A method for determining the content of catechin in a standard kiwi root decoction and a method for constructing a characteristic chromatogram

Abstract

This invention belongs to the field of pharmaceutical analysis technology, and specifically relates to a method for determining the content of catechins in a standard decoction of kiwifruit root and a method for constructing a characteristic chromatogram. High-performance liquid chromatography (HPLC) is used for determination. The HPLC conditions include: an octadecylsilane-bonded silica column as the packing material; methanol as mobile phase A; and a 0.05–0.2% (v / v) formic acid aqueous solution as mobile phase B, with gradient elution. This method has strong specificity, good repeatability and robustness, and high stability and accuracy. The constructed characteristic chromatogram and detection method can rapidly and comprehensively achieve the joint control of multiple components in the standard decoction of kiwifruit root, realizing quality control of kiwifruit root from raw material to preparation, which is beneficial to ensuring the effectiveness of clinical medication.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, and in particular relates to a method for determining the content of catechins in a standard decoction of kiwi root and a method for constructing a characteristic spectrum. Background Technology

[0002] Kiwi root is the dried root of *Actinidia chinensis* Planch, a plant in the Actinidiaceae family. It has excellent health benefits and medicinal value. Kiwi root is distributed in Hunan, Sichuan, Guizhou, and Yunnan provinces, with Hunan being one of the main producing areas, primarily Zhangjiajie, western Hunan, and Shaoyang. Kiwi root contains ursolic acid, oleanolic acid, succinic acid, carotene, as well as arginine, lysine, leucine, alanine, flavanoids, coumarins, and other components. Modern pharmacological studies have shown that this root has therapeutic effects on hepatitis, edema, rheumatoid arthritis, traumatic injuries, dysentery, filariasis, lymph node tuberculosis, carbuncles and boils, chyluria, leukorrhea, gastric cancer, and breast cancer.

[0003] Existing technologies disclose methods for detecting catechin content in kiwifruit roots. However, it is worth noting that these existing technologies all focus on kiwifruit root medicinal materials. In contrast, the standard kiwifruit root decoction is a freeze-dried powder made from kiwifruit root slices through water extraction, concentration, and drying. Compared to the medicinal materials and slices, it has lost its inherent form, and the corresponding active ingredients have also changed accordingly.

[0004] Therefore, it is necessary to establish an HPLC method for the determination of the content and characteristic chromatograms of kiwifruit root standard decoction, so as to provide a basis for effectively controlling and comprehensively evaluating the quality of kiwifruit root standard decoction. Summary of the Invention

[0005] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes a method for determining the content of catechins in a standard decoction of kiwifruit root and a method for constructing a characteristic spectrum, thereby providing assurance for the quality and consistency evaluation of the standard decoction of kiwifruit root.

[0006] In one aspect, the present invention provides a method for determining the content of catechins in a standard decoction of kiwifruit root, comprising: using an external standard method and determining by high performance liquid chromatography (HPLC), wherein the conditions of the HPLC method include:

[0007] The chromatographic column was packed with octadecylsilane-bonded silica gel.

[0008] Methanol was used as mobile phase A, and formic acid aqueous solution with a volume percentage of 0.05-0.2% was used as mobile phase B for gradient elution.

[0009] The conditions for gradient elution are as follows:

[0010]

[0011]

[0012] The percentage sign in the table means volume percentage.

[0013] According to some embodiments of the present invention, the mobile phase B is an aqueous solution of formic acid with a volume percentage of 0.1%.

[0014] According to some embodiments of the present invention, the chromatographic column is an ACQUITY UPLC BEH C18.

[0015] According to some embodiments of the present invention, the chromatographic column has dimensions of 2.1 mm × 100 mm and 1.7 μm.

[0016] According to some embodiments of the present invention, the conditions for the high performance liquid chromatography method further include: the flow rate of the mobile phase is 0.2 to 0.4 mL / min; preferably 0.2 mL / min.

[0017] According to some embodiments of the present invention, the conditions for the high performance liquid chromatography method further include: the column temperature of the chromatographic column is 30-35°C; preferably 30°C.

[0018] According to some embodiments of the present invention, the conditions for the high performance liquid chromatography method further include: an injection volume of 1 to 5 μL.

[0019] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: a detection wavelength of 278 nm.

[0020] According to some embodiments of the present invention, the detector in the high-performance liquid chromatography is an ultraviolet detector.

[0021] According to some embodiments of the present invention, the theoretical plate number of the high performance liquid chromatography, calculated based on the catechin peak, is not less than 2000.

[0022] According to some embodiments of the present invention, the content determination method further includes the preparation of a test solution; the preparation step includes: mixing a standard decoction of kiwi root with a solvent and sonicating to obtain the solution.

[0023] According to some embodiments of the present invention, the mass-to-volume ratio of the kiwi root standard decoction to the solvent is 1-10 mg: 1 mL.

[0024] According to some embodiments of the present invention, the mass concentration of the test solution is 0.1 to 10 mg / mL.

[0025] According to some embodiments of the present invention, the solvent of the test solution is an alcohol solvent or an alcohol-water mixture.

[0026] Further, the solvent is a methanol aqueous solution with a volume percentage of 50-70% or an ethanol aqueous solution with a volume percentage of 50-70%; preferably, the solvent is a methanol aqueous solution with a volume percentage of 50-70%; more preferably, the solvent is a methanol aqueous solution with a volume percentage of 50%.

[0027] According to some embodiments of the present invention, the ultrasonic conditions described herein include at least one of the following i-iii:

[0028] i. Power: 400~600W;

[0029] ii. Frequency: 30–50 kHz;

[0030] iii. Time: 20–40 min.

[0031] According to some embodiments of the present invention, the content determination method further includes the preparation of a reference solution, wherein the preparation step includes: mixing the reference solution with a solvent to obtain the solution.

[0032] According to some embodiments of the present invention, the reference standard is catechin.

[0033] According to some embodiments of the present invention, the mass concentration of the reference solution is 0.05 to 0.5 mg / mL.

[0034] Furthermore, the solvent of the reference solution is an alcohol solvent or an alcohol-water mixture.

[0035] Furthermore, the solvent is methanol, a methanol aqueous solution with a volume percentage of 50-70%, or an ethanol aqueous solution with a volume percentage of 50-70%.

[0036] Another aspect of the present invention provides a method for constructing a characteristic chromatogram of a standard decoction of kiwifruit root, comprising determining the common peak by high performance liquid chromatography of a reference solution, a reference medicinal material solution, and a test solution, respectively;

[0037] The conditions for the high-performance liquid chromatography method include:

[0038] The chromatographic column was packed with octadecylsilane-bonded silica gel.

[0039] Methanol was used as mobile phase A, and formic acid aqueous solution with a volume percentage of 0.05-0.2% was used as mobile phase B for gradient elution.

[0040] The conditions for gradient elution are as follows:

[0041]

[0042] The percentage sign in the table means volume percentage.

[0043] According to some embodiments of the present invention, the external standard method is used, and the determination is performed by high performance liquid chromatography.

[0044] According to some embodiments of the present invention, the mobile phase B is an aqueous solution of formic acid with a volume percentage of 0.1%.

[0045] According to some embodiments of the present invention, the chromatographic column is Ultimate AQ-C18.

[0046] According to some embodiments of the present invention, the chromatographic column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm.

[0047] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: the flow rate of the mobile phase is 0.5 to 1.0 mL / min; preferably 0.8 mL / min.

[0048] According to some embodiments of the present invention, the conditions for the high performance liquid chromatography method further include: the column temperature of the chromatographic column is 30-35°C; preferably 30°C.

[0049] According to some embodiments of the present invention, the conditions for the high performance liquid chromatography method further include: an injection volume of 5 to 20 μL; preferably 10 μL.

[0050] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: a detection wavelength of 280 nm.

[0051] According to some embodiments of the present invention, the detector in the high-performance liquid chromatography is an ultraviolet detector.

[0052] According to some embodiments of the present invention, the theoretical plate number of the high performance liquid chromatography, calculated based on the catechin peak, is not less than 2000.

[0053] According to some embodiments of the present invention, the characteristic spectral method further includes the preparation of a test solution; the preparation step includes: mixing a standard decoction of kiwi root with a solvent and sonicating to obtain the solution.

[0054] According to some embodiments of the present invention, the mass-to-volume ratio of the kiwi root standard decoction to the solvent is 2-20 mg: 1 mL.

[0055] According to some embodiments of the present invention, the mass concentration of the test solution is 0.1 to 10 mg / mL.

[0056] Furthermore, the solvent of the test solution is an alcohol solvent or an alcohol-water mixture.

[0057] Furthermore, the solvent is a methanol aqueous solution with a volume percentage of 50-70% or an ethanol aqueous solution with a volume percentage of 50-70%; preferably, the solvent is a methanol aqueous solution with a volume percentage of 50-70%; more preferably, the solvent is a methanol aqueous solution with a volume percentage of 50%.

[0058] According to some embodiments of the present invention, the method for constructing the characteristic spectrum further includes the preparation of a reference solution, wherein the preparation step includes: mixing the reference solution with a solvent to obtain the solution.

[0059] According to some embodiments of the present invention, the reference standard is catechin.

[0060] According to some embodiments of the present invention, the mass concentration of the reference solution is 0.05 to 0.5 mg / mL.

[0061] Furthermore, the solvent of the reference solution is an alcohol solvent or an alcohol-water mixture.

[0062] Furthermore, the solvent is a methanol aqueous solution with a volume percentage of 50-70% or an ethanol aqueous solution with a volume percentage of 50-70%; preferably, the solvent is a methanol aqueous solution with a volume percentage of 50-70%; more preferably, the solvent is a methanol aqueous solution with a volume percentage of 50%.

[0063] According to some embodiments of the present invention, the method for constructing the feature spectrum further includes the preparation of a control medicinal material solution, wherein the preparation step includes: mixing the control medicinal material with a solvent and sonicating.

[0064] According to some embodiments of the present invention, the mass-to-volume ratio of the reference medicinal material to the solvent is 10-100 mg: 1 mL.

[0065] According to some embodiments of the present invention, the mass concentration of the control medicinal material solution is 0.5 to 50 mg / mL.

[0066] Furthermore, the solvent of the reference medicinal material solution is an alcohol solvent or an alcohol-water mixture.

[0067] Furthermore, the solvent is a methanol aqueous solution with a volume percentage of 50-70% or an ethanol aqueous solution with a volume percentage of 50-70%; preferably, the solvent is a methanol aqueous solution with a volume percentage of 50-70%; more preferably, the solvent is a methanol aqueous solution with a volume percentage of 50%.

[0068] According to some embodiments of the present invention, the ultrasonic conditions described herein include at least one of the following i-iii:

[0069] i. Power: 400~600W;

[0070] ii. Frequency: 30–50 kHz;

[0071] iii. Time: 20–40 min.

[0072] According to some embodiments of the present invention, the characteristic spectrum includes four characteristic peaks, wherein peak 3 (catechin) is used as a reference peak, and the relative retention times of the other characteristic peaks and the reference peak are calculated. The relative retention times should be within ±10% of a specified value, wherein the specified value is: peak 1: 0.60, peak 2: 0.89, and peak 4: 1.38.

[0073] In another aspect, the present invention provides the application of the above-described content determination method or construction method in detecting the quality of a standard decoction of kiwifruit root.

[0074] Beneficial effects:

[0075] This invention provides a characteristic chromatogram and content determination method for a standard decoction of kiwifruit root. It identifies four common peaks, including catechins. The constructed HPLC characteristic chromatogram shows good separation of chromatographic peaks, rich characteristic chromatographic information, and good peak shape. This allows for overall control of the characteristic components in the standard decoction of kiwifruit root, effectively ensuring the overall quality stability of the decoction. Furthermore, this method is simple to operate and has advantages such as high precision, good stability, good repeatability, and high accuracy, providing an effective basis for the quality identification of traditional Chinese medicinal materials.

[0076] definition:

[0077] The kiwi root mentioned in this invention refers to the dried root of the kiwi fruit.

[0078] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0079] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0080] Figure 1 The chromatogram for investigating the extraction of the solvent in Example 1;

[0081] Figure 2 This is a chromatogram of the mobile phase used in Example 1.

[0082] Figure 3 This is a chromatogram of the elution gradient investigated in Example 1;

[0083] Figure 4 The chromatogram is the result of the investigation of the chromatographic column in Example 1;

[0084] Figure 5 This is the chromatogram used in Example 1 to investigate the specificity of the characteristic chromatograms.

[0085] Figure 6 This is a superimposed image of the characteristic spectra of 15 batches of standard decoctions in Example 1;

[0086] Figure 7 This is a characteristic chromatogram of the standard decoction control in Example 1;

[0087] Figure 8 The characteristic chromatogram of the catechin reference standard in Example 1;

[0088] Figure 9 This is a characteristic spectrum of the control medicinal material in Example 1;

[0089] Figure 10 This refers to the maximum absorption wavelength of catechins in Example 2;

[0090] Figure 11 This is a chromatogram of the elution gradient in Example 2;

[0091] Figure 12 This is the chromatogram for the specificity assessment of Example 2;

[0092] Figure 13 This is the standard curve for catechin reference standard in Example 2. Detailed Implementation

[0093] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0094] The lyophilized powder of kiwi root standard decoction described in this invention is prepared as follows: Kiwi root medicinal material is taken, impurities are removed, it is washed, coarsely sliced, and dried to obtain kiwi root slices. 100g of kiwi root slices is placed in an electric ceramic kettle and decocted twice with water: For the first decoction, 9 times the amount of water (by mass) is added and soaked for 30 minutes. After boiling over high heat, the decoction is simmered for 30 minutes. The decoction is then filtered through a 200-mesh sieve while hot and set aside. For the second decoction, 7 times the amount of water (by mass) is added, and the decoction is boiled over high heat and simmered for 15 minutes. The decoction is then filtered through a 200-mesh sieve while hot. The two decoctions are combined and concentrated under reduced pressure at 65°C to approximately 200g of extract. This extract is then dispensed into 6mL–14mL containers, with each bottle containing 2mL–3mL. After dispensing, the extract is lyophilized, removed, and capped with an aluminum cap to obtain the final product. Properties of the freeze-dried powder of kiwi root standard decoction: light reddish-brown to brown powder; faint odor, bland taste. The preparation process complies with the relevant provisions under the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules".

[0095] Example 1: Establishment of UPLC characteristic spectrum method for kiwi root standard decoction

[0096] 1. Instruments and reagents

[0097] 1.1 Instruments

[0098] LC-40D X3 High Performance Liquid Chromatograph (Shimadzu Corporation);

[0099] KM-500DB Ultrasonic Cleaner (Kunshan Meimei Ultrasonic Instrument Co., Ltd.);

[0100] T-214 electronic balance with a strength of 0.01% (Beijing Sartorius Instrument Systems Co., Ltd.);

[0101] XPE105 electronic balance with a strength of 0.00001 (Mettler-Toledo Instruments Ltd.);

[0102] Column: Ultimate AQ-C18 (5μm, 4.6*250mm).

[0103] 1.2 Reagents

[0104] Methanol was of analytical grade (Hunan Huihong Reagent Co., Ltd., batch number: 202111253);

[0105] Methanol was of chromatographic grade (Tianhex Corporation, USA, batch number: 21115198);

[0106] Formic acid was of chromatographic grade (Tianjin Kemeio Chemical Reagent Co., Ltd., batch number: 20201110);

[0107] The water is Yibao Water (China Resources Yibao Beverage Co., Ltd., batch number: 20220113).

[0108] 1.3 Drug Test

[0109] Catechins (98%, Chengdu Kloma Biotechnology Co., Ltd., CHB210106); Kiwi root reference material (batch number: 2012564, Zhuhai Anzhe Biotechnology Co., Ltd.); Kiwi root standard decoction (lyophilized powder), batch numbers: (MHTG2021072901, MHTG2021072902, MHTG2021072903, MHTG2021072904, MHTG2021072905, MHTG202107) 2906, MHTG2021072907, MHTG2021072908, MHTG2021072909, ZS-PF-YL-202-210801, ZS-PF-YL-202-21 0802, ZS-PF-YL-202-210803, ZS-PF-YL-202-210804, ZS-PF-YL-202-210805, ZS-PF-YL-202-210806).

[0110] 2. Chromatographic conditions and system suitability test

[0111] 2.1 Proposed chromatographic conditions

[0112] The column was packed with octadecylsilane-bonded silica gel (4.6 × 250 mm, 5 μm); methanol was used as mobile phase A and 0.1% formic acid aqueous solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the column temperature was 30 °C; the flow rate was 0.8 mL per minute; and the detection wavelength was 280 nm. The theoretical plate number, calculated based on the catechin peak, should not be less than 2000.

[0113]

[0114] The percentage sign in the table means volume percentage.

[0115] 2.2 Solution Preparation

[0116] (1) Preparation of reference solution: Take an appropriate amount of catechin reference standard, weigh it accurately, and add 50% methanol aqueous solution to prepare a solution containing 0.15 mg per 1 mL.

[0117] (2) Preparation of reference herb solution: Take 1.0g of kiwi root reference herb powder, place it in a stoppered conical flask, accurately add 20mL of 50% methanol aqueous solution, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 30min, cool, weigh again, replenish the lost weight with 50% methanol aqueous solution, shake well, filter, and take the filtrate to obtain the solution.

[0118] (3) Preparation of test solution: Take about 0.2g of this product, place it in a stoppered conical flask, accurately add 20mL of 50% methanol aqueous solution, stopper tightly, weigh, sonicate (power 500W, frequency 40kHz) for 30min, cool, weigh again, make up the lost weight with 50% methanol aqueous solution, shake well, filter, and take the filtrate to obtain the test solution.

[0119] Preparation of blank solution: Take 50% methanol solution, filter, and measure.

[0120] 2.3 Determination Method

[0121] Take 10 μL each of the obtained reference solution, reference medicinal material solution, and test solution, inject them into a high-performance liquid chromatograph, and determine the results.

[0122] 3. Investigation of chromatographic conditions

[0123] 3.1 Investigation of Optimal Absorption Wavelength

[0124] Based on the proposed experimental conditions, the test solution of the standard decoction of kiwi root was scanned at all wavelengths using an ultraviolet detector. The results showed that the chromatographic peak information was greater, the chromatographic baseline was more stable, and the response values ​​of each characteristic peak were larger at a detection wavelength of 280 nm. Therefore, the detection wavelength was determined to be 280 nm.

[0125] 3.2 Investigation of extraction solvent

[0126] The effect of different extraction solvents on the chromatogram of the standard decoction was investigated. 0.2 g of sample was weighed and added to 20 mL of methanol, 50% methanol, ethanol, 50% ethanol, and water, respectively. The mixtures were sonicated for 30 minutes. The results showed that 50% methanol had a better peak shape and larger peak area. Therefore, 50% methanol was ultimately selected as the extraction solvent. Figure 1 As shown (the baseline was slightly uneven during the gradient process, but after multiple experiments, the spectrum response did not affect the detection results).

[0127] 3.3 Investigation of the mobile phase

[0128] With other conditions remaining constant, the effect of different mobile phases on the chromatogram of the standard decoction was investigated, specifically: A: methanol-0.1% acetic acid; B: methanol-0.1% phosphoric acid; C: methanol-0.1% formic acid; D: acetonitrile-0.1% formic acid. The detection results are shown below. Figure 2 .

[0129] Depend on Figure 2 It can be seen that when methanol-0.1% formic acid is used as the mobile phase, the peak shape is better, the separation is better, and the baseline is more stable. Therefore, methanol-0.1% formic acid is selected as the mobile phase for the characteristic chromatogram.

[0130] 3.4 Examination of Gradients

[0131] With other conditions remaining constant, the effects of different gradients on the chromatogram of the standard decoction were investigated. The gradients are shown below:

[0132] Gradient 1

[0133]

[0134] Gradient 2

[0135]

[0136]

[0137] Gradient 3

[0138]

[0139] Depend on Figure 3 It can be seen that when the gradient method is 3, the peak shape is better (especially peak 4, which has a better peak shape and is better separated from other peaks), the separation degree is better, and the baseline is more stable. Therefore, the gradient method for the feature map is 3.

[0140] 3.5 Examination of the chromatographic column

[0141] With other conditions unchanged, the effect of different chromatographic columns on the chromatogram of the standard decoction was investigated. The columns were: A: Agilent 5TC-C18(2); B: GL Science InertSustain AQ-C18; C: Ultimate AQ-C18. The detection results are shown in the figure. Figure 4 .

[0142] Depend on Figure 4 It can be seen that when the chromatographic column is Agilent 5 TC-C18(2) or GL Science InertSustain AQ-C18, the relative retention time of peak 4 and peak S exceeds the specified limit. Therefore, the chromatographic column Ultimate AQ-C18 is selected.

[0143] 4. Validation of the Feature Mapping Methodology

[0144] 4.1 Specificity Examination

[0145] In this validation, specificity experiments were conducted using reagent reference materials, reference standards, test solutions, and blank solutions. The results are as follows: Figure 5 As shown.

[0146] The results showed that the retention times of the control medicinal material and the test solution were consistent, and the reagent blank solution and the control material did not interfere with each other near the retention time of the target substance, indicating that the specificity met the requirements for method validation.

[0147] 4.2 Precision Test

[0148] Weigh approximately 0.2 g of lyophilized kiwi root standard decoction powder (batch number DGZS-PF-YL-202-210806), prepare it according to the test sample method, inject it 6 times consecutively and record the chromatogram. Using peak 3 as the reference peak, calculate the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Tables 1 and 2 below. The relative retention time of each characteristic peak is less than 2%, and the RSD of the relative peak area is less than 17%. The experiment shows that the precision of this method is good.

[0149] Table 1. Precision data (relative retention time) of standard kiwi root decoction

[0150]

[0151]

[0152] Table 2 Precision data (relative peak area) of kiwi root standard decoction

[0153]

[0154] 4.3 Repeatability Test

[0155] Weigh approximately 0.2 g of lyophilized kiwi root standard decoction powder (batch number DGZS-PF-YL-202-210806), prepare 6 parallel samples according to the test sample method, inject each sample, record the chromatogram and integrate, using peak 3 as the reference peak, calculate the relative retention time and relative peak area of ​​each characteristic peak, and the results are shown in Tables 3 and 4 below. The relative retention time of each characteristic peak is less than 1%, and the RSD of the relative peak area is less than 17%. The experiment shows that the repeatability of this method is good.

[0156] Table 3. Repeatability data of kiwi root standard decoction (relative retention time)

[0157]

[0158] Table 4. Repeatability data of kiwi root standard decoction (relative peak area)

[0159]

[0160] 4.4 Stability Test

[0161] Weigh approximately 0.2 g of lyophilized kiwi root standard decoction powder (batch number DGZS-PF-YL-202-210806), prepare it according to the test sample method, and inject it at different time points within 12 hours. Record the chromatogram and integrate it. Using peak 3 as the reference peak, calculate the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Tables 5 and 6 below. The relative retention time of each characteristic peak is less than 1%, and the RSD of the relative peak area is less than 15%. The experiment shows that the test sample solution has good stability within 12 hours.

[0162] Table 5. Stability data (relative retention time) of standard kiwi root decoction.

[0163]

[0164] Table 6. Stability data (relative peak area) of standard kiwi root decoction

[0165]

[0166] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective, and the above four characteristic peaks will be included in subsequent tests.

[0167] 5. Characteristic Peak Identification and Selection

[0168] Through the above series of optimization experiments, under the optimal chromatographic conditions, the test sample solution should exhibit four characteristic peaks in its chromatogram. Using the reference standard for localization, peak 3 was identified as catechin. Catechin has a relatively high peak area and is the main active ingredient; therefore, peak 3 (catechin) was selected as the S peak. The HPLC fingerprint chromatograms of 15 batches of kiwifruit root standard decoction were imported into the "Traditional Chinese Medicine Chromatographic Characteristic Chromatographic Similarity Evaluation System Software (2012 version)" to generate overlay diagrams and reference characteristic chromatograms. Four common peaks with good resolution, large peak area, and good peak shape were selected as characteristic peaks. Through comparison with the reference standard, peak 3 was identified as catechin. Catechin is the main active ingredient of kiwifruit root and has stable chemical properties. Its chromatographic peak has good resolution and a large peak area among all chromatographic peaks; therefore, peak 3 (catechin) was selected as the reference peak for the characteristic chromatogram. The relative retention times of peaks 1, 2, and 4 were calculated, and the results are shown in Table 7 below. The overlay diagram of the characteristic chromatograms of 15 batches of kiwifruit root standard decoction is shown below. Figure 6 The characteristic chromatogram of the standard decoction of kiwi root is shown in [reference needed]. Figure 7 The characteristic chromatogram of catechin reference standard is shown in [reference image]. Figure 8 See the characteristic atlas of the reference medicinal materials. Figure 9 The specified values ​​are 0.60 (peak 1), 0.89 (peak 2), and 1.38 (peak 4).

[0169] Table 7 shows the relative retention times of standard kiwi root decoctions.

[0170]

[0171] 6. Establishment of Feature Maps

[0172] The chromatogram of the test solution should show four characteristic peaks, one of which should have a retention time corresponding to the retention time of the corresponding reference peak. The peak corresponding to the catechin reference peak is called the S peak. Calculate the relative retention times of each characteristic peak and the S peak. The relative retention times should be within ±10% of the specified values, which are 0.60 (peak 1), 0.89 (peak 2), and 1.38 (peak 4).

[0173] Example 2: Establishment of UPLC method for determining the content of kiwi root standard decoction

[0174] Based on the method for constructing the UPLC characteristic spectrum of the kiwifruit root standard decoction in Example 1, Example 2 also conducted the following methodological investigation and established a method for determining the UPLC content of the kiwifruit root standard decoction, specifically including:

[0175] 1. Instruments and reagents

[0176] 1.1 Instruments

[0177] Agilent 1290 high performance liquid chromatograph (Agilent Technologies);

[0178] Column: ACQUITY UPLC BEH C18 (2.1×100mm, 1.7μm);

[0179] Other items are the same as "1.1 Instruments" under Example 1.

[0180] 1.2 Reagents

[0181] Same as "1.2 Reagents" under Example 1.

[0182] 1.3 Drug Test

[0183] Same as "1.3 Test Drug" under Example 1.

[0184] 2. Chromatographic conditions and system suitability test

[0185] 2.1 Proposed chromatographic conditions

[0186] The column was packed with octadecylsilane-bonded silica gel (2.1 × 100 mm, 1.7 μm); methanol was used as mobile phase A and 0.1% formic acid aqueous solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the column temperature was 30 °C; the flow rate was 0.2 mL per minute; and the detection wavelength was 278 nm. The theoretical plate number, calculated based on the catechin peak, should be no less than 2000.

[0187]

[0188] 2.2 Preparation of the test solution

[0189] Take about 0.1g of the lyophilized powder of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of 50% methanol, weigh it, sonicate it (500W, 40KHz) for 30 minutes, cool it, weigh it, make up the weight loss with 50% methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0190] 2.3 Preparation of reference solution

[0191] Take an appropriate amount of catechin reference standard, weigh it accurately, and add methanol to prepare a solution containing 100 μg per ml.

[0192] 2.4 Determination Method

[0193] Accurately pipette 1 μl of the reference solution and 2 μL of the test solution into the liquid chromatograph and determine the result.

[0194] 3. Chromatographic conditions investigation

[0195] 3.1 Selection of Analytical Components

[0196] Catechins were selected as the evaluation index to verify the chromatographic conditions and system adaptability for the determination of kiwi root decoction content, and the sample pretreatment method was investigated to determine the test sample preparation method.

[0197] 3.2 Wavelength Selection

[0198] The chromatograms of kiwifruit roots at different wavelengths were examined, and the maximum absorption wavelength of catechins, 278 nm, was ultimately selected as the detection wavelength for content determination. The results are as follows: Figure 10 As shown.

[0199] 3.3 Selection of Ultrasound Time

[0200] The effect of different ultrasonic times on the chromatogram of the standard decoction was investigated. 0.1g of the sample was added to 20ml of 50% methanol and ultrasonicated for 20 minutes, 30 minutes and 40 minutes respectively. The results showed that the catechin content was 1.32mg / g, 4.67mg / g and 3.52mg / g respectively. The catechin content was the highest when ultrasonicated for 30 minutes. Therefore, ultrasonic treatment for 30 minutes was finally selected.

[0201] Table 8. Catechin content of kiwi root standard decoction under different ultrasound times.

[0202]

[0203] 3.4 Selection of Solvent Amount

[0204] The effect of different solvent dosages on the catechin content in the standard decoction was investigated. Samples of 0.1g, 0.2g, and 0.4g were weighed and added to 20ml of 50% methanol, and sonicated for 30 minutes. The catechin contents were 4.67mg / g, 4.64mg / g, and 1.14mg / g, respectively. The highest content was found at 0.1g. Therefore, a sample weight of approximately 0.1g was selected and 20ml of 50% methanol was added.

[0205] Table 9. Catechin content of kiwi root standard decoction under different solvent dosages.

[0206]

[0207] 3.5 Gradient Selection

[0208] The effects of different elution gradients on the chromatograms of the standard decoction were investigated, as shown below:

[0209] Gradient 1

[0210]

[0211] Gradient 2

[0212]

[0213] Gradient 3

[0214]

[0215] The percentage sign in the table means volume percentage.

[0216] The results are as follows Figure 11 As shown: When the gradient method is 3, the peak shape is better, the separation is better, and the baseline is more stable.

[0217] 4. Validation of content determination method

[0218] 4.1 Specificity Examination

[0219] A standard decoction of kiwifruit root (batch number DGZS-PF-YL-202-210806) was used to prepare a test solution. Chromatographic analysis was performed under the conditions described above, and chromatograms of the kiwifruit root standard decoction, catechin reference standard, test blank (50% methanol), and its excipient (dextrin) were plotted. The results are shown in the figure below. In the chromatogram of the test sample, a peak with the same retention time as the reference standard was observed. No interference was observed from the blank and the excipient (dextrin). The experiment shows that this method has good specificity, as indicated by the results. Figure 12 As shown.

[0220] 4.2 Linear Relationship

[0221] Accurately weigh an appropriate amount of catechin reference standard, add methanol to prepare a solution containing 770.28 μg per ml, which serves as the stock solution of the reference standard. Dilute and determine the standard sequentially, record the chromatograms and integrate them. Using catechin as the reference peak, calculate the peak area. The results are shown in the table below. Plot a standard curve with concentration on the x-axis and peak area on the y-axis. The results are shown in the table below. Figure 13 The results, y = 3400.2x + 15278, r = 0.9999, indicate that catechins exhibit good linearity in the range of 15.41 μg / mL to 770.28 μg / mL.

[0222] Table 10. Catechin Concentration and Peak Area Data

[0223]

[0224] 4.3 Repeatability Test

[0225] Weigh approximately 0.1g of freeze-dried kiwi root standard decoction powder (batch number DGZS-PF-YL-202-210806), prepare 6 parallel samples according to the test sample method, determine the content according to the method, record the chromatograms and integrate them, calculate the catechin content, and the results are shown in the table below. The RSD values ​​of the contents are all less than 3%, and the experiment shows that the method has good repeatability.

[0226] Table 11 Repeatability data of standard decoction of kiwi root

[0227]

[0228] 4.4 Precision Test

[0229] Weigh approximately 0.1 g of lyophilized kiwi root standard decoction powder (batch number DGZS-PF-YL-202-210806), prepare the test solution, inject the sample 6 times consecutively, determine the sample according to the method, record the chromatogram and integrate the results. Using catechin as the reference peak, calculate the peak area. The results are shown in the table below. The RSD of the reference peak area is less than 1%, indicating that the method has good precision.

[0230] Table 12 Precision Peak Area Data of Kiwi Root Standard Decoction

[0231]

[0232] 4.5 Stability Test

[0233] Weigh approximately 0.1 g of lyophilized kiwi root standard decoction powder (batch number DGZS-PF-YL-202-210806), prepare the test solution, and inject the sample at different time points within 12 hours. Determine the peak area according to the method, record the chromatogram and integrate it, and calculate the peak area of ​​catechin. The results are shown in the table below. The reference peak area RSD is less than 1%, indicating that the peak area of ​​the test solution is basically stable within 12 hours.

[0234] Table 13 Stability Peak Area Data of Kiwi Root Standard Decoction

[0235]

[0236] 4.6 Intermediate Precision Test

[0237] Weigh approximately 0.1 g of lyophilized kiwi root standard decoction powder (batch number DGZS-PF-YL-202-210806), prepare 6 parallel samples according to the test sample method, determine according to the method, record the chromatograms and integrate, calculate the peak area and content of catechins. The results are shown in the table below. The RSD of the content with the repeatability determination results is within 6%, indicating that the intermediate precision of this method is good.

[0238] Table 14 Intermediate precision content data of standard kiwi root decoction

[0239]

[0240]

[0241] 4.7 Recovery Test

[0242] Six lyophilized portions of the kiwifruit root standard decoction powder (batch number DGZS-PF-YL-202-210806), each approximately 0.05 g, were accurately weighed. Appropriate amounts of catechin were added to each portion in parallel, and the solution was prepared according to the test sample preparation method. The determination was performed according to the prescribed method, and the chromatograms were recorded and integrated. The results are shown in the table below. The recovery rate was within the specified range of 90%–108%, and the RSD was less than 2%. The experiment shows that the spiked recovery rate of this method is good.

[0243] Table 15 Accuracy Data of Kiwi Root Standard Decoction

[0244]

[0245] 4.8 Durability

[0246] Weigh approximately 0.1 g of lyophilized kiwi root standard decoction powder (batch number DGZS-PF-YL-202-210806) and prepare a test solution. Determine the content according to the prescribed method, record the chromatogram, and integrate the results. Using catechin as a reference peak, calculate the content. The results are shown in Table 16 below. Under different chromatographic conditions, the RSD value of catechin content was less than 1%, indicating that this method has good robustness.

[0247] Table 16 Durability data of standard kiwi root decoction

[0248]

[0249] 5. Sample content determination

[0250] The content of 15 standard decoction batches was determined using the aforementioned method, and the results are shown in Table 17 below.

[0251] Table 17 Results of content determination in 15 batches of kiwifruit root standard decoction

[0252]

[0253]

[0254] Conclusion: (1) Based on the distribution of data from 15 batches of standard decoctions, the upper limit was the mean + 3SD, and the lower limit was the lowest value of the measured values. The catechin content of the standard decoctions was finally determined to be 1.5 mg / g to 6.9 mg / g; the transfer rate range was 15.6% to 48.5%.

[0255] (2) The content of 15 batches of standard decoctions met the requirements.

Claims

1. A method for determining the catechin content in a standard decoction of kiwi root, characterized in that, The determination was performed by high-performance liquid chromatography (HPLC), and the conditions for the HPLC method included: The chromatographic column was an ACQUITY UPLC BEH C18, with dimensions of 2.1 mm × 100 mm and a diameter of 1.7 µm. Methanol was used as mobile phase A, and formic acid aqueous solution with a volume percentage of 0.05~0.2% was used as mobile phase B for gradient elution. The conditions for gradient elution are as follows: The detection wavelength is 278nm; The solvent for both the test solution and the reference solution is a methanol-water solution with a volume percentage of 50-70%. The kiwi root mentioned is the dried root of the Chinese kiwifruit plant, which belongs to the Actinidiaceae family.

2. The content determination method according to claim 1, characterized in that, The conditions for the high-performance liquid chromatography method also include at least one of the following i-ⅳ: i. Flow rate: 0.2~0.4 mL / min; ii. Column temperature: 30~35℃; iii. Injection volume: 1~5μL; iv. The theoretical plate number, calculated based on the catechin peak, shall not be less than 2000.

3. The content determination method according to claim 1, characterized in that, The mass concentration of the test solution is 0.1~10 mg / mL.

4. The content determination method according to claim 1, characterized in that, The mass concentration of the reference solution is 0.05~0.5 mg / mL.

5. A method for constructing a characteristic spectrum of a standard decoction of kiwifruit root, characterized in that, The reference solution, reference medicinal material solution, and test solution were determined by high performance liquid chromatography, and the common peak was identified to obtain the final product. The conditions for the high-performance liquid chromatography method include: The chromatographic column was an Ultimate AQ-C18, with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm. Methanol was used as mobile phase A, and formic acid aqueous solution with a volume percentage of 0.05~0.2% was used as mobile phase B for gradient elution. The conditions for gradient elution are as follows: The detection wavelength is 280nm; The solvent for the test solution, reference solution, and reference medicinal material solution is a methanol aqueous solution with a volume percentage of 50-70%. The kiwi root mentioned is the dried root of the Chinese kiwifruit plant, which belongs to the Actinidiaceae family.

6. The construction method according to claim 5, characterized in that, The conditions for the high-performance liquid chromatography method also include at least one of the following i-ⅳ: i. Flow rate: 0.5~1.0 mL / min; ii. Column temperature: 25~35℃; iii. Injection volume: 5~20μL; iv. The theoretical plate number for catechin peak calculation should be no less than 2000.

7. The construction method according to claim 5, characterized in that, The mass concentration of the test solution is 0.1~10 mg / mL.

8. The construction method according to claim 5, characterized in that, The mass concentration of the reference solution is 0.05~0.5 mg / mL.

9. The construction method according to claim 5, characterized in that, The mass concentration of the reference medicinal material solution is 0.5~50 mg / mL.

10. The construction method according to any one of claims 5 to 9, characterized in that, The characteristic spectrum includes four characteristic peaks. Taking peak 3 (catechin) as the reference peak, the relative retention times of the other characteristic peaks and the reference peak are calculated. The relative retention times should be within ±10% of a specified value. The specified values ​​are: peak 1: 0.60, peak 2: 0.89, and peak 4: 1.

38.

11. The application of the content determination method according to any one of claims 1 to 4 or the construction method according to any one of claims 5 to 10 in detecting the quality of kiwifruit root standard decoction.

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