Establishment of HPLC characteristic chromatogram of snakegourd seed, root, standard decoction and dispensing granules

Abstract

The application provides a kind of HPLC characteristic chromatogram establishment method of snake six valley medicinal materials, decoction pieces, standard decoction and its formula granules, comprising the following steps: S 1) snake six valley raw materials are extracted using solvent to obtain the test solution;The snake six valley raw material is snake six valley medicinal materials, decoction pieces, standard decoction or formula granules;S2) the test solution is detected by high performance liquid chromatography, and the HPLC characteristic chromatogram of snake six valley raw material is obtained.The HPLC characteristic chromatogram method of snake six valley medicinal materials, decoction pieces, standard decoction and formula granules constructed in the application is stable, reliable, and can control the quality of snake six valley medicinal materials, decoction pieces, standard decoction and formula granules.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and particularly to a method for establishing HPLC characteristic fingerprints of Pinelliae Rhizoma Pedatisectae drugs, cut pieces, standard decoctions and their formula granules. Background Art

[0002] Pinelliae Rhizoma Pedatisectae is the dried tuber of the Araceae plant Amorphophallus sinensis Belval or Amorphophallus rivieri Durieu. It is pungent, bitter, cold in nature and toxic. Pinelliae Rhizoma Pedatisectae has the effects of resolving phlegm and dissipating accumulation, detoxifying and resolving masses, promoting blood circulation and relieving pain. The standard decoction of Pinelliae Rhizoma Pedatisectae is the freeze-dried powder prepared by decocting the Pinelliae Rhizoma Pedatisectae medicinal materials after processing according to a fixed preparation process.

[0003] The characteristic fingerprint is a quality evaluation method that reflects the overall characteristics of the chemical components of traditional Chinese medicines, and can effectively detect and control the authenticity, quality consistency and stability of traditional Chinese medicines and their preparations. Therefore, establishing an HPLC fingerprint is of great significance for the identification of Pinelliae Rhizoma Pedatisectae and its preparations. Summary of the Invention

[0004] The technical problem solved by the present invention is to provide a method for establishing HPLC characteristic fingerprints of Pinelliae Rhizoma Pedatisectae medicinal materials, cut pieces, standard decoctions and formula granules. The HPLC characteristic fingerprint method of Pinelliae Rhizoma Pedatisectae medicinal materials, cut pieces, standard decoctions and formula granules constructed in this application is stable and reliable, and can control the quality of Pinelliae Rhizoma Pedatisectae medicinal materials, cut pieces, standard decoctions and formula granules.

[0005] In view of this, this application provides a method for establishing HPLC characteristic fingerprints of Pinelliae Rhizoma Pedatisectae medicinal materials, cut pieces, standard decoctions and their formula granules, including the following steps:

[0006] S1) Extract the Pinelliae Rhizoma Pedatisectae raw material with a solvent to obtain a test solution; the Pinelliae Rhizoma Pedatisectae raw material is Pinelliae Rhizoma Pedatisectae medicinal material, cut piece, standard decoction or formula granule;

[0007] S2) Detect the test solution by high performance liquid chromatography to obtain the HPLC characteristic fingerprint of the Pinelliae Rhizoma Pedatisectae raw material;

[0008] The conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column, the mobile phase A is methanol, the mobile phase B is water, and gradient elution is used.

[0009] Preferably, it also includes preparing a reference substance solution of the reference substance and a reference substance solution of the control medicinal material;

[0010] The preparation of the reference substance solution of the control medicinal material is specifically as follows: dissolve the control medicinal material of Pinelliae Rhizoma Pedatisectae with 30% methanol and perform ultrasonic treatment to obtain the reference substance solution of the control medicinal material;

[0011] The preparation of the reference solution specifically involves dissolving guanosine, uridine, and adenosine in 30% methanol to obtain the reference solution.

[0012] The reference solution of the reference standard and the reference medicinal material were determined by high performance liquid chromatography, and chromatograms of the reference standard and the reference medicinal material were obtained respectively.

[0013] The components of the HPLC characteristic chromatograms of the reference standard and reference medicinal materials were qualitatively determined based on the chromatograms of the reference standard and reference medicinal materials.

[0014] Preferably, in step S1), the solvent is 30% methanol, and the ratio of the snake grain raw material to the solvent is 1g:(20-30)ml.

[0015] Preferably, in step S1), the extraction method is ultrasonic extraction or reflux extraction, and the extraction time is 10-45 min.

[0016] Preferably, the gradient elution specifically comprises:

[0017] 0–5 min, Phase A: 0%, Phase B: 100%;

[0018] 5–6 min, Phase A: 0–3%, Phase B: 100–97%;

[0019] 6–10 min, Phase A: 3%, Phase B: 97%;

[0020] 10–15 min, Phase A: 3–5%, Phase B: 97–95%;

[0021] 15–17 min, Phase A: 5%, Phase B: 95%;

[0022] 17–19 min, Phase A: 5–78%, Phase B: 95–22%;

[0023] 19–24 min, Phase A: 78%, Phase B: 22%.

[0024] Preferably, the mobile phase flow rate is 0.3 ml / min, the detection wavelength is 260 nm, and the injection volume is 1 μL.

[0025] Preferably, the chromatographic column has a length of 100 nm, an inner diameter of 2.1 mm, a particle size of 1.6 μm, and a column temperature of 30 °C.

[0026] Preferably, the similarity of the HPLC characteristic chromatograms of Sheliugu medicinal materials, processed slices, standard decoctions and their formula granules is evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system. The HPLC standard characteristic chromatograms of Sheliugu medicinal materials, processed slices, standard decoctions and their formula granules are obtained, consisting of 6 characteristic peaks, where peak 1 is uridine peak, peak 4 (S1) is guanosine peak, and peak 5 (S2) is adenosine peak.

[0027] Preferably, in the characteristic spectrum of the Snake Six Valley Standard Decoction, guanosine is used as the reference peak S1 and adenosine is used as the reference peak S2. The relative retention times of each characteristic peak with respect to peaks S1 and S2 are calculated. The relative retention times are within ±10% of a specified value. The specified values ​​are 0.53 (peak 2), 0.61 (peak 3), and 1.12 (peak 6).

[0028] In the characteristic spectrum of Sheliugu formula granules, with guanosine as the reference peak S1 and adenosine as the reference peak S2, the relative retention times of each characteristic peak with respect to peaks S1 and S2 are calculated. The relative retention times are within ±10% of the specified values, which are 0.53 (peak 2), 0.61 (peak 3), and 1.12 (peak 6).

[0029] Preferably, in the characteristic spectrum of the snake gu medicinal material and decoction pieces, guanosine is used as the reference peak S1 and adenosine is used as the reference peak S2. The relative retention time of each characteristic peak with S1 and S2 peaks is calculated. The relative retention time is within ±10% of a specified value. The specified values ​​are 0.53 (peak 2), 0.61 (peak 3), and 1.12 (peak 6).

[0030] This application provides a method for establishing HPLC characteristic chromatograms of *Smilax china* medicinal materials, processed slices, standard decoctions, and their formulated granules. The method first extracts *Smilax china* raw materials using a solvent to obtain a test solution, and then uses high-performance liquid chromatography (HPLC) to detect the test solution, thereby obtaining the HPLC characteristic chromatogram of the *Smilax china* raw materials. The method provided in this application, using HPLC with methanol-water as the mobile phase and guanosine and adenosine as references, establishes HPLC characteristic chromatograms of *Smilax china* medicinal materials, processed slices, standard decoctions, and their formulated granules. This method features good repeatability, high accuracy, stability, and reliability, and can be used to control the quality of *Smilax china* medicinal materials, processed slices, standard decoctions, and their formulated granules. Attached Figure Description

[0031] Figure 1 This is the uridine spectrum in Example 1 of the present invention;

[0032] Figure 2 This is the adenosine spectrum in Example 1 of the present invention;

[0033] Figure 3 The spectrum of guanosine in Example 1 of this invention;

[0034] Figure 4 The chromatograms of the Snake Six Valley Standard Decoction in Example 1 of this invention at different wavelengths are shown.

[0035] Figure 5 Chromatograms of She Liu Gu standard decoction at different column temperatures;

[0036] Figure 6 Chromatograms of Sheliugu standard decoction at different flow rates;

[0037] Figure 7 Chromatograms of Sheliugu standard decoction under different extraction methods;

[0038] Figure 8 Chromatograms of different extraction solvents for the standard decoction of Sheliugu;

[0039] Figure 9 Chromatograms of Sheliugu standard decoction at different extraction times;

[0040] Figure 10 Chromatograms of Sheliugu standard decoction with different amounts of extraction solvent added;

[0041] Figure 11 Chromatographic peak identification chromatogram for Sheliugu standard decoction;

[0042] Figure 12 Chromatograms of the standard decoction of Snake Six Valley on different instruments;

[0043] Figure 13 Chromatograms of Sheliugu standard decoction on different chromatographic columns;

[0044] Figure 14 Chromatograms of 16 batches of She Liu Gu standard decoction;

[0045] Figure 15 This is a reference spectrum for the characteristic spectrum of the She Liu Gu standard decoction;

[0046] Figure 16 Chromatograms of *Sherrylium sibiricum* medicinal material or processed *Sherrylium sibiricum* slices at different wavelengths;

[0047] Figure 17 Chromatograms of *Smilax china* medicinal material or *Smilax china* processed slices at different column temperatures;

[0048] Figure 18 Chromatograms of *Smilax china* medicinal material or *Smilax china* processed slices at different flow rates;

[0049] Figure 19 Chromatograms of *Smilax china* medicinal material or *Smilax china* processed slices extracted using different methods;

[0050] Figure 20 Chromatograms of *Smilax china* medicinal material and *Smilax china* processed slices under different extraction solvents;

[0051] Figure 21 Chromatograms of *Sherrylium sibiricum* medicinal material or *Sherrylium sibiricum* processed slices at different extraction times;

[0052] Figure 22 Chromatograms of *Sherryum tsao-ko* medicinal material or *Sherryum tsao-ko* processed slices with different solvent additions;

[0053] Figure 23 The chromatographic peak identification chromatogram for characteristic chromatograms of *Sherrylium sibiricum* medicinal material or processed *Sherrylium sibiricum* slices;

[0054] Figure 24 Chromatograms of *Smilax china* medicinal material or processed *Smilax china* slices on different instruments;

[0055] Figure 25 Chromatograms of *Smilax china* medicinal material and *Smilax china* processed slices under different chromatographic columns;

[0056] Figure 26 Verification images of characteristic spectra of 16 batches of Sheliugu medicinal materials;

[0057] Figure 27 A reference atlas for the characteristic atlas of *Snake Six Valley* medicinal materials;

[0058] Figure 28 Characteristic chromatograms of 16 batches of She Liu Gu medicinal slices;

[0059] Figure 29 A comparative atlas of the characteristic atlases of She Liu Gu medicinal slices;

[0060] Figure 30 The spectrum of the reference uridine;

[0061] Figure 31 The spectrum of uridine in the test sample;

[0062] Figure 32 The spectrum of the reference substance adenosine;

[0063] Figure 33 The spectrum of adenosine in the test sample;

[0064] Figure 34 The spectrum of the reference standard guanosine;

[0065] Figure 35 The spectrum of the test sample guanosine;

[0066] Figure 36 Chromatograms of different wavelengths for Sheliugu formula granules;

[0067] Figure 37 Chromatograms of She Liu Gu formula granules at different column temperatures;

[0068] Figure 38 Chromatograms of Sheliugu formula granules at different flow rates;

[0069] Figure 39 Chromatograms of Sheliugu formula granules extracted using different methods;

[0070] Figure 40 Chromatograms of Sheliugu formula granules under different extraction solvents;

[0071] Figure 41 Chromatograms of Sheliugu formula granules extracted at different times;

[0072] Figure 42 Chromatograms of Sheliugu formula granules with different solvent addition amounts;

[0073] Figure 43 The chromatographic peak identification chromatogram of the characteristic granules of Sheliugu formula;

[0074] Figure 44 Chromatograms of She Liu Gu formula granules under different instruments;

[0075] Figure 45 Chromatogram for column durability testing of Sheliugu formula granules;

[0076] Figure 46 Chromatograms of characteristic chromatograms for three batches of She Liu Gu formula granules;

[0077] Figure 47 A comparison spectrum of the characteristic chromatograms of Sheliugu formula granules;

[0078] Figure 48 The HPLC characteristic chromatogram of snake stagnation in Comparative Example 1;

[0079] Figure 49 The HPLC characteristic chromatogram of snake stagnation in Comparative Example 2;

[0080] Figure 50 The HPLC characteristic chromatogram of snake stagnation in Comparative Example 3;

[0081] Figure 51 The HPLC characteristic chromatogram of snake stagnation in Comparative Example 4 is shown. Detailed Implementation

[0082] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims of the present invention.

[0083] In view of the need for quality control of *Smilax china* medicinal materials, processed slices, standard decoctions, and their formulated granules in the prior art, this application provides a method for establishing HPLC characteristic spectra of *Smilax china* medicinal materials, processed slices, standard decoctions, and their formulated granules. This method can accurately and reliably detect the characteristic spectra of *Smilax china* and its preparations; it can effectively detect and control the authenticity, consistency, and stability of the quality of *Smilax china* and its preparations, providing a basis for effectively controlling and comprehensively evaluating the quality of *Smilax china* medicinal materials, processed slices, standard decoctions, and their formulated granules, ensuring the uniformity and stability of the quality of *Smilax china* and its preparations. Specifically, this invention discloses a method for establishing HPLC characteristic spectra of *Smilax china* medicinal materials, processed slices, standard decoctions, and their formulated granules, including the following steps:

[0084] S1) The raw material of *S. coli* is extracted with solvent to obtain the test solution; the raw material of *S. coli* is *S. coli* medicinal material, decoction pieces, standard decoction or formula granules;

[0085] S2) The test solution was detected by high performance liquid chromatography to obtain the HPLC characteristic spectrum of the snake glutinous rice raw material;

[0086] The conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column, the mobile phase A is methanol, the mobile phase B is water, and gradient elution is used.

[0087] In establishing the HPLC characteristic chromatograms of *Smilax china* medicinal materials, processed slices, standard decoctions, and their formulated granules, this application first extracts the *Smilax china* raw material using a solvent to obtain the test solution; the *Smilax china* raw material is *Smilax china* medicinal materials, processed slices, standard decoctions, or formulated granules. The solvent is 30% methanol. Specifically, for *Smilax china* standard decoctions and *Smilax china* formulated granules, the solvent is selected from 30% methanol; for *Smilax china* medicinal materials and processed slices, the solvent is selected from 30% methanol. The above extraction solvents produce characteristic peaks with good shape and moderate resolution.

[0088] The extraction method is ultrasonic extraction or reflux extraction. Considering the speed and convenience of ultrasonic extraction, ultrasonic extraction is chosen. The ratio of the raw material of *Smilax china* to the solvent is 1g:(20-30)ml, more specifically, the ratio is 1g:25ml. The characteristic chromatographic chromatographic peaks show good separation, moderate peak size, and a relatively stable baseline. The extraction time is 10-45min, specifically, the extraction time for the standard *Smilax china* decoction and the *Smilax china* formula granules is 30min, and the extraction time for the *Smilax china* medicinal materials and the *Smilax china* slices is 30min.

[0089] In this process, a reference solution for photographs and a reference solution for control medicinal materials were also prepared. Specifically, the preparation of the reference solution for control medicinal materials was as follows: the reference medicinal material of *Smilax china* was dissolved in 30% methanol and ultrasonically treated to obtain the reference solution for control medicinal materials.

[0090] The preparation of the reference solution specifically involves dissolving guanosine, uridine, and adenosine in 30% methanol to obtain the reference solution.

[0091] In the above reference solutions for medicinal materials, the ratio of *Smilax china* reference material to methanol was 1 g: 25 ml. In the above reference solutions for standard substances, the ratio of guanosine to 30% methanol was 10 μg: 1 ml, the ratio of uridine to 30% methanol was 10 μg: 1 ml, and the ratio of adenosine to 30% methanol was 10 μg: 1 ml.

[0092] This application then uses high-performance liquid chromatography (HPLC) to detect the test solution and obtain the HPLC characteristic chromatogram of the raw material of *Smilax china*. Specifically, when the raw material of *Smilax china* is *Smilax china* medicinal material, the HPLC characteristic chromatogram of *Smilax china* medicinal material can be obtained; when the raw material of *Smilax china* is *Smilax china* sliced ​​food, the HPLC characteristic chromatogram of *Smilax china* sliced ​​food can be obtained; when the raw material of *Smilax china* is *Smilax china* standard decoction, the HPLC characteristic chromatogram of *Smilax china* standard decoction can be obtained; when the raw material of *Smilax china* is *Smilax china* formula granules, the HPLC characteristic chromatogram of *Smilax china* formula granules can be obtained.

[0093] In this application, the conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column, the mobile phase A is methanol, the mobile phase B is water, and gradient elution is used.

[0094] Specifically, the gradient elution is as follows:

[0095] 0–5 min, Phase A: 0%, Phase B: 100%;

[0096] 5–6 min, Phase A: 0–3%, Phase B: 100–97%;

[0097] 6–10 min, Phase A: 3%, Phase B: 97%;

[0098] 10–15 min, Phase A: 3–5%, Phase B: 97–95%;

[0099] 15–17 min, Phase A: 5%, Phase B: 95%;

[0100] 17–19 min, Phase A: 5–78%, Phase B: 95–22%;

[0101] 19–24 min, Phase A: 78%, Phase B: 22%.

[0102] Under the gradient elution conditions described above, the chromatogram baseline is relatively stable, the resolution is good, and the peak shape is good.

[0103] The chromatographic column has a length of 100 nm, an inner diameter of 2.1 mm, a particle size of 1.6 μm, and a column temperature of 30 °C. At a column temperature of 30 °C, the chromatographic peak shape and resolution are better.

[0104] In high performance liquid chromatography, the mobile phase flow rate is 0.3 ml / min, the detection wavelength is 260 nm, and the injection volume is 1 μL. At the above flow rates, the chromatographic peak shape is good and the resolution is moderate. Similarly, at the above detection wavelength, the overall chromatographic peak shape is good, the proportion of each peak is moderate, and the chromatographic baseline is more stable.

[0105] After the above conditions were determined, while the snake gu and its preparations were being measured by high performance liquid chromatography, the reference solution of the reference standard and the reference medicinal material were also measured by high performance liquid chromatography to obtain chromatograms of the reference standard and the reference medicinal material, respectively.

[0106] The components of the HPLC characteristic chromatograms of the reference standard and reference medicinal materials were qualitatively determined based on the chromatograms of the reference standard and reference medicinal materials.

[0107] Based on the above methods, this application has determined the HPLC characteristic chromatographic method for Sheliugu standard decoction:

[0108] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition):

[0109] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in Table 10; flow rate 0.3 ml / min, column temperature 30 °C; detection wavelength 260 nm; and theoretical plate number calculated based on the adenosine peak should be no less than 5000.

[0110] Preparation of reference solution: Take 1g of snake stagnation reference material, add 25ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0111] Take an appropriate amount of adenosine reference standard, weigh it accurately, and add 30% methanol to prepare a solution containing 10 μg per ml, which will be used as the reference solution.

[0112] Preparation of the test solution: Take about 1.0 g of the powder, accurately weigh it, place it in a stoppered conical flask, add 25 ml of 30% methanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 30% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0113] The determination method involves precisely pipetting 1 μL each of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0114] The chromatogram of the test sample should show 6 characteristic peaks, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1, 4, and 5 should correspond to the retention times of the reference peaks, respectively. The peak corresponding to the guanosine reference is peak S1; calculate the relative retention times of peaks 1-3 with peak S1. The peak corresponding to the adenosine reference is peak S2; calculate the relative retention time of peak 6 with peak S2. The relative retention times should be within ±10% of the specified value. (Peak 2: 0.53, Peak 3: 0.61, Peak 6: 1.12). The characteristic chromatogram of the Sheliugu standard decoction is shown below. Figure 15 As shown in the figure, peak 1: uridine; peak 4 (S1): guanosine; peak 5 (S2): adenosine.

[0115] HPLC characteristic chromatographic method for *Smilax china* medicinal materials and processed *Smilax china* slices:

[0116] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in Table 21; flow rate 0.3 ml / min, column temperature 30 °C; detection wavelength 260 nm. The theoretical plate number, calculated based on the adenosine peak, should be no less than 5000.

[0117] Preparation of reference solution: Take 1g of snake stagnation reference material, add 25ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0118] Take appropriate amounts of guanosine, uridine, and adenosine reference standards, add 30% methanol to prepare a solution containing 10 μg of each per ml, and use it as a reference solution.

[0119] Preparation of the test solution: Take about 1.0 g of the powder (passed through a No. 3 sieve), place it in an Erlenmeyer flask, add 25 ml of 30% methanol, seal tightly, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0120] The determination method involves precisely pipetting 1 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0121] The chromatogram of the test sample should show 6 characteristic peaks, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1, 4, and 5 should correspond to the retention times of the reference peaks, respectively. The peak corresponding to the guanosine reference is designated as peak S1. Calculate the relative retention times of peaks 1-3 with peak S1. The peak corresponding to the adenosine reference is designated as peak S2. Calculate the relative retention time of peak 6 with peak S2. The relative retention times should be within ±10% of the specified value. (Peak 2: 0.53, Peak 3: 0.61, Peak 6: 1.12). The characteristic chromatogram of *Smilax china* medicinal material is shown below. Figure 27 As shown, the comparative characteristic spectrum of She Liu Gu medicinal slices is as follows: Figure 29 As shown in the figure, peak 1: uridine; peak 4 (S1): guanosine; peak 5 (S2): adenosine.

[0122] HPLC Characteristic Chromatography Method for Sheliugu Formula Granules:

[0123] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition):

[0124] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in Table 30; flow rate 0.3 ml / min, column temperature 30 °C; detection wavelength 260 nm. The theoretical plate number, calculated based on the adenosine peak, should be no less than 5000.

[0125] Preparation of reference solution: Take 1g of snake stagnation reference material, add 25ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0126] Take appropriate amounts of guanosine, uridine, and adenosine reference standards, add 30% methanol to prepare a solution containing 10 μg of each per ml, and use it as a reference solution.

[0127] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, accurately weigh about 1.0 g, place it in a stoppered conical flask, add 25 ml of 30% methanol, stopper tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, replenish the lost weight with 30% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0128] The determination method involves precisely pipetting 1 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0129] The chromatogram of the test sample should show 6 characteristic peaks, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1, 4, and 5 should correspond to the retention times of the reference peaks, respectively. The peak corresponding to the guanosine reference is peak S1; calculate the relative retention times of peaks 1-3 with peak S1. The peak corresponding to the adenosine reference is peak S2; calculate the relative retention time of peak 6 with peak S2. The relative retention times should be within ±10% of the specified value. (Peak 2: 0.53, Peak 3: 0.61, Peak 6: 1.12). The characteristic chromatogram of the Sheliugu formula granules is shown below. Figure 47 As shown, the characteristic spectrum of Sheliugu formula granules is as follows: Figure 47 As shown in the figure, peak 1: uridine; peak 4 (S1): guanosine; peak 5 (S2): adenosine.

[0130] The HPLC characteristic chromatogram establishment method provided by this invention controls the material groups of snake-eating herbs, decoction pieces, standard decoctions and their formulation particles by fingerprint chromatogram under liquid chromatography conditions, and uses guanosine and adenosine to locate the fingerprint chromatogram; it can greatly reduce the detection cost and achieve qualitative detection.

[0131] The similarity of HPLC characteristic chromatograms of Sheliugu medicinal materials, processed slices, standard decoctions and their formulation granules was evaluated using a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. The HPLC standard characteristic chromatograms of Sheliugu medicinal materials, processed slices, standard decoctions and their formulation granules were obtained, consisting of 6 characteristic peaks, of which peak 5: guanosine peak, peak 6(S): adenosine peak, and peak 7: guanosine peak.

[0132] In the characteristic spectrum of the Snake Six Valley Standard Decoction, guanosine was used as the reference peak S1 and adenosine was used as the reference peak S2. The relative retention times of each characteristic peak with respect to peaks S1 and S2 were calculated. The relative retention times were within ±10% of the specified values, which were 0.53 (peak 2), 0.61 (peak 3), and 1.12 (peak 6).

[0133] In the characteristic spectrum of Sheliugu formula granules, with guanosine as the reference peak S1 and adenosine as the reference peak S2, the relative retention times of each characteristic peak with S1 and S2 peaks are calculated. The relative retention times are within ±10% of the specified values, which are 0.53 (peak 2), 0.61 (peak 3), and 1.12 (peak 6).

[0134] In the characteristic spectrum of the medicinal materials and decoction pieces of Sheliugu, guanosine is used as the reference peak S1 and adenosine is used as the reference peak S2. The relative retention times of each characteristic peak with S1 and S2 are calculated. The relative retention times are within ±10% of the specified values, which are 0.53 (peak 2), 0.61 (peak 3), and 1.12 (peak 6).

[0135] Quality judgment criteria: Take samples of Sheliugu medicinal materials, processed slices, standard decoctions and their formula granules, and operate according to the same method as above to obtain the characteristic chromatograms of Sheliugu medicinal materials, processed slices, standard decoctions and their formula granules. Use the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" of the National Pharmacopoeia Commission to analyze the standard characteristic chromatograms of Sheliugu medicinal materials, processed slices, standard decoctions and their formula granules and the sample characteristic chromatograms. The similarity is greater than 0.90.

[0136] The method provided by this invention can effectively monitor the quality of different batches of snake-eating herbs, decoction pieces, standard decoctions and their formula granules, ensuring their quality stability. The method has the characteristics of high precision and good reproducibility, which is conducive to comprehensive monitoring of product quality.

[0137] The characteristic chromatograms of snake-eating herbs, processed slices, standard decoctions and their formulation granules established in this invention use guanosine, uridine and adenosine as references, and focus on the order of each characteristic peak and its correlation with the herbs and intermediate products. This method can comprehensively evaluate the overall quality characteristics of the products and is scientific and reliable.

[0138] The newly developed characteristic chromatographic method of this invention has a simple and easy-to-operate sample preparation method and a relatively large number of identifying characteristic peaks; it can accurately and reliably detect the characteristic chromatographic features of She Liu Gu and its preparations; it can effectively detect and control the authenticity, quality consistency and stability of She Liu Gu and its preparations; it provides a basis for effectively controlling and comprehensively evaluating the quality of She Liu Gu standard decoction, and ensures the uniformity and stability of the quality of She Liu Gu and its standard decoction.

[0139] To further understand the present invention, the following detailed description, in conjunction with embodiments, illustrates the method for establishing HPLC characteristic spectra of Sheliugu medicinal materials, processed slices, standard decoctions, and their formulation granules provided by the present invention. The scope of protection of the present invention is not limited by the following embodiments.

[0140] Example 1: Method for Establishing the HPLC Characteristic Chromatography of She Liu Gu Standard Decoction

[0141] 1.1 Experimental Instruments and Materials

[0142] High performance liquid chromatographs: Agilent ultra-high performance liquid chromatographs, Thermo Fisher ultra-high performance liquid chromatographs;

[0143] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0144] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0145] Ultrasonic cleaner: KQ5200DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0146] Chromatographic column: C18;

[0147] Methanol was of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0148] Adenosine (China National Institutes for Food and Drug Control, batch number: 110879-201703, content calculated as 99.7%);

[0149] Uric acid (China National Institutes for Food and Drug Control, batch number: 110887-202305, content calculated as 99.6%);

[0150] Guanosine (China National Institutes for Food and Drug Control, batch number: 111977-202202, content calculated as 88.6%);

[0151] Snake-six-grain reference medicinal material (Shandong Bokang Fine Chemical Co., Ltd., batch number: 380184-202306);

[0152] Sheliugu Standard Decoction Freeze-Dried Powder (Prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., Batch Nos.: SLG-BT-230801, SLG-BT-230802, SLG-BT-230803, SLG-BT-230804, SLG-BT-230805, SLG-BT-230806, SLG-BT-230807, SLG-BT-230808, SLG-BT-230809, SLG-BT-230810, SLG-BT-230811, SLG-BT-230812, SLG-BT-230813, SLG-BT-230814, SLG-BT-230815, SLG-BT-230816).

[0153] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in Table 1; flow rate 0.3 ml / min, column temperature 30 °C; detection wavelength 260 nm; theoretical plate number calculated based on adenosine peak should not be less than 5000;

[0154] Table 1 Initial data of gradient elution of Sheliugu standard decoction

[0155] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 0 100 5～6 0→3 100→97 6～10 3 97 10～15 3→5 97→95 15～17 5 95 17～19 5→78 95→22 19～24 78 22

[0156] Preparation of reference solution: Take 1g of snake stagnation reference material, add 25ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0157] Take appropriate amounts of guanosine, uridine, and adenosine reference standards, weigh them accurately, and add 30% methanol to prepare a solution containing 10 μg per ml, which will serve as the reference solution.

[0158] Preparation of the test solution: Take about 1.0 g of the powder, accurately weigh it, place it in a stoppered conical flask, add 25 ml of 30% methanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 30% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0159] The assay involves precisely pipetting 1 μL each of the reference solution and the test solution into the liquid chromatograph and measuring the concentration. 1.2 Chromatographic conditions and system suitability test

[0160] 1.2.1 Wavelength Selection

[0161] 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 at wavelengths of 220 nm, 240 nm, 260 nm, 280 nm, and 300 nm were extracted respectively; as follows Figures 1-4 As shown, Figure 1 This is a spectrum of uridine. Figure 2 This is the spectrum of adenosine. Figure 3 This is the spectrum of guanosine. Figure 4 The results show that the chromatograms of Sheliugu standard decoction at different wavelengths are obtained. The results indicate that the chromatographic peak information is greater and the chromatographic baseline is more stable at a detection wavelength of 260 nm. Therefore, the detection wavelength was determined to be 260 nm.

[0162] 1.2.2 Column Temperature Investigation

[0163] Based on the above-specified experimental conditions, the effects were investigated at column temperatures of 25℃, 30℃, and 35℃; for example... Figure 5 As shown, Figure 5 The results show that the chromatograms of Sheliugu standard decoction at different column temperatures are as follows: the results show that the chromatogram peaks are better and the separation is better at a column temperature of 30℃, so the column temperature of 30℃ is selected.

[0164] 1.2.3 Flow velocity investigation

[0165] Based on the above-established experimental conditions, the flow rates of 0.2 ml / min, 0.25 ml / min, and 0.3 ml / min were investigated respectively. Figure 6 As shown, Figure 6 The results show that the peak shape of the chromatogram of Sheliugu standard decoction is good and the resolution is moderate at a flow rate of 0.3 ml / min. Therefore, the flow rate is tentatively set at 0.3 ml / min.

[0166] In summary, the chromatographic conditions for the characteristic chromatogram of Sheliugu standard decoction are as follows: Octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol as mobile phase A, water as mobile phase B, gradient elution according to the specifications in Table 2; flow rate 0.3 ml / min, column temperature 30℃; detection wavelength 260 nm; theoretical plate number calculated based on the adenosine peak should not be less than 5000.

[0167] Table 2 Gradient elution data of Sheliugu standard decoction

[0168] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 0 100 5～6 0→3 100→97 6～10 3 97 10～15 3→5 97→95 15～17 5 95 17～19 5→78 95→22 19～24 78 22

[0169] 1.3 Preparation of the test solution

[0170] 1.3.1 Extraction Method

[0171] Take approximately 1.0 g of this product (batch number: SLG-BT-230801), place it in a stoppered conical flask, add 25 ml of 30% methanol, seal tightly, heat under reflux and then sonicate (600 W, 40 kHz) for 30 minutes each, cool, shake well, filter, and collect the filtrate; if... Figure 7 As shown, Figure 7 The chromatograms are shown for different extraction methods of the Sheliugu standard decoction. The results show that there is little difference in the effect of reflux and ultrasonic extraction on the test sample. Moreover, the ultrasonic method is fast and simple, so the ultrasonic extraction method was determined to be the test sample extraction method.

[0172] 1.3.2 Investigation of Extraction Solvents

[0173] Take approximately 1.0 g of this product (batch number: SLG-BT-230801), place it in a stoppered conical flask, add 25 ml each of water, 30% methanol, 70% methanol, methanol, and 70% ethanol, seal tightly, sonicate (600W, 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product; Figure 8 As shown, Figure 8 Chromatograms of different extraction solvents for Sheliugu standard decoction are shown. The results indicate that when the extraction solvent is 30% methanol, the characteristic peaks have good shapes and moderate separation. Considering all factors, methanol is the best extraction solvent.

[0174] 1.3.3 Examination of extraction time

[0175] Take approximately 1.0 g of this product (batch number: SLG-BT-230801), place it in a stoppered conical flask, add 25 ml of 30% methanol, seal tightly, and sonicate (600 W, 40 kHz) for 15 minutes, 30 minutes, and 45 minutes respectively. Cool, shake well, filter, and collect the filtrate; [The remaining text appears to be incomplete and possibly contains errors. A more accurate translation would require the full context.] Figure 9 As shown, Figure 9 The images show chromatograms of the Sheliugu standard decoction at different extraction times. The results indicate that extraction times of 15 minutes, 30 minutes, and 45 minutes yielded consistent results. Therefore, an extraction time of 30 minutes was selected for the test sample.

[0176] 1.3.4 Investigation on the amount of extraction solvent added

[0177] Take approximately 1.0 g of this product (batch number: SLG-BT-230801), place it in a stoppered conical flask, and add 25 ml, 50 ml, and 100 ml of 30% methanol respectively for testing. Seal tightly, sonicate (600 W, 40 kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product. Figure 10 As shown, Figure 10 The chromatograms of Sheliugu standard decoction with different extraction solvent additions are shown. The results show that when the extraction solvent addition is 25 ml, the peak shape and resolution of each chromatographic peak are good and the peak size is moderate. Therefore, the solvent volume is selected as 25 ml.

[0178] In summary, the preparation method of the test solution for the characteristic chromatogram of Sheliugu standard decoction is determined as follows: Take about 1.0g of this product, accurately weigh it, place it in an Erlenmeyer flask, add 25ml of 30% methanol, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0179] 1.4 Methodological Examination

[0180] 1.4.1 Chromatographic Peak Identification

[0181] Preparation of test solution: Prepare the test solution of Sheliugu standard decoction according to the experimental conditions proposed above.

[0182] Take 1g of snake-six-grain reference material, add 25ml of 30% methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution for the reference material.

[0183] Take appropriate amounts of uridine, guanosine, and adenosine reference standards and prepare a mixed solution containing 10 μg of each per 1 ml as the reference solution.

[0184] Preparation of negative control solution: Prepare negative control solution of snake-deficient Liugu standard decoction according to the experimental conditions proposed above;

[0185] Locating the characteristic peaks of the standard decoction of snake six grains, such as... Figure 11 As shown, Figure 11 The chromatographic peak identification chromatogram of the She Liu Gu standard decoction.

[0186] 1.4.2 Repeatability Test

[0187] Six portions of the Snake Six Valley Standard Decoction (batch number: SLG-BT-230801) were accurately weighed and prepared and measured according to the proposed experimental method, as shown in Table 3.

[0188] Table 3. Repeatability Tests - Relative Retention Time Data

[0189]

[0190] The results show that the relative retention times of each characteristic peak are consistent, with a relative retention time RSD of 0.01–0.03%; therefore, the method has good repeatability.

[0191] 1.4.3 Intermediate Precision Examination

[0192] 1.4.3.1 Investigation with different instruments

[0193] Based on the above-planned experimental conditions, the test solution was taken and measured using Agilent and Shimadzu high-performance liquid chromatographs, respectively; Figure 12 As shown in Table 4, Figure 12 Chromatograms of the standard decoction of Snake Six Valley on different instruments;

[0194] Table 4. Instrument Durability Assessment - Relative Retention Time Data

[0195]

[0196] The results showed that when the test samples were detected using the two instruments mentioned above, the RSD of the relative retention times of each characteristic peak was 2.73% to 7.95%.

[0197] 1.4.3.2 Investigations by different personnel and at different times

[0198] Based on the experimental conditions proposed above, different personnel (A, B) accurately weighed Sheliugu standard decoction (batch number: SLG-BT-230801) at different times (T1, T2) to prepare test samples and conduct determinations; as shown in Table 5;

[0199] Table 5. Personnel and Time Survey - Relative Retention Time Data Table

[0200]

[0201] The results showed that when different personnel measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 2.24% to 6.53%.

[0202] 1.4.4 Durability Assessment

[0203] 1.4.4.1 Column robustness test

[0204] Based on the above-planned experimental conditions, chromatographic columns of the same model but different batches were used respectively. C18 2.1*100mm, 1.6μm was analyzed and investigated; such as Figure 13 As shown in Table 6, Figure 13 Chromatograms of Sheliugu standard decoction on different chromatographic columns;

[0205] Table 6. Column Robustness Study - Relative Retention Time Data

[0206]

[0207] Depend on Figure 13 It can be seen that when the above three chromatographic columns are used to detect the sample, the RSD of the relative retention time of the characteristic peak is between 0.00% and 0.02%.

[0208] 1.4.5 Stability Assessment

[0209] Based on the above-planned experimental conditions, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h, respectively; see Table 7.

[0210] Table 7. Stability Study - Retention Time Data

[0211]

[0212] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.22% and 0.54%, and the sample solution was relatively stable within 24 hours.

[0213] In summary, the RSD of the retention time / relative retention time of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective.

[0214] 1.5 Determination of characteristic peaks and establishment of reference spectra

[0215] 1.5.1 Verification results of 16 batches of She Liu Gu standard decoction

[0216] Using this method, characteristic spectral analysis was performed on 16 batches of samples to calculate relative retention times; for example... Figure 14 As shown in Table 8, Figure 14The image shows the chromatograms of 16 batches of Sheliugu standard decoction. From bottom to top, the batch numbers are: SLG-BT-230801, SLG-BT-230802, SLG-BT-230803, SLG-BT-230804, SLG-BT-230805, SLG-BT-230806, SLG-BT-230807, SLG-BT-230808, SLG-BT-230809, SLG-BT-230810, SLG-BT-230811, SLG-BT-230812, SLG-BT-230813, SLG-BT-230814, SLG-BT-230815, and SLG-BT-230816. ；

[0217] Table 8. Relative retention time of 16 batches of Sheliugu standard decoction

[0218]

[0219] Based on the principles of stable relative retention times, detectability in all batches of samples, and relatively high peak values, six peaks with good repeatability were selected as characteristic peaks. The specifications are as follows: the chromatogram of the test sample should show six characteristic peaks, and their retention times should correspond to the six characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1, 4, and 5 should correspond to the retention times of the reference material peaks, respectively. The peak corresponding to the guanosine reference material is designated as peak S1, and the relative retention times of peaks 1-3 with peak S1 are calculated. The peak corresponding to the adenosine reference material is designated as peak S2, and the relative retention time of peak 6 with peak S2 is calculated. These relative retention times should be within ±10% of the specified values; 0.55 (peak 2), 0.61 (peak 3), and 1.12 (peak 6).

[0220] 1.5.2 Establishment of Limits for Relative Retention Time

[0221] Table 9 summarizes the methodological examination items and validation results.

[0222] Table 9 Summary of Methodological Results RSD (%) – Relative Retention Time Data Table

[0223] Methodology Peak 1 Peak 2 Peak 3 Peak 4 (S1) Peak 5 (S2) Peak 6 Repeatability 0.07 0.03 0.05 0.04 0.00 0.02 stability 0.34 0.22 0.35 0.54 0.33 0.31 Different people 6.53 2.91 3.69 0.00 0.00 2.24 Durability 0.04 0.02 0.03 0.01 0.00 0.01

[0224] The results show that the retention time or RSD value of the relative retention time of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective.

[0225] The final stipulation is that the chromatogram of the test sample should show 6 characteristic peaks, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1, 4, and 5 should correspond to the retention times of the reference peaks, respectively. The peak corresponding to the guanosine reference is designated as peak S1, and the peak corresponding to the adenosine reference is designated as peak S2. Using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), 16 batches of Sheliugu standard decoction were synthesized, and a reference chromatogram of the characteristic chromatogram of Sheliugu standard decoction was established. Figure 15 As shown.

[0226] 1.6 Determination of the Characteristic Spectrum of She Liu Gu Standard Decoction

[0227] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in Table 10; flow rate 0.3 ml / min, column temperature 30 °C; detection wavelength 260 nm; theoretical plate number calculated based on adenosine peak should not be less than 5000;

[0228] Table 10 Final Data of Gradient Elution of Sheliugu Standard Decoction

[0229] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 0 100 5～6 0→3 100→97 6～10 3 97 10～15 3→5 97→95 15～17 5 95 17～19 5→78 95→22 19～24 78 22

[0230] Preparation of reference solution: Take 1g of snake stagnation reference material, add 25ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0231] Take appropriate amounts of uridine, guanosine, and adenosine reference standards, weigh them accurately, and add 30% methanol to prepare a solution containing 10 μg per ml, which is used as the reference solution.

[0232] Preparation of the test solution: Take about 1.0 g of the powder, accurately weigh it, place it in a stoppered conical flask, add 25 ml of 30% methanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 30% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0233] The assay involves precisely pipetting 1 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0234] Example 2: Method for establishing HPLC characteristic chromatograms of *Smilax china* medicinal materials and processed medicinal slices

[0235] 2.1 Experimental Instruments and Materials

[0236] High performance liquid chromatographs: Agilent ultra-high performance liquid chromatographs, Thermo Fisher ultra-high performance liquid chromatographs;

[0237] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0238] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0239] Ultrasonic cleaner: KQ5200DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0240] Chromatographic column: C18;

[0241] 2.2 Reagents and reagents

[0242] Methanol was of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0243] Adenosine (China National Institutes for Food and Drug Control, batch number: 110879-201703, content calculated as 99.7%);

[0244] Uric acid (China National Institutes for Food and Drug Control, batch number: 110887-202305, content calculated as 99.6%);

[0245] Guanosine (China National Institutes for Food and Drug Control, batch number: 111977-202202, content calculated as 88.6%);

[0246] Snake-six-grain reference medicinal material (Shandong Bokang Fine Chemical Co., Ltd., batch number: 380184-202306);

[0247] Snake Valley Medicinal Herbs (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: XXLS2023071169, XXLS2023071170, XXLS2023071171, XXLS2023071172, XXLS2023071173, XXLS2023071174, XXLS2023071175, XXLS2023071176, XXLS2023071177, XXLS2023071178, XXLS2023071179, XXLS2023071180, XXLS2023071181, XXLS2023071182, XXLS2023071183, 010404-2308001).

[0248] 2.3 Chromatographic conditions

[0249] Determined by high performance liquid chromatography (General Chapter 0512, Part IV, Chinese Pharmacopoeia 2020 Edition).

[0250] Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol was used as mobile phase A and water as mobile phase B, with gradient elution performed according to the specifications in Table 11; the flow rate was 0.3 ml / min, the column temperature was 30 °C; the detection wavelength was 260 nm; the theoretical plate number, calculated based on the adenosine peak, should not be less than 5000.

[0251] Table 11 Initial data for gradient elution of *Smilax china* medicinal materials or processed *Smilax china* slices

[0252] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 0 100 5～6 0→3 100→97 6～10 3 97 10～15 3→5 97→95 15～17 5 95 17～19 5→78 95→22 19～24 78 22

[0253] Preparation of reference solution: Take 1g of snake stagnation reference material, add 25ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0254] Take appropriate amounts of guanosine, uridine, and adenosine reference standards, add 30% methanol to prepare a solution containing 10 μg of each per ml, and use it as a reference solution.

[0255] Preparation of the test solution: Place about 1.0 g of Sheliugu medicinal material or Sheliugu powder (passed through a No. 3 sieve) in an Erlenmeyer flask, add 25 ml of 30% methanol, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0256] For the determination method, accurately pipette 1 μl of each of the above solutions, inject it into the liquid chromatograph, and measure it to obtain the result.

[0257] 2.4 Chromatographic conditions and system suitability test

[0258] 2.4.1 Wavelength Selection

[0259] Based on the above-planned experimental conditions, a diode array detector was used to perform full-band scanning of adenosine and the test solution, and chromatograms of the test solution at wavelengths of 220 nm, 240 nm, 260 nm, 280 nm, and 300 nm were extracted, as shown below. Figure 16 As shown, Figure 16 The results show that the peak shape is better overall and the proportion of each peak is moderate when the detection wavelength is 260 nm. Therefore, the detection wavelength is determined to be 260 nm.

[0260] 2.4.2 Column Temperature Investigation

[0261] Based on the above-specified experimental conditions, investigations were conducted at column temperatures of 25℃, 30℃, and 35℃, respectively. Figure 17 As shown, Figure 17 The chromatograms of *Symplocos edulis* medicinal materials or *Symplocos edulis* slices at different column temperatures are shown. The results indicate that the chromatogram peaks are better and the separation is better at a column temperature of 30℃. Therefore, a column temperature of 30℃ is selected.

[0262] 2.4.3 Flow velocity investigation

[0263] Based on the above-established experimental conditions, the flow rates of 0.0 ml / min, 0.25 ml / min, and 0.3 ml / min were investigated respectively. Figure 18 As shown, Figure 18 The chromatograms of *Sherrylium sibiricum* medicinal material or *Sherrylium sibiricum* slices at different flow rates are shown. The results indicate that the peak shape of the chromatogram is better and the resolution is moderate at a flow rate of 0.3 ml / min. Therefore, the flow rate is tentatively set at 0.3 ml / min.

[0264] In summary, the chromatographic conditions for the characteristic chromatograms of *Smilax china* medicinal material or *Smilax china* processed slices are as follows: Octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol as mobile phase A, water as mobile phase B, gradient elution according to the specifications in Table 12; flow rate 0.3 ml / min, column temperature 30℃; detection wavelength 260 nm; theoretical plate number calculated based on the adenosine peak should not be less than 5000.

[0265] Table 12 Gradient elution data of *Smilax china* medicinal materials or processed *Smilax china* slices

[0266] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 0 100 5～6 0→3 100→97 6～10 3 97 10～15 3→5 97→95 15～17 5 95 17～19 5→78 95→22 19～24 78 22

[0267] 2.5 Preparation of the test solution

[0268] 2.5.1 Examination of Extraction Methods

[0269] Take approximately 1.0 g of the powder (passed through a No. 3 sieve) (batch number: 010404-2308001), place it in an Erlenmeyer flask, add 25 ml of 30% methanol, seal tightly, heat under reflux and then sonicate (600 W power, 40 kHz frequency) for 30 minutes each, cool, shake well, filter, and collect the filtrate. The result is as follows. Figure 19 As shown, Figure 19 The chromatograms of *Smilax china* medicinal material or *Smilax china* processed slices under different extraction methods are shown. The results indicate that there is little difference in the effectiveness of reflux and ultrasonic extraction of the test sample, and the ultrasonic method is rapid and simple. Therefore, ultrasonic extraction was determined as the extraction method for the test sample.

[0270] 2.5.2 Investigation of Extraction Solvents

[0271] Take approximately 1.0 g of the powder (passed through a No. 3 sieve) (batch number: 010404-2308001), place it in an Erlenmeyer flask, and add 25 ml each of water, 30% methanol, 70% methanol, methanol, and 70% ethanol. Seal the flask tightly, sonicate (600W, 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate. This is the product. Figure 20 As shown, Figure 20 Chromatograms of *Sherrylium sibiricum* medicinal material and *Sherrylium sibiricum* slices under different extraction solvents were shown. The results showed that when the extraction solvent was 30% methanol, the peak shapes of each characteristic peak were good and the separation was moderate. Considering all factors, 30% methanol was selected as the extraction solvent.

[0272] 2.5.3 Examination of extraction time

[0273] Take approximately 1.0 g of the powder (passed through a No. 3 sieve) (batch number: 010404-2308001), place it in an Erlenmeyer flask, add 25 ml of 30% methanol, seal tightly, and sonicate (600 W, 40 kHz) for 15 minutes, 30 minutes, and 45 minutes respectively. Cool, shake well, filter, and collect the filtrate to obtain the product. Figure 21 As shown, Figure 21 The chromatograms of *Smilax china* medicinal material or *Smilax china* processed slices at different extraction times are shown. The results indicate that the extraction time for the test sample is consistent when the extraction time is 15 minutes, 30 minutes, and 45 minutes, respectively. Therefore, the extraction time for the test sample is selected as 30 minutes.

[0274] 2.5.3 Investigation of Solvent Addition Amount

[0275] Take approximately 1.0 g of the powder (passed through a No. 3 sieve) (batch number: 010404-2308001), place it in an Erlenmeyer flask, and add 25 ml, 50 ml, and 100 ml of 30% methanol respectively for testing. Seal the flasks tightly, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product. Figure 22 As shown, Figure 22 The figures show the chromatograms of *Sherryum tsao-ko* medicinal materials or *Sherryum tsao-ko* slices with different solvent additions. As can be seen from the figures, when the solvent volume of the test sample is 25 ml, the peak height and peak area of ​​the characteristic chromatogram are more suitable, and the baseline is more stable. Therefore, the solvent volume of the test sample is 25 ml.

[0276] In summary, the preparation method of the test solution of the characteristic spectrum of Sheliugu medicinal material is determined as follows: Take about 1.0g of the powder (passed through a No. 3 sieve), place it in an Erlenmeyer flask, add 25ml of 30% methanol, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0277] 2.6 Methodological Examination

[0278] 2.6.1 Chromatographic Peak Identification

[0279] Preparation of the test solution: Prepare the test solution of Sheliugu medicinal material according to the experimental conditions proposed above;

[0280] Preparation of reference solution for reference medicinal materials: Take 1g of Sheliugu reference medicinal material, add 25ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution for reference medicinal materials.

[0281] Preparation of reference solutions: Take appropriate amounts of guanosine, uridine, and adenosine reference standards, add 30% methanol to prepare a solution containing 10 μg of each per ml, which is used as the reference solution.

[0282] Preparation of negative control solution: Prepare a negative control solution for snake-deficient Liugu medicinal material according to the experimental conditions proposed above;

[0283] Locating the characteristic spectral peaks of *Sherrylula spp.* medicinal materials, such as... Figure 23 As shown, Figure 23 The chromatographic peak identification chromatograms of *Smilax china* medicinal material or processed *Smilax china* slices showed that peak 1 was uridine, peak 4 was guanosine, and peak 5 was adenosine. In the following methodological investigation, six characteristic peaks in the sample were investigated.

[0284] 2.6.2 Repeatability Test

[0285] Six test solutions were prepared according to the proposed experimental method. The relative retention times of each characteristic peak were measured and calculated, as shown in Table 13.

[0286] Table 13 Repeatability Test—Relative Retention Time Data of Characteristic Peaks

[0287]

[0288] The results showed that the relative retention time (RSD) of the six samples ranged from 0.03% to 0.04%, indicating that the method had good repeatability.

[0289] 2.6.3 Intermediate Precision Examination

[0290] 2.6.3.1 Investigations by different personnel and at different times

[0291] Based on the experimental conditions proposed above, two samples of this product (batch number: 010404-2308001) were precisely taken by different personnel (A, B) at different times (T1, T2) to prepare test samples and conduct determination; as shown in Table 14;

[0292] Table 14 Data on the ratio of relative retention time of characteristic peaks for different personnel and time periods.

[0293]

[0294] The results show that when different personnel, at different times, and using different instruments measure the same sample, the RSD of the relative retention time of each characteristic peak is 0.01% to 0.04%, indicating that the method has good intermediate precision.

[0295] 2.6.3.2 Investigation with different instruments

[0296] Based on the above-planned experimental conditions, two portions of this product (batch number: 010404-2308001) were taken and test solutions were prepared. These solutions were then analyzed using Agilent and Thermo Fisher high-performance liquid chromatographs, respectively. (See Table 15.) Figure 24 As shown, Figure 24 Chromatograms of *Smilax china* medicinal material or processed *Smilax china* slices on different instruments;

[0297] Table 15 Instrumental Examination—Relative Retention Time Data of Characteristic Peaks

[0298]

[0299]

[0300] The results showed that the peak elution times of the characteristic peaks differed significantly when the test samples were detected using the two instruments mentioned above.

[0301] 2.6.4 Durability Assessment

[0302] 2.6.4.1 Column robustness test

[0303] Based on the above-planned experimental conditions, chromatographic columns of the same model but different batches were used respectively. C18 2.1*100mm, 1.6μm were analyzed and investigated; as shown in Table 16. Figure 25 As shown, Figure 25 Chromatograms of *Smilax china* medicinal material and *Smilax china* processed slices under different chromatographic columns;

[0304] Table 16 Column Robustness Study—Relative Retention Time Data of Characteristic Peaks

[0305]

[0306] The results showed that the RSD of the relative retention time of the characteristic peak of the same type of column from different batches was 0.01% to 0.04%, indicating that the column had good robustness.

[0307] 2.6.4.2 Stability Assessment

[0308] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h and 24h, as shown in Table 17.

[0309] Table 17 24-hour stability study—retention time of characteristic peaks

[0310]

[0311] As shown in the table, the RSD of the retention time of the characteristic peak is 0.22% to 0.52%, and the sample solution is stable within 24 hours.

[0312] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The above six characteristic peaks will be included in subsequent investigations.

[0313] 2.7 Determination of characteristic peaks and establishment of reference spectra

[0314] 2.7.1 Verification results of 16 batches of Sheliugu medicinal materials

[0315] The characteristic spectra of 16 batches of this product were determined using the proposed method, and the relative retention times were calculated; Figure 26 As shown in Table 18, Figure 26 These are the characteristic chromatograms for 16 batches of Sheliugu medicinal materials; the batch numbers represented by each chromatogram are S1-S16: XXLS2023071169, XXLS2023071170, XXLS2023071171, XXLS2023071172, XXLS2023071173, XXLS2023071174, XXLS2023071175, XXLS2023071176, XXLS2023071177, XXLS2023071178, XXLS2023071179, XXLS2023071180, XXLS2023071181, XXLS2023071182, XXLS2023071183, 010404-2308001;

[0316] Table 18. Relative retention time data of 16 batches of Sheliugu medicinal materials

[0317]

[0318]

[0319] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peaks, a total of 6 peaks with good repeatability were selected as characteristic peaks.

[0320] 2.7.2 Establishment of Limits for Relative Retention Time

[0321] Table 19 summarizes the methodological examination items and validation results:

[0322] Table 19 Summary of RSD% for Methodological Results—Relative Retention Time (Retention Time)

[0323] Methodology Peak 1 Peak 2 Peak 3 Peak 4 (S1) Peak 5 (S2) Peak 6 Repeatability 0.04 0.04 0.03 0.00 0.00 0.04 stability 0.35 0.22 0.36 0.52 0.32 0.32 intermediate precision 0.04 0.01 0.01 0.00 0.00 0.03 Different instruments 7.75 3.05 4.19 0.00 0.00 2.92 Different chromatographic columns 0.02 0.02 0.01 0.00 0.00 0.04 16 batches of verification 0.82 0.29 0.80 0.00 0.00 0.12

[0324] The relative retention times of each characteristic peak are stable and within ±10% of the average value. Therefore, the specified range of relative retention times for each peak is tentatively set at ±10%.

[0325] The chromatogram of the test sample (Sheliugu medicinal material) should show 6 characteristic peaks, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1, 4, and 5 should correspond to the retention times of the reference peaks, respectively. The peak corresponding to the guanosine reference is designated as peak S1. Calculate the relative retention times of peaks 1-3 with peak S1. The peak corresponding to the adenosine reference is designated as peak S2. Calculate the relative retention time of peak 6 with peak S2. The relative retention times should be within ±10% of the specified value. (Peak 2: 0.53, Peak 3: 0.61, Peak 6: 1.12)

[0326] The characteristic chromatograms of 21 batches of *Smilax china* medicinal materials were synthesized using the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version), and a reference chromatogram of the characteristic chromatograms of *Smilax china* medicinal materials was established; for example... Figure 27 As shown in the figure, peak 1: uridine; peak 4 (S1): guanosine; peak 5 (S2): adenosine.

[0327] 2.7.3 Verification of the characteristic chromatogram of She Liu Gu medicinal slices

[0328] According to the results of "2.5 Preparation and Investigation of Test Solution", 16 batches of test solutions of medicinal slices were prepared, and the samples were injected and analyzed according to the results of "2.3 Chromatographic Conditions and System Suitability Test". The relative retention times were calculated. Figure 28 As shown in Table 20, Figure 28 The characteristic chromatograms of 16 batches of She Liu Gu medicinal slices are shown, with each chromatogram representing batch numbers S1-S16: SLG-230801, SLG-230802, SLG-230803, SLG-230804, SLG-230805, SLG-230806, SLG-230807, SLG-230808, SLG-230809, SLG-230810, SLG-230811, SLG-230812, SLG-230813, SLG-230814, SLG-230815, and SLG-230816.

[0329] Table 20: Relative Retention Time Data of Characteristic Peaks in Sheliugu Herbal Slices

[0330]

[0331]

[0332] Based on the principles of stable relative retention times, detectability across all batches of samples, and relatively high peak values, six peaks with good repeatability were selected as characteristic peaks. The results showed that the relative retention times of each characteristic peak were stable and within ±10% of the average value. Therefore, the specified range for the relative retention time of each peak was tentatively set at ±10%.

[0333] The final specification stipulates that the chromatogram of the test sample (Sheliugu slices) should show 6 characteristic peaks, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1, 4, and 5 should correspond to the retention times of the reference peaks, respectively. The peak corresponding to the guanosine reference is designated as peak S1, and the relative retention times of peaks 1-3 with peak S1 should be calculated. The peak corresponding to the adenosine reference is designated as peak S2, and the relative retention time of peak 6 with peak S2 should be calculated. The relative retention times should be within ±10% of the specified value. (Peak 2: 0.53, Peak 3: 0.61, Peak 6: 1.12)

[0334] The characteristic chromatograms of 16 batches of She Liu Gu medicinal slices were synthesized using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and a reference chromatogram of the characteristic chromatograms of She Liu Gu medicinal slices was established, such as... Figure 29 As shown.

[0335] 2.8 Method for determining the characteristic atlas of Sheliugu medicinal materials and processed slices

[0336] Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol was used as mobile phase A and water as mobile phase B, with gradient elution performed according to the specifications in Table 21; the flow rate was 0.3 ml / min, the column temperature was 30 °C; the detection wavelength was 260 nm; the theoretical plate number, calculated based on the adenosine peak, should not be less than 5000.

[0337] Table 21. Gradient elution data for determining the composition of *She Liu Gu* medicinal materials and processed *She Liu Gu* slices.

[0338] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 0 100 5～6 0→3 100→97 6～10 3 97 10～15 3→5 97→95 15～17 5 95 17～19 5→78 95→22 19～24 78 22

[0339] Preparation of reference solution: Take 1g of snake stagnation reference material, add 25ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0340] Take appropriate amounts of guanosine, uridine, and adenosine reference standards, add 30% methanol to prepare a solution containing 10 μg of each per ml, and use it as a reference solution.

[0341] Preparation of the test solution: Take about 1.0 g of the powder (passed through a No. 3 sieve), place it in an Erlenmeyer flask, add 25 ml of 30% methanol, seal tightly, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0342] The determination method involves precisely pipetting 1 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0343] Example 3: Method for Establishing HPLC Characteristic Chromatography of Sheliugu Formula Granules

[0344] 3.1 Experimental Instruments and Materials

[0345] High performance liquid chromatographs: Agilent ultra-high performance liquid chromatographs, Thermo Fisher ultra-high performance liquid chromatographs;

[0346] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0347] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0348] Ultrasonic cleaner: KQ5200DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0349] Chromatographic column: C18;

[0350] 3.2 Reagents and reagents

[0351] Methanol was of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0352] Adenosine (China National Institutes for Food and Drug Control, batch number: 110879-201703, content calculated as 99.7%);

[0353] Uric acid (China National Institutes for Food and Drug Control, batch number: 110887-202305, content calculated as 99.6%);

[0354] Guanosine (China National Institutes for Food and Drug Control, batch number: 111977-202202, content calculated as 88.6%);

[0355] Snake-six-grain reference medicinal material (Shandong Bokang Fine Chemical Co., Ltd., batch number: 380184-202306);

[0356] Snake Six Valley Formula Granules: 2309057, 2309058, 2309059.

[0357] 3.3 Chromatographic conditions

[0358] Through the previous review of the relevant literature on Saururus chinensis, the chromatographic conditions and system suitability test for the characteristic chromatogram of Saururus chinensis formula granules were preliminarily formulated as follows: using octadecylsilyl silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); using methanol as mobile phase A and water as mobile phase B, with a flow rate of 0.3 ml / min and a column temperature of 30°C; performing gradient elution according to the regulations in Table 22 below; the detection wavelength was 260 nm;

[0359] Table 22 Initial data table of gradient elution for Saururus chinensis formula granules

[0360] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 0 100 5～6 0→3 100→97 6～10 3 97 10～15 3→5 97→95 15～17 5 95 17～19 5→78 95→22 19～24 78 22

[0361] Preparation of reference substance solution: Take appropriate amounts of guanosine, uridine, and adenosine reference substances, and make a solution containing 10 μg of each in 1 ml with 30% methanol as the reference substance solution for control;

[0362] Preparation of control crude drug solution: Take 1 g of Saururus chinensis control crude drug, add 25 ml of 30% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate as the control crude drug reference substance solution;

[0363] Preparation of test solution: Take an appropriate amount of this product, grind it finely, take about 1.0 g, place it in a stoppered conical flask, add 25 ml of 30% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0364] Determination method: Precisely pipette 1 μl of each of the above solutions, inject them into the liquid chromatograph for determination to obtain the results.

[0365] 3.4 Chromatographic conditions and system suitability test

[0366] A 3.4.1 Wavelength selection

[0367] Based on the above formulated experimental conditions, use a diode array detector to perform a full wavelength scan on the test solution, and separately extract the chromatograms of the test solution at wavelengths of 220 nm, 240 nm, 260 nm, 280 nm, and 300 nm. As Figures 30-36 shown, Figure 30 is the spectral diagram of uridine as the reference substance, Figure 31 is the spectral diagram of uridine in the test solution, Figure 32 is the spectral diagram of adenosine as the reference substance, Figure 33 is the spectral diagram of adenosine in the test solution, Figure 34 is the spectral diagram of guanosine as the reference substance, Figure 35 is the spectral diagram of guanosine in the test solution, Figure 36The results show that the peak shape is better overall and the proportion of each peak is moderate when the detection wavelength is 260 nm. The baseline of the chromatogram is more stable. Therefore, the detection wavelength is determined to be 260 nm.

[0368] 3.4.2 Column Temperature Investigation

[0369] Based on the above-specified experimental conditions, investigations were conducted at column temperatures of 25℃, 30℃, and 35℃, respectively. Figure 37 As shown, Figure 37 The chromatograms of She Liu Gu formula granules at different column temperatures are shown. The results indicate that the chromatogram peaks are better and the separation is better at a column temperature of 30℃, so a column temperature of 30℃ is selected.

[0370] 3.4.3 Flow velocity investigation

[0371] Based on the above-specified experimental conditions, the flow rates of 0.2 ml / min, 0.25 ml / min, and 0.3 ml / min were investigated respectively; for example... Figure 38 As shown, Figure 38 The chromatograms of Sheliugu formula granules at different flow rates are shown. The results show that the peak shape of the chromatogram is better and the resolution is moderate when the flow rate is 0.3 ml / min, so the flow rate is tentatively set at 0.3 ml / min.

[0372] 3.5 Preparation of the test solution

[0373] 3.5.1 Examination of Extraction Methods

[0374] Take an appropriate amount of this product (batch number: 2309057), grind it into a fine powder, take 1g, place it in a stoppered conical flask, add 25ml of 30% methanol, seal tightly, heat under reflux and sonicate (power 600W, frequency 40kHz) for 30 minutes respectively, cool, shake well, filter, and collect the filtrate to obtain the product. Figure 39 As shown, Figure 39 The chromatograms of Sheliugu formula granules under different extraction methods are shown. The results indicate that there is little difference in the effects of reflux and ultrasonic extraction on the test sample, and the ultrasonic method is rapid and simple. Therefore, ultrasonic extraction was determined to be the extraction method for the test sample.

[0375] 3.5.2 Investigation of Extraction Solvents

[0376] Take an appropriate amount of this product (batch number: 2309057), grind it into a fine powder, take 1g, place it in a stoppered conical flask, add 25ml each of water, 30% methanol, 70% methanol, methanol, and 70% ethanol, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product; Figure 40 As shown, Figure 40The chromatograms of Sheliugu formula granules under different extraction solvents show that when the extraction solvent is 30% methanol or methanol, the peak shapes of each characteristic peak are good and the separation is moderate. Considering all factors, methanol is the best extraction solvent.

[0377] 3.5.3 Examination of extraction time

[0378] Take an appropriate amount of this product (batch number: 2309057), grind it into a fine powder, take 1g, place it in a stoppered conical flask, add 25ml of 30% methanol, seal tightly, and sonicate (power 600W, frequency 40kHz) for 15 minutes, 30 minutes, and 45 minutes respectively. Cool, shake well, filter, and collect the filtrate to obtain the product. Figure 41 As shown, Figure 41 The chromatograms of Sheliugu formula granules at different extraction times are shown. The results indicate that the extraction time for the test sample was consistent at 15 minutes, 30 minutes, and 45 minutes. Therefore, the extraction time for the test sample is tentatively set at 30 minutes.

[0379] 3.5.4 Investigation of Solvent Addition Amount

[0380] Take an appropriate amount of this product (batch number: 2309057), grind it into a fine powder, take 1g, place it in a stoppered conical flask, add 25ml, 50ml, and 100ml of 30% methanol respectively for testing, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product; Figure 42 As shown, Figure 42 The chromatograms of Sheliugu formula granules with different solvent addition amounts are shown. The results indicate that when the extraction solvent addition amount is 25 ml, the peak shape and resolution of each chromatographic peak are good, and the peak size is moderate. Therefore, the solvent amount is selected as 25 ml.

[0381] In summary, the preparation method of the test solution of the Sheliugu formula granules characteristic spectrum is determined as follows: Take an appropriate amount of this product, grind it into a fine powder, take 1g, place it in a stoppered conical flask, add 25ml of 30% methanol, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0382] 3.6 Methodological Examination

[0383] 3.6.1 Chromatographic Peak Identification

[0384] Preparation of the test solution: Prepare the test solution of Sheliugu formula granules according to the experimental conditions proposed above;

[0385] Preparation of reference solution: Take 1g of snake stagnation reference material, add 25ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0386] Take appropriate amounts of uridine, adenosine, and guanosine reference standards and prepare solutions containing 10 μg of each per 1 ml as reference solutions.

[0387] Preparation of negative control solution: Under the experimental conditions outlined above, prepare a negative control solution for the *She Liu Gu* formula granules. Locate the characteristic peaks in the spectral diagram of the *She Liu Gu* formula granules, such as... Figure 43 As shown, Figure 43 The chromatographic peak identification chromatogram of the She Liu Gu formula granules.

[0388] In the following methodological investigation, peak 1 is uridine, peak 4 is guanosine, and peak 5 is adenosine. In the following methodological investigation, six characteristic peaks in the sample were investigated.

[0389] 3.6.2 Repeatability Test

[0390] Accurately weigh 6 portions of Sheliugu formula granules (batch number: 2309057), and prepare and measure them according to the proposed experimental method; as shown in Figure 23;

[0391] Table 23 Repeatability Test—Relative Retention Time Data of Characteristic Peaks

[0392]

[0393] The results showed that the relative retention time RSD of the six samples was 0.00% to 0.06%, indicating that the method had good reproducibility.

[0394] 3.6.3 Intermediate Precision Examination

[0395] 3.6.3.1 Investigations by different personnel and at different times

[0396] Based on the experimental conditions proposed above, different personnel (A, B) weighed Sheliugu formula granules (batch number: 2309057) at different times (T1, T2) to prepare test samples and conduct determinations, as shown in Table 24;

[0397] Table 24 Data on the ratio of relative retention time of characteristic peaks for different personnel and time periods.

[0398]

[0399] The results showed that when different personnel measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.00% to 0.15%, indicating good method stability.

[0400] 3.6.3.2 Investigation with different instruments

[0401] Based on the above-specified experimental conditions, the test solution was taken and analyzed using Agilent and Thermo Fisher high-performance liquid chromatographs, respectively; as shown in Table 25. Figure 44 As shown, Figure 44 Chromatograms of She Liu Gu formula granules under different instruments;

[0402] Table 25 Instrumental Examination—Relative Retention Time Data of Characteristic Peaks

[0403]

[0404] The results showed that the peak elution times of the characteristic peaks differed significantly when the test samples were detected using the two instruments mentioned above.

[0405] 3.6.4 Durability Assessment

[0406] 3.6.4.1 Column robustness test

[0407] Based on the above-planned experimental conditions, chromatographic columns of the same model but different batches were used respectively. C18 2.1*100mm, 1.6μm were analyzed and investigated; as shown in Table 26. Figure 45 As shown, Figure 45 Chromatogram for column durability testing of Sheliugu formula granules;

[0408] Table 26 Column Robustness Study—Relative Retention Time Data of Characteristic Peaks

[0409]

[0410] The results showed that the RSD of the relative retention times of characteristic peaks of chromatographic columns of the same model but different batches ranged from 0.00% to 0.08%.

[0411] 3.6.4.2 Stability Assessment

[0412] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 6h, 8h, 12h, 16h, and 24h, as shown in Table 27.

[0413] Table 27 Stability Study—Characteristic Peak Retention Time Data Table

[0414]

[0415] The results showed that the RSD of the corresponding characteristic peak retention time was 0.24%-0.55%, and the sample solution was relatively stable within 24 hours.

[0416] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The above six characteristic peaks will be included in subsequent investigations.

[0417] 5.7 Determination of characteristic peaks and establishment of reference spectra

[0418] 5.7.1 Validation results of three batches of Sheliugu formula granules

[0419] The characteristic spectra of three batches of this product were determined using the proposed method, and the relative retention times were calculated; Figure 46 As shown in Table 28, Figure 46 Chromatograms of characteristic chromatograms for three batches of She Liu Gu formula granules;

[0420] Table 28. Relative retention time data for three batches of Sheliugu formula granules.

[0421]

[0422]

[0423] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peaks, a total of 6 peaks with good repeatability were selected as characteristic peaks.

[0424] 3.7.2 Establishment of Limits for Relative Retention Time

[0425] Table 29 summarizes the methodological examination items and validation results:

[0426] Table 29 Summary of RSD% of Methodological Results—Relative Retention Time Data Table

[0427] Methodology Peak 1 Peak 2 Peak 3 Peak 4 (S1) Peak 5 (S2) Peak 6 Flow rate 6.52 8.57 6.05 0.00 0.00 0.41 Column temperature 3.38 4.71 3.56 0.00 0.00 1.13 Repeatability 0.06 0.05 0.03 0.00 0.00 0.02 stability 0.36 0.24 0.37 0.55 0.33 0.30 Different people 0.01 0.01 0.15 0.00 0.00 0.05 Different instruments 7.83 3.26 4.22 0.00 0.00 2.75 Different chromatographic columns 0.03 0.08 0.04 0.00 0.00 0.02

[0428] The relative retention times of each characteristic peak are stable and within ±10% of the average value. Therefore, the specified range of relative retention times for each peak is tentatively set at ±10%.

[0429] The chromatogram of the test sample should show 6 characteristic peaks, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1, 4, and 5 should correspond to the retention times of the reference peaks, respectively. The peak corresponding to the guanosine reference is designated as peak S1. Calculate the relative retention times of peaks 1-3 with peak S1. The peak corresponding to the adenosine reference is designated as peak S2. Calculate the relative retention time of peak 6 with peak S2. The relative retention times should be within ±10% of the specified value. (Peak 2: 0.53, Peak 3: 0.61, Peak 6: 1.12)

[0430] The characteristic chromatograms of three batches of She Liu Gu formula granules were synthesized using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and a reference chromatogram of the characteristic chromatograms of She Liu Gu formula granules was established; for example... Figure 47 As shown, Figure 47 Mid-peak 1: uridine; peak 4 (S1): guanosine; peak 5 (S2): adenosine.

[0431] 5.8 Determination of the Characteristic Spectrum of Sheliugu Formula Granules

[0432] Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol was used as mobile phase A and water as mobile phase B, with gradient elution as specified in Table 30; the flow rate was 0.3 ml / min, the column temperature was 30 °C; the detection wavelength was 260 nm; the theoretical plate number, calculated based on the adenosine peak, should not be less than 5000.

[0433] Table 30 Gradient Elution Data of Sheliugu Formula Granules

[0434] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 0 100 5～6 0→3 100→97 6～10 3 97 10～15 3→5 97→95 15～17 5 95 17～19 5→78 95→22 19～24 78 22

[0435] Preparation of reference solution: Take 1g of snake stagnation reference material, add 25ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0436] Take appropriate amounts of guanosine, uridine, and adenosine reference standards, add 30% methanol to prepare a solution containing 10 μg of each per ml, and use it as a reference solution.

[0437] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, weigh about 1.0 g accurately, place it in a stoppered conical flask, add 25 ml of 30% methanol, stopper tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0438] For the determination method, accurately pipette 1 μl of each of the above solutions, inject it into the liquid chromatograph, and measure it to obtain the result.

[0439] Comparative Example 1

[0440] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in Table 31; flow rate 0.3 ml / min, column temperature 30 °C; detection wavelength 260 nm; theoretical plate number calculated based on adenosine peak should not be less than 5000.

[0441] Table 31 Gradient elution data of Sheliugu formula granules

[0442] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 0 100 5～15 0→10 100→90 15～16 10→80 90→20 16～18 80 20

[0443] Prepare the test solution by grinding an appropriate amount of Sheliugu formula granules into a fine powder (about 1.0 g), placing it in an Erlenmeyer flask, adding 25 ml of 30% methanol, sealing tightly, and sonicating (600 W power, 40 kHz frequency) for 30 minutes. After cooling, shake well, filter, and collect the filtrate to obtain the test solution.

[0444] The assay involves accurately pipetting 1 μl of the test solution and injecting it into the liquid chromatograph for determination; the result is as follows. Figure 48 As shown, Figure 48 The HPLC characteristic chromatogram of *Symplocos spp.* is shown.

[0445] Compared with the feature maps, the feature peaks of this method have poor shape and poor separation.

[0446] Comparative Example 2

[0447] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in Table 32; flow rate 0.3 ml / min, column temperature 30 °C; detection wavelength 260 nm; and a theoretical plate number calculated based on the adenosine peak of not less than 5000.

[0448] Table 32 Gradient elution data of Sheliugu formula granules

[0449] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 0 100 5～6 0→3 100→97 6～10 3 97 10～15 3→5 97→95 15～20 5 95 20～22 5→78 95→22 22～25 78 22

[0450] Prepare the test solution by grinding an appropriate amount of Sheliugu formula granules into a fine powder (about 1.0 g), placing it in an Erlenmeyer flask, adding 25 ml of 30% methanol, sealing tightly, and sonicating (600 W power, 40 kHz frequency) for 30 minutes. After cooling, shake well, filter, and collect the filtrate to obtain the test solution.

[0451] The assay involves accurately pipetting 1 μl of the test solution and injecting it into the liquid chromatograph for determination; the result is as follows. Figure 49 As shown, Figure 49 The HPLC characteristic chromatogram of *Symplocos spp.* is shown.

[0452] Compared with the feature maps, the feature peaks of this method have poor shape and poor separation.

[0453] Comparative Example 3

[0454] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile as mobile phase A and water as mobile phase B, with gradient elution as specified in Table 33; flow rate 0.3 ml / min, column temperature 30 °C; detection wavelength 260 nm; theoretical plate number calculated based on adenosine peak should not be less than 5000.

[0455] Table 33 Gradient elution data of Sheliugu formula granules

[0456] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 0 100 5～15 0→10 100→90 15～16 10→80 90→20 16～18 80 20

[0457] Prepare the test solution by grinding an appropriate amount of Sheliugu formula granules into a fine powder (about 1.0 g), placing it in an Erlenmeyer flask, adding 25 ml of 30% methanol, sealing tightly, and sonicating (600 W power, 40 kHz frequency) for 30 minutes. After cooling, shake well, filter, and collect the filtrate to obtain the test solution.

[0458] The assay involves accurately pipetting 1 μl of the test solution and injecting it into the liquid chromatograph for determination; the result is as follows. Figure 50 As shown, Figure 50 The HPLC characteristic chromatogram of *Symplocos spp.* is shown.

[0459] Compared with the feature maps, the feature peaks of this method have poor shape, are enclosing peaks, and have poor separation.

[0460] Comparative Example 4

[0461] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile as mobile phase A and 0.1% phosphoric acid as mobile phase B, with gradient elution as specified in Table 34; flow rate 0.3 mL / min, column temperature 30 °C; detection wavelength 260 nm. The theoretical plate number, calculated based on the adenosine peak, should be no less than 5000.

[0462] Table 34 Gradient elution data of Sheliugu formula granules

[0463] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 0 100 5～15 0→10 100→90 15～16 10→80 90→20 16～18 80 20

[0464] Prepare the test solution by grinding an appropriate amount of Sheliugu formula granules into a fine powder (about 1.0 g), placing it in an Erlenmeyer flask, adding 25 ml of 30% methanol, sealing tightly, and sonicating (600 W power, 40 kHz frequency) for 30 minutes. After cooling, shake well, filter, and collect the filtrate to obtain the test solution.

[0465] The assay involves accurately pipetting 1 μl of the test solution and injecting it into the liquid chromatograph for determination; the result is as follows. Figure 51 As shown, Figure 51 The HPLC characteristic chromatogram of *Symplocos spp.* is shown.

[0466] Compared with the feature maps, the feature peaks of this method have poor shape, are enclosing peaks, and have poor separation.

[0467] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0468] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for establishing HPLC characteristic spectra of *Smilax china* medicinal materials, processed slices, standard decoctions, and their formulated granules, comprising the following steps: S1) Extract the raw material of *S. coli* using a solvent to obtain the test solution; the raw material of *S. coli* is *S. coli* medicinal material, processed slices, standard decoction, or formula granules; the solvent is 30% methanol; S2) The test solution was detected by high performance liquid chromatography to obtain the HPLC characteristic spectrum of the snake glutinous rice raw material; The conditions for the high-performance liquid chromatography (HPLC) method are as follows: the chromatographic column is a C18 column, mobile phase A is methanol, mobile phase B is water, gradient elution is used; the detection wavelength is 260 nm; the column length is 100 nm, the inner diameter is 2.1 mm, and the particle size is 1.6 μm. The gradient elution specifically refers to: 0~5min, Phase A: 0%, Phase B: 100%; 5~6 min, Phase A: 0~3%, Phase B: 100~97%; 6~10min, Phase A: 3%, Phase B: 97%; 10~15min, Phase A: 3~5%, Phase B: 97~95%; 15-17 min, Phase A: 5%, Phase B: 95%; 17~19min, Phase A: 5~78%, Phase B: 95~22%; 19~24min, Phase A: 78%, Phase B: 22%.

2. The method for establishing according to claim 1, characterized in that, It also includes the preparation of reference solutions for reference standards and reference solutions for reference medicinal materials; The preparation of the reference solution of the control medicinal material is as follows: the reference medicinal material of *Smilax china* is dissolved in 30% methanol and ultrasonically treated to obtain the reference solution of the control medicinal material; The preparation of the reference solution specifically involves dissolving guanosine, uridine, and adenosine in 30% methanol to obtain the reference solution. The reference solution of the reference standard and the reference medicinal material were determined by high performance liquid chromatography, and chromatograms of the reference standard and the reference medicinal material were obtained respectively. The components of the HPLC characteristic chromatograms of the reference standard and reference medicinal materials were qualitatively determined based on the chromatograms of the reference standard and reference medicinal materials.

3. The method for establishing according to claim 1 or 2, characterized in that, In step S1), the ratio of the snake glutinous rice raw material to the solvent is 1g: (20~30)ml.

4. The method for establishing according to claim 1 or 2, characterized in that, In step S1), the extraction method is ultrasonic extraction or reflux extraction, and the extraction time is 10~45 min.

5. The method for establishing according to claim 1 or 2, characterized in that, The mobile phase flow rate was 0.3 ml / min, and the injection volume was 1 μL.

6. The method for establishing according to claim 1 or 2, characterized in that, The column temperature of the chromatographic column is 30℃.

7. The method for establishing according to claim 1 or 2, characterized in that, The similarity of HPLC characteristic chromatograms of Sheliugu medicinal materials, processed slices, standard decoctions and their formula granules was evaluated using a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. The HPLC standard characteristic chromatograms of Sheliugu medicinal materials, processed slices, standard decoctions and their formula granules were obtained, consisting of 6 characteristic peaks, of which peak 1: uridine peak, peak 4: guanosine peak, and peak 5: adenosine peak.

8. The method for establishing according to claim 7, characterized in that, In the characteristic spectrum of the Snake Six Valley Standard Decoction, the peak corresponding to the guanosine reference is taken as the S1 peak, and the relative retention time of peaks 1-3 with the S1 peak is calculated. The peak corresponding to the adenosine reference is taken as the S2 peak, and the relative retention time of peak 6 with the S2 peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are 0.53 (peak 2), 0.61 (peak 3), and 1.12 (peak 6). In the characteristic spectrum of Sheliugu formula granules, the peak corresponding to the guanosine reference is taken as the S1 peak, and the relative retention time of peaks 1-3 with the S1 peak is calculated. The peak corresponding to the adenosine reference is taken as the S2 peak, and the relative retention time of peak 6 with the S2 peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are 0.53 (peak 2), 0.61 (peak 3), and 1.12 (peak 6).

9. The method for establishing according to claim 8, characterized in that, In the characteristic chromatogram of the medicinal material and decoction pieces of snake liugu, the peak corresponding to the guanosine reference is taken as the S1 peak, and the relative retention time of peaks 1-3 with the S1 peak is calculated. The peak corresponding to the adenosine reference is taken as the S2 peak, and the relative retention time of peak 6 with the S2 peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are 0.53 (peak 2), 0.61 (peak 3), and 1.12 (peak 6).

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