Construction method and application of characteristic map of gandine and its pharmaceutical preparation

Abstract

The application provides a construction method and application of a characteristic map of Rhamnus davurica Pall and a pharmaceutical preparation thereof, and the construction method of the characteristic map of the Rhamnus davurica Pall and the pharmaceutical preparation thereof provided by the application uses octadecylsilane bonded silica gel as a filler, acetonitrile as mobile phase A, formic acid solution as mobile phase B, and a specific elution procedure, so that the separation effect of multiple effective components is obviously improved while the detection time is shortened, more characteristic peaks are contained in the characteristic map, the map information is greatly enriched, the effective separation of characteristic peaks such as gallic acid, gentisic acid 5-O-(6'-O-galloyl)-beta-D-glucopyranoside, ellagic acid, 3,3',4-tri-O-methyl ellagic acid 4'-glucoside and 3'-O-methyl-3,4-O,O-methylene ellagic acid is achieved, the characteristic map obtained is obviously characteristic, fully reflects the integrity and characteristic of the pharmaceutical preparation of the Rhamnus davurica Pall, and the baseline is smooth and the peak shape is good, thereby providing a basis for comprehensively detecting the quality of the pharmaceutical preparation of the Rhamnus davurica Pall.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection, specifically to a method for constructing and applying characteristic spectra of Gynostemma pentaphyllum and its pharmaceutical preparations. Background Technology

[0002] The main chemical components of *Rhizoma Cynanchi* are phenolic acids, etc. It is slightly astringent and neutral in nature. It enters the liver and kidney meridians. It soothes the liver, unblocks the meridians, and relieves pain. It is used for chest and rib pain due to liver qi stagnation, rheumatic bone pain, and lumbar muscle strain.

[0003] Gangren is a non-pharmacopoeia variety of traditional Chinese medicine. Some literature describes the specific chemical components in Gangren. However, most of the literature discloses the content determination or identification of single components, which cannot detect and control its quality as a whole. Determining the content of single components and then identifying Gangren or its drug preparations in combination with other components is time-consuming and laborious, and it is difficult to apply it widely in production practice. Summary of the Invention

[0004] The problem this invention aims to solve is that there is no publicly available method for effectively detecting Rhizoma Cynanchi or its formulations in the prior art; therefore, this invention provides a characteristic chromatogram of Rhizoma Cynanchi formulation particles, its construction method, and its application.

[0005] Therefore, the present invention provides a method for constructing the characteristic spectrum of *Rhizoma Cynanchi* and its pharmaceutical preparations, comprising the following steps:

[0006] Preparation of the test solution;

[0007] The test solution was analyzed by high performance liquid chromatography (HPLC). The chromatographic conditions included using octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and 0.5 vol% formic acid solution as mobile phase B, with gradient elution performed according to the following procedure:

[0008] From 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B was 2%:98%.

[0009] Over 5-8 minutes, the volume ratio of mobile phase A to mobile phase B changes from 2%:98% to 5%:95%.

[0010] Over 8-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 5%:95% to 17%:83%.

[0011] Over 30-70 minutes, the volume ratio of mobile phase A to mobile phase B changed from 17%:83% to 25%:75%.

[0012] 70-75 min, the volume ratio of mobile phase A to mobile phase B is 25%:75%;

[0013] Preferably, the gradient elution further includes the following procedure:

[0014] The process takes 70-75 minutes, with the volume ratio of mobile phase A to mobile phase B being 25%:75%.

[0015] In some embodiments, the chromatographic conditions further satisfy any one or more of the following (1)-(6):

[0016] (1) A chromatographic column with a specification of 250mm×4.6mm and a particle size of 5μm was used;

[0017] (2) The concentration of the formic acid solution is 0.1-1 vol%.

[0018] (3) The injection volume of the test solution is 5-20 μL;

[0019] (4) The detection wavelength is 220-280nm;

[0020] (5) The column temperature is 20-35℃;

[0021] (6) The flow rate of the eluent is 0.5-1.5 mL / min.

[0022] In some embodiments, the preparation method of the test solution is as follows: weigh the test sample, add solvent to extract, obtain the extract, separate the solid and liquid, and take the liquid, which is the test solution;

[0023] Preferably, the method for preparing the test solution further satisfies any one or more of the following A:

[0024] A. The ratio of the mass of the test sample to the volume of the solvent is 0.05-0.2:10-30; the relationship between mass and volume is g / mL;

[0025] B. The extraction method is either reflux extraction or ultrasonic extraction;

[0026] C. The extraction time is ≥10 min, preferably 20-40 min;

[0027] D. The solid-liquid separation is selected from centrifugation or membrane filtration;

[0028] E. The solvent is selected from at least one of water, methanol and ethanol, preferably an aqueous methanol solution.

[0029] In some embodiments, the construction method further includes the step of preparing a reference solution by adding at least one of gallic acid, gentic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside, ellagic acid, 3,3′,4-tri-O-methylellagic acid 4′-glucoside and 3′-O-methyl-3,4-O,O-methyleneellagic acid as a solvent, and the step of detecting the reference solution with high performance liquid chromatography according to any one of the construction methods of claims 1-3 to obtain a reference chromatogram;

[0030] Preferably, the solvent is selected from at least one of methanol, ethanol, and water;

[0031] More preferably, 1 mL of the reference solution contains 0.005 to 200 μg of each reference standard, and even more preferably 50 to 100 μg.

[0032] In some embodiments, the characteristic spectrum of the Gangren pharmaceutical preparation has 7 common characteristic peaks, including peak 1 being gallic acid, peak 3 being gentic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside, peak 4 being ellagic acid, peak 6 being the characteristic peak corresponding to 3′-O-methyl-3,4-O,O-methylene ellagic acid, and peak 7 being 3,3′,4-tri-O-methylellagic acid 4′-glucopyranoside;

[0033] Preferably, peak 4 is designated as peak S, and the relative retention times of other characteristic peaks relative to peak 4 are within ±10% of a specified value. The specified values ​​for each characteristic peak are as follows:

[0034] Peak 1: 0.25, Peak 2: 0.43, Peak 3: 0.68, Peak 4: 1.0, Peak 5: 1.17, Peak 6: 1.39, Peak 7: 1.48.

[0035] Secondly, this application also provides a method for testing the content of a drug preparation containing *Gynostemma pentaphyllum*, comprising the following steps:

[0036] Preparation of the test solution;

[0037] A reference solution was prepared by adding at least one of gallic acid, gentic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside, ellagic acid, 3,3′,4-tri-O-methylellagic acid 4′-glucopyranoside and 3′-O-methyl-3,4-O,O-methyleneellagic acid to a solvent.

[0038] High-performance liquid chromatography (HPLC) was used to detect the test solution and the reference solution. The chromatographic conditions included using octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and formic acid solution as mobile phase B, with gradient elution performed under the following conditions:

[0039] From 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B was 2%:98%.

[0040] Over 5-8 minutes, the volume ratio of mobile phase A to mobile phase B changes from 2%:98% to 5%:95%.

[0041] Over 8-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 5%:95% to 17%:83%.

[0042] Over 30-70 minutes, the volume ratio of mobile phase A to mobile phase B changed from 17%:83% to 25%:75%.

[0043] Preferably, the gradient elution further includes the following procedure:

[0044] The process takes 70-75 minutes, with the volume ratio of mobile phase A to mobile phase B being 25%:75%.

[0045] In some embodiments, a chromatographic column with dimensions of 250 mm × 4.6 mm and a diameter of 5 μm is used; and / or, the injection volume of the test solution is 5-20 μL; and / or, the concentration of the formic acid solution is 0.1-1 vol%, the detection wavelength is 220-280 nm, the column temperature is 20-35 °C, and the flow rate is 0.5-1.0 mL / min.

[0046] In some embodiments, the preparation method of the test solution is as follows: weigh the test sample, extract with solvent to obtain the extract, separate the solid and liquid, and take the liquid, which is the test solution. Preferably, the preparation method of the test solution further satisfies any one or more of the following A:

[0047] A. The ratio of the mass of the test sample to the volume of the solvent is 0.05-0.2:10-30; the relationship between mass and volume is g / mL;

[0048] B. The extraction method is either reflux extraction or ultrasonic extraction;

[0049] C. The extraction time is ≥10 min, preferably 20-40 min;

[0050] D. The solid-liquid separation is selected from centrifugation or membrane filtration;

[0051] E. The solvent is selected from at least one of water, methanol and ethanol, preferably an aqueous methanol solution.

[0052] In some embodiments, the solvent used in the preparation of the reference solution is selected from at least one of methanol, ethanol and water;

[0053] Preferably, 1 mL of the reference solution contains 0.005–200 μg of each reference standard, more preferably 50–100 μg.

[0054] Thirdly, this application also provides a quality testing method for a Gynostemma pentaphyllum pharmaceutical preparation, including the step of comparing the characteristic chromatogram of the Gynostemma pentaphyllum product to be tested with the characteristic chromatogram of the Gynostemma pentaphyllum pharmaceutical preparation.

[0055] And / or, the step of determining the content of the *Gynostemma pentaphyllum* product to be tested using the content testing method of the *Gynostemma pentaphyllum* drug preparation described above; the characteristic chromatogram of the *Gynostemma pentaphyllum* product to be tested is constructed using the *Gynostemma pentaphyllum* product to be tested according to the construction method described above, and the control characteristic chromatogram of the *Gynostemma pentaphyllum* drug preparation is selected from any one of the following (1)-(3):

[0056] (1) It has 7 common characteristic peaks. Peaks 1, 3, 4, 5 and 6 correspond to the retention times of gallic acid, gentic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside, ellagic acid, 3,3′,4-tri-O-methylellagic acid 4′-glucoside and 3′-O-methyl-3,4-O,O-methyleneellagic acid reference standards, respectively. The peak corresponding to the ellagic acid reference standard peak is S1 peak. The relative retention times of other characteristic peaks with S1 peak are within ±10% of the specified values. The specified values ​​of each characteristic peak are: peak 1: 0.25, peak 2: 0.43, peak 3: 0.68, peak 4: 1.0, peak 5: 1.17, peak 6: 1.39, peak 7: 1.48;

[0057] (2) Characteristic spectra of Gynostemma pentaphyllum drug formulations obtained by using single or multiple batches of Gynostemma pentaphyllum or its formulations according to any one of the construction methods described in claims 1-5;

[0058] (3) Using multiple batches of Gangren drug preparations, the characteristic spectra obtained according to any one of the construction methods described in claims 1-5 are used to prepare a control characteristic spectra by means of average value or median.

[0059] The similarity of the characteristic chromatograms of the Gangren formula granules is greater than 0.90.

[0060] The technical solution of this invention has the following advantages:

[0061] 1. The method for constructing characteristic spectra of *Gynostemma pentaphyllum* and its pharmaceutical preparations provided by this invention uses octadecylsilane-bonded silica gel as a filler, acetonitrile as mobile phase A, formic acid solution as mobile phase B, and a specific elution procedure. This method significantly improves the separation of multiple active ingredients while shortening the detection time, resulting in a more abundant characteristic spectrum with richer spectral information. It effectively separates characteristic peaks including gallic acid, gentic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside, ellagic acid, 3,3′,4-tri-O-methylellagic acid 4′-glucoside, and 3′-O-methyl-3,4-O,O-methyleneellagic acid. The resulting characteristic spectra are highly distinctive, fully reflecting the integrity and characteristics of *Gynostemma pentaphyllum* pharmaceutical preparations, with a stable baseline and good peak shape, providing a basis for comprehensive quality testing of *Gynostemma pentaphyllum* pharmaceutical preparations.

[0062] 2. The method for content determination of *Gynostemma pentaphyllum* pharmaceutical preparations provided by this invention uses the content of gallic acid and the system adaptability of indicator components as evaluation criteria. Experiments have shown that using octadecylsilane-bonded silica gel as the filler, acetonitrile as mobile phase A, formic acid solution as mobile phase B, and a specific elution procedure, the contents of gallic acid, gentic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside, ellagic acid, 3,3′,4-tri-O-methylellagic acid 4′-glucoside, and 3′-O-methyl-3,4-O,O-methyleneellagic acid can be determined simultaneously. The separation of each component is good, and the construction method exhibits high precision, stability, and repeatability. Therefore, this application can comprehensively and rapidly detect the active ingredients and their contents in *Gynostemma pentaphyllum* pharmaceutical preparations.

[0063] 3. The method for constructing the characteristic spectrum of the drug preparation of *Gynostemma pentaphyllum* provided by the present invention can select S-peak ellagic acid as the internal reference peak in the characteristic spectrum, and can determine the seven common characteristic peaks of the drug preparation of *Gynostemma pentaphyllum*, and calculate the relative retention time of each common characteristic peak based on S-peak ellagic acid, which is beneficial to the comprehensive quality detection and overall quality control of the drug preparation of *Gynostemma pentaphyllum*, thereby helping to improve the safety and stability of the drug.

[0064] 4. The quality detection method for the characteristic chromatogram of the pharmaceutical preparation of *Rhizophora stylosa* provided by the present invention can simultaneously construct the characteristic chromatogram of the *Rhizophora stylosa* to be tested and perform content determination. The method is simple and facilitates comprehensive quality control of *Rhizophora stylosa*. Attached Figure Description

[0065] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0066] Figure 1 These are the characteristic chromatograms of 16 batches of *Gynostemma pentaphyllum* formulation granules in Example 1 of this invention;

[0067] Figure 2 This is the comparative feature map in Embodiment 1 of the present invention;

[0068] Figure 3 This is the localization map of the reference standard in Example 1 of the present invention;

[0069] Figure 4 This is the chromatogram of gradient condition 1 in Experiment Example 1 of the present invention;

[0070] Figure 5 This is the chromatogram of gradient condition 2 in Experiment Example 1 of the present invention;

[0071] Figure 6 This is the chromatogram of gradient condition 3 in Experiment Example 1 of the present invention;

[0072] Figure 7 This is the chromatogram of Experiment 1 of the present invention at a wavelength of 254 nm;

[0073] Figure 8 This is the chromatogram of Experiment 1 of the present invention at a wavelength of 290 nm;

[0074] Figure 9 This is the chromatogram of Experiment Example 1 of the present invention at a wavelength of 325 nm;

[0075] Figure 10 This is the chromatogram of Experiment Example 1 of the present invention at a wavelength of 360 nm;

[0076] Figure 11 Chromatogram of 0.1% formic acid in Experimental Example 1 of this invention;

[0077] Figure 12 Chromatogram of 0.3% formic acid in Experimental Example 1 of this invention;

[0078] Figure 13 Chromatogram of 0.5% formic acid in Experimental Example 1 of this invention;

[0079] Figure 14 Chromatogram of Experimental Example 1 of this invention at a flow rate of 0.9 mL / min;

[0080] Figure 15 Chromatogram of Experimental Example 1 of this invention at a flow rate of 1.0 mL / min;

[0081] Figure 16 Chromatogram of Experimental Example 1 of this invention at a flow rate of 1.1 mL / min;

[0082] Figure 17 Comparison chart of Experiment 1 of this invention under column temperature of 28°C;

[0083] Figure 18 Comparison chart of Experiment 1 of this invention under column temperature of 30°C;

[0084] Figure 19 Chromatogram of Experimental Example 1 of this invention at a column temperature of 32°C;

[0085] Figure 20 Chromatogram of the Shim-pack GIST ODS column in Experimental Example 1 of this invention;

[0086] Figure 21 The chromatogram of the XSelect HSS T3 5μm column in Experimental Example 1 of this invention;

[0087] Figure 22The chromatogram of the Polaris 5C18-A column in Experimental Example 1 of this invention;

[0088] Figure 23 Chromatogram of negative blank control in Experimental Example 3 of this invention;

[0089] Figure 24 Chromatogram of negative blank control in Experimental Example 4 of this invention;

[0090] Figure 25 Linearity graph of gallic acid reference standard at different concentrations in Experiment Example 4 of this invention. Detailed Implementation

[0091] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0092] In this application, percentage content refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures, unless otherwise specified.

[0093] In this application, unless otherwise specified, percentage concentrations refer to final concentrations. The final concentration refers to the percentage of the added component in the system after its addition.

[0094] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) or vol% refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0095] Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products. Percentages not specified in this invention are volume percentages.

[0096] The pharmaceutical preparation of *Gynostemma pentaphyllum* described in this invention is prepared by the following method:

[0097] Harvest *Rhodiola rosea*, and extract under reflux at least once, adding 8-14 times its weight of water each time for at least 0.5 hours. Filter, combine the filtrates, and concentrate the filtrate to a relative density of 1.05-1.10 g / mL at 60°C. Add conventional excipients and follow conventional processes to prepare clinically acceptable tablets, capsules, pills, granules, honey-processed pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations, or injectable preparations. The pharmaceutically acceptable excipients include: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrix, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silica, etc.; suspending agents include: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch paste, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavoring agents include: sweeteners and various flavorings; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorethidium acetate, eucalyptus oil, etc.; matrix includes: PEG6000, PEG4000, insect wax, etc.

[0098] The instruments and reagents used in this invention are as follows:

[0099] 1. Instruments and reagents

[0100] 1.1 The instruments are shown in Table 1-3

[0101] Table 1. Chromatographic Instrument Parameters

[0102]

[0103] Table 2. Column Parameters

[0104]

[0105] 1.2 Test Drugs

[0106] Reference material for Rhododendron chinense: purchased from Lemeitian Pharmaceutical | Desite Biotechnology, batch number DSTYG006001;

[0107] Gallic acid reference standard: purchased from the National Institutes for Food and Drug Control, batch number 110831-201906, purity: 91.5%;

[0108] Ellagic acid reference standard: purchased from the National Institutes for Food and Drug Control, batch number 111959-201903, purity: 88.8%.

[0109] Fifteen batches of Gangren formula granule samples: 1902002W, 1904002S, 1901001S, 1903001W, 1905001W, 1910002S, 1912001W, 2004001S, 2011002W, 2105001S, 2108002S, 2112002S, 2203001W, 2209003S, 2212001S.

[0110] 1.3 Reagents

[0111] Methanol and acetonitrile were of chromatographic grade (TEDIA), and water was ultrapure water; all other reagents were of analytical grade.

[0112] Example 1

[0113] A fingerprint spectrum and a method for determining the content of a pharmaceutical preparation of *Rhodiola rosea*, comprising:

[0114] (1) Preparation of test solution: Take about 0.1g of the powder of Gynostemma pentaphyllum, accurately weigh it, place it in a stoppered conical flask, accurately add 20mL of 50% methanol, stopper tightly, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0115] The test sample used in this embodiment is Rhodomyrtus tomentosa formula granules. The specific preparation method of Rhodomyrtus tomentosa formula granules is as follows: Rhodomyrtus tomentosa (Ait.) Hassk., a plant of the Myrtaceae family, is taken and extracted twice by heating and reflux. For the first extraction, 9 times its weight of water is added and soaked for 30 min, and then heated and refluxed for 0.5 h. After filtration, 7 times its weight of water is added and extracted for 0.5 h. After filtration, the filtrates are combined and concentrated to a relative density of 1.05 g / mL at 60 °C. The filtrates are then spray-dried, and the dry powder is mixed with the excipient maltodextrin (the amount of maltodextrin added is 0% to 2.5% of the weight of the dry powder). After mixing evenly, the mixture is dry-granulated to produce granules.

[0116] Preparation of reference solution: Take 1g of *Rhodiola rosea* reference material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 1h, filter, evaporate the filtrate to dryness, add 10ml of 50% methanol to the residue, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, filter, and take the filtrate as the reference solution for the reference material.

[0117] Separately, take appropriate amounts of gallic acid reference standard, gentic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside reference standard, ellagic acid reference standard, 3,3′,4-tri-O-methylellagic acid 4′-glucoside reference standard and 3′-O-methyl-3,4-O,O-methyleneellagic acid reference standard, accurately weigh them, and add 50% methanol to prepare a solution containing 50 μg of each in 1 ml, as the reference solution.

[0118] (2) High-performance liquid chromatography (HPLC) analysis conditions

[0119] Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, and 0.5 vol% formic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 1.0 mL / min, the column temperature was 30 °C, and the detection wavelength was 254 nm. The theoretical plate number, calculated based on gallic acid, should not be less than 3000. Gradient elution was performed according to the specifications in Table 4 below:

[0120] Table 4 Gradient elution conditions for the examples

[0121]

[0122]

[0123] The common pattern of the characteristic fingerprint spectrum of Gangren formula granules was established using the method described above.

[0124] Fifteen batches of *Gynostemma pentaphyllum* formula granules were taken as test solutions, and the fingerprint chromatograms of the *Gynostemma pentaphyllum* formula granules were obtained by high-performance liquid chromatography (HPLC). Figure 1 As shown, Figure 1 In the above, S1 to S15 correspond to: 1902002W, 1904002S, 1901001S, 1903001W, 1905001W, 1910002S, 1912001W, 2004001S, 2011002W, 2105001S, 2108002S, 2112002S, 2203001W, 2209003S, and 2212001S. The results are shown in Tables 5 to 8.

[0125] Table 5. Relative retention time results of characteristic fingerprint spectroscopy determination for 15 batches of Gangren formulation granules.

[0126]

[0127] Table 6. Relative retention time of fingerprint spectra of Gangren formulation granules

[0128]

[0129] Table 7. Comparison of relative peak areas in the chromatograms of Gangren formula granules.

[0130]

[0131]

[0132] Table 8. Similarity results of characteristic fingerprint spectra of 15 batches of Gangren formulation granules

[0133]

[0134] Using the fingerprint similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission, a control characteristic fingerprint spectrum was generated, such as... Figure 2 As shown, the reference standard positioning diagram is as follows: Figure 3 As shown. S1: Reference spectrum; S2: Gallic acid; S3: Gentianic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside; S4: 3′-O-methyl-3,4-O,O-methylene ellagic acid; S5: 3,3′,4-tri-O-methylellagic acid 4′-glucoside; S6: Ellagic acid.

[0135] pass Figure 1 , Figure 2 and Figure 3 It is known that the HPLC characteristic fingerprint of Gangren formula granules contains 7 chromatographic peaks. Through reference standard localization and identification, peaks 1, 3, 4, 6, and 7 should correspond to the retention times of the reference peaks of gallic acid, gentic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside, ellagic acid, 3′-O-methyl-3,4-O,O-methylene ellagic acid, and 3,3′,4-tri-O-methylellagic acid 4′-glucopyranoside, respectively. The peak corresponding to the ellagic acid reference peak is the S peak. The relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are 0.25 (peak 1), 0.43 (peak 2), 0.68 (peak 3), 1.17 (peak 5), 1.39 (peak 6), and 1.48 (peak 7). The similarity of the characteristic chromatograms of Gangren formula granules to the control group is greater than 0.90.

[0136] according to Figure 2 The comparative characteristic fingerprint spectrum shown can be used to analyze and compare the detection results of the characteristic spectrum of the formulation granules, and is used for the quality control of the formulation granules.

[0137] Fifteen batches of *Gynostemma pentaphyllum* formula granules were used as test solutions. The gallic acid content in the *Gynostemma pentaphyllum* formula granules was obtained by high performance liquid chromatography, and the results are shown in Table 9.

[0138] Table 9. Results of gallic acid content in 15 batches of Gangren formula granules

[0139]

[0140]

[0141] The detection method described in this embodiment can effectively obtain fingerprint spectra with good separation of various characteristic peaks, and can also simultaneously determine the content of gallic acid. Furthermore, by selecting the S peak (ellagic acid) as the internal reference peak in the fingerprint spectrum, the relative retention times of the common characteristic peaks 1-3 and 5-7 of Gangren formula granules can be determined. Therefore, it can comprehensively and rapidly detect Gangren formula granules, which is beneficial for the comprehensive quality testing and overall quality control of Gangren formula granules, thereby helping to improve the safety and stability of the drug.

[0142] Investigation of chromatographic conditions in Experimental Example 1

[0143] In this experimental example, both the Gangren formula granules and the test solution were prepared according to the method in Example 1. The test solution was prepared from Gangren formula granules (1903001W).

[0144] Octadecylsilane-bonded silica gel was used as the filler (column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm).

[0145] 1.1 Selection of elution conditions

[0146] Using acetonitrile as mobile phase A and 0.5 vol% formic acid water as mobile phase B, gradient elution was performed according to the specifications in Table 10-12; the detection wavelength was 254 nm, the column temperature was 30 °C, and the flow rate was 1.0 mL per minute.

[0147] Table 10 Gradient Condition 1

[0148]

[0149] Table 11 Gradient Condition 2

[0150]

[0151] Table 12 Gradient Condition 3

[0152]

[0153]

[0154] Test results as follows Figure 4-6 And Table 13.

[0155] Table 13 System suitability parameters for each gradient chromatographic peak

[0156]

[0157] By comparing the chromatograms of samples measured under different elution programs, the gradient with richer chromatographic information, better resolution of major chromatographic peaks, more stable baseline, and more reasonable analysis time was selected. The results showed that the chromatographic peak separation improved with increasing elution time. The chromatographic peaks of gradient 3 were similar to those of gradient 1, but the baseline of gradient 3 was more stable, and all chromatographic peaks achieved better baseline separation. Therefore, gradient 3 was selected as the preferred mobile phase gradient for subsequent condition screening to achieve better separation. Since no characteristic peaks with good resolution were observed after 70 minutes, the elution time was preferably below 70 minutes.

[0158] 1.2 Selection of detection wavelength

[0159] Acetonitrile was used as mobile phase A, and 0.5 vol% formic acid aqueous solution was used as mobile phase B. The column temperature was 30℃, and the flow rate was 1.0 mL per minute. The test solution was injected into the liquid chromatograph, and elution was performed under gradient conditions of gradient 3. A full-wavelength scan was performed, and the chromatograms at four different absorption wavelengths (254 nm, 290 nm, 325 nm, and 360 nm) were compared based on the amount of chromatographic information. The number of detected chromatographic peaks, response value, and retention time were used as evaluation criteria to screen the detection wavelengths. The detection results are as follows: Figure 7-10 And Table 14.

[0160] Table 14 System adaptability parameters for chromatographic peaks at different absorption wavelengths

[0161]

[0162]

[0163] The main active ingredients in *Gynostemma pentaphyllum* are phenolic acids, with an effective detection wavelength range of 190-360 nm. Results showed that the chromatogram at 254 nm exhibited more peaks, greater information content, and a stable baseline. Therefore, 254 nm was selected as the preferred absorption wavelength for detecting the characteristic chromatogram of *Gynostemma pentaphyllum* formulation particles, given its relatively rich peak information and superior system adaptability parameters, for further investigation.

[0164] 1.3 Investigation of mobile phase composition

[0165] Acetonitrile was used as mobile phase A, formic acid solution as mobile phase B, the detection wavelength was 254 nm, the column temperature was 30 °C, and the flow rate was 1.0 mL per minute. The test solution was injected into the liquid chromatograph, and elution was performed under gradient conditions (gradient 3). The effect of different formic acid concentrations on the separation of characteristic chromatograms of Gangren formulation particles was investigated, and comparisons were made between the 0.1 vol% formic acid system, the 0.3 vol% formic acid system, and the 0.5 vol% formic acid system. Results are shown below. Figure 11-13 See Table 15.

[0166] Table 15. Applicability parameters of the system for different formic acid concentrations.

[0167]

[0168] The results showed that different formic acid concentrations had little effect on each chromatographic peak. Among them, the chromatograms eluted with 0.3 vol% to 0.5 vol% formic acid solutions showed better peak shapes and resolutions for the main chromatographic peaks. Considering that peak 1 (gallic acid) detected under the 0.5 vol% formic acid system had the largest response, it could be used for the determination of gallic acid content. Therefore, the 0.5 vol% formic acid solution was preferred for subsequent condition screening.

[0169] 1.4 Investigation of different flow velocities

[0170] Acetonitrile was used as mobile phase A, and 0.5 vol% formic acid solution was used as mobile phase B. The detection wavelength was 254 nm, and the column temperature was 30 °C. The test solution was injected into the liquid chromatograph, and elution was performed under gradient conditions of gradient 3. The determination was carried out at flow rates of 0.9 mL / min, 1.0 mL / min, and 1.1 mL / min, respectively. The effect of different flow rates on the separation effect of the characteristic chromatogram of Gangren formulation particles was investigated. The results are shown in […]. Figure 14-16 See Tables 16-18.

[0171] Table 16 Parameters for assessing system suitability at a flow rate of 0.9 mL / min

[0172]

[0173]

[0174] Table 18 Parameters for assessing system suitability at a flow rate of 1.1 mL / min

[0175]

[0176] The results showed that different flow rates had a significant impact on the retention time of some chromatographic peaks, and also on the resolution and asymmetry of the peaks. The elution time was generally delayed at a flow rate of 0.9 mL / min, the characteristic peak system adaptability parameters were poor at a flow rate of 1.1 mL / min, and the chromatographic peak separation effect was relatively good at a flow rate of 1.0 mL / min. Therefore, a flow rate of 1.0 mL / min was selected as the preferred flow rate for subsequent screening and evaluation.

[0177] 1.5 Investigation at different column temperatures

[0178] Acetonitrile was used as mobile phase A, and 0.5 vol% formic acid solution was used as mobile phase B. The detection wavelength was 254 nm, and the flow rate was 1.0 mL per minute. The test solution was injected into the liquid chromatograph, and elution was performed under gradient conditions of gradient 3. The column temperatures of 28℃, 30℃, and 32℃ were investigated. The results are shown in the figure. Figure 17-19 See Tables 19-21.

[0179] Table 19 Parameters for System Suitability Testing at Column Temperature 28℃

[0180]

[0181] Table 20 Parameters for System Suitability Testing at 30℃ Column Temperature

[0182]

[0183] Table 21 Parameters for System Suitability Testing at Column Temperature of 32℃

[0184]

[0185] The results showed that different column temperatures had a significant impact on the retention time of chromatographic peaks. At a lower column temperature of 28℃, the elution time was prolonged, while at column temperatures of 30℃ and 32℃, the elution times were shorter. At 30℃, the system adaptability parameters for each peak were relatively better. Considering all factors, a column temperature of 30℃ was selected as the optimal temperature for subsequent condition screening.

[0186] 1.6 Investigation of different chromatographic columns

[0187] Acetonitrile was used as mobile phase A, and 0.5 vol% formic acid solution was used as mobile phase B. The detection wavelength was 254 nm, the column temperature was 30 ℃, and the flow rate was 1.0 mL per minute. The test solution was injected into the liquid chromatograph, and elution was performed under gradient conditions of gradient 3. The effect of different brands of chromatographic columns on the characteristic chromatogram of the lyophilized powder of Gangren formulation granules was investigated. The different brands of chromatographic columns investigated were: Shim-pack GIST ODS (4.6*250 mm, 5 μm); XSelect HSS T3 5 μm (4.6*250 mm, 5 μm) column; and Polaris 5C18-A (4.6*250 mm, 5 μm) column. The detection results are as follows. Figure 20-22 See Tables 22-24.

[0188] Table 22 Shim-pack GIST ODS Column Testing System Suitability Parameters

[0189]

[0190] Table 23 XSelect HSS T3 5μm Column: System Suitability Parameters

[0191]

[0192] Table 24. Suitability parameters for Polaris 5C18-A (4.6*250mm, 5μm) chromatographic column.

[0193]

[0194] The results showed that different brands of chromatographic columns had a significant impact on the retention time of each chromatographic peak. The Shim-pack GIST ODS column showed the best separation effect for each chromatographic peak, while the other two brands performed poorly in terms of resolution, symmetry factor, and other parameters. Therefore, the preferred column brand is the Shim-pack GIST ODS (4.6*250mm, 5μm) column.

[0195] 1.7 Determination of chromatographic conditions

[0196] Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the stationary phase (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); acetonitrile was used as mobile phase A, and 0.5 vol% formic acid solution was used as mobile phase B; the flow rate was 1.0 mL / min, and the column temperature was 30 °C; the detection wavelength was 254 nm. The theoretical plate number, calculated based on gallic acid, should be no less than 3000. Gradient elution was performed according to the following procedure:

[0197] From 0 to 5 minutes, the volume ratio of mobile phase A to mobile phase B was 2%:98%.

[0198] Over 5-8 minutes, the volume ratio of mobile phase A to mobile phase B changes from 2%:98% to 5%:95%.

[0199] Over 8-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 5%:95% to 17%:83%.

[0200] Over 30-70 minutes, the volume ratio of mobile phase A to mobile phase B changed from 17%:83% to 25%:75%.

[0201] Experimental Example 2: Investigation of the preparation method of the test sample solution

[0202] The chromatographic conditions for HPLC detection in this experimental example are as defined in 1.7.

[0203] 2.1 Investigation of extraction solvent

[0204] The effects of different extraction solvents (30 vol% methanol, 50 vol% methanol, 70 vol% methanol, and methanol) on the characteristic chromatograms of *Gynostemma pentaphyllum* formulation granules were investigated. Approximately 0.1 g of each *Gynostemma pentaphyllum* formulation granule was accurately weighed and placed in a stoppered conical flask. 20 ml of each different extraction solvent was accurately added, the flask was sealed, and the mixture was sonicated (250 W, 40 kHz) for 30 minutes. After cooling, the mixture was shaken well, filtered, and the filtrate was collected. The results are shown in Table 25.

[0205] Table 25. Parameters for Adaptability of Chromatographic Peaks Based on Methanol Concentration as Extraction Solvent

[0206]

[0207] Based on the chromatographic parameters of each peak, the results showed that methanol extraction was better than ethanol extraction. The total peak area of ​​the characteristic chromatographic peaks obtained by extraction with different methanol concentrations was different. The peak area of ​​the chromatographic peaks obtained by extraction with 50% methanol was significantly larger than that obtained by extraction with other methanol concentrations. Considering all factors, 50% methanol is the preferred extraction solvent.

[0208] 2.2 Examination of extraction time

[0209] The extraction effects of different extraction times (20, 30, and 40 minutes) on the main characteristic peaks of the *Gynostemma pentaphyllum* formula granules were investigated. Approximately 0.1 g of *Gynostemma pentaphyllum* formula granules were accurately weighed and placed in stoppered conical flasks. 20 ml of 50% methanol was accurately added, and the flasks were sealed tightly. The flasks were then sonicated (250 W, 40 kHz) for 20, 30, and 40 minutes respectively. After cooling, the flasks were shaken well, filtered, and the filtrate was collected. The results are shown in Table 26 below.

[0210] Table 1. Extraction time parameters for chromatographic peak system adaptability.

[0211]

[0212]

[0213] Results Analysis: A comprehensive comparison of the results of the three different extraction times showed that different extraction times did not affect the retention time of each peak. There was no significant difference in peak area after ultrasonic treatment for 20 minutes, 30 minutes and 40 minutes, indicating that ultrasonic treatment for 30 minutes resulted in better separation, asymmetry and theoretical plate number of each peak. Therefore, the optimal extraction time is 30 minutes.

[0214] 2.3 Sampling Quantity Examination

[0215] The effect of different sampling amounts on the characteristic chromatogram of *Gynostemma pentaphyllum* formulation granules was investigated. Sample amounts of 0.05 g, 0.1 g, and 0.2 g were examined: approximately 0.05 g, 0.1 g, and 0.2 g of *Gynostemma pentaphyllum* formulation granules were accurately weighed, placed in stoppered conical flasks, and 20 ml of 50% methanol was accurately added. The flasks were then sealed tightly and sonicated (250 W, 40 kHz) for 30 minutes. After cooling, the flasks were shaken well, filtered, and the filtrate was collected. The results are shown in Table 27 below.

[0216] Table 27 Sampling quantity parameters for evaluating the suitability of the chromatographic peak system

[0217]

[0218] Experimental results show that different sample amounts have no effect on the retention time of each characteristic peak, nor on the resolution, symmetry factor, or theoretical plate number. At a sample amount of 0.1 g, the peak areas of the characteristic peaks in the chromatogram of the Gangren formulation particles are relatively moderate. Therefore, 0.1 g is the preferred sample amount for this method.

[0219] 2.4 Investigation of different solvent volumes

[0220] The effect of different solvent volumes (10 ml, 20 ml, and 30 ml) on the characteristic chromatogram of *Gynostemma pentaphyllum* formulation granules was investigated. Approximately 0.1 g of *Gynostemma pentaphyllum* formulation granules were accurately weighed and placed in stoppered conical flasks. 10 ml, 20 ml, and 30 ml of 50% methanol were accurately added respectively. The flasks were then sealed tightly and sonicated (250 W, 40 kHz) for 30 minutes. After cooling, the flasks were shaken well, filtered, and the filtrate was collected. The results are shown in Table 28.

[0221] Table 2 Solvent volume analysis of chromatographic peak system adaptability parameters

[0222]

[0223] Based on the information content and separation effect of the chromatographic peaks as the main evaluation indicators, the experimental results show that the main effect of different solvent volumes on the chromatogram is the peak area. When the solvent volume is 30 ml, the solution concentration is low, the peak area is too small, and it is easy to cause integration error. When the solvent volume is 20 ml, the peak area is larger, and the system adaptability parameters are relatively better. Therefore, 20 ml is preferred as the extraction solvent volume.

[0224] 2.5 Validation of the method for preparing the test solution

[0225] Based on the above results, the preparation method of the test solution for Gangren formula granules is determined as follows: Take 0.1g of Gangren formula granule powder, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of 50% methanol solution, weigh it, sonicate it (power 250W, frequency 40kHz) for 30 minutes, remove it, let it cool, weigh it again, replenish the lost weight with 50% methanol solution, shake it well, filter it, and take the filtrate to obtain the solution.

[0226] Experimental Example 3: Analytical Method Validation

[0227] 3.1 System Suitability Test

[0228] In this experimental example, both the Gangren formula granules and the test solution were prepared according to the method in Example 1, and the chromatograms were recorded under the chromatographic conditions determined in 1.7.

[0229] Octadecylsilane-bonded silica gel was used as the filler (Shim-pack GIST ODS, column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm).

[0230] 3.1.1 Instrument precision test

[0231] Take the same sample solution of Gangren formula granules (batch number: 1903001W), prepare the test solution according to the method confirmed in 2.5, repeat the injection 6 times, record the chromatogram according to the chromatographic conditions determined in 1.7, determine the relative retention time of 7 characteristic peaks, and perform analysis.

[0232] The results show that the RSD of the relative retention time of each characteristic peak and the reference S peak (peak 4) is less than 2%, indicating that the instrument has good precision.

[0233] 3.1.2 Method repeatability test

[0234] Six samples of the same batch of Gangren formula granules (1903001W) were taken, and the relative retention times of seven common peaks were determined and analyzed. The results showed that the RSD values ​​of the relative retention times of each characteristic peak and the reference S peak (peak 4) were all less than 2%, indicating that the method has good repeatability.

[0235] 3.1.3 Intermediate Precision (Different Operators)

[0236] Three inspectors, at different times and using the same equipment, measured the relative retention times of the seven common peaks in the same batch of formula granules and analyzed them. The results showed that the RSD values ​​of the relative retention times of each characteristic peak and the reference peak S (peak 4) were all less than 2%, indicating good intermediate precision (by different personnel) of this method.

[0237] 3.2 Specificity Test

[0238] Take an appropriate amount of negative granules, and follow the preparation and determination methods described in the main text for the test sample. Inject the test sample solution and blank solvent separately, record the chromatograms, and investigate the effect of the blank solvent. The results are shown in […]. Figure 23 Specificity test results showed that the negative blank sample had no interference.

[0239] 3.3 Durability

[0240] 3.3.1 Stability Assessment

[0241] The same test sample was injected at 0, 2, 4, 6, 8, 10, 14, 18, and 24 hours after preparation. The relative retention times of the seven common peaks were measured and analyzed to determine the stability of the test sample solution.

[0242] The results showed that the RSD values ​​of the relative retention times of each characteristic peak and the reference S peak were all less than 2%, indicating that the test solution was stable within 24 hours and met the determination requirements.

[0243] 3.3.3 Investigation of different flow velocities

[0244] Take the same batch of Gangren formula granules test solution and measure it at flow rates of 0.9 mL / min, 1.0 mL / min and 1.1 mL / min respectively. Investigate the relative retention time of each characteristic peak of Gangren formula granules and the reference S peak (peak No. 4) when the flow rate changes slightly, and analyze it.

[0245] The relative retention times of each characteristic peak and the reference peak S (peak 4) were examined when the flow rate changed slightly. The results showed that the relative retention times RSD% were 2.45%, 2.58%, 1.03%, 0.00%, 0.11%, 0.16%, and 0.36%, respectively. The results indicate that the relative retention times of each characteristic peak vary to some extent (within ±10%). Therefore, the preferred flow rate is 1.0 ml per minute.

[0246] 3.3.4 Investigation at different column temperatures

[0247] Take the same Gangren formula granule test sample (1903001W), set the column temperature to 28℃, 30℃ and 32℃ respectively, inject the sample and determine the chromatogram, and analyze the relative retention time of the characteristic peaks.

[0248] The relative retention times of each characteristic peak and the reference peak S (peak 4) were examined when the column temperature varied. The results showed that the RSD% of the relative retention times were 2.99%, 0.71%, 0.63%, 0.00%, 0.34%, 0.42%, and 0.73%, respectively, indicating that different column temperatures have a certain impact on the relative retention of each characteristic peak in the characteristic spectrum. Therefore, it is recommended that the optimal column temperature for this method be 30℃.

[0249] 3.3.5 Investigation of different chromatographic columns

[0250] Different brands of chromatographic columns with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm were selected: Shim-pack GIST 5 μm C18-AQ (column 1); XSelect HSS T3 5 μm (column 2); and Polaris 5C18-A (column 3). The samples were injected and the chromatograms were recorded. The relative retention times of the characteristic peaks were analyzed.

[0251] The relative retention times of each characteristic peak and the reference peak (S peak) were investigated using different brands of chromatographic columns with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm: Shim-pack GIST 5 μm C18-AQ (column 1); XSelect HSS T3 5 μm (column 2); and Polaris 5C18-A (column 3). The RSD% of the relative retention times were 2.37%, 6.89%, 4.57%, 0.00%, 5.10%, 3.43%, and 3.41%, respectively, indicating that different brands of chromatographic columns have a certain influence on the relative retention times of the characteristic chromatograms. The preferred column is the Shim-pack GIST 5 μm C18-AQ.

[0252] Based on the above methodological investigation results, among the seven characteristic peaks of the established chromatographic chromatogram of Gangren formulation granules, each chromatographic peak is affected to a certain extent by different liquid chromatography instruments, chromatographic columns, column temperatures, and flow rates, and the relative retention time values ​​are in the range of -10% to 10%.

[0253] The above experiments determined that octadecylsilane-bonded silica gel should be used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile should be used as mobile phase A, and 0.5 vol% formic acid solution should be used as mobile phase B, with gradient elution performed according to the specifications in Gradient 3 of Table 12; the flow rate should be 1.0 ml per minute, the column temperature should be 30 °C, and the detection wavelength should be 254 nm. The theoretical plate number, calculated based on ellagic acid, should not be less than 3000.

[0254] Preparation of the reference solution: Take 1g of *Rhodiola rosea* reference material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, add 10ml of 50% methanol to the residue, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of gallic acid reference standard and ellagic acid reference standard, accurately weigh them, add 50% methanol to prepare a solution containing 50μg of each per ml, as the reference solution.

[0255] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0256] The chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 1 and 4 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the ellagic acid reference peak is the S peak. Calculate the relative retention times of the remaining characteristic peaks with respect to the S peak. The relative retention times should be within ±10% of the specified values, which are: 0.25 (peak 1), 0.43 (peak 2), 0.68 (peak 3), 1.17 (peak 5), 1.39 (peak 6), and 1.48 (peak 7).

[0257] Test Example 4

[0258] Using gallic acid content and the system adaptability of indicator components as evaluation indicators, the chromatographic conditions and system adaptability of the determination of the content of the formula granules were verified, and the sample pretreatment method was investigated to determine the test sample preparation method. Through methodological investigation of the determination method, a method for determining the content of indicator components in the freeze-dried powder of Gangren formula granules was established, and the content of multiple batches of Gangren formula granules was determined.

[0259] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, and 0.5 vol% formic acid solution was used as mobile phase B, with gradient elution as specified in Table 12; the flow rate was 1.0 mL / min, the column temperature was 30 °C, and the detection wavelength was 254 nm. The theoretical plate number, calculated based on gallic acid, should be no less than 3000.

[0260] Preparation of the reference solution: Take an appropriate amount of gallic acid reference standard, accurately weigh it, and add 50% methanol to prepare a solution containing 0.1 mg per ml.

[0261] Preparation of the test solution: Take 0.1g of Gangren formula granule powder, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of 50% methanol solution, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 50% methanol solution, shake well, filter, and take the filtrate to obtain the test solution.

[0262] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0263] The results of the chromatographic peak system adaptability are shown in Table 29:

[0264] Table 29 Chromatographic Peak System Adaptability Parameters

[0265]

[0266] 4.1 Investigation of the preparation method of the test sample

[0267] The following experiments only change the preparation method of the test solution.

[0268] 4.1.1 Examination of Sampling Size

[0269] The effect of different sampling amounts on the extraction efficiency of *Gynostemma pentaphyllum* formulation granules was investigated.

[0270] Accurately weigh 0.05, 0.1, and 0.2 g of the same *Gynostemma pentaphyllum* formula granules, place them in a stoppered conical flask, accurately add 20 ml of 50% methanol solution, weigh again, sonicate (power 250 W, frequency 40 kHz) for 30 minutes, remove, cool, weigh again, replenish the lost weight with 50% methanol solution, shake well, filter, and collect the filtrate. Perform HPLC analysis; the results are shown in Table 30 below.

[0271] Table 30 Results of the study with different sampling sizes

[0272]

[0273] The experimental results show that the gallic acid content is higher when the sample size is 0.1g, so the preferred sample size is 0.1g.

[0274] 4.1.2 Investigation of Extraction Solvent Concentration

[0275] The effect of different extraction solvents on the extraction efficiency of *Gynostemma pentaphyllum* formulation granules was investigated. 0.1 g of *Gynostemma pentaphyllum* formulation granules were accurately weighed and placed in a stoppered conical flask. 20 ml of 30%, 50%, 70%, and methanol solutions were accurately added respectively. The flasks were sealed tightly, weighed, and subjected to ultrasonic treatment (250 W, 40 kHz) for 30 minutes. After removal and cooling, the flasks were weighed again. The lost weight was replenished with 50% methanol solution, shaken well, filtered, and the filtrate was collected as the final product. HPLC analysis was performed under the above chromatographic conditions, and the results are shown in Table 31 below.

[0276] Table 31 Results of content analysis for different extraction solvents

[0277]

[0278]

[0279] The experimental results show that the gallic acid content obtained by extraction with 30% and 50% methanol as the extraction solvent is similar, indicating that the extraction is relatively thorough; the system adaptability is better when the extraction solvent is 50% methanol, so the extraction solvent of 50% methanol is preferred.

[0280] 4.1.3 Examination of extraction time

[0281] The effect of different extraction times on the extraction efficiency of *Gynostemma pentaphyllum* formula granules was investigated. 0.1 g of *Gynostemma pentaphyllum* formula granules were accurately weighed and placed in a stoppered conical flask. 20 ml of 50% methanol was accurately added, the flask was sealed, and the weight was measured. The flask was then sonicated for 20, 30, and 40 minutes respectively. After removal and cooling, the weight was measured again. The lost weight was replenished with 50% methanol, the flask was shaken well, filtered, and the filtrate was collected as the final product. HPLC analysis was performed under the above chromatographic conditions, and the results are shown in Table 32 below.

[0282] Table 32 Results of content analysis at different extraction times

[0283]

[0284] The experimental results showed that the gallic acid content obtained from extraction at 30 minutes and 40 minutes was similar, indicating that extraction was complete at 30 minutes. From the perspective of saving time, the optimal extraction time is 30 minutes.

[0285] 4.1.4 Investigation on the amount of extraction solvent used

[0286] The effect of different extraction solvent volumes on the extraction efficiency of *Gynostemma pentaphyllum* formulation granules was investigated. 0.1 g of *Gynostemma pentaphyllum* formulation granules were accurately weighed and placed in a stoppered conical flask. 10, 20, and 30 ml of 50% methanol were accurately added, the flask was sealed tightly, and the flask was weighed. The flask was sonicated for 30 minutes, removed, cooled, and weighed again. The weight loss was replenished with 50% methanol, the flask was shaken well, filtered, and the filtrate was collected. HPLC analysis was performed under the above chromatographic conditions, and the results are shown in Table 33 below.

[0287] Table 33 Results of content analysis for different extraction solvent volumes

[0288]

[0289] The experimental results show that gallic acid was completely extracted when the extraction solvent was 20 ml, so the preferred extraction solvent volume is 20 ml.

[0290] 4.2 Methodological Validation

[0291] 4.2.1 Specificity Examination

[0292] Take an appropriate amount of negative granules and prepare a blank sample solution according to the solution preparation method of the test sample solution. Perform HPLC analysis and record the chromatogram as shown. Figure 24 The negative control test showed no interference.

[0293] 4.2.3 Examination of Linear Relationships

[0294] Accurately weigh gallic acid reference standard and dissolve it in 50% methanol to prepare solutions containing 0.0110 mg, 0.0207 mg, 0.0401 mg, 0.0785 mg, 0.1588 mg, 0.3209 mg, and 0.6418 mg per mL, respectively. Accurately inject 10 μL of each of these gallic acid reference standard solutions into the liquid chromatograph and measure the peak area. Plot a standard curve with the injected gallic acid concentration on the x-axis and the peak area integral on the y-axis. The results are shown in the table below. The regression equation is: y = 27917x + 75.434, R0 2 =0.9993. The results are shown in Table 34 and Figure 25 .

[0295] Table 34 Results of linear relationship investigation for different concentrations of gallic acid

[0296]

[0297]

[0298] Experimental results show that the linear relationship of gallic acid injection concentration is good in the range of 0.0110 to 0.6418 mg / ml.

[0299] 4.2.4 Instrument precision test

[0300] The peak area of ​​gallic acid was determined by injecting gallic acid granules (batch number 1903001W) six times consecutively. The results are shown in Table 35 below.

[0301] Table 35 Precision Test Results

[0302]

[0303] The results showed that the RSD of the peak area of ​​gallic acid chromatographic peak after six consecutive injections was 0.13%, indicating that the instrument precision was good.

[0304] 4.3 Repeatability Test

[0305] Six samples of the same batch of Gangren formula granules (batch number 1903001W) were prepared according to the method for preparing the test solution. HPLC determination was performed, and the content was calculated. The results are shown in Table 36 below. The results show that the RSD of gallic acid content was 1.7%, indicating that the method has good repeatability.

[0306] Table 36 Results of Method Repeatability Tests

[0307]

[0308] 4.4 Accuracy Assessment

[0309] The recovery test was conducted by spiking. Approximately 0.05 g of sample (batch number 1903001W, accompanying sample content 17.40 mg / g) was accurately weighed and placed in separate stoppered conical flasks. 10 ml of gallic acid reference solution (mass concentrations of 0.0692 mg / ml, 0.0855 mg / ml, and 0.1028 mg / ml, respectively) was accurately added to each flask. The samples were prepared according to the test sample preparation method. The recovery rate was calculated using the accompanying sample content. The recovery rate results are shown in Table 37 below. The results showed that the average recovery rate of gallic acid was 99.9%, with an RSD of 1.5%, indicating good accuracy of the method.

[0310] Table 37 Results of Gallic Acid Accuracy Test

[0311]

[0312] 4.5 Durability Test

[0313] 4.5.1 Solution stability

[0314] The same sample solution was injected at 0, 2, 4, 6, 8, 14, 18, and 24 hours to determine the peak area and assess the stability of the solution. The results are shown in Table 38 below. The experimental results indicate that the sample solution is basically stable within 24 hours and meets the requirements for the assay.

[0315] Table 38 Results of Solution Stability Tests

[0316]

[0317]

[0318] 4.5.2 Durability Test at Different Flow Rates

[0319] Accurately weigh approximately 0.1 g of *Gynostemma pentaphyllum* granules (1903001W) and process them according to the test sample preparation method. Chromatographic conditions were adjusted only by changing the elution flow rate. Tests were conducted at different flow rates of 0.9 mL / min, 1.0 mL / min, and 1.1 mL / min. The peak area of ​​gallic acid was measured, and its content was calculated. The results are shown in Table 39 below.

[0320] Table 39 Results of measurements at different flow velocities

[0321]

[0322] The results showed that the content of *Gynostemma pentaphyllum* formulation particles varied little with different flow rates, indicating that the method is robust to small fluctuations in flow rate.

[0323] 4.5.3 Durability test at different column temperatures

[0324] Take approximately 0.1g of the *Gynostemma pentaphyllum* formula granules, accurately weigh them, and process them according to the preparation method of the test sample. The chromatographic conditions were changed only by the column temperature. The test was conducted at different column temperatures of 28℃, 30℃, and 32℃. The peak area of ​​gallic acid was measured, and the content was calculated. The results are shown in Table 40 below.

[0325] Table 40 Results of tests at different column temperatures

[0326]

[0327] The results show that small fluctuations in column temperature have little effect on the content of the detected Gangren formulation particles, indicating that the method is robust to small fluctuations in column temperature.

[0328] The above methodological results indicate that this method meets the requirements for content determination and can be used to determine the gallic acid content in Gangren formula granules.

[0329] Based on the above research results, the optimal solution is determined to be:

[0330] Preparation of the test solution: Weigh approximately 0.1 g of the powdered preparation of *Gynostemma pentaphyllum*, place it in a stoppered conical flask, accurately add 20 mL of 50% methanol, stopper tightly, weigh, sonicate (power 250 W, frequency 40 kHz) for 30 minutes, remove, cool, weigh again, replenish the lost weight with 50% methanol, shake well, filter, and collect the filtrate to obtain the test solution;

[0331] Preparation of reference solution: Take 1g of *Rhodiola rosea* reference material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 1h, filter, evaporate the filtrate to dryness, add 10ml of 50% methanol to the residue, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, filter, and take the filtrate as the reference solution for the reference material.

[0332] Preparation of reference solutions: Accurately weigh appropriate amounts of gallic acid reference standard, gentic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside reference standard, ellagic acid reference standard, 3,3′,4-tri-O-methylellagic acid 4′-glucoside reference standard and 3′-O-methyl-3,4-O,O-methyleneellagic acid reference standard, and add 50% methanol to prepare a solution containing 50 μg of each per ml, which is used as the reference solution.

[0333] High performance liquid chromatography (HPLC) analysis conditions

[0334] The column was packed with octadecylsilane-bonded silica gel (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); acetonitrile was used as mobile phase A, and 0.5 vol% formic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 12; the flow rate was 1.0 mL / min, the column temperature was 30 °C, and the detection wavelength was 254 nm. The theoretical plate number, calculated based on gallic acid, should be no less than 3000.

[0335] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0336] The above experiments show that the method for constructing characteristic spectra of *Rhizoma Cynanchi* and its pharmaceutical preparations provided by this invention not only shortens the detection time but also significantly improves the separation effect of multiple active ingredients, resulting in more characteristic peaks in the characteristic spectra, greatly enriching the spectral information. It achieves effective separation of characteristic peaks including gallic acid, gentic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside, ellagic acid, 3,3′,4-tri-O-methylellagic acid 4′-glucoside, and 3′-O-methyl-3,4-O,O-methyleneellagic acid. The obtained characteristic spectra are highly distinctive, fully reflecting the integrity and characteristics of *Rhizoma Cynanchi* pharmaceutical preparations, with stable baselines and good peak shapes. The separation of each component is good, and the construction method has high precision, stability, and repeatability. Therefore, it can comprehensively and rapidly detect the active ingredients and their content in *Rhizoma Cynanchi* and its pharmaceutical preparations. The method is simple and facilitates comprehensive quality control of *Rhizoma Cynanchi* and its pharmaceutical preparations.

[0337] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic spectrum of *Gynostemma pentaphyllum* formulation particles, characterized in that, Includes the following steps: Preparation of the test solution; the method for preparing the test solution is as follows: weigh the test sample, add solvent to extract, obtain the extract, separate the solid and liquid, and take the liquid, which is the test solution; the solvent is selected from methanol or 30-50% methanol aqueous solution; the construction method further includes the step of preparing the reference solution by adding solvent to the reference standard using gallic acid, gentic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside, ellagic acid, 3,3′,4-tri-O-methylellagic acid 4′-glucoside and 3′-O-methyl-3,4-O,O-methyleneellagic acid as reference standards; The test solution and reference solution were detected by high performance liquid chromatography (HPLC). The chromatographic conditions included using octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and formic acid solution as mobile phase B, with gradient elution performed according to the following procedure: For 0-5 min, the volume ratio of mobile phase A to mobile phase B was 2% : 98%. After 5-8 minutes, the volume ratio of mobile phase A to mobile phase B changed from 2%:98% to 5%:95%. Over 8-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 5%:95% to 17%:83%. The volume ratio of mobile phase A to mobile phase B was 17%:83% → 25%:75% for 30-70 min; the concentration of formic acid solution was 0.1-1 vol%; and the detection wavelength was 254 nm.

2. The construction method according to claim 1, characterized in that, The chromatographic conditions also satisfy any one or more of the following (1)-(4): (1) A chromatographic column with a specification of 250mm×4.6mm and a particle size of 5μm was used; (2) The injection volume of the test solution is 5-20 µL; (3) The column temperature is 20-35℃; (4) The flow rate of the eluent is 0.5-1.5 mL / min.

3. The construction method according to claim 1 or 2, characterized in that, The method for preparing the test solution also satisfies any one or more of the following AD: A. The ratio of the mass of the test sample to the volume of the solvent is 0.05-0.2:10-30; the relationship between mass and volume is g / mL; B. The extraction method is either reflux extraction or ultrasonic extraction; C. Extraction time is ≥10 min; D. The solid-liquid separation is selected from centrifugation or membrane filtration.

4. The construction method according to claim 3, characterized in that, The extraction time is 20-40 minutes.

5. The construction method according to claim 1 or 2, characterized in that, In the reference solution, the solvent is selected from at least one of methanol, ethanol, and water.

6. The construction method according to claim 5, characterized in that, 1 mL of the reference solution contains 0.005–200 µg of each reference standard.

7. The construction method according to claim 6, characterized in that, Each 1 mL of the reference solution contains 50-100 µg of each reference standard.

8. The construction method according to claim 1 or 2, characterized in that, The characteristic spectrum of the Gangren formula granules has 7 common characteristic peaks, including peak 1, which is gallic acid; peak 3, which is gentic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside; peak 4, which is ellagic acid; peak 6, which is 3′-O-methyl-3,4-O,O-methylene ellagic acid; and peak 7, which is the characteristic peak corresponding to 3,3′,4-tri-O-methylellagic acid 4′-glucopyranoside.

9. The construction method according to claim 8, characterized in that, Taking peak 4 as the S peak, the relative retention times of other characteristic peaks relative to peak 4 are within ±10% of the specified value. The specified values ​​for each characteristic peak are: Peak 1: 0.25, Peak 2: 0.43, Peak 3: 0.68, Peak 4: 1.0, Peak 5: 1.17, Peak 6: 1.39, Peak 7: 1.

48.

10. A method for testing the content of *Gynostemma pentaphyllum* formulation granules, characterized in that, Includes the following steps: Preparation of the test solution: The test solution is prepared by weighing the test sample, adding solvent to extract the sample, obtaining the extract, separating the solid and liquid components, and taking the liquid, which is the test solution; the solvent is selected from methanol or 30-50% methanol aqueous solution; A reference solution was prepared by adding gallic acid, gentic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside, ellagic acid, 3,3′,4-tri-O-methylellagic acid 4′-glucopyranoside and 3′-O-methyl-3,4-O,O-methyleneellagic acid to a solvent; High-performance liquid chromatography (HPLC) was used to detect the test solution and the reference solution. The chromatographic conditions included using octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and formic acid solution as mobile phase B, with gradient elution performed under the following conditions: For 0-5 min, the volume ratio of mobile phase A to mobile phase B was 2% : 98%. After 5-8 minutes, the volume ratio of mobile phase A to mobile phase B changed from 2%:98% to 5%:95%. Over 8-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 5%:95% to 17%:83%. The volume ratio of mobile phase A to mobile phase B was 17%:83% → 25%:75% for 30-70 min; the concentration of formic acid solution was 0.1-1 vol%; and the detection wavelength was 254 nm.

11. The method for testing the content of *Gynostemma pentaphyllum* formulation granules according to claim 10, characterized in that, A chromatographic column with dimensions of 250 mm × 4.6 mm and a diameter of 5 μm was used; and / or the injection volume of the test solution was 5-20 µL; the column temperature was 20-35 °C, and the flow rate was 0.5-1.0 mL / min.

12. The method for testing the content of *Gynostemma pentaphyllum* formulation granules according to claim 10 or 11, characterized in that, The method for preparing the test solution also satisfies any one or more of the following AD: A. The ratio of the mass of the test sample to the volume of the solvent is 0.05-0.2:10-30; the relationship between mass and volume is g / mL; B. The extraction method is either reflux extraction or ultrasonic extraction; C. Extraction time is ≥10 min; D. The solid-liquid separation is selected from centrifugation or membrane filtration.

13. The method for testing the content of *Gynostemma pentaphyllum* formulation granules according to claim 12, characterized in that, The extraction time is 20-40 minutes.

14. The method for testing the content of *Gynostemma pentaphyllum* formulation granules according to claim 10 or 11, characterized in that, In the reference solution, the solvent is selected from at least one of methanol, ethanol, and water.

15. The method for testing the content of *Gynostemma pentaphyllum* formulation granules according to claim 10 or 11, characterized in that, 1 mL of the reference solution contains 0.005–200 µg of each reference standard.

16. The method for testing the content of *Gynostemma pentaphyllum* formulation granules according to claim 15, characterized in that, Each 1 mL of the reference solution contains 50-100 µg of each reference standard.

17. A method for quality testing of *Gynostemma pentaphyllum* formulation granules, characterized in that, This includes the step of comparing the characteristic spectrum of the product to be tested with the characteristic spectrum of the formulated granules of *Gynostemma pentaphyllum*. And / or, the step of determining the content of the *Gynostemma pentaphyllum* product to be tested using the content testing method of *Gynostemma pentaphyllum* formulation granules as described in any one of claims 10-16; the characteristic spectrum of the *Gynostemma pentaphyllum* product to be tested is constructed using the *Gynostemma pentaphyllum* product to be tested according to the construction method as described in any one of claims 1-9, and the reference characteristic spectrum of the *Gynostemma pentaphyllum* formulation granules is selected from any one of the following (1)-(3): (1) It has 7 common characteristic peaks. Peaks 1, 3, 4, 6, and 7 correspond to the retention times of reference peaks of gallic acid, gentic acid 5-O-(6′-O-galloyl)-β-D-glucopyranoside, ellagic acid, 3′-O-methyl-3,4-O,O-methylene ellagic acid, and 3,3′,4-tri-O-methylellagic acid 4′-glucopyranoside, respectively. The peak corresponding to the ellagic acid reference peak is designated as peak S1. The relative retention times of the other characteristic peaks with peak S1 are within ±10% of the specified values. The specified values ​​for each characteristic peak are as follows: peak 1: 0.25, peak 2: 0.43, peak 3: 0.68, peak 4: 0.68, peak 5: 0.68, peak 6: 0.68, peak 7: 0.68, peak 8: 0.68, peak 9: 0.68, peak 10 ... 1.0, Peak 5: 1.17, Peak 6: 1.39, Peak 7: 1.48; (2) Characteristic chromatograms of *Gynostemma pentaphyllum* formulation particles obtained by using a single batch or multiple batches of *Gynostemma pentaphyllum* or its formulation according to any one of the construction methods described in claims 1-9; (3) Using multiple batches of Gangren formula granules, the characteristic spectrum obtained by any of the construction methods described in claims 1-9 is used to prepare a control characteristic spectrum by the average value or median method.

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