A method for constructing a fingerprint spectrum of Youjing seed soup

Abstract

The application discloses a kind of Yujingzi decoction high performance liquid chromatography fingerprint construction methods.The construction method includes: the preparation of test solution;Reference solution preparation;Respectively, 10 μl of Yujingzi decoction solution and reference solution are precisely taken, injected into high performance liquid chromatograph, and the fingerprint is recorded;Yujingzi decoction fingerprint establishment: the liquid chromatogram of Yujingzi decoction obtained in step (3) is introduced into traditional Chinese medicine chromatographic fingerprint similarity evaluation system for data processing, and the common mode of the fingerprint of Yujingzi decoction is obtained;Yujingzi decoction fingerprint common peak attribution analysis.The high performance liquid chromatogram of single herb preparation, single herb negative preparation and Yujingzi decoction preparation is compared, 23 common peaks can be attributed, so as to judge whether the quality of Yujingzi decoction raw material is qualified.The method is simple and sensitive, and has good repeatability and stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine detection, and particularly relates to a fingerprint spectrum construction method of Youjing Seed Decoction. BACKGROUND

[0002] Youjing Seed Decoction is composed of 20 traditional Chinese medicines, such as Rehmannia glutinosa, Radix Rehmanniae Preparata, Rhizoma Polygonati Odorati, Radix Euonymi, Radix Dipsaci, Radix Cyathulae, Semen Lagenariae, etc. The decoction takes Rehmannia glutinosa and Radix Rehmanniae Preparata as the monarch, which can benefit essence and marrow, nourish yin and generate fluid, and tonify qi and kidney. Radix Euonymi, Radix Dipsaci and Radix Cyathulae can tonify liver and kidney and strengthen bones and muscles. Fructus Lycii can nourish liver and kidney and benefit essence and eyesight. Fructus Morus can nourish yin and generate fluid. Semen Lagenariae, Fructus Rubi and Fructus Rosae Multiflorae can tonify liver and kidney, consolidate essence and reduce urine. Fructus Rosae Multiflorae can also tonify kidney and assist yang. Fructus Schisandrae Chinensis can benefit qi and generate fluid, tonify kidney and calm heart, and astringe and contract. The above-mentioned medicinal ingredients can enhance the function of tonifying kidney essence deficiency, and are all ministerial medicinal ingredients. Radix Adenophorae, Radix Ophiopogonis and Radix Ophiopogonis can nourish yin and clear lung. Radix Salviae Miltiorrhizae and Cortex Moutan can clear heat and cool blood, and activate blood and resolve stasis. The decoction can not be greasy, and is all auxiliary medicinal ingredients. The whole decoction is mainly for nourishing liver and kidney, and is supplemented by clearing heat and cooling blood, so that the decoction can not be greasy, and can moisten without stagnation, and can generate essence and nourish yin.

[0003] The Youjing Seed Decoction is composed of 20 medicinal ingredients. A series of quality standards have been established in the early stage, including thin layer chromatography identification and content determination. However, the traditional Chinese medicine has complex components, and the single index of one or several traditional Chinese medicines cannot meet the overall quality control of the preparation. Therefore, an analysis method for comprehensively reflecting the Youjing Seed Decoction preparation is needed. The traditional Chinese medicine fingerprint spectrum is an analysis method for comprehensively reflecting the components of the traditional Chinese medicine. The single medicinal ingredient preparation, the single medicinal ingredient negative preparation and the finished product preparation are detected under the same chromatographic analysis condition, so that the fingerprint spectrum peaks can be attributed, and the quality of the raw medicinal ingredients and the finished product preparation of the Youjing Seed Decoction can be evaluated. SUMMARY

[0004] The present application aims at the defects of the prior art, and provides a high performance liquid chromatography fingerprint spectrum construction method of Youjing Seed Decoction and a standard fingerprint spectrum. The test solution of the Youjing Seed Decoction is analyzed, and is compared with the reference fingerprint spectrum and the common fingerprint spectrum mode, so that the multiple components in the preparation can be qualitatively analyzed, and the quality of the raw medicinal ingredients and the finished product preparation of the Youjing Seed Decoction can be evaluated.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme: a fingerprint spectrum construction method of Youjing Seed Decoction, comprising the following steps:

[0006] (1) Preparation of the test solution: the Youjing Seed Decoction is taken, and is subjected to ultrasonic treatment, centrifugation and filtration with methanol and water. The filtered solution is taken as the test solution;

[0007] (2) Preparation of the reference solution: the control sample is taken, and is dissolved with methanol to obtain the reference solution;

[0008] (3) precisely pipette 10 μl of the Yujingzi decoction solution and the reference solution respectively, inject into the high performance liquid chromatograph, and record the fingerprint spectrum;

[0009] (4) Establishment of the Yujingzi decoction fingerprint spectrum: introduce the liquid chromatogram of the Yujingzi decoction obtained in step (3) into the traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system for data processing, to obtain the common mode of the Yujingzi decoction fingerprint spectrum;

[0010] (5) Analysis of the attribution of the common peaks of the Yujingzi decoction fingerprint spectrum: inject the preparations prepared from single traditional Chinese medicine, the negative preparations prepared from single traditional Chinese medicine, and the Yujingzi decoction test solution prepared from all the components into the high performance liquid chromatograph, to perform the attribution of the common peaks.

[0011] Further, the Yujingzi decoction comprises the following components in mass parts: 8-12 parts of Rehmannia glutinosa, Radix Rehmanniae Preparata, Eucommia ulmoides Oliv., Dipsacus asper, Cyathula officinalis, Achyranthes bidentata, Taxillus sutchuenensis, Lycium barbarum, Morus alba, Schisandra chinensis, Adenophora stricta, Hedysarum polybotrys, Salvia miltiorrhiza, Radix Asparagi, Ophiopogon japonicus, and Paeonia suffruticosa in 8-12 parts each, and 13-17 parts of Rhizoma Polygonati, Semen Astragali Complanati, Semen Trichosanthis, and Rubus chingii in each.

[0012] Further, the Yujingzi decoction comprises the following components in mass parts: 8-12 parts of Rehmannia glutinosa, Radix Rehmanniae Preparata, Eucommia ulmoides Oliv., Dipsacus asper, Cyathula officinalis, Achyranthes bidentata, Taxillus sutchuenensis, Lycium barbarum, Morus alba, Schisandra chinensis, Adenophora stricta, Hedysarum polybotrys, Salvia miltiorrhiza, Radix Asparagi, Ophiopogon japonicus, and Paeonia suffruticosa in 8-12 parts each, and 13-17 parts of Rhizoma Polygonati, Semen Astragali Complanati, Semen Trichosanthis, and Rubus chingii in each.

[0013] Further, the reference solution is prepared with methanol as the solvent, and each mL of the solution comprises 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 astragalin, 40-80 μg of rosmarinic acid, and 20-80 μg of salvianolic acid B.

[0014] Further, the high performance liquid chromatography conditions are as follows: the mobile phase A is acetonitrile, and the mobile phase B is 0.1%-0.2% phosphoric acid water, gradient elution, the detection wavelength of the diode array detector is 200-400 nm, the chromatographic column is filled with octadecylsilane-bonded silica gel, the column temperature is 25-35 ℃, the flow rate is 0.8-1.2 mL per minute, and the detection wavelength is 254 nm.

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

[0016] Further, the gradient elution conditions of high performance liquid chromatography are as follows: 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] Further, the data processing in step (4) comprises: peak selection, multi-point correction and Mark peak matching, so that the common mode of the fingerprint spectrum of the Yujing seed decoction is obtained.

[0018] Further, the fingerprint spectrum of the Yujing seed decoction obtained in step (4) comprises 23 common peaks, and the reference material corresponding chromatographic peaks are peak 2 (5-hydroxymethyl furfural), peak 8 (syringin), peak 9 (chlorogenic acid), peak 13 (paeoniflorin), peak 16 (ellagic acid), peak 18 (shuyuansu glycoside), peak 19 (rosmarinic acid), and peak 23 (danshensan acid B), and the peak 16 is taken as a reference peak S, and the specified values of the relative retention time of the 23 common peaks of the Yujing seed decoction are respectively 0.16 (peak 1), 0.20 (peak 2), 0.21 (peak 3), 0.27 (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), 1.32 (peak 23).

[0019] Further, the peak 1 in the common peak in the Yujing seed decoction belongs to cortex moutan, raspberry, shuyuansu, cuscuta, rehmannia, the peak 2 belongs to vinegar schisandra, liquor turmeric, rehmannia, raspberry, the peak 3 belongs to liquor turmeric, rehmannia, vinegar schisandra, viscum, shuyuansu, raspberry, the peak 4 belongs to liquor turmeric, vinegar schisandra, rehmannia, raspberry, shuyuansu, the peak 5 belongs to mulberry, continue to break, vinegar schisandra, danshen, shuyuansu, the peak 6, the peak 7, the peak 11, the peak 12, the peak 21, the peak 22 belong to continue to break, the peak 8 belongs to cortex moutan and viscum, the peak 9 belongs to continue to break, cuscuta, salt eucommia, the peak 10 belongs to viscum, continue to break, the peak 13 belongs to cortex moutan, the peak 14, the peak 18 belong to shuyuansu, the peak 15 belongs to cortex moutan, shuyuansu, the peak 16 belongs to raspberry, cuscuta, cortex moutan and atractylodes, the peak 17 belongs to cuscuta, atractylodes, the peak 19, the peak 20, the peak 23 belong to danshen.

[0020] The present application has the following beneficial effects:

[0021] 1. The common mode of the fingerprint was constructed by the fingerprint of 15 batches of Youjing seed decoction. The quality of Youjing seed decoction can be judged objectively and comprehensively by similarity evaluation and comparison with the common mode spectrum.

[0022] 2. The 23 common peaks can be attributed by comparing the high performance liquid chromatogram of single herb preparation, negative preparation lacking single herb and Youjing seed decoction preparation, so as to judge whether the raw material quality of Youjing seed decoction is qualified.

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

[0024] Figure 1 is the liquid chromatogram of the mixed standard solution of the example and the test solution of Youjing seed decoction (S1 is the test solution of Youjing seed decoction, and S2 is the mixed standard solution. The chromatographic peaks in the mixed standard solution from left to right are adenosine (1), 5-hydroxymethylfurfural (2), geniposidic acid (3), syringin (4), chlorogenic acid (5), rosinol diglucoside (6), paeoniflorin (7), verbascoside (8), rutin (9), ellagic acid (10), ferulic acid (11), shuyanzisu glycoside (12), rosmarinic acid (13), salvianolic acid B (14), chuanxudan saponin VI (15), paeonol (16), schisandrol A (17)).

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

[0026] Figure 3 is the chromatogram of durability investigation (different column temperature) of the example.

[0027] Figure 4 is the chromatogram of durability investigation (different flow rate) of the example.

[0028] Figure 5 is the common mode chromatogram of Youjing seed decoction fingerprint of the example.

[0029] Figure 6 is the superimposed chromatogram of 15 batches of Youjing seed decoction fingerprint of the example.

[0030] Figure 7 is the liquid chromatogram comparison of reference (a) and test solution (b) of Youjing seed decoction of the example.

[0031] Figure 8 is the chromatographic peak attribution of Rehmannia glutinosa in Youjing seed decoction of the example (from bottom to top, single herb preparation (S1), negative preparation lacking single herb (S2) and Youjing seed decoction preparation (S3)).

[0032] Figure 9is the chromatographic peak attribution chart of Radix Rehmanniae Preparata in the example Youjingzisu Decoction (from bottom to top, single herb preparation (SI), negative preparation lacking single herb (S2), and finished product preparation of Youjingzisu Decoction (S3), respectively).

[0033] Figure 10 is the chromatographic peak attribution chart of Rhizoma Polygonati Preparata in the example Youjingzisu Decoction (from bottom to top, single herb preparation (SI), negative preparation lacking single herb (S2), and finished product preparation of Youjingzisu Decoction (S3), respectively).

[0034] Figure 11 is the chromatographic peak attribution chart of Radix Euonymi in the example Youjingzisu Decoction (from bottom to top, single herb preparation (SI), negative preparation lacking single herb (S2), and finished product preparation of Youjingzisu Decoction (S3), respectively).

[0035] Figure 12 is the chromatographic peak attribution chart of Radix Dipsaci in the example Youjingzisu Decoction (from bottom to top, single herb preparation (SI), negative preparation lacking single herb (S2), and finished product preparation of Youjingzisu Decoction (S3), respectively).

[0036] Figure 13 is the chromatographic peak attribution chart of Radix et Rhizoma Cyathulae in the example Youjingzisu Decoction (from bottom to top, single herb preparation (SI), negative preparation lacking single herb (S2), and finished product preparation of Youjingzisu Decoction (S3), respectively).

[0037] Figure 14 is the chromatographic peak attribution chart of Radix et Rhizoma Anemarrhenae in the example Youjingzisu Decoction (from bottom to top, single herb preparation (SI), negative preparation lacking single herb (S2), and finished product preparation of Youjingzisu Decoction (S3), respectively).

[0038] Figure 15 is the chromatographic peak attribution chart of Taxillus delavayi in the example Youjingzisu Decoction (from bottom to top, single herb preparation (SI), negative preparation lacking single herb (S2), and finished product preparation of Youjingzisu Decoction (S3), respectively).

[0039] Figure 16 is the chromatographic peak attribution chart of Semen Luffae in the example Youjingzisu Decoction (from bottom to top, single herb preparation (SI), negative preparation lacking single herb (S2), and finished product preparation of Youjingzisu Decoction (S3), respectively).

[0040] Figure 17 is the chromatographic peak attribution chart of Fructus Lycii in the example Youjingzisu Decoction (from bottom to top, single herb preparation (SI), negative preparation lacking single herb (S2), and finished product preparation of Youjingzisu Decoction (S3), respectively).

[0041] Figure 18is the chromatographic peak attribution chart of mulberry fruit in the example Youjingzisu decoction (from bottom to top, single herb preparation (S1), negative preparation without single herb (S2), and finished preparation of Youjingzisu decoction (S3)).

[0042] Figure 19 is the chromatographic peak attribution chart of raspberry in the example Youjingzisu decoction (from bottom to top, single herb preparation (S1), negative preparation without single herb (S2), and finished preparation of Youjingzisu decoction (S3)).

[0043] Figure 20 is the chromatographic peak attribution chart of vinegar five-flavor fruit in the example Youjingzisu decoction (from bottom to top, single herb preparation (S1), negative preparation without single herb (S2), and finished preparation of Youjingzisu decoction (S3)).

[0044] Figure 21 is the chromatographic peak attribution chart of desert medick in the example Youjingzisu decoction (from bottom to top, single herb preparation (S1), negative preparation without single herb (S2), and finished preparation of Youjingzisu decoction (S3)).

[0045] Figure 22 is the chromatographic peak attribution chart of north adenosma in the example Youjingzisu decoction (from bottom to top, single herb preparation (S1), negative preparation without single herb (S2), and finished preparation of Youjingzisu decoction (S3)).

[0046] Figure 23 is the chromatographic peak attribution chart of south adenosma in the example Youjingzisu decoction (from bottom to top, single herb preparation (S1), negative preparation without single herb (S2), and finished preparation of Youjingzisu decoction (S3)).

[0047] Figure 24 is the chromatographic peak attribution chart of red sage root in the example Youjingzisu decoction (from bottom to top, single herb preparation (S1), negative preparation without single herb (S2), and finished preparation of Youjingzisu decoction (S3)).

[0048] Figure 25 is the chromatographic peak attribution chart of wintergreen in the example Youjingzisu decoction (from bottom to top, single herb preparation (S1), negative preparation without single herb (S2), and finished preparation of Youjingzisu decoction (S3)).

[0049] Figure 26 is the chromatographic peak attribution chart of ophiopogon in the example Youjingzisu decoction (from bottom to top, single herb preparation (S1), negative preparation without single herb (S2), and finished preparation of Youjingzisu decoction (S3)).

[0050] Figure 27The chromatograms of the peaks of peony bark in the Youjing Seed Decoction are shown in the example (from bottom to top, they are single-herb preparation (S1), negative preparation without single herb (S2), and Youjing Seed Decoction finished product preparation (S3)). Detailed Implementation

[0051] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application.

[0052] It should be noted that the terms "comprising" and "having" and any variations thereof in the specification, claims and accompanying drawings of this application are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product or device.

[0053] The instruments and materials used in this 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) column; LC-UC-150 ultrasonic instrument; HC-2066 high-speed centrifuge; NewClassic MS 204S balance; METTLER TOLEDO XS205 balance.

[0055] Materials: Phosphoric acid (chromatographic grade, Shanghai Aladdin Company), formic acid (chromatographic grade, Sinopharm Chemical Reagent Co., Ltd.), methanol (chromatographic grade, TEDIA Corporation, USA), acetonitrile (chromatographic grade, TEDIA Corporation, USA), methanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); ultrapure water was obtained from a KZ-60L laboratory ultrapure water system; 15 batches of Youjing Seed Decoction, batch numbers Y1~Y15, all made from 20 herbs including Rehmannia glutinosa, prepared Rehmannia glutinosa, salt-processed Eucommia ulmoides, Dipsacus asper, and Achyranthes bidentata. The Chinese herbs used were provided by Suzhou Tianling Chinese Herbal Medicine Factory.

[0056] Example 1: Screening of Reference Index Components

[0057] (1) Preparation of reference solution: Take 5-hydroxymethyl furfural, syringin, chlorogenic acid, paeoniflorin, ellagic acid, astragalin, rosmarinic acid, salvianolic acid B reference substance, add appropriate amount of methanol to 5 mL volumetric flask, ultrasonic dissolution, and then cool down, add methanol to constant volume. Take adenosine, geniposidic acid, pinoresinol diglucoside, verbascoside, rutin, ferulic acid, asperosaponin VI, paeonol, schisandrol A reference substance, add appropriate amount of methanol to 5 mL volumetric flask, ultrasonic dissolution, and then cool down, add methanol to constant volume. Take 1 mL of standard stock solution 1 and standard stock solution 2 respectively, mix well, and then obtain the 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%), ultrasonic treatment for 60 min (power 40 kHz, frequency 760 w), cool down, shake well, high-speed centrifugation (12000 r / min, 10 min), take supernatant, pass through 0.22 μm microporous filter membrane, and then take the filtrate, and then obtain the test solution.

[0059] (3) Chromatographic conditions: Kromasil 100-5-C18 (w) chromatographic column (4.6 mm x 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. Precisely take 20 μl of test solution and 10 μl of mixed standard solution, and then inject into liquid chromatograph.

[0060] (4) The compound consistent with the retention time and absorption spectrum of the standard substance is identified as the target component. The chromatograms of test solution and mixed standard solution are shown in Figure 1 The following components can be detected in Youjing seed decoction: 5-hydroxymethyl furfural, syringin, chlorogenic acid, paeoniflorin, ellagic acid, astragalin, rosmarinic acid, salvianolic acid B, asperosaponin VI, paeonol, and schisandrol A. Schisandrol A and paeonol have late peak time and low response, so they are not detected as target peaks.

[0061] Therefore, the reference index components are selected as 5-hydroxymethyl furfural, syringin, chlorogenic acid, paeoniflorin, ellagic acid, astragalin, rosmarinic acid, and salvianolic acid B.

[0062] Example 2: Comparison of mobile phase acidity

[0063] (1) Preparation of test solution: 1 mL of Yujing seed soup was precisely taken into a 25 mL volumetric flask, washed with appropriate amount of water, and then added with methanol to a total volume of 20 mL, so that the methanol concentration was 60%, and then shaken uniformly, ultrasonic treatment for 90 min (ultrasonic power 40 kHz, frequency 760 w), and then cooled, supplemented with 60% methanol to the calibration line, shaken uniformly, high-speed centrifugation for 10 min (12000 r / min), and then the supernatant was taken, filtered through a 0.22 μm microporous filter membrane, and then the filtrate was taken as the test solution.

[0064] (2) Chromatographic conditions: Kromasil 100-5-C18 (w) chromatographic column (4.6 mm x 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. Acetonitrile (mobile phase A)-0.1% formic acid water (mobile phase B), acetonitrile (mobile phase A)-0.1% phosphoric acid water (mobile phase B), acetonitrile (mobile phase A)-0.2% phosphoric acid water (mobile phase B) were used as mobile phases, respectively, 10 μl of test solution was precisely taken and injected into the liquid chromatograph.

[0065] Results are shown in Figure 2 , acetonitrile-0.1% formic acid water as mobile phase, the separation effect of multiple chromatographic peaks is poor, acetonitrile-0.1% phosphoric acid water and acetonitrile-0.2% phosphoric acid have small difference in separation effect, but the separation effect of peak 4 and peak 5 is poor, so acetonitrile-0.1% phosphoric acid water is selected as the final mobile phase.

[0066] Example 3: Test product preparation condition optimization

[0067] (1) Preparation of test solution: 1 mL of Yujing seed soup was precisely taken into 3 25 mL volumetric flasks, and then added with appropriate amount of water and methanol to 20 mL, so that the methanol concentration was 90%, 60% and 30% respectively, ultrasonic extraction for 60 min (40 kHz, 760 w), cooled, supplemented with corresponding concentration of methanol to the calibration line, shaken uniformly, high-speed centrifugation (12000 r / min, 10 min), the supernatant was taken, filtered through a 0.22 μm microporous filter membrane, and then the filtrate was taken as the test solution of different methanol extraction systems. In addition, 4 25 mL volumetric flasks were taken, 1 mL of Yujing seed soup was precisely taken into each, added with appropriate amount of water and methanol to 20 mL, so that the methanol concentration was 60%, and then ultrasonic treatment for 30 min, 60 min, 90 min and 120 min respectively, cooled, supplemented with 60% methanol to the calibration line, shaken uniformly, high-speed centrifugation (12000 r / min, 10 min), the supernatant was taken, filtered through a 0.22 μm microporous filter membrane, and then the filtrate was taken as the test solution of different ultrasonic time.

[0068] (2) Chromatographic conditions were optimized according to the conditions in step (2) of Example 2.

[0069] (3) The peak areas of each chromatographic peak of the above test solution were recorded and the total peak area was calculated. The results are shown in Tables 1 and 2, and the total peak area of the extraction solvent is 30% methanol < 60% methanol ≈ 90% methanol. Preferably, the 60% methanol and 90% methanol extracts have more chromatographic peak information and the highest total peak area than other concentration extracts, and further preferably 60% methanol is used as the extraction system for the Yujing seed decoction fingerprint. Preferably, the peak area reaches the maximum when the ultrasonic extraction time reaches 90 min and 120 min, and the peak area increases by a smaller amount when the ultrasonic extraction time is 120 min, and further preferably the ultrasonic extraction time is 90 min to maximize the characterization of the chemical components in the Yujing seed decoction.

[0070] (4) The preferred conditions for preparing the Yujing seed decoction test solution are as follows: 1 mL of Yujing seed decoction is accurately transferred to a 25 mL volumetric flask, and an appropriate amount of water and methanol is added to 20 mL, so that the methanol concentration is 60%, ultrasonic extraction is carried out for 90 min (40 kHz, 760 w), and then the solution is cooled, 60% methanol is added to the mark, shaken well, high-speed centrifugation (12000 r / min, 10 min), the supernatant is taken, filtered through a 0.22 μm microporous filter, and the filtrate is taken, which is obtained.

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

[0072]

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

[0074]

[0075] Example 4: Methodology verification

[0076] 1. Precision: 1 mL of Yujing seed decoction sample (batch number Y1) was prepared as a test sample, and 6 injections were continuously injected according to the above optimized chromatographic conditions, the retention time and peak area of the target peak were recorded, and the chromatographic peak with better separation and larger peak area (ellagic acid) was taken as the S peak, and the relative retention time and relative peak area of each common peak were calculated. The results (Tables 3 and 4) show 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: Precision test results of Yujing seed decoction (relative retention time)

[0078]

[0079] Table 4: Precision test results of Yujing seed decoction (relative peak area)

[0080]

[0081] 2. Repeatability: 6 samples of Yuxing seed decoction were prepared according to the optimized preparation method, and 6 samples of test product were prepared in parallel. The retention time and peak area of common peaks were recorded, and the relative retention time and relative peak area of each common peak were calculated with S peak of tannic acid. The results showed (Tables 5 and 6) that the RSD of the relative retention time of each common peak was less than 0.09%, and the RSD of the relative peak area was less than 4.17%, indicating that the method had good repeatability.

[0082] Table 5: Repeatability test results of Yuxing seed decoction (relative retention time)

[0083]

[0084] Table 6: Repeatability test results of Yuxing seed decoction (relative peak area)

[0085]

[0086] 3. Stability: The test product prepared in the precision experiment was analyzed at 0h, 3h, 6h, 9h, 12h, 21h, and 24h after preparation according to the above optimized chromatographic conditions. The retention time and peak area of the target peak were recorded, and the relative retention time and relative peak area of each common peak were calculated with S peak of tannic acid. The results showed (Tables 7 and 8) that the RSD of the relative retention time of each common peak was less than 0.18%, and the RSD of the relative peak area was less than 5.08%, indicating that the test product solution of Yuxing seed decoction was stable within 24h.

[0087] Table 7: Repeatability test results of Yuxing seed decoction (relative retention time)

[0088]

[0089] Table 8: Repeatability test results of Yuxing seed decoction (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 investigate the influence 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 were as follows Figure 3 、 Figure 4As shown, the separation of peak 6, peak 9, peak 10 and peak 16 is poor at 25℃, the separation of peak 4, peak 5, peak 9 and peak 16 is poor at 35℃, and the separation of peak 8 is poor at a column temperature other than 30℃, so the column temperature is set to 30℃. If the flow rate is lower than 1 mL / min, the separation of peak 6 and peak 19 will be affected, so the flow rate should not be lower than 1 mL / min.

[0092] Example 5: Establishment of the fingerprint of Youjing and similarity evaluation

[0093] Fifteen batches of Youjing seed decoction (batch numbers Y1-Y15) were prepared, and the test solutions were prepared according to the optimized method and the high performance liquid chromatograms were collected. The chromatograms were introduced into the “Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System”, and Y1 was used as the reference chromatogram to generate the control by the average method, with the time window width set to 0.1. After multi-point correction and Mark peak matching, the fingerprint common mode was generated (see Figure 5 ), and the superimposed chromatograms of the 15 batches of Youjing seed decoction fingerprints are shown in Figure 6 The similarity of the HPLC fingerprints of the 15 batches of Youjing seed decoction and the fingerprint common mode (Table 9) was greater than 0.997, and the similarity results between batches were above 0.992, indicating that the similarity of the 15 batches of Youjing seed decoction was good. The number of common peaks was 23, of which peak 2, 8, 9, 13, 16, 18, 19 and 23 were 5-hydroxymethylfurfural, syringin, chlorogenic acid, paeoniflorin, ellagic acid, astragalin, rosmarinic acid and salvianolic acid B, respectively. The reference containing the above 8 components and the HPLC fingerprint of Youjing seed decoction are shown in Figure 7 .

[0094] Table 9 Similarity of the fingerprints of the 15 batches of Youjing seed decoction

[0095]

[0096] Example 6: Attribution of each common peak

[0097] According to the prescription of Youjing seed decoction, 20 negative preparations of 20 medicines such as Radix Rehmanniae Glutinosae, Radix Rehmanniae Preparata and Rhizoma Polygonati Nudati were prepared, and the preparation containing only a single medicine was prepared according to the prescription. The test solution was injected and analyzed, and the results are shown in Figures 8-27Peaks 1~5, 8~10, 15~17 are contributed peaks of multi-taste traditional Chinese medicine, wherein peak 1 belongs to rehmannia root, cuscuta, raspberry, milk vetch seed and peony root, and the main contribution comes from peony root; peak 2 belongs to rehmannia root, rhizoma polygonati, raspberry and schisandra chinensis; peak 3 belongs to rehmannia root, rhizoma polygonati, viscum, raspberry, schisandra chinensis and milk vetch seed; peak 4 belongs to rehmannia root, rhizoma polygonati, raspberry, schisandra chinensis and milk vetch seed; peak 5 belongs to eucommia ulmoides, mulberry, schisandra chinensis, milk vetch seed and salvia miltiorrhiza; peak 8 belongs to viscum and peony root; peak 9 belongs to salt eucommia ulmoides, eucommia ulmoides and cuscuta; peak 10 belongs to eucommia ulmoides and viscum; peak 15 belongs to milk vetch seed and peony root; peak 16 belongs to achyranthes bidentata, cuscuta, raspberry and peony root, and the main contribution comes from raspberry; and peak 17 belongs to achyranthes bidentata and cuscuta. Some traditional Chinese medicines have exclusive peaks: peaks 6, 7, 11, 12, 21 and 22 are exclusive peaks of eucommia ulmoides, peaks 14 and 18 are exclusive peaks of milk vetch seed, peaks 19, 20 and 23 are exclusive peaks of salvia miltiorrhiza, and peak 13 is an exclusive peak of peony root.

[0098] The peak area of the exclusive peak in the fingerprint spectrum is analyzed, so as to provide an experimental basis for judging the quality of eucommia ulmoides, milk vetch seed, salvia miltiorrhiza and peony root as raw materials of Youjingzishen Decoction.

[0099] The method for constructing the fingerprint spectrum of Youjingzishen Decoction provided by the application is sensitive and has good repeatability, and can more objectively and comprehensively evaluate the quality of Youjingzishen Decoction.

[0100] The above describes preferred embodiments of the application in detail. It should be understood that those skilled in the art can make many modifications and changes without creative effort based on the concept of the application. Therefore, any technical solution obtained by logical analysis, reasoning or limited experiment based on the prior art according to the concept of the application should be within the protection scope defined by the claims.

Claims

1. A method for constructing a fingerprint spectrum of a high-quality seed decoction, characterized in that: Includes the following steps: (1) Preparation of test solution: Take the seed soup of high-quality seed, add methanol and water, sonicate, centrifuge, filter, and take the filtrate as test solution; (2) Preparation of reference solution: Take the reference standard, dissolve it in methanol to obtain the reference solution; (3) Accurately pipette 10 μl each of the superior seed decoction solution and the reference solution, inject them into the high performance liquid chromatograph, and record the fingerprint spectrum; (4) Establishment of fingerprint spectrum of Youjing Seed Decoction: The liquid chromatogram of Youjing Seed Decoction obtained in step (3) is imported into the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system for data processing to obtain the common pattern of fingerprint spectrum of Youjing Seed Decoction. (5) Analysis of common peaks in fingerprint spectrum of Youjing Seed Decoction: The preparations made from single Chinese herbs, the negative preparations made without single Chinese herbs, and the test solution of Youjing Seed Decoction made from all the ingredients were injected into the high performance liquid chromatograph and the common peaks were assigned. The superior seed decoction comprises the following components in parts by weight: 8-12 parts each of Rehmannia glutinosa, prepared Rehmannia glutinosa, salt-processed Eucommia ulmoides, Dipsacus asper, Achyranthes bidentata, Achyranthes bidentata, Loranthus parasiticus, Lycium barbarum, Morus alba, vinegar-processed Schisandra chinensis, Adenophora stricta, Adenophora stricta, Salvia miltiorrhiza, Asparagus cochinchinensis, Ophiopogon japonicus, and Paeonia suffruticosa; and 13-17 parts each of Polygonatum sibiricum, Astragalus complanatus, Cuscuta chinensis, and Rubus idaeus. The reference standards include 5-hydroxymethylfurfural, syringin, chlorogenic acid, paeoniflorin, ellagic acid, astragaloside, rosmarinic acid, and salvianolic acid B; The high performance liquid chromatography conditions were as follows: Kromasil 100-5-C18 (w) 4.6 mm × 250 mm, 5 μm column; mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid water, gradient elution, column temperature 30 °C, flow rate 1-1.2 mL per minute, and detection wavelength 254 nm. The high-performance liquid chromatography gradient elution conditions are: 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.

2. The method for constructing the fingerprint spectrum of superior seed soup according to claim 1, characterized in that: The superior seed decoction comprises the following components in parts by weight: 10 parts each of Rehmannia glutinosa, prepared Rehmannia glutinosa, salt-processed Eucommia ulmoides, Dipsacus asper, Achyranthes bidentata, Achyranthes bidentata, Loranthus parasiticus, Lycium barbarum, Morus alba, vinegar-processed Schisandra chinensis, Adenophora stricta, Adenophora stricta, Salvia miltiorrhiza, Asparagus cochinchinensis, Ophiopogon japonicus, and Paeonia suffruticosa; and 15 parts each of Polygonatum sibiricum, Astragalus complanatus, Cuscuta chinensis, and Rubus idaeus.

3. The method for constructing the fingerprint spectrum of superior seed soup according to claim 1, characterized in that: The reference solution was prepared using methanol as a solvent, and each mL of the solution contained 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 astragaloside, 40–80 μg rosmarinic acid, and 20–80 μg salvianolic acid B.

4. The method for constructing the fingerprint spectrum of superior 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, thereby obtaining the fingerprint spectrum common pattern of Youjing Seed Soup.

5. The method for constructing the fingerprint spectrum of superior seed soup according to claim 1, characterized in that: The fingerprint chromatogram of the superior seed decoction obtained in step (4) includes 23 common peaks. The chromatographic peaks corresponding to the reference substances are peak 2 (5-hydroxymethylfurfural), peak 8 (syringin), peak 9 (chlorogenic acid), peak 13 (paeoniflorin), peak 16 (ellagic acid), peak 18 (astragalin), peak 19 (rosmarinic acid), and peak 23 (tanshinone B). With peak 16 as the reference peak S, the specified values ​​for the relative retention times of the 23 common peaks of the superior seed decoction are 0.16 (peak 1), 0.20 (peak 2), 0.21 (peak 3), and 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), 1.32 (peak 23).

6. The method for constructing the fingerprint spectrum of superior seed soup according to claim 5, characterized in that: The aforementioned superior seed decoction contains peak 1 belonging to Moutan bark, raspberry, psoralea corylifolia seed, dodder seed, and prepared rehmannia root; peak 2 belonging to vinegar-processed schisandra fruit, wine-processed polygonatum, prepared rehmannia root, and raspberry; peak 3 belonging to wine-processed polygonatum, prepared rehmannia root, vinegar-processed schisandra fruit, mistletoe, psoralea corylifolia seed, and raspberry; peak 4 belonging to wine-processed polygonatum, vinegar-processed schisandra fruit, prepared rehmannia root, raspberry, and psoralea corylifolia seed; peak 5 belonging to mulberry, dipsacus root, vinegar-processed schisandra fruit, salvia miltiorrhiza, and psoralea corylifolia seed; and peaks 6, 7, and 11. Peaks 12, 21, and 22 belong to Dipsacus asper; peak 8 belongs to Paeonia suffruticosa and Loranthus parasiticus; peak 9 belongs to Dipsacus asper, Cuscuta chinensis, and Eucommia ulmoides; peak 10 belongs to Loranthus parasiticus and Dipsacus asper; peak 13 belongs to Paeonia suffruticosa; peaks 14 and 18 belong to Astragalus complanatus; peak 15 belongs to Paeonia suffruticosa and Astragalus complanatus; peak 16 belongs to Rubus idaeus, Cuscuta chinensis, Paeonia suffruticosa, and Achyranthes bidentata; peak 17 belongs to Cuscuta chinensis and Achyranthes bidentata; and peaks 19, 20, and 23 belong to Salvia miltiorrhiza.

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