Establishment method of fingerprint spectrum of Wenxiao depression-relieving granules and fingerprint spectrum of Wenxiao depression-relieving granules

Abstract

The invention provides an establishment method of a finger-print of Wenxiuqi granules and the finger-print, and relates to the technical field of traditional Chinese medicine preparation detection. The establishment method of the fingerprint spectrum adopts a high performance liquid chromatography to detect Wenxiao depression-relieving particles, and the chromatographic conditions in the method are as follows: Agilent 5TC-C18 (2) (4.6 mm * 250nm, 5 [mu] m) is a chromatographic column; acetonitrile and a 0.1% formic acid aqueous solution are used as a mobile phase; the flow velocity is 1mL. Min <-1 >; the column temperature is 45 DEG C; the sample size is 15 [mu] L; the detection wavelength is 300nm. The fingerprint spectrum detection method for the Wenxiuqianqi granules, established by the invention, is simple to operate, reliable in result, high in stability and precision and good in repeatability; the quality of the preparation can be comprehensively and effectively controlled by utilizing the fingerprint spectrum of the warm laughing and depression relieving granules provided by the invention, and the stability, reliability and safety of the preparation are ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of detection of traditional Chinese medicine preparations, and in particular to a method for establishing a fingerprint spectrum of Wenxiao Jieyu granules and a fingerprint spectrum thereof. Background Art

[0002] Traditional Chinese medicine fingerprint is an important means of using modern analytical technology to identify the authenticity of Chinese medicinal materials or Chinese patent medicines, and to evaluate the consistency and stability of the quality of raw materials, semi-finished products and finished products. Compared with the existing identification of single active ingredients or index ingredients, as a comprehensive and quantifiable quality control method, traditional Chinese medicine fingerprint can comprehensively and effectively reflect the quality of traditional Chinese medicines and their preparations. Traditional Chinese medicine fingerprint technology integrates various effective chemical information to make the results more scientific and reasonable. In recent years, modern traditional Chinese medicine fingerprint analysis technology established in combination with computer software analysis has been widely used in various fields of traditional Chinese medicine research and is currently an internationally recognized traditional Chinese medicine quality control model. The research and establishment of fingerprints is of great significance to improving the quality of traditional Chinese medicine compound prescriptions and promoting the modernization of traditional Chinese medicine.

[0003] "Wenxiao Jieyu Granules" is the first in-hospital preparation that has obtained the preparation registration number of Henan Provincial Hospital of Traditional Chinese Medicine. It is developed and formulated based on the experience formula summarized by Professor Xu Erping, a famous Chinese medicine doctor in Henan Province, in the clinical treatment of depression for many years. Wenxiao Jieyu Granules are made of 15 kinds of Chinese medicinal materials, including Pinellia tuber, Bupleurum root, White Peony Root, Rhizoma Anemarrhenae, Fructus Aurantii Immaturus, Stir-fried Atractylodes Macrocephalae, Angelica Sinensis, Gardenia Fructus, Tangerine Peel, Poria, Peony Root, Roasted Licorice, Mint, Dragon Bone, and Oyster. The combination of these medicines has the effects of regulating the liver and spleen, removing dampness and resolving phlegm, regulating the middle and calming the mind, balancing cold and heat, and nourishing at the same time. It can be used for depression caused by liver depression and spleen deficiency, gallbladder and stomach disharmony, and phlegm and dampness stagnation. A large amount of clinical data shows that Wenxiao Jieyu Granules have a significant anti-depressant effect.

[0004] Wenxiao Jieyu Granules have complex ingredients and multiple medicinal flavors. Qualitative analysis of only one or two of the ingredients is difficult to reflect the comprehensive information of the preparation. Currently, there is no report on using traditional Chinese medicine fingerprint as a quality control method for Wenxiao Jieyu Granules. Therefore, it is of great significance to establish the fingerprint of Wenxiao Jieyu Granules to comprehensively control its product quality. Summary of the invention

[0005] 1. Technical issues to be resolved

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

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] In a first aspect, the present invention provides a method for establishing a fingerprint of Wenxiao Jieyu Granules, wherein the method uses high performance liquid chromatography to detect Wenxiao Jieyu Granules, wherein the chromatographic conditions are:

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

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

[0012] Gradient elution program: 0~10min 5% A; 95% B

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

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

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

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

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

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

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

[0020] Flow rate: 1 mL min -1 ; Column temperature: 45℃; Injection volume: 15μL; Detection wavelength: 300nm.

[0021] The method further comprises the following steps:

[0022] (1) Preparation of test solution

[0023] Weigh 10.0 g of Wenxiao Jieyu Granules into a beaker, add an appropriate amount of distilled water to dissolve, transfer to a 10 mL volumetric flask, add distilled water to make up to volume, shake well, and filter through a 0.22 μm microporous filter membrane to obtain the test solution;

[0024] (2) Preparation of mixed reference solution

[0025] Weigh appropriate amounts of ferulic acid, glycyrrhizin, naringin, naringin, hesperidin, and neohesperidin reference substances, dissolve them in methanol, mix well, and filter through a 0.22 μm microporous filter membrane to obtain a mixed reference substance solution.

[0026] (3) Determination

[0027] Accurately 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, and use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2021 Edition)" to process the chromatogram data to obtain the fingerprint of Wenxiao Jieyu Granules.

[0028] In the second aspect, the present invention provides a fingerprint spectrum of Wenxiao Jieyu Granules obtained by the above-mentioned method for establishing the fingerprint spectrum of Wenxiao Jieyu Granules. There are 13 common peaks in the spectrum, among which common peak No. 10 is a reference peak, and the retention time of each common peak is: common peak 1: 0.309-0.317min; common peak 2: 0.357-0.364min; common peak 3: 0.399-0.407min; common peak 4: 0.421-0.430min; common peak 5: 0 .496-0.505min; common peak 6: 0.637-0.648min; common peak 7: 0.680-6.85min; common peak 8: 0.751-0.761min; common peak 9: 0.821-0.827min; common peak 10: 1.000min; common peak 11: 1.055-1.061min; common peak 12: 1.106-1.113min; common peak 13: 1.156-1.165min.

[0029] (III) Beneficial effects

[0030] The Wenxiao Jieyu Granule fingerprint spectrum detection method established by the present invention is simple to operate, has reliable results, high stability and precision, and good repeatability; the Wenxiao Jieyu Granule fingerprint spectrum provided by the present invention can be used to comprehensively and effectively control the quality of the preparation, thereby ensuring the stability, reliability and safety of the preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the chromatogram of the Wenxiao Jieyu Granule sample solution under gradient elution program 1.

[0032] Figure 2 This is the chromatogram of the Wenxiao Jieyu Granule sample solution under gradient elution program 2.

[0033] Figure 3 This is the chromatogram of the Wenxiao Jieyu Granule sample solution under gradient elution program 3.

[0034] Figure 4 Chromatograms of the test solution (upper) and mixed reference solution (lower).

[0035] Figure 5 This is an overlay of fingerprint spectra of 10 batches of Wenxiao Jieyu Granule sample solutions. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the 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.

[0037] Example 1

[0038] Selection of gradient elution program in chromatographic conditions

[0039] Wenxiao Jieyu Granules is a compound preparation with complex chemical components and large differences in polarity. Therefore, we use gradient elution to detect it and make judgments on the impact of different gradient elution procedures on the test results.

[0040] 1. Preparation of test solution

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

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

[0043] Chromatographic conditions:

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

[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 program is shown in Table 1:

[0048] Table 1 Different gradient elution programs

[0049]

[0050] 3 Results

[0051] The chromatograms of Wenxiao Jieyu Granules under different gradient elution programs are shown in Figure 2. Figure 1-3As shown, the results show that when the gradient elution program 3 is adopted, the number of chromatographic peaks is the largest and the separation of each chromatographic peak is the best. Therefore, this gradient elution program is the optimal condition of the present invention.

[0052] Example 2

[0053] Establishment of fingerprint of Wenxiao Jieyu granules by high performance liquid chromatography

[0054] 1 Materials

[0055] LC-20A Shimadzu HPLC, Agilent 5TC-C column 18 column (4.6 mm × 250 nm, 5 μm); Wenxiao Jieyu granules were provided by Henan Provincial Hospital of Traditional Chinese Medicine, ferulic acid, glycyrrhizin, naringin, naringin, hesperidin, and neohesperidin reference substances were purchased from Shanghai Yuanye Biotechnology Co., Ltd., acetonitrile (analytical grade) was purchased from Tianjin Komiou Chemical Reagent Co., Ltd., and formic acid was purchased from Merck, Germany.

[0056] 2 Methods and Results

[0057] 2.1 Chromatographic conditions

[0058] The chromatographic column is Agilent 5TC-C 18 (2) (4.6 mm × 250 nm, 5 μm), mobile phase is acetonitrile (A)-0.1% formic acid aqueous solution (B), gradient elution program 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. Flow rate: 1 mL min -1 , column temperature: 45°C, injection volume: 15 μL, detection wavelength: 300 nm.

[0059] 2.2 Solution preparation

[0060] 2.2.1 Preparation of test solution

[0061] Weigh 10.0 g of Wenxiao Jieyu Granules into a beaker, add appropriate amount of distilled water to dissolve, transfer to a 10 mL volumetric flask, add distilled water to make up to volume, shake well, filter through a 0.22 μm microporous filter membrane to obtain the test solution.

[0062] 2.2.2 Preparation of mixed reference solution

[0063] Weigh appropriate amounts of ferulic acid, glycyrrhizin, naringin, naringin, hesperidin, and neohesperidin reference substances, dissolve them in methanol, mix well, and filter through a 0.22 μm microporous filter membrane to obtain a mixed reference substance solution.

[0064] 2.3 Fingerprint establishment and similarity evaluation

[0065] According to the preparation method of the test solution, 10 different batches of test solution were prepared and sampled for analysis. The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2021 Edition)" was used to process the chromatogram data, and the common pattern of the fingerprint of Wenxiaosan decoction was established. The S4 spectrum was used as the reference spectrum, and the average method was used. The time window width was 0.3min, multi-point correction and Maker peak automatic matching were added to generate the common pattern of the fingerprint spectrum, and 13 common peaks were calibrated in the fingerprint spectrum. By comparing the retention time and ultraviolet absorption spectrum of the mixed reference substance, six common peaks were identified, including ferulic acid (peak 8), glycyrrhizin (peak 9), naringin (peak 10), naringin (peak 11), hesperidin (peak 12), and neohesperidin (peak 13). The chromatograms of the test solution and the mixed reference solution are shown in the following figure. Figure 4 The relative retention time and relative peak area of ​​each common peak were calculated, and the results are shown in Table 2 and Table 3. The common pattern was used as the reference fingerprint to evaluate the similarity of the chromatograms of the 10 batches of test solutions. The results showed that the similarity of the 10 batches of test solutions was > 0.95. This shows that the similarity between the 10 batches of test solutions is good. Overlay of the fingerprints of the 10 batches of test solutions (see Figure 5 ), the similarity results are shown in Table 4.

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

[0067]

[0068] Table 3 Relative peak area results of fingerprints 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 method for establishing the fingerprint spectrum of Wenxiao Jieyu granules provided by the present invention

[0075] 1. Precision inspection

[0076] Take the same batch of test solution, repeat the injection 6 times, collect the chromatogram, select the chromatographic peak No. 10 as the reference peak, and calculate the RSD of the relative retention time and relative peak area of ​​each common peak. The results are shown in Table 6-7. The RSD of the relative retention time of each common peak is <0.3%, and the RSD value of the relative peak area is <1%, indicating that the instrument has good precision.

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

[0078]

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

[0080]

[0081]

[0082] 2. Stability investigation

[0083] The same batch of test solution was injected at 0, 3, 6, 9, 12, and 24 hours after sample preparation, and the chromatogram was collected. 10 chromatographic peaks were selected as reference peaks, and the RSD values ​​of the relative retention time and relative peak area of ​​each common peak were calculated. The results are shown in Table 8-9. The RSD values ​​of the relative retention time of each common peak were all <0.2%, and the RSD values ​​of the relative peak area were all <0.6%, indicating that the sample had good stability within 24 hours.

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

[0085]

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

[0087]

[0088]

[0089] 3 Repeatability study

[0090] According to the preparation method of the test solution, 6 samples were prepared in parallel, and the samples were injected continuously in sequence, the chromatograms were collected, 10 chromatographic peaks were selected as reference peaks, and the RSD values ​​of the relative retention time and relative peak area of ​​each common peak were calculated. The results showed that the RSD values ​​of the relative retention time of each common peak were all <0.5%, and the RSD values ​​of the relative peak area were all <5%, indicating that the Wenxiao Jieyu Granule fingerprint detection method established by the present invention has good repeatability.

[0091] Table 9 Relative retention time results of test solution repeatability (n=6)

[0092]

[0093] Table 10 Relative peak area results of test solution repeatability (n=6)

[0094]

[0095]

[0096] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for establishing a fingerprint of Wenxiao Jieyu Granules, wherein the method uses high performance liquid chromatography to detect Wenxiao Jieyu Granules, characterized in that: The chromatographic conditions in the method are: Chromatographic column: Agilent 5TC-C 18 (2) (4.6 mm × 250 nm, 5 μm); Mobile phase: acetonitrile (A)-0.1% formic acid aqueous solution (B); Gradient elution program: 0~10min 5%A; 95%B 10~35min 5%~10%A; 95~90%B 35~60min 10%~16%A; 90~84%B 60~75min 16%~18%A; 84~82%B 75~100min 18%~30%A; 82~70%B 100~110min 30%~48%A; 70~52%B 110~120min 48%~50%A; 52~50%B 120~130min 50%A;50%B Flow rate: 1 mL min -1 ; Column temperature: 45°C; injection volume: 15 μL; detection wavelength: 300 nm.

2. The method for establishing the fingerprint spectrum of Wenxiao Jieyu Granules according to claim 1, characterized in that: The method further comprises the following steps: (1) Preparation of test solution Weigh 10.0 g of Wenxiao Jieyu Granules into a beaker, add an appropriate amount of distilled water to dissolve, transfer to a 10 mL volumetric flask, add distilled water to make up to volume, shake well, and filter through a 0.22 μm microporous filter membrane to obtain the test solution; (2) Preparation of mixed reference solution Weigh appropriate amounts of ferulic acid, glycyrrhizin, naringin, naringin, hesperidin, and neohesperidin reference substances, dissolve them in methanol, mix well, and filter through a 0.22 μm microporous filter membrane to obtain a mixed reference substance solution. (3) Determination Accurately 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, and use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2021 Edition)" to process the chromatogram data to obtain the fingerprint of Wenxiao Jieyu Granules.

3. The fingerprint of Wenxiao Jieyu Granules obtained by the method of claim 1 is characterized in that: There are 13 common peaks in the spectrum, among which common peak No. 10 is the reference peak, and the retention times of the common peaks are: common peak 1: 0.309-0.317min; common peak 2: 0.357-0.364min; common peak 3: 0.399-0.407min; common peak 4: 0.421-0.430min; common peak 5: 0.496-0.505min; common peak 6: 0.6 37-0.648min; common peak 7: 0.680-6.85min; common peak 8: 0.751-0.761min; common peak 9: 0.821-0.827min; common peak 10: 1.000min; common peak 11: 1.055-1.061min; common peak 12: 1.106-1.113min; common peak 13: 1.156-1.165min.

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