Construction method and identification method of UPLC specific chromatogram of tetrapanax papyriferus standard decoction and detection method of chemical component content

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a construction method and an identification method of a UPLC specific chromatogram of a tetrapanax papyriferus standard decoction and a detection method of the content of chemical components. The construction method of the UPLC specific chromatogram of the tetrapanax papyriferus standard decoction comprises the following steps: taking a tetrapanax papyriferus reference medicinal material, and preparing a reference medicinal material reference solution; taking one or more of protocatechuic acid and syringaldehyde to prepare a reference solution; taking a tetrapanax papyriferus standard decoction, and preparing a test solution; and respectively carrying out ultra-high performance liquid chromatography detection on the reference substance solution of the reference medicinal material, the reference substance solution and the test solution, and constructing the UPLC characteristic spectrum of the tetrapanax papyriferus standard decoction. The method disclosed by the invention has the advantages of strong specificity, good repeatability, good stability and the like, the constructed UPLC characteristic spectrum has seven characteristic peaks, the spectrum information is rich, and a relatively comprehensive and effective evaluation method can be provided for quality control of the tetrapanax papyriferus standard decoction.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a method for constructing a UPLC characteristic fingerprint of Tongcao standard decoction, a method for identification, and a method for detecting the content of chemical components. Background Art

[0002] It is recorded in the Chinese Pharmacopoeia (2020 Edition) that Tongcao is the dried pith of the stem of Tetrapanax papyrifer (Hook.) K. Koch of the Araliaceae family. In autumn, cut the stem into sections, take out the pith while it is fresh, straighten it, and dry it in the sun.

[0003] At present, the identification of Tongcao under the Chinese Pharmacopoeia (2020 Edition) only includes character identification, microscopic identification, and routine inspection items, and there is no method for identifying and quantifying related chemical components. Research by scholars at home and abroad shows that Tongcao mainly contains triterpenoids and their triterpenoid saponin compounds. In addition, it also contains steroidal glycosides, flavonoids, benzene derivatives, and ceramide components. For the content determination method of Tongcao, it is mainly based on polysaccharides and monosaccharides (D-mannose and fructose). There are also reports on the determination of total flavonoids in Tongcao using rutin as a reference substance. However, the above methods lack specificity. Qiu Zhijie et al. studied using the acid hydrolysis components of Tongcao to reflect the quality of Tongcao medicinal materials, but this cannot well reflect the prototype components in Tongcao. Zhang Rui et al. established a UPLC fingerprint of the chloroform fraction of the water extract of Tongcao. However, their main research is on the chloroform fraction, and the components in the fingerprint are mainly non-polar components.

[0004] None of the above methods can well control the quality of Tongcao. Summary of the Invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a method for constructing a UPLC characteristic fingerprint of Tongcao standard decoction. This method has the advantages of strong specificity, good repeatability, and good stability. The UPLC characteristic fingerprint constructed by the present invention has rich fingerprint information, can realize the identification of Tongcao standard decoction and the determination of the content of chemical components, and can fully reflect the water-soluble components in the water extract of Tongcao, thereby providing a more comprehensive and effective evaluation method for the quality control of Tongcao standard decoction.

[0006] In the first aspect of the present invention, a method for constructing a UPLC characteristic fingerprint of Tongcao standard decoction is provided, including the following steps:

[0007] Take the reference medicinal material of Tongcao and prepare a reference substance solution of the reference medicinal material;

[0008] Take one or more of protocatechuic acid and syringaldehyde and prepare a reference substance solution;

[0009] Take the Tongcao standard decoction and prepare a test solution;

[0010] Perform ultra-high performance liquid chromatography (UPLC) detection on the reference medicinal material reference solution, the reference substance solution, and the test sample solution respectively to construct the UPLC characteristic chromatogram of Tongcao standard decoction;

[0011] The chromatographic conditions for the ultra-high performance liquid chromatography detection include:

[0012] Use acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B for gradient elution.

[0013] In some embodiments, the program for the gradient elution includes:

[0014] From 0 to 10 min, the volume percentage of mobile phase A is 3%;

[0015] From 10 min to 25 min, the volume percentage of mobile phase A changes from 3% to 7%;

[0016] From 25 min to 32 min, the volume percentage of mobile phase A changes from 7% to 10%;

[0017] From 32 min to 40 min, the volume percentage of mobile phase A is 10%;

[0018] From 40 min to 42 min, the volume percentage of mobile phase A changes from 10% to 80%;

[0019] From 42 min to 45 min, the volume percentage of mobile phase A is 80%.

[0020] In some embodiments, in the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.08% - 0.12%.

[0021] In some embodiments, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: using a chromatographic column filled with octadecylsilane-bonded silica gel.

[0022] In some embodiments, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the flow rate is 0.55 mL / min - 0.65 mL / min.

[0023] In some embodiments, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the column temperature is 28°C - 32°C.

[0024] In some embodiments, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the detection wavelength is 210 nm - 230 nm.

[0025] In some embodiments, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the injection volume is 1 μL - 3 μL.

[0026] In some of these embodiments, the steps for preparing the control medicinal material solution include:

[0027] Performing a first water extraction treatment and a first organic solvent extraction treatment on the Tongcao control medicinal material.

[0028] In some of these embodiments, the first water extraction treatment includes the following steps:

[0029] Mixing the Tongcao control medicinal material with water, performing heating under reflux for extraction, concentrating, mixing the residue with water for dissolution, and preparing a first extract;

[0030] The first organic solvent extraction treatment includes the following steps;

[0031] Mixing the first extract with ethyl acetate, extracting, and preparing a second extract;

[0032] Concentrating the second extract, using methanol as the re-dissolution solvent, dissolving the residue, filtering, and taking the filtrate;

[0033] The methanol is an aqueous methanol solution with a volume fraction of 45% - 55%.

[0034] In some of these embodiments, in the first water extraction treatment step, the mixing ratio of the Tongcao control medicinal material to water is 1 g : (90 - 100) mL.

[0035] In some of these embodiments, the heating reflux time is 55 min - 65 min.

[0036] In some of these embodiments, in the first organic solvent extraction step, the volume ratio of the first extract to ethyl acetate is 1 : (2 - 2.5).

[0037] In some of these embodiments, the number of extractions with ethyl acetate is 3 - 4 times.

[0038] In some of these embodiments, the steps for preparing the reference substance solution include:

[0039] Using methanol as the extraction solvent to prepare the reference substance solution;

[0040] In the reference substance solution, the concentration of protocatechuic acid is 8 μg / mL - 12 μg / mL, and the concentration of syringaldehyde is 8 μg / mL - 12 μg / mL.

[0041] In some of these embodiments, the steps for preparing the test sample solution include:

[0042] Performing a second water extraction treatment and a second organic solvent extraction treatment on the Tongcao standard decoction test sample.

[0043] In some of these embodiments, the second water extraction treatment includes the following steps:

[0044] Mix the prepared D. officinale standard decoction sample with water and perform heating under reflux for extraction to prepare a third extract;

[0045] The second organic solvent extraction treatment includes the following steps;

[0046] Mix the third extract with ethyl acetate to prepare a fourth extract;

[0047] Concentrate the fourth extract, use methanol as the reconstitution solvent, dissolve the residue, filter, and take the filtrate;

[0048] The methanol is an aqueous solution of methanol with a volume fraction of 45% - 55%.

[0049] In some of these embodiments, in the second water extraction step, the mixing ratio of the D. officinale standard decoction sample to water is 1 g:(20 - 25) mL.

[0050] In some of these embodiments, the heating reflux time is 15 min - 45 min.

[0051] In some of these embodiments, in the second organic solvent extraction step, the volume ratio of the third extract to ethyl acetate for mixing is 1:(2 - 2.5).

[0052] In some of these embodiments, the number of extractions with ethyl acetate is 3 - 4 times.

[0053] In some of these embodiments, the characteristic chromatogram presents 7 characteristic peaks, which correspond to the retention times of 7 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 1 and peak 6 are respectively consistent with the retention times of the reference peaks of the reference substances, the peak corresponding to the protocatechuic acid reference substance peak is peak S1, calculate the relative retention times of peaks 2 - 4 with respect to peak S1, the peak corresponding to the syringaldehyde reference substance peak is peak S2, calculate the relative retention times of peaks 5 and 7 with respect to peak S2, and their relative retention times are within the range of ±10% of the specified values. The specified values are: 1.44 (peak 2), 2.93 (peak 3), 3.39 (peak 4), 0.96 (peak 5), 1.07 (peak 7).

[0054] In some of these embodiments, the construction method further includes a step of performing mass spectrometric identification on the characteristic peaks in the characteristic chromatogram by using chromatography - mass spectrometry;

[0055] The chromatographic conditions for the identification process include: using acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B for gradient elution;

[0056] The conditions for the mass spectrometry method include:

[0057] The parameters of the HESI ion source include: the sheath gas flow rate is 60 arb - 65 arb, the auxiliary gas flow rate is 18 arb - 22 arb, the spray voltage is 2.2 kV - 2.8 kV, the S-lens voltage is 48 V - 52 V, the heating temperature is 280 °C - 320 °C, and the capillary temperature is 480 °C - 520 °C;

[0058] The mass spectrometry scanning parameters include: the scanning mode is positive ion mode and / or negative ion mode, the scanning range is 120 m / z - 1200 m / z, the normalized collision energy is 20 V, and the mass spectrometry graph type is a peak graph.

[0059] In the second aspect of the present invention, a method for identifying Tongcao standard decoction is also provided, including the following steps:

[0060] Prepare a test solution from the test sample;

[0061] Perform ultra-high performance liquid chromatography detection on the test solution;

[0062] Compare the chromatogram of the obtained test solution with the characteristic chromatogram constructed in any of the above embodiments, and identify whether the test sample is Tongcao standard decoction according to the comparison result;

[0063] The chromatographic conditions for the ultra-high performance liquid chromatography detection include:

[0064] Use acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B for gradient elution.

[0065] In some embodiments of the identification method, the program of the gradient elution includes:

[0066] 0 - 10 min, the volume percentage of mobile phase A is 3%;

[0067] 10 min - 25 min, the volume percentage of mobile phase A changes from 3% to 7%;

[0068] 25 min - 32 min, the volume percentage of mobile phase A changes from 7% to 10%;

[0069] 32 min - 40 min, the volume percentage of mobile phase A is 10%;

[0070] 40 min - 42 min, the volume percentage of mobile phase A changes from 10% to 80%;

[0071] 42 min - 45 min, the volume percentage of mobile phase A is 80%.

[0072] In some embodiments of the identification method, in the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.08% to 0.12%.

[0073] In some embodiments of the identification method, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: using a chromatographic column filled with octadecylsilane-bonded silica gel.

[0074] In some embodiments of the identification method, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the flow rate is 0.55 mL / min to 0.65 mL / min.

[0075] In some embodiments of the identification method, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the column temperature is 28°C to 32°C.

[0076] In some embodiments of the identification method, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the detection wavelength is 210 nm to 230 nm.

[0077] In some embodiments of the identification method, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the injection volume is 1 μL to 3 μL.

[0078] In some embodiments of the identification method, the steps of preparing the test solution include:

[0079] Performing a third water extraction treatment and a third organic solvent extraction treatment on the test sample.

[0080] In some embodiments of the identification method, the third water extraction treatment includes the following steps:

[0081] Mixing the test sample and water and performing heating and reflux extraction to prepare a fifth extract;

[0082] The third organic solvent extraction is as follows:

[0083] Mixing the fifth extract with ethyl acetate to prepare a sixth extract;

[0084] Concentrating the sixth extract, using methanol as a reconstitution solvent, dissolving the residue, filtering, and taking the filtrate; the methanol is an aqueous methanol solution with a volume fraction of 45% to 55%.

[0085] In some embodiments of the identification method, in the third water extraction treatment step, the mixing ratio of the test sample and water is 1 g:(20 - 25) mL.

[0086] In some embodiments of the identification method, the heating and reflux time is 15 min to 45 min.

[0087] In some embodiments of the identification method, in the third organic solvent extraction step, the volume ratio of the fifth extract to ethyl acetate is 1:(2 - 2.5).

[0088] In some embodiments of the identification method, the number of extractions with ethyl acetate is 3 - 4 times.

[0089] The third aspect of the present invention provides a method for detecting the content of chemical components in a standard decoction of Tetrapanax papyriferus, comprising the following steps:

[0090] Take one or more of protocatechuic acid and syringaldehyde to prepare reference substance solutions with different concentrations;

[0091] Perform ultra - high performance liquid chromatography (UHPLC) detection on the reference substance solutions with different concentrations, and construct a standard curve based on the peak area and concentration;

[0092] Take the test sample of the standard decoction of Tetrapanax papyriferus to prepare a test solution;

[0093] Perform ultra - high performance liquid chromatography detection and analysis on the test solution, and combine with the standard curve to determine the content of chemical components in the test sample;

[0094] The chromatographic conditions for the ultra - high performance liquid chromatography detection include:

[0095] Use acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B for gradient elution.

[0096] In some embodiments of the detection method, the gradient elution program includes:

[0097] 0 - 10 min, the volume percentage of mobile phase A is 3%;

[0098] 10 min - 25 min, the volume percentage of mobile phase A changes from 3% to 7%;

[0099] 25 min - 32 min, the volume percentage of mobile phase A changes from 7% to 10%;

[0100] 32 min - 40 min, the volume percentage of mobile phase A is 10%;

[0101] 40 min - 42 min, the volume percentage of mobile phase A changes from 10% to 80%;

[0102] 42 min - 45 min, the volume percentage of mobile phase A is 80%.

[0103] In some embodiments of the detection method, in the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.08% - 0.12%.

[0104] In some embodiments of the detection method, the chromatographic conditions for the ultra-high performance liquid chromatography (UPLC) detection further include: using a chromatographic column filled with octadecylsilyl silica gel as the filler.

[0105] In some embodiments of the detection method, the chromatographic conditions for the UPLC detection further include: the flow rate is 0.55 mL / min to 0.65 mL / min.

[0106] In some embodiments of the detection method, the chromatographic conditions for the UPLC detection further include: the column temperature is 28 °C to 32 °C.

[0107] In some embodiments of the detection method, the chromatographic conditions for the UPLC detection further include: the detection wavelength is 210 nm to 230 nm.

[0108] In some embodiments of the detection method, the chromatographic conditions for the UPLC detection further include: the injection volume is 1 μL to 3 μL.

[0109] In some embodiments of the detection method, the steps for preparing the reference substance solution include:

[0110] Using methanol as the extraction solvent to prepare the reference substance solution.

[0111] In some embodiments of the detection method, the linear regression equation for protocatechuic acid is: y1 = 73993x1 - 10642, r = 1, where x1 represents the concentration of protocatechuic acid and y1 represents the peak area. Protocatechuic acid has a good linear relationship between concentration and peak area in the range of 0.196 μg / mL to 97.993 μg / mL.

[0112] In some embodiments of the detection method, the linear regression equation for syringaldehyde is: y2 = 6590.8x2 + 1963.4, r = 1, where x2 represents the concentration of syringaldehyde and y2 represents the peak area. Syringaldehyde has a good linear relationship between concentration and peak area in the range of 0.516 μg / mL to 103.194 μg / mL.

[0113] Compared with the traditional technology, the present invention has the following beneficial effects:

[0114] The present invention provides a method for constructing the UPLC characteristic fingerprint of Tetrapanacis Medulla. This method has the advantages of strong specificity, good repeatability, and good stability. The UPLC characteristic fingerprint constructed by the present invention has 7 characteristic peaks, rich fingerprint information, can realize the identification of Tetrapanacis Medulla standard decoction and the determination of the content of chemical components, and can fully reflect the water-soluble components in the water extract of Tetrapanacis Medulla, thereby being able to provide a more comprehensive and effective evaluation method for the quality control of Tetrapanacis Medulla standard decoction. Description of the Drawings

[0115] Figure 1 It is the control characteristic chromatogram of the standard decoction of Tetrapanax papyriferus (Hook.) K. Koch. Peak 1 (S1): protocatechuic acid; Peak 6 (S2): syringaldehyde;

[0116] Figure 2 It is the characteristic chromatogram of the control medicinal material of Tetrapanax papyriferus (Hook.) K. Koch. Peak 1 (S1): protocatechuic acid; Peak 6 (S2): syringaldehyde;

[0117] Figure 3 It is the optimized chromatogram of the elution gradient (gradient 1);

[0118] Figure 4 It is the optimized chromatogram of the elution gradient (gradient 2);

[0119] Figure 5 It is the optimized chromatogram of the elution gradient (gradient 3);

[0120] Figure 6 It is the optimized chromatogram of the elution gradient (gradient 4);

[0121] Figure 7 It is the comparative chromatogram of different brands of chromatographic columns;

[0122] Figure 8 It is the common pattern of the characteristic chromatograms of 15 batches of the standard decoction of Tetrapanax papyriferus (Hook.) K. Koch;

[0123] Figure 9 It is the total ion current chromatogram of the test solution of the standard decoction of Tetrapanax papyriferus (Hook.) K. Koch (positive ion mode);

[0124] Figure 10 It is the total ion current chromatogram of the test solution of the standard decoction of Tetrapanax papyriferus (Hook.) K. Koch (negative ion mode);

[0125] Figure 11 It is the ultraviolet absorption chromatogram of the test solution of the standard decoction of Tetrapanax papyriferus (Hook.) K. Koch;

[0126] Figure 12 It is the first-order mass spectrometry scan chromatogram of Peak 1 (positive ion mode);

[0127] Figure 13 It is the first-order mass spectrometry scan chromatogram of Peak 1 (negative ion mode);

[0128] Figure 14 It is the first-order mass spectrometry scan chromatogram of Peak 6 (positive ion mode);

[0129] Figure 15 It is the first-order mass spectrometry scan chromatogram of Peak 6 (negative ion mode);

[0130] Figure 16 It is the specificity chromatogram of the standard decoction of Tetrapanax papyriferus (Hook.) K. Koch;

[0131] Figure 17 It is the linear regression equation chromatogram of protocatechuic acid;

[0132] Figure 18 It is the linear regression equation diagram of syringaldehyde. Specific implementation manners

[0133] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. The preferred embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0134] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0135] Regarding the content determination methods of Tetrapanax papyriferus, they mainly focus on polysaccharides and monosaccharides (D-mannose and fructose). There are also reports on determining the total flavonoids in it using rutin as a reference substance. However, the above methods all lack specificity. There are few studies on the characteristic / fingerprint chromatograms of Tetrapanax papyriferus in the existing literature. The characteristic chromatogram established by Qiu Zhijie et al. reflects the acid hydrolysis components of Tetrapanax papyriferus and cannot well reflect the prototype components in Tetrapanax papyriferus. Moreover, the chromatographic conditions only reflect 4 characteristic peaks and lack sufficient chromatographic peak information to reflect the main components in the standard decoction and formula granules of Tetrapanax papyriferus. Zhang Rui et al. established the UPLC fingerprint chromatogram of the chloroform fraction of the water extract of Tetrapanax papyriferus. However, their main research is on the chloroform fraction, and the components in the fingerprint chromatogram are mainly non-polar components.

[0136] The method for constructing the UPLC characteristic chromatogram of the standard decoction of Tetrapanax papyriferus constructed by the present invention selects a total of 7 prototype component common peaks, among which 2 components, protocatechuic acid and syringaldehyde, are identified, and the contents of protocatechuic acid and syringaldehyde are determined under the same chromatographic conditions. The construction method of the present invention can realize the content detection of water-soluble index components through the control of chromatographic conditions, can better reflect the components in the standard decoction of Tetrapanax papyriferus extracted with water as a solvent, and can better control its quality.

[0137] In the first aspect of the present invention, a method for constructing a UPLC characteristic chromatogram of the standard decoction of Tetrapanax papyriferus is provided, including the following steps:

[0138] Take the control medicinal material of Tetrapanax papyriferus and prepare a reference substance solution of the control medicinal material;

[0139] Take one or more of protocatechuic acid and syringaldehyde and prepare a reference substance solution;

[0140] Take the standard decoction of Tetrapanax papyriferus, and prepare the test solution;

[0141] Perform ultra-high performance liquid chromatography (UPLC) detection on the reference solution of the control medicinal material, the reference substance solution, and the test solution respectively, and construct the UPLC characteristic fingerprint of the standard decoction of Tetrapanax papyriferus;

[0142] The chromatographic conditions for the ultra-high performance liquid chromatography detection include:

[0143] Use acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B for gradient elution.

[0144] In some examples, the program of the gradient elution includes:

[0145] 0 - 10 min, the volume percentage of mobile phase A is 3%;

[0146] 10 min - 25 min, the volume percentage of mobile phase A changes from 3% to 7%;

[0147] 25 min - 32 min, the volume percentage of mobile phase A changes from 7% to 10%;

[0148] 32 min - 40 min, the volume percentage of mobile phase A is 10%;

[0149] 40 min - 42 min, the volume percentage of mobile phase A changes from 10% to 80%;

[0150] 42 min - 45 min, the volume percentage of mobile phase A is 80%.

[0151] In some examples, in the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.08% - 0.12%, for example, it can be 0.08%, 0.09%, 0.1%, 0.11% or 0.12%. Preferably, in the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.1%.

[0152] In some examples, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: using a chromatographic column filled with octadecylsilane chemically bonded silica gel.

[0153] In some specific examples, the specifications of the chromatographic column are: column length 150 mm, inner diameter 3.0 mm, particle size 2.5 μm.

[0154] In some examples, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the flow rate is 0.55 mL / min - 0.65 mL / min, for example, it can be 0.55 mL / min, 0.6 mL / min or 0.65 mL / min. Preferably, the flow rate is 0.6 mL / min.

[0155] In some of these examples, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the column temperature is 28°C to 32°C. For example, it can be 28°C, 29°C, 30°C, 31°C, or 32°C. Preferably, the column temperature is 30°C.

[0156] In some of these examples, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the detection wavelength is 210 nm to 230 nm. For example, it can be 210 nm, 215 nm, 220 nm, 225 nm, or 230 nm. Preferably, the detection wavelength is 220 nm.

[0157] In some of these examples, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the injection volume is 1 μL to 3 μL. For example, it can be 1 μL, 2 μL, or 3 μL. Preferably, the injection volume is 2 μL.

[0158] In some of these examples, the steps for preparing the reference crude drug solution include:

[0159] Performing a first water extraction treatment and a first organic solvent extraction treatment on the Tetrapanacis Medulla reference crude drug.

[0160] In some of these examples, the first water extraction treatment includes the following steps:

[0161] Mixing the Tetrapanacis Medulla reference crude drug and water, heating under reflux for extraction, concentrating, mixing the residue with water for dissolution, and preparing a first extract;

[0162] The first organic solvent extraction treatment includes the following steps;

[0163] Mixing the first extract with ethyl acetate to prepare a second extract;

[0164] Concentrating the second extract, using methanol as the re-dissolution solvent, dissolving the residue, filtering, and taking the filtrate;

[0165] The methanol is an aqueous methanol solution with a volume fraction of 45% to 55%.

[0166] In some of these examples, in the first water extraction treatment step, the mixing ratio of the Tetrapanacis Medulla reference crude drug and water is 1 g : (90 - 100) mL.

[0167] In some specific examples, the mixing ratio of the Tetrapanacis Medulla reference crude drug and water is 1 g : 90 mL.

[0168] In some of these examples, the heating reflux time is 55 min to 65 min, preferably 60 min.

[0169] In some of these examples, in the first organic solvent extraction step, the volume ratio of the first extract to ethyl acetate is 1:(2 - 2.5), preferably 1:2.

[0170] In some of these examples, the number of extractions with ethyl acetate is 3 - 4 times, preferably 3 times.

[0171] It can be understood that the volume ratio of the first extract to ethyl acetate being 1:(2 - 2.5) means that for each extraction, ethyl acetate is added according to the volume ratio of 1:(2 - 2.5) for extraction.

[0172] In some of these examples, using methanol as the re - dissolution solvent, the methanol is an aqueous methanol solution with a volume fraction of 45% - 55%, preferably 50% aqueous methanol solution.

[0173] In some of these examples, the steps for preparing the reference substance solution include:

[0174] Using methanol as the extraction solvent to prepare the reference substance solution;

[0175] In the reference substance solution, the concentration of protocatechuic acid is 8 μg / mL - 12 μg / mL, and the concentration of syringaldehyde is 8 μg / mL - 12 μg / mL.

[0176] In some specific examples, in the reference substance solution, the concentration of protocatechuic acid is 10 μg / mL; the concentration of syringaldehyde is 10 μg / mL.

[0177] In some of these examples, the steps for preparing the test sample solution include: subjecting the Tongcao standard decoction test sample to a second water extraction treatment and a second organic solvent extraction treatment.

[0178] In some of these examples, the second water extraction treatment includes the following steps:

[0179] Mixing the Tongcao standard decoction test sample and water and performing heating under reflux for extraction to prepare a third extract;

[0180] The second organic solvent extraction treatment includes the following steps;

[0181] Mixing the third extract with ethyl acetate to prepare a fourth extract;

[0182] Concentrating the fourth extract, using methanol as the re - dissolution solvent, dissolving the residue, filtering, and taking the filtrate;

[0183] The methanol is an aqueous methanol solution with a volume fraction of 45% - 55%.

[0184] In some of these examples, in the second water extraction step, the mixing ratio of the prepared test sample of Tetrapanacis Medulla and water is 1 g : (20 - 25) mL.

[0185] In some of these specific examples, the mixing ratio of the prepared test sample of Tetrapanacis Medulla and water is 1 g : 20 mL.

[0186] In some of these examples, the heating reflux time is 15 min to 45 min, preferably 30 min.

[0187] In some of these examples, in the second organic solvent extraction step, the volume ratio of the third extract to ethyl acetate is 1 : (2 - 2.5), preferably 1 : 2.

[0188] In some of these examples, the number of extractions with ethyl acetate is 3 to 4 times, preferably 3 times.

[0189] It can be understood that the volume ratio of the third extract to ethyl acetate being 1 : (2 - 2.5) means that for each extraction, ethyl acetate is added according to the volume ratio of 1 : (2 - 2.5) for extraction.

[0190] In some of these examples, the characteristic chromatogram presents 7 characteristic peaks, and corresponds to the retention times of 7 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 1 and peak 6 are consistent with the retention times of the reference peaks of the reference substance, the peak corresponding to the protocatechuic acid reference substance peak is peak S1, calculate the relative retention times of peak 2 to peak 4 and peak S1, the peak corresponding to the syringaldehyde reference substance peak is peak S2, calculate the relative retention times of peak 5 and peak 7 and peak S2, and their relative retention times are within the range of ±10% of the specified value. The specified values are: 1.44 (peak 2), 2.93 (peak 3), 3.39 (peak 4), 0.96 (peak 5), 1.07 (peak 7).

[0191] In some of these examples, the construction method further includes the step of performing mass spectrometric identification on the characteristic peaks in the characteristic chromatogram by using chromatography - mass spectrometry coupling method;

[0192] The chromatographic conditions for the identification process include: using acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B for gradient elution;

[0193] The conditions for the mass spectrometry method include:

[0194] The HESI ion source parameters include: sheath gas flow rate is 60 arb to 65 arb, auxiliary gas flow rate is 18 arb to 22 arb, spray voltage is 2.2 kV to 2.8 kV, S - lens voltage is 48 V to 52 V, heating temperature is 280 °C to 320 °C, capillary temperature is 480 °C to 520 °C;

[0195] The mass spectrometry scanning parameters include: the scanning mode is positive ion mode and / or negative ion mode, the scanning range is 120 m / z to 1200 m / z, the normalized collision energy is 20 V, and the type of mass spectrometry graph is a peak graph.

[0196] In some examples of the identification process, the gradient elution program includes:

[0197] From 0 to 10 min, the volume percentage of mobile phase A is 3%;

[0198] From 10 min to 25 min, the volume percentage of mobile phase A changes from 3% to 7%;

[0199] From 25 min to 32 min, the volume percentage of mobile phase A changes from 7% to 10%;

[0200] From 32 min to 40 min, the volume percentage of mobile phase A is 10%;

[0201] From 40 min to 42 min, the volume percentage of mobile phase A changes from 10% to 80%;

[0202] From 42 min to 45 min, the volume percentage of mobile phase A is 80%.

[0203] In some examples of the identification process, in the formic acid aqueous solution, the volume percentage of formic acid is 0.08% to 0.12%. For example, it can be 0.08%, 0.09%, 0.1%, 0.11% or 0.12%. Preferably, in the formic acid aqueous solution, the volume percentage of formic acid is 0.1%.

[0204] In some examples of the identification process, the chromatographic conditions further include: using a chromatographic column filled with octadecylsilane-bonded silica gel.

[0205] In some specific examples of the identification process, the specifications of the chromatographic column are: column length 150 mm, inner diameter 3.0 mm, particle size 2.5 μm.

[0206] In some examples of the identification process, the chromatographic conditions further include: the flow rate is 0.55 mL / min to 0.65 mL / min. Preferably, the flow rate is 0.6 mL / min.

[0207] In some examples of the identification process, the chromatographic conditions further include: the column temperature is 28 °C to 32 °C. Preferably, the column temperature is 30 °C.

[0208] In some examples of the identification process, the chromatographic conditions further include: the injection volume is 1 μL to 3 μL. Preferably, the injection volume is 2 μL.

[0209] In the second aspect of the present invention, a method for identifying the standard decoction of Tetrapanax papyriferus is further provided, including the following steps:

[0210] Prepare a test solution from the test sample;

[0211] Perform ultra-high performance liquid chromatography (UHPLC) detection on the test solution, compare the chromatogram of the obtained test solution with the characteristic chromatogram constructed in any of the above examples, and identify whether the test sample is the standard decoction of Tetrapanax papyriferus according to the comparison result;

[0212] The chromatographic conditions for the UHPLC detection include:

[0213] Use acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B for gradient elution.

[0214] In some examples of the identification method, the program of the gradient elution includes:

[0215] 0 - 10 min, the volume percentage of mobile phase A is 3%;

[0216] 10 min - 25 min, the volume percentage of mobile phase A changes from 3% to 7%;

[0217] 25 min - 32 min, the volume percentage of mobile phase A changes from 7% to 10%;

[0218] 32 min - 40 min, the volume percentage of mobile phase A is 10%;

[0219] 40 min - 42 min, the volume percentage of mobile phase A changes from 10% to 80%;

[0220] 42 min - 45 min, the volume percentage of mobile phase A is 80%.

[0221] In some examples of the identification method, in the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.08% - 0.12%, for example, it can be 0.08%, 0.09%, 0.1%, 0.11% or 0.12%. Preferably, in the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.1%.

[0222] In some examples of the identification method, the chromatographic conditions for the UHPLC detection further include: using a chromatographic column filled with octadecylsilane-bonded silica gel.

[0223] In some specific examples of the identification method, the specifications of the chromatographic column are: column length 150 mm, inner diameter 3.0 mm, particle size 2.5 μm.

[0224] In some examples of the identification method, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the flow rate is 0.55 mL / min to 0.65 mL / min. For example, it can be 0.55 mL / min, 0.6 mL / min, or 0.65 mL / min. Preferably, the flow rate is 0.6 mL / min.

[0225] In some examples of the identification method, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the column temperature is 28°C to 32°C. For example, it can be 28°C, 29°C, 30°C, 31°C, or 32°C. Preferably, the column temperature is 30°C.

[0226] In some examples of the identification method, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the detection wavelength is 210 nm to 230 nm. For example, it can be 210 nm, 215 nm, 220 nm, 225 nm, or 230 nm. Preferably, the detection wavelength is 220 nm.

[0227] In some examples of the identification method, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: the injection volume is 1 μL to 3 μL. For example, it can be 1 μL, 2 μL, or 3 μL. Preferably, the injection volume is 2 μL.

[0228] In some examples of the identification method, the steps for preparing the solution to be tested include:

[0229] Performing a third water extraction treatment and a third organic solvent extraction treatment on the product to be tested.

[0230] In some examples of the identification method, the third water extraction treatment includes mixing the product to be tested and water and performing heating under reflux for extraction to prepare a fifth extract;

[0231] The third organic solvent extraction treatment includes the following steps;

[0232] Mixing the fifth extract with ethyl acetate to prepare a sixth extract;

[0233] Concentrating the sixth extract, using methanol as a reconstitution solvent to dissolve the residue, filtering, and taking the filtrate;

[0234] The methanol is an aqueous methanol solution with a volume fraction of 45% to 55%.

[0235] In some examples of the identification method, in the third water extraction treatment step, the mixing ratio of the product to be tested and water is 1 g : (20 - 25) mL.

[0236] In some examples of the identification method, the heating under reflux time is 15 min to 45 min.

[0237] In some examples of the identification method, in the third organic solvent extraction step, the volume ratio of the fifth extract to ethyl acetate is 1:(2 - 2.5).

[0238] In some examples of the identification method, the number of extractions with ethyl acetate is 3 - 4 times.

[0239] The third aspect of the present invention provides a method for detecting the content of chemical components in a standard decoction of Tetrapanax papyriferus, comprising the following steps:

[0240] S110: Take one or more of protocatechuic acid and syringaldehyde to prepare reference substance solutions with different concentrations;

[0241] S210: Perform ultra - high performance liquid chromatography detection on the reference substance solutions with different concentrations, and construct a standard curve based on the peak area and concentration;

[0242] S310: Take the test sample of the standard decoction of Tetrapanax papyriferus and prepare a test solution;

[0243] S410: Perform ultra - high performance liquid chromatography detection and analysis on the test solution, and determine the content of chemical components in the test sample in combination with the standard curve.

[0244] In some specific examples of the detection method, in step S110, methanol is used as the extraction solvent to prepare a mixed reference substance stock solution of protocatechuic acid and syringaldehyde;

[0245] Dilute the mixed reference substance stock solution into 6 mixed reference substance solutions with different concentrations.

[0246] In some more specific examples of the detection method, in the mixed reference substance stock solution, the concentration of protocatechuic acid is 97.993 μg / mL, and the concentration of syringaldehyde is 103.194 μg / mL.

[0247] In some examples of the detection method, in step S210, perform ultra - high performance liquid chromatography detection on the mixed reference substance stock solution prepared in S110 and 6 mixed reference substance solutions with different concentrations.

[0248] In some examples of the detection method, the chromatographic conditions for the ultra - high performance liquid chromatography detection include: using acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B for gradient elution;

[0249] In some examples of the detection method, the program of the gradient elution includes:

[0250] 0 - 10 min, the volume percentage of mobile phase A is 3%;

[0251] 10 min - 25 min, the volume percentage of mobile phase A changes from 3% to 7%;

[0252] From 25 min to 32 min, the volume percentage of mobile phase A changes from 7% to 10%.

[0253] From 32 min to 40 min, the volume percentage of mobile phase A is 10%.

[0254] From 40 min to 42 min, the volume percentage of mobile phase A changes from 10% to 80%.

[0255] From 42 min to 45 min, the volume percentage of mobile phase A is 80%.

[0256] In some examples of the detection method, in the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.08% - 0.12%. For example, it can be 0.08%, 0.09%, 0.1%, 0.11% or 0.12%. Preferably, in the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.1%.

[0257] In some examples of the detection method, the chromatographic conditions for the ultra - performance liquid chromatography detection further include: using a chromatographic column filled with octadecylsilane - bonded silica gel.

[0258] In some more specific examples, the specifications of the chromatographic column are: column length 150 mm, inner diameter 3.0 mm, particle size 2.5 μm.

[0259] In some examples of the detection method, the chromatographic conditions for the ultra - performance liquid chromatography detection further include: the flow rate is 0.55 mL / min - 0.65 mL / min. For example, it can be 0.55 mL / min, 0.6 mL / min or 0.65 mL / min. Preferably, the flow rate is 0.6 mL / min.

[0260] In some examples of the detection method, the chromatographic conditions for the ultra - performance liquid chromatography detection further include: the column temperature is 28°C - 32°C. For example, it can be 28°C, 29°C, 30°C, 31°C or 32°C. Preferably, the column temperature is 30°C.

[0261] In some examples of the detection method, the chromatographic conditions for the ultra - performance liquid chromatography detection further include: the detection wavelength is 210 nm - 230 nm. For example, it can be 210 nm, 215 nm, 220 nm, 225 nm or 230 nm. Preferably, the detection wavelength is 220 nm.

[0262] In some examples of the detection method, the chromatographic conditions for the ultra - performance liquid chromatography detection further include: the injection volume is 1 μL - 3 μL. For example, it can be 1 μL, 2 μL or 3 μL. Preferably, the injection volume is 2 μL.

[0263] In some examples of the detection method, in step S310, preparing the solution to be tested includes the following steps:

[0264] Perform a fourth water extraction treatment and a fourth organic solvent extraction treatment on the sample to be tested of the Tongcao standard decoction.

[0265] In some examples of the detection method, the fourth water extraction treatment includes the following steps:

[0266] Mix the sample to be tested of the Tongcao standard decoction and water, and perform heating under reflux extraction to prepare a seventh extract;

[0267] The fourth organic solvent extraction treatment includes the following steps:

[0268] Mix the seventh extract with ethyl acetate to prepare an eighth extract;

[0269] Concentrate the eighth extract, use methanol as the reconstitution solvent, dissolve the residue, filter, and take the filtrate; the methanol is an aqueous methanol solution with a volume fraction of 45% - 55%.

[0270] In some examples of the detection method, in the fourth water extraction treatment step, the mixing ratio of the sample to be tested of the Tongcao standard decoction and water is 1 g : (20 - 25) mL.

[0271] In some examples of the detection method, the heating under reflux time is 15 min - 45 min.

[0272] In some examples of the detection method, in the fourth organic solvent extraction step, the volume ratio of the seventh extract to ethyl acetate is 1 : (2 - 2.5).

[0273] In some examples of the detection method, the number of extractions with ethyl acetate is 3 - 4 times.

[0274] It can be understood that the chromatographic conditions for performing ultra - high performance liquid chromatography detection and analysis on the solution to be tested are the same as those for the reference solution in ultra - high performance liquid chromatography detection.

[0275] In some examples of the detection method, the linear regression equation of protocatechuic acid is: y1 = 73993x1 - 10642, r = 1, where x1 represents the concentration of protocatechuic acid, y1 represents the peak area, and within the concentration range of 0.196 μg / mL - 97.993 μg / mL of protocatechuic acid, the linear relationship between the concentration and the peak area is good.

[0276] In some examples of the detection method, the linear regression equation of syringaldehyde is: y2 = 6590.8x2 + 1963.4, r = 1, where x2 represents the concentration of syringaldehyde and y2 represents the peak area. The linear relationship between the concentration and the peak area of syringaldehyde is good in the range of 0.516 μg / mL to 103.194 μg / mL.

[0277] The embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following examples, the guidance given in the present invention is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturers, or referring to the experimental methods known in the art.

[0278] In the following specific examples, regarding the measurement parameters of the raw material components, without special instructions, there may be slight deviations within the weighing accuracy range. Regarding the temperature and time parameters, acceptable deviations caused by the instrument test accuracy or operation accuracy are allowed.

[0279] Example 1

[0280] This example provides a method for constructing the UPLC characteristic fingerprint of Tetrapanax papyrifer standard decoction.

[0281] 1 Instruments, reagents and test drugs

[0282] Instruments: Waters ultra-high performance liquid chromatograph (Waters H-Class, Waters Corporation), Agilent ultra-high performance liquid chromatograph (model 1290, Agilent Technologies Co., Ltd.), Waters BEH C18 chromatographic column (3×150 mm, 2.5 μm, serial number: JS-194); Waters Cortecs T3 chromatographic column (3×150 mm, 2.7 μm, serial number: JS-342); Waters HSS T3 chromatographic column (4.6×150 mm, 5 μm, serial number: JS-153); Waters Cortecs T3 Phenyl chromatographic column (3×150 mm, 2.7 μm, serial number: JS-349); ten-thousandth balance (ME204E, Mettler-Toledo GmbH), millionth balance (XP26, Mettler-Toledo GmbH), numerically controlled ultrasonic cleaner (KQ500D, Kunshan Ultrasonic Instruments Co., Ltd.), constant temperature water bath (model HWS28, Shanghai Yiheng Scientific Co., Ltd.), ultra-pure water system (Milli-Q Direct, Merck KGaA).

[0283] Reagents: Ethanol (Tianjin Yongda Chemical Reagent Co., Ltd.) and methanol (Guangdong Guanghua Sci-Tech Co., Ltd.) are of analytical grade, methanol and acetonitrile (Merck KGaA) are of chromatographic grade, and water is ultrapure water (self-made in the laboratory).

[0284] Test drugs: Protocatechuic acid (batch number: 110809 - 201906, content: 97.7%, manufacturer: National Institutes for Food and Drug Control); Syringaldehyde (batch number: R09J6Y1, content: 98%, manufacturer: Shanghai Yuanye Bio-Technology Co., Ltd.); Tongcao reference crude drug (batch number: 125029 - 202006A, verified by Guangdong Institute for Drug Control); The batch number information of 15 batches of Tongcao standard decoctions is shown in Table 1.

[0285] Table 1 Information Table of Tongcao Standard Decoctions

[0286]

[0287] 2 Chromatographic Conditions and Preparation of Test Solution

[0288] 2.1 Chromatographic Conditions and System Suitability Test

[0289] Using octadecylsilane chemically bonded silica gel as the filler (column length 150 mm, inner diameter 3.0 mm, particle size 2.5 μm); Using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution was carried out according to the regulations in Table 2 below; The flow rate was 0.60 mL per minute; The column temperature was 30 °C; The detection wavelength was 220 nm.

[0290] Table 2 Gradient Elution Table

[0291]

[0292] 2.2 Preparation of Test Solution:

[0293] Preparation of test solution for Tongcao standard decoction: Take an appropriate amount of this product, grind it finely, take about 0.5 g, weigh it accurately, place it in a stoppered conical flask, add 10 mL of water, weigh it, heat under reflux for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with water, extract with ethyl acetate by shaking 3 times, 20 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 50% methanol and transfer it to a 2 mL volumetric flask, add 50% methanol to the scale, shake well, filter, and take the subsequent filtrate, that is obtained.

[0294] 2.3 Preparation of Reference Solution and Reference Substance Solution:

[0295] Take 10 g of the control crude drug of Tetrapanacis Medulla, place it in a flask, add 900 mL of water, heat under reflux for 60 minutes, filter by suction, evaporate the filtrate to dryness, add 15 mL of water to the residue to dissolve it, extract with ethyl acetate by shaking 3 times, 30 mL each time. Combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 50% methanol and transfer it to a 2-mL volumetric flask, add 50% methanol to the scale, shake well, filter, and take the subsequent filtrate as the reference solution of the control crude drug.

[0296] Weigh accurately appropriate amounts of protocatechuic acid reference substance and syringaldehyde reference substance, dissolve them in methanol to prepare a solution containing 10 μg of protocatechuic acid reference substance and 10 μg of syringaldehyde reference substance per 1 mL, which is used as the reference solution for content determination and the reference solution of the reference substance for the characteristic chromatogram.

[0297] 2.4 Determination method:

[0298] Precisely pipette 2 μL each of the reference solution and the test solution, inject them into the liquid chromatograph for determination, and thus obtain the results.

[0299] 2.5 Drafting of the characteristic chromatogram

[0300] Drafting of the characteristic chromatogram of the standard decoction of Tetrapanacis Medulla: Match the UPLC characteristic chromatograms of 15 batches of the standard decoction of Tetrapanacis Medulla in Table 1 using the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints", generate a reference chromatogram by the average method, and establish the reference characteristic chromatogram of the standard decoction of Tetrapanacis Medulla. For example, Figure 1 , this characteristic chromatogram has a total of 7 characteristic peaks, which correspond to the 7 characteristic peaks of the control crude drug of Tetrapanacis Medulla. The characteristic chromatogram of the control crude drug of Tetrapanacis Medulla can be seen in Figure 2 .

[0301] 3 Optimization of chromatographic conditions and determination of the preparation of the test solution

[0302] 3.1 Optimization of the elution gradient:

[0303] Take an appropriate amount of the standard decoction of Tetrapanacis Medulla (batch number is BTS13), grind it finely, take about 0.5 g, and prepare the test solution according to the method under "2.2". Except for different elution gradients, other analyses are carried out according to the regulations under "2.1".

[0304] Gradient 1:

[0305]

[0306] Gradient 2:

[0307]

[0308] Gradient 3:

[0309]

[0310] Gradient 4:

[0311]

[0312] The results showed that by comparing the chromatograms of different gradient elutions ( Figures 3 - 6 ), it was found that when gradient 4 was selected, the baseline of the chromatogram was stable and the separation effect of each chromatographic peak was better. Therefore, gradient 4 was selected as the final elution gradient.

[0313] 3.2 Column investigation

[0314] An appropriate amount of the standard decoction of Tetrapanax papyriferus (batch number: BTS13) was taken, ground finely, about 0.5 g was taken, accurately weighed, and the test solution was prepared according to the preparation method of the test solution determined in item "2.2". Except that the chromatographic columns were Waters BEH C18 column (3×150 mm, 2.5 μm, serial number: JS-194); Waters Cortecs T3 column (3×150 mm, 2.7 μm, serial number: JS-342); Waters HSS T3 column (4.6×150 mm, 5 μm, serial number: JS-153); Waters Cortecs T3 Phenyl column (3×150 mm, 2.7 μm, serial number: JS-349), other chromatographic conditions were the same as those specified in item "2.1", and injection analysis was carried out. The experimental results were as Figure 7 .

[0315] The results showed that except for the Waters BEH C18 (3 mm×150 mm, 2.5 μm) column, there were significant differences in the separation effect and retention time of the standard decoction of Tetrapanax papyriferus on the other three different types of columns, and some chromatographic peaks were missing, indicating that different types of columns had a great influence on the relative retention time of each characteristic peak. The Waters BEH C18 (3 mm×150 mm, 2.5 μm) column provided more abundant chromatographic information. Therefore, in order to more fully reflect the components of Tetrapanax papyriferus, it was recommended to fix the chromatographic column as the Waters BEH C18 (3 mm×150 mm, 2.5 μm) column.

[0316] 3.3 Investigation on the preparation of test solution

[0317] 3.3.1 Investigation on the number of shaking extractions

[0318] In this experiment, the influence of the number of shaking extractions on the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus was investigated. The main investigation was on shaking extraction 2 times, 3 times, and 4 times. By observing the peak shapes and resolutions of 7 characteristic peaks, and calculating the "total peak area / sample weight" of the 7 characteristic peaks and the contents of protocatechuic acid and syringaldehyde, the influence of different extraction times on the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus was compared.

[0319] Take an appropriate amount of the standard decoction of Tetrapanax papyriferus, grind it finely, take about 0.5 g, weigh it accurately, in parallel for 3 groups, 2 portions in each group, place in a stoppered conical flask, add 10 mL of water, weigh, perform ultrasonic extraction (power 300 W, frequency 40 kHz) for 30 minutes, take out, let it cool, weigh again, make up the lost weight with water, extract with ethyl acetate by shaking 2, 3, and 4 times respectively, 20 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue with 50% methanol and transfer it to a 2-mL volumetric flask, add 10% methanol to the scale, shake well, filter, take the continued filtrate, and you will get it. Others are analyzed by injection according to the chromatographic conditions under item "2.1", as shown in Table 3.

[0320] Table 3 Results of the investigation on the number of shaking extractions for the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus

[0321]

[0322] The results showed that there was little change in the values of "total peak area / sample weight" and the contents of protocatechuic acid and syringaldehyde for each characteristic peak in the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus after shaking extraction 3 times and 4 times, and the extraction was basically complete. To ensure complete extraction and save solvents, the number of shaking extractions was therefore selected as 3 times.

[0323] 3.3.2 Investigation on the re-dissolving solvent

[0324] In this experiment, the effects of different re-dissolving solvents on the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus were investigated. Using methanol, 70% methanol, 50% methanol, and 10% methanol as re-dissolving solvents, by observing the peak shapes and resolution of 7 characteristic peaks, and calculating the "total peak area / sample weight" of 7 characteristic peaks and the contents of protocatechuic acid and syringaldehyde, the effects of different re-dissolving solvents on the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus were compared.

[0325] Take an appropriate amount of the standard decoction of Tetrapanax papyriferus, grind it finely, take about 0.5 g, weigh it accurately, in parallel for 4 groups, 2 portions in each group, place in a stoppered conical flask, add 10 mL of water, weigh, perform ultrasonic extraction (power 300 W, frequency 40 kHz) for 30 minutes, take out, let it cool, weigh again, make up the lost weight with water, extract with ethyl acetate by shaking 3 times respectively, 20 mL each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue with methanol, 70% methanol, 50% methanol, and 10% methanol respectively and transfer it to a 2-mL volumetric flask, add methanol, 70% methanol, 50% methanol, and 10% methanol respectively to the scale, shake well, filter, take the continued filtrate, and you will get it. Others are analyzed by injection according to the chromatographic conditions under item "2.1", as shown in Table 4.

[0326] Table 4 Results of the investigation on the re-dissolving solvent for the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus

[0327]

[0328] The results showed that there was no significant difference in the "total peak area / sample weight" when methanol, 70% methanol, and 50% methanol were used for dissolution and transfer in the preparation of the test solution for the characteristic chromatogram of Tetrapanacis Medulla standard decoction. The contents of protocatechuic acid and syringaldehyde were the highest in 50% methanol. Therefore, 50% methanol was selected as the transfer and re-dissolution solvent for more complete extraction.

[0329] 3.3.3 Investigation of extraction solvents

[0330] In this experiment, the effects of different extraction solvents on the characteristic chromatogram of Tetrapanacis Medulla standard decoction were investigated. Dichloromethane, diethyl ether, and ethyl acetate were used as extraction solvents. By observing the peak shapes and resolutions of 7 characteristic peaks, and calculating the "total peak area / sample weight" of the 7 characteristic peaks as well as the contents of protocatechuic acid and syringaldehyde, the effects of different extraction solvents on the characteristic chromatogram of Tetrapanacis Medulla standard decoction were compared.

[0331] Take an appropriate amount of Tetrapanacis Medulla standard decoction, grind it finely, take about 0.5 g, weigh it precisely, in parallel for 3 groups, 2 portions in each group, place it in a stoppered conical flask, add 10 mL of water, weigh it, ultrasonically extract (power 300 W, frequency 40 kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with water, shake and extract 3 times with ethyl acetate, diethyl ether, and dichloromethane respectively, 20 mL each time. Combine the ethyl acetate layer, diethyl ether solution, and dichloromethane solution respectively, evaporate to dryness, dissolve the residue in 50% methanol and transfer it to a 2 mL volumetric flask, add 50% methanol to the scale, shake well, filter, and take the continuous filtrate to obtain the test solution. Other samples were analyzed by injection according to the chromatographic conditions under "2.1" as shown in Table 5.

[0332] Table 5 Results of the investigation of extraction solvents for the characteristic chromatogram of Tetrapanacis Medulla standard decoction

[0333]

[0334] The results showed that among the 3 extraction solvents, dichloromethane failed to show 7 characteristic peaks. For the characteristic chromatogram extracted by ethyl acetate, the values of "total peak area / sample weight" and the contents of protocatechuic acid and syringaldehyde were the highest, and its extraction efficiency was the best. Therefore, ethyl acetate was selected as the extraction solvent.

[0335] 3.3.4 Investigation of extraction methods

[0336] In this experiment, the effects of different extraction methods on the characteristic chromatogram of Tetrapanacis Medulla standard decoction were investigated. Ultrasonic extraction and reflux extraction were mainly investigated. By observing the peak shapes and resolutions of 7 characteristic peaks, and calculating the "total peak area / sample weight" of the 7 characteristic peaks as well as the contents of protocatechuic acid and syringaldehyde, the effects of different extraction methods on the characteristic chromatogram of Tetrapanacis Medulla standard decoction were compared.

[0337] Take an appropriate amount of the standard decoction of Tetrapanax papyriferus, grind it finely, take about 0.5 g, weigh it accurately, prepare 4 parallel groups, with 2 portions in each group. Place them in a stoppered conical flask, add 10 mL of water, weigh, and ultrasonically extract (power 300 W, frequency 40 kHz) for 30 minutes and reflux under heating for 30 minutes respectively. Take out, let it cool, weigh again, make up the lost weight with water, extract with ethyl acetate by shaking 3 times, 20 mL each time. Combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 50% methanol and transfer it to a 2-mL volumetric flask, add 50% methanol to the scale. And ultrasonically extract (power 300 W, frequency 40 kHz) for 30 minutes and reflux under heating for 30 minutes respectively. Take out, let it cool, weigh again, make up the lost weight with water, shake well, filter, and take the continuous filtrate to obtain the sample solution. Inject and analyze under the chromatographic conditions in item "2.1" as shown in Table 6.

[0338] Table 6 Results of the investigation on the extraction methods of the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus

[0339]

[0340] The results showed that with different extraction methods, the baselines of the characteristic chromatograms of the samples extracted by direct ultrasonic extraction and reflux under heating were relatively noisy; and the extraction efficiencies of the samples extracted by ultrasonic treatment followed by extraction were all relatively low, with little difference. While for the extraction method of reflux under heating followed by shaking extraction, the extraction efficiencies of each characteristic peak, as well as the contents of protocatechuic acid and syringaldehyde, were the best. Therefore, to ensure sufficient extraction, the extraction method of reflux under heating followed by shaking extraction was selected.

[0341] 3.3.5 Investigation on extraction time

[0342] In this experiment, the effect of reflux time under heating on the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus was investigated. By observing the peak shapes and resolutions of the 7 temporarily determined characteristic peaks, and calculating the "total peak area / sample weight" of the 7 characteristic peaks, as well as the contents of protocatechuic acid and syringaldehyde, the effects of different reflux times under heating on the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus were compared.

[0343] Take an appropriate amount of the standard decoction of Tetrapanax papyriferus, grind it finely, take about 0.5 g, weigh it accurately, prepare 3 parallel groups, with 2 portions in each group. Place them in a stoppered conical flask, add 10 mL of water, weigh, and reflux under heating for 15 minutes, 30 minutes, and 45 minutes respectively. Take out, let it cool, weigh again, make up the lost weight with water, extract with ethyl acetate by shaking 3 times, 20 mL each time. Combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 50% methanol and transfer it to a 2-mL volumetric flask, add 50% methanol to the scale, shake well, filter, and take the continuous filtrate to obtain the sample solution. Inject and analyze under the chromatographic conditions in item "2.1" as shown in Table 7.

[0344] Table 7 Results of the investigation on the extraction time of the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus

[0345]

[0346] The results showed that there were no significant differences in the "total peak area / sample weight" of the 7 characteristic peaks, as well as the contents of protocatechuic acid and syringaldehyde, at different heating reflux times. Complete extraction could be achieved after heating reflux for 30 minutes. Considering the influence of the experimental environment, in order to ensure the durability of the method, the ultrasonic extraction time was selected as 30 minutes.

[0347] 3.3.6 Determination of the preparation method of the test solution

[0348] According to the above experimental results, the pretreatment method of the characteristic chromatogram sample of Tetrapanacis Medulla standard decoction can be determined as follows:

[0349] Take an appropriate amount of this product, grind it finely, take about 0.5 g, weigh it precisely, place it in a stoppered conical flask, add 10 mL of water, weigh it, heat under reflux for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with water, extract it 3 times with ethyl acetate by shaking, 20 mL each time. Combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 50% methanol and transfer it to a 2-mL volumetric flask, add 50% methanol to the scale, shake well, filter, and take the subsequent filtrate, which is the test solution.

[0350] 4 Determination of the common peaks:

[0351] Taking Tetrapanacis Medulla standard decoction as an example, the determination of the common peaks of this characteristic chromatogram method was elaborated, and the similarity evaluation of Tetrapanacis Medulla standard decoction was carried out as follows:

[0352] Take 15 batches of Tetrapanacis Medulla standard decoction samples, prepare the test solutions according to the preparation method of the test solution determined under item "2.2", precisely pipette the above-mentioned test solutions and the reference solution of the control crude drug respectively, inject the samples for determination according to the chromatographic conditions determined under item "2.1", use the "Similarity Evaluation Software for Traditional Chinese Medicine Chromatographic Fingerprints" to identify the common peaks of the characteristic chromatograms of 15 batches of Tetrapanacis Medulla standard decoction, and select 7 common peaks with known components, larger peak areas, better peak shapes and resolutions, and higher purities as the characteristic peaks of Tetrapanacis Medulla standard decoction. As Figure 8 shown, select peak 1 protocatechuic acid as the reference peak S1, and calculate the relative retention time and relative peak area of peaks 2 - 4 with respect to peak S1; select peak 6 syringaldehyde as the reference peak S2, and calculate the relative retention time and relative peak area of peaks 5 and 7 with respect to peak S2; calculate the RSD value, and use the relative retention time to locate the characteristic peaks.

[0353] 5 Mass spectrometric identification of the characteristic peaks:

[0354] Referring to the liquid chromatography conditions of the characteristic chromatogram of Tetrapanacis Medulla standard decoction, the compounds in the test solution of Tetrapanacis Medulla standard decoction were analyzed by mass spectrometry, and the secondary fragment ions were matched with the data in the local mass spectrometry database and relevant literature was referred to identify the compounds. Finally, reference substances were used, and through the comparative analysis of the retention time of liquid chromatography, ultraviolet absorption spectrum, accurate molecular weight of mass spectrometry, and fragment ions, it was finally confirmed that peak 1 (S1) in the characteristic chromatogram of Tetrapanacis Medulla standard decoction was protocatechuic acid, and peak 6 (S2) was syringaldehyde. See the total ion current chromatogram of the test solution in Figure 9 、 Figure 10 , and see the ultraviolet absorption chromatogram in Figure 11 . The compound information is shown in Table 10.

[0355] 5.1 Chromatography - Mass Spectrometry Conditions:

[0356] Chromatography conditions: The filler is octadecylsilane - bonded silica gel (column length 150 mm, inner diameter 3.0 mm, particle size 2.5 μm); acetonitrile is used as mobile phase A, and 0.1% formic acid solution is used as mobile phase B, and gradient elution is carried out according to the regulations in Table 8 below; the flow rate is 0.60 mL per minute; the column temperature is 30 °C.

[0357] Table 8 Gradient Elution Table

[0358]

[0359] Mass spectrometry conditions: As shown in Table 9.

[0360] Table 9 Mass Spectrometry Parameters (Shimadzu LCMS - 9030)

[0361]

[0362] 5.2 Determination method: Precisely pipette 2 μL of the test solution under item 2.2, inject it into the liquid chromatograph or liquid chromatography - mass spectrometry hyphenated instrument for determination to obtain the result.

[0363] Table 10 Identification Results of Compounds in Tetrapanacis Medulla Standard Decoction

[0364]

[0365] Mass spectrometry analysis of peak 1:

[0366] Extract the primary chromatogram of peak 1 (peak 1), and the time period is 8.12 - 8.43 min. The result is shown in Figure 12~ Figure 13 .

[0367] In the primary chromatogram extraction of peak 1, the peak with m / z = 155.0335 in the positive ion mode should be the [M + H]+ peak, see Figure 12, its molecular formula was speculated to be C7H6O4 from its exact molecular weight; the second-order spectrum of the peak with m / z = 153.0181 at a collision energy of 20 in the negative ion mode was extracted. The results showed that the main ion fragments included 109.0281, etc. For the detailed results, see Figure 13 .

[0368] By matching with the local mass spectrometry database and combining with the identification results of reference substances, it was speculated that peak 1 might be protocatechuic acid, and its structural formula was Formula 1.

[0369]

[0370] Formula 1

[0371] The possible cleavage modes are as follows:

[0372]

[0373] Mass spectrometry analysis of peak 6:

[0374] The first-order chromatogram of peak 11 (peak 6) was extracted, and the time period was 39.57 - 40.28 min. The results are shown in Figures 14 - 15 :

[0375] In the extracted first-order chromatogram of peak 11, the response value of the peak with m / z = 183.0651 in the positive ion mode was the highest, which was the [M + H]+ peak, as shown in Figure 14 , its molecular formula was speculated to be C9H 10 O4 from its exact molecular weight; the second-order spectrum of the peak with m / z = 181.0496 at a collision energy of 20 in the negative ion mode was extracted, as shown in Figure 15 , and the results showed that the main ion fragments included 166.0260, etc. By matching with the local mass spectrometry database and combining with the comparison results of reference substances, peak 11 was confirmed to be syringaldehyde, and its structural formula was Formula 2.

[0376]

[0377] Formula 2

[0378] 6 Methodological verification

[0379] 6.1 Specificity investigation

[0380] Precisely pipette 2 μL of the test solution of the prepared standard decoction of Tetrapanax papyriferus, the reference solution, and the blank solvent respectively, inject them into the liquid chromatograph, and determine according to the chromatographic conditions under item "2.1". The results are as Figure 16 . The results showed that the test solution chromatogram had the same chromatographic peaks at the retention times corresponding to the reference solution chromatogram, and the blank solvent had no interference, indicating that the method had good specificity.

[0381] 6.2 Precision investigation

[0382] Take an appropriate amount of the standard decoction of Tetrapanax papyriferus, grind it finely, take about 0.5 g, weigh it accurately, prepare the test solution according to the preparation method of the test solution determined under item "2.2", and inject and analyze it under the chromatographic conditions in item "2.1". The same test solution was injected continuously for 6 times. The results showed that in the precision test, the same test solution was injected continuously for 6 times. Using the protocatechuic acid chromatographic peak as the reference peak S1 and the syringaldehyde chromatographic peak as the reference peak S2, the RSD values of the relative retention times of each characteristic peak to the S peak were in the range of 0.04% - 0.32%, and the RSD values of the relative peak areas were in the range of 0.50% - 2.63%, both less than 3.0%, indicating that the instrument precision was good.

[0383] 6.3 Repeatability test

[0384] Take an appropriate amount of the standard decoction of Tetrapanax papyriferus, grind it finely, take about 0.5 g, weigh 6 parallel portions accurately, prepare 6 test solutions according to the preparation method of the test solution determined under item "2.2", and inject and analyze them under the chromatographic conditions in item "2.1". The results showed that the same batch of samples was repeatedly determined 6 times. Using the protocatechuic acid chromatographic peak as the reference peak S1 and the syringaldehyde chromatographic peak as the reference peak S2, the RSD values of the relative retention times of each characteristic peak to the S peak were in the range of 0.05% - 0.21%, and the RSD values of the relative peak areas were in the range of 0.64% - 2.97%, both less than 3.0%, indicating that the repeatability of this method was good.

[0385] 6.4 Intermediate precision

[0386] Determined by different experimenters at different times on different high-performance liquid chromatographs. Take an appropriate amount of the standard decoction of Tetrapanax papyriferus, grind it finely, take about 0.5 g, weigh 6 parallel portions accurately, prepare 6 test solutions according to the preparation method of the test solution determined under item "2.2", and inject and analyze them under the chromatographic conditions in item "2.1". The results showed that for the 6 test solutions, using the protocatechuic acid chromatographic peak as the reference peak S1 and the syringaldehyde chromatographic peak as the reference peak S2, the RSD values of the relative retention times of each characteristic peak to the S peak were in the range of 0.07% - 0.28%, and the RSD values of the relative peak areas were in the range of 0.44% - 2.57%, both less than 3.0%. Calculated together with the 6 test solutions in the repeatability test, the RSD values of the relative retention times of the 12 test solutions were in the range of 0.12% - 0.38%, and the RSD values of the relative peak areas were in the range of 0.24% - 2.01%, both less than 3.0%. This indicated that the intermediate precision of this method was good.

[0387] 6.5 Stability test

[0388] Take an appropriate amount of the standard decoction of Tetrapanax papyriferus, grind it finely, take about 0.5 g, weigh it accurately, prepare the test solution according to the test solution preparation method determined under item "2.2", and inject and analyze it at 0, 2, 4, 6, 10, 12, and 20 hours respectively according to the chromatographic conditions under item "2.1". The results show that the same test solution is analyzed at 0, 2, 4, 6, 10, 12, and 20 hours respectively. Taking the protocatechuic acid chromatographic peak as the reference peak S1 and the syringaldehyde chromatographic peak as the reference peak S2, the RSD values of the relative retention times of each characteristic peak to the S peak are in the range of 0.04% - 0.31%, and the RSD values of the relative peak areas are in the range of 0.53% - 2.10%, both less than 3.0%, indicating that the test solution is relatively stable within 24 hours.

[0389] Example 2

[0390] This example provides a method for determining the contents of protocatechuic acid and syringaldehyde.

[0391] 1. The instruments, reagents and test drugs are the same as those in "Example 1"

[0392] 2. The chromatographic conditions and the preparation of the test solution are the same as those in "Example 1"

[0393] 3. Methodological verification

[0394] 3.1 Linearity

[0395] Accurately weigh 2.006 mg of protocatechuic acid reference substance and 2.106 mg of syringaldehyde reference substance, place them in a 20 mL volumetric flask, dissolve them with formaldehyde and dilute to the mark to prepare a reference substance stock solution containing 97.993 μg of protocatechuic acid and 103.194 μg of syringaldehyde per 1 mL. Accurately pipette the above reference substance stock solution respectively and dilute it into 6 linear reference substance solutions with different concentrations. Then accurately pipette the above 6 reference substance solutions with different concentrations and the stock solution respectively, inject and determine according to the chromatographic conditions under item "2.1", and record the chromatographic peak areas. Taking the peak area as the ordinate (y) and the reference substance concentration as the abscissa (x), draw the standard curves respectively, as shown in Table 11 below and Figure 17 、 Figure 18 shown. The results show that the linear regression equation of protocatechuic acid is: y = 73993x - 10642, r = 1, indicating that in the concentration range of 0.196 μg / mL - 97.993 μg / mL, the linear relationship between the concentration and the peak area is good; the linear regression equation of syringaldehyde is: y = 6590.8x + 1963.4, r = 1, indicating that in the concentration range of 0.516 μg / mL - 103.194 μg / mL, the linear relationship between the concentration and the peak area is good.

[0396] Table 11 Results of linear investigation of protocatechuic acid and syringaldehyde

[0397]

[0398] 3.2 Repeatability

[0399] Take the repeatability test sample solution of "6.3", and determine it according to the chromatographic conditions of "2.1". Calculate the contents of protocatechuic acid and syringaldehyde, and calculate the RSD of 6 parallel samples. As shown in Table 12 below, the results show that the RSDs of the repeatability contents of protocatechuic acid and syringaldehyde are 3.0% and 2.9% respectively, indicating that the repeatability of the method is good.

[0400] Table 12 Results of the repeatability investigation of the contents of protocatechuic acid and syringaldehyde

[0401]

[0402] 3.3 Intermediate precision

[0403] Take the intermediate precision test sample solution of "6.4", and determine it according to the chromatographic conditions of "2.1". Calculate the contents of protocatechuic acid and syringaldehyde, and calculate the RSD of 6 parallel samples. The results are 1.2% and 3.6% respectively; and calculate together with the 6 samples of repeatability. The RSDs of the contents of protocatechuic acid and syringaldehyde in 12 samples are 3.3% and 3.4% respectively. As shown in Table 13 below, it indicates that the intermediate precision of the method is good.

[0404] Table 13 Results of the intermediate precision investigation of the contents of protocatechuic acid and syringaldehyde

[0405]

[0406] 3.4 Recovery

[0407] Accurately weigh 2.005 mg of protocatechuic acid reference substance and 2.756 mg of syringaldehyde reference substance, dissolve them with methanol and prepare a mixed reference substance solution containing 4.897 μg of protocatechuic acid and 13.504 μg of syringaldehyde per 1 mL.

[0408] Take an appropriate amount of Tongcao standard decoction, grind it finely, weigh 0.25 g, accurately weigh it, and accurately add 1 mL of the above mixed reference substance solution respectively. According to the preparation method of the test sample solution determined under "2.2", prepare the test sample solution. According to the chromatographic conditions under "2.1", determine the contents of protocatechuic acid and syringaldehyde in the test sample solution, and calculate the sample addition recovery rate. As shown in Table 14 below, the experimental results show that the average recovery rate of protocatechuic acid is 100.8%, and the recovery rate of syringaldehyde is 91.6%. According to the "Guidelines for Validation of Analytical Methods" in the 2020 Edition of the Chinese Pharmacopoeia, when the content of the component to be determined in the sample is in the range of 0.1% - 1%, the recovery limit is 85% - 110%, indicating that the recovery rate is good.

[0409] Table 14 Results of the sample addition recovery test for protocatechuic acid and syringaldehyde

[0410]

[0411] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0412] The above-described embodiments only represent several implementation manners of the present invention, which are convenient for understanding the technical solutions of the present invention specifically and in detail, but should not be construed as a limitation on the protection scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of this invention patent shall be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for constructing the UPLC characteristic fingerprint of Tetrapanax papyrifer standard decoction, characterized in that, It includes the following steps: Take the Caulis Tetrapanacis control medicinal material and prepare a reference substance solution of the control medicinal material; Take one or more of protocatechuic acid and syringaldehyde and prepare a reference substance solution; Take the standard decoction of Caulis Tetrapanacis and prepare a test solution; Perform ultra-high performance liquid chromatography detection on the reference substance solution of the control medicinal material, the reference substance solution, and the test solution respectively, and construct the UPLC characteristic fingerprint of the standard decoction of Caulis Tetrapanacis; The chromatographic conditions for the ultra-high performance liquid chromatography detection include: Use acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B for gradient elution.

2. The construction method according to claim 1, characterized in that, The program for the gradient elution includes: From 0 to 10 min, the volume percentage of mobile phase A is 3%; From 10 min to 25 min, the volume percentage of mobile phase A changes from 3% to 7%; From 25 min to 32 min, the volume percentage of mobile phase A changes from 7% to 10%; From 32 min to 40 min, the volume percentage of mobile phase A is 10%; From 40 min to 42 min, the volume percentage of mobile phase A changes from 10% to 80%; From 42 min to 45 min, the volume percentage of mobile phase A is 80%.

3. The construction method according to claim 1, characterized in that, In the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.08% - 0.12%.

4. The construction method according to claim 1, characterized in that, The chromatographic conditions for the ultra-high performance liquid chromatography detection also include one or more of the following (1) - (5): (1) Use a chromatographic column filled with octadecylsilane chemically bonded silica gel; (2) The flow rate is 0.55 mL / min - 0.65 mL / min; (3) The column temperature is 28°C - 32°C; (4) The detection wavelength is 210 nm - 230 nm; (5) The injection volume is 1 μL - 3 μL.

5. The construction method according to any one of claims 1 to 4, characterized in that, The steps for preparing the control medicinal material solution include: Perform the first water extraction treatment and the first organic solvent extraction treatment on the Caulis Tetrapanacis control medicinal material.

6. The construction method according to claim 5, characterized in that, The first water extraction treatment includes the following steps: Mix the Caulis Tetrapanacis control medicinal material and water, perform heating under reflux for extraction, concentrate, mix the residue with water for dissolution, and prepare the first extract; The first organic solvent extraction treatment includes the following steps: Mix the first extract with ethyl acetate for extraction and prepare the second extract; Concentrate the second extract, use methanol as the re-dissolution solvent, dissolve the residue, filter, and take the filtrate; The methanol is a methanol aqueous solution with a volume fraction of 45% - 55%.

7. The construction method according to claim 6, characterized in that, Meet one or more of the following (1) - (4): (1) In the first water extraction treatment step, the mixing ratio of the Caulis Tetrapanacis control medicinal material and water is 1 g:(90 - 100) mL; (2) The heating reflux time is 55 min - 65 min; (3) In the first organic solvent extraction step, the mixing volume ratio of the first extract and ethyl acetate is 1:(2 - 2.5); (4) The number of times of extraction with ethyl acetate is 3 - 4 times.

8. The construction method according to any one of claims 1 to 4, characterized in that, The steps for preparing the reference substance solution include: Use methanol as the extraction solvent to prepare the reference substance solution; In the reference substance solution, the concentration of protocatechuic acid is 8 μg / mL - 12 μg / mL, and the concentration of syringaldehyde is 8 μg / mL - 12 μg / mL.

9. The construction method according to any one of claims 1 to 4, characterized in that, The steps for preparing the test solution include: The test sample of the prepared Herba Tetrapanacis standard decoction is subjected to a second water extraction treatment and a second organic solvent extraction treatment.

10. The construction method according to claim 9, characterized in that, The second water extraction treatment includes the following steps: Mix the test sample of the Herba Tetrapanacis standard decoction with water and perform heating under reflux extraction to prepare a third extract. The second organic solvent extraction treatment includes the following steps: Mix the third extract with ethyl acetate, extract, and prepare a fourth extract. Concentrate the fourth extract, use methanol as the reconstitution solvent to dissolve the residue, filter, and take the filtrate. The methanol is an aqueous methanol solution with a volume fraction of 45% - 55%.

11. The construction method according to claim 10, characterized in that, Meet one or more of the following (1) - (4): (1) In the second water extraction step, the mixing ratio of the test sample of the Herba Tetrapanacis standard decoction to water is 1 g : (20 - 25) mL; (2) The heating reflux time is 15 min - 45 min; (3) In the second organic solvent extraction step, the volume ratio of the third extract to ethyl acetate is 1 : (2 - 2.5); (4) The number of extractions with ethyl acetate is 3 - 4 times.

12. The construction method according to any one of claims 1 to 4, characterized in that, The characteristic chromatogram shows 7 characteristic peaks, which correspond to the retention times of 7 characteristic peaks in the chromatogram of the reference medicinal material as the reference. Among them, peak 1 and peak 6 are consistent with the retention times of the reference peaks of the reference medicinal material respectively. The peak corresponding to the protocatechuic acid reference medicinal material peak is peak S1. Calculate the relative retention times of peak 2 - peak 4 with peak S1. The peak corresponding to the syringaldehyde reference medicinal material peak is peak S2. Calculate the relative retention times of peak 5 and peak 7 with peak S2. The relative retention times are within the range of ±10% of the specified values. The specified values are: 1.44 (peak 2), 2.93 (peak 3), 3.39 (peak 4), 0.96 (peak 5), 1.07 (peak 7).

13. The construction method according to any one of claims 1 to 4, characterized in that, The construction method further includes a step of performing mass spectrometry identification on the characteristic peaks in the characteristic chromatogram by using chromatography - mass spectrometry coupling method; The chromatographic conditions for the identification process include: using acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B for gradient elution; The conditions of the mass spectrometry method include: The HESI ion source parameters include: sheath gas flow rate is 60 arb - 65 arb, auxiliary gas flow rate is 18 arb - 22 arb, spray voltage is 2.2 kV - 2.8 kV, S - lens voltage is 48 V - 52 V, heating temperature is 280 °C - 320 °C, capillary temperature is 480 °C - 520 °C; The mass spectrometry scanning parameters include: scanning mode is positive ion mode and / or negative ion mode, scanning range is 120 m / z - 1200 m / z, normalized collision energy is 20 V, and the mass spectrometry graph type is a peak shape graph.

14. A method for identifying a standard decoction of Tetrapanax papyriferus, characterized in that, It includes the following steps: Take the test sample to prepare a test solution; Perform ultra - high performance liquid chromatography detection on the test solution; Compare the chromatogram of the obtained test solution with the characteristic chromatogram constructed according to any one of claims 1 - 13, and identify whether the test sample is the Herba Tetrapanacis standard decoction according to the comparison result; The chromatographic conditions for the ultra - high performance liquid chromatography detection include: Using acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B for gradient elution.

15. The identification method according to claim 14, wherein The program of the gradient elution includes: 0 - 10 min, the volume percentage of mobile phase A is 3%; From 10 min to 25 min, the volume percentage of mobile phase A changes from 3% to 7%; From 25 min to 32 min, the volume percentage of mobile phase A changes from 7% to 10%; From 32 min to 40 min, the volume percentage of mobile phase A is 10%; From 40 min to 42 min, the volume percentage of mobile phase A changes from 10% to 80%; From 42 min to 45 min, the volume percentage of mobile phase A is 80%.

16. The identification method according to claim 14, wherein In the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.08% - 0.12%.

17. The identification method according to claim 14, wherein The chromatographic conditions for the ultra - performance liquid chromatography detection further include one or more of the following (1) - (5): (1) Use a chromatographic column filled with octadecylsilane - bonded silica gel; (2) The flow rate is 0.55 mL / min - 0.65 mL / min; (3) The column temperature is 28 °C - 32 °C; (4) The detection wavelength is 210 nm - 230 nm; (5) The injection volume is 1 μL - 3 μL.

18. The identification method according to any one of claims 14 to 17, wherein The steps for preparing the test solution include: Perform the third water extraction treatment and the third organic solvent extraction treatment on the test sample.

19. The identification method according to claim 18, wherein The third water extraction treatment includes the following steps: Mix the test sample and water and perform heating under reflux extraction to prepare the fifth extract; The third organic solvent extraction treatment includes the following steps; Mix the fifth extract with ethyl acetate, extract to prepare the sixth extract; Concentrate the sixth extract, use methanol as the re - dissolution solvent, dissolve the residue, filter, and take the filtrate; The methanol is a methanol aqueous solution with a volume fraction of 45% - 55%.

20. The identification method according to claim 19, wherein Meet one or more of the following (1) - (4): (1) In the third water extraction step, the mixing ratio of the test sample and water is 1 g:(20 - 25) mL; (2) The heating under reflux time is 15 min - 45 min; (3) In the third organic solvent extraction step, the volume ratio of the fifth extract to ethyl acetate is 1:(2 - 2.5); (4) The number of extractions with ethyl acetate is 3 - 4 times.

21. A method for detecting the content of chemical components in a standard decoction of Tetrapanax papyriferus, wherein Include the following steps: Take one or more of protocatechuic acid and syringaldehyde, and prepare reference substance solutions with different concentrations; Perform ultra - performance liquid chromatography detection on the reference substance solutions with different concentrations, and construct a standard curve based on the peak area and concentration; Take the Tongcao standard decoction test sample and prepare the test solution; Perform ultra - performance liquid chromatography detection and analysis on the test solution, and combine with the standard curve to determine the content of chemical components in the test sample; The chromatographic conditions for the ultra - performance liquid chromatography detection include: Use acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B for gradient elution.

22. The detection method according to claim 21, wherein, The program of the gradient elution includes: From 0 to 10 min, the volume percentage of mobile phase A is 3%; From 10 min to 25 min, the volume percentage of mobile phase A changes from 3% to 7%; From 25 min to 32 min, the volume percentage of mobile phase A changes from 7% to 10%; From 32 min to 40 min, the volume percentage of mobile phase A is 10%; From 40 min to 42 min, the volume percentage of mobile phase A changes from 10% to 80%; From 42 min to 45 min, the volume percentage of mobile phase A is 80%.

23. The detection method according to claim 21, wherein, In the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.08% to 0.12%.

24. The detection method according to claim 21, wherein, The chromatographic conditions for the ultra-high performance liquid chromatography detection further include one or more of the following (1) to (5): (1) Use a chromatographic column filled with octadecylsilane-bonded silica gel; (2) The flow rate is 0.55 mL / min to 0.65 mL / min; (3) The column temperature is 28 °C to 32 °C; (4) The detection wavelength is 210 nm to 230 nm; (5) The injection volume is 1 μL to 3 μL.

25. The detection method according to any one of claims 21 to 24, wherein, The steps for preparing the reference solution include: Use methanol as the extraction solvent to prepare the reference solution.

26. The detection method according to any one of claims 21 to 24, wherein, Meet one or more of the following (1) to (2): (1) The linear regression equation of protocatechuic acid is: y1 = 73993x1 - 10642, r = 1, where x1 represents the concentration of protocatechuic acid and y1 represents the peak area. The linear relationship between the concentration and the peak area of protocatechuic acid is good in the concentration range of 0.196 μg / mL to 97.993 μg / mL; (2) The linear regression equation of syringaldehyde is: y2 = 6590.8x2 + 1963.4, r = 1, where x2 represents the concentration of syringaldehyde and y2 represents the peak area. The linear relationship between the concentration and the peak area of syringaldehyde is good in the concentration range of 0.516 μg / mL to 103.194 μg / mL.

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