Method for constructing fingerprint spectrum of formula of radix puerariae, rhizoma coptidis, radix astragali and ginseng

Abstract

The invention relates to the technical field of construction of fingerprint spectrums, in particular to a construction method of a fingerprint spectrum of a formula of kudzuvine root, coptis root, astragalus root and ginseng. The preparation raw materials of the radix puerariae, radix salviae miltiorrhizae, roasted radix puerariae, rhizoma coptidis, rhizoma alismatis stir-fried with bran, semen cassiae, raw fructus crataegi, raw malt and lotus leaves are prepared, and the construction method comprises the following steps: (1) preparation of a reference sample test solution: decocting the preparation raw materials of the radix puerariae, radix salviae miltiorrhizae, radix astragali and radix scrophulariae formula with water to serve as a reference sample, and adding a solvent for extraction to obtain the reference sample test solution; (2) preparing a mixed reference substance solution; (3) injecting the reference sample test solution and the mixed reference substance solution into a high performance liquid chromatograph for determination to obtain a fingerprint spectrum; chromatographic conditions comprise that acetonitrile is adopted as a mobile phase A, and an ammonium acetate-triethylamine solution is adopted as a mobile phase B. The construction method disclosed by the invention has the advantages of relatively high precision, repeatability and stability and relatively high similarity.

Description

Technical Field

[0001] This invention relates to the field of fingerprint pattern construction technology, and specifically to a method for constructing a fingerprint pattern of the Ge Lian Qi Shen formula. Background Technology

[0002] Ge Lian Qi Shen Fang is an in-house formula of Kunshan Traditional Chinese Medicine Hospital in Jiangsu Province. It consists of nine herbs, including Astragalus membranaceus, Salvia miltiorrhiza, Pueraria lobata, Coptis chinensis, Cassia tora, and Nelumbo nucifera. It is a modified version of the hospital's lipid-lowering granules (Suzhou Pharmaceutical Manufacturing License Z04000676). While Lipid-lowering granules have been used to treat non-alcoholic fatty liver disease (NAFLD) for many years with definite efficacy, their application in NAFLD patients with diabetes is limited by dosage form and manufacturing process. Ge Lian Qi Shen Fang adds Pueraria lobata and Coptis chinensis to Lipid-lowering granules. Studies have reported that Pueraria lobata and Coptis chinensis have excellent lipid-lowering and blood sugar-lowering effects. Ge Lian Qi Shen Fang has the effects of invigorating qi and strengthening the spleen, soothing the liver and removing blood stasis, and promoting diuresis and eliminating turbidity, thus having a wider range of indications for clinical use in NAFLD.

[0003] To further improve the overall quality control of the Ge Lian Qi Shen formula and establish comprehensive quality control standards, it is necessary to construct a fingerprint spectrum for the formula to quantitatively detect multiple components. Currently, there are no relevant studies on the construction of a fingerprint spectrum for the Ge Lian Qi Shen formula.

[0004] Therefore, it is essential to develop a method for constructing the fingerprint spectrum of Ge Lian Qi Shen formula that can solve the above-mentioned technical problems. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for constructing a fingerprint spectrum of Ge Lian Qi Shen Fang (a traditional Chinese medicine formula) with high precision, repeatability, stability, and similarity.

[0006] This invention is achieved through the following technical solutions: This invention provides a method for constructing a fingerprint spectrum of a formula containing Astragalus membranaceus, Salvia miltiorrhiza, roasted Pueraria lobata, Coptis chinensis, stir-fried Alisma plantago-aquatica, Cassia tora, raw hawthorn, raw malt, and lotus leaf. The construction method includes the following steps: (1) Preparation of reference sample test solution: The raw materials for the preparation of Ge Lian Qi Shen Fang were decocted in water and used as reference samples. The solution was extracted with solvent to obtain the reference sample test solution. (2) Preparation of mixed reference solution: Take the reference standards 3'-hydroxypuerarin, puerarin-6''-O-xyloside, puerarin apigenin, 3'-methoxypuerarin, puerarin, daidzein, palmatine hydrochloride, stigmosiderin, auranthiazines, sennae, tanshinone, quercetin-3-O-β-D-glucopyranoside, lotus leaf alkaloid, berberine hydrochloride, sennae, cassia seed, and cassia anthraquinone, dissolve them in solvent to obtain mixed reference solution; (3) Inject the reference sample test solution and the mixed reference solution into the high performance liquid chromatograph for determination to obtain the fingerprint spectrum; the chromatographic conditions include: using acetonitrile as mobile phase A and using ammonium acetate-triethylamine solution as mobile phase B.

[0007] In one embodiment of the present invention, the solvent in steps (1) and (2) is methanol, preferably anhydrous methanol.

[0008] In one embodiment of the present invention, the molar concentration of ammonium acetate in the mobile phase B is 0.01-0.03 mol / L, and the mass concentration of triethylamine is 0.1-0.5%.

[0009] In a preferred embodiment of the present invention, the molar concentration of ammonium acetate in the mobile phase B is 0.02 mol / L, and the mass concentration of triethylamine is 0.3%.

[0010] Preferably, the pH of the mobile phase B is 5.5-6.5.

[0011] More preferably, the pH of the mobile phase B is 6.0.

[0012] More preferably, the mobile phase B uses glacial acetic acid to adjust the pH.

[0013] In one embodiment of the present invention, the chromatographic conditions further include gradient elution, with the following specific procedure: 0-20 min, mobile phase A 5%-13%; 20-35 min, mobile phase A 13%-17%; 35-60 min, mobile phase A 17%-35%; 60-70 min, mobile phase A 35%-40%; 70-80 min, mobile phase A 40%-80%; 80-85 min, mobile phase A 80%-5%; 85-105 min, mobile phase A 5%.

[0014] As one embodiment of the present invention, the chromatographic conditions further include any one or more of the following conditions: a) Column temperature: 30-40℃, preferably 35℃; b) Flow rate: 0.8-1.2 mL / min, preferably 1.0 mL / min; c) Detection wavelength: 250-300nm, preferably 280nm; d) Injection volume: 5-12 μL, preferably 10 μL; e) Chromatographic column: C18 chromatographic column.

[0015] As one embodiment of the present invention, the decoction is prepared by the following process: mixing the raw materials, soaking them in 8-12 times the amount of water for 0.5-1.5 hours, decocting them 1-3 times for 1-2 hours each time, and combining the decoctions.

[0016] As a preferred embodiment of the present invention, the decoction process is as follows: the raw materials are mixed, soaked in 10 times the amount of water for 1 hour, decocted twice for 1 hour each time, and the decoctions are combined.

[0017] In a preferred embodiment of the present invention, the decoction liquid is further concentrated after being combined. More preferably, it is concentrated to 15-25% of the volume of the decoction liquid.

[0018] Preferably, the construction method further includes preparing a single-ingredient decoction sample test solution and a negative control sample test solution, and determining the chromatograms of the single-ingredient decoction sample test solution and the negative control sample test solution; The preparation of the single-decocted sample test solution includes: weighing out 9 medicinal materials, namely Astragalus membranaceus, Salvia miltiorrhiza, roasted Pueraria lobata, Coptis chinensis, stir-fried Alisma plantago-aquatica, Cassia tora, raw hawthorn, raw malt, and lotus leaf, and preparing them according to the prescription process of water decoction to obtain a single-decocted sample of each medicinal material, and extracting them with solvent to obtain a single-decocted sample test solution of each medicinal material. The preparation of the negative control sample test solution includes: weighing the other 8 medicinal materials that are missing the corresponding medicinal materials according to the prescription amount, preparing them according to the decoction prescription process, and obtaining 9 negative control samples that are missing Astragalus membranaceus, Salvia miltiorrhiza, roasted Pueraria lobata, Coptis chinensis, stir-fried Alisma plantago-aquatica, Cassia tora, raw Crataegus pinnatifida, raw malt, and lotus leaf, respectively, extracting them with solvent, and obtaining negative control sample test solutions that are missing Astragalus membranaceus, Salvia miltiorrhiza, roasted Pueraria lobata, Coptis chinensis, stir-fried Alisma plantago-aquatica, Cassia tora, raw Crataegus pinnatifida, raw malt, and lotus leaf, respectively.

[0019] More preferably, the construction method further includes importing the chromatograms obtained from the determination of multiple batches of test sample solutions into the chromatographic fingerprint similarity evaluation system of traditional Chinese medicine to determine the common fingerprint characteristic peaks.

[0020] More preferably, the construction method further includes comparing the common fingerprint characteristic peaks with the chromatograms of the single decoction sample test solution and the negative control sample test solution to assign the common fingerprint characteristic peaks.

[0021] Particularly preferred, the fingerprint spectrum exhibits 26 chromatographic peaks, wherein peak 2 is 3'-hydroxypuerarin, peak 3 is puerarin, peak 4 is puerarin-6''-O-xyloside, peak 5 is 3'-methoxypuerarin, peak 6 is puerarin apigenin, peak 8 is salvianolic acid B, peak 11 is quercetin-3-O-β-D-glucopyranoside, peak 15 is stigmosiderin, peak 16 is daidzein, peak 18 is palmatine hydrochloride, peak 19 is berberine hydrochloride, peak 22 is cassia seed extract, peak 23 is nuciferine, peak 24 is cassia seed extract, peak 25 is cassia seed extract, and peak 26 is cassia anthraquinone.

[0022] In one embodiment of the present invention, the volume ratio of the reference sample to the solvent in step (1) is 1:1-4, preferably 1:1.

[0023] As one embodiment of the present invention, the extraction method in step (1) is ultrasonic extraction, and the extraction time is 15-60 min, preferably 30 min.

[0024] As one embodiment of the present invention, after the extraction in step (1) is completed, filtration is also performed using a microporous membrane.

[0025] As one embodiment of the present invention, the raw materials for preparing the Ge Lian Qi Shen formula, by weight, include 12-21 parts of Astragalus membranaceus, 7-15 parts of Salvia miltiorrhiza, 11-22 parts of roasted Pueraria lobata, 4-7 parts of Coptis chinensis, 8-14 parts of stir-fried Alisma plantago-aquatica, 7-15 parts of Cassia tora, 8-15 parts of raw Crataegus pinnatifida, 10-23 parts of raw malt, and 8-15 parts of lotus leaf.

[0026] In a preferred embodiment of the present invention, the raw materials for preparing the Ge Lian Qi Shen formula, by weight, include 15 parts of Astragalus membranaceus, 10 parts of Salvia miltiorrhiza, 15 parts of roasted Pueraria lobata, 5 parts of Coptis chinensis, 10 parts of stir-fried Alisma plantago-aquatica, 10 parts of Cassia tora, 10 parts of raw Crataegus pinnatifida, 15 parts of raw malt, and 10 parts of lotus leaf.

[0027] In one embodiment of the present invention, the multiple batches of test solution consist of 20 batches, and the similarity of the chromatograms is all >0.960.

[0028] As an embodiment of the present invention, in step (2), each 1 mL of the mixed reference solution contains 225.30 μg of 3'-hydroxy puerarin, 433.00 μg of puerarin-6''-O-xyloside, 507.50 μg of puerarin apioside, 736.20 μg of 3'-methoxy puerarin, 1870.40 μg of puerarin, 125.60 μg of daidzin, 93.00 μg of palmatine hydrochloride, 278.70 μg of formononetin, 105.90 μg of aurantio-obtusin, 800.80 μg of salvianolic acid B, 516.30 μg of quercetin-3-O-β-D-glucuronide, 277.50 μg of nuciferine, 192.00 μg of berberine hydrochloride, 86.30 μg of chryso-obtusin, 23.40 μg of obtusin, and 93.30 μg of chryso-obtusin anthraquinone.

[0029] The beneficial effects of the present invention are as follows: (1) The present invention provides a method for constructing the fingerprint of the reference sample of Ge-Lian-Qi-Shen formula, and establishes the HPLC fingerprints of 20 batches of reference samples of Ge-Lian-Qi-Shen formula, with a similarity greater than 0.960. Each batch of reference samples has good uniformity and stability. The fingerprint has a relatively rich number of chromatographic peaks and good resolution, and a total of 26 common peaks are determined, which comprehensively reflects the chemical composition information of Ge-Lian-Qi-Shen formula.

[0030] (2) Among the 26 common peaks of the fingerprint of the present invention, the chemical components represented by 16 chromatographic peaks are identified. It covers the main chemical components of the monarch drugs Astragalus membranaceus and Salvia miltiorrhiza, the ministerial drugs Pueraria lobata and Coptis chinensis, the adjuvant drug Cassia obtusifolia, and the guiding drug Nelumbo nucifera in the prescription. Among them, peak 2 is 3'-hydroxy puerarin, peak 3 is puerarin, peak 4 is puerarin-6''-O-xyloside, peak 5 is 3'-methoxy puerarin, peak 6 is puerarin apioside, peak 8 is salvianolic acid B, peak 11 is quercetin-3-O-β-D-glucuronide, peak 15 is formononetin, peak 16 is daidzin, peak XVIII is palmatine hydrochloride, peak XIX is berberine hydrochloride, peak XXII is aurantio-obtusin, peak XXIII is nuciferine, peak XXIV is chryso-obtusin, peak XXV is obtusin, and peak XXVI is chryso-obtusin anthraquinone.

[0031] (3) The HPLC fingerprint established by the present invention can be used for the quality control of Ge-Lian-Qi-Shen formula to ensure the safety and effectiveness of clinical medication.

[0032] (4) By optimizing the composition, pH and elution program of the mobile phase, the present invention improves the separation effect of each peak, which is more conducive to accurately reflecting the chemical composition information of Ge-Lian-Qi-Shen formula. [[ID=十六]]Description of the Drawings

[0033] Figure 1 It is the HPLC fingerprint of 20 batches of reference samples of Ge-Lian-Qi-Shen formula in Example 1; Figure 2 The HPLC chromatograms are of the Ge Lian Qi Shen Fang standard sample (A) and the mixed reference standard (B); Figure 3 The HPLC chromatograms are of the reference sample of Ge Lian Qi Shen Fang, each negative control sample and each single decoction sample; Figure 4 HPLC chromatogram of the Ge Lian Qi Shen Fang reference sample with acetonitrile-0.2% formic acid aqueous solution as the mobile phase; Figure 5 HPLC chromatogram of the Ge Lian Qi Shen Fang reference sample with acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase; Figure 6 The HPLC chromatogram of the Ge Lian Qi Shen Fang standard sample in mobile phase B at pH 4.3 is shown. Figure 7 The HPLC chromatogram of the Ge Lian Qi Shen Fang reference sample under gradient elution program (a) is shown. Figure 8 The HPLC chromatogram of the Ge Lian Qi Shen Fang reference sample under gradient elution program (b) is shown. Figure 9 The HPLC chromatogram of the Ge Lian Qi Shen Fang reference sample under gradient elution program (c) is shown. Figure 10 The HPLC chromatogram of the Ge Lian Qi Shen Fang standard sample in mobile phase B at pH 5.5 is shown. Figure 11 The HPLC chromatogram is of the Ge Lian Qi Shen Fang reference sample in mobile phase B at pH 6.5. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0035] Example 1 1. Instruments and Materials 1.1 Instruments LC-20AD High Performance Liquid Chromatograph (Shimadzu Corporation, Japan); BT 25S 0.0001g electronic balance (Sartorius Scientific Instruments, Germany); TGL-16G benchtop high-speed centrifuge (Shanghai Anting Scientific Instrument Factory); LC-PTHW-2L temperature-controlled electric heater (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); N-1200B rotary evaporator (Shanghai Ailang Instrument Co., Ltd.); KQ500E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Milli-Q ultrapure water system (Merck, USA).

[0036] 1.2 Medicinal Materials The prescription for Ge Lian Qi Shen Fang includes Astragalus membranaceus, Salvia miltiorrhiza, roasted Pueraria lobata, Coptis chinensis, stir-fried Alisma plantago-aquatica, Cassia tora, raw hawthorn, raw malt, and lotus leaf. The origin and batch number are shown in Table 1. All the above medicinal materials were identified as qualified processed medicinal slices by Chief Pharmacist Li Qingsong of the Pharmacy Department of Kunshan Traditional Chinese Medicine Hospital according to the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0037] 1.3 Reference Standards and Main Reagents Reference standards: puerarin (batch number MUST-24051602), 3'-hydroxypuerarin (batch number MUST-25051219), puerarin-6''-O-xyloside (batch number MUST-24032919), puerarin apigenin (batch number MUST-25012503), 3'-methoxypuerarin (batch number MUST-25010418), salvianolic acid B (batch number MUST-240912190), daidzein (batch number MUST-24051506), and palmatine hydrochloride (batch number MUST-24041414). The following reagents were purchased from Chengdu Manster Biotechnology Co., Ltd.: styracin (batch number MUST-21080311), quercetin-3-O-β-D-glucopyranoside (batch number MUST-21072805), nuciferine (batch number MUST-20032204), berberine hydrochloride (batch number MUST-22010607), cassia seed extract (MUST-19111312), cassia seed extract (MUST-19060107), cassia seed anthraquinone (MUST-19070608), and orange-yellow cassia seed extract (batch number MUST-22062303). Acetonitrile and triethylamine were of chromatographic grade; water was ultrapure water; all other reagents were of analytical grade.

[0038] Table 1 Information on medicinal materials in prescriptions containing Ge Lian Qi Shen (Ge Lian Qi Shen).

[0039] 2. Methods and Results 2.1 Preparation of reference samples for Ge Lian Qi Shen formula The single herbs were randomly sorted and combined using a random number table method to obtain the batch numbers of each herb corresponding to 20 batches of the Ge-Lian-Qi-Shen formula reference samples. Twenty batches of Ge-Lian-Qi-Shen formula reference samples were prepared, and the information of each batch of herbs is shown in Table 2 and Table 3. For each batch, 15 g of Astragalus membranaceus, 10 g of Salvia miltiorrhiza, 15 g of Radix Puerariae (processed with bran), 5 g of Coptis chinensis, 10 g of Alisma orientale (processed with bran), 10 g of Cassia obtusifolia, 10 g of Crataegus pinnatifida, 15 g of Hordeum vulgare germinatum, and 10 g of Folium Nelumbinis were weighed, soaked in 10 times the amount of water for 1 h, decocted twice for 1 h each time, the decoction liquids were combined, and concentrated to 400 mL to obtain the product.

[0040] Weigh the prescribed amounts of the 9 herbs, namely Astragalus membranaceus, Salvia miltiorrhiza, Radix Puerariae (processed with bran), Coptis chinensis, Alisma orientale (processed with bran), Cassia obtusifolia, Crataegus pinnatifida, Hordeum vulgare germinatum, and Folium Nelumbinis, respectively, and obtain the single decoction samples of each herb according to the above preparation method of the reference samples; weigh the other 8 herbs lacking the corresponding herb according to the prescription amount, and obtain 9 negative control samples lacking Astragalus membranaceus, Salvia miltiorrhiza, Radix Puerariae (processed with bran), Coptis chinensis, Alisma orientale (processed with bran), Cassia obtusifolia, Crataegus pinnatifida, Hordeum vulgare germinatum, and Folium Nelumbinis respectively in the same way.

[0041] 2.2 Preparation of the mixed reference substance solution and the test solution of the reference sample Mixed reference substance solution: Weigh appropriate amounts of each reference substance accurately, dissolve in methanol to prepare a mixed reference substance solution containing 225.30 μg of 3'-hydroxy puerarin, 433.00 μg of puerarin-6''-O-xyloside, 507.50 μg of puerarin apiin, 736.20 μg of 3'-methoxy puerarin, 1870.40 μg of puerarin, 125.60 μg of daidzin, 93.00 μg of palmatine hydrochloride, 278.70 μg of formononetin, 105.90 μg of aurantio-obtusin, 800.80 μg of salvianolic acid B, 516.30 μg of quercetin-3-O-β-D-glucuronide, 277.50 μg of nuciferine, 192.00 μg of berberine hydrochloride, 86.30 μg of chryso-obtusin, 23.40 μg of obtusin, and 93.30 μg of chryso-obtusone per 1 mL, shake well to obtain the solution.

[0042] Test solution of the reference sample: Pipette 10 mL of the Ge-Lian-Qi-Shen formula reference sample accurately into a stoppered conical flask, add 10 mL of methanol accurately, weigh, extract by ultrasonic wave for 30 min, cool, make up the reduced weight with 50% methanol, shake well, after centrifugation, take the supernatant and filter through a 0.22 μm microporous filter membrane to obtain the test solution of the reference sample. The test solutions of the single decoction samples and the negative control samples are prepared in the same way.

[0043] Table 2 Information of the batch numbers of the cut crude drugs corresponding to 20 batches of the Ge-Lian-Qi-Shen formula reference samples (1)

[0044] Table 3 Information of the batch numbers of the cut crude drugs corresponding to 20 batches of the Ge-Lian-Qi-Shen formula reference samples (2)

[0045] 2.3 Investigation of Fingerprint Mapping Methodology 2.3.1 Chromatographic conditions Agilent C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile (mobile phase A) and 0.02 mol / L ammonium acetate + 0.3% triethylamine (mobile phase B, pH adjusted to 6.0 with glacial acetic acid); column temperature: 35 °C; flow rate: 1.0 mL / min -1 Detection wavelength: 280nm; Injection volume: 10μL.

[0046] Gradient elution: 0-20 min, acetonitrile 5%-13%; 20-35 min, acetonitrile 13%-17%; 35-60 min, acetonitrile 17%-35%; 60-70 min, acetonitrile 35%-40%; 70-80 min, acetonitrile 40%-80%; 80-85 min, acetonitrile 80%-5%; 85-105 min, acetonitrile 5%.

[0047] 2.3.2 Precision Examination The S2 reference sample was prepared according to the test sample method. Under the chromatographic conditions in section “2.3.1”, it was injected 6 times consecutively. With peak 3 (puerarin) as the reference peak, the relative retention time RSD of each common peak was in the range of 0.01%-0.06%, and the relative peak area RSD was in the range of 0.72%-2.03%, indicating that the instrument precision was good.

[0048] 2.3.3 Repeatability Test Six samples of the S2 reference sample were prepared in parallel according to the test sample method. Under the chromatographic conditions in section “2.3.1”, the samples were injected and analyzed. With peak 3 (puerarin) as the reference peak, the relative retention time RSD of each common peak was in the range of 0.02%-0.10%, and the relative peak area RSD was in the range of 0.77%-2.77%, indicating that the method has good repeatability.

[0049] 2.3.4 Stability Test The S2 reference sample was prepared according to the method for the test sample. Under the chromatographic conditions specified in section "2.3.1", the sample was injected and analyzed at 0, 2, 4, 6, 8, 10, 12, and 24 hours after preparation. Peak 3 (puerarin) was used as the reference peak. The relative retention time RSD of each common peak was in the range of 0.01%-0.06%, and the relative peak area RSD was in the range of 0.78%-2.61%, indicating that the test sample has good stability.

[0050] 2.4 Fingerprint mapping and similarity evaluation Take 20 batches of reference samples of Ge-Lian-Qi-Shen formula test samples, inject and detect them according to the chromatographic conditions under "2.3.1", and record the chromatograms. Using the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" evaluation software, with the S2 reference sample chromatogram as the reference, set the time width to 0.1 min, use the median method, perform multi-point calibration and full-peak matching, and generate the fingerprint of the reference sample of Ge-Lian-Qi-Shen formula and its reference fingerprint (R). The results are shown in Figure 1 .

[0051] A total of 26 chromatographic peaks were calibrated in the fingerprint. 16 chromatographic peaks were identified according to the mixed reference substances ( Figure 2 , where A is the HPLC chromatogram of the S2 reference sample, and B is the HPLC chromatogram of the mixed reference substances): Peak 2 is 3'-hydroxy puerarin, Peak 3 is puerarin, Peak 4 is puerarin-6''-O-xyloside, Peak 5 is 3'-methoxy puerarin, Peak 6 is puerarin apiin, Peak 8 is salvianolic acid B, Peak 11 is quercetin-3-O-β-D-glucuronide, Peak 15 is formononetin, Peak 16 is daidzin, Peak 18 is palmatine hydrochloride, Peak 19 is berberine hydrochloride, Peak 22 is aurantio-obtusin, Peak 23 is nuciferine, Peak 24 is chryso-obtusin, Peak 25 is obtusin, and Peak 26 is chrysophanol anthraquinone.

[0052] Taking the reference fingerprint as the reference, calculate the similarity of the chromatograms of 20 batches of reference samples of Ge-Lian-Qi-Shen formula. The similarity evaluation results are shown in Table 4. The similarities are all > 0.960, indicating that the quality of the fingerprint of each batch of reference samples established is stable.

[0053] Table 4 Similarities of Fingerprints of 20 Batches of Reference Samples

[0054] 2.5 Identification of Common Peaks and Their Medicinal Material Attribution <M The HPLC chromatograms of the reference sample of Ge-Lian-Qi-Shen formula, the single decoction samples of each medicinal material (referred to as single decoction), and the negative control sample (referred to as negative) are shown in Figure 3 . Through comparative identification, it can be known that Peak 8 (salvianolic acid B) belongs to Salvia miltiorrhiza, Peaks 2 (3'-hydroxy puerarin), 3 (puerarin), 4 (puerarin-6''-O-xyloside), 5 (3'-methoxy puerarin), 6 (puerarin apiin), 7, 9, 10, 16 (daidzin) belong to Pueraria lobata, Peaks 18 (palmatine hydrochloride), 19 (berberine hydrochloride) belong to Coptis chinensis, Peaks 14, 20, 21, 22 (aurantio-obtusin), 24 (chryso-obtusin), 25 (obtusin), 26 (chrysophanol anthraquinone) belong to Cassia obtusifolia, Peaks 11 (quercetin-3-O-β-D-glucuronide), 17, 23 (nuciferine) belong to Nelumbo nucifera; Peak 1 is a common peak of Pueraria lobata, Crataegus pinnatifida, and Hordeum vulgare, Peak 12 is a common peak of Pueraria lobata and Nelumbo nucifera, and Peak 15 (formononetin) is a common peak of Astragalus membranaceus and Pueraria lobata.

[0055] 2.6 Determination of the content of major components and the transfer rate of indicative components in reference samples 2.6.1 Examination of Linear Relationships Accurately weigh appropriate amounts of 3'-hydroxypuerarin, puerarin, puerarin-6''-O-xyloside, 3'-methoxypuerarin, puerarin apigenin, salvianolic acid B, quercetin-3-O-β-D-pyranoside, gentiopicroside, daidzein, palmatine hydrochloride, berberine hydrochloride, cassia seed extract, nuciferine, cassia seed extract, cassia seed extract, and cassia anthraquinone, and place them in a volumetric flask. Dissolve in methanol and dilute to the mark to prepare a mixed reference stock solution. Accurately transfer appropriate volumes to different volumetric flasks, dilute with methanol, and obtain a series of reference solutions with different mass concentrations. Inject and analyze according to the chromatographic conditions in section "2.3.1". The reference concentration is C (μg·ml). -1 Using α as the x-axis and peak area A as the y-axis, a standard curve was plotted, and the linear regression equation results are shown in Table 5.

[0056] Table 5 Results of linear relationship investigation of main components

[0057] 2.6.2 Precision test Take the same reference sample test solution (S2), and inject it six times consecutively under the chromatographic conditions in section "2.3.1". Record the peak areas of 3'-hydroxypuerarin, puerarin, puerarin-6''-O-xyloside, 3'-methoxypuerarin, puerarin apigenin, salvianolic acid B, quercetin-3-O-β-D-pyranoside, gentiopicroside, daidzein, palmatine hydrochloride, berberine hydrochloride, cassia seed extract, nuciferine, cassia seed extract, cassia seed extract, and cassia anthraquinone. Calculate their RSDs as 0.93%, 0.95%, 0.91%, 0.97%, 0.89%, 0.73%, 0.97%, 2.03%, 1.88%, 1.79%, 0.96%, 1.61%, 1.19%, 0.94%, 1.29%, and 1.35%, respectively, indicating good instrument precision.

[0058] 2.6.3 Repeatability Test Take the same reference sample and prepare 6 parallel solutions according to the test solution preparation method. Inject and determine the chromatographic conditions under section "2.3.1", recording the following: 3'-hydroxypuerarin, puerarin, puerarin-6''-O-xyloside, 3'-methoxypuerarin, puerarin apigenin, salvianolic acid B, quercetin-3-O-β-D-pyranoside, stigmosiderin, daidzein, palmatine hydrochloride, berberine hydrochloride, and citrinin. The peak areas of cassia seed extract, lotus leaf alkaloid, cassia seed extract, cassia seed extract, and cassia anthraquinone were calculated, and their RSDs were 0.96%, 0.87%, 0.77%, 0.89%, 0.95%, 1.06%, 1.12%, 1.08%, 0.96%, 0.82%, 1.67%, 1.73%, 2.77%, 2.28%, 1.68%, and 0.83%, respectively, indicating that the method has good repeatability.

[0059] 2.6.4 Stability Test Take the same reference sample test solution (S2), and perform chromatographic analysis under the conditions described in section "2.3.1" at 0, 2, 4, 6, 8, 10, 12, and 24 hours after preparation. Record the results for 3'-hydroxypuerarin, puerarin, puerarin-6''-O-xyloside, 3'-methoxypuerarin, puerarin apigenin, salvianolic acid B, quercetin-3-O-β-D-pyranoside, gentiopicroside, daidzein, palmatine hydrochloride, and berberine hydrochloride. The peak areas of cassia aurantium, nuciferine, cassia scabra, cassia scabra, and cassia anthraquinone were calculated, and their RSDs were 1.12%, 0.86%, 0.79%, 0.95%, 0.90%, 1.09%, 1.52%, 1.79%, 1.85%, 1.75%, 1.43%, 1.50%, 1.14%, 1.90%, 1.16%, and 1.21%, respectively. The results indicate that the test solution has good stability within 24 hours.

[0060] 2.6.5 Determination of recovery rate Accurately measure a standard sample solution with known content of major components, add a reference standard with the same content as each index component, and inject for analysis according to the test sample solution preparation method. The calculated recoveries of 3'-hydroxypuerarin, puerarin, puerarin-6''-O-xyloside, 3'-methoxypuerarin, puerarin apigenin, salvianolic acid B, quercetin-3-O-β-D-pyranoside, gentiopicroside, daidzein, palmatine hydrochloride, berberine hydrochloride, cassia seed extract, nuciferine, cassia seed extract, cassia seed extract, and cassia seed anthraquinone were 99.69%, 100.98%, 99.58%, 102.15%, 101.93%, 98.81%, and 97.66%, respectively. The percentages were 101.10%, 95.24%, 97.85%, 99.01%, 97.59%, 102.46%, 97.11%, 95.47%, and 95.06%, with RSDs of 1.92%, 1.74%, 1.92%, 2.20%, 2.80%, 1.88%, 1.46%, 2.54%, 1.60%, 2.92%, 1.50%, 1.38%, 2.55%, 1.85%, 2.19%, and 2.71%, respectively, indicating that the method has good accuracy.

[0061] 2.6.6 Determination of the content of major components and the transfer rate of indicative components in each batch of reference samples Twenty batches of Ge Lian Qi Shen Fang reference samples were taken, and test solutions were prepared according to the method in section "2.2". The samples were injected and analyzed under the chromatographic conditions in section "2.3.1" to detect the mass concentration of the index components in the test solutions. The content of each index component in the reference samples was calculated, as shown in Tables 6 and 7. From the common peak assignments in section "2.5", it can be seen that: tanshinone B is the index component of Tanshinone; 3'-hydroxypuerarin, puerarin, puerarin-6''-O-xyloside, 3'-methoxypuerarin, puerarin apigenin, and daidzein are the index components of Pueraria lobata; palmatine hydrochloride and berberine hydrochloride are the index components of Coptis chinensis; cassia seed extract, cassia seed extract, cassia seed extract, and cassia seed anthraquinone are the index components of Cassia tora seed; and lotus leaf alkaloid is the index component of lotus leaf. The content of indicative components in 20 batches of Ge Lian Qi Shen formula reference samples and corresponding decoction pieces was determined, and the transfer rate of indicative components was calculated. The results are shown in Tables 8-11. Transfer rate % = (content of indicative component in reference sample / content of indicative component in corresponding decoction piece) * 100%.

[0062] As shown in Tables 8-11, the transfer rates of the following components from the reference samples in the 20 batches were as follows: Tanshinone B, puerarin, 3'-hydroxypuerarin, puerarin-6''-O-xyloside, 3'-methoxypuerarin, puerarin apigenin, daidzein, palmatine hydrochloride, berberine hydrochloride, auranthiazines, cassia seed extract, cassia seed extract, cassia seed anthraquinone, and nuciferine from the medicinal slices to the reference samples were 27.92%–49.91%, 64.71%–96.60%, 56.17%–90.98%, 36.39%–64.36%, 55.00%–98.30%, 53.68%–96.23%, and 36.25%, respectively. The percentages of the constituent components were as follows: 24.05%–63.07%, 24.05%–43.82%, 20.73%–34.40%, 47.99%–78.92%, 41.17%–73.47%, 33.66%–56.62%, 27.53%–46.39%, and 52.35%–88.98%, respectively; the average transfer rates were 38.42%, 83.84%, 74.59%, 49.98%, 78.15%, 74.68%, 48.60%, 33.07%, 27.24%, 64.54%, 57.15%, 43.76%, 36.06%, and 67.92%, respectively. The content of the constituent components in each batch was generally within ±30% of the mean, indicating that the preparation process of the reference sample was stable and that the constituent components could be stably transferred from the medicinal slices to the reference sample.

[0063] Table 6 Results of determination of the content of main components (1)

[0064] Table 7 Results of determination of the content of main components (2)

[0065] Table 8. Transfer rate of indicative components (1)

[0066] Table 9. Transfer rate of indicative components (2)

[0067] Table 10. Transfer rate of indicative components (3)

[0068] Table 11. Transfer rate of indicative components (4)

[0069] 3. Discussion 3.1 Selection of chromatographic conditions Due to the complex chemical components of the Ge-Lian-Qi-Shen formula reference sample, the chromatographic peaks are prone to overlap, increasing the difficulty of establishing the fingerprint. In the preliminary experiment, the elution of the chemical components of the Ge-Lian-Qi-Shen formula in the S2 reference sample with different mobile phases was investigated, such as acetonitrile-0.2% formic acid aqueous solution, pH = 2.5 ( Figure 4 ), acetonitrile-0.1% phosphoric acid aqueous solution, pH = 2.1 ( Figure 5 ), etc. The results showed that the number of chromatographic peaks was small and the resolution was poor. Specifically, Figure 4 the number of chromatographic peaks decreased and the peak shapes were poor from 30 to 60 min. Figure 5 In [reference 5], the resolution of peaks 4 and 5 (puerarin-6''-O-xyloside and 3'-methoxypuerarin) was poor, the number of chromatographic peaks decreased from 30 to 60 min, and the peak shapes were poor.

[0070] The experiment used acetonitrile-ammonium acetate + triethylamine (adjusted pH with glacial acetic acid) as the mobile phase for detection. Considering the actual separation of the components of the Ge-Lian-Qi-Shen formula, the information content of the chromatogram, the response levels of the chromatographic peaks, and the separation effect, the composition of the mobile phase was adjusted. Finally, 0.02 mol / L ammonium acetate + 0.3% triethylamine (adjusted pH to 6.0 with glacial acetic acid) was selected as mobile phase B for establishing the fingerprint. The pre-experiment also investigated the chromatogram of the Ge-Lian-Qi-Shen formula under different wavelength conditions (250 nm, 280 nm, 320 nm). The results showed that the resolution of each chromatographic peak at 320 nm was poor, and the chromatographic peaks at 250 and 280 nm were well separated. However, due to the excessive response value of peak 3 (puerarin) at 250 nm, it caused a certain masking effect on the chromatogram information of other chromatographic peaks with relatively lower response values. Therefore, the detection wavelength of 280 nm was finally selected for the fingerprint. Under the selected chromatographic conditions, the separation effect of each chromatographic peak was good, the peak shapes were excellent, and the number of chromatographic peaks was rich.

[0071] After investigating the pH of mobile phase B, it was found that using acetonitrile as mobile phase A and 0.02 mol / L ammonium acetate + 0.3% triethylamine (adjusted pH to 4.3 with glacial acetic acid) as mobile phase B, with the remaining conditions the same as in "2.3.1 Chromatographic conditions", the chromatogram of the S2 reference sample was as shown in Figure 6 . The resolution of peaks 4 and 5 (puerarin-6''-O-xyloside and 3'-methoxypuerarin) was poor, the peak shape of peak 8 (salvianolic acid B, at about 35 min) was poor, and the peak shape of peak 19 (berberine hydrochloride, at about 56 min) was slightly poor.

[0072] Using acetonitrile as mobile phase A and 0.02 mol / L ammonium acetate + 0.3% triethylamine as mobile phase B, adjusted pH to 5.5 or 6.5 with glacial acetic acid, with the remaining conditions the same as in "2.3.1 Chromatographic conditions", the chromatograms of the S2 reference sample were as shown in Figure 10 and Figure 11As shown in the figure, when the pH of mobile phase B is 5.5 or 6.5, the separation effect of each chromatographic peak is also better, the peak shape is better, and the number of chromatographic peaks is abundant.

[0073] In addition to investigating the composition of the mobile phase, the gradient elution program was also investigated, for example, program (a): 0~35 min, acetonitrile 5%~18%; 35~50 min, acetonitrile 18%~35%; 50~60 min, acetonitrile 35%~40%; 60~70 min, acetonitrile 40%~80%; 70~75 min, acetonitrile 80%~5%; 75~90 min, acetonitrile 5%; Program (b): 0-20 min, acetonitrile 5%-13%; 20-35 min, acetonitrile 13%-16%; 35-60 min, acetonitrile 16%-35%; 60-70 min, acetonitrile 35%-40%; 70-80 min, acetonitrile 40%-80%; 80-85 min, acetonitrile 80%-5%; 85-105 min, acetonitrile 5%; Program (c): 0~20min, acetonitrile 5%~13%; 20~35min, acetonitrile 13%~18%; 35~60min, acetonitrile 18%~35%; 60~70min, acetonitrile 35%~40%; 70~80min, acetonitrile 40%~80%; 80~85min, acetonitrile 80%~5%; 85~105min, acetonitrile 5%; The remaining conditions for procedures (a) to (c) are the same as in "2.3.1 Chromatographic Conditions". The chromatograms obtained from the S2 reference sample are as follows: Figures 7-9 As shown. Figure 7 The mid-peaks 4 and 5 (puerarin-6''-O-xyloside and 3'-methoxypuerarin) showed poor separation, while peak 19 (berberine hydrochloride) had a tailing effect. Figure 8 Mid-peak 8 (tanshinone B) merges with peak 9, peak 15 overlaps with the preceding chromatographic peaks, and peak 17 shows a split peak. Figure 9 Peak 8 (tanshinone B) merged with peak 9, peak 10 was not separated from the preceding chromatographic peak, and peak 15 overlapped with the preceding chromatographic peak.

[0074] 3.2 Determination of multiple component contents and identification of index components The fingerprint chromatogram similarity of 20 batches of Ge Lian Qi Shen formula reference samples was all greater than 0.960, indicating that the properties were stable among batches. A total of 26 common peaks were identified in the fingerprint chromatograms. By comparing with the reference standard, the chemical components represented by 16 chromatographic peaks were identified. These components are the main active ingredients of Pueraria lobata (puerarin, 3'-hydroxypuerarin, puerarin-6''-O-xyloside, 3'-methoxypuerarin, puerarin apigenin, daidzein, argentin), Salvia miltiorrhiza (tanshinone B), Astragalus membranaceus (argentin), Coptis chinensis (palmatine hydrochloride, berberine hydrochloride), Cassia tora (auroside, cassia scabra, cassia anthraquinone), and Nelumbo nucifera (nelumbo nucifera alkaloid, quercetin-3-O-β-D-glucopyranoside), covering the pharmacodynamic substances of the principal herbs Astragalus membranaceus and Salvia miltiorrhiza, the assistant herbs Pueraria lobata and Coptis chinensis, the adjuvant herbs Cassia tora and the guiding herbs Nelumbo nucifera. The results also show that a large portion of the chromatographic peak information comes from kudzu root, indicating that the effective components of kudzu root are easy to extract by decoction, and that the chromatographic conditions are conducive to the elution and detection of the main components in kudzu root.

[0075] The experiment determined the content of 16 components. The results showed that the reference sample had relatively high levels of puerarin and its derivatives, salvianolic acid B, berberine hydrochloride, and quercetin-3-O-β-D-glucopyranoside, while the content of indicative components in cassia seeds was relatively low. This may be related to the highly lipid-soluble chemical properties of cassia seed extract, cassia seed extract, cassia seed extract, and cassia seed anthraquinone. The content of most components varied little between batches, but the content of some components fluctuated significantly between batches. For example, the content of lotus leaf alkaloids varied significantly between different batches, indicating that lotus leaf alkaloids are greatly affected by the place of origin and batch, consistent with literature reports. Referring to the quantitative determination of medicinal materials as specified in the pharmacopoeia and relevant reports on the indicative components of medicinal materials in the literature, 14 components, excluding argentin and quercetin-3-O-β-D-glucopyranoside, were selected as indicative components of the Ge Lian Qi Shen formula. These components are derived from the principal, assistant, adjuvant, and guiding herbs in the prescription (Salvia miltiorrhiza, Pueraria lobata, Coptis chinensis, Cassia tora, and Nelumbo nucifera), and can comprehensively characterize the chemical composition information of the Ge Lian Qi Shen formula.

[0076] 3.3 Value Transfer Analysis Generally, the research on the value transfer of traditional Chinese medicine reference samples uses the transfer rate of index components within ±30% of the mean as the measurement standard. In this study, except for individual batches of palmatine hydrochloride (batches 8 and 10) and nuciferine (batch 18) slightly exceeding the range, the transfer rates of the index components in other batches are all within the range of ±30% of the mean, indicating that the preparation process of the reference sample is relatively stable and feasible, and can provide a basis for the subsequent preparation and production of Ge-Lian-Qi-Shen formula. The study found that the transfer rates of components such as salvianolic acid B, berberine hydrochloride, and palmatine hydrochloride are relatively low, which may be related to the poor thermal stability of these components. During the long-term high-temperature decocting and concentration processes, chemical structure hydrolysis, oxidation and other reactions are likely to occur; moreover, traditional Chinese medicine compound prescriptions have many chemical components and a complex system, and different acidic and basic components may interact with each other, resulting in complexation reactions or co-precipitation phenomena, thus leading to component loss and reducing the transfer rate.

[0077] 4 Conclusions Through the establishment of HPLC fingerprint and multi-component content determination methods for Ge-Lian-Qi-Shen formula reference samples, this study analyzed the transfer of key index components from Chinese herbal pieces to reference samples, ensuring the stability of the preparation process and the consistency of index components as a whole, and laying an experimental foundation for the development of hospital preparations of this formula and the establishment of comprehensive quality control standards.

[0078] The above detailed description is for a specific feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or change without departing from the present invention shall be included within the scope of the technical solution of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of a formula containing Ge Lian Qi Shen, characterized in that, The raw materials for preparing the Ge Lian Qi Shen formula include Astragalus membranaceus, Salvia miltiorrhiza, roasted Pueraria lobata, Coptis chinensis, stir-fried Alisma plantago-aquatica, Cassia tora, raw hawthorn, raw malt, and lotus leaf. The construction method includes the following steps: (1) Preparation of reference sample test solution: The raw materials for the preparation of Ge Lian Qi Shen Fang were decocted in water and used as reference samples. The solution was extracted with solvent to obtain the reference sample test solution. (2) Preparation of mixed reference solution: Take the reference standards 3'-hydroxypuerarin, puerarin-6''-O-xyloside, puerarin apigenin, 3'-methoxypuerarin, puerarin, daidzein, palmatine hydrochloride, stigmosiderin, auranthiazines, sennae, tanshinone, quercetin-3-O-β-D-glucopyranoside, lotus leaf alkaloid, berberine hydrochloride, sennae, cassia seed, and cassia anthraquinone, dissolve them in solvent to obtain mixed reference solution; (3) Inject the reference sample test solution and the mixed reference solution into the high performance liquid chromatograph for determination to obtain the fingerprint spectrum; The chromatographic conditions included: using acetonitrile as mobile phase A and ammonium acetate-triethylamine solution as mobile phase B.

2. The construction method according to claim 1, characterized in that, The solvent mentioned in steps (1) and (2) is anhydrous methanol.

3. The construction method according to claim 1, characterized in that, The mobile phase B has a molar concentration of ammonium acetate of 0.01-0.03 mol / L and a mass concentration of triethylamine of 0.1-0.5%; preferably, the mobile phase B has a molar concentration of ammonium acetate of 0.02 mol / L and a mass concentration of triethylamine of 0.3%; more preferably, the mobile phase B has a pH of 5.5-6.5; even more preferably, the mobile phase B uses glacial acetic acid to adjust the pH.

4. The construction method according to claim 1, characterized in that, The chromatographic conditions also include gradient elution, with the following specific procedure: 0–20 min, mobile phase A 5%–13%; 20–35 min, mobile phase A 13%–17%; 35–60 min, mobile phase A 17%–35%; 60–70 min, mobile phase A 35%–40%; 70–80 min, mobile phase A 40%–80%; 80–85 min, mobile phase A 80%–5%; 85–105 min, mobile phase A 5%.

5. The construction method according to claim 1, characterized in that, The chromatographic conditions also include one or more of the following conditions: a) Column temperature: 30-40℃, preferably 35℃; b) Flow rate: 0.8-1.2 mL / min, preferably 1.0 mL / min; c) Detection wavelength: 250-300nm, preferably 280nm; d) Injection volume: 5-12 μL, preferably 10 μL; e) Chromatographic column: C18 chromatographic column.

6. The construction method according to claim 1, characterized in that, The decoction process is as follows: Mix the raw materials, soak them in 8-12 times the amount of water for 0.5-1.5 hours, decoct 1-3 times, 1-2 hours each time, combine the decoctions, and concentrate.

7. The construction method according to claim 6, characterized in that, The construction method further includes preparing a single-ingredient decoction sample test solution and a negative control sample test solution, and determining the chromatograms of the single-ingredient decoction sample test solution and the negative control sample test solution. The preparation of the single-decocted sample test solution includes: weighing out 9 medicinal materials, namely Astragalus membranaceus, Salvia miltiorrhiza, roasted Pueraria lobata, Coptis chinensis, stir-fried Alisma plantago-aquatica, Cassia tora, raw hawthorn, raw malt, and lotus leaf, and preparing them according to the prescription process of water decoction to obtain a single-decocted sample of each medicinal material, and extracting them with solvent to obtain a single-decocted sample test solution of each medicinal material. The preparation of the negative control sample test solution includes: weighing the other 8 medicinal materials that are missing the corresponding medicinal materials according to the prescription amount, preparing them according to the decoction prescription process, and obtaining 9 negative control samples that are missing Astragalus membranaceus, Salvia miltiorrhiza, roasted Pueraria lobata, Coptis chinensis, stir-fried Alisma plantago-aquatica, Cassia tora, raw Crataegus pinnatifida, raw malt, and lotus leaf, respectively, extracting them with solvent, and obtaining negative control sample test solutions that are missing Astragalus membranaceus, Salvia miltiorrhiza, roasted Pueraria lobata, Coptis chinensis, stir-fried Alisma plantago-aquatica, Cassia tora, raw Crataegus pinnatifida, raw malt, and lotus leaf, respectively.

8. The construction method according to claim 1, characterized in that, In step (1), the volume ratio of the reference sample to the solvent is 1:1-4, preferably 1:1; the extraction method in step (1) is ultrasonic extraction, and the extraction time is 15-60 min, preferably 30 min; after the extraction in step (1) is completed, filtration is also performed using a microporous membrane. In step (2), each 1 mL of the mixed reference solution contains 225.30 μg of 3'-hydroxypuerarin, 433.00 μg of puerarin-6''-O-xyloside, 507.50 μg of puerarin apigenin, 736.20 μg of 3'-methoxypuerarin, 1870.40 μg of puerarin, 125.60 μg of daidzein, and 93.00 μg of palmatine hydrochloride. The following ingredients were present: 278.70 μg of argentin, 105.90 μg of cassia seed extract, 800.80 μg of salvianolic acid B, 516.30 μg of quercetin-3-O-β-D-glucopyranoside, 277.50 μg of nuciferine, 192.00 μg of berberine hydrochloride, 86.30 μg of cassia seed extract, 23.40 μg of cassia seed extract, and 93.30 μg of anthraquinone.

9. The construction method according to claim 1, characterized in that, The raw materials for preparing the Ge Lian Qi Shen formula, by weight, include 12-21 parts of Astragalus membranaceus, 7-15 parts of Salvia miltiorrhiza, 11-22 parts of roasted Pueraria lobata, 4-7 parts of Coptis chinensis, 8-14 parts of stir-fried Alisma plantago-aquatica, 7-15 parts of Cassia tora, 8-15 parts of raw Crataegus pinnatifida, 10-23 parts of raw malt, and 8-15 parts of lotus leaf. Preferably, the ingredients include 15 parts Astragalus membranaceus, 10 parts Salvia miltiorrhiza, 15 parts roasted Pueraria lobata, 5 parts Coptis chinensis, 10 parts stir-fried Alisma plantago-aquatica, 10 parts Cassia tora, 10 parts raw Crataegus pinnatifida, 15 parts raw malt, and 10 parts lotus leaf.

10. The construction method according to claim 7, characterized in that, The construction method further includes importing the chromatograms obtained from multiple batches of test solutions into a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine to determine common fingerprint characteristic peaks. Preferably, the multiple batches of test solutions are 20 batches, and the similarity of the fingerprint chromatograms of each batch is >0.

960.

11. The construction method according to claim 10, characterized in that, The construction method further includes comparing the common fingerprint characteristic peaks with the chromatograms of the single decoction sample test solution and the negative control sample test solution to assign the common fingerprint characteristic peaks.

12. The construction method according to claim 11, characterized in that, The fingerprint spectrum showed 26 chromatographic peaks, including peak 2 (3'-hydroxypuerarin), peak 3 (puerarin), peak 4 (puerarin-6''-O-xyloside), peak 5 (3'-methoxypuerarin), peak 6 (puerarin apigenin), peak 8 (tanshinone B), peak 11 (quercetin-3-O-β-D-glucopyranoside), peak 15 (stigmosiderin), peak 16 (daidzein), peak 18 (palmatine hydrochloride), peak 19 (berberine hydrochloride), peak 22 (auroside), peak 23 (nuciferine), peak 24 (yellow cassiaside), peak 25 (cassiaside), and peak 26 (cassia anthraquinone).

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