Construction method and application of characteristic spectrum of tongfengqingxiao preparation

Abstract

The application discloses a construction method and application of a characteristic chromatogram of a gout clearing and eliminating preparation, and comprises the following steps: (1) taking a control sample, adding a dissolving solvent to prepare a control sample solution; (2) using an extraction solvent to extract the gout clearing and eliminating preparation to prepare a sample solution; (3) respectively performing high performance liquid chromatography detection on the sample solution and the control sample solution to respectively obtain a fingerprint chromatogram and a control chromatogram of the gout clearing and eliminating preparation, identifying common peaks of the fingerprint chromatogram of the gout clearing and eliminating preparation according to the control chromatogram, and constructing a characteristic chromatogram of the gout clearing and eliminating preparation. The characteristic chromatogram construction method provided by the application has 22 characteristic peaks, 6 identified characteristic peaks, good stability and repeatability, small differences between different groups of batches, good uniformity between batches, high stability, can more comprehensively control the product quality of the gout clearing and eliminating preparation, and strengthens the specificity identification and overall quality control of the gout clearing and eliminating preparation.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a method for constructing and applying a characteristic fingerprint of Tongfeng Qingxiao preparation. Background Art

[0002] Tongfeng Qingxiao formula is a classic prescription for treating gout. Its prescription is an agreed formula formed based on the ancient formula "Simiao Powder" according to clinical applications, and is composed of fifteen herbs including Atractylodis Macrocephalae Rhizoma, Atractylodis Rhizoma Praeparatum, Coicis Semen Praeparatum, Phellodendri Chinensis Cortex Praeparatum, Amomi Fructus Rotundus, Cyathulae Radix, Smilacis Glabrae Rhizoma, Lysimachiae Herba, Plantaginis Herba, Cynanchi Wilfordii Radix, Taraxaci Herba, Dioscoreae Hypoglaucae Rhizoma, Paris Polyphylla Smith, Cremastrae seu Pleiones Pseudobulbus, and Sinomenii Caulis. It is used for acute gouty arthritis with the syndrome of damp-heat accumulation. Symptoms include redness, swelling, and pain in muscles or joints, refusal to be pressed, local burning sensation upon touching, and relief when getting cool. There is a feeling of heaviness, difficulty in walking, fever, thirst, restlessness, and yellow urine. The tongue is red, the tongue coating is yellow and greasy, and the pulse is slippery and rapid, etc. This formula has been used in hospitals clinically for decades. According to the feedback from clinical patients and pharmacological experimental data, after two courses of treatment, the symptoms of gouty arthritis improved significantly, and the inflammatory factors and uric acid levels in patients also decreased significantly; pharmacological research shows that this formula mainly treats gouty arthritis by increasing uric acid metabolism and enhancing the body's immunity. The Guzhong Qingxiao Granules newly prepared and registered by the applicant's team are also prepared with Tongfeng Qingxiao as the basic formula and are hospital preparations developed in accordance with the "Regulations on the Registration and Administration of Hospital Preparations" (Jiangxi Medicine Preparation No. Z20250013000).

[0003] Regarding Tongfeng Qingxiao preparation, the current quality control mainly relies on thin-layer chromatography identification and single content determination of sinomenine, which is difficult to meet the overall quality evaluation requirements of the multi-component synergistic effect of compound preparations. In view of the complex characteristics of the components of traditional Chinese medicine compounds, the traditional Chinese medicine fingerprint technology can establish a quality evaluation model containing multiple characteristic peaks by systematically analyzing chemical substance groups. It can not only reflect the unique chemical characteristic pattern of the preparation, but also monitor the batch-to-batch stability through similarity analysis.

[0004] In order to more comprehensively and effectively control the quality of Tongfeng Qingxiao preparation in clinical use and ensure its safety and effectiveness, it is necessary to establish a fingerprint to more comprehensively control the overall quality of Tongfeng Qingxiao preparation. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing gout-clearing preparations, which rely solely on single-component quality control and lack characteristic chromatograms, making it difficult to comprehensively control the quality standards of the preparations. This invention provides a method and application for constructing characteristic chromatograms of gout-clearing preparations. This method exhibits good stability and repeatability, minimal differences between different batches, and good uniformity and high stability within the same batch. It enables more comprehensive control of the quality of gout-clearing preparations, strengthening the specific identification and overall quality control of these preparations. Furthermore, this invention optimizes the extraction method, extraction solvent, extraction time, and high-performance liquid chromatography (HPLC) detection wavelength during the preparation of the gout-clearing preparation test solution. Elution is performed using a specific gradient program to obtain fingerprint chromatograms of gout-clearing preparations with relatively good peak separation and peak shape.

[0006] The technical solution of the present invention is as follows:

[0007] The method for constructing the characteristic spectrum of a gout-clearing preparation includes the following steps:

[0008] (1) Take the reference standard and add it to the dissolving solvent to prepare a reference standard solution;

[0009] (2) Use an extraction solvent to extract the gout-clearing preparation and prepare a test solution;

[0010] (3) The test solution and the reference solution were subjected to high performance liquid chromatography to obtain the fingerprint spectrum and reference spectrum of the gout-clearing preparation, respectively. Based on the reference spectrum, the chromatographic peaks of the fingerprint spectrum of the gout-clearing preparation were identified as common peaks, and the characteristic spectrum of the gout-clearing preparation was constructed.

[0011] The conditions for high performance liquid chromatography (HPLC) detection include: a detection wavelength of 280 nm, acetonitrile as mobile phase A, and phosphoric acid aqueous solution or formic acid aqueous solution as mobile phase B, with gradient elution.

[0012] The gradient elution conditions are as follows:

[0013] From 0 to 5 minutes, the volume percentage changes of mobile phase A and mobile phase B were 5%-5% and 95%-95%, respectively.

[0014] Over 5-10 minutes, the volume percentage changes of mobile phase A and mobile phase B were 5%-7% and 95%-93%, respectively.

[0015] Over 10-25 minutes, the volume percentage changes of mobile phase A and mobile phase B were 7%-9% and 93%-91%, respectively.

[0016] Between 25 and 35 minutes, the volume percentage changes of mobile phase A and mobile phase B were 9%-12% and 91%-88%, respectively.

[0017] Over 35-50 minutes, the volume percentage changes of mobile phase A and mobile phase B were 12%-15% and 88%-85%, respectively.

[0018] Over 50-55 minutes, the volume percentage changes of mobile phase A and mobile phase B were 15%-18% and 85%-82%, respectively.

[0019] From 55 to 70 minutes, the volume percentage changes of mobile phase A and mobile phase B were 18%-20% and 82%-80%, respectively.

[0020] Over 70-80 minutes, the volume percentage changes of mobile phase A and mobile phase B were 20%-25% and 80%-75%, respectively.

[0021] Over 80-85 minutes, the volume percentage changes of mobile phase A and mobile phase B were 25%-30% and 75%-70%, respectively.

[0022] Over 85-90 minutes, the volume percentage changes of mobile phase A and mobile phase B were 30%-30% and 70%-70%, respectively.

[0023] Preferably, the gout-clearing preparation is gout-clearing granules or gout-clearing decoction.

[0024] Preferably, in step (1), the reference standard includes any one or more of sinomenine, caffeic acid, magnoflorine, astilbene, chicoric acid, and berberine hydrochloride. The concentration of the reference standard solution is 0.1-0.5 mg / ml, and the dissolving solvent is methanol. More preferably, the concentration of the reference standard solution is: sinomenine 0.312 mg / ml, caffeic acid 0.31 mg / ml, magnoflorine 0.292 mg / ml, astilbene 0.38 mg / ml, chicoric acid 0.1993 mg / ml, and berberine hydrochloride 0.318 mg / ml.

[0025] Preferably, in step (2), the extraction method is ultrasonic extraction or reflux extraction, the extraction solvent is water, methanol, or a methanol solution, the volume concentration of the methanol solution is 30-60%, and the extraction time is 15-60 min. More preferably, the extraction method is ultrasonic extraction, the ultrasonic power is 200-400W, the ultrasonic frequency is 30-50kHz, the extraction solvent is a 60% methanol aqueous solution, and the extraction time is 15 min.

[0026] Preferably, in step (3), the conditions for high-performance liquid chromatography (HPLC) detection further include: a chromatographic column packed with octadecylsilane-bonded silica gel, with a column size of 4.6 × 250 mm and a particle size of 5 μm; a column temperature of 30-38℃, a flow rate of 0.8-1.2 ml / min, and an injection volume of 10-30 μL. More preferably, the column temperature is 35℃, the flow rate is 1 ml / min, and the injection volume is 20 μL.

[0027] Preferably, the volume concentration of the phosphoric acid aqueous solution or formic acid aqueous solution is 0.1-0.5%. More preferably, the mobile phase B is a phosphoric acid aqueous solution with a volume concentration of 0.1%.

[0028] Preferably, the construction method further includes: taking the single herb of the gout-clearing preparation according to the prescription, extracting and evaporating it with water, and then extracting it with an extraction solvent (the extraction method is the same as the extraction method of the gout-clearing preparation in step (2)), preparing a test solution of the single herb, and performing high performance liquid chromatography detection to determine the peak assignment of the fingerprint spectrum of the gout-clearing preparation.

[0029] Preferably, the fingerprint spectrum of the gout-clearing preparation includes 22 common peaks, and the 6 identified characteristic peaks include: peak 6 is sinomenine, peak 7 is caffeic acid, peak 8 is magnoflorine, peak 15 is astilbene, peak 16 is chicoric acid, and peak 22 is berberine hydrochloride.

[0030] Among them, peaks 6 and 8 belong to *Sinomenium acutum*, peak 7 belongs to *Achyranthes bidentata*, *Taraxacum mongolicum*, and *Plantago asiatica*, peak 15 belongs to *Smilax glabra*, peak 16 belongs to *Taraxacum mongolicum*, and peak 22 belongs to *Achyranthes bidentata* and *Phellodendron chinense*.

[0031] Using magnoflorin peak 8 as a reference peak, the relative retention times and relative peak areas of the six characteristic peaks are as follows:

[0032] The relative retention time of peak 6 is 0.5988–0.6019, and the relative peak area is 0.3578–0.4502.

[0033] The relative retention time of peak 7 is 0.7875-0.7905, and the relative peak area is 0.2504-0.3045.

[0034] The relative retention time of peak 8 is 1.0000, and the relative peak area is 1.0000.

[0035] The relative retention time of peak 15 is 1.5610–1.5730, and the relative peak area is 0.4877–0.6120.

[0036] The relative retention time of peak 16 is 1.5953-1.6014, and the relative peak area is 0.3634-0.4664.

[0037] The relative retention time of peak 22 is 2.0608-2.0699, and the relative peak area is 0.2754-0.3275.

[0038] The present invention also provides a characteristic spectrum of the gout-clearing preparation obtained by the above construction method.

[0039] The present invention also provides the application of the method for constructing the characteristic spectrum of the gout-clearing preparation in the quality control of the gout-clearing preparation.

[0040] The beneficial effects of this invention are:

[0041] 1. The method for constructing the characteristic spectrum of the gout-clearing preparation provided by the present invention yields 22 characteristic peaks, of which 6 are identified. This method has good stability and repeatability, small differences between different batches, and good uniformity and high stability among the same batch. It can more comprehensively control the quality of the gout-clearing preparation product and strengthen the specific identification and overall quality control of the gout-clearing preparation.

[0042] 2. This invention optimizes the extraction method, extraction solvent, extraction time, and high-performance liquid chromatography detection wavelength during the preparation of the gout-clearing preparation test solution, and uses a specific gradient program for elution to obtain a fingerprint spectrum of the gout-clearing preparation with relatively good chromatographic peak separation effect and peak shape.

[0043] 3. The method for constructing the characteristic spectrum of the gout-clearing preparation provided by this invention yields a fingerprint spectrum with a large amount of information and rich peak information. Six common peaks are identified in the fingerprint spectrum, effectively separating most of the components in the gout-clearing preparation. It can provide comprehensive information on the overall medicinal components of the gout-clearing preparation. Therefore, the fingerprint spectrum can reflect the overall quality attributes of the gout-clearing preparation, which not only helps to formulate specific quality standards for the gout-clearing preparation, but also lays the foundation for the identification of the components of the gout-clearing preparation and the discovery of pharmacodynamic substances. Attached Figure Description

[0044] Figure 1 Peak attribution for the fingerprint spectrum of the Gout-Clearing Granules of the present invention (Note: S1: Cynanchum paniculatum, S2: Achyranthes bidentata, S3: Lysimachia christinae, S4: Atractylodes macrocephala, S5: Atractylodes lancea, S6: Dioscorea hypoglauca, S7: Paris polyphylla, S8: Cremastra appendiculata, S9: Taraxacum mongolicum, S10: Phellodendron chinense, S11: Plantago asiatica, S12: Amomum villosum, S13: Sinomenium acutum, S14: Coix lacryma-jobi, S15: Smilax glabra, S16: Gout-Clearing Granules);

[0045] Figure 2 To confirm the characteristic chromatogram of the gout-relieving granules of the present invention (Note: S1: gout-relieving granules, S2: reference standard);

[0046] Figure 3 The fingerprint spectrum of the gout-clearing granules under different detection wavelengths of the present invention (Note: S1: 220nm, S2: 254nm, S3: 280nm, S4: 310nm, S5: 340nm, S6: 370nm).

[0047] Figure 4The fingerprint spectrum of the gout-relieving granules under different mobile phase conditions of the present invention is shown below (Note: S1: acetonitrile-water, S2: acetonitrile-0.1% formic acid aqueous solution, S3: acetonitrile-0.05% phosphoric acid aqueous solution, S4: acetonitrile-0.1% phosphoric acid aqueous solution).

[0048] Figure 5 This study compares the similarity of fingerprint spectra of 15 batches of Gout Relief Granules of the present invention. Detailed Implementation

[0049] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0051] The gout-clearing formula of this invention consists of: Atractylodes macrocephala, stir-fried Atractylodes lancea, stir-fried Coix lacryma-jobi, Phellodendron chinense, Amomum villosum, Achyranthes bidentata, Smilax glabra, Lysimachia christinae, Plantago asiatica, Cynanchum paniculatum, Taraxacum mongolicum, Dioscorea hypoglauca, Paris polyphylla, Cremastra appendiculata, and Sinomenium acutum.

[0052] Example 1: Establishment of a detection method for Gout-Clearing Granules

[0053] 1. Instruments and Preparations

[0054] Instruments: Shimadzu LC-20AT high performance liquid chromatograph, AL-104 analytical balance (Mettler Toledo Shanghai Co., Ltd.), ultrasonic cleaner KQ-250DB (Kunshan Ultrasonic Instrument Co., Ltd.).

[0055] Preparation of Gout-Clearing Granules: Weigh out 15 medicinal materials according to the prescription dosage, including Atractylodes macrocephala (stir-fried with wheat bran), Coix lacryma-jobi (stir-fried with wheat bran), Phellodendron chinense (stir-fried with salt), Amomum villosum, Achyranthes bidentata, Smilax glabra, Lysimachia christinae, Plantago asiatica, Cynanchum paniculatum, Taraxacum mongolicum, Dioscorea hypoglauca, Paris polyphylla, Cremastra appendiculata, and Sinomenium acutum. Add water and decoct twice. For the first decoction, add 10 times the volume of water and decoct for 1 hour. For the second decoction, add 8 times the volume of water and decoct for 1 hour. Combine the decoctions, filter, concentrate the filtrate to a clear paste with a relative density of 1.15-1.25 (55-60℃), dry, pulverize, add an appropriate amount of dextrin and sucralose, mix well, make into granules, and dry to obtain Gout-Clearing Granules.

[0056] 2. Method Establishment

[0057] (1) Preparation of test solution

[0058] The proposed method for preparing the test sample is as follows: Grind the gout-clearing granules into a fine powder, take 2g, place it in a stoppered conical flask, accurately add 50ml of 60% (volume concentration) methanol aqueous solution, weigh it, sonicate for 30 minutes, cool it, weigh it again, make up the lost weight with 60% methanol aqueous solution, shake well, filter, take the filtrate, filter it through a microporous membrane, and the test sample is obtained.

[0059] (2) Preparation of reference solution

[0060] The reference solutions were prepared as follows: Berberine, caffeic acid, magnoflorine, astilbene, chicoric acid, and berberine hydrochloride were dissolved in methanol to prepare reference solutions of 0.312 mg / ml berberine, 0.31 mg / ml caffeic acid, 0.292 mg / ml magnoflorine, 0.38 mg / ml astilbene, 0.1993 mg / ml chicoric acid, and 0.318 mg / ml berberine hydrochloride, respectively.

[0061] (3) High performance liquid chromatography detection conditions

[0062] Taking into account factors such as baseline, number of peaks, and resolution of each chromatographic peak, and after gradual exploration, the proposed chromatographic conditions are as follows: using a Shimadzu LC-20AT high-performance liquid chromatograph, with Diamonsil C4 stationary phase. 18 A chromatographic column (4.6×250 mm, 5 μm) was used, with gradient elution using acetonitrile (phase A) - 0.1% phosphoric acid aqueous solution (phase B) as the mobile phase.

[0063] The gradient elution conditions are as follows:

[0064] 0-5 min, B phase 95-95 vol%, A phase 5-5 vol%;

[0065] 5-10 min, B phase 95-93 vol%, A phase 5-7 vol%;

[0066] 10-25 min, B phase 93-91 vol%, A phase 7-9 vol%;

[0067] 25-35 min, B phase 91-88 vol%, A phase 9-12 vol%;

[0068] 35-50 min, B phase 88-85 vol%, A phase 12-15 vol%;

[0069] 50-55 min, B phase 85-82 vol%, A phase 15-18 vol%;

[0070] 55-70 min, B phase 82-80 vol%, A phase 18-20 vol%;

[0071] 70-80 min, B phase 80-75 vol%, A phase 20-25 vol%;

[0072] 80-85 min, B phase 75-70 vol%, A phase 25-30 vol%;

[0073] 85-90 min, B phase 70-70 vol%, A phase 30-30 vol.

[0074] Column temperature 35℃, flow rate 1ml / min, wavelength 280nm.

[0075] Example 2: Fingerprint construction of Gout-Clearing Granules

[0076] 1. Peak attribution

[0077] Weigh out the following herbs according to the prescription proportions: Atractylodes macrocephala (Bai Zhu), stir-fried Atractylodes lancea (Cang Zhu), stir-fried Coix lacryma-jobi (Yi Yi Ren), salt-processed Phellodendron chinense (Huang Bai), Amomum villosum (Dou Zi), Achyranthes bidentata (Chuan Niu Xi), Smilax glabra (Tu Fu Ling), Lysimachia christinae (Jin Qian Cao), Plantago asiatica (Che Qian Cao), Cynanchum paniculatum (Xu Chang Qing), Taraxacum mongolicum (Pu Gong Ying), Dioscorea hypoglauca (Mian Bi Xie), Paris polyphylla (Jie Lou), Cremastra appendiculata (Shan Ci Gu), and Sinomenium acutum (Qing Feng Teng). Add 25 ml of water to each herb, decoct for 30 minutes, filter, and evaporate the decoctions to dryness. Then, prepare the test samples of each herb sequentially using the same method as in Example 1. Inject and detect the samples according to the determined chromatographic conditions. The results are shown in the figure. Figure 1 See Table 1.

[0078] Peak assignment results for the 22 identified chromatographic peaks: Peak 1 is a common peak for 12 medicinal herbs (Atractylodes macrocephala, Atractylodes lancea stir-fried with wheat bran, Coix lacryma-jobi stir-fried with wheat bran, Achyranthes bidentata, Smilax glabra, Lysimachia christinae, Cynanchum paniculatum, Taraxacum mongolicum, Dioscorea hypoglauca, Paris polyphylla, Cremastra appendiculata, and Sinomenium acutum); Peak 2 is a common peak for 9 medicinal herbs (Atractylodes macrocephala, Atractylodes lancea stir-fried with wheat bran, Coix lacryma-jobi stir-fried with wheat bran, Achyranthes bidentata, Smilax glabra, Cynanchum paniculatum, Paris polyphylla, Cremastra appendiculata, and Sinomenium acutum); Peak 3 is a common peak for 10 medicinal herbs (Coix lacryma-jobi stir-fried with wheat bran, Amomum villosum, Smilax glabra, Lysimachia christinae, Plantago asiatica, Taraxacum mongolicum, Dioscorea hypoglauca, Paris polyphylla, Cremastra appendiculata, and Sinomenium acutum). Peak 4 is a common peak for three medicinal ingredients (stir-fried coix seed, Paris polyphylla, and Atractylodes macrocephala); peaks 6, 8, and 12 belong to Sinomenium acutum; peak 7 is a common peak for three medicinal ingredients (Achyranthes bidentata, Taraxacum mongolicum, and Plantago asiatica); peak 10 belongs to stir-fried coix seed; peak 11 belongs to Lysimachia christinae; peaks 13 and 17 belong to Plantago asiatica; peaks 9, 14, 15, 18, 19, 20, and 21 belong to Smilax glabra; peaks 5 and 16 belong to Taraxacum mongolicum; and peak 22 is a common peak for Achyranthes bidentata and Phellodendron chinense.

[0079] Table 1 Peak assignments in fingerprint spectroscopy

[0080]

[0081] 2. Chromatographic peak identification

[0082] Following the chromatographic method determined above, the test solution and reference solution (berberine, caffeic acid, magnoflorine, astilbene, chicoric acid, and berberine hydrochloride) were detected respectively. The results are shown in the figure. Figure 2 .

[0083] By comparing the peak times and the spectra of the reference sample and the test sample, it was determined that peak 6 in the figure was sinomenine, peak 7 was caffeic acid, peak 8 was magnoflorine, peak 15 was astilbin, peak 16 was chicoric acid, and peak 22 was berberine hydrochloride.

[0084] Example 3: Parameter Optimization and Method Validation in Fingerprint Map Construction

[0085] 1. Parameter optimization

[0086] (1) Selection of detection wavelength

[0087] The test solution prepared in Example 1 was compared with the chromatograms at wavelengths of 220 nm, 254 nm, 280 nm, 310 nm, 340 nm, and 370 nm. The results are shown in the figure. Figure 3 The results showed that by using a diode array detector (DAD) for full scanning in the wavelength range of 190–400 nm, and based on factors such as the number of chromatographic peaks, peak response values ​​and their proportions, peak resolution and tailing factor, and baseline stability, the fingerprint spectrum of Gout Clearing Granules was determined at a wavelength of 280 nm.

[0088] (2) Selection of mobile phase type

[0089] The mobile phases investigated included acetonitrile-water, acetonitrile-0.1% formic acid aqueous solution, acetonitrile-0.1% phosphoric acid aqueous solution, and acetonitrile-0.05% phosphoric acid aqueous solution. The results are shown in [Figure number missing]. Figure 4 The results showed that when acetonitrile-0.1% phosphoric acid aqueous solution was used as the mobile phase, the fingerprint chromatogram showed better separation of chromatographic peaks and peak shapes compared to other mobile phases. Therefore, acetonitrile-0.1% phosphoric acid aqueous solution with a lower acid concentration was selected as the mobile phase.

[0090] (3) Examination of extraction methods

[0091] Grind the Gout-Clearing Granules into a fine powder, take 2g of each (two portions in total), accurately weigh them, and place them in a stoppered conical flask. Accurately add 50ml of 60% methanol aqueous solution, and treat with ultrasound (300W power, 40kHz frequency) and reflux (80℃) for 30 minutes each, respectively. Remove and cool, then weigh again. Make up the lost weight with 60% methanol aqueous solution, shake well, filter, and collect the filtrate. Measure the fingerprint chromatogram according to the established chromatographic conditions. The results are shown in Table 2. The results show that the extraction effects of the six characteristic peaks in the fingerprint chromatograms of the test sample are similar in both ultrasound and reflux extraction. Considering the convenience of operation, ultrasound extraction was chosen as the extraction method for Gout-Clearing Granules.

[0092] Table 2. Results of the investigation of characteristic peak areas using different extraction methods

[0093]

[0094] (4) Investigation of extraction solvent

[0095] Grind the Gout-Clearing Granules into a fine powder, take 2g of each, and divide into four portions. Accurately weigh each portion and place them in stoppered conical flasks. Add 50ml each of water, 30% methanol aqueous solution, 60% methanol aqueous solution, and methanol to each flask. Sonicate (300W power, 40kHz frequency) for 30 minutes. Remove and cool, then weigh again. Make up the lost weight with the respective extraction solvents, shake well, filter, and collect the filtrate. Measure the fingerprint under the established chromatographic conditions. The results are shown in Table 3. The results show that the sample treated with 60% methanol aqueous solution has a better overall peak shape, and the characteristic peak area is slightly larger than that of the samples treated with the other solvents. Therefore, 60% methanol aqueous solution is selected as the extraction solvent.

[0096] Table 3. Results of the investigation of characteristic peak areas for different extraction solvents

[0097]

[0098] (5) Extraction time

[0099] Grind the granules for gout relief into a fine powder, take 2g of each, and divide into four portions. Accurately weigh each portion and place them in a stoppered conical flask. Accurately add 50ml of 60% methanol aqueous solution. Sonicate (300W power, 40kHz frequency) for 15 minutes, 30 minutes, 45 minutes, and 60 minutes respectively. Remove and cool, then weigh again. Make up the lost weight with 60% methanol aqueous solution, shake well, filter, and collect the filtrate. Measure the fingerprint under the established chromatographic conditions. The results are shown in Table 4. The results show that the peak areas of the characteristic peaks in the sample are relatively close at different extraction times. Sonication for 15 minutes is sufficient for complete extraction. Considering time and energy saving, a sonication time of 15 minutes is selected.

[0100] Table 4. Results of characteristic peak area investigation at different extraction times

[0101]

[0102] 2. The fingerprint spectrum detection and analysis method of this invention is established.

[0103] After extensive investigation and research, the analytical method for Gout-Clearing Granules was determined as follows:

[0104] Chromatographic conditions: Diamonsil C18 column (4.6×250mm, 5μm), flow rate 1.0mL / min, column temperature 30℃, wavelength 280nm, gradient elution with acetonitrile (A)-0.1% phosphoric acid aqueous solution (B) as mobile phase.

[0105] Preparation of the test solution: Grind the granules of Gout Clearing and Relieving into a fine powder, weigh 2 g accurately, place it in a stoppered conical flask, accurately add 50 mL of 60% methanol aqueous solution, seal tightly, weigh, sonicate for 15 minutes (300 W, 40 kHz), remove and cool, weigh again, replenish the lost weight with 60% methanol aqueous solution, shake well, filter, and collect the filtrate to obtain the test solution.

[0106] Assay: Accurately pipette 20 μl of the test solution and inject it into the liquid chromatograph. Measure and record the chromatogram to obtain the result.

[0107] Example 4: Validation of the detection method for Gout-Clearing Granules in fingerprint pattern construction

[0108] 1. Repeatability test

[0109] Six test solutions were prepared according to the established analytical method and injected sequentially for analysis to assess repeatability. Using peak 8 (magnolianine) as the reference peak, the relative retention times and relative peak areas of the other characteristic peaks were calculated. The results are shown in Tables 5 and 6. The relative retention time RSD of the six characteristic peaks did not exceed 1.0%, and the relative peak area RSD did not exceed 5.0%, indicating good method repeatability.

[0110] Table 5. Results of Relative Retention Time for Fingerprint Repeatability

[0111]

[0112] Table 6. Relative peak area results of fingerprint repeatability

[0113]

[0114] 2. Stability test

[0115] One test solution was prepared according to the established analytical method, and fingerprint spectra were measured at 0h, 8h, 16h, 24h, 32h, and 48h to assess stability. Using peak 8 (magnolianine) as a reference peak, the relative retention times and relative peak areas of other characteristic peaks were calculated. The results are shown in Tables 7 and 8. The relative retention time RSD of the six characteristic peaks did not exceed 1.0%, and the relative peak area RSD did not exceed 5.0%, indicating that the solution exhibited good stability within 50 hours.

[0116] Table 7. Results of relative retention time for fingerprint spectrum stability

[0117]

[0118] Table 8. Relative peak area results of fingerprint spectrum stability

[0119]

[0120] 3. Precision test

[0121] Prepare one sample solution according to the established analytical method, and inject it six times consecutively to test the precision. Using peak 8 (magnolia alkaloid) as the reference peak, calculate the relative retention time and relative peak area of ​​the other characteristic peaks. The results are shown in Tables 9 and 10. The RSD of the relative retention time of the six characteristic peaks does not exceed 1.0%, and the RSD of the relative peak area does not exceed 5.0%, indicating that the instrument precision is good.

[0122] Table 9. Results of fingerprint precision relative retention time

[0123]

[0124] Table 10 Relative Peak Area Results of Fingerprint Precision

[0125]

[0126] 4. Similarity Evaluation

[0127] The results of the analysis, calculated using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012A version software, are shown in Tables 11-13. Figure 5 (The number 22 in parentheses after sample numbers S1 to S15 in the figure represents a total of 22 chromatographic peaks in the figure, as shown by the vertical dotted line in the figure.) The RSD of the relative retention time of the common peaks in the chromatograms of the fifteen batches of Gout Clearing Granules were all less than 2%, the RSD of the relative peak area was all less than 5%, and the chromatogram similarity was all >0.98, indicating that the differences between batches were small, the preparation process was reliable and stable, and the method was accurate.

[0128] Table 11 Relative retention times of characteristic peaks in fifteen batches of particle fingerprint spectra

[0129]

[0130] Table 12 Relative peak area results of characteristic peaks in the fingerprint spectra of fifteen batches of particles

[0131]

[0132] Table 13 Comparison Results of Similarity of Fifteen Batches of Particle Fingerprint Spectra

[0133]

[0134] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing the characteristic spectrum of a gout-clearing preparation, characterized in that: The construction method includes the following steps: (1) Take the reference standard and add it to the dissolving solvent to prepare the reference standard solution; the reference standards include sinomenine, caffeic acid, magnoflorine, astilbene, chicoric acid, and berberine hydrochloride; (2) Use an extraction solvent to extract the gout-clearing preparation and prepare a test solution; the extraction solvent is water, methanol or methanol solution; (3) The test solution and the reference solution were subjected to high performance liquid chromatography to obtain the fingerprint spectrum and reference spectrum of the gout-clearing preparation. Based on the reference spectrum, the chromatographic peaks of the fingerprint spectrum of the gout-clearing preparation were identified as common peaks, and the characteristic spectrum of the gout-clearing preparation was constructed. The conditions for high-performance liquid chromatography (HPLC) detection include: a column packed with octadecylsilane-bonded silica gel, a detection wavelength of 280 nm, a column temperature of 30-38℃, a flow rate of 0.8-1.2 ml / min, an injection volume of 10-30 μL, acetonitrile as mobile phase A, and an aqueous solution of phosphoric acid or formic acid as mobile phase B, with gradient elution. The volume concentration of the aqueous solution of phosphoric acid or formic acid is 0.1-0.5%, and the gradient elution conditions are as follows: From 0 to 5 minutes, the volume percentage changes of mobile phase A and mobile phase B were 5%-5% and 95%-95%, respectively. Over 5-10 minutes, the volume percentage changes of mobile phase A and mobile phase B were 5%-7% and 95%-93%, respectively. Over 10-25 minutes, the volume percentage changes of mobile phase A and mobile phase B were 7%-9% and 93%-91%, respectively. Between 25 and 35 minutes, the volume percentage changes of mobile phase A and mobile phase B were 9%-12% and 91%-88%, respectively. Over 35-50 minutes, the volume percentage changes of mobile phase A and mobile phase B were 12%-15% and 88%-85%, respectively. Over 50-55 minutes, the volume percentage changes of mobile phase A and mobile phase B were 15%-18% and 85%-82%, respectively. From 55 to 70 minutes, the volume percentage changes of mobile phase A and mobile phase B were 18%-20% and 82%-80%, respectively. Over 70-80 minutes, the volume percentage changes of mobile phase A and mobile phase B were 20%-25% and 80%-75%, respectively. Over 80-85 minutes, the volume percentage changes of mobile phase A and mobile phase B were 25%-30% and 75%-70%, respectively. Over 85-90 minutes, the volume percentage changes of mobile phase A and mobile phase B were 30%-30% and 70%-70%, respectively. The raw materials of the gout-clearing preparation include: Atractylodes macrocephala, stir-fried Atractylodes lancea, stir-fried Coix lacryma-jobi, salt-processed Phellodendron chinense, Amomum villosum, Achyranthes bidentata, Smilax glabra, Lysimachia christinae, Plantago asiatica, Cynanchum paniculatum, Taraxacum mongolicum, Dioscorea hypoglauca, Paris polyphylla, Cremastra appendiculata, and Sinomenium acutum.

2. The method for constructing the characteristic spectrum of the gout-clearing preparation according to claim 1, characterized in that: The gout-clearing preparation is either gout-clearing granules or gout-clearing decoction.

3. The method for constructing the characteristic spectrum of the gout-clearing preparation according to claim 1, characterized in that: In step (1), the concentration of the reference solution is 0.1-0.5 mg / ml.

4. The method for constructing the characteristic spectrum of the gout-clearing preparation according to claim 1, characterized in that: In step (2), the extraction method is ultrasonic extraction or reflux extraction, the volume concentration of the methanol solution is 30-60%, and the extraction time is 15-60 min.

5. The method for constructing the characteristic spectrum of the gout-clearing preparation according to claim 1, characterized in that: The construction method further includes: extracting and evaporating the single herb of the gout-clearing preparation according to the prescription, then extracting it again using an extraction solvent to prepare a test solution of the single herb, and performing high-performance liquid chromatography to determine the peak assignment of the fingerprint spectrum of the gout-clearing preparation.

6. The method for constructing the characteristic spectrum of the gout-clearing preparation according to claim 1, characterized in that: In step (3), the conditions for high performance liquid chromatography detection also include: column temperature 35℃, flow rate 1ml / min, injection volume 20μL; mobile phase B is a 0.1% phosphoric acid aqueous solution.

7. The method for constructing the characteristic spectrum of the gout-clearing preparation according to claim 4, characterized in that: The extraction method was ultrasonic extraction, with an ultrasonic power of 200-400W and an ultrasonic frequency of 30-50kHz. The extraction solvent was a 60% (v / v) methanol aqueous solution, and the extraction time was 15 min.

8. The method for constructing the characteristic spectrum of the gout-clearing preparation according to any one of claims 1-7, characterized in that: The fingerprint spectrum of the gout-clearing preparation includes 22 common peaks, and the identified characteristic peaks include: peak 6 is sinomenine, peak 7 is caffeic acid, peak 8 is magnoflorine, peak 15 is astilbene, peak 16 is chicoric acid, and peak 22 is berberine hydrochloride.

9. The application of a method for constructing a characteristic spectrum of the gout-clearing preparation according to claim 1 in the quality control of the gout-clearing preparation.

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