Fingerprint establishment method and detection method of traditional Chinese medicine composition

Abstract

The present invention relates to a fingerprint establishment method and detection method of a traditional Chinese medicine composition. The raw materials of the traditional Chinese medicine composition include: ginseng, astragalus, atractylodes, tuckahoe, oriental rhizome, pinellia tuber, notopterygium, angelica dahurica, saposhnikovia divaricata, bupleurum, coptis root, white peony root, tangerine peel, licorice root, ginger and jujube; the fingerprint establishment method includes the preparation of a reference medicinal material solution and a test solution; the reference medicinal material solution and the test solution are detected by high performance liquid chromatography; the mobile phases used in the high performance liquid chromatography are acetonitrile and water, and the elution method is gradient elution. The fingerprint of the traditional Chinese medicine composition constructed by the above method can effectively characterize the traditional Chinese medicine composition, provide richer chemical information and more comprehensive basis for its quality evaluation, and better ensure the safety of medication and therapeutic effect of patients.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, and in particular to a fingerprint establishment method and a detection method of a traditional Chinese medicine composition. Background Art

[0002] Traditional Chinese medicine compositions contain botanicals with multiple sources and complex ingredients. The efficacy of traditional Chinese medicine compositions does not come from a single active ingredient, but is the result of the combined action of the raw materials of the entire traditional Chinese medicine composition. Differences in the content and composition ratio of active ingredients in traditional Chinese medicine compositions will affect the efficacy. Due to the complexity of the ingredients of traditional Chinese medicine compositions, it is difficult to effectively evaluate the quality of traditional Chinese medicines with any single active ingredient or index ingredient. When evaluating the quality of traditional Chinese medicine compositions and their preparations, appropriate detection methods that can provide rich identification information should be used. Traditional microscopic identification, physical and chemical identification, and content determination methods are not sufficient to solve this problem. The establishment of traditional Chinese medicine fingerprints will be able to more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicines and their preparations, and thus provide an overall description and evaluation of the quality of the medicines.

[0003] Traditional Chinese medicine fingerprint refers to the chromatogram or spectrum that can indicate the chemical characteristics of Chinese medicinal materials or Chinese medicinal preparations after appropriate treatment using certain analytical methods. It is a key technology for achieving overall relevant quality evaluation of multiple components. Traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method. It is based on the systematic research on the chemical components of traditional Chinese medicine. It is mainly used to evaluate the authenticity, quality and stability of the quality of traditional Chinese medicinal materials and semi-finished Chinese medicinal preparations. It specifically includes two aspects: (1) The authenticity of traditional Chinese medicine can be effectively identified through the characteristic characteristics of the fingerprint spectrum; (2) The quality of traditional Chinese medicine can be effectively controlled through the main characteristic peak area and ratio of the fingerprint spectrum.

[0004] At present, the fingerprint technology of traditional Chinese medicine has involved many methods, among which the chromatographic method is the mainstream method, especially high performance liquid chromatography (HPLC), thin layer scanning (TLCS) and gas chromatography (GC) have become the three recognized conventional analytical methods. HPLC has the characteristics of high separation efficiency, high selectivity, high detection sensitivity, fast analysis speed, wide application range, etc. It is not limited by the volatility and thermal stability of the sample. Most components in traditional Chinese medicine samples can be analyzed and detected on a high performance liquid chromatograph. Therefore, HPLC has become the preferred method of traditional Chinese medicine fingerprint technology.

[0005] Chinese patent CN100500206C discloses a Chinese medicine composition for treating chronic nephritis and its preparation method. The Chinese medicine composition is composed of 16 herbs including ginseng, astragalus, atractylodes, tuckahoe, oriental rhizome, pinellia tuber, notopterygium, angelica dahurica, saposhnikovia divaricata, bupleurum, coptis root, white peony root, dried orange peel, licorice root, ginger and jujube. It has the functions of promoting yang and tonifying spleen, benefiting kidney and removing dampness, promoting diuresis and reducing swelling. It can treat proteinuria caused by spleen deficiency and dampness in chronic glomerulonephritis (renal function: SCr<2mg / dL), concurrent with edema, fatigue, fear of cold and cold limbs, and poor appetite. At present, there is little research on this prescription, and the existing information cannot fully reflect the chemical characteristics and quality of this prescription, making it difficult to guarantee the quality and therapeutic effect of this prescription. Summary of the invention

[0006] Based on this, the present invention provides a method for establishing and detecting a fingerprint of a Chinese medicine composition that can comprehensively reflect the chemical characteristics and quality of the above-mentioned Chinese medicine composition.

[0007] The specific technical solutions are as follows:

[0008] The first aspect of the present invention provides a method for establishing a fingerprint of a traditional Chinese medicine composition, wherein the raw materials of the traditional Chinese medicine composition include: ginseng, astragalus, atractylodes, tuckahoe, oriental rhizome, pinellia tuber, notopterygium, angelica dahurica, siler, radix scutellariae, bupleurum, coptis root, white peony root, tangerine peel, roasted liquorice, ginger and jujube;

[0009] The fingerprint establishment method comprises the following steps:

[0010] Weigh the Chinese medicine composition, extract it with an extraction solvent, filter it, take the filtrate, and prepare a test solution;

[0011] The test solution is tested by high performance liquid chromatography to establish a fingerprint;

[0012] The mobile phases used in the high performance liquid chromatography method are acetonitrile and water, and the elution method is gradient elution.

[0013] In one embodiment, the gradient elution comprises the following procedure:

[0014] 0-10min, the volume percentage of acetonitrile increases from 5% to 15%;

[0015] 10 to 30 minutes, the volume percentage of acetonitrile increases from 15% to 20%;

[0016] 30-50 min, the volume percentage of acetonitrile increases from 20% to 30%;

[0017] 50-80 min, the volume percentage of acetonitrile increases from 30% to 90%;

[0018] From 80 to 95 min, the volume percentage of acetonitrile is maintained at 90%.

[0019] In one embodiment, the high performance liquid chromatography method further comprises the following detection conditions:

[0020] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0021] Flow rate: 0.1~0.5mL / min;

[0022] Column temperature: 30-45°C;

[0023] UV detection wavelength: 200~320nm.

[0024] In one embodiment, the column temperature of the HPLC is 35-45° C., and the UV detection wavelength is 200-300 nm.

[0025] In one embodiment, the extraction solvent is a methanol aqueous solution with a volume fraction of 40-60%.

[0026] In one embodiment, the extraction method is ultrasonic extraction for 20 to 40 minutes.

[0027] In one embodiment, the fingerprint spectrum includes 17 characteristic peaks. Taking peak 6 as a reference, the relative retention times of the 17 characteristic peaks are:

[0028] Peak 1: 0.353 ± 0.019%; Peak 2: 0.491 ± 0.018%; Peak 3: 0.582 ± 0.021%;

[0029] Peak 4: 0.758 ± 0.032%; Peak 5: 0.908 ± 0.016%; Peak 6: 1.000 ± 0.000%;

[0030] Peak 7: 1.103 ± 0.036%; Peak 8: 1.273 ± 0.026%; Peak 9: 1.354 ± 0.032%;

[0031] Peak 10: 1.540 ± 0.049%; Peak 11: 1.585 ± 0.054%; Peak 12: 1.720 ± 0.047%;

[0032] Peak 13: 1.770 ± 0.038%; Peak 14: 1.875 ± 0.023%; Peak 15: 1.890 ± 0.021%;

[0033] Peak 16: 1.982±0.046%; Peak 17: 2.435±0.035%.

[0034] The second aspect of the present invention provides a method for detecting a Chinese medicine composition, wherein the raw materials of the Chinese medicine composition include: ginseng, astragalus, atractylodes, tuckahoe, oriental rhizome, pinellia tuber, notopterygium, angelica dahurica, siler, radix scutellariae, bupleurum, coptis root, white peony root, tangerine peel, roasted liquorice, ginger and jujube;

[0035] The detection method of the Chinese medicine composition comprises the following steps:

[0036] Weigh the Chinese medicine composition to be tested, extract it with an extraction solvent, filter it, take the filtrate, and prepare a solution of the product to be tested;

[0037] The test solution is tested by high performance liquid chromatography;

[0038] The mobile phases used in the high performance liquid chromatography method are acetonitrile and water, and the elution method is gradient elution.

[0039] In one embodiment, the gradient elution comprises the following procedure:

[0040] 0-10min, the volume percentage of acetonitrile increases from 5% to 15%;

[0041] 10 to 30 minutes, the volume percentage of acetonitrile increases from 15% to 20%;

[0042] 30-50 min, the volume percentage of acetonitrile increases from 20% to 30%;

[0043] 50-80 min, the volume percentage of acetonitrile increases from 30% to 90%;

[0044] From 80 to 95 min, the volume percentage of acetonitrile is maintained at 90%.

[0045] In one embodiment, the high performance liquid chromatography method further comprises the following detection conditions:

[0046] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0047] Flow rate: 0.1~0.5mL / min;

[0048] Column temperature: 30-45°C;

[0049] UV detection wavelength: 200~320nm.

[0050] In one embodiment, the column temperature of the HPLC is 35-45° C., and the UV detection wavelength is 200-300 nm.

[0051] In one embodiment, the extraction solvent is a methanol aqueous solution with a volume fraction of 40-60%.

[0052] In one embodiment, the extraction method is ultrasonic extraction for 20 to 40 minutes.

[0053] The fingerprint establishment method provided by the present invention adopts high performance liquid chromatography and combines with appropriate mobile phase settings to obtain a fingerprint that is systematic, characteristic and stable, which is specifically embodied in:

[0054] 1. Systematicity: The raw materials of the Chinese medicine composition include: ginseng, astragalus, tuckahoe, jujube, atractylodes, oriental rhizome, pinellia tuber, notopterygium, angelica pubescens, saposhnikovia divaricata, bupleurum, coptis root, white peony root, tangerine peel, licorice root and ginger. The fingerprint spectrum established by the present invention includes 17 characteristic peaks, and the attribution of the 17 characteristic peaks in the raw medicinal materials of the Chinese medicine composition is as follows, see Table 1.

[0055] Table 1 Chromatographic peak attribution table of fingerprint of traditional Chinese medicine composition

[0056] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Atractylodes macrocephala ● ● ● ● ● ● ● Poria ● Roasted Licorice ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● White Peony Root ● ● ● ● ● ● Tangerine peel ● ● ● ● ● ● ● ● ● ● ● ● Pinellia tuber ● ● ● ● ● ● ● ● ● ● ● Ginger ● ● ● ● ● ● ● ● Windproof ● ● ● ● ● ● Astragalus ● ● ● ● ● ● ● ● ● Angelica dahurica ● ● ● ● ● ● ● ● ● ● ● Notopterygium wilfordii ● ● ● ● ● ● ● ● ● Jujube ● Bupleurum ● ● ● ● ● ● ● ● Ginseng ● ● ● ● ● ● ● Alisma orientalis ● ● ● ● ● Coptis chinensis ● ● ● ● ● ● ● ● ● ●

[0057] 2. Characteristic: The fingerprint established by the method of the present invention is based on the specific raw materials of the Chinese medicine composition of the present invention and the interaction between the chemical components in the preparation process. Taking peak 6 as a reference, the relative retention time and relative peak area of ​​each characteristic peak are fixed within a certain range, and will produce differences when the medicinal taste composition changes (such as the use of fake and inferior medicinal materials). Therefore, the authenticity identification and quality control of the Chinese medicine composition can be well performed.

[0058] 3. Stability: The method of the present invention can ensure that the fingerprints produced by different operators and different laboratories are within the allowable error range, which reflects the versatility and practicality of the method.

[0059] In summary, the fingerprint spectrum established by the method for establishing the fingerprint spectrum of the traditional Chinese medicine composition provided by the present invention can effectively characterize the traditional Chinese medicine composition of the present invention, provide richer chemical information and more comprehensive basis for its quality evaluation, and better ensure the safety of patient medication and therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The chromatograms of the Chinese medicine composition in Example 1 at different ultraviolet detection wavelengths (S1: 203nm; S2: 254nm; S3: 280nm; S4: 320nm);

[0061] Figure 2 The chromatograms of the Chinese medicine composition in Example 1 at different column temperatures (S1: 30°C; S2: 35°C; S3: 40°C);

[0062] Figure 3The chromatograms of different extraction solvents of the Chinese medicine composition in Example 1 (S1: 30% methanol aqueous solution; S2: 50% methanol aqueous solution; S3: 70% methanol aqueous solution);

[0063] Figure 4 This is an overlay chromatogram of the instrument precision investigation in Example 1;

[0064] Figure 5 This is an overlay chromatogram of the reproducibility study of the method in Example 1;

[0065] Figure 6 The superimposed chromatograms of the stability study of the method in Example 1 (S1: 0h; S2: 4.5h; S3: 8h; S4: 24h; S5: 48h);

[0066] Figure 7 The superimposed fingerprints of 10 batches of Yishen Huashi Granules in Example 1;

[0067] Figure 8 Number the characteristic peaks of the fingerprints of 10 batches of Yishen Huashi Granules in Example 1;

[0068] Fig. 9 The fingerprint of Yishen Huashi Granule in Example 1 is superimposed with the fingerprint of each medicinal material in the raw material (1. Yishen Huashi Granule, 2. Atractylodes macrocephala, 3. Poria, 4. Radix Glycyrrhizae Preparata, 5. White Peony Root, 6. Tangerine Peel, 7. Pinellia Tuber, 8. Ginger, 9. Saposhnikovia divaricata, 10. Astragalus, 11. Angelica dahurica, 12. Notopterygium incisum, 13. Jujube, 14. Bupleurum, 15. Ginseng, 16. Alisma orientalis, 17. Coptis chinensis);

[0069] Fig.10 This is the superimposed fingerprint of 6 batches of Yishen Huashi Granules in Example 2. DETAILED DESCRIPTION

[0070] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the embodiments, and preferred embodiments of the present invention are given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0072] The optional scope of the terms "and / or", "or / and", and "and / or" used in this article includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, said any and all combinations include any combination of any two related listed items, any more related listed items, or all related listed items.

[0073] In the present invention, "the first aspect", "the second aspect", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "the first", "the second", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0074] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0075] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0076] The percentage contents involved in the present invention, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

[0077] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0078] The present invention provides a method for establishing a fingerprint spectrum of a traditional Chinese medicine composition. The raw materials of the traditional Chinese medicine composition include: ginseng, astragalus, atractylodes, tuckahoe, oriental rhizome, pinellia tuber, notopterygium, angelica dahurica, siler, radix scutellariae, bupleurum, coptis root, white peony root, tangerine peel, roasted liquorice root, ginger and jujube;

[0079] The fingerprint establishment method comprises the following steps:

[0080] Weigh the Chinese medicine composition, extract it with an extraction solvent, filter it, and take the filtrate to prepare a test solution;

[0081] The test solution was tested by high performance liquid chromatography to establish a fingerprint;

[0082] The mobile phases used in the HPLC method were acetonitrile and water, and the elution method was gradient elution.

[0083] In one example, gradient elution includes the following procedure:

[0084] From 0 to 10 min, the volume percentage of acetonitrile increased from 5% to 15%;

[0085] From 10 to 30 min, the volume percentage of acetonitrile increased from 15% to 20%;

[0086] From 30 to 50 min, the volume percentage of acetonitrile increased from 20% to 30%;

[0087] From 50 to 80 min, the volume percentage of acetonitrile increased from 30% to 90%;

[0088] From 80 to 95 min, the volume percentage of acetonitrile was maintained at 90%.

[0089] In one example, the high performance liquid chromatography method further includes the following detection conditions: the chromatographic column uses octadecylsilane bonded silica gel as a filler. Furthermore, the chromatographic column is an AdvanceBio Peptide Mapping (2.1×250 mm, 2.7 μm) chromatographic column.

[0090] In one example, the high performance liquid chromatography method further includes the following detection conditions: a flow rate of 0.1 to 0.5 mL / min. Further, the flow rate includes but is not limited to: 0.1 mL / min, 0.15 mL / min, 0.2 mL / min, 0.25 mL / min, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min.

[0091] In one example, the high performance liquid chromatography method further includes the following detection conditions: the column temperature is 30-45° C. Further, the column temperature includes but is not limited to: 30° C., 35° C., 37° C., 39° C., 40° C., 41° C., 45° C.

[0092] In one example, the high performance liquid chromatography method further includes the following detection conditions: the ultraviolet detection wavelength is 200-320 nm. Further, the ultraviolet detection wavelength includes but is not limited to: 200 nm, 203 nm, 205 nm, 210 nm, 230 nm, 258 nm, 280 nm, 300 nm.

[0093] Specifically, in one example, the high performance liquid chromatography method further includes the following detection conditions:

[0094] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0095] Flow rate: 0.1~0.5mL / min;

[0096] Column temperature: 30-45°C;

[0097] The UV detection wavelength is 200-320nm.

[0098] In one example, the extraction solvent is a methanol aqueous solution with a volume fraction of 40-60%. Further, the volume fraction of the methanol aqueous solution includes but is not limited to: 40%, 45%, 47%, 50%, 53%, 55%, 60%.

[0099] In one example, the extraction method is ultrasonic extraction for 20 to 40 minutes. Further, the time of ultrasonic extraction includes but is not limited to: 20 minutes, 25 minutes, 29 minutes, 30 minutes, 31 minutes, 35 minutes, and 40 minutes.

[0100] In one example, the ultrasonic power of the ultrasonic extraction is 300w and the frequency is 40kHz.

[0101] In one example, the fingerprint of the Chinese medicine composition established according to the method includes 17 characteristic peaks. Taking peak 6 as a reference, the relative retention times of the 17 characteristic peaks are:

[0102] Peak 1: 0.353 ± 0.019%; Peak 2: 0.491 ± 0.018%; Peak 3: 0.582 ± 0.021%;

[0103] Peak 4: 0.758 ± 0.032%; Peak 5: 0.908 ± 0.016%; Peak 6: 1.000 ± 0.000%;

[0104] Peak 7: 1.103 ± 0.036%; Peak 8: 1.273 ± 0.026%; Peak 9: 1.354 ± 0.032%;

[0105] Peak 10: 1.540 ± 0.049%; Peak 11: 1.585 ± 0.054%; Peak 12: 1.720 ± 0.047%;

[0106] Peak 13: 1.770 ± 0.038%; Peak 14: 1.875 ± 0.023%; Peak 15: 1.890 ± 0.021%;

[0107] Peak 16: 1.982±0.046%; Peak 17: 2.435±0.035%.

[0108] In one example, the steps of the fingerprint establishment method include:

[0109] (1) Take 0.5 g of the Chinese medicine composition, accurately weigh it, add 45% to 55% methanol aqueous solution to make it up to 100 mL, perform ultrasonic extraction for 25 to 35 min, leave it at room temperature, add 45% to 55% methanol aqueous solution to make it up to 100 mL, filter it with a microporous membrane, take the filtrate, and prepare the test solution;

[0110] (2) The test solution is tested by high performance liquid chromatography, the test method being as follows: 5 μL of the test solution is accurately drawn, injected into a high performance liquid chromatograph, measured, and the chromatogram is recorded for 100 min;

[0111] The detection steps include the following chromatographic conditions:

[0112] Chromatographic column: AdvanceBio Peptide Mapping (2.1×250mm, 2.7μm) chromatographic column, with octadecylsilane bonded silica as filler;

[0113] Mobile phase: Use acetonitrile-water solution as the mobile phase and perform gradient elution according to the following procedure:

[0114] 0-10min, the volume percentage of acetonitrile increases from 5% to 15%;

[0115] 10 to 30 minutes, the volume percentage of acetonitrile increases from 15% to 20%;

[0116] 30-50 min, the volume percentage of acetonitrile increases from 20% to 30%;

[0117] 50-80 min, the volume percentage of acetonitrile increases from 30% to 90%;

[0118] From 80 to 95 min, the volume percentage of acetonitrile is maintained at 90%.

[0119] Flow rate: 0.1~0.3mL / min;

[0120] Column temperature: 35-45°C;

[0121] UV detection wavelength: 200~220nm.

[0122] In one of the examples, the Chinese medicine composition, measured in parts by mass, includes the following raw materials: 170-171 parts of ginseng, 340-342 parts of astragalus, 51-52 parts of atractylodes, 51-52 parts of poria, 51-52 parts of oriental rhizome, 170-171 parts of pinellia, 85-86 parts of notopterygium, 85-86 parts of angelica dahurica, 85-86 parts of saposhnikovia divaricata, 51-52 parts of bupleurum, 34-35 parts of coptis chinensis, 85-86 parts of white peony root, 68-69 parts of tangerine peel, 170-171 parts of roasted liquorice, 49-51 parts of ginger, and 99-101 parts of jujube.

[0123] In one example, the dosage form of the Chinese medicine composition is granules.

[0124] In one example, the steps of preparing the granules include:

[0125] (1) taking various medicinal materials according to the raw material composition of the Chinese medicine composition;

[0126] (2) Add water to 12 herbs including Atractylodes macrocephala, Rhizoma Alismatis, Pinellia ternata, Notopterygium wilfordii, Angelica dahurica, Saposhnikovia divaricata, Radix Bupleuri, Rhizoma Coptidis, Paeonia lactiflora, Pericarpium Citri Reticulatae, Radix Glycyrrhizae, and Ginger, and decoct for 2 to 4 times. For the first decoction, add 10 times the amount of water and decoct for 1 to 3 hours. At the same time, extract the volatile oil and store it in a separate container. For the second and third decoctions, add 8 times the amount of water and decoct for 0.5 to 1.5 hours each. Filter the decoction, and decompress the filtrate to a thick paste with a density of about 1.15 (60-65°C). After cooling, add ethanol to make the alcohol content 55-65%, stir evenly, and let it stand for 12-72 hours. Take the supernatant and decompress it to a thick paste with a relative density of about 1.2 (60-65°C) 1;

[0127] Add water to decoct the four medicinal materials of ginseng, astragalus, poria and jujube for 2 to 4 times, add 10 times the amount of water for the first time and decoct for 0.5 to 1.5 hours, add 8 times the amount of water for the second and third times and decoct for 0.5 to 1.5 hours respectively, filter the decoction, and concentrate the filtrate under reduced pressure into a thick paste 2 with a relative density of about 1.2 (60-65°C);

[0128] The thick paste 1 and the thick paste 2 are combined, granulated, volatile oil is added, and the mixture is sealed and stored to prepare granules.

[0129] The present invention also provides a method for detecting a Chinese medicine composition, wherein the raw materials of the Chinese medicine composition include: ginseng, astragalus, atractylodes, tuckahoe, oriental rhizome, pinellia tuber, notopterygium, angelica dahurica, siler, radix scutellariae, bupleurum, coptis root, white peony root, tangerine peel, roasted liquorice, ginger and jujube;

[0130] The detection method of the Chinese medicine composition comprises the following steps:

[0131] Weigh the Chinese medicine composition to be tested, extract it with an extraction solvent, filter it, and take the filtrate to prepare a test product solution;

[0132] The solution of the test product is tested by high performance liquid chromatography;

[0133] The mobile phases used in the HPLC method were acetonitrile and water, and the elution method was gradient elution.

[0134] It can be understood that the spectrum obtained by using high performance liquid chromatography to detect the solution of the test product can be analyzed as needed. For example, a single test product is tested and compared with the fingerprint spectrum established by the above fingerprint spectrum establishment method to obtain the quality of the single test product; or multiple batches of test products are tested and the spectra of the multiple batches of test products are compared to obtain the similarity between the multiple batches of test products.

[0135] It can be understood that the technical scheme of the above-mentioned detection method of the Chinese medicine composition is similar to the aforementioned method for establishing the fingerprint of the Chinese medicine composition, wherein the preparation of the test solution is the same as the preparation of the test solution, which will not be repeated here.

[0136] The present invention is further described in detail below in conjunction with specific examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal raw materials, auxiliary materials, reagents, etc. used in the following examples are all commercially available products unless otherwise specified.

[0137] Example 1 Method for establishing fingerprint of Chinese medicine composition

[0138] 1. Instruments, reagents and test drugs

[0139] (1) Instrument

[0140] AgiLent 1260 high performance liquid chromatograph;

[0141] AdvanceBio Peptide Mapping (2.1×250mm, 2.7μm) column;

[0142] Ultrasonic cleaning machine (Shenzhen Jiemeng Cleaning Equipment Co., Ltd., JP-060);

[0143] BS224S electronic balance (Sartorius);

[0144] Synergy ultrapure water machine (Guangzhou Dongrui Technology).

[0145] (2) Reagents

[0146] Acetonitrile (chromatographic grade, Merck);

[0147] Ultrapure water (self-made in the laboratory);

[0148] Methanol (analytical grade, Guangzhou Chemical Reagent Factory).

[0149] (3) Drug testing

[0150] Yishen Huashi Granules (Guangzhou Kangchen Pharmaceutical Research Co., Ltd., batch numbers: 20180303, 20180301, 20180302, 20180401, 20180402, 20180403, 20180404, 20180501, 20180502, 20180503). The preparation method of the Yishen Huashi Granules is as follows:

[0151] 1) Prepare the following medicinal materials: 170.5g ginseng, 341g astragalus, 51.1g atractylodes, 51.1g tuckahoe, 51.1g orientale, 170.5g pinellia, 85.2g notopterygium, 85.2g angelica pubescens, 85.2g siler, 51.1g bupleurum, 34.1g coptis, 85.2g white peony root, 68.2g tangerine peel, 170.5g roasted liquorice, 50g ginger, 100g jujube;

[0152] 2) 12 herbs, including Atractylodes macrocephala, Rhizoma Alismatis, Rhizoma Pinelliae, Radix Notopterygii, Radix Angelicae Pubescentis, Radix Saposhnikoviae, Radix Bupleuri, Rhizoma Coptidis, Radix Paeoniae Alba, Pericarpium Citri Reticulatae, Radix Glycyrrhizae, and Ginger, are decocted with water for 3 times: the first time, 10 times the amount of water is added and decocted for 2 hours, and volatile oil is extracted at the same time and stored in a separate container; the second and third times, 8 times the amount of water are added and decocted for 1 hour each, the decoction is filtered, and the filtrate is concentrated under reduced pressure to a thick paste with a relative density of about 1.15 (60-65°C), cooled, ethanol is added to make the alcohol content 60%, stirred evenly, left to stand for 24 hours, the supernatant is taken, and reduced pressure is concentrated to a thick paste with a relative density of about 1.2 (60-65°C) 1;

[0153] Add water to decoct the four medicinal materials of ginseng, astragalus, poria and jujube for 3 times: add 10 times the amount of water for 1 hour for the first time, add 8 times the amount of water for the second and third times and decoct for 1 hour each, filter the decoction, and concentrate the filtrate under reduced pressure into a thick paste 2 with a relative density of about 1.2 (60-65°C);

[0154] The thick paste 1 and the thick paste 2 are heated at 60°C to be combined, the paste is collected, dextrin is added to the dosage prescribed by the preparation, spray granulation is performed, drying is performed, whole granulation is performed, granules are prepared, the total mixing is performed, volatile oil is sprayed in, the granules are sealed for storage, and the granules are packaged according to the dosage.

[0155] 2. Fingerprint map construction

[0156] 2.1 Establishment of chromatographic conditions

[0157] (1) Determination of UV detection wavelength

[0158] Weigh 0.5g of Yishen Huashi Granules accurately, add 50% methanol aqueous solution to 100mL, extract by ultrasonic for 30min, place at room temperature, add 50% methanol aqueous solution to 100mL, filter with microporous membrane, and take the filtrate. For injection determination, use acetonitrile-water solution as the mobile phase, perform gradient elution according to the provisions in Table 2, the flow rate is 0.2mL / min; the column temperature is 40℃; the injection volume is 5μL.

[0159] Table 2 Gradient elution

[0160]

[0161] The chromatograms at UV wavelengths of 203nm, 258nm, 280nm and 300nm were examined respectively, and the results are shown in Figure 1 .

[0162] The results show that there are more chromatographic peaks at a wavelength of 203 nm, the apparent kurtosis is higher, and the baseline is relatively stable, so 203 nm is selected as the ultraviolet detection wavelength of this embodiment.

[0163] (2) Determination of column temperature

[0164] For the injection determination, acetonitrile-water solution was used as the mobile phase, and gradient elution was performed according to the provisions in Table 2, with a flow rate of 0.2 mL / min; the ultraviolet detection wavelength was 203 nm; and the injection volume was 5 μL.

[0165] The chromatograms at column temperatures of 30°C, 35°C and 40°C were examined respectively. The results are shown in Figure 2 .

[0166] The results show that when the temperature is 40°C, the column pressure is relatively low, relatively safe, and the separation is better, so 40°C is selected as the column temperature of this example.

[0167] (3) Determination of chromatographic conditions

[0168] Chromatographic column: AdvanceBio Peptide Mapping (2.1×250mm, 2.7μm) chromatographic column, with octadecylsilane bonded silica as filler;

[0169] Mobile phase: Use acetonitrile-water solution as the mobile phase and perform gradient elution according to the requirements in Table 2;

[0170] Flow rate: 0.2mL / min

[0171] Column temperature: 40°C;

[0172] UV detection wavelength: 203nm;

[0173] Determination method: Accurately pipette 5 μL of the test solution, inject it into the liquid chromatograph, measure, and record the chromatogram for 100 minutes.

[0174] 2.2 Preparation of test samples

[0175] (1) Investigation of extraction solvent

[0176] This experiment investigated the effects of different extraction solvents on the characteristic spectra. Aqueous methanol solutions with volume fractions of 30%, 50% and 70% were selected as extraction solvents, and the chromatogram results using different extraction solvents were compared.

[0177] Take 0.5g of Yishen Huashi Granules, weigh accurately, add 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution to 100mL, extract by ultrasonic for 30min, place at room temperature, add corresponding solvent to 100mL, filter with microporous membrane, take the filtrate, and obtain. For injection determination, use acetonitrile-water solution as mobile phase, perform gradient elution according to the provisions in Table 2, flow rate is 0.2mL / min; column temperature is 40℃; UV detection wavelength is 203nm; injection volume is 5μL. Results are shown in Figure 3 .

[0178] from Figure 3 It can be seen that when 50% methanol aqueous solution is used as the extraction solvent, the peak area of ​​the characteristic peak is the largest, so 50% methanol aqueous solution is selected as the extraction solvent.

[0179] (2) Determination of the test sample preparation method

[0180] Accurately weigh 0.5 g of Yishen Huashi Granules, add 50% methanol aqueous solution to make up to 100 mL, perform ultrasonic extraction for 30 min, leave at room temperature, add 50% methanol aqueous solution to make up to 100 mL, filter with a microporous membrane, and obtain the filtrate.

[0181] 3. Methodological validation

[0182] (1) Precision inspection

[0183] About 0.5 g of Yishen Huashi Granules (batch number: 20180301) was taken to prepare the test solution, and the sample was injected 6 times continuously for determination. The fingerprint software was used for similarity evaluation. The experimental results are shown in Table 3. Figure 4 .

[0184] Table 3 Precision test results

[0185]

[0186] The results showed that the similarities of the chromatograms were all greater than 0.95, indicating that the precision of the experimental method was good.

[0187] (2) Reproducibility study

[0188] Six portions of Yishen Huashi Granules (batch number: 20180301) were accurately weighed, 0.5 g each, and six test solutions were prepared in parallel. The samples were injected and tested separately, and the similarity was evaluated using fingerprint software. The experimental results are shown in Table 4. Figure 5 .

[0189] Table 4 Reproducibility test results

[0190]

[0191] The results showed that the similarities of the chromatograms were all greater than 0.95, indicating that the experimental method had good reproducibility.

[0192] (3) Stability investigation

[0193] About 0.5 g of Yishen Huashi Granules (batch number: 20180301) was taken to prepare the test solution, which was placed at room temperature. The samples were injected and measured at 0 h, 4 h, 8 h, 24 h, and 48 h, respectively. The fingerprint software was used for similarity evaluation. The experimental results are shown in Table 5. Figure 6 .

[0194] Table 5 Stability test results

[0195]

[0196] The results showed that the similarities of the chromatograms were all greater than 0.95, indicating that the test solution was stable within 48 h.

[0197] 4. Fingerprint determination

[0198] 4.1 Experimental procedures

[0199] (1) Take 10 batches of Yishen Huashi Granules, prepare the test solution according to the provisions under "Construction of Fingerprint", inject the sample for measurement, and use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, 2004A Edition" for matching to establish the control fingerprint of the preparation, such as Figure 7 By comparing and analyzing the fingerprints, 17 common characteristic peaks were identified, such as Figure 8 As shown; Peak 6 has the largest area, better separation, moderate and stable retention time, so Peak 6 is selected as the reference.

[0200] (2) The retention time and peak area of ​​Peak 6 were set to 1, and the relative retention times and relative peak areas of other chromatographic peaks were calculated. The results are shown in Tables 6 and 7.

[0201] Table 6 Relative retention time of 17 common peaks of 10 batches of Yishen Huashi Granules

[0202]

[0203] Table 7 Relative peak areas of 17 common peaks of 10 batches of Yishen Huashi Granules

[0204]

[0205] (3) The fingerprints of 10 batches of Yishen Huashi Granules were matched with common peaks using the “Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine, 2004A Edition”, and the similarity between the fingerprints of different batches of Yishen Huashi Granules was calculated. The results are shown in Table 8.

[0206] Table 8 Similarity of fingerprint spectra of 10 batches of Yishen Huashi Granules

[0207]

[0208] The results showed that the similarities between the chromatograms of different batches of Yishen Huashi Granules were all greater than 0.950, indicating good similarity.

[0209] (4) Correlation between Yishen Huashi Granules and the 16 medicinal materials contained in the raw materials and attribution of characteristic peaks

[0210] Take the sixteen medicinal materials contained in the raw materials, accurately weigh the amount of each medicinal material corresponding to the prescription, add 50% methanol aqueous solution to make up to 100mL, ultrasonic extraction for 30 minutes, after leaving at room temperature, add 50% methanol aqueous solution to make up to 100mL, filter with a microporous filter membrane, take the filtrate to obtain the reference solution of each medicinal material, and perform sampling and determination as specified under the "Determination of chromatographic conditions" to obtain the fingerprint of each medicinal material.

[0211] The fingerprint of Yishen Huashi Granules was compared with the fingerprint of each medicinal material in the raw material. Fig. 9 Among the 17 characteristic peaks contained in the fingerprint of the obtained Yishen Huashi Granules, 17 characteristic peaks identical to the fingerprints of the medicinal materials in the raw materials were detected. The characteristic peaks of the fingerprint of the Yishen Huashi Granules were assigned according to the fingerprints of the medicinal materials in the raw materials, and the results are shown in Table 1.

[0212] The results show that the fingerprint of the traditional Chinese medicine composition established by the present invention reflects the chemical components of each medicinal material in the raw materials and has a good correlation with the medicinal materials.

[0213] Through the above research, this application has established the fingerprint method of the Chinese medicine composition and established the fingerprint of the Chinese medicine composition. The determination experiments on multiple batches of samples have proved that this method has good precision, reproducibility and stability, and provides a deeper quality evaluation basis for the Chinese medicine composition than the conventional quality standards.

[0214] Example 2 Detection method of Chinese medicine composition

[0215] Take the Yishen Huashi Granules to be tested (Guangzhou Kangchen Pharmaceutical Research Co., Ltd., batch numbers: 20201207, 20201208, 20201209, 20201218, 20201219, 20201220) and prepare the samples to be tested according to the method under "Determination of the test sample preparation method" in Example 1.

[0216] According to the method under "Determination of Chromatographic Conditions" in Example 1, the fingerprint of the tested Yishen Huashi Granules was obtained. The fingerprints of the six batches of Yishen Huashi Granules were matched with common peaks using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine, 2004A Edition" to calculate the similarity between the fingerprints of the six batches of Yishen Huashi Granules. The results are shown in Fig.10 , Table 9.

[0217] Table 9 Similarity of fingerprints of 6 batches of Yishen Huashi Granules

[0218]

[0219] The results showed that the similarities of the fingerprint spectra of the six batches of Yishen Huashi Granules were all greater than 0.990, indicating that the six batches of Yishen Huashi Granules had good repeatability and small differences between batches.

[0220] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0221] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for establishing a fingerprint of a traditional Chinese medicine composition, characterized in that: The raw materials of the Chinese medicine composition are: ginseng, astragalus, atractylodes, tuckahoe, oriental rhizome, pinellia tuber, notopterygium, angelica dahurica, saposhnikovia divaricata, bupleurum, coptis root, white peony root, tangerine peel, roasted liquorice root, ginger and jujube; The fingerprint establishment method comprises the following steps: Weigh the Chinese medicine composition, extract it with an extraction solvent, filter it, take the filtrate, and prepare a test solution; The test solution is tested by high performance liquid chromatography to establish a fingerprint; The mobile phase used in the high performance liquid chromatography method is acetonitrile and water, and the elution method is gradient elution; The gradient elution includes the following procedures: 0-10min, the volume percentage of acetonitrile increases from 5% to 15%; 10min~30min, the volume percentage of acetonitrile increases from 15% to 20%; 30min~50min, the volume percentage of acetonitrile increases from 20% to 30%; 50min~80min, the volume percentage of acetonitrile increases from 30% to 90%; 80min~95min, the volume percentage of acetonitrile is maintained at 90%; The high performance liquid chromatography method includes the following detection conditions: flow rate: 0.1 mL / min~0.5 mL / min; UV detection wavelength: 203 nm; The extraction method is ultrasonic extraction for 20min~40min, the ultrasonic power of ultrasonic extraction is 300w, and the frequency is 40kHz; Chromatographic column: Octadecylsilane bonded silica gel as filler, specification 2.1×250mm, 2.7μm; The fingerprint spectrum includes 17 characteristic peaks. Taking peak 6 as a reference, the relative retention times of the 17 characteristic peaks are: Peak 1: 0.353 ± 0.019%; Peak 2: 0.491 ± 0.018%; Peak 3: 0.582 ± 0.021%; Peak 4: 0.758 ± 0.032%; Peak 5: 0.908 ± 0.016%; Peak 6: 1.000 ± 0.000%; Peak 7: 1.103 ± 0.036%; Peak 8: 1.273 ± 0.026%; Peak 9: 1.354 ± 0.032%; Peak 10: 1.540 ± 0.049%; Peak 11: 1.585 ± 0.054%; Peak 12: 1.720 ± 0.047%; Peak 13: 1.770 ± 0.038%; Peak 14: 1.875 ± 0.023%; Peak 15: 1.890 ± 0.021%; Peak 16: 1.982 ± 0.046%; Peak 17: 2.435 ± 0.035%; The extraction solvent is a methanol aqueous solution with a volume fraction of 45% to 55%.

2. The fingerprint establishment method according to claim 1, characterized in that: The column temperature of the high performance liquid chromatography is 30°C to 45°C.

3. The fingerprint establishment method according to claim 2, characterized in that: The column temperature of the high performance liquid chromatography is 35°C to 45°C.

4. The fingerprint establishment method according to claim 1, characterized in that: The extraction solvent is a 50% by volume methanol aqueous solution.

5. The fingerprint establishment method according to claim 1, characterized in that: The flow rate of the HPLC was 0.2 mL / min.

6. A method for detecting a Chinese medicine composition, characterized in that: The raw materials of the Chinese medicine composition are: ginseng, astragalus, atractylodes, tuckahoe, oriental rhizome, pinellia tuber, notopterygium, angelica dahurica, saposhnikovia divaricata, bupleurum, coptis root, white peony root, tangerine peel, roasted liquorice root, ginger and jujube; The detection method of the Chinese medicine composition comprises the following steps: Weigh the Chinese medicine composition to be tested, extract it with an extraction solvent, filter it, take the filtrate, and prepare a solution of the product to be tested; The test solution is tested by high performance liquid chromatography; The mobile phase used in the high performance liquid chromatography method is acetonitrile and water, and the elution method is gradient elution; The gradient elution includes the following procedures: 0-10min, the volume percentage of acetonitrile increases from 5% to 15%; 10min~30min, the volume percentage of acetonitrile increases from 15% to 20%; 30min~50min, the volume percentage of acetonitrile increases from 20% to 30%; 50min~80min, the volume percentage of acetonitrile increases from 30% to 90%; 80min~95min, the volume percentage of acetonitrile is maintained at 90%; The high performance liquid chromatography method includes the following detection conditions: flow rate: 0.1 mL / min~0.5 mL / min; UV detection wavelength: 203 nm; The extraction method is ultrasonic extraction for 20min~40min, the ultrasonic power of ultrasonic extraction is 300w, and the frequency is 40kHz; Chromatographic column: Octadecylsilane bonded silica gel as filler, specification 2.1×250mm, 2.7μm; The extraction solvent is a methanol aqueous solution with a volume fraction of 45% to 55%.

7. The method for detecting the Chinese medicine composition according to claim 6, characterized in that: The column temperature of the high performance liquid chromatography is 30°C to 45°C.

8. The method for detecting the Chinese medicine composition according to claim 7, characterized in that: The column temperature of the high performance liquid chromatography is 35°C to 45°C.

9. The method for detecting the Chinese medicine composition according to claim 6, characterized in that: The extraction solvent is a 50% by volume methanol aqueous solution.

10. The method for detecting the Chinese medicine composition according to claim 6, characterized in that: The flow rate of the HPLC was 0.2 mL / min.

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