Quality detection method of uncaria antihypertensive prescription preparation

Abstract

The invention relates to a quality detection method of an uncaria antihypertensive prescription preparation, which belongs to the technical field of traditional Chinese medicine component detection, and comprises a specific chromatogram establishment method and / or a multi-component content determination method. According to the method, stable chromatographic conditions including elution gradient, column temperature, detection wavelength and the like are determined, and a specific chromatogram which is stable in relative retention time, good in chromatographic peak shape and good in separation degree is established by utilizing a high performance liquid chromatography technology; according to the method, a quantitative analysis of multi-components by single marker is adopted, puerarin is taken as an internal reference substance, content determination of the 11 components of the uncaria hypotensive prescription is rapidly and conveniently realized, and the method has the advantages of high detection efficiency and high accuracy. The quality detection method of the uncaria antihypertensive prescription provides a basis for quality control of the uncaria antihypertensive prescription.

Description

Technical Field

[0001] The present invention relates to the field of detection of traditional Chinese medicine components, and particularly to a quality detection method for a Gouteng Jiangya Fang preparation. Background Art

[0002] The Gouteng Jiangya Fang is composed of 9 herbs, namely Uncaria rhynchophylla, Cortex Moutan, Radix Scutellariae, Cortex Phellodendri, Rhizoma Coptidis, Pueraria lobata, Hirudo, Alisma orientale, and Ligustrum lucidum, and is a clinical experience formula of Professor Ding Shuwen, a famous cardiovascular disease expert in traditional Chinese medicine in Shandong Province. All the herbs used in this formula have heat-clearing effects to varying degrees. When combined with herbs for ascending yang, activating blood circulation, and dredging collaterals, it has been clinically proven to have significant curative effects on the treatment of hypertension of the hyperactive liver-yang type. At present, no quality standard for the formula granules of the Gouteng Jiangya Fang has been established. In order to better control the production quality, it is imperative to establish a quality detection method. Summary of the Invention

[0003] The present invention aims to make up for the deficiencies of the prior art and provides a quality detection method for a Gouteng Jiangya Fang preparation, solving the problems existing in the prior art.

[0004] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0005] A quality detection method for a Gouteng Jiangya Fang preparation, wherein the Gouteng Jiangya Fang preparation is Gouteng Jiangya Fang formula granules or freeze-dried powder of a standard decoction, and includes a method for establishing a characteristic chromatogram and / or a method for determining the contents of multiple components;

[0006] The method for establishing the characteristic chromatogram includes the following steps:

[0007] (1.1) Prepare a test solution, and prepare a mixed reference substance solution of 11 components including puerarin, 3'-methoxypuerarin, puerarin apiose glucoside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside, and benzoylpaeoniflorin at different concentrations;

[0008] (1.2) Determine the liquid chromatography conditions;

[0009] (1.3) According to the chromatographic conditions in step (1.2), collect the chromatograms of the mixed reference substance solution and multiple batches of test solutions, identify the common peaks in each test solution chromatogram, select 21 common peaks with stable peak emergence, good peak shape, and resolution as characteristic peaks, and compare with the reference substance chromatogram to identify 15 components;

[0010] The method for determining the contents of multiple components includes the following steps:

[0011] (2.1) Prepare the test solution and the mixed reference solution of 11 components including puerarin, 3'-methoxypuerarin, puerarin apiose glucoside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside and benzoylpaeoniflorin at different concentrations.

[0012] (2.2) Determine the liquid chromatography conditions.

[0013] (2.3) Determine the relative retention time and relative correction factor:

[0014] Use chromatographic columns of different brands and types, collect the chromatograms of the mixed reference solution and the test solution according to the chromatographic conditions in step (2.2), take puerarin as the internal reference substance, and calculate the relative retention times of 3'-methoxypuerarin, puerarin apiose glucoside, magnoflorine, paeoniflorin, and daidzin respectively; take berberine as the internal reference substance, and calculate the relative retention times of baicalin, palmatine, wogonoside and benzoylpaeoniflorin respectively.

[0015] According to the chromatographic conditions in step (2.2), collect the chromatograms of the mixed reference solution at different concentrations, take puerarin as the internal reference substance, and calculate the relative correction factors of 3'-methoxypuerarin, puerarin apiose glucoside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside and benzoylpaeoniflorin respectively.

[0016] (2.4) Determination:

[0017] According to the chromatographic conditions in step (2.2), collect the chromatograms of the mixed reference solution and the test solution. With the concentrations and chromatographic peak areas of puerarin and berberine reference substances as references, locate the chromatographic peaks of each component to be measured according to the relative retention time determined in step (2.3), and calculate the contents of 3'-methoxypuerarin, puerarin apiose glucoside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside and benzoylpaeoniflorin according to the relative correction factor determined in step (2.3).

[0018] Preferably, the liquid chromatography conditions in steps (1.2) and (2.2) are as follows: Use a C 18 chromatographic column, use acetonitrile as mobile phase A, use 0.2% phosphoric acid solution as mobile phase B, and the gradient elution conditions are: 0 - 20 min, 5% - 13% (A); 20 - 40 min, 13% - 16% (A); 40 - 50 min, 16% - 25% (A); 50 - 83 min, 25% - 37% (A); the flow rate is 1.0 mL·min -1 , the column temperature is 30 °C, the wavelength is 240 nm, and the injection volume is 10 μL.

[0019] Preferably, in step (1.3), the characteristic chromatogram of the Gouteng Jiangya Fang preparation contains 21 common characteristic peaks. The relative retention times of peaks 1-5, 7-13 with respect to peak 6, and peaks 14-18, 20-21 with respect to peak 19 are 0.14, 0.28, 0.57, 0.65, 0.79, 1.07, 1.11, 1.12, 1.18, 1.22, 1.28, 1.33, 2.00, 0.92, 0.94, 0.97, 0.98, 1.06, 1.11, respectively.

[0020] Preferably, in the characteristic chromatogram of the Gouteng Jiangya Fang preparation in step (1.3), the 2nd, 3rd, 5th, 6th, 7th, 8th, 9th, 10th, 13th, 15th, 16th, 18th, 19th, 20th, and 21st characteristic peaks are gallic acid, salidroside, 3'-hydroxy puerarin, puerarin, 3'-methoxy puerarin, puerarin apioside, magnoflorine, paeoniflorin, daidzin, baicalin, ligustroside G13, palmatine, berberine, wogonoside, benzoylpaeoniflorin, respectively.

[0021] Preferably, in steps (1.1) and (2.1), the preparation method of the mixed reference substance solutions with different concentrations is as follows: Weigh appropriate amounts of puerarin, 3'-methoxy puerarin, puerarin apioside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside, benzoylpaeoniflorin, and prepare single reference substance solutions with certain concentrations in methanol solutions respectively;

[0022] Take appropriate amounts of each single reference substance solution, add methanol to prepare a mixed reference substance solution containing 119.065, 46.821, 34.458, 12.659, 56.257, 36.653, 372.296, 41.587, 215.605, 459.015, 17.150 μg of reference substances per 1 mL respectively, and then obtain mixed reference substance solutions with different concentrations by the multiple dilution method.

[0023] Preferably, in steps (1.1) and (2.1), the preparation method of the test solution is as follows: Take an appropriate amount of the Gouteng Jiangya Fang preparation, grind it finely, take about 0.15 g, weigh it precisely, place it in a stoppered conical flask, precisely add 25 mL of 70% methanol, reflux for 30 min, cool, weigh, supplement the lost mass with 70% methanol, filter, and take the subsequent filtrate to obtain the solution.

[0024] Preferably, in step (2.3), the relative retention times of puerarin, 3'-methoxy puerarin, puerarin apioside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside, and benzoylpaeoniflorin are 1.00, 1.07, 1.11, 1.12, 1.18, 1.32, 1.00, 0.92, 0.98, 1.06, 1.11, respectively.

[0025] Preferably, in step (2.3), the relative correction factors of 3'-methoxypuerarin, puerarin apioglucoside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside, and benzoylpaeoniflorin are 1.75, 1.46, 1.81, 4.28, 1.39, 3.25, 1.24, 1.08, 4.08, and 3.22, respectively.

[0026] The advantages of the present invention with the above structure are as follows:

[0027] 1. For the quality detection method of the Uncaria rhynchophylla antihypertensive formula preparation provided by the present invention, a characteristic chromatogram is established by high performance liquid technology, and the content determination of 11 components in the Uncaria rhynchophylla antihypertensive formula is realized by the multiple components by single marker method with puerarin and berberine as internal references. Specifically, in the process of establishing the chromatogram and performing the content determination, stable chromatographic conditions including elution gradient, column temperature, detection wavelength, etc. are determined, so that the detection results of the components to be measured are stable on different instruments and chromatographic columns, the relative retention time of the chromatographic peaks is stable, the chromatographic peaks have good shapes and good separation degrees; and the detection efficiency and accuracy are relatively high, providing a basis for the quality control of the Uncaria rhynchophylla antihypertensive formula.

[0028] When the detection and analysis time is too long, in a complex sample matrix, other substances may produce peaks similar to the target peak, resulting in misjudgment. The present invention selects two reference peaks, which can effectively improve the accuracy of peak positioning.

[0029] 2. The present invention can quickly detect the contents of puerarin, puerarin apioglucoside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside, and benzoylpaeoniflorin in the Uncaria rhynchophylla antihypertensive formula preparation, providing an experimental basis for the quality control and clinical application rationality of the Uncaria rhynchophylla antihypertensive formula granules, providing a research direction for process optimization, and also providing a reference for the scientific development of the formula granules. Description of the Drawings

[0030] Figure 1 is the characteristic chromatogram of the Uncaria rhynchophylla antihypertensive formula of the present invention;

[0031] Figure 2 is the characteristic chromatogram of the present invention at different wavelengths;

[0032] Figure 3 is the characteristic chromatogram of the present invention at different concentrations of mobile phase B and different column temperatures;

[0033] Figure 4 is the characteristic chromatogram of the traditional decoction freeze-dried powder and formula granules of the Uncaria rhynchophylla antihypertensive formula of multiple batches of the present invention. Detailed Embodiments

[0034] The present invention will be further described in detail hereinafter. It should be noted, however, that the following specific embodiments are only exemplary examples of the specific operations of the present invention, but the protection scope of the present invention is not limited thereto. The protection scope of the present invention is only defined by the claims. Those skilled in the art can obviously think that various other improvements and substitutions can be made to the embodiments described in the present invention within the protection scope defined by the claims of the present invention, and still be able to achieve the same technical effects and reach the ultimate technical purpose of the present invention.

[0035] Unless otherwise specified, all raw materials in this specification are purchased through commercial channels.

[0036] 1. Instruments and Materials

[0037] 1.1 Instruments

[0038] Agilent 1260 DAD / VWD high performance liquid chromatograph (purchased from Agilent Corporation, USA), TS8606 freeze dryer (purchased from Fevik Corporation, Germany), BSA224S-CW 1 / 10,000 electronic balance (purchased from Sartorius Group, Germany), QY-300 high-speed reciprocating cutting machine for medicine (purchased from Kanghua Pharmaceutical Machinery Co., Ltd.), XS105 1 / 100,000 electronic balance (purchased from Mettler Toledo Group), precision forced air drying oven (purchased from Shanghai Yiheng Scientific Instruments Co., Ltd.), TLCX-600 ultrasonic sample processor (purchased from Jining Tianzhilan Biotechnology Co., Ltd.), YRE2000A rotary evaporator (purchased from Yuhua Instruments Co., Ltd., Gongyi City).

[0039] 1.2 Materials

[0040] Gallic acid, puerarin, paeoniflorin, and ligustroside G13 (batch numbers 130011-202112, 070018-202106, 190001-202205, 140086-202311, with purities all ≥98%) were purchased from Shanghai Hongyong Biotechnology Co., Ltd.; salidroside, magnoflorine, daidzin, baicalin, palmatine, berberine, and wogonoside (batch numbers 110818-202210, 112090-202201, 111739-202305, 110715-202223, 110713-201913, 110713-202316, 112002-202303, with purities 99.7%, ≥98%, ≥98%, 97.20%, ≥98%, ≥98%, 98.50% respectively) were purchased from the National Institutes for Food and Drug Control; 3'-hydroxy puerarin and puerarin apiin (batch numbers Q390877 and Q404487, with purities 99.52% and 98.37% respectively) were purchased from Shandong Waldson Biotechnology Co., Ltd.; 3'-methoxy puerarin (batch number CFS202202, with purity ≥98%) was purchased from Wuhan Tianzhi Biotechnology Co., Ltd.; benzoyl paeoniflorin (batch number CFS202301, with purity ≥98%) was purchased from Suzhou Yani Biotechnology Co., Ltd.

[0041] Water was ultrapure water (purchased from Guangzhou Watson Food and Beverage Co., Ltd.), and acetonitrile (purchased from Yuexu Technology (Shanghai) Co., Ltd.), methanol (purchased from Tianjin Fuyu Fine Chemical Co., Ltd.), and phosphoric acid (purchased from Tianjin Kemiou Chemical Reagent Co., Ltd.) were of chromatographic purity.

[0042] The 9 batches of formula granules of Uncaria rhynchophylla, Cortex Moutan, Scutellaria baicalensis, Phellodendron amurense, Coptis chinensis, Pueraria lobata, Alisma orientale, and Ligustrum lucidum all met the national formula granule standards, with specifications of 1 g of formula granules equivalent to 8.0, 2.0, 4.5, 2.2, 5.0, 4.0, 3.3, 2.5 g of the corresponding crude drugs respectively. The 5 batches of formula granules of Hirudo all met the Shandong formula granule standards, with a specification of 1 g of formula granules equivalent to 4.0 g of the crude drug. Multiple batches of single-herb formula granules were randomly combined to prepare the formula granules of Gouteng Jiangya Decoction (S16-S24). The formula granule information and combination details are shown in Table 1.

[0043] Table 1 Formula granule information and combination details

[0044]

[0045] Note: A—Shandong Hongjitang Pharmaceutical Group Co., Ltd.; B—Shandong Yifang Pharmaceutical Co., Ltd.; C—Anhui Xiehecheng Pharmaceutical Co., Ltd.; D—Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0046] 2. Determination method

[0047] 2.1 Sample preparation

[0048] 2.1.1 Preparation of freeze-dried powder and formula granules of traditional decoction of Gouteng Jiangya Prescription

[0049] Weigh a total of 100.5 g of cut crude drugs at 1-fold clinical dose (Uncaria rhynchophylla, Cortex Moutan, Scutellaria baicalensis, Phellodendron amurense, Coptis chinensis, Pueraria lobata, Hirudo, Alisma orientale, Ligustrum lucidum are 30, 15, 9, 9, 9, 9, 9, 9, 1.5 g respectively), put them into a casserole, add 9 times the amount of water, soak for 30 min to fully moisten the cut crude drugs, bring to a boil over high heat and then decoct over low heat for 30 min, filter while it is hot (200 mesh), add 7 times the amount of water to the residue, bring to a boil over high heat and then decoct over low heat for 20 min, filter (200 mesh), combine the two filtrates, concentrate at 60 °C, and freeze-dry to obtain the freeze-dried powder of the traditional decoction of Gouteng Jiangya Prescription (numbered S1 - S15).

[0050] According to the dosages of each medicinal material in Gouteng Jiangya Prescription and the specifications of single-flavor formula granules, the amounts of Uncaria rhynchophylla, Cortex Moutan, Coptis chinensis, Scutellaria baicalensis, Phellodendron amurense, Alisma orientale, Ligustrum lucidum, Pueraria lobata, Hirudo formula granules required to prepare 1 equivalent dose of Gouteng Jiangya Prescription formula granules are 3.7500, 7.5000, 2.0000, 4.0909, 1.8000, 2.2500, 2.7273, 3.6000, 0.3750 g respectively. Weigh accurately the above-mentioned single-flavor formula granules of the above quality to prepare Gouteng Jiangya Prescription formula granules (numbered S16 - S24).

[0051] 2.1.2 Preparation of mixed reference substance solution

[0052] Weigh accurately appropriate amounts of puerarin, 3'-methoxypuerarin, puerarin apiose glucoside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside and benzoylpaeoniflorin reference substances respectively, dissolve them with methanol, and prepare single-reference substance solutions with mass concentrations of 0.618, 1.352, 0.669, 0.644, 0.837, 0.890, 0.458, 1.385, 1.350, 0.644, 0.954 mg·mL -1 as the mother liquor of the reference substance solution.

[0053] Accurately measure appropriate amounts of the mother liquor of puerarin, 3'-methoxypuerarin, puerarin apiose glucoside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside and benzoylpaeoniflorin reference substance solutions, add methanol to prepare mixed reference substance solutions with mass concentrations of 119.065, 46.821, 34.458, 12.659, 56.257, 36.653, 372.296, 41.587, 215.605, 459.015, 17.150 μg·mL -1 respectively.

[0054] 2.1.3 Preparation of Test Solution

[0055] 2.1.3.1 Selection of Extraction Method

[0056] Take about 0.1 g of the Gouteng Jiangya Fang preparation, and carry out heating under reflux and ultrasonic treatment respectively. Measure the peak areas of each labeled peak. The results show (see Table 2) that compared with the ultrasonic extraction method, the peak areas of each peak obtained by the reflux extraction method are larger, and the compounds in the sample can be extracted more effectively. Therefore, the reflux extraction method is selected.

[0057] Table 2 Results of Investigation on Extraction Methods (Peak Areas)

[0058]

[0059] 2.1.3.2 Selection of Extraction Time

[0060] Set the reflux times to 15 min, 30 min, and 45 min respectively, and investigate the peak areas of each labeled peak. The results show (see Table 3) that the peak area of the chromatogram obtained by ultrasonic treatment for 15 min is slightly smaller, and the substance information presented in the characteristic chromatograms obtained by 30 min and 45 min is basically the same. Therefore, the ultrasonic time is selected as 30 min.

[0061] Table 3 Results of Investigation on Extraction Time (Peak Areas)

[0062]

[0063] 2.1.3.3 Selection of Extraction Solvent

[0064] Investigate the peak areas of each labeled peak when the extraction solvents are 10% methanol, 30% methanol, 50% methanol, 70% methanol, and pure methanol respectively. The results show (see Table 4) that the peak area of the labeled peak extracted by 70% methanol is larger. Finally, the extraction solvent is selected as 70% methanol.

[0065] Table 4 Results of Investigation on Extraction Solvent (Peak Areas)

[0066]

[0067] 2.1.3.4 Selection of Sampling Amount

[0068] Investigate the peak areas of each labeled peak when the sampling amounts are 0.05, 0.10, 0.15, and 0.20 g respectively. The results show (see Table 5) that after eliminating the difference in sampling amount, the peak area of the labeled peak at 0.15 g is larger. Finally, the sampling amount is selected as 0.15 g.

[0069] Table 5 Results of Investigation on Sampling Amount (Peak Areas)

[0070]

[0071]

[0072] In summary, take an appropriate amount of the Gouteng Jiangya Fang preparation, grind it finely, take about 0.15 g, weigh it precisely, place it in a stoppered conical flask, precisely add 25 mL of 70% methanol, reflux for 30 min, cool, weigh, supplement the lost mass with 70% methanol, filter, and take the subsequent filtrate to obtain the solution.

[0073] 2.1.4 Preparation of Negative Samples

[0074] The amounts of the formula granules of Uncaria rhynchophylla, Alisma orientale, Ligustrum lucidum, and Hirudo needed are 3.7500, 2.2500, 2.7273, and 0.3750 g respectively. Precisely weigh the above-mentioned amounts of single-flavor formula granules to prepare negative samples.

[0075] 2.2 Determination of Chromatographic Conditions

[0076] Use a YMC-Triart C 18 chromatographic column (4.6×250 mm, 5 μm, SN: 127XB20269), with acetonitrile as mobile phase A and 0.2% phosphoric acid solution as mobile phase B, gradient elution: 0 - 20 min, 5% - 13% (A); 20 - 40 min, 13% - 16% (A); 40 - 50 min, 16% - 25% (A); 50 - 83 min, 25% - 37% (A), flow rate 1.0 mL·min -1 , column temperature 30 °C, wavelength 240 nm, injection volume 10 μL.

[0077] 2.2.1 Selection of Detection Wavelength

[0078] Prepare the test solution according to the method in 2.1.3. Precisely weigh 1 μL of each of 8 portions of the test solution and inject them into the ultra-high performance liquid chromatograph to detect the chromatograms at wavelengths of 192 nm, 210 nm, 240 nm, 260 nm, 280 nm, 300 nm, 320 nm, and 360 nm respectively. The results are shown in Figure 2 . Based on the principles such as the number of chromatographic peaks, the response value of chromatographic peaks, and the stability of the baseline, finally select 240 nm as the detection wavelength for the Gouteng Jiangya Fang preparation.

[0079] 2.2.1 Selection of Mobile Phase

[0080] Prepare the test solution according to the method in 2.1.3. Precisely weigh 1 μL of each of 2 portions of the test solution and inject them into the ultra-high performance liquid chromatograph to detect the chromatograms using 0.2% phosphoric acid solution and 0.1% phosphoric acid solution as mobile phase B respectively. The results are shown in Figure 3According to the principles such as the number of chromatographic peaks, the response value of chromatographic peaks, and whether the baseline is stable, 0.2% phosphoric acid solution was finally selected as mobile phase B for the Gouteng Jiangya Fang preparation.

[0081] 2.2.1 Selection of column temperature

[0082] Prepare the test solution according to the method in 2.1.3. Precisely weigh 1 μL of each of the 3 portions of the test solution and inject it into the ultra-high performance liquid chromatograph to detect the chromatograms at column temperatures of 28 °C, 30 °C, and 32 °C respectively. The results are shown in Figure 3 According to the principles such as the number of chromatographic peaks, the response value of chromatographic peaks, and whether the baseline is stable, 30 °C was finally selected as the column temperature for the Gouteng Jiangya Fang preparation.

[0083] 2.3 HPLC characteristic fingerprints of the standard decoction and formula granules of Gouteng Jiangya Fang

[0084] 2.3.1 Method for establishing characteristic fingerprints:

[0085] Prepare the test solutions from the freeze-dried powders of 15 batches of the standard decoction of Gouteng Jiangya Fang and the formula granules of 9 batches of Gouteng Jiangya Fang, and determine them according to the chromatographic conditions in 2.2. Import the chromatograms into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints", as shown in Figure 4 .

[0086] Taking the characteristic fingerprint of the S1 batch of the standard decoction of Gouteng Jiangya Fang as a reference, identify the common peaks in the characteristic fingerprints of 15 batches of the standard decoction of Gouteng Jiangya Fang and 9 batches of formula granules. Select 21 common peaks with stable peak emergence, good peak shape and resolution as characteristic peaks. After comparing with the chromatogram of the reference substance, 15 components were identified, namely gallic acid (peak 2), salidroside (peak 3), 3'-hydroxy puerarin (peak 5), puerarin (peak 6), 3'-methoxy puerarin (peak 7), puerarin apiose glycoside (peak 8), magnoflorine (peak 9), paeoniflorin (peak 10), daidzin (peak 13), baicalin (peak 15), ligustroside G13 (peak 16), palmatine (peak 18), berberine (peak 19), wogonoside (peak 20), benzoylpaeoniflorin (peak 21).

[0087] Perform multi-point calibration on the characteristic fingerprints of 15 batches of the standard decoction of Gouteng Jiangya Fang and conduct Mark matching for the common peaks to generate a reference characteristic fingerprint (see Figure 1 ). Compare the similarity between the chromatograms of 15 batches of the standard decoction of Gouteng Jiangya Fang and the reference characteristic fingerprint. The similarity > 0.90 indicates that the different batches of the standard decoction of Gouteng Jiangya Fang prepared have a high similarity.

[0088] Taking peak 6 (S1) and peak 19 (S2) as reference peaks, the relative retention times RSD of each common peak were calculated to be 0.0% - 2.00%. The relative retention times of the 21 characteristic peaks had small differences and were all within the range of the average value ±2%. The average value of the relative retention time was selected as the specified value. The specified values are: 0.14 (peak 1), 0.28 (peak 2), 0.57 (peak 3), 0.65 (peak 4), 0.79 (peak 5), 1.07 (peak 7), 1.11 (peak 8), 1.12 (peak 9), 1.18 (peak 10), 1.22 (peak 11), 1.28 (peak 12), 1.33 (peak 13), 2.00 (peak 14), 0.92 (peak 15), 0.94 (peak 16), 0.97 (peak 17), 0.98 (peak 18), 1.06 (peak 20), 1.11 (peak 21), and the allowable error is ±10%.

[0089] Table 6 Results of relative retention time

[0090]

[0091] The average value of the total peak areas of the characteristic peaks of 15 batches of traditional decoctions was set as 1 (100%). The relative values of the total peak areas of the characteristic peaks of 9 batches of Uncaria rhynchophylla formula granules for lowering blood pressure were calculated with respect to it, which were 1.05, 1.08, 1.02, 1.06, 1.20, 1.07, 1.10, 1.07, 1.06 respectively. Similarly, the relative values of the total peak areas of all peaks in the characteristic chromatograms of 9 batches of formula granules with respect to the average value of the total peak areas of all peaks in the traditional decoction were calculated to be 1.08, 1.07, 1.01,

[0092] 1.04, 1.24, 1.05, 1.10, 1.08, 1.06. The above data indicate that the characteristic peaks and the total peak areas of all peaks of Uncaria rhynchophylla formula granules for lowering blood pressure are basically consistent with those of the traditional decoction.

[0093] 2.3.2 Methodology investigation

[0094] 2.3.2.1 Repeatability investigation

[0095] Take the Uncaria rhynchophylla formula granules for lowering blood pressure under item "2.1.1" (No.: S1), prepare 6 portions of test samples in parallel according to item "2.1.3", detect them under the chromatographic conditions in item "2.2", and record the chromatographic data. Calculate the relative standard deviations (RSD) of the relative peak areas and relative retention times of peaks 1 - 5, 7 - 13 with respect to peak 6 (S1 peak), and peaks 14 - 18, 20 - 21 with respect to peak 19 (S2 peak). The results show (see Tables 7 and 8) that the RSD of the relative peak areas of each common peak is <2.83%, and the RSD of the relative retention times is <1.73%, indicating that the method has good repeatability.

[0096] Table 7 Results of repeatability investigation (relative peak area)

[0097]

[0098]

[0099] Table 8 Results of Repeatability Test (Relative Retention Time)

[0100]

[0101] 2.3.2.2 Stability Test

[0102] Take the formula granules under item "2.1.1" and prepare samples according to item "2.1.3". Detect them under the chromatographic conditions in item "2.2" at 0, 2, 4, 8, 12, and 24 h respectively, and record the chromatographic data. The results show (see Tables 9 and 10) that the RSDs of the relative peak areas of each common peak are all < 2.25%, and the RSDs of the relative retention times are all < 0.69%, indicating that the test solution has good stability within 24 h.

[0103] Table 9 Results of Stability Test (Relative Peak Area)

[0104]

[0105] Table 10 Results of Stability Test (Relative Retention Time)

[0106]

[0107] 2.3.2.3 Intermediate Precision Test

[0108] Different experimenters used another high-performance liquid chromatograph to conduct the test again according to item "2.3.2.1", and record the chromatographic data. Analysis of the results of the two tests shows (see Tables 11 and 12) that the RSDs of the relative peak areas of each common peak are all < 2.84%, and the RSDs of the relative retention times are all < 1.83%, indicating that the method has good intermediate precision.

[0109] Table 11 Results of Stability Test (Relative Peak Area)

[0110]

[0111] Table 12 Results of Stability Test (Relative Retention Time)

[0112]

[0113]

[0114] 2.4 Determination of Multi-component Contents in Standard Decoction and Formula Granules of Gouteng Jiangya Prescription

[0115] 2.4.1 Determination Method for Multi-component Content

[0116] 2.4.1.1 Obtaining Relative Retention Time

[0117] Using 3 instruments and 4 chromatographic columns of different brands and types, collect the chromatograms of the mixed reference substances according to the chromatographic conditions in 2.2. Locate the chromatographic peaks through the retention time of the reference substances to obtain the actual retention time of each characteristic peak. Calculate the average value of the actual retention times as the standard retention time tSRT. Using puerarin and berberine as internal reference substances, calculate the relative retention times of each target component, which are 1.07, 1.11, 1.12, 1.18, 1.32, 0.92, 0.98, 1.06, 1.11 respectively.

[0118] 2.4.1.2 Obtaining Relative Correction Factor (f)

[0119] Inject and analyze the mixed reference substance solutions at 5 different concentrations under item "2.1.2", and detect according to the chromatographic conditions under item "2.2". Using puerarin as the internal reference substance, according to the formula (A s is the internal standard peak area, C s is the internal standard concentration; A i is the peak area of the analyte, C i is the concentration of the analyte) calculate the f of 10 target components, and the results are shown in Table 13.

[0120] Table 13 Relative Correction Factors of Each Target Component under Mixed Reference Substance Solutions at Different Concentrations

[0121]

[0122] 2.4.1.3 Determination

[0123] According to the chromatographic conditions in 2.2, collect the chromatograms of the mixed reference substance solution and the test solution. Taking the concentration and chromatographic peak area of the puerarin reference substance solution as a reference, locate the chromatographic peaks of each analyte according to the relative retention time determined in step (2.3), and calculate the contents of 3'-methoxypuerarin, puerarin apioside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside, benzoylpaeoniflorin according to the relative correction factor determined in 2.4.1.2.

[0124] 2.4.2 Methodology Investigation

[0125] 2.4.2.1 Specificity Investigation

[0126] Take the formula granules of the Gouteng Jiangya Prescription under item "2.1.1" and the negative samples under item "2.1.4", prepare the test solution and the negative sample solution respectively according to item "2.1.3", and detect according to the chromatographic method under item "2.2". The results show that there is no interference from the negative samples and the method has good specificity.

[0127] 2.4.2.2 Investigation of linear relationship

[0128] Precisely pipette the mixed reference substance solution under item "2.1.2" and use the multiple dilution method to dilute it by 1.25, 2.5, 5, and 10 times respectively. Detect the 5 mixed reference substance solutions with different concentrations according to the chromatographic method under item "2.2", and record the peak areas of 11 marker components such as puerarin. Using the mass concentration (C, μg·mL -1 ) of the component to be measured as the abscissa and the peak area (A) as the ordinate for linear regression. The results show that the linear relationship of each marker component is good within a certain concentration range. The regression equations and linear ranges are shown in Table 14.

[0129] Table 14 Regression equations and linear ranges

[0130]

[0131] 2.4.2.3 Repeatability investigation

[0132] Take the formula granules of the Gouteng Jiangya Prescription under item "2.1.1" and prepare 6 test solutions in parallel according to item "2.1.3", and detect according to the chromatographic conditions under item "2.2". The results show (see Table 15) that the RSD of the contents of 11 marker components ≤ 2.10%, indicating that the method has good repeatability.

[0133] Table 15 Results of repeatability investigation (mg·g -1 )

[0134]

[0135] 2.4.2.4 Stability investigation

[0136] Detect the test solution of the formula granules of the same Gouteng Jiangya Prescription at 0, 2, 4, 8, 12, and 24 h after preparation according to the chromatographic conditions under item "2.2". The results show (see Table 16) that the RSD of the contents of 11 marker components ≤ 1.19%, indicating that the test sample has good stability within 24 h.

[0137] Table 16 Results of stability investigation (mg·g -1 )

[0138]

[0139] 2.4.2.5 Intermediate precision investigation

[0140] Another experimenter used another high performance liquid chromatograph to conduct the test again according to the method under item "2.4.1.2", and recorded the chromatographic data. Analyze the results of the two tests (see Table 17). The RSD of the contents of 11 index components is ≤2.13%, indicating that this method has good intermediate precision.

[0141] Table 17 Results of intermediate precision investigation (mg·g -1 )

[0142]

[0143]

[0144] 2.4.2.6 Spiked recovery rate

[0145] Take 0.75 g of the formulated granules under item "2.1.1", add a reference substance solution equivalent to the content of the sample, prepare 6 portions of the test solution in parallel according to item "2.1.3", and detect according to the chromatographic conditions under item "2.2". The results show (see Table 18) that the average spiked recovery rates (RSD) of puerarin, 3'-methoxypuerarin, puerarin apiose glucoside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside, and benzoylpaeoniflorin are 97.27% (0.43%), 101.54% (0.72%), 102.00% (0.68%), 100.99% (0.67%), 100.81% (1.01%), 99.86% (0.88%), 99.45% (0.36%), 101.64% (0.90%), 100.84% (0.30%), 99.29% (0.77%), and 100.83% (1.11%) respectively, indicating that this method has good accuracy.

[0146] Table 18 Results of spiked recovery rate

[0147]

[0148]

[0149] 2.4.2.7 Durability investigation of relative correction factor f

[0150] Investigate the influence of different liquid chromatographs and chromatographic columns on f of each compound. The RSD of f is ≤2.75% (Table 19), indicating that different instruments and chromatographic columns have no significant influence on f of each component to be measured. Investigate the influence of different mobile phase volume flow rates and column temperatures on f of each compound. The RSD of f is ≤2.53% (Table 20), indicating that different mobile phase volume flow rates and column temperatures have no significant influence on f of each component to be measured.

[0151] Table 19 Relative correction factors of each component under different instruments and chromatographic column conditions

[0152]

[0153] Table 20 Relative correction factors of each component under different mobile phase volume flow rates and column temperatures

[0154]

[0155] 2.4.2.8 Chromatographic peak localization of target components

[0156] Calculate the relative retention times of each target component with respect to puerarin and berberine, and investigate the influence of different liquid chromatographs and chromatographic columns on the relative retention times of the chromatographic peaks of each target component. The results show (Table 21) that the relative retention time RSD < 1.38%. It indicates that these instruments and chromatographic columns can be used for characteristic peak localization.

[0157] Table 21 Relative retention times of each component under different instruments and chromatographic column conditions

[0158]

[0159]

[0160] 2.4.3 Comparative study on content determination between QAMS and external standard method (ESM)

[0161] Use QAMS and external standard method (ESM) to calculate the contents of each component in the sample respectively, calculate their relative deviation (RD / %), and use SPSS 24.0 software to conduct t-test analysis on the data calculated by the two methods. The results show that RD < 3% and P > 0.05, indicating that the QAMS method has good accuracy. The data are shown in Tables 22 - 23.

[0162] Table 22 Comparison of the contents of 6 components determined by ESM and QAMS (mg·g -1 )

[0163]

[0164] Table 23 Comparison of the contents of the remaining 5 components determined by ESM and QAMS (mg·g -1 )

[0165]

[0166] The above specific embodiments cannot be used to limit the protection scope of the present invention. For those skilled in the art of the present invention, any alternative improvement or transformation made to the embodiments of the present invention falls within the protection scope of the present invention. Where the present invention is not described in detail, it is all well-known technology in the technical field of the present invention.

Claims

1. A quality inspection method for a Uncaria rhynchophylla antihypertensive prescription preparation, wherein the Uncaria rhynchophylla antihypertensive prescription preparation is Uncaria rhynchophylla antihypertensive prescription granules or standard decoction freeze-dried powder, characterized in that: Including methods for establishing characteristic profiles and / or methods for determining the contents of multiple components; The method for establishing the characteristic spectrum comprises the following steps: (1.1) Prepare test sample solutions and mixed reference sample solutions of 11 ingredients, including puerarin, 3'-methoxypuerarin, puerarin apiglycoside, magnolamine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin, and benzoylpeoniflorin, at different concentrations; (1.2) Determine the liquid chromatography conditions; (1.3) According to the chromatographic conditions in step (1.2), collect chromatograms of the mixed reference solution and multiple batches of test solution, identify the common peaks of each test solution chromatogram, select 21 common peaks with stable peaks, good peak shape and separation as characteristic peaks, compare with the reference solution chromatogram, and identify 15 components; The multi-component content determination method comprises the following steps: (2.1) Prepare test sample solutions and mixed reference sample solutions of 11 ingredients, including puerarin, 3'-methoxypuerarin, puerarin apiglycoside, magnolamine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin, and benzoylpeoniflorin, at different concentrations; (2.2) Determine the liquid chromatography conditions; (2.3) Determine the relative retention time and relative correction factor: Using different instruments, different brands and types of chromatographic columns, collect chromatograms of the mixed reference solution and the test solution according to the chromatographic conditions in step (2.2), use puerarin as the internal reference, calculate the relative retention times of 3'-methoxypuerarin, puerarin apiglycoside, magnoliavine, paeoniflorin, and daidzein, respectively, use berberine as the internal reference, calculate the relative retention times of baicalin, bamipine, wogonin, and benzoylpeoniflorin, respectively; According to the chromatographic conditions in step (2.2), the chromatograms of the mixed reference solution of different concentrations were collected, and puerarin was used as the internal reference to calculate the relative correction factors of 3'-methoxypuerarin, puerarin apiglycoside, magnolamine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin and benzoylpeoniflorin respectively; (2.4) Determination: According to the chromatographic conditions in step (2.2), the chromatograms of the mixed reference solution and the test solution are collected. The concentrations and peak areas of the chromatographic peaks of the puerarin and berberine reference substances are used as references. The chromatographic peaks of the components to be tested are located according to the relative retention times determined in step (2.3). The contents of 3'-methoxypuerarin, puerarin apiglycoside, magnoliavine, paeoniflorin, daidzein, baicalin, bamipine, berberine, wogonin, and benzoylpeoniflorin are calculated according to the relative correction factors determined in step (2.3).

2. The quality detection method of the Uncaria rhynchophylla antihypertensive prescription according to claim 1, characterized in that: The liquid chromatography conditions in steps (1.2) and (2.2) are: using C 18 Chromatographic column, acetonitrile as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution conditions: 0-20min, 5%-13% (A); 20-40min, 13%-16% (A); 40-50min, 16%-25% (A); 50-83min, 25%-37% (A); flow rate 1.0mL·min -1 , column temperature 30°C, wavelength 240nm, injection volume 10μL.

3. The quality detection method of the Uncaria rhynchophylla antihypertensive prescription according to claim 1, characterized in that: In step (1.3), the characteristic spectrum of the Uncaria rhynchophylla antihypertensive prescription preparation contains 21 common characteristic peaks, and the relative retention times of peaks 1-5, 7-13 to peak 6, and peaks 14-18, 20-21 to peak 19 are calculated to be 0.14, 0.28, 0.57, 0.65, 0.79, 1.07, 1.11, 1.12, 1.18, 1.22, 1.28, 1.33, 2.00, 0.92, 0.94, 0.97, 0.98, 1.06, and 1.11, respectively.

4. The quality detection method of the Uncaria rhynchophylla antihypertensive prescription according to claim 1, characterized in that: In step (1.3), in the characteristic spectrum of the Uncaria rhynchophylla antihypertensive prescription preparation, the 2nd, 3rd, 5th, 6th, 7th, 8th, 9th, 10th, 13th, 15th, 16th, 18th, 19th, 20th, and 21st characteristic peaks are gallic acid, salidroside, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin apiglycoside, magnolamine, paeoniflorin, daidzin, baicalin, privet glycoside G13, palmatine, berberine, baicalin, and benzoylpeoniflorin, respectively.

5. The quality detection method of the Uncaria rhynchophylla antihypertensive prescription according to claim 1, characterized in that: The preparation method of mixed reference substance solutions of different concentrations in steps (1.1) and (2.1) is as follows: Weigh appropriate amounts of puerarin, 3'-methoxypuerarin, puerarin apiglycoside, magnolamine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin, and benzoylpeoniflorin, respectively, and add methanol solution to prepare single reference substance solutions of certain concentrations; Take an appropriate amount of each single reference substance solution, add methanol to prepare a mixed reference substance solution containing 119.065, 46.821, 34.458, 12.659, 56.257, 36.653, 372.296, 41.587, 215.605, 459.015, and 17.150 μg of the reference substance per 1 mL, and then use the multiple dilution method to obtain mixed reference substance solutions of different concentrations.

6. The quality detection method of the Uncaria rhynchophylla antihypertensive prescription according to claim 1, characterized in that: The preparation method of the test solution in steps (1.1) and (2.1) is as follows: take an appropriate amount of Uncaria rhynchophylla antihypertensive prescription, grind it into powder, take about 0.15 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 70% methanol, reflux for 30 minutes, cool, weigh it, make up for the lost mass with 70% methanol, filter it, and take the filtrate to obtain the solution.

7. The quality detection method of the Uncaria rhynchophylla antihypertensive prescription according to claim 1, characterized in that: In step (2.3), the relative retention times of puerarin, 3'-methoxypuerarin, puerarin apiglycoside, magnolamine, paeoniflorin, daidzein, baicalin, bamipine, berberine, wogonin and benzoylpeoniflorin are 1.00, 1.07, 1.11, 1.12, 1.18, 1.32, 1.00, 0.92, 0.98, 1.06 and 1.11, respectively.

8. The quality detection method of the Uncaria rhynchophylla antihypertensive prescription according to claim 1, characterized in that: In step (2.3), the relative correction factors of 3'-methoxypuerarin, puerarin apiglycoside, magnolamine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin and benzoylpeoniflorin are 1.75, 1.46, 1.81, 4.28, 1.39, 3.25, 1.24, 1.08, 4.08 and 3.22, respectively.

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