Methods for constructing UPLC characteristic spectra, identification methods, and detection methods for chemical component content of standard decoction of Tetrapanax papyriferus.

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a method for constructing, identifying, and detecting the chemical composition content of a standard decoction of *Tetrapanax papyriferus* (Tongcao). The method for constructing the UPLC characteristic spectrum of the standard decoction of *Tetrapanax papyriferus* includes the following steps: taking *Tetrapanax papyriferus* as a reference herb and preparing a reference solution; taking one or more of protocatechuic acid and syringaldehyde to prepare a reference solution; taking the standard decoction of *Tetrapanax papyriferus* and preparing a test solution; and performing ultra-high performance liquid chromatography (UHPLC) on the reference solution, the reference solution, and the test solution to construct the UPLC characteristic spectrum of the standard decoction of *Tetrapanax papyriferus*. The method of this invention has advantages such as high specificity, good repeatability, and good stability. The constructed UPLC characteristic spectrum has seven characteristic peaks and rich spectral information, providing a comprehensive and effective evaluation method for the quality control of the standard decoction of *Tetrapanax papyriferus*.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for constructing, identifying, and detecting the content of chemical components in a standard decoction of Tetrapanax papyriferus. Background Technology

[0002] According to the Chinese Pharmacopoeia (2020 edition), Tetrapanax papyrifer (Hook.) K. Koch is the dried pith of the stem of the plant Tetrapanax papyrifer (Hook.) K. Koch, belonging to the Araliaceae family. The stems are harvested in autumn, cut into sections, and the pith is extracted while fresh, straightened, and then sun-dried.

[0003] Currently, the identification of Tetrapanax papyriferus (2020 edition) in the Chinese Pharmacopoeia only includes morphological, microscopic, and routine examination items, and does not yet include identification and quantitative methods for related chemical components. Studies by domestic and international scholars have shown that Tetrapanax papyriferus mainly contains triterpenoids and their triterpenoid saponins, as well as steroidal glycosides, flavonoids, benzene derivatives, and ceramides. Methods for determining the content of Tetrapanax papyriferus mainly focus on polysaccharides and monosaccharides (D-mannose and fructose), and some reports use rutin as a reference standard to determine total flavonoids; however, all of these methods lack specificity. Qiu Zhijie et al. studied the use of acid-hydrolyzed components of Tetrapanax papyriferus to reflect the quality of the herbal material, but this does not accurately reflect the original components. Zhang Rui et al. established a UPLC fingerprint of the chloroform fraction of the water extract of Tetrapanax papyriferus; however, their main study focused on the chloroform fraction, and the components in the fingerprint were mainly non-polar.

[0004] None of the above methods can effectively control the quality of Tetrapanax papyriferus. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a method for constructing a UPLC characteristic spectrum of a standard decoction of Tetrapanax papyriferus. This method has advantages such as high specificity, good repeatability, and good stability. The UPLC characteristic spectrum constructed by the present invention contains rich spectral information, enabling the identification of the standard decoction of Tetrapanax papyriferus and the determination of its chemical components. It can fully reflect the water-soluble components in the water extract of Tetrapanax papyriferus, thus providing a more comprehensive and effective evaluation method for the quality control of the standard decoction of Tetrapanax papyriferus.

[0006] In a first aspect, the present invention provides a method for constructing a UPLC characteristic spectrum of a standard decoction of Tetrapanax papyriferus, comprising the following steps:

[0007] Take Tetrapanax papyriferus as a reference material and prepare a reference solution;

[0008] Prepare a reference solution by taking one or more of protocatechuic acid and eugenol;

[0009] Take the standard decoction of Tetrapanax papyriferus and prepare the test solution;

[0010] The reference solution of the control medicinal material, the reference solution and the test solution were respectively subjected to ultra-high performance liquid chromatography (UPLC) to construct the UPLC characteristic spectrum of the standard decoction of Tetrapanax papyriferus.

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

[0012] Gradient elution was performed using acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B.

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

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

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

[0016] Between 25 and 32 minutes, the volume percentage of mobile phase A changed from 7% to 10%.

[0017] From 32 to 40 minutes, the volume percentage of mobile phase A was 10%.

[0018] From 40 to 42 minutes, the volume percentage of mobile phase A changed from 10% to 80%.

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

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

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

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

[0023] In some embodiments, the chromatographic conditions for ultra-high performance liquid chromatography detection also include a column temperature of 28°C to 32°C.

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

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

[0026] In some embodiments, the step of preparing the control herbal solution includes:

[0027] The reference herb, Tetrapanax papyriferus, underwent a first water extraction treatment and a first organic solvent extraction treatment.

[0028] In some embodiments, the first water extraction process includes the following steps:

[0029] The reference herb Tetrapanax papyrifer was mixed with water and heated under reflux for extraction. The mixture was then concentrated, and the residue was dissolved in water to prepare the first extract.

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

[0031] The first extract was mixed with ethyl acetate and extracted to prepare the second extract.

[0032] The second extract was concentrated, and the residue was dissolved in methanol as a redissolution solvent. The residue was then filtered and the filtrate was collected.

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

[0034] In some embodiments, in the first water extraction step, the ratio of the Tetrapanax papyrifer reference herb to water is 1g:(90~100)mL.

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

[0036] In some 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 embodiments, the extraction is performed with ethyl acetate 3 to 4 times.

[0038] In some embodiments, the step of preparing the reference solution includes:

[0039] The reference solution was prepared using methanol as the extraction solvent.

[0040] The concentration of protocatechuic acid in the reference solution is 8 μg / mL to 12 μg / mL, and the concentration of syringaldehyde is 8 μg / mL to 12 μg / mL.

[0041] In some embodiments, the step of preparing the test solution includes:

[0042] The standard decoction of Tetrapanax papyriferus was subjected to a second water extraction and a second organic solvent extraction.

[0043] In some embodiments, the second water extraction process includes the following steps:

[0044] The standard decoction of Tetrapanax papyriferus was mixed with water and subjected to reflux extraction to prepare a third extract.

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

[0046] The third extract was mixed with ethyl acetate to prepare the fourth extract;

[0047] The fourth extract was concentrated, and the residue was dissolved in methanol as a redissolution solvent. The residue was then filtered and the filtrate was collected.

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

[0049] In some embodiments, in the second water extraction step, the ratio of the standard decoction of Tetrapanax papyriferus to water is 1g:(20~25)mL.

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

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

[0052] In some embodiments, the extraction is performed with ethyl acetate 3 to 4 times.

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

[0054] In some embodiments, the construction method further includes the step of mass spectrometric identification of characteristic peaks in the characteristic spectrum using chromatography-mass spectrometry.

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

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

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

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

[0059] A second aspect of the present invention also provides a method for identifying a standard decoction of Tetrapanax papyriferus, comprising the following steps:

[0060] Prepare the test solution from the sample;

[0061] The test solution was analyzed by ultra-high performance liquid chromatography.

[0062] Compare the obtained chromatogram of the test solution with the characteristic chromatogram constructed in any of the above embodiments, and identify whether the test sample is a standard decoction of Tetrapanax papyriferus based on the comparison results;

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

[0064] Gradient elution was performed using acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B.

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

[0066] From 0 to 10 minutes, the volume percentage of mobile phase A is 3%.

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

[0068] Between 25 and 32 minutes, the volume percentage of mobile phase A changed from 7% to 10%.

[0069] From 32 to 40 minutes, the volume percentage of mobile phase A was 10%.

[0070] From 40 to 42 minutes, the volume percentage of mobile phase A changed from 10% to 80%.

[0071] From 42 to 45 minutes, the volume percentage of mobile phase A was 80%.

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

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

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

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

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

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

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

[0079] The sample to be tested was subjected to a third water extraction treatment and a third organic solvent extraction treatment.

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

[0081] The sample to be tested is mixed with water and subjected to reflux extraction to prepare the fifth extract;

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

[0083] The fifth extract was mixed with ethyl acetate to prepare the sixth extract;

[0084] The sixth extract was concentrated, and the residue was dissolved in methanol as a redissolver. The residue was then filtered, and the filtrate was collected. The methanol was a methanol-water solution with a volume fraction of 45% to 55%.

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

[0086] In some embodiments of the identification method, the heating 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 extraction is performed with ethyl acetate 3 to 4 times.

[0089] A 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 solutions of different concentrations;

[0091] The reference solutions of different concentrations were detected by ultra-high performance liquid chromatography, and a standard curve was constructed based on the peak area and concentration.

[0092] Take the standard decoction of Tetrapanax papyriferus and prepare the test solution;

[0093] The solution to be tested is analyzed by ultra-high performance liquid chromatography, and the content of chemical components in the sample to be tested is determined by combining the standard curve.

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

[0095] Gradient elution was performed using acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B.

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

[0097] From 0 to 10 minutes, the volume percentage of mobile phase A is 3%.

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

[0099] Between 25 and 32 minutes, the volume percentage of mobile phase A changed from 7% to 10%.

[0100] From 32 to 40 minutes, the volume percentage of mobile phase A was 10%.

[0101] From 40 to 42 minutes, the volume percentage of mobile phase A changed from 10% to 80%.

[0102] From 42 to 45 minutes, the volume percentage of mobile phase A was 80%.

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

[0104] In some embodiments of the detection method, the chromatographic conditions for the ultra-high performance liquid chromatography detection further include: using a chromatographic column packed with octadecylsilane-bonded silica gel.

[0105] In some embodiments of the detection method, the chromatographic conditions for ultra-high performance liquid chromatography detection further include a flow rate of 0.55 mL / min to 0.65 mL / min.

[0106] In some embodiments of the detection method, the chromatographic conditions for ultra-high performance liquid chromatography detection also include: a column temperature of 28℃~32℃.

[0107] In some embodiments of the detection method, the chromatographic conditions for ultra-high performance liquid chromatography detection further include a detection wavelength of 210 nm to 230 nm.

[0108] In some embodiments of the detection method, the chromatographic conditions for ultra-high performance liquid chromatography detection further include: an injection volume of 1 μL to 3 μL.

[0109] In some embodiments of the detection method, the step of preparing the reference solution includes:

[0110] The reference solution was prepared using methanol as the extraction solvent.

[0111] In some implementations 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. The concentration of protocatechuic acid shows a good linear relationship with the peak area in the range of 0.196 μg / mL to 97.993 μg / mL.

[0112] In some implementations 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. Within the concentration range of 0.516 μg / mL to 103.194 μg / mL, the concentration and peak area of ​​syringaldehyde show a good linear relationship.

[0113] Compared with traditional technologies, the present invention has the following advantages:

[0114] This invention provides a method for constructing a UPLC characteristic spectrum of a standard decoction of Tetrapanax papyriferus. This method has the advantages of strong specificity, good repeatability, and good stability. The UPLC characteristic spectrum constructed by this invention has 7 characteristic peaks and rich spectral information, which can realize the identification of the standard decoction of Tetrapanax papyriferus and the determination of the content of chemical components. It can fully reflect the water-soluble components in the water extract of Tetrapanax papyriferus, thus providing a more comprehensive and effective evaluation method for the quality control of the standard decoction of Tetrapanax papyriferus. Attached Figure Description

[0115] Figure 1 The characteristic chromatogram of the standard decoction of Tetrapanax papyriferus is as follows: Peak 1 (S1): protocatechuic acid; Peak 6 (S2): syringaldehyde;

[0116] Figure 2 The characteristic spectrum of the reference herb Tetrapanax papyriferus is shown. Peak 1 (S1): protocatechuic acid; Peak 6 (S2): syringaldehyde.

[0117] Figure 3 Optimize the chromatogram for the elution gradient (gradient 1);

[0118] Figure 4 Optimize the chromatogram for the elution gradient (gradient 2);

[0119] Figure 5 Optimize the chromatogram for the elution gradient (gradient 3);

[0120] Figure 6 Optimize the chromatogram for the elution gradient (gradient 4);

[0121] Figure 7 Comparative chromatograms of different brands of chromatographic columns;

[0122] Figure 8 The common pattern of characteristic chromatograms of 15 batches of standard decoction of Tetrapanax papyriferus;

[0123] Figure 9 Total ion chromatogram (positive ion mode) of the test solution of the standard decoction of Tetrapanax papyriferus.

[0124] Figure 10 Total ion chromatogram (negative ion mode) of the test solution of the standard decoction of Tetrapanax papyriferus.

[0125] Figure 11 The ultraviolet absorption chromatogram of the test solution of the standard decoction of Tetrapanax papyriferus;

[0126] Figure 12 This is the first-order mass spectrum scan of peak 1 (positive ion mode).

[0127] Figure 13 This is the first-order mass spectrum scan of peak 1 (negative ion mode).

[0128] Figure 14 The mass spectrum scan of peak 6 (positive ion mode) is shown.

[0129] Figure 15 The mass spectrum scan of peak 6 (negative ion mode) is shown.

[0130] Figure 16 A specific diagram for the standard decoction of Tetrapanax papyriferus;

[0131] Figure 17 The graph shows the linear regression equation for protocatechuic acid.

[0132] Figure 18 The graph shows the linear regression equation for syringaldehyde. Detailed Implementation

[0133] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0134] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0135] Methods for determining the content of Tetrapanax papyriferus (Tetrapanax papyriferus) mainly focus on polysaccharides and monosaccharides (D-mannose and fructose). Some reports use rutin as a reference standard to determine total flavonoids, but these methods lack specificity. Existing literature has limited research on the characteristic / fingerprint chromatograms of Tetrapanax papyriferus. The characteristic chromatogram established by Qiu Zhijie et al. reflects the acid-hydrolyzed components of Tetrapanax papyriferus and does not accurately reflect the original components. Furthermore, the chromatographic conditions only reflect four characteristic peaks, lacking sufficient chromatographic peak information to represent the main components in standard decoctions and granules of Tetrapanax papyriferus. Zhang Rui et al. established a UPLC fingerprint chromatogram of the chloroform fraction of the water extract of Tetrapanax papyriferus; however, their main study focused on the chloroform fraction, and the components in the fingerprint chromatogram were primarily non-polar.

[0136] The UPLC characteristic chromatographic method for the standard decoction of Tetrapanax papyriferus constructed in this invention selected common peaks of seven prototype components, identifying protocatechuic acid and syringaldehyde as two components. The contents of protocatechuic acid and syringaldehyde were determined under the same chromatographic conditions. The construction method of this invention, through the control of chromatographic conditions, can achieve the content detection of water-soluble indicator components, better reflecting the components in the standard decoction of Tetrapanax papyriferus extracted with water as the solvent, and thus better controlling its quality.

[0137] In a first aspect, the present invention provides a method for constructing a UPLC characteristic spectrum of a standard decoction of Tetrapanax papyriferus, comprising the following steps:

[0138] Take Tetrapanax papyriferus as a reference material and prepare a reference solution;

[0139] Prepare a reference solution by taking one or more of protocatechuic acid and eugenol;

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

[0141] The reference solution of the control medicinal material, the reference solution and the test solution were respectively subjected to ultra-high performance liquid chromatography (UPLC) to construct the UPLC characteristic spectrum of the standard decoction of Tetrapanax papyriferus.

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

[0143] Gradient elution was performed using acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B.

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

[0145] From 0 to 10 minutes, the volume percentage of mobile phase A is 3%.

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

[0147] Between 25 and 32 minutes, the volume percentage of mobile phase A changed from 7% to 10%.

[0148] From 32 to 40 minutes, the volume percentage of mobile phase A was 10%.

[0149] From 40 to 42 minutes, the volume percentage of mobile phase A changed from 10% to 80%.

[0150] From 42 to 45 minutes, the volume percentage of mobile phase A was 80%.

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

[0152] In some of these examples, the chromatographic conditions for the ultra-high performance liquid chromatography detection also include the use of a column packed with octadecylsilane-bonded silica gel.

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

[0154] In some examples, the chromatographic conditions for ultra-high performance liquid chromatography detection further include a flow rate of 0.55 mL / min to 0.65 mL / min, for example, 0.55 mL / min, 0.6 mL / min or 0.65 mL / min, preferably 0.6 mL / min.

[0155] In some examples, the chromatographic conditions for ultra-high performance liquid chromatography detection also include: a column temperature of 28°C to 32°C, for example, 28°C, 29°C, 30°C, 31°C or 32°C, preferably 30°C.

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

[0157] In some examples, the chromatographic conditions for ultra-high performance liquid chromatography detection also include: an injection volume of 1 μL to 3 μL, for example, 1 μL, 2 μL or 3 μL, preferably 2 μL.

[0158] In some of these examples, the steps for preparing the control herbal solution include:

[0159] The reference herb, Tetrapanax papyriferus, underwent a first water extraction treatment and a first organic solvent extraction treatment.

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

[0161] The reference herb Tetrapanax papyrifer was mixed with water and heated under reflux for extraction. The mixture was then concentrated, and the residue was dissolved in water to prepare the first extract.

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

[0163] The first extract was mixed with ethyl acetate to prepare the second extract;

[0164] The second extract was concentrated, and the residue was dissolved in methanol as a redissolution solvent. The residue was then filtered and the filtrate was collected.

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

[0166] In some of these examples, in the first water extraction step, the ratio of the Tetrapanax papyrifer control herb to water is 1 g: (90~100) mL.

[0167] In some specific examples, the ratio of the Tetrapanax papyrifer control herb to water was 1g:90mL.

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

[0169] In some 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 extraction was performed with ethyl acetate 3 to 4 times, preferably 3 times.

[0171] Understandably, the volume ratio of the first extract to ethyl acetate is 1:(2~2.5), which means that each extraction is performed by adding ethyl acetate at a volume ratio of 1:(2~2.5).

[0172] In some of these examples, methanol is used as the redissolving solvent, wherein the methanol is an aqueous solution with a volume fraction of 45% to 55%, preferably a 50% aqueous solution.

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

[0174] The reference solution was prepared using methanol as the extraction solvent.

[0175] The concentration of protocatechuic acid in the reference solution is 8 μg / mL to 12 μg / mL, and the concentration of syringaldehyde is 8 μg / mL to 12 μg / mL.

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

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

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

[0179] The standard decoction of Tetrapanax papyriferus was mixed with water and subjected to reflux extraction to prepare a third extract.

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

[0181] The third extract was mixed with ethyl acetate to prepare the fourth extract;

[0182] The fourth extract was concentrated, and the residue was dissolved in methanol as a redissolution solvent. The residue was then filtered and the filtrate was collected.

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

[0184] In some of these examples, during the second water extraction step, the ratio of the standard decoction of Tetrapanax papyriferus to water is 1 g: (20~25) mL.

[0185] In some specific examples, the ratio of the test sample of the standard decoction of Tetrapanax papyriferus to water is 1g:20mL.

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

[0187] In some examples, during 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 extraction was performed with ethyl acetate 3 to 4 times, preferably 3 times.

[0189] Understandably, the volume ratio of the third extract to ethyl acetate being 1:(2~2.5) means that each extraction is performed by adding ethyl acetate at a volume ratio of 1:(2~2.5).

[0190] In some examples, 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. Peaks 1 and 6 are consistent with the retention times of the reference material peaks. The peak corresponding to the protocatechuic acid reference material peak is peak S1. The relative retention times of peaks 2 to 4 with peak S1 are calculated. The peak corresponding to the syringaldehyde reference material peak is peak S2. The relative retention times of peaks 5 and 7 with peak S2 are calculated. The relative retention times are within ±10% of the specified values, which are: 1.44 (peak 2), 2.93 (peak 3), 3.39 (peak 4), 0.96 (peak 5), and 1.07 (peak 7).

[0191] In some examples, the construction method further includes the step of mass spectrometric identification of characteristic peaks in the characteristic spectrum using chromatography-mass spectrometry;

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

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

[0194] HESI ion source parameters include: sheath gas flow rate of 60arb~65arb, auxiliary gas flow rate of 18arb~22arb, spray voltage of 2.2kV~2.8kV, S-lens voltage of 48V~52V, heating temperature of 280℃~320℃, and capillary temperature of 480℃~520℃.

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

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

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

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

[0199] Between 25 and 32 minutes, the volume percentage of mobile phase A changed from 7% to 10%.

[0200] From 32 to 40 minutes, the volume percentage of mobile phase A was 10%.

[0201] From 40 to 42 minutes, the volume percentage of mobile phase A changed from 10% to 80%.

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

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

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

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

[0206] In some examples of the designation process, the chromatographic conditions further include a flow rate of 0.55 mL / min to 0.65 mL / min, preferably 0.6 mL / min.

[0207] In some examples of the designation process, the chromatographic conditions further include a column temperature of 28°C to 32°C, preferably 30°C.

[0208] In some examples of the designation process, the chromatographic conditions further include an injection volume of 1 μL to 3 μL, preferably 2 μL.

[0209] A second aspect of the present invention also provides a method for identifying a standard decoction of Tetrapanax papyriferus, comprising the following steps:

[0210] Prepare the test solution from the sample;

[0211] The test solution is subjected to ultra-high performance liquid chromatography (UHPLC) detection. The chromatogram of the obtained test solution is compared with the characteristic chromatogram constructed in any of the above examples. Based on the comparison results, it is determined whether the test sample is a standard decoction of Tetrapanax papyriferus.

[0212] The chromatographic conditions for ultra-high performance liquid chromatography detection include:

[0213] Gradient elution was performed using acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B.

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

[0215] From 0 to 10 minutes, the volume percentage of mobile phase A is 3%.

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

[0217] Between 25 and 32 minutes, the volume percentage of mobile phase A changed from 7% to 10%.

[0218] From 32 to 40 minutes, the volume percentage of mobile phase A was 10%.

[0219] From 40 to 42 minutes, the volume percentage of mobile phase A changed from 10% to 80%.

[0220] From 42 to 45 minutes, the volume percentage of mobile phase A was 80%.

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

[0222] In some examples of the identification method, the chromatographic conditions for the ultra-high performance liquid chromatography detection also include: using a chromatographic column packed with octadecylsilane-bonded silica gel.

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

[0224] In some examples of the identification method, the chromatographic conditions for ultra-high performance liquid chromatography detection further include a flow rate of 0.55 mL / min to 0.65 mL / min, for example, 0.55 mL / min, 0.6 mL / min or 0.65 mL / min, preferably 0.6 mL / min.

[0225] In some examples of the identification method, the chromatographic conditions for ultra-high performance liquid chromatography detection also include: a column temperature of 28°C to 32°C, for example, 28°C, 29°C, 30°C, 31°C or 32°C, preferably 30°C.

[0226] In some examples of the identification method, the chromatographic conditions for ultra-high performance liquid chromatography detection further include: a detection wavelength of 210 nm to 230 nm, for example, 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 ultra-high performance liquid chromatography detection further include: an injection volume of 1 μL to 3 μL, for example, 1 μL, 2 μL or 3 μL, preferably 2 μL.

[0228] In some examples of identification methods, the steps for preparing the test solution include:

[0229] The sample to be tested was subjected to a third water extraction treatment and a third organic solvent extraction treatment.

[0230] In some examples of identification methods, the third water extraction process includes mixing the test sample with water and heating and refluxing to extract the fifth extract;

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

[0232] The fifth extract was mixed with ethyl acetate to prepare the sixth extract;

[0233] The sixth extract was concentrated, and the residue was dissolved in methanol as a redissolution solvent. The mixture was then filtered, and the filtrate was collected.

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

[0235] In some examples of the identification method, in the third water extraction step, the mixing ratio of the test sample and water is 1 g: (20~25) mL.

[0236] In some examples of the identification method, the heating 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 methods, ethyl acetate was used for extraction 3 to 4 times.

[0239] A 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 solutions of different concentrations;

[0241] S210: The reference solutions of different concentrations are detected by ultra-high performance liquid chromatography, and a standard curve is constructed based on the peak area and concentration;

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

[0243] S410: Perform ultra-high performance liquid chromatography (UHPLC) analysis on the test solution, and determine the content of chemical components in the test sample by combining the standard curve.

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

[0245] The mixed reference stock solution was diluted to prepare six mixed reference solutions of different concentrations.

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

[0247] In some examples of the detection method, step S210 involves performing ultra-high performance liquid chromatography (UHPLC) on the mixed reference stock solution prepared in S110 and six mixed reference solutions of different concentrations.

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

[0249] In some examples of detection methods, the gradient elution procedure includes:

[0250] From 0 to 10 minutes, the volume percentage of mobile phase A is 3%.

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

[0252] Between 25 and 32 minutes, the volume percentage of mobile phase A changed from 7% to 10%.

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

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

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

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

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

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

[0259] In some examples of the detection method, the chromatographic conditions for ultra-high performance liquid chromatography detection further include a flow rate of 0.55 mL / min to 0.65 mL / min, for example, 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 ultra-high performance liquid chromatography detection also include: a column temperature of 28℃~32℃, for example, 28℃, 29℃, 30℃, 31℃ or 32℃, preferably, the column temperature is 30℃.

[0261] In some examples of the detection method, the chromatographic conditions for ultra-high performance liquid chromatography detection further include: a detection wavelength of 210 nm to 230 nm, for example, 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 ultra-high performance liquid chromatography detection further include: an injection volume of 1 μL to 3 μL, for example, 1 μL, 2 μL or 3 μL, preferably 2 μL.

[0263] In some examples of the detection method, step S310, preparing the test solution includes the following steps:

[0264] The standard decoction of Tetrapanax papyriferus was subjected to a fourth water extraction treatment and a fourth organic solvent extraction treatment.

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

[0266] The test sample of the standard decoction of Tetrapanax papyriferus was mixed with water and heated under reflux to extract the seventh extract.

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

[0268] The seventh extract was mixed with ethyl acetate to prepare the eighth extract;

[0269] The eighth extract was concentrated, and the residue was dissolved in methanol as a redissolver. The residue was then filtered, and the filtrate was collected. The methanol was a methanol-water solution with a volume fraction of 45% to 55%.

[0270] In some examples of the detection method, in the fourth water extraction step, the ratio of the test sample of the Tetrapanax papyriferus standard decoction to water is 1g:(20~25)mL.

[0271] In some examples of the detection method, the heating reflux time is 15 min to 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 methods, ethyl acetate is used for extraction 3 to 4 times.

[0274] Understandably, the chromatographic conditions for detecting and analyzing the test solution using ultra-high performance liquid chromatography (UHPLC) are the same as those for detecting the reference solution using UHPLC.

[0275] In some examples 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. The concentration of protocatechuic acid shows a good linear relationship with the peak area in the range of 0.196 μg / mL to 97.993 μg / mL.

[0276] In some examples 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. 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 with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0278] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0279] Example 1

[0280] This embodiment provides a method for constructing the UPLC characteristic spectrum of a standard decoction of Tetrapanax papyriferus.

[0281] 1. Instruments, reagents and reagents

[0282] Instruments: Waters UHPLC (Waters H-Class, Waters Corporation), Agilent UHPLC (Model 1290, Agilent Technologies), Waters BEH C18 column (3×150mm, 2.5μm, serial number: JS-194); Waters Cortecs T3 column (3×150mm, 2.7μm, serial number: JS-342); Waters HSS T3 column (4.6×150mm, 5μm, serial number: JS-153); Waters Cortecs T3 Phenyl column (3×150mm, 2.7μm, serial number: JS-349); 0.001 g balance (ME204E, Mettler Toledo), 0.1 mg balance (XP26, Mettler Toledo), CNC ultrasonic cleaner (KQ500D, Kunshan Ultrasonic Instrument Co., Ltd.), constant temperature water bath (HWS28, Shanghai Yiheng Technology Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck KGaA).

[0283] Reagents: Ethanol (Tianjin Yongda Chemical Reagent Co., Ltd.) and methanol (Guangdong Guanghua Science & Technology Co., Ltd.) were analytical grade; methanol and acetonitrile (Merck, Inc.) were chromatographic grade; and water was ultrapure water (prepared in the laboratory).

[0284] Test drugs: protocatechuic acid (batch number: 110809-201906, content: 97.7%, manufacturer: China National Institutes for Food and Drug Control); eugenol (batch number: R09J6Y1, content: 98%, manufacturer: Shanghai Yuanye Biotechnology Co., Ltd.); Tetrapanax papyrifer reference material (batch number: 125029-202006A, tested by Guangdong Provincial Institute for Drug Control); Batch number information of 15 batches of Tetrapanax papyrifer standard decoction is shown in Table 1.

[0285] Table 1 Information on Standard Decoctions of Tetrapanax papyriferus

[0286]

[0287] 2. Chromatographic conditions and preparation of test solution

[0288] 2.1 Chromatographic conditions and system suitability test

[0289] Octadecylsilane-bonded silica gel was used as the packing material (column length 150 mm, inner diameter 3.0 mm, particle size 2.5 μm); acetonitrile was used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the specifications in Table 2; the flow rate was 0.60 mL per minute; the column temperature was 30 °C; and the detection wavelength was 220 nm.

[0290] Table 2 Gradient Elution Table

[0291]

[0292] 2.2 Preparation of the test solution:

[0293] Preparation of the test solution of standard decoction of Tetrapanax papyriferus: Take an appropriate amount of this product, grind it into a fine powder, take about 0.5g, accurately weigh it, place it in a stoppered conical flask, add 10mL of water, weigh it, heat under reflux for 30 minutes, remove it, let it cool, weigh it again, make up the weight loss with water, extract it three times with ethyl acetate, 20mL each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 50% methanol and transfer it to a 2mL volumetric flask, add 50% methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0294] 2.3 Preparation of reference solution and standard solution:

[0295] Take 10g of Tetrapanax papyriferus reference material, place it in a flask, add 900mL of water, heat under reflux for 60 minutes, filter, evaporate the filtrate to dryness, add 15mL of water to the residue to dissolve it, extract with ethyl acetate three times, 30ml each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 50% methanol and transfer it to a 2mL volumetric flask, add 50% methanol to the mark, shake well, filter, and take the filtrate as the reference solution for the reference material.

[0296] Take appropriate amounts of protocatechuic acid reference standard and eugenol reference standard, accurately weigh them, and add methanol to prepare a solution containing 10 µg of protocatechuic acid reference standard and 10 µg of eugenol reference standard per 1 mL. This solution will be used as the reference solution for content determination and the reference solution for characteristic chromatogram.

[0297] 2.4 Determination Method:

[0298] Accurately pipette 2 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0299] 2.5 Formulation of Feature Map

[0300] Characteristic chromatograms of standard decoctions of *Tetrapanax papyriferus* were constructed by matching the UPLC characteristic chromatograms of 15 batches of standard decoctions of *Tetrapanax papyriferus* in Table 1 using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine". A control chromatogram was generated using the mean method to establish a control characteristic chromatogram for the standard decoctions of *Tetrapanax papyriferus*. Figure 1 The characteristic spectrum has 7 characteristic peaks, which correspond to the 7 characteristic peaks of the Tetrapanax papyrifer (Tongcao) reference material. The characteristic spectrum of the Tetrapanax papyrifer (Tongcao) reference material can be found here. Figure 2 .

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

[0302] 3.1 Optimization of the elution gradient:

[0303] Take an appropriate amount of standard decoction of Tetrapanax papyriferus (batch number BTS13), grind it into a fine powder, take about 0.5g, and prepare the test solution according to the method in section "2.2". Except for different elution gradients, all other analyses are performed according to the provisions in section "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 elution gradients ( Figures 3-6 It can be seen that when gradient 4 is selected, the baseline of the chromatogram is stable and the separation of each chromatographic peak is good. Therefore, gradient 4 is selected as the final elution gradient.

[0313] 3.2 Column Investigation

[0314] Take an appropriate amount of standard decoction of Tetrapanax papyriferus (batch number BTS13), grind it into a fine powder, and accurately weigh about 0.5g. Prepare the test solution according to the method specified in section "2.2". Except for the chromatographic columns (Waters BEH C18 column (3×150mm, 2.5μm, serial number: JS-194); Waters Cortecs T3 column (3×150mm, 2.7μm, serial number: JS-342); Waters HSS T3 column (4.6×150mm, 5μm, serial number: JS-153); and Waters Cortecs T3 Phenyl column (3×150mm, 2.7μm, serial number: JS-349), all other chromatographic conditions are the same as specified in section "2.1". Inject and analyze. The experimental results are as follows: Figure 7 .

[0315] The results showed that, except for the Waters BEH C18 (3mm×150mm, 2.5μm) column, the other three different column types exhibited significant differences in separation efficiency and retention time for the standard decoction of Tetrapanax papyriferus, with some peaks missing. This indicates that different column types have a significant impact on the relative retention time of each characteristic peak, with the Waters BEH C18 (3mm×150mm, 2.5μm) column providing richer chromatographic information. Therefore, to more fully represent the components of Tetrapanax papyriferus, it is recommended to consistently use the Waters BEH C18 (3mm×150mm, 2.5μm) column.

[0316] 3.3 Investigation on the preparation of the test solution

[0317] 3.3.1 Examination of the number of shaking extraction times

[0318] This experiment investigated the effect of the number of shaking extractions on the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus. The main focus was on shaking extractions of 2, 3, and 4 times. The peak shape and resolution of the 7 characteristic peaks were observed, and the "total peak area / sample weight" of the 7 characteristic peaks, as well as the contents of protocatechuic acid and eugenol, were calculated to compare the effects of different extraction times on the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus.

[0319] Take an appropriate amount of standard decoction of Tetrapanax papyriferus, grind it into a fine powder, take about 0.5g, accurately weigh it, and perform three parallel groups, two portions per group. Place each group in a stoppered conical flask, add 10mL of water, weigh it, and perform ultrasonic extraction (300W power, 40kHz frequency) for 30 minutes. Remove it, let it cool, weigh it again, and replenish the lost weight with water. Extract with ethyl acetate 2, 3, and 4 times, 20mL each time. Combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 50% methanol and transfer it to a 2mL volumetric flask, add 10% methanol to the mark, shake well, filter, and collect the filtrate. Other analyses are performed according to the chromatographic conditions in section "2.1", as shown in Table 3.

[0320] Table 3. Characteristics of Standard Tetrapanax papyriferus Decoction: Results of Shaking Extraction Times

[0321]

[0322] The results showed that the values ​​of "total peak area / sample weight" and the contents of protocatechuic acid and syringaldehyde did not change much in the characteristic spectrum of the standard decoction of Tetrapanax papyriferus after three and four shake extractions, indicating that the extraction was basically complete. To ensure complete extraction and save solvent, three shake extractions were selected.

[0323] 3.3.2 Investigation of Reconstitution Solvents

[0324] This experiment investigated the effects of different resolvation solvents on the characteristic chromatograms of the standard decoction of Tetrapanax papyriferus. Methanol, 70% methanol, 50% methanol, and 10% methanol were used as resolvation solvents. The peak shape and resolution of the seven characteristic peaks were observed, and the "total peak area / sample weight" of the seven characteristic peaks, as well as the contents of protocatechuic acid and syringaldehyde, were calculated to compare the effects of different resolvation solvents on the characteristic chromatograms of the standard decoction of Tetrapanax papyriferus.

[0325] Take an appropriate amount of standard decoction of Tetrapanax papyriferus, grind it into a fine powder, take about 0.5g, accurately weigh it, and divide it into 4 parallel groups, 2 portions per group. Place each group in a stoppered conical flask, add 10mL of water, weigh it, and extract it ultrasonically (300W power, 40kHz frequency) for 30 minutes. Remove it, let it cool, weigh it again, and make up the weight loss with water. Extract it three times with ethyl acetate, 20mL each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in methanol, 70% methanol, 50% methanol, and 10% methanol respectively, and transfer them to 2mL volumetric flasks. Add methanol, 70% methanol, 50% methanol, and 10% methanol respectively to the mark, shake well, filter, and collect the filtrate. Other chromatographic analyses are performed according to the chromatographic conditions in section "2.1", as shown in Table 4.

[0326] Table 4. Results of the investigation on the reconstitution solvent of the standard decoction of Tetrapanax papyriferus.

[0327]

[0328] The results showed that the difference in "total peak area / sample weight" between methanol, 70% methanol and 50% methanol was not significant when the test sample solution was prepared using the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus. However, the contents of protocatechuic acid and syringaldehyde were highest in 50% methanol. Therefore, 50% methanol was chosen as the transfer and reconstitution solvent in order to achieve more complete extraction.

[0329] 3.3.3 Investigation of Extraction Solvents

[0330] This experiment investigated the effects of different extraction solvents on the characteristic chromatograms of the standard decoction of Tetrapanax papyriferus. Dichloromethane, diethyl ether, and ethyl acetate were used as extraction solvents. The effects of different extraction solvents on the characteristic chromatograms of the standard decoction of Tetrapanax papyriferus were compared by observing the peak shape and resolution of seven characteristic peaks, calculating the "total peak area / sample weight" of the seven characteristic peaks, and calculating the contents of protocatechuic acid and eugenol.

[0331] Take an appropriate amount of standard decoction of Tetrapanax papyriferus, grind it into a fine powder, take about 0.5g, accurately weigh it, and perform three parallel groups, two portions per group. Place each group in a stoppered conical flask, add 10mL of water, weigh it, and extract ultrasonically (300W power, 40kHz frequency) for 30 minutes. Remove it, let it cool, weigh it again, and replenish the lost weight with water. Extract it three times with ethyl acetate, ether, and dichloromethane, 20mL each time. Combine the ethyl acetate layer, ether solution, and dichloromethane solution, evaporate to dryness, dissolve the residue in 50% methanol, transfer it to a 2mL volumetric flask, add 50% methanol to the mark, shake well, filter, and collect the filtrate. Other chromatographic analyses are performed according to the chromatographic conditions in section "2.1", as shown in Table 5.

[0332] Table 5. Results of the investigation of extraction solvents for the characteristic chromatograms of the standard decoction of Tetrapanax papyriferus.

[0333]

[0334] The results showed that among the three extraction solvents, dichloromethane failed to exhibit all seven characteristic peaks, while the characteristic spectrum of ethyl acetate extraction showed the highest values ​​for "total peak area / sample weight" and the contents of protocatechuic acid and syringaldehyde, indicating the best extraction efficiency. Therefore, ethyl acetate was selected as the extraction solvent.

[0335] 3.3.4 Examination of Extraction Methods

[0336] This experiment investigated the effects of different extraction methods on the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus. The main focus was on two extraction methods: ultrasonic and reflux. The effects of different extraction methods on the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus were compared by observing the peak shape and resolution of the seven characteristic peaks, calculating the "total peak area / sample weight" of the seven characteristic peaks, and calculating the contents of protocatechuic acid and eugenol.

[0337] Take an appropriate amount of standard decoction of Tetrapanax papyriferus, grind it into a fine powder, take about 0.5g, accurately weigh it, and divide it into 4 parallel groups, 2 portions per group. Place each group in a stoppered conical flask, add 10mL of water, weigh it, and extract it by ultrasonication (300W power, 40kHz frequency) for 30 minutes and by reflux for 30 minutes. Remove the flask, let it cool, weigh it again, and make up the weight loss with water. Extract it three times with ethyl acetate, 20mL each time. Combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 50% methanol and transfer it to a 2mL volumetric flask, add 50% methanol to the mark, and extract it by ultrasonication (300W power, 40kHz frequency) for 30 minutes and by reflux for 30 minutes. Remove the flask, let it cool, weigh it again, make up the weight loss with water, shake well, filter, and collect the filtrate. Other chromatographic analyses are performed according to the chromatographic conditions in section "2.1", as shown in Table 6.

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

[0339]

[0340] The results showed that the baseline of the characteristic spectrum of the samples extracted by direct ultrasound and heating reflux was relatively rough; and the extraction efficiency of extraction after ultrasound treatment was also low, with little difference. However, the extraction efficiency of each characteristic peak and the content of protocatechuic acid and syringaldehyde were the best when the extraction method of heating reflux treatment followed by shaking was the best. Therefore, the extraction method of heating reflux treatment followed by shaking was selected to ensure sufficient extraction.

[0341] 3.3.5 Examination of Extraction Time

[0342] This experiment investigated the effect of heating reflux time on the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus. By observing the peak shape and resolution of seven tentatively determined characteristic peaks, and calculating the "total peak area / sample weight" of the seven characteristic peaks, as well as the contents of protocatechuic acid and eugenol, the effects of different heating reflux times on the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus were compared.

[0343] Take an appropriate amount of standard decoction of Tetrapanax papyriferus, grind it into a fine powder, take about 0.5g, accurately weigh it, and divide it into 3 parallel groups, 2 portions per group. Place each group in a stoppered conical flask, add 10mL of water, weigh it, and heat under reflux for 15 minutes, 30 minutes, and 45 minutes respectively. Remove it, let it cool, weigh it again, and make up the weight loss with water. Extract it three times with ethyl acetate, 20mL each time. Combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 50% methanol and transfer it to a 2mL volumetric flask, add 50% methanol to the mark, shake well, filter, and collect the filtrate. Other chromatographic analyses are performed according to the chromatographic conditions in section "2.1", as shown in Table 7.

[0344] Table 7. Results of the investigation on the extraction time of the characteristic chromatograms 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 seven characteristic peaks, as well as the contents of protocatechuic acid and syringaldehyde, with different heating reflux times. Complete extraction was achieved by heating reflux for 30 minutes. Considering the influence of the experimental environment, the ultrasonic extraction time was selected as 30 minutes to ensure the robustness of the method.

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

[0348] Based on the above experimental results, the sample pretreatment method for the characteristic chromatogram of the standard decoction of Tetrapanax papyriferus can be determined as follows:

[0349] Take an appropriate amount of this product, grind it into a fine powder, accurately weigh about 0.5g, place it in a stoppered conical flask, add 10mL of water, weigh it, heat under reflux for 30 minutes, remove it, let it cool, weigh it again, make up the weight loss with water, extract it three times with ethyl acetate, 20mL each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 50% methanol and transfer it to a 2mL volumetric flask, add 50% methanol to the mark, shake well, filter, and take the filtrate to obtain the product.

[0350] 4. Determination of common peaks:

[0351] Taking the standard decoction of Tetrapanax papyriferus as an example, this paper illustrates the determination of common peaks using the characteristic spectral method and evaluates the similarity of the standard decoction of Tetrapanax papyriferus, as detailed below:

[0352] Fifteen batches of standard decoction samples of *Tetrapanax papyriferus* were taken. The test solutions were prepared according to the method specified in section "2.2". The test solutions and reference solutions were precisely pipetted and injected according to the chromatographic conditions specified in section "2.1". The common peaks of the characteristic chromatograms of the 15 batches of standard decoction of *Tetrapanax papyriferus* were identified using the *Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software*. Seven common peaks with known components, large peak areas, good peak shape and resolution, and high purity were selected as the characteristic peaks of the standard decoction of *Tetrapanax papyriferus*. Figure 8 As shown, peak 1, protocatechuic acid, was selected as the reference peak S1, and the relative retention times and relative peak areas of peaks 2-4 with peak S1 were calculated; peak 6, eugenol, was selected as the reference peak S2, and the relative retention times and relative peak areas of peaks 5 and 7 with peak S2 were calculated; the RSD values ​​were calculated, and the characteristic peaks were located using the relative retention times.

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

[0354] Referring to the liquid chromatography conditions of the characteristic chromatogram of the standard decoction of *Tetrapanax papyriferus*, the compounds in the test sample solution of the standard decoction were analyzed by mass spectrometry. Secondary fragment ion matching was performed with data from the local mass spectrometry database, and the compounds were identified by referring to relevant literature. Finally, using a reference standard, through comparative analysis of retention time in liquid chromatography, ultraviolet absorption spectrum, and precise molecular weight and fragment ion count in mass spectrometry, peak 1 (S1) in the characteristic chromatogram of the standard decoction of *Tetrapanax papyriferus* was confirmed to be protocatechuic acid, and peak 6 (S2) to be syringaldehyde. The total ion chromatogram of the test sample solution is shown below. Figure 9 , Figure 10 The ultraviolet absorption chromatogram is shown below. Figure 11 Compound information is shown in Table 10.

[0355] 5.1 Chromatographic-mass spectrometry conditions:

[0356] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the packing material (column length 150 mm, inner diameter 3.0 mm, particle size 2.5 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 8; the flow rate was 0.60 mL per minute; and the column temperature was 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: Accurately pipette 2 μL of the test solution under section 2.2, inject it into the liquid chromatograph or liquid chromatography-mass spectrometry instrument, and determine the result.

[0363] Table 10. Compound identification results in the standard decoction of Tetrapanax papyriferus.

[0364]

[0365] Mass spectrometry analysis of peak 1:

[0366] The primary chromatogram of peak 1 (peak 1) was extracted over a period of 8.12–8.43 min. The results are shown in Figure 12. Figure 13 .

[0367] In the primary chromatographic extraction chromatogram of peak 1, the peak at m / z = 155.0335 in positive ion mode should be the [M+H]+ peak, see [image / image]. Figure 12Based on its precise molecular weight, its molecular formula is presumably C7H6O4. A secondary chromatogram of the peak at m / z=153.0181 with a collision energy of 20 Ω was extracted in negative ion mode. The results show that the main ion fragments include 109.0281, etc. Detailed results can be found in [link to relevant documentation]. Figure 13 .

[0368] Based on the matching results of the local mass spectrometry database and the identification results of the reference standard, it is speculated that peak 1 may be protocatechuic acid, with the structural formula shown in Formula 1.

[0369]

[0370] Formula 1

[0371] The possible pyrolysis mechanisms are as follows:

[0372]

[0373] Mass spectrometry analysis of peak 6:

[0374] The primary chromatogram of peak 11 (peak 6) was extracted over a time interval of 39.57–40.28 min. Results are shown below. Figures 14-15 :

[0375] In the primary chromatographic extraction chromatogram of peak 11, the peak with m / z = 183.0651 in positive ion mode has the highest response value, which is the [M+H]+ peak. (See...) Figure 14 Based on its precise molecular weight, its molecular formula is likely C9H. 10 O4; Secondary plot of peak collision energy of m / z=181.0496 with 20 ohms in negative ion mode, see [link to plot]. Figure 15 The results showed that the main ion fragments included 166.0260, etc. By matching the local mass spectrometry database and comparing with the reference standard, peak 11 was confirmed to be syringaldehyde, with the structural formula of formula 2.

[0376]

[0377] Formula 2

[0378] 6. Methodological Validation

[0379] 6.1 Specificity Examination

[0380] Accurately pipette 2 μL each of the test solution, reference solution, and blank solvent of the standard decoction of Tetrapanax papyriferus, and inject them into the liquid chromatograph. Determine the results under the chromatographic conditions described in section "2.1". The results are as follows: Figure 16 The results showed that the chromatogram of the test sample had the same chromatographic peak at the corresponding retention time as that of the reference sample, and there was no interference from the blank solvent, indicating that the method has good specificity.

[0381] 6.2 Precision test

[0382] Take an appropriate amount of standard decoction of Tetrapanax papyriferus, grind it into a fine powder, and accurately weigh about 0.5g. Prepare the test solution according to the method specified in section "2.2". Inject and analyze the sample under the chromatographic conditions in section "2.1". Inject the same test solution six times consecutively. The results show that in the precision test, when the same test solution was injected six times consecutively, with the protocatechuic acid chromatographic peak as the reference peak S1 and the syringaldehyde chromatographic peak as the reference peak S2, the relative retention time RSD values ​​of each characteristic peak and the S peak were in the range of 0.04%~0.32%, and the relative peak area RSD values ​​were in the range of 0.50%~2.63%, all less than 3.0%, indicating good instrument precision.

[0383] 6.3 Repeatability Test

[0384] Take an appropriate amount of standard decoction of Tetrapanax papyriferus, grind it into a fine powder, take about 0.5g, and accurately weigh 6 parallel portions. Prepare 6 test solutions according to the test solution preparation method determined in section "2.2". Inject and analyze according to the chromatographic conditions in section "2.1". The results show that when the same batch of samples is repeatedly measured 6 times, with the protocatechuic acid chromatographic peak as the reference peak S1 and the syringaldehyde chromatographic peak as the reference peak S2, the relative retention time RSD values ​​of each characteristic peak and the S peak are in the range of 0.05%~0.21%, and the relative peak area RSD values ​​are in the range of 0.64%~2.97%, all less than 3.0%, indicating that the method has good repeatability.

[0385] 6.4 Intermediate Precision

[0386] Different researchers conducted measurements at different times using different high-performance liquid chromatographs (HPLC). Approximately 0.5g of a suitable amount of the standard decoction of *Tetrapanax papyriferus* was finely ground and prepared in six parallel portions. These portions were accurately weighed and prepared according to the method specified in section "2.2". The samples were then analyzed under the chromatographic conditions described in section "2.1". The results showed that, with protocatechuic acid as the reference peak (S1) and syringaldehyde as the reference peak (S2), the relative retention time (RSD) values ​​of each characteristic peak relative to peak S2 were within the range of 0.07%–0.28%, and the relative peak area (RSD) values ​​were within the range of 0.44%–2.57%, all less than 3.0%. Combined with the six samples from the repeatability test, the relative retention time (RSD) values ​​of the 12 sample solutions were within the range of 0.12%–0.38%, and the relative peak area (RSD) values ​​were within the range of 0.24%–2.01%, all less than 3.0%. This indicates that the method has good intermediate precision.

[0387] 6.5 Stability Test

[0388] Take an appropriate amount of standard decoction of Tetrapanax papyriferus, grind it into a fine powder, and accurately weigh about 0.5g. Prepare the test solution according to the test solution preparation method determined in section "2.2". Analyze the sample under the chromatographic conditions in section "2.1" at 0, 2, 4, 6, 10, 12, and 20 hours. The results show that when the same test solution is analyzed at 0, 2, 4, 6, 10, 12, and 20 hours, with the protocatechuic acid chromatographic peak as reference peak S1 and the syringaldehyde chromatographic peak as reference peak S2, the relative retention time RSD values ​​of each characteristic peak and peak S are in the range of 0.04%~0.31%, and the relative peak area RSD values ​​are in the range of 0.53%~2.10%, all less than 3.0%, indicating that the test solution is relatively stable within 24 hours.

[0389] Example 2

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

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

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

[0393] 3. Methodological Validation

[0394] 3.1 Linear

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

[0396] Table 11 Results of linearity study of protocatechuic acid and eugenol

[0397]

[0398] 3.2 Repeatability

[0399] Take the repeatability test solution from “6.3” and determine it according to the chromatographic conditions in “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 repeatability RSDs of protocatechuic acid and syringaldehyde are 3.0% and 2.9% respectively, indicating that the method has good repeatability.

[0400] Table 12 Results of repeatability study of protocatechuic acid and eugenol content

[0401]

[0402] 3.3 Intermediate Precision

[0403] The intermediate precision test solution (6.4) was taken and determined according to the chromatographic conditions (2.1). The contents of protocatechuic acid and syringaldehyde were calculated, and the RSDs of 6 parallel samples were calculated. The results were 1.2% and 3.6%, respectively. Together with the 6 repeatability samples, the RSDs of protocatechuic acid and syringaldehyde contents of the 12 samples were 3.3% and 3.4%, respectively, as shown in Table 13 below, indicating that the intermediate precision of the method is good.

[0404] Table 13 Results of intermediate precision study on protocatechuic acid and eugenol content

[0405]

[0406] 3.4 Recovery rate

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

[0408] Take an appropriate amount of the standard decoction of Tetrapanax papyriferus, grind it into a fine powder, weigh 0.25g accurately, and add 1mL of the above mixed reference solution. Prepare the test solution according to the test solution preparation method determined in section "2.2". Determine the contents of protocatechuic acid and syringaldehyde in the test solution according to the chromatographic conditions in section "2.1". Calculate the 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 "Guiding Principles for Analytical Method Validation" of the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is in the range of 0.1% to 1%, the recovery limit is 85% to 110%, indicating a good recovery rate.

[0409] Table 14 Results of the recovery test of protocatechuic acid and eugenol

[0410]

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

[0412] The embodiments described above are merely illustrative of several implementations of the present invention, designed to facilitate a detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this invention patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing a UPLC characteristic spectrum of a standard decoction of Tetrapanax papyriferus, characterized in that, Includes the following steps: Take Tetrapanax papyriferus as a reference material and prepare a reference solution; Prepare a reference solution by taking protocatechuic acid and syringaldehyde; Take the standard decoction of Tetrapanax papyriferus and prepare the test solution; The steps for preparing the test solution include: subjecting the standard decoction of Tetrapanax papyriferus to a second water extraction and a second organic solvent extraction; the second water extraction includes the following steps: mixing the standard decoction of Tetrapanax papyriferus with water and heating under reflux to extract, thereby preparing a third extract; the second organic solvent extraction includes the following steps: mixing the third extract with ethyl acetate and extracting, thereby preparing a fourth extract; concentrating the fourth extract, using methanol as a redissolving solvent to dissolve the residue, filtering, and collecting the filtrate; wherein the methanol is a methanol-water solution with a volume fraction of 45%~55%; The reference solution of the control medicinal material, the reference solution and the test solution were respectively subjected to ultra-high performance liquid chromatography (UPLC) to construct the UPLC characteristic spectrum of the standard decoction of Tetrapanax papyriferus. The chromatographic conditions for ultra-high performance liquid chromatography detection include: A Waters BEH C18 column packed with octadecylsilane-bonded silica gel was used. The column specifications were: column length 150 mm, inner diameter 3 mm, and particle size of the packing material 2.5 μm. Gradient elution was performed using acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B. The detection wavelength was 210 nm to 230 nm. The gradient elution procedure includes: From 0 to 10 minutes, the volume percentage of mobile phase A is 3%. From 10 min to 25 min, the volume percentage of mobile phase A changed from 3% to 7%; Between 25 and 32 minutes, the volume percentage of mobile phase A changed from 7% to 10%. From 32 to 40 minutes, the volume percentage of mobile phase A was 10%. From 40 to 42 minutes, the volume percentage of mobile phase A changed from 10% to 80%. From 42 to 45 minutes, the volume percentage of mobile phase A was 80%. In the aqueous phosphoric acid solution, the volume percentage of phosphoric acid is 0.08% to 0.12%.

2. The construction method according to claim 1, characterized in that, The chromatographic conditions for ultra-high performance liquid chromatography detection also include one or more of the following (1) to (3): (1) The flow rate is 0.55 mL / min to 0.65 mL / min; (2) The column temperature is 28℃~32℃; (3) The injection volume is 1 μL to 3 μL.

3. The construction method according to any one of claims 1 to 2, characterized in that, The steps for preparing the control herbal solution include: The reference herb, Tetrapanax papyriferus, underwent a first water extraction treatment and a first organic solvent extraction treatment.

4. The construction method according to claim 3, characterized in that, The first water extraction process includes the following steps: The reference herb Tetrapanax papyrifer was mixed with water and heated under reflux for extraction. The mixture was then concentrated, and the residue was dissolved in water to prepare the first extract. The first organic solvent extraction process includes the following steps: The first extract was mixed with ethyl acetate and extracted to prepare the second extract. The second extract was concentrated, and the residue was dissolved in methanol as a redissolution solvent. The residue was then filtered and the filtrate was collected. The methanol is an aqueous solution of methanol with a volume fraction of 45% to 55%.

5. The construction method according to claim 4, characterized in that, Satisfy one or more of the following conditions (1) to (4): (1) In the first water extraction treatment step, the mixing ratio of the reference herb Tetrapanax papyriferus and water is 1g: (90~100)mL; (2) The heating and reflux time is 55 min to 65 min; (3) In the first organic solvent extraction step, the mixing volume ratio of the first extract to ethyl acetate is 1:(2~2.5). (4) The extraction with ethyl acetate was performed 3 to 4 times.

6. The construction method according to any one of claims 1 to 2, characterized in that, The steps for preparing the reference solution include: The reference solution was prepared using methanol as the extraction solvent. The concentration of protocatechuic acid in the reference solution is 8 μg / mL to 12 μg / mL, and the concentration of syringaldehyde is 8 μg / mL to 12 μg / mL.

7. The construction method according to claim 6, characterized in that, Satisfy one or more of the following conditions (1) to (4): (1) In the second water extraction step, the mixing ratio of the standard decoction of Tetrapanax papyriferus test sample and water is 1g: (20~25)mL; (2) The heating and reflux time is 15 min to 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 extraction with ethyl acetate was performed 3 to 4 times.

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

07.

9. The construction method according to any one of claims 1 to 2, characterized in that, The construction method further includes the step of mass spectrometry identification of characteristic peaks in the characteristic spectrum using chromatography-mass spectrometry. The chromatographic conditions for the identification process include gradient elution using acetonitrile as mobile phase A and formic acid aqueous solution as mobile phase B; The conditions for the mass spectrometry include: HESI ion source parameters include: sheath gas flow rate of 60arb~65arb, auxiliary gas flow rate of 18arb~22arb, spray voltage of 2.2kV~2.8kV, S-lens voltage of 48V~52V, heating temperature of 280℃~320℃, and capillary temperature of 480℃~520℃. The mass spectrometry scanning parameters include: scanning mode is positive ion mode and / or negative ion mode, scanning range is 120 m / z to 1200 m / z, normalized collision energy is 20 V, and mass spectrum type is peak shape.

10. A method for identifying a standard decoction of Tetrapanax papyriferus, characterized in that, Includes the following steps: The sample to be tested undergoes a third water extraction and a third organic solvent extraction to prepare a test solution. The third water extraction includes the following steps: mixing the sample to be tested with water and heating under reflux to extract a fifth extract. The third organic solvent extraction includes the following steps: mixing the fifth extract with ethyl acetate and extracting to prepare a sixth extract. The sixth extract is concentrated, and methanol is used as a redissolving solvent to dissolve the residue. The residue is then filtered, and the filtrate is collected. The methanol is a 45%~55% (v / v) methanol-water solution. The test solution was analyzed by ultra-high performance liquid chromatography. Compare the obtained chromatogram of the test solution with the characteristic chromatogram constructed according to any one of claims 1 to 9, and identify whether the test sample is a standard decoction of Tetrapanax papyriferus based on the comparison result; The chromatographic conditions for ultra-high performance liquid chromatography detection include: A Waters BEH C18 column packed with octadecylsilane-bonded silica gel was used. The column specifications were: column length 150 mm, inner diameter 3 mm, and particle size of the packing material 2.5 μm. Gradient elution was performed using acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B. The detection wavelength was 210 nm to 230 nm. The gradient elution procedure includes: From 0 to 10 minutes, the volume percentage of mobile phase A is 3%. From 10 min to 25 min, the volume percentage of mobile phase A changed from 3% to 7%; Between 25 and 32 minutes, the volume percentage of mobile phase A changed from 7% to 10%. From 32 to 40 minutes, the volume percentage of mobile phase A was 10%. From 40 to 42 minutes, the volume percentage of mobile phase A changed from 10% to 80%. From 42 to 45 minutes, the volume percentage of mobile phase A was 80%. In the aqueous phosphoric acid solution, the volume percentage of phosphoric acid is 0.08% to 0.12%.

11. The identification method according to claim 10, characterized in that, The chromatographic conditions for ultra-high performance liquid chromatography detection also include one or more of the following (1) to (3): (1) The flow rate is 0.55 mL / min to 0.65 mL / min; (2) The column temperature is 28℃~32℃; (3) The injection volume is 1 μL to 3 μL.

12. The identification method according to claim 11, characterized in that, Satisfy one or more of the following conditions (1) to (4): (1) In the third water extraction step, the mixing ratio of the test sample and water is 1g: (20~25)mL; (2) The heating and reflux time is 15 min to 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 extraction with ethyl acetate was performed 3 to 4 times.

13. A method for detecting the content of chemical components in a standard decoction of Tetrapanax papyriferus, characterized in that, Includes the following steps: Protocatechuic acid and syringaldehyde were used to prepare reference solutions of different concentrations. The reference solutions of different concentrations were analyzed by ultra-high performance liquid chromatography, and a standard curve was constructed based on the peak area and concentration. The standard decoction of Tetrapanax papyriferus was subjected to a third water extraction and a third organic solvent extraction to prepare a test solution. The third water extraction included the following steps: mixing the standard decoction of Tetrapanax papyriferus with water and heating under reflux to extract, thus preparing a fifth extract. The third organic solvent extraction included the following steps: mixing the fifth extract with ethyl acetate and extracting to prepare a sixth extract; concentrating the sixth extract, using methanol as a redissolving solvent to dissolve the residue, filtering, and collecting the filtrate; wherein the methanol was a methanol-water solution with a volume fraction of 45% to 55%. The solution to be tested is analyzed by ultra-high performance liquid chromatography, and the content of chemical components in the sample to be tested is determined by combining the standard curve. The chromatographic conditions for ultra-high performance liquid chromatography detection include: A Waters BEH C18 column packed with octadecylsilane-bonded silica gel was used. The column specifications were: column length 150 mm, inner diameter 3 mm, and particle size of the packing material 2.5 μm. Gradient elution was performed using acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B. The detection wavelength was 210 nm to 230 nm. The gradient elution procedure includes: From 0 to 10 minutes, the volume percentage of mobile phase A is 3%. From 10 min to 25 min, the volume percentage of mobile phase A changed from 3% to 7%; Between 25 and 32 minutes, the volume percentage of mobile phase A changed from 7% to 10%. From 32 to 40 minutes, the volume percentage of mobile phase A was 10%. From 40 to 42 minutes, the volume percentage of mobile phase A changed from 10% to 80%. From 42 to 45 minutes, the volume percentage of mobile phase A was 80%. In the aqueous phosphoric acid solution, the volume percentage of phosphoric acid is 0.08% to 0.12%.

14. The detection method according to claim 13, characterized in that, The chromatographic conditions for ultra-high performance liquid chromatography detection also include one or more of the following (1) to (3): (1) The flow rate is 0.55 mL / min to 0.65 mL / min; (2) The column temperature is 28℃~32℃; (3) The injection volume is 1 μL to 3 μL.

15. The detection method according to any one of claims 13-14, characterized in that, The steps for preparing the reference solution include: The reference solution was prepared using methanol as the extraction solvent.

16. The detection method according to any one of claims 13-14, characterized in that, Satisfy one or more of the following conditions (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 concentration of protocatechuic acid in the range of 0.196 μg / mL to 97.993 μg / mL shows a good linear relationship with the peak area. (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 concentration of syringaldehyde in the range of 0.516 μg / mL to 103.194 μg / mL shows a good linear relationship with the peak area.

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