A method for constructing a characteristic chromatogram of a pharmaceutical preparation of tylophora and a method for quality detection

Abstract

The application belongs to the technical field of traditional Chinese medicine detection, and specifically provides a construction method and a quality detection method for characteristic chromatograms of Sarcandra glabra medicinal preparation, wherein octadecylsilane bonded silica gel is used as a filler, acetonitrile is used as mobile phase A, and a water solution containing potassium dihydrogen phosphate is used as mobile phase B, a gradient elution program is continuously optimized, the column temperature is controlled to be 23-27 DEG C, 11 common characteristic peaks are finally obtained, 9 common characteristic peaks are effectively separated, the characteristic chromatogram obtained has a smooth baseline, the characteristic peaks have good peak shapes, and the detection time is short, thereby providing a scientific basis for comprehensively establishing the quality control standard of Sarcandra glabra formula granules. Moreover, the peak positions of tryptophan, syringin, isovanillin and magnoflorine can be accurately positioned, the known information of the characteristic peaks is increased, and the integrity and characteristics of Sarcandra glabra medicinal preparation (for example, formula granules and other preparations) are fully reflected.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for constructing a characteristic chromatogram and a quality detection method for a Tinospora sinensis drug preparation. Background Art

[0002] Tinospora sinensis is the dried stem of the plant Tinospora sinensis (Lour.) Merr. of the Menispermaceae family. Tinospora sinensis is mainly produced in southeastern Tibet, Hainan, Yunnan, Guangdong, Guangxi and other places in China. Tinospora sinensis has pharmacological effects such as anti-inflammatory, hepatoprotective, antioxidant, and immunomodulatory effects. The main chemical components include sesquiterpenoids, diterpenoids, lignans, phenylpropanoids, alkaloids and other compounds. Most of the research on Tinospora sinensis stays at the identification of chemical components.

[0003] Common preparation types of Tinospora sinensis preparations include powder, formula granules, etc. For example, formula granules are obtained by steps such as extraction, concentration, drying, and preparation from Tinospora sinensis decoction pieces, and there are significant differences in the material basis between them and the material basis of the medicinal materials. At present, the literature research report "Study on the UPLC Characteristic Chromatogram of Tinospora sinensis Medicinal Materials", authors Chi Senshen, Han Dan, etc. This literature mainly focuses on the research of Tinospora sinensis medicinal materials. It can be directly seen from the comparison chromatograms of multiple batches that the peak emergence times of the characteristic peaks in the established chromatogram are relatively late and relatively dense. Moreover, the resolution of most chromatographic peaks is poor and there is no effective separation. In addition, since traditional Chinese medicine formula granules no longer have the characteristics of medicinal material character identification, the above method is also not suitable for the quality detection of preparations made from Tinospora sinensis drugs such as Tinospora sinensis formula granules, and there are defects such as fewer characteristic peaks and poor separation effect. The characteristic chromatogram of Tinospora sinensis (Tinospora sinensis) formula granules promulgated by the Guangdong Province Traditional Chinese Medicine Formula Granule Standard Yue PFKL20240016 also has problems of poor separation effect of characteristic peaks and fewer characteristic peaks. Summary of the Invention

[0004] Therefore, the purpose of the present invention is to provide a method for constructing a characteristic chromatogram and a quality detection method for a Tinospora sinensis drug preparation. According to the characteristics of the Tinospora sinensis drug preparation, this method establishes the characteristic chromatogram of this variety, realizes the effective separation of each characteristic peak, increases the number of characteristic peaks, and provides a scientific basis for comprehensively establishing the quality control standard of Tinospora sinensis formula granules.

[0005] For this purpose, the present invention provides a method for constructing a characteristic chromatogram of a Tinospora sinensis drug preparation, including the following steps,

[0006] (1) Preparation of the test solution;

[0007] (2) The test solution was analyzed by high performance liquid chromatography (HPLC). Octadecylsilane-bonded silica gel was used as the stationary phase, acetonitrile was used as the mobile phase A, and an aqueous solution containing potassium dihydrogen phosphate was used as the mobile phase B. The column temperature was 23-27℃, and gradient elution was performed according to the following procedure:

[0008] From 0 to 15 min, the volume ratio of mobile phase A to mobile phase B was 6%:94% to 10%:90%.

[0009] 15→20min, the volume ratio of mobile phase A to mobile phase B is 10%:90%;

[0010] 20→40min, the volume ratio of mobile phase A to mobile phase B is 10%:90%→19-21%:79-81%.

[0011] Furthermore, step (2) also satisfies at least one of the following 1)-5):

[0012] 1) The detection wavelength is 220-300nm, preferably 280nm;

[0013] 2) The flow rate is 0.9-1.1 mL / min, preferably 1.0 mL / min;

[0014] 3) The injection volume is 3-10 μl;

[0015] 4) The concentration of the aqueous solution containing potassium dihydrogen phosphate is 0.03-0.08 mol / L, and the pH of the aqueous solution containing potassium dihydrogen phosphate is 3.0-4.0; preferably, phosphoric acid is used to adjust the pH of the aqueous solution containing potassium dihydrogen phosphate to 3.0-4.0; more preferably, phosphoric acid is used to adjust the pH of the aqueous solution containing potassium dihydrogen phosphate to 3.7.

[0016] 5) During the high performance liquid chromatography detection process, a chromatographic column with a specification of 4.6mm×150mm and a diameter of 2.7~3.0μm is used.

[0017] Further, step (1) includes weighing the test sample, adding solvent to extract it, obtaining the extract, separating the solid and liquid, and taking the liquid, which is the test sample solution.

[0018] Furthermore, step (1) also satisfies any one or more of the following AEs:

[0019] A. The ratio of the mass of the test sample to the volume of the solvent is 0.1-0.4:5-25; the relationship between mass and volume is g / mL.

[0020] B. The extraction method is either reflux extraction or ultrasonic extraction;

[0021] C. The extraction time is ≥10 min, preferably 15-40 min;

[0022] D. The solid-liquid separation is selected from centrifugation or membrane filtration;

[0023] E. The solvent is selected from one or more of methanol, ethanol, and water; preferably, it is a methanol aqueous solution with a volume percentage of 50-70%.

[0024] Further, step (1) includes weighing 0.1-0.4g of the granules of the *Tinospora sinensis* formula, placing them in a stoppered conical flask, adding 5-25ml of 50%-70% methanol, sealing tightly, sonicating for 15-40 minutes, cooling, filtering, and taking the filtrate to obtain the test solution.

[0025] Furthermore, the construction method further includes the step of preparing a reference solution by adding at least one of tryptophan, syringin, isovanillin and magnoflorine as a solvent, and the step of detecting the reference solution by high performance liquid chromatography according to any of the above construction methods to obtain a reference chromatogram.

[0026] Further, each 1 mL of the reference solution contains at least one of 1-100 μg tryptophan, 1-100 μg syringin, 1-100 μg isovanillin and 1-100 μg magnoflorine; and / or, the solvent used in the preparation of the reference solution is selected from methanol or an aqueous methanol solution.

[0027] Furthermore, the construction method further includes the steps of preparing a reference drug solution by extracting the reference drug *Tinospora sinensis* with water according to the preparation method of the test solution in any of the above-described construction methods, and detecting the reference drug solution by high performance liquid chromatography in any of the above-described construction methods to obtain a reference drug reference spectrum.

[0028] Preferably, after water extraction of the reference herb *Tinospora sinensis*, the solution is filtered, dried, and then prepared as a reference herb solution according to the preparation method of the test solution in any of the above-described construction methods.

[0029] Further, take 1.0-3.0g of *Tinospora sinensis* reference material and 1-3g of *Tinospora sinensis* reference material, place them in a stoppered conical flask, add 50-200ml of water, heat under reflux for 20-50min, filter, evaporate the filtrate to dryness, add 5-25ml of 50%-70% methanol to the residue, seal tightly, sonicate for 15-40min, cool, shake well, filter, and take the filtrate to obtain the reference material solution.

[0030] Furthermore, the characteristic chromatogram of the *Tinospora sinensis* drug preparation has 9 common characteristic peaks. Peaks 1 and 7 correspond to the retention times of the reference peaks of tryptophan and magnoflorine, respectively. The peak corresponding to the reference peak of magnoflorine is the S peak. The relative retention times of peaks 2-6 and peaks 8-9 with the S peak are within ±10% of the specified values. The specified values ​​of peaks 2-6 and peaks 8-9 are 0.53, 0.68, 0.71, 0.81, 0.94, 1.34, and 1.47, respectively.

[0031] In this invention, the medicinal preparation of *Tinospora sinensis* can be *Tinospora sinensis* formula granules, or *Tinospora sinensis* standard decoction freeze-dried powder, or other common preparations obtained by water extraction of *Tinospora sinensis*.

[0032] In some embodiments, the method further includes constructing a reference characteristic chromatogram of the *Tinospora sinensis* drug preparation. This involves generating a reference characteristic chromatogram of the *Tinospora sinensis* drug preparation by using a similarity evaluation system for chromatographic characteristic chromatograms of traditional Chinese medicine from the characteristic chromatograms obtained from multiple batches of *Tinospora sinensis* drug preparation samples. At least three batches of *Tinospora sinensis* formula granules are used, for example, two batches, ten batches, fifteen batches, or eighteen batches of *Tinospora sinensis* formula granules.

[0033] In some embodiments, after generating the control characteristic spectrum of the *Tinospora sinensis* drug preparation using the software for evaluating the similarity of chromatographic characteristic spectra of traditional Chinese medicine, the method further includes the step of marking common characteristic peaks.

[0034] The present invention also provides an application of the method for constructing the characteristic spectrum of the *Cistanche deserticola* drug preparation described in any one of the above claims in the quality detection of *Cistanche deserticola* drug preparations.

[0035] This invention also provides a quality testing method for a *Cephalotaxus fortunei* pharmaceutical preparation, comprising the step of comparing the characteristic spectrum of the *Cephalotaxus fortunei* product to be tested with the control characteristic spectrum of the *Cephalotaxus fortunei* pharmaceutical preparation; wherein the characteristic spectrum of the *Cephalotaxus fortunei* product to be tested is constructed using the *Cephalotaxus fortunei* product to be tested according to any of the construction methods described above, and the control characteristic spectrum of the *Cephalotaxus fortunei* pharmaceutical preparation is selected from any one of the following (1)-(3):

[0036] (1) It has 9 common characteristic peaks. Peaks 1 and 7 correspond to the retention times of the reference peaks of tryptophan and magnoflorine, respectively. The peak corresponding to the reference peak of magnoflorine is the S peak. The relative retention times of peaks 2-6 and peaks 8-9 with the S peak are within ±10% of the specified values. The specified values ​​of peaks 2-6 and peaks 8-9 are 0.53, 0.68, 0.71, 0.81, 0.94, 1.34, and 1.47, respectively.

[0037] (2) Characteristic spectrum of Tinospora fasciata obtained by using single or multiple batches of Tinospora fasciata drug preparations according to any of the above-described construction methods;

[0038] (3) The characteristic fingerprint obtained by using the multi-batch Tinospora sinensis drug preparation according to the construction method described in any one of the above is made into a control characteristic fingerprint by the average value or median method.

[0039] % methanol represents the volume percentage of methanol in the methanol aqueous solution.

[0040] Tinospora sinensis (Tinospora sinensis) represents the Tinospora sinensis medicinal material with the botanical origin of Tinospora sinensis. What is inside the brackets represents the botanical origin of the medicinal material.

[0041] The technical solution of the present invention has the following advantages:

[0042] 1. For the construction method of the characteristic fingerprint of the Tinospora sinensis drug preparation described in the present invention, octadecylsilyl silica gel is used as the filler, acetonitrile is used as mobile phase A, and an aqueous solution containing potassium dihydrogen phosphate is used as mobile phase B. By continuously optimizing the gradient elution program, gradient elution is carried out according to the following program: 0 → 15 min, the volume ratio of mobile phase A to mobile phase B is 6%:94% → 10%:90%; 15 → 20 min, the volume ratio of mobile phase A to mobile phase B is 10%:90%; 20 → 40 min, the volume ratio of mobile phase A to mobile phase B is 10%:90% → 19 - 21%:79 - 81%. Combining with controlling the column temperature at 23 - 27°C, finally 11 common characteristic peaks are obtained, and effective separation of 9 common characteristic peaks is achieved. Moreover, the obtained characteristic fingerprint has a stable baseline, good peak shapes of the characteristic peaks, and a short detection time, providing a scientific basis for comprehensively establishing the quality control standard of Tinospora sinensis formula granules. Moreover, the peak positions of tryptophan, syringin, isovanillin, and magnoflorine can also be accurately located, increasing the known information of the characteristic peaks, and fully reflecting the integrity and characteristics of the Tinospora sinensis drug preparation (such as formula granules and other preparations).

[0043] 2. For the construction method of the characteristic fingerprint of the Tinospora sinensis drug preparation described in the present invention, by investigating extraction conditions such as optimizing chromatographic conditions (pH of mobile phase B, wavelength, flow rate, etc.), types of extraction solvents, and amounts of extraction solvents, the optimal extraction process and chromatographic conditions are determined, resulting in higher peak areas, better separation effects and peak shapes, and enabling more comprehensive quality monitoring of Tinospora sinensis formula granules.

[0044] 3. For the quality detection method of the Tinospora sinensis drug preparation described in the present invention, by comparing the characteristic fingerprint of the tested Tinospora sinensis formula granule product with the control characteristic fingerprint of the Tinospora sinensis formula granule, the quality of the Tinospora sinensis formula granule can be comprehensively, clearly, and effectively detected. Description of the Drawings

[0045] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0046] Figure 1 The chromatograms at 220 nm are from the different wavelengths tested in Experiment Example 1.

[0047] Figure 2 The chromatograms at 240 nm are from the different wavelengths tested in Experiment Example 1.

[0048] Figure 3 The chromatograms at 260 nm are from the different wavelengths tested in Experiment Example 1.

[0049] Figure 4 The chromatograms at 280 nm are from the different wavelengths tested in Experiment Example 1.

[0050] Figure 5 The chromatograms at 300 nm are from the different wavelengths tested in Experiment Example 1.

[0051] Figure 6 The chromatogram of gradient two in the first gradient elution procedure of Experiment Example 1 is examined.

[0052] Figure 7 The chromatogram of gradient three in the first gradient elution procedure of Experiment Example 1 is examined.

[0053] Figure 8 The chromatograms at 25°C are from the different column temperatures tested in Experiment Example 1.

[0054] Figure 9 The chromatogram at 35℃ is shown in Experiment Example 1, which was used to investigate different column temperatures.

[0055] Figure 10 The chromatogram for pH 3.0 in the experiment investigating the pH of mobile phase B in Experiment Example 1;

[0056] Figure 11 The chromatogram for pH 3.4 in the experiment investigating the pH of mobile phase B in Experiment Example 1;

[0057] Figure 12 The chromatogram for pH 3.7 in the experiment investigating the pH of mobile phase B in Experiment Example 1;

[0058] Figure 13 The chromatogram for pH 4.0 in the experiment investigating the pH of mobile phase B in Experiment Example 1;

[0059] Figure 14 This is a comparison diagram of the mixed control solution and the test solution in Experiment Example 1; S2(2) is the mixed control solution, and S1(2) is the test solution;

[0060] Figure 15 This is a comparison diagram of the syringin reference solution and the test solution in Experiment Example 1; S2(1) is the syringin reference solution, and S1(1) is the test solution;

[0061] Figure 16 This is a comparison diagram of the isovanillin reference solution and the test solution in Experiment Example 1; S2(1) is the isovanillin reference solution, and S1(1) is the test solution.

[0062] Figure 17 Characteristic chromatograms of 18 batches of standard decoction (freeze-dried powder) of *Tinospora sinensis* (Kuanjinteng) slices and 3 batches of formulation granules;

[0063] Figure 18 For comparison of feature maps;

[0064] Figure 19 This is the negative control chromatogram used in the method validation of Experiment Example 2;

[0065] Figure 20 The chromatogram of the control medicinal material obtained in Example 1;

[0066] Figure 21 The characteristic spectrum of the *Vitis thunbergii* (kuanjinteng) formulation granules obtained in Example 1;

[0067] Figure 22 The chromatograms are of the tryptophan and magnoflorine reference standards constructed in Example 1. Detailed Implementation

[0068] The following embodiments are provided to better understand the present invention and are not intended to limit the scope of the invention. They do not constitute a limitation on the content or scope of protection of the present invention. Any product identical or similar to the present invention derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art falls within the scope of protection of the present invention. Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in the art. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products. % methanol refers to the volume percentage of methanol in an aqueous methanol solution.

[0069] Examination of the construction method in Experiment Example 1

[0070] 1. Instruments, reagents and reagents

[0071] (1) Instruments and Equipment: Chromatograph 1: Waters ACQUITY Arc chromatography system, including a quaternary gradient pump (QSM-R), a 96-position high-performance autosampler (FTN-R), an original imported column oven, a Waters 2998 diode array UV detector, and an Empower chromatography management system; Chromatograph 2: Agilent 1260 Infinity II chromatography system, including a G7111B quaternary pump (1260 Quat Pump), a G7129A autosampler (1260 Vialsampler), a G7115A PDA diode array detector (1260 DAD WR), a G7116A column oven (1260 MCT), and an OpenLAB CDS chromatography workstation; Chromatograph 3: UltiMate The 3000 chromatography system includes an LPG-3400A quaternary pump, a WPS-3000TSL autosampler, a PDA diode array detector, and a chromatography workstation; an XSR304 part per ten thousand balance (Mettler, Switzerland), a ME36S part per million balance (Sartorius), an ultrasonic instrument (Shanghai Kedao Ultrasonic Instrument Co., Ltd.), and a water bath.

[0072] (2) Chromatographic columns: 1) Agilent Poroshell 120EC-C18, 4.6mm×150mm, 2.7μm; 2) SHIMADZUShim-pack GIST C18-AQ, 4.6mm×150mm, 3.0μm; 3) CORTECS T3, 4.6mm×150mm, 2.7μm.

[0073] (3) Reagents: Acetonitrile (Fisher Chemical, chromatographic grade), water is ultrapure water, potassium dihydrogen phosphate, phosphoric acid, methanol and other reagents are all analytical grade.

[0074] (4) Drug testing:

[0075] * *Tinospora sinensis* reference material (batch number: 121391-202102; purchased from the National Institutes for Food and Drug Control); magnoflorine reference standard (batch number: 15998; purchased from Shanghai Shidander Biotechnology Co., Ltd., purity 93.9%); tryptophan reference standard (batch number: 140686-202205; purchased from the National Institutes for Food and Drug Control, purity 100%); syringin reference standard (batch number: 111574-202106; purchased from the National Institutes for Food and Drug Control, purity 94.3%); *Tinospora sinensis* (*Tinospora sinensis*) formula granules (2304001Y, 2304002Y, 2304003Y).

[0076] The freeze-dried powder of *Tinospora sinensis* can be prepared using conventional methods in the field. For example, the present invention is prepared according to the following steps: Take about 100g of *Tinospora sinensis* slices, add 10 times the amount of water to the slices for 30 minutes for the first decoction, bring to a boil over high heat, then simmer over low heat for 30 minutes, and filter through a 200-mesh filter. For the second decoction, add 8 times the amount of water to the slices, bring to a boil over high heat, then simmer over low heat for 20 minutes, and filter through a 200-mesh filter. Combine the filtrates; concentrate under reduced pressure (50-65℃) to a concentrated extract with a relative density of 1.04-1.12g / ml, and freeze-dry to obtain freeze-dried powder of *Tinospora sinensis* (multiples refer to weight multiples).

[0077] The granules of *Vitis thunbergii* (kuanjinteng) can be prepared using conventional methods in the field. For example, the present invention is prepared according to the following steps: Take *Vitis thunbergii* slices, add water and decoct twice. For the first decoction, add 10 times the amount of water to soak the slices for 30 minutes, decoct for 30 minutes, and filter. For the second decoction, add 8 times the amount of water to decoct for 20 minutes, filter, combine the filtrates, concentrate at 60℃~80℃, spray dry, and granulate by dry method to obtain *Vitis thunbergii* (kuanjinteng) granules (multiples refer to mass multiples).

[0078] 2. Preparation of the test solution

[0079] Accurately weigh the granules of the *Vitis thunbergii* formula, grind them finely, take about 0.2g, place them in a stoppered conical flask, add 10ml of 70% methanol, seal tightly, sonicate (power 350W, frequency 40kHz) for 30 minutes, cool, filter, and collect the filtrate to obtain the final product.

[0080] 3. Optimization of chromatographic conditions

[0081] In the process of continuously optimizing the elution gradient, a more reasonable wavelength and gradient are selected based on indicators such as the information content of the chromatographic peaks and the resolution of the chromatographic peaks.

[0082] (1) Investigation of different wavelengths

[0083] The test solution prepared according to item 2 of this experimental example was detected by high performance liquid chromatography. An Agilent Poroshell 120EC-C18 column, 4.6 mm × 150 mm, 2.7 μm, was used. Acetonitrile was used as mobile phase A, and 0.05 mol / L potassium dihydrogen phosphate (adjusted to pH 3.7 with phosphoric acid) was used as mobile phase B. Gradient elution was performed according to the gradient one specification. The column temperature was 30 °C, the injection volume was 5 μl, the flow rate was 1.0 ml / min, and the wavelength was full wavelength scan.

[0084] The spectra at detection wavelengths of 220nm, 240nm, 260nm, 280nm, and 300nm are shown below. Figure 1-5As shown in the results from different wavelengths, the chromatographic peaks at 280 nm exhibit more information and more uniform characteristic peaks. Therefore, 280 nm is tentatively selected as the wavelength for subsequent gradient investigation and optimization. The chromatogram at 280 nm shows poor separation of characteristic peaks at 10-15 minutes; therefore, further gradient optimization is needed.

[0085] (2) Examination of a single gradient elution procedure

[0086] The test solution prepared according to item 2 of this experimental example was detected by high performance liquid chromatography. An Agilent Poroshell 120EC-C18 column, 4.6 mm × 150 mm, 2.7 μm, was used. Acetonitrile was used as mobile phase A, and 0.05 mol / L potassium dihydrogen phosphate (adjusted to pH 3.7 with phosphoric acid) was used as mobile phase B. Gradient elution was performed according to the specifications of gradient two and three, respectively. Column temperature: 30 °C, injection volume: 5 μl, flow rate: 1.0 ml / min, wavelength: 280 nm.

[0087] See results Figure 6 and 7 As shown, through with Figure 4 In comparison, using gradient two resulted in poor separation of the characteristic peaks at 10-15 minutes (separation less than 1.5), while using gradient three resulted in less information in the characteristic peaks and some overlapping of the characteristic peaks. Therefore, adjusting the gradient did not improve the separation of the characteristic peaks.

[0088] Table 1 Gradient conditions for gradients one through three.

[0089]

[0090] (3) Investigation at different column temperatures

[0091] Based on gradient 1, column temperatures of 25℃ and 30℃ were investigated. Specifically, high performance liquid chromatography (HPLC) was used to detect the test solution prepared according to item 2 of this experimental example. An Agilent Poroshell 120EC-C18 column, 4.6 mm × 150 mm, 2.7 μm, was used. Acetonitrile was used as mobile phase A, and 0.05 mol / L potassium dihydrogen phosphate (pH adjusted to 3.7 with phosphoric acid) was used as mobile phase B. Gradient elution was performed according to the specifications of gradient 1. The column temperatures were 25℃ and 35℃, respectively. The injection volume was 5 μl, the flow rate was 1.0 ml / min, and the wavelength was 280 nm.

[0092] See results Figure 8 and 9 As shown, through with Figure 4Compared to conditions at 30℃ and 35℃, the resolution and peak shape of the chromatographic peaks were significantly improved at 25℃. Therefore, it is recommended to optimize the gradient with a column temperature of 25℃.

[0093] (4) Investigation of the secondary gradient elution procedure

[0094] The test solution prepared according to item 2 of this experimental example was detected by high performance liquid chromatography. An Agilent Poroshell 120EC-C18 column, 4.6 mm × 150 mm, 2.7 μm, was used. Acetonitrile was used as mobile phase A, and 0.05 mol / L potassium dihydrogen phosphate (adjusted to pH 3.7 with phosphoric acid) was used as mobile phase B. Gradient elution was performed according to gradient one, gradient two, and gradients four to seven as specified in Tables 2 and 3, respectively. Column temperature: 25 °C, injection volume: 5 μl, flow rate: 1.0 ml / min, wavelength: 280 nm.

[0095] The results are shown in Table 4. Using gradient one, the characteristic peak symmetry of number 3 is poor. Using gradient four, the separation degree between the characteristic peak of number 3 and surrounding impurities is less than 1.5, and the characteristic peaks of numbers 5 and 9 also have poor symmetry, exhibiting peak encirclement. Using gradient five, the separation degree between the characteristic peak of number 3 and surrounding impurities is less than 1.5, and the characteristic peak of number 8 also has poor symmetry, exhibiting peak encirclement. Therefore, adjusting the gradient does not improve the separation degree of the characteristic peaks. Using gradients six and seven, the characteristic peak symmetry is better, with gradient seven exhibiting the best characteristic peak symmetry. Therefore, gradient seven is recommended as the preferred gradient.

[0096] Table 2 Gradient conditions for gradients four to five

[0097]

[0098] Table 3 Gradient conditions for gradients 6 to 7

[0099]

[0100] Table 4 Peak Results

[0101]

[0102]

[0103] (5) Experiment investigating the pH of mobile phase B

[0104] The test solution of *Tinospora sinensis* granules was prepared according to the method described in section 2 of this experimental example. The test solution was then analyzed using the chromatographic conditions of gradient six in this experimental example, with the only difference being the pH of mobile phase B. Mobile phase B consisted of 0.05 mol / L potassium dihydrogen phosphate, adjusted to different pH values ​​with phosphoric acid. The different pH values ​​were 3.0, 3.4, 3.7, and 4.0.

[0105] See results Figure 10-13 As shown, the separation effect of each chromatographic peak is better under the condition of pH 3.0-4.0, and the preferred pH is 3.0-3.7, with pH 3.7 being the best choice.

[0106] 4. Preparation of the test solution

[0107] (1) Examination of extraction methods

[0108] The effects of different extraction methods—ultrasonic treatment (350W, 40kHz) for 30 minutes and reflux for 30 minutes—on the extraction efficiency of *Tinospora sinensis* (kuanjinteng) formulation granules were investigated. *Tinospora sinensis* formulation granules were finely ground, and approximately 0.2g was placed in a stoppered conical flask. 10ml of 70% methanol was added, and the flask was sealed tightly. The mixture was then subjected to ultrasonic treatment (350W, 40kHz) for 30 minutes or reflux for 30 minutes, respectively. After cooling, the solution was filtered, and the filtrate was collected to obtain the test solution. Chromatographic analysis was performed under the conditions described in Example 1. The information content of the chromatographic peaks and system suitability parameters were used as the main evaluation indicators. The results showed that the extraction method did not significantly affect the information content of the chromatographic peaks, system suitability parameters, or the total peak area. Considering the simplicity of the method, ultrasonic treatment was chosen as the extraction method for further investigation.

[0109] Table 5. Chromatographic parameters of *Tinospora sinensis* (Kuaijin Teng) formulation granules under different treatment methods

[0110]

[0111] (2) Investigation on the amount of extraction solvent used

[0112] Based on the extraction conditions determined above, the separation effect of extraction times of 15 min, 30 min, and 45 min was further investigated. The *Tinospora sinensis* formula granules were finely ground, and approximately 0.2 g was placed in a stoppered conical flask. 10 ml of 70% methanol was added, and the flask was sealed tightly. The flask was then ultrasonically treated (350 W power, 40 kHz frequency) for 15 min or 45 min, respectively. After cooling, the flask was filtered, and the filtrate was collected to obtain the test solution. Chromatographic analysis was performed according to the conditions in Example 1. The results were compared with those in Table 5 where the ultrasonic extraction time was 30 min. The experimental results showed that the total peak area of ​​the characteristic peaks did not differ significantly with the extension of extraction time, indicating that within this range, all characteristic components could be completely extracted. Considering the extraction efficiency, 30 min was selected as the extraction time.

[0113] Table 6. Chromatographic peak system adaptability parameters at different extraction times.

[0114]

[0115]

[0116] (3) Selection of extraction solvent

[0117] Taking all factors into consideration, water, methanol, and ethanol were tested separately. The *Tinospora sinensis* formula granules were finely ground, and approximately 0.2 g was placed in a stoppered conical flask. 10 ml of methanol, water, or ethanol were added to each flask, which was then sealed tightly. The flasks were ultrasonically treated (350 W, 40 kHz) for 30 min, cooled, filtered, and the filtrate was collected to obtain the test solution. Chromatographic analysis was performed under the conditions described in Example 1. The results showed that the total peak area of ​​methanol and water as solvents was relatively better than that of ethanol, and the system suitability parameters of most chromatographic peaks were also relatively better.

[0118] Table 7. Chromatographic parameters of *Tinospora sinensis* (Kuaijin Teng) formulation particles under different extraction solvents.

[0119]

[0120] The extraction effects of 50% methanol solution and 70% methanol solution on the granules of *Tinospora sinensis* (kuanjinteng) were further investigated. *Tinospora sinensis* granules were finely ground, and approximately 0.2g was placed in a stoppered conical flask. 10ml of 50% methanol solution and 10ml of 70% methanol solution were added respectively. The flasks were sealed tightly and ultrasonically treated (350W power, 40kHz frequency) for 30min respectively. After cooling, the flasks were filtered, and the filtrate was collected to obtain the test solution. Chromatographic analysis was performed under the conditions described in Example 1.

[0121] Table 8. Chromatographic parameters of *Tinospora sinensis* (Kuaijin Teng) formulation particles under different extraction solvents.

[0122]

[0123]

[0124] By comparing the chromatograms, system adaptability parameters of chromatographic peaks and total peak area obtained by extraction with various solvents, the chromatographic peak response values ​​obtained by extraction with 70% methanol and 50% methanol solutions were larger. Considering the overall extraction efficiency, 70% methanol was selected as the extraction solvent for this experiment.

[0125] (4) Selection of solvent addition amount

[0126] Based on the extraction method and solvent determined above, the effect of extraction solvent volumes of 5 ml, 10 ml, 15 ml, and 25 ml on the extraction effect of *Tinospora sinensis* (kuanjinteng) formulation granules was further investigated. *Tinospora sinensis* formulation granules were finely ground, and approximately 0.2 g was placed in a stoppered conical flask. 5 ml, 15 ml, and 25 ml of 70% methanol solution were added respectively. The flasks were sealed tightly and ultrasonically treated (350 W, 40 kHz) for 30 min. After cooling, the flasks were filtered, and the filtrate was collected to obtain the test solution. Chromatographic analysis was performed under the conditions described in Example 1. The results were compared with those obtained by adding 10 ml of 70% methanol in Table 8. The results showed that when the extraction solvent was 5-25 ml, there was no significant difference in the total peak area of ​​the characteristic peaks multiplied by the corresponding solvent volume, indicating that the components could be completely extracted within this range. Considering the moderate peak area response value, an extraction solvent volume of 10 ml was selected.

[0127] Table 9 Comparison of chromatographic peak system adaptability parameters for different extraction solvent volumes of *Tinospora sinensis* (kuanjinteng) formulation granules.

[0128]

[0129]

[0130] (5) Selection of sampling size

[0131] Based on the extraction conditions determined above, the effects of different sample amounts of *Tinospora sinensis* (kuanjinteng) formula granules (0.1g, 0.2g, 0.3g, and 0.4g) on ​​the extraction efficiency of *Tinospora sinensis* (kuanjinteng) formula granules were further investigated. The information content of chromatographic peaks and system adaptability parameters were used as the main evaluation indicators. *Tinospora sinensis* formula granules were finely ground, and approximately 0.1g, 0.3g, and 0.4g were taken respectively and placed in stoppered conical flasks. 10ml of 70% methanol solution was added to each flask, and the flasks were sealed tightly. The flasks were then sonicated (350W power, 40kHz frequency) for 30min, cooled, filtered, and the filtrate was collected to obtain the test solution. The chromatographic conditions of Example 1 were used for determination. The results were compared with those in Table 8 when 0.2g of 70% methanol was added. The above experimental results show that the system adaptability parameters of the chromatographic peaks do not differ significantly under different sampling amounts. However, when the sampling amount is 0.2g, the peak height and peak width of the chromatographic peaks are relatively moderate. Therefore, 0.2g was selected as the sampling amount for this experiment.

[0132] Table 10 Comparison of chromatographic peak system adaptability parameters for different sampling amounts of *Tinospora sinensis* (kuanjinteng) formulation granules.

[0133]

[0134]

[0135] 5. Determination of characteristic peaks and establishment of reference spectra

[0136] (1) Identification of characteristic peaks

[0137] Nine characteristic peaks of the formula granules of *Tinospora sinensis* (kuanjinteng) were identified and designated using HPLC.

[0138] Accurately weigh appropriate amounts of magnoflorine and tryptophan reference standards, and dissolve them in methanol to prepare a solution containing 50 μg of magnoflorine and 50 μg of tryptophan per ml, thus obtaining a mixed reference solution. Accurately weigh appropriate amounts of isovanillin reference standard, and dissolve them in methanol to prepare a solution containing 20 μg of isovanillin per ml, thus obtaining an isovanillin reference solution. Accurately weigh appropriate amounts of syringin reference standard, and dissolve them in methanol to prepare a solution containing 20 μg of syringin per ml, thus obtaining a syringin reference solution. Grind an appropriate amount of *Tinospora sinensis* granules into a fine powder, take about 0.2 g, place it in a stoppered conical flask, add 10 ml of 70% methanol, seal tightly, sonicate (350 W, 40 kHz) for 30 minutes, cool, filter, and collect the filtrate to obtain the test solution.

[0139] Accurately pipette 5 μl each of the above-mentioned reference solution and test solution, inject them into the liquid chromatograph, and determine the results according to the chromatographic conditions of Example 1. Figure 14-16 Peak 1 was identified as tryptophan, peak 2 as syringin, peak 5 as isovanillin, and peak 7 as magnoflorine.

[0140] By comparing and analyzing the liquid chromatography-ultraviolet absorption spectra of the test solution and the reference solution, and by using LC / MS / MS (liquid chromatography-mass spectrometry) to identify and characterize the nine characteristic peaks of the Kuanjinteng (Kuanjinteng) formulation granules, it was confirmed that peak 1 is tryptophan, peak 2 is syringin, peak 5 is isovanillin, and peak 7 is magnoflorine.

[0141] (2) Construction of characteristic spectra of multiple batches of Tinospora sinensis drug preparations

[0142] Eighteen batches of freeze-dried *Tinospora sinensis* powder and three batches of formulation granules were taken and tested according to the method in Example 1. The solutions were then analyzed by high-performance liquid chromatography (HPLC). The results are as follows: Figure 17 As shown, where Figure 17 In the figure, S1(9)~S21(9) are the characteristic spectra of 18 batches of freeze-dried powder of *Tinospora sinensis* and 3 batches of formulation granules of *Tinospora sinensis*.

[0143] Table 11. Results of relative retention time analysis of characteristic spectra of 18 batches of lyophilized powder and 3 batches of formulation granules.

[0144]

[0145]

[0146] Table 12. Detection results of relative peak areas in the characteristic spectra of 18 batches of lyophilized powder and 3 batches of formulation granules.

[0147]

[0148]

[0149] (3) Construction of characteristic maps and definition of characteristic peaks

[0150] The fingerprint chromatograms of multiple batches of test samples were analyzed. The "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" software, compiled by the Pharmacopoeia Commission, was used for fitting and generating a reference characteristic chromatogram using the "multi-point correction, MARK peak matching" mode. The reference characteristic chromatograms of the obtained *Tinospora sinensis* (kuanjinteng) lyophilized powder and *Tinospora sinensis* formulation granules showed a total of 9 chromatographic peaks. (See attached image). Figure 18As shown, based on the results of different chromatographic columns and instruments, it is recommended to use peak 7 (magnoliaine) as the reference peak to calculate the relative retention time and relative peak area of ​​each characteristic peak, which can better evaluate the relative retention time and relative peak area of ​​each characteristic peak. Considering that the retention time of peak 1 (tryptophan) is close to that of the solvent peak and is easily affected by various factors, it is recommended to use the reference standard for matching. The relative retention times of the remaining characteristic peaks with peak S are 0.53 (peak 2), 0.68 (peak 3), 0.71 (peak 4), 0.81 (peak 5), 0.94 (peak 6), 1.34 (peak 8), and 1.47 (peak 9). The characteristic chromatogram results of 18 batches of lyophilized *Tinospora sinensis* powder and 3 batches of *Tinospora sinensis* formulation granules show that the 9 characteristic peaks should correspond to the reference medicinal material chromatograms. The 9 common peaks have good transferability, and the relative retention times of each characteristic peak in the 21 batches of samples are all within ±10% of the specified value.

[0151] Therefore, it is stipulated that the characteristic chromatogram of *Tinospora sinensis* (kuanjinteng) formula granules has 9 characteristic peaks, which should correspond to the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peaks 1 and 7 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the magnoflorine reference peak is the S peak. The relative retention times of each characteristic peak are calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are 0.53 (peak 2), 0.68 (peak 3), 0.71 (peak 4), 0.81 (peak 5), 0.94 (peak 6), 1.34 (peak 8), and 1.47 (peak 9).

[0152] Table 13 Relative retention times of the comparative atlases

[0153]

[0154] Table 14: Relative Peak Areas of Comparative Spectra

[0155]

[0156] Experiment Example 2: Methodological Validation

[0157] 1. System adaptability

[0158] The negative control solution (70% methanol solution) was tested according to the high-performance liquid chromatography method of Example 1. The results showed that... Figure 19 As shown, the negative control did not interfere with the characteristic chromatogram, and the chromatographic method has good system adaptability and specificity, and can be used as a method for detecting the characteristic chromatogram of *Tinospora sinensis* (kuanjinteng) formulation granules.

[0159] 2. Instrument precision

[0160] The same sample solution of *Tinospora sinensis* granules prepared according to the method in Example 1 was tested using the high-performance liquid chromatography method described in Example 1. The injection was repeated six times, and the RSDs of the relative retention times and relative peak areas of the nine characteristic peaks were all less than 2% (see Table 15). This indicates good instrument precision.

[0161] Table 15 Results of Instrument Precision and Relative Retention Time Tests

[0162]

[0163] Table 16 Results of Instrument Precision Relative Peak Area Test

[0164]

[0165] 3. Repeatability

[0166] Six replicates of the same sample of *Vitis thunbergii* granules were prepared using the method described in Example 1. The relative retention times and relative peak areas of nine characteristic peaks were determined under chromatographic conditions. The results showed that the RSDs of the relative retention times of the nine characteristic peaks and the reference peak were all less than 2% (see Table 17). This indicates that the method has good repeatability.

[0167] Table 17 Results of the method repeatability relative retention time test

[0168]

[0169]

[0170] Table 18 Results of the method repeatability test for relative peak area

[0171]

[0172] 4. Intermediate precision (for different operators)

[0173] Three inspectors, at different times, took the same sample of *Tinospora sinensis* (kuanjinteng) granules and prepared test solutions according to the preparation method in Example 1. Using the same equipment, they tested the relative retention times and relative peak areas of each common peak according to the high-performance liquid chromatography method in Example 1. The results showed that the relative average deviation of the relative retention times of the nine characteristic peaks from the reference peak was less than 2% (see Table 19). This indicates that the method has good intermediate precision.

[0174] Table 19 Intermediate Precision Relative Retention Time Test Results (Different Operators)

[0175]

[0176] Table 20 Results of Intermediate Precision Relative Peak Area Tests (Different Operators)

[0177]

[0178] 5. Durability

[0179] (1) Stability test

[0180] The same sample solution of *Vitis thunbergii* granules prepared according to the method in Example 1 was injected into the sample at 0, 4, 8, 12, 16, and 24 hours using the high-performance liquid chromatography (HPLC) method described in Example 1. The relative retention times and relative peak areas of each common peak were measured. The results showed that the deviations of the relative retention times of each chromatographic peak from the reference peak were all less than 2% (see Table 21). This indicates that the sample solution was stable within 24 hours and met the measurement requirements.

[0181] Table 21 Results of the relative retention time test for stability

[0182]

[0183]

[0184] Table 22 Results of Stability Relative Peak Area Test

[0185]

[0186] (2) Investigation at different column temperatures

[0187] The same sample solution of *Vitis thunbergii* granules prepared according to the method in Example 1 was tested by high performance liquid chromatography according to Example 1. The column temperatures were set to 23℃, 25℃, and 27℃, respectively. The effects of different column temperatures on the relative retention time and relative peak area of ​​each characteristic peak were investigated. The results showed that when the column temperature changed, the deviation of the relative retention time of the nine characteristic peaks was within 3% (see Table 23), indicating that the column temperature was robust and could be used at 23-27℃.

[0188] Table 23 Comparison of relative retention times at different column temperatures

[0189]

[0190] Table 24 Comparison of relative peak areas at different column temperatures

[0191]

[0192] (3) Investigation of different flow velocities

[0193] The same sample solution of *Vitis thunbergii* granules prepared according to the method in Example 1 was tested by high performance liquid chromatography according to Example 1. Flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min were used to investigate the effect of slight changes in flow rate on the relative retention time and relative peak area of ​​each characteristic peak. The results showed that when the flow rate changed slightly, the deviation of the relative retention time of each characteristic chromatographic peak was within 4.0% (see Table 25), indicating good flow rate robustness. A flow rate of 0.9-1.1 ml / min is considered suitable, with 1.0 ml / min being preferred.

[0194] Table 25 Comparison of Relative Retention Times for Different Flow Rates

[0195]

[0196] Table 26 Comparison of relative peak area results for different flow velocities

[0197]

[0198] (4) Investigation with different instruments

[0199] The same sample solution of *Vitis thunbergii* granules prepared according to the method in Example 1 was tested by high performance liquid chromatography (HPLC) according to Example 1. Different instruments (Waters, Agilent, Thermo) were used for injection analysis to investigate the effects of different instruments on the relative retention time and relative peak area of ​​each characteristic peak. The results showed that the use of instruments from different manufacturers had a certain impact on the relative retention time of each characteristic chromatographic peak. However, within ±10% of the specified value, the separation effect and peak shape of the nine characteristic peaks were good under different instruments. Therefore, it is recommended to combine the results of the investigation of different instruments and fix the specified value of the relative retention time of each characteristic peak within ±10%.

[0200] Table 27 Comparison of relative retention time results from different instruments

[0201]

[0202] Table 28 Comparison of relative peak area results from different instruments

[0203]

[0204]

[0205] (5) Investigation of different chromatographic columns

[0206] The same sample solution of *Vitis thunbergii* granules prepared according to the method in Example 1 was tested using high-performance liquid chromatography (HPLC) as described in Example 1. Columns were set as follows: Agilent Poroshell 120EC-C18 (4.6 mm × 150 mm, 2.7 μm), SHIMADZU Shim-pack GIST C18-AQ (4.6 mm × 150 mm, 3.0 μm), and Waters CORTECS T3 (4.6 mm × 150 mm, 2.7 μm). The relative retention times and relative peak areas of each characteristic peak compared to the reference peak were investigated for different columns. The results showed that different types of columns had a significant impact on the relative retention times of the characteristic peaks. The separation effect and peak shape of the nine characteristic peaks were good under different columns. The preferred column was Agilent Poroshell 120EC-C18 (4.6 mm × 150 mm, 2.7 μm).

[0207] Table 29 Comparison of relative retention times for different chromatographic columns

[0208]

[0209] (6) Investigation of different pH values ​​of mobile phase

[0210] The same sample solution of *Vitis thunbergii* granules prepared according to the method in Example 1 was tested using the high-performance liquid chromatography (HPLC) method described in Example 1. The samples were analyzed using 0.05 mol / L potassium dihydrogen phosphate (adjusted to pH 3.5 with phosphoric acid), 0.05 mol / L potassium dihydrogen phosphate (adjusted to pH 3.7 with phosphoric acid), and 0.05 mol / L potassium dihydrogen phosphate (adjusted to pH 3.9 with phosphoric acid). The effect of slight pH changes on the relative retention time and relative peak area of ​​each characteristic peak was investigated. The results showed that when the pH changed slightly, different mobile phase pH values ​​had little effect on the relative retention time of the characteristic peaks. The deviation of the relative retention time of each characteristic chromatographic peak was within 3.0%, and the pH range of 3.5-3.9 was suitable. Therefore, this method recommends using 0.05 mol / L potassium dihydrogen phosphate (adjusted to pH 3.7 with phosphoric acid) for the determination.

[0211] Based on the above methodological investigation results, a method for establishing the characteristic chromatogram of *Tinospora sinensis* (kuanjinteng) formulation granules was developed. The overall results indicate that this method is simple to operate, yields accurate results, and has good reproducibility.

[0212] Example 1

[0213] This embodiment provides a method for constructing the characteristic spectrum of *Vitis thunbergii* formulation granules, including the following steps:

[0214] Preparation of the test solution: Take an appropriate amount of the test sample, grind it into a fine powder, take about 0.2g, place it in a stoppered conical flask, add 10ml of 70% methanol, seal tightly, sonicate (power 350W, frequency 40kHz) for 30 minutes, cool, filter, and take the filtrate to obtain the test solution.

[0215] Preparation of reference solution: Take about 2g of *Tinospora sinensis* reference material, place it in a stoppered conical flask, add 100ml of water, heat under reflux for 30min, filter, evaporate the filtrate to dryness, add 10ml of 70% methanol to the residue, seal tightly, sonicate (350W, 40kHz) for 30min, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of tryptophan reference standard and magnoflorine reference standard, accurately weigh them, add methanol to prepare a solution containing 50μg of each per ml, as the reference solution.

[0216] High-performance liquid chromatography (HPLC) test: Accurately pipette 5 μl each of the reference solution and the test solution into the HPLC system and determine the result. The chromatographic conditions are as follows: octadecylsilane-bonded silica gel is used as the stationary phase (column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm); acetonitrile is used as mobile phase A, and 0.05 mol / L potassium dihydrogen phosphate (adjusted to pH 3.7 with phosphoric acid) is used as mobile phase B, with gradient elution as specified in the table below; column temperature is 25℃; flow rate is 1.0 ml / min; detection wavelength is 280 nm. The theoretical plate number, calculated based on the magnoflorine peak, should not be less than 5000.

[0217] Table 30 Gradient Elution Procedure

[0218]

[0219] Table 31 Results of peak comparison of medicinal materials

[0220]

[0221] Table 32 Results of Formulation Particle Peaks

[0222]

[0223] Table 33 Results of control standard peaks

[0224]

[0225] From the above table and Figure 20-22 It can be seen that, Figure 21The nine characteristic peaks in the sample were well separated, exhibited good symmetry, had high peak areas, and required short detection and analysis times. The test sample chromatogram should show nine characteristic peaks, which should correspond to the nine characteristic peaks in the reference chromatogram of the medicinal material. Peaks 1 and 7 correspond to the retention times of their respective reference peaks. The peak corresponding to the magnoflorine reference peak is designated as the S peak. The relative retention times of each characteristic peak should be calculated and should be within ±10% of the specified values. The specified values ​​are 0.53 (peak 2), 0.68 (peak 3), 0.71 (peak 4), 0.81 (peak 5), 0.94 (peak 6), 1.34 (peak 8), and 1.47 (peak 9).

[0226] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this application.

Claims

1. A method for constructing a characteristic map of a wideleaf fig formula granule, characterized by, Includes the following steps, (1) Preparation of test solution: Weigh the test sample, add extraction solvent to extract, obtain extract, separate solid and liquid, take the liquid, which is the test solution; the extraction method is heating reflux extraction or ultrasonic extraction; the extraction solvent is selected from one or more of methanol, ethanol, and water; the construction method also includes the step of preparing reference solution by adding solvent to tryptophan, syringin, isovanillin and magnoflorine; (2) The test solution and reference solution were analyzed by high performance liquid chromatography. Octadecylsilane-bonded silica gel was used as the stationary phase, acetonitrile was used as the mobile phase A, and an aqueous solution containing potassium dihydrogen phosphate was used as the mobile phase B. The column temperature was 23-27℃, and gradient elution was performed according to the following procedure: From 0 to 15 min, the volume ratio of mobile phase A to mobile phase B was 6%:94% to 10%:90%. 15→20min, the volume ratio of mobile phase A to mobile phase B is 10%:90%; The flow rate was 20-40 min, with the volume ratio of mobile phase A to mobile phase B being 10%:90%-20%:80%; the detection wavelength was 280 nm; the flow rate was 1.0 mL / min; the concentration of the aqueous solution containing potassium dihydrogen phosphate was 0.03-0.08 mol / L, and the pH of the aqueous solution containing potassium dihydrogen phosphate was adjusted to 3.7 using phosphoric acid; a chromatographic column with dimensions of 4.6 mm × 150 mm and a diameter of 2.7-3.0 µm was used.

2. The construction method of claim 1, wherein, The injection volume is 3-10 μl.

3. The construction method of claim 1, wherein, Step (1) also satisfies any one or more of the following AD: A. The ratio of the mass of the test sample to the volume of the solvent is 0.1-0.4:5-25; the relationship between mass and volume is g / mL. B. Extraction time is ≥10 min; C. The solid-liquid separation is selected from centrifugation or membrane filtration; D. The extraction solvent is selected from a methanol aqueous solution with a volume percentage of 50-70%.

4. The construction method of claim 1, wherein, The extraction time is 15-40 minutes.

5. The construction method according to claim 1, characterized in that, Each 1 mL of the reference solution contains at least one of 1-100 µg tryptophan, 1-100 µg syringin, 1-100 µg isovanillin and 1-100 µg magnoflorine; and / or, the solvent used in the preparation of the reference solution is selected from methanol or an aqueous methanol solution.

6. The construction method according to any one of claims 1-5, characterized in that, The construction method further includes the steps of preparing a reference solution of reference medicinal material by extracting the reference medicinal material of *Tinospora sinensis* with water according to the preparation method of the test solution in any of the construction methods of claims 1-5, and detecting the reference solution of reference medicinal material by high performance liquid chromatography according to any of the construction methods of claims 1-5 to obtain the reference medicinal material reference spectrum.

7. The construction method according to claim 6, characterized in that, After water extraction of the *Tinospora sinensis* reference material, the solution is filtered, dried, and then prepared into a reference solution according to the preparation method of the test solution in any of the construction methods described in claims 1-5.

8. The construction method according to any one of claims 1-5, characterized in that, The characteristic spectrum of the *Vitis thunbergii* formula granules has 9 common characteristic peaks. Peaks 1 and 7 correspond to the retention times of the reference peaks of tryptophan and magnoflorine, respectively. The peak corresponding to the reference peak of magnoflorine is the S peak. The relative retention times of peaks 2-6 and peaks 8-9 with the S peak are within ±10% of the specified values. The specified values ​​of peaks 2-6 and peaks 8-9 are 0.53, 0.68, 0.71, 0.81, 0.94, 1.34, and 1.47, respectively.

9. The application of the method for constructing the characteristic spectrum of the *Tinospora sinensis* formula granules according to any one of claims 1-8 in the quality inspection of *Tinospora sinensis* formula granules.

10. A method for quality testing of *Vitis thunbergii* (a type of vine) formulation granules, characterized in that... The method includes a step of comparing the characteristic spectrum of the product to be tested, *Tinospora sinensis*, with the control characteristic spectrum of *Tinospora sinensis* granules; the characteristic spectrum of the product to be tested is constructed using the product to be tested according to any one of the construction methods described in claims 1-8, and the control characteristic spectrum of the *Tinospora sinensis* granules is selected from any one of the following (1)-(3): (1) It has 9 common characteristic peaks. Peaks 1 and 7 correspond to the retention times of the reference peaks of tryptophan and magnoflorine, respectively. The peak corresponding to the reference peak of magnoflorine is the S peak. The relative retention times of peaks 2-6 and peaks 8-9 with the S peak are within ±10% of the specified values. The specified values ​​of peaks 2-6 and peaks 8-9 are 0.53, 0.68, 0.71, 0.81, 0.94, 1.34, and 1.47, respectively. (2) The characteristic spectrum of the *Tinospora sinensis* formulation granules obtained by using single or multiple batches of the formulation granules according to any one of the construction methods described in claims 1-7; (3) Using multiple batches of *Vitis thunbergii* formulation granules, the characteristic spectrum obtained by any of the construction methods described in claims 1-7 is used to prepare a control characteristic spectrum by means of the average or median method.

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