Method for determining content of effective components in Kunxinning preparation

Abstract

The invention relates to a method for determining the content of effective components in a Kunxinning preparation. Comprising the following steps: step 1, preparing a reference substance solution, namely respectively taking a proper amount of one or two or more of catalpol, rehmannia glutinosa liboside D, paeoniflorin, calycosin-7-glucoside, (-)-syringinol-4-O-beta-D-furanselinosyl-(1-2)-beta-D-glucopyranoside, curculigoside, icariin and baohuoside I reference substances; preparing a reference substance solution; step 2, preparation of a test solution: taking a Kunxinning preparation, weighing the Kunxinning preparation, placing the Kunxinning preparation in a measuring flask, adding methanol to dilute the Kunxinning preparation to a scale, filtering the Kunxinning preparation, and taking a subsequent filtrate; step 3, a determination method: respectively and precisely sucking the reference substance solution and the test solution, injecting the solutions into a high performance liquid chromatograph, and determining component types and / or component contents; the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, water is used as a mobile phase A, acetonitrile is used as a mobile phase B, gradient elution is performed, the flow velocity is 0.5-1.5 mL / min, the column temperature is 25-35 DEG C, the detection wavelength is 200-300 nm, and the sample size is 10-30 [mu] L.

Description

TECHNICAL FIELD

[0001] The present application relates to a detection method of traditional Chinese medicine preparation, in particular to a multi-index component detection method of a traditional Chinese medicine composition for treating female climacteric syndrome, and particularly relates to a content determination method of effective components in Kunxinning preparation. BACKGROUND

[0002] Menopausal syndrome, also known as perimenopausal syndrome (PMS), is a group of symptoms characterized by hot flashes, irritability, fatigue, insomnia, osteoporosis, etc. occurring in women before and after menopause. There are many theories about the etiology and pathogenesis of PMS in traditional Chinese medicine, among which kidney qi deficiency is considered to be the core pathogenesis of the disease. The characteristics of multi-component, multi-target and multi-pathway synergy of traditional Chinese medicine not only can improve the symptoms of PMS more comprehensively, but also are more and more applied to the treatment of PMS due to its mild nature, less adverse reactions and diversified treatment options.

[0003] Kunxinning preparation is composed of seven traditional Chinese medicines, including rehmannia, astragalus, herba rhizoma, epimedium, red shaddock bauhinia bark and concha margaritifera usta, which has the effects of warming yang and nourishing yin, and benefiting kidney and calming liver. It is a clinical experience prescription for treating PMS of kidney yin and yang deficiency type. According to the approved product specification, the function and indication are as follows: warming yang and nourishing yin, benefiting kidney and calming liver, for female menopausal syndrome of kidney yin and yang deficiency type, with symptoms of hot flashes, cold pain in waist and back, alternating heat and cold, irritability, cold limbs, vaginal dryness, depression, dizziness, tinnitus, insomnia and dreaminess, pale tongue, thin white fur and deep and fine pulse. As a unique product for the treatment of female menopausal syndrome (kidney yin and yang deficiency type), the prescription of Kunxinning granules takes rehmannia and astragalus as the monarch, nourishing blood and tonifying yin, and tonifying qi and spleen to supplement qi and yin deficiency; assisted by herba rhizoma and epimedium, warming and tonifying kidney yang, and used with the monarch to achieve the effect of tonifying yin and yang of kidney; supplemented by red shaddock bauhinia bark and concha margaritifera usta, clearing liver heat and calming liver yang; bauhinia bark soothing the mind and relieving depression; seven ingredients are used together to achieve the effects of warming yang and nourishing yin, benefiting kidney and calming liver.

[0004] The compatibility and composition of the prescription are complex, and the existing research only conducts qualitative identification (Zhao Guiling, Miao Xinglong, Song Zhaohui, et al. UPLC-Q-TOF / MS method for chemical component analysis of Kunyining granules [J]. Chinese patent medicine, 2020, 42(04): 1074-1082), lacking content determination. It is of great significance to establish a multi-index content determination method of Kunxinning granules which covers more comprehensively, has strong drug material specificity and high correlation with drug effectiveness, to ensure the efficacy and safety of the drug. SUMMARY

[0005] The purpose of the present application is to provide a content determination method of effective components in Kunxinning preparation, which comprises the following steps:

[0006] Step 1 preparation of reference solution: take one or two or more of catalpol, rehmatann D, paeoniflorin, calycosin-7-glucoside, (-)-syringaresinol-4-O-β-D-apiosyl-(1→2)-β-D-glucopyranoside, curculigoside, icariin, baohuoside I reference substance, and prepare into reference solution;

[0007] Step 2 preparation of test solution: take Kunxinning preparation, weigh and place in a flask, dilute to the mark with methanol, filter, and take the filtrate;

[0008] Step 3 determination method: respectively take the reference solution and the test solution, inject into the high performance liquid chromatograph, and obtain the chromatogram, according to the chromatogram, calculate the content of the effective component in the test solution;

[0009] Among them, the chromatographic conditions are: octadecylsilane bonded silica gel as the filler, water as the mobile phase A, acetonitrile as the mobile phase B, gradient elution, the flow rate is 0.5-1.5 mL / min, the column temperature is 25-35℃, the detection wavelength is 200-300 nm, the injection amount is 10-30 μL; the gradient elution program is:

[0010]

[0011] Among them, in the step 1 preparation of reference solution, the catalpol reference substance concentration is 1.0000-3.0000 mg / mL; the rehmatann D reference substance concentration is 1.0000-3.0000 mg / mL; the paeoniflorin reference substance concentration is 2.0000-4.5000 mg / mL; the calycosin-7-glucoside reference substance concentration is 0.0500-0.2000 mg / mL; the (-)-syringaresinol-4-O-β-D-apiosyl-(1→2)-β-D-glucopyranoside reference substance concentration is 0.1200-0.3600 mg / mL; the curculigoside reference substance concentration is 0.05-0.2 mg / mL; the icariin reference substance concentration is 0.5000-2.0000 mg / mL; the baohuoside I reference substance concentration is 0.1000-0.3000 mg / mL.

[0012] Preferably, the step 1 preparation of the control solution is as follows: accurately weigh appropriate amounts of catalpol, rehmaplatin D, paeoniflorin, calycosin-7-glucoside, (-)-syringaresinol-4-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside, curculigoside, and icariin control samples, and add methanol to prepare control stock solutions with a mass concentration of 2.0000 mg / mL, 2.0000 mg / mL, 4.1250 mg / mL, 0.1000 mg / mL, 0.2400 mg / mL, 0.1000 mg / mL, and 1.0000 mg / mL, respectively; accurately weigh an appropriate amount of baohuoside I control sample, and add acetonitrile to prepare a control stock solution with a mass concentration of 0.1800 mg / mL; accurately take 0.50 mL of each of the above control stock solutions, mix them uniformly, and obtain a mixed control sample solution.

[0013] Preferably, the step 2 preparation of the test sample solution is as follows: accurately weigh about 1 g of the Kunxinning preparation, add about 40 mL of methanol to a 50-mL volumetric flask, and ultrasonically treat for 30 min at an ultrasonic power of 300 W and an ultrasonic frequency of 50 kHz, take out the solution, cool it, dilute to the mark with methanol, shake it uniformly, filter it, and take the filtrate, thereby obtaining the test sample solution.

[0014] Preferably, the step 2 preparation of the test sample solution is as follows: accurately weigh about 1 g of the Kunxinning preparation, add about 40 mL of methanol to a 50-mL volumetric flask, and ultrasonically treat for 30 min at an ultrasonic power of 300 W and an ultrasonic frequency of 50 kHz, take out the solution, cool it, dilute to the mark with methanol, shake it uniformly, filter it, and take the filtrate, thereby obtaining the test sample solution.

[0015] Preferably, in the step 3 determination method, the flow rate is 1.0 mL / min, the column temperature is 28°C, the detection wavelength is 210 nm, the injection volume is 20 μL, and the chromatographic column is NX-C18 4.6 mm x 250 mm, 5 μm.

[0016] Most preferably, the method of the present application comprises the following steps:

[0017] Step 1, preparation of the control sample solution

[0018] Take appropriate amount of lognannoside, rehmatannoside D, paeoniflorin, calycosin-7-glucoside, (-)-syringaresinol-4-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside, curculigoside, icariin reference substance, accurately weigh, add methanol to prepare reference substance stock solution with mass concentration of 2.0000 mg / mL, 2.0000 mg / mL, 4.1250 mg / mL, 0.1000 mg / mL, 0.2400 mg / mL, 0.1000 mg / mL, 1.0000 mg / mL respectively; take appropriate amount of baohuogoside I reference substance, accurately weigh, add acetonitrile to prepare reference substance stock solution with mass concentration of 0.1800 mg / mL; accurately take 0.50 mL of each of the above reference substance stock solution, mix uniformly to obtain a mixed reference substance solution;

[0019] Step 2, preparation of test sample solution

[0020] Take Kunxinning granules, finely grind, accurately weigh about 1 g, place in a 50 mL volumetric flask, add about 40 mL of methanol, ultrasonic for 30 min, ultrasonic power 300 W, ultrasonic frequency 50 kHz, take out, cool, dilute to the mark with methanol, shake uniformly, filter, take the filtrate, and obtain;

[0021] Step 3, detection method

[0022] Accurately pipette the reference substance solution and the test sample solution respectively, inject into the high performance liquid chromatograph, determine according to the chromatographic conditions, and quantitate by external standard method;

[0023] The chromatographic conditions are as follows: NX-C18 Chromatographic column, 4.6 mm*250 mm, 5 μm, mobile phase A: water, mobile phase B: acetonitrile, gradient elution: 0-20 min, 97%-90% A, 3%-10% B; 20-25 min, 90%-80% A, 10%-20% B; 25-45 min, 80%-60% A, 20%-40% B; 45-55 min, 60%-50% A, 40%-50% B; 55-65 min, 50%-30% A, 50%-70% B; 65-70 min, 30%-15%, 70%-85% B; 70-72 min, 15%-0% A, 85%-100% B; injection volume: 20 μL; detection wavelength: 210 nm; volume flow rate: 1.0 mL / min; column temperature: 28 °C.

[0024] The Kunxinning preparation in the application is selected from one or more of Kunxinning granules, Kunxinning capsules and Kunxinning tablets.

[0025] The content of each of the following components in 1 g of the Kunxinning granules detected by the method of the present application is in the range of 3.5854-4.9141 mg of catalpol, 0.3869-0.6765 mg of rehmatannin D, 6.6162-8.2455 mg of paeoniflorin, 0.2890-0.6520 mg of calycosin-7-glucoside, 0.0802-0.1552 mg of (-)-syringaresinol-4-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside, 0.3647-0.5908 mg of curculigoside, 1.9886-2.9467 mg of turpinorin, and 0.0894-0.1289 mg of baohuoside I.

[0026] The Kunxinning preparation of the present application is a known product and is commercially available, such as Kunxinning granules with the State Drug Standard Number Z20210006, which is prepared from the following Chinese medicinal raw materials in the weight parts: Rehmannia glutinosa 10-15 parts, Concha Margaritiferae 15-20 parts, Albizia julibrissin 8-12 parts, Radix Paeoniae Rubra 10-15 parts, Astragalus membranaceus 16-24 parts, Epimedium brevicornu 10-15 parts, and Curculigo orchioides 10-15 parts. Preferably, it is prepared from the following Chinese medicinal raw materials in the weight parts: Rehmannia glutinosa 4 parts, Concha Margaritiferae 5 parts, Albizia julibrissin 3 parts, Radix Paeoniae Rubra 4 parts, Astragalus membranaceus 6 parts, Epimedium brevicornu 4 parts, and Curculigo orchioides 4 parts.

[0027] The preparation method of the Kunxinning preparation or Kunxinning granules is as follows:

[0028] Step (1): Take Concha Margaritiferae and decoct it;

[0029] Step (2): Add Rehmannia glutinosa, Albizia julibrissin, Radix Paeoniae Rubra, Astragalus membranaceus, Epimedium brevicornu, and Curculigo orchioides, and decoct them with water;

[0030] Step (3): Filter;

[0031] Step (4): Concentrate the filtrate to a relative density of 1.01-1.02;

[0032] Step (5): High-speed centrifuge the concentrated solution, and concentrate it again to a relative density of 1.15-1.20;

[0033] Step (6): Add lactose, dextrin, and sugar powder, and perform boiling granulation;

[0034] Preferably, in the step (1), the Concha Margaritiferae is decocted for 30 minutes.

[0035] In the step (2), after adding Rehmannia glutinosa, Albizia julibrissin, Radix Paeoniae Rubra, Astragalus membranaceus, Epimedium brevicornu, and Curculigo orchioides, decoct them with water for 2 times, each time for 1.5 hours.

[0036] The following are the explanations and descriptions of the terms of the present application:

[0037] "Multiple" refers to the volume / weight ratio, such as L / kg or mL / g. For example, adding 1 kg of medicinal materials to 10 L of water is 10 times the volume.

[0038] The full name of the C18 column is octadecylsilane bonded silica gel column.

[0039] The decoction method in preparation generally refers to boiling with water.

[0040] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0041] Beneficial technical effects of the present invention:

[0042] This invention outperforms existing technologies in many technical indicators, including detection accuracy, sensitivity, separation, ease of operation, cost, and operation time. Attached Figure Description

[0043] Figure 1 Graphs showing the effects of mixed reference standards under different wavelengths;

[0044] Figure 2 Graphs showing the effects of Kunxinning granule extract (10mg / mL) on different wavelengths.

[0045] Figure 3 Graphs showing the effects of mixed reference standards under different mobile phase conditions;

[0046] Figure 4 Figure 1: Evaluation of Kunxinning Granule Extract (10 mg / mL) under different mobile phase conditions;

[0047] Figure 5 Chromatograms of the mixed reference standard and each single component reference standard, with the following chromatographic peaks in sequence: catalpol, rehinoside D, paeoniflorin, verbenafiloside, (-)-eugenol-4-O-β-D-furanoside-(1→2)-β-D-pyranoside, curculigoside, icariin and baicalin I reference standard;

[0048] Figure 6 Figure 1 shows the content determination of Kunxinning granules; Figure A is the HPLC chromatogram of 15 batches of Kunxinning granules, where a to h are the chromatographic peaks of catalpol, rehinoside D, paeoniflorin, verrucoside, (-)-eugenol-4-O-β-D-furanoside-(1→2)-β-D-glucopyranoside, curculigoside, icariin and baicalin I, respectively; Figure B is the content of components in 15 batches of Kunxinning granules.

[0049] Figure 7Typical HPLC chromatogram of Kunxinning granules, in which the serial numbers a to h are catalpol, rehinoside D, paeoniflorin, verbascoside, (-)-eugenol-4-O-β-D-furanoside-(1→2)-β-D-glucopyranoside, curculigoside, icariin and baicalin I;

[0050] Figure 8 Chemical structures of: citronol, rehmannia glycoside D, paeoniflorin, verrucoside, (-)-eugenol-4-O-β-D-furanoside-(1→2)-β-D-pyranoside, curculigoside, epimedium glycoside and baicalin I. Detailed Implementation

[0051] The present invention is further illustrated by the following examples, but these are not intended to limit the invention.

[0052] Instruments: LC-20AT high performance liquid chromatograph, Shimadzu Corporation, Japan; SPD-20A detector, Shimadzu Corporation, Japan; XS105DU microbalance, Mettler Toledo, USA.

[0053] Drugs and reagents: 15 batches of Kunxinning granules, either self-made or commercially available, 6g / bag; Reference standards: citronellol (ST03400120), rehmannia glutinosa D (ST11190120), verrucoside isoflavone glucoside (ST08820120), curculigoside (ST02910120), icariin (ST00800120) and (-)-eugenol-4-O-β-D-furanoside-(1→2)-β-D-glucopyranoside (ST16670120) were purchased from Shanghai Shidande Standard Technical Service Co., Ltd., baicalin I (MB6526) was purchased from Dalian Meilun Biotechnology Co., Ltd., and paeoniflorin (S31585) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; methanol (chromatographic grade) and acetonitrile (chromatographic grade) were purchased from Tianjin Kangked Technology Co., Ltd.

[0054] Experimental Example 1: Wavelength Investigation

[0055] Chromatographic conditions: water (A) - acetonitrile (B), gradient elution: 0–15 min, 5%–10% B; 15–35 min, 10%–20% B; 35–55 min, 20%–40% B; 55–65 min, 40%–50% B; 65–75 min, 50%–70% B; 75–80 min, 70%–85% B; 80–85 min, 85%–100% B.

[0056] Wavelength studies were conducted on a mixed reference standard and Kunxinning granule extract (10 mg / mL), respectively. Figure 1In the mixed control drugs shown, the chromatographic peaks were observed at 210 nm (black) and 254 nm (red). It was found that the chromatographic peaks at 210 nm were more numerous and higher; therefore, 210 nm was selected. Figure 2 The Kunxinning granule extract (10 mg / mL) shown was examined under conditions of 210 nm (black) and 254 nm (red). It was found that there were more and higher chromatographic peaks at 210 nm, so 210 nm was selected.

[0057] Experimental Example 2: Investigation of the Mobile Phase

[0058] Chromatographic conditions: Gradient elution: 0–15 min, 5%–10% B; 15–35 min, 10%–20% B; 35–55 min, 20%–40% B; 55–65 min, 40%–50% B; 65–75 min, 50%–70% B; 75–80 min, 70%–85% B; 80–85 min, 85%–100% B.

[0059] The mobile phase was investigated using a mixed reference standard and Kunxinning granule extract (10 mg / mL), respectively. Figure 3 In the mixed reference standard shown, the baseline of the formic acid-acetonitrile mixture was found to be unstable under both formic acid-water-acetonitrile (red) and water-acetonitrile (black) conditions. Therefore, water-acetonitrile was selected as the mobile phase. Figure 4 The Kunxinning granule extract (10 mg / mL) was investigated under formic acid-water-acetonitrile (red) and water-acetonitrile (black) conditions. It was found that the baseline of formic acid-water-acetonitrile was not stable, so water-acetonitrile was selected as the mobile phase.

[0060] Experimental Example 3: Preparation of Kunxinning Granules

[0061] Kunxinning granules can be purchased commercially or prepared using the steps described in the specific embodiments of patent CN200410053184.4.

[0062] Example 1: Content determination

[0063] 1. Preparation of reference solution

[0064] Accurately weigh appropriate amounts of reference standards including citronellol, rehmannia glycoside D, paeoniflorin, verbascoside, (-)-eugenol-4-O-β-D-furanoside-(1→2)-β-D-glucopyranoside (hereinafter referred to as eugenol glycoside), curculigoside, and icariin, and prepare reference stock solutions with mass concentrations of 2.0000 mg / mL, 2.0000 mg / mL, 4.1250 mg / mL, 0.1000 mg / mL, 0.2400 mg / mL, 0.1000 mg / mL, and 1.0000 mg / mL, respectively, by adding methanol. Accurately weigh an appropriate amount of cymosin I reference standard and prepare a reference stock solution with a mass concentration of 0.1800 mg / mL by adding acetonitrile. Accurately measure 0.50 mL of each of the above reference stock solutions, mix them thoroughly, and obtain a mixed reference solution.

[0065] 2. Preparation of the test solution

[0066] Take Kunxinning granules, grind them into a fine powder, accurately weigh about 1g, place it in a 50mL volumetric flask, add about 40mL of methanol, sonicate (power 300W, frequency 50kHz) for 30min, remove, cool, dilute with methanol to the mark, shake well, filter, and take the filtrate to obtain the solution.

[0067] 3. Testing

[0068] Accurately pipette the reference solution and the test solution separately, inject them into the high-performance liquid chromatograph, determine them according to the chromatographic conditions, quantify them using the external standard method, and calculate the results.

[0069] Chromatographic conditions: Shimadzu LC-20AT high performance liquid chromatograph. NX-C18 Chromatographic column (4.6 mm × 250 mm, 5 μm), mobile phase: water (A) - acetonitrile (B), gradient elution: 0–20 min, 3%–10% B; 20–25 min, 10%–20% B; 25–45 min, 20%–40% B; 45–55 min, 40%–50% B; 55–65 min, 50%–70% B; 65–70 min, 70%–85% B; 70–72 min, 85%–100% B; injection volume: 20 μL; detection wavelength: 210 nm; flow rate: 1.0 mL / min; column temperature: 28 °C.

[0070] 4. Test Results

[0071] The specific test results of the eight components in 15 batches of Kunxinning granules are shown in Table 1.

[0072] Table 1 Content of different batches of Kunxinning granules

[0073]

[0074] Conclusion: Under the same chromatographic conditions, eight components in Kunxinning granules, including catalpol, rehmannia glycoside D, and verbascoside, can be well separated and determined. Figure 7 This is a typical HPLC chromatogram of Kunxinning preparation. Figure 5 The chromatograms are of the mixed reference standard and each single component reference standard. The peaks numbered a to h are, in order, the chromatographic peaks of the reference standards: catalpol, rehinoside D, paeoniflorin, verbascoside, eugenol, curculigoside, icariin, and baicalin I. The content determination results of the eight components are shown below. Figure 6 In A-6B, the content of catalpol, rehmannia glycoside D, paeoniflorin, verbascoside, eugenol, curculigoside, icariin, and baicalin I per 1g of Kunxinning granules ranges from 3.5854 to 4.9141 mg, 0.3869 to 0.6765 mg, 6.6162 to 8.2455 mg, 0.2890 to 0.6520 mg, 0.0802 to 0.1552 mg, 0.3647 to 0.5908 mg, 1.9886 to 2.9467 mg, and 0.0894 to 0.1289 mg, respectively. Among them, the content of catalpol, paeoniflorin, and icariin is relatively high, while the content of eugenol and baicalin I is relatively low. Figure 8 The chemical structures of the eight isolated components are shown.

[0075] Example 2: Examination of Linear Relationships

[0076] The mixed reference solution from Example 1, "1. Preparation of Reference Solution," was prepared into a series of concentration solutions using chromatographic methanol. These solutions were filtered through a 0.22 μm microporous membrane before injection. Based on the HPLC results, the peak areas of each reference standard at different concentrations were integrated, and linear regression analysis was performed using the peak area (Y) of each chromatographic peak against the concentration (X) of each component. The mixed reference solution and stock solutions of each single-component reference standard were analyzed under the chromatographic conditions of Example 1. The eight components showed good separation and moderate retention times, suitable for subsequent analysis. The results are shown in Table 2. The mass concentration and peak area of ​​catalpol, rehmannia glycoside D, paeoniflorin, verbascoside, eugenol, curculigoside, icariin, and baicalin I showed good linearity within a certain range.

[0077] Table 2 Linear Relationships

[0078]

[0079] Conclusion: The established HPLC method for content determination was validated, and the results are shown in Table 2. The mass concentrations and peak areas of catalpol, rehmannia glycoside D, paeoniflorin, verbascoside, eugenol, curculigoside, icariin, and baicalin I showed good linearity within a certain range.

[0080] Example 3 Precision Examination

[0081] Six parallel samples were prepared according to the method under "2. Preparation of test solution" in Example 1, and tested according to the detection method in Example 1. The relative standard deviation (RSD) of each content in the samples was calculated, and the results are shown in Table 3.

[0082] Table 3. Precision (Repeatability) Test Results

[0083]

[0084]

[0085] Example 4 Accuracy

[0086] Six parallel samples were prepared according to the method described in "2. Preparation of test solution" in Example 1. 25 mL of test solution was accurately measured, and 1.00 mL of mixed reference solution with the same content as the eight quality markers in the sample was accurately added to each sample. The samples were detected according to the detection method in Example 1. The peak area was recorded, and the recovery rate of each quality marker was calculated. The results are shown in Table 4.

[0087] Table 4. Statistical Table of Accuracy Test Results

[0088]

[0089]

[0090] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for determining the content of active ingredients in a Kunxinning preparation, characterized in that, Includes the following steps: Step 1: Preparation of reference solution: Take one or more of the following reference standards: catalpol, rehmannia glycoside D, paeoniflorin, verbenafiloside, (-)-eugenol-4-O-β-D-furanoside-(1→2)-β-D-glucopyranoside, curculigoside, icariin, and baicalin I, and prepare a reference solution. Step 2: Preparation of test solution: Weigh the Kunxinning preparation and place it in a volumetric flask. Dilute with methanol to the mark, filter, and take the filtrate. Step 3 Determination method: Pipe the reference solution and the test solution separately and inject them into the high performance liquid chromatograph to obtain the chromatogram. Calculate the content of the active ingredient in the test solution based on the chromatogram. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel as the stationary phase; water as mobile phase A; acetonitrile as mobile phase B; gradient elution; flow rate of 0.5-1.5 mL / min; column temperature of 25℃-35℃; detection wavelength of 200 nm-300 nm; and injection volume of 10 μL-30 μL. The gradient elution program was as follows:

2. The method according to claim 1, characterized in that, In the preparation of the reference solution in step 1, the concentrations of the following reference standards are as follows: 1.0000-3.0000 mg / mL for catalpol; 1.0000-3.0000 mg / mL for rehmannia glycoside D; 2.0000-4.5000 mg / mL for paeoniflorin; 0.0500-0.2000 mg / mL for verbascoside; 0.1200-0.3600 mg / mL for (-)-eugenol-4-O-β-D-furanoside-(1→2)-β-D-glucopyranoside; 0.0500-0.2000 mg / mL for curculigoside; 0.5000-2.0000 mg / mL for icariin; and 0.1000-0.3000 mg / mL for cymosiderin I.

3. The method according to claim 2, characterized in that, The preparation of the reference solution in step 1 is as follows: Accurately weigh appropriate amounts of the following reference standards: catalpol, rehmannia glycoside D, paeoniflorin, verbascoside, (-)-eugenol-4-O-β-D-furanoside-(1→2)-β-D-pyranoside, curculigoside, and icariin. Add methanol to prepare reference stock solutions with concentrations of 2.0000 mg / mL, 2.0000 mg / mL, 4.1250 mg / mL, 0.1000 mg / mL, 0.2400 mg / mL, 0.1000 mg / mL, and 1.0000 mg / mL, respectively. Accurately weigh an appropriate amount of cymosin I reference standard and add acetonitrile to prepare a reference stock solution with a concentration of 0.1800 mg / mL. Accurately measure 0.50 mL of each of the above reference stock solutions, mix thoroughly, and obtain a mixed reference solution.

4. The method according to claim 1, characterized in that, The preparation method of the test solution in step 2 is as follows: Take Kunxinning preparation, grind it into a fine powder, accurately weigh 0.5-2g, place it in a volumetric flask, add methanol, sonicate for 15-60min, cool, dilute with methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

5. The method according to claim 4, characterized in that, The preparation method of the test solution in step 2 is as follows: Take Kunxinning preparation, grind it into a fine powder, accurately weigh about 1g, place it in a 50mL volumetric flask, add about 40mL of methanol, sonicate for 30min, ultrasonic power 300W, ultrasonic frequency 50kHz, take it out, let it cool, dilute it with methanol to the mark, shake it well, filter it, and take the filtrate to obtain the test solution.

6. The method according to claim 1, characterized in that, In the determination method of step 3, the flow rate is 1.0 mL / min, the column temperature is 28℃, the detection wavelength is 210 nm, and the injection volume is 20 μL; the chromatographic column is... NX-C18 4.6mm×250mm, 5μm.

7. The method according to claim 1, characterized in that, Includes the following steps: Step 1: Preparation of the reference solution Accurately weigh appropriate amounts of the following reference standards: ziziphus jujuba var. spinosa, rehmannia glutinosa D, paeoniflorin, verrucoside, (-)-eugenol-4-O-β-D-furanoside-(1→2)-β-D-pyranoside, curculigoside, and icariin. Dissolve them in methanol to prepare stock solutions with concentrations of 2.0000 mg / mL, 2.0000 mg / mL, 4.1250 mg / mL, 0.1000 mg / mL, 0.2400 mg / mL, 0.1000 mg / mL, and 1.0000 mg / mL, respectively. Accurately weigh an appropriate amount of cymosin I reference standard and dissolve it in acetonitrile to prepare a stock solution with a concentration of 0.1800 mg / mL. Accurately measure 0.50 mL of each of the above stock solutions and mix thoroughly to obtain a mixed reference solution. Step 2: Preparation of the test solution Take Kunxinning granules, grind them into a fine powder, accurately weigh about 1g, place them in a 50mL volumetric flask, add about 40mL of methanol, sonicate for 30min, ultrasonic power 300W, ultrasonic frequency 50kHz, remove, cool, dilute with methanol to the mark, shake well, filter, and take the filtrate to obtain the product. Step 3, Detection Method Accurately pipette the reference solution and the test solution separately, inject them into the high performance liquid chromatograph, and determine them according to the chromatographic conditions. Quantify using the external standard method. The chromatographic conditions are as follows: NX-C18 Chromatographic column, 4.6 mm × 250 mm, 5 μm; mobile phase A: water; mobile phase B: acetonitrile; gradient elution: 0–20 min, 97%–90% A, 3%–10% B; 20–25 min, 90%–80% A, 10%–20% B; 25–45 min, 80%–60% A, 20%–40% B; 45–55 min, 60%–50% A, 40%–50% B; 55–65 min, 50%–30% A, 50%–70% B; 65–70 min, 30%–15%, 70%–85% B; 70–72 min, 15%–0% A, 85%–100% B; injection volume: 20 μL; detection wavelength: 210 nm; flow rate: 1.0 mL / min. Column temperature: 28℃.

8. The method according to any one of claims 1-2, characterized in that, The Kunxinning preparation mentioned therein is selected from one or more of Kunxinning granules, Kunxinning capsules, and Kunxinning tablets.

9. The method according to claim 7, characterized in that, The detection results of the method are as follows: the content ranges of catalpol, rehmannia glycoside D, paeoniflorin, verbascoside, (-)-eugenol-4-O-β-D-furanoside-(1→2)-β-D-glucopyranoside, curculigoside, icariin and baicalin I in each 1g of Kunxinning granules are 3.5854~4.9141mg, 0.3869~0.6765mg, 6.6162~8.2455mg, 0.2890~0.6520mg, 0.0802~0.1552mg, 0.3647~0.5908mg, 1.9886~2.9467mg, and 0.0894~0.1289mg, respectively.

10. The method according to claim 1, characterized in that, The Kunxinning preparation is made from the following Chinese herbal raw materials in parts by weight: 10-15 parts Rehmannia glutinosa, 15-20 parts Abalone shell, 8-12 parts Albizia julibrissin bark, 10-15 parts Paeonia lactiflora, 16-24 parts Astragalus membranaceus, 10-15 parts Epimedium brevicornu, and 10-15 parts Curculigo orchioides; preferably, 4 parts Rehmannia glutinosa, 5 parts Abalone shell, 3 parts Albizia julibrissin bark, 4 parts Paeonia lactiflora, 6 parts Astragalus membranaceus, 4 parts Epimedium brevicornu, and 4 parts Curculigo orchioides.

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