A quality testing method for a preparation of Uncaria rhynchophylla antihypertensive formula

Abstract

The present application relates to a kind of gouteng antihypertensive prescription quality detection method, belong to traditional Chinese medicine component detection technical field, the quality detection method of the gouteng antihypertensive prescription, it includes the establishment method of characteristic map and / or the content determination method of multiple components.The former determines the stable chromatographic condition including elution gradient, column temperature, detection wavelength etc., establishes the characteristic map with relative retention time stability, good chromatographic peak shape, good separation degree using high performance liquid chromatography technology;The latter adopts one measurement multiple evaluation method and uses puerarin as internal standard to quickly and conveniently realize the content determination of 11 kinds of components of gouteng antihypertensive prescription, with the advantages of high detection efficiency, high accuracy.The quality detection method of the gouteng antihypertensive prescription provides basis for the quality control of gouteng antihypertensive prescription.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine component detection, and in particular to a quality detection method for a preparation of Uncaria rhynchophylla antihypertensive formula. Background Technology

[0002] The Uncariae Antihypertensive Formula consists of nine herbs: Uncariae, Moutan Cortex, Scutellariae Radix, Phellodendri Cortex, Coptise Rhizoma, Puerariae Radix, Hirudo Radix, Alisma Rhizoma, and Ligustrum Lucidum Fructus. It is a clinical experience formula developed by Professor Ding Shuwen, a renowned TCM cardiovascular disease expert in Shandong Province. All the herbs in this formula have varying degrees of heat-clearing effects, and when combined with herbs that promote Yang and invigorate blood circulation, it has been clinically proven to be significantly effective in treating hypertension of the liver Yang hyperactivity type. Currently, there is no established quality standard for the Uncariae Antihypertensive Formula granules. To better control production quality, establishing quality testing methods is imperative. Summary of the Invention

[0003] To overcome the shortcomings of the prior art, this invention provides a quality testing method for Uncaria rhynchophylla antihypertensive formula, which solves the problems existing in the prior art.

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

[0005] A quality testing method for a preparation of Uncaria rhynchophylla antihypertensive formula, wherein the preparation is granules of Uncaria rhynchophylla antihypertensive formula or freeze-dried powder of standard decoction, including a method for establishing characteristic chromatograms and / or a method for determining the content of multiple components;

[0006] The method for establishing the feature map includes the following steps:

[0007] (1.1) Prepare test solution and prepare mixed reference solution of 11 components with different concentrations, including puerarin, 3'-methoxypuerarin, puerarin apigenin, magnoflorine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin and benzoyl paeoniflorin.

[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 solution and multiple batches of test solution, identify the common peaks of each test sample chromatogram, select 21 common peaks with stable peaks, good peak shape and resolution as characteristic peaks, compare with the reference chromatogram, and identify 15 components;

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

[0011] (2.1) Prepare test solution and prepare mixed reference solution of 11 components with different concentrations, including puerarin, 3'-methoxypuerarin, puerarin apigenin, magnoflorine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin and benzoyl paeoniflorin.

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

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

[0014] Using different brands and types of chromatographic columns, chromatograms of the mixed reference solution and the test solution were collected according to the chromatographic conditions in step (2.2). With puerarin as an internal reference, the relative retention times of 3'-methoxypuerarin, puerarin apigenin, magnoflorine, paeoniflorin, and daidzein were calculated. With berberine as an internal reference, the relative retention times of baicalin, palmatine, wogonin, and benzoyl paeoniflorin were calculated.

[0015] Following the chromatographic conditions in step (2.2), chromatograms of mixed reference solutions of different concentrations were collected. Using puerarin as an internal reference, the relative correction factors for 3'-methoxypuerarin, puerarin apigenin, magnoflorine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin, and benzoyl paeoniflorin were calculated respectively.

[0016] (2.4) Measurement:

[0017] Chromatograms of the mixed reference solution and the test solution were collected according to the chromatographic conditions in step (2.2). The concentrations and peak areas of the chromatographic peaks of puerarin and berberine reference standards were used as references. The chromatographic peaks of each analyte were located according to the relative retention times determined in step (2.3). The contents of 3'-methoxypuerarin, puerarin apigenin, magnoflorine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin, and benzoyl paeoniflorin were calculated according to the relative correction factors determined in step (2.3).

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

[0019] Preferably, in step (1.3), the characteristic spectrum of the Uncaria rhynchophylla antihypertensive formula contains 21 common characteristic peaks. The relative retention times of peaks 1-5 and 7-13 relative to peak 6, and peaks 14-18 and 20-21 relative 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.

[0020] Preferably, in step (1.3), the characteristic peaks of the Uncaria rhynchophylla antihypertensive formula preparation, numbered 2, 3, 5, 6, 7, 8, 9, 10, 13, 15, 16, 18, 19, 20, and 21, are gallic acid, rhodioloside, 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin apigenin, magnoflorine, paeoniflorin, daidzein, baicalin, privetin G13, palmatine, berberine, wogonin, and benzoyl paeoniflorin, respectively.

[0021] Preferably, the method for preparing the mixed reference solutions of different concentrations in steps (1.1) and (2.1) is as follows: weigh appropriate amounts of puerarin, 3'-methoxypuerarin, puerarin apigenin, magnoflorine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin, and benzoyl paeoniflorin, and add methanol solution to prepare single reference solutions of a certain concentration.

[0022] Take appropriate amounts of each single reference solution and add methanol to prepare mixed reference solutions 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 reference standard per mL, respectively. Then, use the dilution method to obtain mixed reference solutions of different concentrations.

[0023] Preferably, 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 formula, grind it into a fine powder, take about 0.15g, weigh it accurately, place it in a stoppered conical flask, add 25mL of 70% methanol accurately, reflux for 30min, cool, weigh it, replenish the lost mass with 70% methanol, filter it, and take the filtrate to obtain the test solution.

[0024] Preferably, in step (2.3), the relative retention times of puerarin, 3'-methoxypuerarin, puerarin apigenin, magnoflorine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin, and benzoylpaeoniflorin 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.

[0025] Preferably, in step (2.3), the relative correction factors for 3'-methoxypuerarin, puerarin apigenin, magnoflorin, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin, 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 present invention employs the above-described structure and has the following advantages:

[0027] 1. The quality detection method for the Uncaria rhynchophylla antihypertensive formula provided by this invention uses high-performance liquid chromatography (HPLC) to establish characteristic chromatograms and employs a one-test-multiple-evaluation method with puerarin and berberine as internal references to determine the content of 11 components in the Uncaria rhynchophylla antihypertensive formula. Specifically, in the process of establishing the chromatograms and performing the content determination, stable chromatographic conditions, including elution gradient, column temperature, and detection wavelength, were determined, ensuring stable detection results of the analytes on different instruments and chromatographic columns, stable relative retention times of chromatographic peaks, good peak shapes, and good resolution; and high detection efficiency and accuracy, providing a basis for the quality control of the Uncaria rhynchophylla antihypertensive formula.

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

[0029] 2. This invention can rapidly detect the content of puerarin, puerarin apigenin, magnoflorine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin, and benzoyl paeoniflorin in the Uncaria rhynchophylla antihypertensive formula. It provides experimental basis for the quality control and rational clinical application of the Uncaria rhynchophylla antihypertensive formula granules, provides research direction for process optimization, and can also provide a reference for the scientific development of formula granules. Attached Figure Description

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

[0031] Figure 2 These are the characteristic spectra of the present invention at different wavelengths;

[0032] Figure 3 These are characteristic spectra of mobile phase B at different concentrations and column temperatures according to the present invention;

[0033] Figure 4 These are characteristic chromatograms of multiple batches of the traditional decoction powder and formula granules of the Uncaria rhynchophylla antihypertensive formula of this invention. Detailed Implementation

[0034] The present invention will be further described in detail below. However, it should be noted that the following specific embodiments are merely exemplary examples of the invention, and the scope of protection of the invention is not limited thereto. The scope of protection of the invention is defined only by the claims. It will be apparent to those skilled in the art that various other modifications and substitutions can be made to the embodiments of the invention within the scope of protection defined by the claims, and the same technical effects can still be achieved, thus achieving the ultimate technical objective of the invention.

[0035] Unless otherwise specified, all raw materials in this manual were obtained through commercial channels.

[0036] 1. Instruments and Materials

[0037] 1.1 Instruments

[0038] Agilent 1260 DAD / VWD high-performance liquid chromatograph (purchased from Agilent Technologies, USA), TS8606 lyophilizer (purchased from Fevik GmbH, Germany), BSA224S-CW 1 / 10,000 electronic balance (purchased from Sartorius Group, Germany), QY-300 high-speed reciprocating cutting machine (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 Gongyi Yuhua Instrument Co., Ltd.).

[0039] 1.2 Materials

[0040] Gallic acid, puerarin, paeoniflorin, and privetin G13 (batch numbers 130011-202112, 070018-202106, 190001-202205, and 140086-202311, all with a purity ≥98%) were purchased from Shanghai Hongyong Biotechnology Co., Ltd.; rhodioloside, magnoflorin, daidzein, baicalin, palmatine, berberine, and wogonin (batch numbers 110818-202210, 112090-202201, 111739-202305, 110715-202223, 110713-201913, 110713-202316, and 11200) were purchased from Shanghai Hongyong Biotechnology Co., Ltd. Product 2-202303 (with purities of 99.7%, ≥98%, ≥98%, 97.20%, ≥98%, ≥98%, and 98.50% respectively) was purchased from the China National Institutes for Food and Drug Control; 3'-hydroxypuerarin and puerarin apigenin (batch numbers Q390877 and Q404487 respectively, with purities of 99.52% and 98.37% respectively) were purchased from Shandong Wodesen Biotechnology Co., Ltd.; 3'-methoxypuerarin (batch number CFS202202, with a purity ≥98%) was purchased from Wuhan Tianzhi Biotechnology Co., Ltd.; and benzoyl paeoniflorin (batch number CFS202301, with a purity ≥98%) was purchased from Suzhou Yani Biotechnology Co., Ltd.

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

[0042] Nine batches of formula granules containing Uncaria rhynchophylla, Paeonia suffruticosa, Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, Pueraria lobata, Alisma plantago-aquatica, and Ligustrum lucidum all met the national standards for formula granules. The specifications were 1g of formula granules equivalent to 8.0, 2.0, 4.5, 2.2, 5.0, 4.0, 3.3, and 2.5g of the corresponding medicinal slices, respectively. Five batches of formula granules containing Hirudo medicinalis all met the Shandong Province standards for formula granules. The specification was 1g of formula granules equivalent to 4.0g of medicinal slices. Multiple batches of single-herb formula granules were randomly combined to prepare Uncaria rhynchophylla antihypertensive formula granules (S16-S24). The formula granule information and combination details are shown in Table 1.

[0043] Table 1. Formulation Granule Information and Combination

[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. Measurement Method

[0047] 2.1 Sample Preparation

[0048] 2.1.1 Preparation of freeze-dried powder and granules of traditional decoction for lowering blood pressure using Uncaria rhynchophylla

[0049] Weigh out 100.5g of the decoction pieces at one clinical dose (30g of Uncaria rhynchophylla, 30g of Paeonia suffruticosa, 30g of Scutellaria baicalensis, 30g of Phellodendron chinense, 30g of Coptis chinensis, 30g of Pueraria lobata, 30g of Hirudo medicinalis, 30g of Alisma plantago-aquatica, 30g of Ligustrum lucidum, 15g of Paeonia suffruticosa, 9g of Hirudo medicinalis, 9g of Hirudo medicinalis, 9g of Alisma plantago-aquatica, and 1.5g of Ligustrum lucidum), place them in a clay pot, add 9 times the amount of water, soak for 30 minutes to fully moisten the decoction pieces, bring to a boil over high heat, then simmer over low heat for 30 minutes, sieve while hot (200 mesh), add 7 times the amount of water to the residue, bring to a boil over high heat, then simmer over low heat for 20 minutes, filter (200 mesh), combine the two filtrates, concentrate at 60℃, freeze dry to obtain the freeze-dried powder of the traditional decoction of Uncaria rhynchophylla for lowering blood pressure (numbered S1-S15).

[0050] Based on the dosage of each herb in the Uncaria rhynchophylla antihypertensive formula and the specifications of the single-herb granules, the amounts of Uncaria rhynchophylla, Paeonia suffruticosa, Coptis chinensis, Scutellaria baicalensis, Phellodendron chinense, Alisma plantago-aquatica, Ligustrum lucidum, Pueraria lobata, and Hirudo medicinalis granules required to prepare one dose of the Uncaria rhynchophylla antihypertensive formula granules are calculated to be 3.7500, 7.5000, 2.0000, 4.0909, 1.8000, 2.2500, 2.7273, 3.6000, and 0.3750 g respectively. The above-mentioned amounts of single-herb granules are accurately weighed to prepare the Uncaria rhynchophylla antihypertensive formula granules (numbered S16-S24).

[0051] 2.1.2 Preparation of mixed reference solution

[0052] Accurately weigh appropriate amounts of puerarin, 3'-methoxypuerarin, puerarin apigenin, magnoflorine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin, and benzoyl paeoniflorin reference standards, dissolve them in methanol, and prepare 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, and 0.954 mg·mL, respectively. -1 A single reference solution was used as the reference solution stock solution.

[0053] Accurately measure appropriate amounts of the stock solutions of puerarin, 3'-methoxypuerarin, puerarin apigenin, magnoflorine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin, and benzoylpaeoniflorin reference solutions, and dissolve them in methanol to prepare 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, and 17.150 μg·mL, respectively. -1 A mixed reference solution.

[0054] 2.1.3 Preparation of the test solution

[0055] 2.1.3.1 Selection of Extraction Method

[0056] Approximately 0.1g of the Uncaria rhynchophylla antihypertensive formula was taken and subjected to reflux heating and ultrasonic treatment respectively. The peak areas of each labeled peak were measured. The results showed (see Table 2) that, compared with ultrasonic extraction, reflux extraction yielded larger peak areas and was more effective in extracting compounds from the sample. Therefore, reflux extraction was selected.

[0057] Table 2. Results of the extraction method investigation (peak area)

[0058]

[0059] 2.1.3.2 Selection of extraction time

[0060] The reflux times were set to 15 min, 30 min, and 45 min, and the peak areas of each marker peak were examined. The results (see Table 3) showed that the peak area of ​​the chromatogram obtained by sonication at 15 min was slightly smaller, while the characteristic chromatograms obtained at 30 min and 45 min presented basically the same material information. Therefore, the sonication time was selected as 30 min.

[0061] Table 3. Results of Extraction Time Examination (Peak Area)

[0062]

[0063] 2.1.3.3 Selection of Extraction Solvent

[0064] The peak areas of each marker peak were examined when the extraction solvents were 10% methanol, 30% methanol, 50% methanol, 70% methanol and pure methanol. The results showed (see Table 4) that the peak area of ​​the marker peak was larger when extracted with 70% methanol, and 70% methanol was finally selected as the extraction solvent.

[0065] Table 4. Results of Extraction Solvent Investigation (Peak Area)

[0066]

[0067] 2.1.3.4 Selection of Sample Size

[0068] The peak areas of each labeled peak were examined when the sample weights were 0.05, 0.10, 0.15 and 0.20 g. The results (see Table 5) showed that after eliminating the difference in sample weight, the peak area of ​​the labeled peak was larger at 0.15 g, and the final sample weight was selected as 0.15 g.

[0069] Table 5. Results of Sample Size Analysis (Peak Area)

[0070]

[0071]

[0072] In summary, take an appropriate amount of the Uncaria rhynchophylla antihypertensive formula, grind it into a fine powder, take about 0.15g, weigh it accurately, place it in a stoppered conical flask, add 25mL of 70% methanol accurately, reflux for 30min, cool, weigh it, replenish the lost mass with 70% methanol, filter it, and take the filtrate to obtain the final product.

[0073] 2.1.4 Preparation of negative samples

[0074] The required amounts of the formula granules containing Uncaria rhynchophylla, Alisma plantago-aquatica, Ligustrum lucidum, and Hirudo medicinalis are 3.7500, 2.2500, 2.7273, and 0.3750 g, respectively. Accurately weigh the above-mentioned amounts of single-herb formula granules to prepare a negative sample.

[0075] 2.2 Determining chromatographic conditions

[0076] YMC-Triart C 18 The chromatographic column (4.6×250mm, 5μm, SN: 127XB20269) was used with acetonitrile as mobile phase A and 0.2% phosphoric acid solution as mobile phase B. Gradient elution was performed as follows: 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 was 1.0 mL / min. -1 The column temperature was 30℃, the wavelength was 240nm, and the injection volume was 10μL.

[0077] 2.2.1 Selection of detection wavelength

[0078] The test solution was prepared according to method 2.1.3. Eight 1 μL portions of the test solution were accurately weighed and injected into an ultra-high performance liquid chromatograph (UHPLC) to detect the spectra at wavelengths of 192 nm, 210 nm, 240 nm, 260 nm, 280 nm, 300 nm, 320 nm, and 360 nm. The results are shown in the figure. Figure 2 Based on the principles of the number of chromatographic peaks, peak response values, and baseline stability, 240 nm was ultimately selected as the detection wavelength for the Uncaria rhynchophylla antihypertensive formula.

[0079] 2.2.1 Selection of mobile phase

[0080] The test solution was prepared according to method 2.1.3. Two 1 μL portions of the test solution were accurately weighed and injected into an ultra-high performance liquid chromatograph. The chromatograms were detected using 0.2% phosphoric acid solution and 0.1% phosphoric acid solution as mobile phase B, respectively. The results are shown in [Figure 1]. Figure 3Based on the principles of the number of chromatographic peaks, peak response values, and baseline stability, 0.2% phosphoric acid solution was ultimately selected as the mobile phase B for the Uncaria rhynchophylla antihypertensive formula.

[0081] 2.2.1 Selection of column temperature

[0082] The test solution was prepared according to method 2.1.3. Three 1 μL portions of the test solution were accurately weighed and injected into an ultra-high performance liquid chromatograph (UHPLC). The chromatograms at column temperatures of 28℃, 30℃, and 32℃ were detected respectively. The results are shown in [Figure 1]. Figure 3 Based on the principles of the number of chromatographic peaks, peak response values, and baseline stability, 30℃ was ultimately selected as the column temperature for the Uncaria rhynchophylla antihypertensive formula.

[0083] 2.3 HPLC Characteristic Chromatography of Standard Decoction and Granules of Uncaria Rhizoma Antihypertensive Formula

[0084] 2.3.1 Method for establishing feature maps:

[0085] The test solutions prepared from 15 batches of freeze-dried powder of the standard decoction of Uncaria rhynchophylla and 9 batches of granules of Uncaria rhynchophylla were analyzed according to the chromatographic conditions in 2.2. The chromatograms were then imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System". Figure 4 .

[0086] Using the characteristic chromatogram of the S1 batch of Uncaria rhynchophylla antihypertensive formula standard decoction as a reference, common peaks were identified in the characteristic chromatograms of 15 batches of Uncaria rhynchophylla antihypertensive formula standard decoction and 9 batches of formula granules. Twenty-one common peaks with stable peaks, good peak shape and resolution were selected as characteristic peaks. After comparison with the chromatogram of the reference standard, 15 components were identified, namely gallic acid (peak 2), rhodioloside (peak 3), 3'-hydroxypuerarin (peak 5), puerarin (peak 6), 3'-methoxypuerarin (peak 7), puerarin apigenin (peak 8), magnoflorin (peak 9), paeoniflorin (peak 10), daidzein (peak 13), baicalin (peak 15), privetin G13 (peak 16), palmatine (peak 18), berberine (peak 19), wogonin (peak 20), and benzoylpaeoniflorin (peak 21).

[0087] Multi-point calibration and Mark matching were performed on the characteristic spectra of 15 batches of Uncaria rhynchophylla antihypertensive formula standard decoction to generate control characteristic spectra (see Figure 1 Comparing the chromatograms of 15 batches of the Uncaria rhynchophylla antihypertensive formula standard decoction with the control characteristic chromatogram, the similarity was >0.90, indicating that the different batches of the Uncaria rhynchophylla antihypertensive formula standard decoction prepared had a high degree of similarity.

[0088] Using peaks 6 (S1) and 19 (S2) as reference peaks, the relative retention time (RSD) of each common peak was calculated to be 0.0%–2.00%. The relative retention times of the 21 characteristic peaks showed little difference, all within ±2% of the average value. The average 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), and 1.11 (peak 21), with an allowable error of ±10%.

[0089] Table 6 Relative Retention Time Results

[0090]

[0091] The average total peak area of ​​the characteristic peaks in 15 batches of traditional decoctions was set as 1 (100%). The relative values ​​of the total peak area of ​​the characteristic peaks in 9 batches of Uncaria rhynchophylla antihypertensive formula granules to the average total peak area of ​​the traditional decoction were calculated, and were 1.05, 1.08, 1.02, 1.06, 1.20, 1.07, 1.10, 1.07, and 1.06, respectively. Similarly, the relative values ​​of the total peak area of ​​all peaks in the characteristic spectrum of the 9 batches of formula granules to the average total peak area of ​​all peaks in the traditional decoction were calculated, and were 1.08, 1.07, 1.01, and 1.06, respectively.

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

[0093] 2.3.2 Methodological Examination

[0094] 2.3.2.1 Repeatability Test

[0095] Six parallel test samples were prepared using the Uncaria rhynchophylla antihypertensive formula granules (No.: S1) from section "2.1.1" according to section "2.1.3". The samples were analyzed under the chromatographic conditions described in section "2.2", and the chromatographic data were recorded. The relative standard deviations (RSDs) of the relative peak areas and relative retention times of peaks 1-5 and 7-13 relative to peak 6 (S1 peak), and peaks 14-18 and 20-21 relative to peak 19 (S2 peak) were calculated. The results (see Tables 7 and 8) show that the RSDs of the relative peak areas for each common peak were all <2.83%, and the RSDs of the relative retention times were all <1.73%, indicating that the method has good repeatability.

[0096] Table 7. Repeatability test results (relative peak area)

[0097]

[0098]

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

[0100]

[0101] 2.3.2.2 Stability Assessment

[0102] Samples were prepared using the formulation granules described in section "2.1.1" according to section "2.1.3". Chromatographic data were recorded at 0, 2, 4, 8, 12, and 24 hours under the chromatographic conditions described in section "2.2". The results (see Tables 9 and 10) show that the relative peak area RSD of each common peak was <2.25%, and the relative retention time RSD was <0.69%, indicating that the test solution exhibits good stability within 24 hours.

[0103] Table 9. Stability test results (relative peak area)

[0104]

[0105] Table 10. Stability Study Results (Relative Retention Time)

[0106]

[0107] 2.3.2.3 Intermediate Precision Examination

[0108] Different researchers used a different high-performance liquid chromatograph to repeat the experiment according to section "2.3.2.1" and recorded the chromatographic data. Analysis of the results of the two experiments showed (see Tables 11 and 12) that the relative peak area RSD of each common peak was <2.84% and the relative retention time RSD was <1.83%, indicating that the method has good intermediate precision.

[0109] Table 11 Stability test results (relative peak area)

[0110]

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

[0112]

[0113]

[0114] 2.4 Determination of multiple components in standard decoction and granules of Uncaria rhynchophylla antihypertensive formula

[0115] 2.4.1 Methods for determining the content of multiple components

[0116] 2.4.1.1 Obtaining the relative retention time

[0117] Three instruments with four columns of different brands and types were used to collect chromatograms of the mixed reference standards under the chromatographic conditions described in section 2.2. The chromatographic peaks were located by the retention times of the reference standards, and the actual retention times of each characteristic peak were obtained. The average value of the actual retention times was calculated as the standard retention time (tSRT). Puerarin and berberine were used as internal controls, and the relative retention times of each index component were calculated to be 1.07, 1.11, 1.12, 1.18, 1.32, 0.92, 0.98, 1.06, and 1.11, respectively.

[0118] 2.4.1.2 Obtaining the relative correction factor (f)

[0119] Five mixed reference solutions of different concentrations as described in section "2.1.2" were injected for analysis. The chromatographic conditions as described in section "2.2" were followed, with puerarin as an internal reference, and the analysis was performed according to the formula. (A s C represents the internal standard peak area. s Internal standard concentration; A i C represents the peak area of ​​the analyte. i The f values ​​for 10 index components were calculated (to be the concentration of the analyte), and the results are shown in Table 13.

[0120] Table 13 Relative correction factors for each indicator component in mixed reference solutions of different concentrations.

[0121]

[0122] 2.4.1.3 Measurement

[0123] According to the chromatographic conditions in 2.2, collect the chromatograms of the mixed reference solution and the test solution. Using the concentration and peak area of ​​the puerarin reference solution as references, locate the chromatographic peaks of each analyte according to the relative retention time determined in step (2.3). Calculate the contents of 3'-methoxypuerarin, puerarin apigenin, magnoflorine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin, and benzoyl paeoniflorin according to the relative correction factor determined in 2.4.1.2.

[0124] 2.4.2 Methodological Examination

[0125] 2.4.2.1 Specificity Examination

[0126] Take the Uncaria rhynchophylla antihypertensive formula granules under section "2.1.1" and the negative sample under section "2.1.4", and prepare the test solution and negative sample solution respectively according to section "2.1.3". Detect them according to the chromatographic method under section "2.2". The results show that there is no interference from the negative sample and the method has good specificity.

[0127] 2.4.2.2 Examination of Linear Relationships

[0128] Accurately pipette the mixed reference solution from section "2.1.2" and dilute it by 1.25, 2.5, 5, and 10 times, respectively. Analyze the five different concentrations of the mixed reference solution according to the chromatographic method described in section "2.2," and record the peak areas of 11 index components, including puerarin. The concentration of the analyte (C, μg·mL⁻¹) is then recorded. -1 Using the peak area (A) as the ordinate and the horizontal axis as the vertical axis, a linear regression was performed. The results showed that the linear relationship of each index component was good within a certain concentration range. The regression equation and linear range are shown in Table 14.

[0129] Table 14 Regression Equation and Linear Range

[0130]

[0131] 2.4.2.3 Repeatability Test

[0132] Six test solutions were prepared in parallel using the Uncaria rhynchophylla antihypertensive formula granules under section “2.1.1” and under section “2.1.3”. The solutions were then analyzed under the chromatographic conditions under section “2.2”. The results (see Table 15) showed that the RSD of the contents of the 11 index components was ≤2.10%, indicating that the method had good repeatability.

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

[0134]

[0135] 2.4.2.4 Stability Assessment

[0136] The test solution of the same Uncaria rhynchophylla antihypertensive formula granules was tested at 0, 2, 4, 8, 12 and 24 h after preparation under the chromatographic conditions in section “2.2”. The results showed (see Table 16) that the RSD of the contents of 11 index components was ≤1.19%, and the test sample had good stability within 24 h.

[0137] Table 16 Stability test results (mg·g) -1 )

[0138]

[0139] 2.4.2.5 Intermediate Precision Examination

[0140] Different researchers conducted the experiment again using a separate high-performance liquid chromatograph according to the method described in section "2.4.1.2", and recorded the chromatographic data. Analysis of the two experimental results (see Table 17) showed that the RSD of the 11 index components was ≤2.13%, indicating that this method has good intermediate precision.

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

[0142]

[0143]

[0144] 2.4.2.6 Recovery rate

[0145] Take 0.75 g of the formulation granules under section "2.1.1", add an equal volume of reference solution to the sample content, and prepare 6 parallel test solutions according to section "2.1.3". Detect the solutions under the chromatographic conditions under section "2.2". The results (see Table 18) show that the average recoveries (RSD) of puerarin, 3'-methoxypuerarin, puerarin apigenin, magnoflorine, paeoniflorin, daidzein, baicalin, palmatine, berberine, wogonin, and benzoylpaeoniflorin were 97.27%. The results (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%) indicate that the method has good accuracy.

[0146] Table 18 Recovery Results

[0147]

[0148]

[0149] 2.4.2.7 Durability assessment of the relative correction factor f

[0150] The effects of different liquid chromatographs and columns on the analyte f were investigated. The RSD of f was ≤2.75% (Table 19), indicating that different instruments and columns had no significant effect on the analyte f. The effects of different mobile phase flow rates and column temperatures on the analyte f were investigated. The RSD of f was ≤2.53% (Table 20), indicating that different mobile phase flow rates and column temperatures had no significant effect on the analyte f.

[0151] Table 19 Relative correction factors for each component under different instrument and column conditions

[0152]

[0153] Table 20 Relative correction factors for each component under different mobile phase volumetric flow rates and column temperatures.

[0154]

[0155] 2.4.2.8 Localization of chromatographic peaks of index components

[0156] The relative retention times of each index component with respect to puerarin and berberine were calculated, and the effects of different liquid chromatographs and columns on the relative retention times of the chromatographic peaks of each index component were investigated. The results (Table 21) showed that the relative retention time RSD < 1.38%, indicating that these instruments and columns can be used for characteristic peak localization.

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

[0158]

[0159]

[0160] 2.4.3 Comparative Study of QAMS and External Standard Method (ESM) for Content Determination

[0161] The contents of each component in the sample were calculated using both QAMS and external standard method (ESM). The relative deviation (RD / %) between the two methods was calculated, and t-tests were performed on the data calculated by SPSS 24.0 software. The results showed that RD < 3%, P > 0.05, indicating that the QAMS method had good accuracy. Detailed data are shown in Tables 22-23.

[0162] Table 22 Comparison of the contents of six 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 specific embodiments described above should not be construed as limiting the scope of protection of this invention. Any alternative modifications or variations made to the embodiments of this invention by those skilled in the art will fall within the scope of protection of this invention. All aspects not detailed in this invention are well-known to those skilled in the art.

Claims

1. A quality detection method of Gouteng Jiangya Decoction preparation, the Gouteng Jiangya Decoction preparation is Gouteng Jiangya Decoction dispensing granules or standard decoction freeze-dried powder, characterized in that, The method comprises the following steps: The method for establishing the characteristic spectrum comprises the following steps: (1.1) preparing a test sample solution, and preparing mixed control sample solutions of 11 components including puerarin, 3'-methoxypuerarin, puerarin apioside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside and benzoylpaeoniflorin at different concentrations; (1.2) determining the liquid chromatography conditions; (1.3) collecting the chromatograms of the mixed control sample solutions and the test sample solutions according to the chromatography conditions in step (1.2), identifying the common peaks in the chromatograms of the test samples, selecting 21 common peaks with stable peaks, good peak shape and separation degree as characteristic peaks, and identifying the 11 components by comparing with the chromatogram of the control sample; The method for determining the contents of the multiple components comprises the following steps: (2.1) preparing a test sample solution, and preparing mixed control sample solutions of 11 components including puerarin, 3'-methoxypuerarin, puerarin apioside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside and benzoylpaeoniflorin at different concentrations; (2.2) determining the liquid chromatography conditions; (2.3) determining the relative retention time and the relative correction factor: collecting the chromatograms of the mixed control sample solutions and the test sample solutions according to the chromatography conditions in step (2.2), taking puerarin as the internal reference substance to calculate the relative retention time of 3'-methoxypuerarin, puerarin apioside, magnoflorine, paeoniflorin and daidzin, and taking berberine as the internal reference substance to calculate the relative retention time of baicalin, palmatine, wogonoside and benzoylpaeoniflorin; collecting the chromatograms of the mixed control sample solutions at different concentrations according to the chromatography conditions in step (2.2), and taking puerarin as the internal reference substance to calculate the relative correction factor of 3'-methoxypuerarin, puerarin apioside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside and benzoylpaeoniflorin; (2.4) determining the contents of the components: collecting the chromatograms of the mixed control sample solutions and the test sample solutions according to the chromatography conditions in step (2.2), taking the concentrations and the peak areas of the chromatographic peaks of the puerarin and berberine control samples as the references, locating the chromatographic peaks of the components to be determined according to the relative retention time determined in step (2.3), and calculating the contents of 3'-methoxypuerarin, puerarin apioside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside and benzoylpaeoniflorin according to the relative correction factor determined in step (2.3); The liquid chromatography conditions in steps (1.2) and (2.2) are as follows: using C 18 chromatographic column, acetonitrile as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution conditions: 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 -30 ℃, wavelength 240 nm, injection volume 10 μL; The preparation method of the test sample solution in steps (1.1) and (2.1) is as follows: taking a proper amount of Gouteng Antihypertensive Prescription preparation, grinding it into powder, accurately taking about 0.15 g, accurately weighing, placing it in a conical flask with a plug, accurately adding 25 mL of 70% methanol, refluxing for 30 min, cooling, weighing, supplementing the lost mass with 70% methanol, filtering, and taking the filtrate, thereby obtaining the test sample solution.

2. The method for detecting the quality of Gouteng Hypotensive Decoction preparation according to claim 1, characterized in that, In step (1.3), the characteristic chromatogram of Gouteng Jiangya Decoction 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 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.

3. The method for detecting the quality of Gouteng Hypotensive Formula according to claim 1, characterized in that, In step (1.3), the characteristic chromatogram of Gouteng Jiangya Decoction Preparation, characteristic peaks 6, 7, 8, 9, 10, 13, 15, 18, 19, 20, and 21 are puerarin, 3'-methoxypuerarin, puerarin apioside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside, and benzoylpaeoniflorin, respectively.

4. The method for detecting the quality of Gouteng Hypotensive Formula according to claim 1, characterized in that, In steps (1.1) and (2.1), the preparation method of mixed control solution with different concentrations is as follows: A certain amount of puerarin, 3'-methoxypuerarin, puerarin apioside, magnoflorine, paeoniflorin, daidzin, baicalin, palmatine, berberine, wogonoside, and benzoylpaeoniflorin was weighed, and methanol was added to prepare single control solution with a certain concentration; A certain amount of each single control solution was taken, and methanol was added to prepare a mixed control 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 control per 1 mL, respectively. Then, a mixed control solution with different concentrations was obtained by multiple dilution method.

5. The method for detecting the quality of Gouteng Hypotensive Formula according to claim 1, characterized in that, In step (2.3), the relative retention times of puerarin, 3'-methoxypuerarin, 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, and 1.11, respectively.

6. The method for detecting the quality of Gouteng Hypotensive Formula according to claim 1, characterized in that, In step (2.3), the relative correction factors of 3'-methoxypuerarin, puerarin apioside, 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.

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