water-acetonitrile, 0.1% formic acid water-acetonitrile, 0.2% phosphoric acid water-methanol, water-acetonitrile) on the fingerprint spectrum of the ether-ethyl acetate extracted sample (340nm) in Example 3. Detailed Implementation

[0076] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and experimental embodiments. The following embodiments illustrate in detail the implementation methods of various technical solutions covered by the claims of this invention and provide sufficient methodological verification data. It should be understood that these embodiments are only for explaining the invention and not for limiting the invention. Experimental methods not specifying specific conditions are generally performed according to the General Rules, Part IV of the Chinese Pharmacopoeia (2025 Edition) or conventional conditions in the art.

[0077] The materials and instruments used in the embodiments are described below:

[0078] Samples: Extract powder of Tanyu Tongbi Formula (batch number: GZ-TYT251015-01); and several negative control samples prepared according to the prescription ratio, which lacked licorice, ginseng, coptis, turmeric, and vinegar-processed corydalis.

[0079] Reference standards and reference medicinal materials: glycyrrhizin (111610-202209), ginsenoside Rb1 (110704-202330), Re (110754-202328), Rf (111737-202206), Rg1 (110703-202335), berberine hydrochloride (110713-202316), corydaline (110726-202421), chlorogenic acid (110753-202220), salvianolic acid B (111562-202216), and ammonium glycyrrhizate (110731-202223) were all purchased from the National Institutes for Food and Drug Control. Turmeric reference medicinal material batch number: 120949-202109.

[0080] Main reagents: Methanol and acetonitrile were of chromatographic grade; ethyl acetate, formic acid, phosphoric acid, potassium dihydrogen phosphate, glacial acetic acid, triethylamine, n-hexane, cyclohexane, isopropanol, toluene, acetone, diethyl ether, anhydrous ethanol, and ammonia solution were of analytical grade; water was ultrapure water.

[0081] Main instruments:

[0082] Liquid chromatography: Ultra-high performance liquid chromatograph (Waters ACQUITY UPLC H-Class), equipped with a quaternary pump, autosampler, column oven and PDA detector; chromatography workstation (Empower 3).

[0083] Thin-layer chromatography system: silica gel G thin-layer plate, high-performance silica gel G thin-layer plate (Merck); 1% sodium hydroxide silica gel G thin-layer plate (self-made); dual-groove developing tank (10cm×10cm); CAMAG Automatic TLC Sampler 4 spotter; CAMAG TLCVisualizer 2 imaging system.

[0084] Sample processing equipment: electronic balance (METTLER TOLEDO, sensitivity 0.01mg and 0.1mg); KQ-500DE CNC ultrasonic cleaner; rotary evaporator (BUCHI R-300); constant temperature water bath.

[0085] Example 1

[0086] This embodiment illustrates the most comprehensive application mode of the present invention, which is based on the construction of characteristic chromatograms and further employs two techniques: "thin-layer chromatography identification" (extended to licorice and ginseng) and "multi-index content determination" to conduct systematic quality control analysis on the same batch of phlegm-stasis-relieving and pain-relieving formula extract (dry powder, as an intermediate sample).

[0087] 1.1 Characteristic Spectrum Determination and Analysis

[0088] 1.1.1 Preparation of test solution

[0089] Weigh 2.0g of the dry extract powder, add 25ml of methanol and extract by ultrasonication. Evaporate the filtrate to dryness. Dissolve the residue in 10ml of water, transfer to a separatory funnel, and extract sequentially:

[0090] Ether extraction (3 times × 10 ml): Discard the ether layer (to remove fat-soluble impurities).

[0091] Ethyl acetate extraction (3 times × 10 ml): Combine the ester layers, evaporate to dryness, and dilute to 5 ml with methanol to obtain the ethyl acetate fraction solution (V). Filter the solution through a 0.22 μm filter membrane.

[0092] 1.1.2 Chromatographic Analysis Conditions

[0093] Instruments and columns: Waters ACQUITY UPLC, Waters CORIECS T3 column (2.1×100 mm, 1.6 μm).

[0094] Detection wavelength: 280 nm; column temperature: 30℃; flow rate: 0.3 mL / min; injection volume: 1 μL.

[0095] The elution gradient settings are shown in Table 1 below.

[0096]

[0097] 1.1.3 Measurement and Spectral Analysis

[0098] Accurately pipette 1 μL of the test solution and inject it for analysis under the conditions described above. Record the chromatograms. The results are as follows: Figure 6 As shown, characteristic spectral information was obtained. The similarity of this spectral data with the control characteristic spectral data established from 10 batches of qualified samples was evaluated (cosine similarity method). The similarity was 0.992 (>0.90), and the relative retention times of the 15 calibrated common peaks were stable, indicating that the overall chemical characteristics of this batch of samples were consistent with those of qualified products, and the batch-to-batch stability was good.

[0099] 1.2 Thin-layer chromatography (TLC) identification

[0100] 1.2.1 Identification of Licorice

[0101] (1) Test solution: Take 2.0 g of the dry extract powder and place it in a round-bottom flask. Add 20 ml of ether, heat under reflux for 1 hour, filter, discard the ether liquid, and evaporate the ether from the residue. Add 15 ml of methanol to the residue, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve the residue, and transfer to a separatory funnel. Extract three times with 10 ml of water-saturated n-butanol each time. Combine the n-butanol solutions, wash twice with 10 ml of ammonia solution each time, and then wash twice with 10 ml of water each time. Discard the water washing liquid, evaporate the n-butanol solution to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution.

[0102] (2) Reference solution: Take about 1.0 mg of glycyrrhizin reference standard and place it in a 1 ml volumetric flask. Dissolve and dilute with methanol to the mark, shake well, and you will get (1 mg / ml).

[0103] (3) Negative control solution: Take 2.0g of negative sample lacking licorice and prepare the solution in the same way.

[0104] TLC Procedure and Results: Apply 10 μl of each of the three solutions to a high-performance silica gel G thin-layer plate. Use dichloromethane:methanol:formic acid:water (8:3:0.1:1) as the developing solvent, pre-equilibrate for 15 minutes, develop to approximately 8 cm, remove, and air dry. Spray with 10% sulfuric acid in ethanol, heat at 105°C for 5 minutes, and examine under a 365 nm UV lamp.

[0105] The results are as follows Figure 2 As shown, in the chromatogram of the test sample, a bright blue fluorescent spot appears at the same position as the glycyrrhizin reference standard, while the negative control lacking licorice does not have this spot.

[0106] 1.2.2 Identification of Ginseng

[0107] (1) Test solution: Take 3.0 g of the dry extract powder and place it in a round-bottom flask. Add 40 ml of chloroform and heat under reflux for 1 hour. Filter and discard the chloroform solution. Evaporate the solvent from the residue. Add 0.5 ml of water and stir to moisten. Accurately add 10 ml of water-saturated n-butanol, weigh, sonicate (300 W, 40 kHz) for 30 minutes, cool, add weight, shake well, and centrifuge. Accurately pipette 5 ml of the supernatant, add 15 ml of ammonia test solution, shake, allow to stand for separation, take the upper layer and evaporate to dryness. Dissolve the residue in 1 ml of methanol.

[0108] (2) Reference solution: Take about 2.0 mg of each of ginsenoside Rb1, Re, Rf and Rg1 reference standards, put them in a 1 ml volumetric flask, add methanol to dissolve and dilute to the mark to prepare a mixed reference solution (2 mg / ml each).

[0109] (3) Negative control solution: Take 3.0g of negative sample lacking ginseng and prepare the solution in the same way.

[0110] TLC Procedure and Results: Spot 20 μl of each of the three solutions described above. Develop with ethyl acetate:acetone:water (40:25:10) as the developing solvent, and air dry after development. Spray with 10% sulfuric acid in ethanol, heat at 105°C for 10 minutes, and examine under sunlight.

[0111] The results are as follows Figure 3 As shown, in the chromatogram of the test sample, the same purplish-red spots appeared at multiple positions corresponding to the mixed reference standard, while the negative control lacking ginseng did not have corresponding spots.

[0112] 1.2.3 Identification of Coptis chinensis

[0113] (1) Test solution: Take 0.25g of the dry extract powder of this product, add 25ml of methanol, sonicate for 30 minutes, filter, and take the filtrate.

[0114] (2) Reference standard and negative control: Berberine hydrochloride reference solution (0.5 mg / ml) and negative control solution lacking Coptis chinensis were prepared by the same method.

[0115] TLC Procedure and Results: Spot 1 μl each of the test sample, control solution, and negative control solution onto a silica gel G plate. Develop with petroleum ether:acetone:methanol:ammonia (3:4:2:0.5) at 60–90 °C and examine under a 365 nm UV lamp.

[0116] The results are as follows Figure 4 The test sample showed bright yellow fluorescent spots at the same positions as the reference sample, while the negative control showed no interference.

[0117] 1.2.4 Identification of Curcuma longa

[0118] (1) Test solution: Take 2.0g of the dry extract powder of this product, add 25ml of anhydrous ethanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 1ml of ethanol.

[0119] (2) Control medicinal materials and negative control solutions: Curcuma longa control medicinal materials were prepared in the same way; and negative control solutions lacking Curcuma longa were prepared in the same way.

[0120] TLC procedure and results: Spot 5 μl of each solution. Develop with petroleum ether:chloroform (84:16) at 60-90℃, spray with 10% sulfuric acid ethanol solution, heat to 105℃ and examine under sunlight.

[0121] The results are as follows Figure 5 The test sample showed purplish-red spots at the same positions as the main spots of the control medicinal material, while the negative control did not have these spots.

[0122] 1.2.5 Identification of Vinegar-Processed Corydalis Rhizome

[0123] (1) Test solution: Take 1.0g of the dry extract powder of this product, add 50ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 10ml of water, adjust the pH to 9 with concentrated ammonia test solution, extract with ether 3 times (10ml each time), combine the ether solutions, evaporate to dryness, and dissolve the residue in 1ml of methanol.

[0124] (2) Reference standard and negative control solution: Corydalis yanhusuo reference standard solution (0.5 mg / ml) and negative control solution lacking vinegar were prepared by the same method.

[0125] TLC Procedure and Results: Apply 6 μl each of the test sample and negative control solution, and 2 μl of the control solution to a 1% sodium hydroxide silica gel G plate (use silica gel G thin-layer plate, which has been soaked or sprayed with 1% sodium hydroxide solution and dried before use). Develop with petroleum ether:ethyl acetate:triethylamine (7:2:1) at 60–90 °C. After evaporating the iodine for 3 minutes, examine under a 365 nm UV lamp.

[0126] The results are as follows Figure 6 The test sample showed bright yellow-green fluorescent spots at the same positions as the reference sample, while the negative control showed no interference.

[0127] Conclusion: All five TLC identification methods showed strong specificity, clear target spots, and no interference from the negative control, indicating that the five medicinal materials contained in this batch of samples all met the requirements.

[0128] 1.3 Determination of the content of multiple components

[0129] 1.3.1 Preparation of the test solution

[0130] Determination of chlorogenic acid and berberine hydrochloride: Take about 0.2g of the dry extract powder of this product, add 25ml of 50% methanol, sonicate for 30 minutes, filter, and obtain solution C1.

[0131] Determination of salvianolic acid B and glycyrrhizic acid: Take about 0.5g of the dry extract powder of this product, add 25ml of 70% methanol, and treat in the same way to obtain solution C2.

[0132] 1.3.2 Preparation of reference solution

[0133] Chlorogenic acid: 30 μg / mL (50% methanol).

[0134] A mixed solution of salvianolic acid B and ammonium glycyrrhizate: the concentration of salvianolic acid B is 0.1 mg / mL, and the concentration of ammonium glycyrrhizate is 40 μg / mL (70% methanol).

[0135] Berberine hydrochloride: 90 μg / mL (50% methanol).

[0136] 1.3.3 Chromatographic conditions and determination results

[0137] (1) Chlorogenic acid determination:

[0138] Chromatographic conditions: Waters BEH C18 column (2.1×100 mm, 1.7 μm); mobile phase: 0.2% phosphoric acid aqueous solution-methanol (93:7); flow rate: 0.3 mL / min; column temperature: 30℃; detection wavelength: 327 nm; injection volume: 1 μL.

[0139] Results: Calculated using the external standard method, this product contained 1.40 mg / g of chlorogenic acid (n=4, RSD=0.6%). The chromatogram is shown below. Figure 7 .

[0140] (2) Determination of salvianolic acid B and glycyrrhizic acid:

[0141] Chromatographic conditions: column as above; mobile phase: 0.2% phosphoric acid aqueous solution (A)-acetonitrile (B), gradient: 0-15 min (18% B), 15-20 min (18% to 35% B), 20-30 min (35% B); flow rate: 0.3 mL / min; column temperature: 30℃; detection wavelength: salvianolic acid B 286 nm, glycyrrhizic acid 237 nm; injection volume: 1 μL.

[0142] Results: This product contained 23.74 mg / g of salvianolic acid B and 2.32 mg / g of glycyrrhizic acid (calculated as ammonium salt) (n=4, RSDs were 1.3% and 1.4%, respectively).

[0143] (3) Berberine hydrochloride determination:

[0144] Chromatographic conditions: column as above; mobile phase: 0.05 mol / L potassium dihydrogen phosphate (pH 3.0)-methanol (70:30); flow rate: 0.2 mL / min; column temperature: 30℃; detection wavelength: 345 nm; injection volume: 1 μL.

[0145] Results: This product contains 1.38 mg / g of berberine hydrochloride (n=4, RSD=0.1%).

[0146] 1.4 Conclusion

[0147] This embodiment conducted a complete analysis of the same batch of phlegm-stasis-relieving and pain-relieving formula extract (GZ-TYT251015-01) using three methods: TLC identification, characteristic chromatogram, and content determination. The following conclusions were drawn:

[0148] Qualitative results were satisfactory: all five TLC identification parameters showed strong specificity, and the characteristics of the target medicinal material were detected.

[0149] Consistent overall characteristics: The characteristic spectrum is highly similar to that of qualified products, and the overall chemical profile is stable.

[0150] Quantitative compliance: The content of all four active ingredients was accurately measured, and the results met the preset standards.

[0151] The results comprehensively and reliably demonstrate that the quality of this batch of products meets the requirements, and also verify the effectiveness and feasibility of the quality control scheme proposed in this invention.

[0152] Example 2

[0153] This embodiment is used to demonstrate the effectiveness of the method provided by the present invention (including the specificity of TLC identification, the precision and stability of characteristic spectra, the accuracy of content determination, etc.) through systematic experimental data.

[0154] 2.1 Specificity Validation of Thin-Layer Chromatography Identification Method

[0155] Following the method described in section 1.1 of Example 1, test solutions, reference solutions (medicinal materials), and corresponding single-flavor-deficient negative control solutions (a total of 5) of licorice, ginseng, coptis, turmeric, and vinegar-processed corydalis were prepared, and compared with the whole formula test solutions on the same thin-layer plate.

[0156] Results: In all TLC identifications, the target spots in the chromatograms of the whole formula test sample were clearly located. However, in the corresponding positions of the negative control chromatograms lacking any flavor, no spots appeared that matched the color, fluorescence, and Rf value of the reference standard / reference herb, but other non-target spots were still present. For example, in the negative chromatogram lacking Coptis chinensis, the berberine hydrochloride spot showed no fluorescence; in the negative chromatogram lacking Glycyrrhiza uralensis, the glycyrrhizin spot showed no fluorescence, etc.

[0157] Conclusion: The TLC identification methods of the present invention are highly specific and can accurately indicate the presence of target medicinal materials without being affected by other components in the compound.

[0158] 2.2 Methodological Validation of the Feature Mapping Method

[0159] 2.2.1 Precision Test

[0160] Take the same ethyl acetate fraction test solution (V) and autosample it six times consecutively under the conditions described in Example 1. Select eight stable major common peaks in the spectrum and calculate their relative retention times (relative to the S peak) and relative peak areas (RSDs).

[0161] Results: The relative retention time RSD ranged from 0.12% to 0.45%; the relative peak area RSD ranged from 1.05% to 2.89%.

[0162] Conclusion: The precision is good and meets the requirements of the analytical method.

[0163] 2.2.2 Repeatability Test

[0164] Six portions of the same batch of samples (GZ-TYT251015-01) were taken and independently prepared into test solutions (V) according to the method described in section 1.1.1 of Example 1 for analysis. The relative retention times of the 15 common peaks were calculated.

[0165] Results: The relative retention time RSD of all 15 common peaks was less than 2.0% (n=6).

[0166] Conclusion: The method of the present invention has good repeatability throughout the entire process from sample processing to instrumental analysis.

[0167] 2.2.3 Stability Test

[0168] Take the same sample solution (V) and inject it for analysis at room temperature at 0, 2, 4, 8, 12, and 24 hours. Examine the changes in the relative retention time and relative peak area of ​​the eight major common peaks.

[0169] Results: Within 24 hours, the relative retention time RSD was <1.5%, and the relative peak area RSD was <3.0%.

[0170] Conclusion: The test solution prepared by the method of the present invention has good stability at room temperature for 24 hours, which is sufficient for routine testing.

[0171] 2.3 Methodological validation of content determination methods (taking chlorogenic acid determination as an example)

[0172] 2.3.1 Repeatability Test

[0173] Take 6 samples from the same batch, prepare test solutions (C1) independently and determine the chlorogenic acid content.

[0174] Results: The average content was 1.51 mg / g, and the RSD was 1.5%.

[0175] Conclusion: The method has good reproducibility.

[0176] 2.3.2 Accuracy Test

[0177] The spiking recovery method was used. Nine sample powders with known content were taken and divided into three spiking levels: low (80%), medium (100%), and high (120%). Three samples were added to each group, and chlorogenic acid reference standard was added before the sample was processed and determined in the same way.

[0178] Results: The average recoveries for the low, medium and high levels were 99.2%, 100.1% and 98.8%, respectively, with an overall average recovery of 99.4% and an RSD of 1.8% (n=9).

[0179] Conclusion: The method provided by this invention has high accuracy and ideal recovery rate.

[0180] Example 3

[0181] This embodiment is used to illustrate the superiority of the pretreatment and chromatographic strategies in the feature map construction method provided by the present invention.

[0182] 3.1 Screening of Preprocessing Methods

[0183] Experimental group: The pretreatment method was optimized, namely, the test solution D was obtained by two-stage fractional extraction with ether-ethyl acetate. The specific steps were as follows: 2g of Tanyu Tongbi powder was weighed, 25ml of methanol was added and sonicated for 30 minutes, the residue was weighed, filtered and evaporated to dryness; the residue was redissolved in 10ml of water, and extracted 3 times with ether at a ratio of 1:1 (v:v). The residue was then extracted 3 times with ethyl acetate. The ethyl acetate layer was evaporated to dryness and redissolved in methanol to 5ml.

[0184] Control group: Four groups with different pretreatment methods were set up for comparison, namely:

[0185] Control 1 (Test Solution A): After extraction with methanol, it was first extracted three times with dichloromethane, and the residue was then extracted three times with ethyl acetate. The ethyl acetate layer was evaporated to dryness and then reconstituted with methanol to 5 ml.

[0186] Control 2 (Test Solution B): After extraction with methanol, it was extracted three times with dichloromethane as the sole solvent, evaporated to dryness, and then reconstituted with methanol to 5 ml.

[0187] Control 3 (test solution C): After extraction with methanol, it was extracted three times with only diethyl ether as a single solvent, evaporated to dryness, and then reconstituted with methanol to 5 ml;

[0188] Control 4 (Test Solution E): After extraction with methanol, the solution was extracted three times each with diethyl ether, ethyl acetate, and water-saturated n-butanol. The n-butanol layer was evaporated to dryness and then redissolved in methanol to 5 ml.

[0189] The composition and gradient of the mobile phase are shown in Table 2:

[0190]

[0191] Evaluation metrics: total number of peaks in the chromatogram, peak shape (symmetry factor) of key characteristic peaks, resolution, baseline noise, and overall information content of the chromatogram.

[0192] Comparison results:

[0193] Comparison 1 ( Figure 11 Curve B): The solvent polarity is low, the amount of target compound obtained is small, the extraction efficiency is low, there are many impurity peaks in the spectrum, and the separation effect is poor.

[0194] Comparison 2 ( Figure 11 Curve C): Similar to dichloromethane, but with insufficient polarity adaptability, it cannot effectively enrich the target component, and there is significant interference from impurity peaks;

[0195] Comparison 3 ( Figure 11 Curve A): Although some lipid interference was removed by segmented extraction, a large number of highly polar compounds remained after dichloromethane pretreatment, and the subsequent separation efficiency was lower than that of the experimental group.

[0196] Compare with 4 ( Figure 11 (Curve E): Although it can further obtain some effective components, the number of characteristic peaks is reduced and there is overlap of chromatographic peaks. The peak tailing is obvious and the separation is poor, which is not conducive to subsequent condition optimization.

[0197] Experimental group (diethyl ether-ethyl acetate fractional extraction): Diethyl ether pretreatment can effectively remove lipid-soluble impurities and some highly polar interfering substances. Ethyl acetate, as a moderately polar solvent, can efficiently enrich the target components. The final spectrum has the most characteristic peaks, the most symmetrical peak shapes, the least interference from impurity peaks, and the best separation effect.

[0198] 3.2 Detection Wavelength Screening

[0199] Experimental group: The optimized detection wavelength of 280 nm was used, and the analysis was performed using the preferred "ethyl ether-ethyl acetate" pretreatment method and gradient program 1 chromatographic conditions in Example 1.

[0200] Control group: Five different detection wavelengths were selected for comparison, namely 203nm, 254nm, 270nm, 340nm and 430nm, and the same pretreatment method and chromatographic conditions as the experimental group were used.

[0201] Evaluation indicators: number of characteristic peaks, peak shape symmetry, target peak response value, degree of interference from extraneous peaks, and comprehensiveness of component coverage. Results are as follows: Figure 12 As shown.

[0202] Comparison 1 (203nm): The spectrum contains many impurity peaks, some target peaks are masked, the response value is low, and the separation is insufficient;

[0203] Control 2 (254nm): Severe interference from impurity peaks made it difficult to distinguish between the target peak and the impurity peak, resulting in poor separation.

[0204] The number of characteristic peaks in control 3 (270nm) was moderate and the peak shape was acceptable, but the response value of the target peak was lower than that of the experimental group, and the response of some weakly polar components was not obvious.

[0205] Control 4 (340nm) only responded to specific types of compounds and could not fully cover the multiple active ingredients in the Tanyu Tongbi formula, resulting in incomplete component characterization.

[0206] The control group (430 nm) showed even fewer response compounds, with only a few strongly conjugated structural components detected, which could not meet the requirements for comprehensive characterization of the characteristic spectra.

[0207] Experimental group (280nm): The number of characteristic peaks is moderate and the response value is high. The peak shape is sharp and symmetrical, which can effectively distinguish the target component from the impurity peak. It can fully cover the main active components in the compound and meet the requirements of characteristic spectrum detection.

[0208] 3.3 Screening of mobile phase system and gradient elution program

[0209] Experimental group: A 0.1% formic acid-acetonitrile system was used, and the gradient elution program is shown in Table 3.

[0210]

[0211] Control group: Five groups of different mobile phase systems and gradient programs were set up for comparison: Control 1 (0.2% phosphoric acid water-acetonitrile system) selected elution program 1 as the mobile phase gradient; Control 2 (0.2% phosphoric acid-acetonitrile system), Control 3 (0.2% phosphoric acid-methanol system) and Control 4 (water-acetonitrile system) had the same gradient elution program as the experimental group.

[0212] Evaluation metrics: characteristic peak resolution, peak shape symmetry, tailing factor, retention time stability, baseline noise and drift, and column robustness.

[0213] Comparison results:

[0214] Control 1 (0.1% phosphoric acid solution-acetonitrile) Figure 13 (Figure A): The target compound peaks have a long detection time and some compounds have insufficient separation.

[0215] Control 2 (0.2% phosphate-acetonitrile, Figure 13 (Figure B): The separation effect of the target compound is generally poor, and long-term use of phosphoric acid will accelerate the aging of silica gel matrix columns, resulting in limited method durability;

[0216] Control 3 (0.2% phosphoric acid-methanol) Figure 14 The retention time of the target component is significantly delayed, resulting in low analytical efficiency and broadening of peak shapes for some components.

[0217] Control 4 (water-acetonitrile, Figure 14 The peak shape is poor, the tailing factor is large, the baseline noise and drift are obvious, and the separation is insufficient.

[0218] Experimental group (0.1% formic acid water - acetonitrile + elution gradient 2, Figure 14 Although the separation of the target compound was not optimal in this method, the peak shape was symmetrical, the tailing factor was low, the baseline was stable with low noise, and the retention time was stable. Furthermore, the formic acid system caused minimal damage to the chromatographic column, the method was robust, and the gradient reproducibility was good, meeting the requirements for detection sensitivity and accuracy. The optimal mobile phase system was determined to be 0.1% formic acid-acetonitrile.

[0219] Conclusion: For systems like the Tanyu Tongbi Formula, which have extremely complex chemical components and a wide polarity range, a single chromatographic mode without fractional extraction cannot obtain high-quality characteristic spectra with discriminative power and practical value. The "fractional extraction" analytical strategy adopted in this invention solves the problems of insufficient information coverage and poor separation under complex matrices by specifically removing interferences, enriching the target, and optimizing conditions according to polarity segments. It is an important technological innovation for obtaining a comprehensive and accurate chemical characteristic spectrum of the compound.

[0220] The above embodiments illustrate in detail the specific implementation methods, methodological verification basis, flexible application scenarios, and technical advantages of the technical solution of the present invention. Those skilled in the art can make appropriate changes or equivalent substitutions to the details in the above embodiments without departing from the concept and scope of the present invention, and such changes or substitutions should also be considered as included within the protection scope of the present invention.

Claims

1. A method for identifying and detecting phlegm-stasis-relieving and pain-clearing formulas, characterized in that, The method includes: (1) Construction of feature maps, which includes: (1.1) Preparation of test solution V: Take the powder of the phlegm-stasis-relieving and pain-relieving formula, extract it with methanol and concentrate it, dissolve the residue in water, and perform two-stage liquid-liquid extraction with ether and ethyl acetate in sequence. Collect and combine the ethyl acetate extract fraction to obtain test solution V. (1.2) Chromatographic analysis: The test solution V was injected into an ultra-high performance liquid chromatograph, and gradient elution was performed using 0.1% formic acid aqueous solution and acetonitrile as the mobile phase. Detection was performed at a wavelength of 280 nm. The ultra-high performance liquid chromatograph uses a C18 column bonded with surface-charged hybrid silica particles. The column size is 2~3 mm × 80~120 mm, and the packing particle size is 1.5~2 μm. (2) Thin-layer chromatography identification of medicinal materials, including: (2.1) Identification of Coptis chinensis: Take the powder of the phlegm-stasis-relieving and pain-relieving formula, add methanol and extract by ultrasonication, and use the filtrate as test solution I; use berberine hydrochloride reference solution as control; use silica gel G thin layer plate, develop with petroleum ether: acetone: methanol: ammonia water at 60~90℃ as developing solvent, and examine under ultraviolet light at 365nm. (2.2) Identification of Curcuma longa: Take the powder of the phlegm-stasis-relieving and pain-relieving formula, add anhydrous ethanol for ultrasonic extraction, evaporate the filtrate to dryness, dissolve the residue in ethanol to prepare the test solution II; use Curcuma longa as the reference material and prepare the solution in the same way as the control; use silica gel G thin layer plate, develop with petroleum ether: chloroform at 60~90℃, spray with sulfuric acid ethanol solution, heat to develop color, and examine under sunlight; (2.3) Identification of Corydalis rhizome in vinegar: Take the powder of the phlegm-stasis-relieving and pain-relieving formula, add methanol for ultrasonic extraction, evaporate the filtrate to dryness, dissolve the residue in water, alkalize it, extract it with ether, evaporate the ether solution to dryness, dissolve the residue in methanol to prepare test solution III; use the Corydalis rhizome standard solution as a control; use alkaline silica gel G thin layer plate, develop with petroleum ether: ethyl acetate: triethylamine at 60~90℃ as the developing solvent, and examine under ultraviolet light at 365nm after iodine fuming. (3) Determination of the content of active ingredients, including: (3.1) Preparation of test solution IV: Take the powder of the phlegm-stasis-relieving and pain-relieving formula, add methanol aqueous solution for ultrasonic extraction, and filter; (3.2) Chlorogenic acid determination: elution was performed using 0.2% phosphoric acid aqueous solution-methanol as the mobile phase, and the detection wavelength was 327 nm; (3.3) Determination of salvianolic acid B and glycyrrhizic acid: Gradient elution was performed using 0.2% phosphoric acid aqueous solution-acetonitrile as the mobile phase, and the detection wavelengths were 286 nm for salvianolic acid B and 237 nm for glycyrrhizic acid. (3.4) Berberine hydrochloride determination: elution was performed with phosphate buffer-methanol as the mobile phase, and the detection wavelength was 345 nm.

2. The method according to claim 1, wherein, The gradient elution procedure in step (1.2) is as follows: 0~9 min: The volume percentage of acetonitrile increases from 5% to 15%; 9-11 min: The volume percentage of acetonitrile remains at 15%; 11-15 min: The volume percentage of acetonitrile increases from 15% to 20%; 15~25 min: The volume percentage of acetonitrile is maintained at 20%; 25-26 min: The volume percentage of acetonitrile increases from 20% to 21%; 26-30 min: The volume percentage of acetonitrile remains at 21%.

3. The method according to claim 1, wherein, The thin-layer chromatography identification of the medicinal materials also includes the identification of licorice and / or ginseng, wherein: (2.4) Identification of licorice: Take the powder of the phlegm-stasis-relieving and pain-relieving formula, defatt it with ether, add ethyl acetate for ultrasonic extraction, evaporate the extract to dryness, dissolve the residue in methanol to prepare the test solution; use glycyrrhizin as the reference standard, use dichloromethane:methanol:formic acid:water as the developing solvent, spray with sulfuric acid ethanol solution and heat to develop color, and examine under ultraviolet light at 365nm; (2.5) Identification of ginseng: Take the powder of the phlegm-stasis-relieving and pain-relieving formula, defatt it with chloroform, moisten the residue with water, add water-saturated n-butanol for ultrasonic extraction, purify the extract with ammonia solution, take the upper layer of liquid and evaporate it to dryness, dissolve the residue in methanol to use as the test solution; use ginsenosides Rb1, Re, Rf, and Rg1 as reference standards, use ethyl acetate: acetone: water as the developing solvent, spray with sulfuric acid ethanol solution and heat to develop color, and examine under sunlight.

4. The method according to claim 1, wherein, In the identification of Coptis chinensis in (2.1), the volume ratio of petroleum ether:acetone:methanol:ammonia in the developing solvent is (2.5~3.5):(3~5):(1.5~2.5):(0.2~0.8); the concentration of the berberine hydrochloride reference solution is 0.4~0.6 mg / ml; and / or In the identification of Curcuma longa in (2.2), the volume ratio of petroleum ether to chloroform in the developing solvent is (88~80):(18~14); the concentration of the sulfuric acid ethanol solution is 8%~12%; and / or In the identification of Corydalis rhizome in (2.3), the volume ratio of petroleum ether: ethyl acetate: triethylamine in the developing solvent is (6~8):(1.5~2.5):(0.5~1.5); the alkaline silica gel G thin-layer plate is prepared using a 1% sodium hydroxide solution.

5. The method according to claim 1, wherein, In the determination of the effective component content, the isocratic elution mobile phase ratio for the determination of chlorogenic acid in step (3.2) is: 0.2% phosphoric acid aqueous solution: methanol = (88~94):(12~6).

6. The method according to claim 1, wherein, In the determination of the content of the active ingredient, the gradient elution procedure for the determination of salvianolic acid B and glycyrrhizic acid in step (3.3) is as follows: 0~15 min: Acetonitrile volume percentage remains at 18%; 15~20 min: The volume percentage of acetonitrile increases from 18% to 35%; 20-30 min: The volume percentage of acetonitrile remains at 35%.

7. The method according to claim 1, wherein, In the determination of the effective component content, in the mobile phase of the berberine hydrochloride determination in step (3.4), the phosphate buffer is a 0.05 mol / L potassium dihydrogen phosphate solution, and the pH is adjusted to 3.0 with phosphoric acid, and its volume ratio with methanol is 70:30.

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