Fingerprint construction method and quality detection method of liver regulating decoction compound preparation

Abstract

The invention belongs to the technical field of quality detection of traditional Chinese medicine preparations, and particularly provides a fingerprint spectrum construction method and a quality detection method of a liver regulating decoction compound preparation. According to the fingerprint spectrum construction method of the liver regulating decoction compound preparation, octadecylsilane chemically bonded silica is used as a filling agent; an acetonitrile-phosphoric acid-containing aqueous solution is used as a mobile phase for gradient elution, an elution procedure is obtained through repeated tests, under the elution condition of the method, the number of common peaks (up to 22) is remarkably increased, the common peaks are good in peak shape and separation effect, a base line is stable, and the method is suitable for large-scale industrial production. Therefore, the quality of the liver regulating decoction compound preparation can be detected more comprehensively, clearly and effectively.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection of traditional Chinese medicine preparations, and particularly relates to a fingerprint spectrum construction method and a quality detection method of a Tiaogan Decoction compound preparation. Background Art

[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 comes from Fu Shan's "Fu Qing Nu Zhu Ke" in the Qing Dynasty. "Fu Qing Nu Zhu Ke" records: "Women who have lower abdominal pain after menstruation... should use Tiaogan Decoction." Tiaogan Decoction can replenish kidney water and regulate liver qi; it is used for women with symptoms such as soreness of waist and knees and lower abdominal pain caused by insufficient kidney water and uncomfortable liver qi.

[0003] At present, there is no literature report on the comprehensive analysis and fingerprint study of the chemical components of the Tiaogan Decoction granules claimed in the present invention. In the prior art, there is no comprehensive and systematic quality control method to reflect the quality status of the main components in the finished granules of the present invention, and it is impossible to effectively control its production process and product quality, and it is impossible to better guarantee its clinical efficacy. Therefore, it is of great significance to develop a method for detecting the fingerprint of the Tiaogan Decoction compound preparation, especially to determine its fingerprint information by high performance liquid chromatography, and comprehensively control its key quality.

[0004] Tiaogan Decoction is made of 7 Chinese herbs: yam, donkey-hide gelatin, angelica, white peony root, cornus fruit, Morinda officinalis, and licorice. Each herb contains key ingredients that can effectively exert the efficacy of Tiaogan Decoction. Therefore, controlling each ingredient in the prescription can more comprehensively evaluate the overall quality of the prescription. The compound preparation has lost the morphological characteristics of the original pieces, and the simple qualitative identification and quantitative analysis of the index components are difficult to reflect the quality of the compound preparation.

[0005] Therefore, developing a new method to effectively, accurately and comprehensively detect the quality of Tiaogan Decoction compound preparations has become an urgent problem to be solved. Summary of the invention

[0006] Therefore, the object of the present invention is to provide a fingerprint spectrum construction method and a quality detection method for a Tiaogan Decoction compound preparation. The method establishes a fingerprint spectrum of the variety according to the characteristics of the Tiaogan Decoction compound preparation, realizes effective separation of each characteristic peak, increases the number of characteristic peaks, improves the separation effect, and can effectively, accurately and comprehensively detect the Tiaogan Decoction compound preparation.

[0007] Specifically, the present invention discloses a method for constructing a fingerprint spectrum of a compound preparation of Tiaogan Decoction, comprising the following steps:

[0008] (1) Preparation of test solution;

[0009] (2) The sample solution was tested by high performance liquid chromatography, using octadecylsilane bonded silica gel as filler and acetonitrile-phosphoric acid aqueous solution as mobile phase for gradient elution; the gradient elution program included: 0→10 min→20 min→40 min→60 min→90 min→95 min→115 min, and 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) comprises: weighing the compound preparation of Tiaogan Decoction, extracting with a solvent, separating the solid from the liquid, and taking the liquid, which is the test solution.

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

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

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

[0014] C. The ratio of the mass of the compound preparation of Tiaogan Decoction to the volume 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 a methanol aqueous solution with a volume percentage of not less than 50%; more preferably a methanol aqueous solution with a volume percentage of 80%.

[0017] According to any one of the construction methods of the present invention, the compound preparation of Tiaogan Decoction is a compound preparation prepared by conventional technical means using Chinese yam, donkey-hide gelatin, Chinese angelica, white peony root, cornus officinalis, Morinda officinalis, and licorice as raw materials. According to weight parts, the compound preparation of Tiaogan Decoction includes the following raw materials: 10-20 parts of Chinese yam, 6-12 parts of donkey-hide gelatin, 6-12 parts of Chinese angelica, 6-12 parts of white peony root, 6-12 parts of cornus officinalis, 2-4 parts of Morinda officinalis, and 2-4 parts of licorice. The method of the present invention can be used to construct fingerprints for these compound preparations.

[0018] The compound preparation of Tiaogan Decoction is preferably a solid preparation or a liquid preparation of Tiaogan Decoction, more preferably at least one of Tiaogan Decoction decoction, Tiaogan Decoction granules, Tiaogan Decoction capsules and Tiaogan Decoction tablets. The compound preparation of Tiaogan Decoction can be prepared by conventional technical means, such as but not limited to the methods specified in the Chinese Pharmacopoeia.

[0019] Preferably, the compound preparation of Tiaogan Decoction is granules obtained by decocting 7 Chinese medicinal herbs including yam, donkey-hide gelatin, angelica, white peony root, cornus fruit, Morinda officinalis and licorice in water and then drying and squeezing them.

[0020] According to any one of the construction methods of the present invention, the step (1) comprises: taking the Tiaogan Decoction compound preparation, using 50% to 100% methanol as an 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 100-500W, for example, about 250W.

[0022] According to any one 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 one of the construction methods of the present invention, the ultrasonication time 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) to 6):

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

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

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

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

[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 chromatographic peak of paeoniflorin is not less than 10,000.

[0032] In certain preferred embodiments, the chromatographic column used is an Inert Sustain C18 chromatographic column.

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

[0034] In certain preferred embodiments, the construction method further includes the step of preparing a reference solution using at least one of liquiritin, ammonium glycyrrhizinate, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloyl glucose, benzoylpeoniflorin, ferulic acid, ligusticum chuanxiong lactone H, loganin, morroniside, and crystalline orchid glycoside, and the step of detecting the reference solution by high performance liquid chromatography according to any of the above construction methods to obtain a reference fingerprint.

[0035] The reference substance solution can be prepared by using any one or more of the reference substances selected from the group consisting of grass glycoside, ammonium glycyrrhizinate, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloyl glucose, benzoylpeoniflorin, ferulic acid, ligusticum chuanxiong lactone H, loganin, morroniside, and crystal orchid glycoside. When using two or more reference substances, the reference substances can be mixed to prepare a mixed solution, or the reference substance solution can be prepared separately.

[0036] According to the construction method described in any one of the present invention, the preparation method of the reference solution comprises the following steps: taking glycyrrhizin, ammonium glycyrrhizinate, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloyl glucose, benzoylpeoniflorin, ferulic acid, ligusticum chuanxiong lactone H, loganin, morroniside, and crystalline orchid reference substances, and adding a solvent to prepare a reference solution containing 1 to 100 μg of each reference substance per 1 ml; preferably, the solvent is selected from methanol or methanol aqueous solution; the volume fraction of methanol in the methanol aqueous solution is not less than 10%.

[0037] In certain preferred embodiments, the control fingerprint of the Tiaogan Decoction compound preparation is constructed, and the fingerprints obtained by testing multiple batches of the Tiaogan Decoction compound preparation samples are imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" calculation software to form a common pattern diagram and generate the control fingerprint of the Tiaogan Decoction compound preparation. In certain preferred embodiments, after the control fingerprint of the Tiaogan Decoction compound preparation is generated using the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, the step of marking the common characteristic peaks is also included.

[0038] At least 10 batches of the compound preparation of Tiaogan Decoction are used to obtain the control spectrum, for example, 10 batches, 15 batches, 20 batches, and 30 batches of the compound preparation of Tiaogan Decoction are used.

[0039] In certain preferred embodiments, the fingerprint of the compound preparation of Tiaogan Decoction contains 22 common characteristic peaks, and the relative standard deviation RSD values ​​of their retention times are all less than 10%; among them, the average retention time of peak 1 is: 7.637min; the average retention time of peak 2 is: 11.845min; the average retention time of peak 3 is: 24.337min; the average retention time of peak 4 is: 24.922min; the average retention time of peak 5 is: 26.741min; the average retention time of peak 6 is: 27.558min; the average retention time of peak 7 is: 28.413min; the average retention time of peak 8 is: 31.496min; the average retention time of peak 9 is: 34.935min; the average retention time of peak 10 is: 40.118 min; the average retention time of peak 11 is 52.807 min; the average retention time of peak 12 is 56.134 min; the average retention time of peak 13 is 57.682 min; the average retention time of peak 14 is 58.867 min; the average retention time of peak 15 is 62.534 min; the average retention time of peak 16 is 67.797 min; the average retention time of peak 17 is 71.537 min; the average retention time of peak 18 is 78.719 min; the average retention time of peak 19 is 82.982 min; the average retention time of peak 20 is 89.872 min; the average retention time of peak 21 is 97.081 min; the average retention time of peak 22 is 97.531 min.

[0040] In certain preferred embodiments, the fingerprint of the Tiaogan Decoction compound preparation comprises 22 common characteristic peaks, peak 10 is the paeoniflorin peak, the paeoniflorin peak is used as the reference peak, and the relative retention time of each characteristic peak and the reference peak is within the range of ±10% of the specified value; the specified values ​​of peaks 1 to 22 are, respectively: 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 chloranthus glycoside, peak 2 is gallic acid, peak 4 is morroniside, 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 ligustrin H, peak 18 is benzoylpeoniflorin, and peak 19 is ammonium glycyrrhizinate.

[0042] In certain preferred embodiments, peak 1 comes from Morinda officinalis, peak 2 comes from Angelica sinensis, Cornus officinalis and white peony root, peak 3 comes from Cornus officinalis, peak 4 comes from Cornus officinalis, peak 5 comes from white peony root, peak 6 comes from Angelica sinensis, peak 7 comes from Cornus officinalis, peak 8 comes from Cornus officinalis, peak 9 comes from white peony root, peak 10 comes from white peony root, peak 11 comes from Angelica sinensis, peak 12 comes from white peony root, peak 13 comes from Licorice, peak 14 comes from Licorice, peak 15 comes from Angelica sinensis, peak 16 comes from white peony root, peak 17 comes from Angelica sinensis, peak 18 comes from white peony root, peak 19 comes from Licorice, peak 20 comes from Angelica sinensis, peak 21 comes from Angelica sinensis, and peak 22 comes from Angelica sinensis.

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

[0044] Specifically, it includes the step of comparing the fingerprint of the Tiaogan Decoction compound preparation to be tested with the control fingerprint of the Tiaogan Decoction compound preparation; the fingerprint of the Tiaogan Decoction compound preparation to be tested is obtained using the Tiaogan Decoction compound preparation to be tested according to any construction method described in the present invention, and the control fingerprint of the Tiaogan Decoction compound preparation is the control fingerprint of the Tiaogan Decoction compound preparation described in the present invention.

[0045] If it meets the requirements, it is a qualified product; if it does not meet the requirements, it is an unqualified product.

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

[0047] (1) The fingerprint of the tested Tiaogan Decoction compound preparation presents 22 characteristic peaks, and the retention time of each characteristic peak is within ±10% of the retention time value of the corresponding chromatographic peak in the control fingerprint of the Tiaogan Decoction compound preparation;

[0048] (2) With the paeoniflorin peak as the S peak, the relative retention time of each characteristic chromatographic peak in the fingerprint of the tested Tiaogan Decoction compound preparation and the S peak is within ±10% of the relative retention time value of each characteristic peak in the reference fingerprint of the Tiaogan Decoction compound preparation;

[0049] (3) If the similarity between the fingerprint of the tested Tiaogan Decoction compound preparation and the control fingerprint of the Tiaogan Decoction compound preparation 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 using the Chinese medicine chromatographic fingerprint similarity evaluation software.

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

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

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

[0053] 2. The fingerprint spectrum construction method of the Tiaogan Decoction compound preparation described in the present 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 the classic Chinese medicine prescriptions.

[0054] 3. The fingerprint spectrum construction method of the Tiaogan Decoction compound preparation described in the present invention confirmed 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 basis and quality reference for the quality control of subsequent compound preparations.

[0055] 4. The quality detection method of the Tiaogan Decoction compound preparation described in the present invention is to construct the fingerprint of the Tiaogan Decoction compound preparation to be tested according to the construction method of the fingerprint of the Tiaogan Decoction compound preparation of the present invention, and perform quality detection on the characteristic effective ingredients of the Tiaogan Decoction compound preparation, so that the quality detection of the preparations related to the Tiaogan Decoction compound preparation is more comprehensive, and the effectiveness and controllability of the quality of the preparations related to the Tiaogan Decoction compound preparation are guaranteed. The fingerprint of each effective ingredient in the Tiaogan Decoction compound preparation of the present invention is regarded as a whole, and the identification of the characteristic peak groups contained in each medicinal taste in the composition is emphasized, which avoids the one-sidedness of evaluating the Chinese medicine compound as a whole by measuring only a few chemical components, and reduces the possibility of artificial processing for quality compliance, and provides a new method and means for comprehensively and accurately evaluating the quality of the 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0057] Figure 1 is the fingerprint of Tiaogan Decoction Granules when gradient 1 is used in Example 1;

[0058] Figure 2 is the fingerprint of Tiaogan Decoction Granules when gradient 2 is used in Example 1;

[0059] Figure 3 is the fingerprint of Tiaogan Decoction Granules when gradient 3 is used in Example 1;

[0060] Figure 4 is the fingerprint of Tiaogan Decoction Granules when gradient 4 is used in Example 1;

[0061] Figure 5 is the fingerprint of Tiaogan Decoction Granules when gradient 5 is used in Example 1;

[0062] Figure 6 is the fingerprint of Tiaogan Decoction Granules when the gradient is 6 in Example 1;

[0063] Figure 7 is the fingerprint of Tiaogan Decoction Granules when the gradient is 7 in Example 1;

[0064] Figure 8 It is the fingerprint spectrum of Tiaogantang granules when using different wavelengths in Example 1;

[0065] Fig. 9 is the fingerprint of Tiaogantang granules at different flow rates in Example 1;

[0066] Fig.10 It is the fingerprint spectrum of Tiaogan Decoction Granules at different column temperatures in Example 1;

[0067] Fig.11 is the fingerprint of Tiaogantang granules when different extraction methods are used in Example 2;

[0068] Fig.12 is the fingerprint of Tiaogantang granules when different solvents are used in Example 2;

[0069] Fig.13 It is the chromatographic peak identification overlay of the fingerprint of Tiaogan Decoction Granules in Example 3; wherein, S1 to S14 are respectively solvent blank, chloranthin reference substance, gallic acid reference substance, morroniside reference substance, catechin reference substance, loganin reference substance, paeoniflorin reference substance, ferulic acid reference substance, licorice reference substance, 1,2,3,4,6-pentagalloylglucose reference substance, ligusticum chuanxiong lactone H, benzoylpeoniflorin, ammonium glycyrrhizate, and Tiaogan Decoction test substance;

[0070] Fig.14 It is the superposition diagram of the chromatographic peaks attribution of the fingerprint spectrum of Tiaogan Decoction Granules in Example 4;

[0071] Fig.15 It is the similarity matching diagram of the instrument precision test of the fingerprint spectrum of Tiaogan Decoction Granules in Example 4;

[0072] Fig.16 It is the similarity matching diagram of the repeatability test of the fingerprint of Tiaogan Decoction Granules in Example 4;

[0073] Fig.17 It is the similarity matching diagram of the solvent stability test of the fingerprint spectrum of Tiaogan Decoction Granules in Example 4;

[0074] Fig.18 This is the reference fingerprint of Tiaogantang Granules. DETAILED DESCRIPTION

[0075] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0076] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0077] The present invention provides a method for constructing a fingerprint spectrum of a compound preparation of Tiaogan Decoction, comprising the following steps:

[0078] Preparation of the test solution: weigh an appropriate amount of Tiaogantang granules, place in a volumetric flask, add the first solvent, plug, ultrasonically treat, cool, shake, filter, and take the filtrate to obtain the test solution;

[0079] Preparation of reference solution: weigh appropriate amounts of glycyrrhizin, ammonium glycyrrhizinate, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloyl glucose, benzoylpeoniflorin, ferulic acid, ligusticolide H, loganin, morroniside, and crystalline orchid reference substances, and add 10% to 100% by volume, for example, 80% by volume, of methanol to prepare a reference solution of glycyrrhizin, ammonium glycyrrhizinate, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloyl glucose, benzoylpeoniflorin, ferulic acid, ligusticolide H, loganin, morroniside, and crystalline orchid with a concentration of 1 μg to 100 μg;

[0080] According to the results of high performance liquid chromatography detection of the test solution and the reference solution, a fingerprint spectrum of Tiaogantang granules is obtained;

[0081] The detection conditions of the high performance liquid chromatography are as follows: a chromatographic column whose filler is octadecylsilane bonded silica gel is used, a mobile phase A is selected from one or more of acetonitrile and methanol, a mobile phase B is an acid aqueous solution, an alkali aqueous solution and / or a buffered saline solution, a 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, a flow rate of 0.5-1.5 ml / min, a column temperature of 20-50° C., a detection wavelength of 200-400 nm, and an injection volume of 1-20 μl.

[0082] 1. Instruments and test drugs

[0083] Instruments: High performance liquid chromatograph Thermo Fisher U3000 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 cleaning machine: KQ-500 Kunshan Ultrasonic Instrument Co., Ltd.; intelligent constant temperature heating table: BY-2020, Bangyuan Electronics Co., Ltd.

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

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

[0086] Reference substance information: Crystalline orchid glycoside (batch number 111870-202004, content 96.5%, China Food and Drug Inspection Institute); Gallic acid (batch number 110831-201906, content 91.5%, China Food and Drug Inspection Institute); Morroniside (batch number 111998-202205, content 98.4%, China Food and Drug Inspection Institute); Catechins (batch number 110877-201604, content 99.2%, China Food and Drug Inspection Institute); Loganin (batch number 111870-202004, content 99.0%, China Food and Drug Inspection Institute); Paeoniflorin (batch number 110736-202145, content 94.6%, China Food and Drug Inspection Institute); Ferulic acid ( Batch number 110773-201614, content 99.0%, China Food and Drug Inspection Institute); glycyrrhizin (batch number 111610-201908, content 95.0%, China Food and Drug Inspection Institute); 1,2,3,4,6-pentagalloyl glucose (batch number 9618, content 95.9%, Shanghai Shidande Standard Technology Service Co., Ltd.); ligusticum chuanxiong lactone H (batch number 13866, content 95.2%, Shanghai Shidande Standard Technology Service Co., Ltd.); benzoylpeonidin (batch number MUST-14112704, content 99.28%, Chengdu Mansite Biotechnology Co., Ltd.); ammonium glycyrrhizinate (batch number 110731-202122, content 94.4%, China Food and Drug Inspection Institute).

[0087] The preparation method of the Tiaogan Decoction granules used in the following examples, embodiments and comparative examples is as follows:

[0088] Weigh 15g of Chinese yam, 9g of donkey-hide gelatin, 9g of Chinese angelica, 9g of white peony root, 9g of cornus officinalis, 3g of Morinda officinalis, and 3g of liquorice, add 9 times the weight of the raw material medicine, and soak for 30 minutes. Boil over high heat and then simmer for 60 minutes, filter out the liquid medicine, and set aside. Second decoction, add 7 times the weight of the raw material medicine, simmer over high heat until boiling and then simmer for 60 minutes, filter out the liquid medicine. Combine the two liquid medicines, dry after concentration, add appropriate amount of auxiliary materials and extrude granules to use as liver-regulating soup granules.

[0089] Example 1 Investigation of chromatographic conditions

[0090] 1. Preparation of test solution: Take Tiaogan Decoction granules, grind them into powder, weigh about 0.5 g, accurately weigh, put into a stoppered conical flask, accurately add 25 ml of 50% methanol aqueous solution by volume, plug it tightly, and treat it with ultrasound (power 250 W, frequency 40 kHz) for 30 minutes. Take it out, cool it, shake it well, filter it, and take the filtrate to obtain the solution.

[0091] 2. Optimization of chromatographic conditions

[0092] The test solution prepared in Item 1 of this Example was tested by HPLC according to the following method.

[0093] Octadecylsilane bonded silica gel is used as filler (chromatographic column is Inert Sustain C18; column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); acetonitrile is used as mobile phase A, 0.1% phosphoric acid is used as mobile phase B; gradient elution is performed according to the provisions in Table 1; detection wavelength is 190-400 nm. The theoretical plate number calculated based on the paeoniflorin peak should not be less than 10,000.

[0094] Table 1 Gradient elution program (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 show that under this condition, the chromatographic peaks are too concentrated within 20 to 30 minutes and 45 to 55 minutes, and the separation is poor. Therefore, in order to make each main chromatographic peak in the chromatogram have better separation parameters and present better chromatographic behavior, the test solution prepared in item 1 of this embodiment is taken, and the Shimadzu Inert Sustain C18 chromatographic column (column length 250mm, inner diameter 4.6mm, particle size 5μm) is selected; the detection wavelength is 240nm, acetonitrile is used as mobile phase A; 0.1% phosphoric acid solution is used as mobile phase B; the flow rate is 0.8ml per minute; the column temperature is 35°C, and the elution gradient is optimized. The optimized gradient program is as shown in the following table.

[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 program (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 program (Gradient 4)

[0102]

[0103]

[0104] Table 5 Gradient elution program table (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 table (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 program table (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] Results Figure 1-7 As shown, compared with gradient 1, under the optimized elution procedure of gradient 7, the number of chromatographic peaks increased from 100 to 111, the separation degree between the chromatographic peak where S peak (peonidin) is located and the surrounding peaks increased from 3.03 (gradient 1) to 3.39, the separation degree between peak 9 and the surrounding peaks under gradient 1 was only 1.35, which was increased to 2.27 under gradient 7, the separation degree between peak 1 (crystal orchid glycoside) and the surrounding peaks increased from 2.05 (gradient 1) to 3.16, and the number of plates increased from 13645 to 16823. Under gradient 1, the separation degree between peak 6 and peak 7 was only 1.35 (less than 1.5), which was increased to 2.27. Under gradient 1, peak 11 (ferulic acid) was not visible, and the separation degree between peak 12 and peak 13 (liquiritin) was only 1.21 (less than 1.5), which was increased to 2.03 under the optimized gradient 7. Therefore, it was determined that the chromatographic conditions of gradient 7 were selected for fingerprint determination.

[0111] 3. Selection of detection wavelength

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

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

[0114] 4. Investigation of flow rate

[0115] This section investigates the flow rate of the mobile phase, and the test solution prepared in Item 1 of this Example is tested by HPLC. The effects of flow rates of 0.7 ml per minute, 0.8 ml per minute, and 0.9 ml per minute on the fingerprint of Tiaogan Decoction were investigated respectively. A Shimadzu Inert Sustain C18 chromatographic column (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm) was selected, acetonitrile was used as mobile phase A; 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 7, the column temperature was 35°C, and the detection wavelength was 240 nm; the results are shown in Fig. 9 .

[0116] The results showed that when the flow rate was 0.7 ml / min, 21 chromatographic peaks appeared, of which peak 12 was merged with peak 13, and peaks 5, 11 and 19 were merged with the original surrounding peaks, resulting in a large difference in peak area; when the flow rate was 0.9 ml / min, 22 chromatographic peaks appeared, and the peak shapes of some peaks were poor, of which peak 3 was merged with the surrounding peaks, and peak 14 was split; while at 0.8 ml / min, there were no such problems. Therefore, in order to ensure the good reproducibility of the method, the recommended optimal flow rate was 0.8 ml / min.

[0117] 5. Investigation of column temperature

[0118] The test solution prepared in item 1 of this example was tested by HPLC. The column temperature was investigated, and the effects of column temperatures of 30°C, 35°C, and 40°C on the fingerprint of Tiaogan Decoction were investigated. A Shimadzu Inert Sustain C18 chromatographic column (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm) was selected, acetonitrile was used as mobile phase A; 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 7, the flow rate was 0.8 ml / min, and the detection wavelength was 240 nm. The results are shown in Table 7. Fig.10 .

[0119] The results showed that different chromatographic column temperatures had no significant effect on the fingerprint of Tiaogan Decoction, indicating that this method can adapt to certain changes in column temperature. However, considering the chromatographic peak shape, peak separation, baseline and other factors, the preferred chromatographic column temperature for the fingerprint study of Tiaogan Decoction in this experiment was 35°C.

[0120] 6. Determination of optimal chromatographic conditions

[0121] Based on the above experiments, the chromatographic conditions were determined as follows: octadecylsilane bonded silica gel as filler (InertSustain C18, column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B; gradient elution according to the provisions in Table 7; flow rate 0.8 ml per minute; column temperature 35°C; detection wavelength 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. Investigation of extraction methods

[0124] Different extraction methods will affect the extraction efficiency of each chromatographic peak. Therefore, the extraction efficiency of each chromatographic peak of ultrasonic extraction and heating reflux extraction is compared, and the appropriate extraction method is determined in combination with the peak area of ​​each characteristic peak; take about 0.5g of Tiaogantang granules, grind them into powder, accurately weigh them, put them in a stoppered conical flask, accurately add 25ml of 50% methanol aqueous solution by volume, plug it, and use ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes or reflux extraction for 30 minutes, respectively, take it out, cool, shake well, filter, and take the filtrate. Use high performance liquid chromatography to detect under the optimal chromatographic conditions of Example 1, see Fig.11 shown.

[0125] The results showed that 22 characteristic peaks appeared when ultrasonic or reflux extraction was used. There was no difference in the ratio of peak area to sampling volume. Considering the convenience of sample preparation, ultrasonic extraction was the preferred extraction method for the sample.

[0126] 2. Investigation of extraction solvent

[0127] Different proportions of solvents will affect the extraction efficiency of each chromatographic peak. Therefore, the extraction efficiency of each chromatographic peak extracted by 50% methanol, 80% methanol and methanol is compared, and the appropriate extraction solvent is determined in combination with the peak area of ​​each characteristic peak. Specifically, about 0.5g of Tiaogantang granules is taken, ground, accurately weighed, placed in a stoppered conical flask, accurately added with 50% or 80% methanol aqueous solution or 25ml of methanol by volume percentage, plugged, and ultrasonically treated (power 250W, frequency 40kHz) for 30 minutes respectively, taken out, cooled, shaken, filtered, and the filtrate is obtained. Detection by high performance liquid chromatography under the optimal chromatographic conditions of Example 1, see Fig.12 shown.

[0128] Analysis of the results showed that 50% methanol, 80% methanol and methanol all had 22 chromatographic peaks. Comparing the peak area / sampling volume results of each characteristic peak, the peak area was higher when the solvent was 80% methanol, so the preferred solvent for preparing the test sample was 80% methanol.

[0129] 3. Determine the preparation method of the test solution

[0130] Take about 0.5 g of the test sample, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of 80% methanol, stopper it tightly, and treat it with ultrasound (power 250 W, frequency 40 kHz) for 30 minutes. Take it out, cool it, shake it well, filter it, and take the filtrate to obtain the product.

[0131] Example 3 Peak attribution and selection of references

[0132] (1) Construction of reference fingerprint

[0133] Fifteen batches of samples of Tiaogan Decoction were taken respectively, and the sample solutions prepared by the preparation method of the sample solution determined in Example 2 were measured under the optimal chromatographic conditions of Example 1 to construct multiple batches of fingerprints. The fingerprint detection AIA data was imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" calculation software, and a total of 22 peaks were calibrated to form a common pattern diagram, and a control spectrum was established. Fig.18 Peak 10 was selected, and the paeoniflorin peak was used as the reference peak, which was marked as Peak S.

[0134] There are 22 common characteristic peaks in the control fingerprint, namely: Peak 1, average retention time: 7.637min, RSD: 0.45%; Peak 2, average retention time: 11.845min, RSD: 0.31%; Peak 3, average retention time: 24.337min, RSD: 0.12%; Peak 4, average retention time: 24.922min, RSD: 0.11%; Peak 5, average retention time: 26.741min, RSD: 0.10%; Peak 6 , the average retention time is: 27.558min, RSD is 0.10%; peak 7, the average retention time is: 28.413min, RSD is 0.10%; peak 8, the average retention time is: 31.496min, RSD is 0.12%; peak 9, the average retention time is: 34.935min, RSD is 0.11%; peak 10, the average retention time is: 40.118min, RSD is 0.10%; peak 11, the average retention time is: 52.807min, RSD is 0.04%; Peak 12, average retention time: 56.134min, RSD: 0.05%; Peak 13, average retention time: 57.682min, RSD: 0.03%; Peak 14, average retention time: 58.867min, RSD: 0.03%; Peak 15, average retention time: 62.534min, RSD: 0.02%; Peak 16, average retention time: 67.797min, RSD: 0.01%; Peak 17, average retention time: The average retention time of peak No. 18 was 78.719 min, and the RSD was 0.01%. The average retention time of peak No. 19 was 82.982 min, and the RSD was 0.01%. The average retention time of peak No. 20 was 89.872 min, and the RSD was 0.01%. The average retention time of peak No. 21 was 97.081 min, and the RSD was 0.01%. The average retention time of peak No. 22 was 97.531 min, and the RSD was 0.01%.

[0135] Taking the paeoniflorin peak as the reference peak, marked as Peak S, the relative retention time of each characteristic peak and Peak S is within the range of ±10% of the specified value; the specified values ​​of Peak 1 to Peak 30 are: 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, respectively.

[0136] According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints, the similarity was calculated based on the common peaks. The similarities between the test sample fingerprint and the control fingerprint were both higher than 0.900. The differences between the fingerprints of each batch of particles were small based on the similarity evaluation.

[0137] (2) Characteristic peak identification

[0138] Take appropriate amount of glycyrrhizin, ammonium glycyrrhizinate, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloylglucose, benzoylpeoniflorin, ferulic acid, ligusticum lactone H, loganin, morroniside and crystalline orchid, weigh accurately, 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 reference solution and the test solution prepared by the preparation method of the test solution determined in Example 2 were measured under the optimal chromatographic conditions of Example 1, and the fingerprint of the test sample of Tiaogan Decoction was compared with the chromatogram of the reference solution. The results are shown in Fig.13 As shown, peak 1 is chloranthus glycoside, peak 2 is gallic acid, peak 4 is morroniside, 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 ligustrin H, peak 18 is benzoylpeoniflorin, and peak 19 is ammonium glycyrrhizinate.

[0140] Example 4 Methodology Verification

[0141] (1) Exclusivity

[0142] ① Preparation of negative sample solution lacking yam: weigh other medicinal ingredients except yam according to the prescription of Tiaogan Decoction Granules, prepare negative sample lacking yam according to the preparation method of Tiaogan Decoction Granules, and prepare negative sample solution lacking yam according to the "preparation method of determined test solution" in Example 2;

[0143] ② Preparation of negative sample solution lacking donkey-hide gelatin: weigh the other medicinal ingredients except donkey-hide gelatin according to the prescription of Tiaogan Decoction Granules, prepare a negative sample lacking donkey-hide gelatin according to the preparation method of Tiaogan Decoction Granules, and prepare a negative sample solution lacking donkey-hide gelatin according to the "preparation method of the determined test 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 the determined test 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 a negative sample lacking white peony root according to the preparation method of Tiaogan Decoction Granules, and prepare a negative sample solution lacking white peony root according to the "preparation method of the determined test solution" in Example 2;

[0146] ⑤ Preparation of negative sample solution lacking cornus: weigh the other medicinal ingredients except cornus according to the prescription of Tiaogan Decoction Granules, prepare negative sample lacking cornus according to the preparation method of Tiaogan Decoction Granules, and prepare negative sample solution lacking cornus according to the "preparation method of the determined test solution" in Example 2;

[0147] ⑥ Preparation of negative sample solution lacking Morinda officinalis: weigh the other medicinal ingredients 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 the determined test solution" in Example 2;

[0148] ⑦ Preparation of negative sample solution lacking licorice: According to the prescription of Tiaogan Decoction Granules, weigh the other medicinal ingredients except licorice, prepare the negative sample lacking licorice according to the preparation method of Tiaogan Decoction Granules, and prepare the negative sample solution lacking licorice according to Example 2 "Determined preparation method of test solution".

[0149] The test solution prepared by the preparation method of the test solution determined in Example 2 and the negative sample solution were accurately taken and measured according to the high performance liquid chromatography under the optimal chromatographic conditions of Example 1. The 22 fingerprint peaks were assigned as follows: Fig.14 As shown in Table 8, there was no negative interference and the method had good specificity.

[0150] As shown in Table 8, peak 1 comes from Morinda officinalis, peak 2 comes from Angelica sinensis, Cornus officinalis and white peony root, peak 3 comes from Cornus officinalis, peak 4 comes from Cornus officinalis, peak 5 comes from white peony root, peak 6 comes from Angelica sinensis, peak 7 comes from Cornus officinalis, peak 8 comes from Cornus officinalis, peak 9 comes from white peony root, peak 10 comes from white peony root, peak 11 comes from Angelica sinensis, peak 12 comes from white peony root, peak 13 comes from Licorice, peak 14 comes from Licorice, peak 15 comes from Angelica sinensis, peak 16 comes from white peony root, peak 17 comes from Angelica sinensis, peak 18 comes from white peony root, peak 19 comes from Licorice, 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 test solution prepared by the preparation method of the test solution determined in Example 2, inject it continuously 6 times under the optimal chromatographic conditions of Example 1, record the chromatogram, and calculate the RSD of the relative retention time and relative peak area of ​​each fingerprint peak.

[0156] like Fig.15 As shown, the results show that the RSD of this method meets the requirements and the similarity of 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 samples from the same batch, 0.5 g each, accurately weigh, prepare the test sample according to the preparation method of the test solution determined in Example 2, measure it according to the high performance liquid chromatography under the optimal chromatographic conditions of Example 1, record the chromatogram, and calculate the relative retention time and relative peak area RSD of each fingerprint peak.

[0159] like Fig.16 As shown, the results show that the RSD of its repeatability meets the requirements and the similarity results are all greater than 0.90, which is in line with the regulations.

[0160] (4) Solvent stability test

[0161] Take the test solution prepared by the same preparation method of the test solution determined in Example 2, inject at 0h, 6h, 12h, 24h, 30h and 36h, and measure under the optimal chromatographic conditions of Example 1. Record the chromatogram, calculate the relative retention time and relative peak area RSD of each fingerprint peak, and investigate the stability of the test solution.

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

[0163] According to the results of the above methodological investigation, among the 22 common peaks of the established fingerprint, each chromatographic peak is less affected by factors such as different column temperatures and flow rates, and the influence of other chromatographic conditions does not change much, indicating that the method has good durability, is stable and reliable, and can be used for fingerprint determination.

[0164] Chromatographic conditions and system suitability: octadecylsilane bonded silica gel as filler; acetonitrile as mobile phase A; 0.1% phosphoric acid solution as mobile phase B; gradient elution as specified in Table 7; flow rate of 0.8 ml per minute; column temperature of 35°C; detection wavelength of 240 nm. The theoretical plate number calculated based on the paeoniflorin peak should not be less than 10,000.

[0165] Preparation of reference solution: Take appropriate amount of glycyrrhizin, ammonium glycyrrhizinate, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloyl glucose, benzoylpeoniflorin, ferulic acid, ligusticum chuanxiong lactone H, loganin, morroniside, and crystalline orchid, weigh accurately, add 50% methanol to make a mixed solution containing 0.05 mg of each per ml, as the reference solution.

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

[0167] Determination method: Accurately aspirate 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0168] According to the above-mentioned fingerprint detection method, three batches of Tiaogantang Granules samples were tested. The results showed that the RSD of the relative retention time of each common peak was 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%, all less than 5%. The similarity results of the three batches of samples were greater than 0.90, which was in compliance with the regulations.

[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 method of Tiaogan Decoction and has guiding significance for the quality detection and evaluation of the variety.

[0170] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for constructing a fingerprint spectrum of a compound preparation of Tiaogan Decoction, characterized in that: The following steps are included: (1) Preparation of test solution; (2) The sample solution was tested by high performance liquid chromatography, using octadecylsilane bonded silica gel as filler and acetonitrile-phosphoric acid aqueous solution as mobile phase for gradient elution; the gradient elution program included: 0→10 min→20 min→40 min→60 min→90 min→95 min→115 min, and the volume percentage of acetonitrile in the mobile phase was: 2%→5%→12%→12%→18%→60%→60%→75%.

2. The method for constructing the fingerprint of the Tiaogan Decoction compound preparation according to claim 1, characterized in that: Step (1) comprises: weighing the compound preparation of Tiaogan Decoction, extracting with a solvent, separating the solid from the liquid, and taking the liquid, which is the test solution.

3. The method for constructing the fingerprint of the Tiaogan Decoction compound preparation according to claim 2, characterized in that: The step (1) also satisfies any one or more of the following AE: A. The extraction is ultrasonic extraction or thermal reflux extraction; B. Extraction time is 10-60 minutes; C. The ratio of the mass of the compound preparation of Tiaogan Decoction to the volume of the solvent is 0.3-0.6g:20-50ml; D. The solid-liquid separation is selected from centrifugation or filtration; E. The solvent is selected from at least one of methanol, water and ethanol; preferably methanol or a methanol aqueous solution with a volume percentage of not less than 50%; more preferably a methanol aqueous solution with a volume percentage of 80%.

4. The method for constructing the fingerprint of the Tiaogan Decoction compound preparation according to any one of claims 1 to 3, characterized in that: Step (2) also satisfies any one or more of the following items 1) to 6): 1) The gradient elution program also includes: 115min→120min, the volume percentage of acetonitrile in the mobile phase is: 75%→75%; 2) The volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid is 0.05%-0.15%; 3) The detection wavelength is 200nm-400nm, preferably 240nm, 4) The column temperature is 30°C-50°C, preferably 35°C; 5) The flow rate is 0.5 ml / min-1.5 ml / min, preferably 0.8 ml / min; 6) The injection volume is 1 μl-20 μl, preferably 10 μl.

5. The method for constructing the fingerprint of the Tiaogan Decoction compound preparation according to any one of claims 1 to 4, characterized in that: The construction method also includes the step of preparing a reference solution using at least one of liquiritin, ammonium glycyrrhizinate, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloyl glucose, benzoylpeoniflorin, ferulic acid, ligusticum chuanxiong lactone H, loganin, morroniside, and quartz orchid glycoside, and the step of detecting the reference solution by high performance liquid chromatography according to the construction method described in any one of claims 1 to 4 to obtain a reference fingerprint.

6. The method for constructing the fingerprint of the Tiaogan Decoction compound preparation according to claim 5, characterized in that: The preparation method of the reference solution comprises the following steps: taking glycyrrhizin, ammonium glycyrrhizinate, gallic acid, catechin, paeoniflorin, 1,2,3,4,6-pentagalloyl glucose, benzoylpeoniflorin, ferulic acid, ligusticum chuanxiong lactone H, loganin, morroniside, and crystalline orchid reference substances, and adding a solvent to prepare a reference solution containing 1 to 100 μg of each reference substance per 1 ml; preferably, the solvent is selected from methanol or methanol aqueous solution; the volume fraction of methanol in the methanol aqueous solution is not less than 10%.

7. The method for constructing the fingerprint of the Tiaogan Decoction compound preparation according to any one of claims 1 to 6, characterized in that: The fingerprint of the compound preparation of Tiaogan Decoction contains 22 common characteristic peaks, and the relative standard deviation RSD values ​​of their retention times are all less than 10%; among them, the average retention time of peak 1 is: 7.637min; the average retention time of peak 2 is: 11.845min; the average retention time of peak 3 is: 24.337min; the average retention time of peak 4 is: 24.922min; the average retention time of peak 5 is: 26.741min; the average retention time of peak 6 is: 27.558min; the average retention time of peak 7 is: 28.413min; the average retention time of peak 8 is: 31.496min; the average retention time of peak 9 is: 34.935min; the average retention time of peak 10 is: 40.118min; The average retention time of Peak 1 is 52.807 min; the average retention time of Peak 12 is 56.134 min; the average retention time of Peak 13 is 57.682 min; the average retention time of Peak 14 is 58.867 min; the average retention time of Peak 15 is 62.534 min; the average retention time of Peak 16 is 67.797 min; the average retention time of Peak 17 is 71.537 min; the average retention time of Peak 18 is 78.719 min; the average retention time of Peak 19 is 82.982 min; the average retention time of Peak 20 is 89.872 min; the average retention time of Peak 21 is 97.081 min; the average retention time of Peak 22 is 97.531 min.

8. The method for constructing the fingerprint of the Tiaogan Decoction compound preparation according to any one of claims 1 to 6, characterized in that: The fingerprint of the compound preparation of Tiaogan Decoction contains 22 common characteristic peaks, peak 10 is the paeoniflorin peak, the paeoniflorin peak is used as the reference peak, and the relative retention time of each characteristic peak and the reference peak is within the range of ±10% of the specified value; the specified values ​​of peaks 1 to 22 are: 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 respectively.

9. The method for constructing the fingerprint of the Tiaogan Decoction compound preparation according to claim 7 or 8, characterized in that: Peak 1 is quartz orchid glycoside, peak 2 is gallic acid, peak 4 is morroniside, peak 5 is catechin, peak 8 is loganin, peak 10 is paeoniflorin, peak 11 is ferulic acid, peak 13 is liquiritigenin, peak 16 is 1,2,3,4,6-pentagalloylglucose, peak 17 is ligustrin H, peak 18 is benzoylpeoniflorin, and peak 19 is ammonium glycyrrhizinate; Preferably, peak 1 comes from Morinda officinalis, peak 2 comes from Angelica sinensis, Cornus officinalis and white peony root, peak 3 comes from Cornus officinalis, peak 4 comes from Cornus officinalis, peak 5 comes from white peony root, peak 6 comes from Angelica sinensis, peak 7 comes from Cornus officinalis, peak 8 comes from Cornus officinalis, peak 9 comes from white peony root, peak 10 comes from white peony root, peak 11 comes from Angelica sinensis, peak 12 comes from white peony root, peak 13 comes from Licorice, peak 14 comes from Licorice, peak 15 comes from Angelica sinensis, peak 16 comes from white peony root, peak 17 comes from Angelica sinensis, peak 18 comes from white peony root, peak 19 comes from Licorice, peak 20 comes from Angelica sinensis, peak 21 comes from Angelica sinensis, and peak 22 comes from Angelica sinensis.

10. A quality detection method for a compound preparation of Tiaogan Decoction, characterized in that: The method comprises taking the Tiaogan Decoction compound preparation to be tested and constructing a fingerprint of the Tiaogan Decoction compound preparation to be tested according to the fingerprint construction method of the Tiaogan Decoction compound preparation according to any one of claims 1 to 9.

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