Construction method and quality detection method of specific chromatogram of tinospora sinensis merr medicinal preparation

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly provides a construction method and a quality detection method of a specific chromatogram of a tinospora sinensis merr medicinal preparation, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, an aqueous solution containing monopotassium phosphate is used as a mobile phase B, and a gradient elution procedure is continuously optimized; according to the method, the column temperature is controlled to be 23-27 DEG C, 11 common characteristic peaks are finally obtained, effective separation of 9 common characteristic peaks is achieved, the obtained characteristic chromatogram is stable in base line, good in characteristic peak shape and short in detection time, and a scientific basis is provided for comprehensively establishing the quality control standard of the tinospora sinensis merr formula granules. And moreover, the peak positions of tryptophan, syringin, isovanillin and magnoflorine can be accurately positioned, the known information of characteristic peaks is increased, and the integrity and characteristics of the tinospora sinensis merr medicinal preparations (such as preparations such as formula granules) are fully reflected.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and specifically relates to a method for constructing a characteristic fingerprint 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, liver protection, antioxidant, and immune regulation. 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 extracting, concentrating, drying, and preparing 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, there is a literature research report "Study on the UPLC Characteristic Fingerprint 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 fingerprints of multiple batches that the elution times of the characteristic peaks in the fingerprint established by it 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 prepared from Tinospora sinensis drugs such as Tinospora sinensis formula granules, and there are defects such as few characteristic peaks and poor separation effect. The characteristic fingerprint of Tinospora sinensis (Tinospora sinensis) formula granules promulgated by the Guangdong Provincial Traditional Chinese Medicine Formula Granule Standard Yue PFKL20240016 also has problems of poor separation effect of characteristic peaks and few characteristic peaks. Summary of the Invention

[0004] Therefore, the purpose of the present invention is to provide a method for constructing a characteristic fingerprint 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 fingerprint 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] To this end, the present invention provides a method for constructing a characteristic fingerprint of a Tinospora sinensis drug preparation, including the following steps,

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

[0007] (2) The test solution is detected by high performance liquid chromatography. Octadecylsilane chemically bonded 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. The column temperature is 23 - 27 °C, and gradient elution is carried out according to the following procedure:

[0008] 0 → 15 min, the volume ratio of mobile phase A to mobile phase B is 6%:94% → 10%:90%;

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

[0010] 20 → 40 min, 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 - 300 nm, preferably 280 nm;

[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 detection by high performance liquid chromatography, a chromatographic column with a specification of 4.6 mm × 150 mm, 2.7 - 3.0 μm is used.

[0017] Furthermore, step (1) includes weighing the test sample, extracting with a solvent to obtain an extract, separating the solid and liquid, and taking the liquid as the test solution.

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

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

[0020] B. The extraction method is heating 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, an aqueous methanol solution with a volume percentage of 50 - 70%.

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

[0025] Further, the construction method further includes the steps of preparing a reference solution with at least one of tryptophan, syringin, isovanillin, and magnoflorine plus a solvent, and detecting the reference solution by the high-performance liquid chromatography method in any of the above-mentioned construction methods to obtain a reference spectrum of the reference substance.

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

[0027] Further, the construction method further includes the steps of preparing a reference solution of the reference medicinal material of Tinospora sinensis according to the preparation method of the test solution in any of the above-mentioned construction methods with the extract obtained by water extraction of the reference medicinal material of Tinospora sinensis, and detecting the reference solution of the reference medicinal material by the high-performance liquid chromatography method in any of the above-mentioned construction methods to obtain a reference spectrum of the reference medicinal material.

[0028] Preferably, after water extraction of the reference medicinal material of Tinospora sinensis, it is filtered, the liquid is taken and dried, and then the reference solution of the reference medicinal material is prepared according to the preparation method of the test solution in any of the above-mentioned construction methods.

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

[0030] Furthermore, the characteristic chromatogram of the Tinospora sinensis drug preparation has 9 common characteristic peaks. The retention times of peak 1 and peak 7 correspond to those of the tryptophan and magnoflorine reference substance peaks respectively. The peak corresponding to the magnoflorine reference substance peak is the S peak. The relative retention times of peaks 2 - 6 and peaks 8 - 9 with respect to the S peak are within the range of ±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 in sequence.

[0031] In the present invention, the Tinospora sinensis drug preparation can be Tinospora sinensis formula granules, or freeze-dried powder of Tinospora sinensis standard decoction, etc., which are common preparations prepared by water extraction of Tinospora sinensis.

[0032] In certain embodiments, it further includes the construction of the control characteristic chromatogram of the Tinospora sinensis drug preparation. For the characteristic chromatograms obtained by testing the test samples of multiple batches of Tinospora sinensis drug preparations, the control characteristic chromatogram of the Tinospora sinensis drug preparation is generated using the similarity evaluation system for traditional Chinese medicine chromatographic characteristic chromatograms. At least 3 batches of Tinospora sinensis formula granules are used, for example, 2 batches, 10 batches, 15 batches, 18 batches of Tinospora sinensis formula granules.

[0033] In certain embodiments, after generating the control characteristic chromatogram of the Tinospora sinensis drug preparation using the similarity evaluation software for traditional Chinese medicine chromatographic characteristic chromatograms, it further includes the step of marking the common characteristic peaks.

[0034] The present invention also provides the application of the method for constructing the characteristic chromatogram of the Tinospora sinensis drug preparation described in any one of the above in the quality inspection of the Tinospora sinensis drug preparation.

[0035] The present invention also provides a quality inspection method for the Tinospora sinensis drug preparation, including the step of comparing the characteristic chromatogram of the Tinospora sinensis product to be tested with the control characteristic chromatogram of the Tinospora sinensis drug preparation; the characteristic chromatogram of the Tinospora sinensis product to be tested is constructed using the Tinospora sinensis product to be tested according to the construction method described in any one of the above, and the control characteristic chromatogram of the Tinospora sinensis drug preparation is selected from any one of the following (1) - (3):

[0036] (1) It has 9 common characteristic peaks. The retention times of peak 1 and peak 7 correspond to those of the tryptophan and magnoflorine reference substance peaks respectively. The peak corresponding to the magnoflorine reference substance peak is the S peak. The relative retention times of peaks 2 - 6 and peaks 8 - 9 with respect to the S peak are within the range of ±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 in sequence;

[0037] (2) The characteristic chromatogram of the Tinospora sinensis drug preparation obtained by using a single batch or multiple batches of Tinospora sinensis drug preparations according to the construction method described in any one of the above;

[0038] (3) Using multiple batches of Caulis Spatholobi drug preparations and obtaining characteristic spectra according to any of the construction methods described above, a control characteristic spectra is prepared by using the average value or median method.

[0039] %Methanol represents the volume percentage of methanol in the methanol-water solution.

[0040] The base source of the medicinal material is the plant Rhizoma Coptidis. The base source of the medicinal material is indicated in the brackets.

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

[0042] 1. The method for constructing the characteristic spectrum of the Herba Lycopodii var. ilicotii pharmaceutical preparation described in the present invention comprises the following steps: using octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and an aqueous solution containing potassium dihydrogen phosphate as a mobile phase B, and performing gradient elution according to the following procedure by continuously optimizing the gradient elution program: 0→15min, the volume ratio of mobile phase A to mobile phase B is 6%:94%→10%:90%; 15→20min, the volume ratio of mobile phase A to mobile phase B is 10%:90%; 20→40min, the volume ratio of mobile phase A to mobile phase B is 10%:90%→19-21%:79-81%; and combining with controlling the column temperature to be 23-27°C, 11 common characteristic peaks are finally obtained, and effective separation of 9 common characteristic peaks is achieved, and the obtained characteristic spectrum has a stable baseline, good characteristic peak shape, and short detection time, which provides a scientific basis for comprehensively establishing the quality control standard of Herba Lycopodii var. ilicotii formula granules. Moreover, the peak positions of tryptophan, syringin, isovanillin and magnolia alkaloids can be accurately located, the known information of characteristic peaks can be increased, and the integrity and characteristic of the drug preparations of the broad-tendon vine (such as the formulation granules and other preparations) can be fully reflected.

[0043] 2. The method for constructing the characteristic spectrum of the Caulis Schizonepetae drug preparation described in the present invention is to investigate the extraction conditions such as the optimization of chromatographic conditions (pH, wavelength, flow rate of mobile phase B, etc.), type of extraction solvent, and amount of extraction solvent, and determine the optimal extraction process and chromatographic conditions, so that the peak area is higher, the separation effect and peak shape are better, and the quality of the Caulis Schizonepetae formula granules can be more comprehensively monitored.

[0044] 3. The quality detection method for the radix strychnifolia pharmaceutical preparation described in the present invention can comprehensively, clearly and effectively detect the quality of the radix strychnifolia formula granules by comparing the characteristic spectrum of the radix strychnifolia formula granules to be tested with the control characteristic spectrum of the radix strychnifolia formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0046] Figure 1 It is the chromatogram at 220 nm in the investigation and test of different wavelengths in Experimental Example 1;

[0047] Figure 2 It is the chromatogram at 240 nm in the investigation and test of different wavelengths in Experimental Example 1;

[0048] Figure 3 It is the chromatogram at 260 nm in the investigation and test of different wavelengths in Experimental Example 1;

[0049] Figure 4 It is the chromatogram at 280 nm in the investigation and test of different wavelengths in Experimental Example 1;

[0050] Figure 5 It is the chromatogram at 300 nm in the investigation and test of different wavelengths in Experimental Example 1;

[0051] Figure 6 It is the chromatogram of gradient two in the investigation and test of the first gradient elution program in Experimental Example 1;

[0052] Figure 7 It is the chromatogram of gradient three in the investigation and test of the first gradient elution program in Experimental Example 1;

[0053] Figure 8 It is the chromatogram at 25 °C in the investigation and test of different column temperatures in Experimental Example 1;

[0054] Figure 9 It is the chromatogram at 35 °C in the investigation and test of different column temperatures in Experimental Example 1;

[0055] Figure 10 It is the chromatogram at pH 3.0 in the investigation experiment of the pH of mobile phase B in Experimental Example 1;

[0056] Figure 11 It is the chromatogram at pH 3.4 in the investigation experiment of the pH of mobile phase B in Experimental Example 1;

[0057] Figure 12 It is the chromatogram at pH 3.7 in the investigation experiment of the pH of mobile phase B in Experimental Example 1;

[0058] Figure 13 It is the chromatogram at pH 4.0 in the investigation experiment of the pH of mobile phase B in Experimental Example 1;

[0059] Figure 14 It is the comparison chart of the mixed reference substance solution and the test solution in Experimental Example 1; S2(2) is the mixed reference substance solution, and S1(2) is the test solution;

[0060] Figure 15 It is the comparison chart of the syringin reference substance solution and the test solution in Experimental Example 1; S2(1) is the syringin reference substance solution, and S1(1) is the test solution;

[0061] Figure 16 It is the comparison chart of the isovanillin reference substance solution and the test solution in Experimental Example 1; S2(1) is the isovanillin reference substance solution, and S1(1) is the test solution;

[0062] Figure 17 It is the characteristic chromatogram of the standard decoction (freeze-dried powder) of 18 batches of Tinospora sinensis (Lour.) Merr. decoction pieces and 3 batches of formula granules;

[0063] Figure 18 It is the reference characteristic chromatogram;

[0064] Figure 19 It is the negative control chromatogram in the methodological verification of Experimental Example 2;

[0065] Figure 20 It is the chromatogram of the control crude drug constructed in Example 1;

[0066] Figure 21 It is the characteristic chromatogram of the Tinospora sinensis (Lour.) Merr. formula granules constructed in Example 1;

[0067] Figure 22 It is the chromatogram of the tryptophan and magnoflorine reference substances constructed in Example 1. Detailed implementation mode

[0068] The following examples are provided to better further understand the present invention. It is not limited to the described best implementation mode, and does not constitute a limitation to the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior art features falls within the protection scope of the present invention. For those examples where specific experimental steps or conditions are not indicated, the operations or conditions of the conventional experimental steps described in the literature in this field can be carried out. For the reagents or instruments whose manufacturers are not indicated, they are all conventional reagent products that can be obtained through commercial purchase. % methanol refers to the volume percentage of methanol in the methanol aqueous solution.

[0069] Investigation on the construction method of Experimental Example 1

[0070] 1. Instruments, reagents and test drugs

[0071] (1) Instrument and equipment: Chromatograph 1: Waters ACQUITY Arc chromatographic system, including a quaternary gradient infusion pump (QSM-R), a 96-position high-performance automatic sampler (FTN-R), an imported column oven, a Waters 2998 diode array UV detector, and an Empower chromatographic management system; Chromatograph 2: Agilent 1260 lnfinityⅡ chromatographic system, including a G7111B type quaternary pump (1260Quat Pump), a G7129A type automatic sampler (1260Vialsampler), a G7115A type PDA diode array detector (1260DAD WR), a G7116A type column oven (1260MCT), and an OpenLAB CDS chromatographic workstation; Chromatograph 3: UltiMate 3000 chromatographic system, including an LPG-3400A quaternary pump, a WPS-3000TSL automatic sampler, a PDA diode array detector, and a chromatographic workstation; XSR304 one in ten-thousandth balance (Mettler, Switzerland), ME36S one in millionth balance (Sartorius), ultrasonic instrument (Shanghai Kedao Ultrasonic Instrument Co., Ltd.), water bath.

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

[0073] (3) Reagents: Acetonitrile (Fisher Chemical, chromatographically pure), water is ultrapurified water, and other reagents such as potassium dihydrogen phosphate, phosphoric acid, and methanol are all of analytical purity.

[0074] (4) Test drugs:

[0075] Control crude drug of Tinospora sinensis (Lot No.: 121391-202102; purchased from National Institutes for Food and Drug Control); Magnoflorine reference substance (Lot No.: 15998; purchased from Shanghai Standard Biotech Co., Ltd., purity 93.9%); Tryptophan reference substance (Lot No.: 140686-202205; purchased from National Institutes for Food and Drug Control, purity 100%); Syringin reference substance (Lot No.: 111574-202106; purchased from 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 (Lour.) Merr. can be prepared by conventional methods in the art. For example, the present invention is prepared according to the following steps: Take about 100 g of the cut Tinospora sinensis (Lour.) Merr., soak it in 10 times the amount of water for 30 minutes for the first decoction, boil it over high heat, and then simmer for 30 minutes, filter through a 200-mesh sieve. For the second decoction, add 8 times the amount of water, boil it over high heat, and simmer for 20 minutes, filter through a 200-mesh sieve, and combine the filtrates; Concentrate under reduced pressure (50 - 65 °C) to a thick extract with a relative density of 1.04 - 1.12 g / ml, and then perform freeze-drying to obtain the freeze-dried powder of Tinospora sinensis (Lour.) Merr. (The multiple is the mass multiple).

[0077] The formula granules of Tinospora sinensis (Lour.) Merr. can be prepared by conventional methods in the art. For example, the present invention is prepared according to the following steps: Take the cut Tinospora sinensis (Lour.) Merr., decoct it with water twice. For the first time, soak it in 10 times the amount of water for 30 minutes, decoct for 30 minutes, and filter. For the second time, add 8 times the amount of water, decoct for 20 minutes, and filter. Combine the filtrates, concentrate at 60 °C - 80 °C, perform spray drying, and granulate by dry granulation to obtain the formula granules of Tinospora sinensis (Lour.) Merr. (The multiple is the mass multiple).

[0078] 2. Preparation of the test solution

[0079] Precisely weigh the formula granules of Tinospora sinensis (Lour.) Merr., grind them finely, take about 0.2 g, place it in a stoppered conical flask, add 10 ml of 70% methanol, tightly stopper it, ultrasonically treat it (power 350 W, frequency 40 kHz) for 30 minutes, let it cool, filter, and take the subsequent filtrate to obtain the solution.

[0080] 3. Optimization of the chromatographic conditions

[0081] During the continuous optimization of the elution gradient, select a more reasonable wavelength and gradient 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] Use high-performance liquid chromatography to detect the test solution prepared according to item 2 of this experimental example. Use an Agilent Poroshell 120EC-C18, 4.6 mm × 150 mm, 2.7 μm chromatographic column, use acetonitrile as mobile phase A, 0.05 mol / L potassium dihydrogen phosphate (adjust the pH to 3.7 with phosphoric acid) as mobile phase B, perform gradient elution according to the regulations of gradient one, column temperature: 30 °C, injection volume: 5 μl, flow rate: 1.0 ml / min, wavelength: full wavelength scanning.

[0084] The chromatograms with detection wavelengths of 220 nm, 240 nm, 260 nm, 280 nm, and 300 nm are respectively shown in Figures 1-5As shown, the results of inspections at different wavelengths indicate that under the condition of a wavelength of 280 nm, the chromatographic peaks have more information and the characteristic peaks are relatively uniform. The tentative wavelength is 280 nm, which is used for subsequent gradient inspections and optimizations. The chromatogram at a wavelength of 280 nm shows that the resolution of the characteristic peaks at 10 - 15 minutes is poor. Therefore, the gradient is further optimized.

[0085] (2) Inspection of the primary gradient elution program

[0086] The test solution prepared according to item 2 of this experimental example was detected by high performance liquid chromatography using an Agilent Poroshell 120 EC - C18 column, 4.6 mm × 150 mm, 2.7 μm. 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 carried out according to the regulations of gradient two and three respectively. Column temperature: 30 °C, injection volume: 5 μl, flow rate: 1.0 ml / min, wavelength: 280 nm.

[0087] The results are shown in Figure 6 and 7 As shown, by comparison with Figure 4 using gradient two, the resolution of the characteristic peaks at 10 - 15 minutes is poor (resolution is less than 1.5), while using gradient three, the information content of the characteristic peaks is less and some characteristic peaks overlap. Therefore, adjusting the gradient does not improve the resolution of the characteristic peaks.

[0088] Table 1 Gradient conditions under gradients one to three

[0089]

[0090] (3) Inspection of different column temperatures

[0091] On the basis of gradient 1, inspections were carried out at column temperatures of 25 °C and 30 °C. Specifically: The test solution prepared according to item 2 of this experimental example was detected by high performance liquid chromatography using an Agilent Poroshell 120 EC - C18 column, 4.6 mm × 150 mm, 2.7 μm. 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 carried out according to the regulations of gradient one, with column temperatures of 25 °C and 35 °C respectively, injection volume: 5 μl, flow rate: 1.0 ml / min, wavelength: 280 nm.

[0092] The results are shown in Figure 8 and 9 As shown, by comparison with Figure 4Comparison. Compared with the conditions at 30 °C and 35 °C, the resolution and peak shape of the chromatographic peaks were significantly improved at 25 °C. Therefore, it is recommended to optimize the gradient at a column temperature of 25 °C.

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

[0094] The test solution prepared according to item 2 of this experimental example was detected by high performance liquid chromatography. An Agilent Poroshell 120 EC-C18, 4.6 mm × 150 mm, 2.7 μm chromatographic column 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 carried out according to the regulations of gradient one, gradient two, and gradients four to seven 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. For gradient one, the symmetry of the characteristic peak of No. 3 was poor; for gradient four, the resolution between the characteristic peak of No. 3 and the surrounding impurity peaks was less than 1.5, and the symmetry of the characteristic peaks of No. 5 and No. 9 was poor, showing the phenomenon of peak wrapping; for gradient five, the resolution between the characteristic peak of No. 3 and the surrounding impurity peaks was less than 1.5, and the symmetry of the characteristic peak of No. 8 was poor, showing the phenomenon of peak wrapping. Therefore, adjusting the gradient could not improve the resolution of the characteristic peaks. For gradients six and seven, the symmetry of the characteristic peaks was good, and among them, the symmetry of the characteristic peaks of gradient seven was the best. Therefore, it is recommended to preferably use gradient seven.

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

[0097]

[0098] Table 3 Gradient conditions for gradients six to seven

[0099]

[0100] Table 4 Peak results

[0101]

[0102]

[0103] (5) Investigation experiment on the pH of mobile phase B

[0104] Take the Ligustri Pedunculati Radix formula granules to prepare the test solution of Ligustri Pedunculati Radix formula granules according to the method under item 2 of this experimental example. The test solution of Ligustri Pedunculati Radix formula granules was detected respectively under the chromatographic conditions of gradient six in this experimental example, with the only difference being the different pH values of mobile phase B. 0.05 mol / L potassium dihydrogen phosphate was adjusted to different pH values with phosphoric acid and used as mobile phase B, and the different pH values were 3.0, 3.4, 3.7, and 4.0 respectively.

[0105] The results are shown in Figures 10-13 As shown, the separation effect of each chromatographic peak is better under the conditions of pH 3.0 - 4.0. The preferred pH is 3.0 - 3.7, and the best choice is pH 3.7.

[0106] 4. Preparation of the test solution

[0107] (1) Investigation of extraction methods

[0108] Investigate different extraction methods: ultrasonic treatment (power 350 W, frequency 40 kHz) for 30 minutes and heating under reflux for 30 minutes on the extraction effect of Ligustri Pedunculati Radix formula granules. Take the Ligustri Pedunculati Radix formula granules, grind them finely, take about 0.2 g, place them in a stoppered conical flask, add 10 ml of 70% methanol, stopper tightly, and perform ultrasonic treatment (power 350 W, frequency 40 kHz) for 30 minutes or heating under reflux for 30 minutes respectively. Let it cool, filter, and take the subsequent filtrate to obtain the test solution, and determine it according to the chromatographic conditions in Example 1. The information content of chromatographic peaks and system suitability parameters were used as the main investigation indicators. The results showed that there were no obvious differences in the information content of chromatographic peaks, system suitability parameters, and the total peak area among different extraction methods. Considering the simplicity of the method, ultrasonic extraction was selected for subsequent investigation.

[0109] Table 5 Chromatogram parameters of Ligustri Pedunculati Radix formula granules under different treatment methods

[0110]

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

[0112] According to the above determined extraction conditions, further investigate the extraction time of the test solution: the separation effects at 15 min, 30 min, and 45 min. Take the Ligustri Nuciferae formula granules, grind them finely, take about 0.2 g, place them in a stoppered conical flask, add 10 ml of 70% methanol, tightly stopper, and ultrasonically treat (power 350 W, frequency 40 kHz) for 15 min or 45 min respectively. Let it cool, filter, take the continuous filtrate to obtain the test solution, and determine it according to the chromatographic conditions in Example 1. And compare it with the results of ultrasonic extraction time of 30 minutes in Table 5. The test results show that with the extension of the extraction time, the total peak area of the characteristic peaks has little difference, indicating that within this range, each characteristic component can be completely extracted. Considering the extraction efficiency, 30 min is selected as the extraction time.

[0113] Table 6 Chromatographic peak system suitability parameters under different extraction times

[0114]

[0115]

[0116] (3) Selection of extraction solvent

[0117] Taking everything into consideration, three solvents, namely water, methanol, and ethanol, were investigated respectively. Take the Ligustri Nuciferae formula granules, grind them finely, take about 0.2 g, place them in a stoppered conical flask, add 10 ml of methanol, water, or ethanol respectively, tightly stopper, ultrasonically treat (power 350 W, frequency 40 kHz) for 30 min respectively, let it cool, filter, take the continuous filtrate to obtain the test solution, and determine it according to the chromatographic conditions in Example 1. The results show that the total peak areas of methanol and water as solvents are relative to ethanol, and the system suitability parameters of most chromatographic peaks are relatively good.

[0118] Table 7 Chromatogram parameters of Ligustri Nuciferae (Ligustri Nuciferae) formula granules under different extraction solvents

[0119]

[0120] Further investigate the extraction effects of 50% methanol solution and 70% methanol solution on Ligustri Nuciferae (Ligustri Nuciferae) formula granules. That is, take the Ligustri Nuciferae formula granules, grind them finely, take about 0.2 g, place them in a stoppered conical flask, add 10 ml of 50% methanol solution and 70% methanol solution respectively, tightly stopper, ultrasonically treat (power 350 W, frequency 40 kHz) for 30 min respectively, let it cool, filter, take the continuous filtrate to obtain the test solution, and determine it according to the chromatographic conditions in Example 1.

[0121] Table 8 Chromatogram parameters of Ligustri Nuciferae (Ligustri Nuciferae) formula granules under different extraction solvents

[0122]

[0123]

[0124] By comparing the chromatograms, the system suitability parameters of the chromatographic peaks, and the total peak areas obtained by extraction with various solvents, the chromatographic peak response values obtained by extraction with 70% methanol and 50% methanol solution are relatively large. Considering the overall extraction efficiency, 70% methanol was selected as the extraction solvent for this experiment.

[0125] (4) Selection of the amount of solvent added

[0126] According to the extraction method and extraction solvent determined above, the effects of extraction solvent volumes of 5 ml, 10 ml, 15 ml, and 25 ml on the extraction effect of the formula granules of Tinospora sinensis (Lour.) Merr. were further investigated. Take the formula granules of Tinospora sinensis (Lour.) Merr., grind them finely, take about 0.2 g, place them in a stoppered conical flask, add 5 ml, 15 ml, and 25 ml of 70% methanol solution respectively, stopper tightly, ultrasonically treat (power 350 W, frequency 40 kHz) for 30 min respectively, let it cool, filter, take the continuous filtrate to obtain the test solution, and determine it according to the chromatographic conditions in Example 1. And compare with the results of adding 10 ml of 70% methanol in Table 8. The results show that the extraction solvent for the test sample is 5 - 25 ml, and there is no obvious difference in the total peak area of the characteristic peaks in the characteristic chromatogram multiplied by the corresponding solvent amount, indicating that within this range, the substance components can be completely extracted. Considering the moderate peak area response value comprehensively, the extraction solvent amount of 10 ml was selected.

[0127] Table 9 Comparison of system suitability parameters of chromatographic peaks of formula granules of Tinospora sinensis (Lour.) Merr. with different extraction solvent volumes

[0128]

[0129]

[0130] (5) Selection of the sampling amount

[0131] According to the above determined extraction conditions, further investigate the effects of different sampling amounts of Tinospora sinensis (Lour.) Merr. formula granules: 0.1 g, 0.2 g, 0.3 g, 0.4 g on the extraction effect of Tinospora sinensis (Lour.) Merr. formula granules. Take the information content of chromatographic peaks and system suitability parameters as the main investigation indexes. Take Tinospora sinensis (Lour.) Merr. formula granules, grind them finely, take about 0.1 g, 0.3 g, 0.4 g respectively, place them in a stoppered conical flask, add 10 ml of 70% methanol solution respectively, stopper tightly, ultrasonically treat (power 350 W, frequency 40 kHz) for 30 min respectively, let it cool, filter, take the continuous filtrate, and obtain the test solution. Determine according to the chromatographic conditions in Example 1. And compare with the results of adding 0.2 g of 70% methanol sampling amount in Table 8. From the above test results, it shows that under each sampling amount, the differences in the system suitability parameters of chromatographic peaks are not obvious, and when the sampling amount is 0.2 g, the peak height and peak width of chromatographic peaks are relatively moderate. Therefore, select 0.2 g as the sampling amount for this test.

[0132] Table 10 Comparison of system suitability parameters of chromatographic peaks of Tinospora sinensis (Lour.) Merr. formula granules with different sampling amounts

[0133]

[0134]

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

[0136] (1) Identification of characteristic peaks

[0137] Use HPLC to identify and assign 9 characteristic peaks of Tinospora sinensis (Lour.) Merr. formula granules.

[0138] Take appropriate amounts of magnoflorine and tryptophan reference substances, weigh them accurately, add methanol to make a solution containing 50 μg of magnoflorine and 50 μg of tryptophan per 1 ml, and obtain the mixed reference substance solution. Take an appropriate amount of isovanillin reference substance, weigh it accurately, add methanol to make a solution containing 20 μg of isovanillin per 1 ml, and obtain the isovanillin reference substance solution. Take an appropriate amount of syringin reference substance, weigh it accurately, add methanol to make a solution containing 20 μg of syringin per 1 ml, and obtain the syringin reference substance solution. Take an appropriate amount of Tinospora sinensis (Lour.) Merr. formula granules, grind them finely, take about 0.2 g, place it in a stoppered conical flask, add 10 ml of 70% methanol, stopper tightly, ultrasonically treat (power 350 W, frequency 40 kHz) for 30 minutes, let it cool, filter, take the continuous filtrate, and obtain the test solution.

[0139] Precisely pipette 5 μl of each of the above reference substance solutions and test solution respectively, inject them into the liquid chromatograph, and determine according to the chromatographic conditions in Example 1 of this example. The results are as Figures 14-16 . Determine that peak 1 is tryptophan, peak 2 is syringin, peak 5 is isovanillin, and peak 7 is magnoflorine.

[0140] The liquid-phase UV absorption spectra of the test solution and the reference solution were compared and analyzed, and LC / MS / MS (liquid-phase tandem mass spectrometry) technology was used to identify and assign 9 characteristic peaks of the Tinospora sinensis (Lour.) Merr. formula granules. It was also confirmed that peak 1 was tryptophan, peak 2 was syringin, peak 5 was isovanillin, and peak 7 was magnoflorine.

[0141] (2) Construction of characteristic fingerprints of multiple batches of Tinospora sinensis (Lour.) Merr. pharmaceutical preparations

[0142] Take 18 batches of freeze-dried powder of Tinospora sinensis (Lour.) Merr. and 3 batches of formula granule samples, and prepare test solutions according to the method of Example 1 and perform high-performance liquid chromatography detection respectively. The results are as Figure 17 shown, among which Figure 17 S1(9)~S21(9) are the characteristic fingerprints of 18 batches of freeze-dried powder of Tinospora sinensis (Lour.) Merr. and 3 batches of Tinospora sinensis (Lour.) Merr. formula granules.

[0143] Table 11 Detection results of relative retention time of characteristic fingerprints of 18 batches of freeze-dried powder and 3 batches of formula granules

[0144]

[0145]

[0146] Table 12 Detection results of relative peak areas of characteristic fingerprints of 18 batches of freeze-dried powder and 3 batches of formula granules

[0147]

[0148]

[0149] (3) Construction of reference characteristic fingerprints and regulations for characteristic peaks

[0150] The detection results of fingerprints of multiple batches of test samples were analyzed. The "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission was used, and the "multi-point calibration and MARK peak matching" mode was used for fitting to generate a reference characteristic fingerprint. The reference characteristic fingerprints of the freeze-dried powder of Tinospora sinensis (Lour.) Merr. and Tinospora sinensis (Lour.) Merr. formula granules were obtained. A total of 9 chromatographic peaks were shown in the fingerprint, see Figure 18As shown in the figure, according to the investigation results of different chromatographic columns and different instruments, it is recommended to use peak 7 (magnoflorine) 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 relatively close to the solvent peak and is easily affected by various factors, it is recommended to use the reference substance for positioning during the analysis. The relative retention times of the remaining characteristic peaks with respect to 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 freeze-dried powder of Tinospora sinensis (Lour.) Merr. and 3 batches of formula granules of Tinospora sinensis (Lour.) Merr. showed that they should correspond to 9 characteristic peaks in the reference chromatogram of the control medicinal material. The 9 common peaks had good transferability, and the relative retention times of the characteristic peaks of the 21 batches of samples were all within the range of ±10% of the specified values.

[0151] Therefore, it is stipulated that: the characteristic chromatogram of the formula granules of Tinospora sinensis (Lour.) Merr. has 9 characteristic peaks, which should correspond to the 9 characteristic peaks in the reference chromatogram of the control medicinal material. Among them, peak 1 and peak 7 should correspond to the retention times of the corresponding reference substance peaks of the reference substances respectively. The peak corresponding to the magnoflorine reference substance peak is peak S. Calculate the relative retention time of each characteristic peak, and its relative retention time should be within the range of ±10% of the specified value. 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 Time of the Reference Chromatogram

[0153]

[0154] Table 14 Relative Peak Area of the Reference Chromatogram

[0155]

[0156] Experimental Example 2 Methodology Validation

[0157] 1. System Suitability

[0158] Take the negative control solution (70% methanol solution) and test it by the high performance liquid chromatography method of Example 1. The results showed that, as shown Figure 19 in the figure, the negative control had no interference on the characteristic chromatogram, and the system suitability and specificity of the chromatographic method were good, which could be used as the detection method for the characteristic chromatogram of the formula granules of Tinospora sinensis (Lour.) Merr.

[0159] 2. Instrument Precision

[0160] Take the same batch of the prepared sample solution of the Tinospora sagittata formula granules obtained by the method of Example 1, and test it according to the high performance liquid chromatography method of Example 1. Inject the sample solution six times repeatedly, and calculate that the RSDs of the relative retention times and relative peak areas of the nine characteristic peaks are all less than 2% (see Table 15). It shows that the precision of the instrument is good. The results show that the precision of the instrument is good.

[0161] Table 15 Results of the relative retention time test for the precision of the instrument

[0162]

[0163] Table 16 Results of the relative peak area test for the precision of the instrument

[0164]

[0165] 3. Repeatability

[0166] Take the same batch of the Tinospora sagittata formula granule samples, prepare six samples in parallel according to the method of Example 1, and determine the relative retention times and relative peak areas of the nine characteristic peaks according to the chromatographic conditions. The results show that the RSDs of the relative retention times of the nine characteristic peaks and the reference substance peak are all less than 2% (see Table 17). It shows that the repeatability of this method is good.

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

[0168]

[0169]

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

[0171]

[0172] 4. Intermediate precision (different operators)

[0173] Three inspectors respectively take the same batch of the Tinospora sagittata formula granules at different times, prepare the test sample solutions according to the preparation method of Example 1, and test the relative retention times and relative peak areas of the common peaks with the same instrument according to the high performance liquid chromatography method of Example 1. The results show that the relative average deviations of the relative retention times of the nine characteristic peaks and the reference substance peak are all less than 2% (see Table 19). It shows that the intermediate precision of this method is good.

[0174] Table 19 Results of the relative retention time test for the intermediate precision (different operators)

[0175]

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

[0177]

[0178] 5. Durability

[0179] (1) Stability Investigation

[0180] Take the same sample solution of the wide tendon rattan formula granules prepared according to the method of Example 1, and inject and test it by the high performance liquid chromatography method of Example 1 at 0, 4, 8, 12, 16, and 24 hours respectively. Determine the relative retention time and relative peak area of each common peak. The results show that the deviation of the relative retention time of each chromatographic peak from the reference substance peak is less than 2% (see Table 21). It shows that the sample solution is stable within 24 hours and meets the determination requirements.

[0181] Table 21 Results of Relative Retention Time in Stability Test

[0182]

[0183]

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

[0185]

[0186] (2) Investigation of Different Column Temperatures

[0187] Take the same sample solution of the wide tendon rattan formula granules prepared according to the method of Example 1, and test it by the high performance liquid chromatography method of Example 1. Set the column temperatures at 23°C, 25°C, and 27°C respectively, and investigate the effects of different column temperatures on the relative retention time and relative peak area of each characteristic peak. The results show that when the column temperature changes, the deviation of the relative retention time of 9 characteristic peaks is within 3% (see Table 23), indicating good durability of the column temperature, and 23 - 27°C can be adopted.

[0188] Table 23 Comparison of Results of Relative Retention Time at Different Column Temperatures

[0189]

[0190] Table 24 Comparison of Results of Relative Peak Area at Different Column Temperatures

[0191]

[0192] (3) Investigation of Different Flow Rates

[0193] Take the same test solution of the Tinospora sagittata formula granules prepared by the method of Example 1, and test it by the high performance liquid chromatography method of Example 1. Determine it at the flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min respectively, and investigate the effects of the small changes in the flow rate on the relative retention time and relative peak area of each characteristic peak. The results show that when the flow rate changes slightly, the deviation of the relative retention time of each characteristic chromatographic peak is within 4.0% (see Table 25), indicating that the flow rate has good durability. Considering the flow rate, it can be 0.9 - 1.1 ml / min, preferably 1.0 ml / min.

[0194] Table 25 Comparison of relative retention time results at different flow rates

[0195]

[0196] Table 26 Comparison of relative peak area results at different flow rates

[0197]

[0198] (4) Investigation of different instruments

[0199] Take the same test solution of the Tinospora sagittata formula granules prepared by the method of Example 1, and test it by the high performance liquid chromatography method of Example 1. Inject and analyze it using different instruments (waters, Agilent, Thermo) respectively, and investigate the effects of different instruments on the relative retention time and relative peak area of each characteristic peak. The results show that when using instruments from different manufacturers, there is a certain impact on the relative retention time of each characteristic chromatographic peak, but within the range of ±10% of the specified value. The separation effect and peak shape of the 9 characteristic peaks are good under different instruments. Therefore, it is recommended to fix the specified value of the relative retention time of each characteristic peak within the range of ±10% in combination with the investigation results of different instruments.

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

[0201]

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

[0203]

[0204]

[0205] (5) Investigation of different chromatographic columns

[0206] Take the same test solution of the Tinospora sinensis formula granules prepared by the method of Example 1, and test it by the high performance liquid chromatography method of Example 1. Set the chromatographic columns Agilent Poroshell 120 EC-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) to investigate the relative retention times and relative peak areas of each characteristic peak and the reference peak when using different chromatographic columns. The results show that different types of chromatographic columns have a greater impact on the relative retention times of the characteristic peaks. The separation effects and peak shapes of the 9 characteristic peaks under different chromatographic columns are all good. The preferred chromatographic column is: Agilent Poroshell 120 EC-C18 (4.6 mm × 150 mm, 2.7 μm).

[0207] Table 29 Comparison of relative retention time results of different chromatographic columns

[0208]

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

[0210] Take the same test solution of the Tinospora sinensis formula granules prepared by the method of Example 1, and test it by the high performance liquid chromatography method of Example 1. Measure it respectively with 0.05 mol / L potassium dihydrogen phosphate (adjust the pH to 3.5 with phosphoric acid), 0.05 mol / L potassium dihydrogen phosphate (adjust the pH to 3.7 with phosphoric acid), and 0.05 mol / L potassium dihydrogen phosphate (adjust the pH to 3.9 with phosphoric acid), and investigate the effects of small changes in the pH value on the relative retention times and relative peak areas of each characteristic peak. The results show that when the pH value changes slightly, different pH values of the mobile phase have little effect on the relative retention times of the characteristic peaks, and the deviations of the relative retention times of each characteristic chromatographic peak are all within 3.0%. The pH can be 3.5 - 3.9. Therefore, this method recommends using 0.05 mol / L potassium dihydrogen phosphate (adjust the pH to 3.7 with phosphoric acid) for determination.

[0211] Based on the results of the above methodological investigations, in the characteristic chromatogram method of the Tinospora sinensis (Tinospora sinensis) formula granules established. The comprehensive above results show that this method is simple to operate, accurate in results, and has good method reproducibility.

[0212] Example 1

[0213] This example provides a method for constructing a characteristic chromatogram of Tinospora sinensis formula granules, including the following steps:

[0214] Preparation of the test solution: Take an appropriate amount of the test sample, grind it finely, take about 0.2 g, place it in a stoppered conical flask, add 10 ml of 70% methanol, stopper tightly, ultrasonically treat (power 350 W, frequency 40 kHz) for 30 minutes, let it cool, filter, and take the subsequent filtrate, which is the test solution.

[0215] Preparation of the reference solution: Take about 2 g of the reference crude drug of Tinospora sinensis (Lour.) Merr., place it in a stoppered conical flask, add 100 ml of water, heat under reflux for 30 min, filter, evaporate the filtrate to dryness, add 10 ml of 70% methanol to the residue, stopper tightly, ultrasonically treat (power 350 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate as the reference solution of the reference crude drug. Separately take appropriate amounts of tryptophan reference substance and magnoflorine reference substance, accurately weigh them, dissolve them in methanol to prepare a solution containing 50 μg of each per 1 ml as the reference solution of the reference substance.

[0216] High performance liquid chromatography test: Accurately pipette 5 μl each of the reference solution and the test solution, inject them into the liquid chromatograph for determination, and the chromatographic conditions are as follows: Using octadecylsilane chemically bonded silica gel as the filler (column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm); Using acetonitrile as mobile phase A and 0.05 mol / L potassium dihydrogen phosphate (adjusted to pH 3.7 with phosphoric acid) as mobile phase B, perform gradient elution according to the regulations in the following table; Column temperature is 25 °C; Flow rate is 1.0 ml / min; Detection wavelength is 280 nm. The number of theoretical plates calculated based on the magnoflorine peak should be not less than 5000.

[0217] Table 30 Gradient elution program

[0218]

[0219] Table 31 Results of the peaks of the reference crude drug

[0220]

[0221] Table 32 Results of the peaks of the formulated granules

[0222]

[0223] Table 33 Results of the peaks of the reference substances

[0224]

[0225] From the above table and Figures 20-22 it can be seen that Figure 21Among the nine characteristic peaks, the separation effect is good, the symmetry is good, the peak area is high, and the detection and analysis time is short. Nine characteristic peaks should appear in the chromatogram of the test sample and correspond to the nine characteristic peaks in the chromatogram of the reference medicinal material. Among them, the retention times of peak 1 and peak 7 correspond to those of the corresponding reference peaks of the reference substances. The peak corresponding to the magnoflorine reference substance peak is the S peak. Calculate the relative retention times of each characteristic peak, and the relative retention times are within the range of ±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 only examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for constructing a characteristic spectrum of a drug preparation of Rhizoma Achyranthis Bidentatae, characterized in that: The following steps are included: (1) Preparation of test solution; (2) Take the test solution and use high performance liquid chromatography to detect it, using octadecylsilane bonded silica gel as the filler, acetonitrile as the mobile phase A, and an aqueous solution containing potassium dihydrogen phosphate as the mobile phase B. The column temperature is 23-27°C, and the gradient elution is performed according to the following procedure: 0→15min, the volume ratio of mobile phase A to mobile phase B was 6%:94%→10%:90%; 15→20min, the volume ratio of mobile phase A to mobile phase B is 10%:90%; 20→40min, the volume ratio of mobile phase A to mobile phase B was 10%:90%→19-21%:79-81%.

2. The construction method according to claim 1, characterized in that: Step (2) also satisfies at least one of the following 1)-5): 1) The detection wavelength is 220-300nm, preferably 280nm; 2) The flow rate is 0.9-1.1 mL / min, preferably 1.0 mL / min; 3) The injection volume is 3-10 μl; 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 of potassium dihydrogen phosphate to 3.0-4.0; more preferably, phosphoric acid is used to adjust the pH of the aqueous solution of potassium dihydrogen phosphate to 3.7; 5) During the high performance liquid chromatography detection process, a chromatographic column with a specification of 4.6 mm×150 mm and a diameter of 2.7 to 3.0 μm was used.

3. The construction method according to claim 1 or 2, characterized in that: Step (1) comprises weighing a test sample, extracting with a solvent to obtain an extract, separating the solid from the liquid, and obtaining a liquid, which is the test sample solution.

4. The construction method according to claim 3, characterized in that: The step (1) also satisfies any one or more of the following AE: 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. The extraction method is heating reflux extraction or ultrasonic extraction; C. Extraction time is ≥10min, preferably 15-40min; D. The solid-liquid separation is selected from centrifugation or membrane filtration; E. The solvent is selected from one or more of methanol, ethanol and water; preferably, the methanol-water solution has a volume percentage of 50-70%.

5. The construction method according to any one of claims 1 to 4, characterized in that: The construction method also includes the step of preparing a reference solution using at least one solubilizing agent selected from tryptophan, syringin, isovanillin, and magnolia alkaloids, and the step of obtaining a reference spectrum by detecting the reference solution using high performance liquid chromatography in the construction method according to any one of claims 1 to 4.

6. The construction method according to claim 5, 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 magnolia alkaloids; and / or, the solvent used in the preparation of the reference solution is selected from methanol or methanol-water solution.

7. The construction method according to any one of claims 1 to 4, characterized in that: The construction method also includes the steps of using the extract obtained by water extraction of the control medicinal material of Codonopsis pilosula to prepare a control medicinal material reference solution according to the preparation method of the test solution in the construction method described in any one of claims 1-4, and detecting the control medicinal material reference solution by high performance liquid chromatography according to the construction method described in any one of claims 1-4 to obtain a reference medicinal material reference atlas. Preferably, after the water extraction of the control medicinal material of Codonopsis pilosula, the control medicinal material is filtered, the liquid is dried, and then the control medicinal material reference solution is prepared according to the preparation method of the test solution in the construction method described in any one of claims 1-4.

8. The construction method according to any one of claims 1 to 7, characterized in that: The characteristic spectrum of the described tricholoma pectinata drug preparation has 9 common characteristic peaks, peak 1 and peak 7 correspond to the retention times of tryptophan and magnolia alkaloid reference substance peaks respectively, the peak corresponding to the magnolia alkaloid reference substance peak is the S peak, and the relative retention times of peaks 2 to 6 and peaks 8 to 9 and the S peak are within the range of ±10% of the specified values; the specified values ​​of peaks 2 to 6 and peaks 8 to 9 are 0.53, 0.68, 0.71, 0.81, 0.94, 1.34 and 1.47 respectively.

9. Application of the method for constructing the characteristic spectrum of the Thunb. Croton tiglium drug preparation according to any one of claims 1 to 8 in the quality inspection of the Thunb. Croton tiglium drug preparation.

10. A quality inspection method for a drug preparation of Rhizoma Achyranthis Bidentatae, characterized in that: The method comprises the steps of comparing the characteristic spectrum of the tested strychnos nucifera product with the control characteristic spectrum of the strychnos nucifera pharmaceutical preparation; the characteristic spectrum of the tested strychnos nucifera product is constructed using the tested strychnos nucifera product according to the construction method described in any one of claims 1 to 8, and the control characteristic spectrum of the strychnos nucifera pharmaceutical preparation is selected from any one of the following (1) to (3): (1) It has 9 common characteristic peaks, peak 1 and peak 7 correspond to the retention times of tryptophan and magnolia alkaloid reference substance peaks, respectively, the peak corresponding to the magnolia alkaloid reference substance peak is the S peak, and the relative retention times of peaks 2 to 6 and peaks 8 to 9 and the S peak are within the range of ±10% of the specified values; the specified values ​​of peaks 2 to 6 and peaks 8 to 9 are 0.53, 0.68, 0.71, 0.81, 0.94, 1.34, and 1.47, respectively; (2) a characteristic spectrum of a Caulis Achyranthis Bidentata drug preparation obtained by using a single batch or multiple batches of Caulis Achyranthis Bidentata drug preparation according to the construction method described in any one of claims 1 to 8; (3) Using multiple batches of Thunbergia scoparia drug preparations and obtaining characteristic spectra according to the construction method described in any one of claims 1 to 8, a control characteristic spectra is prepared by using the average value or median method.

Images