Construction method and application of UPLC (Ultra Performance Liquid Chromatography) fingerprint spectrum of sinew-spreading and blood-activating powder

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and relates to a construction method and application of a UPLC fingerprint spectrum of sinew-spreading and blood-activating powder. The construction method comprises the following steps: preparing a test solution, a reference medicinal material solution and a reference substance solution, performing UPLC analysis, obtaining the UPLC fingerprint spectrum of the tendon-expanding and blood-activating powder by using a traditional Chinese medicine fingerprint spectrum similarity evaluation system, and the like. The UPLC fingerprint spectrum of the sinew-spreading and blood-activating powder is successfully constructed by reasonably controlling chromatographic conditions, the product quality can be effectively represented, the one-sidedness of determining the product quality by measuring individual chemical components is avoided, the possibility of manual treatment for quality standard reaching is reduced, and the method has the advantages of being simple, convenient, rapid and high in accuracy and has a wide application prospect. Meanwhile, the accuracy, precision, repeatability, stability and durability of the method are good, the detection time is short, a scientific basis is provided for quality evaluation of the sinew-spreading and blood-activating powder, and the method has important significance for controlling the product quality and ensuring the clinical curative effect.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and relates to a method for constructing a UPLC fingerprint of Zhanjin Huoxue Powder, and an application of the fingerprint (for example, application in quality detection of Zhanjin Huoxue Powder). Background Art

[0002] Zhanjin Huoxue San is a compound preparation made of ten Chinese medicinal materials including ginseng, amber, myrrh, frankincense, and dragon's blood. It is used to treat joint and muscle swelling and pain caused by trauma, acute soft tissue injury and other chronic tissue injury, lumbar muscle strain, etc. Modern pharmacological studies have shown that Zhanjin Huoxue San targets key targets such as IL-6, TLR4, TNF, CASP3, VEGFA and ESR1 through active ingredients such as ginsenoside Rh2, dragon's blood, soybean yellow, quercetin, kaempferol, phellodendron and coniferyl ferulate to treat soft tissue injuries (Liu Xu, Li Zhe, Wang Leilei, et al., Exploration of the mechanism of action of Zhanjin Huoxue San in treating soft tissue injuries based on network pharmacology molecular docking technology [J], Modern Drugs and Clinics, 2024, 39(09): pp2254-2262).

[0003] In addition to microscopic identification, the quality control method of Zhanjin Huoxue Powder mainly involves thin layer identification of draconite and angelica. Other quality control studies in the prior art mainly focus on thin layer identification of ginseng, Panax notoginseng, and myrrh, HPLC identification of bilirubin, and determination of draconite content by HPLC (Yang Ruirui, Li Xue, Qiao Rongxia, et al., Study on the Quality Standard of Zhanjin Huoxue Powder [J], Chinese Pharmaceutical Standards, 2013, 14(04): pp251-255). The quality control methods in the prior art only focus on a few components in Zhanjin Huoxue Powder and cannot fully reflect the overall quality of the preparation. Therefore, it is crucial to establish a comprehensive, rapid, and objective method for evaluating its intrinsic quality.

[0004] Traditional Chinese medicine fingerprint (or "fingerprint" for short) is a comprehensive and quantifiable identification method that can systematically, holistically and exclusively characterize the intrinsic characteristics of traditional Chinese medicine. It can be used to evaluate the quality of traditional Chinese medicine materials, traditional Chinese medicine preparations and semi-finished products, and is an effective means of overall quality control of traditional Chinese medicine. So far, no research on fingerprints that can better reveal the intrinsic quality of Zhanjin Huoxue Powder has been published. Therefore, it is necessary to establish an analytical method with excellent specificity, stability and repeatability to better control the preparation quality of Zhanjin Huoxue Powder. Summary of the invention

[0005] Problem that the invention aims to solve

[0006] In order to solve the above problems in the prior art, the present invention provides a method for constructing an ultra-high performance liquid chromatography fingerprint of Zhanjin Huoxue Powder, and the application of the fingerprint in the quality detection of Zhanjin Huoxue Powder. The construction method of the present invention enables the quality control of Zhanjin Huoxue Powder to rise from the original identification and content determination of several components to the control of the intrinsic quality of the entire Zhanjin Huoxue Powder, which can more comprehensively reflect the characteristics of Zhanjin Huoxue Powder, provide a new analytical means for the intrinsic quality control of Zhanjin Huoxue Powder, and achieve the purpose of controlling the quality of Zhanjin Huoxue Powder preparations as a whole.

[0007] Solutions for solving problems

[0008] In a first aspect, the present invention provides a method for constructing an ultra-high performance liquid chromatography fingerprint of Zhanjin Huoxue Powder, comprising the following steps:

[0009] (1) Prepare different batches of test solution of Zhanjin Huoxue Powder, control solution of single medicinal material, and control solution of single indicator component;

[0010] (2) performing ultra-high performance liquid chromatography analysis on different batches of test sample solutions, reference medicinal material solutions, and reference substance solutions prepared in step (1) to obtain corresponding ultra-high performance liquid chromatograms; and

[0011] (3) The ultra-high performance liquid chromatography (ULHPLC) of the reference medicinal material solution was used to identify the medicinal materials in the ultra-high performance liquid chromatography (ULHPLC) of the test sample solutions of different batches; the ultra-high performance liquid chromatography (ULHPLC) of the reference solution was used to identify the index components in the ultra-high performance liquid chromatography (ULHPLC) of the test sample solutions of different batches; and the ULHPLC of the test sample solutions of different batches was processed using the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints to obtain the ULHPLC fingerprint of Zhanjin Huoxue Powder.

[0012] In one embodiment, in step (1) of the above construction method, the Zhanjin Huoxue powder is made of ginseng, amber, myrrh, frankincense, dragon's blood, pearl powder, angelica, Panax notoginseng, musk, and bezoar.

[0013] In a specific embodiment, the myrrh in the Zhanjin Huoxue Powder is processed myrrh, i.e., myrrh (processed) or processed myrrh, such as vinegar myrrh.

[0014] In a specific embodiment, the frankincense in the Zhanjin Huoxue Powder is processed frankincense, i.e., frankincense (processed) or processed frankincense, such as vinegar frankincense.

[0015] In a specific embodiment, the musk in the Zhanjin Huoxue Powder is artificially made musk, that is, musk (artificial) or artificial musk.

[0016] In a specific embodiment, the bezoar in the Zhanjin Huoxue powder is artificially made bezoar, i.e. bezoar (artificial) or artificial bezoar.

[0017] In one embodiment, in step (1) of the above construction method, the medicinal material comprises at least one of ginseng, amber, myrrh, frankincense, dragon's blood, pearl powder, angelica, Panax notoginseng, musk and bezoar.

[0018] In one embodiment, in step (1) of the above construction method, the medicinal material comprises at least one of ginseng, myrrh, frankincense, dragon's blood, angelica, Panax notoginseng and musk.

[0019] In one embodiment, in step (1) of the above construction method, the medicinal materials include ginseng, myrrh, frankincense, dragon's blood, angelica, Panax notoginseng and musk, preferably ginseng, myrrh, frankincense, dragon's blood, angelica, Panax notoginseng and musk.

[0020] In a specific embodiment, the myrrh in the medicinal material is processed myrrh, i.e., myrrh (processed) or processed myrrh, such as vinegar myrrh.

[0021] In a specific embodiment, the frankincense in the medicinal material is processed frankincense, i.e., frankincense (processed) or processed frankincense, such as vinegar frankincense.

[0022] In a specific embodiment, the musk in the medicinal material is artificially made musk, that is, musk (artificial) or artificial musk.

[0023] In one embodiment, in step (1) of the above construction method, the indicative component comprises at least one of ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re and dracoside.

[0024] In one embodiment, in step (1) of the above construction method, the indicative component comprises at least one of ginsenoside Rb1, ginsenoside Re and dracoside.

[0025] In a specific embodiment, the indicative components include ginsenoside Rb1, ginsenoside Re and dracoside, preferably ginsenoside Rb1, ginsenoside Re and dracoside.

[0026] In one embodiment, in step (1) of the above construction method, the indicative component comprises at least one of ginsenoside Rg1, ginsenoside Rb1 and dracoside.

[0027] In a specific embodiment, the indicative components include ginsenoside Rg1, ginsenoside Rb1 and dracoside, preferably ginsenoside Rg1, ginsenoside Rb1 and dracoside.

[0028] In one embodiment, in step (1) of the above-mentioned construction method, the preparation method of the test solution includes: extracting different batches of Zhanjin Huoxue Powder with solvent 1 respectively; preferably, the preparation method also includes: shaking the extracted liquid, filtering (preferably microporous membrane filtration), and taking the filtrate.

[0029] In one embodiment, in step (1) of the above construction method, the preparation method of the control medicinal material solution includes: extracting each medicinal material with solvent 1 respectively; preferably, the preparation method also includes: shaking the extracted liquid, filtering (preferably microporous membrane filtration), and taking the filtrate.

[0030] In one embodiment, in step (1) of the above construction method, the method for preparing the control medicinal material solution further comprises: before the extraction step, grinding and sieving each medicinal material separately or drying and crushing and sieving it separately.

[0031] In a specific embodiment, in step (1) of the above-mentioned construction method, the preparation method of the control medicinal material solution also includes: when the medicinal material contains musk and / or bezoar, before the extraction stage, the musk and / or bezoar are ground and sieved separately in advance, and the remaining medicinal materials in the medicinal material are dried, crushed and sieved separately.

[0032] In one embodiment, in step (1) of the above-mentioned construction method, the preparation method of the reference solution includes: dissolving each index component with solvent 1 or solvent 2 respectively; preferably, the preparation method also includes: shaking the dissolved liquid, filtering (preferably microporous membrane filtration), and taking the filtrate.

[0033] In a specific embodiment, the solvent 1 is a fatty alcohol or an aqueous solution thereof, preferably methanol, ethanol or an aqueous solution thereof, more preferably methanol, ethanol or a 30% to 70% v / v methanol aqueous solution or an ethanol aqueous solution, further preferably methanol, ethanol or a 50% to 70% v / v methanol aqueous solution or an ethanol aqueous solution, further preferably a 70% v / v methanol aqueous solution or an ethanol aqueous solution, and most preferably a 70% v / v methanol aqueous solution.

[0034] In a specific embodiment, the solvent 2 is a fatty alcohol solution of an acid, preferably a fatty alcohol solution of an inorganic acid, more preferably a methanol or ethanol solution of phosphoric acid, further preferably a 1% to 5% v / v methanol solution of phosphoric acid or an ethanol solution of phosphoric acid, further preferably a 1% to 3% v / v methanol solution of phosphoric acid or an ethanol solution of phosphoric acid, and most preferably a 3% v / v methanol solution of phosphoric acid.

[0035] In a specific embodiment, the extraction method is ultrasonic extraction or reflux extraction, preferably ultrasonic extraction.

[0036] In a specific embodiment, the extraction time is 5 to 60 min, preferably 10 to 30 min, and more preferably 20 min.

[0037] In a specific embodiment, when preparing the test solution, the dosage ratio of Zhanjin Huoxue Powder to Solvent 1 is 0.01-0.04 g / mL, preferably 0.02-0.03 g / mL, and more preferably 0.025 g / mL. However, it should be understood that when preparing the test solution, the dosage ratio of Zhanjin Huoxue Powder to Solvent 1 can be adjusted according to the operator's routine experience and actual conditions, and is not limited to the exemplary range in this application.

[0038] In one embodiment, when preparing a control medicinal solution, since the weight proportion of a single medicinal material in the preparation is different, there are also multiple situations for the dosage ratio of each medicinal material to solvent 1. For example, based on 1 part by weight, the dosage ratio of each medicinal material to solvent 1 is 0.1 to 1 mg / mL, preferably 0.2 to 0.8 mg / mL, and more preferably 0.3 to 0.5 mg / mL. However, it should be understood that when preparing a control medicinal solution, the dosage ratio of a single medicinal material to solvent 1 can be adjusted according to the operator's routine experience and actual conditions, and is not limited to the exemplary range in this application.

[0039] In a specific embodiment, the medicinal materials include ginseng, myrrh, frankincense, dragon's blood, angelica, Panax notoginseng and musk, preferably ginseng, myrrh, frankincense, dragon's blood, angelica, Panax notoginseng and musk, wherein: ginseng corresponds to 5 parts by weight, myrrh corresponds to 5 parts by weight, frankincense corresponds to 5 parts by weight, dragon's blood corresponds to 20 parts by weight, angelica corresponds to 5 parts by weight, Panax notoginseng corresponds to 5 parts by weight, and musk corresponds to 3 parts by weight.

[0040] In a specific embodiment, the concentration of the reference solution is 0.025-0.4 mg / mL, preferably 0.05-0.2 mg / mL, and more preferably 0.1 mg / mL. However, it should be understood that when preparing the reference solution, the dosage ratio of the single indicator component to the solvent 1 or the solvent 2 can be adjusted according to the operator's routine experience and actual conditions, and is not limited to the exemplary range in this application.

[0041] In one embodiment, when preparing the reference solution, different solvents are required to dissolve the indicative components due to their different properties.

[0042] In a specific embodiment, the indicative components include ginsenoside Rg1, ginsenoside Rb1 and dracoside, preferably ginsenoside Rg1, ginsenoside Rb1 and dracoside, wherein: solvent 1 is used to dissolve ginsenoside Rg1 and / or ginsenoside Rb1, and solvent 2 is used to dissolve dracoside.

[0043] In one embodiment, in step (2) of the above construction method, the conditions for the ultra-high performance liquid chromatography analysis include:

[0044] The chromatographic column is an octadecylsilane bonded silica gel packing chromatographic column (C18 chromatographic column);

[0045] The mobile phase is an aqueous solution of acetonitrile-phosphoric acid or phosphate (e.g. sodium dihydrogen phosphate);

[0046] The elution method was gradient elution.

[0047] In a specific embodiment, the C18 chromatographic column is a Phenomenex Luna Omega (2.1mm×150mm, 1.6μm), Phenomenex Luna Omega Polar C18 100A (2.1mm×100mm, 1.7μm), Waters CORTECS UPLC T3 (2.1mm×150mm, 1.6μm) or Waters CORTECS UPLC T3 (2.1mm×100mm, 1.6μm) chromatographic column, preferably a Waters CORTECS UPLC T3 (2.1mm×150mm, 1.6μm) chromatographic column.

[0048] In a specific embodiment, the mobile phase is acetonitrile-phosphoric acid aqueous solution, preferably acetonitrile-0.05% to 0.2% v / v phosphoric acid aqueous solution, more preferably acetonitrile-0.05% v / v phosphoric acid aqueous solution.

[0049] In a specific embodiment, the mobile phase is acetonitrile-sodium dihydrogen phosphate aqueous solution, preferably acetonitrile-0.02-0.08 M sodium dihydrogen phosphate aqueous solution, more preferably acetonitrile-0.05 M sodium dihydrogen phosphate aqueous solution.

[0050] In a specific embodiment, the procedure of gradient elution is as follows.

[0051]

[0052] In one embodiment, in step (2) of the above construction method, the conditions for the ultra-high performance liquid chromatography analysis further include: the column temperature of the chromatographic column is 25-40°C.

[0053] In a specific embodiment, the column temperature is 35-40°C, preferably 35°C.

[0054] In one embodiment, in step (2) of the above construction method, the conditions for the ultra-high performance liquid chromatography analysis further include: the flow rate of the mobile phase is 0.25 to 0.35 mL / min.

[0055] In a specific embodiment, the flow rate is 0.28-0.32 mL / min, preferably 0.30 mL / min.

[0056] In one embodiment, in step (2) of the above construction method, the conditions for the ultra-high performance liquid chromatography analysis further include: the detector is a UV detector or a UV-Vis detector.

[0057] In a specific embodiment, the detection wavelength of the detector is 190-210 nm, preferably 203 nm.

[0058] In a more specific embodiment, the conditions for the ultra-high performance liquid chromatography analysis include:

[0059] The chromatographic column is a C18 column;

[0060] The mobile phase was acetonitrile-0.05% to 0.2% v / v phosphoric acid aqueous solution;

[0061] The elution method is gradient elution, and the procedure of the gradient elution is as follows:

[0062]

[0063] The detection wavelength is 190~210nm.

[0064] In a more specific embodiment, the conditions for the ultra-high performance liquid chromatography analysis include:

[0065] The chromatographic column was a Waters CORTECS UPLC T3 (2.1 mm × 150 mm, 1.6 μm) column;

[0066] The mobile phase was acetonitrile-0.05% v / v phosphoric acid in water;

[0067] The elution method is gradient elution, and the procedure of the gradient elution is as follows:

[0068]

[0069] The detection wavelength is 203 nm.

[0070] In one embodiment, in step (3) of the above-mentioned construction method, the ultra-high performance liquid chromatography fingerprint contains at least 6, preferably at least 9, more preferably at least 12, further preferably at least 18, and most preferably at least 24 common peaks.

[0071] In a specific embodiment, the ultra-high performance liquid chromatography fingerprint comprises 24 common peaks;

[0072] Furthermore, taking peak 3 as the reference peak, the relative retention times of the 24 common peaks were: peak 1: 0.85±5%, peak 2: 0.99±5%, peak 3: 1.00±5%, peak 4: 1.30±5%, peak 5: 1.39±5%, peak 6: 1.41±5%, peak 7: 1.54±5%, peak 8: 1.61±5%, peak 9: 2.05±5%, peak 10: 2.08±5%, peak 11: 2.13±5%, peak 12: 2. Peak No. 1: 2.16±5%, Peak No. 13: 2.20±5%, Peak No. 14: 2.34±5%, Peak No. 15: 2.40±5%, Peak No. 16: 2.45±5%, Peak No. 17: 3.02±5%, Peak No. 18: 3.36±5%, Peak No. 19: 3.42±5%, Peak No. 20: 3.56±5%, Peak No. 21: 3.70±5%, Peak No. 22: 3.80±5%, Peak No. 23: 3.90±5% and Peak No. 24: 3.93±5%.

[0073] Furthermore, peak 1 among the 24 common peaks belongs to Panax notoginseng and ginseng, peak 2 belongs to musk, peak 3 belongs to Sanguisorba officinalis, peak 4 belongs to Panax notoginseng, peaks 5 to 10 belong to Sanguisorba officinalis, peaks 11 to 12 belong to myrrh, peak 13 belongs to Sanguisorba officinalis, peak 14 belongs to myrrh and Sanguisorba officinalis, peak 15 belongs to myrrh, peaks 16 to 17 belong to Sanguisorba officinalis, peak 18 belongs to frankincense, peak 19 belongs to Sanguisorba officinalis, peak 20 belongs to Angelica sinensis, and peaks 21 to 24 belong to frankincense.

[0074] Furthermore, peak No. 1 among the 24 common peaks corresponds to ginsenoside Rg1 and / or ginsenoside Re, peak No. 3 corresponds to dracoside, and peak No. 4 corresponds to ginsenoside Rb1.

[0075] In a second aspect, the present invention provides an application of the ultra-high performance liquid chromatography fingerprint of Zhanjin Huoxue Powder obtained based on the above-mentioned construction method in the quality detection of Zhanjin Huoxue Powder.

[0076] In one embodiment, the quality detection includes quality evaluation of the Zhanjin Huoxue Powder sample to be tested.

[0077] In a third aspect, the present invention provides a quality detection method for Zhanjin Huoxue Powder, which comprises the following steps:

[0078] (I) obtaining the ultra-high performance liquid chromatography fingerprint of Zhanjin Huoxue Powder based on the above construction method as a standard fingerprint;

[0079] (II) preparing a test solution of the Zhanjin Huoxue Powder sample by the same test solution preparation method and ultra-high performance liquid chromatography analysis conditions as the above-mentioned construction method, and performing ultra-high performance liquid chromatography analysis on the test solution to obtain a corresponding ultra-high performance liquid chromatogram; and

[0080] (III) Using a Chinese medicine chromatographic fingerprint similarity evaluation system, the ultra-high performance liquid chromatogram of the sample to be tested obtained in step (II) and the standard fingerprint obtained in step (I) are evaluated for similarity to complete quality inspection.

[0081] In one embodiment, in step (III) of the above-mentioned quality detection method, the quality detection includes quality evaluation of the Zhanjin Huoxue Powder sample to be tested.

[0082] In a specific embodiment, when the quality detection is quality evaluation, the similarity is above 90%, preferably above 92%, more preferably above 95%, further preferably above 98%, and most preferably above 99%, indicating that the quality uniformity of the samples to be tested is good.

[0083] Effects of the Invention

[0084] (1) The present invention successfully constructed the ultra-high performance liquid chromatography fingerprint of Zhanjin Huoxue Powder by rationally controlling the chromatographic conditions, calibrated a total of 24 common characteristic peaks, and identified 3 common characteristic peaks, namely ginsenoside Rg1 and / or ginsenoside Re, dracoside and ginsenoside Rb1. The fingerprint can effectively characterize the quality of Zhanjin Huoxue Powder, which is conducive to comprehensive control of the quality of the drug.

[0085] (2) The fingerprint spectrum focuses on the order and relationship of the peaks that constitute the fingerprint characteristics, and pays attention to the overall characteristics. It not only avoids the one-sidedness of judging the quality of Zhanjin Huoxue Powder by measuring individual chemical components, but also reduces the possibility of artificial processing to meet quality standards.

[0086] (3) The method of the present invention is based on ultra-high performance liquid chromatography technology, which has the advantages of being simple, rapid and highly accurate. At the same time, the accuracy, precision, repeatability, stability and durability of the method are all good, and the detection time is short, which provides a scientific basis for the quality evaluation of Zhanjin Huoxue Powder and is of great significance for controlling the quality of Zhanjin Huoxue Powder and ensuring the clinical efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 UPLC chromatograms of Zhanjin Huoxue Powder under different elution systems (elution systems 1-3).

[0088] Figure 2 UPLC chromatograms of Zhanjin Huoxue Powder under different elution systems (elution systems 4-6).

[0089] Figure 3 UPLC chromatograms of Zhanjin Huoxue Powder under the conditions of acetonitrile-water binary elution system with different acid concentrations.

[0090] Figure 4 This is the UPLC chromatogram of Zhanjin Huoxue Powder under different elution gradient conditions (elution gradient 1).

[0091] Figure 5 This is the UPLC chromatogram of Zhanjin Huoxue Powder under different elution gradient conditions (elution gradient 2).

[0092] Figure 6 This is the UPLC chromatogram of Zhanjin Huoxue Powder under different elution gradient conditions (elution gradient 3).

[0093] Figure 7 This is the UPLC chromatogram of Zhanjin Huoxue Powder under different elution gradient conditions (elution gradient 4).

[0094] Figure 8 This is the UPLC chromatogram of Zhanjin Huoxue Powder under different elution gradient conditions (elution gradient 5).

[0095] Fig. 9 This is the UPLC chromatogram of Zhanjin Huoxue Powder under different chromatographic column conditions.

[0096] Fig.10 This is the UPLC chromatogram of Zhanjin Huoxue Powder under different column temperature conditions.

[0097] Fig.11 This is the UPLC chromatogram of Zhanjin Huoxue Powder under different flow rate conditions.

[0098] Fig.12 This is the UPLC chromatogram of Zhanjin Huoxue Powder under different extraction solvents (methanol).

[0099] Fig.13 UPLC chromatograms of Zhanjin Huoxue Powder under different extraction solvents (30% v / v methanol in water).

[0100] Fig.14 UPLC chromatograms of Zhanjin Huoxue Powder under different extraction solvents (50% v / v methanol in water).

[0101] Fig.15 UPLC chromatograms of Zhanjin Huoxue Powder under different extraction solvents (70% v / v methanol in water).

[0102] Fig.16 UPLC chromatograms of Zhanjin Huoxue Powder under different extraction solvents (70% v / v ethanol in water).

[0103] Fig.17 This is the UPLC chromatogram of Zhanjin Huoxue Powder under different extraction solvents (ethanol).

[0104] Fig.18 UPLC chromatograms of Zhanjin Huoxue Powder under different extraction solvents (3% v / v methanolic phosphoric acid) conditions.

[0105] Fig.19 This is the UPLC chromatogram of Zhanjin Huoxue Powder under different extraction conditions.

[0106] Fig. 20 This is the UPLC chromatogram of Zhanjin Huoxue Powder under different extraction time conditions.

[0107] Fig.21 The UPLC superimposed chromatograms of 15 batches of Zhanjin Huoxue Powder on the market under the preferred chromatographic conditions of the present invention.

[0108] Fig. 22 These are the UPLC reference fingerprints of 15 batches of Zhanjin Huoxue Powder on the market. DETAILED DESCRIPTION

[0109] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The special word "exemplary" here means "used as an example, embodiment or for illustration". Any embodiment described here as "exemplary" should not be interpreted as being superior or better than other embodiments.

[0110] In addition, in order to better understand the present invention, numerous specific details are given in some of the examples below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0111] Unless otherwise specified, the units used in the present invention are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0112] Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated herein by reference in their entirety.

[0113] Unless otherwise specified, in the present invention, "value %" refers to volume percentage. For example, "70% methanol aqueous solution" means a methanol aqueous solution with a volume percentage of 70%, that is, 70% v / v methanol aqueous solution.

[0114] Definition of terms

[0115] Unless otherwise specified, in the present invention, the term "may" includes both the meanings of performing a certain treatment and not performing a certain treatment.

[0116] Unless otherwise specified, in the present invention, the terms "comprising", "including" or "consisting essentially of..." are open expressions, i.e., including the contents, elements, parts or features specified in the present invention, but do not exclude other contents, elements, parts or features not specified.

[0117] Unless otherwise specified, in the present invention, the term "Zhanjin Huoxue San" refers to a traditional Chinese medicine preparation, which is composed of ginseng (5 parts), amber (5 parts), myrrh (5 parts), frankincense (5 parts), dragon's blood (20 parts), pearl powder (5 parts), angelica (5 parts), Panax notoginseng (5 parts), musk (3 parts) and bezoar (1 part) by weight. These medicinal materials work together to promote blood circulation and remove blood stasis, dredge meridians and stretch tendons, and reduce swelling and relieve pain. It is suitable for treating joint muscle swelling and pain caused by traumatic injuries and various acute and chronic tissue injuries, such as lumbar muscle strain, joint contusion, scapulohumeral periarthritis, cervical spondylosis, lumbar disc herniation, etc.

[0118] Unless otherwise specified, in the present invention, the term "ginseng" refers to the dried root of Panaxginseng CAMey. Ginseng is slightly warm in nature, sweet and slightly bitter in taste, and enters the spleen and lung meridians. It has the effects of greatly replenishing vital energy, tonifying the spleen and lungs, promoting fluid production and quenching thirst, calming the mind and increasing intelligence.

[0119] Unless otherwise specified, in the present invention, the term "amber" refers to the resin of the ancient pine genus of the pine family, which is transformed after being buried in the stratum for many years. Amber is flat in nature, sweet in taste, and enters the heart, liver, and bladder meridians. It has the effects of calming the nerves, promoting blood circulation and removing blood stasis, and promoting diuresis and relieving stranguria.

[0120] Unless otherwise specified, in the present invention, the term "myrrh" refers to the dried resin of Commiphora myrrha Engl., a plant of the genus Commiphora in the family Oleaceae, or other plants of the same genus. Myrrh is flat in nature, slightly bitter in taste, and enters the heart, liver, and spleen meridians, and has the effects of promoting blood circulation, relieving pain, reducing swelling, and promoting tissue regeneration.

[0121] Unless otherwise specified, in the present invention, the term "frankincense" refers to the dry resin of Boswellia carterii Birdw. or other plants of the same genus. Frankincense is warm in nature, pungent and bitter in taste, and enters the heart, liver, and spleen meridians. It has the effects of promoting blood circulation and relieving pain, reducing swelling and promoting tissue regeneration.

[0122] Unless otherwise specified, in the present invention, the term "dragon's blood" refers to the dry resin of Daemonorops draco Bl., a plant of the genus Pteris genus of the palm family, or other plants of the same genus. Dragon's blood is flat in nature, sweet and salty in taste, and enters the heart and liver meridians. It has the effects of stopping bleeding, promoting tissue regeneration, and healing sores when used externally, and promoting blood circulation, dispersing blood stasis, and relieving pain when taken internally.

[0123] Unless otherwise specified, in the present invention, the term "pearl powder" refers to the powder obtained by processing the calcium carbonate-containing mineral beads generated by the stimulation of Pinctada martensii Dunker, Hyriopsis cumingii Lea, or Cristaria plicata Leach. Pearl powder is cold in nature, sweet and salty in taste, and enters the heart and liver meridians. It has the effects of calming the heart and relieving convulsions, clearing the liver and removing cataracts, astringing and promoting tissue regeneration.

[0124] Unless otherwise specified, in the present invention, the term "Danggui" refers to the dried root of Angelica sinensis (Oliv.) Diels of the Umbelliferae family. Danggui is slightly warm in nature, sweet and spicy in taste, and enters the liver, heart, and spleen meridians, and has the effects of nourishing blood, promoting blood circulation, relieving pain, and moistening the intestines.

[0125] Unless otherwise specified, in the present invention, the term "Sanqi" refers to the dried root of Panax notoginseng (Burk.) FHChen ex CHChow, a plant of the Araliaceae family. Sanqi is warm in nature, sweet and slightly bitter in taste, enters the liver and stomach meridians, and has the effects of removing blood stasis and stopping bleeding, promoting blood circulation and relieving pain.

[0126] Unless otherwise specified, in the present invention, the term "musk" refers to the dried secretion in the musk sac of mature male musk deer Moschusberezovskii Flerov, musk deer M.sifanicus Przewalski or musk deer M.moschiferus L. of the family Muskidae, which is called natural musk, while artificial substitutes are called artificial musk. Musk is warm in nature, pungent in taste, and enters the heart and spleen meridians. It has the effects of invigorating the mind, promoting blood circulation and dispersing stagnation, relieving pain, and inducing labor.

[0127] Unless otherwise specified, in the present invention, the term "bezoar" refers to the dried gallstones of Bos taurusdomesticus Gmelin, an animal of the Bovidae family, which is called natural bezoar, while the artificial product extracted and processed from cattle bile or pig bile is called artificial bezoar. Bezoar is cool in nature, bitter in taste, and enters the liver and heart meridians. It has the effects of clearing away heat and detoxifying, calming wind and stopping spasms, and resolving phlegm and opening the mind.

[0128] Abbreviations of technical terms, trade names and their meanings

[0129] Abbreviation or trade name meaning UPLC Ultra-high performance liquid chromatography HPLC High performance liquid chromatography v / v Volume Percent M Mole / liter (mol / L) C18 Column Octadecylsilane bonded silica gel chromatographic column

[0130] Experimental instruments and materials

[0131] 1. Instrument

[0132] Z00QSV-F6BA80606 ultrapure water instrument (Millipore, USA);

[0133] JM-380-40 ultrasonic cleaning machine (Jiemeng Ultrasonic Technology Co., Ltd.);

[0134] XSE205DU electronic balance (METTLER TOLEDO, Switzerland);

[0135] Ultimate 3000 ultra-high performance liquid chromatograph, Vanquish ultra-high performance liquid chromatograph (ThermoFisher, USA);

[0136] Double-row four-hole water bath (Shanghai Suopu Instrument Co., Ltd.).

[0137] 2. Reagents

[0138] Methanol and acetonitrile (chromatographic grade): Beijing Dima Technology Co., Ltd.;

[0139] Phosphoric acid, anhydrous ethanol, sodium dihydrogen phosphate (analytical grade): Sinopharm Chemical Reagent Co., Ltd.;

[0140] Microporous membrane (F membrane, mixed cellulose lipid membrane): Shanghai Bandao Industrial Co., Ltd. Purification Equipment Factory;

[0141] Syringe filter (Nylon): Shanghai Hanyao Instrument Equipment Co., Ltd.

[0142] 3. Reference substances

[0143] Ginsenoside Rg1 (110703-202235), ginsenoside Re (110754-202129), dracoside perchlorate (110811-201105), ginsenoside Rb1 (110704-202230): China Food and Drug Inspection Institutes.

[0144] 4. Comparative medicinal materials

[0145] Ginseng (2113-230401): Anhui Bozhou Medicinal Materials Corporation;

[0146] Myrrh (2161-230601): Anhui WeiBo Chinese Medicine Co., Ltd.;

[0147] Frankincense (2160-230301), Dragon's Blood (2124-230301): Bozhou Jiyang Zhenyuan Chinese Medicine Pieces Co., Ltd.;

[0148] Angelica sinensis (2019-230101): Gansu Foci Chinese Medicinal Materials Management Co., Ltd.;

[0149] Panax notoginseng (2119-230101): Anhui Nabaichuan Chinese Medicine Technology Co., Ltd.;

[0150] Artificial musk (2123-230701): China National Traditional Chinese Medicine Co., Ltd.

[0151] 5. Test sample

[0152] Commercially available Zhanjin Huoxue Powder (20240101018 (S1), 20240101029 (S2), 20240301003 (S3), 20240301015 (S4), 20240302002 (S5), 20240302037 (S6), 20240701013 (S7), 20240701024 (S8), 2 0230601015(S9), 20230701027(S10), 20230702025(S11), 20231001015(S12), 20231001027(S13), 20231101028(S14), 20231101040(S15), a total of 15 batches): Shaanxi Dongke Pharmaceutical Co., Ltd.;

[0153] Self-made Zhanjin Huoxue powder: Take 125g ginseng, 125g amber, 125g myrrh (processed), 125g frankincense (processed), 500g blood clotting, 125g pearl powder, 125g angelica, 125g Panax notoginseng, 75g artificial musk, 25g artificial bezoar. Except for artificial musk and artificial bezoar, the other eight ingredients including ginseng are dried and crushed into fine powder. Sieve. Grind the artificial musk and artificial bezoar through a 120-mesh sieve, and mix and grind with the above powders in an increasing manner. Mix well to obtain.

[0154] Experimental Example 1: Investigation of Chromatographic Conditions of UPLC Fingerprint

[0155] The chromatographic conditions were investigated using self-made Zhanjin Huoxue Powder.

[0156] 1. Investigation of elution system

[0157] Chromatographic column: Waters CORTECS UPLC T3 (2.1mm×100mm, 1.6μm); column temperature: 35℃; gradient elution program: 0~50min, 5%A / 95%B→95%A / 5%B; flow rate: 0.30mL / min; detection wavelength: 203nm. Under the above chromatographic conditions, the effects of different types of elution systems in Table 1 on the UPLC chromatogram of Zhanjin Huoxue Powder were investigated respectively. The results are shown in the figure below. Figure 1 and Figure 2 shown.

[0158] Table 1. Different types of elution systems

[0159]

[0160] Result analysis: When the mobile phase A (organic phase) is methanol, the baseline drift in the chromatogram is serious and the number of chromatographic peaks is relatively small; when acetonitrile-0.1% acetic acid aqueous solution is used, the baseline drift in the chromatogram is also serious. Therefore, acetonitrile-phosphoric acid aqueous solution or acetonitrile-sodium dihydrogen phosphate aqueous solution is selected as the elution system of Zhanjin Huoxue Powder.

[0161] 2. Investigation of acid concentration

[0162] Chromatographic column: Waters CORTECS UPLC T3 (2.1mm×150mm, 1.6μm); column temperature: 35℃; gradient elution program: 0~50min, 5%A / 95%B→95%A / 5%B; mobile phase flow rate: 0.30mL / min; detection wavelength: 203nm. Under the above chromatographic conditions, the effects of different acid concentrations in Table 2 on the UPLC chromatogram of Zhanjin Huoxue Powder were investigated respectively. The results are shown in the figure below. Figure 3 shown.

[0163] Table 2. Different acid concentrations

[0164]

[0165] Results: The binary system with acetonitrile as mobile phase A and 0.05% to 0.2% phosphoric acid aqueous solution as mobile phase B can be used as the elution system of Zhanjin Huoxue Powder. Moreover, when the mobile phase B is 0.05% phosphoric acid aqueous solution, the separation effect of the chromatographic peak with a retention time near 8.7min is better.

[0166] 3. Investigation of elution gradient

[0167] Chromatographic column: Waters CORTECS UPLC T3 (2.1mm×100mm, 1.6μm); column temperature: 35°C; mobile phase A: acetonitrile, mobile phase B: 0.05% phosphoric acid aqueous solution; gradient elution program: as shown in Table 3; mobile phase flow rate: 0.3mL / min; detection wavelength: 203nm. Under the above chromatographic conditions, the UPLC chromatogram of Zhanjin Huoxue Powder was obtained, and the results are as follows Figure 4 shown.

[0168] Table 3. Elution gradient 1

[0169]

[0170] Result analysis: No peak appeared within the first 8 minutes, indicating that the elution efficiency under this condition was poor.

[0171] In order to shorten the detection time, under the premise of fixing other conditions, the initial gradient of mobile phase B was changed to 82%, and gradient elution was performed according to the procedure in Table 4 to obtain the UPLC chromatogram of Zhanjin Huoxue Powder. The results are shown in Figure 4. Figure 5 shown.

[0172] Table 4. Elution gradient 2

[0173]

[0174] Result analysis: There was no peak in the first 5 minutes, the peaks were more dispersed after 30 minutes, but the peaks were too dense between 17 and 20 minutes.

[0175] In order to further shorten the detection time and optimize the separation, under the premise of fixing other conditions, the gradient changes of the two sections from 0 to 5 min and after 30 min were accelerated, and the gradient of the section from 17 to 20 min was slowed down. Gradient elution was performed according to the procedure in Table 5 to obtain the UPLC chromatogram of Zhanjin Huoxue Powder. The results are shown in the figure. Figure 6 shown.

[0176] Table 5. Elution gradient 3

[0177]

[0178] Result analysis: The separation of the chromatographic peaks at 17-19 min did not improve significantly and needed further optimization.

[0179] Under the premise of fixing other conditions, the gradient of the 17-19 min segment was further slowed down, and the post-equilibrium time was increased. Gradient elution was performed according to the procedure in Table 6 to obtain the UPLC chromatogram of Zhanjin Huoxue Powder. The results are shown in Figure 6. Figure 7 shown.

[0180] Table 6. Elution gradient 4

[0181]

[0182] Result analysis: The peak at 16-18 min needs to be further optimized.

[0183] Under the premise of fixing other conditions, we tried to increase the isocratic elution period and perform gradient elution according to the procedure in Table 7 to obtain the UPLC chromatogram of Zhanjin Huoxue Powder. The results are as follows: Figure 8 shown.

[0184] Table 7. Elution gradient 5

[0185]

[0186] Result analysis: The chromatographic peaks were evenly distributed and well separated, so the elution gradient 5 was used as the elution gradient for UPLC analysis of Zhanjin Huoxue Powder.

[0187] 4. Inspection of chromatographic column

[0188] After determining the elution system and elution gradient, the effects of four different types of C18 columns, including Phenomenex Luna Omega (2.1mm×150mm, 1.6μm), Phenomenex Luna Omega Polar C18 100A (2.1mm×100mm, 1.7μm), Waters CORTECS UPLC T3 (2.1mm×150mm, 1.6μm) and Waters CORTECS UPLC T3 (2.1mm×100mm, 1.6μm), on the UPLC chromatogram of Zhanjin Huoxue Powder were investigated. The results are shown in the figure. Fig. 9 shown.

[0189] Results: In the chromatograms obtained using different types of C18 columns, the chromatographic peaks were evenly distributed and well separated. In addition, the Waters CORTECS UPLC T3 (2.1mm×150mm, 1.6μm) column had a better separation effect on the five chromatographic peaks at 17-20min.

[0190] 5. Investigation of column temperature

[0191] After determining the elution system, elution gradient and chromatographic column, the effects of different column temperatures (25°C, 30°C, 35°C, 40°C) on the UPLC chromatogram of Zhanjin Huoxue Powder were investigated. The results are as follows: Fig.10 shown.

[0192] Result analysis: In the chromatograms obtained with different column temperatures, the chromatographic peaks are evenly distributed and well separated. Moreover, when the column temperature is 35℃ or 40℃, the separation effect of the chromatographic peaks is better.

[0193] 6. Investigation of flow rate

[0194] After determining the elution system, elution gradient, chromatographic column and column temperature (35°C), the effects of different flow rates (0.25 mL / min, 0.30 mL / min, 0.35 mL / min) on the UPLC chromatogram of Zhanjin Huoxue Powder were investigated. The results are as follows: Fig.11 shown.

[0195] Result analysis: In the chromatograms obtained with different flow rates, the chromatographic peaks are evenly distributed and well separated. Moreover, when the flow rate is 0.30mL / min, the separation effect of the chromatographic peaks is better.

[0196] Example 2: Investigation of the preparation method of the sample for UPLC fingerprint

[0197] The preparation method of the test sample was investigated using homemade Zhanjin Huoxue Powder.

[0198] The chromatographic conditions used in this example are as follows: chromatographic column: Waters CORTECS UPLC T3 (2.1 mm×150 mm, 1.6 μm); column temperature: 35° C.; mobile phase A: acetonitrile, mobile phase B: 0.05% phosphoric acid aqueous solution; gradient elution program: elution gradient 5 in Table 7; flow rate: 0.30 mL / min; detection wavelength: 203 nm.

[0199] 1. Investigation of extraction solvent

[0200] The chromatographic conditions were fixed, and the effects of different extraction solvents (methanol, 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, 70% ethanol aqueous solution, ethanol, 3% phosphoric acid methanol solution) on the UPLC chromatogram of Zhanjin Huoxue Powder were investigated. The results are shown in Figure 2. Figures 12 to 18 shown.

[0201] Result analysis: Based on comprehensive considerations such as whether the number of chromatographic peaks is appropriate, whether the peak shape is good, whether the separation is good and whether the distribution is reasonable, 50% to 70% methanol aqueous solution was selected as the extraction solvent for Zhanjin Huoxue Powder, among which the chromatographic behavior of the sample extracted with 70% methanol aqueous solution was the best.

[0202] 2. Investigation of extraction methods

[0203] The chromatographic conditions and extraction solvent (70% methanol in water) were fixed, and the effects of different extraction methods (ultrasonic extraction and reflux extraction) on the UPLC chromatogram of Zhanjin Huoxue Powder were investigated. The results are shown in Figure 2. Fig.19 shown.

[0204] Result analysis: There is no obvious difference between the two; considering the convenience and stability of the experiment, ultrasonic extraction was selected as the extraction method of Zhanjin Huoxue Powder.

[0205] 3. Investigation of extraction time

[0206] The chromatographic conditions, extraction solvent (70% methanol aqueous solution) and extraction method (ultrasonic extraction) were fixed to investigate the effects of different extraction times (10min, 20min, 30min) on the UPLC chromatogram of Zhanjin Huoxue Powder. The results are shown in Figure 2. Fig. 20 shown.

[0207] Result analysis: Extraction time of 10 to 30 minutes can achieve effective extraction, and 20 minutes is the preferred extraction time for Zhanjin Huoxue Powder.

[0208] Example 3: Methodological verification of UPLC fingerprint construction method

[0209] The self-made Zhanjin Huoxue powder was used to verify the analytical methodology.

[0210] The chromatographic conditions used in this example are as follows: chromatographic column: Waters CORTECS UPLC T3 (2.1 mm×150 mm, 1.6 μm); column temperature: 35° C.; mobile phase A: acetonitrile, mobile phase B: 0.05% phosphoric acid aqueous solution; gradient elution program: elution gradient 5 in Table 7; flow rate: 0.30 mL / min; detection wavelength: 203 nm.

[0211] 1. Precision inspection

[0212] Take 0.25g of homemade Zhanjin Huoxue powder, prepare the test solution according to the test sample preparation method determined in Example 2, inject 6 times continuously, record the chromatogram, import it into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012.13073 version) for processing, perform multi-point correction and peak matching, and generate a reference fingerprint spectrum at the same time. Calculate the similarity of each chromatogram with the reference fingerprint spectrum.

[0213] The results showed that the similarity between each chromatogram and the control fingerprint was 1.000, indicating good precision.

[0214] 2. Repeatability test

[0215] Take 6 portions of homemade Zhanjin Huoxue powder, prepare the test solution according to the test sample preparation method determined in Example 2, inject and measure respectively, record the chromatogram, import it into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012.13073 version) for processing, perform multi-point correction and peak matching, and generate a control fingerprint spectrum at the same time. Calculate the similarity between each chromatogram and the control fingerprint spectrum.

[0216] The results showed that the similarity between each chromatogram and the control fingerprint was 1.000, indicating good repeatability.

[0217] 3. Stability inspection

[0218] The test solution was prepared according to the test sample preparation method determined in Example 2, and the same Zhanjin Huoxue Powder test solution was accurately drawn, and the measurement was performed at 0, 2, 4, 6, 8, 10, 12, 18, 24, 36, and 48 hours, respectively, and the chromatogram was recorded and imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012.13073 version) for processing, multi-point correction and peak matching were performed, and a control fingerprint was generated at the same time. The similarity of each chromatogram and the control fingerprint was calculated respectively.

[0219] The results showed that the similarity between each chromatogram and the control fingerprint was ≥0.999, indicating that the test results of the test solution were stable within 48 h.

[0220] 4. Instrument durability inspection

[0221] The test sample solution was prepared according to the test sample preparation method determined in Example 2, and the same Zhanjin Huoxue Powder test sample solution was accurately aspirated and injected into different ultra-high performance liquid chromatographs (ThermoUltima 3000, Thermo Vanquish) according to the chromatographic conditions determined in Example 1.

[0222] The results show that, using different instruments, under the conditions of the present invention, the samples can be well separated, indicating that the method of the present invention has good instrument durability.

[0223] 5. Durability inspection of chromatographic columns

[0224] The test sample solution was prepared according to the test sample preparation method determined in Example 2, and the same Zhanjin Huoxue Powder test sample solution was accurately aspirated. According to the chromatographic conditions determined in Example 1, different chromatographic columns (chromatographic column 1 (SN: 1183103415410), chromatographic column 2 (SN: 01173022515454)) of Waters CORTECS UPLC T3 (2.1 mm × 100 mm, 1.6 μm) were used for analysis.

[0225] The results show that, using different chromatographic columns, under the conditions of the present invention, the samples can be well separated, indicating that the method of the present invention has good chromatographic column durability.

[0226] 6. Column temperature durability inspection

[0227] The test sample solution was prepared according to the test sample preparation method determined in Example 2, and the same Zhanjin Huoxue Powder test sample solution was accurately aspirated. According to the chromatographic conditions determined in Example 1, different column temperatures (35° C., 37° C.) were used for analysis.

[0228] The results showed that the samples were well separated at different column temperatures, indicating that the method of the present invention has good column temperature durability.

[0229] (7) Flow rate durability investigation

[0230] The test sample solution was prepared according to the test sample preparation method determined in Example 2, and the same Zhanjin Huoxue Powder test sample solution was accurately aspirated and analyzed using different flow rates (0.28 mL / min, 0.30 mL / min, 0.32 mL / min) according to the chromatographic conditions determined in Example 1.

[0231] The results showed that the samples were well separated at different flow rates, indicating that the method of the present invention has good flow rate durability.

[0232] Example 4: Construction of UPLC fingerprint of Zhanjin Huoxue Powder

[0233] 1. Preparation of reference solution:

[0234] Weigh ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 respectively, add 70% methanol aqueous solution to prepare a reference solution with a concentration of 0.1 mg / mL; weigh dracoside perchlorate separately, add 3% methanol phosphoric acid solution to prepare a reference solution with a concentration of 0.1 mg / mL; shake each reference solution, filter with a 0.22 μm microporous filter membrane, take the subsequent filtrate (middle filtrate) to obtain a reference solution.

[0235] 2. Preparation of reference medicinal material solution:

[0236] Weigh 21.18 mg of ginseng, 21.18 mg of myrrh, 21.18 mg of frankincense, 84.74 mg of blood clotting, 21.18 mg of angelica, 21.18 mg of Panax notoginseng, and 12.71 mg of artificial musk respectively, put them in a stoppered conical flask, add 10 mL of 70% methanol aqueous solution, extract by ultrasonic (power 250 W, frequency 40 kHz) for 20 min, cool, shake well, filter with 0.22 μm microporous filter membrane, take the filtrate (middle filtrate), and obtain the control medicinal material solution.

[0237] 3. Preparation of test solution:

[0238] Weigh about 0.25 g of commercially available Zhanjin Huoxue Powder from different batches respectively, accurately weigh, place in a stoppered conical flask, accurately add 10 mL of 70% methanol aqueous solution, ultrasonically extract (power 250 W, frequency 40 kHz) for 20 min, cool, shake well, filter with a 0.22 μm microporous filter membrane, take the filtrate (middle filtrate) to obtain the test solution.

[0239] 4. Chromatographic conditions

[0240] Chromatographic column: Waters CORTECS UPLC T3 (2.1 mm×150 mm, 1.6 μm); column temperature: 35°C; mobile phase A: acetonitrile, mobile phase B: 0.05% phosphoric acid aqueous solution; gradient elution program: elution gradient 5 in Table 7; flow rate: 0.30 mL / min; detection wavelength: 203 nm.

[0241] 5. Establishment of fingerprint

[0242] Accurately pipette 2 μL of each reference solution, reference medicinal material solution and test solution, inject into ultra-high performance liquid chromatography, measure according to the above chromatographic conditions, and record the chromatogram.

[0243] The UPLC chromatograms of the test sample solutions made from 15 batches (S1-S15) of commercially available Zhanjin Huoxue Powder (purchased from Shaanxi Dongke Pharmaceutical Co., Ltd.) were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012.13073 version) for processing, setting S1 as the reference spectrum, the average method, and the time window width of 0.1. The chromatograms of other samples were matched with the reference spectrum for Mark peaks, and the control fingerprint spectrum was generated at the same time. The UPLC superimposed chromatograms of 15 batches of commercially available Zhanjin Huoxue Powder are shown in Figure 1. Fig.21 As shown, the control fingerprint is as Fig. 22 The similarity between the chromatograms of each batch of samples and the reference fingerprint was calculated respectively, and the similarity evaluation results are shown in Table 8.

[0244] Table 8. Similarity evaluation results of commercially available Zhanjin Huoxue Powder (15 batches)

[0245]

[0246] Result analysis:

[0247] The fingerprint of Zhanjin Huoxue Powder contains 24 common peaks. Taking peak 3 (dracoside) as the reference peak, the relative retention times of the 24 common peaks are: peak 1: 0.85±5%, peak 2: 0.99±5%, peak 3: 1.00±5%, peak 4: 1.30±5%, peak 5: 1.39±5%, peak 6: 1.41±5%, peak 7: 1.54±5%, peak 8: 1.61±5%, peak 9: 2.05±5%, peak 10: 2.08±5%, peak 11: 2 .13±5%, Peak 12: 2.16±5%, Peak 13: 2.20±5%, Peak 14: 2.34±5%, Peak 15: 2.40±5%, Peak 16: 2.45±5%, Peak 17: 3.02±5%, Peak 18: 3.36±5%, Peak 19: 3.42±5%, Peak 20: 3.56±5%, Peak 21: 3.70±5%, Peak 22: 3.80±5%, Peak 23: 3.90±5% and Peak 24: 3.93±5%.

[0248] By comparing the chromatographic peaks in the UPLC chromatograms of the reference substance and reference medicinal materials, the attribution of the medicinal materials and index components of the chromatographic peaks of the fingerprint of Zhanjin Huoxue Powder was determined as follows: Peak 1 of the 24 common peaks belongs to Panax notoginseng and ginseng, Peak 2 belongs to Musk, Peak 3 belongs to Sanguisorba officinalis, and Peak 4 belongs to Panax notoginseng; Peaks 5 to 10 belong to Sanguisorba officinalis, Peaks 11 to 12 belong to Myrrh, Peak 13 belongs to Sanguisorba officinalis, Peak 14 belongs to Myrrh and Sanguisorba officinalis, Peak 15 belongs to Myrrh, Peaks 16 to 17 belong to Sanguisorba officinalis, Peak 18 belongs to frankincense, Peak 19 belongs to Sanguisorba officinalis, Peak 20 belongs to Angelica sinensis, and Peaks 21 to 24 belong to frankincense; Peak 1 corresponds to ginsenoside Rg1 and / or ginsenoside Re, Peak 3 corresponds to Sanguisorba officinalis, and Peak 4 corresponds to ginsenoside Rb1.

[0249] The similarities of the fingerprints of 15 batches of commercially available Zhanjin Huoxue Powder were all greater than the prescribed lower limit of 0.900, and were all greater than 0.990, indicating that the fingerprints can be used as common features for quality control of Zhanjin Huoxue Powder.

[0250] The above is only an exemplary embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, improvements and optimizations can be made to the above exemplary embodiments based on the prior art, and these improvements and optimizations also fall within the scope of protection of the present invention.

Claims

1. A method for constructing an ultra-high performance liquid chromatography fingerprint of Zhanjin Huoxue Powder, comprising the following steps: (1) Prepare different batches of test solution of Zhanjin Huoxue Powder, control solution of single medicinal material, and control solution of single indicator component; (2) performing ultra-high performance liquid chromatography analysis on different batches of test sample solutions, reference medicinal material solutions, and reference substance solutions prepared in step (1) to obtain corresponding ultra-high performance liquid chromatograms; and (3) The ultra-high performance liquid chromatography (ULHPLC) of the reference medicinal material solution was used to identify the medicinal materials in the ultra-high performance liquid chromatography (ULHPLC) of the test sample solutions of different batches; the ultra-high performance liquid chromatography (ULHPLC) of the reference solution was used to identify the index components in the ultra-high performance liquid chromatography (ULHPLC) of the test sample solutions of different batches; and the ULHPLC of the test sample solutions of different batches was processed using the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints to obtain the ULHPLC fingerprint of Zhanjin Huoxue Powder.

2. The construction method according to claim 1, characterized in that: In step (1), the Zhanjin Huoxue powder is made of ginseng, amber, myrrh, frankincense, dragon's blood, pearl powder, angelica, Panax notoginseng, musk, and bezoar; and / or, In step (1), the medicinal material comprises at least one of ginseng, amber, myrrh, frankincense, dragon's blood, pearl powder, angelica, notoginseng, musk and bezoar; Preferably, the medicinal material comprises at least one of ginseng, myrrh, frankincense, dragon's blood, angelica, notoginseng and musk; More preferably, the medicinal materials include ginseng, myrrh, frankincense, dragon's blood, angelica, notoginseng and musk; More preferably, the medicinal materials are ginseng, myrrh, frankincense, dragon's blood, angelica, notoginseng and musk; and / or, In step (1), the index component comprises at least one of ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re and dracoside; Preferably, the indicative component comprises at least one of ginsenoside Rb1, ginsenoside Re and dracoside; More preferably, the index components include ginsenoside Rb1, ginsenoside Re and dracoside; More preferably, the index components are ginsenoside Rb1, ginsenoside Re and dracoside; or, Preferably, the indicative component comprises at least one of ginsenoside Rg1, ginsenoside Rb1 and dracoside; More preferably, the index components include ginsenoside Rg1, ginsenoside Rb1 and dracoside; More preferably, the indicator components are ginsenoside Rg1, ginsenoside Rb1 and dracoside.

3. The construction method according to claim 1 or 2, characterized in that: In step (1), the method for preparing the test solution comprises: extracting different batches of Zhanjin Huoxue Powder with solvent 1 respectively; Preferably, the preparation method further comprises: shaking the extracted liquid, filtering, and taking a filtrate; and / or, In step (1), the method for preparing the control medicinal material solution comprises: extracting each medicinal material with solvent 1 respectively; Preferably, the preparation method further comprises: shaking the extracted liquid, filtering, and taking a filtrate; and / or, In step (1), the preparation method of the reference solution comprises: dissolving each index component with solvent 1 or solvent 2 respectively; Preferably, the preparation method further comprises: shaking the dissolved liquid, filtering, and taking a filtrate.

4. The construction method according to claim 3, characterized in that: The solvent 1 is a fatty alcohol or an aqueous solution thereof, preferably methanol, ethanol or an aqueous solution thereof, more preferably methanol, ethanol or a 30% to 70% v / v methanol aqueous solution or an ethanol aqueous solution, further preferably methanol, ethanol or a 50% to 70% v / v methanol aqueous solution or an ethanol aqueous solution, further preferably a 70% v / v methanol aqueous solution or an ethanol aqueous solution, most preferably a 70% v / v methanol aqueous solution; and / or, The solvent 2 is a fatty alcohol solution of an acid, preferably a fatty alcohol solution of an inorganic acid, more preferably a methanol or ethanol solution of phosphoric acid, further preferably a 1% to 5% v / v methanol phosphoric acid solution or an ethanol phosphoric acid solution, further preferably a 1% to 3% v / v methanol phosphoric acid solution or an ethanol phosphoric acid solution, and most preferably a 3% v / v methanol phosphoric acid solution.

5. The construction method according to any one of claims 1 to 4, characterized in that: In step (2), the conditions for the ultra-high performance liquid chromatography analysis include: The chromatographic column is an octadecylsilane bonded silica gel packing column; The mobile phase is acetonitrile-phosphoric acid or an aqueous solution of phosphate; The elution mode is gradient elution; Preferably, the octadecylsilane bonded silica gel filler chromatographic column is a Phenomenex Luna Omega (2.1 mm × 150 mm, 1.6 μm), Phenomenex Luna Omega Polar C18 100A (2.1 mm × 100 mm, 1.7 μm), Waters CORTECS UPLC T3 (2.1 mm × 150 mm, 1.6 μm) or Waters CORTECS UPLC T3 (2.1 mm × 100 mm, 1.6 μm) chromatographic column, preferably a Waters CORTECS UPLC T3 (2.1 mm × 150 mm, 1.6 μm) chromatographic column; and / or, Preferably, the mobile phase is acetonitrile-phosphoric acid aqueous solution, preferably acetonitrile-0.05% to 0.2% v / v phosphoric acid aqueous solution, more preferably acetonitrile-0.05% v / v phosphoric acid aqueous solution; or, The mobile phase is acetonitrile-sodium dihydrogen phosphate aqueous solution, preferably acetonitrile-0.02-0.08M sodium dihydrogen phosphate aqueous solution, more preferably acetonitrile-0.05M sodium dihydrogen phosphate aqueous solution; and / or, Preferably, the procedure of the gradient elution is as follows:

6. The construction method according to claim 5, characterized in that: In step (2), the ultra-high performance liquid chromatography analysis conditions further include at least one of the following conditions: The column temperature of the chromatographic column is 25-40°C; The flow rate of the mobile phase is 0.25-0.35 mL / min; The detector is a UV detector or a UV-Vis detector; Preferably, the column temperature is 35-40°C, preferably 35°C; and / or, Preferably, the flow rate is 0.28 to 0.32 mL / min, preferably 0.30 mL / min; and / or, Preferably, the detection wavelength of the detector is 190-210 nm, preferably 203 nm.

7. The construction method according to any one of claims 1 to 4, characterized in that: In step (2), the conditions for the ultra-high performance liquid chromatography analysis include: The chromatographic column is a C18 column; The mobile phase was acetonitrile-0.05% to 0.2% v / v phosphoric acid aqueous solution; The elution method is gradient elution, and the procedure of the gradient elution is as follows: The detection wavelength is 190~210nm; Preferably, in step (2), the conditions for the ultra-high performance liquid chromatography analysis include: The chromatographic column was a Waters CORTECS UPLC T3 (2.1 mm × 150 mm, 1.6 μm) column; The mobile phase was acetonitrile-0.05% v / v phosphoric acid in water; The elution method is gradient elution, and the procedure of the gradient elution is as follows: The detection wavelength is 203 nm.

8. The construction method according to any one of claims 1 to 7, characterized in that: In step (3), the ultra-high performance liquid chromatography fingerprint contains at least 6, preferably at least 9, more preferably at least 12, further preferably at least 18, and most preferably at least 24 common peaks; Preferably, the ultra-high performance liquid chromatography fingerprint contains 24 common peaks; More preferably, among the 24 common peaks, taking peak 3 as the reference peak, the relative retention times of the 24 common peaks are: peak 1: 0.85±5%, peak 2: 0.99±5%, peak 3: 1.00±5%, peak 4: 1.30±5%, peak 5: 1.39±5%, peak 6: 1.41±5%, peak 7: 1.54±5%, peak 8: 1.61±5%, peak 9: 2.05±5%, peak 10: 2.08±5%, peak 11: 2.13 ±5%, peak 12: 2.16±5%, peak 13: 2.20±5%, peak 14: 2.34±5%, peak 15: 2.40±5%, peak 16: 2.45±5%, peak 17: 3.02±5%, peak 18: 3.36±5%, peak 19: 3.42±5%, peak 20: 3.56±5%, peak 21: 3.70±5%, peak 22: 3.80±5%, peak 23: 3.90±5% and peak 24: 3.93±5%; Further preferably, among the 24 common peaks, peak 1 belongs to Panax notoginseng and ginseng, peak 2 belongs to musk, peak 3 belongs to Sanguisorba officinalis, peak 4 belongs to Panax notoginseng, peaks 5 to 10 belong to Sanguisorba officinalis, peaks 11 to 12 belong to myrrh, peak 13 belongs to Sanguisorba officinalis, peak 14 belongs to myrrh and Sanguisorba officinalis, peak 15 belongs to myrrh, peaks 16 to 17 belong to Sanguisorba officinalis, peak 18 belongs to frankincense, peak 19 belongs to Sanguisorba officinalis, peak 20 belongs to Angelica sinensis, and peaks 21 to 24 belong to frankincense; and / or, among the 24 common peaks, peak 1 corresponds to ginsenoside Rg1 and / or ginsenoside Re, peak 3 corresponds to dracoside, and peak 4 corresponds to ginsenoside Rb1.

9. Application of the ultra-high performance liquid chromatography fingerprint of Zhanjin Huoxue Powder obtained by the construction method according to any one of claims 1 to 8 in the quality detection of Zhanjin Huoxue Powder; Preferably, the quality detection includes quality evaluation of the Zhanjin Huoxue Powder sample to be tested.

10. A quality inspection method for Zhanjin Huoxue Powder, comprising the following steps: (I) obtaining an ultra-high performance liquid chromatography fingerprint of Zhanjin Huoxue Powder based on the construction method according to any one of claims 1 to 8 as a standard fingerprint; (II) preparing a test solution of the Zhanjin Huoxue Powder sample by the same test solution preparation method and ultra-high performance liquid chromatography analysis conditions as the construction method according to any one of claims 1 to 8, and performing ultra-high performance liquid chromatography analysis on the test solution to obtain a corresponding ultra-high performance liquid chromatogram; and (III) using a Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity between the ultra-high performance liquid chromatogram of the sample to be tested obtained in step (II) and the standard fingerprint obtained in step (I) to complete the quality inspection; Preferably, in step (III), the quality detection includes quality evaluation of the Zhanjin Huoxue Powder sample to be tested; More preferably, when the quality detection is a quality evaluation, the similarity is above 90%, preferably above 92%, more preferably above 95%, further preferably above 98%, and most preferably above 99%, indicating that the quality uniformity of the samples to be tested is good.

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