Construction method of high performance liquid chromatography characteristic spectrum of pittosporic cortex and determination method of content

Abstract

The application provides a construction method of high performance liquid chromatography characteristic spectrum of Pittospori Cortex medicinal material, decoction piece, standard decoction, intermediate and formula granules, comprising the following steps: S1) extracting Pittospori Cortex raw material by using a solvent to obtain a to-be-tested liquid; S2) determining the to-be-tested liquid by using high performance liquid chromatography to obtain the high performance liquid chromatography characteristic spectrum of the Pittospori Cortex raw material; the chromatography conditions of the high performance liquid chromatography are that a C18 column is used as the chromatographic column; acetonitrile is used as the mobile phase A, 0.02% formic acid solution is used as the mobile phase B, and gradient elution is adopted. Compared with the prior art, the application firstly establishes the high performance liquid chromatography characteristic spectrum method for the detection of Pittospori Cortex (Erythrina variegata) medicinal material, decoction piece, standard decoction, extract, formula granule and related preparations, and the method has good stability, high precision, good reproducibility, convenience and easiness to master, and can control the quality of Pittospori Cortex medicinal material, decoction piece, standard decoction, intermediate and formula granule.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, and in particular relates to a method for constructing high-performance liquid chromatography (HPLC) characteristic spectra of Erythrina bark medicinal materials, decoction pieces, standard decoctions, intermediates and their formulation particles, and a method for determining their content. Background Technology

[0002] The earliest record of Erythrina bark can be found in the *Haiyao Bencao* by Li Xun of the Five Dynasties period, which quotes the *Guangzhi* written by Guo Yigong of the Western Jin Dynasty (or, according to another account, the Eastern Jin Dynasty): "According to the *Guangzhi*, it grows in the valleys of the South China Sea. It resembles the bark of a tung tree, yellowish-white, hence the name. It tastes bitter, is warm, and is non-toxic. It is mainly used for weakness and numbness in the lower back and legs, pain in the legs and knees, cholera, dysentery with red and white stools, bloody dysentery, and scabies." The 1977 edition of the *Chinese Pharmacopoeia* includes Erythrina bark as the dried bark of *Erythrina var. legata* var. *orientalis* (L.) Merr or *Erythrina arborescens* Roxb., belonging to the legume family. It has many other names, including *dingtongpi*, *citongpi*, *gutongpi*, *citong*, and *jieguyao*. It is used to dispel wind and dampness, unblock meridians, and relieve pain. It can be used for pain in the lower back, knees, shoulders, and arms, and externally for eczema. Modern pharmacological studies have shown that Erythrina bark has anti-inflammatory, anti-tumor, antioxidant, antibacterial, anti-caries, analgesic, antirheumatic, and antiviral effects.

[0003] Currently, the medicinal materials circulating in the market are mostly from the Erythrina var. legata var. orientalis (L.) Merr, with very few from the Erythrina arborescens Roxb. Domestic research on the quality control of Erythrina var. legata is limited, and the standard control indicators are simple, making it difficult to effectively control the quality of the medicinal materials. This has led to the long-standing problem of inconsistent quality between the medicinal materials and their finished products.

[0004] However, there are currently few quality studies on Erythrina bark (Coral Tree) and its compound preparations, and there are no relevant reports on high performance liquid chromatography detection and content determination, lacking an effective means for quality control. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing high-performance liquid chromatography (HPLC) characteristic spectra of Erythrina bark medicinal materials, processed slices, standard decoctions, intermediates and their formulation granules, and a method for determining their content. The HPLC characteristic spectra method for Erythrina bark medicinal materials, processed slices, standard decoctions, intermediates and their formulation granules constructed by the present invention is stable and reliable, and can be used to control the quality of Erythrina bark medicinal materials, processed slices, standard decoctions, intermediates and their formulation granules.

[0006] This invention, through the construction of high-performance liquid chromatography (HPLC) characteristic chromatograms and content determination methods for Erythrina variegata bark, identifies nine characteristic peaks, specifies their relative retention times, establishes a control characteristic chromatogram, and includes a content determination method using the total amount of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid as indicators. This fully demonstrates the chemical composition characteristics of Erythrina variegata bark and comprehensively reflects its quality information, thereby enabling comprehensive and effective control of the quality of Erythrina variegata bark medicinal materials, processed slices, and related preparations.

[0007] This invention provides a method for constructing high-performance liquid chromatography (HPLC) characteristic spectra of Erythrina bark, processed medicinal slices, standard decoctions, intermediates, and their formulation particles, comprising the following steps:

[0008] S1) Extract the raw material of Pittosporum tobira using a solvent to obtain the test solution;

[0009] S2) The test solution is analyzed by high performance liquid chromatography to obtain the high performance liquid characteristic spectrum of the Erythrina bark raw material;

[0010] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.02% formic acid solution, and gradient elution is used.

[0011] The present invention provides a method for constructing high-performance liquid chromatography (HPLC) characteristic spectra of Erythrina bark medicinal material, processed slices, standard decoctions, intermediates and their formulation granules. First, Erythrina bark raw material is taken and extracted with a solvent to obtain the test solution. In a specific embodiment of the present invention, the solvent is preferably 50% ethanol. The ratio of the mass (g) of the Erythrina bark raw material to the volume (mL) of the solvent is preferably (0.1–1):(15–50), more preferably (0.1–1):(15–30), even more preferably (0.1–1):(20–30), and most preferably (0.1–1):25.

[0012] In one specific embodiment of the present invention, the raw material of Erythrina bark includes raw medicinal material, processed slices, standard decoction, intermediates and formulation granules of Erythrina bark; wherein, the intermediate of Erythrina bark can be any intermediate well known to those skilled in the art, and there are no special limitations. In the present invention, the intermediate of Erythrina bark includes, but is not limited to, Erythrina bark extract, Erythrina bark paste, etc.

[0013] In a specific embodiment of the present invention, the preparation of the test solution of Erythrina bark / processed medicinal material is as follows: Erythrina bark / processed medicinal material is refluxed with water for 50-60 minutes, then extracted with 50% ethanol using ultrasound, cooled, shaken, and filtered to obtain the final product; wherein, the mass ratio of Erythrina bark / processed medicinal material to water is preferably 1:(40-60), more preferably 1:(45-55), and even more preferably 1:50; after reflux with water, filtration is preferred, and the filtrate is evaporated to dryness and then extracted with 50% ethanol using ultrasound; the mass of the Erythrina bark / processed medicinal material is specified in g. The volume ratio of the ultrasonic to 50% ethanol is preferably (0.1-1):(15-50), more preferably (0.1-1):(15-30), even more preferably (0.5-1):(20-30), and most preferably 1:25; the ultrasonic power is preferably 500-700W, more preferably 600W; the ultrasonic frequency is preferably 30-50kHz, more preferably 40kHz; the extraction time is preferably 20-60min, more preferably 30-50min, and even more preferably 40min.

[0014] In a specific embodiment of the present invention, the preparation of the test solution of the standard decoction, intermediate or formulation granules of Erythrina variegata bark is as follows: the standard decoction, intermediate or formulation granules of Erythrina variegata bark are extracted by ultrasonic extraction with 50% ethanol, cooled, shaken, and filtered to obtain the solution; the mass ratio (g) of the standard decoction, intermediate or formulation granules of Erythrina variegata bark to the volume ratio (mL) of 50% ethanol is preferably (0.1-1):(15-50), more preferably (0.1-1):(15-30), even more preferably (0.1-5):(20-30), and most preferably 0.1:25; the ultrasonic power is preferably 500-700W, more preferably 600W; the ultrasonic frequency is preferably 30-50kHz, more preferably 40kHz; the extraction time is preferably 20-60min, more preferably 30-50min, and even more preferably 40min.

[0015] In one specific embodiment of the present invention, the preparation of a reference standard and a reference medicinal material solution is also included:

[0016] Take chlorogenic acid reference standard, neochlorogenic acid reference standard and cryptochlorogenic acid reference standard respectively, and dissolve them in 50% ethanol to obtain reference standard solutions;

[0017] The reference medicinal material was refluxed with water and then extracted with 50% ethanol by ultrasonic extraction to obtain a reference solution of the reference medicinal material.

[0018] The reference standard and reference medicinal material solutions were determined by high performance liquid chromatography (HPLC) to obtain chromatograms of the reference standard and reference medicinal material, respectively. Based on the chromatograms of the reference standard and reference medicinal material, the components of the HPLC characteristic spectra of Erythrina bark, processed slices, standard decoction, intermediates and their formulation granules were qualitatively determined.

[0019] In this invention, the preparation of the reference solution of the control medicinal material is preferably the same as the preparation of the test solution of Erythrina bark / processed medicinal material.

[0020] In one specific embodiment of the present invention, the high-performance liquid chromatography method uses a C18 column (octadecylsilane-bonded silica gel); the C18 column can be any C18 column well-known to those skilled in the art, and there are no special limitations. Specifically, it can be any type of C18 column. T3C18, Endeavorsil C18, or ZORBAX SB-C18.

[0021] In one specific embodiment of the present invention, the specifications of the chromatographic column are preferably 1.6-1.8 μm and 2.1×100-2.1×150 mm; more preferably 1.6 μm and 2.1×150 mm.

[0022] In one specific embodiment of the present invention, the column temperature of the chromatographic column is preferably 25℃~40℃, specifically 25℃, 30℃, 35℃, or 40℃.

[0023] In the high-performance liquid chromatography method of this invention, mobile phase A is acetonitrile, and mobile phase B is a gradient elution of 0.02% formic acid solution. In a specific embodiment provided by this invention, the gradient elution, by volume percentage, specifically comprises:

[0024] From 0 to 8 min, mobile phase A changed from 10% to 15%, and mobile phase B changed from 90% to 85%.

[0025] 8–15 min, mobile phase A 15% → 19%, mobile phase B 85% → 81%;

[0026] 15–17 min, mobile phase A 19%, mobile phase B 81%;

[0027] Over 17–27 minutes, mobile phase A decreased from 19% to 22%, and mobile phase B decreased from 81% to 78%.

[0028] In a specific embodiment of the present invention, the mobile phase flow rate is preferably 0.1–0.4 mL / min, more preferably 0.2–0.3 mL / min, and even more preferably 0.3 mL / min. The present invention has found that a flow rate of 0.3 mL / min results in better peak shapes for each chromatographic peak, and this is considered a preferred embodiment.

[0029] In a specific embodiment of the present invention, the detection wavelength is preferably 327 nm; when the detection wavelength is 327 nm, the index peak has a large absorption and the chromatogram baseline is more stable, so the detection wavelength is determined to be 327 nm.

[0030] In one specific embodiment of the present invention, the injection volume is preferably 1 μL.

[0031] This invention uses a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of high performance liquid chromatography (HPLC) characteristic spectra of Erythrina bark, processed slices, standard decoctions, intermediates, and their formulation granules. The resulting standard HPLC characteristic spectra of Erythrina bark, processed slices, standard decoctions, intermediates, and their formulation granules consist of 9 characteristic peaks, where peak 1 is the neochlorogenic acid peak, peak 2 (S) is the chlorogenic acid peak, and peak 3 is the cryptochlorogenic acid peak.

[0032] In a specific embodiment of the present invention, in the high performance liquid chromatography characteristic spectrum of the Erythrina bark, the chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the average value, and the average values ​​are: 0.54 (peak 1), 1.20 (peak 3), 1.69 (peak 4), 1.84 (peak 5), 2.13 (peak 6), 2.21 (peak 7), 2.28 (peak 8), and 3.29 (peak 9).

[0033] In a specific embodiment of the present invention, in the high performance liquid chromatography characteristic spectrum of the Erythrina bark slices, the chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the average value, and the average values ​​are: 0.54 (peak 1), 1.20 (peak 3), 1.70 (peak 4), 1.84 (peak 5), 2.12 (peak 6), 2.21 (peak 7), 2.26 (peak 8), and 3.26 (peak 9).

[0034] In a specific embodiment of the present invention, in the high performance liquid chromatography characteristic spectrum of the standard decoction of Erythrina bark, the chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the average value, and the average values ​​are: 0.56 (peak 1), 1.19 (peak 3), 1.73 (peak 4), 1.88 (peak 5), 2.33 (peak 6), 2.41 (peak 7), 2.52 (peak 8), and 3.68 (peak 9).

[0035] In a specific embodiment of the present invention, in the high performance liquid chromatography characteristic spectrum of the intermediate of Erythrina bark, the chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the average value, and the average values ​​are: 0.56 (peak 1), 1.18 (peak 3), 1.68 (peak 4), 1.82 (peak 5), 2.15 (peak 6), 2.25 (peak 7), 2.26 (peak 8), and 3.26 (peak 9).

[0036] In a specific embodiment of the present invention, in the high performance liquid chromatography characteristic spectrum of the Erythrina bark formulation granules, the chlorogenic acid peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the average value, and the average values ​​are: 0.56 (peak 1), 1.18 (peak 3), 1.71 (peak 4), 1.86 (peak 5), 2.22 (peak 6), 2.34 (peak 7), 2.39 (peak 8), and 3.45 (peak 9).

[0037] According to the present invention, most preferably, the high-performance liquid chromatography (HPLC) standard characteristic chromatograms of Erythrina bark, processed slices, standard decoctions, intermediates, and their formulation granules should show nine characteristic peaks, which should correspond to the retention times of nine characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1, 2, and 3 should correspond to the retention times of their respective reference standard peaks, and the peak corresponding to the chlorogenic acid reference standard peak is designated as peak S. The relative retention times of the remaining characteristic peaks and peak S are calculated. These relative retention times should be within ±10% of a specified value, which is: 1.71 (peak 4), 1.86 (peak 5), 2.22 (peak 6), 2.34 (peak 7), 2.39 (peak 8), and 3.45 (peak 9).

[0038] This invention also provides a method for determining the content of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in Erythrina bark medicinal materials, processed slices, standard decoctions, intermediates, and their formulation granules, comprising the following steps:

[0039] A1) Extract the raw material of Pittosporum tobira using a solvent to obtain the test solution;

[0040] A2) Using neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid as references, the test solution was determined by high performance liquid chromatography to obtain the content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the raw material of Erythrina bark;

[0041] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.02% formic acid solution, and gradient elution is used.

[0042] In the method for determining the content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in Erythrina bark medicinal materials, decoction pieces, standard decoctions, intermediates and their formulation granules, Erythrina bark raw material is first taken, solvent extracted, and the test solution is obtained; in a specific embodiment provided by the present invention, the solvent is preferably 50% ethanol; the ratio of the mass g of the Erythrina bark raw material to the volume mL of the solvent is preferably (0.1~1):(15~50), more preferably (0.1~1):(15~30), even more preferably (0.1~1):(20~30), and most preferably (0.1~1):25.

[0043] In one specific embodiment of the present invention, the raw material of Erythrina bark includes raw medicinal material, processed slices, standard decoction, intermediates and formulation granules of Erythrina bark; wherein, the intermediate of Erythrina bark can be any intermediate well known to those skilled in the art, and there are no special limitations. In the present invention, the intermediate of Erythrina bark includes, but is not limited to, Erythrina bark extract, Erythrina bark paste, etc.

[0044] In a specific embodiment of the present invention, the preparation of the test solution of Erythrina bark / processed medicinal material is as follows: Erythrina bark / processed medicinal material is refluxed with water for 50-60 minutes, then extracted with 50% ethanol using ultrasound, cooled, shaken, and filtered to obtain the final product; wherein, the mass ratio of Erythrina bark / processed medicinal material to water is preferably 1:(40-60), more preferably 1:(45-55), and even more preferably 1:50; after reflux with water, filtration is preferred, and the filtrate is evaporated to dryness and then extracted with 50% ethanol using ultrasound; the mass of the Erythrina bark / processed medicinal material is specified in g. The volume ratio of the ultrasonic to 50% ethanol is preferably (0.1-1):(15-50), more preferably (0.1-1):(15-30), even more preferably (0.5-1):(20-30), and most preferably 1:25; the ultrasonic power is preferably 500-700W, more preferably 600W; the ultrasonic frequency is preferably 30-50kHz, more preferably 40kHz; the extraction time is preferably 20-60min, more preferably 30-50min, and even more preferably 40min.

[0045] In a specific embodiment of the present invention, the preparation of the test solution of the standard decoction, intermediate or formulation granules of Erythrina variegata bark is as follows: the standard decoction, intermediate or formulation granules of Erythrina variegata bark are extracted by ultrasonic extraction with 50% ethanol, cooled, shaken, and filtered to obtain the solution; the mass ratio (g) of the standard decoction, intermediate or formulation granules of Erythrina variegata bark to the volume ratio (mL) of 50% ethanol is preferably (0.1-1):(15-50), more preferably (0.1-1):(15-30), even more preferably (0.1-5):(20-30), and most preferably 0.1:25; the ultrasonic power is preferably 500-700W, more preferably 600W; the ultrasonic frequency is preferably 30-50kHz, more preferably 40kHz; the extraction time is preferably 20-60min, more preferably 30-50min, and even more preferably 40min.

[0046] In a specific embodiment of the present invention, using neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid as references is preferably achieved by taking chlorogenic acid reference standard, neochlorogenic acid reference standard and cryptochlorogenic acid reference standard respectively, dissolving them in 50% ethanol to obtain reference standard solution;

[0047] The reference solution and the test solution were determined by high performance liquid chromatography, and chromatograms of the reference solution and the test solution were obtained respectively. The contents of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the raw material of Erythrina bark were obtained according to the peak areas of the reference solution and the test solution.

[0048] In one specific embodiment of the present invention, the high-performance liquid chromatography method uses a C18 column (octadecylsilane-bonded silica gel); the C18 column can be any C18 column well-known to those skilled in the art, and there are no special limitations. Specifically, it can be any type of C18 column. T3C18, Endeavorsil C18, or ZORBAX SB-C18.

[0049] In one specific embodiment of the present invention, the specifications of the chromatographic column are preferably 1.6-1.8 μm and 2.1×100-2.1×150 mm; more preferably 1.6 μm and 2.1×150 mm.

[0050] In one specific embodiment of the present invention, the column temperature of the chromatographic column is preferably 25℃~40℃, specifically 25℃, 30℃, 35℃, or 40℃.

[0051] In the high-performance liquid chromatography method of this invention, mobile phase A is acetonitrile, and mobile phase B is a gradient elution of 0.02% formic acid solution. In a specific embodiment provided by this invention, the gradient elution, by volume percentage, specifically comprises:

[0052] From 0 to 8 min, mobile phase A changed from 10% to 15%, and mobile phase B changed from 90% to 85%.

[0053] 8–15 min, mobile phase A 15% → 19%, mobile phase B 85% → 81%;

[0054] 15–17 min, mobile phase A 19%, mobile phase B 81%;

[0055] Over 17–27 minutes, mobile phase A decreased from 19% to 22%, and mobile phase B decreased from 81% to 78%.

[0056] In a specific embodiment of the present invention, the mobile phase flow rate is preferably 0.1–0.4 mL / min, more preferably 0.2–0.3 mL / min, and even more preferably 0.3 mL / min. The present invention has found that a flow rate of 0.3 mL / min results in better peak shapes for each chromatographic peak, and this is considered a preferred embodiment.

[0057] In a specific embodiment of the present invention, the detection wavelength is preferably 327 nm; when the detection wavelength is 327 nm, the index peak has a large absorption and the chromatogram baseline is more stable, so the detection wavelength is determined to be 327 nm.

[0058] In one specific embodiment of the present invention, the injection volume is preferably 1 μL.

[0059] In one specific embodiment of the present invention, using neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid as references, reference solutions of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid at different concentrations are prepared respectively. High-performance liquid chromatography (HPLC) is used to determine the content of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid, respectively, and standard curves are obtained. The test solution is then analyzed by HPLC, and the content of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in the Erythrina bark raw material can be obtained based on the standard curves.

[0060] In one specific embodiment of the present invention, the neochlorogenic acid exhibits a linear relationship in the range of 11.8384–295.96 μg / mL; the linear relationship is y = 9062.5253x – 24223.0471, R 2 =0.9999.

[0061] In one specific embodiment of the present invention, the chlorogenic acid exhibits a linear relationship in the range of 11.753224–293.8305600 μg / mL; the linear relationship is y = 10193.5303x – 38248.3215, R 2 =0.9997.

[0062] In one specific embodiment of the present invention, the cryptochlorogenic acid exhibits a linear relationship in the range of 11.956–298.900 μg / mL; the linear relationship is y = 8566.2003x – 5442.2014, R 2 =0.9995.

[0063] The present invention also provides a quality control method for Erythrina bark medicinal material, decoction pieces, standard decoction, intermediates and formulation granules, which uses the above-mentioned content determination method, with chlorogenic acid, chlorogenic acid and cryptochlorogenic acid as indicator components to achieve quality control.

[0064] This invention provides a method for determining the content of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in Erythrina bark (also known as coral tree) medicinal materials, processed slices, standard decoctions, intermediates, and formulated granules. This method enables effective detection of these components in Erythrina bark samples.

[0065] This invention provides a method for constructing high-performance liquid chromatography (HPLC) characteristic spectra of Erythrina variegata bark, processed medicinal materials, standard decoctions, intermediates, and their formulations, comprising the following steps: S1) extracting Erythrina variegata bark raw material with a solvent to obtain a test solution; S2) determining the test solution using HPLC to obtain the HPLC characteristic spectra of Erythrina variegata bark raw material; the chromatographic conditions of the HPLC method are: a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.02% formic acid solution, and gradient elution. Compared with the prior art, this invention establishes for the first time an HPLC characteristic spectra method for the detection of Erythrina variegata bark (Erythrina variegata) raw material, processed medicinal materials, standard decoctions, extracts, formulations, and related preparations. Furthermore, this method exhibits good stability, high precision, good reproducibility, convenience, and ease of mastery, and can control the quality of Erythrina variegata bark raw material, processed medicinal materials, standard decoctions, intermediates, and their formulations.

[0066] In establishing the characteristic spectrum of Erythrina bark, this invention identified nine common characteristic peaks, identified three components, and studied their relative retention times and relative peak areas, ensuring the stability of its chemical composition and the safety of its use.

[0067] This invention establishes a method for determining the content of Erythrina bark (Coralia erythrorhizon), using the total amount of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid as indicators. This method can control the intrinsic quality of Erythrina bark (Coralia erythrorhizon) and its related preparations from an overall and macroscopic perspective, ensuring the efficacy of the drugs and enabling more standardized quality control of the medicinal materials and their related preparations. Attached Figure Description

[0068] Figure 1 Select the mobile phase diagram;

[0069] Figure 2 This is the UV absorption spectrum of neochlorogenic acid reference standard;

[0070] Figure 3 The UV absorption spectrum of neochlorogenic acid in the test sample;

[0071] Figure 4 This is the UV absorption spectrum of chlorogenic acid reference standard;

[0072] Figure 5 The UV absorption spectrum of chlorogenic acid in the test sample;

[0073] Figure 6 This is the UV absorption spectrum of cryptochlorogenic acid reference standard;

[0074] Figure 7 The UV absorption spectrum of cryptochlorogenic acid in the test sample;

[0075] Figure 8 The graph shows the results of the flow velocity investigation.

[0076] Figure 9 The graph shows the results of the column temperature investigation.

[0077] Figure 10 A diagram illustrating the specificity of Erythrina bark (Coralia) formulation granules;

[0078] Figure 11 This is a standard curve of neochlorogenic acid;

[0079] Figure 12 This is a standard curve for chlorogenic acid.

[0080] Figure 13 This is a standard curve for cryptochlorogenic acid.

[0081] Figure 14 Chromatograms were examined using different instruments.

[0082] Figure 15 Chromatograms for column robustness testing;

[0083] Figure 16 The results of the extraction method examination are shown in the figure;

[0084] Figure 17 Figure showing the results of the solvent extraction investigation;

[0085] Figure 18 A graph showing the amount of extraction solvent added;

[0086] Figure 19 For extracting time-based observation graphs;

[0087] Figure 20 For chromatographic peak identification;

[0088] Figure 21 This is the spectrum of neochlorogenic acid reference standard;

[0089] Figure 22 The spectrum of neochlorogenic acid in the test sample;

[0090] Figure 23 This is the spectrum of chlorogenic acid reference standard;

[0091] Figure 24 Spectrum of chlorogenic acid in the test sample - Spectrum of the test sample;

[0092] Figure 25 The spectrum of cryptochlorogenic acid reference standard;

[0093] Figure 26 The spectrum of cryptochlorogenic acid in the test sample;

[0094] Figure 27 The chromatogram is for intermediate precision testing;

[0095] Figure 28 A comparison chromatogram of different brands of chromatographic columns;

[0096] Figure 29 This is a comparison chart of chromatographic columns from different batches;

[0097] Figure 30 Characteristic atlas of Erythrina bark (Coralia erythrorhizon);

[0098] Figure 31 Characteristic atlas of Erythrina bark (Zealoides) slices;

[0099] Figure 32 Characteristic chromatograms of standard decoction of Erythrina bark (Coralia galbana);

[0100] Figure 33 Characteristic atlas of intermediates of Erythrina bark;

[0101] Figure 34 Characteristic chromatograms of three batches of Erythrina bark (Coralia) formulation granules were used for verification.

[0102] Figure 35 A comparative characteristic atlas of Erythrina bark (Coral Tree) medicinal material;

[0103] Figure 36 A comparative characteristic atlas of Erythrina bark (Zealoides) slices;

[0104] Figure 37 A comparative characteristic spectrum of the standard decoction of Erythrina bark (Coralia galbana);

[0105] Figure 38 Characteristic spectrum of Erythrina bark extract (Coralia galbana) as a reference;

[0106] Figure 39 A comparative characteristic spectrum of Erythrina bark (Coral Tree) formulation granules. Detailed Implementation

[0107] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0108] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for constructing high-performance liquid chromatography (HPLC) characteristic spectra of Erythrina bark medicinal material, processed slices, standard decoctions, intermediates, and their formulation particles provided by the present invention.

[0109] All reagents used in the following examples are commercially available.

[0110] Experimental instruments and materials

[0111] Liquid Chromatographs: Waters Acquity UPLCH-Class PLUS ultra-high performance liquid chromatograph, Shimadzu L-40AB X3 high performance liquid chromatograph;

[0112] Electronic balances: ME204E, XPE26 (Mettler-Toledo Instruments Ltd.);

[0113] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0114] Ultrasonic cleaner: KQ-600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0115] Erythrina bark (also known as coral tree) reference material (Chengdu Pusi Biotechnology Co., Ltd., batch number: PS030195);

[0116] Chlorogenic acid (China National Institutes for Food and Drug Control, batch number: 110753-202119, purity: 96.3%);

[0117] New chlorogenic acid (Chengdu Desite Biotechnology Co., Ltd., batch number: DSDDX001504, purity: 98%);

[0118] Cryptochlorogenic acid (Chengdu Desite Biotechnology Co., Ltd., batch number: DST220104-035, purity: 98%);

[0119] Acetonitrile and formic acid were of chromatographic grade; water was ultrapure water; all other reagents were of analytical grade.

[0120] Erythrina bark (Coral Tree) medicinal material batch numbers: 20101, 20102, 20103, 20104, 20105, 20106, 20107, 20108, 20109, 20110, 20111, 20112, 20113, 20114, 20115, 20116;

[0121] Erythrina bark (Zyrtustoides) processed slices batch numbers: ET101, ET102, ET103, ET104, ET105, ET106, ET107, ET108, ET109, ET110, ET111, ET112, ET113, ET114, ET115, ET116;

[0122] Standard decoction of Erythrina bark (Coral Tree) (Sichuan New Green Pharmaceutical Technology Development Co., Ltd.) Batch numbers: AX101, AX102, AX103, AX104, AX105, AX106, AX107, AX108, AX109, AX110, AX111, AX112, AX113, AX114, AX115, AX116;

[0123] Erythrina bark (Coralia) intermediate (extract) (Sichuan New Green Pharmaceutical Technology Development Co., Ltd.) Batch numbers: ZJT01, ZJT02, ZJT03;

[0124] Erythrina bark (Zanthoxylum bungeanum) formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd.) Batch numbers: KL101, KL102, KL103.

[0125] Example 1: Chromatographic conditions and system suitability test

[0126] 1.1 Using octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); using acetonitrile as mobile phase A and 0.02% formic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.3 ml / min; the column temperature was 30℃; the detection wavelength was 327 nm; and the theoretical plate number, calculated based on the chlorogenic acid peak, should not be less than 5000.

[0127]

[0128] Preparation of reference solution

[0129] Preparation of reference solution: Take 1g of Erythrina bark (Coralia erythrorhizon) reference material, add 50mL of water, heat under reflux for 60 minutes, filter, evaporate the filtrate to dryness, add 25mL of 50% ethanol to the residue, sonicate (600W power, 40KHz frequency) for 40 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of chlorogenic acid reference standard, neochlorogenic acid reference standard, and cryptochlorogenic acid reference standard, accurately weigh them, add 50% ethanol to prepare a mixed solution containing 50μg of each per 1mL, as the reference solution.

[0130] Preparation of test solution

[0131] Medicinal material test solution: Take about 1.0 g of the powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of water, heat under reflux for 60 minutes, filter, wash the container and residue with 10 mL of water, combine the filtrate and washings, evaporate to dryness, accurately add 25 mL of 50% ethanol to the residue, stopper tightly, weigh, sonicate (600 W, 40 kHz) for 40 minutes, cool, weigh again, make up the lost weight with 50% ethanol, shake well, filter, and take the filtrate to obtain the solution.

[0132] Test solution of medicinal slices: Weigh approximately 1.0 g of the powder (passed through a No. 3 sieve) accurately, place it in a stoppered conical flask, add 50 ml of water accurately, heat under reflux for 60 minutes, filter, wash the container and residue with 10 mL of water, combine the filtrate and washings, evaporate to dryness, add 25 mL of 50% ethanol accurately to the residue, stopper tightly, weigh, sonicate (600 W, 40 kHz) for 40 minutes, cool, weigh again, replenish the lost weight with 50% ethanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0133] Standard decoction test solution: Weigh approximately 0.1 g of this product accurately, place it in a stoppered conical flask, accurately add 25 mL of 50% ethanol, seal tightly, weigh, sonicate (power 600 W, frequency 40 kHz) for 40 minutes, cool, weigh again, replenish the lost weight with 50% ethanol, shake well, filter, and collect the filtrate to obtain the solution.

[0134] Intermediate test solution: Weigh approximately 0.1 g of this product accurately, place it in a stoppered conical flask, accurately add 25 mL of 50% ethanol, seal tightly, weigh, sonicate (600 W power, 40 kHz frequency) for 40 minutes, cool, weigh again, replenish the lost weight with 50% ethanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0135] Formula granule test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.1g, weigh it accurately, place it in a stoppered conical flask, accurately add 25mL of 50% ethanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 40 minutes, cool it, weigh it again, make up the lost weight with 50% ethanol, shake well, filter, and take the filtrate to obtain the solution.

[0136] Determination method

[0137] Accurately pipette 1 μL of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0138] 1.2 Establishment of content determination method

[0139] The content determination method was established by using the test solution of Erythrina bark (Coral Tree) formula granules.

[0140] Chromatographic conditions and system suitability test

[0141] Mobile phase selection

[0142] Based on the above-specified experimental conditions, the separation effects of two different mobile phases were investigated. Except for the mobile phases, all other conditions were the optimal screening conditions. The mobile phases were acetonitrile-0.02% phosphoric acid and acetonitrile-0.02% formic acid, respectively. Figure 1 Table 1 Figure 1 Select the mobile phase diagram.

[0143] Table 1. Analysis results of different mobile phases

[0144]

[0145] The results showed that the peak shape and separation effect of the target peak were better when acetonitrile-0.02% formic acid was used as the mobile phase. Therefore, acetonitrile-0.02% formic acid solution was selected as the mobile phase for further investigation.

[0146] Wavelength selection

[0147] Based on the above-specified experimental conditions (except for wavelength, all other conditions are optimal for screening), a diode array detector was used to perform full-band scanning of the neochlorogenic acid reference solution, chlorogenic acid reference solution, cryptochlorogenic acid reference solution, and the test solution. See... Figures 2-7 , Figure 2 This is the UV absorption spectrum of neochlorogenic acid reference standard; Figure 3 The UV absorption spectrum of neochlorogenic acid in the test sample;

[0148] Figure 4 This is the UV absorption spectrum of chlorogenic acid reference standard; Figure 5 The UV absorption spectrum of chlorogenic acid in the test sample; Figure 6This is the UV absorption spectrum of cryptochlorogenic acid reference standard; Figure 7 The UV absorption spectrum of cryptochlorogenic acid in the test sample is shown.

[0149] By analyzing the maximum absorption wavelengths of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in the spectra and referring to the chromatograms, it was found that the index peaks showed greater absorption at a detection wavelength of 327 nm, and the chromatogram baseline was more stable. Therefore, the detection wavelength was determined to be 327 nm.

[0150] Flow velocity investigation

[0151] Under the above-specified experimental conditions (except for flow rate, all other conditions are optimal for screening), the flow rates of 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, and 0.4 mL / min were investigated. See [link / details]. Figure 8 Table 2 Figure 8 The graph shows the results of the flow velocity investigation.

[0152] Table 2 Analysis results for different flow velocities

[0153]

[0154] The results showed that the peak shapes and separation effects of the chromatograms met the requirements when the flow rates were 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, and 0.4 mL / min. However, the peak shapes of each chromatogram were better when the flow rate was 0.3 mL / min. Therefore, the flow rate of 0.3 mL / min was determined.

[0155] Column temperature investigation

[0156] Based on the above-specified experimental conditions (except for column temperature, all other conditions are optimal for screening), the effects were investigated at column temperatures of 25℃, 30℃, 35℃, and 40℃. See [link / reference]. Figure 9 Table 3 Figure 9 The graph shows the results of the column temperature investigation.

[0157] Table 3. Analytical results at different column temperatures

[0158]

[0159]

[0160] The results showed that the chromatogram peak shape and separation effect met the requirements when the column temperature was 25℃, 30℃, 35℃ and 40℃. 30℃ was selected as the detection column temperature.

[0161] In summary, the chromatographic conditions and system usability test for the chlorogenic acid content assay of Erythrina variegata bark are tentatively set as follows: Octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile as mobile phase A, and 0.02% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 0.3 mL / min; column temperature 30℃; detection wavelength 327 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should not be less than 5000.

[0162]

[0163] 1.3 Investigation on the preparation of test solution

[0164] Extraction method examination

[0165] Take an appropriate amount of this product (batch number: KL101), grind it into a fine powder, accurately weigh about 0.1g, place it in a stoppered conical flask, accurately add 25mL of 50% ethanol, seal tightly, weigh, and sonicate (power 600W, frequency 40KHz) and reflux for 40 minutes respectively. Cool, weigh again, and make up the weight loss with 50% ethanol. Shake well, filter, and collect the filtrate. Accurately pipette 1μL of each test solution and inject it into the liquid chromatograph. Calculate the total amount of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid under different extraction methods. The results are shown in Table 4.

[0166] Table 4. Examination of Extraction Methods

[0167]

[0168] The results showed that when ultrasound was used for extraction, the contents of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid were higher and the extraction was more complete. Therefore, ultrasound was tentatively chosen as the extraction method.

[0169] Extraction solvent investigation

[0170] Take an appropriate amount of this product (batch number: KL101), grind it into a fine powder, accurately weigh about 0.1g, place it in a stoppered conical flask, and accurately add methanol, 50% methanol, 30% methanol, ethanol, 50% ethanol, 30% ethanol, and 25mL of water respectively. Seal tightly, weigh, and sonicate (power 600W, frequency 40kHz) for 40 minutes. Cool, weigh again, and replenish the lost weight with the corresponding solvents. Shake well, filter, and collect the filtrate. Accurately pipette 1μL of each test solution and inject it into the liquid chromatograph. Calculate the total amount of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid under different extraction solvents. The results are shown in Table 5.

[0171] Table 5 Results of the extraction solvent investigation

[0172]

[0173]

[0174] As can be seen from the above, the extraction efficiency is the highest when the extraction solvent is 50% ethanol. Therefore, the extraction solvent is tentatively set to 50% ethanol.

[0175] Investigation on the amount of extraction solvent added

[0176] Take an appropriate amount of this product (batch number: KL101), grind it into a fine powder, accurately weigh about 0.1g, place it in a stoppered conical flask, and accurately add 15mL, 25mL, and 50mL of 50% ethanol respectively. Seal the flask tightly, weigh the contents, and sonicate (600W, 40kHz) for 40 minutes. Cool the flask, weigh it again, and replenish the lost weight with 50% ethanol. Shake well, filter, and collect the filtrate. Accurately pipette 1μL of each test solution and inject it into the liquid chromatograph. Calculate the total amounts of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid under different solvent additions. The results are shown in Table 6.

[0177] Table 6. Investigation of the amount of extraction solvent added.

[0178]

[0179] The results showed that neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid could be fully extracted when the amount of extraction solvent added was 25 mL. Therefore, the amount of solvent added was tentatively set at 25 mL.

[0180] Extraction time examination

[0181] Take an appropriate amount of this product (batch number: KL101), grind it into a fine powder, accurately weigh about 0.1g, place it in a stoppered conical flask, accurately add 25mL of 50% ethanol, seal tightly, weigh, and sonicate (600W, 40KHz) for 20, 40, and 60 minutes. After cooling, weigh again, replenish the lost weight with 50% ethanol, shake well, filter, and collect the filtrate. Accurately pipette 1μL of each test solution and inject it into the liquid chromatograph. Calculate the total amounts of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid at different extraction times. The results are shown in Table 7.

[0182] Table 7. Examination of Extraction Time

[0183]

[0184] The results showed that complete extraction was achieved in 40 minutes, so the extraction time was tentatively set at 40 minutes.

[0185] In summary, the preparation method for the test sample for determining the content of Erythrina bark (Coral Tree) granules is tentatively set as follows: Take an appropriate amount of this product, grind it into a fine powder, take about 0.1g, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 50% ethanol, seal tightly, weigh it, sonicate (600W, 40KHz) for 40 minutes, cool it, weigh it again, make up the lost weight with 50% ethanol, shake well, filter it, and take the filtrate to obtain the product.

[0186] Example 2: Method for determining the content of Erythrina bark (Coral Tree)

[0187] 2.1 Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile was used as mobile phase A, and 0.02% formic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 0.3 mL / min; the column temperature was 30 °C; and the detection wavelength was 327 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should not be less than 5000.

[0188]

[0189] Preparation of the mixed solution of reference standards: Take appropriate amounts of chlorogenic acid reference standard, neochlorogenic acid reference standard and cryptochlorogenic acid reference standard, accurately weigh them, add 50% ethanol to prepare a mixed solution containing 50 μg of each per 1 mL.

[0190] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.1 g, weigh it accurately, place it in a stoppered conical flask, accurately add 25 mL of 50% ethanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 40 minutes, cool it, weigh it again, make up the lost weight with 50% ethanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0191] Determination method: Accurately pipette 1 μL of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0192] 2.2 Methodological Examination

[0193] Exclusivity Examination

[0194] Preparation of the reference standard mixed solution: Prepare the reference standard mixed solution according to the experimental conditions proposed above.

[0195] Preparation of the test solution: Prepare the test solution according to the experimental conditions proposed above.

[0196] Preparation of negative control solution: A negative control solution lacking the Erythrina bark granules was prepared according to the above-described method for preparing the test sample solution. Results are shown below. Figure 10 , Figure 10A diagram illustrating the specificity of Erythrina bark (Coral Tree) formulation granules.

[0197] As shown in the figure, the negative solution does not interfere with the determination of the target peak, indicating that the method has good specificity.

[0198] Precision test

[0199] Six consecutive injections were performed using a mixed solution of neochlorogenic acid reference standard, chlorogenic acid reference standard, and cryptochlorogenic acid reference standard. The peak areas were recorded, and the RSD values ​​were calculated. The results are shown in Table 8.

[0200] Table 8 Precision test results

[0201]

[0202] As can be seen from the above, the peak area RSD of neochlorogenic acid is 0.28%, that of chlorogenic acid is 0.18%, and that of cryptochlorogenic acid is 0.10%, indicating that the instrument has good precision.

[0203] linear relationship

[0204] Take an appropriate amount of neochlorogenic acid reference standard (purity 98.0%), place it in a 25 mL volumetric flask, and dissolve it in 50% ethanol to prepare a solution containing 295.9600 μg of neochlorogenic acid per mL. Then dilute it to concentrations of 177.5760 μg / mL, 118.3840 μg / mL, 59.1920 μg / mL, 29.5960 μg / mL, and 11.8384 μg / mL. Take an appropriate amount of chlorogenic acid reference standard (purity 96.3%), place it in a 25 mL volumetric flask, and dissolve it in 50% ethanol to prepare a solution containing 293.8305600 μg of chlorogenic acid per mL. Then dilute it to concentrations of 176.2983360 μg / mL, 117.5322240 μg / mL, 58.7661120 μg / mL, 29.3830560 μg / mL, and 11.7532224 μg / mL. Take an appropriate amount of cryptochlorogenic acid reference standard (purity 98.0%), place it in a 25 mL volumetric flask, and dissolve it in 50% ethanol to prepare a solution containing 298.900 μg of cryptochlorogenic acid per mL. Then dilute to concentrations of 179.340 μg / mL, 119.560 μg / mL, 59.780 μg / mL, 29.890 μg / mL, and 11.956 μg / mL. Accurately pipette 1 μL of each solution and inject it into the liquid chromatograph. Measure the peak area and plot the response curve with concentration (X, μg / mL) on the x-axis and peak area (Y) on the y-axis. The results are shown in Tables 9-11. Figures 11-13 . Figure 11 This is a standard curve of neochlorogenic acid. Figure 12 This is a standard curve for chlorogenic acid. Figure 13 This is a standard curve for cryptochlorogenic acid.

[0205] Table 9. Results of analysis of neochlorogenic acid standard curve

[0206]

[0207] Table 10. Results of Chlorogenic Acid Standard Curve Analysis

[0208]

[0209] Table 11 Results of Cryptochlorogenic Acid Standard Curve Analysis

[0210]

[0211] The results showed that when the concentration of neochlorogenic acid was between 11.8384 and 295.96 μg / mL, the linear relationship was y = 9062.5253x – 24223.0471, R0 2 =0.9999, indicating a good linear relationship when the injected chlorogenic acid concentration is between 11.8384 and 295.96 μg / mL. When the injected chlorogenic acid concentration is between 11.753224 and 293.8305600 μg / mL, the linear relationship is y = 10193.5303x – 38248.3215, R0. 2 =0.9997. A good linear relationship was observed for chlorogenic acid concentrations ranging from 11.753224 to 293.8305600 μg / mL. For cryptochlorogenic acid concentrations ranging from 11.956 to 298.900 μg / mL, the linear relationship was y = 8566.2003x – 5442.2014, R0. 2 =0.9995, and the cryptochlorogenic acid concentration showed a good linear relationship when the injection concentration was between 11.956 and 298.900 μg / mL.

[0212] Repeatability

[0213] Take an appropriate amount of this product (batch number: KL101), grind it into a fine powder, take about 0.1g, accurately weigh 6 portions, and have the same operator prepare the test solution according to the proposed experimental method. Accurately pipette 1μL of each test solution and inject it into the liquid chromatograph. Calculate the total amount of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the 6 samples. The results are shown in Table 12.

[0214] Table 12 Results of Repeatability Experiments

[0215]

[0216] The results showed that the RSD of the total amount of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the six repeatability test samples was 0.46%, indicating that the method had good repeatability.

[0217] intermediate precision

[0218] Different personnel (A1, A2) prepared the test sample (batch number: KL101) solution at different times (T1, T2) using different instruments (C1: Waters, C2: Shimadzu) according to the prescribed method. 1 μL of each test sample solution was precisely pipetted into the liquid chromatograph, and the total amounts of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in the sample were calculated. The results are shown in Table 13. Figure 14 , Figure 14 Chromatograms were examined using different instruments.

[0219] Table 13 Results of intermediate precision testing

[0220]

[0221] As can be seen from the above, when different personnel conducted tests on different instruments at different times, the RSD value of the sample content determination results was 0.33%, indicating that the intermediate precision of this method is good.

[0222] Recovery rate of spiking

[0223] Take approximately 0.05 g of the test sample (batch number: KL101, with neochlorogenic acid content of 25.3 mg / g, chlorogenic acid content of 30.3 mg / g, and cryptochlorogenic acid content of 30.5 mg / g), accurately weigh it, and prepare 6 portions. Accurately add a certain amount of neochlorogenic acid reference standard, chlorogenic acid reference standard, and cryptochlorogenic acid reference standard to each portion. Prepare and determine the test solution according to the prescribed method, and calculate the recovery rate. The results are shown in Table 14. The calculation formula is as follows:

[0224]

[0225] Table 14 Results of the recovery experiment

[0226]

[0227] As can be seen from the above, the average recovery rate of neochlorogenic acid was 98.3%, the average recovery rate of chlorogenic acid was 97.6%, and the average recovery rate of cryptochlorogenic acid was 98.0%, indicating that the method has good accuracy.

[0228] Durability test

[0229] Column durability study

[0230] Different types of C18 chromatographic columns were used. Column 1 was... Column 3 was a 2.1×150mm, 1.6μm column; column 2 was an Endeavorsil C18, 2.1×150mm, 1.8μm column; column 3 was a ZORBAX SB-C18, 2.1×100mm, 1.8μm column. For the same test sample (batch number: 19010243), 1μL of the test solution was precisely pipetted into the liquid chromatograph, and the total amounts of chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid in the sample were calculated. The results are shown in [Figure number missing]. Figure 15 Table 15. Figure 15 Chromatograms for column durability testing.

[0231] Table 15 Results of column robustness test

[0232]

[0233] As can be seen from the above, the RSD value of the sample content determination results using different types of chromatographic columns is 0.27%, indicating that the chromatographic columns of this method have good durability.

[0234] Stability test

[0235] According to the experimental conditions proposed above, a test solution was prepared and measured at 0h, 4h, 10h, 15h, 20h, and 24h. The results are shown in Table 16.

[0236] Table 16 Stability test results

[0237]

[0238] As can be seen from the above, the RSD value of the peak area of ​​neochlorogenic acid in the sample solution within 24 hours is 0.79%, the RSD value of the peak area of ​​chlorogenic acid is 0.71%, and the RSD value of the peak area of ​​cryptochlorogenic acid is 1.20%, indicating that the test solution has good stability within 24 hours.

[0239] 2.3 Validation of Sample Content Determination

[0240] The proposed methods were used to test 16 batches of raw materials, 16 batches of processed slices, 16 batches of standard decoctions, 3 batches of extracts, and 3 batches of formula granules of Erythrina bark (Zanthoxylum bungeanum). The results are shown in Tables 17-21.

[0241] Table 17 Results of content determination of 16 batches of Erythrina bark (Coralia galbana) medicinal materials

[0242]

[0243] Table 18 Results of content determination of 16 batches of Erythrina bark (Coralia galbana) slices

[0244]

[0245]

[0246] Table 19 Results of content determination in 16 batches of Erythrina bark (Coralia) standard decoction

[0247]

[0248] Table 20. Results of content determination of extract from three batches of Erythrina bark (Coralia galbana).

[0249]

[0250] Table 21 Test Results of Content in Three Batches of Erythrina Bark (Coral Tree) Formula Granules

[0251]

[0252] As can be seen from the above, the method for determining the content of Erythrina bark (Coral Tree) can effectively detect Erythrina bark (Coral Tree) medicinal materials, processed slices, standard decoctions, extracts, and formula granules. This method is stable and feasible.

[0253] Example 3: Establishment of Feature Map Method

[0254] A characteristic chromatographic method was established using the test solution of Erythrina bark (Coral Tree) formulation granules under the chromatographic conditions for content determination.

[0255] 3.1 Investigation on the preparation of the test solution

[0256] Extraction method examination

[0257] Take an appropriate amount of this product (batch number: KL101), grind it into a fine powder, accurately weigh about 0.1g, place it in a stoppered conical flask, accurately add 25mL of 50% ethanol, seal tightly, weigh, and sonicate (600W, 40KHz) and reflux for 40 minutes respectively. Cool, weigh again, and make up the weight loss with 50% ethanol. Shake well, filter, and collect the filtrate to obtain the final product. See results below. Figure 16 , Figure 16 The image shows the results of the extraction method examination.

[0258] The results showed that the peak shapes of each characteristic peak were good when the extraction method was ultrasonic. In order to maintain consistency with the preparation of the test sample for content determination, the extraction method was tentatively set as ultrasonic.

[0259] Test sample weight test

[0260] Take an appropriate amount of this product (batch number: KL101), grind it into a fine powder, accurately weigh about 0.1g, place it in a stoppered conical flask, and accurately add methanol, 50% methanol, 30% methanol, ethanol, 50% ethanol, 30% ethanol, and 25mL of water respectively. Seal the flask tightly, weigh it, and sonicate (600W power, 40kHz frequency) for 40 minutes. Cool, weigh it again, and replenish the lost weight with the corresponding solvents. Shake well, filter, and collect the filtrate. See the results below. Figure 17 , Figure 17 The figure shows the results of the solvent extraction investigation.

[0261] As can be seen from the above, when 50% ethanol is used as the extraction solvent, the amount of chromatographic peak information is large and the peak shape of each chromatographic peak is good. In order to maintain consistency with the preparation of the test sample for content determination, the extraction solvent is determined to be 50% ethanol after comprehensive consideration.

[0262] Investigation on the amount of extraction solvent added

[0263] Take an appropriate amount of this product (batch number: KL101), grind it into a fine powder, accurately weigh about 0.1g, and place it in a stoppered conical flask. Accurately add 15mL, 25mL, and 50mL of 50% ethanol respectively, seal tightly, weigh, and sonicate (600W, 40kHz) for 30 minutes. Cool, weigh again, and replenish the lost weight with 50% ethanol. Shake well, filter, and collect the filtrate. See results below. Figure 18 , Figure 18 The graph shows the amount of extraction solvent added.

[0264] As shown above, when the solvent volume is 25 mL, the peak height and peak area of ​​the characteristic chromatographic peaks are more suitable, and the baseline is more stable. Therefore, the solvent volume for the test sample is determined to be 25 mL.

[0265] Extraction time examination

[0266] Take an appropriate amount of this product (batch number: KL101), grind it into a fine powder, accurately weigh about 0.1g, place it in a stoppered conical flask, accurately add 25mL of 50% ethanol, seal tightly, weigh, and sonicate (600W, 40KHz) for 30, 45, and 60 minutes. After cooling, weigh again, replenish the lost weight with 50% ethanol, shake well, filter, and collect the filtrate. Results are shown in the figure. Figure 19 , Figure 19 To extract the time-based observation graph.

[0267] As shown in the figure, there was no significant difference in the chromatographic peaks when the extraction time was 20 minutes, 40 minutes, and 60 minutes. Based on the results of the content determination, the extraction time of the test sample was determined to be 40 minutes.

[0268] In summary, the preparation method of the test solution of the Erythrina bark (Coral Tree) formulation granules is determined as follows: Take an appropriate amount of this product, grind it into a fine powder, take about 0.1g, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 50% ethanol, seal tightly, weigh, sonicate (600W, 40KHz) for 40 minutes, cool, weigh again, replenish the lost weight with 50% ethanol, shake well, filter, and take the filtrate to obtain the product.

[0269] 3.2 Feature Mapping Method (Tentative)

[0270] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile was used as mobile phase A, and 0.02% formic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 0.3 mL / min; the column temperature was 30 °C; and the detection wavelength was 327 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should not be less than 5000.

[0271]

[0272] Preparation of reference solution: Take 1g of Erythrina bark (Coralia erythrorhizon) reference material, add 50mL of water, heat under reflux for 60 minutes, filter, evaporate the filtrate to dryness, add 25ml of 50% ethanol to the residue, sonicate (600W power, 40KHz frequency) for 40 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of chlorogenic acid reference standard, neochlorogenic acid reference standard, and cryptochlorogenic acid reference standard, accurately weigh them, add 50% ethanol to prepare a mixed solution containing 50μg of each per 1mL, as the reference solution.

[0273] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.1 g, weigh it accurately, place it in a stoppered conical flask, accurately add 25 mL of 50% ethanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 40 minutes, cool it, weigh it again, make up the lost weight with 50% ethanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0274] Determination method: Accurately pipette 1 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0275] Methodological investigation

[0276] Chromatographic peak identification

[0277] Preparation of the test solution: Prepare the test solution of Erythrina bark granules according to the experimental conditions proposed above.

[0278] Preparation of reference solution: Take appropriate amounts of neochlorogenic acid reference standard, chlorogenic acid reference standard and cryptochlorogenic acid reference standard, accurately weigh them, and accurately add 50% ethanol to prepare a solution containing 50 μg of each per 1 mL, which is used as the reference solution.

[0279] Preparation of negative control solution: Prepare negative control solution lacking Erythrina bark granules according to the experimental conditions proposed above.

[0280] The characteristic spectral peaks of the Pittosporum tobira formulation granules were located, see [link / reference needed]. Figures 20-26 . Figure 20 For chromatographic peak identification; Figure 21 This is the spectrum of neochlorogenic acid reference standard; Figure 22 The spectrum of neochlorogenic acid in the test sample; Figure 23 This is the spectrum of chlorogenic acid reference standard; Figure 24 Spectrum of chlorogenic acid in the test sample - Spectrum of the test sample; Figure 25 The spectrum of cryptochlorogenic acid reference standard; Figure 26 The spectrum of cryptochlorogenic acid in the test sample.

[0281] The results showed that among the nine characteristic peaks of the test sample, peak 1 was neochlorogenic acid, peak 2 was chlorogenic acid, and peak 3 was cryptochlorogenic acid. Therefore, the nine characteristic peaks of the test sample were investigated in the following methodological investigation.

[0282] Precision test

[0283] Prepare one sample solution according to the proposed experimental method, inject it six times consecutively, and calculate the retention time and peak area of ​​each characteristic peak. See Tables 22-23.

[0284] Table 22 Precision Examination - Retention Time

[0285]

[0286] Table 23 Precision Examination - Peak Area

[0287]

[0288] The results show that the retention time RSD of each characteristic peak of the sample ranges from 0.19% to 0.50%, and the peak area RSD ranges from 0.23% to 6.60%, indicating that the instrument precision of this method is good.

[0289] Repeated examination

[0290] Six test solutions were prepared according to the proposed experimental method. The relative retention times and relative peak areas of each characteristic peak were measured and calculated. See Tables 24 and 25.

[0291] Table 24 Repeatability Tests - Relative Retention Time Ratios

[0292]

[0293] Table 25 Repeatability Tests - Relative Peak Area Ratio

[0294]

[0295] The results showed that the relative retention time (RSD) of the six samples ranged from 0.00% to 0.87%, indicating that the method had good repeatability.

[0296] Intermediate precision test

[0297] Personnel A1 prepared two test solutions at time T1 according to the proposed experimental method and measured them on instrument C1 (WatersAcquity UPLCH-Class PLUS type); personnel A2 prepared two test solutions at time T2 according to the proposed experimental method and measured them on instrument C2 (Shimadzu L-40AB X); the relative retention times and relative peak areas of each characteristic peak were calculated. See Figure 27 Tables 26-27. Figure 27 The chromatogram is used for intermediate precision testing.

[0298] Table 26 Intermediate Precision Examination - Relative Retention Time Ratio

[0299]

[0300] Table 27 Intermediate Precision Examination - Relative Peak Area Ratio

[0301]

[0302] The results show that when different personnel, at different times, and using different instruments measure the same sample, the RSD of the relative retention time of each characteristic peak is 0.99% to 6.08%, indicating that the method has good intermediate precision.

[0303] Column durability study

[0304] Durability Study of Different Types of Chromatographic Columns

[0305] Based on the above-established experimental conditions, five chromatographic columns of different types were investigated. Column 1 was... Column 3: 2.1 × 150 mm, 1.6 μm; Column 2: Endeavorsil C18, 2.1 × 150 mm, 1.8 μm; Column 3: ZORBAX SB-C18, 2.1 × 100 mm, 1.8 μm; Column 4: InfinityLab Poroshell120Aq-C18, 2.1 × 100 mm, 2.7 μm; Column 5: Shim-pack Scepter C18-120, 2.1 × 150 mm, 1.9 μm. See [link / reference] Figure 28 Tables 28-29. Figure 28 A comparison chart of chromatographic columns from different brands.

[0306] Table 28 Column Robustness Study - Relative Retention Time

[0307]

[0308]

[0309] Table 29 Column Robustness Study - Relative Peak Area

[0310]

[0311] Comparing the different types of chromatographic columns and considering the number of chromatographic peaks, the chromatogram of column 1 shows that peaks 1, 2, and 3 were effectively separated; the chromatograms of columns 2 and 3 show that peaks 7 and 8 were not effectively separated; no peaks were observed in column 4; and peak 2 in column 5 had a poor peak shape. Therefore, column 1 is recommended.

[0312] Durability study of chromatographic columns from different batches

[0313] Based on the experimental conditions outlined above, chromatographic column 1 was used to investigate three columns from different batches (Serial Nos. 01273308718508, 01273308718501, and 01283301618542). See Table 30. Figure 29 , Figure 29 This is a comparison chart of chromatographic columns from different batches.

[0314] Table 30 Column robustness study - relative retention time

[0315]

[0316] As shown in the figure above, all three batches of chromatographic columns exhibited nine common peaks, with the RSD of the relative retention time of each characteristic peak ranging from 0.88% to 2.11%. Therefore, this type of chromatographic column is recommended.

[0317] stability

[0318] Prepare a test solution according to the experimental conditions proposed above, and measure the results at 0h, 4h, 10h, 15h, 20h, and 24h. See Tables 31-32.

[0319] Table 31 Stability Study - Retention Time

[0320]

[0321] Table 32 Stability Study - Peak Area

[0322]

[0323] As shown in the table, the RSD of the retention time of the characteristic peak is 2.07% to 3.92%, and the RSD of the peak area is 0.43% to 9.77%, indicating that the sample solution is stable within 24 hours.

[0324] 3.3 Determination of characteristic peaks and establishment of reference spectra

[0325] Based on the principles of stable relative retention time, detectability in all batches of samples, and relatively high peaks, nine peaks with good repeatability were finally selected as characteristic peaks.

[0326] Sample characteristic spectrum verification

[0327] The proposed method was used to determine the characteristic spectra of 16 batches of Erythrina bark (also known as Coral Tree bark), 16 batches of processed medicinal slices, 16 batches of standard decoctions, 3 batches of extracts, and 3 batches of formulated granules, and the relative retention times were calculated. See [link / reference needed]. Figures 30-34 Tables 33-37. Figure 30 Characteristic atlas of Erythrina bark (Coralia erythrorhizon) (S1-S16 are: 20101, 20102, 20103, 20104, 20105, 20106, 20107, 20108, 20109, 20110, 20111, 20112, 20113, 20114, 20115, 20116 respectively); Figure 31 Characteristic atlas of Erythrina bark (Zyrtus tomentosa) slices (S1-S16 are respectively: ET101, ET102, ET103, ET104, ET105, ET106, ET107, ET108, ET109, ET1010, ET1011, ET1012, ET1013, ET1014, ET1015, ET1016); Figure 32 Characteristic chromatograms of standard decoction of Erythrina bark (S1~S16 are: AX101, AX102, AX103, AX104, AX105, AX106, AX107, AX108, AX109, AX110, AX111, AX112, AX113, AX114, AX1115, AX116); Figure 33 Characteristic atlas of intermediates of Erythrina bark; Figure 34 Characteristic chromatograms of three batches of Erythrina bark (Coralia) formulation granules were used for verification.

[0328] Table 33. Relative retention time of the medicinal characteristics of Erythrina bark (Coral Tree).

[0329]

[0330] Table 34. Relative retention time of characteristic images of Erythrina bark (Zanthoxylum bungeanum) slices.

[0331]

[0332] Table 35. Relative retention time of standard decoction of Erythrina bark (Coral Tree) in 16 batches.

[0333]

[0334] Table 36 Relative retention times of intermediates from three batches of Erythrina bark (Coral bark)

[0335]

[0336]

[0337] Table 37 Relative Retention Time of Three Batches of Erythrina Bark Granules

[0338]

[0339] The results showed that the relative retention times (RSDs) of the nine characteristic peaks of 16 batches of Erythrina bark (also known as coral tree bark) medicinal materials, 16 batches of processed slices, 16 batches of standard decoctions, 3 batches of extracts, and 3 batches of formulation granules were all less than 2.0%.

[0340] The final specification stipulates that the chromatogram of the test sample should show 9 characteristic peaks, which should correspond to the retention times of the 9 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 1, 2, and 3 should correspond to the retention times of their respective reference peaks. The peak corresponding to the chlorogenic acid reference peak is designated as the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated. These relative retention times should be within ±10% of the specified values. The specified values ​​are: 1.71 (peak 4), 1.86 (peak 5), 2.22 (peak 6), 2.34 (peak 7), 2.39 (peak 8), and 3.45 (peak 9).

[0341] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize reference chromatograms for 16 batches of raw materials, 16 batches of processed slices, 16 batches of standard decoctions, 3 batches of extracts, and 3 batches of formulation granules of Erythrina variegata (Coral Tree Bark). See [link to reference]. Figures 35-39 . Figure 35The reference characteristic spectrum of Erythrina bark (Coralia erythrorhizon) is shown, where peak 1: neochlorogenic acid, peak 2 (S): chlorogenic acid, and peak 3: cryptochlorogenic acid; Figure 36 The characteristic chromatogram of Erythrina bark (Zygodium japonicum) slices is as follows: Peak 1: neochlorogenic acid, Peak 2 (S): chlorogenic acid, Peak 3: cryptochlorogenic acid; Figure 37 The characteristic chromatogram of the standard decoction of Erythrina bark (Coralia erythrorhizon) is shown, where peak 1: neochlorogenic acid, peak 2 (S): chlorogenic acid, and peak 3: cryptochlorogenic acid; Figure 38 The characteristic chromatogram of Erythrina bark extract is shown, where peak 1: neochlorogenic acid, peak 2 (S): chlorogenic acid, and peak 3: cryptochlorogenic acid. Figure 39 The characteristic chromatogram of the formula granules of Erythrina bark is shown, in which peak 1: neochlorogenic acid, peak 2 (S): chlorogenic acid, and peak 3: cryptochlorogenic acid.

[0342] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing high performance liquid chromatography characteristic spectrum of Pittospori Cortex, medicinal material, decoction piece, standard decoction, intermediate and its formula granules, characterized in that, It comprises the following steps: S1) The sea-tortoise-bark raw material is extracted by solvent to obtain a test solution; the sea-tortoise-bark is the dried bark of Erythrina variegate; the sea-tortoise-bark raw material is sea-tortoise-bark raw medicinal material, decoction piece, standard decoction, intermediate and its formula granule; the sea-tortoise-bark medicinal material / decoction piece test solution is prepared by adding water to reflux the sea-tortoise-bark medicinal material / decoction piece for 50-60 min, and then adding 50% ethanol for ultrasonic extraction; the sea-tortoise-bark standard decoction, intermediate or formula granule test solution is prepared by ultrasonic extraction of the sea-tortoise-bark standard decoction, intermediate or formula granule with 50% ethanol; S2) The test solution is determined by high performance liquid chromatography to obtain the high performance liquid characteristic spectrum of the sea-tortoise-bark raw material; The chromatographic conditions of the high performance liquid chromatography are that the chromatographic column is a C18 column; the mobile phase A is acetonitrile, and the mobile phase B is 0.02% formic acid solution, and gradient elution is adopted; The gradient elution is specifically as follows in terms of volume percentage: 0-8 min, mobile phase A 10%→15%, mobile phase B 90%→85%; 8-15 min, mobile phase A 15%→19%, mobile phase B 85%→81%; 15-17 min, mobile phase A 19%, mobile phase B 81%; 17-27 min, mobile phase A 19%→22%, mobile phase B 81%→78%; The chromatographic column is a C18 column with a specification of 1.6 μm, 2.1×150 mm; The detection wavelength is 327 nm.

2. The construction method of claim 1, wherein, A control and a control medicinal material reference solution are also prepared: The chlorogenic acid control, neochlorogenic acid control and cryptochlorogenic acid control are respectively dissolved in 50% ethanol to obtain a control reference solution; The control medicinal material is refluxed with water, and then ultrasonically extracted with 50% ethanol to obtain a control medicinal material reference solution; The control and the control medicinal material reference solution are determined by high performance liquid chromatography to obtain the chromatograms of the control and the control medicinal material reference solution respectively; and the components of the high performance liquid characteristic spectrum of the sea-tortoise-bark medicinal material, decoction piece, standard decoction, intermediate and formula granule are qualitatively determined according to the chromatograms of the control and the control medicinal material reference solution.

3. The construction method of claim 1, wherein, The column temperature is 25-40°C.

4. The construction method according to claim 3, characterized in that, The flow rate of the mobile phase is 0.3 mL / min; and the injection amount is 1 μL.

5. The construction method of claim 1, wherein, The similarity of the high performance liquid characteristic spectrum of the sea-tortoise-bark medicinal material, decoction piece, standard decoction, intermediate and formula granule is evaluated by a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain a high performance liquid standard characteristic spectrum of the sea-tortoise-bark medicinal material, decoction piece, standard decoction, intermediate and formula granule composed of 9 characteristic peaks, wherein peak 1 is a neochlorogenic acid peak, peak 2 is a chlorogenic acid peak, and peak 3 is a cryptochlorogenic acid peak.

6. The construction method of claim 5, wherein, In the high performance liquid standard characteristic chromatogram of Pittospori Cortex, the 9 characteristic peaks should be presented, and the retention time of the 9 characteristic peaks in the reference chromatogram of the control material should be corresponding. The retention time of peak 1, peak 2 and peak 3 should be corresponding to the retention time of the corresponding reference peak of the control substance. The peak corresponding to the reference peak of chlorogenic acid control substance is S peak, and the relative retention time of the other characteristic peaks to S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is: 1.71 (peak 4), 1.86 (peak 5), 2.22 (peak 6), 2.34 (peak 7), 2.39 (peak 8), 3.45 (peak 9).

7. The construction method of claim 1, wherein, The preparation of the Pttospori Cortex material / decoction pieces to be tested is as follows: the Pttospori Cortex material / decoction pieces are refluxed with water for 50-60 min, and then extracted with 50% ethanol by ultrasonic extraction. After cooling, shaking and filtering, the to-be-tested solution is obtained. The preparation of the Pttospori Cortex standard decoction, intermediate or formula granules to be tested is as follows: the Pttospori Cortex standard decoction, intermediate or formula granules are extracted with 50% ethanol by ultrasonic extraction. After cooling, shaking and filtering, the to-be-tested solution is obtained. The power of the ultrasonic extraction is 600 W, and the frequency is 40 kHz. The extraction time is 30-50 min.

8. The construction method of claim 1, wherein, The ratio of the mass of the Pttospori Cortex raw material (g) to the volume of the solvent (mL) is (0.1-1):(15-50).

9. A method for determining the content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in Pittospori Cortex, medicinal slices, standard decoction, intermediates and formula granules, characterized in that, The method comprises the following steps: A1) The Pttospori Cortex raw material is extracted with a solvent to obtain a to-be-tested solution. The Pttospori Cortex is the dried bark of Erythrina variegate. The Pttospori Cortex raw material is Pttospori Cortex raw material, decoction pieces, standard decoction, intermediate and formula granules. The preparation of the Pttospori Cortex material / decoction pieces to be tested is as follows: the Pttospori Cortex material / decoction pieces are refluxed with water for 50-60 min, and then extracted with 50% ethanol by ultrasonic extraction. The preparation of the Pttospori Cortex standard decoction, intermediate or formula granules to be tested is as follows: the Pttospori Cortex standard decoction, intermediate or formula granules are extracted with 50% ethanol by ultrasonic extraction. A2) The to-be-tested solution is determined by high performance liquid chromatography with neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid as reference substances to obtain the content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the Pttospori Cortex raw material. The chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, and the mobile phase B is 0.02% formic acid solution, and the gradient elution is performed. The gradient elution is as follows in terms of volume percentage: 0-8 min, mobile phase A 10%→15%, mobile phase B 90%→85%; 8-15 min, mobile phase A 15%→19%, mobile phase B 85%→81%; 15-17 min, mobile phase A 19%, mobile phase B 81%; 17-27 min, mobile phase A 19%→22%, mobile phase B 81%→78%. The chromatographic column is a C18 column with a specification of 1.6 μm, 2.1×150 mm. The detection wavelength is 327 nm.

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