Construction method and application of dehumidification guling decoction fingerprint spectrum

Abstract

This invention provides a method for constructing and applying a fingerprint spectrum of Chushi Weiling Decoction, belonging to the field of drug detection technology. The method involves first preparing a test solution using Chushi Weiling Decoction, then using acetonitrile (A)-0.18~0.22wt% formic acid aqueous solution as the mobile phase, and obtaining the fingerprint spectrum of Chushi Weiling Decoction through high-performance liquid chromatography (HPLC). A standard fingerprint spectrum is then established using the "Similarity Evaluation System for Chromatographic Fingerprint Spectrum of Traditional Chinese Medicine" for quality evaluation or control throughout the research, development, production, and clinical application of Chushi Weiling Decoction. This invention's method for constructing the fingerprint spectrum of Chushi Weiling Decoction enables the detection of the main medicinal components in Chushi Weiling Decoction solely through HPLC, thus providing a more comprehensive reflection of the quality status of Chushi Weiling Decoction.

Description

Technical Field

[0001] This invention relates to drug detection technology, and more particularly to a method for constructing and applying a fingerprint spectrum of the dampness-removing Weiling Decoction. Background Technology

[0002] Chushi Weiling Decoction, from Volume 4 of "Orthodox Manual of Surgery", has the effects of clearing heat and removing dampness, strengthening the spleen and promoting diuresis. It is mainly used to treat erysipelas caused by damp-heat stagnation in the spleen and lung meridians, which is characterized by festering and painful sores. Erysipelas of the waist (commonly known as snake-like sores) is characterized by dampness, with yellowish-white blisters of varying sizes, festering and oozing fluid, and is more painful than the dry type.

[0003] The main ingredients of the Weiling Decoction for Eliminating Dampness are: tangerine peel, gardenia, saposhnikovia root, ginger-processed magnolia bark, stir-fried atractylodes rhizome, polyporus umbellatus, alisma rhizome, red poria cocos, stir-fried atractylodes rhizome, akebia stem, and talc, 1 mace each; licorice root and cinnamon bark, 3 fen each; 2 cups of water; 20 stalks of rush pith (1.5m); decoct until reduced to 8 fen; and take before meals.

[0004] Because the prepared Weiling Decoction for Removing Dampness loses the morphological characteristics of the original medicinal slices, simple qualitative identification and quantitative analysis of indicator components are insufficient to reflect its quality. As a standard reference for measuring whether classic Chinese medicine formulas are basically consistent with clinical decoctions, its quality should be strengthened through specific identification and multi-component, overall quality control.

[0005] Traditional Chinese medicine (TCM) preparation chromatograms refer to chromatograms established by appropriately processing TCM preparation samples and employing suitable analytical methods. These chromatograms reflect information about multiple components and demonstrate their quality characteristics. TCM preparation characteristic chromatograms are of great significance for identifying key quality attributes of TCM preparations, studying the transfer of measurement values, evaluating the uniformity and stability of TCM preparation quality, and improving the overall quality control level of TCM preparations.

[0006] Therefore, by establishing a feature map, the overall quality of Chushi Weiling Decoction can be comprehensively controlled. Summary of the Invention

[0007] To address the above problems, this invention provides a method for constructing and applying the fingerprint spectrum of the dampness-removing Weiling Decoction.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for constructing a fingerprint spectrum of the dampness-removing Weiling Decoction, the method comprising the following steps:

[0010] S1. Prepare the test solution using the dampness-removing Weiling Decoction;

[0011] S2. Take the test solution and perform high performance liquid chromatography to obtain the fingerprint spectrum of the dehumidifying and stomachic decoction.

[0012] The mobile phase A for the high-performance liquid chromatography detection is acetonitrile, and the mobile phase B is an aqueous solution of formic acid with a concentration of 0.18~0.22wt%.

[0013] The elution method for the high-performance liquid chromatography detection is gradient elution;

[0014] The elution conditions for the gradient elution are as follows:

[0015] 0~70min, 8%→20% mobile phase A, 92%→80% mobile phase B;

[0016] 70~105min, 20%→37% mobile phase A, 80%→63% mobile phase B;

[0017] 105~120min, 37%→70% mobile phase A, 63%→30% mobile phase B;

[0018] 120~130 min, 70% mobile phase A, 30% mobile phase B.

[0019] Furthermore, the detection wavelength of the high-performance liquid chromatography is 260~270nm.

[0020] Furthermore, the test solution is prepared by passing the dehumidifying Weiling Decoction liquid through a D101 macroporous adsorption resin column, washing with water until no obvious color is observed, discarding the aqueous solution, eluting with an ethanol aqueous solution, collecting the eluent, evaporating it to dryness in a water bath, dissolving the residue in a methanol aqueous solution, shaking well, filtering, and obtaining the test solution.

[0021] Furthermore, the concentration of the ethanol-water solution used for elution is 90-98 vol%, and the volume is 45-60 mL.

[0022] Furthermore, the concentration of the methanol-water solution used for dissolution is 40-60 vol.

[0023] Furthermore, the liquid form of the dampness-removing Weiling Decoction is made by dissolving the dry powder of the dampness-removing Weiling Decoction in water;

[0024] Alternatively, it can be prepared by diluting the concentrated Weiling Decoction for Dampness Removal with water.

[0025] Alternatively, the decoction of Weiling Decoction for Removing Dampness can be used directly as the liquid form of Weiling Decoction for Removing Dampness;

[0026] When the dry powder of the dehumidifying stomach-soothing decoction is dissolved in water to prepare the liquid dehumidifying stomach-soothing decoction, the weight-to-volume ratio of the dry powder of the dehumidifying stomach-soothing decoction to the prepared test solution is 0.064~0.096g:1mL;

[0027] When the concentrated dehumidifying Weiling Decoction is diluted with water to prepare the dehumidifying Weiling Decoction liquid, the volume ratio of the concentrated dehumidifying Weiling Decoction to the prepared test solution is 0.8~1.2mL:1mL;

[0028] When using the decoction of Weiling Decoction for Dampness Removal directly as the liquid form of Weiling Decoction for Dampness Removal, the volume ratio of the decoction of Weiling Decoction for Dampness Removal to the prepared test solution is 4~6mL:1mL.

[0029] Furthermore, the flow rate for the high-performance liquid chromatography detection is 0.8~1.2 mL / min, the column temperature is 40~50℃, and the injection volume is 5~20 μL;

[0030] The construction method also includes high-performance liquid chromatography detection of a reference solution made of hesperidin.

[0031] The application of a standard fingerprint spectrum of Chushi Weiling Decoction obtained using the above construction method in the quality evaluation or control of the entire process of Chushi Weiling Decoction research / development / production / clinical application.

[0032] Furthermore, the standard fingerprint spectrum of the aforementioned Weiling Decoction for Removing Dampness identified 21 common peaks.

[0033] Among them, peak 1 is genipin-1-gentiopicroside, peak 2 is geniposide, peak 3 is magnolinoside B, peak 4 is glucosylapigenin, peak 5 is cimicifugin glycoside, peak 6 is glycyrrhizin, peak 7 is rutin, peak 8 is cimicifugin, peak 9 is rutin, peak 10 is 5-O-methylvisamidol glycoside, peak 11(S) is hesperidin, and peak 12 is 6'-O-p-coumaryl quercetin gentiopicroside. The peaks are: disaccharide 13 (crocin I), peak 14 (citrus glycoside), peak 15 (piperidin), peak 16 (glucosyl naringin), peak 17 (ammonium glycyrrhizate), peak 18 (3,5,6,7,3',4',5'-heptamethoxyflavone), peak 19 (5,6,7,8,4'-pentamethoxyflavone), peak 20 (honokiol), and peak 21 (honokiol).

[0034] Furthermore, the standard fingerprint spectrum was established using the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine".

[0035] The beneficial effects of the method for constructing the fingerprint spectrum of the dampness-removing Weiling Decoction of the present invention and its application are as follows:

[0036] The method for constructing the fingerprint spectrum of the dampness-removing Weiling Decoction of the present invention can detect the main medicinal components in the dampness-removing Weiling Decoction by high performance liquid chromatography, thereby more comprehensively reflecting the quality status of the dampness-removing Weiling Decoction.

[0037] This invention combines the different physicochemical properties of various effective components in the Chushi Weiling Decoction, and by adjusting the elution conditions, obtains a standard fingerprint spectrum with good separation, a relatively stable baseline, more peaks, and a better reflection of the quality of the Chushi Weiling Decoction; the elution conditions of this invention are significantly better than other elution conditions.

[0038] This invention, by adjusting the mobile phase, obtains a standard fingerprint spectrum with good separation, a relatively stable baseline, more peaks, and a better reflection of the quality of the dampness-removing Weiling Decoction.

[0039] This invention achieves the goal of effectively separating and simultaneously detecting different active ingredients in the dampness-removing and stomach-strengthening decoction by adjusting the detection wavelength; the detection wavelength of this invention is significantly superior to other detection wavelengths.

[0040] This invention, by adjusting the preparation method of the test solution, can more fully extract the medicinal components involved in the dampness-removing Weiling Decoction, and the concentration of each medicinal component in the obtained test solution can be separated and detected under the same high performance liquid chromatography conditions, resulting in good peak elution in the obtained chromatogram.

[0041] This invention effectively optimizes separation conditions by selecting appropriate chromatographic columns, column temperatures, flow rates, and other process conditions, thereby improving the peak stability of the standard fingerprint spectrum of the dampness-removing Weiling Decoction.

[0042] The construction method of the present invention also has good specificity and integrity, precision, stability, repeatability and durability;

[0043] The standard fingerprint spectrum of the dampness-removing Weiling Decoction obtained by this invention can provide effective assurance for the quality monitoring of the dampness-removing Weiling Decoction throughout the entire process of research, development, production and clinical application.

[0044] By utilizing the presence and characteristics of common characteristic peaks in the standard fingerprint spectrum of the dampness-removing Weiling Decoction obtained by this invention, the product quality of the dampness-removing Weiling Decoction can be comprehensively monitored. By comparing the similarity of chromatographic fingerprint characteristics, the quality of the dampness-removing Weiling Decoction can be evaluated, and its stability and uniformity can be examined, thus making up for the deficiencies of the current quality control methods. At the same time, the stability of the production process of the dampness-removing Weiling Decoction can also be monitored to ensure its quality is stable, uniform, and controllable.

[0045] The standard fingerprint spectrum of Chushi Weiling Decoction obtained by this invention improves the quality control standards of Chushi Weiling Decoction finished and semi-finished products, effectively prevents product counterfeiting, and ensures the normal production and circulation order of Chushi Weiling Decoction. Based on this invention, correlation studies between fingerprint spectrum information and pharmacodynamic information can also be carried out to further clarify the correlation between the intrinsic chemical components of Chushi Weiling Decoction and the efficacy of the preparation. Attached Figure Description

[0046] Figure 1 These are the results of the examination of the test solution preparation method in Example 1 of the present invention; wherein, from top to bottom, numbers 1-3 are the characteristic spectra of test solution three, the characteristic spectra of test solution one, and the characteristic spectra of test solution two, respectively;

[0047] Figure 2This is a 3D fingerprint spectrum from the detection wavelength investigation in Embodiment 1 of the present invention;

[0048] Figure 3 This refers to the absorption spectrum, such as the fingerprint spectrum, used in the detection wavelength examination of Embodiment 1 of the present invention.

[0049] Figure 4 This is the HPLC chromatogram at a wavelength of 230 nm used in the detection wavelength study of Example 1 of the present invention;

[0050] Figure 5 This is the HPLC chromatogram at a wavelength of 240 nm used in the detection wavelength study of Example 1 of the present invention;

[0051] Figure 6 This is the HPLC chromatogram at a wavelength of 250 nm used in the detection wavelength study of Example 1 of the present invention;

[0052] Figure 7 This is the HPLC chromatogram at a wavelength of 260 nm used in the detection wavelength study of Example 1 of the present invention;

[0053] Figure 8 This is the HPLC chromatogram at a wavelength of 265 nm used in the detection wavelength study of Example 1 of the present invention;

[0054] Figure 9 This is the HPLC chromatogram at a wavelength of 270 nm used in the detection wavelength study of Example 1 of the present invention;

[0055] Figure 10 This is the HPLC chromatogram at a wavelength of 280 nm used in the detection wavelength study of Example 1 of the present invention;

[0056] Figure 11 This is the HPLC chromatogram at a wavelength of 300 nm used in the detection wavelength study of Example 1 of the present invention;

[0057] Figure 12 This is the chromatographic attribution of each medicinal flavor peak in Example 1 of the present invention. Figure 1 Among them, from top to bottom, numbers 1-6 are the characteristic spectra of licorice, saposhnikovia, magnolia bark, gardenia, tangerine peel, and the test solution, respectively.

[0058] Figure 13 This is the chromatographic attribution of each medicinal flavor peak in Example 1 of the present invention. Figure 2 Among them, from top to bottom, numbers 1-9 are the characteristic chromatograms of stir-fried Atractylodes macrocephala, stir-fried Atractylodes lancea, red Poria cocos, Polyporus umbellatus, Akebia trifoliata, Alisma plantago-aquatica, Juncus effusus, Cinnamomum cassia, and the characteristic chromatogram of the test solution.

[0059] Figure 14This is the reference peak identification chromatogram in Example 1 of the present invention. Figure 1 Among them, from top to bottom, numbers 1-6 are the characteristic spectra of glycyrrhizin, the characteristic spectra of ammonium glycyrrhizate, the characteristic spectra of magnolol, the characteristic spectra of magnolol, the characteristic spectra of the test solution, and the characteristic spectra of the blank solution.

[0060] Figure 15 This is the reference peak identification chromatogram in Example 1 of the present invention. Figure 2 Among them, from top to bottom, numbers 1-6 are the characteristic spectra of hesperidin, geniposide, cimicifugoside, 5-O-methylvisamidol, the characteristic spectra of the test solution, and the characteristic spectra of the blank solution.

[0061] Figure 16 These are the UPLC-UV chromatogram and UPLC-MS total ion chromatogram determined by liquid chromatography-mass spectrometry in Example 1 of this invention; wherein, from top to bottom, they are the positive ion mode TIC chromatogram, the negative ion mode TIC chromatogram, and the UV 265nm HPLC chromatogram;

[0062] Figure 17 These are the fingerprint spectra of the dampness-removing Weiling Decoction and the reference object in Embodiment 2 of the present invention; wherein, from top to bottom, they are the fingerprint spectra of the dampness-removing Weiling Decoction and the characteristic spectra of the reference object;

[0063] Figure 18 This is a common pattern diagram of the fingerprint spectrum of the dampness-removing Weiling Decoction in Embodiment 11 of the present invention; wherein, from top to bottom, are the control fingerprint spectrum and the fingerprint spectra of two parallel samples from the first batch to two parallel samples from the 15th batch;

[0064] Figure 19 This is the standard fingerprint spectrum of the dampness-removing Weiling Decoction in Embodiment 11 of the present invention;

[0065] Figure 20 This is a graph showing the results of the specificity and integrity assessment in Example 12 of the present invention; from top to bottom, it shows the characteristic spectrum of the test sample solution, the characteristic spectrum of the reference solution, and the characteristic spectrum of the blank solvent.

[0066] Figure 21 This is a graph showing the results of the precision test in Example 12 of the present invention; where numbers 1-6 from top to bottom are characteristic spectra of the test solution after 6 consecutive injections;

[0067] Figure 22 This is a graph showing the results of the stability test in Embodiment 12 of the present invention; where the numbers 1-7 from top to bottom are the characteristic spectra at 0h, 3h, 6h, 9h, 12h, 18h, and 24h respectively.

[0068] Figure 23 This is a graph showing the results of the repeatability test in Example 12 of the present invention; where numbers 1-6 from top to bottom represent the characteristic spectra of the six test solutions.

[0069] Figure 24 These are fingerprint spectra examined at different flow rates in Example 12 of this invention; from top to bottom, they are fingerprint spectra at 0.8, 1.0, and 1.2 mL / min.

[0070] Figure 25 This is a similarity comparison chart of different flow rates examined in Example 12 of the present invention; from top to bottom, the fingerprint spectra and control spectra are at 0.8, 1.0, and 1.2 mL / min respectively.

[0071] Figure 26 These are fingerprint spectra obtained from different column temperatures in Example 12 of the present invention; the fingerprint spectra from top to bottom are at 40, 45, and 50°C.

[0072] Figure 27 This is a similarity comparison chart of different column temperatures examined in Example 12 of the present invention; from top to bottom, the fingerprint spectra and the control spectra are at 40, 45, and 50°C respectively.

[0073] Figure 28 These are fingerprint spectra examined using different chromatographic columns in Example 12 of this invention; where numbers 1-3 from top to bottom represent the columns used in the chromatographic study. Fingerprint patterns at 250 mm and 5 μm, using Welch Ultimate AQ-C18 (4.6) Fingerprint patterns at 250mm, 5μm, using Shimadzu ODS-SP (4.6) Fingerprint pattern at 250mm, 5μm;

[0074] Figure 29 This is a similarity comparison chart of different chromatographic columns examined in Example 12 of the present invention; from top to bottom, the columns used are: Phenomen Gemini 5u C18 110A (4.6). Fingerprint patterns at 250mm, 5μm, using Welch Ultimate AQ-C18 (4.6) Fingerprint patterns at 250mm, 5μm, using Shimadzu ODS-SP (4.6) Fingerprint patterns and control patterns at 250 mm and 5 μm;

[0075] Figure 30These are fingerprint spectra examined by different instruments in Embodiment 12 of the present invention; from top to bottom, they are fingerprint spectra obtained using Shimadzu A20 and fingerprint spectra obtained using Agilent 1260;

[0076] Figure 31 This is a similarity comparison chart of different instruments examined in Embodiment 12 of the present invention; from top to bottom, it shows the fingerprint spectrum when using Shimadzu A20, the fingerprint spectrum when using Agilent 1260, and the control spectrum. Detailed Implementation

[0077] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0078] Example 1: Screening of a method for constructing the fingerprint spectrum of the dampness-removing Weiling Decoction

[0079] In this embodiment, the dampness-removing Weiling Decoction material standard is used as the sample for the dampness-removing Weiling Decoction. The preparation method of the dampness-removing Weiling Decoction material standard is as follows:

[0080] Take one mace each of dried tangerine peel, gardenia, saposhnikovia root, ginger-processed magnolia bark, stir-fried atractylodes rhizome, poria cocos, alisma rhizome, red poria cocos, stir-fried atractylodes rhizome, akebia stem, and talc, three fen each of licorice root and cinnamon bark, two cups of water, add twenty 1.5m pieces of rush pith, decoct until eight-tenths remains, filter, and freeze-dry the resulting decoction to obtain the dry powder of the dampness-removing stomach-strengthening decoction, which is the material basis of the dampness-removing stomach-strengthening decoction.

[0081] In this embodiment, the material standard of the dampness-removing Weiling Decoction was used as the sample for screening the fingerprint spectrum construction method of the dampness-removing Weiling Decoction, as follows:

[0082] I. Investigation of the preparation method of the test solution

[0083] Accurately weigh 2.0g of the dampness-removing Weiling Decoction standard and place it in a stoppered conical flask. Accurately add 25mL each of methanol and 75vol% ethanol aqueous solution. Sonicate for 30min (power 800W, frequency 40KHz), remove, cool, shake well, and filter to obtain test solution one (solvent is methanol) and test solution two (solvent is 75vol% ethanol aqueous solution).

[0084] Accurately weigh 2.0g of the dampness-removing Weiling Decoction standard, add 25mL of water, sonicate to dissolve, and pass the entire solution through a D101 macroporous adsorption resin column (inner diameter 1.5cm, column height 12cm). Wash with water until no obvious color is observed, discard the aqueous solution, and continue eluting with 50mL of 95vol% ethanol aqueous solution. Collect the eluent, evaporate to dryness in a water bath, dissolve the residue in 50vol% methanol aqueous solution, transfer to a 25mL volumetric flask, add 50vol% methanol aqueous solution to the mark, shake well, filter, and obtain the test solution three.

[0085] Accurately pipette 10 μL of each of the above test solutions and inject them into the liquid chromatograph. Measure and record the chromatograms. The results are as follows: Figure 1 As shown.

[0086] The high-performance liquid chromatography (HPLC) conditions are as follows:

[0087] Octadecylsilane-bonded silica gel is used as a filler;

[0088] Flow rate 1.0 mL / min;

[0089] The column temperature is 45℃;

[0090] The detection wavelength is 265nm;

[0091] Acetonitrile was used as mobile phase A, and 0.2 wt% formic acid aqueous solution was used as mobile phase B;

[0092] The elution method is gradient elution, and the elution conditions are:

[0093] 0~70min, 8%→20% mobile phase A, 92%→80% mobile phase B;

[0094] 70~105min, 20%→37% mobile phase A, 80%→63% mobile phase B;

[0095] 105~120min, 37%→70% mobile phase A, 63%→30% mobile phase B;

[0096] 120~130 min, 70% mobile phase A, 30% mobile phase B.

[0097] The theoretical plate number, calculated based on the hesperidin peak, should be no less than 2000.

[0098] Depend on Figure 1It can be seen that, through the investigation of the different test solution preparation methods, the method of elution with macroporous resin yielded more chromatographic peak information and had a moderate response value. Furthermore, after impurity removal treatment, the impurity peaks in the first 15 minutes of the chromatogram of test solution three were improved. Therefore, the preparation method of test solution three was finally determined as the final test solution preparation method, namely, elution with macroporous adsorption resin column, collection of 95 vol% ethanol aqueous solution eluent, evaporation to dryness and reconstitution to prepare the test solution.

[0099] The following test solutions were prepared by eluting with macroporous adsorption resin columns, collecting the eluent in 95 vol% ethanol aqueous solution, evaporating to dryness and reconstituted. These solutions were used to investigate the construction method.

[0100] II. Investigation of Detection Wavelength

[0101] A diode array detector was used to scan the test solution at wavelengths of 190–400 nm. Results were obtained through 3D visualization and isoabsorption spectroscopy. (See attached image). Figures 2-3 .Depend on Figure 2 and Figure 3 It can be seen that the baseline is stable and the information content is relatively large in the wavelength range of 230~300nm. The results of the analysis in this wavelength range are shown below. Figures 4-11 By comparison Figures 4-11 As can be seen, the response values ​​of each chromatographic peak are most uniform and contain the most information at a wavelength of 265nm. Therefore, 265nm is the preferred detection wavelength.

[0102] Meanwhile, since the chromatographic peak response values ​​at wavelengths of 260nm and 270nm are relatively uniform, the 21 characteristic peaks required for the final detection can also be detected, so they can also be used as detection wavelengths.

[0103] III. Peak Attribution and Selection of Reference Frames

[0104] 1) Attribution of medicinal flavor peak

[0105] The material basis for the Chushi Weiling Decoction is a mixture of 14 medicinal slices prepared by decoction in accordance with the prescribed proportions. Its chemical components mainly include polysaccharides, saponins, and flavonoids. Considering the need for multi-component and overall quality control, the medicinal slices were decocted separately. The decoction was then used to prepare corresponding solutions of the medicinal slices according to the fingerprint chromatogram test sample preparation method. The chromatographic conditions used in the test sample solution preparation method in step one were employed for single-herb peak assignment. The results are shown in [Figure Number]. Figures 12-13 See Table 1. Talc is a mineral drug, mainly inorganic, and its peaks could not be detected by liquid chromatography, therefore its attribution was not studied.

[0106] Table 1. Overview of chromatographic peak attribution to medicinal herbs

[0107]

[0108] From Table 1 and Figures 12-13 It can be seen that the characteristic chromatogram of Chu Si Wei Ling Tang can be attributed to the chromatographic peaks of Gardenia, Tangerine Peel, Saposhnikovia, Licorice, and Magnolia officinalis, while the other herbs have no specific components or low chromatographic peak response values.

[0109] 2) Chromatographic peak identification

[0110] Based on literature review, the main components related to each herb were selected as reference standards: geniposide, cimicifugin, 5-O-methylvisamidol, hesperidin, glycyrrhizin, glycyrrhizic acid, and magnolol and honokiol. Corresponding reference solutions were prepared, and peak localization was studied using the chromatographic conditions described in step one of the test solution preparation method investigation. Simultaneously, a 50 vol% methanol aqueous solution was used as a blank solution for determination. The results are as follows: Figures 14-15 The preparation method for each reference solution is as follows: take an appropriate amount of the corresponding reference standard, accurately weigh it, add methanol to prepare a solution containing 0.2 mg of the corresponding reference standard per 1 mL.

[0111] Simultaneously, the chromatographic peaks in the fingerprint spectrum of the dehumidifying stomach-soothing decoction were identified by detecting the material reference of the dehumidifying stomach-soothing decoction using liquid chromatography-mass spectrometry (LC-MS). The obtained UPLC-UV chromatogram and total ion chromatogram (positive mode) are shown below. Figure 16 Since the liquid phase of the liquid chromatography-mass spectrometry (LC-MS) instrument is UPLC, its chromatogram is somewhat different from the fingerprint spectrum obtained by high performance liquid chromatography in this embodiment. The specific peak correspondence is shown in Table 2.

[0112] The chromatographic conditions for the liquid chromatography-mass spectrometry (LC-MS) detection process were as follows: acetonitrile as mobile phase A and 0.1 wt% formic acid aqueous solution as mobile phase B, with gradient elution; flow rate 0.4 mL / min; column temperature 45℃; detection wavelength 265 nm; injection volume 3 μL; and gradient elution conditions as follows:

[0113] 0~15min, 8%→20% mobile phase A, 92%→80% mobile phase B;

[0114] 15~22 min, 20%→37% mobile phase A, 80%→63% mobile phase B;

[0115] 22~27 min, 37%→70% mobile phase A, 63%→30% mobile phase B;

[0116] 27~29 min, 70%→8% mobile phase A, 30%→92% mobile phase B.

[0117] Mass spectrometry conditions were as follows: mass spectrometry detection was performed in both positive and negative ion modes, with a drying gas temperature of 350℃, a drying gas flow rate of 10 L / min, a nebulizer gas pressure of 35 psi, a sheath gas temperature of 350℃, a sheath gas flow rate of 12 L / min, and capillary voltages of 3500 V (positive mode) and 3500 V (negative mode). The primary mass spectrometry used MS mode with a mass scan range of 100–1200 m / z. The secondary mass spectrometry used Auto MSMS, with the collision voltage adjusted appropriately for different compounds. The obtained LC-MS data were acquired using Agilent MassHunter (B.08.00) software. Data processing was performed using Agilent software Qualitative Navigator (B.08.00) and Qualitative Workflows (B.08.00).

[0118] Table 2 Summary of Chromatographic Peak Identification Results

[0119]

[0120] By comparing with reference standards of eight known components (geniposide, cimicifugin, 5-O-methylvisamidol, hesperidin, glycyrrhizin, glycyrrhizic acid, and magnolol and honokiol), the corresponding compounds of the eight chromatographic peaks were identified, and the analytical results were consistent with the mass spectrometry results.

[0121] Since the hesperidin chromatographic peak has moderate absorption intensity and retention time and stable response among the identified chromatographic peaks, achieving baseline separation, hesperidin was selected as the reference peak and labeled as peak S. At the same time, the hesperidin standard was used as the reference.

[0122] Example 2: A method for constructing a fingerprint spectrum of the dampness-removing Weiling Decoction

[0123] In this embodiment, the material standard of the dampness-removing and stomach-strengthening decoction is used as the sample for the dampness-removing and stomach-strengthening decoction. The specific method for constructing the fingerprint spectrum of the dampness-removing and stomach-strengthening decoction is as follows:

[0124] S1, Solution Preparation

[0125] S11. Preparation of reference solution

[0126] Take an appropriate amount of hesperidin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.2 mg per 1 mL, which is the reference solution.

[0127] S12. Preparation of the test solution

[0128] Accurately weigh 2.0 g of the dampness-removing Weiling Decoction standard (i.e., the dry powder of dampness-removing Weiling Decoction), add 25 mL of water, and sonicate to dissolve. Pass the resulting dampness-removing Weiling Decoction liquid through a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column height 12 cm), wash with water until no obvious color is visible, discard the water, and continue eluting with 50 mL of 95 vol% ethanol aqueous solution. Collect the eluent, evaporate to dryness in a water bath, dissolve the residue in 50 vol% methanol aqueous solution, transfer to a 25 mL volumetric flask, add 50 vol% methanol aqueous solution to the mark, shake well, filter, and obtain the test solution.

[0129] S2, High Performance Liquid Chromatography Detection

[0130] Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph, and determine the chromatographic conditions as described in step one of Example 1 for the preparation of the test solution. Record the chromatograms to obtain the corresponding characteristic spectra. The results are shown in [Figure 1]. Figure 17 Among them, the characteristic chromatogram obtained by detecting the test solution is the fingerprint chromatogram of Chushi Weiling Decoction.

[0131] That is, the high performance liquid chromatography conditions are:

[0132] Octadecylsilane-bonded silica gel is used as a filler;

[0133] Flow rate 1.0 mL / min;

[0134] The column temperature is 45℃;

[0135] The detection wavelength is 265nm;

[0136] Acetonitrile was used as mobile phase A, and 0.2 wt% formic acid aqueous solution was used as mobile phase B;

[0137] The elution method is gradient elution, and the elution conditions are:

[0138] 0~70min, 8%→20% mobile phase A, 92%→80% mobile phase B;

[0139] 70~105min, 20%→37% mobile phase A, 80%→63% mobile phase B;

[0140] 105~120min, 37%→70% mobile phase A, 63%→30% mobile phase B;

[0141] 120~130 min, 70% mobile phase A, 30% mobile phase B.

[0142] The theoretical plate number, calculated based on the hesperidin peak, should be no less than 2000.

[0143] Meanwhile, in the process of preparing the test solution in step S12, the concentrated solution of the dampness-removing stomach-soothing decoction can be diluted with water to prepare the dampness-removing stomach-soothing decoction liquid, and the test solution can be prepared; or, the decoction of the dampness-removing stomach-soothing decoction can be used as the dampness-removing stomach-soothing decoction liquid to prepare the test solution.

[0144] Accurately weigh 5 mL of the concentrated dehumidifying Weiling Decoction, add 25 mL of water, sonicate to dissolve, and then pass it through a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column height 12 cm). Wash with water until no obvious color is visible, discard the aqueous solution, and continue eluting with 50 mL of 95 vol% ethanol aqueous solution. Collect the eluent, evaporate to dryness in a water bath, dissolve the residue in 50 vol% methanol aqueous solution, transfer to a 5 mL volumetric flask, add 50 vol% methanol aqueous solution to the mark, shake well, filter, and obtain the test solution.

[0145] Alternatively, take 25 mL of the decoction for removing dampness and pass it directly through a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column height 12 cm), wash with water until there is no obvious color, discard the water, and continue to elute with 50 mL of 95 vol% ethanol aqueous solution. Collect the eluent, evaporate it to dryness in a water bath, dissolve the residue in 50 vol% methanol aqueous solution, transfer it to a 5 mL volumetric flask, add 50 vol% methanol aqueous solution to the mark, shake well, filter, and obtain the test solution.

[0146] Examples 3-10: Methods for constructing fingerprint patterns of the Weiling Decoction for Eliminating Dampness

[0147] Examples 3-10 represent methods for constructing fingerprint spectra of the dampness-removing Weiling Decoction. Based on the optimal construction conditions for the dampness-removing Weiling Decoction fingerprint spectra given in Example 2, the range of construction conditions that can achieve good dampness-removing Weiling Decoction fingerprint spectra was further explored, resulting in the construction methods in Examples 3-10. The construction methods for the dampness-removing Weiling Decoction fingerprint spectra in Examples 3-10 are basically the same as those in Example 2, differing only in some process parameters, as detailed in Tables 3 and 4.

[0148] Table 3. Summary of process parameters in Examples 3-6

[0149]

[0150] Table 4. Summary of process parameters in Examples 7-10

[0151]

[0152] The contents and results of other parts of Examples 3 to 10 are the same as those of Example 2, and will not be repeated here (since other embodiments of the present invention have sufficiently demonstrated that the method can construct the fingerprint spectrum of the dampness-removing Weiling Decoction, no additional figures are added here).

[0153] Example 11 Standard fingerprint spectrum and application of Chushi Weiling Decoction

[0154] 1) Calibration of common peaks

[0155] The fingerprint chromatogram construction method described in Example 2 was used to determine the fingerprints of 15 batches of the Chushi Weiling Decoction reference material (two parallel samples were measured for each batch, totaling 30 samples). The obtained fingerprint chromatograms were analyzed, and the chromatographic peaks with good stability and suitable response values ​​in the fingerprint chromatograms of the 15 batches of Chushi Weiling Decoction reference material (two parallel samples were measured for each batch) were selected as common peaks. A total of 21 common peaks were identified. The results are shown in […]. Figure 18 .

[0156] 2) Establishment of standard fingerprint patterns

[0157] Using the "Software System for Similarity Evaluation of Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" issued by the National Pharmacopoeia Commission, the chromatographic peaks of fingerprints from 15 batches of Chushi Weiling Decoction (two parallel samples per batch) were automatically matched to form a common pattern diagram, and a standard fingerprint was established. The results are shown in [Figure Number]. Figure 19 The established standard fingerprint spectrum was then used as a control fingerprint spectrum for subsequent experiments.

[0158] Simultaneously, based on the chromatographic peak identification results, peak 1 is genipin-1-gentiopicroside, peak 2 is geniposide, peak 3 is magnolinoside B, peak 4 is glucosylapigenin, peak 5 is cimicifugin glycoside, peak 6 is glycyrrhizin, peak 7 is rutin, peak 8 is cimicifugin, peak 9 is rutin, peak 10 is 5-O-methylvisamidol glycoside, peak 11 (S) is hesperidin, and peak 12 is 6'-O-p-coumaroyl... Quercetin gentiopicroside, peak 13 is crocin I, peak 14 is limonin, peak 15 is limonin, peak 16 is glucosyl naringin, peak 17 is ammonium glycyrrhizate, peak 18 is 3,5,6,7,3',4',5'-heptamethoxyflavone, peak 19 is 5,6,7,8,4'-pentamethoxyflavone, peak 20 is magnolol, and peak 21 is honokiol.

[0159] 3) Similarity calculation

[0160] The similarity evaluation software system for chromatographic fingerprints of traditional Chinese medicine was used to calculate the similarity between the fingerprint spectra of 15 batches of Chushi Weiling Decoction (two parallel samples per batch, totaling 30 samples) and the control fingerprint spectra, based on common peaks. The similarity calculated from the fingerprint spectra showed that the actual range of similarity between the 15 batches of Chushi Weiling Decoction and the control fingerprint spectra was 0.986–1.000, all greater than 0.90. Therefore, the fingerprint spectra were selected as the evaluation standard for Chushi Weiling Decoction. The Chushi Weiling Decoction fingerprint spectra should show chromatographic peaks with the same retention times as the reference peaks. The Chushi Weiling Decoction chromatogram should be basically consistent with the control fingerprint spectra, with 21 corresponding common peaks. Using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the Chushi Weiling Decoction fingerprint spectra and the control fingerprint spectra should not be lower than 0.90.

[0161] 4) Application of standard fingerprint spectrum

[0162] The standard fingerprint spectrum of the dampness-removing Weiling Decoction obtained in this embodiment can be used for quality evaluation or control throughout the entire process of research, development, production, and clinical application of the dampness-removing Weiling Decoction.

[0163] Example 12: Investigation of Fingerprint Mapping Methodology

[0164] This embodiment examines the methodological aspects of constructing the fingerprint chromatogram of the dampness-removing Weiling Decoction in Example 2, primarily focusing on the specificity, integrity, precision, stability, repeatability, and robustness of the method. In the methodological examination, both the reference solution and the test solution were prepared according to the method in Example 2, and the chromatographic conditions used for high-performance liquid chromatography (HPLC) detection were also those in Example 2.

[0165] I. Examination of Exclusivity and Holistic Nature

[0166] To investigate whether the blank solvent interfered with the fingerprint chromatogram of the dampness-removing Weiling Decoction, a 50 vol% methanol aqueous solution was used as the blank solvent. Precisely pipettes of the 50 vol% methanol aqueous solution, reference solution, and test solution were used for determination, maintaining the final gradient of the elution program with a 30-minute extended detection time. Chromatograms were recorded, and their overall integrity was examined. The results are shown in [Figure number missing]. Figure 20 .Depend on Figure 20 It can be seen that, through specificity and integrity tests, the blank solvent has no interference and good specificity; and there are no chromatographic peaks within the extended 30 minutes, indicating good integrity.

[0167] II. Precision Test

[0168] Accurately weigh approximately 2.0 g of the dampness-removing Weiling Decoction reference material and prepare a test solution according to the method in Example 2. Perform the determination according to the method in Example 2, injecting the sample six times consecutively. Using peak S as a reference, calculate the relative retention times of the remaining peaks. Compare this with the reference chromatogram R (using the "Similarity Evaluation Software System for Chromatographic Fingerprints of Traditional Chinese Medicine" promulgated by the National Pharmacopoeia Commission in 2012), automatically matching the chromatographic peaks of the repeatability characteristic chromatogram to form a common pattern diagram and establishing the reference chromatogram R. Calculate the similarity; see the specific determination results below. Figure 21 Tables 5 and 6.

[0169] Table 5. Summary of Precision Similarity Results

[0170]

[0171] Table 6. Summary of Relative Retention Time Results for Precision Testing

[0172]

[0173] Depend on Figure 7 Tables 4 and 5 show that the fingerprint chromatogram of Chushi Weiling Decoction shows chromatographic peaks with the same retention time as the reference chromatographic peaks, and presents 21 main chromatographic peaks with a relative retention time RSD of less than 2%. The similarity results are all greater than 0.95, indicating that the instrument precision is good.

[0174] III. Stability Test

[0175] Accurately weigh approximately 2.0 g of the dampness-removing Weiling Decoction reference material and prepare a test solution according to the method in Example 2. Perform the determination according to the method in Example 2, injecting and measuring at 0, 3, 6, 9, 12, 18, and 24 hours. Using peak S as a reference, calculate the relative retention times of the remaining peaks. Calculate the similarity with the reference chromatogram R (using the "Similarity Evaluation Software System for Chromatographic Fingerprints of Traditional Chinese Medicine" promulgated by the National Pharmacopoeia Commission in 2012, which automatically matches repeatable HPLC chromatographic peaks to form a common pattern diagram and establish a reference chromatogram R). See the specific measurement results below. Figure 22 Tables 7 and 8.

[0176] Table 7 Summary of Precision Similarity Results

[0177]

[0178] Table 8 Summary of Relative Retention Time Results for Precision Study

[0179]

[0180] Depend on Figure 22As shown in Tables 7 and 8, the fingerprint chromatogram of Chushi Weiling Decoction exhibits chromatographic peaks with the same retention times as the reference chromatographic peaks, and presents 21 main chromatographic peaks. Its relative retention time RSD is less than 2%, and the similarity results are all greater than 0.95, indicating that the test solution prepared in this invention has good stability within 24 hours.

[0181] IV. Repeatability Test

[0182] Six portions (approximately 2.0 g each) of the dampness-removing Weiling Decoction standard were accurately weighed and prepared according to the method in Example 2. The solutions were then analyzed according to the method in Example 2, with peak S as a reference, and the relative retention times of the remaining peaks were calculated. The results were compared with the reference chromatogram R (using the "Similarity Evaluation Software System for Chromatographic Fingerprints of Traditional Chinese Medicine" issued by the National Pharmacopoeia Commission in 2012, which automatically matched repeatable HPLC chromatographic peaks to form a common pattern diagram and establish a reference chromatogram R). The specific results are shown in [link to specific results]. Figure 23 Tables 9 and 10.

[0183] Table 9 Summary of Repeatability Similarity Results

[0184]

[0185] Table 10 Summary of Relative Retention Time Results for Repeatability Testing

[0186]

[0187] Depend on Figure 23 Tables 9 and 10 show that the fingerprint chromatogram of Chushi Weiling Decoction exhibits chromatographic peaks with the same retention times as the reference chromatographic peaks, and presents 21 main chromatographic peaks. The relative retention time RSD is less than 2%, and the similarity results are all greater than 0.95, indicating good repeatability.

[0188] V. Durability Test

[0189] The robustness of different flow rates (0.8, 1.0, 1.2 mL / min), column temperatures (40, 45, 50℃), columns (Shimadzu Inertsil ODS-SP, Pheromon Gemini C18), and instruments (Shimadzu 20A, Agilent 1260) under the chromatographic conditions described in Example 2 was investigated. Similarity was calculated by comparing the results with the reference chromatogram R (using the "Similarity Evaluation Software System for Chromatographic Fingerprints of Traditional Chinese Medicine" promulgated by the National Pharmacopoeia Commission in 2012, which automatically matched repeatable HPLC chromatographic peaks to form a common pattern diagram and established a reference chromatogram R). The results for different flow rates are shown below. Figures 24-25 See Table 11; results of measurements at different column temperatures are shown in Table 11. Figures 26-27 See Table 12; results for different chromatographic columns are shown in Table 12. Figures 28-29 See Table 13; results measured by different instruments are shown in Table 13. Figures 30-31 And Table 14.

[0190] Table 11 Summary of Similarity Results for Different Flow Velocities

[0191]

[0192] Table 12 Summary of Similarity Results at Different Column Temperatures

[0193]

[0194] Table 13 Summary of Similarity Results for Different Chromatographic Columns

[0195]

[0196] Table 14 Summary of Similarity Results for Different Instruments

[0197]

[0198] Depend on Figures 24-31 As shown in Tables 11-14, the results obtained under different column temperatures, flow rates, chromatographic columns, and instrument conditions are basically consistent. The common peaks in the chromatograms are sharp, symmetrical, and well-resolved, with similarities not less than 0.90. The fingerprint chromatograms of the Chushi Weiling Decoction show 21 main chromatographic peaks, which correspond to the characteristic peaks in the reference standard chromatograms. This indicates that the method of this invention has good robustness under different column temperatures, flow rates, chromatographic columns, and instrument conditions.

[0199] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of the dampness-removing Weiling Decoction, characterized in that, The construction method includes the following steps: S1. Pass the dehumidifying Weiling Decoction liquid through a macroporous adsorption resin column, wash with water, discard the water, continue eluting with an ethanol-water solution, collect the eluent, evaporate to dryness in a water bath, dissolve the residue in a methanol-water solution, shake well, filter, and prepare the test solution; the dehumidifying Weiling Decoction is prepared from tangerine peel, gardenia, saposhnikovia root, ginger magnolia bark, stir-fried atractylodes rhizome, polyporus umbellatus, alisma rhizome, red poria cocos, stir-fried atractylodes macrocephala, akebia stem, talc, licorice root, cinnamon, and rush pith. S2. Take the test solution and perform high performance liquid chromatography to obtain the fingerprint spectrum of the dehumidifying and stomachic decoction. The mobile phase A for the high-performance liquid chromatography detection is acetonitrile, and the mobile phase B is an aqueous solution of formic acid with a concentration of 0.18~0.22wt%. The elution method for the high-performance liquid chromatography detection is gradient elution; The elution conditions for the gradient elution are as follows: 0~70min, 8%→20% mobile phase A, 92%→80% mobile phase B; 70~105min, 20%→37% mobile phase A, 80%→63% mobile phase B; 105~120min, 37%→70% mobile phase A, 63%→30% mobile phase B; 120~130 min, 70% mobile phase A, 30% mobile phase B; The detection wavelength for the high-performance liquid chromatography is 260~270nm; The high-performance liquid chromatography (HPLC) detection was performed at a flow rate of 0.8–1.2 mL / min, a column temperature of 40–50 °C, and an injection volume of 5–20 μL. The chromatographic column was packed with octadecylsilane-bonded silica gel. The construction method also includes high-performance liquid chromatography detection of a reference solution made of hesperidin. The fingerprint spectrum of the dehumidifying Weiling Decoction showed 21 common peaks. Among them, peak 1 is genipin-1-gentiopicroside, peak 2 is geniposide, peak 3 is magnolinoside B, peak 4 is glucosylapigenin, peak 5 is cimicifugin, peak 6 is glycyrrhizin, peak 7 is rutin, peak 8 is cimicifugin, peak 9 is rutin, peak 10 is 5-O-methylvisamidol, peak 11 is hesperidin, peak 12 is 6'-O-p-coumaryl quercetin gentiopicroside, peak 13 is crocin I, peak 14 is limonin, peak 15 is limonin, peak 16 is glucosyl naringin, peak 17 is ammonium glycyrrhizate, peak 18 is 3,5,6,7,3',4',5'-heptamethoxyflavone, peak 19 is 5,6,7,8,4'-pentamethoxyflavone, peak 20 is magnolol, and peak 21 is honokiol.

2. The method for constructing the fingerprint spectrum of the dampness-removing Weiling Decoction according to claim 1, characterized in that, The concentration of the ethanol-water solution used for elution is 90-98 vol%, and the volume is 45-60 mL.

3. The method for constructing the fingerprint spectrum of the dampness-removing Weiling Decoction according to claim 1, characterized in that, The concentration of the methanol-water solution used for dissolution is 40-60 vol.

4. The method for constructing the fingerprint spectrum of the dampness-removing Weiling Decoction according to claim 1, characterized in that, The liquid form of the dampness-removing Weiling Decoction is made by dissolving the dry powder of the dampness-removing Weiling Decoction in water. Alternatively, it can be prepared by diluting the concentrated Weiling Decoction for Dampness Removal with water. Alternatively, the decoction of Weiling Decoction for Removing Dampness can be used directly as the liquid form of Weiling Decoction for Removing Dampness; When the dry powder of the dehumidifying stomach-soothing decoction is dissolved in water to prepare the liquid dehumidifying stomach-soothing decoction, the weight-to-volume ratio of the dry powder of the dehumidifying stomach-soothing decoction to the prepared test solution is 0.064~0.096g:1mL; When the concentrated dehumidifying Weiling Decoction is diluted with water to prepare the dehumidifying Weiling Decoction liquid, the volume ratio of the concentrated dehumidifying Weiling Decoction to the prepared test solution is 0.8~1.2mL:1mL; When the decoction of Weiling Decoction is used directly as the liquid of Weiling Decoction, the volume ratio of the decoction of Weiling Decoction to the prepared test solution is 4~6mL:1mL.

5. The application of a standard fingerprint spectrum of Chushi Weiling Decoction obtained by the construction method according to any one of claims 1-4 in the quality evaluation or control of Chushi Weiling Decoction throughout the research / development / production / clinical application process.

6. The application according to claim 5, characterized in that, The standard fingerprint spectrum of the dampness-removing Weiling Decoction identified 21 common peaks. Among them, peak 1 is genipin-1-gentiopicroside, peak 2 is geniposide, peak 3 is magnolinoside B, peak 4 is glucosylapigenin, peak 5 is cimicifugin, peak 6 is glycyrrhizin, peak 7 is rutin, peak 8 is cimicifugin, peak 9 is rutin, peak 10 is 5-O-methylvisamidol, peak 11 is hesperidin, peak 12 is 6'-O-p-coumaryl quercetin gentiopicroside, peak 13 is crocin I, peak 14 is limonin, peak 15 is limonin, peak 16 is glucosyl naringin, peak 17 is ammonium glycyrrhizate, peak 18 is 3,5,6,7,3',4',5'-heptamethoxyflavone, peak 19 is 5,6,7,8,4'-pentamethoxyflavone, peak 20 is magnolol, and peak 21 is honokiol.

7. The application according to claim 5 or 6, characterized in that, The standard fingerprint spectrum was established using the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine".

Images