A method for detecting the characteristic spectra of anti-inflammatory granules or their intermediate extracts and its application

Abstract

This invention relates to a method for detecting characteristic chromatograms of anti-inflammatory granules or their intermediate extracts, and its application. The anti-inflammatory granules or their intermediate extracts are prepared from the following medicinal ingredients: Astragalus membranaceus, Panax notoginseng, Salvia miltiorrhiza, Paeonia lactiflora, Stephania tetrandra, Alisma plantago-aquatica, Coix lacryma-jobi, Poria cocos, and Achyranthes bidentata. The detection method is high-performance liquid chromatography (HPLC). The HPLC conditions are: an octadecylsilane-bonded silica gel column, acetonitrile as mobile phase A, and phosphoric acid aqueous solution as mobile phase B, with gradient elution. The method of this invention can obtain characteristic chromatograms with high information content and high peak response values. This method can effectively control the key quality attributes of anti-inflammatory granules and is conducive to industrial promotion, which is of great significance for the quality control standards of anti-inflammatory granules.

Description

Technical Field

[0001] This invention relates to the field of quality control of traditional Chinese medicine, specifically to a method for detecting the characteristic chromatograms of anti-inflammatory granules or their intermediate extracts and its application. Background Technology

[0002] Knee osteoarthritis is a chronic disease primarily caused by the wear and tear and degeneration of articular cartilage. Its clinical manifestations include joint stiffness, swelling, pain, deformity, and limited range of motion, resulting in a high degree of disability and making it the most common type of osteoarthritis in clinical practice. Traditional Chinese medicine considers this disease to be a type of "Bi syndrome," and its causes may be due to aging or long-term labor, leading to deficiency of liver and kidney, and insufficient Qi and blood; on the other hand, it may be due to wind, cold, and dampness obstructing the meridians, causing the tendons and bones to lose nourishment and thus leading to the disease. Therefore, the treatment of this disease should primarily focus on promoting blood circulation and removing blood stasis. In recent years, with the improvement and development of traditional Chinese medicine preparation technology, the application of external Chinese medicine preparations in the treatment of knee osteoarthritis has become increasingly widespread, but oral preparations are less common.

[0003] The prescription for Xiaozhong Granules consists of nine traditional Chinese medicines: Astragalus membranaceus, Panax notoginseng, Salvia miltiorrhiza, Paeonia lactiflora, Stephania tetrandra, Alisma plantago-aquatica, Coix lacryma-jobi, Poria cocos, and Achyranthes bidentata. This herbal composition is described in patents CN112402565B and CN112402566B. Xiaozhong Granules have the effects of promoting blood circulation, diuresis, reducing swelling, and relieving pain. They are used for knee osteoarthritis, and their clinical efficacy is definite and their safety is high.

[0004] Due to the complexity of traditional Chinese medicine (TCM) components, qualitative and quantitative analysis of single indicator components is insufficient to comprehensively reflect the quality of the tested products. Therefore, holistic quality control of multiple components should be strengthened. TCM characteristic chromatograms are a multi-indicator quality control model that can comprehensively display the characteristic information of the chemical components contained in TCM, thereby comprehensively reflecting and monitoring the quality of TCM and enabling holistic control and evaluation of TCM products. However, a comprehensive characteristic chromatogram detection method for anti-inflammatory granules has not yet been developed in this field.

[0005] Therefore, there is an urgent need in this field to develop a characteristic spectrum detection method for anti-inflammatory granules, which can obtain a characteristic spectrum with a large amount of information and identify multiple components, thereby improving the quality control level of anti-inflammatory granules. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for detecting the characteristic spectra of anti-inflammatory granules or their intermediate extracts. This method has the advantages of being comprehensive, rapid, and reliable, and can obtain relatively comprehensive characteristic spectra information of multiple components. This method provides a basis for the quality control research of anti-inflammatory granules, provides a method for the comprehensive and scientific evaluation of the quality of anti-inflammatory granules, and is of great significance to the quality control standards of anti-inflammatory granules.

[0007] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a method for detecting the characteristic spectrum of swelling-reducing granules or their intermediate extracts, wherein the swelling-reducing granules or their intermediate extracts are prepared from the following medicinal ingredients: Astragalus membranaceus, Panax notoginseng, Salvia miltiorrhiza, Paeonia lactiflora, Stephania tetrandra, Alisma plantago-aquatica, Coix lacryma-jobi, Poria cocos, and Achyranthes bidentata.

[0009] The detection method is high performance liquid chromatography;

[0010] The conditions for the high-performance liquid chromatography (HPLC) are as follows: the chromatographic column is an octadecylsilane-bonded silica column, acetonitrile is used as mobile phase A, and phosphoric acid aqueous solution is used as mobile phase B, with gradient elution.

[0011] This invention utilizes high-performance liquid chromatography (HPLC) with an octadecylsilane-bonded silica column and a specific combination of mobile phase A (acetonitrile) and mobile phase B (aqueous phosphoric acid solution) to obtain characteristic spectra with high information content and peak response values. Nine common characteristic peaks were identified, allowing for the identification of four medicinal herbs: Danshen, Chishao, Fangji, and Huangqi. Simultaneously, the characteristic peaks of three substances—paeoniflorin, verbascoside, and salvianolic acid B—were successfully identified. This avoids the limitations and biases of quality control, effectively controlling the key quality attributes of compound preparations. It offers advantages such as speed, simplicity, and comprehensiveness, high reproducibility and stability, and is conducive to industrial application. It is of significant importance for the quality control standards of anti-inflammatory granules.

[0012] Furthermore, the present invention demonstrates through methodological verification that the feature map detection method has good repeatability and high feasibility.

[0013] In this invention, the intermediate extract refers to the intermediate product obtained by extracting, concentrating, and drying the prescription medicinal ingredients, including dry extract powder.

[0014] Preferably, the detection method specifically includes the following steps:

[0015] (1) Prepare a test solution of anti-swelling granules or their intermediate extracts;

[0016] (2) Take the test solution, determine it according to the high performance liquid chromatography method, record the chromatogram, and obtain the result;

[0017] The conditions for the high-performance liquid chromatography (HPLC) are as follows: the chromatographic column is an octadecylsilane-bonded silica column, acetonitrile is used as mobile phase A, and phosphoric acid aqueous solution is used as mobile phase B, with gradient elution.

[0018] Preferably, step (1) includes: mixing the swelling-reducing granules or its intermediate extract with the extraction solvent, extracting, filtering, and obtaining the test solution.

[0019] Preferably, the extraction solvent comprises methanol or an aqueous solution thereof, more preferably methanol, a 50% aqueous methanol solution, and even more preferably a 50% aqueous methanol solution. Wherein, 50% is a volume ratio.

[0020] In the preferred embodiment of this invention, methanol is selected as the extraction solvent, resulting in a large amount of information in the final characteristic chromatogram, which can identify more medicinal flavors and is more conducive to the quality control of the swelling-reducing granules. Further preferred methods include methanol and a 50% methanol aqueous solution, which, compared to methanol aqueous solutions of other concentrations, produce chromatograms with higher resolution and more balanced peak shapes and proportions.

[0021] Preferably, the extraction method includes ultrasonic extraction, reflux extraction or shaking extraction, and the weight loss is replenished with an extraction solvent after extraction. Ultrasonic extraction is the preferred extraction method.

[0022] Preferably, the ultrasonic extraction power is 250W and the frequency is 40kHz.

[0023] Preferably, the extraction time is 15-45 minutes, such as 15 minutes, 30 minutes, 45 minutes, etc., with 30 minutes being the most preferred.

[0024] Preferably, the sample to be tested is swelling-reducing granules. Step (1) specifically includes: reducing swelling granules, grinding them into a fine powder, taking about 1g, accurately weighing it, placing it in a stoppered conical flask, accurately adding 50ml of 50% methanol, sealing it tightly, weighing it, sonicating it (power 250W, frequency 40kHz) for 30 minutes, taking it out, cooling it, weighing it again, replenishing the lost weight with 50% methanol, shaking it well, filtering it, and taking the filtrate to obtain the product.

[0025] Alternatively, the sample to be tested is an intermediate extract of anti-inflammatory granules, and the intermediate extract is a dry powder. Step (1) specifically includes: accurately weighing about 0.5g of the intermediate extract of anti-inflammatory granules, placing it in a stoppered conical flask, accurately adding 50ml of 50% methanol, sealing tightly, weighing, sonicating (power 250W, frequency 40kHz) for 30 minutes, taking it out, cooling, weighing it again, replenishing the lost weight with 50% methanol, shaking well, filtering, and taking the filtrate to obtain the final product.

[0026] Preferably, the gradient elution procedure of the high-performance liquid chromatography is as follows:

[0027] Within 0 to 10 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 5% to 11% at a constant rate.

[0028] Within 10–25 minutes, the volume ratio of mobile phase A to the total mobile phase gradually and uniformly changes from 11% to 16%.

[0029] Within 25–50 minutes, the volume ratio of mobile phase A to the total mobile phase gradually and uniformly changes from 16% to 21%.

[0030] The present invention further preferably uses the above-mentioned specific gradient for elution, resulting in a more stable baseline and better separation of peaks in the chromatogram.

[0031] Preferably, the concentration of the phosphoric acid aqueous solution is 0.05%-0.15%, for example, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, etc. In this invention, the concentration of the phosphoric acid solution refers to the volume concentration.

[0032] Preferably, the chromatographic column has a length of 100 mm and a diameter of 2.1 mm.

[0033] Preferably, the particle size of the octadecylsilane-bonded silica gel in the chromatographic column is 1.8 μm.

[0034] Preferably, the chromatographic column is selected from Agilent ZORBAX Eclipse Plus-C18 or Waters ACQVITYUPLC HSS T3.

[0035] Preferably, the chromatographic column is selected from the following models: Agilent ZORBAX Eclipse Plus-C18 (SN:USDAZ12398), Agilent ZORBAX Eclipse Plus-C18 (SN:USDAZ12801), Agilent ZORBAX Eclipse Plus-C18 (SN:USDAZ17859) or Waters ACQVITY UPLC HSS T3.

[0036] The present invention preferably uses the above-mentioned chromatographic column, which yields chromatographic peaks with greater information content and better resolution.

[0037] Preferably, the detection wavelength of the high-performance liquid chromatography is 200nm-300nm, such as 200nm, 220nm, 260nm, 280nm, 300nm, 320nm, etc., and more preferably 235nm.

[0038] The present invention further optimizes the detection wavelength to 200nm-300nm, which can obtain chromatograms with more information. Among them, the response values ​​of each chromatographic peak are the highest and the information content is the largest when the wavelength is 235nm.

[0039] Preferably, the flow rate of the high-performance liquid chromatography is 0.25-0.35 ml / min, such as 0.25 ml / min, 0.30 ml / min, 0.35 ml / min, etc.

[0040] The present invention further preferably uses a flow rate of 0.25-0.35 ml / min, which can improve the chromatographic resolution. When the flow rate is higher than 0.35 ml / min, the resolution will decrease.

[0041] Preferably, the column temperature of the high performance liquid chromatography is 30-40℃, such as 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, etc.

[0042] Preferably, the injection volume for the high-performance liquid chromatography is 2 μL.

[0043] Preferably, the chromatogram obtained by the detection method can identify the characteristic peaks of paeoniflorin, verbascoside, and salvianolic acid B.

[0044] Preferably, in the chromatogram obtained by the detection method, paeoniflorin is used as a reference peak and is taken as the S peak. The relative retention time of each peak is calculated using the reference peak. The relative retention time of the characteristic peak of the verrucoside isoflavone glucoside is 1.28-1.56, and the relative retention time of the characteristic peak of the salvianolic acid B is 2.76-3.38.

[0045] Preferably, the chromatogram obtained by the detection method can identify the characteristic peaks of Danshen, Chishao, Fangji, and Huangqi.

[0046] Preferably, in the chromatogram obtained by the detection method, paeoniflorin is used as a reference peak as the S peak, and the relative retention time of each peak is calculated using the reference peak. The relative retention times of the characteristic peaks of Salvia miltiorrhiza include 0.19–0.23, 0.34–0.42, 1.71–2.09, 2.05–2.51, and 2.76–3.38; the relative retention times of the characteristic peaks of Paeonia lactiflora include 1.00 and 1.04–1.28; the relative retention times of the characteristic peaks of Stephania tetrandra include 1.17–1.43; and the relative retention times of the characteristic peaks of Astragalus membranaceus include 1.28–1.56.

[0047] Preferably, the characteristic spectrum detection method for the swelling-reducing granules or their intermediate extracts specifically includes the following steps:

[0048] (1) Mix the swelling-reducing granules or their intermediate extract with the extraction solvent, extract, filter, and obtain the test solution;

[0049] (2) Take the test solution, determine it according to the high performance liquid chromatography method, record the chromatogram, and obtain the result;

[0050] The conditions for the high-performance liquid chromatography (HPLC) method include: an octadecylsilane-bonded silica column, a detection wavelength of 200 nm-300 nm, a flow rate of 0.25-0.35 mL / min, a column temperature of 30-40 °C, an injection volume of 2 μL, acetonitrile as mobile phase A, and 0.05%-0.15% phosphoric acid aqueous solution as mobile phase B, with elution performed according to the following gradient:

[0051] Within 0 to 10 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 5% to 11% at a constant rate.

[0052] Within 10–25 minutes, the volume ratio of mobile phase A to the total mobile phase gradually and uniformly changes from 11% to 16%.

[0053] Within 25–50 minutes, the volume ratio of mobile phase A to the total mobile phase gradually and uniformly changes from 16% to 21%.

[0054] In the preferred embodiment of the present invention, by comprehensively considering and analyzing the combined effects of conditions such as the gradient elution program of the mobile phase, the flow rate, and the column temperature on the separation and detection, the detection results are optimized, and finally a feature spectrum with a large amount of information and a high peak response value is obtained.

[0055] A second objective of this invention is to provide a quality control method for anti-inflammatory granules, wherein the quality control method includes the feature spectrum detection method described in the first objective.

[0056] Preferably, the quality control method specifically includes: using the characteristic spectrum detection method described in one of the objectives to perform quality control on the swelling-reducing granules. The obtained characteristic spectrum of the test sample should show 9 characteristic peaks corresponding to the control characteristic spectrum, and their relative retention times should be within specified values, which are: 0.19–0.23, 0.34–0.42, 1.00, 1.04–1.28, 1.17–1.43, 1.28–1.56, 1.71–2.09, 2.05–2.51, and 2.76–3.38. Products meeting these conditions are considered qualified. For example, as shown... Figure 6 As shown.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] (1) This invention utilizes high performance liquid chromatography, selects a specific combination of mobile phase A and mobile phase B, obtains characteristic spectra with large information content and high peak response values, identifies 9 common characteristic peaks, can identify four medicinal herbs: Danshen, Chishao, Fangji, and Huangqi, and successfully identifies the characteristic peaks of three substances: paeoniflorin, verbascoside, and salvianolic acid B. This avoids the singleness and one-sidedness of quality control, can effectively control the key quality attributes of swelling-reducing granules, has the advantages of being fast, simple, and comprehensive, and has high reproducibility and stability. It is also conducive to industrial promotion and has important significance for the quality control standards of swelling-reducing granules.

[0059] (2) Furthermore, the present invention has demonstrated through method verification that the characteristic spectrum detection method has good repeatability, high feasibility, and good durability for different instruments, different column temperatures, different flow rates, and different chromatographic columns. Attached Figure Description

[0060] Figure 1 This is the UPLC spectrum of Comparative Example 1.

[0061] Figure 2 This is the UPLC spectrum of Comparative Example 2.

[0062] Figure 3 This is the UPLC spectrum of Example 1.

[0063] Figure 4 This is the UPLC spectrum of Example 2.

[0064] Figure 5 This is the UPLC spectrum of Example 3.

[0065] Figure 6 This is the UPLC spectrum of Example 4.

[0066] Figure 7 This is a comparison of UPLC chromatograms of different brands of chromatographic columns in Example 5.

[0067] Figure 8 This is the UPLC spectrum (200 nm) of Example 6.

[0068] Figure 9 This is the UPLC spectrum (220 nm) of Example 7.

[0069] Figure 10 This is the UPLC spectrum (260 nm) of Example 8.

[0070] Figure 11 This is the UPLC spectrum (280 nm) of Example 9.

[0071] Figure 12 This is the UPLC spectrum (300 nm) of Example 10.

[0072] Figure 13 This is the UPLC spectrum (320 nm) of Example 11.

[0073] Figure 14 These are comparison diagrams of UPLC spectra of different extraction solvents in Examples 4, 12-13.

[0074] Figure 15 These are comparison images of UPLC spectra obtained using different extraction methods in Examples 4 and 14-15.

[0075] Figure 16 These are comparison charts of UPLC spectra from different extraction times in Examples 4 and 16-17.

[0076] Figure 17 This is the UPLC spectrum of the medicinal flavor peaks in Example 18.

[0077] Figure 18 This is the UPLC chromatogram of the reference standard from Example 18.

[0078] Figure 19 The images show the UPLC-UV chromatogram and UPLC-MS total ion current of the swelling-reducing granules from Example 18.

[0079] Figure 20 This is a UPLC comparison chart of the test sample, blank solvent, blank excipient, and reference standard in Example 19.

[0080] Figure 21 This is a superimposed graph of the instrument precision results in Example 19.

[0081] Figure 22 This is a superimposed graph of the instrument stability results in Example 19.

[0082] Figure 23 This is a superimposed graph of repeatability results from Example 19.

[0083] Figure 24 This is a superimposed UPLC diagram at different column temperatures in Example 19.

[0084] Figure 25 This is a superimposed UPLC diagram of different flow rates in Example 19.

[0085] Figure 26 This is a UPLC overlay image of different SN columns in Example 19.

[0086] Figure 27 This is a superimposed UPLC image of different instruments in Example 19.

[0087] Figure 28 This is a common pattern diagram of the characteristic spectra of 10 batches of samples in Example 19.

[0088] Figure 29 This is the comparative feature map in Example 19. Detailed Implementation

[0089] The present invention will be further described in detail below through embodiments. These embodiments are only used to illustrate the present invention and do not limit the scope of the present invention.

[0090] The test samples for the following examples and comparative examples are anti-inflammatory granules, and their formulations and preparation methods are as follows:

[0091] Prescription: Astragalus membranaceus 417g, Panax notoginseng 125g, Salvia miltiorrhiza 250g, Paeonia lactiflora 250g, Stephania tetrandra 417g, Alisma plantago-aquatica 250g, Coix lacryma-jobi 417g, Poria cocos 250g, Achyranthes bidentata 250g.

[0092] Preparation method: Take the above nine ingredients, crush Panax notoginseng, and decoct them twice with water, one hour each time. Add 10 times the amount of water for the first decoction and 8 times the amount of water for the second decoction. Filter the decoction and concentrate it to a clear extract with a relative density of 1.10-1.15 (60℃). Spray dry the extract, add 1g of sucralose and an appropriate amount of maltodextrin, mix well, and make 1000g of the product.

[0093] The instruments and reagents used in the following examples and comparative examples are as follows:

[0094] Ultra-high performance liquid chromatograph: Agilent 1290 ultra-high performance liquid chromatograph (DAD detector).

[0095] Electronic analytical balances: Tianjin Tianma Hengji Instrument Co., Ltd. TD5002C; Mettler Toledo MS204TS; Mettler Toledo XPE105.

[0096] Chromatographic columns: Agilent ZORBAX Eclipse Plus-C18 (2.1×100mm, 1.8μm) SN: USDAZ12398, Agilent ZORBAX Eclipse Plus-C18 (2.1×100mm, 1.8μm) SN: USDAZ12801, Agilent ZORBAX Eclipse Plus-C18 (2.1×100mm, 1.8μm) SN: USDAZ17859 or Waters ACQVITYUPLC HSS T3 (2.1×100mm, 1.8μm).

[0097] Reagents: Methanol was chromatographically pure and manufactured by Concord; acetonitrile was chromatographically pure and manufactured by Concord; phosphoric acid was chromatographically pure and manufactured by Tianjin Damao Chemical Reagent Co., Ltd.; water was Wahaha purified water.

[0098] Reference standard:

[0099] Tetrandrine reference standard (batch number: 110711-201810, purity: 99.6%);

[0100] Tetrandrine reference standard (batch number: 110793-202108, purity: 96.3%);

[0101] Verbena isoflavone glucoside reference standard (batch number: 111920-201907, purity: 96.8%);

[0102] Amarantrol reference standard (batch number: 111804-202206, purity: 97.2%);

[0103] Paeoniflorin reference standard (batch number: 110736-202246, purity: 96.7%);

[0104] Tanshinone B reference standard (batch number: 111562-201917, purity: 96.6%)

[0105] All the reference standards mentioned above were purchased from the National Institutes for Food and Drug Control.

[0106] Comparative Example 1

[0107] This comparative example provides a characteristic spectrum detection method for anti-inflammatory granules, as detailed below:

[0108] (1) Preparation of the anti-edema granule test solution:

[0109] Take 1g of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 50% methanol, seal tightly, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, remove it, cool it, weigh it again, make up the lost weight with 50% methanol, shake well, filter it, and take the filtrate to obtain the test sample.

[0110] (2) Determination by high performance liquid chromatography:

[0111] Chromatographic conditions: An Agilent ZORBAX Eclipse Plus-C18 column (2.1×100mm, 1.8μm) was used; acetonitrile was used as mobile phase A, and 0.2% phosphoric acid aqueous solution containing 0.4% triethylamine was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the detection wavelength was 235nm; the theoretical plate number calculated based on the paeoniflorin peak should not be less than 5000; the flow rate was 0.4mL / min; the column temperature was 35℃; and the injection volume was 2μL.

[0112] Time (min) Mobile phase A (%) Mobile phase B (%) 0～5 5→9 95→91 5～14 9 91 14～28 9→17 91→83 28～35 17→25 83→75 35～55 25 75

[0113] The chromatogram obtained in Example 1 is as follows: Figure 1 As shown, under these chromatographic conditions, the chromatographic peak resolution is poor, and no chromatographic peaks are observed after 34 minutes.

[0114] Comparative Example 2

[0115] This comparative example provides a characteristic spectrum detection method for anti-inflammatory granules, as detailed below:

[0116] (1) Preparation of the anti-edema granule test solution:

[0117] Take 1g of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 50% methanol, seal tightly, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, remove it, cool it, weigh it again, make up the lost weight with 50% methanol, shake well, filter it, and take the filtrate to obtain the test sample.

[0118] (2) Determination by high performance liquid chromatography:

[0119] Chromatographic conditions: A Waters ACQVITYUPLC BEH C18 (2.1×100mm, 1.7μm) column was used; acetonitrile was used as mobile phase A, and 0.2% phosphoric acid solution containing 0.4% triethylamine was used as mobile phase B, with gradient elution as specified in the table below; the detection wavelength was 235nm; the theoretical plate number calculated based on the paeoniflorin peak should not be less than 5000; the flow rate was 0.4mL / min; the column temperature was 35℃; and the injection volume was 2μL.

[0120] Time (min) Mobile phase A (%) Mobile phase B (%) 0～5 4→10 96→90 5～14 10 90 14～28 10→16 90→84 28～35 16→27 84→73

[0121] The chromatogram obtained from Comparative Example 2 is as follows: Figure 2 As shown, the peak resolution is poor under these chromatographic conditions.

[0122] Example 1

[0123] The only difference from Comparative Example 1 is that in step (2), mobile phase B was replaced with a 0.1% aqueous solution of phosphoric acid containing 0.4% triethylamine instead of 0.2% phosphoric acid. The chromatogram obtained in Example 1 is shown below. Figure 3 As shown.

[0124] By comparing the chromatograms of Example 1 and Comparative Example 1, it can be seen that high performance liquid chromatography using acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B can produce chromatograms with more information and higher resolution, thereby improving the quality control level of anti-inflammatory granules.

[0125] Example 2

[0126] The only difference from Example 1 is that the elution gradient is as shown in the table below:

[0127] Time (min) Mobile phase A (%) Mobile phase B (%) 0～5 5→9 95→91 5～14 9 91 14～17 9→16 91→84 17～30 16→25 84→75

[0128] The chromatogram obtained in Example 2 is as follows Figure 4As shown, the chromatogram contains a large amount of information and has a higher resolution in the 26-40 min range compared to Example 1.

[0129] Example 3

[0130] The only difference from Example 2 is that the flow rate in step (2) is replaced with 0.3 ml / min instead of 0.4 ml / min.

[0131] The chromatogram obtained in Example 3 is as follows: Figure 5 As shown, the chromatogram contains a large amount of information and has a high peak response value. Compared with Example 2, the resolution is further improved in the range of 22-30 min.

[0132] Example 4

[0133] The only difference from Example 3 is that the elution gradient is as shown in the table below:

[0134]

[0135]

[0136] The chromatogram obtained in Example 4 is as follows Figure 6 As shown, compared with Examples 1-3, the spectrum has a more stable baseline and better separation of peaks. Therefore, the present invention further prefers to use the elution gradient of Example 4.

[0137] Example 5

[0138] The only difference from Example 4 is that the chromatographic column was replaced from an Agilent ZORBAX Eclipse Plus-C18 (2.1×100mm, 1.8μm) to a Waters ACQVITY UPLC HSS T3 (2.1×100mm, 1.8μm).

[0139] Chromatograms obtained by comparing Examples 4 and 5 ( Figure 7 As can be seen, all of the above chromatographic columns can obtain spectra with rich chromatographic information and good resolution.

[0140] Examples 6-11

[0141] Based on Example 4, chromatograms obtained by different detection wavelengths were compared. The detection wavelengths were 200nm (Example 6), 220nm (Example 7), 260nm (Example 8), 280nm (Example 9), 300nm (Example 10), and 320nm (Example 11).

[0142] By comparing the chromatograms of Examples 4 and 6-11 ( Figure 6 , 8-13) It can be seen that setting the detection wavelength in the range of 200-300 nm yields a spectrum with more information, among which, when the wavelength is 235 nm ( Figure 6 Each chromatographic peak has the highest response value and the largest amount of information.

[0143] Examples 12-13

[0144] The only difference from Example 4 is that the extraction solvent in step (1) was replaced by methanol (Example 12) and 80% methanol (Example 13) instead of 50% methanol.

[0145] Comparative chromatograms obtained from Examples 4 and 12-13 Figure 14 As can be seen, compared with using 80% methanol aqueous solution as solvent, the test samples prepared with methanol or 50% methanol aqueous solution as solvent have better resolution of each chromatographic peak and more balanced peak shape and proportion. Therefore, the present invention further prefers methanol or 50% methanol as solvent to prepare the test sample solution to obtain characteristic chromatograms with better resolution and more information.

[0146] Examples 14-15

[0147] The only difference from Example 4 is that the extraction method in step (1) is changed from ultrasonic extraction to reflux extraction (Example 14) and shaking extraction (Example 15).

[0148] The chromatograms obtained by comparing Examples 4 and 14-15 ( Figure 15 As can be seen, there is little difference between different extraction methods. To facilitate operation, this invention further optimizes ultrasonic extraction.

[0149] Examples 16-17

[0150] The only difference from Example 4 is that the extraction time in step (1) is replaced by 15 minutes (Example 16) and 45 minutes (Example 17) instead of 30 minutes.

[0151] The chromatograms obtained by comparing Examples 4 and 16-17 ( Figure 16 As can be seen, there is no significant difference between different extraction times. To save time while ensuring complete extraction of the sample, this invention further optimizes the extraction time to 30 minutes.

[0152] Example 18: Characteristic Map Analysis of Anti-Swelling Granules

[0153] The following analysis is performed on the feature map obtained in Example 4:

[0154] 1. Assignment of chromatographic peaks of medicinal herbs

[0155] (1) Weigh out 0.5004g of Astragalus membranaceus, 0.5007g of Panax notoginseng, 0.5014g of Salvia miltiorrhiza, 0.5009g of Paeonia lactiflora, 0.5005g of Stephania tetrandra, 0.5015g of Alisma plantago-aquatica, 0.5006g of Coix lacryma-jobi, 0.5007g of Poria cocos, and 0.5014g of Achyranthes bidentata. Add 30ml of water to each herb, decoct for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 50% methanol, dilute to 50ml in a volumetric flask, filter, and collect the filtrate to obtain the decoction of each herb, which is used for single herb identification.

[0156] (2) Inject 2 μl of each of the above-mentioned medicinal slices solutions into an ultra-high performance liquid chromatograph and perform detection under the same chromatographic conditions as in Example 4 to obtain the chromatogram of the single-herb test sample solution.

[0157] (3) Compare the chromatogram of the single herb slices with the chromatogram of the test sample obtained in Example 4, such as... Figure 17 As shown in the chromatogram, Danshen, Chishao, Fangji, and Huangqi all have corresponding chromatographic peaks in the test sample. Among them, peaks 1, 2, 7, 8, and 9 belong to Danshen; peaks 3 and 4 belong to Chishao; peak 5 belongs to Fangji; and peak 6 belongs to Huangqi.

[0158] 2. Chromatographic peak identification

[0159] (1) Take appropriate amounts of each reference standard (tetracycline, tetracycline, and salvianolic acid B) and add methanol to prepare a solution containing 50 μg per ml; take appropriate amounts of the reference standard (verrucine isoflavone glucoside) and add methanol to prepare a solution containing 20 μg per ml; take appropriate amounts of the reference standards (amarantrolone and paeoniflorin) and add methanol to prepare a solution containing 10 μg per ml, thus obtaining the solutions of each reference standard.

[0160] (2) Inject 2 μl of the above reference solution into an ultra-high performance liquid chromatograph and perform detection under the same chromatographic conditions as in Example 4 to obtain the chromatogram of the reference solution.

[0161] (3) Compare the chromatograms of each reference solution with the chromatogram of the test sample obtained in Example 6, such as... Figure 18 As shown, after comparing the retention time with the reference standard, peak 3 in the characteristic spectrum of the swelling-reducing granules is paeoniflorin, peak 6 is salvia miltiorrhiza glucoside, and peak 9 is salvianolic acid B.

[0162] (4) Qualitative analysis of the chemical components of the swelling-reducing granules was performed using UPLC-Q-TOF-MS, and the characteristic chromatographic peaks were identified and deduced. UPLC-UV chromatograms and total ion chromatograms (positive mode) are shown below. Figure 19 Since the liquid phase of the liquid chromatography-mass spectrometry (LC-MS) instrument is UPLC, its chromatogram differs somewhat from that of the UPLC fingerprint spectrum. The specific peak correspondence is shown in Table 1.

[0163] Table 1. Characteristic spectrum of Xiaozhong Granules: Herb composition and peak identification.

[0164]

[0165] The results showed that, after comparison with the reference standard and each medicinal herb slice, each medicinal herb slice and its corresponding actual substance (test sample) had corresponding chromatographic peaks. The chromatographic peaks with good stability and suitable response values ​​in the characteristic chromatograms were selected as characteristic peaks, and a total of 9 characteristic peaks were identified. Among them, peaks 1, 2, and 7-9 are characteristic peaks of Salvia miltiorrhiza, peaks 3 and 4 are characteristic peaks of Paeonia lactiflora, peak 5 is characteristic peak of Stephania tetrandra, and peak 6 is characteristic peak of Astragalus membranaceus.

[0166] 3. Selection of reference point

[0167] Compared with the reference standard, the chromatographic peak at 15.543 min (peak 3) was paeoniflorin, the chromatographic peak at 21.925 min (peak 5) was verbascoside glucoside, and the chromatographic peak at 43.159 min (peak 9) was salvianolic acid B. Among them, the paeoniflorin chromatographic peak had moderate absorption intensity and retention time, stable response, and achieved baseline separation. Therefore, paeoniflorin was selected as the reference peak and labeled as peak S. The relative retention of each peak was calculated using the reference peak for evaluation.

[0168] Example 19 Methodological Investigation

[0169] The following methodological investigations were conducted based on the sample preparation method and chromatographic conditions of Example 4:

[0170] 1. Examination of specificity and integrity

[0171] To investigate whether the blank solvent interfered with the characteristic chromatogram of the anti-inflammatory granules, the test solution, reference solution, blank solvent (50% methanol), and excipient blank were precisely pipetted and analyzed according to the chromatographic conditions of Example 4. Simultaneously, the integrity of the sample was examined by extending the sampling time by 30 minutes (using the highest organic phase concentration in the gradient table, isocratic elution). The results showed that the common peak positions of the blank solvent and excipient blank did not interfere with each other, and there were essentially no chromatographic peaks after 50 minutes. The results generally met the principle of maximizing information content. Figure 20 As shown.

[0172] 2. Instrument precision test

[0173] Take the test sample, grind it finely, and accurately weigh approximately 1 g. Perform the determination under the chromatographic conditions described in Example 4, injecting the sample six times consecutively. The characteristic chromatogram of the test sample should show a peak with the same retention time as the reference peak. Using the S peak as a reference, calculate the relative retention times of the remaining peaks. See [link to specific determination results] for details. Figure 21 (The numbers 1-6 in the figure represent the numbering of 6 injections), Table 2.

[0174] Table 2. Results of relative retention time in instrument precision test

[0175]

[0176] The results showed that the relative retention times were basically consistent, RSD < 5.0%, and the instrument precision was good.

[0177] 3. Stability test

[0178] Take the test sample, grind it finely, and accurately weigh approximately 1 g. Perform the determination under the chromatographic conditions described in Example 4, injecting the sample at 0, 2, 4, 8, 12, and 24 hours. The characteristic chromatogram of the test sample should show a peak with the same retention time as the reference peak. Using the S peak as a reference, calculate the relative retention times of the remaining peaks. See [link to specific results] for details. Figure 22 Table 3.

[0179] Table 3. Results of relative retention times in stability tests

[0180]

[0181] The results showed that the relative retention times were basically consistent, with RSD < 5.0%, indicating that the test solution had good stability within 24 hours.

[0182] 4. Repeatability test

[0183] Take the test sample, grind it finely, and accurately weigh approximately 1g (6 portions in total). Perform the determination under the chromatographic conditions described in Example 4. The characteristic chromatogram of the test sample should show a peak with the same retention time as the reference peak. Using the S peak as a reference, calculate the relative retention times of the remaining peaks. See [link to specific determination results] for details. Figure 23 (Numbers 1-6 in the figure represent the numbers of the 6 samples), Table 4.

[0184] Table 4. Results of Relative Retention Times in Repeatability Tests

[0185]

[0186] The results show that the relative retention times are basically consistent, with RSD < 5.0%, indicating that the method has good repeatability.

[0187] 5. Durability test

[0188] The robustness of the test sample to the chromatographic conditions was investigated under different column temperatures, flow rates, different SN numbers of the same type of column, and two different instruments. The characteristic chromatogram of the test sample should show a peak with the same retention time as the reference peak. Using the S peak as a reference, the relative retention times of the remaining peaks were calculated. The specific results are shown in Tables 5 to 8. Figures 24-27 ,in, Figure 24 This is a superimposed graph of UPLC at different column temperatures. Figure 25 UPLC overlay plots at different flow rates Figure 26 UPLC overlays for columns with different serial numbers (1-Agilent ZORBAX Eclipse Plus-C18 (2.1×100mm, 1.8μm) SN: USDAZ12398; 2-Agilent ZORBAX Eclipse Plus-C18 (2.1×100mm, 1.8μm) SN: USDAZ12801; 3-Agilent ZORBAX Eclipse Plus-C18 (2.1×100mm, 1.8μm) SN: USDAZ17859). Figure 27 This is a superimposed UPLC image from different instruments.

[0189] Table 5. Results of relative retention times at different column temperatures

[0190]

[0191] The results show that the relative retention times are basically consistent at different column temperatures, with RSD < 10.0%, indicating that the method has good robustness.

[0192] Table 6. Results of relative retention time for different flow velocities

[0193]

[0194] The results show that the relative retention time is basically the same for different flow rates, with RSD < 10.0%, indicating that the method has good robustness.

[0195] Table 7. Relative retention times of chromatographic columns of the same model but different SN numbers.

[0196]

[0197] The results showed that the relative retention times of chromatographic columns of the same model but different SNs were basically consistent, with RSD < 10.0%, indicating that the method had good robustness.

[0198] Table 8. Results of relative retention times for different instruments

[0199]

[0200] The results show that the relative retention times of different instruments are basically consistent, with RSD < 10.0%, indicating that the method has good robustness.

[0201] 6. Method Validation

[0202] The chromatographic method described in Example 4 was used to determine the chromatographic values ​​of 10 batches of samples. Using the peak corresponding to the paeoniflorin reference as the S peak, the relative retention times of each characteristic peak and the S peak in the 10 batches of samples (230318-01, 230318-02, 230318-03, 240201, 240202, 240203, 231226-01, 241226-02, 241226-03, 230601) were calculated. Using the "Software System for Similarity Evaluation of Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" issued by the National Pharmacopoeia Commission, the AIA files of the 10 batches of UPLC chromatograms were exported. Chromatographic peaks were automatically matched to form a common pattern diagram, and a reference chromatogram was generated. The results are shown in [Figure Number]. Figure 28 (R-comparison feature maps, S1-230318-01, S2-230318-02, S3-230318-03, S4-231226-01, S5-231226-02, S6-231226-03, S7-240201, S8-240202, S9-240203, S10-230601) and Figure 29 (Peak 3(S): paeoniflorin, peak 6: verbascoside, peak 9: salvianolic acid B), and Table 9.

[0203] Table 9. Relative retention time results for 10 batches of samples.

[0204]

[0205] Conclusion: The relative retention times of the nine characteristic peaks and the S peak in the 10 batches of samples were all within the mean ±10%, indicating that the detection method provided by this invention is highly feasible.

[0206] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for detecting the characteristic chromatogram of an anti-inflammatory granule or its intermediate extract, wherein the anti-inflammatory granule or its intermediate extract is prepared from the following medicinal ingredients: Astragalus membranaceus, Panax notoginseng, Salvia miltiorrhiza, Paeonia lactiflora, Stephania tetrandra, Alisma plantago-aquatica, Coix lacryma-jobi, Poria cocos, and Achyranthes bidentata; Its features The detection method is high performance liquid chromatography; The conditions for the high-performance liquid chromatography (HPLC) are as follows: the chromatographic column is an octadecylsilane-bonded silica column, acetonitrile is used as mobile phase A, and phosphoric acid aqueous solution is used as mobile phase B, with gradient elution.

2. The feature map detection method according to claim 1, characterized in that, The detection method specifically includes the following steps: (1) Prepare a test solution of anti-swelling granules or their intermediate extracts; (2) Take the test solution, determine it according to the high performance liquid chromatography method, record the chromatogram, and obtain the result; The conditions for the high-performance liquid chromatography (HPLC) are as follows: the chromatographic column is an octadecylsilane-bonded silica column, acetonitrile is used as mobile phase A, and phosphoric acid aqueous solution is used as mobile phase B, with gradient elution.

3. The feature map detection method according to claim 2, characterized in that, Step (1) includes: mixing the swelling-reducing granules or its intermediate extract with the extraction solvent, extracting, filtering, and obtaining the test solution; Preferably, the extraction solvent includes methanol or an aqueous solution thereof, more preferably methanol, a 50% aqueous methanol solution, and even more preferably a 50% aqueous methanol solution; Preferably, the extraction method includes ultrasonic extraction, reflux extraction or shaking extraction, and the weight loss is replenished with extraction solvent after extraction. The preferred extraction method is ultrasonic extraction. Preferably, the ultrasonic extraction power is 250W and the frequency is 40kHz; Preferably, the extraction time is 15-45 minutes.

4. The feature map detection method according to claim 1 or 2, characterized in that, The gradient elution procedure for the high-performance liquid chromatography is as follows: Within 0 to 10 minutes, the volume ratio of mobile phase A to the total mobile phase gradually changes from 5% to 11% at a constant rate. Within 10–25 minutes, the volume ratio of mobile phase A to the total mobile phase gradually and uniformly changes from 11% to 16%. Within 25–50 minutes, the volume ratio of mobile phase A to the total mobile phase gradually and uniformly changes from 16% to 21%.

5. The feature map detection method according to claim 1 or 2, characterized in that, The chromatographic column is 100 mm long and 2.1 mm in diameter; Preferably, the particle size of the octadecylsilane-bonded silica gel in the chromatographic column is 1.8 μm; Preferably, the chromatographic column is selected from Agilent ZORBAX Eclipse Plus-C18 or Waters ACQVITYUPLCHSS T3.

6. The feature map detection method according to claim 1 or 2, characterized in that, The detection wavelength of the high-performance liquid chromatography is 200nm-300nm, preferably 235nm.

7. The feature map detection method according to claim 1 or 2, characterized in that, The flow rate for the high-performance liquid chromatography method is 0.25-0.35 ml / min.

8. The feature map detection method according to claim 1 or 2, characterized in that, The column temperature for the high-performance liquid chromatography method is 30–40 °C.

9. The feature map detection method according to claim 1 or 2, characterized in that, The characteristic peaks of paeoniflorin, verbascoside, and salvianolic acid B can be identified in the chromatogram obtained by the detection method. Preferably, in the chromatogram obtained by the detection method, paeoniflorin is used as a reference peak and as the S peak. The relative retention time of each peak is calculated using the reference peak. The relative retention time of the characteristic peak of the verbascoside isoflavone glucoside is 1.28-1.56, and the relative retention time of the characteristic peak of salvianolic acid B is 2.76-3.

38. Preferably, the chromatogram obtained by the detection method can identify the characteristic peaks of Danshen, Chishao, Fangji, and Huangqi; Preferably, in the chromatogram obtained by the detection method, paeoniflorin is used as a reference peak as the S peak, and the relative retention time of each peak is calculated using the reference peak. The relative retention times of the characteristic peaks of Salvia miltiorrhiza include 0.19–0.23, 0.34–0.42, 1.71–2.09, 2.05–2.51, and 2.76–3.38; the relative retention times of the characteristic peaks of Paeonia lactiflora include 1.00 and 1.04–1.28; the relative retention times of the characteristic peaks of Stephania tetrandra include 1.17–1.43; and the relative retention times of the characteristic peaks of Astragalus membranaceus include 1.28–1.

56.

10. A quality control method, characterized in that, The quality control method includes the feature map detection method according to any one of claims 1-9; Preferably, the quality control method specifically includes: using the characteristic spectrum detection method according to any one of claims 1-9 to perform quality control on the swelling-reducing granules. The obtained characteristic spectrum of the test sample should show 9 characteristic peaks corresponding to the control characteristic spectrum, and their relative retention times should be within the specified values, which are: 0.19-0.23, 0.34-0.42, 1.00, 1.04-1.28, 1.17-1.43, 1.28-1.56, 1.71-2.09, 2.05-2.51, and 2.76-3.

38. Products that meet the above conditions are qualified products.

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