A method for constructing fingerprint profiles and a method for quality detection of a compound preparation of liver-regulating decoction.

Abstract

This invention belongs to the field of quality testing technology for traditional Chinese medicine preparations. Specifically, it provides a method for constructing a fingerprint spectrum and a quality testing method for a Tiaogan Decoction compound preparation. The fingerprint spectrum construction method of the Tiaogan Decoction compound preparation described in this invention uses octadecylsilane-bonded silica gel as the packing material and acetonitrile-phosphoric acid aqueous solution as the mobile phase for gradient elution. The elution program was obtained through repeated experiments. Under the elution conditions of this invention, not only is the number of common peaks significantly increased (up to 22), but each common peak also has a good peak shape, good separation effect, and stable baseline. This allows for a more comprehensive, clear, and effective quality testing of the Tiaogan Decoction compound preparation.

Description

Technical Field

[0001] This invention belongs to the field of quality testing technology for traditional Chinese medicine preparations, specifically relating to a method for constructing a fingerprint spectrum and a quality testing method for a compound preparation of Tiaogan Decoction. Background Technology

[0002] Tiaogan Decoction is the 70th prescription in the "Catalogue of Ancient Classic Prescriptions (Second Batch) - Han Medicine" published by the State Administration of Traditional Chinese Medicine. The prescription originates from "Fu Qing Nü Zhu Ke" by Fu Shan of the Qing Dynasty. "Fu Qing Nü Zhu Ke" records: "For women who experience lower abdominal pain after menstruation... use Tiaogan Decoction." The main functions of Tiaogan Decoction are to replenish kidney water and regulate liver qi; it is used for symptoms such as soreness and weakness of the lower back and knees and lower abdominal pain caused by insufficient kidney water and stagnation of liver qi in women.

[0003] Currently, no literature reports a comprehensive analysis of the chemical composition and fingerprint spectral studies of the Tiaogan Tang granules claimed in this invention. Existing technologies lack comprehensive and systematic quality control methods to reflect the quality status of the main components in the finished granules, making it impossible to effectively control the production process and product quality, and thus failing to adequately guarantee its clinical efficacy. Therefore, developing a method for detecting the fingerprint spectral data of the Tiaogan Tang compound preparation, particularly through high-performance liquid chromatography (HPLC), to comprehensively control its key quality aspects, is of great significance.

[0004] The Liver-Regulating Decoction is made from seven Chinese medicinal herbs: yam, donkey-hide gelatin, angelica sinensis, white peony root, cornus officinalis, morinda officinalis, and licorice. Each herb contains key components that effectively exert the efficacy of the Liver-Regulating Decoction. Therefore, controlling each component in this formula allows for a more comprehensive evaluation of its overall quality. However, compound preparations lose the morphological characteristics of the original medicinal herbs, and simple qualitative and quantitative analysis of indicator components is insufficient to reflect their quality.

[0005] Therefore, researching a new, effective, accurate, and comprehensive method for detecting the quality of Tiaogan Decoction compound preparations has become an urgent problem to be solved. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a method for constructing a fingerprint spectrum and a quality detection method for a compound preparation of Tiaogan Decoction. Based on the characteristics of the compound preparation of Tiaogan Decoction, this method establishes a fingerprint spectrum of the product, achieves effective separation of each characteristic peak, increases the number of characteristic peaks, improves the separation effect, and can effectively, accurately and comprehensively detect the compound preparation of Tiaogan Decoction.

[0007] Specifically, this invention discloses a method for constructing the fingerprint spectrum of a compound preparation of liver-regulating decoction, including the following steps:

[0008] (1) Preparation of the test solution;

[0009] (2) The test solution was analyzed by high performance liquid chromatography. Octadecylsilane-bonded silica gel was used as the stationary phase, and acetonitrile-phosphoric acid aqueous solution was used as the mobile phase for gradient elution. The gradient elution program included: 0→10min→20min→40min→60min→90min→95min→115min. The volume percentage of acetonitrile in the mobile phase was: 2%→5%→12%→12%→18%→60%→60%→75%.

[0010] According to any one of the construction methods of the present invention, step (1) includes: weighing the Tiaogan Decoction compound preparation, extracting it with a solvent, separating the solid and liquid, and taking the liquid, which is the test solution.

[0011] According to any one of the construction methods of the present invention, step (1) further satisfies any one or more of the following AE:

[0012] A. The extraction is either ultrasonic extraction or hot reflux extraction;

[0013] B. Extraction time is 10-60 minutes;

[0014] C. The mass ratio of the compound preparation of Tiaogan Decoction to the volume ratio of the solvent is 0.3-0.6g: 20-50ml;

[0015] D. The solid-liquid separation is selected from centrifugation or filtration;

[0016] E. The solvent is selected from at least one of methanol, water and ethanol; preferably methanol or an aqueous methanol solution with a volume percentage of not less than 50%; more preferably an aqueous methanol solution with a volume percentage of 80%.

[0017] According to any one of the construction methods of the present invention, the Tiaogan Tang compound preparation is a compound preparation made from yam, donkey-hide gelatin, angelica, white peony root, cornus officinalis, morinda officinalis, and licorice as raw materials using conventional techniques. By weight, the Tiaogan Tang compound preparation comprises the following raw materials: 10-20 parts yam, 6-12 parts donkey-hide gelatin, 6-12 parts angelica, 6-12 parts white peony root, 6-12 parts cornus officinalis, 2-4 parts morinda officinalis, and 2-4 parts licorice. The fingerprint spectrum of these compound preparations can be constructed using the method of the present invention.

[0018] The preferred formulations of the liver-regulating decoction are solid or liquid preparations of the liver-regulating decoction, and more preferably at least one of the following: liver-regulating decoction, liver-regulating decoction granules, liver-regulating decoction capsules, and liver-regulating decoction tablets. The liver-regulating decoction formulations can be prepared using conventional techniques, such as, but not limited to, the methods specified in the Chinese Pharmacopoeia.

[0019] Preferably, the liver-regulating decoction compound preparation is made from 7 Chinese herbs: yam, donkey-hide gelatin, angelica, white peony root, cornus officinalis, morinda officinalis, and licorice. The decoction is then dried and extruded into granules.

[0020] According to any one of the construction methods of the present invention, step (1) includes: taking the Tiaogan Decoction compound preparation, using 50% to 100% methanol as the extraction solvent, ultrasonically extracting, filtering, and obtaining the test solution.

[0021] According to any one of the construction methods of the present invention, the power of the ultrasound is 100 to 500 W, for example, about 250 W.

[0022] According to any of the construction methods of the present invention, the frequency of the ultrasound is 30 to 50 kHz, for example, about 40 kHz.

[0023] According to any of the construction methods of the present invention, the ultrasound duration is 20 to 60 minutes, for example, about 30 minutes.

[0024] According to any one of the construction methods of the present invention, step (2) further satisfies any one or more of the following items 1)-6):

[0025] 1) The gradient elution program further includes: 115 min → 120 min, and the volume percentage of acetonitrile in the mobile phase is: 75% → 75%;

[0026] 2) The volume percentage of phosphoric acid in the phosphoric acid-containing aqueous solution is 0.05%-0.15%;

[0027] 3) The detection wavelength is 200nm-400nm, preferably 240nm.

[0028] 4) The column temperature is 30℃-50℃, preferably 35℃;

[0029] 5) The flow rate is 0.5 ml / min - 1.5 ml / min, preferably 0.8 ml / min;

[0030] 6) The injection volume is 1 μl-20 μl, preferably 10 μl.

[0031] According to any one of the construction methods of the present invention, the theoretical plate number of the paeoniflorin chromatographic peak is not less than 10,000.

[0032] In some preferred embodiments, an Inert Sustain C18 column is used.

[0033] In some preferred embodiments, the chromatographic column used has the following specifications: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0034] In some preferred embodiments, the construction method further includes the step of preparing a reference solution using at least one of glycyrrhizin, ammonium glycyrrhizate, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloglucoside, benzoylpaeoniflorin, ferulic acid, ligustilide H, loganin, monoglucoside, and crystal blue glycoside, and the step of detecting the reference solution using high performance liquid chromatography according to any of the above-described construction methods to obtain a reference fingerprint chromatogram.

[0035] Reference solutions can be prepared using any one or more of the following reference standards: glycyrrhizin, glycyrrhizic acid, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloglucoside, benzoylpaeoniflorin, ferulic acid, ligustrazine lactone H, loganin, monoglucoside, and crystal blue glycoside. When using two or more reference standards, they can be mixed to prepare a mixed solution or prepared separately.

[0036] According to any one of the construction methods of the present invention, the preparation method of the reference solution includes the following steps: taking glycyrrhizin, ammonium glycyrrhizate, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloglucoside, benzoylpaeoniflorin, ferulic acid, ligustrazine lactone H, loganin, monoglucoside, and crystal blue glycoside reference standards, and adding solvent to prepare a reference solution containing 1-100 μg of each reference standard per 1 ml; preferably, the solvent is selected from methanol or methanol-water solution; the volume fraction of methanol in the methanol-water solution is not less than 10%.

[0037] In some preferred embodiments, the method further includes constructing a control fingerprint chromatogram of the Tiaogan Decoction compound preparation. Fingerprint chromatograms obtained from multiple batches of Tiaogan Decoction compound preparation test samples are imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" calculation software to form a common pattern diagram, thereby generating a control fingerprint chromatogram of the Tiaogan Decoction compound preparation. In some preferred embodiments, after generating the control fingerprint chromatogram of the Tiaogan Decoction compound preparation using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software, the method further includes a step of marking common characteristic peaks.

[0038] At least 10 batches of Tiaogan Tang compound preparations were used to obtain a control spectrum, for example, 10 batches, 15 batches, 20 batches, and 30 batches of Tiaogan Tang compound preparations were used.

[0039] In certain preferred embodiments, the fingerprint spectrum of the liver-regulating decoction compound preparation contains 22 common characteristic peaks, all of which have a relative standard deviation (RSD) of retention time of less than 10%. Specifically, the average retention time of peak 1 is 7.637 min; peak 2 is 11.845 min; peak 3 is 24.337 min; peak 4 is 24.922 min; peak 5 is 26.741 min; peak 6 is 27.558 min; peak 7 is 28.413 min; peak 8 is 31.496 min; peak 9 is 34.935 min; and peak 10 is 40.118 min. The average retention times for peaks are as follows: Peak 11: 52.807 min; Peak 12: 56.134 min; Peak 13: 57.682 min; Peak 14: 58.867 min; Peak 15: 62.534 min; Peak 16: 67.797 min; Peak 17: 71.537 min; Peak 18: 78.719 min; Peak 19: 82.982 min; Peak 20: 89.872 min; Peak 21: 97.081 min; Peak 22: 97.531 min.

[0040] In certain preferred embodiments, the fingerprint spectrum of the liver-regulating decoction compound preparation contains 22 common characteristic peaks, with peak 10 being the paeoniflorin peak. Using the paeoniflorin peak as a reference peak, the relative retention time of each characteristic peak and the reference peak is within ±10% of a specified value. The specified values ​​for peaks 1-22 are as follows: 0.19, 0.30, 0.61, 0.62, 0.67, 0.69, 0.71, 0.79, 0.87, 1.00, 1.32, 1.40, 1.44, 1.47, 1.56, 1.69, 1.78, 1.96, 2.07, 2.24, 2.42, and 2.43.

[0041] In certain preferred embodiments, peak 1 is crystalloside, peak 2 is gallic acid, peak 4 is monoglobulin, peak 5 is catechin, peak 8 is loganin, peak 10 is paeoniflorin, peak 11 is ferulic acid, peak 13 is glycyrrhizin, peak 16 is 1,2,3,4,6-pentagalloylglucose, peak 17 is ligustilide H, peak 18 is benzoylpaeoniflorin, and peak 19 is ammonium glycyrrhizate.

[0042] In certain preferred embodiments, peak 1 is derived from Morinda officinalis, peak 2 from Angelica sinensis, Cornus officinalis, and Paeonia lactiflora, peak 3 from Cornus officinalis, peak 4 from Cornus officinalis, peak 5 from Paeonia lactiflora, peak 6 from Angelica sinensis, peak 7 from Cornus officinalis, peak 8 from Cornus officinalis, peak 9 from Paeonia lactiflora, peak 10 from Paeonia lactiflora, peak 11 from Angelica sinensis, peak 12 from Paeonia lactiflora, peak 13 from Glycyrrhiza uralensis, peak 14 from Glycyrrhiza uralensis, peak 15 from Angelica sinensis, peak 16 from Paeonia lactiflora, peak 17 from Angelica sinensis, peak 18 from Paeonia lactiflora, peak 19 from Glycyrrhiza uralensis, peak 20 from Angelica sinensis, peak 21 from Angelica sinensis, and peak 22 from Angelica sinensis.

[0043] The present invention also provides a method for quality testing of a Tiaogan Decoction compound preparation, comprising taking the Tiaogan Decoction compound preparation to be tested and constructing a fingerprint spectrum of the Tiaogan Decoction compound preparation to be tested according to the fingerprint spectrum construction method of any one of the above-mentioned methods.

[0044] Specifically, the process includes comparing the fingerprint spectrum of the liver-regulating decoction compound preparation to be tested with the control fingerprint spectrum of the liver-regulating decoction compound preparation; the fingerprint spectrum of the liver-regulating decoction compound preparation to be tested is obtained by using the liver-regulating decoction compound preparation to be tested according to any of the construction methods described in this invention, and the control fingerprint spectrum of the liver-regulating decoction compound preparation is the control fingerprint spectrum of the liver-regulating decoction compound preparation described in this invention.

[0045] Products that meet the requirements are considered qualified; those that do not meet the requirements are considered unqualified.

[0046] Furthermore, the compliance requirement includes one or more of the following:

[0047] (1) The fingerprint spectrum of the test compound preparation of Tiaogantang showed 22 characteristic peaks, and the retention time of each characteristic peak was within ±10% of the retention time value of the corresponding chromatographic peak in the fingerprint spectrum of the control compound preparation of Tiaogantang.

[0048] (2) Taking paeoniflorin peak as S peak, the relative retention time of each characteristic chromatographic peak in the fingerprint spectrum of the liver-regulating decoction compound preparation to be tested and the S peak is within ±10% of the relative retention time value of each characteristic peak in the fingerprint spectrum of the liver-regulating decoction compound preparation.

[0049] (3) If the similarity between the fingerprint spectrum of the test compound preparation of Tiaogan Decoction and the control fingerprint spectrum of Tiaogan Decoction is not less than 0.90, the quality is qualified; if it is less than 0.90, it is unqualified. Specifically, the similarity is obtained by the software for evaluating the similarity of chromatographic fingerprint spectrum of traditional Chinese medicine.

[0050] In this invention, 0.1% or 0.05% phosphoric acid refers to an aqueous solution containing 0.1% or 0.05% phosphoric acid by volume, respectively.

[0051] The technical solution of this invention has the following advantages:

[0052] 1. The fingerprint chromatogram construction method of the Tiaogan Decoction compound preparation of the present invention uses octadecylsilane-bonded silica gel as the packing material and acetonitrile-phosphoric acid aqueous solution as the mobile phase for gradient elution. The elution program was obtained through repeated experiments. Under the elution conditions of the present invention, not only is the number of common peaks significantly increased (up to 22), but each common peak also has a good peak shape, good separation effect, and stable baseline, thereby enabling more comprehensive, clear, and effective quality detection of the Tiaogan Decoction compound preparation.

[0053] 2. The fingerprint spectrum construction method of the Tiaogan Decoction compound preparation described in this invention overcomes the defect that it is difficult to monitor the overall quality under the same conditions by detecting a single content. It can more comprehensively reflect the overall characteristics of the Chinese medicine compound, scientifically evaluate the quality of the Chinese medicine compound, improve the consistency and safety of the Chinese medicine compound preparation products, and realize the inheritance and secondary development of classic Chinese medicine formulas.

[0054] 3. The fingerprint spectrum construction method of the liver-regulating decoction compound preparation described in this invention identified 22 common characteristic peaks, and studied their relative retention time, relative peak area and similarity, ensuring the chemical composition stability and safety of the particles, and providing important reference and quality standard for the subsequent quality control of the compound preparation.

[0055] 4. The quality testing method for the Tiaogan Tang compound preparation of the present invention constructs a fingerprint spectrum of the Tiaogan Tang compound preparation according to the fingerprint spectrum construction method of the present invention. This method then performs quality testing on the characteristic effective components of the Tiaogan Tang compound preparation, making the quality testing of related preparations more comprehensive and ensuring the effectiveness and controllability of the quality of the related preparations. By considering the fingerprint spectrum of each effective component in the Tiaogan Tang compound preparation of the present invention as a whole, and focusing on the identification of characteristic peak groups contained in each herb in the composition, the method avoids the one-sidedness of evaluating the entire traditional Chinese medicine compound preparation by only measuring a few chemical components, and reduces the possibility of artificial manipulation to meet quality standards. This provides a new method and means for comprehensively and accurately evaluating the quality of the traditional Chinese medicine compound granules of the present invention. The method of the present invention has good stability, high precision, convenience, and is easy to master. Attached Figure Description

[0056] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0057] Figure 1 This is the fingerprint spectrum of the liver-regulating decoction granules when gradient 1 was used in Example 1;

[0058] Figure 2 This is the fingerprint spectrum of the liver-regulating decoction granules using gradient 2 in Example 1;

[0059] Figure 3 This is the fingerprint spectrum of the liver-regulating decoction granules using gradient 3 in Example 1;

[0060] Figure 4 This is the fingerprint spectrum of the liver-regulating decoction granules using gradient 4 in Example 1;

[0061] Figure 5 This is the fingerprint spectrum of the liver-regulating decoction granules using gradient 5 in Example 1;

[0062] Figure 6 This is the fingerprint spectrum of the liver-regulating decoction granules using gradient 6 in Example 1;

[0063] Figure 7 This is the fingerprint spectrum of the liver-regulating decoction granules using gradient 7 in Example 1;

[0064] Figure 8 This is the fingerprint spectrum of the liver-regulating decoction granules at different wavelengths in Example 1;

[0065] Figure 9 This is the fingerprint spectrum of the liver-regulating decoction granules when different flow rates were used in Example 1;

[0066] Figure 10 This is the fingerprint spectrum of the liver-regulating decoction granules when using different column temperatures in Example 1;

[0067] Figure 11 This is the fingerprint spectrum of the liver-regulating decoction granules when different extraction methods were used in Example 2;

[0068] Figure 12 This is the fingerprint spectrum of the liver-regulating decoction granules when using different solvents in Example 2;

[0069] Figure 13 This is the superimposed chromatographic peak identification diagram of the fingerprint spectrum of Tiaogantang granules in Example 3; wherein, S1 to S14 are solvent blank, crystallizing blue glycoside reference, gallic acid reference, monosodium glutamate reference, catechin reference, loganin reference, paeoniflorin reference, ferulic acid reference, glycyrrhizin reference, 1,2,3,4,6-pentagalloyl glucose reference, ligustrazine lactone H, benzoyl paeoniflorin, ammonium glycyrrhizate, and Tiaogantang test sample, respectively.

[0070] Figure 14 This is the overlay diagram of the chromatographic peak assignments of the fingerprint chromatogram of the liver-regulating decoction granules in Example 4;

[0071] Figure 15 This is the similarity matching diagram of the fingerprint spectrum instrument precision test of the liver-regulating decoction granules in Example 4;

[0072] Figure 16 This is the similarity matching diagram of the fingerprint repeatability test of the liver-regulating decoction granules in Example 4;

[0073] Figure 17 This is the similarity matching diagram of the solvent stability test of the fingerprint spectrum of the liver-regulating decoction granules in Example 4;

[0074] Figure 18 Fingerprint spectrum for comparison of Tiaogantang granules. Detailed Implementation

[0075] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0076] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0077] This invention provides a method for constructing the fingerprint spectrum of a compound preparation of liver-regulating decoction, comprising the following steps:

[0078] Preparation of the test solution: Weigh an appropriate amount of Tiaogantang granules, place them in a volumetric flask, add the first solvent, seal tightly, sonicate, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0079] Preparation of reference solutions: Weigh appropriate amounts of glycyrrhizin, glycyrrhizic acid ammonium, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloglucoside, benzoylpaeoniflorin, ferulic acid, ligustrazine lactone H, loganin, monoglucoside, and crystallizing blue glycoside reference standards, and add 10% to 100% (e.g., 80%) of methanol to prepare a reference solution with a concentration of 1 μg to 100 μg for glycyrrhizin, glycyrrhizic acid ammonium, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloglucoside, benzoylpaeoniflorin, ferulic acid, ligustrazine lactone H, loganin, monoglucoside, and crystallizing blue glycoside.

[0080] Based on the results of high performance liquid chromatography detection of the test solution and the reference solution, the fingerprint spectrum of Tiaogantang granules was obtained.

[0081] The detection conditions for this high-performance liquid chromatography (HPLC) are as follows: a column packed with octadecylsilane-bonded silica gel is used; mobile phase A is selected from one or more of acetonitrile and methanol; mobile phase B is an aqueous solution of acid, an aqueous solution of alkaline solution, and / or an aqueous solution of buffer salt; the gradient elution program is as follows: 0–10 min, 98% B → 95% B; 10–20 min, 95% B → 88% B; 20–40 min, 88% B; 40–60 min, 88% B → 82% B; 60–90 min, 82% B → 40% B; 90–95 min, 40% B; 95–115 min, 40% B → 25% B; 115–120 min, 25% B; the flow rate is 0.5–1.5 mL / min; the column temperature is 20–50 °C; the detection wavelength is 200–400 nm; and the injection volume is 1–20 μL.

[0082] 1. Instruments and reagents

[0083] Instruments: Thermo Fisher U3000 high-performance liquid chromatograph system, including LPG-3400RS quaternary pump, WPS-3000 autosampler, TCC-3x00(RS) column oven, DAD-3000(RS) detector, Chameleon 7 chromatography workstation; Electronic analytical balance: METTLER TOLEDO AL204, XS105; Ultrasonic cleaner: KQ-500 model, Kunshan Ultrasonic Instrument Co., Ltd.; Intelligent constant temperature heating stage: BY-2020, Bangyuan Electronics Co., Ltd.

[0084] Column: Shimadzu Inert Sustain C18 (4.6×250mm, 5μm) SN: 22B0291765

[0085] Acetonitrile was chromatographic grade, phosphoric acid was HPLC grade, methanol and ethanol were analytical grade, and water was Wahaha purified water.

[0086] Reference Standard Information: Crystallized blue glycoside (batch number 111870-202004, content 96.5%, China National Institutes for Food and Drug Control); Gallic acid (batch number 110831-201906, content 91.5%, China National Institutes for Food and Drug Control); Monoglycoside (batch number 111998-202205, content 98.4%, China National Institutes for Food and Drug Control); Catechin (batch number 110877-201604, content 99.2%, China National Institutes for Food and Drug Control); Loganin (batch number 111870-202004, content 99.0%, China National Institutes for Food and Drug Control); Paeoniflorin (batch number 110736-202145, content 94.6%, China National Institutes for Food and Drug Control); Ferulic acid ( Batch No. 110773-201614, content 99.0%, China National Institutes for Food and Drug Control; Glycyrrhizin (Batch No. 111610-201908, content 95.0%, China National Institutes for Food and Drug Control); 1,2,3,4,6-Pentagalloglucoside (Batch No. 9618, content 95.9%, Shanghai Shidander Standard Technical Service Co., Ltd.); Ligusticum lactone H (Batch No. 13866, content 95.2%, Shanghai Shidander Standard Technical Service Co., Ltd.); Benzoylpaeoniflorin (Batch No. MUST-14112704, content 99.28%, Chengdu Mansite Biotechnology Co., Ltd.); Ammonium glycyrrhizate (Batch No. 110731-202122, content 94.4%, China National Institutes for Food and Drug Control).

[0087] The preparation methods of the liver-regulating decoction granules used in the following examples, embodiments, and comparative examples are as follows:

[0088] Weigh out 15g of yam, 9g of donkey-hide gelatin, 9g of angelica sinensis, 9g of white peony root, 9g of cornus officinalis, 3g of morinda officinalis, and 3g of licorice root. Add water equal to 9 times the weight of the raw materials and soak for 30 minutes. Bring to a boil over high heat, then simmer over low heat for 60 minutes. Filter the liquid and set aside. For the second decoction, add water equal to 7 times the weight of the raw materials, bring to a boil over high heat, then simmer over low heat for 60 minutes. Filter the liquid. Combine the two decoctions, concentrate, dry, add appropriate excipients, and granulate to produce Liver-Regulating Decoction granules.

[0089] Example 1: Investigation of chromatographic conditions

[0090] 1. Preparation of the test solution: Take Tiaogantang granules, grind them into a fine powder, weigh about 0.5g accurately, place them in a stoppered conical flask, accurately add 25ml of 50% methanol aqueous solution, seal tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, remove, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0091] 2. Optimization of chromatographic conditions

[0092] The test solution prepared in the first step of this embodiment was analyzed by HPLC according to the following method.

[0093] The chromatographic column was an Inert Sustain C18 column (250 mm long, 4.6 mm inner diameter, 5 μm particle size) packed with octadecylsilane-bonded silica gel (250 mm long, 4.6 mm inner diameter, 5 μm particle size). Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid as mobile phase B. Gradient elution was performed according to the specifications in Table 1. The detection wavelength was 190-400 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should be no less than 10,000.

[0094] Table 1 Gradient elution procedure (Gradient 1)

[0095] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 2→5 98→95 10～20 5→12 95→88 20～30 12 88 30～40 12→18 88→82 40～60 18 82 60～80 18→60 82→40 80～95 60 40 95～110 60→75 40→25 110～120 75 25

[0096] The results showed that under these conditions, the chromatographic peaks were too concentrated and the resolution was poor within the ranges of 20–30 minutes and 45–55 minutes. Therefore, in order to ensure that each major chromatographic peak in the chromatogram has good separation parameters and exhibits better chromatographic behavior, the test solution prepared in the first step of this example was used. A Shimadzu Inert Sustain C18 column (250 mm in length, 4.6 mm in inner diameter, and 5 μm in particle size) was selected; the detection wavelength was 240 nm; acetonitrile was used as mobile phase A; 0.1% phosphoric acid solution was used as mobile phase B; the flow rate was 0.8 mL per minute; and the column temperature was 35 °C. The elution gradient was optimized, and the optimized gradient program is shown in the table below.

[0097] Table 2 Gradient elution program (Gradient 2)

[0098] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 2→5 98→95 10～20 5→10 95→90 20～30 10 90 30～40 10→16 90→84 40～60 16 84 60～80 16→58 84→42 80～95 58 42 95～110 58→75 42→25 110～120 75 25

[0099] Table 3 Gradient elution procedure (Gradient 3)

[0100] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 2→5 98→95 10～20 5→12 95→88 20～40 12 88 40～60 12→18 88→82 60～80 18→60 82→40 80～95 60 40 95～110 60→75 40→25 110～120 75 25

[0101] Table 4 Gradient elution procedure (Gradient 4)

[0102]

[0103]

[0104] Table 5 Gradient elution procedure (gradient 5)

[0105] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 2→5 98→95 10～20 5→12 95→88 20～25 12 88 25～30 12→5 88→95 30～40 5→12 95→88 40～60 12→18 88→82 60～80 18→60 82→40 80～95 60 40 95～110 60→75 40→25 110～120 75 25

[0106] Table 6 Gradient elution program (Gradient 6)

[0107] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 2→5 98→95 10～20 5→12 95→88 20～40 12 88 40～60 12→18 88→82 60～80 18→46 82→54 80～95 46→60 54→40 95～100 60 40 100～115 60→75 40→25 115～120 75 25

[0108] Table 7 Gradient elution procedure (Gradient 7)

[0109] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 2→5 98→95 10～20 5→12 95→88 20～40 12 88 40～60 12→18 88→82 60～90 18→60 82→40 90～95 60 40 95～115 60→75 40→25 115～120 75 25

[0110] See results Figure 1-7 As shown, compared to gradient 1, under the optimized gradient 7 elution program, the number of chromatographic peaks increased from 100 to 111. The resolution between the peak containing peak S (paeoniflorin) and its surrounding peaks improved from 3.03 (gradient 1) to 3.39. Under gradient 1, the resolution between peak 9 and its surrounding peaks was only 1.35, which improved to 2.27 under gradient 7. The resolution between peak 1 (monochlorophyll) and its surrounding peaks improved from 2.05 (gradient 1) to 3.16, and the plate number increased from 13645 to 16823. Under gradient 1, the resolution between peaks 6 and 7 was only 1.35 (less than 1.5), which improved to 2.27. Under gradient 1, peak 11 (ferulic acid) was not visible, and the resolution between peaks 12 and 13 (glycyrrhizin) was only 1.21 (less than 1.5), which improved to 2.03 under the optimized gradient 7. Therefore, gradient 7 chromatographic conditions were selected for fingerprint determination.

[0111] 3. Selection of detection wavelength

[0112] The test solution prepared in step 1 of this embodiment was analyzed by HPLC, and the absorption wavelengths were investigated. The absorption wavelengths were 200 nm, 240 nm, 280 nm, and 300 nm. A Shimadzu Inert Sustain C18 column (250 mm length, 4.6 mm inner diameter, 5 μm particle size) was used, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. Gradient elution was performed according to the specifications in Table 7. The flow rate was 0.8 mL per minute, and the column temperature was 35 °C. The results are shown in Table 7. Figure 8 .

[0113] By comparing fingerprint spectra at different wavelengths of 200nm, 240nm, 280nm and 300nm, it can be seen that the baseline wavelength is relatively large at 200nm, some chromatographic peak information is missing at 280nm and 300nm, and the chromatographic peak information is rich at 240nm with balanced response of each chromatographic peak. Therefore, the detection wavelength of Tiaogan Decoction fingerprint spectrum is determined to be 240nm.

[0114] 4. Flow velocity assessment

[0115] This section investigates the flow rate of the mobile phase. The test solution prepared in step 1 of this example was analyzed by HPLC. The effects of flow rates of 0.7 ml / min, 0.8 ml / min, and 0.9 ml / min on the fingerprint chromatogram of Tiaogan Decoction were investigated. A Shimadzu Inert Sustain C18 column (250 mm length, 4.6 mm inner diameter, 5 μm particle size) was used, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. Gradient elution was performed according to the specifications in Table 7. The column temperature was 35 °C, and the detection wavelength was 240 nm. The results are shown in Table 7. Figure 9 .

[0116] The results showed that at a flow rate of 0.7 ml / min, 21 chromatographic peaks appeared, of which peaks 12 and 13 merged, and peaks 5, 11, and 19 merged with their original surrounding peaks, resulting in significant differences in peak area. At a flow rate of 0.9 ml / min, 22 chromatographic peaks appeared, with some peaks having poor shapes. Peak 3 merged with its surrounding peaks, and peak 14 split. The above problems did not occur at a flow rate of 0.8 ml / min. Therefore, to ensure better reproducibility of this method, it is recommended to optimize the flow rate to 0.8 ml / min.

[0117] 5. Examination of column temperature

[0118] The test solution prepared in step 1 of this embodiment was analyzed by HPLC. The column temperature was investigated, and the effects of column temperatures of 30℃, 35℃, and 40℃ on the fingerprint chromatogram of Tiaogan Decoction were examined. A Shimadzu Inert Sustain C18 column (250 mm length, 4.6 mm inner diameter, 5 μm particle size) was used, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. Gradient elution was performed according to the specifications in Table 7, with a flow rate of 0.8 ml / min and a detection wavelength of 240 nm. The results are shown in [Table 7]. Figure 10 .

[0119] The results showed that different column temperatures had no significant effect on the fingerprint chromatogram of Tiaogan Decoction, indicating that the method can adapt to certain column temperature variations. However, considering factors such as peak shape, peak resolution, and baseline, the optimal column temperature for this study of Tiaogan Decoction fingerprint chromatograms was 35℃.

[0120] 6. Determination of optimal chromatographic conditions

[0121] Based on the above experiments, the chromatographic conditions were determined as follows: Octadecylsilane-bonded silica gel was used as the packing material (InertSustain C18, column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid was used as mobile phase B; gradient elution was performed according to the specifications in Table 7; the flow rate was 0.8 ml / min; the column temperature was 35℃; and the detection wavelength was 240 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should not be less than 10,000.

[0122] Example 2: Investigation of the preparation method of the test solution

[0123] 1. Examination of extraction methods

[0124] Different extraction methods affect the extraction efficiency of each chromatographic peak. Therefore, the extraction efficiencies of ultrasonic extraction and reflux extraction were compared, and the appropriate extraction method was determined based on the peak area of ​​each characteristic peak. Approximately 0.5g of Tiaogantang granules were finely ground, accurately weighed, and placed in a stoppered conical flask. 25ml of a 50% (v / v) methanol aqueous solution was accurately added, and the flask was tightly sealed. The flask was then subjected to ultrasonic treatment (250W, 40kHz) for 30 minutes or reflux extraction for 30 minutes, respectively. After removal, cooling, and mixing, the flask was filtered, and the filtrate was collected as the final product. High-performance liquid chromatography (HPLC) was used for detection under the optimal chromatographic conditions of Example 1, as shown in [reference needed]. Figure 11 As shown.

[0125] The results showed that 22 characteristic peaks appeared when ultrasonic or reflux extraction was used. The peak area / sample amount ratio was found to be no different. Considering the convenience of sample preparation, ultrasonic extraction was the preferred extraction method for the sample.

[0126] 2. Investigation of extraction solvents

[0127] Different solvent ratios affect the extraction efficiency of each chromatographic peak. Therefore, the extraction efficiencies of 50% methanol, 80% methanol, and methanol for each chromatographic peak were compared, and the appropriate extraction solvent was determined based on the peak area of ​​each characteristic peak. Specifically, approximately 0.5g of Tiaogantang granules were finely ground, accurately weighed, and placed in a stoppered conical flask. 25ml of 50% or 80% methanol aqueous solution or methanol (by volume) was accurately added, the flask was tightly sealed, and the mixture was ultrasonically treated (250W power, 40kHz frequency) for 30 minutes. After treatment, the flask was removed, cooled, shaken well, filtered, and the filtrate was collected. The sample was then detected using high-performance liquid chromatography (HPLC) under the optimal chromatographic conditions described in Example 1. Figure 12 As shown.

[0128] The results analysis showed that 22 chromatographic peaks could be observed in 50% methanol, 80% methanol, and methanol. Comparing the peak area / sample amount of each characteristic peak, the peak area was higher in 80% methanol. Therefore, 80% methanol is the preferred solvent for preparing the test sample.

[0129] 3. Determined method for preparing the test solution

[0130] Take approximately 0.5 g of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 80% methanol, seal tightly, sonicate (power 250 W, frequency 40 kHz) for 30 minutes, remove, cool, shake well, filter, and collect the filtrate to obtain the test sample.

[0131] Example 3 Peak Assignment and Reference Selection

[0132] (1) Construction of comparative fingerprint patterns

[0133] Fifteen batches of Tiaogan Decoction samples were prepared according to the method specified in Example 2. The test solutions were then analyzed under the optimal chromatographic conditions of Example 1 to construct multiple batches of fingerprint chromatograms. The AIA data from these fingerprint chromatograms were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software. A total of 22 common peaks were identified, forming a common pattern diagram, and a control chromatogram was established. Figure 18 As shown. Peak 10, the paeoniflorin peak, was selected as the reference peak and labeled as peak S.

[0134] The fingerprint spectrum showed 22 common characteristic peaks, namely: Peak 1, with an average retention time of 7.637 min and an RSD of 0.45%; Peak 2, with an average retention time of 11.845 min and an RSD of 0.31%; Peak 3, with an average retention time of 24.337 min and an RSD of 0.12%; Peak 4, with an average retention time of 24.922 min and an RSD of 0.11%; Peak 5, with an average retention time of 26.741 min and an RSD of 0.10%; Peak 6... The average retention time for peak 1 was 27.558 min, with an RSD of 0.10%; peak 7 had an average retention time of 28.413 min, with an RSD of 0.10%; peak 8 had an average retention time of 31.496 min, with an RSD of 0.12%; peak 9 had an average retention time of 34.935 min, with an RSD of 0.11%; peak 10 had an average retention time of 40.118 min, with an RSD of 0.10%; and peak 11 had an average retention time of 52.807 min, with an RSD of [missing value]. 0.04%; Peak 12, average retention time: 56.134 min, RSD: 0.05%; Peak 13, average retention time: 57.682 min, RSD: 0.03%; Peak 14, average retention time: 58.867 min, RSD: 0.03%; Peak 15, average retention time: 62.534 min, RSD: 0.02%; Peak 16, average retention time: 67.797 min, RSD: 0.01%; Peak 17, average retention time: Peak 18 had an average retention time of 71.537 min with an RSD of 0.01%; Peak 19 had an average retention time of 82.982 min with an RSD of 0.01%; Peak 20 had an average retention time of 89.872 min with an RSD of 0.01%; Peak 21 had an average retention time of 97.081 min with an RSD of 0.01%; Peak 22 had an average retention time of 97.531 min with an RSD of 0.01%.

[0135] Using paeoniflorin as a reference peak, labeled as peak S, the relative retention times of each characteristic peak and peak S are within ±10% of the specified values. The specified values ​​for peaks 1 to 30 are as follows: 0.19, 0.30, 0.61, 0.62, 0.67, 0.69, 0.71, 0.79, 0.87, 1.00, 1.32, 1.40, 1.44, 1.47, 1.56, 1.69, 1.78, 1.96, 2.07, 2.24, 2.42, and 2.43.

[0136] According to the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity was calculated based on common peaks. The similarity between the fingerprint chromatogram of the test sample and the fingerprint chromatogram of the control sample was higher than 0.900. The similarity evaluation showed that the differences between the fingerprint chromatograms of different batches of particles were small.

[0137] (2) Characteristic peak identification

[0138] Take appropriate amounts of glycyrrhizin, glycyrrhizic acid ammonium, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloglucoside, benzoylpaeoniflorin, ferulic acid, ligustrazine lactone H, loganin, monoglucoside, and crystallizing blue glycoside, accurately weigh them, and add 50% methanol to prepare reference solutions containing 0.05 mg of each per ml.

[0139] The solvent blank (50% methanol aqueous solution), the above-mentioned reference solution, and the test solution prepared according to the method determined in Example 2 were analyzed under the optimal chromatographic conditions of Example 1. The fingerprint chromatogram of the Tiaogan Decoction test solution was compared with the chromatogram of the reference solution. The results are shown in […]. Figure 13 As shown, peak 1 is crystalloside, peak 2 is gallic acid, peak 4 is monoglobulin, peak 5 is catechin, peak 8 is loganin, peak 10 is paeoniflorin, peak 11 is ferulic acid, peak 13 is glycyrrhizin, peak 16 is 1,2,3,4,6-pentagalloylglucose, peak 17 is ligustilide H, peak 18 is benzoylpaeoniflorin, and peak 19 is ammonium glycyrrhizate.

[0140] Example 4 Methodological Validation

[0141] (1) Exclusivity

[0142] ① Preparation of negative sample solution lacking yam: Weigh the other medicinal ingredients except yam according to the prescription of Tiaogantang granules, prepare negative sample lacking yam according to the preparation method of Tiaogantang granules, and prepare negative sample solution lacking yam according to the preparation method of test sample solution in Example 2.

[0143] ② Preparation of negative sample solution lacking donkey-hide gelatin: Weigh out the other medicinal ingredients except donkey-hide gelatin according to the prescription of Tiaogan Decoction Granules, prepare negative sample lacking donkey-hide gelatin according to the preparation method of Tiaogan Decoction Granules, and prepare negative sample solution lacking donkey-hide gelatin according to the preparation method of test sample solution in Example 2.

[0144] ③ Preparation of negative sample solution lacking Angelica sinensis: Weigh the other medicinal ingredients except Angelica sinensis according to the prescription of Tiaogan Decoction Granules, prepare negative sample lacking Angelica sinensis according to the preparation method of Tiaogan Decoction Granules, and prepare negative sample solution lacking Angelica sinensis according to the preparation method of test sample solution in Example 2.

[0145] ④ Preparation of negative sample solution lacking white peony root: Weigh the other medicinal ingredients except white peony root according to the prescription of Tiaogan Decoction Granules, prepare negative sample lacking white peony root according to the preparation method of Tiaogan Decoction Granules, and prepare negative sample solution lacking white peony root according to the preparation method of test sample solution in Example 2.

[0146] ⑤ Preparation of negative sample solution without Cornus officinalis: Weigh the other medicinal ingredients except Cornus officinalis according to the prescription of Tiaogan Decoction Granules, prepare negative sample without Cornus officinalis according to the preparation method of Tiaogan Decoction Granules, and prepare negative sample solution without Cornus officinalis according to the preparation method of Tiaogan Decoction Granules in Example 2 "Preparation method of test solution".

[0147] ⑥ Preparation of negative sample solution lacking Morinda officinalis: Weigh the other herbs except Morinda officinalis according to the prescription of Tiaogan Decoction Granules, prepare a negative sample lacking Morinda officinalis according to the preparation method of Tiaogan Decoction Granules, and prepare a negative sample solution lacking Morinda officinalis according to the preparation method of Tiaogan Decoction Granules in Example 2 "Determined preparation method of test solution".

[0148] ⑦ Preparation of negative sample solution lacking licorice: Weigh the other medicinal ingredients except licorice according to the prescription of Tiaogantang granules, prepare negative sample lacking licorice according to the preparation method of Tiaogantang granules, and prepare negative sample solution lacking licorice according to the preparation method of test sample solution in Example 2.

[0149] The test solution prepared according to the method specified in Example 2 and the above-mentioned negative sample solution were precisely pipetted and analyzed by high performance liquid chromatography under the optimal chromatographic conditions of Example 1. The 22 fingerprint peaks were assigned as follows: Figure 14 As shown in Table 8, negative results showed no interference, indicating good method specificity.

[0150] As shown in Table 8, peak 1 comes from Morinda officinalis, peak 2 comes from Angelica sinensis, Cornus officinalis, and Paeonia lactiflora, peak 3 comes from Cornus officinalis, peak 4 comes from Cornus officinalis, peak 5 comes from Paeonia lactiflora, peak 6 comes from Angelica sinensis, peak 7 comes from Cornus officinalis, peak 8 comes from Cornus officinalis, peak 9 comes from Paeonia lactiflora, peak 10 comes from Paeonia lactiflora, peak 11 comes from Angelica sinensis, peak 12 comes from Paeonia lactiflora, peak 13 comes from Glycyrrhiza uralensis, peak 14 comes from Glycyrrhiza uralensis, peak 15 comes from Angelica sinensis, peak 16 comes from Paeonia lactiflora, peak 17 comes from Angelica sinensis, peak 18 comes from Paeonia lactiflora, peak 19 comes from Glycyrrhiza uralensis, peak 20 comes from Angelica sinensis, peak 21 comes from Angelica sinensis, and peak 22 comes from Angelica sinensis.

[0151] Table 8. Characteristic Peak Attribution Table

[0152]

[0153]

[0154] (2) Instrument precision test

[0155] Take the same test solution prepared according to the method determined in Example 2, inject it 6 times consecutively under the optimal chromatographic conditions in Example 1, record the chromatogram, and calculate the relative retention time and relative peak area RSD of each fingerprint peak.

[0156] like Figure 15 As shown, the results indicate that the RSD of this method meets the requirements, and the similarity of the instrument precision is greater than 0.90, indicating that the instrument precision of this method is good.

[0157] (3) Method repeatability test

[0158] Take 6 portions of the same batch of samples, each weighing 0.5g, and prepare the test sample according to the preparation method of the test sample solution determined in Example 2. Perform the determination by high performance liquid chromatography under the optimal chromatographic conditions in Example 1, record the chromatogram, and calculate the relative retention time and relative peak area RSD of each fingerprint peak.

[0159] like Figure 16 As shown, the results indicate that the RSD of repeatability meets the requirements, and the similarity results are all greater than 0.90, which complies with the regulations.

[0160] (4) Solvent stability test

[0161] The same test solution prepared according to the method determined in Example 2 was injected at 0h, 6h, 12h, 24h, 30h, and 36h, and the determination was performed under the optimal chromatographic conditions of Example 1. The chromatograms were recorded, and the relative retention time and relative peak area RSD of each fingerprint peak were calculated to examine the stability of the test solution.

[0162] like Figure 17 As shown, the results indicate that the RSD meets the requirements and the similarity result is greater than 0.90, which complies with the regulations, indicating that it is stable within 36 hours.

[0163] Based on the above methodological investigation results, among the 22 common peaks of the established fingerprint spectrum, each chromatographic peak is less affected by factors such as different column temperatures and flow rates, and the other chromatographic conditions have little effect on the changes. This indicates that the method has good robustness, is stable and reliable, and can be used for fingerprint spectrum determination.

[0164] Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A; 0.1% phosphoric acid solution was used as mobile phase B; gradient elution was performed according to the specifications in Table 7; the flow rate was 0.8 mL / min; the column temperature was 35℃; and the detection wavelength was 240 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should be no less than 10,000.

[0165] Preparation of reference solution: Take appropriate amounts of glycyrrhizin, glycyrrhizic acid ammonium, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloglucoside, benzoylpaeoniflorin, ferulic acid, ligustrazine lactone H, loganin, monoglucoside, and crystal blue glycoside, accurately weigh them, and add 50% methanol to prepare a mixed solution containing 0.05 mg of each per ml, which is used as the reference solution.

[0166] Preparation of the test solution: Weigh approximately 0.5 g of the test sample accurately, place it in a stoppered conical flask, add 25 ml of 80% methanol accurately, stopper tightly, sonicate (power 250 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0167] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0168] Following the fingerprint spectral detection method determined above, three batches of Tiaogantang granule samples were tested. The results showed that the relative retention times (RSDs) of the common peaks were 0.54%, 0.31%, 0.07%, 0.07%, 0.05%, 0.05%, 0.04%, 0.04%, 0.02%, 0.00%, 0.08%, 0.08%, 0.07%, 0.09%, 0.09%, 0.09%, 0.11%, 0.11%, 0.11%, 0.11%, 0.11%, 0.11%, and 0.11%, all less than 5%. The similarity results of the three batches of samples were greater than 0.90, which met the requirements.

[0169] The fingerprint spectrum construction method of the present invention has good system applicability, stability and reliability, high specificity, high instrument precision and good repeatability, which makes up for the shortcomings of the existing quality control methods of Tiaogan Decoction and has guiding significance for the quality detection and evaluation of this product.

[0170] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing the fingerprint spectrum of a compound preparation of liver-regulating decoction, characterized in that, Includes the following steps, (1) Preparation of test solution; Step (1) includes: weighing the Tiaogan Decoction compound preparation, extracting it with solvent A, separating the solid and liquid, and taking the liquid, which is the test solution; the extraction is ultrasonic extraction or hot reflux extraction; solvent A is methanol or methanol aqueous solution with a volume percentage of not less than 50%; the construction method also includes the step of preparing a reference solution using glycyrrhizin, glycyrrhizic acid ammonium, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloglucoside, benzoylpaeoniflorin, ferulic acid, ligustrolactone H, loganin, monoglucoside and crystal blue glycoside; (2) The test solution and the reference solution were detected by high performance liquid chromatography. Octadecylsilane bonded silica gel was used as the stationary phase and acetonitrile-phosphoric acid aqueous solution was used as the mobile phase for gradient elution. The gradient elution program included: 0→10min→20min→40min→60min→90min→95min→115min. The volume percentage of acetonitrile in the mobile phase was: 2%→5%→12%→12%→18%→60%→60%→75%. The detection wavelength was 240nm.

2. The method for constructing the fingerprint spectrum of the liver-regulating decoction compound preparation according to claim 1, characterized in that, Step (1) also satisfies any one or more of the following AD: A. Extraction time is 10-60 minutes; B. The mass ratio of the compound preparation of Tiaogan Decoction to the volume ratio of the solvent is 0.3-0.6g: 20-50ml; C. The solid-liquid separation is selected from centrifugation or filtration; D. Solvent A is an 80% (by volume) aqueous methanol solution.

3. The method for constructing the fingerprint spectrum of the liver-regulating decoction compound preparation according to claim 1, characterized in that, Step (2) also satisfies any one or more of the following items 1)-5): 1) The gradient elution program further includes: 115 min → 120 min, with the volume percentage of acetonitrile in the mobile phase being 75% → 75%; 2) The volume percentage of phosphoric acid in the phosphoric acid-containing aqueous solution is 0.05%-0.15%; 3) Column temperature: 30℃-50℃; 4) The flow rate is 0.5 ml / min - 1.5 ml / min; 5) The injection volume is 1μl-20μl.

4. The method for constructing the fingerprint spectrum of the liver-regulating decoction compound preparation according to claim 3, characterized in that, The column temperature is 35℃.

5. The method for constructing the fingerprint spectrum of the liver-regulating decoction compound preparation according to claim 3, characterized in that, The flow rate was 0.8 ml / min.

6. The method for constructing the fingerprint spectrum of the liver-regulating decoction compound preparation according to claim 3, characterized in that, The injection volume was 10 μl.

7. The method for constructing the fingerprint spectrum of the liver-regulating decoction compound preparation according to claim 1, characterized in that, The preparation method of the reference solution includes the following steps: taking glycyrrhizin, glycyrrhizic acid ammonium, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloglucoside, benzoylpaeoniflorin, ferulic acid, ligustrazine lactone H, loganin, monoglucoside, and crystal blue glycoside reference standards, and adding solvent B to prepare a reference solution containing 1~100µg of each reference standard per 1ml.

8. The method for constructing the fingerprint spectrum of the liver-regulating decoction compound preparation according to claim 7, characterized in that, Solvent B is selected from methanol or an aqueous methanol solution; the volume fraction of methanol in the aqueous methanol solution is not less than 10%.

9. The method for constructing the fingerprint spectrum of the liver-regulating decoction compound preparation according to claim 1, characterized in that, The fingerprint spectrum of the liver-regulating decoction compound preparation contained 22 common characteristic peaks, all with relative standard deviations (RSD) of retention times less than 10%. Specifically, the average retention time of peak 1 was 7.637 min; peak 2 was 11.845 min; peak 3 was 24.337 min; peak 4 was 24.922 min; peak 5 was 26.741 min; peak 6 was 27.558 min; peak 7 was 28.413 min; peak 8 was 31.496 min; peak 9 was 34.935 min; and peak 10 was 40.118 min. The average retention time of peak 1 was 52.807 min; the average retention time of peak 12 was 56.134 min; the average retention time of peak 13 was 57.682 min; the average retention time of peak 14 was 58.867 min; the average retention time of peak 15 was 62.534 min; the average retention time of peak 16 was 67.797 min; the average retention time of peak 17 was 71.537 min; the average retention time of peak 18 was 78.719 min; the average retention time of peak 19 was 82.982 min; the average retention time of peak 20 was 89.872 min; the average retention time of peak 21 was 97.081 min; and the average retention time of peak 22 was 97.531 min.

10. The method for constructing the fingerprint spectrum of the liver-regulating decoction compound preparation according to claim 1, characterized in that, The fingerprint spectrum of the liver-regulating decoction compound preparation contains 22 common characteristic peaks. Peak 10 is the paeoniflorin peak. Using the paeoniflorin peak as a reference peak, the relative retention time of each characteristic peak and the reference peak is within ±10% of the specified value. The specified values ​​of peaks 1-22 are as follows: 0.19, 0.30, 0.61, 0.62, 0.67, 0.69, 0.71, 0.79, 0.87, 1.00, 1.32, 1.40, 1.44, 1.47, 1.56, 1.69, 1.78, 1.96, 2.07, 2.24, 2.42 and 2.

43.

11. The method for constructing the fingerprint spectrum of the liver-regulating decoction compound preparation according to claim 9 or 10, characterized in that, Peak 1 is crystalloside, peak 2 is gallic acid, peak 4 is monoglobulin, peak 5 is catechin, peak 8 is loganin, peak 10 is paeoniflorin, peak 11 is ferulic acid, peak 13 is glycyrrhizin, peak 16 is 1,2,3,4,6-pentagalloylglucose, peak 17 is ligustilide H, peak 18 is benzoylpaeoniflorin, and peak 19 is ammonium glycyrrhizate.

12. The method for constructing the fingerprint spectrum of the liver-regulating decoction compound preparation according to claim 9 or 10, characterized in that, Peak 1 comes from Morinda officinalis, Peak 2 comes from Angelica sinensis, Cornus officinalis, and Paeonia lactiflora, Peak 3 comes from Cornus officinalis, Peak 4 comes from Cornus officinalis, Peak 5 comes from Paeonia lactiflora, Peak 6 comes from Angelica sinensis, Peak 7 comes from Cornus officinalis, Peak 8 comes from Cornus officinalis, Peak 9 comes from Paeonia lactiflora, Peak 10 comes from Paeonia lactiflora, Peak 11 comes from Angelica sinensis, Peak 12 comes from Paeonia lactiflora, Peak 13 comes from Glycyrrhiza uralensis, Peak 14 comes from Glycyrrhiza uralensis, Peak 15 comes from Angelica sinensis, Peak 16 comes from Paeonia lactiflora, Peak 17 comes from Angelica sinensis, Peak 18 comes from Paeonia lactiflora, Peak 19 comes from Glycyrrhiza uralensis, Peak 20 comes from Angelica sinensis, Peak 21 comes from Angelica sinensis, and Peak 22 comes from Angelica sinensis.

13. A quality testing method for a compound preparation of liver-regulating decoction, characterized in that, The method includes constructing a fingerprint spectrum of the liver-regulating decoction compound preparation to be tested according to any one of claims 1-12.

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