Construction method and application of fingerprint spectrum of Dangui Yinzi

Abstract

The application provides a construction method and application of a fingerprint spectrum of Danggui Yinzi, and belongs to the technical field of drug detection. The construction method is as follows: first, a test sample solution of Danggui Yinzi is prepared; then, 0.08-0.12 wt% aqueous phosphoric acid (A)-acetonitrile (B) is used as a mobile phase, high performance liquid chromatography detection is performed, the fingerprint spectrum of Danggui Yinzi is obtained, and a standard fingerprint spectrum is established, which is used for quality evaluation or control in the whole process of research / development / production / clinical application of Danggui Yinzi. The construction method of the fingerprint spectrum of Danggui Yinzi can detect the main medicinal material components in Danggui Yinzi through high performance liquid chromatography, so as to more comprehensively reflect the quality status of Danggui Yinzi.

Description

TECHNICAL FIELD

[0001] The present application relates to drug detection technology, in particular to a construction method and application of Angelica flos drink fingerprint. BACKGROUND

[0002] Angelica flos drink is out of "revised strict Ji Sheng Fang", which is a compound preparation prepared from eleven kinds of traditional Chinese medicines, such as Angelica flos, white peony root, Chuanxiong, Rehmannia glutinosa, Tribulus terrestris, windproof, schizonepeta tectorium, radix polygoni multiflori, Astragalus, licorice and ginger. It is suitable for blood stasis, internal wind-heat, skin scabies, or swelling or itching, or pus water immersion, or red rash. According to its composition, four things, radix polygoni multiflori nourish yin and blood, which is suitable for blood deficiency and dryness, so for various skin diseases for a long time, Yin blood, or swelling or itching, can consider this prescription.

[0003] Because the prepared Angelica flos drink loses the morphological characteristics of the original decoction pieces, the qualitative identification and quantitative analysis of the simple index component can not reflect the quality of the product. As a standard reference for measuring whether the classic Chinese medicine is basically consistent with the clinical decoction, the quality should be strengthened in the specificity identification and multi-component, overall quality control.

[0004] The traditional Chinese medicine preparation spectrum refers to the spectrum of traditional Chinese medicine preparation sample after appropriate treatment, using appropriate analysis method, which can reflect the multi-component information and embody the quality characteristics. The characteristic spectrum of traditional Chinese medicine preparation has important significance for identifying the key quality attributes of traditional Chinese medicine preparation, studying the value transmission, evaluating the uniformity and stability of traditional Chinese medicine preparation quality, and improving the overall quality control level of traditional Chinese medicine preparation.

[0005] Therefore, by establishing the characteristic spectrum, the overall quality of Angelica flos drink can be comprehensively controlled. SUMMARY

[0006] In view of the above problems, the present application provides a construction method and application of Angelica flos drink fingerprint.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0008] A construction method of Angelica flos drink fingerprint, the construction method comprises the following steps:

[0009] S1, taking Angelica flos drink to prepare test solution;

[0010] S2, taking the test solution to carry out high performance liquid chromatography detection, and obtaining the Angelica flos drink fingerprint;

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

[0012] In which, the elution program is:

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

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

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

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

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

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

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

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

[0021] 43-53min: 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] 65-70min: 60%→40% mobile phase A, 40%→60% mobile phase B;

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

[0026] Further, in the high performance liquid chromatography detection process, the chromatographic column is an Agilent ZORBAX Eclipse Plus C18 column.

[0027] Further, in the high performance liquid chromatography detection process, the detection wavelength is 205-215nm, 300-310nm.

[0028] Further, the test sample solution is prepared by taking the Angelica drink and methanol aqueous solution, ultrasonic treatment, and filtration.

[0029] Further, in the preparation of the test sample solution, the concentration of the methanol aqueous solution is 65-75vol%.

[0030] Further, during the preparation of the test sample solution, the volume ratio of the Angelica Decoction to the test sample solution is 0.8-1.2:10.

[0031] Further, the flow rate of the high performance liquid chromatography detection is 0.8-1.2 mL / min, the column temperature is 30-40 DEG C, and the injection amount is 5-20 mu L.

[0032] Further, the construction method further comprises preparing a mixed control solution by using paeoniflorin, ammonium glycyrrhizinate, glycyrrhizin, hesperidin, 6-gingerol, epimedoside, 5-O-methylvisamminol, ferulic acid and 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside, and performing high performance liquid chromatography detection.

[0033] The application of the standard fingerprint of the Angelica Decoction obtained by the construction method in the quality evaluation or control of the Angelica Decoction in the whole process of research / development / production / clinical application of the Angelica Decoction.

[0034] Further, under the wavelength of 210 nm, 20 common peaks are marked in the standard fingerprint of the Angelica Decoction, wherein the peak No. 8 is paeoniflorin, the peak No. 9 is epimedoside, the peak No. 10 is glycyrrhizin, the peak No. 11 is 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside, the peak No. 13 is 5-O-methylvisamminol, the peak No. 14 is hesperidin, and the peak No. 19 is 6-gingerol.

[0035] Under the wavelength of 305 nm, 13 common peaks are marked in the standard fingerprint of the Angelica Decoction, wherein the peak No. 4 is epimedoside, the peak No. 5 is ferulic acid, the peak No. 6 is glycyrrhizin, the peak No. 7 is 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside, and the peak No. 9 is 5-O-methylvisamminol.

[0036] The construction method and application of the Angelica Decoction fingerprint have the following beneficial effects:

[0037] The construction method of the Angelica Decoction fingerprint can detect the main medicinal material components in the Angelica Decoction by high performance liquid chromatography, so that the quality status of the Angelica Decoction can be more comprehensively reflected.

[0038] The construction method of the Angelica Decoction fingerprint combines the characteristics of different physicochemical properties of different effective components in the Angelica Decoction, adjusts the elution conditions, and obtains the standard fingerprint of the Angelica Decoction with good separation degree, relatively stable baseline, more peaks and better reflecting the quality of the Angelica Decoction.

[0039] The construction method of the Angelica Decoction fingerprint adjusts the mobile phase, and obtains the standard fingerprint of the Angelica Decoction with a more stable baseline, better peak shape and separation effect.

[0040] The application can separate different effective components in Angelica drink by adjusting the chromatographic column, and the response value and separation degree of the chromatographic peak are better;

[0041] The application can extract the medicinal material components in Angelica drink more fully by adjusting the preparation method of the test sample solution, and the concentrations of the medicinal material components in the obtained test sample solution can be separated and detected under the same high performance liquid chromatography condition, and the chromatogram is good;

[0042] The application effectively optimizes the separation condition by selecting appropriate process conditions, thereby improving the linearity, precision, stability, repeatability and specificity of Angelica drink;

[0043] The standard fingerprint spectrum of Angelica drink obtained by the application can provide effective guarantee for the quality monitoring of Angelica drink in the whole process of research, development, production and clinical application;

[0044] The presence or absence of common characteristic peaks and characteristics in the standard fingerprint spectrum of Angelica drink obtained by the application can comprehensively monitor the product quality of Angelica drink, and through the comparison of the similarity degree of the chromatographic fingerprint characteristics, the advantages and disadvantages of Angelica drink, the stability and uniformity are evaluated, which makes up for the shortcomings of the current quality control method, and at the same time, the stability of the production process of Angelica drink can be monitored, so as to ensure the stability, uniformity and controllability of the quality;

[0045] The standard fingerprint spectrum of Angelica drink obtained by the application improves the quality monitoring standard of Angelica drink finished product and semi-finished product, effectively prevents the occurrence of product counterfeiting events, and ensures the normal production and circulation order of Angelica drink; on the basis of the application, the correlation between the fingerprint spectrum information and the pharmacodynamic activity information can be studied, so as to further clarify the correlation between the internal chemical components of Angelica drink and the curative effect of the preparation. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 is the chromatogram of Angelica drink detected by different chromatographic columns in the chromatographic column screening process of the embodiment 1 of the application; wherein, A is the chromatogram of Angelica drink detected by Waters Sunfire C18 column, B is the chromatogram of Angelica drink detected by Agilent ZORBAX Eclipes Plus C18 column, and C is the chromatogram of Angelica drink detected by Phenomenex Luna Su C18 column;

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

[0048] Figure 3 is the chromatogram of Angelica drink detected by different elution programs in the elution program screening process of Example 1 of the present application; wherein, the chromatogram of Angelica drink detected by elution program one is shown in A, and the chromatogram of Angelica drink detected by elution program two is shown in B;

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

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

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

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

[0053] Figure 8 is the chromatogram of fifteen batches of Angelica drink detected at 210nm wavelength in Example 2 of the present application;

[0054] Figure 9 is the standard fingerprint chromatogram at 210nm wavelength in Example 2 of the present application;

[0055] Figure 10 is the chromatogram of fifteen batches of Angelica drink detected at 305nm wavelength in Example 2 of the present application;

[0056] Figure 11 is the standard fingerprint chromatogram at 305nm wavelength in Example 2 of the present application;

[0057] Figure 12 is the precision investigation fingerprint chromatogram result at 210nm wavelength in Example 7 of the present application;

[0058] Figure 13 is the precision investigation fingerprint chromatogram result at 305nm wavelength in Example 7 of the present application;

[0059] Figure 14 is the repeatability investigation fingerprint result of the present application example 7 at 210 nm wavelength;

[0060] Figure 15 is the repeatability investigation fingerprint result of the present application example 7 at 305 nm wavelength;

[0061] Figure 16 is the stability investigation fingerprint result of the present application example 7 at 210 nm wavelength;

[0062] Figure 17 is the stability investigation fingerprint result of the present application example 7 at 305 nm wavelength. DETAILED DESCRIPTION

[0063] The technical solutions in the embodiments of the present application are described clearly and completely below. In the following description, a lot of specific details are set forth in order to fully understand the present application, but the present application can also be implemented in other ways different from those described herein, and a person skilled in the art can make similar extensions without departing from the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0064] Example 1 Screening of a construction method of Angelica Decoction fingerprint

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

[0066] The prescription is: Angelica 5.84g, White Peony Root 5.84g, Chuanxiong 5.84g, Rehmannia 5.84g, Tribulus terrestris 5.84g, Saposhnikovia divaricata 5.84g, Schizonepeta 5.84g, Radix Polygoni Multiflori 2.92g, Astragalus 2.92g, Fried Licorice 2.92g and Ginger 15g.

[0067] The whole medicinal material is crushed / pulverized into the coarsest powder (5mm).

[0068] Take 16.52g of the coarsest powder of medicinal materials (Angelica, White Peony Root, Chuanxiong, Rehmannia, White Tribulus terrestris, Saposhnikovia divaricata, Schizonepeta, each 1.95g, Radix Polygoni Multiflori, Astragalus, Fried Licorice, each 0.97g), add 450mL of water, 5g of ginger, boil with strong fire, simmer with weak fire to 80% of the original volume (240mL), filter out the residue while hot, and the obtained medicinal liquid is concentrated to the extract with a density of 1.15-1.22 at 60°C under reduced pressure, to obtain Angelica Decoction extract.

[0069] In this embodiment, Angelica Decoction extract is used as the sample to be tested, and the preparation method of Angelica Decoction extract is as follows:

[0070] 1) Preparation method of test solution

[0071] Precisely pipette Angelica Decoction 1 mL (i.e. v = 1 mL) into a 10 mL volumetric flask (i.e. V = 10 mL), dilute to the calibration mark with 70 vol% methanol aqueous solution, shake well, ultrasonically treat for 20 min, filter, and take the filtrate to obtain the test sample solution.

[0072] 2) Column screening

[0073] Precisely pipette 10 μL of the test sample solution into a high performance liquid chromatograph, and respectively investigate the chromatograms when a Waters Sunfire C18 column, an Agilent ZORBAX Eclipes Plus C18 column, and a Phenomenex Luna Su C18 column are used, and the results are shown in FIGS. 1-3. Figure 1

[0074] In which, other conditions of the high performance liquid chromatograph are as follows:

[0075] Column temperature: 35℃;

[0076] Flow rate: 1.0 mL / min;

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

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

[0079] 0-5 min: 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] 38-43 min: 80% mobile phase A, 20% mobile phase B;

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

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

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

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

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

[0091] By Figure 1 It can be seen that the response value and resolution of the chromatographic peaks are better using Agilent ZORBAX Eclipes Plus C18 column, so the column is selected to continue to investigate the construction method.

[0092] 3) Mobile phase screening

[0093] C18 (Agilent ZORBAX Eclipse Plus C18, 4.6x250mm Column, 5μm, P.N.959990-902, S.N.USUXA32938) is used as the chromatographic column, and the chromatographic conditions in step 2) chromatographic column screening (i.e. only the type of mobile phase is changed, and other analysis parameters are unchanged) are further investigated to investigate the influence of 0.1wt% formic acid aqueous solution (A)-acetonitrile (B), 0.1wt% acetic acid aqueous solution (A)-acetonitrile (B), 0.1wt% phosphoric acid aqueous solution (A)-acetonitrile (B) on the resolution of the fingerprint, and the results are shown in Figure 2 . By Figure 2 It can be seen that the baseline is more stable, and the peak shape and separation effect are better when 0.1wt% phosphoric acid aqueous solution (A)-acetonitrile (B) is used as the mobile phase, so 0.1wt% phosphoric acid aqueous solution (A)-acetonitrile (B) is finally selected as the mobile phase.

[0094] 4) Elution program screening

[0095] 0.1wt% phosphoric acid aqueous solution (A)-acetonitrile (B) is used as the mobile phase, and the chromatographic conditions in step 3) mobile phase screening (i.e. only the type of mobile phase is changed, and other analysis parameters are unchanged) are further investigated to investigate the separation effect of different elution programs under the condition that the detection wavelength is 210nm, and the results are shown in Figure 3 .

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

[0097] Elution program one:

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

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

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

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

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

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

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

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

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

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

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

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

[0110] The elution procedure two is the elution procedure in the step 1) chromatographic column screening.

[0111] From Figure 3 It can be seen that the chromatographic peaks are densely distributed under the elution procedure one (i.e. the proportion of the mobile phase with high polarity), and the elution procedure two is obtained by optimizing the elution procedure one, wherein the chromatographic peaks are uniformly distributed and the resolution is good, and the elution procedure two is determined as the final elution procedure.

[0112] Example 2: a method for constructing a fingerprint of Angelica drink, a standard fingerprint of Angelica drink constructed by the method and application

[0113] In this example, the extract of Angelica drink is used as the sample of Angelica drink, and the method for constructing the fingerprint of Angelica drink is as follows:

[0114] S1, solution preparation

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

[0116] S12. Accurately weigh appropriate amounts of paeoniflorin, glycyrrhizin, hesperidin, 6-gingerol, cimicifugain, 5-O-methylvisamiloside, ferulic acid, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference standards, place them in a brown volumetric flask, and add methanol to prepare a mixed solution containing 90 μg paeoniflorin, 70 μg glycyrrhizin, 90 μg hesperidin, 105 μg 6-gingerol, 70 μg cimicifugain, 80 μg 5-O-methylvisamiloside, 70 μg ferulic acid, and 90 μg 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside per mL.

[0117] S2, Qualitative analysis by high performance liquid chromatography

[0118] The test solution and the mixed reference solution were subjected to high performance liquid chromatography (HPLC) to obtain the corresponding HPLC chromatograms and generate a reference chromatogram (i.e., fingerprint chromatogram).

[0119] The chromatographic conditions for detecting the mixed reference solution are as follows:

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

[0121] Column temperature: 35℃;

[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), and the elution method was gradient elution. The elution program was as follows:

[0126] 0–5 min: 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] 38-43 min: 80% mobile phase A, 20% mobile phase B;

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

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

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

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

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

[0138] The chromatogram of the mixed control solution is shown in Figure 5 , and the chromatogram of the test solution is shown in Figure 4 ; at a wavelength of 305 nm, the chromatogram of the mixed control solution is shown in Figure 6 , and the chromatogram of the test solution is shown in Figure 7 .

[0139] S3, Fingerprint Establishment and Similarity Results

[0140] Fifteen batches of Angelica decoction extract were used to prepare test solution, and the fingerprint detection was performed. The common peaks, similarity and standard fingerprint were obtained by Chinese medicine chromatographic fingerprint similarity evaluation system (2012, 130723 version), and the detection results are shown in Figure 8-11 and Tables 1-2.

[0141]

[0142]

[0143] S4, Common Peak Calibration

[0144] Combined with Figure 4 , Figure 5 and Figure 9It can be seen that 20 common peaks are co-designated at 210 nm wavelength, see Table 3 for details, wherein peak No. 8 is paeoniflorin, peak No. 9 is cimifugin, peak No. 10 is glycyrrhizin, peak No. 11 is 2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-D-glucoside, peak No. 13 is 5-O-methylvisamminol, peak No. 14 is hesperidin, and peak No. 19 is 6-shogaol.

[0145] Table 3: Common peak results at 210 nm wavelength

[0146] Peak number 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] In combination Figure 6 , Figure 7 and Figure 11 It can be seen that 13 common peaks are co-designated at 305 nm wavelength, see Table 4 for details, wherein peak No. 4 is cimifugin, peak No. 5 is ferulic acid, peak No. 6 is glycyrrhizin, peak No. 7 is 2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-D-glucoside, and peak No. 9 is 5-O-methylvisamminol.

[0148] Table 4: Common peak results at 305 nm wavelength

[0149]

[0150]

[0151] S4, Application of standard fingerprint

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

[0153] Embodiments 3-6 are methods for constructing a fingerprint of Angelica drink, which further explore the range of conditions that can achieve a better fingerprint of Angelica drink, based on the optimal fingerprint construction conditions given in embodiment 2. The fingerprint construction methods in embodiments 3-6 are basically the same as in embodiment 2, except for the differences in some process parameters, as shown in Table 5:

[0154] Table 5: List of process parameters in embodiments 3-6

[0155] Table 5: List of process parameters in embodiments 3-6

[0156]

[0157] The contents and results of other parts of Examples 3-6 are the same as those of Example 2, which will not be repeated here (as other embodiments of the present application have been able to sufficiently prove that the method can better construct the fingerprint of Angelica drink, additional figures will not be added here).

[0158] Example 7: Fingerprint methodology investigation

[0159] I. Precision investigation

[0160] The same test solution was taken, and high performance liquid chromatography detection was carried out according to the chromatographic conditions of Example 2. Angelica glycoside was taken as the reference peak at 210 nm, and ferulic acid was taken as the reference peak at 305 nm. The relative retention time and relative peak area of each common peak were calculated, the corresponding RSD value was obtained, and the similarity was obtained through the traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012, version 130723). The results are shown in Table 6-Table 11. Among them, the standard fingerprint was used as the control fingerprint. Figure 12-13

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

[0162] S1 S2 S3 S4 S5 S6 Control 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 Control fingerprint 0.995 0.998 0.998 0.998 0.995 0.997 1.000

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

[0164] S1 S2 S3 S4 S5 S6 Control 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 Control fingerprint 0.995 0.998 0.997 0.997 0.997 0.997 1.000

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

[0166]

[0167]

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

[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 (210 nm)

[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 (305 nm)

[0173]

[0174]

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

[0176] II. Reproducibility Investigation

[0177] The same person used the same method to prepare 6 test solution samples in parallel, and the high performance liquid chromatography was detected according to the chromatographic conditions of Example 2, and the relative retention time and relative peak area of each common peak were calculated under the condition of 210 nm with the reference peak of rhizomavladimirin and under the condition of 305 nm with the reference peak of ferulic acid, the corresponding RSD value was obtained, and the similarity was obtained through the traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012, version 130723), and the results are shown in Table 12-Table 17. Figure 14-15 and Table 12-Table 17. Among them, the standard fingerprint is used as the control fingerprint.

[0178] Table 12 Reproducibility Similarity Results Table (210 nm)

[0179] S1 S2 S3 S4 S5 S6 Control 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 Control fingerprint 0.997 0.973 0.997 0.997 0.998 0.997 1.000

[0180] Table 13 Reproducibility Similarity Results Table (305 nm)

[0181] S1 S2 S3 S4 S5 S6 Control 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 Control fingerprint 0.997 0.997 0.997 0.997 0.998 0.997 1.000

[0182] Table 14 Reproducibility Relative Retention Time Results Table (210 nm)

[0183]

[0184]

[0185] Table 15 Reproducibility Relative Retention Time Results Table (305 nm)

[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 Reproducibility Relative Peak Area Results Table (210 nm)

[0188]

[0189]

[0190] Table 17 Reproducibility Relative Peak Area Results Table (305 nm)

[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 application has good reproducibility.

[0193] III. Stability Investigation

[0194] The same test sample solution was injected at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h respectively, and was detected by high performance liquid chromatography according to the chromatographic conditions of Example 2, and the relative retention time and relative peak area of each common peak were calculated under the condition that the peak of Cimicifugoside was used as a reference peak at 210 nm and the peak of Ferulic acid was used as a reference peak at 305 nm, and the corresponding RSD value was obtained, and the similarity was obtained through the traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012, version 130723), and the results are shown in Tables 18-23. Figure 16-17 and Tables 18-23. Among them, the standard fingerprint is used as the control fingerprint.

[0195] Table 18 Stability Similarity Result List (210 nm)

[0196]

[0197]

[0198] Table 19 Stability Similarity Result List (305 nm)

[0199] S1 S2 S3 S4 S5 S6 Control 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 Control fingerprint 0.997 0.997 0.997 0.998 0.998 0.996 1.000

[0200] Table 20 Stability Relative Retention Time Result List (210 nm)

[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 Result List (305 nm)

[0203]

[0204]

[0205] Table 22 Stability Relative Peak Area Result List (210 nm)

[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 Result List (305 nm)

[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] From the above, it can be seen that the stability of the construction method of the present application is good.

[0210] Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

Claims

1. A method for constructing a fingerprint of Angelica sinensis Diels, characterized in that, The construction method comprises the following steps: S1, taking Angelica Decoction and a methanol aqueous solution with a concentration of 65-75 vol%, performing ultrasonic treatment, and filtering to prepare a test sample solution; S2, taking the test sample solution to perform high performance liquid chromatography detection to obtain the Angelica Decoction fingerprint; In the high performance liquid chromatography detection process, the chromatographic column is an Agilent ZORBAX Eclipse Plus C18 column; the detection wavelength is 205-215 nm and 300-310 nm; 0.08-0.12 wt% phosphoric acid aqueous solution is used as a mobile phase A, and acetonitrile is used as a mobile phase B to perform elution in a gradient elution mode; The elution program is as follows: 0-5 min: 95% mobile phase A and 5% mobile phase B; 5-7 min: 95%→88% mobile phase A and 5%→12% mobile phase B; 7-15 min: 88% mobile phase A and 12% mobile phase B; 15-23 min: 88%→85% mobile phase A and 12%→15% mobile phase B; 23-28 min: 85%→83% mobile phase A and 15%→17% mobile phase B; 28-38 min: 83%→80% mobile phase A and 17%→20% mobile phase B; 38-43 min: 80% mobile phase A and 20% mobile phase B; 43-53 min: 80%→70% mobile phase A and 20%→30% mobile phase B; 53-60 min: 70%→60% mobile phase A and 30%→40% mobile phase B; 60-65 min: 60% mobile phase A and 40% mobile phase B; 65-70 min: 60%→40% mobile phase A and 40%→60% mobile phase B; 70-80 min: 40%→0% mobile phase A and 60%→100% mobile phase B; In the construction method, a mixed control sample solution is prepared by using paeoniflorin, ammonium glycyrrhizinate, liquiritin, hesperidin, 6-gingerol, epimedoside, 5-O-methylvisamminol, ferulic acid, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, and high performance liquid chromatography detection is performed.

2. The method for constructing the fingerprint spectrum of Angelica sinensis decoction according to claim 1, characterized in that, In the preparation of the test sample solution, the volume ratio of Angelica Decoction to the test sample solution is 0.8-1.2:

10.

3. The method for constructing the fingerprint spectrum of Angelica sinensis decoction according to claim 1 or 2, 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 amount is 5-20 μL.

4. Application of a standard fingerprint of Angelica Decoction obtained by the construction method in claim 1-3 in the whole process of research / development / production / clinical application of the Angelica Decoction for quality evaluation or control.

5. Use according to claim 4, characterized in that, In the standard fingerprint of the Angelica Decoction, 20 common peaks are labeled, wherein peak No. 8 is paeoniflorin, peak No. 9 is epimedoside, peak No. 10 is liquiritin, peak No. 11 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, peak No. 13 is 5-O-methylvisamminol, peak No. 14 is hesperidin, and peak No. 19 is 6-gingerol; The standard fingerprint spectrum of the Angelicae Gomme et Radix et Rhizoma Decoction has 13 common peaks at 305 nm wavelength, wherein peak 4 is threoceine, peak 5 is ferulic acid, peak 6 is glycyrrhizin, peak 7 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, and peak 9 is 5-O-methylvisamminol glycoside.

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