Construction method of gynostemma pentaphyllum hplc fingerprint and application thereof

Abstract

The present application relates to the construction method of Gengnining HPLC fingerprint and its application, which can comprehensively evaluate and control the quality of Gengnining, is beneficial to stabilize the product quality, ensure the stability and uniformity of its inherent quality, ensure the safety and effectiveness of clinical medication, and the technical solution thereof is that the construction method of Gengnining HPLC fingerprint comprises the following steps: 1) preparation of reference solution; 2) preparation of medicinal material solution; 3) preparation of test sample solution; 4) determination of HPLC chromatographic conditions; 5) calculation and determination of the relative retention time and the ratio of relative peak area of 9 main characteristic peaks with large peak area; the method is simple, fast, reproducible, stable, and through the comparison of characteristic peaks in the standard fingerprint, the quality of Gengnining can be comprehensively evaluated and controlled, which is beneficial to stabilize the product quality, ensure the stability and uniformity of its inherent quality, ensure the safety and effectiveness of clinical medication, and is an innovation in the quality detection method of Gengnining product.

Description

I. Technical Field

[0001] This invention relates to the field of traditional Chinese medicine preparation analysis, and in particular to a method for constructing an HPLC fingerprint of menopause-relieving medicine and its application. II. Background Technology

[0002] Gengnianning is a compound preparation composed of 22 Chinese herbs, including Bupleurum, Scutellaria, and Ginseng. It is available in pill form, including water-honey pills and large honey pills. It soothes the liver and relieves depression, invigorates qi and nourishes blood, strengthens the spleen and calms the mind. It is used for symptoms caused by menopause, such as palpitations, shortness of breath, irritability, dizziness, insomnia, hot flashes, night sweats, breast tenderness, and menstrual irregularities. The prescription is as follows: Bupleurum 9g, Scutellaria 9g, White Peony Root 12g, Eclipta 12g, Ginseng 3g, Codonopsis 9g, Curcuma 9g, Cyperus (vinegar-processed) 12g, Angelica 12g, Peppermint 3g, Ligusticum chuanxiong 6g, Scrophularia 12g, Poria 9g, Pinellia 9g, Acorus 9g, Moutan Bark 12g, Tangerine Peel 9g, Dried Ginger 3g, Atractylodes (wheat bran-fried) 9g, Salvia miltiorrhiza 9g, Vaccaria (fried) 9g, Ligustrum lucidum (wine-processed) 12g. Preparation: Grind the above twenty-two ingredients into a fine powder, sift, and mix well. Add 55-60g of refined honey and an appropriate amount of water to every 100g of powder, form into pills, and dry; or add 120-130g of refined honey to make large honey pills. The current standard for Gengnianning is the "Drug Standards of the Ministry of Health of the People's Republic of China" for Traditional Chinese Medicine Compound Preparations (Volume 6) WS3-B-1138-92. This standard has simple testing items, only including appearance, pill examination, microscopic identification of seven medicinal materials, and thin-layer identification of Eclipta prostrata. It cannot effectively control product quality, which may lead to unscrupulous manufacturers using insufficient or no ingredients, or using inferior medicinal materials, resulting in decreased efficacy, affecting the product's effectiveness and safety, and harming patients' interests. Currently, there is a lack of comprehensive quality testing methods for Gengnianning. Reports mostly focus on qualitative identification of single components and quantitative analysis of several components. For a large prescription preparation containing 22 medicinal materials, other active ingredients are unclear, making it difficult to comprehensively evaluate the authenticity and quality of Gengnianning.

[0003] Traditional Chinese medicine (TCM) fingerprinting, based on systematic research of the chemical components of TCM, can comprehensively control drug quality. As a quality control and evaluation technique, it possesses characteristics such as systematicity, integrity, and stability. Therefore, establishing fingerprint profiles for TCM preparations is of great significance for improving TCM quality and promoting the modernization of TCM. Currently, there is no method for controlling the quality of Gengnianning (a TCM preparation) using fingerprinting. Therefore, there is an urgent need to provide a method for establishing a fingerprint profile for Gengnianning to ensure product quality, provide a reliable basis for the internal quality control and authenticity identification of Gengnianning, and provide a reliable analytical method and technical means to ensure the stability, safety, and efficacy of product quality. III. Summary of the Invention

[0004] In view of the above situation and to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a method for constructing the HPLC fingerprint of Gengnianning and its application, which can comprehensively evaluate and control the quality of Gengnianning, help stabilize product quality, ensure the stability and uniformity of its intrinsic quality, and ensure the safety and efficacy of clinical use.

[0005] The technical solution provided by this invention is that the method for constructing the HPLC fingerprint of menopause includes the following steps:

[0006] 1) Preparation of reference solution

[0007] Take appropriate amounts of 5-hydroxymethylfurfural, paeoniflorin, hesperidin, baicalin, salvianolic acid B, wogonin, paeonol, baicalin, wogonin, and ligustilide reference standards, accurately weigh them, and prepare a solution containing 10-70 μg of each in 1 ml with methanol to obtain the reference solution.

[0008] 2) Preparation of medicinal material solution

[0009] Weigh out 0.2-0.8g each of Bupleurum chinense, Scutellaria baicalensis, Paeonia lactiflora, Eclipta prostrata, Panax ginseng, Codonopsis pilosula, Curcuma longa, Cyperus rotundus (vinegar-processed), Angelica sinensis, Mentha haplocalyx, Ligusticum chuanxiong, Scrophularia ningpoensis, Poria cocos, Pinellia ternata (processed), Acorus tatarinowii, Paeonia suffruticosa, Citrus reticulata, Zingiber officinale (dried), Atractylodes macrocephala (wheat bran-fried), Salvia miltiorrhiza, Vaccaria segetalis (fried), and Ligustrum lucidum (wine-processed). Add 15-50ml of methanol to each herb, sonicate for 15-60min, filter, and collect the filtrate to obtain the herbal solution.

[0010] 3) Preparation of the test solution

[0011] Take 1.5-2.5g of genistein, grind it into a fine powder, place it in a stoppered conical flask, accurately add 15-50ml of methanol, sonicate for 15-60min, filter, and take the filtrate to obtain the test solution.

[0012] 4) Determination of HPLC chromatographic conditions

[0013] Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and 0.05%–0.2% phosphoric acid solution was used as mobile phase B. The flow rate was 1 ml / min, the column temperature was 30 °C, and gradient elution was performed according to the following mobile phase ratios:

[0014]

[0015] The detection wavelengths are as follows:

[0016]

[0017] 5) Take 10 batches of Gengnianning samples, prepare test solutions according to the method in step 3), and perform detection and analysis according to the chromatographic conditions in step 4) to obtain chromatograms of 10 batches of samples. Import the HPLC fingerprint chromatograms of the 10 batches of Gengnianning into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) for chromatographic peak matching and fitting to generate standard fingerprint chromatograms. The obtained standard fingerprint chromatograms have a total of 27 common characteristic peaks. Using peak 18 (baicalin) as the reference peak, calculate and determine the ratio of the relative retention time and relative peak area of ​​the 9 main characteristic peaks with larger peak areas.

[0018] Of the 27 common characteristic peaks, with peak 18 (baicalin) as the reference peak, nine common peaks with relatively large peak areas were identified as the main characteristic peaks of Gengnianning, with relative retention times of 0.155 (peak 4), 0.450 (peak 10), 0.640 (peak 14), 0.784 (peak 16), 1.000 (peak 18), 1.068 (peak 19), 1.253 (peak 22), 1.317 (peak 23), and 1.913 (peak 27).

[0019] Peak 4 is 5-hydroxymethylfurfural, peak 10 is paeoniflorin, peak 16 is hesperidin, peak 18 is baicalin, peak 19 is salvianolic acid B, peak 22 is wogonin, peak 23 is paeonol, peak 24 is baicalein, peak 25 is wogonin, and peak 27 is ligustilide.

[0020] Peak 4 is derived from refined honey, peak 6 from Ligusticum chuanxiong, peaks 9 and 10 are components of Paeonia lactiflora, peaks 14 and 23 are components of Paeonia suffruticosa, peak 16 is a component of Citrus reticulata peel, peak 17 is a component of Scrophularia ningpoensis, peaks 18, 20, 21, 22, 24, and 25 are components of Scutellaria baicalensis, peak 19 is a component of Salvia miltiorrhiza, peak 26 is a component of Acorus tatarinowii, and peak 27 is a shared component of Angelica sinensis and Ligusticum chuanxiong.

[0021] The application of the method for constructing the HPLC fingerprint of menopause described in this invention in the quality evaluation standard of menopause.

[0022] The method of this invention is simple and quick to operate, has good reproducibility and high stability. By comparing the characteristic peaks in the standard fingerprint spectrum, the quality of Gengnianning can be comprehensively evaluated and controlled, which is conducive to stabilizing product quality, ensuring the stability and uniformity of its intrinsic quality, and ensuring the safety and efficacy of clinical use. The fingerprint spectrum of Gengnianning was established by gradient elution and multi-wavelength switching of HPLC, which can reflect its overall chemical characteristics. This provides a scientific basis for comprehensively improving the quality of Gengnianning products and is an innovation in the quality detection method of Gengnianning products. IV. Description of the attached drawings

[0023] Figure 1 This is an HPLC chromatogram of the test solution under different mobile phase gradient conditions according to the present invention.

[0024] Figure 2 This is an HPLC chromatogram of the test solution under different detection wavelengths according to the present invention.

[0025] Figure 3 This is a superimposed HPLC fingerprint image of 10 batches of menopausal samples from this invention.

[0026] Figure 4 This is a standard reference fingerprint spectrum for menopause relief in this invention.

[0027] Figure 5 This is a comparison of the HPLC chromatograms of the reference solution and the test solution of this invention.

[0028] Figure 6 This is a comparison of the HPLC chromatograms of the medicinal material solution and the test sample solution belonging to this invention.

[0029] Figure 7 This is a comparison of the HPLC chromatograms of solutions of unattributed medicinal materials and test sample solutions in this invention. V. Detailed Implementation Methods

[0030] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples.

[0031] Example 1

[0032] In a specific implementation of this invention, the method for constructing the HPLC fingerprint of menopause-relieving drugs may include the following steps:

[0033] 1) Preparation of reference solution

[0034] Take appropriate amounts of 5-hydroxymethylfurfural, paeoniflorin, hesperidin, baicalin, salvianolic acid B, wogonin, paeonol, baicalein, wogonin, and ligustilide as reference standards, accurately weigh them, and add methanol to prepare solutions containing 10.012 μg, 40.056 μg, 20.015 μg, 25.056 μg, 10.567 μg, 56.075 μg, 60.056 μg, 25.017 μg, 65.065 μg, and 58.076 μg of each per ml, which are the reference standard solutions.

[0035] 2) Preparation of medicinal material solution

[0036] Weigh out 0.5g each of Bupleurum chinense, Scutellaria baicalensis, Paeonia lactiflora, Eclipta prostrata, Panax ginseng, Codonopsis pilosula, Curcuma longa, Cyperus rotundus (vinegar-processed), Angelica sinensis, Mentha haplocalyx, Ligusticum chuanxiong, Scrophularia ningpoensis, Poria cocos, Pinellia ternata (processed), Acorus tatarinowii, Paeonia suffruticosa, Citrus reticulata, Zingiber officinale (dried), Atractylodes macrocephala (wheat bran-fried), Salvia miltiorrhiza, Vaccaria segetalis (fried), and Ligustrum lucidum (wine-processed). Add 25ml of methanol to each herb, sonicate for 30min, filter, and collect the filtrate to obtain

[0037] 3) Preparation of the test solution

[0038] Take 2.0g of Gengnianning, grind it into a fine powder, place it in a stoppered conical flask, accurately add 25ml of methanol, sonicate for 30min, filter, and take the filtrate to obtain the test solution.

[0039] 4) Determination of HPLC chromatographic conditions

[0040] Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the mobile phase ratios in Table 1; the detection wavelength is shown in Table 2; the flow rate was 1 ml / min; and the column temperature was 30℃.

[0041] Table 1. Characteristic Spectrum of Geng Nianning Mobile Phase Ratio

[0042]

[0043] Table 2. Detection wavelengths of Geng Nianning's characteristic spectrum.

[0044]

[0045]

[0046] 5) Take 10 batches of Gengnianning samples, prepare test solutions according to the method in step 3), and perform detection and analysis according to the chromatographic conditions in step 4) to obtain chromatograms of 10 batches of samples. Import the HPLC fingerprint chromatograms of the 10 batches of Gengnianning into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) for chromatographic peak matching and fitting to generate standard fingerprint chromatograms. The obtained standard fingerprint chromatograms have a total of 27 common characteristic peaks. Using peak 18 (baicalin) as the reference peak, calculate and determine the ratio of the relative retention time and relative peak area of ​​the 9 main characteristic peaks with larger peak areas.

[0047] This invention, through extensive experimentation, determined optimal gradient elution conditions for liquid chromatography. Liquid chromatography revealed the specific peak positions of 10 reference solutions: 5-hydroxymethylfurfural, paeoniflorin, hesperidin, baicalin, salvianolic acid B, wogonin, paeonol, baicalein, wogonin, and ligustilide. These 10 reference solutions can be injected in combination or individually, depending on the specific conditions. Then, under the same gradient elution conditions, menopause-releasing hormone (GnRH) was injected into the liquid chromatograph to obtain a complete chemical fingerprint of GnRH. This allows for more accurate quality control and ensures the stability of GnRH during subsequent sample analysis. Relevant experimental data are as follows:

[0048] Establishment of HPLC standard fingerprint of menopausal drugs

[0049] 1. Instruments and reagents

[0050] 1.1 Instruments

[0051] Waters 2695 high performance liquid chromatograph, Waters Corporation, USA; Wates 2998 PDA detector, Waters Corporation, USA.

[0052] 1.2 Test Drugs

[0053] The menopausal granules were supplied by four manufacturers; the sample sources and batch numbers are shown in Table 3. Acetonitrile was of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0054] Table 3. Source and batch number of Geng Nianning samples

[0055]

[0056] 2. Methods and Results

[0057] 2.1 Selection of chromatographic conditions

[0058] 2.1.1 Selection of mobile phase

[0059] Column: Agilent ZORBAX SB-C 18 (250×4.6mm, 5μm) column; acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. Due to the complex composition of the formulation, five groups of different mobile phase ratios were set up for gradient condition optimization to better ensure chromatographic peak resolution and peak shape. Specific conditions are shown in Tables 4 to 8. Flow rate: 1ml / min, column temperature: 30℃.

[0060] Table 4 Gradient elution conditions for Geng Nianning fingerprint spectroscopy 1

[0061]

[0062] Table 5 Gradient elution conditions for Geng Nianning fingerprint spectroscopy 2

[0063]

[0064] Table 6 Gradient elution conditions for Geng Nianning fingerprint spectroscopy 3

[0065]

[0066] Table 7 Gradient elution conditions for Geng Nianning fingerprint 4

[0067]

[0068] Table 8 Gradient elution conditions for Geng Nianning fingerprint spectroscopy 5

[0069]

[0070] By employing different mobile phase ratios to perform gradient elution of the test sample solution, the resulting spectra are shown below. Figure 1 As can be seen from the figure, using condition 5 for elution yields better chromatograms with a stable baseline, good peak separation, a large number of peaks, and moderate peak height. Therefore, condition 5 is selected as the mobile phase elution gradient.

[0071] 2.1.2 Selection of detection wavelength

[0072] The sample solution was analyzed using HPLC, with detection wavelengths between 210-400 nm. Referring to the detection wavelengths of the quality control components of each herb in the Gengnianning prescription included in the 2020 edition of the Chinese Pharmacopoeia, several optimal wavelengths were ultimately selected. The same sample was injected at wavelengths of 230 nm, 254 nm, 280 nm, and 326 nm. Evaluation was based on response value, number of chromatographic peaks, and resolution. The results are shown in [Figure number missing]. Figure 2 As can be seen from the figure, single-wavelength detection cannot meet the detection requirements of large prescription preparations. Therefore, this invention adopts multi-wavelength switching detection technology. Based on the elution time of different components, the detection wavelength of the Gengnianning fingerprint spectrum is determined as shown in Table 9. This method has a large number of chromatographic peaks in the chromatogram, the main chromatographic peak area is high, the impurity peak area is relatively low, and the fingerprint characteristics are obvious.

[0073] Table 9. Multiwavelength switching table of Geng Nianning fingerprint spectrum

[0074]

[0075]

[0076] 2.2 Preparation of reference solution, medicinal material solution and test solution

[0077] Accurately weigh appropriate amounts of 5-hydroxymethylfurfural, paeoniflorin, hesperidin, baicalin, salvianolic acid B, wogonin, paeonol, baicalein, wogonin, and ligustilide reference standards, and prepare reference solutions with methanol containing 10.012 μg, 40.056 μg, 20.015 μg, 25.056 μg, 10.567 μg, 56.075 μg, 60.056 μg, 25.017 μg, 65.065 μg, and 58.076 μg of each per ml.

[0078] Weigh out 0.5g each of Bupleurum chinense, Scutellaria baicalensis, Paeonia lactiflora, Eclipta prostrata, Panax ginseng, Codonopsis pilosula, Curcuma longa, Cyperus rotundus (processed with vinegar), Angelica sinensis, Mentha haplocalyx, Ligusticum chuanxiong, Scrophularia ningpoensis, Poria cocos, Pinellia ternata (processed), Acorus tatarinowii, Paeonia suffruticosa, Citrus reticulata, Zingiber officinale (dried), Atractylodes macrocephala (stir-fried with wheat bran), Salvia miltiorrhiza, Vaccaria segetalis (stir-fried), and Ligustrum lucidum (processed with wine). Add 25ml of methanol to each herb, sonicate for 30 minutes, filter, and collect the filtrate to obtain the herbal solution.

[0079] Take an appropriate amount of Gengnianning, grind it into a fine powder, accurately weigh 2.0g, place it in a stoppered conical flask, accurately add 25ml of methanol, sonicate for 30 minutes, filter, take the filtrate, and filter it through a 0.45μm microporous membrane to obtain the test solution.

[0080] 2.3 Establishment of fingerprint patterns

[0081] Ten batches of Gengnianning samples were taken and prepared into test solutions according to the method in section "2.2". The solutions were then analyzed according to the method in section "2.1", yielding the chromatograms of the ten batches of samples. Figure 3 Ten batches of Gengnianning HPLC fingerprints were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) for chromatographic peak matching and fitting to generate standard fingerprints. Figure 4 The obtained standard fingerprint spectrum has 27 common characteristic peaks. Using peak 18 (baicalin) as the reference peak, the ratio of the relative retention time and relative peak area of ​​the nine main characteristic peaks with larger peak areas was calculated and determined, as shown in Tables 10 and 11.

[0082] Table 10. Relative retention time ratios of the main characteristic peaks in 10 batches of Gengnianning.

[0083]

[0084] Table 11. Relative peak area (%) of the main characteristic peaks of 10 batches of Gengnianning.

[0085]

[0086]

[0087] 2.4 Similarity Evaluation

[0088] The chromatograms of 10 batches of Gengnianning were compared with the standard fingerprint chromatograms. The similarity calculation results were: 0.960, 0.945, 0.946, 0.955, 0.990, 0.985, 0.915, 0.945, 0.950, and 0.930. All similarities were above 0.900, indicating good quality stability and uniformity among samples from different companies.

[0089] 2.5 Attribution and Identification of Common Peaks

[0090] Take the reference solution and analyze it according to the chromatographic conditions under section "2.1". The results are as follows: Figure 5 As shown. Based on retention time localization and chromatographic peak analysis of the reference standard, peak 4 was identified as 5-hydroxymethylfurfural, peak 10 as paeoniflorin, peak 16 as hesperidin, peak 18 as baicalin, peak 19 as salvianolic acid B, peak 22 as wogonin, peak 23 as paeonol, peak 24 as baicalein, peak 25 as wogonin, and peak 27 as ligustilide.

[0091] The medicinal material solution was analyzed under the chromatographic conditions described in section "2.1". Based on the retention time and peak analysis of each medicinal material, peak 4 was identified as originating from refined honey; peak 6 from Ligusticum chuanxiong; peaks 9 and 10 as components of Paeonia lactiflora; peaks 14 and 23 as components of Paeonia suffruticosa; peak 16 as a component of Citrus reticulata peel; peak 17 as a component of Scrophularia ningpoensis; peaks 18, 20, 21, 22, 24, and 25 as components of Scutellaria baicalensis; peak 19 as a component of Salvia miltiorrhiza; peak 26 as a component of Acorus tatarinowii; and peak 27 as a shared component of Angelica sinensis and Ligusticum chuanxiong. The overlapping chromatograms of the attributed medicinal materials and the test solution are shown below. Figure 6 As shown, the overlapping chromatograms of the unassigned medicinal materials and the test sample solution are as follows: Figure 7 As shown.

[0092] 2.6 Methodological Examination

[0093] 2.6.1 Precision Test

[0094] Take a sample (batch number: 21030536), prepare the test solution according to the method in section 2.2, and inject it 6 times consecutively. Using peak 18 as the reference peak, the RSD of the relative retention time and relative peak area of ​​the common peaks 1 to 27 were all less than 3.0%, indicating that the instrument precision was good.

[0095] 2.6.2 Repeatability Test

[0096] Take a sample (batch number: 21030536), accurately weigh 6 portions, prepare the test solution according to the procedure in section 2.2, and inject the sample according to the procedure in section 2.1. Using peak 18 as the reference peak, the RSD of the relative retention time and relative peak area of ​​the common peaks 1 to 27 are all less than 3.0%, indicating that the method has good repeatability.

[0097] 2.6.3 Stability Test

[0098] Take a sample (batch number: 21030536), prepare the test solution according to the method in section 2.2, and inject and measure at 0, 2, 4, 6, 8, 10 and 12 h respectively. Using peak 18 as the reference peak, the RSD of the relative retention time and relative peak area of ​​the common peaks 1 to 27 were all less than 3.0%, indicating that the test solution has good stability within 12 h.

[0099] In summary, compared with the prior art, the present invention has the following beneficial technical effects:

[0100] 1) The method for establishing the HPLC fingerprint of Gengnianning in this invention optimizes the HPLC conditions based on the composition characteristics of the prescription components of Gengnianning, solves the problem of difficult separation of characteristic peaks, and obtains fingerprints with high resolution and good peak shape. All characteristic chromatographic peaks achieve good baseline separation, and the fingerprints have many characteristic peaks and good stability, which can comprehensively and accurately evaluate the quality of Gengnianning.

[0101] 2) The detection method of this invention uses baicalin as a reference peak and identifies 27 common peaks as the common peaks constituting the fingerprint spectrum of Gengnianning, making the fingerprint spectrum more abundant and comprehensive. Ten chemical components, namely 5-hydroxymethylfurfural, paeoniflorin, hesperidin, baicalin, salvianolic acid B, wogonin, paeonol, baicalein, wogonin, and ligustilide, were detected from the common peaks and their components were assigned, further clarifying the chemical basis of Gengnianning.

[0102] 3) The HPLC fingerprint of menopause established by this invention overcomes the shortcomings of existing technologies, which have single detection indicators and cannot reflect intrinsic quality. The method for establishing the HPLC fingerprint of menopause provided has high precision, good stability and repeatability. By comparing the presence or absence of common peaks in the obtained fingerprint, the quality of menopause can be comprehensively evaluated, and the quality of the finished product can be more effectively guaranteed.

[0103] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any modifications or alterations made by those skilled in the art without departing from the scope of the present invention to create equivalent embodiments based on the disclosed technical content shall fall within the protection scope of the present invention.

Claims

1. A method for constructing a menopausal HPLC fingerprint, characterized in that, The method for constructing the HPLC fingerprint of menopause includes the following steps: 1) Preparation of reference solution Take appropriate amounts of 5-hydroxymethylfurfural, paeoniflorin, hesperidin, baicalin, salvianolic acid B, wogonin, paeonol, baicalin, wogonin, and ligustilide reference standards, accurately weigh them, and add methanol to prepare a solution containing 10μg-70μg of each in 1ml to obtain the reference solution. 2) Preparation of medicinal material solution Weigh out 0.2-0.8g each of Bupleurum chinense, Scutellaria baicalensis, Paeonia lactiflora, Eclipta prostrata, Panax ginseng, Codonopsis pilosula, Curcuma longa, Cyperus rotundus (processed with vinegar), Angelica sinensis, Mentha haplocalyx, Ligusticum chuanxiong, Scrophularia ningpoensis, Poria cocos, Pinellia ternata (processed), Acorus tatarinowii, Paeonia suffruticosa, Citrus reticulata, Zingiber officinale (dried), Atractylodes macrocephala (stir-fried with wheat bran), Salvia miltiorrhiza, Vaccaria segetalis (stir-fried), and Ligustrum lucidum (processed with wine). Add 15-50ml of methanol to each ingredient, sonicate for 15-60min, filter, and collect the filtrate to obtain the medicinal solution. 3) Preparation of the test solution Take 1.5-2.5g of genistein, grind it into a fine powder, place it in a stoppered conical flask, accurately add 15-50ml of methanol, sonicate for 15-60min, filter, and take the filtrate to obtain the test solution. 4) Determination of HPLC chromatographic conditions Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and 0.05%–0.2% phosphoric acid solution was used as mobile phase B. The flow rate was 1 ml / min, the column temperature was 30 °C, and gradient elution was performed according to the following mobile phase ratios: The detection wavelengths are as follows: 5) Take 10 batches of Gengnianning samples, prepare test solutions according to the method in step 3), and perform detection and analysis according to the chromatographic conditions in step 4) to obtain chromatograms of 10 batches of samples. Import the HPLC fingerprint chromatograms of the 10 batches of Gengnianning into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" for chromatographic peak matching and fitting to generate standard fingerprint chromatograms. The obtained standard fingerprint chromatograms have a total of 27 common characteristic peaks. Using peak 18 as the reference peak, calculate and determine the ratio of the relative retention time and relative peak area of ​​the 9 main characteristic peaks with larger peak areas.

2. The method for constructing the HPLC fingerprint of menopause according to claim 1, characterized in that, Of the 27 common characteristic peaks, with peak 18 as the reference peak, nine common peaks with relatively large peak areas were identified as the main characteristic peaks of Gengnianning. Their relative retention times were 0.155 for peak 4, 0.450 for peak 10, 0.640 for peak 14, 0.784 for peak 16, 1.000 for peak 18, 1.068 for peak 19, 1.253 for peak 22, 1.317 for peak 23, and 1.913 for peak 27.

3. The method for constructing the HPLC fingerprint of menopause according to claim 1, characterized in that, Peak 4 is 5-hydroxymethylfurfural, peak 10 is paeoniflorin, peak 16 is hesperidin, peak 18 is baicalin, peak 19 is salvianolic acid B, peak 22 is wogonin, peak 23 is paeonol, peak 24 is baicalein, peak 25 is wogonin, and peak 27 is ligustilide.

4. The method for constructing the HPLC fingerprint of menopause according to claim 1, characterized in that, Peak 4 is derived from refined honey, peak 6 from Ligusticum chuanxiong, peaks 9 and 10 are components of Paeonia lactiflora, peaks 14 and 23 are components of Paeonia suffruticosa, peak 16 is a component of Citrus reticulata peel, peak 17 is a component of Scrophularia ningpoensis, peaks 18, 20, 21, 22, 24, and 25 are components of Scutellaria baicalensis, peak 19 is a component of Salvia miltiorrhiza, peak 26 is a component of Acorus tatarinowii, and peak 27 is a shared component of Angelica sinensis and Ligusticum chuanxiong.

5. The method for constructing the HPLC fingerprint of menopause according to claim 1, characterized in that, Includes the following steps: 1) Preparation of reference solution Take appropriate amounts of 5-hydroxymethylfurfural, paeoniflorin, hesperidin, baicalin, salvianolic acid B, wogonin, paeonol, baicalein, wogonin, and ligustilide reference standards, accurately weigh them, and add methanol to prepare solutions containing 10.012 μg, 40.056 μg, 20.015 μg, 25.056 μg, 10.567 μg, 56.075 μg, 60.056 μg, 25.017 μg, 65.065 μg, and 58.076 μg of each per ml, which are the reference standard solutions. 2) Preparation of medicinal material solution Weigh out 0.5g each of Bupleurum chinense, Scutellaria baicalensis, Paeonia lactiflora, Eclipta prostrata, Panax ginseng, Codonopsis pilosula, Curcuma longa, Cyperus rotundus (processed with vinegar), Angelica sinensis, Mentha haplocalyx, Ligusticum chuanxiong, Scrophularia ningpoensis, Poria cocos, Pinellia ternata (processed), Acorus tatarinowii, Paeonia suffruticosa, Citrus reticulata, Zingiber officinale (dried), Atractylodes macrocephala (stir-fried with wheat bran), Salvia miltiorrhiza, Vaccaria segetalis (stir-fried), and Ligustrum lucidum (processed with wine). Add 25ml of methanol to each herb, sonicate for 30min, filter, and collect the filtrate to obtain the herbal solution. 3) Preparation of the test solution Take 2.0g of Gengnianning, grind it into a fine powder, place it in a stoppered conical flask, accurately add 25ml of methanol, sonicate for 30min, filter, and take the filtrate to obtain the test solution. 4) Determination of HPLC chromatographic conditions Octadecylsilane-bonded silica gel is used as a filler; Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the mobile phase ratios in Table 1. The detection wavelengths are shown in Table 2. The flow rate was 1 ml / min, and the column temperature was 30℃. Table 1. Characteristic Spectrum of Geng Nianning Mobile Phase Ratio Table 2. Detection wavelengths of Geng Nianning's characteristic spectrum. 5) Take 10 batches of Gengnianning samples, prepare test solutions according to the method in step 3), and perform detection and analysis according to the chromatographic conditions in step 4) to obtain chromatograms of 10 batches of samples. Import the HPLC fingerprint chromatograms of the 10 batches of Gengnianning into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" for chromatographic peak matching and fitting to generate standard fingerprint chromatograms. The obtained standard fingerprint chromatograms have a total of 27 common characteristic peaks. Using peak 18 as the reference peak, calculate and determine the ratio of the relative retention time and relative peak area of ​​the 9 main characteristic peaks with larger peak areas.

6. The application of the method for constructing the HPLC fingerprint of menopause according to any one of claims 1-5 in the quality evaluation standard of menopause.

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