Establishment method of fingerprint spectrum of Wenxiao Jieyu granules and fingerprint spectrum thereof

Abstract

The application provides a method for establishing a fingerprint spectrum of Wenshoujieyu granules and the fingerprint spectrum, and relates to the technical field of traditional Chinese medicine preparation detection. The method for establishing the fingerprint spectrum adopts high performance liquid chromatography to detect the Wenshoujieyu granules, and in the method, the chromatographic conditions are as follows: Agilent 5TC-C 18 (2) (4.6mm*250nm, 5um) is used as a chromatographic column; acetonitrile and 0.1% formic acid aqueous solution are used as mobile phases; the flow rate is 1mL*min ‑1 ; the column temperature is 45 DEG C; the injection amount is 15ul; and the detection wavelength is 300nm. The method for detecting the fingerprint spectrum of the Wenshoujieyu granules established by the application is simple in operation, reliable in result, high in stability and precision, and good in repeatability; the fingerprint spectrum provided by the application can be used to comprehensively and effectively control the quality of the preparation, and ensure the stability, reliability and safety of the preparation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine preparation detection, in particular to a method for establishing a fingerprint spectrum of Wenxiao Jieyu Granules and the fingerprint spectrum thereof. BACKGROUND

[0002] Traditional Chinese medicine fingerprint spectrum is an important means for identifying the authenticity of traditional Chinese medicinal materials or Chinese patent medicines, and evaluating the consistency and stability of raw materials, semi-finished products and finished products. Compared with the existing identification of single effective component or index component, as a comprehensive and quantifiable quality control method, traditional Chinese medicine fingerprint spectrum can comprehensively and effectively reflect the quality of traditional Chinese medicinal materials and their preparations. Traditional Chinese medicine fingerprint spectrum technology integrates various effective chemical information, making the results more scientific and reasonable. In recent years, modern traditional Chinese medicine fingerprint spectrum analysis technology established by combining computer software analysis has been widely applied in various fields of traditional Chinese medicine research, and is currently internationally recognized as a quality control mode of traditional Chinese medicine. The research and establishment of fingerprint spectrum are of great significance for improving the quality of traditional Chinese medicine compounds and promoting the modernization of traditional Chinese medicine.

[0003] "Wenxiao Jieyu Granules" is the first in-hospital preparation with a preparation record number in Henan Provincial Hospital of Traditional Chinese Medicine. It is an experience formula summarized by Professor Xu Erping, a famous traditional Chinese medicine doctor in Henan Province, through years of clinical treatment of depression. Wenxiao Jieyu Granules is made of fifteen kinds of traditional Chinese medicinal materials, including Qingbanxia, Chaihu, Baishao, Zhuru, Zhishi, Chao Baishu, Danggui, Zhizi, Chenpi, Fuling, Mudanpi, Zhigancao, Bohe, Longgu and Muli. The combination of these medicines has the effects of regulating liver and spleen, eliminating dampness and phlegm, regulating the middle and calming the mind, and applying cold and warm together. It can be used for treating depression caused by liver stagnation, spleen deficiency, disharmony of gallbladder and stomach, and phlegm and blood stasis. A large amount of clinical data shows that Wenxiao Jieyu Granules has significant anti-depression effect.

[0004] Wenxiao Jieyu Granules has complex components and multiple flavors. Qualitative analysis of only one or two components cannot reflect the overall information of the preparation. At present, there is no report on using traditional Chinese medicine fingerprint spectrum as a quality control method for Wenxiao Jieyu Granules. Therefore, it is of great significance to establish the fingerprint spectrum of Wenxiao Jieyu Granules for comprehensive control of the quality of the product. SUMMARY

[0005] (I) Technical problems solved

[0006] In view of the deficiencies of the prior art, the present application provides a method for establishing a fingerprint spectrum of Wenxiao Jieyu Granules and the fingerprint spectrum thereof.

[0007] (II) Technical solutions

[0008] To achieve the above object, the present application is implemented by the following technical solutions:

[0009] In the first aspect, the present application provides a fingerprint spectrum establishing method of Wenxiao Jieyu granules, which adopts high performance liquid chromatography to detect the Wenxiao Jieyu granules, wherein the chromatographic conditions are as follows:

[0010] Chromatographic column: Agilent 5TC-C 18 (2) (4.6 mm x 250 nm, 5 μm);

[0011] Mobile phase: acetonitrile (A) - 0.1% formic acid aqueous solution (B);

[0012] The gradient elution program is as follows: 0-10 min 5% A; 95% B

[0013] 10-35 min 5%-10% A; 95-90% B

[0014] 35-60 min 10%-16% A; 90-84% B

[0015] 60-75 min 16%-18% A; 84-82% B

[0016] 75-100 min 18%-30% A; 82-70% B

[0017] 100-110 min 30%-48% A; 70-52% B

[0018] 110-120 min 48%-50% A; 52-50% B

[0019] 120-130 min 50% A; 50% B

[0020] Flow rate: 1 mL·min -1 ; column temperature: 45°C; injection volume: 15 μL; detection wavelength: 300 nm.

[0021] The method further comprises the following steps:

[0022] (1) Preparation of test solution

[0023] 10.0 g of Wenxiao Jieyu granules were weighed in a beaker, dissolved by adding an appropriate amount of distilled water, then transferred to a 10 mL volumetric flask, added with distilled water to constant volume, shaken uniformly, and filtered through a 0.22 μm microporous filter to obtain the test solution;

[0024] (2) Preparation of mixed reference solution

[0025] An appropriate amount of ferulic acid, glycyrrhizin, narirutin, naringin, hesperidin and neohesperidin reference substances were weighed, dissolved in methanol, mixed uniformly, and filtered through a 0.22 μm microporous filter to obtain the mixed reference solution.

[0026] (3)Determination

[0027] Accurately pipette 15 μL of the control solution and the test solution respectively, inject into the high performance liquid chromatograph, record the chromatogram for 130 min, process the chromatogram data by "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2021 version)", and the fingerprint spectrum of Wenxiao Jieyu Granules is obtained.

[0028] In a second aspect, the present application provides the fingerprint spectrum of Wenxiao Jieyu Granules obtained by the above-mentioned fingerprint spectrum establishment method of Wenxiao Jieyu Granules, wherein there are 13 common peaks in the spectrum, and the No. 10 common peak is the reference peak, and the retention time of each common peak is: common peak 1: 0.309-0.317 min; common peak 2: 0.357-0.364 min; common peak 3: 0.399-0.407 min; common peak 4: 0.421-0.430 min; common peak 5: 0.496-0.505 min; common peak 6: 0.637-0.648 min; common peak 7: 0.680-6.85 min; common peak 8: 0.751-0.761 min; common peak 9: 0.821-0.827 min; common peak 10: 1.000 min; common peak 11: 1.055-1.061 min; common peak 12: 1.106-1.113 min; common peak 13: 1.156-1.165 min.

[0029] (III)Beneficial effects

[0030] The fingerprint spectrum detection method of Wenxiao Jieyu Granules established by the present application is simple in operation, reliable in result, high in stability and precision, and good in repeatability; the fingerprint spectrum of Wenxiao Jieyu Granules provided by the present application can comprehensively and effectively control the quality of the preparation, and ensure the stability, reliability and safety of the preparation. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is the chromatogram of the sample solution of Wenxiao Jieyu Granules under the condition of gradient elution program 1.

[0032] Figure 2 It is the chromatogram of the sample solution of Wenxiao Jieyu Granules under the condition of gradient elution program 2.

[0033] Figure 3 It is the chromatogram of the sample solution of Wenxiao Jieyu Granules under the condition of gradient elution program 3.

[0034] Figure 4 It is the chromatogram of the test solution (upper) and the mixed control solution (lower).

[0035] Figure 5 It is the superimposed chromatogram of the sample solution of 10 batches of Wenxiao Jieyu Granules. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. 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 those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1

[0038] Selection of gradient elution program in chromatographic conditions

[0039] Wenxiao Jieyu Granules are a compound preparation with complex chemical components and large differences in polarity. Therefore, we used gradient elution for detection and made judgments on the impact of different gradient elution procedures on the detection results.

[0040] 1. Preparation of the test solution

[0041] Weigh 10.0g of Wenxiao Jieyu Granules into a beaker, add an appropriate amount of distilled water to dissolve, transfer to a 10mL volumetric flask, add distilled water to make up to volume, shake well, and filter through a 0.22μm microporous membrane to obtain the test solution.

[0042] 2. Effects of different gradient elution programs on the chromatogram of Wenxiao Jieyu Granules

[0043] Chromatographic conditions:

[0044] Column: Agilent 5TC-C 18 (2) (4.6mm×250nm, 5μm);

[0045] Mobile phase: Acetonitrile (A) - 0.1% formic acid aqueous solution (B);

[0046] Flow rate: 1 mL·min -1 Column temperature: 45℃; injection volume: 15μL; detection wavelength: 300nm.

[0047] The gradient elution procedure is shown in Table 1:

[0048] Table 1 Elution procedures with different gradients

[0049]

[0050] 3 Results

[0051] Chromatograms of Wenxiao Jieyu Granules under different gradient elution programs are shown below. Figures 1-3As shown in the figure, the results show that the number of chromatographic peaks is the largest and the separation of each chromatographic peak is the best when the gradient elution procedure 3 is used, and therefore, the gradient elution procedure is the best condition of the present application.

[0052] Example 2

[0053] Establishment of Fingerprint of Wenshoujieyu Granules by High Performance Liquid Chromatography

[0054] 1 Materials

[0055] LC-20A Shimadzu high performance liquid chromatograph, chromatographic column is Agilent 5TC-C 18 column (4.6 mm x 250 nm, 5 μm); Wenshoujieyu granules are provided by Henan Provincial Hospital of Traditional Chinese Medicine, ferulic acid, glycyrrhizin, narirutin, naringin, hesperidin, neohesperidin reference substances are purchased from Shanghai Yuan Ye Biological Technology Co., Ltd., acetonitrile (analytical grade) is purchased from Tianjin Kemio Chemical Reagent Co., Ltd., formic acid is purchased from Germany Merck.

[0056] 2 Methods and results

[0057] 2.1 Chromatographic conditions

[0058] The chromatographic column is Agilent 5TC-C 18 (2) (4.6 mm x 250 nm, 5 μm), the mobile phase is acetonitrile (A) - 0.1% formic acid aqueous solution (B), the gradient elution procedure is: 0-10 min, 5% A; 10-35 min, 5%-10% A; 35-60 min, 10%-16% A; 60-75 min, 16%-18% A; 75-100 min, 18%-30% A; 100-110 min, 30%-48% A; 110-120 min, 48%-50% A; 120-130 min, 50% A. The flow rate is 1 mL·min -1 , the column temperature is 45°C, the injection amount is 15 μL, and the detection wavelength is 300 nm.

[0059] 2.2 Preparation of solutions

[0060] 2.2.1 Preparation of test solution

[0061] 10.0 g of Wenshoujieyu granules was weighed in a beaker, dissolved after adding appropriate amount of distilled water, transferred to a 10 mL volumetric flask, added with distilled water to constant volume, shaken, and filtered with a 0.22 μm microporous filter to obtain the test solution.

[0062] 2.2.2 Preparation of mixed reference solution

[0063] Accurately weigh ferulic acid, glycyrrhizin, rutinoside, naringin, hesperidin, neohesperidin reference substance, dissolve with methanol, mix well, filter with 0.22 μm microporous filter, and the mixed reference solution is obtained.

[0064] 2.3 Fingerprint spectrum establishment and similarity evaluation

[0065] Prepare 10 different batches of test solution according to the preparation method of test solution, and analyze the sample. Use the“Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2021 Edition)” to process the chromatogram data, establish the common mode of Wenxiao San decoction fingerprint spectrum, take S4 spectrum as the reference spectrum, use the average method, the time window width is 0.3 min, add multiple point correction and Maker peak automatic matching, generate the common mode of fingerprint spectrum, and mark out 13 common peaks in the fingerprint spectrum. Through the comparison of the retention time and ultraviolet absorption spectrum of the mixed reference substance, 6 common peaks of ferulic acid (peak 8), glycyrrhizin (peak 9), rutinoside (peak 10), naringin (peak 11), hesperidin (peak 12) and neohesperidin (peak 13) are identified, and the chromatograms of the test solution and the mixed reference solution are as shown in Figure 4 The relative retention time and relative peak area of each common peak are calculated, and the results are shown in Tables 2 and 3. Taking the common mode as the control fingerprint spectrum, the similarity of the chromatograms of 10 batches of test solution is evaluated, and the results show that the similarity of 10 batches of test solution is greater than 0.95. It is shown that the similarity of 10 batches of test solution is good. The fingerprint spectrum superimposed graph of 10 batches of test solution (see Figure 5 ), and the similarity results are shown in Table 4.

[0066] Table 2 Relative retention time results of common peaks in fingerprint spectrum of 10 batches of test solution (n=10)

[0067]

[0068] Table 3 Relative peak area results of fingerprint spectrum of 10 batches of test solution (n=10)

[0069]

[0070]

[0071] Table 4 Similarity evaluation results of 10 batches of test solution

[0072]

[0073] Example 3

[0074] Methodological investigation on the establishment method of Wenxiao Jieyu Granules fingerprint spectrum provided by the application

[0075] 1 Precision investigation

[0076] Take the same batch of test solution, continuously repeated 6 times, the chromatogram was collected, 10 chromatographic peak as reference peak, the relative retention time and relative peak area of each common peak RSD. Results as shown in Tables 6-7, the relative retention time of each common peak RSD <0.3%, the relative peak area RSD <1%, indicating that the instrument precision is good.

[0077] Table 5 test solution precision relative retention time results (n = 6)

[0078]

[0079] Table 6 test solution precision relative peak area results (n = 6)

[0080]

[0081]

[0082] 2 Stability

[0083] The same batch of test solution, respectively, after 0, 3, 6, 9, 12, 24h sample, the chromatogram was collected, 10 chromatographic peak as reference peak, the relative retention time and relative peak area of each common peak RSD value. Results as shown in Tables 8-9, the relative retention time of each common peak RSD <0.2%, the relative peak area RSD <0.6%, indicating that the sample within 24h stability is good.

[0084] Table 7 test solution stability relative retention time results (n = 6)

[0085]

[0086] Table 8 test solution stability relative peak area results (n = 6)

[0087]

[0088]

[0089] 3 repeatability

[0090] According to the preparation method of test solution, 6 samples were prepared in parallel, in turn, continuously sample, the chromatogram was collected, 10 chromatographic peak as reference peak, the relative retention time and relative peak area of each common peak RSD value. Results show that the relative retention time of each common peak RSD <0.5%, the relative peak area RSD <5%, indicating that the fingerprint of Wenxiao Jieyu particles detection method established by the present application repeatability is good.

[0091] Table 9 Test solution repeatability relative retention time results (n=6)

[0092]

[0093] Table 10 Test solution repeatability relative peak area results (n=6)

[0094]

[0095]

[0096] The above examples are only used to illustrate the technical solutions of the present application, rather than limit the same; although the present application has been described in detail with reference to the foregoing examples, those ordinarily skilled in the art should understand: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for establishing the fingerprint of Wenxiao Jieyu granules, wherein the method detects the Wenxiao Jieyu granules by high performance liquid chromatography, characterized in that, The chromatographic conditions in the method are as follows: Column: Agilent 5TC-C 18 (2), 4.6 x 250 mm, 5 μm; The mobile phase A is acetonitrile; the mobile phase B is 0.1% formic acid aqueous solution; The gradient elution program is as follows: 0-10 min 5% A; 95% B 10-35 min 5%-10% A; 95-90% B 35-60 min 10%-16% A; 90-84% B 60-75 min 16%-18% A; 84~82%B 75-100 min 18%-30% A; 82-70% B 100-110 min 30%-48% A; 70~52%B 110-120 min 48%-50% A; 52-50% B 120-130 min 50% A; 50% B Flow rate: 1 mL•min -1 ; The column temperature is 45°C; the injection volume is 15 μL; the detection wavelength is 300 nm; the method further comprises the following steps: (1) Preparation of the test solution Transfer 10.0 g of Wenxiao Jieyu granules into a beaker, dissolve by adding appropriate amount of distilled water, transfer into a 10 mL volumetric flask, add distilled water to constant volume, shake well, filter through a 0.22 µm microporous filter, and the test solution is obtained; (2) Preparation of the mixed reference solution Dissolve the ferulic acid, glycyrrhizin, narirutin, naringin, hesperidin, and neohesperidin reference substances in methanol, mix well, filter through a 0.22 µm microporous filter, and the mixed reference solution is obtained; (3) Determination Precisely pipette 15 μL of the reference solution and the test solution respectively, inject into the high performance liquid chromatograph, record the chromatogram for 130 min, process the chromatogram data using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2021 Edition)", and the fingerprint spectrum of Wenxiao Jieyu granules is obtained.

2. The fingerprint spectrum of Wenxiao Jieyu Granules obtained by using the establishment method of claim 1, characterized in that, There are 13 common peaks in the fingerprint spectrum of the Wenxiao Jieyu granules, and the No. 10 common peak is the reference peak. The retention times of the common peaks are as follows: common peak 1: 0.309-0.317 min; common peak 2: 0.357-0.364 min; common peak 3: 0.399-0.407 min; common peak 4: 0.421-0.430 min; common peak 5: 0.496-0.505 min; common peak 6: 0.637-0.648 min; common peak 7: 0.680-0.685 min; common peak 8: 0.751-0.761 min; common peak 9: 0.821-0.827 min; common peak 10: 1.000 min; common peak 11: 1.055-1.061 min; common peak 12: 1.106-1.113 min; common peak 13: 1.156-1.165 min.

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