Construction method and application of fingerprint spectrum of angelica sinensis potion

Abstract

The invention provides a construction method and application of an angelica sinensis potion fingerprint spectrum, and belongs to the technical field of drug detection.The construction method includes the steps that firstly, an angelica sinensis potion is used for preparing a test solution, then 0.08-0.12 wt% phosphoric acid aqueous solution (A)-acetonitrile (B) serves as a mobile phase, and the angelica sinensis potion fingerprint spectrum is obtained through high performance liquid chromatography detection, a standard fingerprint spectrum is established and is used for quality evaluation or control in the whole process of research / development / production / clinical application of the angelica sinensis potion. According to the construction method of the fingerprint spectrum of the angelica sinensis potion, main medicinal material components in the angelica sinensis potion can be detected only through high performance liquid chromatography, so that the quality condition of the angelica sinensis potion is reflected more comprehensively.

Description

Technical Field

[0001] The present invention relates to drug detection technology, and in particular to a method for constructing a fingerprint spectrum of Dangguiyinzi and its application. Background Art

[0002] Danggui Yinzi comes from the Revised Yan's Jisheng Prescription. It is a compound preparation made of 11 Chinese herbs, including Danggui, White Peony, Chuanxiong, Raw Rehmannia, Tribulus, Saposhnikovia, Nepeta Spike, Polygonum Multiflorum, Astragalus, Roasted Licorice and Ginger. It is suitable for stagnation of blood in the heart, internal wind-heat, skin sores and scabies, or swelling or itching, or pus infiltration, or red rashes and tumors. Looking at its composition, the four herbs and Polygonum Multiflorum nourish yin and blood, and are suitable for those with blood deficiency and wind dryness. Therefore, all kinds of skin diseases that have been suffered for a long time, damage yin and blood, or swelling or itching can be considered this prescription.

[0003] Since the prepared Danggui Yinzi has lost the morphological characteristics of the original pieces, the simple qualitative identification and quantitative analysis of the index components are difficult to reflect its quality. As a standard reference to measure whether the classic Chinese medicine prescription is basically consistent with the clinical decoction, its quality should strengthen the specific identification and multi-component and overall quality control.

[0004] The spectrum of Chinese medicine preparations refers to the spectrum that can reflect the information of multiple components and reflect its quality characteristics after the samples of Chinese medicine preparations are properly processed and analyzed by appropriate methods. The characteristic spectrum of Chinese medicine preparations is of great significance for identifying the key quality attributes of Chinese medicine preparations, studying the transfer of quantity values, evaluating the uniformity and stability of the quality of Chinese medicine preparations, and improving the overall quality control level of Chinese medicine preparations.

[0005] Therefore, by establishing a characteristic map, the overall quality of Danggui Yinzi can be comprehensively controlled. Summary of the invention

[0006] In view of the above problems, the present invention provides a method for constructing a fingerprint spectrum of Dangguiyinzi and its application.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is:

[0008] A method for constructing a fingerprint of Dangguiyinzi, the method comprising the following steps:

[0009] S1. Prepare the test solution by taking Dangguiyinzi;

[0010] S2, taking the test solution and performing high performance liquid chromatography to obtain the fingerprint of Dangguiyinzi;

[0011] During the high performance liquid chromatography detection process, 0.08-0.12 wt % phosphoric acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and elution is performed in a gradient elution manner;

[0012] The elution procedure is:

[0013] The mobile phase is, the elution mode is gradient elution, and the elution program is:

[0014] 0-5min: 95% mobile phase A, 5% mobile phase B;

[0015] 5-7 min: 95% → 88% mobile phase A, 5% → 12% mobile phase B;

[0016] 7-15 min: 88% mobile phase A, 12% mobile phase B;

[0017] 15-23 min: 88% → 85% mobile phase A, 12% → 15% mobile phase B;

[0018] 23-28 min: 85% → 83% mobile phase A, 15% → 17% mobile phase B;

[0019] 28-38 min: 83% → 80% mobile phase A, 17% → 20% mobile phase B;

[0020] 38-43min: 80% mobile phase A, 20% mobile phase B;

[0021] 43-53 min: 80% → 70% mobile phase A, 20% → 30% mobile phase B;

[0022] 53-60min: 70%→60% mobile phase A, 30%→40% mobile phase B;

[0023] 60-65min: 60% mobile phase A, 40% mobile phase B;

[0024] 65-70 min: 60% → 40% mobile phase A, 40% → 60% mobile phase B;

[0025] 70-80min: 40%→0% mobile phase A, 60%→100% mobile phase B.

[0026] 2 Further, in the HPLC detection process, the chromatographic column is an Agilent ZORBAX Eclipse Plus C18 column.

[0027] Furthermore, during the high performance liquid chromatography detection process, the detection wavelengths are 205-215 nm and 300-310 nm.

[0028] Furthermore, the test solution is prepared by adding Danggui Yinzi to a methanol aqueous solution, ultrasonically treating the solution, and filtering the solution.

[0029] Furthermore, during the preparation of the test solution, the concentration of the methanol aqueous solution was 65-75 vol 1%.

[0030] Furthermore, in the process of preparing the test solution, the volume ratio of Danggui Yinzi to the test solution was 0.8-1.2:10.

[0031] Furthermore, the flow rate of the HPLC detection is 0.8-1.2 mL / min, the column temperature is 30-40° C., and the injection volume is 5-20 μL.

[0032] Furthermore, the construction method also includes preparing a mixed reference solution using paeoniflorin, ammonium glycyrrhizinate, liquiritin, hesperidin, 6-gingerol, cimicifuga glycoside, 5-O-methylvisaminol glycoside, ferulic acid, and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside, and performing high performance liquid chromatography detection.

[0033] An application of the standard fingerprint of Danggui Yinzi obtained by the above-mentioned construction method in the quality evaluation or control of the whole process of research / development / production / clinical application of Danggui Yinzi.

[0034] Further, at a wavelength of 210 nm, a total of 20 common peaks were calibrated in the standard fingerprint of Danggui Yinzi, of which peak 8 was paeoniflorin, peak 9 was cimicifuga glycoside, peak 10 was liquiritigenin, peak 11 was 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, peak 13 was 5-O-methylvisaminol glycoside, peak 14 was hesperidin, and peak 19 was 6-gingerol;

[0035] At a wavelength of 305 nm, 13 common peaks were calibrated in the standard fingerprint of Danggui Yinzi, of which peak 4 was cimicifuga glycoside, peak 5 was ferulic acid, peak 6 was liquiritigenin, peak 7 was 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, and peak 9 was 5-O-methylvisaminol glycoside.

[0036] The beneficial effects of the method for constructing the Dangguiyinzi fingerprint spectrum and its application are as follows:

[0037] The method for constructing the fingerprint of Danggui Yinzi of the present invention can detect the main medicinal ingredients in Danggui Yinzi by high performance liquid chromatography, thereby more comprehensively reflecting the quality of Danggui Yinzi.

[0038] The present invention combines the different physical and chemical properties of different effective ingredients in Danggui Yinzi, and by adjusting the elution conditions, obtains a standard fingerprint spectrum with good separation, a relatively stable baseline, more peaks, and better ability to reflect the quality of Danggui Yinzi;

[0039] The present invention obtains a standard fingerprint spectrum of Dangguiyinzi quality with a more stable baseline, better peak shape and separation effect by adjusting the mobile phase;

[0040] The present invention can separate different effective components in Danggui Yinzi well by adjusting the chromatographic column, and the response value and separation degree of the chromatographic peak are good;

[0041] The present invention can more fully extract the medicinal ingredients involved in Danggui Yinzi by adjusting the preparation method of the test solution, and the concentration of each medicinal ingredient in the obtained test solution can be separated and detected under the same high performance liquid chromatography conditions, and the peaks in the obtained chromatogram are good;

[0042] The present invention effectively optimizes the separation conditions by selecting appropriate process conditions, thereby improving the linearity, precision, stability, repeatability and specificity of Dangguiyinzi;

[0043] The standard fingerprint spectrum of Danggui Yinzi obtained by the present invention can provide effective guarantee for the quality monitoring of Danggui Yinzi in the whole process of research, development, production and clinical application.

[0044] By using the presence and characteristics of the common characteristic peaks in the standard fingerprint spectrum of Danggui Yinzi obtained by the present invention, the product quality of Danggui Yinzi can be fully monitored. By comparing the similarity of the chromatographic fingerprint characteristics, the quality of Danggui Yinzi can be evaluated, and the stability and uniformity of Danggui Yinzi can be examined, which makes up for the shortcomings of the current quality control method. At the same time, the stability of the production process of Danggui Yinzi can also be monitored to ensure that its quality is stable, uniform and controllable.

[0045] The standard fingerprint spectrum of Danggui Yinzi obtained by the present invention improves the quality monitoring standards of Danggui Yinzi finished products and semi-finished products, effectively prevents the occurrence of product counterfeiting incidents, and ensures the normal production and circulation order of Danggui Yinzi; on the basis of the present invention, the correlation study between fingerprint spectrum information and pharmacodynamic activity information can also be carried out, so as to deeply clarify the correlation between the intrinsic chemical components of Danggui Yinzi and the efficacy of the preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 These are chromatograms of Danggui Yinzi detected by different chromatographic columns during the chromatographic column screening process in Example 1 of the present invention; wherein, Figure A is a chromatogram of Danggui Yinzi detected by a Waters Sunfire C18 column, Figure B is a chromatogram of Danggui Yinzi detected by an AgilentZORBAX Eclipes Plus C18 column, and Figure C is a chromatogram of Danggui Yinzi detected by a Phenomenex Luna Su C18 column;

[0047] Figure 2It is a chromatogram of Danggui Yinzi detected by different mobile phases in the mobile phase screening process of Example 1 of the present invention; wherein, Figure A is a chromatogram of Danggui Yinzi detected by 0.1wt% formic acid aqueous solution (A)-acetonitrile (B), Figure B is a chromatogram of Danggui Yinzi detected by 0.1wt% acetic acid aqueous solution (A)-acetonitrile (B), and Figure C is a chromatogram of Danggui Yinzi detected by 0.1wt% phosphoric acid aqueous solution (A)-acetonitrile (B);

[0048] Figure 3 It is a chromatogram of Danggui Yinzi detected at a wavelength of 210 nm using different elution programs during the elution program screening process of Example 1 of the present invention; wherein Figure A is a chromatogram of Danggui Yinzi detected using elution program one, and Figure B is a chromatogram of Danggui Yinzi detected using elution program two;

[0049] Figure 4 is the high performance liquid chromatography of the mixed reference solution at a wavelength of 210 nm in Example 2 of the present invention;

[0050] Figure 5 is the fingerprint of the test solution at a wavelength of 210 nm in Example 2 of the present invention;

[0051] Figure 6 is the high performance liquid chromatography of the mixed reference solution at a wavelength of 305 nm in Example 2 of the present invention;

[0052] Figure 7 is the fingerprint of the test solution at a wavelength of 305 nm in Example 2 of the present invention;

[0053] Figure 8 is a chromatogram of fifteen batches of Danggui Yinzi detected at a wavelength of 210 nm in Example 2 of the present invention;

[0054] Fig. 9 It is the standard fingerprint spectrum at a wavelength of 210 nm in Example 2 of the present invention;

[0055] Fig.10 is a chromatogram of fifteen batches of Danggui Yinzi detected at a wavelength of 305 nm in Example 2 of the present invention;

[0056] Fig.11 It is the standard fingerprint spectrum at a wavelength of 305 nm in Example 2 of the present invention;

[0057] Fig.12 It is the result of the precision inspection fingerprint spectrum at a wavelength of 210nm in Example 7 of the present invention;

[0058] Fig.13 It is the result of the precision inspection fingerprint spectrum at a wavelength of 305 nm in Example 7 of the present invention;

[0059] Fig.14 It is the fingerprint result of the repeatability inspection at a wavelength of 210nm in Example 7 of the present invention;

[0060] Fig.15 It is the fingerprint result of the repeatability inspection at a wavelength of 305 nm in Example 7 of the present invention;

[0061] Fig.16 It is the stability investigation fingerprint result at a wavelength of 210 nm in Example 7 of the present invention;

[0062] Fig.17 This is the fingerprint result of the stability investigation at a wavelength of 305 nm in Example 7 of the present invention. DETAILED DESCRIPTION

[0063] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0064] Example 1 Screening of a method for constructing a fingerprint of Dangguiyinzi

[0065] In this embodiment, Dangguiyinzi extract is used as the sample to be tested, wherein the preparation method of Dangguiyinzi extract is as follows:

[0066] The prescription is: Angelica 5.84g, White Peony 5.84g, Ligusticum chuanxiong 5.84g, Rehmannia glutinosa 5.84, Tribulus terrestris 5.84g, Saposhnikovia divaricata 5.84g, Schizonepeta tenuifolia 5.84g, Polygonum multiflorum 2.92g, Astragalus 2.92g, Roasted Licorice 2.92g and Ginger 15g.

[0067] Crush / powder all the herbs into the coarsest powder (5mm).

[0068] Take 16.52g of the coarsest powder of medicinal materials (1.95g each of angelica, white peony root, chuanxiong, raw rehmannia, white tribulus, siler, and schizonepeta spicate, 0.97g each of polygonum multiflorum, astragalus, and licorice), add 450mL of water and 5g of ginger, boil over high heat, and simmer until the original volume is 80% (240mL). Filter out the residue while it is hot, and concentrate the resulting medicinal solution under reduced pressure at 60°C to an extract with a density of 1.15-1.22, thereby obtaining the angelica decoction extract.

[0069] In this example, Dangguiyinzi extract was used as the Dangguiyinzi sample to screen the Dangguiyinzi fingerprint construction method, as follows:

[0070] 1) Preparation method of test solution

[0071] Accurately measure 1 mL of Dangguiyinzi extract (i.e., v = 1 mL), place it in a 10 mL volumetric flask (i.e., V = 10 mL), add 70 vol% methanol aqueous solution to dilute to the scale, shake well, ultrasonically treat for 20 min, filter, and take the filtrate to obtain the test solution.

[0072] 2) Chromatographic column screening

[0073] Accurately pipette 10 μL of the test solution and inject it into a high performance liquid chromatograph to examine the chromatograms using a Waters Sunfire C18 column, an Agilent ZORBAX Eclipes Plus C18 column, and a Phenomenex Luna Su C18 column. The results are as follows: Figure 1 shown.

[0074] Among them, other conditions of high performance liquid chromatography are:

[0075] Column temperature: 35°C;

[0076] Flow rate: 1.0 mL / min;

[0077] Detection wavelength: 210nm and 305nm;

[0078] The mobile phase was 0.1 wt% phosphoric acid aqueous solution (A)-acetonitrile (B), the elution mode was gradient elution, and the elution program was:

[0079] 0-5min: 95% mobile phase A, 5% mobile phase B;

[0080] 5-7 min: 95% → 88% mobile phase A, 5% → 12% mobile phase B;

[0081] 7-15 min: 88% mobile phase A, 12% mobile phase B;

[0082] 15-23 min: 88% → 85% mobile phase A, 12% → 15% mobile phase B;

[0083] 23-28 min: 85% → 83% mobile phase A, 15% → 17% mobile phase B;

[0084] 28-38 min: 83% → 80% mobile phase A, 17% → 20% mobile phase B;

[0085] 38-43min: 80% mobile phase A, 20% mobile phase B;

[0086] 43-53 min: 80% → 70% mobile phase A, 20% → 30% mobile phase B;

[0087] 53-60min: 70%→60% mobile phase A, 30%→40% mobile phase B;

[0088] 60-65min: 60% mobile phase A, 40% mobile phase B;

[0089] 65-70 min: 60% → 40% mobile phase A, 40% → 60% mobile phase B;

[0090] 70-80min: 40%→0% mobile phase A, 60%→100% mobile phase B.

[0091] Depend on Figure 1 It can be seen that the response value and separation of the chromatographic peaks are good when using the Agilent ZORBAX Eclipes Plus C18 column, so this column was selected to continue the construction of the method.

[0092] 3) Mobile phase screening

[0093] C18 (Agilent ZORBAX Eclipse Plus C18, 4.6×250mm Column, 5μm, PN959990-902, SNUSUXA32938) was used as the chromatographic column. According to the chromatographic conditions in step 2) of the chromatographic column screening (i.e., only the type of mobile phase was changed, and other analysis parameters remained unchanged), the effects of three mobile phases, 0.1wt% formic acid aqueous solution (A)-acetonitrile (B), 0.1wt% acetic acid aqueous solution (A)-acetonitrile (B), and 0.1wt% phosphoric acid aqueous solution (A)-acetonitrile (B), on the fingerprint separation degree were further investigated at detection wavelengths of 210nm and 305nm. The results are shown in Figure 2 .Depend on Figure 2 It can be seen that when 0.1 wt % phosphoric acid aqueous solution (A)-acetonitrile (B) is used as the mobile phase, the baseline is more stable, the peak shape and separation effect are better, so 0.1 wt % phosphoric acid aqueous solution (A)-acetonitrile (B) is finally selected as the mobile phase.

[0094] 4) Elution program screening

[0095] Using 0.1wt% phosphoric acid aqueous solution (A)-acetonitrile (B) as the mobile phase, according to the chromatographic conditions in step 3) mobile phase screening (i.e. only the type of mobile phase was changed, and other analytical parameters remained unchanged), the separation effect of different elution procedures under the condition of a detection wavelength of 210nm was further investigated. The results are shown in Figure 3 .

[0096] Among them, the different elution procedures are as follows:

[0097] Elution procedure 1:

[0098] 0-10min: 95%→85% mobile phase A, 5%→15% mobile phase B;

[0099] 10-20 min: 85% mobile phase A, 15% mobile phase B;

[0100] 20-30 min: 85% → 80% mobile phase A, 15% → 20% mobile phase B;

[0101] 30-35 min: 80% mobile phase A, 80% mobile phase B;

[0102] 35-45 min: 80% → 70% mobile phase A, 20% → 30% mobile phase B;

[0103] 45-55 min: 70% mobile phase A, 30% mobile phase B;

[0104] 55-60min: 70%→60% mobile phase A, 30%→40% mobile phase B;

[0105] 60-70 min: 60% mobile phase A, 40% mobile phase B;

[0106] 70-72 min: 60% → 40% mobile phase A, 40% → 60% mobile phase B;

[0107] 72-80 min: 40% mobile phase A, 60% mobile phase B;

[0108] 80-82 min: 40% → 20% mobile phase A, 60% → 80% mobile phase B;

[0109] 82-100min: 20%→0% mobile phase A, 80%→100% mobile phase B.

[0110] Elution procedure 2 is the elution procedure in step 1) chromatographic column screening.

[0111] Depend on Figure 3 It can be seen that the chromatographic peaks are densely distributed under elution procedure one (i.e., the proportion of high-polarity mobile phase). Elution procedure one was optimized to obtain the chromatogram of elution procedure two, in which the chromatographic peaks are evenly distributed and the separation is good. Elution procedure two was determined as the final elution procedure.

[0112] Example 2 A method for constructing a fingerprint of Danggui Yinzi and a standard fingerprint of Danggui Yinzi constructed and its application

[0113] In this example, Dangguiyinzi extract was used as the Dangguiyinzi sample, and the specific method for constructing the Dangguiyinzi fingerprint was as follows:

[0114] S1. Solution preparation

[0115] S11. Prepare the test solution according to the test solution preparation method in step 1 of Example 1, chromatographic condition screening.

[0116] S12. Take appropriate amounts of paeoniflorin, liquiritin, hesperidin, 6-gingerol, cimicifuga glycoside, 5-O-methylvisaminol glycoside, ferulic acid, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference substances, accurately weigh them, place them in a brown volumetric flask, and add methanol to make a mixed solution containing 90 μg of paeoniflorin, 70 μg of liquiritin, 90 μg of hesperidin, 105 μg of 6-gingerol, 70 μg of cimicifuga glycoside, 80 μg of 5-O-methylvisaminol glycoside, 70 μg of ferulic acid, and 90 μg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside per 1 mL to obtain a mixed reference substance solution.

[0117] S2. High performance liquid chromatography qualitative analysis

[0118] The test solution and the mixed reference solution are respectively taken for high performance liquid chromatography detection to obtain the corresponding high performance liquid chromatogram and generate a reference spectrum (i.e., fingerprint spectrum).

[0119] Among them, the chromatographic conditions for detecting the mixed reference solution are:

[0120] Chromatographic column: C18 (Agilent ZORBAX Eclipse Plus C18, 4.6×250mm Column, 5μm, PN959990-902, SNUSUXA32938);

[0121] Column temperature: 35 °C;

[0122] Flow rate: 1.0 mL / min;

[0123] Detection wavelength: 210nm or 305nm;

[0124] Injection volume: 10 μL;

[0125] The mobile phase was 0.1 wt% phosphoric acid aqueous solution (A)-acetonitrile (B), the elution mode was gradient elution, and the elution program was:

[0126] 0-5min: 95% mobile phase A, 5% mobile phase B;

[0127] 5-7 min: 95% → 88% mobile phase A, 5% → 12% mobile phase B;

[0128] 7-15 min: 88% mobile phase A, 12% mobile phase B;

[0129] 15-23 min: 88% → 85% mobile phase A, 12% → 15% mobile phase B;

[0130] 23-28 min: 85% → 83% mobile phase A, 15% → 17% mobile phase B;

[0131] 28-38 min: 83% → 80% mobile phase A, 17% → 20% mobile phase B;

[0132] 38-43min: 80% mobile phase A, 20% mobile phase B;

[0133] 43-53 min: 80% → 70% mobile phase A, 20% → 30% mobile phase B;

[0134] 53-60min: 70%→60% mobile phase A, 30%→40% mobile phase B;

[0135] 60-65min: 60% mobile phase A, 40% mobile phase B;

[0136] 65-70 min: 60% → 40% mobile phase A, 40% → 60% mobile phase B;

[0137] 70-80min: 40%→0% mobile phase A, 60%→100% mobile phase B.

[0138] At a wavelength of 210 nm, the chromatogram of the mixed reference solution is as follows: Figure 5 The chromatogram of the test solution is as follows: Figure 4 As shown; at a wavelength of 305nm, the chromatogram of the mixed reference solution is as follows Figure 6 The chromatogram of the obtained test solution is shown in Figure 7 shown.

[0139] S3. Fingerprint establishment and similarity results

[0140] Fifteen batches of Dangguiyinzi extract were used to prepare the test solution, and the fingerprint spectrum was tested. The common peaks, similarities and standard fingerprints were obtained through the Chinese medicine chromatographic fingerprint similarity evaluation system (2012, 130723 version). The test results are as follows: Figure 8 to Figure 11 As shown in Tables 1 and 2.

[0141]

[0142]

[0143] S4. Common peak calibration

[0144] Combination Figure 4 , Figure 5 and Fig. 9It can be seen that at a wavelength of 210 nm, a total of 20 common peaks were identified, as shown in Table 3, among which peak 8 was paeoniflorin, peak 9 was cimioglucoside, peak 10 was liquiritigenin, peak 11 was 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, peak 13 was 5-O-methylvisaminol glycoside, peak 14 was hesperidin, and peak 19 was 6-gingerol.

[0145] Table 3 List of common peak results at 210nm wavelength

[0146] Peak Retention time / min Standard fingerprint peak area Retention time RSD (%) 1 5.738 1688.073 0.21 2 9.035 162.733 0.37 3 12.684 757.644 0.62 4 13.391 97.516 0.57 5 14.041 225.878 0.72 6 14.694 338.399 0.92 7 18.521 185.885 0.82 8 24.704 792.509 0.46 9 26.745 620.746 0.22 10 32.010 2236.185 0.49 1 1 33.237 725.048 0.32 12 35.071 384.881 0.84 13 39.564 855.621 0.07 14 42.905 347.872 0.30 15 47.974 374.969 0.77 16 59.315 140.683 0.09 17 67.612 64.412 0.16 18 70.437 98.912 0.08 19 71.327 452.986 0.02 20 73.073 128.285 0.04

[0147] Combination Figure 6 , Figure 7 and Fig.11 It can be seen that 13 common peaks were identified at a wavelength of 305 nm, as shown in Table 4, among which peak 4 was cimicifuga glycoside, peak 5 was ferulic acid, peak 6 was liquiritigenin, peak 7 was 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, and peak 9 was 5-O-methylvisaminol glycoside.

[0148] Table 4 List of common peak results at 305nm wavelength

[0149]

[0150]

[0151] S4. Application of standard fingerprint

[0152] The standard fingerprint of Danggui Yinzi obtained in this example can be used for quality evaluation or control in the whole process of research / development / production / clinical application of Danggui Yinzi.

[0153] Embodiments 3 to 6 Method for constructing the fingerprint of Dangguiyinzi

[0154] Examples 3 to 6 are respectively methods for constructing a fingerprint of Danggui Yinzi. According to the optimal Danggui Yinzi fingerprint construction conditions given in Example 2, the range of conditions for constructing a better Danggui Yinzi fingerprint is further explored to obtain the construction methods in Examples 3 to 6. Among them, the construction methods of Danggui Yinzi fingerprint in Examples 3 to 6 are basically the same as those in Example 2, and the only difference is that some process parameters are different, as shown in Table 5 for details:

[0155] Table 5 List of process parameters in Examples 3 to 6

[0156]

[0157] The contents and results of other parts of Examples 3 to 6 are the same as those of Example 2 and will not be repeated here (since other embodiments of the present invention have been able to fully prove that this method can better construct the fingerprint spectrum of Danggui Yinzi, no additional drawings will be added here).

[0158] Example 7 Fingerprint Methodology Study

[0159] 1. Precision inspection

[0160] The same test solution was taken and HPLC was performed according to the chromatographic conditions of Example 2. The sample was injected six times continuously. The relative retention time and relative peak area of ​​each common peak were calculated under the conditions of 210 nm and 305 nm, respectively. The corresponding RSD values ​​were obtained, and the similarity was obtained by the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012, 130723 version). The results are shown in Figure 12-13 And Table 6 to Table 11. Among them, the standard fingerprint is used as the reference fingerprint.

[0161] Table 6 Precision similarity results list (210nm)

[0162] S1 S2 S3 S4 S5 S6 Comparison fingerprint S1 1.000 0.988 0.988 0.989 0.999 0.993 0.995 S2 0.988 1.000 1.000 1.000 0.988 0.994 0.998 S3 0.988 1.000 1.000 1.000 0.988 0.993 0.998 S4 0.989 1.000 1.000 1.000 0.989 0.993 0.998 S5 0.999 0.988 0.988 0.989 1.000 0.993 0.995 S6 0.993 0.994 0.993 0.993 0.993 1.000 0.997 Comparison fingerprint 0.995 0.998 0.998 0.998 0.995 0.997 1.000

[0163] Table 7 Precision similarity results list (305nm)

[0164] S1 S2 S3 S4 S5 S6 Comparison fingerprint S1 1.000 0.999 0.990 0.992 0.990 0.998 0.998 S2 0.999 1.000 0.991 0.991 0.990 0.999 0.998 S3 0.990 0.991 1.000 0.998 0.999 0.991 0.997 S4 0.992 0.991 0.998 1.000 0.998 0.989 0.997 S5 0.990 0.990 0.999 0.998 1.000 0.989 0.997 S6 0.998 0.999 0.991 0.989 0.989 1.000 0.997 Comparison fingerprint 0.995 0.998 0.997 0.997 0.997 0.997 1.000

[0165] Table 8 Precision relative retention time results (210nm)

[0166]

[0167]

[0168] Table 9 Precision relative retention time results (305nm)

[0169] Relative retention time S1 S2 S3 S4 S5 S6 RSD% 1 0.577 0.576 0.576 0.577 0.576 0.575 0.11 2 0.613 0.613 0.613 0.613 0.613 0.612 0.06 3 0.859 0.859 0.858 0.859 0.859 0.858 0.07 4 0.877 0.878 0.879 0.878 0.879 0.879 0.09 5 1.000 1.000 1.000 1.000 1.000 1.000 0.00 6 1.056 1.057 1.057 1.057 1.057 1.057 0.03 7 1.100 1.100 1.100 1.100 1.100 1.100 0.01 8 1.132 1.133 1.135 1.132 1.134 1.135 0.13 9 1.294 1.296 1.298 1.294 1.298 1.299 0.17 10 1.439 1.441 1.443 1.440 1.442 1.443 0.12 1 1 1.550 1.550 1.551 1.550 1.549 1.550 0.03 12 1.633 1.636 1.638 1.634 1.636 1.638 0.13 13 1.943 1.948 1.951 1.945 1.949 1.951 0.16

[0170] Table 10 Precision relative peak area results (210nm)

[0171] Relative peak area S1 S2 S3 S4 S5 S6 RSD% 1 3.077 3.152 3.163 3.173 3.263 3.161 1.87 2 0.247 0.258 0.259 0.256 0.260 0.261 2.01 3 1.157 1.202 1.221 1.203 1.200 1.222 1.95 4 0.164 0.169 0.165 0.171 0.169 0.170 1.69 5 0.355 0.371 0.366 0.374 0.369 0.369 1.77 6 0.478 0.490 0.497 0.498 0.523 0.500 2.99 7 0.293 0.302 0.300 0.309 0.298 0.298 1.78 8 1.344 1.31 1 1.304 1.345 1.307 1.304 1.52 9 1.000 1.000 1.000 1.000 1.000 1.000 0.00 10 3.534 3.618 3.597 3.697 3.612 3.613 1.44 1 1 1.164 1.189 1.198 1.222 1.202 1.189 1.60 12 0.613 0.618 0.640 0.631 0.640 0.643 2.01 13 1.296 1.353 1.348 1.351 1.354 1.362 1.79 14 0.587 0.585 0.558 0.577 0.555 0.559 2.57 15 0.527 0.535 0.531 0.544 0.531 0.528 1.14 16 0.209 0.212 0.219 0.220 0.218 0.217 2.15 17 0.134 0.136 0.138 0.138 0.136 0.139 1.40 18 0.152 0.156 0.157 0.160 0.156 0.155 1.76 19 0.702 0.733 0.732 0.731 0.735 0.738 1.84 20 0.204 0.205 0.211 0.211 0.212 0.207 1.64

[0172] Table 11 Precision relative peak area results (305nm)

[0173]

[0174]

[0175] In summary, it can be seen that the construction method of the present invention has good precision.

[0176] 2. Repeatability Study

[0177] The same person prepared 6 test solutions in parallel using the same method, and performed HPLC detection according to the chromatographic conditions of Example 2. Under the condition of 210 nm, cimicifuga glycosides were used as the reference peak, and under the condition of 305 nm, ferulic acid was used as the reference peak. The relative retention time and relative peak area of ​​each common peak were calculated to obtain the corresponding RSD value, and the similarity was obtained by the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012, 130723 version). The results are shown in Figure 14-15 and Table 12 to Table 17. Wherein, the standard fingerprint is used as the reference fingerprint.

[0178] Table 12 Repeatability similarity results (210nm)

[0179] S1 S2 S3 S4 S5 S6 Comparison fingerprint S1 1.000 0.953 0.991 0.999 0.991 0.999 0.997 S2 0.953 1.000 0.972 0.956 0.973 0.956 0.973 S3 0.991 0.972 1.000 0.991 1.000 0.991 0.997 S4 0.999 0.956 0.991 1.000 0.991 0.999 0.997 S5 0.991 0.973 1.000 0.991 1.000 0.991 0.998 S6 0.999 0.956 0.991 0.999 0.991 1.000 0.997 Comparison fingerprint 0.997 0.973 0.997 0.997 0.998 0.997 1.000

[0180] Table 13 Repeatability similarity results (305nm)

[0181] S1 S2 S3 S4 S5 S6 Comparison fingerprint S1 1.000 0.999 0.990 0.989 0.991 0.999 0.997 S2 0.999 1.000 0.989 0.989 0.990 0.998 0.997 S3 0.990 0.989 1.000 0.999 0.999 0.990 0.997 S4 0.989 0.989 0.999 1.000 0.999 0.989 0.997 S5 0.991 0.990 0.999 0.999 1.000 0.992 0.998 S6 0.999 0.998 0.990 0.989 0.992 1.000 0.997 Comparison fingerprint 0.997 0.997 0.997 0.997 0.998 0.997 1.000

[0182] Table 14 Reproducibility relative retention time results (210nm)

[0183]

[0184]

[0185] Table 15 Reproducibility relative retention time results (305nm)

[0186] Relative retention time S1 S2 S3 S4 S5 S6 RSD% 1 0.575 0.575 0.576 0.575 0.575 0.576 0.09 2 0.612 0.612 0.612 0.612 0.611 0.612 0.04 3 0.858 0.858 0.859 0.858 0.857 0.859 0.07 4 0.878 0.879 0.879 0.879 0.879 0.877 0.10 5 1.000 1.000 1.000 1.000 1.000 1.000 0.00 6 1.057 1.057 1.057 1.057 1.058 1.057 0.03 7 1.100 1.100 1.099 1.099 1.100 1.100 0.03 8 1.134 1.136 1.134 1.135 1.136 1.133 0.10 9 1.297 1.298 1.297 1.299 1.300 1.296 0.12 10 1.442 1.444 1.442 1.443 1.445 1.441 0.10 11 1.550 1.551 1.550 1.549 1.553 1.551 0.08 12 1.637 1.639 1.637 1.638 1.640 1.636 0.11 13 1.949 1.953 1.950 1.952 1.954 1.947 0.13

[0187] Table 16 Repeatability relative peak area results (210nm)

[0188]

[0189]

[0190] Table 17 Repeatability relative peak area results (305nm)

[0191] Relative peak area S1 S2 S3 S4 S5 S6 RSD% 1 0.363 0.366 0.362 0.364 0.365 0.359 0.68 2 0.456 0.460 0.458 0.458 0.459 0.455 0.35 3 0.118 0.113 0.119 0.113 0.114 0.119 2.58 4 0.688 0.679 0.689 0.679 0.681 0.691 0.77 5 1.000 1.000 1.000 1.000 1.000 1.000 0.00 6 1.052 1.049 1.054 1.046 1.050 1.057 0.35 7 2.136 2.133 2.136 2.130 2.133 2.131 0.12 8 0.120 0.119 0.121 0.119 0.120 0.121 0.83 9 1.067 1.096 1.068 1.092 1.098 1.104 1.45 10 0.222 0.227 0.221 0.227 0.224 0.213 2.31 11 0.188 0.188 0.191 0.187 0.189 0.197 2.02 12 0.283 0.283 0.282 0.283 0.280 0.278 0.64 13 0.088 0.087 0.088 0.088 0.087 0.087 0.43

[0192] In summary, it can be seen that the construction method of the present invention has good repeatability.

[0193] 3. Stability inspection

[0194] The same test solution was injected at 0h, 2h, 4h, 8h, 12h, and 24h, respectively, and HPLC detection was performed according to the chromatographic conditions of Example 2. Under the condition of 210nm, cimicifuga glycosides were used as the reference peak, and under the condition of 305nm, ferulic acid was used as the reference peak. The relative retention time and relative peak area of ​​each common peak were calculated to obtain the corresponding RSD value, and the similarity was obtained by the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012, 130723 version). The results are shown in Figure 16-17 and Table 18 to Table 23. Among them, the standard fingerprint is used as the reference fingerprint.

[0195] Table 18 Stability similarity results (210nm)

[0196]

[0197]

[0198] Table 19 Stability similarity results (305nm)

[0199] S1 S2 S3 S4 S5 S6 Comparison fingerprint S1 1.000 1.000 0.998 0.991 0.990 0.989 0.997 S2 1.000 1.000 0.998 0.990 0.990 0.989 0.997 S3 0.998 0.998 1.000 0.990 0.991 0.988 0.997 S4 0.991 0.990 0.990 1.000 1.000 0.997 0.998 S5 0.990 0.990 0.991 1.000 1.000 0.997 0.998 S6 0.989 0.989 0.988 0.997 0.997 1.000 0.996 Comparison fingerprint 0.997 0.997 0.997 0.998 0.998 0.996 1.000

[0200] Table 20 Stability relative retention time results (210nm)

[0201] Relative retention time S1 S2 S3 S4 S5 S6 RSD% 1 0.217 0.217 0.218 0.219 0.219 0.217 0.41 2 0.339 0.339 0.340 0.341 0.341 0.338 0.26 3 0.476 0.476 0.477 0.477 0.478 0.476 0.13 4 0.505 0.505 0.506 0.506 0.506 0.505 0.12 5 0.529 0.528 0.529 0.529 0.530 0.528 0.10 6 0.555 0.555 0.556 0.557 0.558 0.555 0.22 7 0.698 0.697 0.699 0.699 0.700 0.697 0.19 8 0.927 0.927 0.928 0.928 0.928 0.927 0.08 9 1.000 1.000 1.000 1.000 1.000 1.000 0.00 10 1.203 1.203 1.204 1.206 1.206 1.203 0.11 11 1.252 1.252 1.253 1.255 1.256 1.251 0.16 12 1.319 1.319 1.320 1.321 1.321 1.319 0.08 13 1.476 1.478 1.476 1.476 1.476 1.478 0.07 14 1.607 1.609 1.609 1.610 1.610 1.608 0.06 15 1.803 1.803 1.805 1.807 1.807 1.803 0.12 16 2.218 2.218 2.218 2.217 2.217 2.220 0.04 17 2.529 2.529 2.528 2.528 2.527 2.532 0.06 18 2.634 2.634 2.634 2.633 2.633 2.637 0.06 19 2.665 2.665 2.664 2.663 2.662 2.668 0.09 20 2.732 2.732 2.731 2.730 2.729 2.735 0.08

[0202] Table 21 Stability relative retention time results (305nm)

[0203]

[0204]

[0205] Table 22 Stability relative peak area results (210nm)

[0206] Relative peak area S1 S2 S3 S4 S5 S6 RSD% 1 3.061 3.096 3.047 3.068 3.056 3.040 0.64 2 0.254 0.257 0.252 0.253 0.251 0.258 1.03 3 1.179 1.195 1.179 1.185 1.180 1.186 0.54 4 0.166 0.170 0.168 0.173 0.171 0.168 1.40 5 0.370 0.371 0.367 0.372 0.373 0.370 0.59 6 0.494 0.493 0.490 0.488 0.487 0.489 0.55 7 0.296 0.297 0.302 0.302 0.297 0.294 1.07 8 1.294 1.310 1.321 1.400 1.395 1.289 3.74 9 1.000 1.000 1.000 1.000 1.000 1.000 0.00 10 3.560 3.597 3.648 3.667 3.631 3.582 1.14 11 1.172 1.196 1.213 1.208 1.213 1.174 1.58 12 0.633 0.640 0.621 0.642 0.616 0.637 1.69 13 1.340 1.360 1.335 1.341 1.333 1.354 0.80 14 0.590 0.550 0.565 0.544 0.520 0.551 4.21 15 0.526 0.533 0.531 0.539 0.538 0.520 1.35 16 0.211 0.214 0.212 0.211 0.205 0.206 1.74 17 0.135 0.137 0.136 0.136 0.135 0.134 0.88 18 0.155 0.157 0.162 0.160 0.160 0.154 2.15 19 0.725 0.735 0.722 0.729 0.725 0.731 0.66 20 0.210 0.206 0.209 0.210 0.209 0.206 0.89

[0207] Table 23 Stability relative peak area results (305nm)

[0208] Relative peak area S1 S2 S3 S4 S5 S6 RSD% 1 0.360 0.361 0.359 0.354 0.351 0.364 1.29 2 0.452 0.454 0.422 0.422 0.422 0.454 3.92 3 0.120 0.120 0.120 0.117 0.114 0.120 2.17 4 0.692 0.691 0.694 0.695 0.700 0.691 0.52 5 1.000 1.000 1.000 1.000 1.000 1.000 0.00 6 1.056 1.053 1.060 1.063 1.066 1.054 0.48 7 2.117 2.120 2.119 2.114 2.116 2.114 0.11 8 0.121 0.121 0.122 0.122 0.129 0.121 2.67 9 1.069 1.073 1.111 1.114 1.116 1.102 1.93 10 0.209 0.210 0.206 0.228 0.220 0.215 3.70 11 0.190 0.188 0.196 0.199 0.200 0.186 3.00 12 0.282 0.283 0.279 0.278 0.276 0.286 1.26 13 0.085 0.086 0.085 0.085 0.078 0.084 3.56

[0209] In summary, it can be seen that the construction method of the present invention has good stability.

[0210] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint of Dangguiyinzi, characterized in that: The construction method comprises the following steps: S1. Prepare the test solution by taking Dangguiyinzi; S2, taking the test solution and performing high performance liquid chromatography to obtain the fingerprint of Dangguiyinzi; During the high performance liquid chromatography detection process, 0.08-0.12 wt % phosphoric acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and elution is performed in a gradient elution manner; The elution procedure is: The mobile phase is, the elution mode is gradient elution, and the elution program is: 0-5min: 95% mobile phase A, 5% mobile phase B; 5-7 min: 95% → 88% mobile phase A, 5% → 12% mobile phase B; 7-15 min: 88% mobile phase A, 12% mobile phase B; 15-23 min: 88% → 85% mobile phase A, 12% → 15% mobile phase B; 23-28 min: 85% → 83% mobile phase A, 15% → 17% mobile phase B; 28-38 min: 83% → 80% mobile phase A, 17% → 20% mobile phase B; 38-43min: 80% mobile phase A, 20% mobile phase B; 43-53min: 80%→70% mobile phase A, 20%→30% mobile phase B; 53-60min: 70%→60% mobile phase A, 30%→40% mobile phase B; 60-65min: 60% mobile phase A, 40% mobile phase B; 65-70 min: 60% → 40% mobile phase A, 40% → 60% mobile phase B; 70-80min: 40%→0% mobile phase A, 60%→100% mobile phase B.

2. The method for constructing the fingerprint of Dangguiyinzi according to claim 1, characterized in that: In the high performance liquid chromatography detection process, the chromatographic column is an Agilent ZORBAX Eclipse Plus C18 column.

3. The method for constructing the fingerprint of Dangguiyinzi according to claim 1 or 2, characterized in that: During the high performance liquid chromatography detection process, the detection wavelengths are 205-215 nm and 300-310 nm.

4. The method for constructing the fingerprint of Dangguiyinzi according to claim 1 or 2, characterized in that: The test solution is prepared by adding methanol aqueous solution to Danggui Yinzi, ultrasonically treating the solution, and filtering the solution.

5. The method for constructing the fingerprint of Dangguiyinzi according to claim 4, characterized in that: During the preparation of the test solution, the concentration of the methanol-water solution was 65-75 vol%.

6. The method for constructing the fingerprint of Dangguiyinzi according to claim 4, characterized in that: During the preparation of the test solution, the volume ratio of Danggui Yinzi to the test solution was 0.8-1.2:

10.

7. The method for constructing the fingerprint of Dangguiyinzi according to claim 1, 2, 5 or 6, characterized in that: The flow rate of the high performance liquid chromatography detection is 0.8-1.2 mL / min, the column temperature is 30-40° C., and the injection volume is 5-20 μL.

8. The method for constructing the fingerprint of Dangguiyinzi according to claim 1, 2, 5 or 6, characterized in that: The construction method also includes preparing a mixed reference solution using paeoniflorin, ammonium glycyrrhizinate, liquiritin, hesperidin, 6-gingerol, cimicifuga glycoside, 5-O-methylvisaminol glycoside, ferulic acid, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, and performing high performance liquid chromatography detection.

9. An application of the standard fingerprint of Danggui Yinzi obtained by the construction method described in any one of claims 1 to 8 in the quality evaluation or control of the whole process of research / development / production / clinical application of Danggui Yinzi.

10. The use according to claim 9, characterized in that: At a wavelength of 210 nm, a total of 20 common peaks were calibrated in the standard fingerprint of Danggui Yinzi, of which peak 8 was paeoniflorin, peak 9 was cimicifuga glycoside, peak 10 was liquiritigenin, peak 11 was 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, peak 13 was 5-O-methylvisaminol glycoside, peak 14 was hesperidin, and peak 19 was 6-gingerol; At a wavelength of 305 nm, 13 common peaks were calibrated in the standard fingerprint of Danggui Yinzi, of which peak 4 was cimicifuga glycoside, peak 5 was ferulic acid, peak 6 was liquiritigenin, peak 7 was 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, and peak 9 was 5-O-methylvisaminol glycoside.

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