A construction method for determining the content of areca seed by HPLC

Abstract

This invention provides a method for determining the content of *Zanthoxylum bungeanum* seeds using HPLC, comprising: A) dissolving and extracting the sample raw material using a solvent to obtain a test solution; B) determining the test solution using high-performance liquid chromatography (HPLC) to obtain an HPLC chromatogram of *Zanthoxylum bungeanum* seeds; the HPLC conditions are as follows: a C18 column; mobile phase A is acetonitrile solution, mobile phase B is 0.1% phosphoric acid aqueous solution, and gradient elution is performed; specifically, the gradient elution is as follows: 0–20 min, phase A: 10% → 11%, phase B: 90% → 89%; 20–25 min, phase A: 11% → 18%, phase B: 89% → 82%; 25–40 min, phase A: 18% → 40%, phase B: 82% → 60%. This invention employs high-performance liquid chromatography (HPLC) with acetonitrile-0.1% phosphoric acid solution as the mobile phase for gradient elution. Using chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid as reference standards, an HPLC method for determining the content of *Zanthoxylum bungeanum* seeds was established, providing more scientific technical means for controlling the medicinal quality of *Zanthoxylum bungeanum* seeds.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a method for constructing an HPLC method for determining the content of *Gnaphalium affine* seeds. Background Technology

[0002] The name Nandina domestica was first recorded in Volume 6, "Wood Section," of the Compendium of Materia Medica Supplement. It records that Nandina domestica is "the tree of the Yang Tong, which people plant in their courtyards today. The red fruit that grows in winter is used as ornaments and is not Nan Zhu." "The fruit is called Hong Pa Zi, which can treat eight-cornered lice. It can be pounded with mercury and applied. It can also be soaked in wine to treat wind-dampness."

[0003] Currently, there are no relevant standards for the determination of the content of *Bamboo Sinensis* (Tianzhuzi) medicinal materials and processed slices. Related literature mainly focuses on the study of flavonoids and alkaloids, with a lack of qualitative and quantitative research on *Bamboo Sinensis* granules and related preparations. Our research found that after decoction, the levels of flavonoids and alkaloids in *Bamboo Sinensis* are low. Using these content limits as quality control indicators is insufficient for evaluating the quality of *Bamboo Sinensis* medicinal materials, processed slices, standard decoctions, extracts, and granules. Modern research indicates that phenolic acids such as chlorogenic acid have antibacterial, antiviral, white blood cell-increasing, hepatoprotective, choleretic, antitumor, blood pressure-lowering, blood lipid-lowering, free radical scavenging, and central nervous system stimulating effects.

[0004] Therefore, the present invention aims to construct a high-performance liquid chromatography method for the determination of the content of *Zanthoxylum bungeanum* seeds, which can comprehensively reflect the quality level of *Zanthoxylum bungeanum* seeds and related preparations, and provide guidance for the quality control of *Zanthoxylum bungeanum* medicinal materials, decoction pieces and related preparations. 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 determining the content of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid by HPLC. The method of the present invention is accurate and reliable for the detection of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid.

[0006] This invention discloses the construction and application of a high-performance liquid chromatography (HPLC) method for the determination of the content of *Zanthoxylum bungeanum* seeds. This method establishes a content determination method with the total amount of chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid as indicators, which fully demonstrates the chemical composition characteristics of *Zanthoxylum bungeanum* seeds and comprehensively reflects the quality information of *Zanthoxylum bungeanum* seeds, thereby enabling comprehensive and effective control of the quality of *Zanthoxylum bungeanum* seed medicinal materials, decoction pieces, and related preparations.

[0007] A method for determining the content of *Gnaphalium affine* seeds by HPLC includes:

[0008] A) Dissolve and extract the raw material of the test sample using a solvent to obtain the test solution;

[0009] B) The test solution was analyzed by high performance liquid chromatography to obtain the HPLC chromatogram of the seed;

[0010] The chromatographic conditions for the high-performance liquid chromatography (HPLC) are as follows: the chromatographic column is a C18 column; preferably... C18(2)100A250×4.6mm, 5μm; C18 250×4.6mm, 5μm; ZORBAX Eclipse Plus C18 250×4.6mm, 5μm column.

[0011] In this invention, mobile phase A is an acetonitrile solution, and mobile phase B is a 0.1% phosphoric acid aqueous solution, with gradient elution.

[0012] The gradient elution specifically refers to:

[0013] 0–20 min, Phase A: 10% → 11%, Phase B: 90% → 89%;

[0014] 20–25 min, Phase A: 11% → 18%, Phase B: 89% → 82%;

[0015] 25–40 min, Phase A: 18% → 40%, Phase B: 82% → 60%.

[0016] The raw materials for the test sample described in this invention are one or more of the following: Bambusa textilis medicinal material, Bambusa textilis processed slices, Bambusa textilis standard decoction, or Bambusa textilis formula granules.

[0017] This invention provides a method for determining the content of *Zanthoxylum bungeanum* seeds by HPLC. First, the raw material is dissolved and extracted using a solvent to obtain the test solution.

[0018] Specifically, the sample raw material is dissolved in a solvent, extracted, cooled, shaken, and filtered to obtain the final product. This invention does not limit the specific methods and operations for cooling, shaking, and filtering described above; those familiar with these techniques are acceptable.

[0019] The extraction method described in this invention is either reflux extraction or ultrasonic extraction; preferably, ultrasonic treatment is used. The ultrasonic power is 600W, the frequency is 40kHz, and the ultrasonic time is 15–60 min; preferably 30 min.

[0020] According to the present invention, the ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.1-1:25-100;

[0021] The solvent used in this invention is 50% to 70% methanol; specifically, it can be 50% methanol, 60% methanol, or 70% methanol; preferably, it is 50% methanol.

[0022] In one specific embodiment of the present invention, the ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.1 to 1:50;

[0023] In some specific embodiments of the present invention,

[0024] For the test solution of medicinal materials, take 1.0 g of Tianzhuzi medicinal material powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 50% methanol, weigh it, sonicate it (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0025] For the test solution of processed medicinal materials, take 1.0 g of powdered Tianzhuzi (Bamboo Seed) slices, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 50% methanol, weigh it, sonicate it (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0026] For the standard decoction test solution, take an appropriate amount of the standard decoction of Tianzhuzi (approximately 0.1 g), accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 50% methanol, seal tightly, weigh it, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with 50% methanol, shake well, filter it, and collect the filtrate to obtain the test solution.

[0027] For the test solution of the granules, take an appropriate amount of Tianzhuzi granules, grind them into a fine powder, take about 0.1g, weigh it accurately, place it in a stoppered conical flask, accurately add 50ml of 50% methanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0028] All the raw materials mentioned above can be subjected to quality control and qualitative and quantitative detection by the method of the present invention.

[0029] The present invention also includes the preparation of a reference solution: chlorogenic acid reference standard, neochlorogenic acid reference standard and cryptochlorogenic acid reference standard are respectively dissolved in 50% methanol to obtain a reference solution;

[0030] The reference solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the Tianzhu seed were qualitatively identified based on the chromatogram of the reference.

[0031] The preferred concentrations of the reference solutions in this invention are as follows: chlorogenic acid reference standard of 2.6763696 μg / ml to 267.6369600 μg / ml, neochlorogenic acid reference standard of 1.622296 μg / ml to 162.229600 μg / ml, and cryptochlorogenic acid reference standard of 1.03082 μg / ml to 103.08200 μg / ml.

[0032] Specifically, the linear regression equation for neochlorogenic acid is y = 13.2522X - 3.7026, with a correlation coefficient R0. 2 =1.0000, indicating a good linear relationship between the concentration of neochlorogenic acid and the peak area within the concentration range of 1.622296 μg / ml to 162.229600 μg / ml; the linear regression equation for chlorogenic acid is y = 15.4919X - 7.2945, and the correlation coefficient R is 1.0000. 2 =1.0000, indicating a good linear relationship between chlorogenic acid concentration and peak area within the concentration range of 2.6763696 μg / ml to 267.6369600 μg / ml; the linear regression equation for cryptochlorogenic acid is y = 12.242X - 2.0249, with a correlation coefficient R. 2 =1.0000, indicating a good linear relationship between cryptochlorogenic acid concentration and peak area in the concentration range of 1.03082 μg / ml to 103.08200 μg / ml.

[0033] The test solution was analyzed by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of Tianzhuzi.

[0034] The chromatographic conditions for the high-performance liquid chromatography (HPLC) method are as follows: a C18 column with dimensions of 250 × 4.6 mm and a diameter of 5 μm; and a column temperature of 25–35 °C, preferably 25 °C. Under the above column temperatures, the chromatographic peaks of this invention are symmetrical, exhibiting good resolution and complete elution.

[0035] In this invention, mobile phase A is an acetonitrile solution, and mobile phase B is a 0.1% phosphoric acid aqueous solution, with gradient elution.

[0036] Specifically, the gradient elution is as follows:

[0037] 0–20 min, Phase A: 10% → 11%, Phase B: 90% → 89%;

[0038] 20–25 min, Phase A: 11% → 18%, Phase B: 89% → 82%;

[0039] 25–40 min, Phase A: 18% → 40%, Phase B: 82% → 60%.

[0040] The theoretical plate number, calculated based on chlorogenic acid, should be no less than 5000.

[0041] The present invention exhibits good baseline separation, good peak separation, and stable baseline under the above-mentioned elution gradient.

[0042] The flow rate of the mobile phase described in this invention is 0.8 to 1.2 mL / min; more preferably 1.0 mL / min.

[0043] The present invention found that the chromatographic peaks were well separated and the resolution was moderate at the above flow rates, which is the optimal solution.

[0044] The injection volume is 1–5 μL.

[0045] The detection wavelength is 325 nm. The inventors have discovered that at 325 nm, the chromatographic peak information content is greater, the chromatographic baseline is more stable, and the peak areas are larger.

[0046] This invention provides a method for quality evaluation of *Gnaphalium affine* raw materials and their preparations, which involves testing using any one of the methods described above and analyzing the test results.

[0047] (1) The present invention establishes a method for determining the content of Tianzhuzi, using the total amount of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid as an indicator, which can control the intrinsic quality of Tianzhuzi and its related preparations as a whole and macroscopically, ensuring the efficacy of the drug and enabling the medicinal materials and their related preparations to obtain more standardized quality control.

[0048] (2) The method of the present invention has good stability, high precision, good reproducibility, and is convenient and easy to master.

[0049] This invention provides a method for determining the content of *Zanthoxylum bungeanum* seeds using HPLC, comprising: A) dissolving and extracting the sample raw material using a solvent to obtain a test solution; B) determining the test solution using high-performance liquid chromatography (HPLC) to obtain an HPLC chromatogram of *Zanthoxylum bungeanum* seeds; the HPLC conditions are as follows: a C18 column; mobile phase A is acetonitrile solution, mobile phase B is 0.1% phosphoric acid aqueous solution, and gradient elution is performed; specifically, the gradient elution is as follows: 0–20 min, phase A: 10% → 11%, phase B: 90% → 89%; 20–25 min, phase A: 11% → 18%, phase B: 89% → 82%; 25–40 min, phase A: 18% → 40%, phase B: 82% → 60%. This invention employs high-performance liquid chromatography (HPLC) with acetonitrile-0.1% phosphoric acid solution as the mobile phase for gradient elution. Using chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid as reference standards, an HPLC method for determining the content of *Zanthoxylum bungeanum* seeds was established, providing more scientific technical means for controlling the medicinal quality of *Zanthoxylum bungeanum* seeds. Attached Figure Description

[0050] Figure 1 Mobile phase selection diagram;

[0051] Figure 2 Ultraviolet absorption spectrum of neochlorogenic acid-reference standard;

[0052] Figure 3 Neochlorogenic acid - UV absorption spectrum of the test sample;

[0053] Figure 4Chlorogenic acid-reference standard UV absorption spectrum;

[0054] Figure 5 Chlorogenic acid - UV absorption spectrum of the test sample;

[0055] Figure 6 Cryptochlorogenic acid-reference standard UV absorption spectrum;

[0056] Figure 7 Cryptochlorogenic acid - UV absorption spectrum of the test sample;

[0057] Figure 8 Flow velocity study results;

[0058] Figure 9 Column temperature investigation results;

[0059] Figure 10 An investigation into the exclusivity of Indian seeds;

[0060] Figure 11 New chlorogenic acid standard curve;

[0061] Figure 12 chlorogenic acid standard curve;

[0062] Figure 13 Cryptochlorogenic acid standard curve;

[0063] Figure 14 Chromatographic column analysis;

[0064] Figure 15 The chromatogram is for Comparative Example 1;

[0065] Figure 16 The chromatogram for Comparative Example 2 is shown below.

[0066] Figure 17 This is the chromatogram of Comparative Example 3. Detailed Implementation

[0067] This invention provides a method for the HPLC content determination of *Zanthoxylum bungeanum* seeds. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0068] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for determining the content of *Zanthoxylum bungeanum* by HPLC.

[0069] Experimental instruments and materials

[0070] High Performance Liquid Chromatograph (HPLC): Agilent 1260 HPLC system;

[0071] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0072] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0073] Ultrasonic cleaner: KQ600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0074] Chromatographic column: C18(2)100A250×4.6mm, 5μm; C18 250×4.6mm, 5μm; ZORBAX Eclipse Plus C18 250×4.6mm, 5μm

[0075] Chlorogenic acid (China National Institutes for Food and Drug Control, batch number: 110753-202119, purity: 96.3%);

[0076] New chlorogenic acid (Chengdu Desite Biotechnology Co., Ltd., batch number: DSDDX001504, purity: 98.80%)

[0077] Cryptochlorogenic acid (Chengdu Desite Biotechnology Co., Ltd., batch number: DST220104-035, purity: 99.30%)

[0078] Methanol, acetonitrile, and phosphoric acid were of chromatographic grade; water was ultrapure water; and all other reagents were of analytical grade.

[0079] Tianzhuzi (Indian Bamboo Seed) Formula Granules: TZZ-KL-01, TZZ-KL-02, TZZ-KL-03;

[0080] Tianzhuzi medicinal materials: TZZ-YC-01, TZZ-YC-02, TZZ-YC-03, TZZ-YC-04, TZZ-YC-05, TZZ-YC-06, TZZ-YC-07, TZZ-YC-08, TZZ-YC-09, TZZ-YC-10, TZ Z-YC-11, TZZ-YC-12, TZZ-YC-13, TZZ-YC-14, TZZ-YC-15, TZZ-YC-16, TZZ-YC-17, TZZ-YC-18, TZZ-YC-19, TZZ-YC-20, TZZ-YC-21;

[0081] Tianzhuzi decoction pieces: TZZ-YP-01, TZZ-YP-02, TZZ-YP-03, TZZ-YP-04, TZZ-YP-05, TZZ-YP-06, TZZ-YP-07, TZZ-YP-08, TZZ-YP-09, TZZ-YP-10, TZ Z-YP-11, TZZ-YP-12, TZZ-YP-13, TZZ-YP-14, TZZ-YP-15, TZZ-YP-16, TZZ-YP-17, TZZ-YP-18, TZZ-YP-19, TZZ-YP-20, TZZ-YP-21;

[0082] Tianzhuzi standard decoction: TZZ-BT-01, TZZ-BT-02, TZZ-BT-03, TZZ-BT-04, TZZ-BT-05, TZZ-BT-06, TZZ-BT-07, TZZ-BT-08, TZZ-BT-09, TZZ-BT-10, T ZZ-BT-11, TZZ-BT-12, TZZ-BT-13, TZZ-BT-14, TZZ-BT-15, TZZ-BT-16, TZZ-BT-17, TZZ-BT-18, TZZ-BT-19, TZZ-BT-20, TZZ-BT-21.

[0083] Example: Chromatographic Conditions Investigation

[0084] 2.2.1 Chromatographic conditions and system suitability test

[0085] The column was packed with octadecylsilane-bonded silica gel (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 1.0 mL / min; the column temperature was 25 °C; and the detection wavelength was 325 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 5000.

[0086]

[0087] 2.2.2 Preparation of reference solution

[0088] Take appropriate amounts of chlorogenic acid reference standard, neochlorogenic acid reference standard, and cryptochlorogenic acid reference standard, accurately weigh them, and add 50% methanol to prepare a mixed solution containing 50 μg of neochlorogenic acid, 30 μg of chlorogenic acid, and 20 μg of cryptochlorogenic acid per 1 ml.

[0089] 2.2.3 Preparation of the test solution

[0090] For the test solution of medicinal materials, take about 1.0 g of powdered Tianzhuzi (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 50% methanol, weigh it, sonicate it (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0091] For the test solution of processed medicinal materials, take about 1.0g of powdered Tianzhuzi (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 50% methanol, weigh it, sonicate it (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with 50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0092] For the standard decoction test solution, take an appropriate amount of the standard decoction of Tianzhuzi (approximately 0.1 g), accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 50% methanol, seal tightly, weigh it, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with 50% methanol, shake well, filter it, and collect the filtrate to obtain the test solution.

[0093] For the test solution of the granules, take an appropriate amount of Tianzhuzi granules, grind them into a fine powder, take about 0.1g, weigh it accurately, place it in a stoppered conical flask, accurately add 50ml of 50% methanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0094] 2.2.4 Determination Method

[0095] Accurately pipette 5 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0096] 2.3 Establishment of content determination method

[0097] 2.3.1 Chromatographic conditions and system suitability test

[0098] 2.3.1.1 Selection of mobile phase

[0099] Based on the above-planned experimental conditions, acetonitrile-0.1% phosphoric acid was selected for the investigation. (See...) Figure 1 . Figure 1 Mobile phase selection diagram; the results show that when acetonitrile-0.1% phosphoric acid is used as the mobile phase, the peak shape and separation effect of the target peak are good. Therefore, acetonitrile-0.1% phosphoric acid solution was selected as the mobile phase for further investigation.

[0100] 2.3.1.2 Wavelength Selection

[0101] Based on the above-planned experimental conditions, 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 Figure 2-7 . Figure 2 Ultraviolet absorption spectrum of neochlorogenic acid-reference standard; Figure 3 Neochlorogenic acid - UV absorption spectrum of the test sample; Figure 4 Chlorogenic acid-reference standard UV absorption spectrum; Figure 5 Chlorogenic acid - UV absorption spectrum of the test sample; Figure 6 Cryptochlorogenic acid-reference standard UV absorption spectrum; Figure 7 Cryptochlorogenic acid - UV absorption spectrum of the test sample.

[0102] By comparing 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 peak had a larger absorption at a detection wavelength of 325 nm, and the chromatogram baseline was more stable. Therefore, the detection wavelength was determined to be 325 nm.

[0103] 2.3.1.3 Flow velocity investigation

[0104] Under the proposed experimental conditions, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated. (See...) Figure 8 Table 1. Figure 8 Results of flow velocity study.

[0105] Table 1. Analysis results for different flow velocities

[0106]

[0107] The results showed that the peak shape and separation effect of the chromatograms met the requirements when the flow rate was 0.8 ml / min, 1.0 ml / min and 1.2 ml / min. The proposed flow rate was 1.0 ml / min.

[0108] 2.3.1.4 Column Temperature Investigation

[0109] Based on the above-specified experimental conditions, the results were investigated at column temperatures of 25℃, 30℃, and 35℃. (See...) Figure 9 Table 2. Figure 9 Column temperature test results.

[0110] Table 2 Analytical results at different column temperatures

[0111]

[0112]

[0113] The results showed that the peak shape and separation efficiency of the chromatograms met the requirements when the column temperature was 25℃, 30℃, and 35℃. A column temperature of 25℃ was tentatively set.

[0114] The chromatographic conditions and system usability test for the determination of the content of *Tradescantia scutellariae* granules are tentatively set as follows: Octadecylsilane-bonded silica gel is used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile is used as mobile phase A, and 0.1% phosphoric acid solution is used as mobile phase B, with gradient elution as specified in the table below; the flow rate is 1.0 mL per minute; the column temperature is 25℃; and the detection wavelength is 325 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should not be less than 5000.

[0115]

[0116] 2.3.2 Investigation on the preparation of test solution

[0117] 2.3.2.1 Examination of Extraction Methods

[0118] Take an appropriate amount of *Tradescantia sibiricum* granules (batch number: TZZ-KL-01), grind them into a fine powder, accurately weigh approximately 0.1 g, place them in a stoppered conical flask, accurately add 50 ml of 50% methanol, weigh, reflux, and sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, weigh again, replenish the lost weight with 50% methanol, shake well, filter, and collect the filtrate. Accurately pipette 5 μl of each test solution and inject them into the liquid chromatograph. Calculate the total amounts of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid under different extraction methods. The results are shown in Table 3.

[0119] Table 3. Examination of Extraction Methods

[0120]

[0121] The results showed that the extraction method had little effect on the content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid, so ultrasound was chosen as the extraction method because it is simpler to operate.

[0122] 2.3.2.2 Investigation of Extraction Solvents

[0123] Take an appropriate amount of *Tradescantia serratifolia* granules (batch number: TZZ-KL-01), grind them into a fine powder, weigh approximately 0.1g, and place them in a stoppered conical flask. Accurately add 50ml each of water, 10% methanol, 50% methanol, 70% methanol, methanol, and 50% ethanol, weigh each, and sonicate (600W power, 40kHz frequency) for 30 minutes. Cool, weigh again, and replenish the lost weight with the appropriate solvent. Shake well, filter, and collect the filtrate. Accurately pipette 5μl 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 extraction solvents. The results are shown in Table 4.

[0124] Table 4 Results of the extraction solvent investigation

[0125]

[0126]

[0127] As can be seen from the above, the extraction solvents of 50% methanol, 70% methanol, and 50% ethanol have little effect on the content of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid. Therefore, the extraction solvent is tentatively set as 50% methanol.

[0128] 2.3.2.3 Investigation on the amount of extraction solvent added

[0129] Take an appropriate amount of *Tradescantia serratifolia* granules (batch number: TZZ-KL-01), grind them into a fine powder, accurately weigh approximately 0.1g, place them in a stoppered conical flask, accurately add 25ml, 50ml, and 100ml of 50% methanol, weigh them, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, weigh them again, replenish the lost weight with 50% methanol, 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 extraction solvents. The results are shown in Table 5.

[0130] Table 5. Investigation of the amount of extraction solvent added.

[0131]

[0132] The results showed that the content difference was not significant when the solvent addition amount was 25 ml, 50 ml, and 100 ml. Therefore, the amount of sample with a peak area close to that of the reference standard was selected, and the solvent addition amount was determined to be 50 ml.

[0133] 2.3.2.4 Examination of extraction time

[0134] Take an appropriate amount of *Tradescantia serratifolia* granules (batch number: TZZ-KL-01), grind them into a fine powder, accurately weigh approximately 0.1 g, and place them in a stoppered conical flask. Accurately add 50 ml of 50% methanol to each flask, weigh them, and sonicate them (600 W power, 40 kHz frequency) for 15 minutes, 30 minutes, and 60 minutes respectively. After cooling, weigh them again, replenish the lost weight with 50% methanol, shake well, filter, and collect the filtrate. Accurately pipette 5 μl of each test solution and inject them into the liquid chromatograph. Calculate the total amounts of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid under different extraction solvents. The results are shown in Table 6.

[0135] Table 6. Examination of Extraction Time

[0136]

[0137]

[0138] The results showed that the content difference was not significant when the extraction time was 15 minutes, 30 minutes, and 60 minutes, so 30 minutes of extraction was sufficient. Therefore, the extraction time for the test sample was determined to be 30 minutes.

[0139] In summary, the preparation method for the test sample for determining the content of Tianzhuzi is tentatively set as follows: Take an appropriate amount of Tianzhuzi formula granules, grind them into a fine powder, take about 0.1g, weigh it accurately, place it in a stoppered conical flask, accurately add 50ml of 50% methanol, seal tightly, weigh it, sonicate (power 600W, frequency 40KHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain the test sample.

[0140] 2.3.2.5 Method for determining the content of Tianzhu seeds

[0141] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 25 °C; detection wavelength 325 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 5000.

[0142]

[0143] Preparation of reference solution: Take appropriate amounts of chlorogenic acid reference standard, neochlorogenic acid reference standard, and cryptochlorogenic acid reference standard, accurately weigh them, and add 50% methanol to prepare a mixed solution containing 50 μg of neochlorogenic acid, 30 μg of chlorogenic acid and 20 μg of cryptochlorogenic acid per 1 ml.

[0144] Preparation of the test solution: Take an appropriate amount of Tianzhuzi formula granules, grind them into a fine powder, take about 0.1g, weigh accurately, place in a stoppered conical flask, accurately add 50ml of 50% methanol, stopper tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, replenish the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0145] The assay involves precisely pipetting 5 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0146] 2.3.3 Methodological Examination

[0147] 2.3.3.1 Specificity Examination

[0148] 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 add 50% methanol to prepare a solution containing 50 μg of each per ml, as the reference solution.

[0149] Preparation of the test solution: Take an appropriate amount of Tianzhuzi formula granules, grind them into a fine powder, take about 0.1g, weigh it accurately, place it in a stoppered conical flask, accurately add 50ml of 50% methanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0150] Preparation of negative control solution: A negative control solution lacking *Tetracentron sinense* seeds was prepared according to the above-described method for preparing the test sample solution. Results are shown below. Figure 10 . Figure 10 An examination of the exclusivity of Indian seeds; by Figure 10 It can be seen that the negative solution does not interfere with the determination of the target peak, indicating that the method has good specificity.

[0151] 2.3.3.2 Precision Examination

[0152] A mixed solution of neochlorogenic acid reference standard, chlorogenic acid reference standard, and cryptochlorogenic acid reference standard was injected six times consecutively. The peak area was recorded and the RSD value was calculated. The results are shown in Table 7.

[0153] Table 7 Precision test results

[0154]

[0155]

[0156] As can be seen from the above, the peak area RSD value of neochlorogenic acid is 1.0%, the peak area RSD value of chlorogenic acid is 0.1%, and the peak area RSD value of cryptochlorogenic acid is 1.6%, indicating that the instrument has good precision.

[0157] 2.3.3.3 Linear Relationship

[0158] Precisely pipette the mixed mother liquor of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid to prepare reference solutions of different concentrations (for content determination). Inject 5 μl of each solution into the liquid chromatograph and obtain the peak area. Plot the response curve with the reference concentration (X, μg / ml) on the x-axis and the peak area (Y) on the y-axis. The results are shown in Tables 8, 9, and 10. Figure 11 , 12 13. Figure 11 New chlorogenic acid standard curve; Figure 12 chlorogenic acid standard curve; Figure 13 Cryptochlorogenic acid standard curve.

[0159] Table 8. Results of analysis of neochlorogenic acid standard curve

[0160]

[0161] Table 9. Results of Chlorogenic Acid Standard Curve Analysis

[0162]

[0163] Table 10 Results of Cryptochlorogenic Acid Standard Curve Analysis

[0164]

[0165] The results showed that the linear regression equation for neochlorogenic acid was y = 13.2522X - 3.7026, and the correlation coefficient R0 was [value missing]. 2 =1.0000, indicating a good linear relationship between the concentration of neochlorogenic acid and the peak area within the concentration range of 1.622296 μg / ml to 162.229600 μg / ml; the linear regression equation for chlorogenic acid is y = 15.4919X - 7.2945, and the correlation coefficient R is 1.0000. 2 =1.0000, indicating a good linear relationship between chlorogenic acid concentration and peak area within the concentration range of 2.6763696 μg / ml to 267.6369600 μg / ml; the linear regression equation for cryptochlorogenic acid is y = 12.242X - 2.0249, with a correlation coefficient R. 2 =1.0000, indicating a good linear relationship between cryptochlorogenic acid concentration and peak area in the concentration range of 1.03082 μg / ml to 103.08200 μg / ml.

[0166] 2.3.3.4 Repeatability

[0167] Take an appropriate amount of Tianzhuzi formula granules (batch number: TZZ-KL-01), grind them into a fine powder, about 0.1g, accurately weigh 6 portions, and have the same operator prepare the test solution according to the determined method. Accurately pipette 5μl 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 11.

[0168] Table 11 Results of Repeatability Experiments

[0169]

[0170]

[0171] The results showed that the RSD of the total content was 0.7%, indicating that the method had good repeatability.

[0172] 2.3.3.5 Intermediate Precision

[0173] Based on the above-planned experimental conditions, different personnel conducted measurements at different times using different instruments. An appropriate amount of *Tetrapanax papyriferus* granules (batch number: TZZ-KL-01) was accurately weighed, finely ground, and approximately 0.1g was used to prepare the test sample for measurement. The calculated contents of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid are shown in Table 12.

[0174] Table 12 Results of intermediate precision testing

[0175]

[0176] The results showed that the RSD of the total content was 0.8%, indicating good intermediate precision of the method.

[0177] 2.3.3.6 Recovery rate

[0178] Take a known amount of the test sample (batch number: TZZ-KL-01, chlorogenic acid content 10.7 mg / g, neochlorogenic acid content 4.6 mg / g, cryptochlorogenic acid content 5.2 mg / g), grind it finely, about 0.05 g, and divide it into 6 portions. Accurately weigh each portion and add a certain amount of neochlorogenic acid reference standard (4.566536 μg / ml), chlorogenic acid reference standard (10.306026 μg / ml), and cryptochlorogenic acid reference standard (5.256942 μg / ml) to each portion. Prepare the test solution according to the proposed method and determine the recovery rate. The results are shown in Table 13. The calculation formula is as follows:

[0179]

[0180] Table 13 Results of the recovery experiment

[0181]

[0182]

[0183] The results showed that the average recovery rate of neochlorogenic acid was 99.3% with an RSD of 1.3%, the average recovery rate of chlorogenic acid was 101.9% with an RSD of 1.5%, and the average recovery rate of cryptochlorogenic acid was 101.6% with an RSD of 1.3%. The method showed good accuracy.

[0184] 2.3.3.7 Durability Assessment

[0185] 2.3.3.7.1 Column robustness test

[0186] Based on the above-proposed experimental conditions, the following experiments were conducted: C18(2)100A250×4.6mm, 5μm; The study investigated the chromatographic results using a C18 column (250×4.6mm, 5μm) and a ZORBAX Eclipse Plus C18 column (250×4.6mm, 5μm). 5μl of the test 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 14. Figure 14 . Figure 14 Chromatographic column analysis.

[0187] Table 14 Results of column durability study

[0188]

[0189] The results showed that the analytical chromatographic parameters of different chromatographic columns were good.

[0190] 2.3.3.8 Stability Test

[0191] According to the experimental conditions proposed above, a test solution was prepared, and the peak area of ​​uridine was measured at 0h, 2h, 4h, 8h, 16h and 24h respectively. The results are shown in Table 15.

[0192] Table 15 Stability test results

[0193]

[0194]

[0195] The results showed that under the experimental conditions, the RSD values ​​of neochlorogenic acid content were 0.6%, chlorogenic acid content were 0.6%, and cryptochlorogenic acid content were 1.3%, indicating that the test solution had good stability within 24 hours.

[0196] 2.3.4 Verification of Sample Content Determination

[0197] The following tests were conducted on 21 batches of raw materials, 21 batches of processed medicinal slices, 21 batches of standard decoctions, and 3 batches of formula granules of *Zanthoxylum bungeanum* according to the proposed method. The results are shown in Tables 16-20.

[0198] Table 16 Results of content determination in 21 batches of Indian Bambusa textilis medicinal materials

[0199]

[0200] Table 17 Results of content determination in 21 batches of Tianzhuzi (a type of medicinal slice)

[0201]

[0202]

[0203] Table 18 Results of content determination in 21 batches of standard decoction of *Zanthoxylum bungeanum* seeds

[0204]

[0205] Table 19 Test results of the content of granules in three batches of Tianzhuzi formula

[0206]

[0207] As can be seen from the above, the method for determining the content of Tianzhuzi can effectively detect Tianzhuzi medicinal materials, processed slices, standard decoctions, and formula granules, and the method is stable and feasible.

[0208] Comparative Example 1

[0209] The pretreatment was the same as in the previous examples. Octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 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 the table below; the flow rate was 1.0 ml per minute; the column temperature was 25 °C; and the detection wavelength was 325 nm.

[0210]

[0211] The results are as follows Figure 15 , Figure 15 The chromatogram of Comparative Example 1; by Figure 15 It can be seen that the chromatographic peaks of neochlorogenic acid and cryptochlorogenic acid are poorly separated from the adjacent impurity peaks. Further optimization by changing the gradient ratio is necessary.

[0212] Comparative Example 2

[0213] The pretreatment was the same as in the previous examples. Octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 1.0 ml / min; the column temperature was 25°C; and the detection wavelength was 325 nm.

[0214]

[0215] The results are as follows Figure 16 , Figure 16 The chromatogram of Comparative Example 2; by Figure 16 It can be seen that the chromatographic peaks of neochlorogenic acid and cryptochlorogenic acid are poorly separated from the adjacent impurity peaks. Further optimization by changing the gradient ratio is necessary.

[0216] Comparative Example 3

[0217] The pretreatment was the same as in the previous examples. Results from different mobile phases were compared. Octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid, 0.1% formic acid, and 0.1% glacial acetic acid solutions were used as mobile phase B, respectively, for gradient elution according to the specifications in the table below; the flow rate was 1.0 ml / min; the column temperature was 25 °C; and the detection wavelength was 325 nm.

[0218]

[0219] The results are as follows Figure 17 , Figure 17 The chromatogram of Comparative Example 3; by Figure 17 It can be seen that when 0.1% phosphoric acid is used as mobile phase B, the separation of the three chromatographic peaks of the sample is better, and mobile phase B is determined to be 0.1% phosphoric acid.

[0220] 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 an HPLC method for the determination of the content of *Zanthoxylum bungeanum* seeds, comprising: A) The test sample raw material is dissolved and extracted using a solvent to obtain the test solution; the test sample raw material is one or more of the following: Tianzhuzi medicinal material, Tianzhuzi slices, Tianzhuzi standard decoction, or Tianzhuzi formula granules; the solvent is 50%~70% methanol; the extraction is ultrasonic extraction; Preparation of reference solutions: Take chlorogenic acid reference standard, neochlorogenic acid reference standard and cryptochlorogenic acid reference standard respectively, and dissolve them in 50% methanol to obtain reference solutions; B) The test solution was analyzed by high performance liquid chromatography (HPLC) to obtain the HPLC chromatogram of the *Zanthoxylum bungeanum* seeds; the reference solution was analyzed by HPLC to obtain the chromatogram of the reference substance; and the components of the *Zanthoxylum bungeanum* seeds were qualitatively identified based on the chromatogram of the reference substance; the specific concentrations of the reference solutions were: chlorogenic acid reference standard 2.6763696 μg / ml~267.6369600 μg / ml, neochlorogenic acid reference standard 1.622296 μg / ml~162.229600 μg / ml, and cryptochlorogenic acid reference standard 1.03082 μg / ml~103.08200 μg / ml; 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 solution, mobile phase B is 0.1% phosphoric acid aqueous solution, and gradient elution is used. The gradient elution specifically refers to: 0–20 min, Phase A: 10% → 11%, Phase B: 90% → 89%; 20–25 min, Phase A: 11% → 18%, Phase B: 89% → 82%; 25–40 min, Phase A: 18% → 40%, Phase B: 82% → 60%.

2. The method of claim 1, wherein, The chromatographic column is C 18 250 x 4.6 mm 5 μm; the column temperature is 25-35°C; the theoretical plate number calculated according to chlorogenic acid should be not less than 5000.

3. The method of claim 1, wherein, The flow rate of the mobile phase is 0.8~1.2 mL / min; the injection volume is 1~5 μL.

4. The method according to claim 1, characterized in that, The detection wavelength is 325nm.

5. The method of claim 1, wherein, The column temperature was 25℃; the mobile phase flow rate was 1.0 mL / min.

6. The method of claim 1, wherein, Step A) The ultrasonic power is 600W, the frequency is 40kHz, and the ultrasonic time is 15~60min.

7. The method of claim 1, wherein, The ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.1~1:25~100.

8. A method for quality evaluation of *Gnaphalium affine* raw materials and their preparations, comprising testing using the method described in any one of claims 1 to 7 and analyzing the test results.

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