Method for constructing HPLC (High Performance Liquid Chromatography) characteristic chromatograms of pollen typhae medicinal material, fried pollen typhae decoction pieces, standard decoction and formula granules thereof

Abstract

The invention provides a pollen typhae medicinal material, fried pollen typhae decoction pieces, standard decoction and formula granule HPLC characteristic chromatogram construction method, which comprises: A) extracting a pollen typhae raw material and a fried pollen typhae raw material by using a solvent to obtain a liquid to be detected; b) measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain HPLC characteristic chromatograms of the pollen typhae raw material and the fried pollen typhae raw material; chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column is a C18 column; a mobile phase A is methanol, a mobile phase B is 0.1% formic acid, and gradient elution is carried out. According to the method, a high performance liquid chromatography method is adopted, methanol-0. 1% formic acid is selected as a mobile phase for gradient elution, uridine and 5-hydroxymethyl-2-furancarboxylic acid are taken as reference substances, the HPLC characteristic chromatograms of the fried pollen typhae decoction pieces, the standard decoction and the formula granules thereof are established, the repeatability and precision are good, the method is stable and reliable, and the method is suitable for industrial production. And the quality of the pollen typhae medicinal material, the fried pollen typhae decoction pieces, the standard decoction and the formula granules thereof can be controlled.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for constructing HPLC characteristic spectra of Pollen Typhae medicinal materials, stir-fried Pollen Typhae decoction pieces, standard decoctions and their formula particles. Background Art

[0002] Fried Typha angustifolia is made from the dried pollen of Typha angustifolia L., a plant of the Typhaceae family. It can stop bleeding, dissipate blood stasis, and relieve stranguria. It is used for hematemesis, abalone hemorrhage, hemoptysis, metrorrhagia, traumatic bleeding, amenorrhea, dysmenorrhea, stabbing pain in the chest and abdomen, swelling and pain from falls, and hematuria. The standard decoction is a freeze-dried powder made from the medicinal materials after processing according to a fixed preparation process.

[0003] In order to ensure the uniformity and stability of the quality of Puhuang medicinal materials, fried Puhuang medicinal materials, decoction pieces and their standard decoctions, a new characteristic spectrum method was established in this paper to control their quality. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing HPLC characteristic spectra of Puhuang medicinal materials, stir-fried Puhuang decoction pieces, standard decoctions and their formula granules. The HPLC characteristic spectra method of Puhuang medicinal materials, stir-fried Puhuang decoction pieces, standard decoctions and their formula granules constructed by the present invention is stable and reliable, and can control the quality of Puhuang medicinal materials, stir-fried Puhuang decoction pieces, standard decoctions and their formula granules.

[0005] Fried Puhuang is a processed product of Puhuang medicinal material after frying. Its main active ingredients, typhanoside and isorhamnetin-3-O-neohesperidin, are also indicator ingredients for judging the quality of Puhuang medicinal material included in the "Chinese Pharmacopoeia" (2020 edition).

[0006] Literature has shown that cattail glycosides and isorhamnetin-3-O-neohesperidin will decompose and reduce after high-temperature heating. The reduction of old components indicates the increase of new components. It is speculated that new components may be produced during the frying process. Therefore, it is believed that the high-temperature heating method has a certain impact on the quality of the fried pieces of Typha latifolia. The method of controlling the quality of fried pieces based solely on content is too simplistic.

[0007] The HPLC characteristic spectrum method established by this patent is of great significance for the overall macroscopic characterization of chemical components of Puhuang medicinal materials, stir-fried Puhuang decoction pieces, Puhuang charcoal and other related stir-fried decoction pieces. Not only can 5-hydroxymethyl-2-furancarboxylic acid be used as the identification point of Puhuang medicinal materials and stir-fried Puhuang decoction pieces and other processed products to distinguish the two, but also the relative peak area of ​​5-hydroxymethyl-2-furancarboxylic acid can be specified to distinguish stir-fried Puhuang and Puhuang charcoal, two types of commonly used clinical processed products, and at the same time, the quality of Puhuang medicinal materials and stir-fried Puhuang decoction pieces can be controlled as a whole, and the material basis of the entire production process of stir-fried Puhuang decoction pieces formula granules, other related derivatives, and derivative products can be dynamically monitored, providing effective detection means for related products.

[0008] The present invention provides a method for constructing HPLC characteristic spectrum of Typhae Pollen medicinal material, stir-fried Typhae Pollen decoction pieces, standard decoction and formula granules thereof, comprising:

[0009] A) extracting the Pollen Typhae raw material and the stir-fried Pollen Typhae raw material with a solvent to obtain a test solution;

[0010] B) the liquid to be tested is measured by high performance liquid chromatography to obtain HPLC characteristic spectra of the Pollen Typhae raw material and the stir-fried Pollen Typhae raw material;

[0011] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is 0.1% formic acid, and the elution is gradient;

[0012] The gradient elution is specifically:

[0013] 0-4min, phase A: 0%→2%, phase B: 100%→98%;

[0014] 4-19 min, phase A: 2%→10%, phase B: 98%→90%;

[0015] 19-34 min, phase A: 10%→12%, phase B: 90%→88%;

[0016] 34-47min, phase A: 12%→13%, phase B: 88%→87%.

[0017] The invention provides a method for constructing HPLC characteristic spectrum of a Puhuang medicinal material, a stir-fried Puhuang decoction piece, a standard decoction and its formula granules. First, stir-fried Puhuang raw material is taken and solvent extracted to obtain a test solution. The solvent is preferably 30% methanol.

[0018] According to the present invention, the stir-fried pollen raw materials are pollen medicinal materials, stir-fried pollen decoction pieces, standard decoction and formula granules thereof.

[0019] According to the present invention, the preparation of the test solution of the stir-fried pollen and the decoction pieces is as follows: the stir-fried pollen is dissolved in 30% methanol, heated under reflux for extraction, cooled, shaken and filtered to obtain the product;

[0020] The heating reflux time is 60min;

[0021] The ratio of the mass g of the Puhuang medicinal material and the stir-fried Puhuang decoction pieces to the volume mL of the solvent is 0.5-0.7:20-30.

[0022] ; Preferably 0.6g:25mL.

[0023] The preparation of the stir-fried Pollen Typhae standard decoction is specifically as follows: the stir-fried Pollen Typhae is dissolved in 30% methanol, ultrasonically extracted, cooled, shaken, and filtered; the ultrasonic power is 600W and the frequency is 40kHz; the ratio of the mass g of the stir-fried Pollen Typhae standard decoction to the volume mL of the solvent is 0.1-0.3:20-30; preferably 0.2:25.

[0024] The preparation of the stir-fried pollen formula granules is as follows: stir-fried pollen is dissolved in 30% methanol, ultrasonically extracted, cooled, shaken, and filtered; the ultrasonic power is 600W and the frequency is 40kHz. The ratio of the mass g of the stir-fried pollen raw material to the volume mL of the solvent is 0.1-0.3:20-30; preferably 0.2:25.

[0025] The present invention adopts the above 30% methanol extraction solvent, the chromatographic peak information is large, each characteristic peak has a good peak shape, the separation is moderate, and the extraction effect is good. At the same time, the ultrasonic extraction time is 30 minutes and the effect is good when the reflux time is 60 minutes.

[0026] The stir-fried pollen raw material of the present invention is stir-fried pollen medicinal material, decoction pieces, decoction or formula granules. The present invention does not limit it, and the above raw materials can all be subjected to quality control and qualitative detection by the method of the present invention.

[0027] The present invention also includes preparing reference substances and reference medicinal material reference solutions:

[0028] Take uridine and 5-hydroxymethyl-2-furancarboxylic acid reference substances respectively, dissolve them in 30% methanol to obtain reference substance solutions.

[0029] In some specific embodiments, the concentration of the uridine reference substance is 20 μg / mL; the concentration of the 5-hydroxymethyl-2-furancarboxylic acid reference substance is 20 μg / mL;

[0030] Take the stir-fried pollen pollen reference medicinal material and dissolve it in 30% methanol, heat it under reflux for extraction, cool it, and filter it to obtain a reference medicinal material solution;

[0031] The reference substance and reference medicinal material reference substance solutions are determined by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material reference substance, respectively; and based on the chromatograms of the reference substance and the reference medicinal material reference substance, the components of the HPLC characteristic spectra of snake exuviae medicinal materials, medicinal materials, decoction pieces, standard decoctions and their formula granules are qualitatively determined.

[0032] The mobile phase A of the present invention is methanol, the mobile phase B is 0.1% formic acid, and the gradient elution is performed; the gradient elution of the present invention is preferably as follows:

[0033] 0-4min, phase A: 0%→2%, phase B: 100%→98%;

[0034] 4-19 min, phase A: 2%→10%, phase B: 98%→90%;

[0035] 19-34 min, phase A: 10%→12%, phase B: 90%→88%;

[0036] 34-47min, phase A: 12%→13%, phase B: 88%→87%.

[0037] The present invention has good baseline separation under the above elution gradient, good separation of each peak and stable baseline.

[0038] The chromatographic column is a C18 column with specifications of 1.8 μm and 2.1×100 mm; the column temperature is 30° C.

[0039] Preferably, the chromatographic column 1 may be Agilent ZORBAX SB-C18 2.1*100mm, 1.8μm, the chromatographic column 2 may be DIKMA Endeavorsil C18 2.1*100mm, 1.8μm, and the chromatographic column 3 may be Shimadzu Shim-pack Velox C18 2.1*100mm, 1.8μm.

[0040] The chromatographic column of the present invention has symmetrical chromatographic peaks and good separation under the above 30°C condition.

[0041] The mobile phase flow rate is preferably 0.3 ml / min.

[0042] The present invention found that the chromatographic peaks were better separated and the peak shapes were more symmetrical at a flow rate of 0.3 ml / min, which was the most preferred solution.

[0043] The detection wavelength of the present invention is preferably 254 nm.

[0044] The inventors found that at 254 nm, the chromatographic information is rich, each component has good absorption, the response value is moderate, the peak separation is good, and the baseline is stable.

[0045] The injection volume of the present invention is preferably 2 μL.

[0046] The beneficial effect of the present invention is that under one liquid chromatography condition, the substance groups of Typha lachryma-curd medicinal materials, stir-fried Typha lachryma-curd slices, standard decoctions and their formula granules are controlled by fingerprints, and the Typha lachryma-curd glycoside peak is used to locate the fingerprints; the detection cost can be greatly reduced and qualitative detection can be achieved.

[0047] The similarity of the HPLC characteristic spectrum of Typha latifolia was evaluated by the similarity evaluation system of Chinese medicine chromatographic fingerprint, and a HPLC standard characteristic spectrum consisting of 9 characteristic peaks was obtained, including peak 1: uridine; peak 2: p-hydroxybenzoic acid; peak 5: quercetin-3-O-neohesperidin; peak 7 (S): typhaneoglycans; peak 8: kaempferol-3-O-neohesperidin; peak 9: isorhamnetin-3-O-neohesperidin;

[0048] In the Typha lachryma-curcuma medicinal material characteristic spectrum, the relative retention time of each characteristic peak and the S peak is calculated with typhala glycoside as the reference peak, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.49 (peak 2), 0.68 (peak 3), 0.92 (peak 4), 0.96 (peak 5), 0.98 (peak 6), and 0.105 (peak 8).

[0049] The similarity of HPLC characteristic spectra of stir-fried Typhae Pollen slices, standard decoction and its formula granules was evaluated by using the similarity evaluation system of Chinese medicine chromatographic fingerprint, and the HPLC standard characteristic spectra of stir-fried Typhae Pollen slices, standard decoction and its formula granules were obtained, which consisted of 10 characteristic peaks, including peak 1: uridine; peak 2: 5-hydroxymethyl-2-furancarboxylic acid; peak 3: p-hydroxybenzoic acid; peak 6: quercetin-3-O-neohesperidin; peak 8 (S): typhaneoglycans; peak 9: kaempferol-3-O-neohesperidin; peak 10: isorhamnetin-3-O-neohesperidin;

[0050] In the characteristic spectra of the stir-fried Typha Pollen slices, standard decoction and its formula granules, the relative retention time of each characteristic peak and the S peak is calculated with typhala glycoside as the reference peak S peak, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.49 (peak 3), 0.68 (peak 4), 0.92 (peak 5), 0.96 (peak 6), 0.98 (7), and 1.05 (peak 9).

[0051] Quality judgment standard: Take samples of Puhuang medicinal materials, stir-fried Puhuang decoction pieces, standard decoction and its formula granules, and operate according to the same method as above to obtain the characteristic spectra of Puhuang medicinal materials, stir-fried Puhuang decoction pieces, standard decoction and its formula granules. Use the "Similarity Evaluation System of Chinese Medicine Chromatographic Fingerprints" 2012 edition of the National Pharmacopoeia Committee to analyze the standard characteristic spectra and sample characteristic spectra of Puhuang medicinal materials, stir-fried Puhuang decoction pieces, standard decoction and its formula granules, and the similarity is greater than 0.90.

[0052] The method provided by the present invention can effectively monitor the quality of different batches of Puhuang medicinal materials, stir-fried Puhuang decoction pieces, standard decoctions and their formula granules to ensure stable quality. The method has the characteristics of high precision and good reproducibility, and is conducive to comprehensive monitoring of product quality.

[0053] The characteristic spectra of Puhuang medicinal materials, stir-fried Puhuang decoction pieces, standard decoctions and their formula granules established by the present invention take psoralen as a reference, focus on the order of each characteristic peak and the correlation with the medicinal materials and intermediate products, can comprehensively evaluate the overall quality features of the products, and the method is scientific and reliable.

[0054] The newly developed characteristic spectrum method of the present invention can detect components with greater polarity in stir-fried Typhae Pollen and its standard decoction. The test sample preparation method is simple and easy to operate, and relatively more characteristic peaks can be identified. Accurate and reliable characteristic spectrum detection can be performed on stir-fried Typhae Pollen and its preparations. The authenticity, quality consistency and stability of stir-fried Typhae Pollen and its preparations can be effectively detected and controlled. This provides a basis for effectively controlling and more comprehensively evaluating the quality of stir-fried Typhae Pollen standard decoction. This ensures the uniformity and stability of the quality of stir-fried Typhae Pollen and its standard decoction.

[0055] The present invention provides a method for identifying stir-fried Typhae Pollen slices, standard decoctions and formula granules thereof from Typhae Pollen medicinal materials, characterized in that the identification is performed using the method described in the above technical scheme, and the difference is that the stir-fried Typhae Pollen slices, standard decoctions and formula granules thereof contain a characteristic peak of 5-hydroxymethyl-2-furancarboxylic acid, while the Typhae Pollen medicinal materials do not contain 5-hydroxymethyl-2-furancarboxylic acid.

[0056] The present invention provides a method for constructing an HPLC characteristic spectrum of a Puhuang medicinal material, a stir-fried Puhuang decoction piece, a standard decoction and its formula granules, comprising: A) extracting the stir-fried Puhuang with a solvent to obtain a test liquid; B) determining the test liquid with a high performance liquid chromatography method to obtain an HPLC characteristic spectrum of the stir-fried Puhuang raw material; the chromatographic conditions of the high performance liquid chromatography method are: a chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is 0.1% formic acid, and the gradient elution is performed. The present invention adopts high performance liquid chromatography, selects methanol-0.1% formic acid as the mobile phase for gradient elution, and uses uridine and 5-hydroxymethyl-2-furancarboxylic acid as reference substances to establish an HPLC characteristic spectrum of a Puhuang medicinal material, a stir-fried Puhuang decoction piece, a standard decoction and its formula granules, which has good repeatability and precision, and the method is stable and reliable, and can control the quality of the Puhuang medicinal material, the stir-fried Puhuang decoction piece, the standard decoction and its formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Investigation of extraction solvents;

[0058] Figure 2 Extraction time investigation;

[0059] Figure 3 Sampling volume investigation;

[0060] Figure 4 Investigation of extraction methods;

[0061] Figure 5 Chromatographic peak identification result diagram;

[0062] Figure 6 Surveys with different instruments;

[0063] Figure 7 Investigation of different chromatographic columns;

[0064] Figure 8 Fried Typha acuminata (Typha scabra) medicinal material characteristic spectrum;

[0065] Fig. 9 Comparative characteristic spectrum of the medicinal material of stir-fried Typha acuminata (Typha rapa);

[0066] Fig.10 Characteristic spectra of 23 batches of stir-fried Typha Pollen (Typha radix) slices;

[0067] Fig.11 Comparison chart of fried Typha acuminata (Typha scabra) decoction pieces;

[0068] Fig.12 Chromatograms of different wavelengths of stir-fried Puhuang formula granules;

[0069] Fig.13 Column temperature inspection chromatogram;

[0070] Fig.14 Flow rate investigation;

[0071] Fig.15 Different injection volumes;

[0072] Fig.16 Delayed inspection;

[0073] Fig.17 Investigation of extraction solvents;

[0074] Fig.18 Investigation of extraction methods;

[0075] Fig.19 Extraction time investigation;

[0076] Fig. 20 Investigation of solvent addition amount;

[0077] Fig.21 Chromatographic peak identification;

[0078] Fig. 22 Surveys with different instruments;

[0079] Fig.23 Investigation of different chromatographic columns;

[0080] Fig.24 Characteristic spectrum of standard decoction of stir-fried Typha acuminata (Typha rapa);

[0081] Fig.25 Comparative characteristic spectrum of standard decoction of stir-fried Typha acuminata (Typha rapa);

[0082] Fig.26 Chromatograms of different wavelengths of stir-fried Puhuang formula granules;

[0083] Fig. 27 Column temperature inspection chromatogram;

[0084] Fig.28 Flow rate investigation results;

[0085] Fig.29 Results of different injection volumes;

[0086] Fig.30 Delayed inspection;

[0087] Fig.31 Investigation of extraction solvents;

[0088] Fig.32 Investigation of extraction methods;

[0089] Fig.33 Extraction time investigation;

[0090] Fig.34 Sample collection and inspection;

[0091] Fig.35 Chromatographic peak identification;

[0092] Fig.36 Spectrum of uridine reference;

[0093] Fig.37 Granular uridine comparison spelling spectrum;

[0094] Fig.38 Spectrum of 5-hydroxymethylfurancarboxylic acid reference substance;

[0095] Fig.39 Spectrum of granular 5-hydroxymethylfurancarboxylic acid;

[0096] Fig.40 Spectrum of typhala glycoside reference substance;

[0097] Fig.41 Granular Typha neoglycoside spectrum;

[0098] Fig.42 Spectrum of isorhamnetin-3-O-neohesperidin reference substance;

[0099] Fig.43 Spectrum of granular isorhamnetin-3-O-neohesperidin;

[0100] Fig.44 Different types of chromatographic columns;

[0101] Fig.45 Different instruments;

[0102] Fig.46 Characteristic spectrum verification diagram of stir-fried Puhuang formula granules (KL-01);

[0103] Fig.47 Characteristic spectrum verification diagram of stir-fried Puhuang formula granules (KL-02);

[0104] Fig.48 Characteristic spectrum verification diagram of stir-fried Puhuang formula granules (KL-03);

[0105] Fig.49 Comparative characteristic spectrum of stir-fried Puhuang formula granules;

[0106] Fig.50 It is the characteristic spectrum of comparative example 1. DETAILED DESCRIPTION

[0107] The present invention provides a method for constructing HPLC characteristic spectra of Pollen Typhae medicinal materials, stir-fried Pollen Typhae decoction pieces, standard decoctions and their formula particles. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0108] To further illustrate the present invention, the following describes in detail a method for constructing HPLC characteristic spectra of a Puhuang medicinal material, stir-fried Puhuang decoction pieces, a standard decoction and its formula granules provided by the present invention in combination with embodiments.

[0109] Example 1 HPLC characteristic spectrum of medicinal materials and decoction pieces

[0110] 2.2.1.1 Materials, reagents and instruments

[0111] 2.2.1.1.1 Instruments and materials

[0112] High performance liquid chromatography: Agilent, Thermo Fisher;

[0113] Electronic balance: ME204E / 02, XPE26 (Mettler-Toledo Instrument Co., Ltd.);

[0114] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0115] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0116] Chromatographic columns: Column 1 Agilent ZORBAX SB-C18 2.1*100mm, 1.8μm, Column 2 DIKMA Endeavorsil C18 2.1*100mm, 1.8μm, Column 3 Shimadzu Shim-pack Velox C18 2.1*100mm, 1.8μm;

[0117] 2.2.1.1.2 Reagents and test drugs

[0118] Pollen Typhae reference material (China Food and Drug Inspection Institute, 121225-201804);

[0119] Typha cattail (Typha pueraria) control drug (Shanghai Hongyong Biotechnology Co., Ltd., 350020-202105);

[0120] Methanol and formic acid were of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade.

[0121] Uridine (China Food and Drug Inspection Institute, batch number: 110887-202305, purity: 99.6%);

[0122] 5-Hydroxymethyl-2-furancarboxylic acid (Shandong Bokang Fine Chemical Co., Ltd., batch number: CFS202201, purity: ≥98%);

[0123] 4-Hydroxybenzoic acid (China Food and Drug Inspection Institute, batch number: 101149-202204, purity: 100%);

[0124] Vanillic acid (China Food and Drug Inspection Institute, batch number: 110776-201503, purity: 99.8%);

[0125] Quercetin-3-O-neohesperidin (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDH016401, purity: 99.54%); Typhaneoglycans (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq18050902, purity: 98.0%)

[0126] Kaempferol-3-O-neohesperidin (Chengdu Desit Biotechnology Co., Ltd., batch number DSTDK006701, purity: 99.25%);

[0127] Isorhamnetin-3-O-neohesperidin (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq18031905, purity: 98.0%);

[0128] Pollen Puhuang medicinal material (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: PH202301, PH202302, PH202303, PH202304, PH202305, PH202306, PH202307, PH202308, PH202309, PH202310, PH202311, PH202312, PH202313, PH202314, PH202315, PH202316, PH202317, PH202318, PH202319, PH202320, PH202321, PH202322).

[0129] Fried Puhuang slices (Batch number: CPH-202301, CPH-202302, CPH-202303, CPH-202304, CPH-202305, CPH-202306, CPH-202307, CPH-202308, CPH-202309, CPH-202310, CPH-202311, CPH-202 312. CPH-202313, CPH-202314, CPH-202315, CPH-202316, CPH-202317, CPH-20231 8. CPH-202319, CPH-202320, CPH-202321, CPH-202322, CPH-202323, CPH-202324).

[0130] 2.2.1.2 Characteristic spectrum determination method

[0131] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm; methanol was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, gradient elution was performed according to the provisions in the following table, the flow rate was 0.3 ml per minute; column temperature was 30°C; detection wavelength was 254 nm, and the number of theoretical plates calculated based on the typhane glycoside peak should be not less than 3000.

[0132]

[0133] Preparation of reference solution Take 0.6g of Typha typhae (water candle cattail) reference medicinal material, place it in a stoppered conical bottle, add 25ml of 30% methanol, heat and reflux for 60 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take appropriate amounts of isorhamnetin-3-O-neohesperidin reference substance, typhane glycoside, uridine, and 5-hydroxymethyl-2-furancarboxylic acid reference substance, accurately weigh them, add 30% methanol to make a mixed solution containing 20μg of each per 1ml, as the reference solution of the reference substance.

[0134] Preparation of the test solution: Take 0.6 g of Pollen Puhuang powder (passed through No. 3 sieve), place it in a stoppered conical flask, add 25 ml of 30% methanol, heat and reflux for 60 minutes, cool, shake well, filter, and take the filtrate to obtain the solution.

[0135] Determination method: Accurately pipette 2μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0136] 2.2.1.3 Chromatographic conditions and system suitability test

[0137] Chromatographic conditions investigation refers to the results of chromatographic conditions investigation of stir-fried pollen standard decoction:

[0138] Octadecylsilane bonded silica gel is used as the filler (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm); methanol is used as mobile phase A, 0.1% formic acid solution is used as mobile phase B, gradient elution is performed according to the provisions in the following table, and the flow rate is 0.3 ml per minute; the column temperature is 30°C; the detection wavelength is 254 nm, and the theoretical plate number calculated based on the typhalaenoside peak should be not less than 3000.

[0139]

[0140] 2.2.1.4 Preparation of test solution

[0141] 2.2.1.4.1 Investigation of extraction solvent

[0142] Take 0.6g of pollen powder (passed through No. 3 sieve, batch number: PH202301), put it in a stoppered conical flask, and test the extraction solvents of methanol, ethanol, 70% methanol, 50% methanol, 30% methanol, and water 25ml each, heat and reflux for 60 minutes, cool, shake well, filter, and take the filtrate to obtain. Figure 1 .

[0143] Figure 1 Investigation of extraction solvent; the results showed that when the extraction solvent was 30% methanol, the peak shapes of the characteristic peaks were good and the separation was moderate. The extraction solvent was temporarily set to 30% methanol.

[0144] 2.2.1.4.2 Extraction time investigation

[0145] Take 0.6g of pollen powder (passed through No. 3 sieve, batch number: PH202301), put it in a stoppered conical flask, add 25ml of 30% methanol, reflux, and test the extraction time of the test sample for 40 minutes, 60 minutes, and 80 minutes respectively, cool, shake well, filter, and take the filtrate to obtain. Figure 2 . Figure 2The extraction time was investigated; the results showed that the extraction time had little effect on the extraction of each chromatographic peak, so it was consistent with the extraction time of the reference, so the extraction time was determined to be 60 minutes.

[0146] 2.2.1.4.3 Sampling volume investigation

[0147] Take 0.3g, 0.4g and 0.6g of pollen powder (passed through No. 3 sieve, batch number: PH202301), place in a stoppered conical flask, add 25ml of 30% methanol respectively, reflux extraction for 60 minutes, cool, shake well, filter, and take the filtrate to obtain. Figure 3 . Figure 3 Sampling volume inspection.

[0148] The results showed that when the sampling volume was 0.3, 0.4, and 0.6 g, the peak shapes and separations of the chromatographic peaks were better. In order to keep consistent with the reference medicinal materials, the sampling volume was selected as 0.6 g.

[0149] 2.2.1.4.4 Investigation of extraction methods

[0150] Take 0.6g of Puhuang powder (passed through No. 3 sieve, batch number: PH202301), put it in a stoppered conical flask, add 50ml of water, boil for 60 minutes, cool, centrifuge (speed is 2000 revolutions per minute) for 10 minutes, take the supernatant, evaporate to dryness, add 25ml of 30% methanol to the residue to dissolve, shake well, filter, take the filtrate as the test solution; separately, take 0.5g of this product powder, put it in a stoppered conical flask, add 25ml of 30% methanol, seal it, reflux for 60 minutes, cool, shake well, filter, take the filtrate, and get it. Figure 4 . Figure 4 Investigation of extraction methods.

[0151] The results showed that the peaks of the test samples and control herbs after decoction and reflux were most similar, and the peaks of reflux were better separated, so the extraction method of the test samples was determined to be reflux extraction.

[0152] In summary, the preparation method of the test solution of the characteristic spectrum of Typha acuminata (Typha acuminata) medicinal material is determined as follows: take 0.6 g of Typha acuminata (Typha acuminata) medicinal material powder (passed through No. 3 sieve), put it in a stoppered conical flask, add 25 ml of 30% methanol, reflux extraction for 60 minutes, cool, shake well, filter, and take the filtrate as the test solution.

[0153] 2.2.1.5 Methodological investigation

[0154] 2.2.1.5.1 Chromatographic peak identification

[0155] Preparation of test solution: According to the above proposed experimental conditions, prepare the test solution of stir-fried Typha acuminata (Typha rapa).

[0156] Preparation of reference solution: Take 0.6g of Typha typhae (water candle cattail) reference medicinal material, place it in a stoppered conical bottle, add 25ml of 30% methanol, heat and reflux for 60 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take appropriate amounts of isorhamnetin-3-O-neohesperidin reference substance, typhane glycoside reference substance, uridine, and 5-hydroxymethyl-2-furancarboxylic acid reference substance, accurately weigh them, add 30% methanol to make a mixed solution containing 20μg of each per 1ml, as the reference solution of the reference substance.

[0157] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution lacking Typha acuminata (Typha rapa) was prepared.

[0158] The characteristic spectrum peaks of Typha Pollen (Typha pueraria) were located. See Table 5. Figure 5 . Figure 5 Chromatographic peak identification results. The results show that peak 1 is uridine; peak 2 is p-hydroxybenzoic acid; peak 5 is quercetin-3-O-neohesperidin; peak 7 (S) is typhaneoglycans; peak 8 is kaempferol-3-O-neohesperidin; peak 9 isorhamnetin-3-O-neohesperidin. In the following methodological investigation, the 9 characteristic peaks in the sample were investigated.

[0159] 2.2.1.5.2 Precision test

[0160] Take the test solution of Typha typhae (Batch No.: PH202301) and inject it 6 times continuously according to the proposed experimental method, 2 μl each time, and calculate the relative retention time and relative peak area of ​​each characteristic peak. See Table 1-2.

[0161] Table 1 Precision investigation - retention time

[0162]

[0163] Table 2 Precision investigation - peak area

[0164]

[0165]

[0166] The results showed that the RSD of the relative retention time of each characteristic peak of the sample was 0.03% to 0.21%, and the instrument had good precision.

[0167] 2.2.1.5.3 Repeatability study

[0168] Take 6 samples of Typha Pollen (Typha puerariae) (batch number: PH202301) for testing, and prepare and test them according to the proposed experimental method. See Table 32.

[0169] Table 32 Repeatability Study-Relative Retention Time

[0170]

[0171] Table 4 Repeatability study - relative peak area

[0172]

[0173] The results showed that the RSD of the relative retention time of the six samples was 0.29% to 0.79%, indicating that the method had good repeatability.

[0174] 2.2.1.5.4 Intermediate precision study

[0175] 2.2.1.5.4.1 Investigation with different instruments

[0176] Based on the above proposed experimental conditions, two portions of Typha Pollen (Typha puerariae) medicinal material (batch number: PH202301) were weighed to prepare the test solution, which was measured on Waters H-Class Plus and Agilent 1290 ultra-high performance liquid chromatographs, respectively. See Table 5 and Table 6.

[0177] Table 5 Instrument durability investigation - relative retention time

[0178]

[0179] Table 6 Instrument durability investigation - relative peak area

[0180]

[0181] Figure 6 The results showed that when the two instruments were used to detect the test sample, the RSD of the relative retention time of each characteristic peak was 0.00% to 1.23%, indicating that the instrument had good durability.

[0182] 2.2.1.5.4.2 Investigation by different personnel and time

[0183] Based on the above proposed experimental conditions, three portions of Typha Pollen (Typha puerariae) (batch number: PH202301) were weighed by different personnel (A, B) at different times (T1, T2) to prepare the test samples for determination. See Tables 7 and 8.

[0184] Table 7 Personnel and time inspection-relative retention time

[0185]

[0186] Table 8 Personnel and time investigation-relative peak area

[0187]

[0188] The results showed that when different personnel measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.01% to 0.40%, and the method had good stability.

[0189] 2.2.1.5.5 Durability test

[0190] 2.2.1.5.5.1 Study on the durability of chromatographic columns

[0191] On the basis of the above-proposed experimental conditions, the analysis and investigation were carried out using chromatographic column 1 Agilent ZORBAX SB-C18 2.1*100mm, 1.8μm, chromatographic column 2 DIKMA Endeavorsil C18 2.1*100mm, 1.8μm, and chromatographic column 3 Shimadzu Shim-pack Velox C18 2.1*100mm, 1.8μm, and the results are shown in Tables 9 and 10.

[0192] Table 9 Chromatographic column durability investigation-relative retention time

[0193]

[0194] Table 10 Column durability investigation - relative peak area

[0195]

[0196]

[0197] Figure 7 Different chromatographic columns were investigated; the results showed that when the samples were detected using the above three chromatographic columns, the RSD of the relative retention time of the characteristic peaks was between 0.71% and 1.89%, and the chromatographic columns had good durability.

[0198] 2.2.1.5.5.2 Stability study

[0199] Based on the above experimental conditions, take the same test solution and measure it at 0h, 2h, 4h, 8h, 16h and 24h respectively. See Tables 11 and 12.

[0200] Table 11 Stability Study-Retention Time

[0201]

[0202] Table 12 Stability Study-Peak Area

[0203]

[0204] The results showed that the RSD of the characteristic peak retention time was between 0.22% and 0.96%, and the sample solution was stable within 24 hours.

[0205] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 9 characteristic peaks were included in the subsequent investigation.

[0206] 2.2.1.6 Verification of the characteristic spectrum of Puhuang medicinal material

[0207] Characteristic spectrum analysis was performed on 22 Typhae Pollen medicinal materials, and relative retention time and relative peak area were calculated. The results are shown in Figure 8 , Tables 13 and 14. Figure 8 Characteristic spectrum of Typha typhae (water candle) medicinal material; (peak 1: uridine; peak 2: p-hydroxybenzoic acid; peak 5: quercetin-3-O-neohesperidin; peak 7 (S): typhaneoglycans; peak 8: kaempferol-3-O-neohesperidin; peak 9: isorhamnetin-3-O-neohesperidin (S1~S22 are: PH202301, PH202302, PH202303, PH202304, PH202305, PH2 02306, PH202307, PH202308, PH202309, PH202310, PH202311, PH202312, PH202313, PH2023 14. PH202315, PH202316, PH202317, PH202318, PH202319, PH202320, PH202321, PH202322).

[0208] Table 13 Relative retention time of the characteristic spectrum of the herbal material of stir-fried Typha acuminata (Typha scabra)

[0209]

[0210]

[0211] Table 14 Relative peak areas of Typha Pollen (Typha pueraria) medicinal material characteristic spectrum

[0212]

[0213] According to the principle that the relative retention time is stable and each batch of samples can be detected and the peak is relatively high, a total of 9 peaks with good repeatability were selected as characteristic peaks. The relative retention time RSD of the 9 characteristic peaks of 22 batches of Typha acuminatum (Typha pupleura) medicinal materials was less than 1.0%.

[0214] Final regulations: 9 characteristic peaks should be present in the chromatogram of the test sample, and the retention time should correspond to the 9 characteristic peaks in the chromatogram of the reference medicinal material. Peak 1, peak 7, and peak 9 should correspond to the relative retention time of the reference peaks of uridine, typhaneoglycans, and isorhamnetin-3-O-neorhamnetin, respectively. The peak corresponding to the typhaneoglycans reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are 0.49 (peak 2), 0.68 (peak 3), 0.92 (peak 4), 0.96 (peak 5), 0.98 (peak 6), and 0.105 (peak 8).

[0215] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 22 batches of Typha acuminata (water candle typha) medicinal materials, and the reference characteristic spectrum of the characteristic spectrum of stir-fried Typha acuminata (water candle typha) medicinal materials was established. Fig. 9 . Fig. 9 Comparative characteristic spectrum of the medicinal material of stir-fried Typha typha (Typha rapa); Peak 1: uridine; Peak 3: p-hydroxybenzoic acid; Peak 6: quercetin-3-O-neohesperidin; Peak 8 (S): typhaneoglycans; Peak 9: kaempferol-3-O-neohesperidin; Peak 10: isorhamnetin-3-O-neohesperidin; Chromatographic column: Agilent ZORBAX SB-C18, 2.1mm×100mm, 1.8μm.

[0216] 2.2.1.7 Verification of characteristic spectrum of stir-fried pollen slices

[0217] According to the characteristic spectrum determination method, chromatographic conditions and system suitability in 2.2.1.2, 24 batches of test sample solutions of stir-fried Typha lachryma-curd slices were prepared, and characteristic spectrum analysis was performed to calculate the relative retention time and relative peak area ratio. Fig.10 , Table 8. Fig.10Characteristic spectra of 24 batches of stir-fried Typha Pollen slices; Peak 1: uridine; Peak 2: 5-hydroxymethyl-2-furancarboxylic acid; Peak 3: p-hydroxybenzoic acid; Peak 6: quercetin-3-O-neohesperidin; Peak 8 (S): typhaneoglucoside; Peak 9: kaempferol-3-O-neohesperidin; Peak 10: isorhamnetin-3-O-neohesperidin (S1-S22 are: CPH-202301, CPH-202302, CPH-202303, CPH-202304, CPH-202305, CPH-202306, CPH-2023 07. CPH-202308, CPH-202309, CPH-202310, CPH-202311, CPH-202312, CPH-202313, CPH-202314, CPH-202315, CPH- 202316, CPH-202317, CPH-202318, CPH-202319, CPH-202320, CPH-202321, CPH-202322, CPH-202323, CPH-202324).

[0218] Table 15 Relative retention time of characteristic spectra of fried Typha acuminata (Typha scabra) slices

[0219]

[0220] Table 16 Relative peak areas of characteristic spectra of fried Typha acuminata (Typha scabra) slices

[0221]

[0222]

[0223] According to the principle of stable relative retention time and that all batches of samples can be detected and the peaks are relatively high, a total of 10 peaks with good repeatability were selected as characteristic peaks. The results showed that the relative peak area RSD of each characteristic peak of 24 batches of stir-fried Typhae Pollen (Water Candle Typha) pieces was too large, among which the chromatographic peak 2 was the material after the medicinal material was processed, and the relative retention time of the chromatographic peak was stable. Due to certain differences between the processing batches, the relative peak area of ​​the chromatographic peak was regulated, with the average value of its relative peak area + 30% as the upper limit, that is, 0.49 as the upper limit, -3SD as the lower limit, and the relative retention time RSD of 10 characteristic peaks of 24 batches of stir-fried Typhae Pollen (Water Candle Typha) pieces was less than 1.0%.

[0224] Final regulations: There should be 10 characteristic peaks in the chromatogram of the test sample, and the retention times should correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal material. Peak 1, peak 2, peak 8, and peak 10 should correspond to the relative retention times of the reference peaks of uridine, 5-hydroxymethyl-2-furancarboxylic acid, typhaneoglycans, and isorhamnetin-3-O-neohesperidin, respectively. The peak corresponding to the typhaneoglycans reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are 0.49 (peak 3), 0.68 (peak 4), 0.92 (peak 5), 0.96 (peak 6), 0.98 (7), and 1.05 (peak 9). The relative peak area of ​​peak 2 and S peak is calculated, and the relative peak area should be within the specified value range, which is 0.08 to 0.49.

[0225] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 24 batches of stir-fried Typha acuminata slices, and a reference spectrum of the characteristic spectrum of stir-fried Typha acuminata slices was established. Fig.11 . Fig.11 Comparative spectrum of fried Typha typhae (Typha truncatum) slices; Peak 1: uridine; Peak 2: 5-hydroxymethyl-2-furancarboxylic acid; Peak 3: p-hydroxybenzoic acid; Peak 6: quercetin-3-O-neohesperidin; Peak 8 (S): typhaneoglycans; Peak 9: kaempferol-3-O-neohesperidin; Peak 10: isorhamnetin-3-O-neohesperidin, chromatographic column: Agilent ZORBAX SB-C18, 2.1mm×100mm, 1.8μm).

[0226] 2.2.1.8 Determination of the characteristic spectrum of Puhuang medicinal material

[0227] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm; methanol was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, gradient elution was performed according to the provisions in the following table, the flow rate was 0.3 ml per minute; column temperature was 30°C; detection wavelength was 254 nm, and the number of theoretical plates calculated based on the typhane glycoside peak should be not less than 3000.

[0228]

[0229] Preparation of reference solution Take 0.6g of Typha typhae (water candle cattail) reference medicinal material, place it in a stoppered conical bottle, add 25ml of 30% methanol, heat and reflux for 60 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take appropriate amounts of isorhamnetin-3-O-neohesperidin reference substance, typhane glycoside reference substance, uridine, and 5-hydroxymethyl-2-furancarboxylic acid reference substance, accurately weigh them, add 30% methanol to make a mixed solution containing 20μg of each per 1ml, as the reference solution of the reference substance.

[0230] Preparation of test solution: Take 0.6 g of the product powder (passed through No. 3 sieve), place it in a stoppered conical flask, add 25 ml of 30% methanol, heat and reflux for 60 minutes, cool, shake well, filter, and obtain the filtrate.

[0231] Determination method: Accurately pipette 2μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0232] There should be 9 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 9 characteristic peaks in the chromatogram of the reference medicinal material. Peak 1, peak 7, and peak 9 should correspond to the relative retention time of the reference peaks of uridine, typhanoside, and isorhamnetin-3-O-neohesperidin, respectively. The peak corresponding to the typhanoside reference is the S peak. Calculate the relative retention time of each characteristic peak and the S peak, and the relative retention time should be within ±10% of the specified value. The specified values ​​are 0.49 (peak 2), 0.68 (peak 3), 0.92 (peak 4), 0.96 (peak 5), 0.98 (peak 6), and 0.105 (peak 8). See Figure Fig. 9 .

[0233] 2.2.1.9 Determination of the characteristic spectrum of stir-fried pollen slices

[0234] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm; methanol was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, gradient elution was performed according to the provisions in the following table, the flow rate was 0.3 ml per minute; column temperature was 30°C; detection wavelength was 254 nm, and the number of theoretical plates calculated based on the typhane glycoside peak should be not less than 3000.

[0235]

[0236] Preparation of reference solution Take 0.6g of Typha typhae (water candle cattail) reference medicinal material, place it in a stoppered conical bottle, add 25ml of 30% methanol, heat and reflux for 60 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take appropriate amounts of isorhamnetin-3-O-neohesperidin reference substance, typhane glycoside reference substance, uridine, and 5-hydroxymethyl-2-furancarboxylic acid reference substance, accurately weigh them, add 30% methanol to make a mixed solution containing 20μg of each per 1ml, as the reference solution of the reference substance.

[0237] Preparation of the test solution: Take 0.6 g of stir-fried Typha Pollen (water candle Typha) decoction powder (passed through No. 3 sieve), place it in a stoppered conical flask, add 25 ml of 30% methanol, heat and reflux for 60 minutes, cool, shake well, filter, and obtain the filtrate.

[0238] Determination method: Accurately pipette 2μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0239] There should be 10 characteristic peaks in the chromatogram of the test sample. Except for peak 2, the other 9 characteristic peaks should correspond to the retention times of the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 1, peak 2, peak 8, and peak 10 should correspond to the relative retention times of the reference peaks of uridine, 5-hydroxymethyl-2-furancarboxylic acid, typhaneoglycans, and isorhamnetin-3-O-neohesperidin, respectively. The peak corresponding to the typhaneoglycans reference peak is the S peak. Calculate the relative retention time of peaks 3 to 7, peak 9 and S peak. The relative retention time should be within the relative range of ±10% of the specified value. The specified values ​​are: 0.49 (peak 3), 0.68 (peak 4), 0.92 (peak 5), 0.96 (peak 6), 0.98 (7), and 1.05 (peak 9). Calculate the relative peak area of ​​peak 2 and S peak, and the relative peak area should be within the specified value range, which is 0.08 to 0.49.

[0240] Example 2 HPLC characteristic spectrum of stir-fried pollen standard decoction

[0241] 2.2.2.1 Materials, reagents and instruments

[0242] 2.2.2.2 Instruments and materials

[0243] Same as Example 1.

[0244] 2.2.1.1.2 Reagents and test drugs

[0245] The rest is the same as in Example 1.

[0246] Standard decoction of stir-fried pollen: Batch number: CPHBT-202304, CPHBT-202305, CPHBT-202306, CPHBT-202307, CPHBT-202308, CPHBT-202309, CPHBT-202310, CPHBT-202311, CPHBT-202312, CPHBT-202313, CPHBT-202314, CPHBT-202315 2315, CPHBT-202316, CPHBT-202317, CPHBT-202318, CPHBT-202319, CPHBT-202320, CPHBT-2023 21. CPHBT-202322, CPHBT-202323, CPHBT-202324, CPHBT-202301, CPHBT-202302, CPHBT-202303.

[0247] 2.2.2.2 Characteristic spectrum determination method

[0248] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, gradient elution was performed as specified in the table below, flow rate was 0.3 ml per minute; column temperature was 30°C; detection wavelength was 254 nm. The theoretical plate number calculated based on the typhane novoglycoside peak should be no less than 3000.

[0249]

[0250] Preparation of reference solution Take 0.6g of Typha typhae (water candle cattail) reference medicinal material, place it in a stoppered conical bottle, add 25ml of 30% methanol, heat and reflux for 60 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take appropriate amounts of isorhamnetin-3-O-neohesperidin reference substance, typhane glycoside reference substance, uridine, and 5-hydroxymethyl-2-furancarboxylic acid reference substance, accurately weigh them, add 30% methanol to make a mixed solution containing 20μg of each per 1ml, as the reference solution of the reference substance.

[0251] Preparation of test solution Take an appropriate amount of standard decoction of fried Typha acuminatum (water candle Typha acuminatum), take about 0.2g, accurately weigh, put it in a stoppered conical flask, accurately add 25ml of 30% methanol, stopper it, weigh it, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 30% methanol, shake it well, filter it, and take the filtrate to obtain

[0252] The determination method is to accurately pipette 2 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0253] 2.2.2.3 Chromatographic conditions and system suitability test

[0254] 2.2.2.3.1 Wavelength selection

[0255] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning of the test solution, and the chromatograms of the test solution at wavelengths of 190nm, 210nm, 230nm, 254nm, 270nm, and 365nm were extracted respectively. Fig.12 . Fig.12 Chromatograms of stir-fried Puhuang formula granules at different wavelengths; the results show that when the detection wavelength is 254nm, the chromatographic peak has a larger amount of information, better separation, and a more stable chromatogram baseline, so the detection wavelength is determined to be 254nm.

[0256] 2.2.2.3.2 Column temperature investigation

[0257] Based on the experimental conditions proposed above, the column temperatures of 20℃, 30℃ and 40℃ were investigated. Fig.13 , Tables 17-18.

[0258] Table 17 Column temperature investigation-relative retention time

[0259]

[0260] Table 18 Column temperature investigation-relative peak area

[0261]

[0262] Fig.13 Column temperature inspection chromatogram; the results of column temperature inspection showed that when the column temperature was 30℃, the chromatogram peaks were relatively symmetrical and the separation was good, so 30℃ was finally determined as the column temperature for the characteristic spectrum method of Chao Puhuang Formula Granules.

[0263] 2.2.2.3.3 Flow rate investigation

[0264] Based on the experimental conditions proposed above, the flow rates of 0.2ml / min, 0.3ml / min, and 0.4ml / min were investigated. Fig.14 , Tables 19-20.

[0265] Table 19 Flow rate investigation - relative retention time

[0266]

[0267] Table 20 Flow rate investigation - relative peak area

[0268]

[0269] Fig.14 The flow rate investigation results show that when the flow rate is 0.3 ml / min, the chromatogram peak shape is better and the distribution of each characteristic peak is moderate. Therefore, the flow rate is determined to be 0.3 ml / min.

[0270] 2.2.2.3.4 Sample volume investigation

[0271] Based on the experimental conditions proposed above, the flow rates of 1μl, 2μl, and 3μl were investigated. Fig.15 , Tables 21-22.

[0272] Table 21 Injection volume investigation-relative retention time

[0273]

[0274] Table 22 Injection volume investigation - relative peak area

[0275]

[0276] Fig.15 Different injection volumes. The results showed that when the injection volume was 2 μl, the chromatogram peak shape was better and the separation was moderate, so the injection volume was determined to be 2 μl.

[0277] 2.2.2.3.5 Delay Investigation

[0278] Based on the above experimental conditions, the analysis time was extended to 94 minutes to observe whether there was a chromatographic peak after 47 minutes. The results are shown in Fig.16 . Fig.16 Delay investigation; the results showed that there was basically no large chromatographic peak after an extension of 47 minutes, and the analysis time of the characteristic spectrum method of Chao Puhuang Formula Granules was finally determined to be 47 minutes.

[0279] In summary, the chromatographic conditions and system suitability test of the characteristic spectrum of the stir-fried Puhuang formula granules were determined as follows: octadecylsilane bonded silica gel was used as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, and gradient elution was performed according to the regulations in the following table; the flow rate was 0.3 ml per minute; the column temperature was 30°C; and the detection wavelength was 254 nm.

[0280]

[0281] 2.2.2.4 Preparation of test solution

[0282] 2.2.2.4.1 Investigation of extraction solvent

[0283] Take 0.2g of stir-fried Typha typhae (water candle Typha) standard decoction powder (batch number: CPHBT-202320), put it in a stoppered conical bottle, and test the extraction solvents of water, 30% methanol, 50% methanol, 70% methanol, methanol, and ethanol 25ml each, seal it tightly, and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake it well, filter it, and take the filtrate to obtain it. Fig.17 . Fig.17 Investigation of extraction solvents.

[0284] The results showed that when the extraction solvent was 30% methanol, the peak shapes of the characteristic peaks were good and the separation was moderate. The extraction solvent was temporarily set to 30% methanol.

[0285] 2.2.2.4.2 Investigation of extraction methods

[0286] Take 0.2g of stir-fried Typha Pollen (water candle Typha) standard decoction powder (batch number: CPHBT-202320), put it in a stoppered conical bottle, add 25ml of 30% methanol, plug it tightly, and examine the extraction method of the test sample when it is refluxed and ultrasonic respectively. The extraction time is 30 minutes, let it cool, shake well, filter, and take the filtrate to obtain. Fig.18 . Fig.18 Investigation of extraction methods.

[0287] The results showed that there was little difference in the effects of reflux and ultrasonic extraction of the test sample, and the ultrasonic method was fast and simple, so the extraction method of the test sample was determined to be ultrasonic extraction.

[0288] 2.2.2.4.3 Extraction time investigation

[0289] Take 0.2g of stir-fried Typha Pollen (water candle Typha) standard decoction powder (batch number: CPHBT-202320), put it in a stoppered conical bottle, add 25ml of 30% methanol, plug it tightly, and treat it with ultrasound. The extraction time of the test sample is 20 minutes, 30 minutes, and 40 minutes respectively. Cool it, shake it well, filter it, and take the filtrate to obtain it. Fig.19 . Fig.19 The results of the extraction time showed that the peak shape and separation of the chromatogram were better when the extraction time was 30 minutes. Therefore, the extraction time was determined to be 30 minutes.

[0290] 2.2.2.4.4 Sample collection and investigation

[0291] Take 0.1g, 0.2g and 0.3g of stir-fried Typha Pollen (water candle Typha) standard decoction powder (batch number: CPHBT-202320) respectively and put them into stoppered conical bottles, add 25ml of 30% methanol respectively for inspection, seal, ultrasonically treat for 30 minutes, cool, shake well, filter, and take the filtrate to obtain. Fig. 20 . Fig. 20 The amount of solvent added was investigated; the results showed that when the sampling amount was 0.2g, the peak shape and separation of each chromatographic peak were better, so the sampling amount was set at 0.2g.

[0292] In summary, the preparation method of the test solution of the characteristic spectrum of the stir-fried Typha acuminata decoction is determined as follows: take 0.2 g of the product, put it in a stoppered conical flask, add 25 ml of 30% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0293] 2.2.2.5 Methodological investigation

[0294] 2.2.2.5.1 Chromatographic peak identification

[0295] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of stir-fried Typha acuminata (Typha rapa).

[0296] Preparation of control medicinal material solution: Take 0.6 g of Typha Pollen (Typha rapa) control medicinal material, place it in a stoppered conical flask, add 25 ml of 30% methanol, heat and reflux for 60 minutes, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0297] Preparation of reference solution: Take appropriate amount of isorhamnetin-3-O-neohesperidin reference substance, typhaneoglycan reference substance, uridine, and 5-hydroxymethyl-2-furancarboxylic acid reference substance, accurately weigh them, and add 30% methanol to make a mixed solution containing 20 μg of each per 1 ml, which serves as the reference substance solution.

[0298] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution lacking the stir-fried Typha acuminata (Typha rapa) standard decoction was prepared.

[0299] Position the characteristic peaks of the standard decoction of fried Typha acuminata (Typha acuminata). Fig.21 . Fig.21 Chromatographic peak identification; the results show that peak 1 is uridine; peak 2 is 5-hydroxymethyl-2-furancarboxylic acid, peak 3 is p-hydroxybenzoic acid; peak 6 is quercetin-3-O-neohesperidin; peak 8 (S) is typhaneoglycan; peak 9 is kaempferol-3-O-neohesperidin; peak 10 isorhamnetin-3-O-neohesperidin. In the following methodological investigation, 10 characteristic peaks in the sample were investigated.

[0300] 2.2.2.5.2 Precision test

[0301] Take the standard decoction of stir-fried Typha acuminata (batch number: CPHBT-202320) as the test solution, inject 6 times continuously according to the proposed experimental method, 2 μl each time, and calculate the retention time and peak area of ​​each characteristic peak. See Tables 23 and 24.

[0302] Table 23 Precision investigation - retention time

[0303]

[0304] Table 24 Precision investigation - Peak area

[0305]

[0306] The results showed that the RSD of the retention time of each characteristic peak was 0.02% to 0.12%, indicating that the instrument had good precision.

[0307] 2.2.2.5.3 Repeatability study

[0308] Take 6 portions of stir-fried Typha Pollen (Typha pueraria) standard decoction (batch number: CPHBT-202320), prepare and measure according to the proposed experimental method, and calculate the relative retention time and relative peak area of ​​each characteristic peak. See Tables 25 and 26.

[0309] Table 25 Repeatability study - relative retention time

[0310]

[0311] Table 26 Repeatability Study - Relative Peak Area

[0312]

[0313]

[0314] The results showed that the RSD of the relative retention time of the six samples was 0.01% to 0.13%, indicating that the method had good repeatability.

[0315] 2.2.2.5.4 Intermediate precision study

[0316] 2.2.2.5.4.1 Investigation with different instruments

[0317] Based on the above experimental conditions, two portions of stir-fried Typha Pollen (Typha pueraria) standard decoction (batch number: CPHBT-202320) were accurately weighed to prepare the test solution, which was measured on the Thermo Fisher Vanquish Flex ultra-high performance liquid chromatograph and Agilent 1290 ultra-high performance liquid chromatograph, respectively, and the relative retention time and relative peak area of ​​each characteristic peak were calculated. See Tables 27 and 28. Fig. 22 . Fig. 22 Different instrument surveys.

[0318] Table 27 Instrument durability study - relative retention time

[0319]

[0320] Table 28 Instrument durability investigation - relative peak area

[0321]

[0322] The results showed that when the two instruments were used to detect the test samples, the RSD of the relative retention time of each characteristic peak was 0.07% to 1.21%, indicating that the instruments had good durability.

[0323] 2.2.2.5.4.2 Investigation by different personnel and time

[0324] Based on the experimental conditions proposed above, three portions of stir-fried Typha acuminatum (Typha puerariae) standard decoction (batch number: CPHBT-202320) were accurately weighed by different personnel (A, B) at different times (T1, T2) to prepare the test sample, conduct the determination, and calculate the relative retention time and relative peak area of ​​each characteristic peak. See Tables 29 and 30.

[0325] Table 29 Personnel and time inspection - relative retention time

[0326]

[0327] Table 30 Personnel and time investigation-relative peak area

[0328]

[0329] The results showed that when different personnel measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.05% to 2.04%, and the method had good stability.

[0330] 2.2.2.5.5 Durability test

[0331] 2.2.2.5.5.1 Study on the durability of chromatographic columns

[0332] Based on the experimental conditions proposed above, the analysis was carried out using chromatographic column 1 Agilent ZORBAX SB-C18 2.1*100mm, 1.8μm, chromatographic column 2 DIKMA Endeavorsil C18 2.1*100mm, 1.8μm, and chromatographic column 3 Shimadzu Shim-pack Velox C18 2.1*100mm, 1.8μm, and the relative retention time and relative peak area of ​​each characteristic peak were calculated. The results are shown in Tables 31 and 32. Fig.23 , Fig.23 Investigation of different chromatographic columns.

[0333] Table 31 Column durability study - relative retention time

[0334]

[0335] Table 32 Chromatographic column durability investigation - relative peak area

[0336]

[0337] The results showed that the RSD of the relative retention time of the characteristic peaks was between 0.80% and 1.86% when the three chromatographic columns were used to detect the samples, and the chromatographic columns had good durability.

[0338] 2.2.2.5.5.2 Stability study

[0339] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 2h, 4h, 8h, 16h and 24h respectively. See Tables 33 and 34.

[0340] Table 33 Stability Study-Retention Time

[0341]

[0342] Table 34 Stability Study-Peak Area

[0343]

[0344] The results showed that the RSD of the characteristic peak retention time was between 0.04% and 0.13%, and the sample solution was stable within 24 hours.

[0345] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 10 characteristic peaks were included in the subsequent investigation.

[0346] 2.2.2.6 Determination of characteristic peaks and establishment of reference spectrum

[0347] 2.2.2.6.1 Verification results of 24 batches of stir-fried pollen standard decoction

[0348] Using this method, 24 batches of samples were analyzed for characteristic spectra, and relative retention times and relative peak areas were calculated. Fig.24 , Tables 35 and 36. Fig.24 Characteristic spectrum of standard decoction of stir-fried Typha typha (water candle Typha); Peak 1: uridine; Peak 2: 5-hydroxymethyl-2-furancarboxylic acid; Peak 3: p-hydroxybenzoic acid; Peak 6: quercetin-3-O-neohesperidin; Peak 8 (S): typhaneoglycan; Peak 9: kaempferol-3-O-neohesperidin; Peak 10: isorhamnetin-3-O-neohesperidin; S1~S24 are: CPHBT-202304, CPHBT-202305, CPHBT-202306, CPHBT-202307, CPHBT-202308, CPHBT-202309, CPHBT-202310, CPHBT- 202311, CPHBT-202312, CPHBT-202313, CPHBT-202314, CPHBT-202315, CPHBT-202316, CPHBT-202317, CPHBT-202318, CPHBT-2023 19. CPHBT-202320, CPHBT-202321, CPHBT-202322, CPHBT-202323, CPHBT-202324, CPHBT-2023201, CPHBT-202302, CPHBT-202303.

[0349] Table 35 Relative retention time of standard decoction of fried Typha acuminata (Typha acuminata)

[0350]

[0351] Table 36 Relative peak area of ​​standard decoction of fried Typha acuminata (Typha acuminata)

[0352]

[0353]

[0354] Based on the principle of stable relative retention time and the fact that each batch of samples can be detected and the peak is relatively high, a total of 10 peaks with good durability were selected as characteristic peaks. According to the results of the methodological investigation and the verification results of 24 batches of standard soup, the theoretical plate number calculated based on the typhane glycoside peak should be no less than 5000.

[0355] 2.2.2.6.2 Establishment of relative retention time limit values

[0356] The summary of the methodology investigation items and verification results are shown in Tables 37 and 38:

[0357] Table 37 Summary of RSD% results of each item of the methodology - relative retention time / retention time

[0358]

[0359] Table 38 Methodology Results RSD% Summary Standard - Relative Peak Area / Peak Area

[0360]

[0361]

[0362] The relative retention time of each characteristic peak is stable and within the range of ±10% of the average value. The relative peak areas of 24 batches of stir-fried Typha acuminata (water candle Typha acuminata) standard decoctions, among which chromatographic peak 2 is the substance after the medicinal materials are processed, have a stable relative retention time. Due to certain differences between batches of processed products, the relative peak area of ​​the chromatographic peak is regulated, with the average value of the relative peak area +30%, i.e. 0.49, as the upper limit, and the -3SD value is 0.02, which is much lower than the limit of 0.08 for stir-fried Typha acuminata pieces. In order to strictly control the degree of frying, 0.08 is temporarily set as the lower limit.

[0363] Final regulations: There should be 10 characteristic peaks in the chromatogram of the test sample, and the retention times of the 10 characteristic peaks in the chromatogram of the reference medicinal material should correspond. Peak 1, peak 2, peak 8, and peak 10 should correspond to the relative retention times of the reference peaks of uridine, 5-hydroxymethyl-2-furancarboxylic acid, typhaneoglycans, and isorhamnetin-3-O-neohesperidin, respectively. The peak corresponding to the typhaneoglycans reference peak is the S peak. The relative retention times of peaks 3 to 7, peak 9, and S peak are calculated. The relative retention times should be within the relative range of ± of the specified value. The specified values ​​are: 0.49 (peak 3), 0.68 (peak 4), 0.92 (peak 5), 0.96 (peak 6), 0.98 (7), and 1.05 (peak 9). Calculate the relative peak area of ​​peak 2 and S peak, and the relative peak area should be within the specified value range, which is 0.08 to 0.49.

[0364] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 24 batches of standard decoctions of stir-fried Typha acuminata (water candle typha) and a comparison spectrum of the characteristic spectrum of standard decoctions of stir-fried Typha acuminata (water candle typha) was established. Fig.25 . Fig.25 Characteristic spectrum of the standard decoction of fried Typha typhae; Peak 1: uridine; Peak 2: 5-hydroxymethyl-2-furancarboxylic acid; Peak 3: p-hydroxybenzoic acid; Peak 6: quercetin-3-O-neohesperidin; Peak 8 (S): typhaneoglycans; Peak 9: kaempferol-3-O-neohesperidin; Peak 10: isorhamnetin-3-O-neohesperidin. Chromatographic column: Agilent ZORBAX SB-C18, 2.1mm×100mm, 1.8μm.

[0365] 2.2.2.7 Determination of the characteristic spectrum of the standard decoction of stir-fried pollen

[0366] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, gradient elution was performed as specified in the table below, flow rate was 0.3 ml per minute; column temperature was 30°C; detection wavelength was 254 nm. The theoretical plate number calculated based on the typhane novoglycoside peak should be no less than 3000.

[0367]

[0368] Preparation of reference solution Take 0.6g of Typha typhae (water candle cattail) reference medicinal material, place it in a stoppered conical bottle, add 25ml of 30% methanol, heat and reflux for 60 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take appropriate amounts of isorhamnetin-3-O-neohesperidin reference substance, typhane glycoside, uridine, and 5-hydroxymethyl-2-furancarboxylic acid reference substance, accurately weigh them, add 30% methanol to make a mixed solution containing 20μg of each per 1ml, as the reference solution of the reference substance.

[0369] Preparation of test solution: Take an appropriate amount of standard decoction of fried cattail (boiled cattail), take about 0.2g, accurately weigh, put it in a stoppered conical flask, accurately add 25ml of 30% methanol, stopper it, weigh it, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 30% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0370] The determination method is to accurately pipette 2 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0371] There should be 10 characteristic peaks in the chromatogram of the test sample. Except for peak 2, the other 9 characteristic peaks should correspond to the retention times of the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 1, peak 2, peak 8, and peak 10 should correspond to the relative retention times of the reference peaks of uridine, 5-hydroxymethyl-2-furancarboxylic acid, typhaneoglycans, and isorhamnetin-3-O-neohesperidin, respectively. The peak corresponding to the typhaneoglycans reference peak is the S peak. Calculate the relative retention time of peaks 3 to 7, peak 9 and S peak. The relative retention time should be within the relative range of ±10% of the specified value. The specified values ​​are: 0.49 (peak 3), 0.68 (peak 4), 0.92 (peak 5), 0.96 (peak 6), 0.98 (7), and 1.05 (peak 9). Calculate the relative peak area of ​​peak 2 and S peak, and the relative peak area should be within the specified value range, which is 0.08 to 0.49.

[0372] Example 3 HPLC characteristic spectrum of stir-fried Typha lachryma-leaf formula granules

[0373] 2.2.3.1 Materials, reagents and instruments

[0374] 2.2.3.1.1 Instruments and materials

[0375] Same as Example 1

[0376] 2.2.3.1.2 Reagents and test drugs

[0377] Same as Example 1

[0378] Fried Puhuang Formula Granules (Batch No.: KL-01, KL-02, KL-03.)

[0379] 2.2.3.2 Characteristic spectrum determination method

[0380] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, gradient elution was performed as specified in the table below, flow rate was 0.3 ml per minute; column temperature was 30°C; detection wavelength was 254 nm. The theoretical plate number calculated based on the typhane novoglycoside peak should be no less than 3000.

[0381]

[0382] Preparation of reference solution Take 0.6g of Typha typhae (water candle cattail) reference medicinal material, place it in a stoppered conical bottle, add 25ml of 30% methanol, heat and reflux for 60 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take appropriate amounts of isorhamnetin-3-O-neohesperidin reference substance, typhane glycoside reference substance, uridine, and 5-hydroxymethyl-2-furancarboxylic acid reference substance, accurately weigh them, add 30% methanol to make a mixed solution containing 20μg of each per 1ml, as the reference solution of the reference substance.

[0383] Preparation of test solution: Take the powder of stir-fried Typha Pollen (Typha rapa), grind it into powder, take about 0.2 g, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of 30% methanol, stopper it tightly, weigh it, treat it with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 30% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0384] The determination method is to accurately pipette 2 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0385] 2.2.2.3 Chromatographic conditions and system suitability test

[0386] 2.2.2.3.1 Wavelength selection

[0387] Based on the experimental conditions proposed above, the diode array detector was used to perform full-band scanning of the test solution, and the chromatograms of the test solution at wavelengths of 190nm, 210nm, 230nm, 254nm, 270nm, and 365nm were extracted respectively. Fig.26 . Fig.26 Chromatograms of stir-fried Puhuang formula granules at different wavelengths; the results show that when the detection wavelength is 254nm, the chromatographic peak has a larger amount of information, better separation, and a more stable chromatogram baseline, so the detection wavelength is determined to be 254nm.

[0388] 2.2.2.3.2 Column temperature investigation

[0389] Based on the experimental conditions proposed above, the column temperatures of 20℃, 30℃ and 40℃ were investigated. Fig. 27 , Tables 39-40.

[0390] Table 39 Column temperature investigation-relative retention time

[0391]

[0392] Table 40 Column temperature investigation-relative peak area

[0393]

[0394] Fig. 27 Column temperature inspection chromatogram; the results of column temperature inspection showed that when the column temperature was 30℃, the chromatogram peaks were relatively symmetrical and the separation was good, so 30℃ was finally determined as the column temperature for the characteristic spectrum method of Chao Puhuang Formula Granules.

[0395] 2.2.2.3.3 Flow rate investigation

[0396] Based on the experimental conditions proposed above, the flow rates of 0.2ml / min, 0.3ml / min, and 0.4ml / min were investigated. Fig.28 , Tables 41-42.

[0397] Table 41 Flow rate investigation - relative retention time

[0398]

[0399] Table 42 Flow rate investigation - relative peak area

[0400]

[0401] Fig.28 The flow rate investigation results show that when the flow rate is 0.3 ml / min, the chromatogram peak shape is better and the distribution of each characteristic peak is moderate. Therefore, the flow rate is determined to be 0.3 ml / min.

[0402] 2.2.2.3.4 Sample volume investigation

[0403] Based on the experimental conditions proposed above, the flow rates of 1μl, 2μl, and 3μl were investigated. Fig.29 , Tables 43-44.

[0404] Table 43 Injection volume investigation-relative retention time

[0405]

[0406] Table 44 Injection volume investigation - relative peak area

[0407]

[0408] Fig.29 The results of different injection volumes showed that when the injection volume was 2 μl, the chromatogram peak shape was better and the separation was moderate, so the injection volume was determined to be 2 μl.

[0409] 2.2.2.3.5 Delay Investigation

[0410] Based on the above experimental conditions, the analysis time was extended to 94 minutes to observe whether there was a chromatographic peak after 47 minutes. The results are shown in Fig.30 . Fig.30 Delayed inspection.

[0411] The results showed that there were basically no larger chromatographic peaks after an extension of 47 minutes, and the analysis time of the characteristic spectrum method of Chao Puhuang Formula Granules was finally determined to be 47 minutes.

[0412] In summary, the chromatographic conditions and system suitability test of the characteristic spectrum of the stir-fried Puhuang formula granules were determined as follows: octadecylsilane bonded silica gel was used as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, and gradient elution was performed according to the regulations in the following table; the flow rate was 0.3 ml per minute; the column temperature was 30°C; and the detection wavelength was 254 nm.

[0413]

[0414] 2.2.2.4 Preparation of test solution

[0415] 2.2.2.4.1 Investigation of extraction solvent

[0416] Take an appropriate amount of stir-fried Typha acuminatum (batch number: KL-01), grind it into powder, take 0.2g, put it in a stoppered conical bottle, add 25ml of water, ethanol, methanol, 70% methanol, 50% methanol, 30% methanol, respectively, plug it tightly, and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake it well, filter it, and take the filtrate to obtain it. Fig.31 . Fig.31 Investigation of extraction solvents; the results showed that when 30% methanol was used as the extraction solvent, the chromatographic peaks obtained had a large amount of information and good separation, so 30% methanol was selected as the extraction solvent.

[0417] 2.2.2.4.2 Investigation of extraction methods

[0418] Take about 0.2g of stir-fried Typha acuminatum (batch number: KL-01) granules, put them in a stoppered conical flask, add 25ml of 30% methanol, stopper it tightly, reflux and ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes respectively, cool, shake well, filter, and take the filtrate to obtain. Fig.32 . Fig.32 Investigation of extraction methods.

[0419] The results showed that there was no significant difference in the amount of chromatographic peak information when the test sample was subjected to ultrasonic extraction and reflux extraction. Because ultrasonic extraction is more convenient, the extraction method for the test sample was determined to be ultrasonic extraction.

[0420] 2.2.2.4.3 Extraction time investigation

[0421] Take about 0.2g of stir-fried Typha acuminatum (batch number: KL-01) granules, put them in a stoppered conical flask, add 25ml of 30% methanol, seal it tightly, and perform ultrasonic treatment (power 600W, frequency 40kHz), and observe the extraction time of the test sample at 20 minutes, 30 minutes, and 40 minutes respectively, let it cool, shake it well, filter it, and take the filtrate to obtain the result. Fig.33 . Fig.33 Extraction time investigation.

[0422] The results showed that the extraction was complete within 20 minutes, but in order to ensure a more complete extraction, the extraction time of the test sample was set at 30 minutes.

[0423] 2.2.2.4.4 Sample collection and investigation

[0424] Take about 0.1g, 0.2g, and 0.3g of stir-fried Typha acuminatum (water candle Typha acuminatum) granules (batch number: KL-01), 25ml of 30% methanol, plug, weigh, and ultrasonically treat (power 600W, frequency 40kHz). The extraction time of the test sample is 30 minutes, cool, shake, filter, and take the filtrate. Fig.34 . Fig.34 Investigation on sample quantity; the results show that the target peak can be found when the sample quantity is 0.1g, 0.2g and 0.3g respectively, but in order to ensure more sufficient extraction, the sample quantity is determined to be 0.2g.

[0425] In summary, the preparation method of the test solution of the characteristic spectrum of the standard decoction of stir-fried Typha latifolia is determined as follows: take an appropriate amount of the product, grind it into powder, take 0.2 g, put it in a stoppered conical flask, add 25 ml of 30% methanol, seal it, and treat it with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain it.

[0426] 2.2.3.5 Methodological investigation

[0427] 2.2.3.5.1 Chromatographic peak identification

[0428] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of stir-fried Puhuang formula granules.

[0429] Preparation of control medicinal material solution: Take 0.6 g of Typha Pollen (Typha rapa) control medicinal material, place it in a stoppered conical flask, add 25 ml of 30% methanol, heat and reflux for 60 minutes, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0430] Preparation of reference solution: Take appropriate amount of isorhamnetin-3-O-neohesperidin reference substance, typhaneoglycan reference substance, uridine, 5-hydroxymethyl-2-furancarboxylic acid reference substance, p-hydroxybenzoic acid, quercetin-3-O-neohesperidin, and kaempferol-3-O-neohesperidin, accurately weigh them, and add 30% methanol to make a mixed solution containing 20 μg of each substance per 1 ml, which serves as the reference substance solution.

[0431] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution of the formula granules of Typha acuminata (Typha rapa) was prepared.

[0432] Results Figure 35-43 . Fig.35 Chromatographic peak identification; Fig.36 Spectrum of uridine reference; Fig.37 Spectrum of granular uridine reference substance; Figure 38 Spectrum of 5-hydroxymethylfurancarboxylic acid reference substance; Fig.39 Spectrum of granular 5-hydroxymethylfurancarboxylic acid; Fig.40 Spectrum of typhala glycoside reference substance; Fig.41 Granular Typha neoglycoside spectrum;

[0433] Fig.42 Spectrum of isorhamnetin-3-O-neohesperidin reference substance; Fig.43 Spectrum of granular isorhamnetin-3-O-neohesperidin. The results showed that peak 1 was uridine, peak 2 was 5-hydroxymethyl-2-furancarboxylic acid, peak 3 was p-hydroxybenzoic acid, peak 6 was quercetin-3-O-neohesperidin, peak 8 was typhaneoglucoside, peak 9 was kaempferol-3-O-neohesperidin, and peak 10 was isorhamnetin-3-O-neohesperidin.

[0434] 2.2.3.5.2 Precision test

[0435] Take the stir-fried Puhuang formula granules test solution and inject 6 times continuously according to the proposed experimental method, 2 μl each time, and calculate the relative retention time and relative peak area of ​​each characteristic peak. See Tables 45 and 46.

[0436] Table 45 Precision investigation - retention time

[0437]

[0438] Table 46 Precision Investigation-Peak Area

[0439]

[0440] The results showed that the RSD values ​​of the retention times of the characteristic peaks were between 0.16% and 1.64%, indicating that the instrument had good precision.

[0441] 2.2.3.5.3 Repeatability study

[0442] Weigh 6 portions of stir-fried Puhuang formula granules accurately and prepare and measure them according to the proposed experimental method. See Tables 47 and 48.

[0443] Table 47 Repeatability Study - Relative Retention Time

[0444]

[0445] Table 48 Repeatability study - relative peak area ratio

[0446]

[0447] The results showed that the RSD% values ​​of the relative retention times of the characteristic peaks were between 1.30% and 1.97%, indicating that the method had good repeatability.

[0448] 2.2.3.5.4 Stability study

[0449] Based on the above proposed experimental conditions, take the same test solution and measure it at 0h, 2h, 4h, 8h, 16h and 24h respectively. See Tables 49 and 50.

[0450] Table 49 Stability Study - Retention Time

[0451]

[0452] Table 50 Stability Study-Peak Area

[0453]

[0454] The results showed that the RSD of the corresponding characteristic peak retention time was 0.02%-0.48%, and the RSD of the peak area was 0.20%-1.86%, indicating that the sample solution was stable within 24 hours.

[0455] 2.2.3.5.4 Intermediate precision study

[0456] 2.2.3.5.4.1 Investigation of different instruments

[0457] Based on the above proposed experimental conditions, different personnel (A, B) prepared and tested the stir-fried Puhuang formula granules at different times (T1, T2) according to the proposed experimental method. The results are shown in Tables 51 and 52.

[0458] Table 51 Personnel and time inspection - relative retention time

[0459]

[0460] Table 52 Personnel and time investigation-relative peak area

[0461]

[0462] The results showed that when different personnel measured the same sample at different times, the RSD% values ​​of the relative retention times of the characteristic peaks were between 0.00% and 0.52%, indicating that the method had good repeatability.

[0463] 2.2.3.5.4.2 Column durability test

[0464] Based on the experimental conditions proposed above, the chromatographic columns of different manufacturers and fillers were investigated: chromatographic column 1 Agilent ZORBAX SB-C18 2.1*100mm, 1.8μm, chromatographic column 2 DIKMA Endeavorsil C18 2.1*100mm, 1.8μm, chromatographic column 3 Shimadzu Shim-pack Velox C18 2.1*100mm, 1.8μm. See Tables 53-54, Fig.44 .

[0465] Table 53 Chromatographic column durability investigation - relative retention time

[0466]

[0467] Table 54 Chromatographic column durability investigation - relative peak area

[0468]

[0469] Fig.44 Different types of chromatographic columns. The results show that the RSD of the relative retention time of the characteristic peaks is between 1.36% and 2.03% when the above three chromatographic columns are used to detect the samples. The recommended chromatographic columns are Agilent ZORBAX SB-C18 2.1*100mm, 1.8μm, DIKMA Endeavorsil C18 2.1*100mm, 1.8μm, and chromatographic column 3 is Shimadzu Shim-pack Velox C18 2.1*100mm, 1.8μm.

[0470] 2.2.3.5.4.3 Instrument durability inspection

[0471] Based on the experimental conditions proposed above, two portions of stir-fried Puhuang formula granules were accurately weighed to prepare the test solution, which was measured on the Thermo Fisher Vanquish Flex and Agilent 1290 ultra-high performance liquid chromatographs, respectively. See Table 55, Fig.45 .

[0472] Table 55 Instrument durability study - relative retention time ratio

[0473]

[0474] Fig.45 Different instruments. The results showed that when the two instruments were used to detect the test sample, the RSD range of the relative retention time of each characteristic peak was 0.07%-2.51%, indicating that the method and instrument had good durability.

[0475] 2.2.3.6 Determination of characteristic peaks and establishment of reference spectrum

[0476] 2.2.3.6.1 Verification results of three batches of stir-fried Puhuang formula granules

[0477] Using this method, characteristic spectrum analysis was performed on three batches of samples to calculate relative retention time and relative peak area. Figures 46-48 , Tables 56-57. Fig.46 Characteristic spectrum verification diagram of stir-fried Puhuang formula granules (KL-01), where peak 1: uridine; peak 2: 5-hydroxymethyl-2-furancarboxylic acid; peak 3: p-hydroxybenzoic acid; peak 6: quercetin-3-O-neohesperidin; peak 8 (S): typhaneoglycans; peak 9: kaempferol-3-O-neohesperidin; peak 10: isorhamnetin-3-O-neohesperidin; Fig.47 Verification diagram of characteristic spectrum of stir-fried Puhuang formula granules (KL-02), where: Peak 1: uridine; Peak 2: 5-hydroxymethyl-2-furancarboxylic acid; Peak 3: p-hydroxybenzoic acid; Peak 6: quercetin-3-O-neohesperidin; Peak 8 (S): typhaneoglucoside; Peak 9: kaempferol-3-O-neohesperidin; Peak 10: isorhamnetin-3-O-neohesperidin. Fig.48 Characteristic spectrum verification diagram of stir-fried Puhuang formula granules (KL-03), where peak 1: uridine; peak 2: 5-hydroxymethyl-2-furancarboxylic acid; peak 3: p-hydroxybenzoic acid; peak 6: quercetin-3-O-neohesperidin; peak 8 (S): typhaneoglycans; peak 9: kaempferol-3-O-neohesperidin; peak 10: isorhamnetin-3-O-neohesperidin

[0478] Table 56 Relative retention time of three batches of stir-fried Puhuang formula granules

[0479]

[0480]

[0481] Table 57 Relative peak areas of three batches of stir-fried Typha lachryma-curcuma granules

[0482]

[0483] According to the principle of stable relative retention time and the fact that each batch of samples can be detected and the peak is relatively high, a total of 10 peaks with good repeatability were selected as characteristic peaks. Except for peak 2, the other 9 characteristic peaks should correspond to the retention time of the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 1, peak 2, peak 8, and peak 10 should correspond to the relative retention time of the reference peaks of uridine, 5-hydroxymethyl-2-furancarboxylic acid, typhaneoglycans, and isorhamnetin-3-O-neohesperidin, respectively. The peak corresponding to the typhaneoglycans reference peak is the S peak. The relative retention time of peaks 3 to 7, peak 9 and S peak is calculated. The relative retention time should be within the relative range of ±10% of the specified value. The specified values ​​are: 0.49 (peak 3), 0.68 (peak 4), 0.92 (peak 5), 0.96 (peak 6), 0.98 (7), and 1.05 (peak 9).

[0484] 2.2.3.6.2 Establishment of relative retention time limit values

[0485] The summary of the methodology inspection items and verification results are shown in Tables 58-59:

[0486] Table 58 Summary of RSD% results of each item of methodology - relative retention time

[0487]

[0488] Table 59 Summary of RSD% results of each item of methodology - relative peak area

[0489]

[0490] As can be seen from the table above, different chromatographic columns have a great influence on the retention time of chromatographic peaks. In order to increase the reproducibility and applicability of the method, the relative retention time of each peak is temporarily set at ±10%. Since chromatographic peak 2 is a newly generated chemical component after processing and heating, this document standardizes the relative peak area of ​​chromatographic peak 2. The relative peak areas of the remaining batches vary too much to be specified, so only the relative peak area of ​​peak 2 is included in the standard text. The relative retention times of 22 batches of Puhuang (water candle cattail) medicinal materials, 24 batches of fried Puhuang (water candle cattail) decoction pieces, 24 batches of fried Puhuang (water candle cattail) standard soups, 3 batches of fried Puhuang (water candle cattail) intermediates, and 3 batches of fried Puhuang (water candle cattail) formula granules are summarized, and the mean of the relative retention time is calculated and ±10% of the mean is used as the specified value.

[0491] Final regulations: There should be 10 characteristic peaks in the chromatogram of the test sample. Except for peak 2, the other 9 characteristic peaks should correspond to the retention times of the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 1, 2, 8, and 10 should correspond to the relative retention times of the reference peaks of uridine, 5-hydroxymethyl-2-furancarboxylic acid, typhaneoglycans, and isorhamnetin-3-O-neohesperidin, respectively. The peak corresponding to the typhaneoglycans reference peak is the S peak. Calculate the relative retention time of peaks 3 to 7, peak 9 and S peak. The relative retention time should be within the relative range of ±10% of the specified value. The specified values ​​are: 0.49 (peak 3), 0.68 (peak 4), 0.92 (peak 5), 0.96 (peak 6), 0.98 (7), and 1.05 (peak 9). Calculate the relative peak area of ​​peak 2 and S peak, and the relative peak area should be within the specified value range, which is 0.08 to 0.49.

[0492] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize three batches of stir-fried Puhuang formula granules, and a comparison spectrum of the characteristic spectrum of stir-fried Puhuang formula granules was established. Fig.49 . Fig.49 Comparative characteristic spectrum of stir-fried Puhuang formula granules; Peak 1: uridine; Peak 2: 5-hydroxymethyl-2-furancarboxylic acid; Peak 3: p-hydroxybenzoic acid; Peak 6: quercetin-3-O-neohesperidin; Peak 8 (S): typhaneoglycans; Peak 9: kaempferol-3-O-neohesperidin; Peak 10: isorhamnetin-3-O-neohesperidin; Chromatographic column: Agilent ZORBAX SB-C18 2.1mm×100mm, 1.8μm.

[0493] 2.2.3.7 Determination of characteristic spectrum of stir-fried Puhuang formula granules

[0494] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, gradient elution was performed as specified in the table below, flow rate was 0.3 ml per minute; column temperature was 30°C; detection wavelength was 254 nm. The theoretical plate number calculated based on the typhane novoglycoside peak should be no less than 3000.

[0495]

[0496] Preparation of reference solution Take 0.6g of Typha typhae (water candle cattail) reference medicinal material, place it in a stoppered conical bottle, add 25ml of 30% methanol, heat and reflux for 60 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. Take appropriate amounts of isorhamnetin-3-O-neohesperidin reference substance, typhane glycoside, uridine, and 5-hydroxymethyl-2-furancarboxylic acid reference substance, accurately weigh them, add 30% methanol to make a mixed solution containing 20μg of each per 1ml, as the reference solution of the reference substance.

[0497] Preparation of the test solution: Take fried Typha acuminata (water candle Typha acuminata) particles, grind them into powder, take about 0.2 g, weigh accurately, put it in a stoppered conical flask, accurately add 25 ml of 30% methanol, stopper it, weigh it, treat it with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 30% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0498] The determination method is to accurately pipette 2 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0499] There should be 10 characteristic peaks in the chromatogram of the test sample. Except for peak 2, the other 9 characteristic peaks should correspond to the retention times of the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 1, peak 2, peak 8, and peak 10 should correspond to the relative retention times of the reference peaks of uridine, 5-hydroxymethyl-2-furancarboxylic acid, typhaneoglycans, and isorhamnetin-3-O-neohesperidin, respectively. The peak corresponding to the typhaneoglycans reference peak is the S peak. Calculate the relative retention time of peaks 3 to 7, peak 9 and S peak. The relative retention time should be within the relative range of ±10% of the specified value. The specified values ​​are: 0.49 (peak 3), 0.68 (peak 4), 0.92 (peak 5), 0.96 (peak 6), 0.98 (7), and 1.05 (peak 9). Calculate the relative peak area of ​​peak 2 and S peak, and the relative peak area should be within the specified value range, which is 0.08 to 0.49.

[0500] Comparative Example 1

[0501] The same pretreatment conditions as in Example 1 were used, except that the chromatographic conditions were: acetonitrile as the mobile phase, 0.1% formic acid as the mobile phase B, gradient elution, a C18 column (100 mm, inner diameter 2.1 mm, particle size 1.8 μm), gradient elution, a flow rate of 0.3 ml per minute, a column temperature of 30° C., and a detection wavelength of 254 nm.

[0502]

[0503] The results are as follows:

[0504] In the analysis of stir-fried Typha acuminatum (Typha sphaerocephala) granules, the separation of chromatographic peak 1 as a characteristic chromatographic peak for identification was poor. The separation and purity of chromatographic peak 2 as an indicator peak for the degree of stir-frying to be limited by relative peak area were both poor, which affected the calculation of relative peak area. Fig.50 It is the characteristic spectrum of comparative example 1.

[0505] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing HPLC characteristic spectra of Typhae Pollen medicinal materials, stir-fried Typhae Pollen pieces, standard decoctions and their formula particles, comprising: A) extracting the raw material of Typhae Pollen and the stir-fried raw material of Typhae Pollen respectively by using solvents to obtain a solution to be tested; B) the liquid to be tested is measured by high performance liquid chromatography to obtain HPLC characteristic spectra of the Pollen Typhae raw material and the stir-fried Pollen Typhae raw material; The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is 0.1% formic acid, and the elution is gradient; The gradient elution is specifically: 0-4min, phase A: 0%→2%, phase B: 100%→98%; 4-19 min, phase A: 2%→10%, phase B: 98%→90%; 19-34 min, phase A: 10%→12%, phase B: 90%→88%; 34-47min, phase A: 12%→13%, phase B: 88%→87%.

2. The method according to claim 1, characterized in that It also includes the preparation of reference substances and reference medicinal material solutions: Take uridine and 5-hydroxymethyl-2-furancarboxylic acid reference substances respectively, dissolve them in 30% methanol to obtain reference substance solutions; the concentration of the uridine reference substance is 20 μg / mL; the concentration of the 5-hydroxymethyl-2-furancarboxylic acid reference substance is 20 μg / mL; Take the stir-fried pollen pollen reference medicinal material and dissolve it in 30% methanol, heat it under reflux for extraction, cool it, and filter it to obtain a reference medicinal material solution; The reference substance and reference medicinal material reference substance solutions are determined by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material reference substance, respectively; and based on the chromatograms of the reference substance and the reference medicinal material reference substance, the components of the HPLC characteristic spectra of the Puhuang medicinal material, stir-fried Puhuang decoction pieces, standard decoction and its formula granules are qualitatively determined.

3. The method according to claim 1, characterized in that The chromatographic column is a C18 column with specifications of 1.8 μm and 2.1×100 mm; the column temperature is 30° C.

4. The method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.3 mL / min; the detection wavelength is 254 nm; the theoretical plate number calculated based on the typhala glycoside peak should be no less than 3000, and the injection volume is 2 μL.

5. The method according to claim 1, characterized in that Step A) The stir-fried pollen raw materials include stir-fried pollen slices, standard decoctions and formula granules thereof.

6. The method according to claim 5, characterized in that The similarity of the HPLC characteristic spectrum of Typha latifolia was evaluated by the similarity evaluation system of Chinese medicine chromatographic fingerprint, and a HPLC standard characteristic spectrum consisting of 9 characteristic peaks was obtained, including peak 1: uridine; peak 2: p-hydroxybenzoic acid; peak 5: quercetin-3-O-neohesperidin; peak 7 (S): typhaneoglycans; peak 8: kaempferol-3-O-neohesperidin; peak 9: isorhamnetin-3-O-neohesperidin; In the characteristic spectrum of Typha acuminata medicinal material, the relative retention time of each characteristic peak and the S peak is calculated with typha glycoside as the reference peak, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.49 (peak 2), 0.68 (peak 3), 0.92 (peak 4), 0.96 (peak 5), 0.98 (peak 6), and 1.05 (peak 8).

7. The method according to claim 5, characterized in that The similarity of HPLC characteristic spectra of stir-fried Typhae Pollen slices, standard decoction and its formula granules was evaluated by using the similarity evaluation system of Chinese medicine chromatographic fingerprint, and the HPLC standard characteristic spectra of stir-fried Typhae Pollen slices, standard decoction and its formula granules were obtained, which consisted of 10 characteristic peaks, including peak 1: uridine; peak 2: 5-hydroxymethyl-2-furancarboxylic acid; peak 3: p-hydroxybenzoic acid; peak 6: quercetin-3-O-neohesperidin; peak 8 (S): typhaneoglycans; peak 9: kaempferol-3-O-neohesperidin; peak 10: isorhamnetin-3-O-neohesperidin; In the characteristic spectra of the stir-fried Typha Pollen slices, standard decoction and its formula granules, the relative retention time of each characteristic peak and the S peak is calculated with typhala glycoside as the reference peak S peak, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.49 (peak 3), 0.68 (peak 4), 0.92 (peak 5), 0.96 (peak 6), 0.98 (7), and 1.05 (peak 9).

8. The method according to claim 7, characterized in that The preparation of the test solution of the Puhuang medicinal material and the stir-fried Puhuang decoction piece is as follows: dissolving the stir-fried Puhuang in 30% methanol, heating under reflux for extraction, cooling, shaking, and filtering to obtain the solution; The heating reflux time is 60min; The ratio of the mass g of the Puhuang medicinal material and the stir-fried Puhuang decoction pieces to the volume mL of the solvent is 0.5-0.7:20-30.

9. The method according to claim 7, characterized in that: The preparation of the stir-fried pollen standard decoction and formula granules is as follows: the stir-fried pollen is dissolved in 30% methanol, ultrasonically extracted, cooled, shaken, and filtered; the ultrasonic power is 600W and the frequency is 40kHz; The ratio of the mass g of the stir-fried Pollen Typhae standard decoction and the formula granules to the volume mL of the solvent is 0.1-0.3:20-30.

10. A method for identifying stir-fried Typhae Radix Pieces, standard decoctions and formula granules thereof from Typhae Radix Pieces, characterized in that: The identification is performed by the method described in any one of claims 1 to 9, and the difference is that the stir-fried Typhae Pollen slices, standard decoction and its formula granules contain the characteristic peak 5-hydroxymethyl-2-furancarboxylic acid; the Typhae Pollen medicinal material does not contain 5-hydroxymethyl-2-furancarboxylic acid.

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