Method for constructing HPLC (High Performance Liquid Chromatography) fingerprint spectrum of spleen-tonifying, heat-clearing and stasis-dissipating drink and determining content of multiple

Abstract

The invention discloses a method for constructing an HPLC (High Performance Liquid Chromatography) fingerprint spectrum of a spleen-tonifying, heat-clearing and stasis-dissipating drink and determining the content of multi-index components in the HPLC fingerprint spectrum, which comprises the following steps: detecting different batches of samples by taking 11 components such as tanshinol, liquiritin, hesperidin, lobetyolin, alkannic acid and the like as reference substances, adopting liquid chromatography and taking an acetonitrile-0. 3 vol% phosphoric acid aqueous solution as a mobile phase, and determining the content of the multi-index components in the fingerprint spectrum of the spleen-tonifying, heat-clearing and stasis-dissipating drink. And chemical component attribution and linear fitting are carried out on common peaks in the HPLC fingerprint spectrum, so that quantitative detection of the contents of the 11 components is realized. The method can realize qualitative and quantitative research on the spleen-tonifying, heat-clearing and blood-stasis-dissipating drink under the same chromatographic condition, and has a good application prospect in quality standard control of the spleen-tonifying, heat-clearing and blood-stasis-dissipating drink.

Description

Technical Field

[0001] This invention belongs to the field of quality control of traditional Chinese medicine preparations, specifically relating to the construction of HPLC fingerprint of Jianpi Qinghua Sanyu Decoction and the method for determining the content of multiple index components therein. Background Technology

[0002] Jianpi Qinghua Sanyu Yin is a traditional Chinese medicine formula used to treat spleen and stomach diseases. It consists of 14 Chinese herbs, including Codonopsis pilosula, Artemisia capillaris, Carapax Trionycis, Atractylodes macrocephala, Dolichos lablab, Poria cocos, Citrus reticulata, Pinellia ternata, Magnolia officinalis, Coptis chinensis, Curcuma zedoaria, Salvia miltiorrhiza, Amomum villosum, and Glycyrrhiza uralensis. It has the effects of strengthening the spleen and replenishing qi, clearing damp heat, softening and dispersing nodules, and promoting blood circulation and removing blood stasis. It is used to treat abdominal distension, abdominal pain, acid reflux, etc. caused by erosive gastritis, and spleen deficiency with damp heat and blood stasis with the above symptoms.

[0003] The quality of traditional Chinese medicine preparations is affected by various factors, such as the origin of the medicinal materials, the harvesting season, and the preparation process. Therefore, different batches of Jianpi Qinghua Sanyu Decoction may exhibit significant differences in the types and contents of its components. Establishing HPLC fingerprinting and accurately determining the content of the main components helps ensure the stability and consistency of product quality, thereby ensuring its stable efficacy in clinical applications. However, currently, there are no research reports on Jianpi Qinghua Sanyu Decoction. Therefore, to provide a basis for quality evaluation of Jianpi Qinghua Sanyu Decoction, it is necessary to establish an HPLC fingerprinting method for Jianpi Qinghua Sanyu Decoction and a method for determining the content of multiple indicators of its components. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing an HPLC fingerprint of Jianpi Qinghua Sanyu Decoction and determining the content of multiple components. This method not only obtains fingerprints reflecting the characteristic peaks of 52 chemical components of most of the medicinal materials in the Jianpi Qinghua Sanyu Decoction formula, including Codonopsis pilosula, Citrus reticulata peel, Salvia miltiorrhiza, Coptis chinensis, Magnolia officinalis, Curcuma zedoaria, and Glycyrrhiza uralensis, but also, based on a single wavelength (280 nm), achieves the content determination of 11 components in the Jianpi Qinghua Sanyu Decoction, including tanshinone, glycyrrhizin, hesperidin, codonopsis pilosula glycoside, lithospermic acid, berberine hydrochloride, salvianolic acid B, salvianolic acid A, glycyrrhizic acid, magnolol, and honokiol. This helps to achieve comprehensive and effective quality control of Jianpi Qinghua Sanyu Decoction preparations. The proposed method takes a total of 80 minutes and features high analytical efficiency, simplicity, and strong specificity, providing technical support for the quality control of Jianpi Qinghua Sanyu Decoction granules.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for constructing an HPLC fingerprint of a spleen-strengthening, blood-activating, and stasis-dispersing decoction and determining the content of multiple components, comprising the following steps: 1) Preparation of reference solution: Accurately weigh the reference powders of tanshinone, glycyrrhizin, hesperidin, codonopsis pilosula glycoside, lithospermic acid, berberine hydrochloride, salvianolic acid B, salvianolic acid A, glycyrrhizic acid, and honokiol and honokiol, and place them in volumetric flasks respectively. Add methanol to dissolve and dilute to the mark to prepare a single reference stock solution. Then, accurately measure each reference stock solution and place it in the same volumetric flask, add methanol to dilute to the mark, and shake well to obtain a mixed reference solution containing tanshinone, glycyrrhizin, hesperidin, codonopsis pilosula glycoside, lithospermic acid, berberine hydrochloride, salvianolic acid B, salvianolic acid A, glycyrrhizic acid, and honokiol and honokiol. 2) Preparation of test solution: Take Jianpi Qinghua Sanyu Decoction, shake well, accurately measure 10.0 mL and place it in a 25 mL volumetric flask, add 10 mL of 50% methanol solution, sonicate for 10 min, let it stand at room temperature, and then dilute to the mark with 50% methanol solution. Shake well, filter through a 0.22 μm microporous membrane, and take the filtrate to obtain the test solution. 3) Sample determination: Accurately pipette the mixed reference solution and the test solution separately, and perform HPLC to determine the chromatograms of the mixed reference solution and the test solution; 4) Establishment of fingerprint spectrum: The chromatogram of the obtained test sample is imported into the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine 2012 Edition" for evaluation to obtain the corresponding fingerprint spectrum, and the characteristic components are assigned using the chromatogram of the reference standard. 5) Determination of the content of characteristic components: The mixed reference solution was diluted into reference solutions of gradient concentrations, and the samples were injected and determined by HPLC. Then, linear regression was performed with mass concentration as the abscissa and peak area as the ordinate, and standard curves of each characteristic component were plotted to obtain their linear regression equations. The content of each characteristic component in the test solution was then calculated using the linear regression equations.

[0006] Furthermore, the chromatographic conditions for HPLC injection determination were: WATERS XBridge C. 18 The chromatographic column was 4.6 mm × 250 mm with a diameter of 5 μm. Gradient elution was performed using acetonitrile-0.3 vol% phosphoric acid aqueous solution as the mobile phase at a flow rate of 1.0 mL / min and a column temperature of 25 °C. A diode array detector was used with a detection wavelength of 280 nm and an injection volume of 5 μL.

[0007] Furthermore, the gradient elution program is as follows: 0-5 min, acetonitrile volume increases from 5% to 8%; 5-20 min, acetonitrile volume increases from 8% to 13%; 20-35 min, acetonitrile volume increases from 13% to 15%; 35-55 min, acetonitrile volume increases from 15% to 24%; 55-62 min, acetonitrile volume increases from 24% to 30%; 62-75 min, acetonitrile volume increases from 30% to 75%; and 75-80 min, acetonitrile volume increases from 75% to 80%.

[0008] The significant advantages of this invention are: This invention not only obtains fingerprint spectra of 52 characteristic peaks of chemical components in Jianpi Qinghua Sanyu Decoction under the same chromatographic conditions and determines the attribution of the 52 common characteristic peaks in the formula herbs, but also achieves content determination of 11 components in Jianpi Qinghua Sanyu Decoction based on a single wavelength (280 nm), including tanshinone, glycyrrhizin, hesperidin, codonopsis glycoside, lithospermic acid, berberine hydrochloride, salvianolic acid B, salvianolic acid A, glycyrrhizic acid, magnolol, and honokiol, quantifying the common components and better reflecting its intrinsic quality. Furthermore, this method has the advantages of simple operation, high analytical efficiency, and strong specificity, and can be completed in 80 minutes, laying a technical foundation for the comprehensive and effective quality control of Jianpi Qinghua Sanyu Decoction. Attached Figure Description

[0009] Figure 1 HPLC (208 nm) chromatograms at different extraction times.

[0010] Figure 2 HPLC (208 nm) chromatograms for different extraction solvents.

[0011] Figure 3 HPLC (208 nm) chromatograms for different extraction material-to-liquid ratios.

[0012] Figure 4-1 and Figure 4-2 HPLC (208 nm) spectra of different mobile phase systems.

[0013] Figure 5 HPLC (208nm) chromatograms obtained using C18 columns from different brands.

[0014] Figure 6-1 and Figure 6-2 HPLC chromatograms using different detection wavelengths.

[0015] Figure 7 HPLC (280 nm) spectra obtained using different column temperatures.

[0016] Figure 8To establish a control fingerprint spectrum using 15 batches of Jianpi Qinghua Sanyu Decoction samples.

[0017] Figure 9 The chromatogram of Jianpi Qinghua Sanyu Decoction shows the attribution of 52 common peaks in 12 medicinal materials.

[0018] Figure 10 HPLC chromatograms of Jianpi Qinghua Sanyu Decoction and mixed reference standards.

[0019] Figure 11 This is an HPLC chromatogram of the mixed reference standard, test solution, and negative sample.

[0020] Figure 12 Cluster heatmap analysis of the content of 11 components in 15 batches of Jianpi Qinghua Sanyu Decoction. Detailed Implementation

[0021] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0022] Material 1.1 Instruments DIONEX U3000 high-performance liquid chromatograph (Thermo Scientific, USA); KQ-500DE benchtop ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); EX125ZH electronic balance (Ohaus Instruments (Changzhou) Co., Ltd.); ultrapure water system (Sichuan Zhuoyue Water Treatment Equipment Co., Ltd.); H17.5 high-speed centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.).

[0023] 1.2 Drugs and Reagents Reference substances: hesperidin (batch number: 110721-202220), salvianolic acid B (batch number: 111562-202318), glycyrrhizin (batch number: 111610-202209), berberine hydrochloride (batch number: 110713-202015), glycyrrhizic acid (batch number: 110731-202122), magnolol (batch number: 110729-202015), and honokiol (batch number: 110). 730-202416) was purchased from the National Institutes for Food and Drug Control; tanshinone (batch number: 0611-RG-0035), shikonin (batch number: 0611-RG-0034), and salvianolic acid A (batch number: 0305-RF-0102) were purchased from Guangzhou Jiatu Technology Co., Ltd.; and codonopsis glycoside (batch number: 16030704) was purchased from Beijing Century Aoke Biotechnology Co., Ltd. The purity of all the above reference standards was ≥98%.

[0024] Codonopsis pilosula (batch number: 2401102172) and Magnolia officinalis (batch number: 2405070062) were purchased from Bozhou Huqiao Pharmaceutical Co., Ltd.; Coptis chinensis (batch number: 231101) and Amomum villosum (batch number: 230201) were purchased from Anhui Shangde Chinese Herbal Pieces Co., Ltd.; Curcuma zedoaria (batch number: 240101) and Glycyrrhiza uralensis (batch number: 230803) were purchased from Guangdong Rongxiang Pharmaceutical Co., Ltd.; Vinegar-processed turtle shell (batch number: 2300220101) was purchased from Guilu Pharmaceutical Group Co., Ltd.; Atractylodes macrocephala... (Batch No.: C017241104) was purchased from Hebei Hezhou Pharmaceutical Co., Ltd.; Poria cocos (Batch No.: A230402) was purchased from Hubei Jingui Traditional Chinese Medicine Pieces Co., Ltd.; Tangerine peel (Batch No.: 2406012) was purchased from Bozhou Cijitang Traditional Chinese Medicine Pieces Co., Ltd.; Pinellia ternata (Batch No.: A240528) was purchased from Bozhou Yonggang Traditional Chinese Medicine Pieces Co., Ltd.; Salvia miltiorrhiza (Batch No.: 2207251) was purchased from Anhui Puren Traditional Chinese Medicine Pieces Co., Ltd. The above medicinal materials were used to prepare single-herb samples and negative samples. Fifteen batches of Jianpi Qinghua Sanyu Decoction (formula: Codonopsis pilosula 15g, Artemisia capillaris 9g, Trionyx sinensis 18g, Curcuma zedoaria 9g, Atractylodes macrocephala 9g, Dolichos lablab 15g, Poria cocos 15g, Citrus reticulata 9g, Pinellia ternata 9g, Magnolia officinalis 9g, Coptis chinensis 3g, Salvia miltiorrhiza 15g, Amomum villosum 6g, Glycyrrhiza uralensis 3g), numbered S1~S15, were provided by the Pharmaceutical Preparation Center of the Second Affiliated Hospital of Fujian University of Traditional Chinese Medicine. Methanol and acetonitrile were of chromatographic purity, water was ultrapure water, and all other reagents were of analytical purity.

[0025] Investigation of extraction and chromatographic conditions 2.1 Selection of extraction time Take the same batch of Jianpi Qinghua Sanyu Decoction, shake well, accurately measure 4 portions, 10 mL each, and place them in 25 mL volumetric flasks. Add about 10 mL of 50% methanol, and ultrasonically extract for 5 min, 10 min, 15 min, and 20 min respectively. After cooling to room temperature, dilute to the mark with 50% methanol, shake well, filter through a 0.22 μm microporous membrane, collect the filtrate, and inject it into the sample. The optimal extraction time is determined based on the total number of chromatographic peaks and peak area. The results are shown in the figure. Figure 1 Ultimately, ultrasonic extraction was chosen to be 10 min.

[0026] 2.2 Selection of Extraction Solvent Take the same batch of Jianpi Qinghua Sanyu Decoction, shake well, accurately measure 5 portions, 10 mL each, and place them in 25 mL volumetric flasks. Add approximately 10 mL each of 10% methanol, 30% methanol, 50% methanol, 80% methanol, and pure methanol respectively. Sonicate for 10 min, allow to cool to room temperature, and then dilute to the mark with the corresponding solvent concentration. Shake well, filter through a 0.22 μm microporous membrane, collect the filtrate, and inject it into the sample. The optimal extraction solvent is determined based on the total number of chromatographic peaks and peak area. Results are shown below. Figure 2 Ultimately, 50% methanol was chosen as the extraction solvent.

[0027] 2.3 Selection of Extraction Material-Liquid Ratio Take the same batch of Jianpi Qinghua Sanyu Decoction, shake well, and accurately measure 5 portions, each 4.0, 8.0, 10.0, 15.0, and 20.0 mL respectively. Place each portion in a 25 mL volumetric flask, add approximately 10 mL of 50% methanol, sonicate for 10 min, allow to cool to room temperature, and then dilute to the mark with 50% methanol. Shake well, filter through a 0.22 μm microporous membrane, collect the filtrate, and inject it into the sample. The optimal extraction-liquid ratio is determined using the total number of chromatographic peaks and peak area as indicators. Results are shown below. Figure 3 The optimal extraction material-to-liquid ratio was ultimately determined to be 10:25.

[0028] 2.4 Selection of Mobile Phase System Take Jianpi Qinghua Sanyu Decoction, shake well, accurately measure 10.0 mL and place it in a 25 mL volumetric flask, add about 10 mL of 50% methanol, sonicate for 10 min, let it cool to room temperature, and then dilute to the mark with 50% methanol. Shake well, filter through a 0.22 μm microporous membrane, collect the filtrate, and inject it into the sample. Analyze the sample using methanol-0.1% formic acid solution, acetonitrile-0.1% formic acid solution, acetonitrile-0.1% phosphoric acid solution, acetonitrile-0.2% phosphoric acid solution, acetonitrile-0.3% phosphoric acid solution, and acetonitrile-0.4% phosphoric acid solution as mobile phases. The appropriate mobile phase system was determined based on the total number of chromatographic peaks and the resolution. Results are shown below. Figure 4-1 and 4-2 Ultimately, the acetonitrile-0.3% phosphoric acid water mobile phase system was selected.

[0029] 2.5 Selection of Different Chromatographic Columns Take the Jianpi Qinghua Sanyu Decoction, shake well, accurately measure 10.0 mL and place it in a 25 mL volumetric flask, add about 10 mL of 50% methanol, sonicate for 10 min, let it cool to room temperature, then dilute to the mark with 50% methanol, shake well, filter through a 0.22 μm microporous membrane, collect the filtrate, and inject it. Use C18 columns from Agilent, Thermo Fisher Scientific, Nano, and Waters respectively, and determine the appropriate column based on the total number of chromatographic peaks and resolution. Results are shown below. Figure 5 Ultimately, Waters columns were chosen as the preferred chromatographic column.

[0030] 2.6 Determination of Detection Wavelength Take Jianpi Qinghua Sanyu Decoction, shake well, accurately measure 10.0 mL and place it in a 25 mL volumetric flask, add about 10 mL of 50% methanol, sonicate for 10 min, let it cool to room temperature, then dilute to the mark with 50% methanol, shake well, filter through a 0.22 μm microporous membrane, collect the filtrate, and inject it. Using the total number of chromatographic peaks and resolution as indicators, compare the chromatograms at wavelengths of 210, 230, 250, 260, 280, 300, and 320 nm to determine the appropriate detection wavelength. Results are shown below. Figure 6-1 and 6-2 The optimal detection wavelength was determined to be 280 nm.

[0031] 2.7 Selection of Different Column Temperatures Take Jianpi Qinghua Sanyu Decoction, shake well, accurately measure 10.0 mL and place it in a 25 mL volumetric flask, add about 10 mL of 50% methanol, sonicate for 10 min, let it cool to room temperature, then dilute to the mark with 50% methanol, shake well, filter through a 0.22 μm microporous membrane, collect the filtrate, and inject it. Using the total number of chromatographic peaks and resolution as indicators, compare the chromatograms at column temperatures of 20, 25, and 30℃ to determine the appropriate column temperature. Results are shown below. Figure 7 The optimal column temperature was determined to be 25℃.

[0032] Methods and Results 3.1 Preparation of reference solution Accurately weigh appropriate amounts of each reference standard powder and place them separately in volumetric flasks. Dissolve and dilute to the mark with methanol to prepare single reference standard stock solutions containing tanshinone, glycyrrhizin, hesperidin, codonopsis glycoside, shikonin, berberine hydrochloride, salvianolic acid B, salvianolic acid A, glycyrrhizic acid, magnolol, and honokiol, with mass concentrations of 2.33, 1.32, 2.02, 2.10, 1.13, 1.91, 6.19, 3.07, 2.86, 0.79, and 1.41, respectively. mg / mL, then accurately measure appropriate amounts of each reference standard stock solution and place them in the same volumetric flask, add methanol to make up to volume, shake well, and you will get a mixed reference solution with concentrations of tanshinone, glycyrrhizin, hesperidin, codonopsis glycoside, lithospermic acid, berberine hydrochloride, salvianolic acid B, salvianolic acid A, glycyrrhizic acid, and magnolol, magnolol, with concentrations of 93.20, 26.40, 202.00, 42.00, 56.50, 191.00, 619.00, 30.70, 286.00, 7.90, and 14.10 μg / mL, respectively.

[0033] 3.2 Preparation of the test solution Take Jianpi Qinghua Sanyu Decoction, shake well, accurately measure 10.0 mL and place it in a 25 mL volumetric flask, add about 10 mL of 50% methanol, sonicate (power 250 W, frequency 50 kHz) for 10 min, let it cool to room temperature, then dilute to the mark with 50% methanol, shake well, filter through a 0.22 μm microporous membrane, and take the filtrate to obtain the test solution.

[0034] 3.3 Preparation of single medicinal materials and negative sample solutions According to the prescription ratio of Jianpi Qinghua Sanyu Decoction, weigh out each single herb and negative samples that are missing Codonopsis pilosula, Citrus reticulata peel, Salvia miltiorrhiza, Coptis chinensis, Magnolia officinalis, and Glycyrrhiza uralensis. Prepare the solutions of single herbs and negative samples according to the method in section "3.2".

[0035] 3.4 Chromatographic conditions Using WATERS XBridge C 18 The chromatographic column (4.6 mm × 250 mm, 5 μm), the mobile phase acetonitrile (A)-0.3% phosphoric acid aqueous solution (B), gradient elution (0-5 min, 5%→8%A; 5-20 min, 8%→13%A; 20-35 min, 13%→15%A; 35-55 min, 15%→24%A; 55-62 min, 24%→30%A; 62-75 min, 30%→75%A; 75-80 min, 75%→80%A), the column temperature was 25℃, the flow rate was 1.0 mL / min, a diode array detector was used, the detection wavelength was 280 nm, and the injection volume was 5 μL.

[0036] 3.5 Fingerprint Pattern Study 3.5.1 Precision Test Take approximately 10 mL of Jianpi Qinghua Sanyu Decoction (S5) and prepare the test solution according to the method in section "3.2". Inject the sample six times consecutively under the conditions in section "3.4", record the chromatograms, and use peak 34 (hesperidin) as the reference peak to calculate the peak area and relative retention time RSD values ​​of each common peak. The results show that the peak area and relative retention time RSD values ​​of the common peaks in the chromatograms obtained from the six injections are less than 2.99% and 0.21%, respectively, indicating that the method has good precision.

[0037] 3.5.2 Repeatability Test Take approximately 10 mL of Jianpi Qinghua Sanyu Decoction (S5) and prepare six parallel test solutions according to the method described in section "3.2". Inject and analyze the solutions under the conditions described in section "3.4", record the chromatograms, and calculate the peak area and relative retention time (RSD) of each common peak using peak 34 (hesperidin) as the reference peak. The results show that the RSD values ​​of the peak area and relative retention time of the common peaks in the chromatograms obtained from the six test solutions are less than 3.13% and 0.20%, respectively, indicating good repeatability of the method.

[0038] 3.5.3 Stability Test Take approximately 10 mL of Jianpi Qinghua Sanyu Decoction (S5) and prepare the test solution according to section "3.2". Place the solution at room temperature and inject it at 0, 2, 4, 8, 12, 16, and 24 h under the conditions described in section "3.4". Record the chromatograms. Using peak 34 (hesperidin) as the reference peak, calculate the peak area and relative retention time (RSD) of each common peak. The results show that the RSD values ​​of the peak area and relative retention time of the common peaks in the chromatograms obtained from injections at different times are less than 3.54% and 0.32%, respectively, indicating that the solution has good stability within 24 h.

[0039] 3.5.4 Fingerprint pattern establishment and similarity analysis Fifteen batches of Jianpi Qinghua Sanyu Decoction samples (S1~S15) were collected. Test solutions were prepared according to the method described in section "3.2", and analyzed under the conditions described in section "3.4". Chromatograms were recorded. The above chromatograms were processed and analyzed using the "Traditional Chinese Medicine Chromatogram Fingerprint Similarity Evaluation System (2012 Edition)" software. Using batch S5 as the reference chromatogram, the median method was employed with a time window of 0.1 min. Common peaks with strong signals, distinct peak shapes, and good stability were identified, resulting in 52 common peaks. After multi-point correction, a reference fingerprint chromatogram (R) was generated. The results are shown in [Figure 1]. Figure 8 The fingerprint profiles of 15 batches of Jianpi Qinghua Sanyu Decoction samples (S1~S15) were automatically matched with the generated control fingerprint profiles to calculate similarity. The similarity values ​​were 0.996, 0.996, 0.999, 1.000, 0.985, 0.996, 0.995, 0.999, 0.999, 0.986, 0.996, 0.995, 1.000, 1.000, and 0.986, respectively, all greater than 0.980, indicating that the similarity of the produced batches of Jianpi Qinghua Sanyu Decoction samples was good.

[0040] 3.5.5 Attribution and Identification of Common Peaks The mixed reference solution, test solution, and single herbal medicine solution prepared under sections “3.1”, “3.2”, and “3.3” were injected and analyzed under the conditions specified in section “3.4”. Chromatograms were recorded, and the relative retention times and UV absorption spectra of each chromatographic peak in the test solution and single herbal medicine solutions were compared. The attribution of the 52 common peaks of Jianpi Qinghua Sanyu Decoction to the 12 herbs was determined. The results are shown in […]. Figure 9 .like Figure 9 The results showed that peaks 1, 20, and 36 were from Codonopsis pilosula; peaks 4, 11, 12, 17, 22, 30, 34, 35, 44, 46, 49, and 50 were from Citrus reticulata peel; peaks 3, 10, 21, 31, 37, 40, 42, 43, 45, and 47 were from Salvia miltiorrhiza; peaks 14, 27, 33, 38, and 39 were from Coptis chinensis; peaks 16, 18, 28, 32, 51, and 52 were from Magnolia officinalis; peaks 6, 29, and 41 were from Curcuma zedoaria; peaks 23, 24, 25, 26, and 48 were from Glycyrrhiza uralensis; peak 2 was shared by Coptis chinensis, Codonopsis pilosula, and Poria cocos; peak 5 was shared by Citrus reticulata peel, Magnolia officinalis, and Amomum villosum; peaks 7 and 19 were shared by Citrus reticulata peel and Magnolia officinalis; peak 8 was shared by Citrus reticulata peel and Coptis chinensis; peak 9 was shared by Magnolia officinalis and Amomum villosum; peak 13 was shared by Magnolia officinalis and Coptis chinensis; and peak 15 was shared by Magnolia officinalis and Curcuma zedoaria. At the same time, such as Figure 10As shown, by comparing with the mixed reference standard, 11 components were identified, namely peak 3 (tanshinone), peak 25 (glycyrrhizin), peak 34 (hesperidin), peak 36 (codonopsis glycoside), peak 37 (lithocholic acid), peak 39 (berberine hydrochloride), peak 40 (tanshinone B), peak 43 (tanshinone A), peak 48 (glycyrrhizic acid), peak 51 (honokiol), and peak 52 (honokiol).

[0041] 3.6 Determination of the content of 11 components in Jianpi Qinghua Sanyu Decoction 3.6.1 Specificity test Take the mixed reference solution, test solution, and negative solution prepared under sections "3.1", "3.2", and "3.3", and perform analysis under the conditions described in section "3.4". Record the chromatograms, and the results are shown in [see table below]. Figure 11 The results showed that the components were well separated, and the chromatographic peaks of the negative sample showed no interference from other peaks, indicating that the quantitative method has good specificity.

[0042] 3.6.2 Examination of Linear Relationships Take the mixed reference solution from section "3.1", and transfer 0.5, 1.0, 2.0, 2.5, 4.0, and 5.0 mL respectively into 5 mL volumetric flasks. Dilute to the mark with 50% methanol to prepare a gradient concentration reference solution. Inject and determine the 11 components under the conditions described in section "3.4", and record the peak areas of each component. Then, plot the mass concentration on the x-axis (…). X The peak area is the vertical axis ( Y Linear regression was performed, and standard curves for each of the 11 components were plotted. The correlation coefficients and linear ranges were obtained, and the results are shown in Table 1.

[0043] Table 1 Linear Relationships of Each Component

[0044] 3.6.3 Precision test Take the mixed reference solution from section "3.1" and perform six consecutive injections under the conditions described in section "3.4". Record the peak area of ​​each analyte and calculate the precision RSD values ​​for the 11 components. Table 2 shows that the RSDs are all less than 1.58%, indicating good instrument precision.

[0045] 3.6.4 Stability Test Approximately 10 mL of Jianpi Qinghua Sanyu Decoction (S5) was prepared as a test solution according to the method described in section "3.2". After being left at room temperature for 0, 2, 4, 8, 12, and 24 hours, the solution was injected and analyzed under the conditions described in section "3.4". The peak areas of the 11 components were obtained, and their RSD values ​​were calculated. The results in Table 2 show that the RSDs were all less than 2.01%, indicating that the test solution had good stability within 24 hours.

[0046] 3.6.5 Repeatability Test Approximately 10 mL of Jianpi Qinghua Sanyu Decoction (S5) was prepared in parallel to the method described in section "3.2". The samples were analyzed under the conditions described in section "3.4", and the peak areas were recorded. The contents of each component and its RSD value were calculated using the linear equation described in section "3.6.2". The results in Table 2 show that the RSDs of all 11 components were less than 2.87%, indicating good repeatability of the extraction method.

[0047] Table 2. Methodological investigation results (%) of 11 components

[0048] 3.6.6 Recovery Test Take six samples (approximately 5 mL each) from the same repeatability batch (S5) with known content. Add each reference solution in an approximately 1:1 ratio according to the sample content in the repeatability results. Then prepare the test solution according to the method in section "3.2". Inject and determine the sample under the conditions in section "3.4", and calculate the recovery rate. Table 3 shows that the method has high accuracy.

[0049] Table 3 Results of the recovery rate test for 11 components ,

[0050] 3.6.7 Determination of Sample Content Fifteen batches (S1~S15) of Jianpi Qinghua Sanyu Decoction samples were collected. Test solutions were prepared according to section "3.2," and the samples were injected and analyzed under the conditions described in section "3.4." The peak areas were recorded, and then substituted into the linear equation obtained in section "3.6.2" to calculate the content of each component. The results are shown in Table 4. Simultaneously, the content data of 11 components from the 15 batches of samples were imported into Origin 2024 software, and heatmap cluster analysis was performed using the Heat Map Dendrogram.opx plugin. Figure 12 The color difference of the heat map squares can visually reveal the differences in the content of each component between different batches.

[0051] Table 4. Results of content determination of 11 components in 15 batches of Jianpi Qinghua Sanyu Decoction ( n =3, mg / mL)

[0052] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for constructing an HPLC fingerprint of a spleen-strengthening, blood-activating, and stasis-dispersing decoction and determining the content of multiple components, characterized in that... Includes the following steps: 1) Preparation of reference solution: Accurately weigh the reference powders of tanshinone, glycyrrhizin, hesperidin, codonopsis pilosula glycoside, lithospermic acid, berberine hydrochloride, salvianolic acid B, salvianolic acid A, glycyrrhizic acid, and honokiol and honokiol, and place them in volumetric flasks respectively. Add methanol to dissolve and dilute to the mark to prepare a single reference stock solution. Then, accurately measure each reference stock solution and place it in the same volumetric flask, add methanol to dilute to the mark, and shake well to obtain a mixed reference solution containing tanshinone, glycyrrhizin, hesperidin, codonopsis pilosula glycoside, lithospermic acid, berberine hydrochloride, salvianolic acid B, salvianolic acid A, glycyrrhizic acid, and honokiol and honokiol. 2) Preparation of test solution: Take Jianpi Qinghua Sanyu Decoction, shake well, accurately measure 10.0 mL and place it in a 25 mL volumetric flask, add 10 mL of 50% methanol solution, sonicate for 10 min, let it stand at room temperature, and then dilute to the mark with 50% methanol solution. Shake well, filter through a 0.22 μm microporous membrane, and take the filtrate to obtain the test solution. 3) Sample determination: Accurately pipette the mixed reference solution and the test solution separately, and perform HPLC analysis to obtain the chromatograms of the mixed reference solution and the test solution; 4) Establishment of fingerprint spectrum: The chromatogram of the obtained test sample is imported into the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine 2012 Edition" for evaluation to obtain the corresponding fingerprint spectrum, and the characteristic components are assigned using the chromatogram of the reference standard. 5) Determination of the content of characteristic components: The mixed reference solution was diluted into reference solutions of gradient concentrations, and the samples were injected and determined by HPLC. Then, linear regression was performed with mass concentration as the abscissa and peak area as the ordinate, and standard curves of each characteristic component were plotted to obtain their linear regression equations. The content of each characteristic component in the test solution was then calculated using the linear regression equations.

2. The method for constructing the HPLC fingerprint of the Jianpi Qinghua Sanyu Decoction and determining the content of multiple components according to claim 1, characterized in that, The chromatographic conditions for HPLC injection analysis were: WATERS XBridge C. 18 The chromatographic column was 4.6 mm × 250 mm with a diameter of 5 μm. Gradient elution was performed using acetonitrile-0.3 vol% phosphoric acid aqueous solution as the mobile phase at a flow rate of 1.0 mL / min and a column temperature of 25 °C. A diode array detector was used with a detection wavelength of 280 nm and an injection volume of 5 μL.

3. The method for constructing the HPLC fingerprint of the Jianpi Qinghua Sanyu Decoction and determining the content of multiple components according to claim 2, characterized in that, The gradient elution program was as follows: 0-5 min, acetonitrile volume increased from 5% to 8%; 5-20 min, acetonitrile volume increased from 8% to 13%; 20-35 min, acetonitrile volume increased from 13% to 15%; 35-55 min, acetonitrile volume increased from 15% to 24%; 55-62 min, acetonitrile volume increased from 24% to 30%; 62-75 min, acetonitrile volume increased from 30% to 75%; and 75-80 min, acetonitrile volume increased from 75% to 80%.

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