Fingerprint spectrum of compound astragalus membranaceus for invigorating spleen oral liquid and construction method thereof

Abstract

This invention provides a method for constructing a fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid. The specific steps are as follows: (1) Preparation of the test solution: After freeze-drying the Compound Astragalus Spleen-Strengthening Oral Liquid, dissolve and filter it using the initial mobile phase, and take the filtrate to obtain the test solution; (2) Preparation of the fingerprint spectrum: Test the test solution using high performance liquid chromatography, and generate the fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid from the obtained chromatographic data. The fingerprint spectrum method constructed by this invention is simple and easy to implement, and can effectively identify the main medicinal ingredients in FFHQ. The similarity of 10 batches of samples is above 0.960, indicating that the chemical composition of different batches of samples is relatively consistent, and the preparation process and sample quality are relatively stable.

Description

Technical Field

[0001] This invention relates to the fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid and its construction method. Background Technology

[0002] Compound Astragalus Spleen-Strengthening Oral Liquid (FFHQ) is a pure traditional Chinese medicine preparation developed based on years of clinical experience and traditional Chinese medicine theory to treat children with weak spleen and stomach. It is made from seven Chinese herbs: Astragalus membranaceus, Atractylodes macrocephala (fried), Dioscorea opposita (fried), Jujube, Crataegus pinnatifida (fried), Raphanus sativus (fried), and Morus alba leaves, processed using modern technology. It has the effects of invigorating qi and strengthening the exterior, strengthening the spleen and promoting digestion. It is significantly effective in improving recurrent respiratory infections, nutritional anemia, and anorexia in children caused by weak spleen and stomach. The current standard implemented is the National Drug Standard WS-5541(B-0541)-2014Z of the State Drug Administration.

[0003] The efficacy of traditional Chinese medicine (TCM) stems from its specific chemical components. However, the chemical components in TCM compound formulas are complex and diverse. For example, each individual herb in Compound Astragalus Spleen-Strengthening Oral Liquid contains different chemical components, which exert various medicinal effects. Under current standards, apart from routine thin-layer chromatography identification and astragaloside A content determination, quality control indicators are relatively scarce. To further improve the controllability of its quality, research on the FFHQ quality standard is needed. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid and its construction method. This invention employs high-performance liquid chromatography-diode array detector (HPLC-DAD) full-wavelength scanning, using the number of chromatographic peaks and total peak area as indicators to optimize chromatographic conditions, construct its fusion fingerprint spectrum, and identify the common peaks of 10 batches of samples, thereby achieving identification of its medicinal properties and overall characteristics.

[0005] Traditional Chinese medicine (TCM) fingerprinting is a comprehensive and quantifiable identification method. Based on the chemical components of TCM, it constructs fingerprint maps to comprehensively reflect the types and quantities of chemical components in TCM and its preparations. This allows for a holistic description and evaluation of TCM, providing a powerful and comprehensive reference for TCM quality control. Compound Astragalus Spleen-Strengthening Oral Liquid is a TCM compound preparation composed of seven herbs. Its chemical composition is complex and diverse, encompassing flavonoids, saponins, phenolic acids, and polysaccharides. A single wavelength cannot fully reflect its overall information, and the responses of different chemical components at a single wavelength have limitations. Some components may not effectively exhibit their characteristics at a particular wavelength due to absorption differences. Multi-wavelength fusion fingerprinting, by integrating chemical information from different wavelengths, significantly enhances the richness and complementarity of TCM component information, overcoming the limitations of single-wavelength detection. This provides more comprehensive information for quality evaluation, and is particularly suitable for the holistic characterization and quality control of multiple components.

[0006] The first objective of this invention is to provide a method for constructing the fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid, the specific steps of which are as follows:

[0007] (1) Preparation of test solution: After freeze-drying the compound astragalus spleen-strengthening oral liquid, dissolve it in the initial mobile phase and filter it. Take the filtrate to obtain the test solution;

[0008] (2) Preparation of fingerprint spectrum: The test solution was tested by high performance liquid chromatography, and the fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid was generated from the obtained chromatographic data;

[0009] The high-performance liquid chromatography (HPLC) conditions are as follows: Poroshell EC C using octadecylsilane-bonded silica gel as the stationary phase. 18 The column was 4.6 × 250 mm in diameter and 2.7 μm in diameter. The column temperature was 20–40 °C. The injection volume was 20 μL, the injection concentration was 30 mg / mL, and the flow rate was 0.5 mL / min. The detection wavelength was 200–400 nm for full wavelength scanning. The fusion wavelengths were 0–44 min, 280 nm; 45–71 min, 310 nm; and 72–94 min, 254 nm. Gradient elution was performed using acetonitrile as mobile phase A and 0.1% (v / v) formic acid aqueous solution as mobile phase B.

[0010] Preferably, in step (1), the filtration is performed using a microporous membrane with a pore size of 0.22~0.45 μm; preferably, the pore size of the microporous membrane is 0.22 μm.

[0011] Preferably, in step (2), the column temperature is 30°C.

[0012] Preferably, in step (2), the gradient elution procedure is as follows:

[0013] 0-20 min, mobile phase A is 2%, mobile phase B is 98%;

[0014] Over 20-55 minutes, mobile phase A changed from 2% to 15% at a constant rate, and mobile phase B changed from 98% to 85% at a constant rate.

[0015] From 55 to 94 minutes, mobile phase A changed from 15% to 41% at a constant rate, and mobile phase B changed from 85% to 59% at a constant rate.

[0016] All percentages (%) refer to volume percentages.

[0017] Preferably, the method further includes preparing a reference solution, wherein the preparation method of the reference solution includes: taking chlorogenic acid reference standard, verbascoside reference standard, sinapic acid reference standard, sennatin reference standard, and verbascoside reference standard respectively, and adding methanol to prepare solutions of chlorogenic acid 0.687 mg / mL, verbascoside 2.033 mg / mL, sinapic acid 1.637 mg / mL, sennatin 2.449 mg / mL, and verbascoside 2.259 mg / mL respectively.

[0018] The second objective of this invention is to provide a fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid, using verbascoside as a reference peak. The fingerprint spectrum of the Compound Astragalus Spleen-Strengthening Oral Liquid includes 37 common peaks, as detailed below:

[0019] The relative retention times for peak 1 are 0.069-0.070, peak 2 is 0.089, peak 3 is 0.099-0.101, peak 4 is 0.143-0.149, peak 5 is 0.161-0.171, peak 6 is 0.182-0.191, peak 7 is 0.262-0.274, peak 8 is 0.302-0.313, peak 9 is 0.361-0.377, and peak 10 is 0.4. The relative retention times for peaks 03-0.424, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are as follows: 0.726-0.424, 0.431-0.447, 0.500-0.519, 0.511-0.530, 0.516-0.543, 0.547-0.560, 0.633-0.641, 0.653-0.661, 0.718-0.727, and 0.726-0.724. The relative retention times for peaks 0-20 are 0.757-0.765, 21-22, 23-24, 25-26, 27-28 are 0.892-0.825, 28-29-0.892, 29-29-0.825, 29-20, 29-28, and 29-29-0.892, respectively. The relative retention times for peaks 0.896, 29, 30, 31, 32, 33, 34, 35, 36, and 37 are 1.243-1.249. The relative retention times for peaks 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38 are 1.243-1.249.

[0020] As preferred, peak 20 is chlorogenic acid, peak 29 is verrucoside, peak 34 is sinapic acid, peak 35 is stigmoside, and peak 37 is verrucoside.

[0021] As preferred, in the fingerprint spectrum, Astragalus membranaceus has 11 common peaks, namely peaks 1, 2, 3, 5, 6, 7, 13, 29, 31, 35, and 37; Atractylodes macrocephala (fried) has 12 common peaks, namely peaks 2, 5, 7, 8, 11, 16, 18, 20, 22, 23, 28, and 30; Dioscorea opposita (fried) has 25 common peaks, namely peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 21, 22, 23, 24, 25, 28, 30, and 31; and Ziziphus jujuba has 3 common peaks. The peaks are 2, 7, and 10; radish seeds (fried) have 11 shared peaks, namely peaks 2, 14, 15, 19, 24, 26, 27, 28, 31, 34, and 36; hawthorn (fried) has 16 shared peaks, namely peaks 2, 4, 5, 6, 7, 9, 10, 11, 13, 14, 15, 20, 21, 23, 30, and 31; mulberry leaves have 21 shared peaks, namely peaks 1, 2, 3, 5, 7, 8, 11, 13, 16, 17, 18, 20, 21, 22, 23, 24, 27, 30, 31, 32, and 33.

[0022] The third objective of this invention is to provide a quality control method for Compound Astragalus Spleen-Strengthening Oral Liquid. A fingerprint chromatogram of Compound Astragalus Spleen-Strengthening Oral Liquid is constructed according to the above method. When the common peaks P29, 35, and 37 are missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacks Astragalus; when the common peaks P12 and P25 are missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacks Dioscorea (fried); when the common peaks P19, 26, 34, and 36 are missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacks Raphanus sativus (fried); when the common peaks P32 and P33 are missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacks Mulberry Leaf.

[0023] To obtain overall chemical composition information of FFHQ and identify its medicinal components, fingerprint chromatograms of 10 batches of FFHQ were constructed using HPLC-DAD full-wavelength scanning. Chromatographic conditions were investigated, including column selection, mobile phase, column temperature, injection volume, and injection concentration. The optimal column was ultimately determined to be an Agilent Poroshell EC C10. 18(4.6 × 250 mm, 2.7 μm), mobile phase: acetonitrile-0.1% (v / v) formic acid aqueous solution gradient elution, column temperature: 30℃, injection volume: 20 μL, injection concentration: 30 mg / mL. To obtain more chromatographic peaks and total peak area, three wavelengths (254 nm, 280 nm, and 310 nm) were selected for time-segmented fusion to establish a time-segmented multi-wavelength fusion fingerprint of FFHQ. Ten batches of samples contained 37 common peaks. Peak 29 (verrucoside) was used as the reference peak. The relative retention time and relative peak area RSD of each common peak were all less than 3.0%. The 37 common peaks originated from different Chinese medicinal herbs in the formula, including 11 from Astragalus membranaceus, 12 from Atractylodes macrocephala (fried), 25 from Dioscorea opposita (fried), 3 from Ziziphus jujuba, 16 from Crataegus pinnatifida (fried), 11 from Raphanus sativus (fried), and 21 from Morus alba leaves. The fingerprinting method constructed in this invention is simple and easy to implement, effectively identifying the main medicinal components in FFHQ. Furthermore, the similarity of 10 batches of samples was above 0.960, indicating good consistency in chemical composition among different batches and relatively stable preparation process and sample quality. Attached Figure Description

[0024] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0025] Figure 1 These are high-performance liquid chromatograms for different chromatographic columns.

[0026] Figure 2 These are high-performance liquid chromatograms of different mobile phases.

[0027] Figure 3 These are high-performance liquid chromatograms at different column temperatures.

[0028] Figure 4 These are high-performance liquid chromatograms with different injection concentrations.

[0029] Figure 5 High-performance liquid chromatograms with different injection volumes.

[0030] Figure 6 These are high-performance liquid chromatograms at different wavelengths.

[0031] Figure 7 This is a high-performance liquid chromatogram of a multi-wavelength fusion mixture of traditional Chinese medicine samples.

[0032] Figure 8 The time-segmented multi-wavelength fusion fingerprint spectrum of the mixed traditional Chinese medicine test sample and Compound Astragalus Spleen-Strengthening Oral Liquid (S10).

[0033] Figure 9 This is a fusion fingerprint of Compound Astragalus Spleen-Strengthening Oral Liquid and a fusion fingerprint of mixed reference standards.

[0034] Figure 10 This refers to the precision of the fingerprint spectrum.

[0035] Figure 11 For the repeatability of fingerprint patterns.

[0036] Figure 12 To ensure the stability of the fingerprint spectrum.

[0037] Figure 13 Similarity of multi-wavelength fusion fingerprint spectra of 10 batches of Compound Astragalus Spleen-Strengthening Oral Liquid at different time periods.

[0038] Figure 14 Assignment of 37 common peaks in Compound Astragalus Spleen-Strengthening Oral Liquid.

[0039] Figure 15 Cluster analysis diagram of 10 batches of Compound Astragalus Spleen-Strengthening Oral Liquid.

[0040] Figure 16 Cross-validation plot (A) and scatter plot (B) of PCA for 10 batches of Compound Astragalus Spleen-Strengthening Oral Liquid. Detailed Implementation

[0041] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0042] Example 1

[0043] 1. Instruments and Reagents

[0044] 1.1 Instruments and Equipment

[0045] Table 1 Instruments and Equipment

[0046]

[0047] 1.2 Reagents and Materials

[0048] Table 2 Reagents and Materials

[0049]

[0050] The single Chinese herbs used in the Compound Astragalus Spleen-Strengthening Oral Liquid are: Astragalus membranaceus, Atractylodes macrocephala (fried), Dioscorea opposita (fried), Jujube, Crataegus pinnatifida (fried), Raphanus sativus (fried), and Morus alba. These herbs were identified by the chief pharmacist of the Affiliated Hospital of Gansu University of Traditional Chinese Medicine as Astragalus mongholicus, a legume. Astragalus membranaceus (Fisch.) Bge. var. mongholicus The dried root of (Bge.) Hsiao., a plant of the Asteraceae family, Atractylodes macrocephala. Atractylodes macrocephala The dried rhizome of *Dioscorea opposita*, a plant in the Dioscoreaceae family. Dioscorea opposita The dried rhizome of Thunb., a plant in the Rhamnaceae family, jujube. Ziziphus jujuba The dried, ripe fruit of Mill., a plant of the Rosaceae family, hawthorn. Crataegus pinnatifida Bge. var major NE Br., Radish (a cruciferous plant) Raphanus sativus The dried, mature seeds of L., a plant of the Moraceae family, mulberry. Morus alba Dried mature fruit of L. dried leaves.

[0051] Test methods

[0052] 2.1 Fingerprint pattern establishment

[0053] 2.1.1 Preparation of the test solution

[0054] Take 10 mL of Compound Astragalus Spleen-Strengthening Oral Liquid, freeze-dry it, accurately weigh 150 mg, dissolve it in the initial mobile phase and dilute it to 5 mL in a volumetric flask. Filter the solution through a 0.22 μm microporous membrane, and take the filtrate for analysis as the FFHQ test sample.

[0055] Astragalus membranaceus, Atractylodes macrocephala (fried), Dioscorea opposita (fried), Ziziphus jujuba, Raphanus sativus (fried), Crataegus pinnatifida (fried), and Morus alba leaves were ground into powder. The powder was then precisely weighed and mixed according to the proportions of the seven Chinese herbs in the FFHQ prescription. After preparation according to the prescription preparation process, the powder was freeze-dried, dissolved in the initial mobile phase, and diluted to a 5 mL volumetric flask. The mixture was filtered through a 0.22 μm microporous membrane, and the filtrate was collected for analysis as the mixed Chinese herbal medicine test sample.

[0056] 2.1.2 Preparation of single-herb test solutions

[0057] The seven Chinese medicinal herbs of FFHQ—Astragalus membranaceus, Atractylodes macrocephala (fried), Dioscorea opposita (fried), Jujube, Radish seed (fried), Crataegus pinnatifida (fried), and Mulberry leaf—were ground into powder, prepared according to the preparation process in the prescription, and freeze-dried to serve as single-herb test solutions.

[0058] 2.1.3 Preparation of negative test solution

[0059] Negative samples lacking Astragalus membranaceus, Atractylodes macrocephala (fried), Dioscorea opposita (fried), Jujube, Raphanus sativus (fried), Crataegus pinnatifida (fried), and Morus alba leaves were prepared sequentially according to the prescription ratio and preparation process of Compound Astragalus Spleen-Strengthening Oral Liquid. Negative test samples were prepared according to the above-mentioned test sample solution method.

[0060] 2.1.4 Preparation of reference solution

[0061] Accurately weigh appropriate amounts of chlorogenic acid reference standard, verbascoside reference standard, sinapic acid reference standard, scutellarin reference standard, and verbascoside reference standard, and dissolve them in methanol to prepare reference solutions with chlorogenic acid 0.687 mg / mL, verbascoside 2.033 mg / mL, sinapic acid 1.637 mg / mL, scutellarin 2.449 mg / mL, and verbascoside 2.259 mg / mL.

[0062] 2.1.5 Optimization of chromatographic conditions

[0063] In the following chromatographic condition optimization experiments, the mixed Chinese herbal medicine sample prepared in step 2.1.1 was used.

[0064] (1) Investigation of the chromatographic column

[0065] Agilent ZORBAX EclipseXDB-C 18 (4.6×250 mm, 5 μm), Agilent Poroshell ECC 18 Two chromatographic columns (4.6 × 250 mm, 2.7 μm) were used for separation, and the column with better resolution and more peaks was selected. Because the chemical components of traditional Chinese medicine compound preparations are complex, isocratic elution could not achieve good separation results; therefore, gradient elution was employed.

[0066] Table 3 Mobile phase elution procedure

[0067]

[0068] (2) Investigation of the mobile phase

[0069] When selecting the mobile phase, methanol, acetonitrile, and formic acid were investigated. Methanol-water solution, acetonitrile-water solution, and acetonitrile-0.1% (v / v) formic acid water solution were used as mobile phases. The mobile phase with good chromatographic peak resolution, strong response, and large number of peaks was selected for separation.

[0070] (3) Investigation of column temperature

[0071] The effects of different column temperatures (20℃, 30℃, and 40℃) on the chromatograms were investigated, and a suitable column temperature was selected based on the chromatograms.

[0072] (4) Investigation of injection concentration

[0073] Accurately weigh 50, 100, and 150 mg of the freeze-dried mixed Chinese herbal medicine sample, dissolve it in the initial mobile phase and bring the volume to 5 mL to prepare mixed Chinese herbal medicine test solutions with different concentrations of 10, 20, and 30 mg / mL. Select the appropriate injection concentration based on the chromatographic peak resolution, number, and response intensity.

[0074] (5) Examination of injection volume

[0075] The samples were tested with injection volumes of 10, 20, and 30 μL, respectively, and the injection volume was selected based on the number of chromatographic peaks and the response in the chromatogram.

[0076] (6) Wavelength selection

[0077] Compound Astragalus Spleen-Strengthening Oral Liquid is a traditional Chinese medicine compound preparation made from seven Chinese herbs. Its chemical composition is relatively complex, and its detection wavelength cannot be determined based on the wavelength of a single component. Therefore, a full-wavelength scan is required to determine the maximum absorption wavelength of FFHQ. Under the above conditions, a DAD detector is used to perform a full-wavelength scan at wavelengths of 200-400 nm, and the fusion wavelength is selected according to the liquid phase diagram.

[0078] 2.1.6 Methodological Examination

[0079] Prepare the FFHQ test solution according to the preparation method under section 2.1.1, and perform the determination under the chromatographic conditions under section 2.1.5, recording each chromatogram. The precision test was conducted by injecting the same FFHQ (S10) six times consecutively to assess the instrument's precision; the repeatability test was conducted by preparing six parallel FFHQ (S10) samples to assess the repeatability of the fingerprinting method; and the stability test was conducted by injecting the same FFHQ (S10) at room temperature for 0, 2, 4, 8, 12, and 24 hours to assess the sample's stability.

[0080] Fingerprint analysis

[0081] 2.2.1 Similarity evaluation and common peak attribution of 10 batches of Compound Astragalus Spleen-Strengthening Oral Liquid

[0082] Fingerprint chromatograms of different batches of FFHQ were compared through fingerprint similarity analysis to ensure the stability and consistency of their quality. A similarity score closer to 1 indicates a higher similarity between different batches of samples. Ten batches of FFHQ (named S1-S10 sequentially) were tested according to the preparation method in section 2.1.1 and the optimized chromatographic conditions to establish HPLC fingerprint chromatograms. The established fingerprint chromatogram data were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software" (2012 Version A). Using sample S10 as the reference chromatogram, a control fingerprint chromatogram was generated, and similarity was evaluated, common peaks were identified, and their medicinal properties were assigned.

[0083] 2.2.2 Cluster Analysis

[0084] Cluster analysis groups data based on similarity measures, grouping similar samples into the same class and dissimilar samples into different classes. The results are typically displayed as a dendrogram. The peak areas of common peaks from 10 batches of FFHQ fingerprint spectra were imported into SPSS 26.0 software, and inter-group linkage was selected for cluster analysis using the squared Euclidean distance method.

[0085] 2.2.3 Principal Component Analysis

[0086] Principal component analysis (PCA) employs dimensionality reduction principles, grouping numerous indicators into a smaller set of composite indicators. This ensures that each principal component reflects the majority of the information from the original variables, and that the information carried by each principal component is independent and non-repetitive, thereby achieving efficient simplification and refinement of complex data. SIMCA 14.1 data analysis software was used to construct PCA models from 10 batches of identical FFHQ data matrices, and cross-validation was employed to evaluate the model's effectiveness.

[0087] 3 Results and Discussion

[0088] 3.1 Establishment of multi-wavelength fusion fingerprint spectrum by time segment

[0089] 3.1.1 Determination of chromatographic conditions

[0090] (1) Determination of chromatographic column

[0091] Comparison of two different chromatographic columns revealed that Agilent Poroshell EC C 18 The column (4.6 × 250 mm, 2.7 μm) showed better peak resolution, number of peaks, and response intensity, therefore it was chosen as the column for analysis. The results are as follows: Figure 1 .

[0092] Figure 1 These are high-performance liquid chromatograms for different chromatographic columns.

[0093] (2) Determination of the mobile phase

[0094] Depend on Figure 2 It is known that methanol has relatively weak elution ability. When acetonitrile is used as the mobile phase, the number of chromatographic peaks and the resolution increase compared to methanol. After adding 0.1% (v / v) formic acid, the number of chromatographic peaks and the response intensity both increase. Therefore, acetonitrile-0.1% (v / v) formic acid water is selected as the mobile phase.

[0095] Figure 2 These are high-performance liquid chromatograms of different mobile phases.

[0096] (3) Determination of column temperature

[0097] The effect of different column temperatures (20℃, 30℃, 40℃) on the chromatogram is shown in [reference needed]. Figure 3 By comparing the chromatograms obtained under different column temperatures, it was found that the column temperature had little effect on the separation effect and retention time of the experiment. Therefore, the commonly used column temperature of 30℃ was selected as the detection column temperature.

[0098] Figure 3 These are high-performance liquid chromatograms at different column temperatures.

[0099] (4) Determination of injection concentration

[0100] As the injection concentration increased, both the number of chromatographic peaks and the response intensity increased. Therefore, 30 mg / mL was chosen as the injection concentration, and the results are as follows: Figure 4 .

[0101] Figure 4 These are high-performance liquid chromatograms with different injection concentrations.

[0102] (5) Determination of injection volume

[0103] As the injection volume increases, the number of chromatographic peaks and the response intensity both increase. However, excessive injection volume may lead to peak broadening and tailing, affecting the separation effect, and may also overload the chromatographic column, shortening its lifespan. Therefore, 20 μL was chosen as the injection concentration. Results are as follows... Figure 5 .

[0104] Figure 5 High-performance liquid chromatograms with different injection volumes.

[0105] (6) Wavelength selection

[0106] The mixed traditional Chinese medicine test solution was prepared according to the above method. HPLC-DAD full-wavelength scanning was selected. Based on the scanning results, the fusion wavelength was selected using the number of chromatographic peaks and the total peak area at each wavelength as indicators. For example... Figure 6 The baselines at 254 nm, 280 nm, and 310 nm are relatively stable. Compared with other wavelengths, the number and peak area of ​​chromatographic peaks are higher and the response is better. However, a single wavelength cannot fully reflect the chemical composition information, and the chromatographic peaks of the above three wavelengths can complement each other. Therefore, the above three wavelengths were selected for fusion.

[0107] Figure 6 These are high-performance liquid chromatograms at different wavelengths.

[0108] (7) Final chromatographic conditions

[0109] The chromatographic column was packed with octadecylsilane-bonded silica gel, using Poroshell EC C as the packing material. 18(4.6 × 250 mm, 2.7 μm), the mobile phase was acetonitrile-0.1% (v / v) formic acid aqueous gradient elution, the gradient elution program is shown in Table 3, the column temperature was 30℃, the injection volume was 20 μL, the injection concentration was 30 mg / mL, and the flow rate was 0.5 mL / min. Wavelength selection: 0-44 min, 280 nm; 45-71 min, 310 nm; 72-94 min, 254 nm. A multi-wavelength fusion fingerprint spectrum of the mixed traditional Chinese medicine test sample was established. Figure 7 .

[0110] Figure 7 This is a high-performance liquid chromatogram of a multi-wavelength fusion mixture of traditional Chinese medicine samples.

[0111] 3.1.2 Construction of Multi-wavelength Fusion Fingerprint of Compound Astragalus Spleen-Strengthening Oral Liquid in Different Time Segments

[0112] The FFHQ (S10) test sample was prepared according to the preparation method in section 2.1.1. A multi-wavelength fusion fingerprint of Compound Astragalus Spleen-Strengthening Oral Liquid was established under the final chromatographic conditions in step (7) of section 3.1.1. The results are as follows: Figure 8 .

[0113] Figure 8 The time-segmented multi-wavelength fusion fingerprint spectrum of the mixed traditional Chinese medicine test sample and Compound Astragalus Spleen-Strengthening Oral Liquid (S10).

[0114] Depend on Figure 8 It can be seen that there are 28 common peaks at 254 nm, 29 common peaks at 280 nm, and 22 common peaks at 310 nm. After fusion, the number of chromatographic common peaks increased to 37, and the number of chromatographic common peaks and peak areas increased by 28-68% and 37-46%, respectively. After fusion, the fingerprint spectrum shows as much chemical composition information as possible about FFHQ, and can also be used to identify the flavor of prescription medicines.

[0115] 3.1.3 Identification of chromatographic peaks

[0116] Five chromatographic peaks were identified by comparison with the reference standard: P20 was chlorogenic acid, P29 was verrucoside, P34 was sinapic acid, P35 was gentiopicrin, and P37 was verrucoside. Figure 9 .

[0117] Figure 9 The images show the fusion fingerprints of Compound Astragalus Spleen-Strengthening Oral Liquid and the mixed reference standard. Specifically, a) is the fusion fingerprint of Compound Astragalus Spleen-Strengthening Oral Liquid (S10); b) is the fusion fingerprint of the mixed reference standard; c) is the blank solvent chromatogram; P20: chlorogenic acid; P29: verbascoside; P34: sinapic acid; P35: gentianoside; P37: verbascoside.

[0118] 3.1.4 Methodological Examination

[0119] Using peak 29 (which has good separation and moderate peak area, and whose main component isoflavonoid glycoside is the principal ingredient in Astragalus membranaceus and has certain pharmacological activity) as a reference peak, the methodological investigation was conducted on the relative retention time and relative peak area of ​​other peaks.

[0120] (1) Precision test

[0121] The precision of the instrument was investigated. Using chromatographic peak 29 as a reference peak, the RSD values ​​of the relative retention times of the 37 common peaks were all no higher than 1.2%, and the RSD values ​​of the relative peak areas were all no higher than 3.0%, indicating that the instrument has good precision and the test results are stable and reliable. The results are as follows... Figure 10 Tables 4 and 5.

[0122] Figure 10 This refers to the precision of the fingerprint spectrum.

[0123] Table 4 Results of precision relative to retention time

[0124]

[0125] Table 5 Results of precision relative to peak area

[0126]

[0127] (2) Repeated examination

[0128] The repeatability of the fingerprinting method was investigated. Using peak 29 as the reference peak, the RSD values ​​of the relative retention times of the 37 common peaks were all no higher than 2.2%, and the RSD values ​​of the relative peak areas were all no higher than 2.9%. The results are shown in [Figure number missing]. Figure 11 Tables 6 and 7 show that the method of the present invention has good repeatability.

[0129] Figure 11 For the repeatability of fingerprint patterns.

[0130] Table 6 Results of repeatability relative retention time

[0131]

[0132]

[0133] Table 7 Results of Relative Peak Area for Repeatability

[0134] (3) Stability test

[0135] Using chromatographic peak 29 as the reference peak, by Figure 12As shown in Tables 8 and 9, the relative retention time RSD values ​​of the 37 common peaks at different injection times are all no higher than 2.0%, and the relative peak area RSD values ​​are all no higher than 3.0%, indicating that FFHQ has good stability within 24 h and the test results are stable and reliable.

[0136] Figure 12 To ensure the stability of the fingerprint spectrum.

[0137] Table 8 Results of stability relative to retention time

[0138]

[0139] Table 9 Results of relative peak area for stability

[0140]

[0141] 3.2 Fingerprint analysis

[0142] 3.2.1 Similarity evaluation and common peak attribution of 10 batches of Compound Astragalus Spleen-Strengthening Oral Liquid

[0143] Ten batches of FFHQ samples were prepared according to the FFHQ test sample preparation method in section 2.1.1, and determined according to the chromatographic conditions in section 3.1.1. The fused chromatograms were imported into the "Traditional Chinese Medicine Fingerprint Similarity Evaluation System Software" (2012 Version A) to obtain the fingerprint chromatograms of the 10 batches of samples, with a total of 37 common peaks, such as... Figure 13 The common peak area of ​​each sample accounted for more than 80% of the total chromatographic peak area.

[0144] The similarity scores between the 10 batches of FFHQ fingerprint chromatograms and the control fingerprint chromatogram R were 0.974, 0.973, 0.992, 0.997, 0.997, 0.987, 0.964, 0.970, 0.960, and 0.963, respectively. All similarity scores were above 0.960, indicating good similarity among the batches, relatively stable preparation processes, and minimal component differences. The similarity results for the 10 batches are shown in Table 10.

[0145] Figure 13 Similarity of multi-wavelength fusion fingerprint spectra of 10 batches of Compound Astragalus Spleen-Strengthening Oral Liquid at different time periods.

[0146] Table 10. Similarity results of multi-wavelength fusion fingerprint spectra of 10 batches of Compound Astragalus Spleen-Strengthening Oral Liquid across different time periods.

[0147]

[0148] 3.2.2 Assignment of common peaks

[0149] Fusion fingerprints of single Chinese medicinal herbs and negative samples were constructed to assign chromatographic peaks. By comparing retention times and UV spectra, 37 common peaks were assigned to specific herbs. Among them, Astragalus membranaceus had 11 common peaks: P1, 2, 3, 5, 6, 7, 13, 29, 31, 35, and 37; Atractylodes macrocephala (fried) had 12 common peaks: P2, 5, 7, 8, 11, 16, 18, 20, 22, 23, 28, and 30; Dioscorea opposita (fried) had 25 common peaks: P1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 21, 22, 23, 24, 25, 28, 30, and 31; and Ziziphus jujuba had 3 common peaks. The peaks are P2, 7, and 10; Radish seed (fried) has 11 common peaks, namely P2, 14, 15, 19, 24, 26, 27, 28, 31, 34, and 36; Hawthorn (fried) has 16 common peaks, namely P2, 4, 5, 6, 7, 9, 10, 11, 13, 14, 15, 20, 21, 23, 30, and 31; Mulberry leaf has 21 common peaks, namely P1, 2, 3, 5, 7, 8, 11, 13, 16, 17, 18, 20, 21, 22, 23, 24, 27, 30, 31, 32, and 33. The attribution of each individual herb shows that yam (fried) and mulberry leaf contribute significantly to the fingerprint spectrum. For example... Figure 14 See Table 11.

[0150] Figure 14 The assignments of 37 common peaks in Compound Astragalus Spleen-Strengthening Oral Liquid are as follows: a. Sample lacking Astragalus; b. Sample lacking Atractylodes macrocephala (fried); c. Sample lacking Dioscorea opposita (fried); d. Sample lacking Jujube; e. Sample lacking Radish Seed (fried); f. Sample lacking Hawthorn (fried); g. Sample lacking Mulberry Leaf; h. Astragalus; i. Atractylodes macrocephala (fried); j. Dioscorea opposita (fried); k. Jujube; l. Radish Seed (fried); m. Hawthorn (fried); n. Mulberry Leaf; o. S10 sample; p. Blank solvent.

[0151] Quality control of Compound Astragalus Spleen-Strengthening Oral Liquid prepared in actual production was carried out based on the assignment of common peaks: when common peaks P29, 35, and 37 were missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacked Astragalus; when common peaks P12 and P25 were missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacked Dioscorea opposita (fried); when common peaks P19, 26, 34, and 36 were missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacked Raphanus sativus (fried); when common peaks P32 and P33 were missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacked Morus alba leaves.

[0152] Table 11. Attribution of 37 common peaks in Compound Astragalus Spleen-Strengthening Oral Liquid

[0153]

[0154] 3.2.3 Cluster Analysis

[0155] Using the peak area of ​​37 common peaks from 10 batches of FFHQ as variables, cluster analysis was performed using the between-groups linkage method and squared Euclidean distance. The results are shown in […]. Figure 15 When the square Euclidean distance is 15, the 10 batches of Compound Astragalus Spleen-Strengthening Oral Liquid can be divided into two categories: S1, S2, S9, S4, S5, S3, S7 and S8 cluster in one category, and S6 and S10 cluster in another category.

[0156] Figure 15 Cluster analysis diagram of 10 batches of Compound Astragalus Spleen-Strengthening Oral Liquid.

[0157] Principal component analysis

[0158] Constructing a PCA model and cross-validation plot ( Figure 16 A) The results show that R 2 (cum), Q 2 The cum values ​​were 0.898 and 0.955, respectively, both greater than 0.5, indicating that the model has good fitting effect and predictive ability. Based on the OPLS-DA scores, the 10 batches of samples can be divided into two categories ( Figure 16 B), where S6 and S10 are in one class, and S1, S2, S9, S4, S5, S3, S7 and S8 are in another class. This result is consistent with the results of cluster analysis.

[0159] Figure 16 Cross-validation plot (A) and scatter plot (B) of PCA for 10 batches of Compound Astragalus Spleen-Strengthening Oral Liquid.

[0160] summary

[0161] Compound Astragalus Spleen-Strengthening Oral Liquid is composed of seven traditional Chinese medicines: Astragalus membranaceus, Atractylodes macrocephala (fried), Dioscorea opposita (fried), Ziziphus jujuba, Crataegus pinnatifida (fried), Raphanus sativus (fried), and Morus alba. Its chemical composition is complex and diverse, encompassing flavonoids, saponins, phenolic acids, and polysaccharides. A single wavelength is insufficient to fully reflect chromatographic peak information. Therefore, based on the results of full-wavelength scanning (200-400 nm), three wavelengths—254 nm, 280 nm, and 310 nm—were selected for time-segmented fusion to establish a fingerprint spectrum, avoiding the loss of chromatographic peak information under single wavelengths. After fusion, the number of common peaks increased by 28-68% compared to single-wavelength detection, and the total peak area increased by 37-46%, significantly improving component coverage and supplementing the insufficient information from single-wavelength detection.

[0162] This invention optimizes chromatographic conditions by adjusting the chromatographic column, mobile phase, column temperature, injection volume, and injection concentration. The final chromatographic conditions are determined by the number of chromatographic peaks and their response intensity: the chromatographic column is an Agilent Poroshell EC C18 (4.6×250mm, 2.7 μm), the mobile phase is acetonitrile-0.1% formic acid-water gradient elution, the column temperature is 30℃, the injection volume is 20 μL, and the injection concentration is 30 mg / mL.

[0163] The fusion fingerprint chromatograms of 10 batches of samples contained 37 common peaks. Using peak 29 (verrucoside) as the reference peak, the RSDs of the precision, repeatability, stability, relative retention time, and relative peak area of ​​the 37 common peaks were all less than 3.0%. The 37 common peaks originated from different Chinese medicinal herbs in the formula: 11 from Astragalus membranaceus, 12 from Atractylodes macrocephala (fried), 25 from Dioscorea opposita (fried), 3 from Ziziphus jujuba, 16 from Crataegus pinnatifida (fried), 11 from Raphanus sativus (fried), and 21 from Morus alba leaves. The HPLC fingerprint chromatograms of 10 batches of Compound Astragalus Spleen-Strengthening Oral Liquid established in this invention can effectively identify the main medicinal components with a similarity of over 0.960, indicating good consistency in chemical composition among different batches of samples and relatively stable preparation process and sample quality.

[0164] Quality control of Compound Astragalus Spleen-Strengthening Oral Liquid prepared in actual production was carried out based on the assignment of common peaks: when common peaks P29, 35, and 37 were missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacked Astragalus; when common peaks P12 and P25 were missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacked Dioscorea opposita (fried); when common peaks P19, 26, 34, and 36 were missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacked Raphanus sativus (fried); when common peaks P32 and P33 were missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacked Morus alba leaves.

[0165] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing the fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid, characterized in that: The specific steps of the construction method are as follows: (1) Preparation of test solution: After freeze-drying the compound astragalus spleen-strengthening oral liquid, dissolve it in the initial mobile phase and filter it. Take the filtrate to obtain the test solution; (2) Preparation of reference solutions: The preparation method of the reference solutions includes: taking chlorogenic acid reference standard, verbascoside reference standard, sinapic acid reference standard, styracin reference standard, and verbascoside reference standard respectively, and adding methanol to prepare solutions of chlorogenic acid 0.687 mg / mL, verbascoside 2.033 mg / mL, sinapic acid 1.637 mg / mL, styracin 2.449 mg / mL, and verbascoside 2.259 mg / mL respectively; (3) Preparation of fingerprint spectrum: The test solution was tested by high performance liquid chromatography, and the fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid was generated from the obtained chromatographic data; The high-performance liquid chromatography (HPLC) conditions are as follows: Poroshell EC C using octadecylsilane-bonded silica gel as the stationary phase. 18 The column was 4.6 × 250 mm, 2.7 μm in diameter, with a column temperature of 20–40 °C, an injection volume of 20 μL, an injection concentration of 30 mg / mL, and a flow rate of 0.5 mL / min. The detection wavelength was 200–400 nm for full-wavelength scanning; the fusion wavelengths were 280 nm for 0–44 min, 310 nm for 45–71 min, and 254 nm for 72–94 min. Gradient elution was performed using acetonitrile as mobile phase A and 0.1% (v / v) formic acid aqueous solution as mobile phase B. The gradient elution procedure is as follows: 0-20 min, mobile phase A is 2%, mobile phase B is 98%; Over 20-55 minutes, mobile phase A changed from 2% to 15% at a constant rate, and mobile phase B changed from 98% to 85% at a constant rate. From 55 to 94 minutes, mobile phase A changed from 15% to 41% at a constant rate, and mobile phase B changed from 85% to 59% at a constant rate. All "%" values ​​refer to volume percentages.

2. The method for constructing the fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid according to claim 1, characterized in that: In step (1), the filtration is performed using a microporous membrane with a pore size of 0.22~0.45 μm.

3. The method for constructing a fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid according to claim 2, characterized in that: The microporous filter membrane has a pore size of 0.22 μm.

4. The method for constructing the fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid according to claim 1, characterized in that: In step (2), the column temperature is 30°C.

5. The method for constructing the fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid according to any one of claims 1-4, characterized in that: Using verbascoside as a reference peak, the fingerprint spectrum of the compound astragalus spleen-strengthening oral liquid includes 37 common peaks, as detailed below: The relative retention times for peak 1 are 0.069-0.070, peak 2 is 0.089, peak 3 is 0.099-0.101, peak 4 is 0.143-0.149, peak 5 is 0.161-0.171, peak 6 is 0.182-0.191, peak 7 is 0.262-0.274, peak 8 is 0.302-0.313, peak 9 is 0.361-0.377, and peak 10 is 0.

4. The relative retention times for peaks 03-0.424, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are as follows: 0.726-0.424, 0.431-0.447, 0.500-0.519, 0.511-0.530, 0.516-0.543, 0.547-0.560, 0.633-0.641, 0.653-0.661, 0.718-0.727, and 0.726-0.

724. The relative retention times for peaks 0-20 are 0.757-0.765, 21-22, 23-24, 25-26, 27-28 are 0.892-0.825, 28-29-0.892, 29-29-0.825, 29-20, 29-28, and 29-29-0.892, respectively. The relative retention times for peaks 0.896, 29, 30, 31, 32, 33, 34, 35, 36, and 37 are 1.243-1.

249. The relative retention times for peaks 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38 are 1.243-1.

249.

6. The method for constructing the fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid according to claim 5, characterized in that: Peak 20 is chlorogenic acid, peak 29 is verrucoside, peak 34 is sinapic acid, peak 35 is stigmoside, and peak 37 is verrucoside.

7. The method for constructing the fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid according to claim 5, characterized in that: In the fingerprint spectrum, Astragalus membranaceus had 11 common peaks, namely peaks 1, 2, 3, 5, 6, 7, 13, 29, 31, 35, and 37; Atractylodes macrocephala (fried) had 12 common peaks, namely peaks 2, 5, 7, 8, 11, 16, 18, 20, 22, 23, 28, and 30; Dioscorea opposita (fried) had 25 common peaks, namely peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 21, 22, 23, 24, 25, 28, 30, and 31; and Ziziphus jujuba had 3 common peaks. The peaks are 2, 7, and 10; radish seeds (fried) have 11 shared peaks, namely peaks 2, 14, 15, 19, 24, 26, 27, 28, 31, 34, and 36; hawthorn (fried) has 16 shared peaks, namely peaks 2, 4, 5, 6, 7, 9, 10, 11, 13, 14, 15, 20, 21, 23, 30, and 31; mulberry leaves have 21 shared peaks, namely peaks 1, 2, 3, 5, 7, 8, 11, 13, 16, 17, 18, 20, 21, 22, 23, 24, 27, 30, 31, 32, and 33.

8. A quality control method for Compound Astragalus Spleen-Strengthening Oral Liquid, characterized in that: The fingerprint spectrum of Compound Astragalus Spleen-Strengthening Oral Liquid was constructed according to the method of any one of claims 1-7. When the common peaks of P29, 35 and 37 are missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacks Astragalus; when the common peaks of P12 and P25 are missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacks Dioscorea (fried); when the common peaks of P19, 26, 34 and 36 are missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacks Raphanus sativus (fried); when the common peaks of P32 and P33 are missing, the prepared Compound Astragalus Spleen-Strengthening Oral Liquid lacks Mulberry Leaf.

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