Establishment method of vitality-preserving decoction fingerprint spectrum

Abstract

The invention discloses a method for establishing a vitality-preserving decoction fingerprint spectrum. The method comprises the following steps: preparing a test solution, preparing a reference solution, and obtaining the fingerprint spectrum of the vitality-preserving decoction preparation by adopting a high performance liquid chromatography. According to the chromatographic condition and system applicability test, octadecylsilane chemically bonded silica is used as a filler, acetonitrile is used as a mobile phase A, a 0.1% phosphoric acid solution is used as a mobile phase B for gradient elution, the detection wavelength is 210 nm, and the number of theoretical plates is calculated according to a calycosin-7-glucoside peak and should not be lower than 3000. The method disclosed by the invention can be used for controlling the quality of the vigor-preserving decoction preparation, the specificity and effectiveness of the quality control of the vigor-preserving decoction preparation can be remarkably improved, and the safety and reliability of clinical medication are ensured.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical testing technology, specifically relating to a method for establishing the fingerprint spectrum of Baoyuan Decoction. Background Technology

[0002] Baoyuan Decoction originated from "Jianming Yigu" (1629), compiled by Sun Zhihong in the Ming Dynasty. It is a classic ancient prescription, composed of ginseng, astragalus, licorice, and cinnamon. A slice of ginger is added during decoction. In modern clinical practice, it is widely used to treat coronary heart disease, chronic renal failure and nephritis, aplastic anemia, hepatitis B, leukopenia, and other diseases.

[0003] Traditional Chinese medicine (TCM) fingerprinting refers to the chromatograms or spectra obtained by appropriate analysis of certain TCM materials or preparations, which can indicate their chemical characteristics. It can comprehensively reflect the types and quantities of chemical components contained in the medicinal materials, effectively demonstrating the holistic and comprehensive effects of TCM components. Due to its rapid and accurate characteristics, it has been widely used in the analysis, identification, and quality control of TCM and its preparations. The development of classic formulas is currently a hot topic in the TCM industry. Based primarily on the preparation methods recorded in ancient medical books, modern production methods are used to recreate ancient formulas, thus producing modern TCM prescriptions. However, the chemical components of the various medicinal materials in these formulas often differ significantly, especially the determination of the content of indicative components and the methodological validation, which require further in-depth research. Therefore, it is essential to explore a method for establishing a fingerprint spectrum for Baoyuan Decoction, and to establish a quality evaluation method for Baoyuan Decoction by selecting several highly specific chromatographic peak combinations to construct a characteristic spectrum, thereby improving the specificity and effectiveness of quality control for this drug. Summary of the Invention

[0004] The present invention aims to provide a method for establishing a fingerprint spectrum of Baoyuan Decoction, which aims to construct a characteristic fingerprint spectrum by selecting a combination of several highly specific chromatographic peaks, thereby improving the specificity and effectiveness of quality control of Baoyuan Decoction.

[0005] The technical solution of the present invention is to provide a method for establishing a fingerprint spectrum of Baoyuan Decoction, characterized in that the method includes the following steps: 1. Preparation of test solution: Take about 1.0 g of Baoyuan Decoction preparation, place it in a stoppered conical flask, add 25 ml of methanol, sonicate for 30 min, shake well, filter, and the solution is obtained. 2. Preparation of reference solution: Take appropriate amounts of verbenafil glucoside reference standard and verbenafil reference standard, add methanol to prepare a mixed solution containing 50 μg of verbenafil glucoside and 25 μg of verbenafil in 1 ml. 3. Determination: Accurately inject 10 µl each of the test solution and the reference solution into the high-performance liquid chromatograph (HPLC). Perform the determination under the following chromatographic conditions, record the chromatograms, and obtain the fingerprint chromatogram of Baoyuan Decoction. The chromatographic conditions include: a column packed with octadecylsilane-bonded silica gel (4.6 × 250 mm, 5 µm); gradient elution with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B; a flow rate of 1.0 ml / min; a column temperature of 25 °C; a detection wavelength of 210 nm; and a theoretical plate number, calculated based on the peak of verbascoside glucoside, which should be no less than 3000. The gradient elution program is as follows (all are volume percentages): 0-20 min, 15%→23% mobile phase A, 85%→77% mobile phase B; 20-50 min, 23%→40% mobile phase A, 77%→60% mobile phase B; 50-70 min, 40%→52% mobile phase A, 60%→48% mobile phase B; 70-80 min, 52%→70% mobile phase A, 48%→30% mobile phase B; 80-85 min, 70% mobile phase A, 30% mobile phase B; 85-90 min, 70%→15% mobile phase A, 30%→85% mobile phase B.

[0006] Preferably, in the preparation of the test solution, the Baoyuan Decoction preparation is a liquid preparation formed by oral liquid, compound preparation, or syrup, or a solid preparation formed by powder, granules, tablets, or capsules.

[0007] Preferably, in the preparation of the test solution, the ultrasonic treatment power is 500 W and the frequency is 40 kHz.

[0008] Furthermore, the method for establishing the fingerprint spectrum of Baoyuan Decoction also includes: comparing multiple fingerprint spectra of Baoyuan Decoction, selecting common characteristic peaks, and obtaining the characteristic spectrum of Baoyuan Decoction; calculating the similarity based on the common characteristic peaks according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint spectrum, and the similarity between the fingerprint spectrum of the test sample and the control fingerprint spectrum shall not be lower than 0.90.

[0009] Furthermore, the fingerprint spectrum or characteristic spectrum of Baoyuan Decoction contains 18 characteristic peaks, and the relative retention times of each characteristic peak are as follows: peak 1: 0.243, peak 2: 0.435, peak 3: 0.468, peak 4: 0.490, peak 5: 0.830, peak 6: 0.850, peak 7: 0.927, peak 8: 0.960, peak 9: 0.976, peak 10 (S): 1, peak 11: 1.128, peak 12: 1.217, peak 13: 1.423, peak 14: 1.450, peak 15: 1.474, peak 16: 1.519, peak 17: 1.648, peak 18: 1.769.

[0010] Furthermore, in the fingerprint spectrum or characteristic spectrum of Baoyuan Decoction, peak 2 and peak 10 are the control peaks of verbascoside and verbascoside, respectively.

[0011] The beneficial technical effects of this invention are: 1. This invention uses the fingerprint spectrum of Baoyuan Decoction as a quality control method for its preparation. This can avoid the one-sidedness of judging the overall quality of the preparation by only measuring one or two chemical components, and also reduce the possibility of artificial control in order to achieve the quality target.

[0012] 2. In the process of determining the fingerprint spectrum of Baoyuantang using high performance liquid chromatography, this invention reduces the gradient peaks caused by gradient changes by selecting the mobile phase, separation conditions and parameters, so that the target peaks are well separated from the impurity peaks, and at the same time obtains a good response and relatively stable retention time, resulting in a good characteristic spectrum.

[0013] 3. This invention provides an effective method for the complete and accurate evaluation of the quality of Baoyuan Decoction, thereby ensuring the reliable quality of the product and guaranteeing the safety of clinical medication. Attached Figure Description

[0014] Figure 1 The common pattern diagram is shown for the fingerprint spectra of 15 batches of Baoyuantang corresponding physical samples, where R is the material reference control spectrum and S2-S16 are the fingerprint spectra of 15 batches of test samples.

[0015] Figure 2 The reference chromatograms were automatically matched with the material fingerprint chromatograms of 15 batches of Baoyuan Decoction, among which peak 10 (S peak) is the reference peak of verbascoside isoflavone. Detailed Implementation

[0016] The following examples provide further illustration, but the present invention is not limited to these examples.

[0017] Example: Experiment on establishing the HPLC fingerprint of Baoyuan Decoction.

[0018] 1. Chromatographic conditions and system suitability test: The chromatographic column was packed with octadecylsilane-bonded silica gel (Ultimate XB-C18, 4.6 × 250 mm, 5 µm), and gradient elution was performed using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. The flow rate was 1.0 ml / min, the column temperature was 25 °C, the detection wavelength was 210 nm, and the theoretical plate number, calculated based on the vernix caseinate glucoside peak, should not be less than 3000. The gradient elution program is as follows (all are volume percentages): 0-20 min, 15%→23% mobile phase A, 85%→77% mobile phase B; 20-50 min, 23%→40% mobile phase A, 77%→60% mobile phase B; 50-70 min, 40%→52% mobile phase A, 60%→48% mobile phase B; 70-80 min, 52%→70% mobile phase A, 48%→30% mobile phase B; 80-85 min, 70% mobile phase A, 30% mobile phase B; 85-90 min, 70%→15% mobile phase A, 30%→85% mobile phase B.

[0019] 2. Preparation of test solution: Take about 1.0 g of Baoyuan Decoction preparation, place it in a stoppered conical flask, add 25 ml of methanol, sonicate (power 500 W, frequency 40 kHz) for 30 min, shake well, filter, and the solution is obtained.

[0020] 3. Preparation of reference solution: Take appropriate amounts of verbascoside glucoside reference standard and verbascoside reference standard, add methanol to prepare a mixed solution containing 50 μg of verbascoside glucoside and 25 μg of verbascoside per 1 ml.

[0021] 4. Determination: Accurately pipette 10 µl of each of the test solution and the reference solution into the high performance liquid chromatograph (Shimadzu LC-20AT), and perform the determination according to the above chromatographic conditions, and record the chromatogram.

[0022] 5. Establishment of the fingerprint spectrum of Baoyuan Decoction: (1) Common peak identification: 15 batches of Baoyuan Decoction samples (provided by Guangxi Wuzhou Pharmaceutical (Group) Co., Ltd.) were prepared according to the preparation method of the test solution. Under the above liquid chromatography conditions, fingerprint chromatograms of the 15 batches of test samples were detected and analyzed. Chromatographic peaks with good stability and suitable response values ​​in the fingerprint chromatograms of the 15 batches of test samples were selected as common peaks. A total of 18 common peaks were identified, such as... Figure 1 As shown.

[0023] (2) Establishment of reference chromatograms: The similarity evaluation software system for chromatographic fingerprints of traditional Chinese medicine (2012 edition) promulgated by the Chinese Pharmacopoeia Commission was used to automatically match the chromatographic peaks of the liquid chromatography chromatograms of 15 batches of sample material standards to establish reference chromatograms. The results are as follows: Figure 2As shown. The reference chromatogram should present 18 characteristic peaks, of which 2 peaks should have retention times consistent with the corresponding reference peaks. Among the 18 characteristic peaks, peaks 2 and 10 should correspond to the retention times of the chromatographic peaks of verbascoside glucoside and verbascoside reference standard, respectively. Using the verbascoside reference peak as peak S, calculate the relative retention times of each characteristic peak with peak S. According to the comparison spectrum, the relative retention times of the common characteristic peaks are as follows: peak 1: 0.243, peak 2: 0.435, peak 3: 0.468, peak 4: 0.490, peak 5: 0.830, peak 6: 0.850, peak 7: 0.927, peak 8: 0.960, peak 9: 0.976, peak 10 (S): 1, peak 11: 1.128, peak 12: 1.217, peak 13: 1.423, peak 14: 1.450, peak 15: 1.474, peak 16: 1.519, peak 17: 1.648, peak 18: 1.769.

[0024] (3) Similarity calculation: The similarity evaluation software system of chromatographic fingerprint of traditional Chinese medicine was used to calculate the similarity between the common peaks of 15 batches of samples and the reference material chromatogram. The results are shown in Table 1. Based on the lowest similarity value calculated by fingerprint chromatogram, fingerprint chromatogram was selected as the evaluation standard of reference material. It was determined that the similarity between this product and the reference material chromatogram should not be lower than 0.90.

[0025] Table 1. Similarity calculation results for 15 batches of samples (n=15)

[0026] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for establishing a fingerprint spectrum of Baoyuan Decoction, characterized in that, The method includes the following steps: (1) Preparation of test solution: Take about 1.0 g of Baoyuan Decoction preparation, place it in a stoppered conical flask, add 25 ml of methanol, sonicate for 30 min, shake well, filter, and the solution is obtained. (2) Preparation of reference solution: Take appropriate amounts of verbenafil glucoside reference standard and verbenafil reference standard, add methanol to prepare a mixed solution containing 50 μg of verbenafil glucoside and 25 μg of verbenafil in 1 ml. (3) Determination: Accurately inject 10 µl each of the test solution and the reference solution into the high-performance liquid chromatograph, and perform the determination according to the following chromatographic conditions, record the chromatogram, and obtain the fingerprint spectrum of Baoyuan Decoction. The chromatographic conditions include: the chromatographic column is packed with octadecylsilane-bonded silica gel (4.6 × 250 mm, 5 µm), with gradient elution using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, the flow rate is 1.0 ml / min, the column temperature is 25 °C, the detection wavelength is 210 nm, and the theoretical plate number, calculated based on the peak of isoflavone glucoside, should not be less than 3000. The gradient elution program is as follows (all are volume percentages): 0-20 min, 15%→23% mobile phase A, 85%→77% mobile phase B; 20-50 min, 23%→40% mobile phase A, 77%→60% mobile phase B; 50-70 min, 40%→52% mobile phase A, 60%→48% mobile phase B; 70-80 min, 52%→70% mobile phase A, 48%→30% mobile phase B; 80-85 min, 70% mobile phase A, 30% mobile phase B; 85-90 min, 70%→15% mobile phase A, 30%→85% mobile phase B.

2. The method according to claim 1, characterized in that, In the preparation of the test sample solution, the Baoyuan Decoction preparation is a liquid preparation formed by oral liquid, compound preparation, or syrup, or a solid preparation formed by powder, granule, tablet, or capsule.

3. The method according to claim 1, characterized in that, In the preparation of the test solution, the ultrasonic treatment power is 500 W and the frequency is 40 kHz.

4. The method according to claim 1, characterized in that, The method further includes: comparing multiple fingerprint spectra of Baoyuan Decoction, selecting common characteristic peaks, and obtaining characteristic spectra of Baoyuan Decoction; calculating the similarity based on common characteristic peaks according to the similarity evaluation system of chromatographic fingerprint spectra of traditional Chinese medicine, and the similarity between the fingerprint spectra of the test sample and the fingerprint spectra of the control sample shall not be lower than 0.

90.

5. The method according to claim 4, characterized in that, The fingerprint spectrum or characteristic spectrum of Baoyuan Decoction contains 18 characteristic peaks, and the relative retention times of each characteristic peak are as follows: Peak 1: 0.243, Peak 2: 0.435, Peak 3: 0.468, Peak 4: 0.490, Peak 5: 0.830, Peak 6: 0.850, Peak 7: 0.927, Peak 8: 0.960, Peak 9: 0.976, Peak 10 (S): 1, Peak 11: 1.128, Peak 12: 1.217, Peak 13: 1.423, Peak 14: 1.450, Peak 15: 1.474, Peak 16: 1.519, Peak 17: 1.648, Peak 18: 1.

769.

6. The method according to claim 5, characterized in that, In the fingerprint spectrum or characteristic spectrum of Baoyuan Decoction, peak 2 and peak 10 are the control peaks of verbascoside and verbascoside, respectively.

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