Fingerprint spectrum construction method of Youjing seed decoction

Abstract

The invention discloses a high performance liquid chromatography fingerprint construction method of Youjing seed decoction. The construction method comprises the following steps: preparing a test solution; preparing a reference substance solution; respectively and precisely sucking 10 microliters of a superior and sperm seed soup solution and 10 microliters of a reference solution, injecting the solutions into a high performance liquid chromatograph, and recording a fingerprint spectrum; establishing a fingerprint spectrum of the Youjing seed decoction: importing the liquid chromatogram of the Youjing seed decoction obtained in the step (3) into a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system for data processing to obtain a fingerprint spectrum common mode of the Youjing seed decoction; and analyzing common peak attribution of the fingerprint spectrum of the Youjing seed decoction. By comparing high performance liquid chromatograms of a single medicine preparation, a negative preparation lacking a single medicine and a Youjing seed decoction preparation, 23 common peaks can be attributed, so that whether the quality of the Youjing seed decoction raw material is qualified or not is judged. The method is simple, convenient, sensitive and good in repeatability and stability.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine detection, and in particular to a method for constructing a fingerprint spectrum of a superior fine seed soup. Background Art

[0002] Youjing Seed Soup is composed of 20 traditional Chinese medicines, including Rehmannia glutinosa, Rehmannia glutinosa, Polygonatum odoratum, Eucommia ulmoides, Dipsacus asper, Cyathula officinalis, and Cuscuta chinensis. This recipe uses raw and cooked Rehmannia glutinosa with Polygonatum odoratum as the main ingredients, which can replenish essence and marrow, nourish yin and produce body fluid, and replenish qi and benefit the kidney. Eucommia ulmoides, Dipsacus asper, and Cyathula officinalis can replenish liver and kidney, strengthen tendons and bones; Lycium barbarum can nourish liver and kidney, improve essence and improve eyesight; Mulberry can nourish yin and produce body fluid, and moisturize dryness; Cuscuta chinensis, Rubus idaeus, and Astragalus astragali can replenish liver and kidney, consolidate essence and reduce urine, and Astragalus astragali can also replenish kidney and assist yang; Vinegar Schisandra chinensis can replenish qi and produce body fluid, nourish kidney and calm the mind, and astringe and consolidate; the above-mentioned herbs are matched to enhance the effect of replenishing kidney essence deficiency, and they are all ministerial drugs. Adenophora australis, Adenophora australis, Asparagus cochinchinensis, and Ophiopogon japonicus can nourish yin and clear lungs; Salvia miltiorrhiza and Paeonia suffruticosa can clear heat and cool blood, and promote blood circulation and remove blood stasis. They are all adjuvant drugs, which can make the tonic not too greasy. The whole formula is mainly used to nourish the liver and kidneys, and is supplemented by clearing away heat and cooling blood, so that the nourishment is not too greasy, and the moisturization is not stagnant, which can achieve the effect of nourishing yin and producing essence.

[0003] There are as many as 20 ingredients in Youjing Seed Soup, and a series of quality standards have been established in the early stage, including thin-layer chromatography identification and content determination. However, the composition of Chinese medicine is complex, and a single indicator for only one or several Chinese medicines cannot meet the overall quality control of the preparation. Therefore, it is necessary to establish an analytical method that can comprehensively reflect the preparation of Youjing Seed Soup. The fingerprint of Chinese medicine is an analytical method that can comprehensively characterize the component groups of Chinese medicine. By testing single-ingredient preparations, single-ingredient negative preparations, and finished preparations under the same chromatographic analysis conditions, the chromatographic peaks of the fingerprint can be attributed, thereby indirectly reflecting the quality of the raw materials in the preparation. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing a high-performance liquid phase fingerprint of Youjing Seed Decoction and its standard fingerprint in view of the defects of the prior art. The test solution of Youjing Seed Decoction is analyzed and then compared with the fingerprint of the reference and the common pattern of the fingerprint. The multiple components in the preparation can be qualitatively analyzed, and the quality of the Youjing Seed Decoction raw material and finished preparation can be evaluated at the same time.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme: a method for constructing a fingerprint of a high-quality seed soup, comprising the following steps:

[0006] (1) Preparation of test solution: Take the seed soup of high-quality fine plants, add methanol and water, sonicate, centrifuge, filter, and take the filtrate as the test solution;

[0007] (2) Preparation of reference solution: Take the reference substance and dissolve it in methanol to obtain a reference solution;

[0008] (3) Accurately pipette 10 μl of the superior seed soup solution and the reference solution respectively, inject into the high performance liquid chromatograph, and record the fingerprint spectrum;

[0009] (4) Establishment of the fingerprint of Youjing Seed Soup: The liquid chromatogram of Youjing Seed Soup obtained in step (3) is introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system for data processing to obtain the common pattern of the fingerprint of Youjing Seed Soup;

[0010] (5) Analysis of the common peaks in the fingerprint of Youjing Seed Decoction: The preparations made from a single Chinese herbal medicine, the negative preparations made without a single Chinese herbal medicine, and the test solution of Youjing Seed Decoction made from all the ingredients were injected into a high performance liquid chromatograph to identify the common peaks.

[0011] Furthermore, the Youjing seed soup includes the following components in parts by mass: 8 to 12 parts each of Rehmannia root, Rehmannia root prepared, Eucommia bark, Dipsacus asper, Cyathula officinalis, Achyranthes bidentata, Mistletoe, Lycium barbarum, Mulberry, Schisandra chinensis with vinegar, Adenophora australis, Adenophora australis, Salvia miltiorrhiza, Asparagus cochinchinensis, Ophiopogon japonicus, and Paeonia suffruticosa, and 13 to 17 parts each of Polygonatum sibiricum, Astragalus membranaceus, Cuscuta australis, and Rubus idaeus.

[0012] Furthermore, the Youjing seed soup includes the following components in parts by mass: 10 parts each of Rehmannia root, Rehmannia root prepared, Eucommia bark, Dipsacus asper, Cyathula officinalis, Achyranthes bidentata, Mistletoe, Lycium barbarum, Mulberry, Schisandra chinensis with vinegar, Adenophora australis, Adenophora australis, Salvia miltiorrhiza, Asparagus cochinchinensis, Ophiopogon japonicus, and Paeonia suffruticosa, and 15 parts each of Polygonatum sibiricum, Astragalus membranaceus, Cuscuta australis, and Rubus idaeus.

[0013] Furthermore, the reference substances include 5-hydroxymethylfurfural, syringin, chlorogenic acid, paeoniflorin, ellagic acid, complanatin, rosmarinic acid, and salvianolic acid B.

[0014] Furthermore, the reference solution is prepared with methanol as solvent, and each mL of solution includes 800-1200 μg 5-hydroxymethylfurfural, 120-160 μg syringin, 20-80 μg chlorogenic acid, 120-180 μg paeoniflorin, 50-110 μg ellagic acid, 80-120 μg complanatus glycoside, 40-80 μg rosmarinic acid, and 20-80 μg salvianolic acid B.

[0015] Furthermore, the HPLC conditions are as follows: mobile phase A is acetonitrile, B is 0.1% to 0.2% phosphoric acid water, gradient elution, the detection wavelength of the diode array detector is 200 to 400 nm, the chromatographic column is filled with octadecylsilane bonded silica gel, the column temperature is 25 to 35°C, the flow rate is 0.8 to 1.2 mL per minute, and the detection wavelength is 254 nm.

[0016] Furthermore, the HPLC gradient elution conditions were: 0 min: 2% A, 98% B; 60 min: 13% A, 87% B; 70 min: 16% A, 84% B; 100 min: 22% A, 78% B; 125 min: 30% A, 70% B; 130 min: 75% A, 25% B.

[0017] Furthermore, the data processing in step (4) includes: reference peak selection, multi-point calibration, and Mark peak matching, so as to obtain the common pattern of the fingerprint spectrum of the superior seed soup.

[0018] Furthermore, the fingerprint of Youjing Seed Soup obtained in step (4) includes 23 common peaks, and the corresponding chromatographic peaks of the reference substances are peak 2 (5-hydroxymethylfurfural), peak 8 (syringin), peak 9 (chlorogenic acid), peak 13 (peonidin), peak 16 (ellagic acid), peak 18 (complexa glycosides), peak 19 (rosmarinic acid), and peak 23 (salvianolic acid B). Taking peak 16 as the reference peak S, the specified values ​​of the relative retention times of the 23 common peaks of Youjing Seed Soup are 0.16 (peak 1), 0.20 (peak 2), 0.21 (peak 3), and 0.31 (peak 4). .27 (peak 4), 0.29 (peak 5), 0.45 (peak 6), 0.48 (peak 7), 0.55 (peak 8), 0.59 (peak 9), 0.60 (peak 10), 0.63 (peak 11), 0.66 (peak 12), 0.74 (peak 13), 0.89 (peak 14), 0.92 (peak 15), 1.01 (peak 17), 1.15 (peak 18), 1.17 (peak 19), 1.23 (peak 20), 1.26 (peak 21), 1.28 (peak 22), and 1.32 (peak 23).

[0019] Furthermore, in Youjing Seed Soup, there are peaks 1 belonging to Peony Root, Rubus Idaeus, Astragalus Indica, Cuscuta Semen, and Rehmannia Glutinosa; peak 2 belonging to Schisandra Chinensis Fruit, Polygonatum Sinensis, Rehmannia Glutinosa, and Rubus Idaeus; peak 3 belonging to Polygonatum Sinensis, Rehmannia Glutinosa, Schisandra Chinensis Fruit, Viscum Alata, Astragalus Indica, and Raspberry; peak 4 belonging to Polygonatum Sinensis, Schisandra Chinensis Fruit, Rehmannia Glutinosa, Rubus Idaeus, and Astragalus Idaeus; peak 5 belonging to Morus Fructus, Dipsacus Asper, Schisandra Chinensis Fruit, Salvia Miltiorrhiza, and Astragalus Idaeus; peaks 6, 7, and 1 1. Peak 12, peak 21 and peak 22 belong to Dipsacus asper, peak 8 belongs to Paeonia suffruticosa and Viscum album, peak 9 belongs to Dipsacus asper, Cuscuta chinensis and Eucommia ulmoides, peak 10 belongs to Viscum album and Dipsacus asper, peak 13 belongs to Paeonia suffruticosa, peak 14 and peak 18 belong to Astragalus membranaceus, peak 15 belongs to Paeonia suffruticosa and Astragalus membranaceus, peak 16 belongs to Rubus idaeus, Cuscuta chinensis, Paeonia suffruticosa and Achyranthes bidentata, peak 17 belongs to Cuscuta chinensis and Achyranthes bidentata, peaks 19, peak 20 and peak 23 belong to Salvia miltiorrhiza.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. The common pattern of fingerprints was constructed through the fingerprints of 15 batches of high-quality seed soup. Through similarity evaluation and comparison with the common pattern spectra, the quality of high-quality seed soup can be objectively and comprehensively judged.

[0022] 2. By comparing the HPLC chromatograms of single-herb preparations, single-herb-deficient negative preparations, and Youjing Seed Soup preparations, the 23 common peaks can be attributed, thereby judging whether the quality of the Youjing Seed Soup raw materials is qualified.

[0023] 3. This method is simple, sensitive, and has good repeatability and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The liquid chromatogram of the mixed standard solution of the embodiment and the test solution of Youjing Seed Soup (S1 is the test solution of Youjing Seed Soup, and S2 is the mixed standard solution. The chromatographic peaks in the mixed standard solution are from left to right adenosine (1), 5-hydroxymethylfurfural (2), geniposide (3), syringin (4), chlorogenic acid (5), pinoresinol diglucoside (6), paeoniflorin (7), verbascoside (8), rutin (9), ellagic acid (10), ferulic acid (11), complanatine (12), rosmarinic acid (13), salvianolic acid B (14), chuanxiong saponin VI (15), paeonol (16), and schisandrae alcohol A (17)).

[0025] Figure 2 It is the chromatogram of different mobile phase acidity of embodiment.

[0026] Figure 3 It is the chromatogram of the durability investigation (different column temperatures) of the embodiment.

[0027] Figure 4 It is the chromatogram of the durability investigation (different flow rates) of the embodiment.

[0028] Figure 5 It is a chromatogram of the common pattern of the fingerprint of the excellent fine seed soup in the embodiment.

[0029] Figure 6 It is an overlay of fingerprints of the 15th batch of superior seed soup of Example 1.

[0030] Figure 7 It is a liquid chromatogram comparison diagram of the reference substance (a) in the example and the test solution of Youjing Seed Soup (b).

[0031] Figure 8 This is the chromatographic peak attribution diagram of the raw Rehmannia root in the Excellent Fine Seed Soup of the Example (from bottom to top, they are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2), and the finished preparation of the Excellent Fine Seed Soup (S3)).

[0032] Fig. 91. The chromatographic peak attribution diagram of Rehmannia glutinosa in the Excellent Fine Seed Soup of the Example (from bottom to top, they are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2), and the finished preparation of Excellent Fine Seed Soup (S3)).

[0033] Fig.10 This is the chromatographic peak attribution diagram of the wine-brewed Polygonatum sibiricum in the Excellent Fine Seed Soup of the Example (from bottom to top, they are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2), and the finished preparation of the Excellent Fine Seed Soup (S3)).

[0034] Fig.11 This is the chromatographic peak attribution diagram of the salt Eucommia ulmoides in the Excellent Fine Seed Soup of the Example (from bottom to top, they are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2) and the Excellent Fine Seed Soup finished preparation (S3)).

[0035] Fig.12 This is the chromatographic peak attribution diagram of Dipsacus asper in the Excellent Fine Seed Soup of the Example (from bottom to top, they are the single herb preparation (S1), the single herb-deficient negative preparation (S2) and the finished product preparation (S3) of Excellent Fine Seed Soup).

[0036] Fig.13 This is the chromatographic peak attribution diagram of Achyranthes bidentata in the Excellent Fine Seed Soup of Example (from bottom to top, they are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2), and the finished preparation of Excellent Fine Seed Soup (S3)).

[0037] Fig.14 This is the chromatographic peak attribution diagram of Achyranthes bidentata in the Excellent Fine Seed Soup of the Example (from bottom to top, they are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2), and the finished preparation of Excellent Fine Seed Soup (S3)).

[0038] Fig.15 This is the chromatographic peak attribution diagram of mistletoe in the Excellent Fine Seed Soup of the Example (from bottom to top, they are a single-ingredient drug preparation (S1), a single-ingredient drug-deficient negative preparation (S2), and a finished Excellent Fine Seed Soup preparation (S3)).

[0039] Fig.16 This is the chromatographic peak attribution diagram of Cuscuta australis in the Excellent Fine Seed Soup of the Example (from bottom to top, they are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2), and the finished preparation of Excellent Fine Seed Soup (S3)).

[0040] Fig.17 This is the chromatographic peak attribution diagram of wolfberry in the excellent fine seed soup of the embodiment (from bottom to top, they are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2) and the excellent fine seed soup finished preparation (S3)).

[0041] Fig.18This is the chromatographic peak attribution diagram of mulberry in the Excellent Fine Seed Soup of the embodiment (from bottom to top, they are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2) and the Excellent Fine Seed Soup finished preparation (S3)).

[0042] Fig.19 This is the chromatographic peak attribution diagram of raspberry in the excellent seed soup of the embodiment (from bottom to top, they are single-ingredient drug preparation (S1), single-ingredient drug-deficient negative preparation (S2) and excellent seed soup finished preparation (S3)).

[0043] Fig. 20 This is the chromatographic peak attribution diagram of Schisandra chinensis chinensis vinegar in the Example Youjing Seed Soup (from bottom to top, they are a single herb preparation (S1), a single herb-deficient negative preparation (S2), and a finished Youjing Seed Soup preparation (S3)).

[0044] Fig.21 This is the chromatographic peak attribution diagram of Astragalus membranaceus in the Excellent Fine Seed Soup of the Example (from bottom to top, they are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2), and the finished preparation of Excellent Fine Seed Soup (S3)).

[0045] Fig. 22 This is the chromatographic peak attribution diagram of Adenophora radix in the Excellent Fine Seed Soup of the Example (from bottom to top, they are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2), and the finished preparation of Excellent Fine Seed Soup (S3)).

[0046] Fig.23 This is the chromatographic peak attribution diagram of Adenophora australis in the Excellent Fine Seed Soup of the Example (from bottom to top, they are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2), and the finished preparation of Excellent Fine Seed Soup (S3)).

[0047] Fig.24 This is the chromatographic peak attribution diagram of Salvia miltiorrhiza in the Example Youjing Seed Soup (from bottom to top, they are a single herb preparation (S1), a single herb-deficient negative preparation (S2), and a finished Youjing Seed Soup preparation (S3)).

[0048] Fig.25 This is the chromatographic peak attribution diagram of Asparagus cochinchinensis in the Excellent Fine Seed Soup of the Example (from bottom to top, they are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2), and the finished preparation of Excellent Fine Seed Soup (S3)).

[0049] Fig.26 This is the chromatographic peak attribution diagram of Ophiopogon japonicus in the Excellent Fine Seed Soup of the Example (from bottom to top, they are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2), and the finished preparation of Excellent Fine Seed Soup (S3)).

[0050] Fig. 271. The chromatographic peak attribution diagram of the Cortex Moutan in the Excellent Fine Seed Soup of the Example (from bottom to top are the single-ingredient drug preparation (S1), the single-ingredient drug-deficient negative preparation (S2) and the Excellent Fine Seed Soup finished preparation (S3)). DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present application.

[0052] It should be noted that the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or apparatuses.

[0053] The instruments and materials used in the present invention include:

[0054] Instruments: SHIMADIU high performance liquid chromatograph, equipped with LC-40D mobile phase delivery module, SPD-M40 diode array detector, SIL-40 autosampler and CTO-40S column oven; Kromasil C18 (4.6mm×250mm, 5μm) chromatographic column; LC-UC-150 ultrasonic instrument; HC-2066 high-speed centrifuge; NewClassic MS 204S 1 / 10,000 balance; METTLER TOLEDO XS205 1 / 100,000 balance;

[0055] Materials: phosphoric acid (chromatographic grade, Shanghai Aladdin Company), formic acid (chromatographic grade, Sinopharm Chemical Reagent Co., Ltd.), methanol (chromatographic grade, American TEDIA Company), acetonitrile (chromatographic grade, American TEDIA Company), methanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); ultrapure water was prepared by KZ-60L laboratory ultrapure water machine; 15 batches of excellent seed soup, batch numbers Y1~Y15, all made from 20 herbs including Rehmannia root, Rehmannia glutinosa, Eucommia ulmoides, Dipsacus asper, and Cyathula officinalis. The Chinese medicine used was provided by Suzhou Tianling Chinese Medicine Piece Factory.

[0056] Example 1: Screening of reference index components

[0057] (1) Preparation of reference solution: Take appropriate amount of 5-hydroxymethylfurfural, syringin, chlorogenic acid, paeoniflorin, ellagic acid, complanatin, rosmarinic acid, and salvianolic acid B reference substances, add appropriate amount of methanol to a 5 mL volumetric flask, dissolve by ultrasonication, let cool, and add methanol to the mark, which is the standard stock solution 1. Take appropriate amount of adenosine, geniposide, pinoresinol diglucoside, verbascoside, rutin, ferulic acid, chuanxiong saponin VI, paeonol, and schisandrae alcohol A reference substances, add appropriate amount of methanol to a 5 mL volumetric flask, dissolve by ultrasonication, let cool, and add methanol to the mark, which is the standard stock solution 2. Take 1 mL each of standard stock solution 1 and standard stock solution 2 and mix them evenly to obtain a mixed standard solution.

[0058] (2) Preparation of test solution: Take 1 mL of Youjing Seed Decoction, add 14 mL of methanol, and then add appropriate amount of water to 20 mL (methanol concentration is 70%), ultrasonically treat for 60 min (power 40 kHz, frequency 760 w), cool, shake well, centrifuge at high speed (12000 r / min, 10 min), take the supernatant, filter through a 0.22 μm microporous filter membrane, and take the filtrate.

[0059] (3) Chromatographic conditions: Kromasil 100-5-C18 (w) column (4.6 mm × 250 mm, 5 μm), acetonitrile as mobile phase A, 0.1% phosphoric acid water as mobile phase B, column temperature 30 °C, flow rate 1 mL / min, gradient elution: 0-20 min, 5%-25% A, 20-30 min, 25%-45% A, 30-40 min, 45%-48% A, 40-50 min, 48%-80% A. Accurately pipette 20 μl of the test solution and 10 μl of the mixed standard into the liquid chromatograph.

[0060] (4) The compound with the same retention time and absorption spectrum as the standard is identified as the target component. The chromatograms of the test solution and the mixed standard solution are shown in Figure 1 The following components can be detected in Youjing Seed Soup: 5-hydroxymethylfurfural, syringin, chlorogenic acid, paeoniflorin, ellagic acid, complanatin, rosmarinic acid, salvianolic acid B, chuanxiong saponin VI, paeonol, and schisandrin A. Schisandrin A and paeonol have late elution times and low responses, so they are not detected as target peaks.

[0061] Therefore, the reference indicator components were selected as 5-hydroxymethylfurfural, syringin, chlorogenic acid, paeoniflorin, ellagic acid, complanatin, rosmarinic acid, and salvianolic acid B.

[0062] Example 2: Comparison of mobile phase acidity

[0063] (1) Preparation of test solution: Accurately take 1 mL of high-quality seed soup and place it in a 25 mL volumetric flask. Add appropriate amount of water to rinse, then add methanol to a total volume of 20 mL to make the methanol concentration 60%. Shake well, and ultrasonically treat for 90 min (ultrasonic power 40 kHz, frequency 760w). Let cool, add 60% methanol to the scale line, shake well, and centrifuge at high speed for 10 min (12000 r / min). Take the supernatant, filter through a 0.22 μm microporous filter membrane, and take the filtrate as the test solution.

[0064] (2) Chromatographic conditions: Kromasil 100-5-C18 (w) column (4.6 mm × 250 mm, 5 μm), column temperature 30 °C, flow rate 1 mL / min, gradient elution: 0-60 min, 2%-13% A, 60-70 min, 13%-16% A, 70-100 min, 16%-22% A, 100-110 min, 22%-24% A, 110-125 min, 24%-30% A, 125-130 min, 30%-75% A. The mobile phases were acetonitrile (mobile phase A)-0.1% formic acid water (mobile phase B), acetonitrile (mobile phase A)-0.1% phosphoric acid water (mobile phase B), and acetonitrile (mobile phase A)-0.2% phosphoric acid water (mobile phase B), respectively. Accurately pipette 10 μl of the test solution into the liquid chromatograph.

[0065] Results Figure 2 The separation effect of multiple chromatographic peaks was poor when acetonitrile-0.1% formic acid water was used as the mobile phase. The separation effect of acetonitrile-0.1% phosphoric acid water and acetonitrile-0.2% phosphoric acid was slightly different, but the separation effect of peaks 4 and 5 was not good. Therefore, acetonitrile-0.1% phosphoric acid water was selected as the final mobile phase.

[0066] Example 3: Optimization of test sample preparation conditions

[0067] (1) Preparation of test solution: Accurately transfer 1 mL of the superior seed soup into three 25 mL volumetric flasks, add appropriate amount of water and methanol to 20 mL, and make the methanol concentration 90%, 60%, and 30%, respectively. Ultrasonic extraction was performed for 60 min (40 kHz, 760w). The mixture was cooled and filled to the mark with corresponding concentration of methanol. The mixture was shaken and centrifuged at high speed (12000 r / min, 10 min). The supernatant was collected and filtered through a 0.22 μm microporous filter membrane. The filtrate was collected to obtain test solutions of methanol extraction systems with different concentrations. Take another 4 25mL volumetric flasks, accurately transfer 1mL of high-quality seed soup to each flask, add appropriate amount of water and methanol to 20mL, make the methanol concentration 60%, ultrasonicate for 30min, 60min, 90min and 120min respectively, let cool, add 60% methanol to the scale, shake well, centrifuge at high speed (12000r / min, 10min), take the supernatant, pass through a 0.22μm microporous filter membrane, take the filtrate, and obtain the test solution with different ultrasonic times.

[0068] (2) The chromatographic conditions were optimized according to step (2) of Example 2.

[0069] (3) Record the peak area of ​​each chromatographic peak of the above test solution and calculate the sum of the peak areas. The results are shown in Tables 1 and 2. The sum of the peak areas of the extraction solvents is 30% methanol < 60% methanol ≈ 90% methanol. Preferably, the chromatographic peak information of 60% methanol and 90% methanol extracts is richer than that of other concentrations of extracts, and the sum of the peak areas is the highest. It is further preferred that 60% methanol is used as the fingerprint extraction system of Youjing Seed Soup. Preferably, when the ultrasonic extraction time reaches 90min and 120min, the sum of the peak areas reaches the maximum, and the peak area increase decreases when the ultrasonic extraction time is 120min. It is further preferred that the ultrasonic extraction time is 90min to maximize the characterization of the chemical components in Youjing Seed Soup.

[0070] (4) The optimal conditions for the preparation of the test solution of Youjing Seed Soup are as follows: accurately transfer 1 mL of Youjing Seed Soup into a 25 mL volumetric flask, add appropriate amount of water and methanol to 20 mL, make the methanol concentration 60%, ultrasonically extract for 90 min (40 kHz, 760w), let cool, add 60% methanol to the scale, shake well, centrifuge at high speed (12000 r / min, 10 min), take the supernatant, filter through a 0.22 μm microporous membrane, and take the filtrate.

[0071] Table 1: Comparison of different methanol extraction systems

[0072]

[0073] Table 2: Comparison of different ultrasonic extraction times

[0074]

[0075] Example 4: Methodological Validation

[0076] 1. Precision: Take 1mL sample of Youjing Seed Soup (batch number Y1) to prepare the test sample, and inject 6 needles continuously according to the above optimized chromatographic conditions, record the retention time and peak area of ​​the target peak, take the chromatographic peak (ellagic acid) with good separation and large peak area as the S peak, and calculate the relative retention time and relative peak area of ​​each common peak. The results show (Table 3, Table 4) that the relative retention time RSD of each common peak is less than 0.16%, and the relative peak area RSD is less than 5.16%, indicating that the instrument precision is good.

[0077] Table 3: Results of precision test of superior seed soup (relative retention time)

[0078]

[0079] Table 4: Precision test results of high-quality seed soup (relative peak area)

[0080]

[0081] 2. Repeatability: Take 6 samples of 1mL of Youjing Seed Soup, prepare 6 samples in parallel according to the optimized sample preparation method, inject according to the optimized chromatographic conditions, record the retention time and peak area of ​​the common peak, take ellagic acid as the S peak, and calculate the relative retention time and relative peak area of ​​each common peak. The results show (Table 5, Table 6) that the RSD of the relative retention time of each common peak is less than 0.09%, and the RSD of the relative peak area is less than 4.17%, indicating that the method has good repeatability.

[0082] Table 5: Results of repeatability test of superior seed soup (relative retention time)

[0083]

[0084] Table 6: Results of repeatability test of excellent seed soup (relative peak area)

[0085]

[0086] 3. Stability: Take the test sample prepared in the precision experiment, and analyze it at 0h, 3h, 6h, 9h, 12h, 21h, and 24h after preparation according to the above optimized chromatographic conditions, record the retention time and peak area of ​​the target peak, and calculate the relative retention time and relative peak area of ​​each common peak with ellagic acid as the S peak. The results show (Tables 7 and 8) that the RSD of the relative retention time of each common peak is less than 0.18%, and the RSD of the relative peak area is less than 5.08%, indicating that the test solution of Youjing Seed Soup is stable within 24h.

[0087] Table 7: Results of repeatability test of superior seed soup (relative retention time)

[0088]

[0089] Table 8: Results of repeatability test of excellent seed soup (relative peak area)

[0090]

[0091] 4. Durability test: Based on the above optimized chromatographic conditions, the flow rate and column temperature were slightly changed to examine the effect of slight changes in column temperature (25℃~35℃) and flow rate (0.8mL / min~1.2mL / min) on the separation of common peaks in the fingerprint. The results are as follows: Figure 3 , Figure 4As shown in the figure, the separation of peaks 6, 9, 10 and 16 is poor at 25°C column temperature, the separation of peaks 4, 5, 9 and 16 is poor at 35°C column temperature, and the separation of peak 8 is poor when the column temperature is not 30°C, so the column temperature is set to 30°C. When the flow rate is lower than 1 mL / min, the separation of peaks 6 and 19 will be affected, so the flow rate should not be lower than 1 mL / min.

[0092] Example 5: Establishment of high-quality fingerprints and similarity evaluation

[0093] Fifteen batches of excellent seed soup (batch numbers Y1-Y15) were prepared, and the test solution was made according to the optimized method and the HPLC chromatogram was collected. The chromatogram was imported into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System", and Y1 was used as the reference spectrum. The average method was used to generate the control, and the time window width was set to 0.1. After multi-point correction and Mark peak matching, the fingerprint spectrum common pattern was generated (see Figure 5 ), the fingerprint superposition of 15 batches of high-quality seed soup is shown in Figure 6 The similarity of the HPLC fingerprints of 15 batches of Youjing Seed Soup and the common pattern of the fingerprints (Table 9) were all greater than 0.997, and the similarity results between the batches were above 0.992, indicating that the 15 batches of Youjing Seed Soup had good similarity. There were 23 peaks in total, of which peaks 2, 8, 9, 13, 16, 18, 19, and 23 were 5-hydroxymethylfurfural, syringin, chlorogenic acid, paeoniflorin, ellagic acid, complanatin, rosmarinic acid, and salvianolic acid B, respectively. The reference containing the above 8 components and the HPLC fingerprint of Youjing Seed Soup were shown in Table 9. Figure 7 .

[0094] Table 9 Similarity of fingerprints of 15 batches of high-quality seed soup

[0095]

[0096] Example 6: Attribution of each common peak

[0097] According to the prescription of Youjing Seed Soup, 20 negative preparations lacking 20 herbs, including Radix Rehmanniae glutinosae, Radix Rehmanniae glutinosae, and Radix Polygonati Sinensis, were prepared. Preparations containing only a single herb were prepared according to the prescription and made into test solutions for sampling and analysis. The results are shown in Figure 8~Figure 27Peaks 1 to 5, 8 to 10, and 15 to 17 are contributed by multiple Chinese medicines, among which Peak 1 belongs to Rehmannia glutinosa, Cuscuta australis, Rubus idaeus, Astragalus indica, and Paeonia suffruticosa, and the main contribution comes from Paeonia suffruticosa; Peak 2 belongs to Rehmannia glutinosa, Polygonatum sibiricum, Rubus idaeus, and Schisandra chinensis; Peak 3 belongs to Rehmannia glutinosa, Polygonatum sibiricum, Viscum album, Rubus idaeus, Schisandra chinensis, and Astragalus indica; Peak 4 belongs to Rehmannia glutinosa, Polygonatum sibiricum, Rubus idaeus, Schisandra chinensis, and Astragalus indica; Peak 5 belongs to Radix Dipsaci, Morus alba, Radix Dipsaci, Radix Astragali and Radix Salviae Miltiorrhizae; Peak 8 belongs to Viscum album and Cortex Moutan; Peak 9 belongs to Eucommia ulmoides, Radix Dipsaci and Cuscuta; Peak 10 belongs to Radix Dipsaci and Viscum album; Peak 15 belongs to Astragali and Cortex Moutan; Peak 16 belongs to Radix Achyranthis Bidentatae, Cuscuta, Rubus Idaeus and Cortex Moutan, with the main contribution coming from Rubus Idaeus; Peak 17 belongs to Radix Achyranthis Bidentatae and Cuscuta. Some Chinese medicines have exclusive peaks: Peak 6, Peak 7, Peak 11, Peak 12, Peak 21, Peak 22 are exclusive to Radix Dipsaci, Peak 14, Peak 18 are exclusive to Astragali, Peak 19, Peak 20, Peak 23 are exclusive to Salviae Miltiorrhizae, and Peak 13 is exclusive to Cortex Moutan.

[0098] The peak areas of the exclusive peaks in the fingerprint spectrum were analyzed to provide an experimental basis for the quality judgment of the raw materials of Youjing Seed Decoction, including Dipsacus asper, Astragalus membranaceus, Salvia miltiorrhiza and Paeonia suffruticosa.

[0099] The method for constructing the fingerprint of the high-quality seed soup provided by the present invention is sensitive and has good repeatability, and can more objectively and comprehensively evaluate the quality of the high-quality seed soup preparation.

[0100] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for constructing a fingerprint of a high-quality seed soup, characterized in that: The steps include: (1) Preparation of test solution: Take the seed soup of high-quality fine plants, add methanol and water, sonicate, centrifuge, filter, and take the filtrate as the test solution; (2) Preparation of reference solution: Take the reference substance and dissolve it in methanol to obtain a reference solution; (3) Accurately pipette 10 μl of the superior seed soup solution and the reference solution respectively, inject into the high performance liquid chromatograph, and record the fingerprint spectrum; (4) Establishment of the fingerprint of Youjing Seed Soup: The liquid chromatogram of Youjing Seed Soup obtained in step (3) is introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system for data processing to obtain the common pattern of the fingerprint of Youjing Seed Soup; (5) Analysis of the common peaks in the fingerprint of Youjing Seed Decoction: The preparations made from a single Chinese herbal medicine, the negative preparations made without a single Chinese herbal medicine, and the test solution of Youjing Seed Decoction made from all the ingredients were injected into a high performance liquid chromatograph to identify the common peaks.

2. The method for constructing the fingerprint of the high-quality seed soup according to claim 1, characterized in that: The high-quality seed soup includes the following components in parts by weight: 8 to 12 parts each of Rehmannia root, Rehmannia root prepared, Eucommia bark, Dipsacus asper, Cyathula officinalis, Achyranthes bidentata, Mistletoe, Lycium barbarum, Mulberry, Schisandra chinensis with vinegar, Adenophora australis, Adenophora australis, Salvia miltiorrhiza, Asparagus cochinchinensis, Ophiopogon japonicus, and Paeonia suffruticosa, and 13 to 17 parts each of Polygonatum sibiricum, Astragalus complanatus, Cuscuta australis, and Rubus idaeus.

3. The method for constructing the fingerprint of the high-quality seed soup according to claim 1, characterized in that: The high-quality seed soup includes the following components in parts by mass: 10 parts each of Rehmannia root, Rehmannia root prepared, Eucommia bark, Dipsacus asper, Cyathula officinalis, Achyranthes bidentata, Mistletoe, Lycium barbarum, Mulberry, Schisandra chinensis with vinegar, Adenophora australis, Adenophora australis, Salvia miltiorrhiza, Asparagus cochinchinensis, Ophiopogon japonicus, and Paeonia suffruticosa moutan, and 15 parts each of Polygonatum sibiricum, Astragalus complanatus, Cuscuta australis, and Rubus idaeus.

4. The method for constructing the fingerprint of the high-quality seed soup according to claim 1, characterized in that: The reference substances include 5-hydroxymethylfurfural, syringin, chlorogenic acid, paeoniflorin, ellagic acid, complanatin, rosmarinic acid, and salvianolic acid B.

5. The method for constructing the fingerprint of the high-quality seed soup according to claim 1, characterized in that: The reference solution is prepared with methanol as solvent, and each mL of solution includes 800-1200 μg of 5-hydroxymethylfurfural, 120-160 μg of syringin, 20-80 μg of chlorogenic acid, 120-180 μg of paeoniflorin, 50-110 μg of ellagic acid, 80-120 μg of complanatin, 40-80 μg of rosmarinic acid, and 20-80 μg of salvianolic acid B.

6. The method for constructing the fingerprint of the high-quality seed soup according to claim 1, characterized in that: The HPLC conditions are as follows: mobile phase A is acetonitrile, mobile phase B is 0.1% to 0.2% phosphoric acid water, gradient elution, a diode array detector detection wavelength of 200 to 400 nm, a chromatographic column filled with octadecylsilane bonded silica gel, a column temperature of 25 to 35° C., a flow rate of 0.8 to 1.2 mL per minute, and a detection wavelength of 254 nm.

7. The method for constructing the fingerprint of the high-quality seed soup according to claim 6, characterized in that: The HPLC gradient elution conditions are: 0 min: 2% A, 98% B; 60 min: 13% A, 87% B; 70 min: 16% A, 84% B; 100 min: 22% A, 78% B; 125 min: 30% A, 70% B; 130 min: 75% A, 25% B.

8. The method for constructing the fingerprint of the high-quality seed soup according to claim 1, characterized in that: The data processing in step (4) includes: reference peak selection, multi-point correction, and Mark peak matching, so as to obtain the common pattern of the fingerprint spectrum of the superior seed soup.

9. The method for constructing the fingerprint of the high-quality seed soup according to claim 1, characterized in that: The fingerprint of the Youjing Seed Soup obtained in step (4) comprises 23 common peaks, and the corresponding chromatographic peaks of the reference substances are peak 2 (5-hydroxymethylfurfural), peak 8 (syringin), peak 9 (chlorogenic acid), peak 13 (peonidin), peak 16 (ellagic acid), peak 18 (complexa glycosides), peak 19 (rosmarinic acid), and peak 23 (salvianolic acid B). Taking peak 16 as the reference peak S, the prescribed values ​​of the relative retention times of the 23 common peaks of Youjing Seed Soup are 0.16 (peak 1), 0.20 (peak 2), 0.21 (peak 3), 0.2 7 (peak 4), 0.29 (peak 5), 0.45 (peak 6), 0.48 (peak 7), 0.55 (peak 8), 0.59 (peak 9), 0.60 (peak 10), 0.63 (peak 11), 0.66 (peak 12), 0.74 (peak 13), 0.89 (peak 14), 0.92 (peak 15), 1.01 (peak 17), 1.15 (peak 18), 1.17 (peak 19), 1.23 (peak 20), 1.26 (peak 21), 1.28 (peak 22), and 1.32 (peak 23).

10. The method for constructing the fingerprint of the high-quality seed soup according to claim 9, characterized in that: The described Youjing Seed Soup has a total of peaks, among which peak 1 belongs to peony bark, raspberry, astragalus, dodder seed, and rehmannia root; peak 2 belongs to vinegar schisandra, wine polygonatum, rehmannia root, and raspberry; peak 3 belongs to wine polygonatum, rehmannia root, vinegar schisandra, mistletoe, astragalus, and raspberry; peak 4 belongs to wine polygonatum, vinegar schisandra, rehmannia root, raspberry, and astragalus; peak 5 belongs to mulberry, dipsaccharum, vinegar schisandra, salvia miltiorrhiza, and astragalus; peak 6, peak 7, peak 11, Peak 12, peak 21 and peak 22 belong to Dipsacus asper, peak 8 belongs to Paeonia suffruticosa and Viscum album, peak 9 belongs to Dipsacus asper, Cuscuta chinensis and Eucommia ulmoides, peak 10 belongs to Viscum album and Dipsacus asper, peak 13 belongs to Paeonia suffruticosa, peak 14 and peak 18 belong to Astragalus membranaceus, peak 15 belongs to Paeonia suffruticosa and Astragalus membranaceus, peak 16 belongs to Rubus idaeus, Cuscuta chinensis, Paeonia suffruticosa and Achyranthes bidentata, peak 17 belongs to Cuscuta chinensis and Achyranthes bidentata, peaks 19, 20 and peak 23 belong to Salvia miltiorrhiza.

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