Ultraviolet fingerprint spectrum identification technology for traditional Chinese medicine compound containing glossy privet fruit and application of ultraviolet fingerprint spectrum identification technology

Abstract

The invention discloses a construction method of a traditional Chinese medicine compound ultraviolet fingerprint spectrum containing glossy privet fruit. The construction method comprises the following steps: preparing a test solution; preparing a reference substance solution; and obtaining the fingerprint spectrum of the traditional Chinese medicine composition according to results of high performance liquid detection of the test solution and the reference solution. The chemical components of the traditional Chinese medicine compound containing glossy privet fruit are comprehensively and systematically analyzed, and a theoretical basis is provided for deep research on quality control and pharmacodynamic material basis.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly to an ultraviolet fingerprint identification technology for a traditional Chinese medicine compound containing Glossy Privet Fruit and its application. Background Art

[0002] According to the "Traditional Chinese Medicine Law of the People's Republic of China", ancient classic formulas refer to "formulas recorded in ancient traditional Chinese medicine classics that are still widely used, have definite curative effects, and have obvious characteristics and advantages". Ancient classic formulas have a long history and a rich history of human use in China and are still in use today. They are the summary of the clinical practice essence of past generations of medical experts, carrying the profound accumulation of the brilliant civilization of traditional Chinese medicine over thousands of years. They are the refinement of traditional Chinese medicine theory over thousands of years and the summary of clinical experience of past generations, and are the most essential part of the great treasure house of traditional Chinese medicine. Conducting in-depth research and development on traditional Chinese medicine classic formulas is a golden key to exploring the treasure house of traditional Chinese medicine.

[0003] As the most commonly used dosage form for traditional Chinese medicine clinical use, the decoction of traditional Chinese medicine classic formulas has the advantages of reasonable formula, rapid onset, significant curative effect, and easy absorption, and is deeply trusted by the majority of patients. However, due to disadvantages such as being difficult to prepare, carry, decoct temporarily, prone to mildew and deterioration after long-term storage, bitter taste of the decoction, large volume, and difficulty in standardizing, which seriously affect its clinical curative effect, it cannot meet the living requirements of modern people. In order to maintain the advantages of the decoction and overcome its various deficiencies, the "Technical Guidelines for the Pharmaceutical Research of Traditional Chinese Medicine Compound Preparations Managed According to the Ancient Classic Formula Catalogue (Trial)" issued by the Center for Drug Evaluation of the National Medical Products Administration on August 31, 2021, clearly states that the quality of traditional Chinese medicine compound preparations managed according to the ancient classic formula catalogue should be basically consistent with the quality of the reference samples of the classic formulas. The reference samples represent the overall internal quality of the preparations. Except for the shaping process, the remaining quality control indicators of the reference samples are basically the same as those of the preparations. Therefore, the reference samples are the physical controls for the internal quality of the preparations and the reference objects for optimizing the large-scale production process and formulating its quality standards. The reference samples are the benchmarks for the research and development of classic formulas and even all traditional Chinese medicines, and are the controls to ensure the safety and effectiveness of drugs. The formulation of the production process route, optimization of parameters, and formulation of quality standards for traditional Chinese medicine compound preparations of classic formulas should be based on the reference samples of classic formulas. The reference samples are the link connecting clinical practice, enterprises, and scientific research, and are the benchmarks for inheriting, applying, and carrying forward traditional Chinese medicine.

[0004] Fingerprint is a spectral or chromatogram of traditional Chinese medicine chemical components obtained by means of spectroscopy and chromatography based on the understanding of the overall action of the material group of traditional Chinese medicine. It is a feasible mode to realize the authenticity identification of traditional Chinese medicine, evaluate the quality consistency and product stability, and has the characteristics of large amount of information, strong specificity, integrity and ambiguity. The fingerprint of traditional Chinese medicine can comprehensively reflect the relative relationship of chemical components contained in medicinal materials, embody the complexity and correlation of traditional Chinese medicine components, adapt to the traditional theory of traditional Chinese medicine, and can effectively characterize, comprehensively evaluate and overall control the internal quality of traditional Chinese medicine. It is especially suitable for the quality control of Chinese medicinal materials and traditional Chinese medicine products when the active ingredients are not completely clear or do not need to be completely clear. In addition to being used to investigate factors such as the origin, harvesting season, harvesting part, processing, storage time, etc. of Chinese medicinal materials to provide a basis for identifying the authenticity and quality of raw medicinal materials before production, the fingerprint of traditional Chinese medicine can also be used for the quality control of the production process of traditional Chinese medicine: tracking the changes of certain chemical components in the preparation, monitoring the quality consistency and stability between raw medicinal materials and finished products, and between batches of finished products. Compared with the quality analysis method of determining the content of index components, the fingerprint can more comprehensively reflect the types and quantities of chemical components in traditional Chinese medicine. Under the current situation that the active ingredients of traditional Chinese medicine compound preparations have not been fully elucidated, it can realize the comprehensive evaluation of the internal quality of traditional Chinese medicine and the effective control of its overall substances, and is one of the effective means for the quality control of traditional Chinese medicine and its preparations at present.

[0005] At present, there is no literature report on the comprehensive analysis of the chemical components and fingerprint research of the traditional Chinese medicine compound containing Glossy Privet Fruit claimed in the present invention. In the prior art, only the quality analysis of single components in this traditional Chinese medicine compound is carried out, and there is no relatively comprehensive and systematic quality control method to reflect the quality status of the main reference sample components in the traditional Chinese medicine compound and finished products of the present invention, and it is impossible to effectively control its production process and product quality, and it is not possible to better ensure its clinical efficacy. Therefore, it is necessary to adopt a quality control method of fingerprint that can comprehensively control the overall quality of the traditional Chinese medicine compound of the present invention to control its key quality. Summary of the Invention

[0006] Based on this, the present invention provides a method for constructing an ultraviolet fingerprint of a traditional Chinese medicine compound containing Glossy Privet Fruit. The construction method includes the following steps: Preparation of the test solution of the traditional Chinese medicine compound: Take an appropriate amount of the traditional Chinese medicine compound granules containing Glossy Privet Fruit, place them in a container, add a solvent, ultrasonically treat for a period of time, make up the volume, shake well, take the supernatant and filter, and take the continued filtrate to obtain the test solution of the traditional Chinese medicine compound; wherein, the traditional Chinese medicine compound includes Glossy Privet Fruit, Oyster, Mother-of-Pearl, Chinese Wolfberry Fruit, White Peony Root, Caulis Polygoni Multiflori, Poria Cocos, and Atractylodes Macrocephala; Preparation of the reference solution: Weigh appropriate amounts of paeoniflorin, paeonolide glycoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, specnuezhenide, and atractylenolide III reference substances, and add methanol to prepare a reference solution with the concentrations of paeoniflorin, paeonolide glycoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, specnuezhenide, and atractylenolide III being 1-200 μg / ml respectively; According to the results of high performance liquid chromatography (HPLC) detection of the test solution of the traditional Chinese medicine compound and the reference solution, obtain the ultraviolet fingerprint of the traditional Chinese medicine compound; The chromatographic conditions for the HPLC detection are: Use a chromatographic column packed with octadecylsilyl silica gel, mobile phase A is selected from one or more of acetonitrile, methanol, and tetrahydrofuran, mobile phase B is an acidic aqueous solution, an alkaline aqueous solution, and / or a buffered saline solution, and the gradient elution program is: 0-8 min, 95% → 82% B; 8-18 min, 82% B → 78% B; 18-22 min, 78% B; 22-27 min, 78% → 76% B; 27-34 min, 76% → 27% B; 34-39 min, 27% B → 20% B; 39-40 min, 20% → 95% B; 40-55 min, 95% B; The flow rate is 0.5-1.5 ml / min, the column temperature is 10-40 °C, the detection wavelength is 180-300 nm, and the injection volume is 1-20 μl.

[0007] Further, in the preparation of the traditional Chinese medicine compound test solution, the solvent is alcohol, such as methanol. Further, the volume percentage concentration of the methanol is 20% to 80%, preferably 40% to 60%, such as about 50%. Further, the container is a volumetric flask. Further, the mass of the traditional Chinese medicine compound granules is 5 to 30 g, such as about 15 g. Further, the mass / volume ratio between the traditional Chinese medicine compound granules and the solvent is 0.4 to 0.8, such as about 0.6, with the unit of g / ml. Further, the power of the ultrasonic treatment is 400 to 600 W, such as about 500 W. Further, the frequency of the ultrasonic treatment is 30 to 50 kHz, such as about 40 kHz. Further, the time of the ultrasonic treatment is 20 to 40 min, such as about 30 min. Further, the preparation method of the traditional Chinese medicine compound granules is as follows: Weigh 8 to 20 g, such as about 12 g, of Glossy Privet Fruit, 8 to 20 g, such as about 12 g, of Raw Oyster, 5 to 15 g, such as about 10 g, of Mother-of-Pearl, 8 to 20 g, such as about 12 g, of Chinese Wolfberry Fruit, 5 to 15 g, such as about 10 g, of White Peony Root, 8 to 20 g, such as about 12 g, of Caulis Polygoni Multiflori, 8 to 20 g, such as about 15 g, of Poria, 1 to 10 g, such as about 6 g, of Atractylodes Macrocephala Koidz., soak in water, extract by reflux, filter, take the continuous filtrate, concentrate to a clear extract with a relative density of about 1.20, add an appropriate amount of dextrin, make into granules, and dry to obtain the traditional Chinese medicine compound granules. Further, the mass / volume ratio between the traditional Chinese medicine compound and the water is 0.01 to 0.5, such as about 0.125, with the unit of g / ml. Further, the volume of the water is 500 to 1000 ml, such as about 720 ml. Further, the soaking time is 20 to 60 min, such as about 40 min. Further, the reflux extraction time is 1 to 3 h, such as about 2 h. Further, the filtration is filtration through a filter screen. Further, the filter screen is a 200-mesh filter screen. Further, the concentration of albiflorin in the reference solution is 80 to 120 μg / ml, such as about 100 μg / ml or about 109.7 μg / ml. Further, the concentration of paeoniflorin in the reference solution is 15 to 25 μg / ml, such as about 20 μg / ml or about 21.42 μg / ml. Further, the concentration of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in the reference solution is 80 to 120 μg / ml, such as about 100 μg / ml or about 102.5 μg / ml. Further, the concentration of acteoside in the reference solution is 80 to 120 μg / ml, such as about 100 μg / ml or about 102.4 μg / ml. Further, the concentration of atractylenolide III in the reference solution is 2 to 4 μg / ml, such as about 3 μg / ml or about 2.56 μg / ml. Further, the flow rate of the high performance liquid chromatography detection is 0.7 to 1.1 ml / min, such as about 0.9 ml / min. Further, the column temperature is 20 to 30 °C, such as about 25 °C.Further, the detection wavelength is 200 - 240 nm, such as about 224 nm. Further, the sample injection volume is 5 - 15 μl, such as about 10 μl. Further, the theoretical plate number of the chromatographic peak corresponding to paeoniflorin is not less than 2000. Further, the chromatographic column is a ZORBAX SB-C18 chromatographic column. Further, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm. Further, mobile phase A is acetonitrile. Further, the acidic aqueous solution, basic aqueous solution and / or buffer saline solution are selected from one or more of weak acids and their salts, weak bases and their salts with different concentrations. Further, the acidic aqueous solution, basic aqueous solution and / or buffer saline solution are selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid with different concentrations. Further, the acidic aqueous solution is an acidic aqueous solution with a concentration of 0.01% - 1%. Further, the acidic aqueous solution is a phosphoric acid aqueous solution with a concentration of 0.01% - 1%. Further, the acidic aqueous solution is a phosphoric acid aqueous solution with a concentration of about 0.1%. Further, the buffer saline solution is an acetate aqueous solution and / or an acetate aqueous solution. Further, the pH value of the buffer saline solution is not greater than 7.0. Further, when the detection wavelength is 224 nm, the ultraviolet fingerprint spectrum includes peaks 1 - 8, and the retention times of peaks 1 - 8 correspond to about 6.315 min, about 13.234 min, about 14.253 min, about 18.608 min, about 19.024 min, about 19.598 min, about 20.453 min, about 37.394 min respectively. Further, when the detection wavelength is 224 nm, the ultraviolet fingerprint spectrum includes peaks 1 - 8, where peak 3 is used as the reference peak, and the average relative retention times of peaks 1, 2, 4, 5, 6, 7, 8 correspond to about 0.443, about 0.929, about 1.306, about 1.335, about 1.375, about 1.435, about 2.624 respectively. Further, when the detection wavelength is 224 nm, peak 2 is albiflorin, peak 3 is paeoniflorin, peak 4 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, peak 6 is acteoside, and peak 8 is atractylenolide III. Further, when the detection wavelength is 224 nm, peak 1 comes from Caulis Polygoni Multiflori and Radix Paeoniae Alba herbs, peak 2 comes from Radix Paeoniae Alba herb, peak 3 comes from Radix Paeoniae Alba herb, peak 4 comes from Caulis Polygoni Multiflori herb, peak 5 comes from Radix Paeoniae Alba herb, peak 6 comes from Fructus Ligustri Lucidi herb, peak 7 comes from Fructus Lycii herb, and peak 8 comes from Rhizoma Atractylodis Macrocephalae herb.

[0008] According to another aspect of the present invention, there is provided a method for identifying a traditional Chinese medicine compound containing Glossy Privet Fruit, and the identification method includes the following steps: (1) establishing a standard ultraviolet fingerprint spectrum of a reference sample of the traditional Chinese medicine compound according to the above ultraviolet fingerprint spectrum construction method; (2) taking a test solution of the traditional Chinese medicine compound, and performing detection according to the chromatographic conditions in the above ultraviolet fingerprint spectrum construction method to obtain an ultraviolet fingerprint spectrum of the test sample of the traditional Chinese medicine compound; and (3) comparing the ultraviolet fingerprint spectrum of the test sample of the traditional Chinese medicine compound obtained in step (2) with the standard ultraviolet fingerprint spectrum of the reference sample of the traditional Chinese medicine compound obtained in step (1). If it meets the requirements, it is a qualified product; if it does not meet the requirements, it is an unqualified product. Further, the meeting of the requirements includes one or more of the following: (1) 8 characteristic chromatographic peaks appear in the ultraviolet fingerprint spectrum of the test sample of the traditional Chinese medicine compound, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference substance chromatographic peak in the standard ultraviolet fingerprint spectrum of the reference sample of the traditional Chinese medicine compound; (2) taking the 3rd peak as the S peak, the relative retention time of each characteristic chromatographic peak in the ultraviolet fingerprint spectrum of the test sample of the traditional Chinese medicine compound and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard ultraviolet fingerprint spectrum of the reference sample of the traditional Chinese medicine compound; and (3) according to the similarity evaluation system of the ultraviolet fingerprint spectrum of traditional Chinese medicine chromatography, after similarity calculation of the ultraviolet fingerprint spectrum of the test sample of the traditional Chinese medicine compound and the standard ultraviolet fingerprint spectrum of the reference sample of the traditional Chinese medicine compound, the similarity shall not be lower than 0.90.

[0009] According to another aspect of the present invention, there is provided the use of the above construction method or the above identification technique in the quality detection and / or quality evaluation and / or quality control of a traditional Chinese medicine compound containing Glossy Privet Fruit.

[0010] Advantages of the present invention:

[0011] In short, the present invention provides a method for fingerprint determination and quality control of a reference sample of a traditional Chinese medicine composition containing Glossy Privet Fruit. This method confirms 8 common characteristic peaks, has simple conditions and short analysis time, solves the problems of difficult separation of fingerprint characteristic peaks and interference of impurity peaks, ensures the chemical composition stability and use safety of the reference sample, provides an important reference basis for the quality control of subsequent preparations, ensures the quality stability of the product, guarantees the efficacy of the traditional Chinese medicine compound, and enables the traditional Chinese medicine compound to better serve human life and health.

[0012] Specifically, compared with the prior art, the beneficial effects of the present invention are as follows: (1) By establishing the fingerprint of the traditional Chinese medicine compound reference sample of the present invention, it overcomes the defect that it is difficult to reflect the overall content by measuring the content of a single component. It can control the internal quality of the traditional Chinese medicine compound reference sample of the present invention as a whole and macroscopically, ensuring the efficacy of the drug. Using the main means of modern drug research, it realizes the inheritance of classic formulas with high-quality products, enabling classic famous formulas to obtain more formal quality control; (2) The chemical components in the traditional Chinese medicine compound reference sample of the present invention are complex, and it is difficult to separate its characteristic peaks. In the process of establishing the fingerprint, the present invention adopts the method of gradient elution, solving the problems of difficult separation of fingerprint characteristic peaks and interference of impurity peaks; (3) In the process of establishing the fingerprint of the traditional Chinese medicine compound reference sample of the present invention, 8 common characteristic peaks are confirmed, and their relative retention times, relative peak areas and similarities are studied, ensuring the chemical composition stability and use safety of the reference sample, and providing an important reference basis and quality reference for the quality control of subsequent compound preparations; (4) Regarding the fingerprints of each active ingredient in the traditional Chinese medicine compound reference sample of the present invention as a whole, paying attention to the sequence and mutual relationship of each characteristic peak, it not only avoids the one-sidedness of judging the overall quality of the traditional Chinese medicine compound reference sample of the present invention by only measuring one or two chemical components, but also reduces the possibility of artificial treatment for meeting the quality standards, providing a new method and means for comprehensively and accurately evaluating the quality of the traditional Chinese medicine compound reference sample of the present invention; (5) The method of the present invention has good stability, high precision, good reproducibility, is convenient and easy to master. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to these drawings without exceeding the scope of protection required by the present invention.

[0014] Figure 1 It is the ultraviolet fingerprint of the traditional Chinese medicine compound containing Glossy Privet Fruit of the present invention.

[0015] Figure 2 It is the liquid phase map of the mixed control of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0017] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains or in the field in which the term is used. Although any methods, conditions, materials, or materials similar or equivalent to those disclosed herein may be used in the practice of the present invention, the preferred methods, conditions, materials, or materials are described herein.

[0018] The present invention is expected to cover all alternatives, variations, and equivalents, which may be included in the existing art field as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be applied to the practice of the present invention. The present invention is in no way limited to the description of methods and materials.

[0019] The singular forms "a", "an", and "the" as used in the specification and the appended claims include plural referents unless the context clearly dictates otherwise.

[0020] In the present invention, the term "comprising" is synonymous with "including". The terms "comprising", "including", "having", "containing", or any other variation thereof used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0021] As described in the background art section, there has been no literature report on the comprehensive analysis of the chemical components and fingerprint study of the traditional Chinese medicine compound containing Glossy Privet Fruit claimed in the present invention. To solve the above problems, the present invention provides a method for constructing an ultraviolet fingerprint of a traditional Chinese medicine compound containing Glossy Privet Fruit, and the construction method includes the following steps: Preparation of the test solution of the traditional Chinese medicine compound: Take an appropriate amount of the traditional Chinese medicine compound granules containing Glossy Privet Fruit, place them in a container, add a solvent, perform ultrasonic treatment for a period of time, make up the volume, shake well, take the supernatant and filter it, and take the subsequent filtrate to obtain the test solution of the traditional Chinese medicine compound; wherein, the traditional Chinese medicine compound includes Glossy Privet Fruit, Oyster, Mother-of-Pearl, Chinese Wolfberry Fruit, White Peony Root, Caulis Polygoni Multiflori, Poria Cocos, and Atractylodes Macrocephala; Preparation of the reference solution: Weigh an appropriate amount of paeoniflorin, paeonolide, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, specnuezhenide, and atractylenolide III reference substances, and add methanol to prepare a reference solution with the concentrations of paeoniflorin, paeonolide, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, specnuezhenide, and atractylenolide III being 1 to 200 μg / ml respectively; According to the results of high-performance liquid chromatography detection of the test solution of the traditional Chinese medicine compound and the reference solution, obtain the ultraviolet fingerprint of the traditional Chinese medicine compound; the chromatographic conditions for the high-performance liquid chromatography detection are: use a chromatographic column with octadecylsilane chemically bonded silica as the packing material, mobile phase A is selected from one or more of acetonitrile, methanol, and tetrahydrofuran, mobile phase B is an acid aqueous solution, an alkali aqueous solution, and / or a buffer saline solution, and the gradient elution program is: 0 to 8 min, 95% → 82% B; 8 to 18 min, 82% B → 78% B; 18 to 22 min, 78% B; 22 to 27 min, 78% → 76% B; 27 to 34 min, 76% → 27% B; 34 to 39 min, 27% B → 20% B; 39 to 40 min, 20% → 95% B; 40 to 55 min, 95% B; the flow rate is 0.5 to 1.5 ml / min, the column temperature is 10 to 40 °C, the detection wavelength is 180 to 300 nm, and the injection volume is 1 to 20 μl.

[0022] In the present invention, when time, flow rate, temperature, wavelength, sample injection volume, volume percentage concentration, mass, ratio, power, frequency, volume, concentration, or other values or parameters are expressed as a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, it should be understood that all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value are specifically disclosed, regardless of whether the range is disclosed individually. For example, when the range "10 - 40" is disclosed, the described range should be interpreted to include ranges "10 - 40", "10 - 35", "10 - 30", "10 - 25", "10 - 20", "10 - 15", "15 - 40", "15 - 35", "15 - 30", "15 - 25", "15 - 20", "20 - 40", "20 - 35", "20 - 30", "20 - 25", "25 - 40", "25 - 35", "25 - 30", "30 - 40", "30 - 35", "35 - 40", etc. When a numerical range is described herein, unless otherwise specified, the range is intended to include its end values and all integers and fractions within the range.

[0023] In a preferred embodiment, in the preparation of the traditional Chinese medicine compound test sample solution, the solvent is an alcohol, such as methanol. In a preferred embodiment, the volume percentage concentration of the methanol is 20% - 80%, preferably 40% - 60%, for example, about 50%.

[0024] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 50%" includes ±5% of 50%, or from 47.5% to 52.5%.

[0025] In a preferred embodiment, the container is a volumetric flask.

[0026] In a preferred embodiment, the mass of the traditional Chinese medicine compound granules is 5 - 30 g, for example, about 15 g.

[0027] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0028] In a preferred embodiment, the mass / volume ratio between the traditional Chinese medicine compound granules and the solvent is 0.4 - 0.8, for example, about 0.6, with the unit of g / ml.

[0029] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 0.6" includes ±5% of 0.6, or from 0.57 to 0.63.

[0030] In a preferred embodiment, the power of the ultrasonic treatment is 400 - 600 W, for example, about 500 W.

[0031] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 500" includes ±5% of 500, or from 475 to 525.

[0032] In a preferred embodiment, the frequency of the ultrasonic treatment is 30 - 50 kHz, for example, about 40 kHz.

[0033] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0034] In a preferred embodiment, the time of the ultrasonic treatment is 20 - 40 min, for example, about 30 min.

[0035] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0036] In a preferred embodiment, the preparation method of the traditional Chinese medicine compound granules is as follows: Weigh 8 - 20 g of Glossy Privet Fruit, for example, about 12 g, 8 - 20 g of Raw Oyster, for example, about 12 g, 5 - 15 g of Mother-of-Pearl, for example, about 10 g, 8 - 20 g of Chinese Wolfberry Fruit, for example, about 12 g, 5 - 15 g of White Peony Root, for example, about 10 g, 8 - 20 g of Caulis Polygoni Multiflori, for example, about 12 g, 8 - 20 g of Poria, for example, about 15 g, 1 - 10 g of Atractylodes Macrocephala, for example, about 6 g, soak in water, extract by reflux, filter, take the subsequent filtrate, concentrate to a clear paste with a relative density of about 1.20, add an appropriate amount of dextrin, make into granules, and dry to obtain the traditional Chinese medicine compound granules.

[0037] In a preferred embodiment, the mass / volume ratio between the traditional Chinese medicine compound and the water is 0.01 - 0.5, for example, about 0.125, with the unit of g / ml.

[0038] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 0.125" includes ±5% of 0.125, or from 0.11875 to 0.13125.

[0039] In a preferred embodiment, the volume of the water is 500 - 1000 ml, for example, about 720 ml.

[0040] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 720" includes ±5% of 720, or from 684 to 756.

[0041] In a preferred embodiment, the soaking time is 20 to 60 minutes, for example, about 40 minutes.

[0042] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0043] In a preferred embodiment, the reflux extraction time is 1 to 3 hours, for example, about 2 hours.

[0044] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0045] In a preferred embodiment, the filtration is carried out using a filter mesh.

[0046] In a preferred embodiment, the filter mesh is a 200-mesh filter mesh.

[0047] In a preferred embodiment, the concentration of albiflorin in the reference substance solution is 80 to 120 μg / ml, for example, about 100 μg / ml or about 109.7 μg / ml.

[0048] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105; "about 109.7" includes ±5% of 109.7, or from 104.215 to 115.185.

[0049] In a preferred embodiment, the concentration of paeoniflorin in the reference substance solution is 15 to 25 μg / ml, for example, about 20 μg / ml or about 21.42 μg / ml.

[0050] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21; "about 21.42" includes ±5% of 21.42, or from 20.349 to 22.491.

[0051] In a preferred embodiment, the concentration of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in the reference substance solution is 80 to 120 μg / ml, for example, about 100 μg / ml or about 102.5 μg / ml.

[0052] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105; "about 102.5" includes ±5% of 102.5, or from 97.375 to 107.625.

[0053] In a preferred embodiment, the concentration of oleuropein in the reference solution is 80 - 120 μg / ml, such as about 100 μg / ml or about 102.4 μg / ml.

[0054] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105; "about 102.4" includes ±5% of 102.4, or from 97.28 to 107.52.

[0055] In a preferred embodiment, the concentration of atractylenolide III in the reference solution is 2 - 4 μg / ml, such as about 3 μg / ml or about 2.56 μg / ml.

[0056] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15; "about 2.56" includes ±5% of 2.56, or from 2.432 to 2.688.

[0057] In a preferred embodiment, the flow rate for the high performance liquid chromatography detection is 0.7 - 1.1 ml / min, such as about 0.9 ml / min.

[0058] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 0.9" includes ±5% of 0.9, or from 0.855 to 0.945.

[0059] In a preferred embodiment, the column temperature is 20 - 30 °C, such as about 25 °C.

[0060] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 25" includes ±5% of 25, or from 23.75 to 26.25.

[0061] In a preferred embodiment, the detection wavelength is 200 - 240 nm, such as about 224 nm.

[0062] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 224" includes ±5% of 224, or from 212.8 to 235.2.

[0063] In a preferred embodiment, the injection volume is 5 - 15 μl, such as about 10 μl.

[0064] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0065] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to paeoniflorin is not less than 2000.

[0066] In a preferred embodiment, the chromatographic column is a ZORBAX SB-C18 chromatographic column.

[0067] In a preferred embodiment, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0068] In a preferred embodiment, mobile phase A is acetonitrile.

[0069] In a preferred embodiment, the aqueous acid solution, aqueous base solution, and / or buffer saline solution are selected from one or more of weak acids and their salts, and weak bases and their salts with different concentrations. In a preferred embodiment, the aqueous acid solution, aqueous base solution, and / or buffer saline solution are selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid with different concentrations. In a preferred embodiment, the aqueous acid solution is a 0.01% to 1% aqueous acid solution. In a preferred embodiment, the aqueous acid solution is a 0.01% to 1% aqueous phosphoric acid solution. In a preferred embodiment, the aqueous acid solution is an aqueous phosphoric acid solution of about 0.1%.

[0070] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 0.1%" includes ±5% of 0.1%, or from 0.095% to 0.105%.

[0071] In a preferred embodiment, the buffer saline solution is an acetate aqueous solution and / or an acetate aqueous solution.

[0072] In a preferred embodiment, the pH value of the buffer saline solution is not greater than 7.0.

[0073] In a preferred embodiment, when the detection wavelength is 224 nm, the ultraviolet fingerprint spectrum includes peaks No. 1-8, and the retention times of the peaks No. 1-8 correspond to about 6.315 min, about 13.234 min, about 14.253 min, about 18.608 min, about 19.024 min, about 19.598 min, about 20.453 min, and about 37.394 min, respectively.

[0074] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 6.315" includes ±5% of 6.315, or from 5.99925 to 6.63075; "about 13.234" includes ±5% of 13.234, or from 12.5723 to 13.8957; "about 14.253" includes ±5% of 14.253, or from 13.54035 to 14.96565; "about 18.608" includes ±5% of 18.608, or from 17.6776 to 19.5384; "about 19.024" includes ±5% of 19.024, or from 18.0728 to 19.9752; "about 19.598" includes ±5% of 19.598, or from 18.6181 to 20.5779; "about 20.453" includes ±5% of 20.453, or from 19.43035 to 21.47565; "about 37.394" includes ±5% of 37.394, or from 35.5243 to 39.2637.

[0075] In a preferred embodiment, when the detection wavelength is 224 nm, the ultraviolet fingerprint spectrum includes peaks No. 1 - 8, where peak No. 3 is used as the reference peak, and the average relative retention times of peaks No. 1, 2, 4, 5, 6, 7, and 8 correspond to about 0.443, about 0.929, about 1.306, about 1.335, about 1.375, about 1.435, and about 2.624, respectively.

[0076] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 0.443" includes ±5% of 0.443, or from 0.42085 to 0.46515; "about 0.929" includes ±5% of 0.929, or from 0.88255 to 0.97545; "about 1.306" includes ±5% of 1.306, or from 1.2407 to 1.3713; "about 1.335" includes ±5% of 1.335, or from 1.26825 to 1.40175; "about 1.375" includes ±5% of 1.375, or from 1.30625 to 1.44375; "about 1.435" includes ±5% of 1.435, or from 1.36325 to 1.50675; "about 2.624" includes ±5% of 2.624, or from 2.4928 to 2.7552.

[0077] In a preferred embodiment, when the detection wavelength is 224 nm, peak No. 2 is albiflorin, peak No. 3 is paeoniflorin, peak No. 4 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, peak No. 6 is ligustroside, and peak No. 8 is atractylenolide III.

[0078] In a preferred embodiment, when the detection wavelength is 224 nm, peak 1 comes from the medicinal material of Caulis Polygoni Multiflori and the medicinal material of Radix Paeoniae Alba, peak 2 comes from the medicinal material of Radix Paeoniae Alba, peak 3 comes from the medicinal material of Radix Paeoniae Alba, peak 4 comes from the medicinal material of Caulis Polygoni Multiflori, peak 5 comes from the medicinal material of Radix Paeoniae Alba, peak 6 comes from the medicinal material of Fructus Ligustri Lucidi, peak 7 comes from the medicinal material of Fructus Lycii, and peak 8 comes from the medicinal material of Rhizoma Atractylodis Macrocephalae.

[0079] According to another aspect of the present invention, there is provided a method for identifying a traditional Chinese medicine compound containing Fructus Ligustri Lucidi. The identification method includes the following steps: (1) Establishing a standard ultraviolet fingerprint spectrum of the reference sample of the traditional Chinese medicine compound according to the above ultraviolet fingerprint spectrum construction method; (2) Taking the test sample solution of the traditional Chinese medicine compound and performing detection according to the chromatographic conditions in the above ultraviolet fingerprint spectrum construction method to obtain the ultraviolet fingerprint spectrum of the test sample of the traditional Chinese medicine compound; and (3) Comparing the ultraviolet fingerprint spectrum of the test sample of the traditional Chinese medicine compound obtained in step (2) with the standard ultraviolet fingerprint spectrum of the reference sample of the traditional Chinese medicine compound obtained in step (1). Those that meet the requirements are qualified products, and those that do not meet the requirements are unqualified products. In a preferred embodiment, meeting the requirements includes one or more of the following: (1) There are 8 characteristic chromatographic peaks presented in the ultraviolet fingerprint spectrum of the test sample of the traditional Chinese medicine compound, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard ultraviolet fingerprint spectrum of the reference sample of the traditional Chinese medicine compound; (2) Using peak 3 as the S peak, the relative retention time of each characteristic chromatographic peak in the ultraviolet fingerprint spectrum of the test sample of the traditional Chinese medicine compound with respect to the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard ultraviolet fingerprint spectrum of the reference sample of the traditional Chinese medicine compound; and (3) According to the similarity evaluation system for ultraviolet fingerprint spectra of traditional Chinese medicine chromatography, the similarity between the ultraviolet fingerprint spectrum of the test sample of the traditional Chinese medicine compound and the standard ultraviolet fingerprint spectrum of the reference sample of the traditional Chinese medicine compound obtained by similarity calculation shall not be lower than 0.90.

[0080] According to another aspect of the present invention, there is provided the use of the above construction method or the above identification technique in the quality detection and / or quality evaluation and / or quality control of a traditional Chinese medicine compound containing Fructus Ligustri Lucidi.

[0081] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or the conditions recommended by the manufacturer.

[0082] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described herein are only for demonstration purposes.

[0083] The above-mentioned features mentioned in the present invention, or the features mentioned in the embodiments, can be combined arbitrarily. All the features disclosed in this patent specification can be used in combination with any composition form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0084] Example

[0085] 1. Instruments and Reagents

[0086] 1.1 Instruments

[0087] High-performance liquid chromatograph model: Agilent 1290_DAD, Agilent Technologies; High-performance liquid chromatograph model: Waters 2695_2996, Waters Technology Co., Ltd.; Chromatographic column: ZORBAX SB-C18 4.6mm×250mm, 5μm, SN: USCL136677; Chromatographic column: ZORBAX SB-C18 4.6mm×250mm, 5μm, SN: USCL145450; Chromatographic column: ZORBAX SB-C18 4.6mm×250mm, 5μm, SN: USCL143042; Electronic balance model: BCE224I-1CCN / SQP, Sartorius Group, Germany; Electronic balance model: JY2002, Shanghai Puchun Measuring Instrument Co., Ltd.; Digital display constant temperature water bath model: HH-6, Shanghai Boxun Medical and Biological Instrument Co., Ltd.; Numerical control ultrasonic cleaner model: KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.

[0088] 1.2 Reagents and Medicinal Reagents

[0089] Methanol and acetonitrile are chromatographically pure, methanol, ethanol, phosphoric acid, formic acid, and glacial acetic acid are analytically pure, and water is purified water; Ligustroside (batch number: 111926-201906, content: 95.0%, National Institutes for Food and Drug Control); Albiflorin (batch number: AY010005-202409, content: 99.85%, Foshan Aoyu Biotechnology Co., Ltd.); Paeoniflorin (batch number: 110736-202447, content: 98.1%, National Institutes for Food and Drug Control); 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (batch number: 110844-202317, content: 97.9%, National Institutes for Food and Drug Control); Atractylenolide III (batch number: 111978-202302, content: 100.0%, National Institutes for Food and Drug Control).

[0090] 1.3 Sample Information

[0091] Samples of the traditional Chinese medicine compound granules containing Glossy Privet Fruit of the present invention (batch number: 24-1-ZQKL2025021901-L, source: self-made); Samples of the traditional Chinese medicine compound granules containing Glossy Privet Fruit of the present invention (batch number: 24-1-ZQKL2025022801-L, source: self-made); Negative samples of the traditional Chinese medicine compound granules containing Glossy Privet Fruit of the present invention without Glossy Privet Fruit (batch number: 24-1-ZQKL2025021902-L, source: self-made); Negative samples of the traditional Chinese medicine compound granules containing Glossy Privet Fruit of the present invention without Oyster (batch number: 24-1-ZQKL2025021903-L, source: self-made); Negative samples of the traditional Chinese medicine compound granules containing Glossy Privet Fruit of the present invention without Mother-of-Pearl (batch number: 24-1-ZQKL2025022002-L, source: self-made); Negative samples of the traditional Chinese medicine compound granules containing Glossy Privet Fruit of the present invention without Chinese Wolfberry Fruit (batch number: 24-1--ZQKL2025021904-L, source: self-made); Negative samples of the traditional Chinese medicine compound granules containing Glossy Privet Fruit of the present invention without White Peony Root (batch number: 24-1-ZQKL2025022003-L, source: self-made); Negative samples of the traditional Chinese medicine compound granules containing Glossy Privet Fruit of the present invention without Caulis Polygoni Multiflori (batch number: 24-1-ZQKL2025022001-L, source: self-made); Negative samples of the traditional Chinese medicine compound granules containing Glossy Privet Fruit of the present invention without Poria (batch number: 24-1-ZQKL2025022004-L, source: self-made); Negative samples of the traditional Chinese medicine compound granules containing Glossy Privet Fruit of the present invention without Atractylodes Macrocephala (batch number: 24-1-ZQKL2025022101-L, source: self-made); Slices of Glossy Privet Fruit (batch number: 20240902, place of origin: Puyang, Henan, source: Anguo Shenrong Chinese Medicinal Materials Co., Ltd.); Slices of Oyster (batch number: 24102401, place of origin: Weihai City, Shandong Province, source: Hunan Haodonglin Chinese Medicinal Herb Pieces Co., Ltd.); Slices of Mother-of-Pearl (batch number: 23110601, place of origin: Jiangsu, source: Hunan Haodonglin Chinese Medicinal Herb Pieces Co., Ltd.); Slices of Chinese Wolfberry Fruit (batch number: 24011801, place of origin: Ningxia, source: Hunan Haodonglin Chinese Medicinal Herb Pieces Co., Ltd.); Slices of White Peony Root (batch number: 24092401, place of origin: Bozhou, Anhui, source: Hunan Haodonglin Chinese Medicinal Herb Pieces Co., Ltd.); Slices of Caulis Polygoni Multiflori (batch number: 24062401, place of origin: Yunnan, source: Hunan Haodonglin Chinese Medicinal Herb Pieces Co., Ltd.); Slices of Poria (batch number: ZA04724062001, place of origin: Yingshan County, Huanggang City, Hubei Province, source: Changsha Headquarters); Slices of Atractylodes Macrocephala (batch number: 24101801, place of origin: Bozhou, Anhui, source: Hunan Haodonglin Chinese Medicinal Herb Pieces Co., Ltd.).

[0092] The traditional Chinese medicine compound granule sample containing Glossy Privet Fruit: Take the one-day prescription amount of the traditional Chinese medicine compound granule containing Glossy Privet Fruit of the present invention, add 720 ml (8 times the amount) of water, soak for 40 minutes, reflux and extract for 2 hours, filter through a 200-mesh sieve, concentrate to a clear paste with a relative density of about 1.20, add an appropriate amount of dextrin, make into granules, and dry to obtain the product.

[0093] 2. Establishment of characteristic fingerprint and content determination method

[0094] 2.1. Research on chromatographic conditions

[0095] According to the analysis of the results of preliminary pre-experiments, the initial chromatographic conditions were determined as follows:

[0096] Determined by high performance liquid chromatography (General Principles 0512, Volume IV, Chinese Pharmacopoeia 2020 Edition).

[0097] First, gradient elution was carried out with methanol-water according to Gradient 1 in Table 1 to investigate the separation effect of the characteristic fingerprint.

[0098] Table 1 Gradient 1

[0099] Time (min) Methanol (%) Water (%) 0→15 10→40 90→60 15→45 40 60 45→72 40→90 60→10 72→75 90→10 10→90 75→90 10 90

[0100] The results showed that the peak information of the chromatographic peaks of the mobile phase methanol-water was less and the separation effect was poor. Therefore, the mobile phase acetonitrile - 0.1% phosphoric acid was determined as the mobile phase.

[0101] Chromatographic conditions and system suitability test: Using octadecylsilane chemically bonded silica as the filler; using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution was carried out according to Gradient 2 in Table 2; the flow rate was 1 ml per minute; the column temperature was 25°C; full wavelength scanning was performed.

[0102] Table 2 Gradient 2

[0103] Time (min) Acetonitrile (%) 0.1% Phosphoric Acid (%) 0→120 5→95 95→5

[0104] Preparation of test solution: Take about 15 g of the traditional Chinese medicine compound granule sample containing Glossy Privet Fruit of the present invention, accurately weigh, place in a 25-ml volumetric flask, add an appropriate amount of 50% methanol, ultrasonically treat (500 W, 40 kHz) for 30 minutes, cool to room temperature, add 50% methanol to the scale, shake well, take the supernatant and filter, and take the subsequent filtrate to obtain the test solution.

[0105] Determination method: Accurately pipette 10 μl of the test solution, inject it into the liquid chromatograph, and perform the determination to obtain the result.

[0106] Combined with the 3D chromatogram and the liquid chromatograms at various wavelengths, it can be seen that the extract of the traditional Chinese medicine compound granules containing Glossy Privet Fruit mainly has peak absorption within the first 60 minutes. Therefore, it is further optimized to Gradient 3 in Table 3. At low wavelengths (224 nm, 230 nm), the peak information and response values are relatively high. In view of the fact that the detection wavelength for the determination of the content of specnuezhenide in Glossy Privet Fruit, the monarch drug in this formula, in the Chinese Pharmacopoeia (2020 Edition) is 224 nm, following the idea of "one method for multiple evaluations", a method for simultaneously determining the content of related index components and the characteristic chromatogram of the traditional Chinese medicine compound granules containing Glossy Privet Fruit of the present invention is established using HPLC. Therefore, the detection wavelength is determined to be 224 nm.

[0107] Table 3 Gradient 3

[0108]

[0109]

[0110] The results show that under Gradient 3 in Table 3, the chromatographic peaks within 40 - 52 minutes have poor separation and a long running time. Therefore, it is optimized to Gradient 4 in Table 4.

[0111] Table 4 Gradient 4

[0112]

[0113] Under Gradient 4 in Table 4, all peaks are not well separated and the baseline is uneven after 42 minutes. Therefore, Gradient 5 in Table 5 is designed. Table 5 Gradient 5

[0114]

[0115]

[0116] Under Gradient 5 in Table 5, the resolution of all peaks within 32 - 40 minutes is relatively poor; considering optimization with isocratic elution, Gradient 6 in Table 6 is designed.

[0117] Table 6 Gradient 6

[0118] Time (min) Acetonitrile (%) 0.1 Phosphoric Acid (%) 0→10 5→15 95→85 10→16 15→19 85→81 16→21 19→20 81→80 21→26 20→22 80→78 26→30 22 78 30→37 22→33 78→67 37→41 33→35 67→65 41→46 35→72 65→28 46→50 72→77 28→23

[0119] Under Gradient 6 in Table 6, there is a phenomenon of overlapping peaks around 28 minutes; therefore, Gradient 7 in Table 7 is designed.

[0120] Table 7 Gradient 7

[0121] Time (min) Acetonitrile (%) 0.1% Phosphoric Acid (%) 0→8 5→18 95→82 8→18 18→22 82→78 18→22 22 78 22→27 22→24 78→76 27→34 24→73 76→27 34→39 73→80 27→20

[0122] The results show that under Gradient 7 in Table 7, the separation within 17 - 21 minutes is slightly poor. Therefore, considering reducing the flow rate for research, Gradient 8 in Table 8 is designed.

[0123] Chromatographic conditions and system suitability test: Using octadecylsilyl silica gel as the filler; using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, perform gradient elution 8 according to the regulations in Table 8; the flow rate is 0.9 ml per minute; the column temperature is 25 °C; the detection wavelength is 224 nm. The ultraviolet fingerprint spectrum of the traditional Chinese medicine compound containing Glossy Privet Fruit in the present invention is as Figure 1 shown.

[0124] Table 8 Gradient 8

[0125] Time (min) Acetonitrile (%) 0.1% Phosphoric Acid (%) 0→8 5→18 95→82 8→18 18→22 82→78 18→22 22 78 22→27 22→24 78→76 27→34 24→73 76→27 34→39 73→80 27→20 39→40 80→5 20→95 40→55 5 95

[0126] Preparation of test solution: Take about 15 g of the sample, weigh accurately, place it in a 25-ml volumetric flask, add an appropriate amount of 50% methanol, ultrasonically treat it (500 W, 40 kHz) for 30 minutes, let it cool, add 50% methanol to the scale, shake well, take the supernatant and filter it, and take the subsequent filtrate to obtain the test solution.

[0127] Determination method: Accurately pipette 10 μl of the reference solution and the test solution, inject them into the liquid chromatograph, and perform the determination to obtain the results. The results are shown in Table 9.

[0128] Table 9 Summary table of system suitability parameters of the characteristic liquid chromatogram of the traditional Chinese medicine compound granule containing Glossy Privet Fruit in the present invention

[0129]

[0130] The results show that the resolution of the characteristic chromatogram of the traditional Chinese medicine compound granule containing Glossy Privet Fruit in the present invention is good and has met the requirements of the liquid chromatogram. Therefore, a total of 8 characteristic peaks are included in the characteristic chromatogram of the traditional Chinese medicine compound granule containing Glossy Privet Fruit in the present invention; the response value of peak 3 (paeoniflorin) is the strongest, so it is designated as the S peak. According to the content determination of the white peony root medicinal material in Part I of the Chinese Pharmacopoeia 2020 edition, it is determined that the theoretical plate number of this method calculated by the paeoniflorin peak should be not less than 2000; according to the detection wavelength of the Glossy Privet Fruit medicinal material in Part I of the Chinese Pharmacopoeia 2020 edition, it is determined that the characteristic and content detection wavelength of the reference sample of the traditional Chinese medicine compound granule containing Glossy Privet Fruit in the present invention is 224 nm.

[0131] 2.3 Investigation of different acid types

[0132] Using gradient 8, investigate and study under different acid types at a column temperature of 25 °C and a flow rate of 0.9 ml / min as follows.

[0133] The results show that when the mobile phase is 0.1% phosphoric acid, the baseline of its liquid chromatogram and the resolution of the chromatographic peaks are better; under 0.1% formic acid and 0.1% glacial acetic acid, its liquid chromatogram does not meet the liquid chromatogram requirements. It is determined that the mobile phase of the traditional Chinese medicine compound granule containing Glossy Privet Fruit in the present invention is 0.1% phosphoric acid.

[0134] 2.4 Determination of chromatographic conditions

[0135] Determine by high performance liquid chromatography (General Principles 0512 of Chinese Pharmacopoeia 2020 Edition).

[0136] Chromatographic conditions and system suitability test: Use octadecylsilyl silica gel as the filler (ZORBAX SB-C18 4.6 mm×250 mm, 5 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table 8; the flow rate is 0.9 ml per minute; the column temperature is 25 °C; the detection wavelength is 224 nm. The number of theoretical plates calculated based on the paeoniflorin peak should be not less than 2000.

[0137] Preparation of reference substance solution: Weigh appropriate amounts of reference substances of acteoside, albiflorin, paeoniflorin, atractylenolide III, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside accurately, and dissolve them in methanol respectively to prepare reference substance solutions containing 0.1024 mg of acteoside, 0.1097 mg of albiflorin, 21.42 μg of paeoniflorin, 2.56 μg of atractylenolide III, and 0.1025 mg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside per 1 ml as the reference substance solutions of each reference substance.

[0138] Preparation of test solution: Take about 15 g of the sample, weigh accurately, place it in a 25 ml volumetric flask, add appropriate amount of 50% methanol, ultrasonically treat (500 W, 40 kHz) for 30 minutes, cool to room temperature, add 50% methanol to the scale, shake well, take the supernatant and filter, take the subsequent filtrate, and you will get it.

[0139] Determination method: Accurately pipette 10 μl of the reference substance solution and the test solution, inject them into the liquid chromatograph for determination, and you will get it.

[0140] 3. Investigation on the preparation of test samples

[0141] According to the research results of the preparation process of the traditional Chinese medicine compound granules containing Glossy Privet Fruit in the present invention, the samples are stored in the form of extraction solution, so the preparation method of the test solution of the extraction solution is investigated. It is determined to take about 15 g of the extraction solution to carry out the research.

[0142] 3.1 Investigation on the sampling amount

[0143] Take 3 portions of the extraction solution and investigate the effects of sampling amounts of 10 g, 15 g, and 20 g on the determination of the characteristic chromatogram of the traditional Chinese medicine compound granules containing Glossy Privet Fruit in the present invention respectively.

[0144] Take 3 portions of the extraction solution, each about 10 g, 15 g, and 20 g, weigh accurately, place them in 25 ml volumetric flasks, add appropriate amount of 50% methanol, ultrasonically treat (500 W, 40 kHz) for 30 minutes, cool to room temperature, add 50% methanol to the scale, take the supernatant and filter, take the subsequent filtrate, and you will get it. Inject the sample, and the results are shown in Table 10.

[0145] Table 10 Influence of Different Sampling Volumes on the Characteristic Chromatogram of the Traditional Chinese Medicine Compound Granules Containing Glossy Privet Fruit of the Invention

[0146]

[0147]

[0148] The results showed that after different sampling volumes of the extract of the traditional Chinese medicine compound granules containing glossy privet fruit of the invention were treated with 50% methanol, the total peak area showed a proportional change when the sampling volume was 10 - 20 g, indicating that the detection method was stable within the range of 10 - 20 g of the sampling volume. Considering that the content determination and the characteristic chromatogram detection shared the same method, the sampling volume was determined to be 15 g.

[0149] 3.2. Investigation of Extraction Solvents

[0150] Take 2 portions of the extract and investigate the influence of 50% methanol and dilute ethanol as extraction solvents on the determination of the characteristic chromatogram of the traditional Chinese medicine compound granules containing glossy privet fruit of the invention respectively.

[0151] Take 2 portions of the extract, each about 15 g, accurately weigh, place in 25 - ml volumetric flasks, add appropriate amounts of 50% methanol and dilute ethanol respectively, treat with ultrasound (500 W, 40 kHz) for 30 minutes, cool to room temperature, add the corresponding solvent to the mark, shake well, take the supernatant and filter, take the successive filtrate, and obtain the solution. The injection results are shown in Table 11.

[0152] Table 11 Influence of Different Solvents on the Characteristic Chromatogram of the Traditional Chinese Medicine Compound Granules Containing Glossy Privet Fruit of the Invention

[0153]

[0154]

[0155]

[0156] The results showed that after the traditional Chinese medicine compound granules containing glossy privet fruit of the invention were treated with different extraction solvents, the number of characteristic peaks was the same, and the total peak area was slightly larger after treatment with 50% methanol. Therefore, the treatment solvent was 50% methanol; the difference in the total peak area and the value investigated for the sampling volume of 15 g was relatively large, which might be related to the need for light - avoiding operation and storage time of 2,3,5,4’ - tetrahydroxystilbene - 2 - O - β - D - glucoside.

[0157] Based on the above research, the preparation of the test solution was determined as follows: Take about 15 g of the granule sample, accurately weigh, place in a 25 - ml volumetric flask, add appropriate amount of 50% methanol, treat with ultrasound (500 W, 40 kHz) for 30 minutes, cool to room temperature, add 50% methanol to the mark, shake well, take the supernatant and filter, take the successive filtrate, and obtain the solution.

[0158] 4. Methodological Verification

[0159] 4.1 Specificity

[0160] 4.1.1 Preparation of reference substance solution:

[0161] Appropriately weigh the reference substances of acteoside, albiflorin, paeoniflorin, atractylenolide III, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside respectively. Weigh them accurately and dissolve them in methanol respectively to prepare reference substance solutions containing 0.1024 mg of acteoside, 0.1097 mg of albiflorin, 21.42 μg of paeoniflorin, 2.56 μg of atractylenolide III, and 0.1025 mg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside per 1 ml as the reference substance solutions for each reference substance.

[0162] 4.1.2 Preparation of test solution

[0163] Take about 15 g of the sample, weigh it accurately, place it in a 25-ml volumetric flask, add an appropriate amount of 50% methanol, ultrasonically treat it (500 W, 40 kHz) for 30 minutes, let it cool, add 50% methanol to the scale, shake well, take the supernatant and filter it, and take the subsequent filtrate to obtain the test solution.

[0164] 4.1.3 Preparation of negative control solution

[0165] Preparation of negative sample solution without Glossy Privet Fruit: Weigh the other medicinal flavors without Glossy Privet Fruit according to the prescription, prepare the negative sample without Glossy Privet Fruit according to the determined technological preparation method, and prepare the negative control solution without Glossy Privet Fruit according to item (2) below.

[0166] Prepare negative control solutions without Oyster, Mother-of-Pearl, Caulis Polygoni Multiflori, White Peony Root, Atractylodes Macrocephala, Chinese Wolfberry Fruit, and Poria cocos in the same way respectively.

[0167] 4.1.4 Preparation of single-herb reference solution

[0168] Take an appropriate amount of Glossy Privet Fruit, add 50 ml of purified water, heat under reflux for 60 minutes, let it cool, and prepare the single-herb reference solution of Glossy Privet Fruit according to item (2) below.

[0169] Prepare single-herb reference solutions of Oyster, Mother-of-Pearl, Caulis Polygoni Multiflori, White Peony Root, Atractylodes Macrocephala, Chinese Wolfberry Fruit, and Poria cocos in the same way respectively.

[0170] 4.1.5 Blank solvent: Methanol

[0171] The blank solvent and the reference substance solution (the mixed reference liquid chromatogram is as Figure 2Inject and analyze the sample solution of the traditional Chinese medicine compound granules containing Glossy Privet Fruit of the present invention, the negative control solution of each medicinal flavor, and the single-herb control solution. At the proposed characteristic peak elution position, the blank solvent should not cause interference. Additionally, the attribution of the chromatographic peaks needs to be determined based on the test results of the negative control solution and the single-herb control solution.

[0172] Based on the above research, as can be seen from the identification results of the attribution of the chromatographic peaks in the characteristic chromatogram of the traditional Chinese medicine compound granules containing Glossy Privet Fruit of the present invention (as shown in Table 12), there are mainly 8 chromatographic peaks in its chromatogram. Among them, Peak 1 belongs to Caulis Polygoni Multiflori and Radix Paeoniae Alba; Peaks 2, 3, and 5 belong to Radix Paeoniae Alba, where Peak 2 is albiflorin and Peak 3 is paeoniflorin; Peak 4 belongs to Caulis Polygoni Multiflori and is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside; Peak 6 belongs to Glossy Privet Fruit and is acteoside; Peak 7 belongs to Fructus Lycii; Peak 8 belongs to Rhizoma Atractylodis Macrocephalae and is atractylenolide III.

[0173] Inject and analyze the blank solvent, reference solution, the solution of the traditional Chinese medicine compound granules containing Glossy Privet Fruit of the present invention, the negative control solution of each medicinal flavor, and the single-herb control solution. At the proposed characteristic peak elution position, the blank solvent and the negative control solution show no interference. The details of the medicinal flavors attributed to the specific chromatographic peaks in its chromatogram are shown in Table 12.

[0174] Table 12 Summary Table of the Attribution of Chromatographic Peaks in the Characteristic Chromatogram of the Traditional Chinese Medicine Compound Granules Containing Glossy Privet Fruit of the Present Invention

[0175]

[0176]

[0177] 4.2 Precision

[0178] 4.2.1 Repeatability

[0179] Prepare 6 portions of test solution according to the above method for preparing the test solution, inject for detection, record the chromatogram, and calculate the RSD value of the relative retention time of the characteristic peak and the S peak (paeoniflorin); the RSD of the relative retention time ≤ 3%. The results show that the RSD of the relative retention time of each characteristic peak in the repeatability experiment ≤ 3%, indicating that this method has good repeatability.

[0180] 4.2.2 Intermediate Precision

[0181] Two persons prepared 6 test solution samples according to the above defined conditions, and injected the samples into an Agilent high performance liquid chromatograph and a Waters high performance liquid chromatograph respectively for detection. Calculate the relative standard deviation (RSD) value of the relative retention time of each characteristic peak to the S peak (paeoniflorin). If the RSD of the relative retention time ≤ 3%, it indicates good intermediate precision. The results showed that the RSD of the relative retention time of each characteristic peak of the intermediate precision of Waters and Agilent were both ≤ 3%. It shows that this method has good intermediate precision for Waters and Agilent.

[0182] 4.2.3 Instrument Precision

[0183] Prepare one test solution sample according to the above defined conditions, inject the sample continuously for 6 times, record the chromatogram, and calculate the RSD value of the retention time and peak area of the characteristic peak to the S peak (paeoniflorin). If the RSD of the relative retention time ≤ 3% and the RSD of the relative peak area ≤ 5%, the relative peak area of the chromatographic peak with a relatively small total peak area can be appropriately relaxed. It indicates good instrument precision. The results showed that the RSD of the relative retention time of each characteristic peak in the instrument precision experiment was ≤ 3%, indicating good instrument precision.

[0184] 4.3 Robustness

[0185] 4.3.1 Flow Rate Robustness

[0186] Prepare one test solution sample according to the above test solution preparation method, set the mobile phase flow rate to 0.8 ml / min, 0.9 ml / min, and 1.0 ml / min respectively, inject the sample for detection, record the chromatogram, and calculate the relative mean deviation value of the relative retention time of each characteristic peak. If the relative mean deviation of the relative retention time ≤ 3%, it indicates good flow rate robustness. The results showed that the relative mean deviation of the relative retention time of each characteristic peak in the different flow rate experiments was greater than 3%. The liquid chromatogram separation was better at a flow rate of 0.9 ± 0.1 ml / min, indicating good flow rate robustness.

[0187] 4.3.2 Column Temperature Robustness

[0188] Prepare one test solution sample according to the above test solution preparation method, inject the sample for detection at the column temperature of 20 °C, 25 °C, and 30 °C of the chromatographic column, record the chromatogram, and calculate the relative mean deviation of the relative retention time of each characteristic peak. If the relative mean deviation of the relative retention time ≤ 3%, it indicates good flow rate robustness. The results showed that the relative mean deviation of the relative retention time of each characteristic peak in the different column temperature experiments was ≤ 3%, indicating good column temperature robustness.

[0189] 4.3.3 Robustness with Different Acid Concentrations

[0190] Prepare 1 sample solution according to the above method for preparing the test sample solution, inject and detect it under 0.09%, 0.10% and 0.11% phosphoric acid solutions, record the chromatogram, calculate the relative average deviation value of the relative retention time of each characteristic peak. If the relative average deviation of the relative retention time ≤ 3%, it indicates good durability at different acid concentrations. The results show that the relative average deviation of the relative retention time of each characteristic peak in the experiment with different acid concentrations ≤ 3%; indicating good durability at different acid concentrations.

[0191] 4.3.4, Durability of different chromatographic columns

[0192] Prepare 1 sample solution according to the above method for preparing the test sample solution, use three numbered chromatographic columns respectively, record the chromatogram, calculate the relative average deviation value of the relative retention time of each characteristic peak. If the relative average deviation of the relative retention time ≤ 3%, it indicates good durability of different chromatographic columns. The results show that the relative average deviation of the relative retention time of each characteristic peak in the experiment with different chromatographic columns ≤ 3%; indicating good durability of different chromatographic columns.

[0193] 4.4, Stability of the test sample solution

[0194] Prepare 1 sample solution according to the above method for preparing the test sample solution, inject and detect it at the 0th, 2nd, 4th, 8th, 10th, 16th, 18th, 24th, 36th, and 48th hours respectively. Record the chromatogram and calculate the RSD value of the relative retention time of each characteristic peak; it indicates good stability of the test sample solution. The results show that the RSD of the relative retention time of each characteristic peak in the stability experiment of the test sample solution at the 0th, 2nd, 4th, 8th, 10th, 16th, 18th, 24th, 36th, and 48th hours are all ≤ 3%; indicating good stability of the test sample solution within 48h.

[0195] 5. Confirmation of the characteristic chromatogram method

[0196] Based on the above research results, according to the General Principles for Validation of Analytical Methods 9101 in the Chinese Pharmacopoeia (2020 Edition), the specificity, accuracy, precision, column temperature, flow rate, different acid concentrations, different chromatographic columns, and stability results of the test sample solution of this characteristic detection method all meet the requirements. Therefore, this detection method is good, as follows.

[0197] Determine according to the high performance liquid chromatography method (General Rules 0512 in Part IV of the Chinese Pharmacopoeia 2020 Edition).

[0198] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica as the filler (ZORBAX SB-C18 4.6mm×250mm, 5μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution 8 according to the regulations in Table 8; the flow rate is 0.9 ml per minute; the column temperature is 25°C; the detection wavelength is 224 nm. The number of theoretical plates calculated based on the paeoniflorin peak should be not less than 2000.

[0199] Preparation of reference solution: Take appropriate amounts of paeoniflorin, paeoniflorin, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, teruginosalide and atractylodes lactone III reference substances, weigh them accurately, and add methanol to prepare a mixed solution containing 0.10 mg of paeoniflorin, 20 μg of paeoniflorin, 0.10 mg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, 0.10 mg of teruginosalide and 3 μg of atractylodes lactone III per 1 ml, which is used as the reference solution.

[0200] Preparation of test solution: Take about 15g of particle sample, accurately weigh it, place it in a 25ml volumetric flask, add an appropriate amount of 50% methanol, treat it with ultrasound (500W, 40kHz) for 30 minutes, let it cool, add 50% methanol to the scale, shake it well, filter the supernatant, and take the filtrate to obtain the product.

[0201] Determination method: Accurately aspirate 10 μl of reference solution and test solution, inject into liquid chromatograph, and determine.

[0202] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only intended to help understand the method of the present invention and its core concept. At the same time, changes or modifications made by those skilled in the art based on the concept of the present invention, the specific implementation methods of the present invention, and the scope of application are all within the scope of protection of the present invention. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. A method for constructing an ultraviolet fingerprint spectrum of a traditional Chinese medicine compound containing Glossy Privet Fruit, characterized in that, The construction method includes the following steps: Preparation of the traditional Chinese medicine compound test solution: Take an appropriate amount of the traditional Chinese medicine compound granules containing Glossy Privet Fruit, place them in a container, add a solvent, perform ultrasonic treatment for a period of time, make up the volume, shake well, filter the supernatant, and take the subsequent filtrate to obtain the traditional Chinese medicine compound test solution; wherein, the traditional Chinese medicine compound includes Glossy Privet Fruit, Oyster, Mother-of-Pearl, Chinese Wolfberry Fruit, White Peony Root, Caulis Polygoni Multiflori, Poria, and Atractylodes Macrocephala. Preparation of the reference solution: Weigh an appropriate amount of paeoniflorin, paeonolide glycoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, specnuezhenide, and atractylenolide III reference substances, and add methanol to prepare the reference solution with the concentrations of paeoniflorin, paeonolide glycoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, specnuezhenide, and atractylenolide III being 1 - 200 μg / ml respectively. Obtain the ultraviolet fingerprint of the traditional Chinese medicine compound according to the results of high-performance liquid chromatography detection of the traditional Chinese medicine compound test solution and the reference solution. The chromatographic conditions for the high-performance liquid chromatography detection are as follows: Use a chromatographic column packed with octadecylsilyl bonded silica gel. Mobile phase A is selected from one or more of acetonitrile, methanol, and tetrahydrofuran. Mobile phase B is an acidic aqueous solution, a basic aqueous solution, and / or a buffered saline solution. The gradient elution program is: 0 - 8 min, 95% → 82% B; 8 - 18 min, 82% B → 78% B; 18 - 22 min, 78% B; 22 - 27 min, 78% → 76% B; 27 - 34 min, 76% → 27% B; 34 - 39 min, 27% B → 20% B; 39 - 40 min, 20% → 95% B; 40 - 55 min, 95% B; the flow rate is 0.5 - 1.5 ml / min, the column temperature is 10 - 40 °C, the detection wavelength is 180 - 300 nm, and the injection volume is 1 - 20 μl.

2. The construction method according to claim 1, characterized in that In the preparation of the traditional Chinese medicine compound test solution, the solvent is alcohol, such as methanol. Preferably, the volume percentage concentration of the methanol is 20% - 80%, more preferably 40% - 60%, for example, about 50%. Preferably, the container is a volumetric flask. Preferably, the mass of the traditional Chinese medicine compound granules is 5 - 30 g, for example, about 15 g. Preferably, the mass / volume ratio between the traditional Chinese medicine compound granules and the solvent is 0.4 - 0.8, for example, about 0.6, with the unit of g / ml. More preferably, the power of the ultrasonic treatment is 400 - 600 W, for example, about 500 W. More preferably, the frequency of the ultrasonic treatment is 30 - 50 kHz, for example, about 40 kHz. More preferably, the time of the ultrasonic treatment is 20 - 40 min, for example, about 30 min.

3. The construction method according to claim 1, wherein The preparation method of the traditional Chinese medicine compound granules is as follows: Weigh 8 - 20 g of Glossy Privet Fruit, for example, about 12 g, 8 - 20 g of Raw Oyster, for example, about 12 g, 5 - 15 g of Mother-of-Pearl, for example, about 10 g, 8 - 20 g of Chinese Wolfberry Fruit, for example, about 12 g, 5 - 15 g of White Peony Root, for example, about 10 g, 8 - 20 g of Nightshade Vine, for example, about 12 g, 8 - 20 g of Poria, for example, about 15 g, 1 - 10 g of Atractylodes Macrocephala Koidz., for example, about 6 g, soak in water, extract by reflux, filter, take the continuous filtrate, concentrate to a clear paste with a relative density of about 1.20, add an appropriate amount of dextrin, make into granules, and dry to obtain the traditional Chinese medicine compound granules; Preferably, the mass / volume ratio between the traditional Chinese medicine compound and the water is 0.01 - 0.5, for example, about 0.125, with the unit of g / ml; More preferably, the volume of the water is 500 - 1000 ml, for example, about 720 ml; More preferably, the soaking time is 20 - 60 min, for example, about 40 min; More preferably, the reflux extraction time is 1 - 3 h, for example, about 2 h; More preferably, the filtration is filtration through a filter screen; Also preferably, the filter screen is a 200 - mesh filter screen.

4. The construction method according to claim 1, characterized in that, The concentration of albiflorin in the reference substance solution is 80 - 120 μg / ml, for example, about 100 μg / ml or about 109.7 μg / ml; Preferably, the concentration of paeoniflorin in the reference substance solution is 15 - 25 μg / ml, for example, about 20 μg / ml or about 21.42 μg / ml; Preferably, the concentration of 2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside in the reference substance solution is 80 - 120 μg / ml, for example, about 100 μg / ml or about 102.5 μg / ml; Preferably, the concentration of specnuezhenide in the reference substance solution is 80 - 120 μg / ml, for example, about 100 μg / ml or about 102.4 μg / ml; Preferably, the concentration of atractylenolide III in the reference substance solution is 2 - 4 μg / ml, for example, about 3 μg / ml or about 2.56 μg / ml.

5. The construction method according to claim 1, characterized in that The flow rate of the high-performance liquid chromatography detection is 0.7 - 1.1 ml / min, for example, about 0.9 ml / min; Preferably, the column temperature is 20 - 30 °C, for example, about 25 °C; Preferably, the detection wavelength is 200 - 240 nm, for example, about 224 nm; Preferably, the injection volume is 5 - 15 μl, for example, about 10 μl; More preferably, the theoretical plate number of the chromatographic peak corresponding to paeoniflorin is not less than 2000; Also preferably, the chromatographic column is a ZORBAX SB-C18 chromatographic column; Even more preferably, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm.

6. The construction method according to claim 1, characterized in that Mobile phase A is acetonitrile; Preferably, the acidic aqueous solution, basic aqueous solution and / or buffer saline solution are selected from one or more of weak acids and their salts, weak bases and their salts with different concentrations; Preferably, the aqueous acid solution, aqueous base solution, and / or buffered saline solution are selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid at different concentrations; More preferably, the aqueous acid solution is an aqueous acid solution with a concentration of 0.01% to 1%; More preferably, the aqueous acid solution is an aqueous phosphoric acid solution with a concentration of 0.01% to 1%; More preferably, the aqueous acid solution is an aqueous phosphoric acid solution with a concentration of about 0.1%; Also preferably, the buffered saline solution is an acetate aqueous solution and / or an acetate aqueous solution; Also preferably, the pH value of the buffered saline solution is not greater than 7.

0.

7. The construction method according to claim 1, wherein When the detection wavelength is 224 nm, the ultraviolet fingerprint spectrum includes peaks 1 - 8, and the retention times of peaks 1 - 8 correspond to about 6.315 min, about 13.234 min, about 14.253 min, about 18.608 min, about 19.024 min, about 19.598 min, about 20.453 min, and about 37.394 min respectively; Preferably, when the detection wavelength is 224 nm, the ultraviolet fingerprint spectrum includes peaks 1 - 8, where peak 3 is used as the reference peak, and the average relative retention times of peaks 1, 2, 4, 5, 6, 7, and 8 correspond to about 0.443, about 0.929, about 1.306, about 1.335, about 1.375, about 1.435, and about 2.624 respectively; More preferably, when the detection wavelength is 224 nm, peak 2 is albiflorin, peak 3 is paeoniflorin, peak 4 is 2,3,5,4'-tetrahydroxystilbene - 2 - O - β - D - glucoside, peak 6 is acteoside, and peak 8 is atractylenolide III; More preferably, when the detection wavelength is 224 nm, peak 1 comes from Caulis Polygoni Multiflori and Radix Paeoniae Alba herbs, peak 2 comes from Radix Paeoniae Alba herb, peak 3 comes from Radix Paeoniae Alba herb, peak 4 comes from Caulis Polygoni Multiflori herb, peak 5 comes from Radix Paeoniae Alba herb, peak 6 comes from Fructus Ligustri Lucidi herb, peak 7 comes from Fructus Lycii herb, and peak 8 comes from Rhizoma Atractylodis Macrocephalae herb.

8. A method for identifying a traditional Chinese medicine compound containing Glossy Privet Fruit, characterized in that, The identification technique includes the following steps: (1) Establish the standard ultraviolet fingerprint spectrum of the traditional Chinese medicine compound reference sample according to the ultraviolet fingerprint spectrum construction method described in any one of claims 1 to 7; (2) Take the test solution of the traditional Chinese medicine compound, and perform detection according to the chromatographic conditions in the ultraviolet fingerprint spectrum construction method described in any one of claims 1 to 7 to obtain the ultraviolet fingerprint spectrum of the test sample of the traditional Chinese medicine compound; and (3) Compare the ultraviolet fingerprint spectrum of the test sample of the traditional Chinese medicine compound obtained in step (2) with the standard ultraviolet fingerprint spectrum of the traditional Chinese medicine compound reference sample obtained in step (1). Those that meet the requirements are qualified products, and those that do not meet the requirements are unqualified products.

9. The authentication technique according to claim 8, wherein The meeting the requirements includes one or more of the following: (1) Eight characteristic chromatographic peaks are presented in the ultraviolet fingerprint of the traditional Chinese medicine compound sample to be tested, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference substance chromatographic peak in the standard ultraviolet fingerprint of the traditional Chinese medicine compound reference sample; (2) Taking the 3rd peak as the S peak, the relative retention time of each characteristic chromatographic peak in the ultraviolet fingerprint of the traditional Chinese medicine compound sample to be tested and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard ultraviolet fingerprint of the traditional Chinese medicine compound reference sample; And (3) According to the similarity evaluation system for ultraviolet fingerprints of traditional Chinese medicine chromatography, the similarity calculated between the ultraviolet fingerprint of the traditional Chinese medicine compound sample to be tested and the standard ultraviolet fingerprint of the traditional Chinese medicine compound reference sample shall not be less than 0.

90.

10. Use of the construction method according to any one of claims 1 to 7 or the identification technique according to claim 8 or 9 in the quality inspection and / or quality evaluation and / or quality control of traditional Chinese medicine compounds containing Glossy Privet Fruit.

Images