Construction method of UPLC fingerprint spectrum of Zhenshen Huoxuesan and application thereof

Abstract

The application belongs to the technical field of pharmaceutical analysis, and relates to a construction method of a UPLC fingerprint spectrum of Zhanjin Huoxuesan and application thereof. The construction method comprises the following steps of preparation of a test sample solution, a control medicinal material solution and a control sample solution, UPLC analysis, and obtaining the UPLC fingerprint spectrum of Zhanjin Huoxuesan by using a traditional Chinese medicine fingerprint spectrum similarity evaluation system. The UPLC fingerprint spectrum of Zhanjin Huoxuesan is successfully constructed by reasonably controlling chromatographic conditions, can effectively characterize product quality, avoids one-sidedness of determining product quality due to determination of individual chemical components, reduces possibility of artificial processing for quality standardization, has the advantages of simplicity, rapidness, high accuracy, and good accuracy, precision, repeatability, stability and durability, and short detection time, provides a scientific basis for quality evaluation of Zhanjin Huoxuesan, and has important significance for controlling product quality and guaranteeing clinical curative effect.

Description

Technical Field

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

[0002] Zhanjin Huoxue Powder is a compound preparation made from ten traditional Chinese medicinal materials including Ginseng, Succinum, Myrrha, Olibanum, Sanguis Draxonis, etc., and is used for joint and muscle swelling and pain caused by traumatic injury, acute soft tissue injury and other chronic tissue injuries, lumbar muscle strain, etc. Modern pharmacological studies show that Zhanjin Huoxue Powder exerts the therapeutic effect on soft tissue injury by targeting key targets such as IL-6, TLR4, TNF, CASP3, VEGFA and ESR1 through active components such as ginsenoside Rh2, dracorhodin, daidzein, quercetin, kaempferol, phelloside and coniferyl ferulate (Liu Xu, Li Zhe, Wang Leilei, et al., Exploration on the Mechanism of Zhanjin Huoxue Powder in Treating Soft Tissue Injury Based on Network Pharmacology Molecular Docking Technology[J], Drugs & Clinic, 2024, 39(09): pp2254-2262).

[0003] In addition to microscopic identification, the quality standard of Zhanjin Huoxue Powder mainly adopts thin-layer identification for Sanguis Draxonis and Angelica sinensis as the quality control method. Other quality control studies in the prior art mainly focus on thin-layer identification of Ginseng, Notoginseng and Myrrha in Zhanjin Huoxue Powder, HPLC identification of bilirubin, as well as content determination of dracorhodin by HPLC method (Yang Ruirui, Li Xue, Qiao Rongxia, et al., Study on Quality Standard of Zhanjin Huoxue Powder[J], Drug Standards of China, 2013, 14(04): pp251-255). The quality control methods in the prior art only focus on several specific 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 internal quality.

[0004] Traditional Chinese medicine fingerprint (or referred to as "fingerprint" for short) is a comprehensive and quantifiable identification means, which can systematically, integrally and specifically characterize the internal characteristics of traditional Chinese medicines, can be used to evaluate the quality of traditional Chinese medicinal materials, traditional Chinese medicine preparations and semi-finished products, and is an effective means for the overall quality control of traditional Chinese medicines. So far, no public research has been made on the fingerprint that can better reveal the internal quality of Zhanjin Huoxue Powder. Therefore, it is necessary to establish an analysis means with excellent specificity, stability and reproducibility to better control the preparation quality of Zhanjin Huoxue Powder. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] To address the aforementioned problems in the prior art, this invention provides a method for constructing an ultra-high performance liquid chromatography fingerprint of Zhanjin Huoxue San (a traditional Chinese medicine formula), and the application of this fingerprint in the quality detection of Zhanjin Huoxue San. The construction method of this invention elevates the quality control of Zhanjin Huoxue San from the identification and content determination of a few components to the overall control of the intrinsic quality of Zhanjin Huoxue San. It can more comprehensively reflect the characteristics of Zhanjin Huoxue San, providing a new analytical means for the intrinsic quality control of Zhanjin Huoxue San and achieving the goal of controlling the overall quality of Zhanjin Huoxue San preparations.

[0007] Solution for solving the problem

[0008] In a first aspect, the present invention provides a method for constructing an ultra-high performance liquid chromatography fingerprint of a muscle-strengthening and blood-activating powder, comprising the following steps:

[0009] (1) Prepare test solutions of different batches of Zhanjin Huoxue San, reference solutions of single medicinal materials and reference solutions of single index components;

[0010] (2) The different batches of test solution, reference medicinal material solution and reference solution prepared in step (1) were analyzed by ultra-high performance liquid chromatography (UHPLC) to obtain the corresponding UHPLC chromatograms; and

[0011] (3) Using the ultra-high performance liquid chromatogram of the reference medicinal material solution, the medicinal materials in the ultra-high performance liquid chromatograms of different batches of test sample solutions were assigned; using the ultra-high performance liquid chromatogram of the reference solution, the indicative components in the ultra-high performance liquid chromatograms of different batches of test sample solutions were assigned; at the same time, the ultra-high performance liquid chromatograms of different batches of test sample solutions were processed using the Chinese medicine chromatographic fingerprint similarity evaluation system to obtain the ultra-high performance liquid chromatographic fingerprint of Zhanjin Huoxue San.

[0012] In one implementation, in step (1) of the above construction method, the muscle-strengthening and blood-activating powder is made from ginseng, amber, myrrh, frankincense, dragon's blood, pearl powder, angelica, notoginseng, musk, and bezoar.

[0013] In one specific implementation, the myrrh in the muscle-strengthening and blood-activating powder is processed myrrh, i.e., processed myrrh or prepared myrrh, such as vinegar-processed myrrh.

[0014] In one specific implementation, the frankincense in the muscle-strengthening and blood-activating powder is processed frankincense, namely, frankincense (processed) or processed frankincense, such as vinegar-processed frankincense.

[0015] In one specific implementation, the musk in the muscle-strengthening and blood-activating powder is artificially produced musk, namely, musk (artificial) or artificial musk.

[0016] In one specific implementation, the bezoar in the muscle-strengthening and blood-activating powder is artificially produced bezoar, namely bezoar (artificial) or artificial bezoar.

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

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

[0020] In one specific implementation, the myrrh in the medicinal material is processed myrrh, i.e., processed myrrh or prepared myrrh, such as vinegar-processed myrrh.

[0021] In one specific embodiment, the frankincense in the medicinal material is processed frankincense, namely frankincense (processed) or processed frankincense, such as vinegar-processed frankincense.

[0022] In one specific implementation, the musk in the medicinal material is artificially produced musk, namely 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 gentian root extract.

[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 gentian root extract.

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

[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 gentian root extract.

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

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

[0030] In one embodiment, in step (1) of the above construction method, the preparation method of the control medicinal material solution further includes: before the extraction step, grinding and sieving each medicinal material separately or drying, pulverizing and sieving them separately.

[0031] In one specific implementation, in step (1) of the above construction method, the preparation method of the control medicinal material solution further includes: when the medicinal material contains musk and / or bezoar, before the extraction step, the musk and / or bezoar are ground and sieved separately, and the remaining medicinal materials in the medicinal material are dried, pulverized and sieved separately.

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

[0033] In one 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, even more 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 one specific embodiment, the solvent 2 is an acidic fatty alcohol solution, preferably an inorganic acid fatty alcohol solution, more preferably a phosphoric acid methanol or ethanol solution, further preferably a 1% to 5% v / v phosphoric acid methanol solution or phosphoric acid ethanol solution, even more preferably a 1% to 3% v / v phosphoric acid methanol solution or phosphoric acid ethanol solution, and most preferably a 3% v / v phosphoric acid methanol solution.

[0035] In one specific implementation, the extraction method is ultrasonic extraction or reflux extraction, with ultrasonic extraction being preferred.

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

[0037] In a specific embodiment, when preparing the test sample solution, the ratio of the used amount of Zhanjin Huoxue Powder to Solvent 1 is 0.01 to 0.04 g / mL, preferably 0.02 to 0.03 g / mL, more preferably 0.025 g / mL. However, it should be understood that when preparing the test sample solution, the ratio of the used amount of Zhanjin Huoxue Powder to Solvent 1 can be adjusted according to the ordinary experience of the operator and the actual situation, and is not limited by the exemplary ranges in the present application.

[0038] In one embodiment, when preparing the reference medicinal material solution, since the weight proportion of each single medicinal material in the preparation is different, there are multiple situations for the ratio of the used amount of each medicinal material to Solvent 1. For example, based on 1 part by weight, the ratio of the used amount of each medicinal material to Solvent 1 is 0.1 to 1 mg / mL, preferably 0.2 to 0.8 mg / mL, more preferably 0.3 to 0.5 mg / mL. However, it should be understood that when preparing the reference medicinal material solution, the ratio of the used amount of the single medicinal material to Solvent 1 can be adjusted according to the ordinary experience of the operator and the actual situation, and is not limited by the exemplary ranges in the present application.

[0039] In a specific embodiment, the medicinal materials include Ginseng, Myrrha, Olibanum, Sanguis Draxonis, Angelicae Sinensis Radix, Notoginseng Radix et Rhizoma and Moschus, preferably Ginseng, Myrrha, Olibanum, Sanguis Draxonis, Angelicae Sinensis Radix, Notoginseng Radix et Rhizoma and Moschus, wherein: Ginseng corresponds to 5 parts by weight, Myrrha corresponds to 5 parts by weight, Olibanum corresponds to 5 parts by weight, Sanguis Draxonis corresponds to 20 parts by weight, Angelicae Sinensis Radix corresponds to 5 parts by weight, Notoginseng Radix et Rhizoma corresponds to 5 parts by weight, and Moschus corresponds to 3 parts by weight.

[0040] In a specific embodiment, the concentration of the reference substance solution is 0.025 to 0.4 mg / mL, preferably 0.05 to 0.2 mg / mL, more preferably 0.1 mg / mL. However, it should be understood that when preparing the reference substance solution, the ratio of the used amount of a single marker component to Solvent 1 or Solvent 2 can be adjusted according to the ordinary experience of the operator and the actual situation, and is not limited by the exemplary ranges in the present application.

[0041] In one embodiment, when preparing the reference substance solution, different solvents need to be used for dissolution due to the different properties of the marker components.

[0042] In a specific embodiment, the marker components include Ginsenoside Rg1, Ginsenoside Rb1 and Dracorhodin, preferably Ginsenoside Rg1, Ginsenoside Rb1 and Dracorhodin, wherein: Solvent 1 is used to dissolve Ginsenoside Rg1 and / or Ginsenoside Rb1, and Solvent 2 is used to dissolve Dracorhodin.

[0043] In one implementation, 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 column (C18 column);

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

[0046] The elution method is gradient elution.

[0047] In one specific embodiment, the C18 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) column, preferably a Waters CORTECS UPLC T3 (2.1 mm × 150 mm, 1.6 μm) column.

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

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

[0050] In one specific implementation, the gradient elution procedure 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℃.

[0053] In one specific implementation, 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 one specific embodiment, the flow rate is 0.28 to 0.32 mL / min, preferably 0.30 mL / min.

[0056] In one implementation, 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 one 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 is acetonitrile-0.05%–0.2% v / v phosphoric acid aqueous solution;

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

[0062]

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

[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 aqueous solution;

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

[0068]

[0069] The detection wavelength is 203nm.

[0070] In one embodiment, in step (3) of the above construction method, the ultra-high performance liquid chromatography fingerprint spectrum 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 one specific implementation, the ultra-high performance liquid chromatography fingerprint spectrum contains 24 common peaks;

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

[0073] Still further, among the 24 common peaks, the No. 1 peak is attributed to Panax notoginseng and Panax ginseng, the No. 2 peak is attributed to Moschus, the No. 3 peak is attributed to Sanguis Draxonis, the No. 4 peak is attributed to Panax notoginseng, peaks No. 5 to No. 10 are attributed to Sanguis Draxonis, peaks No. 11 to No. 12 are attributed to Myrrha, the No. 13 peak is attributed to Sanguis Draxonis, the No. 14 peak is attributed to Myrrha and Sanguis Draxonis, the No. 15 peak is attributed to Myrrha, peaks No. 16 to No. 17 are attributed to Sanguis Draxonis, the No. 18 peak is attributed to Olibanum, the No. 19 peak is attributed to Sanguis Draxonis, the No. 20 peak is attributed to Angelica sinensis, and peaks No. 21 to No. 24 are attributed to Olibanum.

[0074] Even further, among the 24 common peaks, the No. 1 peak corresponds to ginsenoside Rg1 and / or ginsenoside Re, the No. 3 peak corresponds to dracorhodin, and the No. 4 peak 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 San obtained based on the above construction method in the quality detection of Zhanjin Huoxue San.

[0076] In one embodiment, the quality detection includes quality evaluation of a test sample of Zhanjin Huoxue San.

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

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

[0079] (II) using the same test sample solution preparation method and ultra-high performance liquid chromatography analysis conditions as those in the above construction method to prepare a test sample solution of the Zhanjin Huoxue San test sample, and performing ultra-high performance liquid chromatography analysis thereon to obtain a corresponding ultra-high performance liquid chromatogram; and

[0080] (III) Using the similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine, the similarity between the ultra-high performance liquid chromatogram of the test sample obtained in step (II) and the standard fingerprint obtained in step (I) is evaluated to complete the quality detection.

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

[0082] In a specific embodiment, when the quality detection is quality evaluation, the test samples with a similarity of 90% or more, preferably 92% or more, more preferably 95% or more, further preferably 98% or more, and most preferably 99% or more have good quality uniformity.

[0083] The effects of the invention

[0084] (1) According to the present invention, by reasonably controlling the chromatographic conditions, the ultra-high performance liquid chromatographic fingerprint of Zhanjin Huoxue Powder is successfully constructed, with a total of 24 common characteristic peaks calibrated, and 3 common characteristic peaks identified, which are ginsenoside Rg1 and / or ginsenoside Re, dracorhodin, and ginsenoside Rb1 respectively. This fingerprint can effectively characterize the quality of Zhanjin Huoxue Powder, which is beneficial to comprehensively control the quality of the drug.

[0085] (2) This fingerprint focuses on the sequence and mutual relationship of each constituent fingerprint characteristic peak, and emphasizes the overall characteristics, which 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 treatment for meeting the quality standard.

[0086] (3) The method of the present invention is based on ultra-high performance liquid chromatography technology, and has the advantages of simplicity, rapidness and high accuracy. Meanwhile, the method has good accuracy, precision, repeatability, stability and durability, and short detection time, 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 clinical efficacy. Brief Description of the Drawings

[0087] Figure 1 It is the UPLC chromatogram of Zhanjin Huoxue Powder under the conditions of different elution systems (elution systems 1-3).

[0088] Figure 2 It is the UPLC chromatogram of Zhanjin Huoxue Powder under the conditions of different elution systems (elution systems 4-6).

[0089] Figure 3 It is the UPLC chromatogram of Zhanjin Huoxue Powder under the conditions of acetonitrile-water binary elution systems with different acid concentrations.

[0090] Figure 4 The UPLC chromatograms of Zhanjin Huoxue San under different elution gradients (elution gradient 1) are shown.

[0091] Figure 5 The UPLC chromatograms of Zhanjin Huoxue San under different elution gradients (elution gradient 2) are shown.

[0092] Figure 6 The UPLC chromatograms of Zhanjin Huoxue San under different elution gradients (elution gradient 3) are shown.

[0093] Figure 7 The UPLC chromatograms of Zhanjin Huoxue San under different elution gradients (elution gradient 4) are shown.

[0094] Figure 8 The image shows the UPLC chromatograms of Zhanjin Huoxue San under different elution gradients (elution gradient 5).

[0095] Figure 9 The UPLC chromatograms of Zhanjin Huoxue San under different column conditions.

[0096] Figure 10 The UPLC chromatograms of Zhanjin Huoxue San under different column temperature conditions.

[0097] Figure 11 The UPLC chromatograms of Zhanjin Huoxue San under different flow rate conditions.

[0098] Figure 12 The UPLC chromatograms of Zhanjin Huoxue San under different extraction solvent (methanol) conditions.

[0099] Figure 13 The UPLC chromatograms of Zhanjin Huoxue San under different extraction solvents (30% v / v methanol aqueous solution) are shown.

[0100] Figure 14 The UPLC chromatograms of Zhanjin Huoxue San under different extraction solvents (50% v / v methanol aqueous solution) are shown.

[0101] Figure 15 The UPLC chromatograms of Zhanjin Huoxue San under different extraction solvents (70% v / v methanol aqueous solution) are shown.

[0102] Figure 16 The UPLC chromatograms of Zhanjin Huoxue San under different extraction solvents (70% v / v ethanol aqueous solution) are shown.

[0103] Figure 17 The UPLC chromatograms of Zhanjin Huoxue San under different extraction solvent (ethanol) conditions.

[0104] Figure 18 The UPLC chromatograms of Zhanjin Huoxue San under different extraction solvents (3% v / v phosphate methanol) are shown.

[0105] Figure 19 The UPLC chromatograms of Zhanjin Huoxue San under different extraction conditions are shown.

[0106] Figure 20 The image shows the UPLC chromatograms of Zhanjin Huoxue San under different extraction time conditions.

[0107] Figure 21 The above are UPLC superimposed chromatograms of 15 batches of commercially available Zhanjin Huoxue San under the preferred chromatographic conditions of this invention.

[0108] Figure 22 UPLC fingerprint chromatograms of 15 batches of commercially available Zhanjin Huoxue San (a traditional Chinese medicine formula). Detailed Implementation

[0109] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" should not be construed as superior to or better than other embodiments.

[0110] Furthermore, to better understand the present invention, numerous specific details are set forth in some of the examples below. Those skilled in the art will understand that the present invention can be practiced even without certain specific details. In other examples, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0111] Unless otherwise stated, all units used in this invention are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0112] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0113] Unless otherwise stated, in this invention, "numerical %" refers to volume percentage. For example, "70% methanol aqueous solution" means a methanol aqueous solution with a volume percentage of 70%, i.e., a 70% v / v methanol aqueous solution.

[0114] Terminology Definition

[0115] Unless otherwise stated, in this invention, the term "may" includes both the meaning of performing a certain process and not performing a certain process.

[0116] Unless otherwise stated, in this invention, the terms "comprising," "including," or "consisting substantially of" are open-ended expressions, meaning they include the contents, elements, components, or features specified in this invention, but do not exclude other contents, elements, components, or features not specified.

[0117] Unless otherwise stated, in this invention, the term "Zhanjin Huoxue San" refers to a traditional Chinese medicine preparation, which, by weight, consists 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), notoginseng (5 parts), musk (3 parts), and bezoar (1 part). These medicinal materials work together to promote blood circulation, remove blood stasis, unblock meridians, relax muscles and tendons, reduce swelling, and relieve pain. It is suitable for treating joint and muscle swelling and pain caused by falls and injuries, as well as various acute and chronic tissue injuries, such as lumbar muscle strain, joint contusions, frozen shoulder, cervical spondylosis, and lumbar disc herniation.

[0118] Unless otherwise stated, in this invention, the term "ginseng" refers to the dried root of Panax ginseng CAMey., a plant belonging to the genus Panax in the family Araliaceae. 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 body fluid production and quenching thirst, calming the mind and enhancing intelligence.

[0119] Unless otherwise stated, in this invention, the term "amber" refers to the resin of ancient pine trees (Pinus genus of the Pinaceae family), which has been transformed over many years by being buried in strata. Amber is neutral in nature and 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 urination.

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

[0121] Unless otherwise stated, in this invention, the term "frankincense" refers to the dried resin of Boswellia carterii Birdw., a plant of the genus Boswellia in the family Burseraceae, 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, relieving pain, reducing swelling, and promoting tissue regeneration.

[0122] Unless otherwise stated, in this invention, the term "dragon's blood" refers to the dried resin of *Daemonorops draco* Bl., a plant of the genus *Daemonorops* in the family Arecaceae, or other plants of the same genus. Dragon's blood is neutral 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 astringing sores when used externally, and promoting blood circulation, removing blood stasis, and relieving pain when taken internally.

[0123] Unless otherwise stated, in this invention, the term "pearl powder" refers to the powder obtained by processing the calcium carbonate mineral beads produced by the following species after stimulation: *Pinctada martensii* Dunker (or *Pinctada martensii*), *Hyriopsiscumingii* Lea (or *Cristaria plicata* Leach), all of which are bred in the family Pearl oysters. 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 mind and relieving anxiety, clearing the liver and removing eye obstructions, and astringing and promoting tissue regeneration.

[0124] Unless otherwise stated, in this invention, the term "Angelica sinensis" refers to the dried root of Angelica sinensis (Oliv.) Diels, a plant belonging to the genus Angelica of the family Apiaceae. Angelica sinensis is slightly warm in nature, sweet and pungent in taste, and enters the liver, heart, and spleen meridians. It has the effects of nourishing blood, promoting blood circulation, relieving pain, and moistening the intestines.

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

[0126] Unless otherwise stated, in this invention, the term "musk" refers to the dried secretion from the musk sac of mature male musk deer (Moschus berezovskii Flerov, M. sifanicus Przewalski, or M. moschiferus L.), and is called natural musk. Artificially produced substitutes are called synthetic musk. Musk is warm in nature, pungent in taste, and enters the heart and spleen meridians. It has the effects of opening the orifices and refreshing the mind, promoting blood circulation and dispersing stagnation, relieving pain, and inducing labor.

[0127] Unless otherwise stated, in this invention, the term "bezoar" refers to the dried gallstone of the bovine animal *Bos taurus domesticus* Gmelin, known as natural bezoar, while artificial bezoar refers to an artificial product extracted and processed from bovine or porcine bile. Bezoar is cool in nature and bitter in taste, and enters the liver and heart meridians. It has the effects of clearing heat and detoxifying, calming wind and stopping spasms, resolving phlegm and opening the orifices.

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

[0129] UPLC Ultra-high performance liquid chromatography HPLC High performance liquid chromatography v / v Volume percentage M moles per liter (mol / L) C18 column Octadecylsilane bonded silica gel chromatographic column

[0130] Experimental instruments and materials

[0131] 1. Instruments

[0132] Z00QSV-F6BA80606 Ultrapure Water System (Millipore, USA);

[0133] JM-380-40 Ultrasonic Cleaner (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 Sopu 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 filtration membranes (F-membrane, mixed cellulose lipid membrane): Shanghai Peninsula Industrial Co., Ltd. Purification Equipment Factory;

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

[0142] 3. Reference standards

[0143] Ginsenoside Rg1 (110703-202235), Ginsenoside Re (110754-202129), Bloodwort Perchlorate (110811-201105), Ginsenoside Rb1 (110704-202230): China National Institutes for Food and Drug Control.

[0144] 4. Reference medicinal materials

[0145] Ginseng (2113-230401): Bozhou Pharmaceutical Corporation, Anhui Province;

[0146] Myrrh (2161-230601): Anhui Weibao Pharmaceutical Co., Ltd.;

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

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

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

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

[0151] 5. Test sample

[0152] Commercially available muscle-strengthening and blood-activating powders (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] Homemade Tendon-Strengthening and Blood-Activating Powder: Take 125g of ginseng, 125g of amber, 125g of processed myrrh, 125g of processed frankincense, 500g of dragon's blood, 125g of pearl powder, 125g of angelica, 125g of notoginseng, 75g of artificial musk, and 25g of artificial bezoar. Except for the artificial musk and artificial bezoar, the remaining eight ingredients (including ginseng) are dried and pulverized into a fine powder. Sift the powder. Grind the artificial musk and artificial bezoar into a fine powder and pass it through a 120-mesh sieve. Mix the artificial musk and artificial bezoar with the above powders in an incremental manner. Mix thoroughly to obtain the final product.

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

[0155] The chromatographic conditions were investigated using a self-made muscle-strengthening and blood-activating powder.

[0156] 1. Examination of the elution system

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

[0158] Table 1. Different types of elution systems

[0159]

[0160] Results analysis: When the mobile phase A (organic phase) was methanol, the baseline drift in the chromatogram was severe, and the number of chromatographic peaks was relatively small; the baseline drift in the chromatogram was also severe when acetonitrile-0.1% acetic acid aqueous solution was used. Therefore, acetonitrile-phosphoric acid aqueous solution or acetonitrile-sodium dihydrogen phosphate aqueous solution was selected as the elution system for Zhanjin Huoxue San (a traditional Chinese medicine formula).

[0161] 2. Investigation of acid concentration

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

[0163] Table 2. Different acid concentrations

[0164]

[0165] Results analysis: Both binary systems with acetonitrile as mobile phase A and 0.05%–0.2% phosphoric acid aqueous solution as mobile phase B can be used as elution systems for Zhanjin Huoxue San (a traditional Chinese medicine formula). Furthermore, when mobile phase B is 0.05% phosphoric acid aqueous solution, the separation effect of the chromatographic peak with a retention time around 8.7 min is better.

[0166] 3. Investigation of elution gradient

[0167] Chromatographic column: Waters CORTECS UPLC T3 (2.1 mm × 100 mm, 1.6 μm); column temperature: 35℃; 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: 0.3 mL / min; detection wavelength: 203 nm. Under the above chromatographic conditions, the UPLC chromatogram of Zhanjin Huoxue San was obtained, and the results are as follows: Figure 4 As shown.

[0168] Table 3. Elution gradient 1

[0169]

[0170] Results analysis: No peaks were observed in the first 8 minutes, indicating poor elution efficiency under these conditions.

[0171] To shorten the detection time, while keeping other conditions constant, the initial gradient of mobile phase B was changed to 82%, and gradient elution was performed according to the procedure in Table 4. The UPLC chromatogram of Zhanjin Huoxue San was obtained, and the results are as follows. Figure 5 As shown.

[0172] Table 4. Elution gradient 2

[0173]

[0174] Results analysis: No peaks were observed in the first 5 minutes, and the peaks were more dispersed after 30 minutes, but the peaks were too dense between 17 and 20 minutes.

[0175] To further shorten the detection time and optimize the resolution, under the premise of keeping other conditions constant, the gradient changes in the 0-5 min and 30 min segments were accelerated, while the gradient change in the 17-20 min segment was slowed down. Gradient elution was performed according to the procedure in Table 5, and the UPLC chromatogram of Zhanjin Huoxue San was obtained. The results are as follows. Figure 6 As shown.

[0176] Table 5. Elution gradient 3

[0177]

[0178] Results analysis: The resolution of the chromatographic peaks at 17–19 min showed no significant improvement and requires further optimization.

[0179] Under the condition that other factors remain constant, the gradient was further reduced in the 17-19 min segment, while the post-equilibrium time was increased. Gradient elution was performed according to the procedure in Table 6, and the UPLC chromatogram of Zhanjin Huoxue San was obtained. The results are as follows: Figure 7 As shown.

[0180] Table 6. Elution gradient 4

[0181]

[0182] Results analysis: The peak elution at 16–18 min needs further optimization.

[0183] Under the premise of keeping other conditions constant, we tried to increase the isocratic elution time and performed gradient elution according to the procedure in Table 7. The UPLC chromatogram of Zhanjin Huoxue San was obtained, and the results are as follows: Figure 8 As shown.

[0184] Table 7. Elution gradient 5

[0185]

[0186] Results analysis: The chromatographic peaks were uniformly distributed and well separated. Therefore, elution gradient 5 was used as the elution gradient for UPLC analysis of Zhanjin Huoxue San.

[0187] 4. Examination of the chromatographic column

[0188] After determining the elution system and elution gradient, the effects of four different C18 columns—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 San were investigated. The results are as follows: Figure 9 As shown.

[0189] Results Analysis: In the chromatograms obtained using different types of C18 columns, the chromatographic peaks were all uniformly distributed and well separated. Furthermore, the Waters CORTECS UPLC T3 (2.1mm × 150mm, 1.6μm) column showed better separation of the five chromatographic peaks at 17–20 min.

[0190] 5. Examination of column temperature

[0191] After determining the elution system, elution gradient, and chromatographic column, the effect of different column temperatures (25℃, 30℃, 35℃, 40℃) on the UPLC chromatogram of Zhanjin Huoxue San was investigated. The results are as follows: Figure 10 As shown.

[0192] Results analysis: In the chromatograms obtained using different column temperatures, the chromatographic peaks were uniformly distributed and well separated. Furthermore, the separation effect was even better when the column temperature was 35℃ or 40℃.

[0193] 6. Flow velocity assessment

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

[0195] Results analysis: In the chromatograms obtained using different flow rates, the chromatographic peaks were uniformly distributed and well separated. Furthermore, the separation effect was even better when the flow rate was 0.30 mL / min.

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

[0197] The preparation method of the test sample was investigated using a self-made muscle-strengthening and blood-activating powder.

[0198] The chromatographic conditions used in this embodiment are as follows: 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 solvents

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

[0201] Results analysis: Considering factors such as the appropriate number of chromatographic peaks, good peak shape, good resolution, and reasonable distribution, 50%–70% methanol aqueous solution was selected as the extraction solvent for Zhanjin Huoxue San (a traditional Chinese medicine formula). Among these, the chromatographic behavior of the sample extracted with 70% methanol aqueous solution was the best.

[0202] 2. Examination of extraction methods

[0203] With fixed chromatographic conditions and extraction solvent (70% methanol aqueous solution), the effects of different extraction methods (ultrasonic extraction and reflux extraction) on the UPLC chromatogram of Zhanjin Huoxue San were investigated. The results are as follows: Figure 19 As shown.

[0204] Results analysis: There was no significant difference between the two methods; considering the convenience and stability of the experiment, ultrasonic extraction was chosen as the extraction method for Zhanjin Huoxue San.

[0205] 3. Examination of extraction time

[0206] Under fixed chromatographic conditions, extraction solvent (70% methanol aqueous solution), and extraction method (ultrasonic extraction), the effect of different extraction times (10 min, 20 min, 30 min) on the UPLC chromatogram of Zhanjin Huoxue San was investigated. The results are as follows: Figure 20 As shown.

[0207] Results analysis: Extraction times of 10 to 30 minutes can achieve effective extraction, with 20 minutes being the preferred extraction time for Zhanjin Huoxue San.

[0208] Example 3: Methodological Validation of UPLC Fingerprint Map Construction Method

[0209] The analytical methodology was validated using a self-made muscle-strengthening and blood-activating powder.

[0210] The chromatographic conditions used in this embodiment are as follows: 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 test

[0212] Take 0.25g of the self-made Zhanjin Huoxue San (a traditional Chinese medicine formula), and prepare the test solution according to the test sample preparation method determined in Example 2. Inject the sample six times consecutively, record the chromatograms, and import them into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (version 2012.13073) for processing, performing multi-point correction and peak matching, and simultaneously generating a reference fingerprint chromatogram. Calculate the similarity between each chromatogram and the reference fingerprint chromatogram.

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

[0214] 2. Repeatability testing

[0215] Six portions of the self-made Zhanjin Huoxue San (a traditional Chinese medicine formula) were taken and prepared into test solutions according to the test solution preparation method determined in Example 2. The solutions were injected and analyzed, and the chromatograms were recorded. The chromatograms were then imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (version 2012.13073) for processing, including multi-point correction and peak matching. Simultaneously, a reference fingerprint chromatogram was generated. The similarity between each chromatogram and the reference fingerprint chromatogram was calculated.

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

[0217] 3. Stability test

[0218] The test solution was prepared according to the test solution preparation method determined in Example 2. The same Zhanjin Huoxue San test solution was accurately pipetted and measured at 0, 2, 4, 6, 8, 10, 12, 18, 24, 36, and 48 hours. Chromatograms were recorded and imported into the "Traditional Chinese Medicine Chromatogram Fingerprint Similarity Evaluation System" (version 2012.13073) for processing, including multi-point correction and peak matching. A reference fingerprint chromatogram was also generated. The similarity between each chromatogram and the reference fingerprint chromatogram was calculated.

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

[0220] 4. Instrument durability test

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

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

[0223] 5. Column durability test

[0224] The test solution was prepared according to the test solution preparation method determined in Example 2. The same Zhanjin Huoxue San test solution was accurately pipetted and analyzed using different chromatographic columns (column 1 (SN: 1183103415410) and column 2 (SN: 01173022515454)) of Waters CORTECS UPLC T3 (2.1mm×100mm, 1.6μm) under the chromatographic conditions determined in Example 1.

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

[0226] 6. Column temperature durability test

[0227] The test solution was prepared according to the test solution preparation method determined in Example 2. The same Zhanjin Huoxue San test solution was accurately pipetted and analyzed using different column temperatures (35℃ and 37℃) according to the chromatographic conditions determined in Example 1.

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

[0229] (7) Flow rate durability test

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

[0231] The results show that the samples can be 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 San (a traditional Chinese medicine formula)

[0233] 1. Preparation of reference solution:

[0234] Weigh out ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 separately, and add them to 70% methanol aqueous solution to prepare a reference solution with a concentration of 0.1 mg / mL; separately weigh out strychnine perchlorate, and add it to 3% phosphate methanol solution to prepare a reference solution with a concentration of 0.1 mg / mL; shake each reference solution well, filter it through a 0.22 μm microporous membrane, and collect the filtrate (middle filtrate) to obtain the reference solution.

[0235] 2. Preparation of the control herbal solution:

[0236] Weigh out 21.18 mg of ginseng, 21.18 mg of myrrh, 21.18 mg of frankincense, 84.74 mg of dragon's blood, 21.18 mg of angelica, 21.18 mg of notoginseng, and 12.71 mg of artificial musk, respectively, and place them in a stoppered conical flask. Add 10 mL of 70% methanol aqueous solution, and extract by ultrasonication (power 250 W, frequency 40 kHz) for 20 min. Cool, shake well, and filter through a 0.22 μm microporous membrane. Collect the filtrate (middle filtrate) to obtain the control medicinal material solution.

[0237] 3. Preparation of the test solution:

[0238] Weigh approximately 0.25g of commercially available Zhanjin Huoxue San from different batches, place the weighed amounts in a stoppered conical flask, accurately add 10mL of 70% methanol aqueous solution, and extract by ultrasonication (power 250W, frequency 40kHz) for 20min. After cooling and shaking, filter the solution using a 0.22μm microporous membrane, and collect 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% aqueous phosphoric acid 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 patterns

[0242] Accurately pipette 2 μL of each reference solution, reference medicinal material solution, and test solution into an ultra-high performance liquid chromatograph, determine the chromatograms under the chromatographic conditions described above, and record the chromatograms.

[0243] The UPLC chromatograms of the test solutions prepared from 15 batches (S1-S15) of commercially available Zhanjin Huoxue Powder (purchased from Shaanxi Dongke Pharmaceutical Co., Ltd.) were imported into the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" (Version 2012.13073) for processing. With S1 set as the reference chromatogram, the average method was adopted, the time window width was set to 0.1, the chromatograms of other samples were subjected to Mark peak matching with the reference chromatogram, and a reference fingerprint was generated simultaneously. The overlaid UPLC chromatograms of the 15 batches of commercially available Zhanjin Huoxue Powder are shown in Figure 21 , and the reference fingerprint is shown in Figure 22 . The similarity between the chromatogram of each batch of sample and the reference fingerprint was calculated respectively, and the similarity evaluation results are shown in Table 8.

[0244] Table 8. Similarity Evaluation Results of 15 Batches of Commercially Available Zhanjin Huoxue Powder

[0245]

[0246] Result analysis:

[0247] The fingerprint of Zhanjin Huoxue Powder contains 24 common peaks, with Peak 3 (dracorhodin) as the reference peak. The relative retention times of the 24 common peaks are respectively: 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 standard and reference medicinal materials, the medicinal materials and index components of the fingerprint chromatogram of Zhanjin Huoxue San were 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 dragon's blood; Peak 4 belongs to Panax notoginseng; Peaks 5 to 10 belong to dragon's blood; Peaks 11 to 12 belong to myrrh; Peak 13 belongs to dragon's blood; Peak 14 belongs to myrrh and dragon's blood; Peak 15 belongs to myrrh; Peaks 16 to 17 belong to dragon's blood; Peak 18 belongs to frankincense; Peak 19 belongs to dragon's blood; Peak 20 belongs to Angelica sinensis; Peaks 21 to 24 belong to frankincense; Peak 1 corresponds to ginsenoside Rg1 and / or ginsenoside Re; Peak 3 corresponds to dragon's blood extract; Peak 4 corresponds to ginsenoside Rb1.

[0249] The fingerprint similarity of 15 batches of commercially available Zhanjin Huoxue San was greater than the lower limit of 0.900 and greater than 0.990, indicating that the fingerprint can be used as a common feature for the quality control of Zhanjin Huoxue San.

[0250] The above description is merely an exemplary embodiment of the present invention. It should be noted that those skilled in the art can make improvements and optimizations to the above exemplary embodiments based on existing technology without departing from the principles of the present invention, and these improvements and optimizations also fall within the protection scope of the present invention.

Claims

1. A method for constructing an ultra-high performance liquid chromatography fingerprint of a muscle-strengthening and blood-activating powder, comprising the following steps: (1) Prepare test solutions of different batches of Zhanjin Huoxue San, reference solutions of single medicinal materials, and reference solutions of single index components; (2) Perform ultra-high performance liquid chromatography analysis on different batches of test sample solution, reference medicinal material solution and reference solution prepared in step (1) to obtain the corresponding ultra-high performance liquid chromatograms; and (3) Using the ultra-high performance liquid chromatogram of the reference medicinal material solution, the medicinal materials in the ultra-high performance liquid chromatograms of different batches of test sample solutions were assigned; using the ultra-high performance liquid chromatogram of the reference solution, the indicative components in the ultra-high performance liquid chromatograms of different batches of test sample solutions were assigned; at the same time, the ultra-high performance liquid chromatograms of different batches of test sample solutions were processed using the Chinese medicine chromatographic fingerprint similarity evaluation system to obtain the ultra-high performance liquid chromatographic fingerprint of Zhanjin Huoxue San. in, In step (2), the conditions for the ultra-high performance liquid chromatography analysis include: The chromatographic column is an octadecylsilane-bonded silica gel column; The mobile phase is an aqueous solution of acetonitrile-phosphoric acid or phosphate; The elution method is gradient elution.

2. The construction method according to claim 1, characterized in that, In step (1), the muscle-strengthening and blood-activating powder is made from ginseng, amber, myrrh, frankincense, dragon's blood, pearl powder, angelica, notoginseng, musk, and bezoar; And / or, In step (1), the medicinal materials include at least one of ginseng, amber, myrrh, frankincense, dragon's blood, pearl powder, angelica, notoginseng, musk and bezoar; And / or, In step (1), the indicative component includes at least one of ginsenoside Rg1, ginsenoside Rb1, ginsenoside Re and drosin.

3. The construction method according to claim 1 or 2, characterized in that, In step (1), the preparation method of the test solution includes: extracting different batches of Zhanjin Huoxue San with solvent 1, shaking the extracted liquid, filtering, and taking the filtrate. And / or, In step (1), the preparation method of the reference medicinal material solution includes: extracting each medicinal material with solvent 1, shaking the extracted liquid, filtering, and taking the filtrate; And / or, In step (1), the preparation method of the reference solution includes: dissolving each index component in solvent 1 or solvent 2 respectively, shaking the dissolved liquid, filtering, and taking the filtrate. Solvent 1 is a fatty alcohol or its aqueous solution; solvent 2 is an acidic fatty alcohol solution.

4. The construction method according to claim 3, characterized in that, Solvent 1 is methanol, ethanol, or an aqueous solution thereof; And / or, Solvent 2 is a methanol or ethanol solution of phosphoric acid.

5. The construction method according to claim 1 or 2, characterized in that, In step (2), The octadecylsilane-bonded silica gel packed column is a Phenomenex Luna Omega column (2.1 mm × 150 mm, 1.6 μm), a Phenomenex Luna Omega Polar C18 100A column (2.1 mm × 100 mm, 1.7 μm), a Waters CORTECS UPLC T3 column (2.1 mm × 150 mm, 1.6 μm), or a Waters CORTECS UPLC T3 column (2.1 mm × 100 mm, 1.6 μm). And / or, The mobile phase is an acetonitrile-phosphoric acid aqueous solution; or, The mobile phase is an aqueous solution of acetonitrile-sodium dihydrogen phosphate; And / or, The gradient elution procedure is as follows: 。 6. The construction method according to claim 5, characterized in that, In step (2), the conditions for the ultra-high performance liquid chromatography analysis also include at least one of the following conditions: The column temperature of the chromatographic column is 25~40℃; 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.

7. The construction method according to claim 1 or 2, 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 is acetonitrile-0.05%~0.2% v / v phosphoric acid aqueous solution; The elution method is gradient elution, and the gradient elution procedure is as follows: The detection wavelength is 190~210 nm.

8. The construction method according to claim 1 or 2, characterized in that, In step (3), the ultra-high performance liquid chromatography fingerprint spectrum contains at least 6 common peaks.

9. The construction method according to claim 8, characterized in that, The ultra-high performance liquid chromatography fingerprint spectrum contains at least 9 common peaks.

10. The construction method according to claim 9, characterized in that, The ultra-high performance liquid chromatography fingerprint spectrum contains at least 12 common peaks.

11. The construction method according to claim 10, characterized in that, The ultra-high performance liquid chromatography fingerprint spectrum contains at least 18 common peaks.

12. The construction method according to claim 11, characterized in that, The ultra-high performance liquid chromatography fingerprint spectrum contains at least 24 common peaks.

13. The construction method according to claim 12, characterized in that, The ultra-high performance liquid chromatography fingerprint spectrum contains 24 common peaks.

14. The construction method according to claim 13, characterized in that, Among the 24 common peaks, taking peak 3 as the reference peak, the relative retention times of the 24 common peaks are respectively: 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%.

15. The construction method according to claim 14, characterized in that, Among the 24 common peaks, peak 1 is attributed to Panax notoginseng and Panax ginseng, peak 2 is attributed to Moschus, peak 3 is attributed to Sanguis Draxonis, peak 4 is attributed to Panax notoginseng, peaks 5 to 10 are attributed to Sanguis Draxonis, peaks 11 to 12 are attributed to Myrrha, peak 13 is attributed to Sanguis Draxonis, peak 14 is attributed to Myrrha and Sanguis Draxonis, peak 15 is attributed to Myrrha, peaks 16 to 17 are attributed to Sanguis Draxonis, peak 18 is attributed to Olibanum, peak 19 is attributed to Sanguis Draxonis, peak 20 is attributed to Angelicae Sinensis Radix, and peaks 21 to 24 are attributed to Olibanum; and / or, among the 24 common peaks, peak 1 corresponds to ginsenoside Rg1 and / or ginsenoside Re, peak 3 corresponds to dracorhodin, and peak 4 corresponds to ginsenoside Rb1.

16. Application of the ultra-high performance liquid chromatography fingerprint of Zhanjin Huoxue San obtained based on the construction method according to any one of claims 1 to 15 in the quality detection of Zhanjin Huoxue San.

17. The application according to claim 16, characterized in that, The quality detection includes quality evaluation of the test sample of Zhanjin Huoxue San.

18. A quality detection method for Zhanjin Huoxue San, comprising the following steps: (I) obtaining the ultra-high performance liquid chromatography fingerprint of Zhanjin Huoxue San based on the construction method according to any one of claims 1 to 15, as the standard fingerprint; (II) using the same test sample solution preparation method and ultra-high performance liquid chromatography analysis conditions as those in the construction method according to any one of claims 1 to 15, preparing a test sample solution of the Zhanjin Huoxue San test sample, and performing ultra-high performance liquid chromatography analysis thereon to obtain the corresponding ultra-high performance liquid chromatogram; and (III) using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine to perform similarity evaluation on the ultra-high performance liquid chromatogram of the test sample obtained in step (II) and the standard fingerprint obtained in step (I), so as to complete the quality detection.

19. The quality detection method according to claim 18, characterized in that, In step (III), the quality detection includes the quality evaluation of the test sample of Zhanjin Huoxue Powder.

20. The quality detection method according to claim 19, characterized in that, When the quality detection is quality evaluation, the test samples with a similarity of 90% or above have good quality uniformity.

Images