Construction method and application of fingerprint spectrum of kidney-tonifying, blood-enriching and fetus-consolidating pill

Abstract

The invention provides a construction method and application of a fingerprint spectrum of kidney-tonifying, blood-enriching and fetus-consolidating pills, and belongs to the technical field of medicines. The method comprises the following steps: extracting the kidney-tonifying, blood-enriching and fetus-consolidating pill by adopting a methanol aqueous solution, and taking the kidney-tonifying, blood-enriching and fetus-consolidating pill extracting solution as a test solution; taking a methanol solution of paeoniflorin, ferulic acid, liquiritin, baicalin, quercetin, baicalein and schizandrin as a reference substance solution; respectively carrying out high performance liquid chromatography analysis on the reference substance solution and the test solution, and taking the chromatographic peak of the reference substance solution as the reference to obtain the fingerprint spectrum of the kidney-tonifying, blood-enriching and fetus-consolidating pill. The fingerprint spectrum of the kidney-tonifying, blood-enriching and fetus-consolidating pills is constructed by adopting the high performance liquid chromatography, the quality of the kidney-tonifying, blood-enriching and fetus-consolidating pills can be comprehensively reflected, and the method can be used for quality control of the kidney-tonifying, blood-enriching and fetus-consolidating

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a method for constructing and applying the fingerprint spectrum of a kidney-tonifying, blood-nourishing, and fetal-stabilizing pill. Background Technology

[0002] Recurrent spontaneous abortion (RSA) is clinically defined as two or more consecutive spontaneous abortions. Currently, the main treatment for RSA is medication, including both Western and traditional Chinese medicine. Commonly used Western medications include dydrogesterone, progesterone, heparin, aspirin, as well as hormonal drugs and immunosuppressants. In traditional Chinese medicine, RSA is referred to as "slippery fetus," and years of clinical practice have demonstrated its significant efficacy in treating this condition.

[0003] Yishen Buxue Gutai Pills have the effects of invigorating qi and nourishing blood, tonifying the kidneys and replenishing essence, clearing heat and calming the fetus. They are used for deficiency of both qi and blood, pale or dark complexion, fatigue and weakness, insufficient kidney essence, soreness or weakness of the lower back and knees, lower back pain, abdominal pain, threatened miscarriage, or repeated miscarriages. Yishen Buxue Gutai Pills are made from ginseng, astragalus, prepared rehmannia root, angelica, wolfberry, schisandra, white peony root, dodder seed, raspberry, chuanxiong rhizome, scutellaria root, eclipta, polygala root, and licorice. The formula contains ginseng, astragalus, rehmannia, and angelica as its main ingredients, which invigorate qi and nourish blood, and help stabilize the fetus. Goji berries replenish essence and marrow, and tonify the liver and kidneys. Schisandra chinensis astringes and consolidates, tonifies the kidneys and calms the mind. White peony nourishes and harmonizes blood, working together with the principal herbs to tonify the kidneys, invigorate qi, and nourish blood, thus making qi and blood more abundant. Cuscuta chinensis and raspberry enter the liver and kidneys, consolidating the Chong and Ren meridians, strengthening the fetus, and ensuring its stability. Invigorating qi and nourishing blood requires preventing stagnation. Stagnation within the body damages the Chong and Ren meridians, causing disharmony of qi and blood, and depriving the fetus of nourishment. Therefore, chuanxiong is used in the formula to invigorate blood, ensuring blood is generated without stagnation. Infertile women often consume rich and fatty foods, which can lead to obesity and heat accumulation. Furthermore, the generation of qi and blood is also a source of heat. However, heat forces the blood to flow, causing it to flow erratically due to heat. Therefore, the formula uses Scutellaria baicalensis and Eclipta prostrata to cool the blood, nourish yin, clear heat, and calm the fetus. Polygala tenuifolia helps with sleep and calms the mind, so that the heart is at peace, heat is cleared, and the uterus is at ease. Licorice root harmonizes the effects of the other herbs.

[0004] To guide the production of Yishen Buxue Gutai Pills and ensure their efficacy, quality control is necessary. However, no methods for quality control of Yishen Buxue Gutai Pills have been reported to date. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing and applying the fingerprint spectrum of Yishen Buxue Gutai Pill. This invention uses high performance liquid chromatography to construct the fingerprint spectrum of Yishen Buxue Gutai Pill, which can comprehensively reflect the quality of Yishen Buxue Gutai Pill and can be used for the quality control of Yishen Buxue Gutai Pill.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for constructing the fingerprint spectrum of Yishen Buxue Gutai Pill, comprising the following steps: The kidney-tonifying and blood-nourishing pill was extracted using a methanol-water solution, and the resulting extract was used as the test solution. The raw materials for preparing the kidney-tonifying and blood-nourishing pill were ginseng, astragalus, rehmannia, angelica, wolfberry, schisandra, white peony root, dodder seed, raspberry, chuanxiong, scutellaria, eclipta, polygala and licorice. A methanol solution of paeoniflorin, ferulic acid, glycyrrhizin, baicalin, quercetin, baicalein and schisandrol A was used as a reference solution. The reference solution and the test solution were analyzed by high performance liquid chromatography, and the fingerprint spectrum of Yishen Buxue Gutai Pill was obtained with the chromatographic peak of the reference solution as the reference. The mobile phase used in the high-performance liquid chromatography (HPLC) analysis includes mobile phase A and mobile phase B. Mobile phase A is acetonitrile, and mobile phase B is a 0.02-0.2% (v / v) aqueous solution of formic acid. The flow rate of the mobile phase is 0.7-1.2 mL / min. The column packing material is octadecylsilane-bonded silica gel. A gradient elution method is used, and the gradient elution program is as follows: 0-10 min, mobile phase A volume fraction 10%; 10-25 min, mobile phase A volume fraction increases from 10% to 15%; 25-40 min, mobile phase A volume fraction increases from 15% to 22%; 40-65 min, mobile phase A volume fraction increases from 22% to 40%; 65-75 min, mobile phase A volume fraction increases from 40% to 50%; 75-90 min, mobile phase A volume fraction increases from 50% to 70%.

[0007] Preferably, the chromatographic column is ACE C. 18 The column has the following specifications: length 250mm, inner diameter 4.60mm, filler particle size 5µm; column temperature 20~40℃.

[0008] Preferably, the detector used in the high-performance liquid chromatography analysis is an ultraviolet detector with a detection wavelength of 230~360nm; the injection volume is 5~15μL.

[0009] Preferably, the theoretical plate number of the high-performance liquid chromatography analysis, calculated based on the baicalin peak, is not less than 5000.

[0010] Preferably, the volume fraction of methanol in the methanol-water solution is 50-80%, and the ratio of the amount of the kidney-tonifying and blood-nourishing pill to the methanol-water solution is 0.2-2g:10-100mL.

[0011] Preferably, the extraction is performed under ultrasonic conditions, wherein the ultrasonic power is 100~300W and the frequency is 30~50kHz; and the extraction time is 20~60min.

[0012] Preferably, the fingerprint spectrum of the Yishen Buxue Gutai Pill contains 24 common peaks, among which peaks 6, 7, 9, 15, 16, 19, and 21 are respectively identified as paeoniflorin, ferulic acid, glycyrrhizin, baicalin, quercetin, baicalein, and schisandrol A.

[0013] Preferably, the 24 common peaks are respectively attributed to Paeonia lactiflora, Angelica sinensis, Rubus idaeus, Lycium barbarum, Astragalus membranaceus, Rehmannia glutinosa, Cuscuta chinensis, Schisandra chinensis, Polygala tenuifolia, Ligusticum chuanxiong, Glycyrrhiza uralensis, Scutellaria baicalensis, Ginseng, and Eclipta prostrata; wherein peak 1 is attributed to Paeonia lactiflora, Angelica sinensis, Rubus idaeus, Lycium barbarum, Astragalus membranaceus, Rehmannia glutinosa, Cuscuta chinensis, Schisandra chinensis, and Polygala tenuifolia; peak 2 is attributed to Paeonia lactiflora and Schisandra chinensis; peak 3 is attributed to Cuscuta chinensis; peak 4 is attributed to Schisandra chinensis; peak 5 is attributed to Cuscuta chinensis; peak 6 is attributed to Paeonia lactiflora; peak 7 is attributed to Angelica sinensis and Ligusticum chuanxiong; peak 8 is attributed to Astragalus membranaceus and Cuscuta chinensis; and peak 9 belongs to... The peaks are classified as follows: Glycyrrhiza uralensis; Peak 10 belongs to Rubus idaeus; Peak 11 belongs to Cuscuta chinensis; Peak 12 belongs to Scutellaria baicalensis; Peak 13 belongs to Polygala tenuifolia; Peak 14 belongs to Ligusticum chuanxiong, Angelica sinensis, Eclipta prostrata, and Polygala tenuifolia; Peak 15 belongs to Scutellaria baicalensis; Peak 16 belongs to Scutellaria baicalensis; Peak 17 belongs to Scutellaria baicalensis; Peak 18 belongs to Scutellaria baicalensis; Peak 19 belongs to Scutellaria baicalensis; Peak 20 belongs to Scutellaria baicalensis; Peak 21 belongs to Schisandra chinensis; Peak 22 belongs to Ligusticum chuanxiong; Peak 23 belongs to Ligusticum chuanxiong, Angelica sinensis, Rubus idaeus, Panax ginseng, and Cuscuta chinensis; Peak 24 belongs to Ligusticum chuanxiong, Angelica sinensis, and Rubus idaeus.

[0014] Preferably, the prescription of the Kidney-Nourishing and Blood-Tonifying Pregnancy-Strengthening Pill, by weight parts, is as follows: 22.5 parts ginseng, 37.5 parts astragalus, 37.5 parts prepared rehmannia root, 30 parts angelica, 30 parts wolfberry, 30 parts schisandra, 25 parts white peony root, 50 parts dodder seed, 50 parts raspberry, 22.5 parts chuanxiong rhizome, 25 parts scutellaria root, 30 parts eclipta, 22.5 parts polygala root, and 12.5 parts licorice root; The preparation method of the kidney-tonifying, blood-nourishing, and fetal-stabilizing pills includes the following steps: Ginseng, angelica, chuanxiong, dodder seed and raspberry are crushed to obtain coarse powder with a particle size of 20-40 mesh. Astragalus membranaceus, Rehmannia glutinosa, Lycium barbarum, Schisandra chinensis, Paeonia lactiflora, Scutellaria baicalensis, Eclipta prostrata, Polygala tenuifolia, and Glycyrrhiza uralensis were decocted in water, and the resulting decoction was concentrated to obtain a paste. The coarse powder is mixed with the paste, and then dried and pulverized in sequence to obtain fine powder with a particle size of 100-120 mesh. The fine powder is made into pills with water, and then dried, coated with talc powder, and polished with water to obtain the kidney-tonifying, blood-nourishing, and fetal-stabilizing pills.

[0015] This invention provides the application of the method for constructing the fingerprint spectrum of Yishen Buxue Gutai Pill described in the above technical solution in the quality control of Yishen Buxue Gutai Pill.

[0016] Beneficial Effects: This invention employs high-performance liquid chromatography (HPLC) to construct a fingerprint chromatogram of Yishen Buxue Gutai Pill, which comprehensively reflects the overall chemical composition of the pill and characterizes its quality. This provides an effective means for the overall quality control and evaluation of Yishen Buxue Gutai Pill, facilitating comprehensive monitoring of product quality stability, consistency, and controllability. It overcomes the deficiency of existing technologies that lack reports on fingerprint chromatograms of Yishen Buxue Gutai Pill. The method of this invention is convenient, rapid, stable, highly precise, and reproducible, enabling a more comprehensive, objective, and scientific evaluation of the quality of Yishen Buxue Gutai Pill, thereby ensuring the safety and effectiveness of clinical medication.

[0017] Furthermore, this invention, through systematic component identification and attribution of single medicinal materials with common peaks in different batches of the drug, comprehensively reflects the current status of each component in Yishen Buxue Gutai Pill, providing a reference for the quality of Yishen Buxue Gutai Pill. Specifically, the fingerprint spectrum of Yishen Buxue Gutai Pill constructed by this invention has 24 peaks, including the effective components belonging to fourteen medicinal materials: white peony root, angelica root, raspberry, wolfberry, astragalus root, prepared rehmannia root, dodder seed, schisandra fruit, polygala root, chuanxiong rhizome, licorice root, scutellaria root, ginseng, and eclipta prostrata. This can efficiently characterize the quality of Yishen Buxue Gutai Pill, which is beneficial for comprehensive product quality monitoring. Attached Figure Description

[0018] Figure 1 The HPLC chromatogram of the mixed reference solution; Figure 2 The HPLC chromatogram of the test solution (batch number 240601); Figure 3 Overlay fingerprint images of 10 batches of Yishen Buxue Gutai Pills; Figure 4 This is a comparison chart of sample fingerprint chromatograms and chromatograms of various medicinal materials. Figure 5 Image of Yishen Buxue Gutai Pill at 360nm wavelength; Figure 6 Atlas of Yishen Buxue Gutai Pill at 320nm wavelength; Figure 7 Atlas of Yishen Buxue Gutai Pill at 280nm wavelength; Figure 8 Image of Yishen Buxue Gutai Pill at 254nm wavelength; Figure 9 Image of Yishen Buxue Gutai Pill at 230nm wavelength; Figure 10 Atlas of Yishen Buxue Gutai Pill at 210nm wavelength; Figure 11 Image of Yishen Buxue Gutai Pill at 203nm wavelength. Detailed Implementation

[0019] This invention provides a method for constructing the fingerprint spectrum of Yishen Buxue Gutai Pill, comprising the following steps: The kidney-tonifying and blood-nourishing pill was extracted using a methanol-water solution, and the resulting extract was used as the test solution. The raw materials for preparing the kidney-tonifying and blood-nourishing pill were ginseng, astragalus, rehmannia, angelica, wolfberry, schisandra, white peony root, dodder seed, raspberry, chuanxiong, scutellaria, eclipta, polygala and licorice. A methanol solution of paeoniflorin, ferulic acid, glycyrrhizin, baicalin, quercetin, baicalein and schisandrol A was used as a reference solution. The reference solution and the test solution were analyzed by high performance liquid chromatography, and the fingerprint spectrum of Yishen Buxue Gutai Pill was obtained with the chromatographic peak of the reference solution as the reference. The mobile phase used in the high-performance liquid chromatography (HPLC) analysis includes mobile phase A and mobile phase B. Mobile phase A is acetonitrile, and mobile phase B is a 0.02-0.2% (v / v) aqueous solution of formic acid. The flow rate of the mobile phase is 0.7-1.2 mL / min. The column packing material is octadecylsilane-bonded silica gel. A gradient elution method is used, and the gradient elution program is as follows: 0-10 min, mobile phase A volume fraction 10%; 10-25 min, mobile phase A volume fraction increases from 10% to 15%; 25-40 min, mobile phase A volume fraction increases from 15% to 22%; 40-65 min, mobile phase A volume fraction increases from 22% to 40%; 65-75 min, mobile phase A volume fraction increases from 40% to 50%; 75-90 min, mobile phase A volume fraction increases from 50% to 70%.

[0020] In this invention, unless otherwise specified, all raw materials used are commercially available products well known to those skilled in the art or prepared using methods well known to those skilled in the art.

[0021] This invention uses a methanol-water solution to extract the kidney-tonifying and blood-nourishing formula, and uses the resulting extract as the test solution. In this invention, the raw materials for preparing the kidney-tonifying and blood-nourishing formula are ginseng, astragalus, rehmannia root, angelica, wolfberry, schisandra, white peony root, dodder seed, raspberry, chuanxiong rhizome, scutellaria root, eclipta, polygala root, and licorice. As one embodiment of this invention, the formula of the kidney-tonifying and blood-nourishing formula, by weight, is: ginseng 22.5 parts, astragalus root 37.5 parts, rehmannia root 37.5 parts, angelica root 30 parts, wolfberry fruit 30 parts, schisandra fruit 30 parts, white peony root 25 parts, dodder seed 50 parts, raspberry 50 parts, chuanxiong rhizome 22.5 parts, scutellaria root 25 parts, eclipta, polygala root 22.5 parts, and licorice root 12.5 parts.

[0022] As one embodiment of the present invention, the preparation method of the Kidney-Nourishing and Blood-Tonifying Pregnancy-Strengthening Pill includes the following steps: Ginseng, Angelica sinensis, Ligusticum chuanxiong, Cuscuta chinensis, and Rubus idaeus are pulverized to obtain coarse powder with a particle size of 20-40 mesh; Astragalus membranaceus, Rehmannia glutinosa, Lycium barbarum, Schisandra chinensis, Paeonia lactiflora, Scutellaria baicalensis, Eclipta prostrata, Polygala tenuifolia, and Glycyrrhiza uralensis are decocted in water, and the resulting decoction is concentrated to obtain a paste; the coarse powder and the paste are mixed, and then dried and pulverized sequentially to obtain fine powder with a particle size of 100-120 mesh; the fine powder is made into pills with water, and then sequentially dried, coated with talc powder, and polished with water to obtain the Kidney-Nourishing and Blood-Tonifying Pregnancy-Strengthening Pill. The preparation method of the Kidney-Nourishing and Blood-Tonifying Pregnancy-Strengthening Pill is described in detail below.

[0023] This invention involves pulverizing ginseng, angelica, chuanxiong, dodder seed, and raspberry to obtain coarse powder with a particle size of 20-40 mesh. This invention does not impose any particular limitation on the pulverization method; any method that yields coarse powder of the desired particle size is acceptable.

[0024] This invention involves decocting Astragalus membranaceus, Rehmannia glutinosa, Lycium barbarum, Schisandra chinensis, Paeonia lactiflora, Scutellaria baicalensis, Eclipta prostrata, Polygala tenuifolia, and Glycyrrhiza uralensis in water, and then concentrating the resulting decoction to obtain a paste. In one embodiment, the decoction can be performed twice. After the two decoctions, the resulting liquids are combined and filtered, and the filtrate is the decoction. The first decoction can last for 2.5–3.5 hours, specifically 3 hours; the second decoction can last for 1.5–2.5 hours, specifically 2 hours. The mass of water used in both decoctions can be 7.5–8.5 times the total mass of Astragalus membranaceus, Rehmannia glutinosa, Lycium barbarum, Schisandra chinensis, Paeonia lactiflora, Scutellaria baicalensis, Eclipta prostrata, Polygala tenuifolia, and Glycyrrhiza uralensis, specifically 8 times. In one embodiment, based on the density of water, the relative density of the paste at 60°C can be 1.30–1.35. This invention does not specifically limit the concentration method; any method that yields a paste with the desired relative density is acceptable.

[0025] After obtaining coarse powder and paste, the present invention mixes the coarse powder and paste, and then successively dries and pulverizes them to obtain fine powder with a particle size of 100-120 mesh. The present invention does not have specific limitations on the drying process, as long as the material is sufficiently dried; the present invention also does not have specific limitations on the pulverization method, as long as the desired particle size of fine powder is obtained.

[0026] After obtaining the fine powder, the present invention uses water to form pellets, followed by drying, talc coating, and polishing with water to obtain the kidney-tonifying, blood-nourishing, and fetal-stabilizing pills. In one embodiment of the present invention, the particle size of the talc powder can be 400 mesh, and the mass of the talc powder can be 0.5-2.5% of the mass of the kidney-tonifying, blood-nourishing, and fetal-stabilizing pills, more specifically 1-2%. The present invention does not have any particular limitations on the methods and conditions for water-forming, drying, and polishing; conventional methods and conditions in the art can be used.

[0027] In this embodiment of the invention, using 22.5g of ginseng, 37.5g of astragalus, 37.5g of prepared rehmannia root, 30g of angelica, 30g of wolfberry, 30g of schisandra, 25g of white peony root, 50g of dodder seed, 50g of raspberry, 22.5g of chuanxiong rhizome, 20g of licorice root, 25g of scutellaria root, 30g of eclipta prostrata, 22.5g of polygala root, and 12.5g of licorice root as raw materials, and following the above method, a total of 1000 pills of Yishen Buxue Gutai Wan (Kidney-Nourishing, Blood-tonifying, and Fetal-Strengthening Pills) were prepared.

[0028] After obtaining the Yishen Buxue Gutai Pill, this invention uses a methanol-water solution to extract the pill, and uses the resulting extract as a test solution. In one embodiment of this invention, the volume fraction of methanol in the methanol-water solution can be 50-80%, more preferably 70-78%, specifically 75%, and the ratio of the Yishen Buxue Gutai Pill to the methanol-water solution can be 0.2-2g:10-100mL, specifically 0.5g:25mL. In another embodiment of this invention, the extraction is performed under ultrasonic conditions, the ultrasonic power can be 100-300W, specifically 250W; the frequency can be 30-50kHz, specifically 40kHz; and the extraction time can be 20-60min, specifically 30min. In another embodiment of this invention, the extraction preferably includes filtration, and the filter membrane used for filtration is preferably a 0.45μm microporous membrane. In this embodiment of the invention, specifically, 0.5g of Yishen Buxue Gutai Pills are ground into a fine powder and accurately weighed. The powder is placed in a stoppered conical flask, and 25mL of a 75% methanol aqueous solution is accurately added. The powder is weighed and extracted ultrasonically for 30min at a power of 250W and a frequency of 40kHz. After cooling, the powder is weighed again. The lost weight is replenished with a 75% methanol aqueous solution, and the powder is shaken well. The powder is filtered through a 0.45μm microporous membrane, and the filtrate is used as the test solution.

[0029] This invention uses a methanol solution of paeoniflorin, ferulic acid, glycyrrhizin, baicalin, quercetin, baicalein, and schisandrol A as a reference solution. By using these references, the invention can effectively compare and identify the components in a sample of Yishen Buxue Gutai Pill. Through comparative analysis of the sample and reference chromatograms, the types of each component in the sample can be more accurately determined, thereby achieving control and evaluation of sample quality. If the reference is not selected appropriately, the obtained fingerprint chromatogram may not fully reflect the quality of the drug, thus hindering comprehensive control and evaluation of drug quality. In one embodiment of the present invention, the concentrations of paeoniflorin, ferulic acid, glycyrrhizin, baicalin, schisandrol A, and schisandrol A in the reference solution are 0.0985 mg / mL, 0.00999 mg / mL, 0.020144 mg / mL, 0.09636 mg / mL, 0.010804 mg / mL, 0.01057 mg / mL, and 0.011468 mg / mL. Preferably, methanol is used as the solvent to prepare separate stock solutions of paeoniflorin, ferulic acid, glycyrrhizin, baicalin, quercetin, baicalin, and schisandrol A. Appropriate amounts of each stock solution are then mixed, diluted with methanol, filtered, and the filtrate is used as the reference solution. In this embodiment of the invention, specifically, appropriate amounts of paeoniflorin, ferulic acid, glycyrrhizin, baicalin, quercetin, baicalein, and schisandrol A reference standards are accurately weighed and placed in 10 mL volumetric flasks. Using methanol as the solvent, reference standard stock solutions with mass concentrations of 0.4925 mg / mL, 0.4995 mg / mL, 0.5036 mg / mL, 0.4818 mg / mL, 0.5402 mg / mL, 0.5285 mg / mL, and 0.5734 mg / mL are prepared, respectively. Then, 2 mL of each reference standard stock solution and 0 mL of the flask are accurately pipetted into the flask. 0.2 mL, 0.4 mL, 2 mL, 0.2 mL, 0.2 mL, and 0.2 mL were placed in the same 10 mL volumetric flask, diluted to the mark with methanol, and shaken well to prepare methanol solutions with mass concentrations of 0.0985 mg / mL, 0.00999 mg / mL, 0.020144 mg / mL, 0.09636 mg / mL, 0.010804 mg / mL, 0.01057 mg / mL, and 0.011468 mg / mL, respectively. The solutions were filtered through a 0.45 μm microporous membrane, and the filtrate was used as the reference solution.

[0030] After obtaining the reference solution and the test solution, the present invention performs high performance liquid chromatography analysis on the reference solution and the test solution respectively, and obtains the fingerprint spectrum of Yishen Buxue Gutai Pill based on the chromatographic peak of the reference solution. In this invention, the mobile phase used for high-performance liquid chromatography (HPLC) analysis includes mobile phase A and mobile phase B. Mobile phase A is acetonitrile, and mobile phase B is an aqueous formic acid solution with a volume fraction of 0.02-0.2%, specifically 0.1%. The flow rate of the mobile phase is 0.7-1.2 mL / min, specifically 1 mL / min. The chromatographic column is packed with octadecylsilane-bonded silica gel. A gradient elution method is used, with the following gradient elution program: 0-10 min, mobile phase A volume fraction 10%; 10-25 min, mobile phase A volume fraction increases from 10% to 15%; 25-40 min, mobile phase A volume fraction increases from 15% to 22%; 40-65 min, mobile phase A volume fraction increases from 22% to 40%; 65-75 min, mobile phase A volume fraction increases from 40% to 50%; 75-90 min, mobile phase A volume fraction increases from 50% to 70%. In the gradient elution program described in this invention, the volume fraction of mobile phase A changes independently and uniformly at each stage. This invention, employing the aforementioned gradient elution program, can effectively separate the main components of the Yishen Buxue Gutai Wan formula; however, an improper gradient elution program can affect chromatographic peak separation, leading to significant differences in detection results.

[0031] In one embodiment of the present invention, the chromatographic column is ACE C. 18 The column has the following specifications: length 250 mm, inner diameter 4.60 mm, and packing material particle size 5 µm; the column temperature is 20~40℃, specifically 25~30℃. In one embodiment of the invention, the detector used for the high-performance liquid chromatography (HPLC) analysis is an ultraviolet detector with a detection wavelength of 230~360 nm, specifically 360 nm, 320 nm, 280 nm, 254 nm, or 230 nm, preferably 254 nm; the injection volume is 5~15 μL, specifically 10 μL. In one embodiment of the invention, the theoretical plate number of the HPLC analysis, calculated based on the baicalin peak, is not less than 5000.

[0032] In the present invention, after performing high performance liquid chromatography (HPLC) analysis on the reference solution, a reference chromatogram is obtained; after performing HPLC analysis on the test solution, a sample chromatogram is obtained. Preferably, the present invention imports the sample chromatogram into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints for similarity analysis to obtain the fingerprint of Yishen Buxue Gutai Pills. As an embodiment of the present invention, the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints is specifically the 2012A version of the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints. Preferably, the present invention uses the median method for the similarity analysis, and the time window width can be set to 0.1 min, and a total of 24 common peaks are calibrated. In the examples of the present invention, specifically, HPLC analysis is performed on 10 batches of Yishen Buxue Gutai Pills, and the obtained sample chromatograms are imported into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints for similarity analysis. The similarity of each batch of Yishen Buxue Gutai Pills is greater than 0.90. The present invention compares the sample chromatogram with the reference chromatogram, and the chromatographic peaks at the same retention time as each reference are identified. The results show that among the 24 common peaks, the 6th, 7th, 9th, 15th, 16th, 19th, and 21st peaks are respectively identified as paeoniflorin, ferulic acid, liquiritin, baicalin, quercetin, baicalein, and schisandrin.

[0033] The present invention assigns the components of the 24 common peaks. The 24 common peaks are respectively attributed to Radix Paeoniae Alba, Radix Angelicae Sinensis, Fructus Rubi, Fructus Lycii, Radix Astragali, Radix Rehmanniae Praeparata, Semen Cuscutae, Fructus Schisandrae Chinensis, Radix Polygalae, Rhizoma Chuanxiong, Radix Glycyrrhizae, Radix Scutellariae, Radix Ginseng, and Herba Ecliptae. Among them, the 1st peak is attributed to Radix Paeoniae Alba, Radix Angelicae Sinensis, Fructus Rubi, Fructus Lycii, Radix Astragali, Radix Rehmanniae Praeparata, Semen Cuscutae, Fructus Schisandrae Chinensis, and Radix Polygalae; the 2nd peak is attributed to Radix Paeoniae Alba and Fructus Schisandrae Chinensis; the 3rd peak is attributed to Semen Cuscutae; the 4th peak is attributed to Fructus Schisandrae Chinensis; the 5th peak is attributed to Semen Cuscutae; the 6th peak is attributed to Radix Paeoniae Alba; the 7th peak is attributed to Radix Angelicae Sinensis and Rhizoma Chuanxiong; the 8th peak is attributed to Radix Astragali and Semen Cuscutae; the 9th peak is attributed to Radix Glycyrrhizae; the 10th peak is attributed to Fructus Rubi; the 11th peak is attributed to Semen Cuscutae; the 12th peak is attributed to Radix Scutellariae; the 13th peak is attributed to Radix Polygalae; the 14th peak is attributed to Rhizoma Chuanxiong, Radix Angelicae Sinensis, Herba Ecliptae, and Radix Polygalae; the 15th peak is attributed to Radix Scutellariae; the 16th peak is attributed to Radix Scutellariae; the 17th peak is attributed to Radix Scutellariae; the 18th peak is attributed to Radix Scutellariae; the 19th peak is attributed to Radix Scutellariae; the 20th peak is attributed to Radix Scutellariae; the 2lth peak is attributed to Fructus Schisandrae Chinensis; the 22nd peak is attributed to Rhizoma Chuanxiong; the 23rd peak is attributed to Rhizoma Chuanxiong, Radix Angelicae Sinensis, Fructus Rubi, Radix Ginseng, and Semen Cuscutae; the 24th peak is attributed to Rhizoma Chuanxiong, Radix Angelicae Sinensis, and Fructus Rubi.

[0034] The present invention provides the application of the method for constructing the fingerprint of Yishen Buxue Gutai Pills described in the above technical solution in the quality control of Yishen Buxue Gutai Pills.

[0035] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0036] Preparation Example 1 The prescription for Yishen Buxue Gutai Wan (Kidney-Nourishing, Blood-Tonifying, and Fetal-Stabilizing Pill) is as follows: Ginseng 22.5g, Astragalus 37.5g, Rehmannia glutinosa 37.5g, Angelica sinensis 30g, Lycium barbarum 30g, Schisandra chinensis 30g, Paeonia lactiflora 25g, Cuscuta chinensis 50g, Rubus idaeus 50g, Ligusticum chuanxiong 22.5g, Scutellaria baicalensis 25g, Eclipta prostrata 30g, Polygala tenuifolia 22.5g, and Glycyrrhiza uralensis 12.5g.

[0037] The preparation method of the kidney-tonifying, blood-nourishing, and fetal-stabilizing pills includes the following steps: According to the above prescription, ginseng, angelica, chuanxiong, dodder seed, and raspberry are pulverized to obtain coarse powder with a particle size of 20-40 mesh; astragalus, rehmannia, wolfberry, schisandra, white peony root, scutellaria, eclipta, polygala, and licorice are decocted twice with water. The first decoction time is 3 hours, and the second decoction time is 2 hours. The mass of water used in both decoctions is 8 times the total mass of astragalus, rehmannia, wolfberry, schisandra, white peony root, scutellaria, eclipta, polygala, and licorice. After boiling, the resulting liquids are combined and filtered to obtain a decoction. The decoction is then concentrated to obtain a paste with a relative density of 1.32 (60°C). The coarse powder is mixed with the paste and then dried and pulverized to obtain a fine powder with a particle size of 100-120 mesh. The fine powder is then coated with water to form pills, followed by drying, coating with talc powder (400 mesh, 1% of the weight of the Yishen Buxue Gutai pill), and polishing with water to obtain 1000 pills of the Yishen Buxue Gutai pill.

[0038] Example 1: Detection of fingerprint spectra of different batches of Yishen Buxue Gutai Wan (a traditional Chinese medicine formula). 1. Instruments and reagents 1.1 Agilent 1220 high performance liquid chromatograph (Agilent Technologies, USA); KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); BSA 124S electronic balance (Sartorius); QUINTIX35-1CN (Sartorius).

[0039] 1.2 Wahaha purified water, acetonitrile (chromatographic grade), and other reagents were of analytical grade. Paeoniflorin, ferulic acid, glycyrrhizin, baicalin, quercetin, baicalein, and schisandrol A reference standards (batch numbers 110736-202447, 110773-202316, 111610-202209, 110715-202223, 100081-202411, 111595-202309, and 110857-202316, with purities of 99.1%, 99.3%, 95.2%, 97.2%, 99.3%, 98.6%, and 99.2%, respectively, were all purchased from the China National Institutes for Food and Drug Control). Kidney-Nourishing and Blood-Nourishing Pregnancy-Strengthening Pills (batch numbers 240601, 240602, 240603, 240801, 240802, 240803, 250201, 250202, 250203, and 250204, all prepared according to the method in Preparation Example 1).

[0040] 2. Fingerprint spectroscopy determination 2.1 Chromatographic conditions: The column packing material was octadecylsilane-bonded silica gel, and the column type was ACE C. 18 The column has the following specifications: length 250 mm, inner diameter 4.60 mm, and packing material particle size 5 µm; column temperature 25℃; detector is a UV detector with a detection wavelength of 254 nm; the mobile phase consists of mobile phase A and mobile phase B, where mobile phase A is acetonitrile and mobile phase B is a 0.1% (v / v) formic acid aqueous solution; the mobile phase flow rate is 1.0 mL / min; gradient elution is used, and the gradient elution program is as follows: 0–10 min, mobile phase A (v / v) 10%; 10–25 min, mobile phase A (v / v) 10%; At 25-40 min, the volume fraction of mobile phase A increases from 10% to 15%; at 40-65 min, the volume fraction of mobile phase A increases from 15% to 22%; at 65-75 min, the volume fraction of mobile phase A increases from 40% to 50%; at 75-90 min, the volume fraction of mobile phase A increases from 50% to 70%; the theoretical plate number calculated based on the baicalin peak is not less than 5000; the injection volume is 10 μL.

[0041] 2.2 Preparation of the test solution: Take Yishen Buxue Gutai Pill, grind it into a fine powder, take 0.5g, weigh it accurately, place it in a stoppered conical flask, accurately add 25mL of 75% methanol aqueous solution, weigh it, extract it by ultrasonication for 30min at a power of 250W and a frequency of 40kHz, cool it, weigh it again, make up the lost weight with 75% methanol aqueous solution, shake it well, filter it through a 0.45μm microporous membrane, and take the filtrate as the test solution.

[0042] 2.3 Preparation of reference medicinal materials and reference extracts: Weigh the reference extracts or reference medicinal materials according to the prescription ratio and preparation method, and prepare them according to the preparation method of the test sample.

[0043] 2.4 Preparation of mixed reference solutions: Accurately weigh appropriate amounts of paeoniflorin, ferulic acid, glycyrrhizin, baicalin, quercetin, baicalein, and schisandrol A reference standards, and place them in 10 mL volumetric flasks. Using methanol as the solvent, prepare reference stock solutions with mass concentrations of 0.4925 mg / mL, 0.4995 mg / mL, 0.5036 mg / mL, 0.4818 mg / mL, 0.5402 mg / mL, 0.5285 mg / mL, and 0.5734 mg / mL, respectively. Then, accurately pipette 2 mL of the reference stock solution and 0 mL of the schisandrol A solution into separate volumetric flasks. 0.2 mL, 0.4 mL, 2 mL, 0.2 mL, 0.2 mL, and 0.2 mL were placed in the same 10 mL volumetric flask, diluted to the mark with methanol, and shaken well to prepare methanol solutions with mass concentrations of 0.0985 mg / mL, 0.00999 mg / mL, 0.020144 mg / mL, 0.09636 mg / mL, 0.010804 mg / mL, 0.01057 mg / mL, and 0.011468 mg / mL, respectively. The solutions were filtered through a 0.45 μm microporous membrane, and the filtrate was used as a mixed reference solution.

[0044] 2.5 Determination: Accurately pipette 10 μL each of the mixed reference solution and the test solution into the high-performance liquid chromatograph (HPLC), and record the chromatograms within 90 min. Figure 1 This is the HPLC chromatogram of the mixed reference solution. Figure 2 This is the HPLC chromatogram of the test solution (batch number 240601). The identification of each peak in the chromatogram will be explained in detail later.

[0045] Example 2: Fingerprint analysis of 10 batches of Yishen Buxue Gutai Pills 1. Fingerprint similarity analysis Ten batches of Yishen Buxue Gutai Pills were collected and samples were prepared according to the test solution preparation method in Example 1. The samples were injected and detected under the chromatographic conditions described in Example 1, and the fingerprint chromatograms of Yishen Buxue Gutai Pills were recorded. The similarity analysis of the fingerprint chromatograms of the ten batches of Yishen Buxue Gutai Pills was performed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (Version 2012A)" issued by the National Pharmacopoeia Commission. S1 was set as the reference chromatogram. Using the median method, the time window width was set to 0.1 min, and a total of 24 common peaks were identified. The similarity of the ten batches of Yishen Buxue Gutai Pills was greater than 0.90. The similarity evaluation results are shown in Table 1. The relative retention times of the 24 common peaks were basically consistent, while the relative peak areas showed significant differences. The standard fingerprint chromatogram data are shown in Table 2. Figure 3 Overlay fingerprint images of 10 batches of Yishen Buxue Gutai Pills. Figure 3 S1~S10 correspond to batch numbers 240601, 240602, 240603, 240801, 240802, 240803, 250201, 250202, 250203, and 250204 of Yishen Buxue Gutai Pills, respectively. R is a reference image.

[0046] Table 1. Similarity evaluation results of 10 batches of Yishen Buxue Gutai Pills

[0047] Table 2 Standard fingerprint data

[0048] 2. Common peak identification: The HPLC chromatogram of the mixed reference solution ( Figure 1 ) and sample spectrum ( Figure 2 By comparison, the chromatographic peaks at the same retention time as the reference standards were identified. The results showed that peaks 6, 7, 9, 15, 16, 19, and 21 were identified as paeoniflorin, ferulic acid, glycyrrhizin, baicalin, quercetin, baicalein, and schisandrol A, respectively.

[0049] 3. Assignment of medicinal materials: Assignment of chromatographic peaks according to standard fingerprint chromatographic conditions. Figure 4This is a comparison chart of the sample fingerprint chromatogram and the chromatograms of each medicinal herb, where R represents the sample, A is the chromatogram of Scutellaria baicalensis, B is the chromatogram of Paeonia lactiflora, C is the chromatogram of Ligusticum chuanxiong, D is the chromatogram of Angelica sinensis, E is the chromatogram of Rubus idaeus, F is the chromatogram of Glycyrrhiza uralensis, G is the chromatogram of Lycium barbarum, H is the chromatogram of Astragalus membranaceus, I is the chromatogram of Eclipta prostrata, J is the chromatogram of Panax ginseng, K is the yellow chromatogram of Rehmannia glutinosa, L is the chromatogram of Cuscuta chinensis, M is the chromatogram of Schisandra chinensis, and N is the chromatogram of Polygala tenuifolia. The 24 common peaks are respectively attributed to fourteen medicinal herbs, including white peony root, angelica root, raspberry, wolfberry, astragalus root, prepared rehmannia root, dodder seed, schisandra fruit, polygala root, chuanxiong rhizome, licorice root, scutellaria root, ginseng, and eclipta prostrata. Among them, peak 1 belongs to white peony root, angelica root, raspberry, wolfberry, astragalus root, prepared rehmannia root, dodder seed, schisandra fruit, and polygala root; peak 2 belongs to white peony root and schisandra fruit; peak 3 belongs to dodder seed; peak 4 belongs to schisandra fruit; peak 5 belongs to dodder seed; peak 6 belongs to white peony root; peak 7 belongs to angelica root and chuanxiong rhizome; peak 8 belongs to astragalus root and dodder seed; peak 9 belongs to... The peaks are classified as follows: Glycyrrhiza uralensis; Peak 10 belongs to Rubus idaeus; Peak 11 belongs to Cuscuta chinensis; Peak 12 belongs to Scutellaria baicalensis; Peak 13 belongs to Polygala tenuifolia; Peak 14 belongs to Ligusticum chuanxiong, Angelica sinensis, Eclipta prostrata, and Polygala tenuifolia; Peak 15 belongs to Scutellaria baicalensis; Peak 16 belongs to Scutellaria baicalensis; Peak 17 belongs to Scutellaria baicalensis; Peak 18 belongs to Scutellaria baicalensis; Peak 19 belongs to Scutellaria baicalensis; Peak 20 belongs to Scutellaria baicalensis; Peak 21 belongs to Schisandra chinensis; Peak 22 belongs to Ligusticum chuanxiong; Peak 23 belongs to Ligusticum chuanxiong, Angelica sinensis, Rubus idaeus, Panax ginseng, and Cuscuta chinensis; Peak 24 belongs to Ligusticum chuanxiong, Angelica sinensis, and Rubus idaeus.

[0050] Example 3 Wavelength Selection 220,901 batches of samples were tested, and the overall effect of the chromatograms at different wavelengths (360 nm, 320 nm, 280 nm, 254 nm, 230 nm, 210 nm, and 203 nm) was examined. Other operating procedures and chromatographic conditions were the same as in Example 1. The chromatograms are shown below. Figures 5-11 The results showed that at a wavelength of 360 nm, the number of chromatographic peaks was small, and all peaks could not fully reflect the characteristics of each medicinal material. Figure 5 At a wavelength of 320 nm, the number of chromatographic peaks is relatively small, and all peaks cannot fully reflect the characteristics of each medicinal material. Figure 6 At a wavelength of 280 nm, the number of chromatographic peaks is relatively small, and the peak of baicalin is too large, differing too much from other peaks, thus failing to fully reflect the characteristics of each medicinal material. Figure 7 At a wavelength of 254 nm, there are more chromatographic peaks, and each peak can comprehensively reflect the characteristics of each medicinal material. The baseline of the chromatogram is relatively flat, and the peak shape is better. Therefore, this is the optimal condition. Figure 8 At a wavelength of 230 nm, the number of chromatographic peaks is small, and all peaks cannot fully reflect the characteristics of each medicinal material, and the baseline is unstable. Figure 9At a wavelength of 210 nm, the chromatogram baseline is uneven, and the chromatographic peaks fluctuate greatly. Figure 10 At a wavelength of 203 nm, the chromatogram baseline was not in the standard position. Figure 11 In summary, fingerprint spectra with a detection wavelength of 254 nm best reflect the composition of the formulation and have the best peak shape. Therefore, 254 nm is the preferred detection wavelength.

[0051] Example 4 Methodological Investigation 240601 batches of samples were tested, and the chromatographic conditions and preparation methods of the test solution were the same as in Example 1.

[0052] (1) Precision test A test solution was prepared using Yishen Buxue Gutai Pills. The solution was injected six times consecutively, and the retention times and areas of 24 major chromatographic peaks were recorded. Using baicalin as a reference peak, the relative peak area RSD < 3.0% (n=6) and the relative retention time RSD < 1.0% (n=6) demonstrated good instrument precision. The experimental results are shown in Table 3.

[0053] (2) Stability test A test solution was prepared using Yishen Buxue Gutai Pills. Tests were conducted at 0, 2, 4, 8, 12, and 24 hours, recording the retention times and areas of 24 major chromatographic peaks. Using baicalin as a reference peak, the relative peak area RSD < 3.0% (n=6) and relative retention time RSD < 1.0% (n=6) demonstrated good stability of the sample within 24 hours. The experimental results are shown in Table 3.

[0054] (3) Repeatability test Six test solutions were prepared from Yishen Buxue Gutai Pills, and the retention times and areas of 24 major chromatographic peaks were recorded. Using baicalin as a reference peak, the relative peak area RSD < 3.0% (n=6) and the relative retention time RSD < 1.0% (n=6) demonstrated good repeatability. The experimental results are shown in Table 3.

[0055] Table 3. Results of the Methodological Investigation Experiment

[0056] From the above results, it can be seen that the present invention has at least the following beneficial effects: 1. This invention established HPLC fingerprint chromatograms of 10 batches of Yishen Buxue Gutai Pills, with a similarity greater than 0.90. 24 common peaks were marked, covering 14 kinds of Chinese medicines. This can more comprehensively reflect the chemical information contained in Yishen Buxue Gutai Pills and better characterize its quality.

[0057] 2. Since fingerprinting is not for determining the precise content of a component, but rather to fully reflect the information of the chemical composition, this invention selects to perform the measurement at a wavelength of 254nm, which has more peaks, reflects more complete information, has good absorption values ​​for each peak, and has a stable baseline.

[0058] 3. The high-performance liquid chromatography fingerprinting method established in this invention is the first to achieve quality control of the entire formula of Yishen Buxue Gutai Pill, rather than identifying a single compound or medicinal material. It overcomes the problems of the singularity and one-sidedness of traditional quality control methods, and can more effectively guide the feeding and strictly standardize the production operation to ensure the safety and effectiveness of clinical medication.

[0059] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of a kidney-tonifying, blood-nourishing, and fetal-stabilizing pill, comprising the following steps: The kidney-tonifying and blood-nourishing pill was extracted using a methanol-water solution, and the resulting extract was used as the test solution. The raw materials for preparing the kidney-tonifying and blood-nourishing pill were ginseng, astragalus, rehmannia, angelica, wolfberry, schisandra, white peony root, dodder seed, raspberry, chuanxiong, scutellaria, eclipta, polygala and licorice. A methanol solution of paeoniflorin, ferulic acid, glycyrrhizin, baicalin, quercetin, baicalein and schisandrol A was used as a reference solution. The reference solution and the test solution were analyzed by high performance liquid chromatography, and the fingerprint spectrum of Yishen Buxue Gutai Pill was obtained with the chromatographic peak of the reference solution as the reference. The mobile phase used in the high-performance liquid chromatography (HPLC) analysis includes mobile phase A and mobile phase B. Mobile phase A is acetonitrile, and mobile phase B is a 0.02-0.2% (v / v) aqueous solution of formic acid. The flow rate of the mobile phase is 0.7-1.2 mL / min. The column packing material is octadecylsilane-bonded silica gel. A gradient elution method is used, and the gradient elution program is as follows: 0-10 min, mobile phase A volume fraction 10%; 10-25 min, mobile phase A volume fraction increases from 10% to 15%; 25-40 min, mobile phase A volume fraction increases from 15% to 22%; 40-65 min, mobile phase A volume fraction increases from 22% to 40%; 65-75 min, mobile phase A volume fraction increases from 40% to 50%; 75-90 min, mobile phase A volume fraction increases from 50% to 70%.

2. The method for constructing the fingerprint spectrum of the Kidney-Nourishing, Blood-Replenishing, and Fetal-Strengthening Pill according to claim 1, characterized in that, The chromatographic column was ACE C. 18 The column has the following specifications: length 250mm, inner diameter 4.60mm, filler particle size 5µm; column temperature 20~40℃.

3. The method for constructing the fingerprint spectrum of Yishen Buxue Gutai Pill according to claim 1 or 2, characterized in that, The detector used in the high-performance liquid chromatography analysis is an ultraviolet detector with a detection wavelength of 230~360nm; the injection volume is 5~15μL.

4. The method for constructing the fingerprint spectrum of Yishen Buxue Gutai Pill according to claim 1 or 2, characterized in that, The theoretical plate number for the high-performance liquid chromatography analysis, calculated based on the baicalin peak, shall not be less than 5000.

5. The method for constructing the fingerprint spectrum of the Kidney-Nourishing, Blood-Replenishing, and Fetal-Strengthening Pill according to claim 1, characterized in that, The volume fraction of methanol in the methanol-water solution is 50-80%, and the ratio of the amount of the kidney-tonifying and blood-nourishing pill to the methanol-water solution is 0.2-2g: 10-100mL.

6. The method for constructing the fingerprint spectrum of Yishen Buxue Gutai Pill according to claim 1 or 5, characterized in that, The extraction is performed under ultrasonic conditions, with an ultrasonic power of 100~300W and a frequency of 30~50kHz; the extraction time is 20~60min.

7. The method for constructing the fingerprint spectrum of the Kidney-Nourishing, Blood-Replenishing, and Fetal-Strengthening Pill according to claim 1, characterized in that, The fingerprint spectrum of the Yishen Buxue Gutai Pill contains 24 common peaks, among which peaks 6, 7, 9, 15, 16, 19, and 21 are identified as paeoniflorin, ferulic acid, glycyrrhizin, baicalin, quercetin, baicalein, and schisandrol A, respectively.

8. The method for constructing the fingerprint spectrum of the Kidney-Nourishing, Blood-Replenishing, and Fetal-Strengthening Pill according to claim 7, characterized in that, The 24 common peaks are respectively attributed to white peony root, angelica root, raspberry, wolfberry, astragalus root, prepared rehmannia root, dodder seed, schisandra fruit, polygala root, chuanxiong rhizome, licorice root, scutellaria root, ginseng, and eclipta prostrata. Peak 1 belongs to white peony root, angelica root, raspberry, wolfberry, astragalus root, prepared rehmannia root, dodder seed, schisandra fruit, and polygala root; Peak 2 belongs to white peony root and schisandra fruit; Peak 3 belongs to dodder seed; Peak 4 belongs to schisandra fruit; Peak 5 belongs to dodder seed; Peak 6 belongs to white peony root; Peak 7 belongs to angelica root and chuanxiong rhizome; Peak 8 belongs to astragalus root and dodder seed; Peak 9 belongs to licorice root; Peak 10 belongs to raspberry; Peak 11 belongs to dodder seed; Peak 12 belongs to... Scutellaria baicalensis; Peak 13 belongs to Polygala tenuifolia; Peak 14 belongs to Ligusticum chuanxiong, Angelica sinensis, Eclipta prostrata, and Polygala tenuifolia; Peak 15 belongs to Scutellaria baicalensis; Peak 16 belongs to Scutellaria baicalensis; Peak 17 belongs to Scutellaria baicalensis; Peak 18 belongs to Scutellaria baicalensis; Peak 19 belongs to Scutellaria baicalensis; Peak 20 belongs to Scutellaria baicalensis; Peak 21 belongs to Schisandra chinensis; Peak 22 belongs to Ligusticum chuanxiong; Peak 23 belongs to Ligusticum chuanxiong, Angelica sinensis, Rubus idaeus, Panax ginseng, and Cuscuta chinensis; Peak 24 belongs to Ligusticum chuanxiong, Angelica sinensis, and Rubus idaeus.

9. The method for constructing the fingerprint spectrum of the Kidney-Nourishing, Blood-Replenishing, and Fetal-Strengthening Pill according to claim 1, characterized in that, The prescription for the Kidney-Nourishing and Blood-Tonifying Pregnancy-Strengthening Pill, by weight and parts, is as follows: 22.5 parts ginseng, 37.5 parts astragalus, 37.5 parts prepared rehmannia root, 30 parts angelica, 30 parts wolfberry, 30 parts schisandra, 25 parts white peony root, 50 parts dodder seed, 50 parts raspberry, 22.5 parts chuanxiong rhizome, 25 parts scutellaria root, 30 parts eclipta, 22.5 parts polygala root, and 12.5 parts licorice root; The preparation method of the kidney-tonifying, blood-nourishing, and fetal-stabilizing pills includes the following steps: Ginseng, angelica, chuanxiong, dodder seed and raspberry are crushed to obtain coarse powder with a particle size of 20-40 mesh. Astragalus membranaceus, Rehmannia glutinosa, Lycium barbarum, Schisandra chinensis, Paeonia lactiflora, Scutellaria baicalensis, Eclipta prostrata, Polygala tenuifolia, and Glycyrrhiza uralensis were decocted in water, and the resulting decoction was concentrated to obtain a paste. The coarse powder is mixed with the paste, and then dried and pulverized in sequence to obtain fine powder with a particle size of 100-120 mesh. The fine powder is made into pills with water, and then dried, coated with talc powder, and polished with water to obtain the kidney-tonifying, blood-nourishing, and fetal-stabilizing pills.

10. The application of the method for constructing the fingerprint spectrum of Yishen Buxue Gutai Pill according to any one of claims 1 to 9 in the quality control of Yishen Buxue Gutai Pill.

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