Establishment method of fingerprint spectrum of liver-resolving decoction

Abstract

The invention discloses a method for establishing a fingerprint spectrum of a liver-removing decoction. The method comprises the following steps: preparing a test solution, preparing a reference solution and obtaining the fingerprint spectrum of the liver-dissolving decoction by adopting a high performance liquid chromatography, and detecting by adopting the high performance liquid chromatography under specific conditions to obtain the fingerprint spectrum of the liver-dissolving decoction with 15 common peaks, which can reflect multi-component information of the product. The detection method is simple, has many characteristic peaks, and can ensure the uniformity and stability of product quality. The method disclosed by the invention can be used for controlling the quality of the liver-removing decoction preparation, the specificity and effectiveness of the quality control can be remarkably improved, and a scientific basis is provided for clinical medication safety.

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 Huagan Decoction. Background Technology

[0002] Huagan Decoction is a representative formula for soothing the liver, relieving depression, clearing liver fire, and purging liver stagnation in the "Catalogue of Famous Ancient Prescriptions (First Batch)". It is recorded in "Complete Works of Jingyue" by Zhang Jingyue, a physician of the Ming Dynasty. The whole formula is composed of seven Chinese herbs: green tangerine peel, dried tangerine peel, gardenia (fried), peony root, peony bark, alisma rhizome, and fritillaria bulb. It is used to treat qi stagnation and fire, anger and liver damage, etc. In modern times, it is used for a variety of diseases caused by liver stagnation.

[0003] Huagan Decoction has a long and rich history of use in my country, but due to the large variety of medicinal materials and complex composition, batch-to-batch consistency of its quality is often affected. Furthermore, a single effective component or effective part cannot adequately reflect the overall quality of the traditional Chinese medicine preparation, hindering the stable exertion of its efficacy. To ensure the relative consistency between the quality and efficacy of Huagan Decoction, it is necessary to study quality testing methods to control its quality. Traditional Chinese medicine fingerprinting can comprehensively reflect the types and quantities of chemical components contained in medicinal materials, effectively demonstrating the holistic and comprehensive effects of the components. Therefore, exploring a method for establishing a fingerprint spectrum for Huagan Decoction, constructing its characteristic spectrum by selecting several highly specific chromatographic peak combinations, and thus establishing a quality evaluation standard for Huagan Decoction, thereby improving the specificity and effectiveness of quality control for this drug, is a technical problem that needs to be solved. Summary of the Invention

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

[0005] The technical solution of this invention is a method for establishing a fingerprint spectrum of Huagan Decoction, characterized in that the method includes the following steps: 1. Preparation of test solution: Take about 0.15 g of Huaganjian powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of methanol, stopper tightly, weigh it, sonicate for 30 min, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and take the filtrate to obtain the test solution. 2. Preparation of reference solution: Take an appropriate amount of hesperidin reference standard, accurately weigh it, and add methanol to prepare a reference solution containing 30 μg of hesperidin per 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 Huagan 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; flow rate of 1.0 ml / min; column temperature of 30 °C; detection wavelength of 230 nm; and a theoretical plate number, calculated based on the hesperidin peak, not less than 2000. The gradient elution program is as follows (all are volume percentages): 0-35 min, 8%→30% mobile phase A, 92%→70% mobile phase B; 35-55 min, 30%→80% mobile phase A, 70%→20% mobile phase B; 55-65 min, 80% mobile phase A, 20% mobile phase B; 65-70 min, 80%→8% mobile phase A, 20%→92% mobile phase B; 70-80 min, 8% mobile phase A, 92% mobile phase B.

[0006] Preferably, in the preparation of the test solution, the ultrasonic treatment power is 500 W and the frequency is 40 kHz.

[0007] Furthermore, the method for establishing the fingerprint spectrum of Huagan Decoction also includes: comparing multiple fingerprint spectra of Huagan Decoction, selecting common characteristic peaks, and obtaining the characteristic spectrum of Huagan 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 less than 0.90.

[0008] Furthermore, the fingerprint spectrum or characteristic spectrum of Huagan Decoction contains 15 characteristic peaks, and the relative retention times of each characteristic peak are as follows: peak 1: 0.178, peak 2: 0.213, peak 3: 0.301, peak 4: 0.493, peak 5: 0.557, peak 6: 0.685, peak 7: 0.897, peak 8: 0.927, peak 9 (S): 1, peak 10: 1.161, peak 11: 1.285, peak 12: 1.372, peak 13: 1.483, peak 14: 1.573, peak 15: 1.637.

[0009] Furthermore, in the fingerprint spectrum or characteristic spectrum of Huagan Decoction, peak 9 is the hesperidin control peak.

[0010] The beneficial technical effects of this invention are: 1. This invention uses the fingerprint spectrum of Huagan 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.

[0011] 2. In the process of determining the fingerprint spectrum of Huagan Decoction by 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.

[0012] 3. This invention provides an effective method for completely and accurately evaluating the quality of Huagan Decoction, thereby ensuring the reliable quality of the product. Attached Figure Description

[0013] Figure 1 The chromatograms selected for reference peaks are, from top to bottom, chlorogenic acid reference standard, paeonol reference standard, geniposide reference standard, hesperidin reference standard, paeoniflorin reference standard, test sample, and blank solvent.

[0014] Figure 2 The fingerprint spectra are for 15 batches of Huagan Decoction corresponding to physical samples, where R is the material reference control spectrum and S2-S16 are the fingerprint spectra of the 15 batches of test samples.

[0015] Figure 3 A reference spectrum was automatically established based on the reference fingerprint spectra of 15 batches of Huagan Decoction, with peak 9 being the reference peak for hesperidin. Detailed Implementation

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

[0017] Example: Experiment to establish HPLC fingerprint of Huagan Decoction.

[0018] 1. Chromatographic conditions and system suitability test: The chromatographic column was packed with octadecylsilane-bonded silica gel (Ultimate AQ-C18, 4.6 × 250 mm, 5 µm). 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 30 °C, and the detection wavelength was 230 nm. The gradient elution program was as follows (all are volume percentages): 0-35 min, 8% → 30% mobile phase A, 92% → 70% mobile phase B; 35-55 min, 30% → 80% mobile phase A, 70% → 20% mobile phase B; 55-65 min, 80% mobile phase A, 20% mobile phase B; 65-70 min, 80% → 8% mobile phase A, 20% → 92% mobile phase B; 70-80 min, 8% mobile phase A, 92% mobile phase B.

[0019] 2. Preparation of test solution: Take about 0.15 g of Huaganjian powder, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of methanol, stopper tightly, weigh it, sonicate (power 500 W, frequency 40 kHz) for 30 min, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0020] 3. Preparation of reference solution: Take an appropriate amount of hesperidin reference standard, accurately weigh it, and add methanol to prepare a reference solution containing 30 μg of hesperidin per ml.

[0021] 4. Determination: Accurately pipette 10 µl 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 chromatographic conditions described above. Record the chromatograms. The theoretical plate number, calculated based on the hesperidin peak, should not be less than 2000.

[0022] 5. Establishment of the fingerprint spectrum for Huagan Decoction: (1) Reference peak selection: Take appropriate amounts of paeoniflorin reference standard, hesperidin reference standard, geniposide reference standard, paeonol reference standard, and chlorogenic acid reference standard, accurately weigh them, and add methanol to prepare solutions containing 0.14 mg of paeoniflorin, 0.05 mg of hesperidin, 0.05 mg of geniposide, 0.004 mg of paeonol, and 0.004 mg of chlorogenic acid per ml. Detect the solutions under the above chromatographic conditions. The results are as follows: Figure 1 As shown. By Figure 1 It can be seen that the blank solvent had no interference, and the peak shape and resolution of both the reference and the test sample were good, indicating that the method has good specificity. Since hesperidin is one of the main components in tangerine peel and green peel, and its content in the sample is high and its peak shape is stable, hesperidin was used as the reference peak.

[0023] (2) Common peak identification: 15 batches of Huagan Decoction corresponding physical samples (provided by Guangxi Wuzhou Pharmaceutical (Group) Co., Ltd.) were prepared as test samples according to the preparation method of the test sample solution. Under the above chromatographic conditions, fingerprint chromatograms of the 15 batches of test samples were detected and analyzed. Chromatographic peaks with good stability, suitable response values, and relatively large peak areas in the fingerprint chromatograms of the 15 batches of test samples were selected as common peaks. A total of 15 common peaks were identified, such as... Figure 2 As shown.

[0024] (3) 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 corresponding physical sample material standards to establish reference chromatograms. The results are as follows: Figure 3As shown. The reference spectrum should present 15 characteristic peaks, one of which should have the same retention time as the corresponding reference peak. Among the 15 characteristic peaks, peak 9 has the same retention time as the peak corresponding to the hesperidin reference. Taking the hesperidin reference peak as peak S, the relative retention times of each characteristic peak and peak S are calculated. According to the reference spectrum, the relative retention times of the common characteristic peaks are as follows: peak 1: 0.178, peak 2: 0.213, peak 3: 0.301, peak 4: 0.493, peak 5: 0.557, peak 6: 0.685, peak 7: 0.897, peak 8: 0.927, peak 9 (S): 1, peak 10: 1.161, peak 11: 1.285, peak 12: 1.372, peak 13: 1.483, peak 14: 1.573, peak 15: 1.637.

[0025] (4) Similarity calculation: The similarity evaluation software system for 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 chromatograms. The results are shown in Table 1. The results show that the similarity of the fingerprint chromatograms of the 15 batches of reference materials prepared from different batches of raw materials is higher than 0.90, indicating that the quality is consistent among different batches.

[0026] Table 1. Similarity calculation results for 15 batches of samples (n=15)

[0027] 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 Huagan Decoction, characterized in that, The method includes the following steps: (1) Preparation of test solution: Take about 0.15 g of Huaganjian powder, accurately weigh it, put it in a stoppered conical flask, accurately add 50 ml of methanol, stopper tightly, weigh it, sonicate for 30 min, cool it, weigh it again, make up the lost weight with methanol, shake well, filter it, and take the filtrate to obtain the test solution. (2) Preparation of reference solution: Take an appropriate amount of hesperidin reference standard, accurately weigh it, and add methanol to prepare a reference solution containing 30 μg of hesperidin per 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 according to the following chromatographic conditions, record the chromatogram, and obtain the fingerprint chromatogram of Huagan 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 30 °C, the detection wavelength is 230 nm, and the theoretical plate number, calculated based on the hesperidin peak, should not be less than 2000. The gradient elution program is as follows (all are volume percentages): 0-35 min, 8%→30% mobile phase A, 92%→70% mobile phase B; 35-55 min, 30%→80% mobile phase A, 70%→20% mobile phase B; 55-65 min, 80% mobile phase A, 20% mobile phase B; 65-70 min, 80%→8% mobile phase A, 20%→92% mobile phase B; 70-80 min, 8% mobile phase A, 92% mobile phase B.

2. 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.

3. The method according to claim 1, characterized in that, The method further includes: comparing multiple fingerprint spectra of Huagan Decoction, selecting common characteristic peaks, and obtaining characteristic spectra of Huagan 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 less than 0.

90.

4. The method according to claim 3, characterized in that, The fingerprint spectrum or characteristic spectrum of Huagan Decoction contains 15 characteristic peaks, and the relative retention times of each characteristic peak are as follows: peak 1: 0.178, peak 2: 0.213, peak 3: 0.301, peak 4: 0.493, peak 5: 0.557, peak 6: 0.685, peak 7: 0.897, peak 8: 0.927, peak 9 (S): 1, peak 10: 1.161, peak 11: 1.285, peak 12: 1.372, peak 13: 1.483, peak 14: 1.573, peak 15: 1.

637.

5. The method according to claim 4, characterized in that, In the fingerprint or characteristic spectrum of Huagan Decoction, peak 9 is the hesperidin control peak.

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