Method for constructing HPLC (High Performance Liquid Chromatography) characteristic chromatograms of senecio scandens medicinal materials, decoction pieces, standard decoction and formula granules

Abstract

The invention provides a method for detecting HPLC (high performance liquid chromatography) characteristic chromatograms of senecio scandens medicinal materials, decoction pieces, standard decoction and formula granules, which comprises the following steps: pre-treating raw materials of a test sample to obtain liquid to be detected; the pretreatment mode comprises solvent dissolution and extraction; measuring the liquid to be measured by adopting HPLC (High Performance Liquid Chromatography) to obtain HPLC characteristic chromatograms of the senecio scandens medicinal material, decoction pieces, standard decoction and formula granules; the preparation method comprises the following steps: preparing a reference substance solution, namely dissolving neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, chlorogenic acid methyl ester, rutin, hyperoside, isoquercitrin, chlorogenic acid ethyl ester, isorhamnetin-3-O-galactoside, quercitrin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin and kaempferol by adopting a solvent; obtaining a reference substance solution of the reference substance; the HPLC conditions are as follows: a chromatographic column is a phenyl column; a mobile phase A is an acetonitrile solution, a mobile phase B is a 0.3% phosphoric acid aqueous solution, and gradient elution is carried out; according to the method, a high performance liquid chromatography method is adopted, and an acetonitrile-0. 3% phosphoric acid solution is selected as a mobile phase for gradient elution; the method comprises the following steps: taking neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, chlorogenic acid methyl ester, rutin, hyperoside, isoquercitrin, chlorogenic acid ethyl ester, isorhamnetin-3-O-galactoside, quercitrin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin and kaempferol as reference substances; the HPLC characteristic chromatogram method of the senecio scandens medicinal material, the decoction pieces, the standard decoction and the formula granules is established, and more scientific technical means are provided for controlling the medicinal quality of the senecio scandens medicinal material, the decoction pieces, the standard decoction and the formula granules.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical and formulation analysis and detection technology, and in particular to a method for constructing HPLC characteristic chromatograms of Senecio scandens medicinal materials, decoction pieces, standard decoctions, and formulation granules. Background Technology

[0002] Senecio scandens, the dried aerial parts of the plant Senecio scandens Buch.-Ham. (family Asteraceae), possesses the effects of clearing heat and detoxifying, improving eyesight, and promoting diuresis. It is used for carbuncles, boils, fever due to colds, red and swollen eyes, diarrhea, dysentery, and eczema. The standard decoction is a freeze-dried powder produced from the medicinal material according to a fixed preparation process. To ensure the uniformity and stability of the quality of Senecio scandens medicinal material, processed slices, and its standard decoction, this paper establishes a new characteristic chromatographic method for quality control. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing HPLC characteristic chromatograms of Senecio scandens medicinal materials, decoction pieces, standard decoctions, and formulation granules. The method of the present invention is accurate and reliable.

[0004] The inventors have made a groundbreaking discovery that neochlorogenic acid is a very important identification point for Senecio scandens medicinal materials, processed slices, standard decoctions, and formulated granules.

[0005] Properties and Chemical Structure of Neochlorogenic Acid: Neochlorogenic acid is a monocaffeoylquinic acid ester compound with the molecular formula C6H2O. 16 H 18 O9, with a molecular weight of 354.31, contains one caffeoyl group and one quinic acid group in its chemical structure. Physicochemical properties: In the solid state, it is a white to off-white crystalline powder, readily soluble in polar solvents such as methanol and ethanol, slightly soluble in water, and hygroscopic; it should be stored at 2-8℃.

[0006] Senecio scandens is easily confused with the adulterant, Artemisia capillaris, as their stems are similar in appearance and difficult to distinguish based on looks alone. Neochlorogenic acid, a characteristic component of Senecio scandens, can help differentiate genuine from adulterants by its presence and content, thus improving the accuracy of identification.

[0007] The content of chlorogenic acid can reflect the consistency of the ingredients between the formula granules and the standard decoction, ensuring that "granules and decoction are equivalent".

[0008] Both neochlorogenic acid and chlorogenic acid are caffeoyl quinic acid esters, differing only in the binding position of the caffeoyl group on the quinic acid (at position 5 in neochlorogenic acid and position 3 in chlorogenic acid). Their molecular polarity is very small, making them difficult to separate using conventional methods.

[0009] A method for detecting the characteristic HPLC chromatograms of Senecio scandens medicinal materials, processed slices, standard decoctions, and formulated granules, including:

[0010] The raw materials for testing are pretreated to obtain the test solution; the pretreatment method includes solvent dissolution and extraction; the test solution is analyzed by HPLC to obtain the HPLC characteristic chromatograms of Senecio scandens medicinal material, decoction pieces, standard decoction, and formula granules;

[0011] Preparation of reference solutions: Neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol were dissolved in solvents to obtain reference solutions.

[0012] The HPLC conditions were as follows: a phenyl column; mobile phase A was acetonitrile solution, mobile phase B was 0.3% phosphoric acid aqueous solution, and gradient elution was used.

[0013] The gradient elution specifically refers to:

[0014] 0–10 min, Phase A: 5% → 7%, Phase B: 95% → 93%;

[0015] 10–18 min, Phase A: 7%, Phase B: 93%;

[0016] 18–26 min, Phase A: 7% → 14%, Phase B: 93% → 86%;

[0017] 26–38 min, Phase A: 14% → 16%, Phase B: 86% → 84%;

[0018] 38–50 min, Phase A: 16% → 25%, Phase B: 84% → 75%;

[0019] 50–60 min, Phase A: 25% → 40%, Phase B: 75% → 60%.

[0020] The raw materials for the test sample described in this invention are one or more of the following: Senecio scandens medicinal material, Senecio scandens standard decoction, or Senecio scandens formula granules.

[0021] This invention provides a method for constructing HPLC characteristic chromatograms of Senecio scandens medicinal materials, processed slices, standard decoctions, and formulated granules. First, the raw materials to be tested are pretreated, and the pretreatment method includes solvent dissolution and extraction to obtain the test solution.

[0022] Specifically, the sample raw material is dissolved in a solvent, heated under reflux for extraction, cooled, shaken well, and filtered to obtain the final product. The solvent used in this invention is preferably methanol; the extraction time is 30 minutes.

[0023] In this invention, complete extraction is achieved under the aforementioned heating and reflux conditions. The chromatogram shows good peak shape and resolution.

[0024] The ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent in this invention is 0.5:25.

[0025] When using the extraction solvent and specific solvent addition amounts, the present invention achieves good peak shape and resolution, and moderate peak size for each chromatographic peak.

[0026] All the raw materials mentioned above can be subjected to quality control using the methods of this invention.

[0027] The present invention also includes the preparation of a reference solution: Preparation of the reference solution: Neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol are dissolved in a solvent to obtain a reference solution;

[0028] The reference herb *Senecio scandens* was dissolved in methanol to obtain a reference herb solution.

[0029] This also includes the preparation of reference solutions of control medicinal materials:

[0030] The reference herb Senecio scandens was dissolved in methanol and extracted by heating and reflux to obtain a reference herb solution; the extraction time was 30 minutes.

[0031] In some specific embodiments, the ratio of the mass (g) of the Senecio scandens reference material to the volume (mL) of the solvent is 3:25.

[0032] The reference standard solution and the reference medicinal material solution were injected into a high-performance liquid chromatograph for determination, and the chromatograms of the reference standard and the characteristic chromatograms of the reference medicinal material were obtained respectively. Based on the characteristic parameters of the chromatograms of the reference standard, the corresponding chromatograms in the HPLC characteristic chromatograms of the Senecio scandens test sample, the standard decoction test sample, and the formula granule test sample were compared and identified.

[0033] The preferred concentration of the reference solution in this invention is as follows: the injection concentration of the reference solution is: neochlorogenic acid 20 μg / mL, protocatechuic aldehyde 20 μg / mL, chlorogenic acid 20 μg / mL, caffeic acid 20 μg / mL, cryptochlorogenic acid 20 μg / mL, p-coumaric acid 20 μg / mL, methyl chlorogenic acid 20 μg / mL, rutin 20 μg / mL, gold Hyperoside 20 μg / mL, isoquercitrin 20 μg / mL, ethyl chlorogenic acid 20 μg / mL, isorhamnetin-3-O-galactoside 20 μg / mL, quercetin 20 μg / mL, isochlorogenic acid B 20 μg / mL, isochlorogenic acid A 20 μg / mL, isochlorogenic acid C 20 μg / mL, quercetin 20 μg / mL, kaempferol 20 μg / mL

[0034] The test solution was analyzed by high performance liquid chromatography to obtain the HPLC characteristic chromatograms of Senecio scandens medicinal material, decoction pieces, standard decoction, and formula granules.

[0035] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a phenyl column with a size of 150×4.6mm 2.7μm; the present invention does not limit the specific chromatographic column model, and any C18 chromatographic column that meets the above model is acceptable.

[0036] In some embodiments, the chromatographic column can be a Waters column. Phenyl 4.6×150mm 2.7μm、Welch

[0037] Phenyl-Hexyl4.6×150mm 2.7μm.

[0038] The preferred column temperature in this invention is 25°C. At a column temperature of 25°C, the peak shapes of each chromatogram are better and the resolution is moderate; therefore, 25°C was ultimately determined as the column temperature for the characteristic chromatogram method of the Senecio scandens standard decoction.

[0039] In this invention, mobile phase A is an acetonitrile solution, and mobile phase B is a 0.3% phosphoric acid aqueous solution, with gradient elution.

[0040] Specifically, the gradient elution is as follows:

[0041] 0–10 min, Phase A: 5% → 7%, Phase B: 95% → 93%;

[0042] 10–18 min, Phase A: 7%, Phase B: 93%;

[0043] 18–26 min, Phase A: 7% → 14%, Phase B: 93% → 86%;

[0044] 26–38 min, Phase A: 14% → 16%, Phase B: 86% → 84%;

[0045] 38–50 min, Phase A: 16% → 25%, Phase B: 84% → 75%;

[0046] 50–60 min, Phase A: 25% → 40%, Phase B: 75% → 60%.

[0047] The theoretical plate number, calculated based on hyperoside, should be no less than 8000. This invention innovatively discovers that, under the above elution gradient, baseline separation is good, the separation between peaks is excellent, the distribution is uniform, and the baseline is stable.

[0048] The flow rate of the mobile phase described in this invention is 0.8 mL / min.

[0049] The results showed that the chromatogram peak shape was good and the resolution was moderate at a flow rate of 0.8 ml / min. Therefore, the flow rate was determined to be 0.8 ml / min.

[0050] The injection volume in this invention is 2 μL. The inventors discovered that when the injection volume is 2 μL, the chromatographic peak shape is good and the resolution is moderate. Therefore, the injection volume was determined to be 2 μL.

[0051] The detection wavelength is 360 nm. The inventors have discovered that at a detection wavelength of 360 nm, the chromatographic peak information content is larger, the separation is better, and the chromatographic baseline is more stable.

[0052] This invention is the first to establish a characteristic chromatographic detection method using ultra-high performance liquid chromatography (UHPLC) to distinguish between raw Senecio scandens, processed slices, standard decoctions, and formulated granules. This method is beneficial for comprehensively evaluating the scientific validity and rationality of the related processes of Senecio scandens and its processed extracts and preparations, and can better control the intrinsic quality of Senecio scandens and its processed extracts and preparations, thus ensuring the clinical efficacy of Senecio scandens and its processed extracts and preparations.

[0053] This invention employs a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine to evaluate the similarity of HPLC characteristic chromatograms of *Senecio scandens* medicinal materials, processed slices, standard decoctions, and formulated granules. A standard HPLC characteristic chromatogram consisting of 19 characteristic peaks was constructed, with the chemical composition information corresponding to each peak as follows: Peak 1: Neochlorogenic acid; Peak 2: Protocatechuic aldehyde; Peak 3: Chlorogenic acid; Peak 4: Caffeic acid; Peak 5: Cryptochlorogenic acid; Peak 6: p-Coumaric acid; Peak 7: Methyl chlorogenic acid; Peak 9: Rutin; Peak 10: Hyperoside; Peak 11: Isoquercetin; Peak 12: Ethyl chlorogenic acid; Peak 13: Isorhamnoside-3-O-galactoside; Peak 14: Quercetin; Peak 15: Isochlorogenic acid B; Peak 16: Isochlorogenic acid A; Peak 17: Isochlorogenic acid C; Peak 18: Quercetin; Peak 19: Kaempferol.

[0054] In the characteristic chromatograms of the medicinal materials, processed slices, standard decoctions, and formulated granules of *Senecio scandens*, ginsenoside is used as the reference peak S. The relative retention time of each characteristic peak and the S peak is calculated. The allowable fluctuation range of the relative retention time of each characteristic peak is within ±10% of the specified value. The specified value is 0.24 (peak 1), 0.30 (peak 2), 0.41 (peak 3), 0.48 (peak 5), 0.75 (peak 6), 0.85 (peak 7), 0.95 (peak 8), 0.97 (peak 9), 1.01 (peak 11), 1.10 (peak 12), 1.19 (peak 13), 1.21 (peak 14), 1.27 (peak 15), 1.31 (peak 16), 1.40 (peak 17), 1.56 (peak 18), and 1.72 (peak 19).

[0055] Quality judgment criteria: Take the extracts and preparations of Senecio scandens and its processed products, and operate according to the above method to obtain the above characteristic chromatograms. Use the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" of the National Pharmacopoeia Commission to analyze the characteristic chromatograms of Senecio scandens and its processed products and their preparations, and the sample characteristic chromatograms. The similarity is greater than 0.90.

[0056] This invention provides a method for detecting the HPLC characteristic chromatograms of *Senecio scandens* medicinal materials, processed slices, standard decoctions, and formulated granules, comprising: pretreating the test sample raw material to obtain a test solution; the pretreatment method includes solvent dissolution and extraction; determining the test solution by HPLC to obtain the HPLC characteristic chromatograms of *Senecio scandens* medicinal materials, processed slices, standard decoctions, and formulated granules; and preparing a reference solution: taking neochlorogenic acid, protocatechuic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol, dissolving them in a solvent to obtain a reference solution; the HPLC method further comprises... The LC conditions were as follows: a phenyl column; mobile phase A was acetonitrile solution, mobile phase B was 0.3% phosphoric acid aqueous solution, and gradient elution was performed. This invention employs high-performance liquid chromatography (HPLC), using acetonitrile-0.3% phosphoric acid solution as the mobile phase for gradient elution. Using neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol as references, a method for establishing HPLC characteristic chromatograms of *Senecio scandens* medicinal materials, processed slices, standard decoctions, and formulated granules was established. This provides more scientific technical means for controlling the medicinal quality of *Senecio scandens* medicinal materials, processed slices, standard decoctions, and formulated granules. Attached Figure Description

[0057] Figure 1Isometric absorption diagram of Senecio scandens formulation particles;

[0058] Figure 2 Chromatograms of Senecio scandens formulation particles at different wavelengths;

[0059] Figure 3 Chromatograms were obtained by examining the column temperature.

[0060] Figure 4 Flow velocity study;

[0061] Figure 5 Different injection volumes;

[0062] Figure 6 Delayed investigation;

[0063] Figure 7 Extraction methods were examined;

[0064] Figure 8 Extraction solvent investigation;

[0065] Figure 9 Extraction time consideration;

[0066] Figure 10 Investigation of solvent addition amount;

[0067] Figure 11-a Chromatographic peak identification diagram of Senecio scandens standard decoction;

[0068] Figure 11-b Chromatographic peak identification diagram of Senecio scandens standard decoction;

[0069] Figure 11-c Chromatographic peak identification diagram of Senecio scandens standard decoction;

[0070] Figure 12 Chromatograms were examined using different instruments;

[0071] Figure 13 Durability study of chromatographic column for Senecio scandens standard decoction;

[0072] Figure 14 Characteristic spectrum of standard decoction of Senecio scandens;

[0073] Figure 15 Characteristic spectrum of Senecio scandens standard decoction compared with other preparations;

[0074] Figure 16 Extraction solvent investigation;

[0075] Figure 17 Extraction methods were examined;

[0076] Figure 18 Extraction time consideration;

[0077] Figure 19 Investigation of solvent addition amount;

[0078] Figure 20-a Chromatographic peak identification diagram of Senecio scandens medicinal material;

[0079] Figure 20-b Chromatographic peak identification diagram of Senecio scandens medicinal material;

[0080] Figure 20-c Chromatographic peak identification diagram of Senecio scandens medicinal material;

[0081] Figure 21 Exploration using different instruments;

[0082] Figure 22 Column durability study;

[0083] Figure 23-a Characteristic atlas of Senecio scandens medicinal materials;

[0084] Figure 23–b: Characteristic map of Senecio scandens medicinal material;

[0085] Figure 24 A comparative characteristic atlas of Senecio scandens medicinal materials;

[0086] Figure 25 Isometric absorption diagram of Senecio scandens formulation particles;

[0087] Figure 26 Chromatograms of Senecio scandens formulation particles at different wavelengths;

[0088] Figure 27 Chromatograms were obtained by examining the column temperature.

[0089] Figure 28 Flow velocity study;

[0090] Figure 29 Different injection volumes;

[0091] Figure 30 Delayed investigation;

[0092] Figure 31 Extraction solvent investigation;

[0093] Figure 32 Extraction methods were examined;

[0094] Figure 33 Extraction time consideration;

[0095] Figure 34 Investigation of solvent addition amount;

[0096] Figure 35-a Chromatographic peak identification diagram of Senecio scandens formulation granules;

[0097] Figure 35-b Chromatographic peak identification diagram of Senecio scandens formulation granules;

[0098] Figure 35-cChromatographic peak identification diagram of Senecio scandens formulation granules;

[0099] Figure 36 Different chromatographic columns;

[0100] Figure 37 Different instruments;

[0101] Figure 38 These are the characteristic spectral verification images of three batches of Senecio scandens formulation particles;

[0102] Figure 39 Characteristic spectrum of Senecio scandens formulation granules.

[0103] Figure 40 Comparative Example Characteristics picture Detailed Implementation

[0104] This invention provides a method for constructing HPLC characteristic chromatograms of *Senecio scandens* medicinal materials, processed slices, standard decoctions, and formulated granules. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0105] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for constructing HPLC characteristic spectra of Senecio scandens medicinal materials, processed slices, standard decoctions, and formulated granules provided by the present invention.

[0106] Agilent 1290 UHPLC; Thermo Fisher Vanquish UHPLC; Waters 2695 HPLC

[0107] Cellular 1810A Ultrapure Water System (Shanghai Moler Scientific Instruments Co., Ltd.);

[0108] KQ-600DB Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0109] Chromatographic column: Waters Phenyl4.6×150mm 2.7μm, Welch Phenyl-Hexyl 4.6×150mm2.7μm.

[0110] 2. Reagents and reagents

[0111] Acetonitrile and phosphoric acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade. The mobile phase water was laboratory-prepared ultrapure water, and all other reagents were laboratory-prepared pure water.

[0112] New chlorogenic acid reference standard (Chengdu Pusi Biotechnology Co., Ltd., batch number: PS000974);

[0113] Protocatechuic aldehyde (China National Institutes for Food and Drug Control, batch number: 110810-201909)

[0114] Chlorogenic acid reference standard (China National Institutes for Food and Drug Control, batch number: 110753-202119);

[0115] Caffeic acid (China National Institutes for Food and Drug Control, batch number: 110885-201703)

[0116] Cryptochlorogenic acid reference standard (Chengdu Pusi Biotechnology Co., Ltd., batch number: PS020999);

[0117] p-Coumaric acid (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq18040406);

[0118] Methyl chlorogenic acid (Chengdu Desite Biotechnology Co., Ltd., batch number: DST220725-066);

[0119] Rutin reference standard (China National Institutes for Food and Drug Control, batch number: 100080-202012);

[0120] Hyperoside reference standard (China National Institutes for Food and Drug Control, batch numbers: 111521-201809, 111521-202310);

[0121] Isoquercetin reference standard (China National Institutes for Food and Drug Control, batch number: 111809-202205);

[0122] Ethyl chlorogenic acid (Shandong Bokang Fine Chemical Co., Ltd., batch number: CFS202201);

[0123] Isorhamnetin-3-O-galactoside (Sichuan Pusio Standard Technology Co., Ltd., batch number: M2109002);

[0124] Quercetin reference standard (China National Institutes for Food and Drug Control, batch number: 111538-201105);

[0125] Isochlorogenic acid B reference standard (Chengdu DST Biotechnology Co., Ltd., DST210823-037);

[0126] Isochlorogenic acid A reference standard (Chengdu Dest Biotechnology Co., Ltd., DSTDY054703);

[0127] Isochlorogenic acid C reference standard (Chengdu Dest Biotechnology Co., Ltd., DSTDY003804);

[0128] Quercetin (China National Institutes for Food and Drug Control, batch number: 100081-200907);

[0129] Kaempferol (China National Institutes for Food and Drug Control, batch number: 110861-202214);

[0130] Senecio scandens reference material (China National Institutes for Food and Drug Control, batch number: 120964-202212);

[0131] Preparation of freeze-dried powder of Senecio scandens standard decoction (Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd.): QLG-BT-01, QLG-BT-02, QLG-BT-03, QLG-BT-04, QLG-BT-05, QLG-BT-06, QLG-BT-07, QLG-BT-08, QLG-BT-09, QLG-BT-10, QLG-BT-11, QLG-BT-12, QLG-BT-13, QLG-BT-14, QLG-BT-15, QLG-BT-16.

[0132] Senecio medicinal materials, batch numbers: QLG-YC-01, QLG-YC-02, QLG-YC-03, QLG-YC-04, QLG-YC-05, QLG-YC-06, QLG-YC-07, QLG-Y C-08, QLG-YC-09, QLG-YC-10, QLG-YC-11, QLG-YC-12, QLG-YC-13, QLG-YC-14, QLG-YC-15, QLG-YC-16.

[0133] QLG-YP-01, QLG-YP-02, QLG-YP-03, QLG-YP-04, QLG-YP-05, QLG-YP-06, QLG-YP-07, QLG-Y P-08, QLG-YP-09, QLG-YP-10, QLG-YP-11, QLG-YP-12, QLG-YP-13, QLG-YP-14, QLG-YP-15, QLG-YP-16.

[0134] Preparation of Senecio scandens formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd.): QLG-KL-01, QLG-KL-02, QLG-KL-03.

[0135] Example 1: HPLC Characteristic Chromatography of Senecio scandens Standard Decoction

[0136] Chromatographic conditions and system suitability tests were performed using phenyl-bonded silica gel as the stationary phase (column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm); acetonitrile as mobile phase A and 0.3% phosphoric acid water as mobile phase B, with gradient elution as specified in the table below; flow rate 0.8 mL / min; column temperature 25 °C; detection wavelength 360 nm. The theoretical plate number, calculated based on the hyperoside peak, should be no less than 8000.

[0137]

[0138] Preparation of the reference solution: Take 3g of *Senecio scandens* reference material, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 60 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol reference standards, accurately weigh them, and add methanol to prepare a mixed solution containing 20μg of each per ml, as the reference solution.

[0139] Preparation of the test solution: Grind the Senecio scandens formula granules into a fine powder, weigh about 0.5g of the powder, place it in a stoppered conical flask, add 25ml of methanol, seal tightly, heat under reflux for 60 minutes, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0140] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0141] Based on the above-planned experimental conditions, a diode array detector was used to perform a full-band scan of the test solution, and chromatograms of the test solution were extracted at wavelengths of 230 nm, 250 nm, 280 nm, 300 nm, 330 nm, and 360 nm. See [link to chromatogram]. Figure 1 , 2 . Figure 1 Isometric absorption diagram of Senecio scandens formulation particles; Figure 2 Chromatograms of different wavelengths of Senecio scandens formulation particles.

[0142] The results showed that the chromatographic peak information was greater, the separation was better, and the chromatographic baseline was more stable when the detection wavelength was 360 nm. Therefore, the detection wavelength was determined to be 360 ​​nm.

[0143] Column temperature investigation: Based on the proposed experimental conditions, the column temperatures at 20℃, 25℃, and 30℃ were investigated respectively. (See attached figures.) Figure 3 Table 1.

[0144] Table 1 Column Temperature Study - Relative Retention Time

[0145]

[0146] Figure 3 The column temperature was used to examine the chromatograms. The results showed that the peak shapes of each chromatogram were better and the resolution was moderate when the column temperature was 25℃. Therefore, 25℃ was finally determined as the column temperature for the characteristic chromatogram method of Senecio scandens standard decoction.

[0147] Flow rate investigation: Based on the experimental conditions proposed above, flow rates of 0.8 ml / min, 0.9 ml / min, and 1.0 ml / min were investigated respectively. (See attached figures.) Figure 4 Table 2.

[0148] Table 2 Flow velocity study - relative retention time

[0149]

[0150] Figure 4 Flow rate investigation: The results showed that a flow rate of 0.8 ml / min resulted in good peak shape and moderate resolution. Therefore, the flow rate was determined to be 0.8 ml / min.

[0151] Injection volume investigation: Based on the experimental conditions outlined above, investigations were conducted at injection volumes of 2 μl, 5 μl, and 8 μl. See [link / reference]. Figure 5 . Figure 5 Results from different injection volumes showed that a chromatogram peak shape and moderate resolution were achieved with an injection volume of 2 μl. Therefore, the injection volume was determined to be 2 μl.

[0152] Delayed detection: Based on the experimental conditions proposed above, the analysis time was extended to 120 minutes, and the presence of chromatographic peaks was observed after 60 minutes. The results are shown in […]. Figure 6 . Figure 6 Delayed analysis was conducted; the results showed that after extending the time by 60 minutes, there were basically no large chromatographic peaks, and the analysis time for the characteristic chromatographic method of Senecio scandens formulation particles was finally determined to be 60 minutes.

[0153] In summary, the chromatographic conditions and system suitability test for the characteristic chromatogram of the Senecio scandens standard decoction were determined as follows: phenyl-bonded silica gel as the packing material (column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm); acetonitrile as mobile phase A, and 0.3% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 0.8 mL / min; column temperature 25℃; detection wavelength 360 nm. The theoretical plate number, calculated based on the hyperoside peak, should not be less than 8000.

[0154]

[0155] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0156] Subsequent standard decoctions and medicinal herb slices were examined under these chromatographic conditions.

[0157] 4. Preparation and investigation of the test solution

[0158] 4.1 Examination of Extraction Methods

[0159] Take approximately 0.5g of the standard decoction of *Senecio scandens* and place it in a stoppered conical flask. Add 25ml of methanol, reflux and sonicate (600W, 40kHz) for 60 minutes each, then cool, shake well, filter, and collect the filtrate. Inject and determine the sample according to the above results. See the attached image. Figure 7 . Figure 7 Extraction methods were investigated. The results showed that the characteristic peaks of the test sample were well-shaped and had large peak areas under reflux extraction, with moderate separation. Therefore, reflux was selected as the extraction method for preparing the test sample solution in the determination of the characteristic spectrum of Senecio scandens standard decoction.

[0160] 4.2 Investigation of Extraction Solvents

[0161] Take approximately 0.5g of the standard decoction of *Senecio scandens* and place it in a stoppered conical flask. Add 25ml each of methanol, 75% methanol, 50% methanol, 30% methanol, ethanol, and water. Seal the flask tightly, heat under reflux for 60 minutes, cool, shake well, filter, and collect the filtrate. Inject and determine the chromatographic results according to the above conditions. The results are shown in the figure. Figure 8 . Figure 8 Extraction solvent investigation: The results showed that when methanol was used as the extraction solvent, the peak shapes of each characteristic peak were good, the separation was moderate, and the baseline of the chromatogram was stable. Therefore, methanol was selected as the extraction solvent for the characteristic chromatogram of Senecio scandens standard decoction.

[0162] 4.3 Examination of extraction time

[0163] Take approximately 0.5g of the standard decoction of *Senecio scandens* and place it in a stoppered conical flask. Add 25ml of methanol and heat under reflux for 30, 60, and 90 minutes respectively. After cooling, shake well, filter, and collect the filtrate. Inject and determine the chromatographic results according to the above conditions. The results are shown in the figure. Figure 9 . Figure 9 Extraction time was investigated; the results showed that the characteristic peaks of the test sample had good shape and moderate separation at different extraction times. Taking all factors into consideration, the extraction time was determined to be 30 minutes.

[0164] 4.3 Investigation of Solvent Addition Amount

[0165] Take approximately 0.5g of the standard decoction of *Senecio scandens* and place it in a stoppered conical flask. Add 10ml, 25ml, and 50ml of methanol respectively. Heat under reflux for 60 minutes, cool, shake well, filter, and collect the filtrate. See [link to decoction]. Figure 10 . Figure 10 The solvent addition amount was investigated; the results showed that when the solvent addition amount was 25 ml, the peak shape and resolution of each chromatographic peak were better, so the solvent addition amount was selected as 25 ml.

[0166] In summary, the preparation method of the test solution for the characteristic chromatogram of Senecio scandens standard decoction is determined as follows: Take about 0.5g of the powder, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0167] 5. Methodological Investigation

[0168] 5.1 Chromatographic Peak Identification

[0169] Preparation of the test solution: Prepare the Senecio scandens standard decoction test solution according to the experimental conditions proposed above.

[0170] Preparation of reference solution: Take 3g of Senecio scandens reference material, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0171] Take appropriate amounts of neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol reference standards, accurately weigh them, and add methanol to prepare a solution containing 20 μg of each per ml, as the reference solution.

[0172] Preparation of negative control solution: Prepare negative control solution of Senecio scandens standard decoction according to the experimental conditions proposed above.

[0173] The results are shown in Figure 11. Figure 11-a , 11-b 11-c Senecio scandens standard decoction chromatographic peak identification. The results showed that peak 1 was neochlorogenic acid, peak 2 was protocatechuic aldehyde, peak 3 was chlorogenic acid, peak 4 was caffeic acid, peak 5 was cryptochlorogenic acid, peak 6 was p-coumaric acid, peak 7 was methyl chlorogenic acid, peak 9 was rutin, peak 10 was hyperoside, peak 11 was isoquercitrin, peak 12 was ethyl chlorogenic acid, peak 13 was isorhamnetin-3-O-galactoside, peak 14 was quercetin, peak 15 was isochlorogenic acid B, peak 16 was isochlorogenic acid A, peak 17 was isochlorogenic acid C, peak 18 was quercetin, and peak 19 was kaempferol.

[0174] 5.2 Precision Test

[0175] Prepare a test solution from the standard decoction of Senecio scandens and inject it 6 times consecutively according to the proposed experimental method, 2 μl each time. Calculate the retention time and peak area. The results are shown in Table 4.

[0176] Table 4 Precision Examination—Retention Time of Characteristic Peaks

[0177]

[0178] The results showed that the retention time RSD of each characteristic peak was 0.01% to 0.17%, indicating that the instrument has good precision.

[0179] 5.3 Repeatability Test

[0180] Accurately weigh 6 portions of the freeze-dried powder of Senecio scandens standard decoction, and prepare and determine it according to the proposed experimental method. See Table 5.

[0181] Table 5 Repeatability Tests—Ratio of Relative Retention Time of Characteristic Peaks

[0182]

[0183] The results show that the relative retention time (RSD%) of each characteristic peak is between 0 and 0.15%, indicating that the method has good repeatability.

[0184] 5.4 Intermediate Precision Examination

[0185] The freeze-dried powder of Senecio scandens standard decoction was prepared and measured by different personnel (A, B) at different times (I, II) according to the proposed experimental method. The results are shown in Table 6.

[0186] Table 6 Precision - Relative Retention Time

[0187]

[0188] As shown in Table 6, the RSD of the relative retention time of each characteristic peak is between 0% and 0.18% under different test solution preparation personnel and different test solution preparation times, indicating that the method has good applicability.

[0189] 5.5 Stability Test

[0190] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 19h, and 24h. See Table 7.

[0191] Table 7. 24-hour stability study—relative retention time of characteristic peaks

[0192]

[0193] The results showed that the RSD of the relative retention time of the corresponding characteristic peaks was between 0 and 0.23%, and the sample solution was relatively stable within 24 hours.

[0194] 5.6 Durability Test

[0195] Instrument durability test

[0196] Based on the above-planned experimental conditions, three portions of the Senecio scandens standard decoction were accurately weighed to prepare test solutions, which were then analyzed using Agilent, Thermo Fisher Scientific, and Waters ultra-high performance liquid chromatographs, respectively. See Table 8. Figure 12 . Figure 12 Chromatograms were examined using different instruments.

[0197] Table 8 Instrument Durability Test - Relative Retention Time Ratio

[0198]

[0199] The results show that when the samples are tested using the above three instruments, the RSD of the relative retention time of each characteristic peak is 0.10% to 5.46%, indicating that the instruments have good durability.

[0200] Column durability study

[0201] Based on the above-planned experimental conditions, the Waters chromatography column was subjected to specific tests. Phenyl 4.6×150mm 2.7μm Phenyl-Hexyl 4.6×150mm2.7μm was investigated. See Figure 13 Table 9. Figure 13 Durability study of chromatographic column for Senecio scandens standard decoction.

[0202] Table 9. Column Robustness Study—Relative Retention Times of Characteristic Peaks

[0203]

[0204]

[0205] The table above shows that the RSD of the relative retention times of each characteristic peak on different chromatographic columns ranges from 0% to 6.08%, indicating that both phenyl columns meet the requirements. This experiment will use Waters... The Phenyl column was subsequently validated, and a total of 19 characteristic peaks were included in the validation process.

[0206] 5.7 Establishment of Limits for Relative Retention Time

[0207] Table 10 summarizes the methodological examination items and validation results:

[0208] Table 10 Summary of RSD% of Methodological Results – Retention Time, Relative Retention Time

[0209]

[0210] As shown in the table above, the relative retention times of each characteristic peak are stable and within ±10% of the average value. Therefore, the specified range of the relative retention times of each peak is tentatively set at ±10%.

[0211] 6. Determination of characteristic peaks and establishment of reference spectra

[0212] The proposed method was used to determine the characteristic spectra of 16 batches of Senecio scandens standard decoction, and the relative retention time and relative peak area were calculated. (See attached image) Figure 14 Table 11. Figure 14 Characteristic spectrum of standard decoction of *Senecio scandens*; Peak 1: Neochlorogenic acid; Peak 2: Protocatechuic aldehyde; Peak 3: Chlorogenic acid; Peak 4: Caffeic acid; Peak 5: Cryptochlorogenic acid; Peak 6: p-Coumaric acid; Peak 7: Methyl chlorogenic acid; Peak 9: Rutin; Peak 10: Hyperoside; Peak 11: Isoquercetin; Peak 12: Ethyl chlorogenic acid; Peak 13: Isorhamnetin-3-O-galactoside; Peak 14: Quercetin; Peak 15: Isochlorogenic acid B; Peak 16: Isochlorogenic acid A; Peak 17: Isochlorogenic acid C; Peak 18: Quercetin; Peak 19: Kaempferol; S1: Reference herb; S2-S17 are 16 batches of standard decoction respectively.

[0213] Table 11 Relative retention times of 16 batches of Senecio scandens standard decoction

[0214]

[0215]

[0216] Based on the principles of stable relative retention times, detectability across all batches, and relatively high peak values, 19 peaks with good repeatability were selected as characteristic peaks. Results showed that the relative peak area RSD of the characteristic peaks in 16 batches of the Senecio scandens standard decoction varied too much and therefore were not included in the quality standard text. The relative retention time RSD of all 19 characteristic peaks in the 16 batches of the Senecio scandens standard decoction was less than 2.0%. Final stipulation:

[0217] The chromatogram of the test sample should show 19 characteristic peaks, and the retention times should correspond to the 19 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 should correspond to the retention times of the reference standard peaks. The peak corresponding to the hyperoside reference standard peak is designated as the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated, and their relative retention times should be within ±10% of the specified value. The specified values ​​are 0.24 (peak 1), 0.30 (peak 2), 0.41 (peak 3), 0.48 (peak 5), 0.75 (peak 6), 0.85 (peak 7), 0.95 (peak 8), 0.97 (peak 9), 1.01 (peak 11), 1.10 (peak 12), 1.19 (peak 13), 1.21 (peak 14), 1.27 (peak 15), 1.31 (peak 16), 1.40 (peak 17), 1.56 (peak 18), and 1.72 (peak 19).

[0218] The similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine (2012 version) was used to synthesize chromatograms of 21 batches of Senecio scandens standard decoctions, and a reference chromatogram of the characteristic chromatograms of Senecio scandens standard decoctions was established. (See attached image.) Figure 15 . Figure 15 Characteristic spectrum of standard decoction of *Senecio scandens*; Peak 1: Neochlorogenic acid; Peak 2: Protocatechuic aldehyde; Peak 3: Chlorogenic acid; Peak 4: Caffeic acid; Peak 5: Cryptochlorogenic acid; Peak 6: p-Coumaric acid; Peak 7: Methyl chlorogenic acid; Peak 9: Rutin; Peak 10: Hyperoside; Peak 11: Isoquercetin; Peak 12: Ethyl chlorogenic acid; Peak 13: Isorhamnoside-3-O-galactoside; Peak 14: Quercetin; Peak 15: Isochlorogenic acid B; Peak 16: Isochlorogenic acid A; Peak 17: Isochlorogenic acid C; Peak 18: Quercetin; Peak 19: Kaempferol.

[0219] 7. Method for determining the characteristic chromatogram of standard Senecio scandens decoction

[0220] Chromatographic conditions and system suitability tests were performed using phenyl-bonded silica gel as the packing material (column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm); acetonitrile as mobile phase A and 0.3% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 0.8 mL / min; column temperature 25 °C; detection wavelength 360 nm. The theoretical plate number, calculated based on the hyperoside peak, should be no less than 8000.

[0221]

[0222] Preparation of the reference solution: Take 3g of *Senecio scandens* reference material, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of the following reference standards: neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol. Accurately weigh these and add methanol to prepare a mixed solution containing 20μg of each standard per 1ml, as the reference solution.

[0223] Preparation of the test solution: Take about 0.5g of Senecio scandens standard decoction powder, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0224] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0225] The chromatogram of the test sample should show 19 characteristic peaks, and the retention times should correspond to the 19 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 should correspond to the retention times of the reference standard peaks. The peak corresponding to the hyperoside reference standard peak is designated as the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated, and their relative retention times should be within ±10% of the specified value. The specified values ​​are 0.24 (peak 1), 0.30 (peak 2), 0.41 (peak 3), 0.48 (peak 5), 0.75 (peak 6), 0.85 (peak 7), 0.95 (peak 8), 0.97 (peak 9), 1.01 (peak 11), 1.10 (peak 12), 1.19 (peak 13), 1.21 (peak 14), 1.27 (peak 15), 1.31 (peak 16), 1.40 (peak 17), 1.56 (peak 18), and 1.72 (peak 19). According to the characteristic spectrum; Peak 1: Neochlorogenic acid; Peak 2: Protocatechuic aldehyde; Peak 3: Chlorogenic acid; Peak 4: Caffeic acid; Peak 5: Cryptochlorogenic acid; Peak 6: p-Coumaric acid; Peak 7: Methyl chlorogenic acid; Peak 9: Rutin; Peak 10: Hyperoside; Peak 11: Isoquercetin; Peak 12: Ethyl chlorogenic acid; Peak 13: Isorhamnoside-3-O-galactoside; Peak 14: Quercetin; Peak 15: Isochlorogenic acid B; Peak 16: Isochlorogenic acid A; Peak 17: Isochlorogenic acid C; Peak 18: Quercetin; Peak 19: Kaempferol.

[0226] Example 2: HPLC Characteristic Chromatography of Senecio scandens Herbal and Processed Herbs

[0227] The column was packed with phenyl-bonded silica gel (150 mm column length, 4.6 mm inner diameter, 2.7 μm particle size); acetonitrile was used as mobile phase A, and 0.3% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 0.8 mL / min; the column temperature was 25 °C; and the detection wavelength was 360 nm. The theoretical plate number, calculated based on the hyperoside peak, should be no less than 8000.

[0228]

[0229] Preparation of the reference solution: Take 3g of *Senecio scandens* reference material, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 60 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol reference standards, accurately weigh them, and add methanol to prepare a mixed solution containing 20μg of each per ml, as the reference solution.

[0230] Preparation of the test solution: Take about 3g of Senecio scandens powder (passed through a No. 2 sieve) and prepare the test solution together with the reference solution of the control herb.

[0231] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0232] 4. Preparation and investigation of the test solution

[0233] 4.1 Investigation of Extraction Solvents

[0234] Take approximately 3g of *Senecio scandens* herb and place it in a stoppered conical flask. Add 25ml each of methanol, 75% methanol, 50% methanol, 30% methanol, ethanol, and water. Seal the flask tightly and heat under reflux for 60 minutes. Cool, shake well, filter, and collect the filtrate. See [link to flask description]. Figure 16 . Figure 16 Extraction solvent investigation

[0235] The results showed that when methanol was used as the extraction solvent, the characteristic peaks had good shapes, moderate resolution, and stable chromatographic baselines. Considering all factors, methanol was tentatively chosen as the extraction solvent.

[0236] 4.2 Examination of Extraction Methods

[0237] Take two portions of the herbal material, approximately 3g each, and place them in stoppered conical flasks. Add 25ml of methanol and reflux and sonicate (600W power, 40kHz frequency) for 60 minutes respectively. Cool, shake well, filter, and collect the filtrate. See [link to product]. Figure 17 . Figure 17 Extraction methods were examined.

[0238] The results showed that the characteristic peaks of the test sample were well shaped and the separation was moderate under reflux extraction. Therefore, reflux was tentatively chosen as the extraction method.

[0239] 4.3 Examination of extraction time

[0240] Take approximately 3g of the herbal material, add 25ml of methanol, and heat under reflux for 30 minutes, 60 minutes, and 90 minutes respectively. Cool, shake well, filter, and collect the filtrate. See [link to product description]. Figure 18 18. Extraction time investigation; the results showed that the characteristic peaks of the test sample had good shape and moderate separation at different extraction times. Taking all factors into consideration, the extraction time was determined to be 30 minutes.

[0241] 4.4 Investigation of Solvent Addition Amount

[0242] Take approximately 3g of the herbal material and place it in a stoppered conical flask. Add 10ml, 25ml, and 50ml of methanol respectively. Heat under reflux for 60 minutes, cool, shake well, filter, and collect the filtrate. See [link to flask]. Figure 19 . Figure 19 Investigation of the amount of solvent added.

[0243] The results showed that when the solvent volume was 25 ml, the peak shape and resolution of each chromatographic peak were better, so the solvent volume was selected as 25 ml.

[0244] In summary, the preparation method of the test solution for the characteristic spectrum of Senecio scandens medicinal material is determined as follows: Take 3g of Senecio scandens medicinal material powder (passed through a No. 2 sieve) and place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 30min, cool, shake well, filter, and take the filtrate as the test solution.

[0245] 5. Methodological Examination

[0246] 5.1 Chromatographic Peak Identification

[0247] Preparation of the test solution: Prepare the Senecio scandens test solution according to the experimental conditions proposed above.

[0248] Take 3g of Senecio scandens reference material, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution for the reference material.

[0249] Take appropriate amounts of neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol reference standards, accurately weigh them, and add methanol to prepare a solution containing 20 μg of each per ml, as the reference solution.

[0250] Preparation of negative control solution: Prepare negative control solution for *Senecio scandens* medicinal material according to the experimental conditions proposed above.

[0251] The characteristic spectral peaks of Senecio scandens were located. See Figure 20. Figure 20-a , 20-b Chromatographic peak identification of 20-C Senecio scandens medicinal material.

[0252] The results showed that peak 1 was neochlorogenic acid, peak 2 was protocatechuic aldehyde, peak 3 was chlorogenic acid, peak 4 was caffeic acid, peak 5 was cryptochlorogenic acid, peak 6 was p-coumaric acid, peak 7 was methyl chlorogenic acid, peak 9 was rutin, peak 10 was hyperoside, peak 11 was isoquercitrin, peak 12 was ethyl chlorogenic acid, peak 13 was isorhamnetin-3-O-galactoside, peak 14 was quercetin, peak 15 was isochlorogenic acid B, peak 16 was isochlorogenic acid A, peak 17 was isochlorogenic acid C, peak 18 was quercetin, and peak 19 was kaempferol.

[0253] 5.2 Precision Test

[0254] Take the sample solution of Senecio scandens and inject it 6 times consecutively according to the proposed experimental method, 2 μl each time. Calculate the peak area and relative peak area of ​​each characteristic peak. See Table 12.

[0255] Table 12 Precision Examination - Retention Time

[0256]

[0257] The results show that the retention time RSD of each characteristic peak is 0.02%–0.20%, and the peak area RSD is 0.58%–4.81%. The instrument has good precision.

[0258] 5.3 Repeatability Test

[0259] Six portions of Senecio scandens were accurately weighed and prepared and measured according to the proposed experimental method. See Table 13.

[0260] Table 13 Repeatability Tests - Relative Retention Time Ratios

[0261]

[0262] The results show that the relative retention time RSD of each characteristic peak is 0.00% to 0.48%, and the method has good repeatability.

[0263] 5.4 Stability

[0264] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 18h, and 24h. See Table 14.

[0265] Table 14 Stability Study - Retention Time

[0266]

[0267] The results showed that the RSD of the corresponding characteristic peak retention time was 0.00%–0.76%, and the sample solution was stable within 24 hours.

[0268] 5.5 Intermediate Precision Examination

[0269] Different instruments

[0270] Based on the above-established experimental conditions, three portions of *Senecio scandens* medicinal material were accurately weighed to prepare test solutions, which were then analyzed using Agilent, Thermo Fisher Scientific, and Waters ultra-high performance liquid chromatographs, respectively. See Table 15. Figure 21 Different instruments were used for the investigation.

[0271] Table 15 Instrument Durability Test - Relative Retention Time Ratio

[0272]

[0273] The results showed that when the test samples were tested using the above three instruments, the RSD of the relative retention time of each characteristic peak ranged from 0.15% to 5.40%, indicating that the instruments had good durability.

[0274] 5.6 Investigations by different personnel and at different times

[0275] Based on the above-established experimental conditions, samples were prepared and measured by different personnel at different times. The *Senecio scandens* medicinal material was accurately weighed, test samples were prepared, and measurements were performed, as shown in Table 16. Table 16: Personnel and Time Investigation - Relative Retention Time Ratio

[0276]

[0277] The results showed that when different personnel tested the same sample at different times, the RSD of the relative retention times of each characteristic peak ranged from 0.00% to 0.96%. This indicates that the method has good stability when different personnel test the same sample at different times.

[0278] 5.7 Durability Test

[0279] Column durability study

[0280] Based on the above-planned experimental conditions, the Welch column was used for each experiment. Phenyl-Hexyl4.6mm×150mm, 2.7μm, Waters Phenyl (4.6 mm × 150 mm, 2.7 μm) was used for analysis and investigation. The relative retention time and relative peak area of ​​each characteristic peak were calculated. See Table 17.

[0281] Table 17 Column robustness test - relative retention time ratio

[0282]

[0283] Figure 22 Column robustness study; results showed that the RSD of the relative retention times of each characteristic peak on different columns ranged from 0% to 5.91%, indicating that both phenyl columns met the requirements. This experiment will use Waters... The Phenyl column was subsequently validated, and a total of 19 characteristic peaks were included in the validation process.

[0284] 5.8 Establishment of Limits for Relative Retention Time

[0285] Table 18 summarizes the methodological examination items and validation results:

[0286] Table 18 Summary of RSD% for Methodological Results – Retention Time, Relative Retention Time

[0287]

[0288]

[0289] As shown in the table above, the relative retention times of each characteristic peak are stable and within ±10% of the average value. Therefore, the specified range of the relative retention times of each peak is tentatively set at ±10%.

[0290] 6. Verification of the characteristic atlas of Senecio scandens medicinal materials

[0291] The characteristic spectra of 16 batches of Senecio scandens were determined according to the method for determining characteristic spectra in section 2.3.4.7.3. The characteristic spectra of the 16 batches of Senecio scandens are shown in Figure 23, and the relative retention time and relative peak area of ​​the characteristic peaks are shown in Table 19.

[0292] Figure 23-a Characteristic spectrum of *Senecio scandens* medicinal material; Peak 1: Neochlorogenic acid; Peak 2: Protocatechuic aldehyde; Peak 3: Chlorogenic acid; Peak 4: Caffeic acid; Peak 5: Cryptochlorogenic acid; Peak 6: p-Coumaric acid; Peak 7: Methyl chlorogenic acid; Peak 9: Rutin; Peak 10: Hyperoside; Peak 11: Isoquercetin; Peak 12: Ethyl chlorogenic acid; Peak 13: Isorhamnetin-3-O-galactoside; Peak 14: Quercetin; Peak 15: Isochlorogenic acid B; Peak 16: Isochlorogenic acid A; Peak 17: Isochlorogenic acid C; Peak 18: Quercetin; Peak 19: Kaempferol; (S1: Control material; S2-S17: 16 batches of medicinal material respectively).

[0293] Table 19 Relative Retention Time of Characteristic Images of Senecio scandens Medicinal Material

[0294]

[0295] Based on the principles of stable relative retention times, detectability across all batches of samples, and relatively high peak values, 19 peaks with good repeatability were selected as characteristic peaks. Results showed that the relative peak area RSD of the characteristic peaks from 16 batches of *Senecio scandens* medicinal materials varied too much and therefore were not included in the quality standard text. The relative retention time RSD of the 19 characteristic peaks from the 16 batches of *Senecio scandens* medicinal materials was less than 2.0%. The final stipulation is that the chromatogram of the test sample should present 19 characteristic peaks, corresponding to the retention times of the 19 characteristic peaks in the chromatogram of the reference medicinal material. Specifically, peaks 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 should correspond to the retention times of the reference peak. The peak corresponding to the hyperoside reference peak is designated as the S peak. The relative retention times of the remaining characteristic peaks and the S peak are calculated, and their relative retention times should be within ±10% of the specified value. The specified values ​​are 0.24 (peak 1), 0.30 (peak 2), 0.41 (peak 3), 0.48 (peak 5), 0.75 (peak 6), 0.85 (peak 7), 0.95 (peak 8), 0.97 (peak 9), 1.01 (peak 11), 1.10 (peak 12), 1.19 (peak 13), 1.21 (peak 14), 1.27 (peak 15), 1.31 (peak 16), 1.40 (peak 17), 1.56 (peak 18), and 1.72 (peak 19).

[0296] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize 16 batches of *Senecio scandens* medicinal materials, and a reference characteristic chromatogram for *Senecio scandens* medicinal materials was established. See [link / reference]. Figure 24 . Figure 24 Characteristic spectrum of Senecio scandens medicinal material; Peak 1: Neochlorogenic acid; Peak 2: Protocatechuic aldehyde; Peak 3: Chlorogenic acid; Peak 4: Caffeic acid; Peak 5: Cryptochlorogenic acid; Peak 6: p-Coumaric acid; Peak 7: Methyl chlorogenic acid; Peak 9: Rutin; Peak 10: Hyperoside; Peak 11: Isoquercetin; Peak 12: Ethyl chlorogenic acid; Peak 13: Isorhamnoside-3-O-galactoside; Peak 14: Quercetin; Peak 15: Isochlorogenic acid B; Peak 16: Isochlorogenic acid A; Peak 17: Isochlorogenic acid C; Peak 18: Quercetin; Peak 19: Kaempferol.

[0297] Senecio scandens slices: This is a processed product obtained by removing impurities from Senecio scandens medicinal material, cutting it into 2-4cm segments, and sifting out mud, sand, and medicinal debris.

[0298] The characteristic chromatograms of Senecio scandens slices were verified using the methods described above, and the results are shown in Table 19.

[0299] Table 19 Relative Retention Time of Characteristic Images of Senecio scandens slices

[0300]

[0301] Figure 23-b Overlay images of characteristic slices of *Senecio scandens* (S1-S17 are, in order: reference material, QLG-YP-01, QLG-YP-02, QLG-YP-03, QLG-YP-04, QLG-YP-05, QLG-YP-06, QLG-YP-07, QLG-YP-08, QLG-YP-09, QLG-YP-10, QLG-YP-11, QLG-YP-12, QLG-YP-13).

[0302] QLG-YP-14, QLG-YP-15, QLG-YP-16)

[0303] The results showed that the characteristic spectrum of Senecio scandens slices matched that of the Senecio scandens medicinal material.

[0304] 7. Method for determining the characteristic atlas of Senecio scandens medicinal materials and processed slices

[0305] Chromatographic conditions and system suitability tests were performed using phenyl-bonded silica gel as the stationary phase; acetonitrile as mobile phase A and 0.3% phosphoric acid as mobile phase B; flow rate: 0.8 mL / min; column temperature: 25 °C; detection wavelength: 360 nm. Gradient elution was performed according to the specifications in the table below. The theoretical plate number, calculated based on hyperoside, should be no less than 8000.

[0306]

[0307] Preparation of the reference solution: Take 3g of *Senecio scandens* reference material, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 30min, cool, shake well, filter, and take the filtrate as the reference solution. Accurately weigh appropriate amounts of the following reference standards: neochlorogenic acid, protocatechuic acid, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol. Add methanol to prepare a solution containing 20ug of each standard per 1ml, which is used as the reference solution.

[0308] Preparation of the test solution: Take about 3g of Senecio scandens medicinal material / processed slices powder (passed through a No. 2 sieve) and prepare the test solution together with the reference solution of the control medicinal material.

[0309] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0310] The chromatogram of the test sample should show 19 characteristic peaks, and the retention times should correspond to the 19 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 should correspond to the retention times of the reference standard peaks. The peak corresponding to the hyperoside reference standard peak is designated as the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated, and their relative retention times should be within ±10% of the specified value. The specified values ​​are 0.24 (peak 1), 0.30 (peak 2), 0.41 (peak 3), 0.48 (peak 5), 0.75 (peak 6), 0.85 (peak 7), 0.95 (peak 8), 0.97 (peak 9), 1.01 (peak 11), 1.10 (peak 12), 1.19 (peak 13), 1.21 (peak 14), 1.27 (peak 15), 1.31 (peak 16), 1.40 (peak 17), 1.56 (peak 18), and 1.72 (peak 19). According to the characteristic spectrum; Peak 1: Neochlorogenic acid; Peak 2: Protocatechuic aldehyde; Peak 3: Chlorogenic acid; Peak 4: Caffeic acid; Peak 5: Cryptochlorogenic acid; Peak 6: p-Coumaric acid; Peak 7: Methyl chlorogenic acid; Peak 9: Rutin; Peak 10: Hyperoside; Peak 11: Isoquercetin; Peak 12: Ethyl chlorogenic acid; Peak 13: Isorhamnoside-3-O-galactoside; Peak 14: Quercetin; Peak 15: Isochlorogenic acid B; Peak 16: Isochlorogenic acid A; Peak 17: Isochlorogenic acid C; Peak 18: Quercetin; Peak 19: Kaempferol.

[0311] Example 3: HPLC Characteristic Chromatography of Senecio scandens Formulation Particles

[0312] Chromatographic conditions and system suitability tests were performed using phenyl-bonded silica gel as the packing material (column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm); acetonitrile as mobile phase A and 0.3% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 0.8 mL / min; column temperature 25 °C; detection wavelength 360 nm. The theoretical plate number, calculated based on the hyperoside peak, should be no less than 8000.

[0313]

[0314] Preparation of the reference solution: Take 3g of *Senecio scandens* reference material, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 60 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol reference standards, accurately weigh them, and add methanol to prepare a mixed solution containing 20μg of each per ml, as the reference solution.

[0315] Preparation of the test solution: Take an appropriate amount of Senecio scandens formula granules, grind them into a fine powder, take about 0.5g, place them in a stoppered conical flask, add 25ml of methanol, seal tightly, heat under reflux for 60 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0316] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0317] Based on the above-planned experimental conditions, a diode array detector was used to perform a full-band scan of the Senecio scandens formulation granule test solution, and chromatograms of the test solution were extracted at wavelengths of 230 nm, 250 nm, 280 nm, 300 nm, 330 nm, and 360 nm. (See...) Figure 25 , 26 . Figure 25 Isometric absorption diagram of Senecio scandens formulation particles; Figure 26 Chromatograms of different wavelengths of Senecio scandens formulation particles.

[0318] The results showed that the chromatographic peak information was greater, the separation was better, and the chromatographic baseline was more stable when the detection wavelength was 360 nm. Therefore, the detection wavelength was determined to be 360 ​​nm.

[0319] Column temperature investigation

[0320] Based on the above-specified experimental conditions, the results were investigated at column temperatures of 20℃, 25℃, and 30℃. (See...) Figure 27 Table 20.

[0321] Table 20 Column Temperature Study - Relative Retention Time

[0322]

[0323] Figure 27 The column temperature was used to investigate the chromatograms. The results showed that the peak shapes of each chromatogram were better and the resolution was moderate when the column temperature was 25℃. Therefore, 25℃ was finally determined as the column temperature for the characteristic chromatogram method of Senecio scandens formulation particles.

[0324] Flow velocity investigation

[0325] Based on the above-established experimental conditions, the flow rates of 0.8 ml / min, 0.9 ml / min, and 1.0 ml / min were investigated. (See...) Figure 28 Table 21.

[0326] Table 21 Flow velocity study - relative retention time

[0327]

[0328] Figure 28 The flow rate was investigated, and the results showed that a flow rate of 0.8 ml / min resulted in good peak shape and moderate resolution. Therefore, the flow rate was determined to be 0.8 ml / min.

[0329] Injection volume investigation: Based on the experimental conditions outlined above, investigations were conducted at injection volumes of 2 μl, 5 μl, and 8 μl. See [link / reference]. Figure 29 .

[0330] Figure 29 The results showed that with an injection volume of 2 μl, the chromatogram peak shape was better and the resolution was moderate. Therefore, the injection volume was determined to be 2 μl.

[0331] Delayed detection: Based on the above-planned experimental conditions, the analysis time was extended to 120 minutes, and the presence of chromatographic peaks was observed after 60 minutes. The results are shown in […]. Figure 30 .

[0332] Figure 30 The delay study showed that there were basically no large chromatographic peaks after extending the time by 60 minutes, and the analysis time of the characteristic chromatographic method of Senecio scandens formulation particles was finally determined to be 60 minutes.

[0333] In summary, the chromatographic conditions and system suitability test for the characteristic chromatogram of the Senecio scandens formulation particles were determined as follows: phenyl-bonded silica gel as the packing material (column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm); acetonitrile as mobile phase A, and 0.3% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 0.8 mL / min; column temperature 25℃; detection wavelength 360 nm. The theoretical plate number, calculated based on the hyperoside peak, should not be less than 8000.

[0334]

[0335] Assay: Accurately pipette 2 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0336] 4. Preparation of the test solution

[0337] 4.1 Investigation of Extraction Solvents

[0338] Take an appropriate amount of Senecio scandens granules, grind them finely, and take about 0.5g. Place the 0g in a stoppered conical flask, and add 25ml each of methanol, 75% methanol, 50% methanol, 30% methanol, ethanol, and water. Seal the flask tightly, heat under reflux for 60 minutes, cool, shake well, filter, and collect the filtrate. See [link to flask description]. Figure 31 .

[0339] Figure 31 Extraction solvent investigation. The results showed that when methanol was used as the extraction solvent, the peak shapes of each characteristic peak were good, the separation was moderate, and the chromatographic baseline was stable. Therefore, methanol was selected as the extraction solvent for the characteristic chromatogram of Senecio scandens formulation particles.

[0340] 4.2 Examination of Extraction Methods

[0341] Take an appropriate amount of Senecio scandens granules, grind them finely, and take about 0.5g. Place the granules in a stoppered conical flask, add 25ml of methanol, and reflux and sonicate (600W power, 40kHz frequency) for 60 minutes respectively. Cool, shake well, filter, and collect the filtrate. See [link to product]. Figure 32 . Figure 32 Extraction method examination

[0342] The results showed that the characteristic peaks of the test sample were well shaped and the separation was moderate under reflux extraction. Therefore, reflux was tentatively chosen as the extraction method.

[0343] 4.3 Examination of extraction time

[0344] Take an appropriate amount of Senecio scandens granules, grind them finely, and take about 0.5g. Place the granules in a stoppered conical flask, add 25ml of methanol, and heat under reflux for 30 minutes, 60 minutes, and 90 minutes respectively. Cool, shake well, filter, and collect the filtrate. See [link to flask description]. Figure 33 . Figure 33 Extraction time was investigated; the results showed that the characteristic peaks of the test sample had good shape and moderate separation at different extraction times. Taking all factors into consideration, the extraction time was determined to be 30 minutes.

[0345] 4.4 Examination of extraction time

[0346] Take an appropriate amount of Senecio scandens granules, grind them finely, and take about 0.5g. Place the granules in a stoppered conical flask, add 10ml, 25ml, and 50ml of methanol respectively, heat under reflux for 60 minutes, cool, shake well, filter, and collect the filtrate. See [link to flask]. Figure 34 . Figure 34 Investigation of the amount of solvent added.

[0347] The results showed that when the solvent volume was 25 ml, the peak shape and resolution of each chromatographic peak were better, so the solvent volume was selected as 25 ml.

[0348] 4.5 Determine the method for preparing the test sample

[0349] Take an appropriate amount of Senecio scandens formula, grind it into a fine powder, take about 0.5g, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the final product.

[0350] 5. Feature mapping method is used to determine

[0351] Determined by high performance liquid chromatography (General Chapter 0512, Part IV, Chinese Pharmacopoeia 2020 Edition).

[0352] Chromatographic conditions and system suitability tests were performed using phenyl-bonded silica gel as the packing material (column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm); acetonitrile as mobile phase A and 0.3% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 0.8 mL / min; column temperature 25 °C; detection wavelength 360 nm. The theoretical plate number, calculated based on the hyperoside peak, should be no less than 8000.

[0353]

[0354] Preparation of the reference solution: Take 3g of *Senecio scandens* reference material, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of the following reference standards: neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol. Accurately weigh these and add methanol to prepare a mixed solution containing 20μg of each standard per 1ml, as the reference solution.

[0355] Preparation of the test solution: Take an appropriate amount of Senecio scandens formula granules, grind them into a fine powder, take about 0.5g, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0356] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0357] Methodological investigation

[0358] 5.1 Chromatographic Peak Identification

[0359] Preparation of the test solution: Prepare the Senecio scandens formula granule test solution according to the experimental conditions proposed above.

[0360] Preparation of reference solution: Take 3g of Senecio scandens reference material, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0361] Separately weigh appropriate amounts of neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol reference standards, and dissolve them in methanol to prepare a solution containing 20 μg of each standard per ml. This solution serves as the reference standard solution. See Figure 35. Figure 35-a , 35-b 35-C Senecio scandens formulation granule chromatographic peak identification.

[0362] The results showed that peak 1 was neochlorogenic acid, peak 2 was protocatechuic aldehyde, peak 3 was chlorogenic acid, peak 4 was caffeic acid, peak 5 was cryptochlorogenic acid, peak 6 was p-coumaric acid, peak 7 was methyl chlorogenic acid, peak 9 was rutin, peak 10 was hyperoside, peak 11 was isoquercitrin, peak 12 was ethyl chlorogenic acid, peak 13 was isorhamnetin-3-O-galactoside, peak 14 was quercetin, peak 15 was isochlorogenic acid B, peak 16 was isochlorogenic acid A, peak 17 was isochlorogenic acid C, peak 18 was quercetin, and peak 19 was kaempferol.

[0363] 5.2 Precision Test

[0364] Take the Senecio scandens formula granule test solution and inject it 6 times consecutively according to the proposed experimental method, 2 μl each time. Calculate the relative retention time and relative peak area of ​​each characteristic peak. See Table 21.

[0365] Table 21 Precision Examination - Retention Time

[0366]

[0367]

[0368] The results show that the retention time RSD of each characteristic peak is between 0.02% and 0.58%, and the peak area RSD of each characteristic peak is between 0.57% and 5.34%, indicating that the instrument has good precision.

[0369] 5.3 Repeatability Test

[0370] Six portions of the Senecio scandens formula granules were accurately weighed and prepared and measured according to the proposed experimental method. See Table 22.

[0371] Table 22 Repeatability Tests - Relative Retention Time Ratios

[0372]

[0373] The results show that the relative retention time (RSD%) values ​​of each characteristic peak are between 0.00% and 0.13%, indicating that the method has good repeatability.

[0374] 5.4 Intermediate Precision Examination

[0375] Different personnel and time periods for inspection

[0376] Based on the above-planned experimental conditions, Senecio scandens formulation particles were prepared and measured by different personnel (A, B) at different times (TⅠ, TⅡ) according to the planned experimental method. The results are shown in Table 23.

[0377] Table 23 Personnel and Time Assessment - Ratio of Relative Retention Time

[0378]

[0379] The results show that when different personnel measure the same sample at different times, the RSD of the relative retention time of each characteristic peak is 0.06% to 0.84%, indicating that the method has good applicability.

[0380] 5.5 Stability

[0381] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 18h, and 24h. See Table 24.

[0382] Table 24 Stability Study - Retention Time of Characteristic Peaks

[0383]

[0384] The results showed that the RSD of the corresponding characteristic peak retention time was 0%–0.74%, indicating that the sample solution was stable within 24 hours.

[0385] 5.6 Column robustness test

[0386] Based on the above-planned experimental conditions, the Welch column was used for each experiment. Phenyl-Hexyl4.6mm×150mm, 2.7μm, Waters Phenyl (4.6mm × 150mm) was used for analysis and investigation, and the relative retention time and relative peak area of ​​each characteristic peak were calculated. See Table 25. Figure 36 .

[0387] Table 25 Column robustness study - relative retention time ratio

[0388]

[0389] Figure 36 Different chromatographic columns were used; the results showed that the RSD of the relative retention times of each characteristic peak on different columns ranged from 0.06% to 5.75%, indicating that both phenyl columns met the requirements. Waters columns were used in this study. The Phenyl column was subsequently validated, and a total of 19 characteristic peaks were included in the validation process.

[0390] 5.7 Instrument Durability Test

[0391] Based on the above-specified experimental conditions, one portion of the *Senecio scandens* formula granules was accurately weighed to prepare a test solution, which was then analyzed using an Agilent, Waters, and Thermo Fisher ultra-high performance liquid chromatograph. See Table 26. Figure 37 .

[0392] Table 26 Instrument Durability Test - Relative Retention Time Ratio

[0393]

[0394] Figure 37 Different instruments were used; the results showed that when the test samples were detected using the above three instruments, the RSD of the relative retention time of each characteristic peak ranged from 0.15% to 5.26%, indicating that the instruments had good durability.

[0395] 6. Determination of characteristic peaks and establishment of reference spectra

[0396] Setting limits for relative retention time

[0397] Table 27 summarizes the methodology, the examination items, and the verification results.

[0398] Table 27 Summary of RSD% of Methodological Results - Retention Time - Relative Retention Time

[0399]

[0400] The relative retention times of each characteristic peak are stable, and the RSD% of the relative retention time is within 10%. Therefore, the specified range of relative retention times for each peak is tentatively set at ±10%.

[0401] Validation results of 3 batches of Senecio scandens formulation granules

[0402] The characteristic spectra of three batches of this product were determined using the proposed method, and the relative retention times and relative peak areas were calculated. (See attached image.) Figure 38 Table 28. Figure 38 Characteristic spectrum verification images of three batches of Senecio scandens formulation granules

[0403] Table 28 Relative retention times of three batches of Senecio scandens formulation granules

[0404]

[0405]

[0406] Based on the principles of stable relative retention times, detectability across all batches, and relatively high peak values, 19 peaks with good repeatability were selected as characteristic peaks. Results showed that the relative peak area RSD of the characteristic peaks from the three batches of *Senecio scandens* formulation granules differed too much and therefore were not included in the quality standard text. The relative retention time RSD of all 19 characteristic peaks from the three batches of *Senecio scandens* formulation granules was less than 2.0%. Final stipulation:

[0407] The chromatogram of the test sample should show 19 characteristic peaks, and the retention times should correspond to the 19 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 should correspond to the retention times of the reference standard peaks. The peak corresponding to the hyperoside reference standard peak is designated as the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated, and their relative retention times should be within ±10% of the specified value. The specified values ​​are 0.24 (peak 1), 0.30 (peak 2), 0.41 (peak 3), 0.48 (peak 5), 0.75 (peak 6), 0.85 (peak 7), 0.95 (peak 8), 0.97 (peak 9), 1.01 (peak 11), 1.10 (peak 12), 1.19 (peak 13), 1.21 (peak 14), 1.27 (peak 15), 1.31 (peak 16), 1.40 (peak 17), 1.56 (peak 18), and 1.72 (peak 19).

[0408] Three batches of *Senecio scandens* (Qianliguang) formula granules were synthesized using the 2012 version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, and a reference chromatogram of the characteristic chromatograms of *Senecio scandens* formula granules was established. (See...) Figure 39 . Figure 39 Characteristic spectrum of Senecio scandens formula granules; Peak 1: Neochlorogenic acid; Peak 2: Protocatechuic aldehyde; Peak 3: Chlorogenic acid; Peak 4: Caffeic acid; Peak 5: Cryptochlorogenic acid; Peak 6: p-Coumaric acid; Peak 7: Methyl chlorogenic acid; Peak 9: Rutin; Peak 10: Hyperoside; Peak 11: Isoquercetin; Peak 12: Ethyl chlorogenic acid; Peak 13: Isorhamnoside-3-O-galactoside; Peak 14: Quercetin; Peak 15: Isochlorogenic acid B; Peak 16: Isochlorogenic acid A; Peak 17: Isochlorogenic acid C; Peak 18: Quercetin; Peak 19: Kaempferol.

[0409] 7. Determination of the characteristic spectra of Senecio scandens formulation particles

[0410] Chromatographic conditions and system suitability tests were performed using phenyl-bonded silica gel as the packing material (column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm); acetonitrile as mobile phase A and 0.3% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 0.8 mL / min; column temperature 25 °C; detection wavelength 360 nm. The theoretical plate number, calculated based on the hyperoside peak, should be no less than 8000.

[0411]

[0412] Preparation of the reference solution: Take 3g of *Senecio scandens* reference material, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of the following reference standards: neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol. Accurately weigh these and add methanol to prepare a mixed solution containing 20μg of each standard per 1ml, as the reference solution.

[0413] Preparation of the test solution: Take an appropriate amount of Senecio scandens formula granules, grind them into a fine powder, take about 0.5g, place it in a stoppered conical flask, add 25ml of methanol, heat under reflux for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0414] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0415] The chromatogram of the test sample should show 19 characteristic peaks, and the retention times should correspond to the 19 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 should correspond to the retention times of the reference standard peaks. The peak corresponding to the hyperoside reference standard peak is designated as the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated, and their relative retention times should be within ±10% of the specified value. The specified values ​​are 0.24 (peak 1), 0.30 (peak 2), 0.41 (peak 3), 0.48 (peak 5), 0.75 (peak 6), 0.85 (peak 7), 0.95 (peak 8), 0.97 (peak 9), 1.01 (peak 11), 1.10 (peak 12), 1.19 (peak 13), 1.21 (peak 14), 1.27 (peak 15), 1.31 (peak 16), 1.40 (peak 17), 1.56 (peak 18), and 1.72 (peak 19). Compare with the characteristic chromatograms; Peak 1: Neochlorogenic acid; Peak 2: Protocatechuic aldehyde; Peak 3: Chlorogenic acid; Peak 4: Caffeic acid; Peak 5: Cryptochlorogenic acid; Peak 6: p-Coumaric acid; Peak 7: Methyl chlorogenic acid; Peak 9: Rutin; Peak 10: Hyperoside; Peak 11: Isoquercetin; Peak 12: Ethyl chlorogenic acid; Peak 13: Isorhamnoside-3-O-galactoside; Peak 14: Quercetin; Peak 15: Isochlorogenic acid B; Peak 16: Isochlorogenic acid A; Peak 17: Isochlorogenic acid C; Peak 18: Quercetin; Peak 19: Kaempferol.

[0416] Comparative Example 1

[0417] method:

[0418] Mixed reference solution: Accurately weigh appropriate amounts of reference standards for chlorogenic acid, cryptochlorogenic acid, caffeic acid, rutin, hyperoside, isoquercitrin, isochlorogenic acid A, quercetin, isochlorogenic acid C, and quercetin, and add 75% methanol aqueous solution to prepare mixed solutions with mass concentrations of 87.73, 28.13, 5.29, 15.39, 26.77, 5.22, 19.96, 8.67, 4.55, and 1.68 μg / mL, respectively.

[0419] Test solution: Take about 1g of Senecio scandens powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 75% methanol aqueous solution, stopper tightly, weigh, heat under reflux for 1h, cool, weigh again, replenish the lost amount with 75% methanol aqueous solution, shake well, filter, and the solution is ready.

[0420] Chromatographic column: YMC C18 (250 mm × 4.6 mm, 5 μm); column temperature: 30 ℃; mobile phase: acetonitrile (A) – 0.1% phosphoric acid aqueous solution (B), gradient elution (0–16 min, 11% A; 16–17 min, 11% A → 16% A; 17–56 min, 16% A; 56–58 min, 16% A → 22% A; 58–66 min, 22% A; 66–72 min, 22% A → 28% A; 72–85 min, 28% A); flow rate: 1.0 mL / min －1 Detection wavelength: 350 nm; injection volume: 10 μL; analysis time: 85 min.

[0421] The results are shown in Figure 40 below, where peak 1: chlorogenic acid; peak 2: cryptochlorogenic acid; peak 3: caffeic acid; peak 4: rutin; peak 5: hyperoside; peak 6: isoquercitrin; peak 7: isochlorogenic acid A; peak 8: quercetin; peak 9: isochlorogenic acid C; and peak 10: quercetin. The results of this invention are shown in Figure 11. Figure 12 As shown in -a. The results show that the comparative method failed to identify the new chlorogenic acid, protocatechuic aldehyde, p-coumaric acid, methyl chlorogenic acid, ethyl chlorogenic acid, isochlorogenic acid B, isorhamnetin-3-O-galactoside, and kaempferol in the method of the present invention.

[0422] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting the characteristic HPLC chromatograms of Senecio scandens medicinal materials, processed slices, standard decoctions, and formulated granules, comprising: The raw sample was pretreated to obtain the test solution; The pretreatment method includes solvent dissolution and extraction; The test solution was analyzed by HPLC to obtain the HPLC characteristic chromatograms of Senecio scandens medicinal material, decoction pieces, standard decoction, and formula granules; Preparation of reference solutions: Neochlorogenic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, cryptochlorogenic acid, p-coumaric acid, methyl chlorogenic acid, rutin, hyperoside, isoquercitrin, ethyl chlorogenic acid, isorhamnetin-3-O-galactoside, quercetin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, quercetin, and kaempferol were dissolved in solvents to obtain reference solutions. The HPLC conditions were as follows: a phenyl column; mobile phase A was acetonitrile solution, mobile phase B was 0.3% phosphoric acid aqueous solution, and gradient elution was used. The gradient elution specifically refers to: 0–10 min, Phase A: 5% → 7%, Phase B: 95% → 93%; 10–18 min, Phase A: 7%, Phase B: 93%; 18–26 min, Phase A: 7% → 14%, Phase B: 93% → 86%; 26–38 min, Phase A: 14% → 16%, Phase B: 86% → 84%; 38–50 min, Phase A: 16% → 25%, Phase B: 84% → 75%; 50–60 min, Phase A: 25% → 40%, Phase B: 75% → 60%.

2. The method according to claim 1, characterized in that, This also includes the preparation of reference solutions of control medicinal materials: The reference herb *Senecio scandens* was dissolved in methanol and extracted by reflux to obtain a reference herb solution. Both the reference herb solution and the reference herb solution were injected into a high-performance liquid chromatograph (HPLC) for analysis, and chromatograms of the reference herb and its characteristic chromatograms were obtained. Based on the characteristic parameters of the chromatograms of the reference herb, the corresponding chromatograms in the HPLC characteristic chromatograms of the *Senecio scandens* sample, the standard decoction sample, and the formulated granule sample were compared and qualitatively analyzed.

3. The method according to claim 2, characterized in that, The specific injection concentrations of the reference solutions are as follows: neochlorogenic acid 20 μg / mL, protocatechuic aldehyde 20 μg / mL, chlorogenic acid 20 μg / mL, caffeic acid 20 μg / mL, cryptochlorogenic acid 20 μg / mL, p-coumaric acid 20 μg / mL, methyl chlorogenic acid 20 μg / mL, rutin 20 μg / mL, hyperoside 20 μg / mL, isoquercitrin 20 μg / mL, ethyl chlorogenic acid 20 μg / mL, isorhamnetin-3-O-galactoside 20 μg / mL, quercetin 20 μg / mL, isochlorogenic acid B 20 μg / mL, isochlorogenic acid A 20 μg / mL, isochlorogenic acid C 20 μg / mL, quercetin 20 μg / mL, and kaempferol 20 μg / mL.

4. The method according to claim 1, characterized in that, The chromatographic column has dimensions of 150×4.6mm².7μm; the column temperature is 25℃; and the theoretical plate number, calculated based on hyperoside, should be no less than 8000.

5. The method according to claim 4, characterized in that, The detection wavelength is 360nm.

6. The method according to claim 4, characterized in that, The flow rate of the mobile phase was 0.8 mL / min; the injection volume was 2 μL.

7. The method according to claim 4, characterized in that, The solvent in step A) is methanol; the extraction step is carried out by heating and reflux extraction, wherein the extraction duration is 30 minutes.

8. The method according to claim 1, characterized in that, The ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.5:25; The raw materials for the test sample are one or more of the following: Senecio scandens medicinal material, Senecio scandens processed slices, Senecio scandens standard decoction, or Senecio scandens formula granules.

9. The method according to claim 1, characterized in that, The similarity of HPLC characteristic chromatograms of *Senecio scandens* medicinal material, processed slices, standard decoctions, and formulated granules was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system. A standard HPLC characteristic chromatogram consisting of 19 characteristic peaks was constructed, with the chemical composition information corresponding to each peak as follows: Peak 1: Neochlorogenic acid; Peak 2: Protocatechuic aldehyde; Peak 3: Chlorogenic acid; Peak 4: Caffeic acid; Peak 5: Cryptochlorogenic acid; Peak 6: p-Coumaric acid; Peak 7: Methyl chlorogenic acid; Peak 9: Rutin; Peak 10: Hyperoside; Peak 11: Isoquercetin; Peak 12: Ethyl chlorogenic acid; Peak 13: Isorhamnoside-3-O-galactoside; Peak 14: Quercetin; Peak 15: Isochlorogenic acid B; Peak 16: Isochlorogenic acid A; Peak 17: Isochlorogenic acid C; Peak 18: Quercetin; Peak 19: Kaempferol.

10. The method according to claim 1, characterized in that, In the characteristic chromatograms of the medicinal materials, processed slices, standard decoctions, and formulated granules of *Senecio scandens*, ginsenoside is used as the reference peak S. The relative retention time of each characteristic peak and the S peak is calculated. The allowable fluctuation range of the relative retention time of each characteristic peak is within ±10% of the specified value. The specified value is as follows: 0.24 (peak 1), 0.30 (peak 2), 0.41 (peak 3), 0.48 (peak 5), 0.75 (peak 6), 0.85 (peak 7), 0.95 (peak 8), 0.97 (peak 9), 1.01 (peak 11), 1.10 (peak 12), 1.19 (peak 13), 1.21 (peak 14), 1.27 (peak 15), 1.31 (peak 16), 1.40 (peak 17), 1.56 (peak 18), and 1.72 (peak 19).

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