A quality detection method of a kidney-nourishing, heat-clearing and blood-activating prescription

Abstract

The application discloses a quality detection method of a Yishen Qingli Huoxue recipe, which comprises preparation of a test sample solution, preparation of a standard solution, preparation of a standard curve and content determination. The application screens an optimal test sample preparation method, a preparation method of a control sample and optimal chromatographic conditions and mass spectrometric analysis conditions through a large number of experiments, and establishes a content determination method of the Yishen Qingli Huoxue recipe, which can simultaneously determine the contents of 11 compounds with different structural types, such as resveratrol, rhein, formononetin, aloe-emodin, emodin, chlorogenic acid, polydatin, astilbin, hyperoside, calycosin-7-glucoside and beta-ecdysterone. The method is favorable for comprehensively monitoring the quality of the Yishen Qingli Huoxue recipe and guaranteeing clinical curative effects. Moreover, the method has the advantages of good stability, high accuracy, good repeatability and high precision.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and specifically relates to a quality detection method for Yishen Qingli Huoxue Prescription. Background Technique

[0002] Yishen Qingli Huoxue Prescription is a characteristic hospital preparation in Jiangsu Provincial Hospital of Traditional Chinese Medicine. Professor Sun Wei has been long engaged in the clinical and basic research of chronic kidney disease (CKD). On the basis of inheriting the academic thoughts of Professor Zou Yunxiang, the founder of modern traditional Chinese medicine nephrology, and Professor Zou Yanqin, a national master of traditional Chinese medicine, through clinical efficacy analysis, prescription data mining, epidemiological research, and expert surveys, etc., it is found that "kidney deficiency", "damp-heat", and "blood stasis" are the core pathogenesis of CKD, and the theory of "kidney deficiency, dampness, and blood stasis" in CKD is creatively proposed, advocating the prevention and treatment of CKD with the theory of "protecting the kidney and delaying senility", and on this basis, Yishen Qingli Huoxue Prescription is created. This prescription is composed of astragalus membranaceus, angelica sinensis, polygonum cuspidatum, serissa japonica, smilax glabra, achyranthes bidentata, pyrrosia lingua, rheum officinale, centella asiatica, polygonatum sibiricum, and abelmoschus manihot. It has the effects of invigorating the spleen and kidney, clearing away damp-heat, and removing blood stasis and discharging turbidity. Astragalus membranaceus is slightly warm in nature and is an important medicine for tonifying the kidney, which can greatly tonify primordial qi. Astragalus membranaceus combined with angelica sinensis tonifies the kidney and nourishes blood, astragalus membranaceus combined with polygonatum sibiricum supplements qi and nourishes yin, angelica sinensis combined with achyranthes bidentata promotes blood circulation and nourishes blood, and the supplementation is not stagnant. Centella asiatica, polygonum cuspidatum, pyrrosia lingua, and abelmoschus manihot clear away damp-heat and promote diuresis for treating stranguria; smilax glabra, serissa japonica, and rheum officinale activate blood circulation to break stasis and dredge the bowel to discharge turbidity. All the medicines work together to achieve the effect of tonifying the kidney, clearing away damp-heat, and activating blood circulation, and good clinical effects have been obtained.

[0003] The components of Yishen Qingli Huoxue Prescription are complex. Relying on a single detection method, it is impossible to comprehensively and objectively detect its quality. In order to fully control its clinical effectiveness and safety and safeguard the interests of patients, it is very necessary to research and design a detection method that can comprehensively and accurately detect the active ingredients of Yishen Qingli Huoxue Prescription on the basis of the existing technology, which can provide a reference basis for comprehensively evaluating and controlling the quality and clinical application of Yishen Qingli Huoxue Prescription. Summary of the Invention

[0004] Purpose of the Invention: The purpose of the present invention is to overcome the deficiencies of the existing technology and develop a quality detection method for Yishen Qingli Huoxue Prescription. Through the method of the present invention, the quality of Yishen Qingli Huoxue Prescription can be objectively, correctly, and effectively controlled, which has important guiding significance for ensuring its safety and effectiveness and guaranteeing clinical quality.

[0005] Technical Solution: To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A quality detection method for Yishen Qingli Huoxue Prescription, which includes the following steps:

[0007] Step 1: Preparation of the test solution of Yishen Qingli Huoxue Prescription

[0008] Weigh out the Chinese herbs of Yishen Qingli Huoxue Fang, pulverize them appropriately, dissolve them in methanol solution in a volumetric flask, add methanol to make up to volume, and extract by ultrasonication to obtain Yishen Qingli Huoxue Fang extract; accurately transfer a certain amount of Yishen Qingli Huoxue Fang extract into a volumetric flask, make up to volume with methanol solution, shake well, centrifuge, and take the supernatant to filter through a 0.22μm filter membrane to obtain Yishen Qingli Huoxue Fang test solution;

[0009] Step 2: Preparation of standard solution

[0010] Accurately weigh appropriate amounts of resveratrol, rhein, styracin, aloe-emodin, rhein, chlorogenic acid, polygalactoside, astilbene, hyperoside, verbascoside, and β-ecdysterone reference standards into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well, and obtain a reference stock solution of a certain concentration for later use.

[0011] Accurately pipette appropriate amounts of resveratrol, rhein, styracin, aloe-emodin, rhein, chlorogenic acid, polygalactoside, astilbene, and hyperoside reference stock solutions into the same volumetric flask, add methanol to make up to the mark, shake well, and obtain a mixed reference working solution of a certain concentration.

[0012] Accurately pipette an appropriate amount of the stock solution of verbenaflavonoid glucoside and β-ecdysterone into the same volumetric flask, add methanol to make up to the mark, shake well, and obtain a working solution of a mixed reference standard of verbenaflavonoid glucoside and β-ecdysterone with a certain concentration.

[0013] Step 3: Preparation of the standard curve

[0014] Negative ion mode: The mixed reference working solution of resveratrol, rhein, styracin, aloe-emodin, rhein, chlorogenic acid, polygalactoside, astilbene, and hyperoside prepared in step 2 was serially diluted to prepare mixed reference solutions of different concentrations, and 5 μL of each solution was taken for HPLC-MS / MS analysis.

[0015] Positive ion mode: The mixed reference solution of verbascoside and β-ecdysterone prepared in step 2 was serially diluted to prepare mixed reference solutions of different concentrations, and 5 μL of each solution was analyzed by HPLC-MS / MS.

[0016] Standard curve calculation: Linear regression was performed on the linear concentration of each standard and the detection peak area to obtain the standard curve equation. All calculations were performed using Analyst 1.6.

[0017] Step 4, Content determination

[0018] Under the same chromatographic and mass spectrometric conditions, the test solution of Yishen Qingli Huoxue Fang prepared in step 1 was injected into HPLC-MS / MS for analysis. The peak areas of the detected compounds were substituted into the standard curve equation in step 3 to calculate the content of each compound in Yishen Qingli Huoxue Fang.

[0019] As a preferred embodiment, the quality testing method for the above-described kidney-tonifying, blood-activating, and detoxifying formula, wherein step 1, the preparation method of the kidney-tonifying, blood-activating, and detoxifying formula test solution, is as follows:

[0020] Weigh a certain amount of the herbal medicine from the Yishen Qingli Huoxue formula, pulverize it appropriately, dissolve it in a 25mL volumetric flask with 50% methanol, add 50% methanol to make up the volume, and extract it by sonication for 20min to obtain the extract of the Yishen Qingli Huoxue formula; accurately transfer 5mL of the extract of the Yishen Qingli Huoxue formula into a 100mL volumetric flask, make up the volume with 50% methanol, shake well, centrifuge at 12000r / min for 10min, and filter the supernatant through a 0.22μm filter membrane to obtain the test solution of the Yishen Qingli Huoxue formula.

[0021] As a preferred embodiment, in the quality testing method of the above-described kidney-tonifying, blood-activating, and detoxifying formula, step 2: the preparation method of the standard solution is as follows:

[0022] Accurately weigh appropriate amounts of resveratrol, rhein, styracin, aloe-emodin, rhein, chlorogenic acid, polygalactoside, astilbin, hyperoside, verbascoside, and β-ecdysterone reference standards into 10 mL volumetric flasks. Dissolve and dilute to the mark with methanol, and shake well to obtain concentrations of 2.21, 1.07, 1.32, 1.22, 1.11, 1.29, 1.10, 1.64, 1.22, 1.12, and 1.52 mg / mL. -1 The reference stock solution was stored at -20°C for later use.

[0023] Accurately pipette appropriate amounts of resveratrol, rhein, monazine, aloe-emodin, rhein, chlorogenic acid, polygalactoside, astilbin, and hyperoside reference stock solutions into the same 10 mL volumetric flask. Dilute to the mark with methanol and mix well to obtain concentrations of 256.00, 640.00, 640.00, 640.00, 1120.00, 2080.00, 640.00, 3200.00, and 3680.00 ng / mL. -1 The mixed reference standard working solution;

[0024] Accurately pipette appropriate amounts of verbascoside and β-ecdysterone stock solutions into the same 10 mL volumetric flask, add methanol to the mark, and shake well to obtain concentrations of 1600.00 and 3200.00 ng / mL, respectively. -1 The mixed reference standard working solution.

[0025] As a preferred embodiment, the chromatographic conditions for steps 3 and 4 of the above-described method for quality detection of the Yishen Qingli Huoxue formula are as follows: chromatographic column: Waters Aurashell C18 with specifications of 2.1mm × 150mm and 2.7μm.

[0026] Positive ion mode mobile phase: Phase A 0.1% formic acid / Phase B acetonitrile, isocratic elution for 3 min; flow rate 0.2 mL / min; column temperature: 40℃; ion source: ESI; quantitative mode: MRM; ion source voltage: 5500V; ion source temperature: 400℃;

[0027] Mobile phase in negative ion mode: Phase A 2 mmol / L ammonium formate / Phase B acetonitrile, gradient elution; flow rate 0.2 mL / min; column temperature: 40℃; ion source: ESI; quantitative mode: MRM; ion source voltage: -4000V; ion source temperature: 400℃.

[0028] As a preferred embodiment, the mass spectrometry conditions for steps 3 and 4 of the above-described method for quality detection of a kidney-tonifying, blood-activating, and detoxifying formula are as follows:

[0029] The analytes were detected using an API4000 tandem quadrupole mass spectrometer and an electrospray ionization interface in both positive and negative ion modes. Multiple reaction monitoring (MRM) was employed to monitor the transition from precursor to product ions. The parameters were set as follows: ion spray voltage: 4000 V; turbine heater temperature: 500 °C; collision activation dissociation: 10 psi; curtain gas: 25 psi. Specific mass spectrometry detection conditions are shown in the table below.

[0030] Standard quality spectral testing conditions

[0031] Detection object Quantitative ion pair m / z DP / V) EP / V CE / V CXP / V Resveratrol 226.8→142.8 -100 -10 -33 -14 rhein 238.6→182.7 -100 -10 -27 -14 Brugrosine 266.7→251.9 -100 -10 -30 -14 Aloe-emodin 268.7→239.8 -90 -10 -33 -14 emodin 268.8→225.0 -110 -10 -35 -14 chlorogenic acid 353.2→190.9 -55 -10 -20 -14 Polygonin 389.2→227.4 -100 -10 -20 -14 Astilbene 449.3→285.2 -110 -10 -34 -14 Hyperoside 463.4→300.1 -94 -10 -34 -14 Versicolor isoflavone glucoside 447.4→285.3 70 10 22 14 β-ecdysterone 481.4→371.3 90 10 20 14 .

[0032] As a preferred embodiment, the gradient elution procedure for steps 3 and 4 of the above-described method for quality testing of the kidney-tonifying, blood-activating, and detoxifying formula is as follows:

[0033] The positive ion elution mode is:

[0034] Flow rate / mL / min A / ％ B / ％ 0.2 60 40

[0035] The negative ion elution mode is:

[0036] Time / min Flow rate / mL / min A / ％ B / ％ 0 0.2 62 38 2.5 0.2 62 38 2.6 0.2 20 80 10 0.2 20 80 10.1 0.2 62 38 30 0.2 62 38

[0037] The quality testing method of the kidney-tonifying, blood-activating and detoxifying formula described in this invention comprises the following Chinese herbal composition: 264g of raw Astragalus membranaceus, 88 parts of Angelica sinensis, 132 parts of Polygonum cuspidatum, 264 parts of Serissa japonica, 264 parts of Smilax glabra, 88 parts of Achyranthes bidentata, 176 parts of Pyrrosia lingua, 53 parts of processed Rheum palmatum, 264 parts of Centella asiatica, 176 parts of processed Polygonatum sibiricum, and 264 parts of Hibiscus syriacus.

[0038] Optimization of detection conditions

[0039] 1. Selection of mobile phase

[0040] In positive ion mode, the aqueous phase consisted of pure water, 0.1% formic acid, 0.2% formic acid, and 0.3% formic acid, while the organic phase consisted of methanol and acetonitrile. Different combinations and ratios of the above aqueous and organic phases were investigated, and finally, 0.1% formic acid:acetonitrile = 60:40 was determined as the mobile phase. Under this condition, the chromatographic peaks of each component were well-shaped and well-separated.

[0041] In negative ion mode, the aqueous phase selected was pure water with a concentration of 1 mmol·L⁻¹. -1 Aqueous solution of methyl / ammonium acetate, 2 mmol·L -1 Aqueous solution of methyl / ammonium acetate, 5 mmol·L -1 An aqueous solution of methyl / ammonium acetate was prepared, with methanol and acetonitrile selected as the organic phase. Different combinations and ratios of the above aqueous and organic phases were investigated, and finally, a concentration of 2 mmol·L⁻¹ was determined. -1 Gradient elution with ammonium formate aqueous solution (aqueous phase) and acetonitrile (organic phase) resulted in good peak shapes and separation for each component.

[0042] 2. Selection of column temperature and flow rate

[0043] This study investigated different column temperatures (25, 30, 35, 40 °C) and flow rates (0.2, 0.3, 0.4 mL·min). -1 The effect of the column temperature on the chromatographic peak was investigated, and the results showed that the optimal column temperature was 40℃ and the flow rate was 0.2 mL / min. -1 The obtained chromatographic peaks showed good performance in terms of peak shape, number, and resolution.

[0044] The beneficial effects of this invention are:

[0045] (1) Because the Yishen Qingli Huoxue Formula consists of 11 Chinese medicinal herbs, its chemical composition is very complex, containing compounds of various structural types such as anthraquinones, flavonoids, aromatic acids, and sterones. Conventional chromatographic conditions cannot effectively separate them, and the sensitivity of ultraviolet detectors is not high. This invention, through extensive experimentation, screened the optimal preparation methods for the test sample and the reference sample, as well as the optimal chromatographic separation conditions and highly sensitive mass spectrometry analysis conditions, and established a method for determining the content of the Yishen Qingli Huoxue Formula. This method can simultaneously detect the content of 11 compounds of different structural types, including resveratrol, rhein, styracin, aloe-emodin, rhein, chlorogenic acid, polygalactosidin, astilbin, hyperoside, verbascoside, and β-ecdysterone. The establishment of this method can not only effectively characterize the quality of the Yishen Qingli Huoxue Formula, but also facilitate comprehensive monitoring of the quality of the drug and ensure clinical efficacy.

[0046] (2) The method of the present invention has the advantages of good stability, high precision, high accuracy, and good reproducibility. It can comprehensively, objectively, and accurately evaluate and control the quality of the kidney-tonifying, blood-activating, and detoxifying formula. Attached Figure Description

[0047] Figure 1 The molecular formulas, standard chromatographic peaks, and chromatographic peaks of chlorogenic acid, polydipsia glycoside, astilbene glycoside, and aloe-emodin in the Yishen Qingli Huoxue formula were determined.

[0048] Figure 2 The molecular formulas, standard chromatographic peaks, and chromatographic peaks of the Yishen Qingli Huoxue formula are given for rhein, hyperoside, emodin, and gentianin.

[0049] Figure 3 The graph shows the molecular formulas, standard chromatographic peaks, and peaks of the Yishen Qingli Huoxue formula for resveratrol, isoflavone glucoside, and β-ecdysterone. The horizontal axis represents the elution time of the compounds, and the vertical axis represents the ion inductance. Detailed Implementation

[0050] The embodiments of the present invention will be described in detail below with reference to the examples. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0051] The following are examples of medicines and reagents:

[0052] Resveratrol (B20044, HPLC ≥ 98%), rhein (B20245, HPLC ≥ 98%), styracin (B20836, HPLC ≥ 98%), aloe-emodin (B20772, HPLC ≥ 98%), emodin (B20240, HPLC ≥ 98%), chlorogenic acid (B20782, HPLC ≥ 98%), polygalactoside (B20533, HPLC ≥ 98%), astilbin (B20812, HPLC ≥ 98%), hyperoside (B20631, HPLC ≥ 98%), and β-ecdysterone (B21268, HPLC ≥ 98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; verbascoside (C823639, HPLC ≥ 98%) was purchased from Shanghai Maclean Biotechnology Co., Ltd. Methanol and acetonitrile were purchased from Merck & Co.

[0053] Main instruments used in the experiment: HPLC-MS / MS detection: Agilent 1200 high performance liquid chromatograph (Agilent, USA); SCIEX API4000 triple quadrupole mass spectrometer (ABSCIEX), equipped with electro-spray ionization (ESI); chromatography workstation: Analyst 1.6; Aurashell C18 column 2.1mm*150mm, 2.7μm (Waters, USA); MICRO-17R refrigerated centrifuge (Thermo, USA); Drict-Q5 ultrapure water system (Millipore, USA).

[0054] Example 1

[0055] 1. A quality testing method for a kidney-tonifying, blood-activating, and detoxifying formula, comprising the following steps:

[0056] Step 1: Preparation of the test solution of the kidney-tonifying, blood-activating and detoxifying formula

[0057] Weigh a certain amount of the herbs in the Yishen Qingli Huoxue formula (264 parts raw Astragalus membranaceus, 88 parts Angelica sinensis, 132 parts Polygonum cuspidatum, 264 parts Serissa japonica, 264 parts Smilax glabra, 88 parts Achyranthes bidentata, 176 parts Pyrrosia lingua, 53 parts processed Rheum palmatum, 264 parts processed Centella asiatica, 176 parts processed Polygonatum sibiricum, and 264 parts Hibiscus syriacus). Pulverize appropriately, dissolve in 50% methanol in a 25 mL volumetric flask, add 50% methanol to dilute to volume, and extract by ultrasonication for 20 min to obtain the extract of the Yishen Qingli Huoxue formula. Accurately transfer 5 mL of the extract to a 100 mL volumetric flask, dilute to volume with 50% methanol, shake well, centrifuge at 12000 r / min for 10 min, and filter the supernatant through a 0.22 μm filter membrane to obtain the test solution of the Yishen Qingli Huoxue formula.

[0058] Step 2: Preparation of standard solution

[0059] Accurately weigh appropriate amounts of resveratrol, rhein, styracin, aloe-emodin, rhein, chlorogenic acid, polygalactoside, astilbin, hyperoside, verbascoside, and β-ecdysterone reference standards into 10 mL volumetric flasks. Dissolve and dilute to the mark with methanol, and shake well to obtain concentrations of 2.21, 1.07, 1.32, 1.22, 1.11, 1.29, 1.10, 1.64, 1.22, 1.12, and 1.52 mg / mL. -1 The reference stock solution was stored at -20°C for later use.

[0060] Accurately pipette appropriate amounts of resveratrol, rhein, monazine, aloe-emodin, rhein, chlorogenic acid, polygalactoside, astilbin, and hyperoside reference stock solutions into the same 10 mL volumetric flask. Dilute to the mark with methanol and mix well to obtain concentrations of 256.00, 640.00, 640.00, 640.00, 1120.00, 2080.00, 640.00, 3200.00, and 3680.00 ng / mL. -1 The mixed reference standard working solution;

[0061] Accurately pipette appropriate amounts of verbascoside and β-ecdysterone stock solutions into the same 10 mL volumetric flask, add methanol to the mark, and shake well to obtain concentrations of 1600.00 and 3200.00 ng / mL, respectively. -1 The mixed reference standard working solution.

[0062] Step 3: Preparation of the standard curve

[0063] Negative ion mode: The mixed reference working solution of resveratrol, rhein, styracin, aloe-emodin, rhein, chlorogenic acid, polygalactoside, astilbene, and hyperoside prepared in step 2 was serially diluted to prepare mixed reference solutions of different concentrations, and 5 μL of each solution was taken for HPLC-MS / MS analysis.

[0064] Positive ion mode: The mixed reference solution of verbascoside and β-ecdysterone prepared in step 2 was serially diluted to prepare mixed reference solutions of different concentrations, and 5 μL of each solution was analyzed by HPLC-MS / MS.

[0065] Standard curve calculation: Linear regression was performed on the linear concentration of each standard and the detection peak area to obtain the standard curve equation. All calculations were completed using Analyst 1.6 software.

[0066] The equations of the obtained standard curves are shown in Table 1 below:

[0067] Table 1

[0068] Detection object Standard curve equation Resveratrol y = 0.0482x + 0.00172 rhein y = 0.0449x + 0.068 Brugrosine y = 0.642x + 0.622 Aloe-emodin y = 0.12x + 0.04 emodin y = 0.862x + 0.00368 chlorogenic acid y = 0.353x + 0.18 Polygonin y = 0.0317x + 0.0433 Astilbene y = 0.0129x + 0.123 Hyperoside y = 0.0201x + 0.201 Versicolor isoflavone glucoside y = 0.0795x + 0.132 β-ecdysterone y = 0.0016x + 0.00242

[0069] Step 4, Content determination

[0070] Under the same chromatographic and mass spectrometric conditions, the test solution of Yishen Qingli Huoxue Fang prepared in step 1 was injected into HPLC-MS / MS for analysis. The peak areas of the detected compounds were substituted into the standard curve equation of step 3 to calculate the content of each compound in Yishen Qingli Huoxue Fang, as shown in Table 2 below.

[0071] Table 2. Content of each compound in the Kidney-Nourishing, Blood-Clearing, and Blood-Activating Formula (mg·g) -1 )

[0072] Compound Name <![CDATA[Content (mg·g -1 )]]> Resveratrol 0.145 rhein 0.024 Brugrosine 0.014 Aloe-emodin 0.014 emodin 0.133 chlorogenic acid 0.250 Polygonin 0.515 Astilbene 2.143 Hyperoside 0.543 Versicolor isoflavone glucoside 0.030 β-ecdysterone 0.053 .

[0073] The chromatographic conditions for steps 3 and 4 above are as follows: chromatographic column: Waters Aurashell C18 with dimensions of 2.1 mm × 150 mm and 2.7 μm;

[0074] Positive ion mode mobile phase: 0.1% formic acid / acetonitrile, gradient elution; flow rate: 0.2 mL / min; column temperature: 40℃; ion source: ESI; quantitative mode: MRM; ion source voltage: 5500V; ion source temperature: 400℃;

[0075] Mobile phase in negative ion mode: 2 mmol / L ammonium formate / acetonitrile, gradient elution; flow rate: 0.2 mL / min; column temperature: 40℃; ion source: ESI; quantitative mode: MRM; ion source voltage: -4000V; ion source temperature: 400℃.

[0076] The gradient elution procedures are shown in Tables 3 and 4 below:

[0077] Table 3 Positive ion elution modes

[0078] Flow rate / mL / min A / ％ B / ％ 0.2 60 40

[0079] Table 4 Negative Ion Eluting Modes

[0080] Time / min Flow rate / mL / min A / ％ B / ％ 0 0.2 62 38 2.5 0.2 62 38 2.6 0.2 20 80 10 0.2 20 80 10.1 0.2 62 38 30 0.2 62 38 .

[0081] Mass spectrometry conditions are:

[0082] The analytes were detected using an API4000 tandem quadrupole mass spectrometer and an electrospray ionization interface in both positive and negative ion modes. Multiple reaction monitoring (MRM) was employed to monitor the transition from precursor to product ions. The parameters were set as follows: ion spray voltage: 4000 V; turbine heater temperature: 500 °C; collision activation dissociation: 10 psi; curtain gas: 25 psi. Specific mass spectrometry detection conditions are shown in Table 4 below.

[0083] Table 4 Standard Mass Spectrometry Testing Conditions

[0084] Detection object Quantitative ion pair m / z DP / V) EP / V CE / V CXP / V Resveratrol 226.8→142.8 -100 -10 -33 -14 rhein 238.6→182.7 -100 -10 -27 -14 Brugrosine 266.7→251.9 -100 -10 -30 -14 Aloe-emodin 268.7→239.8 -90 -10 -33 -14 emodin 268.8→225.0 -110 -10 -35 -14 chlorogenic acid 353.2→190.9 -55 -10 -20 -14 Polygonin 389.2→227.4 -100 -10 -20 -14 Astilbene 449.3→285.2 -110 -10 -34 -14 Hyperoside 463.4→300.1 -94 -10 -34 -14 Versicolor isoflavone glucoside 447.4→285.3 70 10 22 14 β-ecdysterone 481.4→371.3 90 10 20 14 .

[0085] 2. Methodological Examination

[0086] (1) Precision test

[0087] Take the sample of Yishen Qingli Huoxue Fang, prepare the test solution according to the method in step 1 above, and then continuously inject and determine it 6 times under the above chromatographic conditions. With resveratrol as a reference, the RSD of the relative retention time is less than 1.2% (n=6) and the RSD of the relative peak area is less than 2.3% (n=6), indicating that the method has good precision.

[0088] (2) Stability test

[0089] Take the sample of Yishen Qingli Huoxue Formula and prepare the test solution according to the method in step 1 above. Place it at room temperature for 0, 2, 4, 8, 12 and 24 h respectively, and inject it for determination under the above chromatographic conditions. With resveratrol as the reference, the RSD of the relative retention time is less than 1.4% (n=6) and the RSD of the calculated relative peak area is less than 1.7% (n=6), indicating that the test solution has good stability within 24 h at room temperature.

[0090] (3) Repeatability test

[0091] Six samples of the Yishen Qingli Huoxue formula were taken and the test solutions were prepared according to the method in step 1 above. The samples were then injected and determined under the above chromatographic conditions. With resveratrol as a reference, the RSD of the relative retention time was less than 1.1% (n=6), and the RSD of the calculated relative peak area was less than 2.60% (n=6), indicating that the method has good repeatability.

[0092] The above experimental results show that the detection method for Yishen Qingli Huoxue Formula established in this invention has good precision, stability and repeatability, and can effectively characterize the quality of Yishen Qingli Huoxue Formula, which is conducive to comprehensive quality monitoring and ensuring clinical efficacy.

Claims

1. A quality testing method for a kidney-tonifying, blood-activating, and detoxifying formula, characterized in that, It includes the following steps: Step 1: Preparation of the test solution of the kidney-tonifying, blood-activating and detoxifying formula Weigh a certain amount of the herbal medicine of Yishen Qingli Huoxue Fang, pulverize it appropriately, dissolve it in 50% methanol in a 25 mL volumetric flask, add 50% methanol to make up the volume, and extract it by ultrasonication for 20 min to obtain the extract of Yishen Qingli Huoxue Fang; accurately transfer 5 mL of the extract of Yishen Qingli Huoxue Fang into a 100 mL volumetric flask, make up the volume with 50% methanol, shake well, centrifuge at 12000 r / min for 10 min, and filter the supernatant through a 0.22 μm filter membrane to obtain the test solution of Yishen Qingli Huoxue Fang; Step 2: Preparation of standard solution Accurately weigh appropriate amounts of resveratrol, rhein, styracin, aloe-emodin, rhein, chlorogenic acid, polygalactoside, astilbene, hyperoside, verbascoside, and β-ecdysterone reference standards into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well, and obtain a reference stock solution of a certain concentration for later use. Accurately pipette appropriate amounts of resveratrol, rhein, styracin, aloe-emodin, rhein, chlorogenic acid, polygalactoside, astilbene, and hyperoside reference stock solutions into the same volumetric flask, add methanol to make up to the mark, shake well, and obtain a mixed reference working solution of a certain concentration. Accurately pipette an appropriate amount of the stock solution of verbenaflavonoid glucoside and β-ecdysterone into the same volumetric flask, add methanol to make up to the mark, shake well, and obtain a working solution of a mixed reference standard of verbenaflavonoid glucoside and β-ecdysterone with a certain concentration. Step 3: Preparation of the standard curve Negative ion mode: The mixed reference working solution of resveratrol, rhein, styracin, aloe-emodin, rhein, chlorogenic acid, polygalactoside, astilbene, and hyperoside prepared in step 2 was serially diluted to prepare mixed reference solutions of different concentrations, and 5 μL of each solution was taken for HPLC-MS / MS analysis. Positive ion mode: The mixed reference solution of verbascoside and β-ecdysterone prepared in step 2 was serially diluted to prepare mixed reference solutions of different concentrations, and 5 μL of each solution was analyzed by HPLC-MS / MS. Standard curve calculation: Linear regression is performed on the linear concentration of each standard and the detection peak area to obtain the standard curve equation; Step 4, Content determination Under the same chromatographic and mass spectrometric conditions, the test solution of Yishen Qingli Huoxue Fang prepared in step 1 was injected into HPLC-MS / MS for analysis. The peak areas of the detected compounds were substituted into the standard curve equation in step 3 to calculate the content of each compound in Yishen Qingli Huoxue Fang. The chromatographic conditions for steps 3 and 4 are as follows: Column: Waters Aurashell C18 with dimensions of 2.1 mm × 150 mm and 2.7 μm; Positive ion mode mobile phase: Phase A 0.1% formic acid / Phase B acetonitrile, isocratic elution; flow rate 0.2 mL / min; column temperature: 40℃; ion source: ESI; quantitative mode: MRM; ion source voltage: 5500 V; ion source temperature: 400℃; The positive ion elution mode is as follows: ； Negative ion mode mobile phase: Phase A 2 mmol / L ammonium formate / Phase B acetonitrile, gradient elution for 3 min; flow rate 0.2 mL / min; column temperature: 40℃; ion source: ESI; quantitative mode: MRM; ion source voltage: -4000V; ion source temperature: 400℃; The negative ion elution mode is as follows: ； The mass spectrometry conditions for steps 3 and 4 are as follows: The analytes were detected using an API 4000 tandem quadrupole mass spectrometer and an electrospray ionization interface in both positive and negative ion modes. Multiple reaction monitoring (MRM) was employed to monitor the transition from precursor to product ions. The parameters were set as follows: ion spray voltage: 4000 V; turbine heater temperature: 500 °C; collision activation dissociation: 10 psi; curtain gas: 25 psi. Specific mass spectrometry detection conditions are shown in the table below. Standard quality spectral testing conditions ； The herbal composition of the kidney-tonifying, blood-activating, and detoxifying formula described in step 1 is as follows: 264 parts raw Astragalus membranaceus, 88 parts Angelica sinensis, 132 parts Polygonum cuspidatum, 264 parts Serissa japonica, 264 parts Smilax glabra, 88 parts Achyranthes bidentata, 176 parts Pyrrosia lingua, 53 parts processed Rheum palmatum, 264 parts Centella asiatica, 176 parts processed Polygonatum sibiricum, and 264 parts Hibiscus syriacus.

2. The quality testing method for the kidney-tonifying, blood-activating, and detoxifying formula according to claim 1, characterized in that, Step 1: The preparation method of the test solution of the Kidney-Nourishing, Blood-Clearing, and Moistening Formula is as follows: Weigh a certain amount of the herbal medicine from the Yishen Qingli Huoxue formula, pulverize it appropriately, dissolve it in a 25 mL volumetric flask with 50% methanol, add 50% methanol to make up the volume, and extract it by sonication for 20 min to obtain the extract of the Yishen Qingli Huoxue formula; accurately transfer 5 mL of the extract of the Yishen Qingli Huoxue formula into a 100 mL volumetric flask, make up the volume with 50% methanol, shake well, centrifuge at 12000 r / min for 10 min, and filter the supernatant through a 0.22 μm filter membrane to obtain the test solution of the Yishen Qingli Huoxue formula.

3. The quality testing method for the kidney-tonifying, blood-activating, and detoxifying formula according to claim 1, characterized in that, Step 2: The method for preparing the standard solution is as follows: Accurately weigh appropriate amounts of resveratrol, rhein, styracin, aloe-emodin, rhein, chlorogenic acid, polygalactoside, astilbin, hyperoside, verbascoside, and β-ecdysterone reference standards into 10 mL volumetric flasks. Dissolve and dilute to the mark with methanol, and shake well to obtain concentrations of 2.21, 1.07, 1.32, 1.22, 1.11, 1.29, 1.10, 1.64, 1.22, 1.12, and 1.52 mg·mL. -1 The reference stock solution was stored at -20°C for later use. Accurately pipette appropriate amounts of resveratrol, rhein, styracin, aloe-emodin, rhein, chlorogenic acid, polygalactoside, astilbin, and hyperoside reference stock solutions into the same 10 mL volumetric flask. Dilute to the mark with methanol and mix well to obtain concentrations of 256.00, 640.00, 640.00, 640.00, 1120.00, 2080.00, 640.00, 3200.00, and 3680.00 ng·mL. -1 The mixed reference standard working solution; Accurately pipette appropriate amounts of verbascoside and β-ecdysterone stock solutions into the same 10 mL volumetric flask, add methanol to the mark, and shake well to obtain concentrations of 1600.00 and 3200.00 ng·mL, respectively. -1 The mixed reference standard working solution.

4. The quality testing method for the kidney-tonifying, blood-activating, and detoxifying formula according to claim 1, characterized in that, The equation of the standard curve is: 。

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