Characteristic spectrum construction, mass spectrometry identification and detection methods and their applications for Pulsatilla chinensis medicinal materials, standard decoctions or traditional Chinese medicine granules

Abstract

The present invention provides a method for constructing a characteristic spectrum of Pulsatilla chinensis medicinal materials, standard decoctions, or traditional Chinese medicine granules, comprising the following steps: preparing a test solution from the Pulsatilla chinensis medicinal materials, standard decoctions, or traditional Chinese medicine granules; and subjecting the test solution to ultra-high performance liquid chromatography analysis. The ultra-high performance liquid chromatography analysis conditions include: mobile phase A being a mixture of acetonitrile and methanol, mobile phase B being an aqueous phosphoric acid solution, and employing gradient elution. The characteristic spectrum constructed by the method described herein is rich in information, highly characteristic, and has good reproducibility.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine quality control, and in particular to a method for constructing a characteristic spectrum, mass spectrometry identification and detection of a Pulsatilla chinensis medicinal material, a standard decoction or traditional Chinese medicine formula granules, and applications thereof. Background Art

[0002] Pulsatilla chinensis is included in the first part of the 2020 edition of the Chinese Pharmacopoeia. It comes from the dried root of Pulsatilla chinensis (Bge.) Regel, a plant of the Ranunculaceae family. Pulsatilla chinensis tastes bitter and has a mild nature. It enters the stomach and large intestine meridians. It is used for heat-toxic bloody dysentery, vaginal itching and leukorrhea. Modern theoretical research shows that in addition to its anti-tumor, antibacterial, antiviral and insecticidal effects, Pulsatilla chinensis also has anti-cancer, liver-protecting and immunity-enhancing effects. It is mainly used clinically to treat gastrointestinal, respiratory, urinary system, obstetrics and gynecology diseases. The most obvious feature of Pulsatilla chinensis is the white hairs on the root head and visible sheath-like petiole residues. Therefore, the same products with residual leaf bases and stem bases, white fluff and cotton wool in the root head are easily confused with Pulsatilla in clinical use, such as Potentilla chinensis and Potentilla discolor of the Rosaceae family, Rhaponticum uniflorum of the Asteraceae family, and Anemone tomentosa of the Ranunculaceae family.

[0003] At present, the Chinese Pharmacopoeia 2020 edition does not make any provisions for characteristic spectra under the entry for Pulsatilla chinensis, and the provisions under the entry for properties have little reference value for distinguishing medicinal pieces that have changed their morphology. There have been relevant literature reports on identification methods for distinguishing Pulsatilla chinensis and its confounding products, but these identification methods mainly focus on property identification, microscopic identification, and DNA identification. For example, Guo Huan et al. distinguished Pulsatilla chinensis and its confounding products by appearance, nature, flavor, meridians, efficacy, and application; Zhang Liming et al. conducted a pharmacognosy identification of Herba Potentillae Discolor and Herba Potentillae; Pei Huirong et al. identified Pulsatilla chinensis and its common counterfeits Potentillae and Herba Lycopodii based on the original plant properties and microscopic characteristics. In summary, the identification methods for Pulsatilla chinensis and its counterfeits reported in the literature have certain limitations.

[0004] Furthermore, according to modern pharmaceutical research, the chemical composition of Pulsatilla scabra is primarily composed of lupane- and oleanane-type triterpenoid saponins, along with triterpenoid acids such as 23-hydroxybetulinic acid, betulinic acid, and anemonitic acid, as well as small molecules such as anemonin, protoanemonin, coumarins, and lignans. However, there are currently no reports on chemically distinguishing Pulsatilla scabra and its adulterants from different species. Furthermore, decoction of Pulsatilla scabra in standard decoctions and traditional Chinese medicine granules can lose the microscopic characteristics of the original medicinal material. Therefore, establishing a chemical composition identification method capable of distinguishing common adulterants is particularly important. Summary of the Invention

[0005] Based on this, the present invention provides a method for constructing a characteristic spectrum of Pulsatilla dahurica medicinal materials, standard decoctions or Chinese medicine formula granules. The constructed characteristic spectrum is rich in information, highly characteristic and has good reproducibility.

[0006] The present invention is achieved through the following technical solutions.

[0007] A method for constructing a characteristic spectrum of a Pulsatilla chinensis medicinal material, a standard decoction, or a Chinese medicine formula granule comprises the following steps:

[0008] Prepare the test solution by taking Pulsatilla chinensis medicinal materials, standard decoction or Chinese medicine formula granules;

[0009] The test solution is subjected to ultra-high performance liquid chromatography analysis;

[0010] The conditions for ultra-high performance liquid chromatography analysis include: mobile phase A is a mixture of acetonitrile and methanol, and mobile phase B is a phosphoric acid aqueous solution; gradient elution is used; the gradient elution program includes: from 0 min to 9 min, the volume percentage of mobile phase A changes from 8% to 10%; from 9 min to 18 min, the volume percentage of mobile phase A changes from 10% to 18%; from 18 min to 30 min, the volume percentage of mobile phase A changes from 18% to 26%; from 30 min to 38 min, the volume percentage of mobile phase A changes from 26% to 31%; from 38 min to 48 min, the volume percentage of mobile phase A changes from 31% to 45%.

[0011] In one embodiment, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.2%.

[0012] In one embodiment, the conditions for ultra-high performance liquid chromatography analysis also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 210nm to 220nm.

[0013] In one embodiment, the following steps are also included:

[0014] Take Pulsatilla chinensis control medicinal material and prepare reference solution;

[0015] The reference solution was subjected to ultra-performance liquid chromatography analysis.

[0016] In one embodiment, preparing the reference solution comprises the following steps:

[0017] The Pulsatilla chinensis control medicinal material was mixed with water, heated under reflux for extraction, and then filtered to obtain a filtrate.

[0018] In one embodiment, the following steps are also included:

[0019] Take chicoric acid reference substance and pulsatilla saponin B4 reference substance to prepare a mixed reference solution;

[0020] The mixed reference solution is subjected to ultra-performance liquid chromatography analysis.

[0021] In one embodiment, preparing the mixed reference solution comprises the following steps:

[0022] The chicoric acid reference substance and pulsatilla saponin B4 reference substance were mixed with methanol.

[0023] In one embodiment, preparing the test solution comprises the following steps:

[0024] Mixing the Pulsatilla chinensis medicinal material with a methanol aqueous solution having a volume fraction of 40% to 60%, performing ultrasonic extraction, and then filtering to obtain a filtrate; or

[0025] Mixing the standard decoction of Pulsatillae with a methanol aqueous solution with a volume fraction of 40% to 60%, performing ultrasonic extraction, and then filtering to obtain a filtrate; or

[0026] The Pulsatillae Chinese medicinal formula granules are mixed with a methanol aqueous solution with a volume fraction of 40% to 60%, subjected to ultrasonic extraction, and then filtered to obtain a filtrate.

[0027] In one embodiment, the characteristic spectrum of the Pulsatilla scabra medicinal material, standard decoction or Chinese medicine formula granules has a total of 9 characteristic peaks; among them, peak 5 is the chromatographic peak of chicoric acid, and peak 8 is the chromatographic peak of Pulsatilla scabra saponin B4.

[0028] The present invention also provides the use of the method for constructing the characteristic spectrum of the above-mentioned Pulsatilla scabra medicinal material, standard decoction or traditional Chinese medicine formula granule in identifying the authenticity of Pulsatilla scabra.

[0029] The present invention also provides a high-resolution mass spectrometry method for identifying characteristic peaks of characteristic spectra of Pulsatilla dahurica medicinal materials, standard decoctions or Chinese medicine formula granules, comprising the following steps:

[0030] Prepare the test solution by taking Pulsatilla chinensis medicinal materials, standard decoction or Chinese medicine formula granules;

[0031] The test solution is analyzed by ultra-high performance liquid chromatography coupled with mass spectrometry;

[0032] The conditions for ultra-high performance liquid chromatography analysis include: mobile phase A is a mixture of acetonitrile and methanol, and mobile phase B is a formic acid aqueous solution; gradient elution is used; the gradient elution program includes: from 0 min to 9 min, the volume percentage of mobile phase A changes from 8% to 10%; from 9 min to 18 min, the volume percentage of mobile phase A changes from 10% to 18%; from 18 min to 30 min, the volume percentage of mobile phase A changes from 18% to 26%; from 30 min to 38 min, the volume percentage of mobile phase A changes from 26% to 31%; and from 38 min to 48 min, the volume percentage of mobile phase A changes from 31% to 45%.

[0033] In one embodiment, the conditions for mass spectrometry analysis include: a sheath gas flow rate of 35arb±5arb, an auxiliary gas flow rate of 10arb±2arb, a spray voltage of 3.8kV±1kV, an S-lens voltage of 50V±5V, a heating temperature of 350℃±10℃, and a capillary temperature of 350℃±10℃; the scanning mode is positive and negative ion mode, the scanning range is 120m / z~1500m / z, and the normalized collision energy is 40%±5%.

[0034] In one embodiment, the volume fraction of formic acid in the formic acid aqueous solution is 0.05% to 0.2%.

[0035] In one embodiment, the conditions for ultra-high performance liquid chromatography analysis also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 210nm to 220nm.

[0036] The present invention also provides a method for detecting Pulsatilla dasyphylla medicinal material, standard decoction or Chinese medicine formula granules, comprising the following steps:

[0037] Take the product to be tested and prepare a solution of the product to be tested;

[0038] The test solution is subjected to ultra-high performance liquid chromatography determination;

[0039] The conditions for ultra-high performance liquid chromatography analysis include: mobile phase A is a mixture of acetonitrile and methanol, and mobile phase B is a phosphoric acid aqueous solution; gradient elution is used; the gradient elution program includes: from 0 min to 9 min, the volume percentage of mobile phase A changes from 8% to 10%; from 9 min to 18 min, the volume percentage of mobile phase A changes from 10% to 18%; from 18 min to 30 min, the volume percentage of mobile phase A changes from 18% to 26%; from 30 min to 38 min, the volume percentage of mobile phase A changes from 26% to 31%; from 38 min to 48 min, the volume percentage of mobile phase A changes from 31% to 45%.

[0040] In one embodiment, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.2%.

[0041] In one embodiment, the conditions for ultra-high performance liquid chromatography analysis also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 210nm to 220nm.

[0042] Compared with the prior art, the method for constructing characteristic maps of Pulsatilla chinensis medicinal materials, standard decoctions or Chinese medicine formula granules of the present invention has the following beneficial effects:

[0043] The present invention establishes fingerprints of Pulsatilla chinensis medicinal materials, standard decoctions, or traditional Chinese medicine granules using UPLC, and defines the mobile phase solvent and gradient elution procedure, thereby achieving the goals of rich characteristic chromatographic information, strong characteristicity, and good reproducibility. The obtained Pulsatilla chinensis characteristic spectrum defines nine characteristic peaks, which not only reflect triterpenoid saponins but also increase the presence of water-soluble phenolic acids, comprehensively reflecting the intrinsic quality of Pulsatilla chinensis samples. This provides a relatively comprehensive and effective evaluation method for the quality control of Pulsatilla chinensis medicinal materials, standard decoctions, and formula granules, ensuring the safety and effectiveness of clinical medication.

[0044] Furthermore, the method for constructing the fingerprint of the Pulsatilla dahurica medicinal material, standard decoction or Chinese medicine formula granule described in the present invention is simple to operate, has a short analysis time, saves detection costs and improves analysis efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is an overlay of characteristic spectra of the standard decoction of Pulsatillae provided by the present invention;

[0046] Figure 2The reference characteristic spectrum of the standard decoction of Pulsatillae provided by the present invention; wherein, peak 1 represents monocaffeoyltartaric acid; peak 3 represents trans-monoferuloyltartaric acid; peak 5 (S1) represents chicoric acid; peak 8 (S2) represents pulsatillae saponin B4; peak 9 represents hederacoside;

[0047] Figure 3 The total ion current diagram and ultraviolet absorption chromatogram of the test solution of the standard decoction of Pulsatilla chinensis provided by the present invention;

[0048] Figure 4 Investigation of the specificity of the characteristic spectra of the Pulsatillae Herba, standard decoction and formula granules provided by the present invention;

[0049] Figure 5 This is a comparative characteristic spectrum of the Pulsatilla formula granules provided by the present invention; wherein, peak 1 represents monocaffeoyltartaric acid; peak 5 (S1) represents chicoric acid; peak 8 (S2) represents pulsatilla saponin B4; peak 9 represents hedera saponin;

[0050] Figure 6 Overlay of characteristic spectra of the Pulsatilla chinensis medicinal material provided by the present invention and its common counterfeits;

[0051] Figure 7 The characteristic spectrum of the medicinal material Pulsatilla dahurica provided by the present invention is an investigation diagram of the extraction solvent;

[0052] Figure 8 A graph showing the extraction solvent characteristics of the standard decoction of Pulsatillae provided by the present invention;

[0053] Figure 9 This is a graph showing the extraction solvents for the characteristic spectrum of the Pulsatilla dasyphylla formula granules provided by the present invention;

[0054] Figure 10 The characteristic spectrum of the standard decoction of Pulsatillae chinensis under the gradient optimization 1 condition provided by the present invention;

[0055] Figure 11 The characteristic spectrum of the standard decoction of Pulsatillae chinensis under the gradient optimization 2 condition provided by the present invention;

[0056] Figure 12 The characteristic spectrum of the standard decoction of Pulsatillae Herba under the gradient optimization 3 conditions provided by the present invention;

[0057] Figure 13 The characteristic spectrum of the standard decoction of Pulsatillae chinensis under the gradient optimization 4 conditions provided by the present invention;

[0058] Figure 14 The characteristic spectrum of the standard decoction of Pulsatillae chinensis under the gradient optimization 5 conditions provided by the present invention;

[0059] Figure 15 The characteristic spectrum of the standard decoction of Pulsatillae chinensis under the gradient optimization 6 conditions provided by the present invention;

[0060] Figure 16 The characteristic spectrum of the standard decoction of Pulsatillae Herba under the gradient optimization 7 conditions provided by the present invention;

[0061] Figure 17 The characteristic spectrum of the standard decoction of Pulsatillae Herba under the gradient optimization 3 conditions provided by the present invention;

[0062] Figure 18 This is the characteristic spectrum of the standard decoction of Pulsatillae under the gradient optimization 5 conditions provided by the present invention. DETAILED DESCRIPTION

[0063] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0064] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. In the description of the present invention, the meaning of "several" is at least one, such as one, two, etc., unless otherwise clearly and specifically defined.

[0065] The terms "preferably," "more preferably," and the like, used herein refer to embodiments of the present invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the present invention.

[0066] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0067] Unless otherwise indicated, all percentages, fractions, and ratios are calculated based on the total weight of the compositions of the present invention. Unless otherwise indicated, all masses relating to listed ingredients are given as active ingredients and therefore do not include solvents or byproducts that may be present in commercially available materials. The term "mass percentage content" may be expressed herein with the symbol "%." All molecular weights herein are weight-average molecular weights expressed in Daltons, unless otherwise indicated. All formulations and testing herein took place at 25°C, unless otherwise indicated. The terms "comprise," "include," "contain," "have," "have," and other variations herein are intended to encompass non-exclusive inclusions, and do not distinguish between these terms. The term "comprising" means that additional steps and ingredients that do not affect the end result may be added. The compositions and methods / processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein. The terms "efficacy," "performance," "effect," and "efficacy" are not used herein to distinguish between.

[0068] The weights of the relevant components mentioned in the description of the embodiments of the present invention may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally increased or decreased according to the description of the embodiments of the present invention, it is within the scope disclosed in the description of the embodiments of the present invention. Specifically, the weights mentioned in the description of the embodiments of the present invention may be mass units known in the chemical industry, such as ug, mg, g, and kg.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0070] The present invention provides a method for constructing a characteristic spectrum of a Pulsatilla chinensis medicinal material, a standard decoction or a Chinese medicine formula granule, comprising the following steps:

[0071] Prepare the test solution by taking Pulsatilla chinensis medicinal materials, standard decoction or Chinese medicine formula granules;

[0072] The test solution was subjected to ultra-high performance liquid chromatography analysis;

[0073] Among them, the conditions for ultra-high performance liquid chromatography analysis include: mobile phase A is a mixture of acetonitrile and methanol, and mobile phase B is a phosphoric acid aqueous solution; gradient elution is adopted; the gradient elution program includes: from 0 min to 9 min, the volume percentage of mobile phase A changes from 8% to 10%; from 9 min to 18 min, the volume percentage of mobile phase A changes from 10% to 18%; from 18 min to 30 min, the volume percentage of mobile phase A changes from 18% to 26%; from 30 min to 38 min, the volume percentage of mobile phase A changes from 26% to 31%; from 38 min to 48 min, the volume percentage of mobile phase A changes from 31% to 45%.

[0074] In a specific example, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.2%.

[0075] It can be understood that in the present invention, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution includes but is not limited to 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, and 0.2%.

[0076] In a specific example, mobile phase A is a mixture of acetonitrile and methanol in a volume ratio of (1.8-2.2): 1. More specifically, mobile phase A is a mixture of acetonitrile and methanol in a volume ratio of 2:1.

[0077] In a specific example, the conditions for ultra-high performance liquid chromatography analysis also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 210nm to 220nm.

[0078] It is understood that in the present invention, column temperatures include but are not limited to 28°C, 29°C, 30°C, 31°C, and 32°C;

[0079] Flow rates include but are not limited to 0.28 mL / min, 0.29 mL / min, 0.30 mL / min, 0.31 mL / min, 0.32 mL / min;

[0080] The wavelengths include but are not limited to 210 nm, 211 nm, 212 nm, 213 nm, 214 nm, 215 nm, 216 nm, 217 nm, 218 nm, 219 nm, and 220 nm.

[0081] In a specific example, the dimensions of the chromatographic column used in ultra-high performance liquid chromatography analysis include: a column length of 100 mm ± 10 mm, an inner diameter of 2.1 mm ± 0.2 mm, and a particle size of 1.8 μm ± 0.2 μm.

[0082] Preferably, the dimensions of the chromatographic column used in ultra-high performance liquid chromatography analysis include: column length of 100 mm, inner diameter of 2.1 mm, and particle size of 1.8 μm.

[0083] In a specific example, the injection volume is 2 μL.

[0084] In a specific example, the following steps are also included:

[0085] Take Pulsatilla chinensis control medicinal material and prepare reference solution;

[0086] The reference solution was subjected to ultra-performance liquid chromatography analysis.

[0087] In a specific example, preparing the reference solution includes the following steps:

[0088] Mix the Pulsatilla chinensis reference medicinal material with water, heat and reflux for extraction, then filter and take the filtrate.

[0089] In a specific example, the heating extraction time is 30 minutes to 45 minutes. Preferably, the heating extraction time is 30 minutes.

[0090] In a specific example, the following steps are also included:

[0091] Take chicoric acid reference substance and pulsatilla saponin B4 reference substance to prepare a mixed reference solution;

[0092] The mixed reference solution was subjected to ultra-performance liquid chromatography analysis.

[0093] In a specific example, preparing a mixed reference solution includes the following steps:

[0094] Mix the chicoric acid reference substance and pulsatilla saponin B4 reference substance with methanol.

[0095] In a specific example, preparing the test solution includes the following steps:

[0096] Mixing the Pulsatilla chinensis medicinal material with a methanol aqueous solution having a volume fraction of 40% to 60%, performing ultrasonic extraction, and then filtering to obtain a filtrate; or

[0097] Mixing the standard decoction of Pulsatillae with a methanol aqueous solution with a volume fraction of 40% to 60%, performing ultrasonic extraction, and then filtering to obtain the filtrate; or

[0098] The Chinese medicinal formula granules of Pulsatilla chinensis are mixed with a methanol aqueous solution with a volume fraction of 40% to 60%, subjected to ultrasonic extraction, and then filtered to obtain a filtrate.

[0099] It can be understood that in the present invention, the volume fraction of methanol in the methanol aqueous solution includes but is not limited to 40%, 42%, 44%, 46%, 48%, 49%, 50%, 51%, 52%, 54%, 56%, 58%, and 60%.

[0100] In a specific example, the ultrasonic extraction time is 30 minutes to 45 minutes. Preferably, the ultrasonic extraction time is 30 minutes.

[0101] In a specific example, the conditions for ultrasonic extraction include: power of 250 W and frequency of 40 kHz.

[0102] In a specific example, the number of characteristic peaks in the characteristic spectrum of Pulsatilla scabra medicinal material, standard decoction or Chinese medicine formula granules is 9; among them, peak 5 is the chromatographic peak of chicoric acid, and peak 8 is the chromatographic peak of Pulsatilla scabra saponin B4.

[0103] The present invention also provides an application of the method for constructing a characteristic spectrum of the above-mentioned Pulsatilla scabra medicinal material, standard decoction or traditional Chinese medicine formula granules in identifying the authenticity of Pulsatilla scabra.

[0104] In a specific example, the fake Pulsatilla products include one or more of Potentilla scabra, Rhizoma Cyperi, Pulsatilla gansuensis, Herba Potentillae discolor and Herba Cyperi.

[0105] The present invention also provides a high-resolution mass spectrometry method for identifying characteristic peaks of characteristic spectra of Pulsatilla dahurica medicinal materials, standard decoctions or Chinese medicine formula granules, comprising the following steps:

[0106] Prepare the test solution by taking Pulsatilla chinensis medicinal materials, standard decoction or Chinese medicine formula granules;

[0107] The test solution was analyzed by ultra-performance liquid chromatography coupled with mass spectrometry;

[0108] Among them, the conditions for ultra-high performance liquid chromatography analysis include: mobile phase A is a mixture of acetonitrile and methanol, and mobile phase B is a formic acid aqueous solution; gradient elution is adopted; the gradient elution program includes: from 0 min to 9 min, the volume percentage of mobile phase A changes from 8% to 10%; from 9 min to 18 min, the volume percentage of mobile phase A changes from 10% to 18%; from 18 min to 30 min, the volume percentage of mobile phase A changes from 18% to 26%; from 30 min to 38 min, the volume percentage of mobile phase A changes from 26% to 31%; from 38 min to 48 min, the volume percentage of mobile phase A changes from 31% to 45%.

[0109] In a specific example, the conditions for mass spectrometry analysis include: sheath gas flow rate of 35arb±5arb, auxiliary gas flow rate of 10arb±2arb, spray voltage of 3.8kV±1kV, S-lens voltage of 50V±5V, heating temperature of 350℃±10℃, capillary temperature of 350℃±10℃; scanning mode is positive and negative ion mode, scanning range is 120m / z~1500m / z, and normalized collision energy is 40%±5%.

[0110] In a specific example, mobile phase A is a mixture of acetonitrile and methanol in a volume ratio of (1.8-2.2): 1. More specifically, mobile phase A is a mixture of acetonitrile and methanol in a volume ratio of 2:1.

[0111] In a specific example, the volume fraction of formic acid in the formic acid aqueous solution is 0.05% to 0.2%.

[0112] It can be understood that in the present invention, the volume fraction of formic acid in the formic acid aqueous solution includes but is not limited to 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, and 0.2%.

[0113] In a specific example, the conditions for ultra-high performance liquid chromatography analysis also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 210nm to 220nm.

[0114] The present invention also provides a method for detecting Pulsatilla dasyphylla medicinal material, standard decoction or Chinese medicine formula granules, comprising the following steps:

[0115] Take the product to be tested and prepare a solution of the product to be tested;

[0116] The test solution is subjected to ultra-high performance liquid chromatography;

[0117] Among them, the conditions for ultra-high performance liquid chromatography analysis include: mobile phase A is a mixture of acetonitrile and methanol, and mobile phase B is a phosphoric acid aqueous solution; gradient elution is adopted; the gradient elution program includes: from 0 min to 9 min, the volume percentage of mobile phase A changes from 8% to 10%; from 9 min to 18 min, the volume percentage of mobile phase A changes from 10% to 18%; from 18 min to 30 min, the volume percentage of mobile phase A changes from 18% to 26%; from 30 min to 38 min, the volume percentage of mobile phase A changes from 26% to 31%; from 38 min to 48 min, the volume percentage of mobile phase A changes from 31% to 45%.

[0118] In a specific example, mobile phase A is a mixture of acetonitrile and methanol in a volume ratio of (1.8-2.2): 1. More specifically, mobile phase A is a mixture of acetonitrile and methanol in a volume ratio of 2:1.

[0119] In a specific example, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.2%.

[0120] In a specific example, the conditions for ultra-high performance liquid chromatography analysis also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 210nm to 220nm.

[0121] The following is a detailed description of the construction method and detection method of the characteristic spectrum of the Pulsatilla chinensis medicinal material, standard decoction or Chinese medicine formula granules of the present invention in conjunction with specific examples. The raw materials used in the following examples, unless otherwise specified, are all commercially available products.

[0122] Example 1

[0123] This embodiment provides a method for constructing characteristic spectra of Pulsatilla dahurica medicinal materials, standard decoctions, and Chinese medicine formula granules, as follows:

[0124] 1. Instruments and Materials

[0125] Instrument: Waters ultra-high performance liquid chromatograph (H-class (PDA), Waters Corporation, USA);

[0126] Reagents: ethanol (Xilong Scientific Co., Ltd.) and methanol (Xilong Scientific Co., Ltd.) of analytical grade; phosphoric acid for liquid phase (Tianjin Kemeiou Chemical Reagent Co., Ltd.) and formic acid for liquid phase (Tianjin Kemeiou Chemical Reagent Co., Ltd.); acetonitrile (Merck Co., Ltd.) and methanol (Merck Co., Ltd.) of HPLC grade; water of ultrapure grade (prepared in the laboratory).

[0127] Test drugs: chicoric acid (batch number: wkq19011605, content: 98.56%, Sichuan Weikeqi Biotechnology Co., Ltd.), pulsatilla saponin B4 (batch number: 111766-201702, content: 94.7%, China Food and Drug Inspection Institute); pulsatilla control medicinal material (batch number: 121615-201603, China Food and Drug Inspection Institute).

[0128] 2 Methods and Results

[0129] 2.1 Chromatographic conditions

[0130] Octadecylsilane bonded silica gel was used as the packing material (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.8 μm); acetonitrile:methanol (2:1) was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, using a gradient elution as specified in Table 1. The flow rate was 0.30 ml / min; the column temperature was 30°C; and the detection wavelength was 215 nm. The number of theoretical plates, calculated based on Pulsatilla saponin B4, should be no less than 5000.

[0131] Table 1 Gradient elution table

[0132]

[0133]

[0134] 2.2 Preparation of reference solution

[0135] Take appropriate amount of chicoric acid reference substance and pulsatilla saponin B4 reference substance, weigh accurately, add methanol to make a mixed solution containing 50 μg of chicoric acid and 400 μg of pulsatilla saponin B4 per 1 mL, which is used as the reference substance solution.

[0136] 2.3 Preparation of reference solution

[0137] Take about 1 g of Pulsatilla chinensis reference medicinal material, add 30 mL of water, weigh the weight, heat and reflux for 30 minutes, take out, let cool, weigh the weight again, add water to make up the lost weight, filter, take 1 mL of the filtrate, dilute to 10 mL with methanol, shake well, filter, and take the filtrate as the reference solution.

[0138] 2.4 Preparation of test solution

[0139] Preparation of test solution of Pulsatilla dahurica medicinal material: Take about 0.2g of the powder of this product (passed through No. 3 sieve), weigh it accurately, put it into a stoppered conical flask, add 25ml of 50% methanol accurately, weigh it, ultrasonicate (power 250W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0140] Preparation of test solution of Pulsatilla Standard Decoction: Take an appropriate amount of this product, grind it into powder, take about 0.1g, accurately weigh it, put it into a stoppered conical flask, accurately add 25ml of 50% methanol, weigh it, ultrasonically treat it (250W, 40kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0141] Preparation of test solution of Pulsatilla formula granules: Take an appropriate amount of this product, grind it into powder, take about 0.1g, accurately weigh it, put it into a stoppered conical flask, accurately add 25ml of 50% methanol, weigh the weight, ultrasonically treat (250W, 40kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0142] 2.5 Assay

[0143] Accurately pipette 2 μl of reference solution, control solution and test solution respectively, inject them into liquid chromatograph and measure to obtain the result.

[0144] 2.6 Determination of characteristic peaks

[0145] Take 22 batches of Pulsatillae standard decoction samples, prepare the test solution according to the test solution preparation method determined under "2.4", accurately aspirate the above-mentioned reference solution, reference substance solution and test solution respectively, and perform sample injection determination according to the chromatographic conditions determined under "2.1". Use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software" to identify the common peaks of the characteristic spectra of the 22 batches of Pulsatillae standard decoction, and select 9 common peaks with known components, large peak areas, good peak shapes and separation, and high purity as the characteristic peaks of Pulsatillae standard decoction, such as Figure 1 As shown, the chicoric acid reference peak is taken as the S1 peak, and the pulsatilla saponin B4 reference peak is taken as the S2 peak. The relative retention times of each characteristic peak and the S1 peak and the S2 peak are calculated, and the characteristic peaks are positioned using the relative retention times.

[0146] The results, as shown in Tables 2 and 3, show that, using the chicoric acid reference peak as S1 and the pulsatilla saponin B4 reference peak as S2, the relative retention time (RSD) values ​​of the characteristic peaks relative to the S1 and S2 peaks for the 22 batches of Pulsatilla truncatula standard decoctions ranged from 0.01% to 0.72%, indicating that the relative retention times of the characteristic peaks were relatively stable. Therefore, the mean relative retention time values ​​could be used to locate the characteristic peaks. The relative peak area (RSD) values ​​of the characteristic peaks relative to the S1 and S2 peaks ranged from 23.46% to 41.78%, indicating significant differences in relative peak areas between samples from different batches. This suggests that the proportions of the chemical components represented by the characteristic peaks in different batches of Pulsatilla truncatula standard decoctions vary significantly. This variation may be related to factors such as origin and age. Furthermore, since the relative peak areas of characteristic peaks are significantly affected by factors such as column temperature, flow rate, chromatographic column, and operator operation, it is not appropriate to set a standard for the relative peak areas of characteristic peaks at this time.

[0147] Table 2 Characteristic spectra of 22 batches of standard decoction of Pulsatillae (relative retention time)

[0148]

[0149]

[0150] Table 3 Characteristic spectra (relative peak areas) of 22 batches of standard decoctions of Pulsatillae

[0151]

[0152] 2.7 Determination of characteristic maps

[0153] The UPLC characteristic spectra of 22 batches of Pulsatillae decoction were matched using the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System", and a reference spectrum was generated by the average method to establish a reference characteristic spectrum of Pulsatillae decoction. Figure 2 Based on the assay results of 22 batches of Pulsatillae Radix decoction samples, the characteristic spectrum of the Pulsatillae Radix decoction was determined as follows: the test sample chromatogram should show nine characteristic peaks, and their retention times should correspond to those of the nine characteristic peaks in the chromatogram of the reference medicinal material. Two of these peaks should correspond to the retention times of the corresponding reference material peaks. The peak corresponding to the chicoric acid reference material is designated as the S1 peak. The relative retention times of Peaks 1, 2, 3, 4, and 6 relative to the S1 peak should be calculated. These relative retention times should be within ±10% of the specified values: 0.23 (Peak 1), 0.37 (Peak 2), 0.49 (Peak 3), 0.63 (Peak 4), and 1.28 (Peak 6). The peak corresponding to the Pulsatillae Radix saponin B4 reference material is designated as the S2 peak. The relative retention times of Peaks 7 and 9 relative to the S2 peak should be within ±10% of the specified values: 0.82 (Peak 7) and 1.02 (Peak 9).

[0154] 2.8 Characteristic Peak Identification

[0155] With reference to the liquid chromatography conditions of the characteristic spectrum of the standard decoction of Pulsatillae, the compounds in the test solution of the standard decoction of Pulsatillae were subjected to mass spectrometry analysis, and the secondary fragment ion matching was performed with the data in the local mass spectrometry database and with reference to relevant literature to identify the compounds. Finally, using the reference substance, through the comparative analysis of the retention time, ultraviolet absorption spectrum, mass spectrometry accurate molecular weight and fragment ions of the liquid chromatography, it was finally confirmed that in the characteristic spectrum of the standard decoction of Pulsatillae, peak 1 was monocaffeoyltartaric acid, peak 3 was trans-monoferuloyltartaric acid, peak 5 (S1) was chicoric acid, peak 8 (S2) was pulsatillae saponin B4, and peak 9 was ivy saponin C. The total ion current and ultraviolet absorption chromatogram of the test solution are shown in Figure 3 , compound information is shown in Table 6.

[0156] Specifically, accurately pipette 2 μl of the test solution, inject it into a liquid chromatograph or liquid chromatography-mass spectrometer, and measure the solution. The chromatography-mass spectrometry conditions are as follows: octadecylsilane bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile-methanol (2:1) as mobile phase A, 0.1% formic acid solution as mobile phase B, and gradient elution as specified in Table 4 below; flow rate 0.30 ml per minute; column temperature 30°C; detection wavelength 215 nm (or 300 nm). The number of theoretical plates calculated based on Pulsatilla saponin B4 should be no less than 5000. The mass spectrometry parameters are shown in Table 5.

[0157] Table 4 Gradient elution table

[0158]

[0159] Table 5 Mass spectrometry parameters

[0160]

[0161] Table 6 Identification results of compounds in Pulsatillae Standard Decoction

[0162]

[0163] 3 Methodological validation

[0164] 3.1 Exclusivity

[0165] Accurately pipette 2 μl of each of the test solution, reference solution and blank solvent of Pulsatilla dasyphylla, standard decoction and formula granules, inject them into the liquid chromatograph, and measure according to the chromatographic conditions under "2.1". Figure 4 .

[0166] The results showed that the test sample chromatogram had the same chromatographic peak at the corresponding retention time as the reference sample chromatogram, and there was no negative interference, indicating that the method had good specificity.

[0167] 3.2 Repeatability

[0168] Grind appropriate amounts of the standard decoction and granules of Pulsatillae Radix. Accurately weigh approximately 0.1 g of each of the Pulsatillae Radix powders. Prepare the test solution according to the test solution preparation method specified in "2.4." Inject and analyze the sample according to the chromatographic conditions in "2.1." Inject the same aliquot of the test solution six times. Determine the peak S1, using the chicoric acid reference peak, and the Pulsatillae Radix saponin B4 reference peak as S2. Calculate the relative retention time and peak area of ​​each characteristic peak relative to the S1 and S2 peaks, and calculate the RSD values. The experimental results are shown in Tables 7 to 12.

[0169] Table 7 Repeatability test results of characteristic spectrum of Pulsatilla dahurica medicinal material (relative retention time)

[0170]

[0171]

[0172] Table 8 Repeatability test results of characteristic spectrum of Pulsatilla dahurica medicinal material (relative peak area)

[0173]

[0174] Table 9 Repeatability test results of characteristic spectrum of standard decoction of Pulsatillae (relative retention time)

[0175]

[0176] Table 10 Repeatability test results of characteristic spectrum of standard decoction of Pulsatillae (relative peak area)

[0177]

[0178] Table 11 Repeatability of characteristic spectra of Pulsatilla formula granules (relative retention time)

[0179]

[0180] Table 12 Repeatability of characteristic spectrum of Pulsatilla formula granules (relative peak area)

[0181]

[0182] The results showed that the same batch of samples were measured six times, with the chicoric acid chromatographic peak as the reference peak S. The RSD values ​​of the relative retention time and relative peak area of ​​each characteristic peak to the S peak were in the range of 0-0.14% and 0.87%-2.87% for Pulsatilla herb, 0-0.45% and 2.11%-2.62% for Pulsatilla standard decoction, and 0-0.06% and 0.50%-2.82% for Pulsatilla formula granules. The RSDs of the relative retention time and relative peak area were all lower than 3.0%, indicating that the method had good repeatability.

[0183] 3.3 Stability

[0184] Grind appropriate amounts of Pulsatilla standard decoction and formulated granules. Accurately weigh approximately 0.1 g of each Pulsatilla powder. Prepare the test solution according to the test solution preparation method specified in "2.4." Inject and analyze the sample according to the chromatographic conditions in "2.1." Inject and analyze the sample at 0, 3, 6, 9, 12, and 14 hours. Use the chicoric acid reference peak as the S1 peak and the Pulsatilla saponin B4 reference peak as the S2 peak. Calculate the relative retention time and peak area of ​​each characteristic peak relative to the S1 and S2 peaks, and calculate the RSD values. The experimental results are shown in Tables 13 to 18.

[0185] Table 13 Results of stability study of characteristic spectrum of Pulsatilla dahurica medicinal material (relative retention time)

[0186]

[0187] Table 14: Results of stability study of characteristic spectrum of Pulsatilla dahurica medicinal material (relative peak area)

[0188]

[0189] Table 15: Results of stability study of characteristic spectrum of standard decoction of Pulsatillae (relative retention time)

[0190]

[0191] Table 16 Characteristic spectrum stability test results of Pulsatilla standard decoction (relative peak area)

[0192]

[0193] Table 17 Stability Study of Characteristic Spectra of Pulsatilla Formula Granules (Relative Retention Time)

[0194]

[0195] Table 18 Stability Study of Characteristic Spectra of Pulsatilla Formula Granules (Relative Peak Area)

[0196]

[0197] The experimental results showed that the same test solution was sampled and analyzed at 0, 3, 6, 9, 12 and 14 hours, respectively. Taking the chicoric acid chromatographic peak as the reference peak S, the RSD values ​​of the relative retention time and relative peak area of ​​each characteristic peak and the S peak were in the range of 0-0.40% and 1.18%-4.03% for Pulsatilla chinensis medicinal material, in the range of 0-0.45% and 1.37%-3.46% for Pulsatilla chinensis standard decoction, and in the range of 0-0.79% and 1.32%-2.66% for Pulsatilla chinensis formula granules, indicating that the test solution had good relative stability within 14 hours.

[0198] Example 2

[0199] This example provides a method for determining the activity of a sample of a Chinese medicinal formula granule of Pulsatilla dasyphylla, as follows:

[0200] Three batches of Pulsatilla scabra granules (batch numbers: KL01, KL02, KL03) were measured according to the following method, and the chicoric acid reference peak was used as the S1 peak, and the Pulsatilla scabra saponin B4 reference peak was used as the S2 peak. The relative retention times of each characteristic peak and the S1 and S2 peaks were calculated.

[0201] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the packing (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.8 μm); acetonitrile-methanol (2:1) was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in Table 1; the flow rate was 0.30 ml / min; the column temperature was 30°C; and the detection wavelength was 215 nm. The number of theoretical plates, calculated based on Pulsatilla saponin B4, should be no less than 5000.

[0202] Preparation of reference solution: Take about 1 g of Pulsatilla chinensis reference medicinal material, add 30 ml of water, weigh the weight, heat and reflux for 30 minutes, take out, let cool, weigh the weight again, add water to make up the lost weight, filter, take 1 ml of the filtrate, dilute to 10 ml with methanol, shake well, filter, and take the filtrate as the reference solution of the control medicinal material.

[0203] Preparation of reference solution: Take appropriate amount of chicoric acid reference substance and pulsatilla saponin B4 reference substance, weigh them accurately, add methanol to make a mixed solution containing 50 μg of chicoric acid and 400 μg of pulsatilla saponin B4 per 1 ml, which is used as the reference solution.

[0204] Preparation of test solution: Take an appropriate amount of this product, grind it into powder, take about 0.1g, accurately weigh it, put it into a stoppered conical flask, accurately add 25ml of 50% methanol, weigh it, and treat it ultrasonically (250W, 40kHz) for 30 minutes. Take it out, let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0205] Determination method: Accurately pipette 2 μl of each of the reference solution, control solution, and test solution, inject into the liquid chromatograph, and determine the results. The results are shown in Table 19.

[0206] Table 19 Characteristic spectrum of Pulsatilla formula granules (relative retention time)

[0207]

[0208] The UPLC characteristic spectra of the three batches of Pulsatilla Root Granules were then matched using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines". Peak 5 chicoric acid was used as the reference peak S1, and peak 8 pulsatilla saponin B4 was used as the reference peak S2. The control spectra were generated using the average method to determine the control characteristic spectra of the Pulsatilla Root Granules, as shown in Figure 2. Figure 5 shown.

[0209] Table 20 Comparison of characteristic spectra of three batches of Pulsatilla striata granules and standard decoction

[0210]

[0211] The above research results show that the characteristic spectra of the three batches of mass-produced Pulsatilla Root Formula Granules share nine characteristic peaks with the standard decoction and the control medicinal material. The relative retention times of each characteristic peak and the S peak all fall within the specified range of the standard decoction characteristic spectra, demonstrating the consistency of the quality of the mass-produced samples and the standard decoction. Referring to the research results of the characteristic spectra of the standard decoction, the characteristic spectra standard of the Pulsatilla Root Formula Granules was determined as follows: the chromatogram of the test sample should show nine characteristic peaks, and their retention times should correspond to those of the nine characteristic peaks in the chromatogram of the control medicinal material. Two of these peaks should correspond to the retention times of the corresponding reference peaks, with the peak corresponding to the chicoric acid reference being the S1 peak. The relative retention times of Peaks 1, 2, 3, 4, and 6 relative to the S1 peak should be calculated and should be within ±10% of the specified values: 0.23 (Peak 1), 0.37 (Peak 2), 0.49 (Peak 3), 0.63 (Peak 4), and 1.28 (Peak 6). The peak corresponding to the reference substance of pulsatilla saponin B4 is the S2 peak. The relative retention times of peaks 7, 9 and S2 are calculated. The relative retention times should be within the range of ±10% of the specified values, which are: 0.82 (peak 7) and 1.02 (peak 9).

[0212] Example 3

[0213] This embodiment provides a method for distinguishing Pulsatilla chinensis from different counterfeit products, as follows:

[0214] Common counterfeit products of Pulsatilla chinensis (Potentilla, Rhizoma Lupulinae, Pulsatilla gansuensis, Herba Dioscoreae, and Herba Pulsatillae) were tested using the following method and compared with the characteristic spectrum of Pulsatilla chinensis:

[0215] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the packing (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.8 μm); acetonitrile-methanol (2:1) was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in Table 1; the flow rate was 0.30 ml / min; the column temperature was 30°C; and the detection wavelength was 215 nm. The number of theoretical plates, calculated based on Pulsatilla saponin B4, should be no less than 5000.

[0216] Preparation of reference solution: Take about 1g of Pulsatilla chinensis reference medicinal material, add 30ml of water, weigh the weight, heat and reflux for 30 minutes, take out, let cool, weigh the weight again, add water to make up the lost weight, filter, take 1ml of the filtrate, dilute to 10ml with methanol, shake well, filter, and take the filtrate as the reference solution.

[0217] Preparation of reference solution: Take appropriate amount of chicoric acid reference substance and pulsatilla saponin B4 reference substance, weigh them accurately, add methanol to make a mixed solution containing 50 μg of chicoric acid and 400 μg of pulsatilla saponin B4 per 1 ml, which is used as the reference solution.

[0218] Preparation of test solution: Take the powder of the medicinal materials of Pulsatilla dahurica and its counterfeit products (Potentilla truncatula, Rhizoma Cynanchum, Pulsatilla gansuensis, Herba Distichum radiatae and Herba Cynanchifoliae), pass through No. 3 sieve, about 0.2 g, accurately weigh, put into a stoppered conical flask, accurately add 25 ml of 50% methanol, weigh the weight, ultrasonically treat (250W, 40kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain.

[0219] Determination method: Accurately pipette 2μl of reference solution, control solution and test solution respectively, inject into liquid chromatograph, and determine. Figure 6 shown.

[0220] The results showed that the characteristic spectra of Potentilla, Lophatherum gracile, Pulsatilla gansuense, Herba Potentillae scabra and Herba Porophyllae did not contain the nine characteristic peaks with the same retention time as the characteristic spectra of Pulsatilla chinensis medicinal materials, indicating that the established characteristic spectra of Pulsatilla chinensis medicinal materials can effectively distinguish its common counterfeits.

[0221] Example 4

[0222] This example provides an investigation of a method for constructing characteristic maps of Pulsatilla dahurica medicinal materials, standard decoctions, and Chinese medicine granules, as follows:

[0223] 1. Investigation of extraction solvent

[0224] The effects of different extraction solvents on the characteristic spectra of Pulsatilla chinensis, standard decoction and formula granules were investigated respectively. Methanol, 50% methanol, 10% methanol and 50% ethanol were selected as extraction solvents. The peak shapes and separations of 9 characteristic peaks were compared and the "total peak area / sample weight" of 9 characteristic peaks were calculated to compare the effects of different extraction solvents on the characteristic spectra of Pulsatilla chinensis, standard decoction and formula granules. The best extraction solvent was selected. The results are shown in Tables 21 to 23 and Figures 7 to 9 shown.

[0225] By comparing the characteristic spectra of four different extraction solvents, it can be seen that among the results of different solvent investigations on Pulsatilla Root medicinal materials, standard decoctions and formula granules, the "total peak area / sample weight" extracted with 50% methanol as the solvent is the highest, indicating that 50% methanol has the best extraction effect. Therefore, 50% methanol was selected as the extraction solvent for Pulsatilla Root medicinal materials, standard decoctions and formula granules.

[0226] Table 21 Results of investigation on extraction solvents of Pulsatilla dahurica medicinal material characteristic spectrum

[0227]

[0228] Table 22 Characteristic spectrum of Pulsatillae standard decoction extraction solvent investigation results

[0229]

[0230] Table 23 Characteristic spectrum of Pulsatilla formula granules extraction solvent investigation results

[0231]

[0232] 2. Investigation of mobile phase composition and elution procedure

[0233] 2.1 In this experiment, acetonitrile-0.1% phosphoric acid solution was selected as the mobile phase, and the gradient of the mobile phase was optimized:

[0234] Take an appropriate amount of Pulsatilla standard decoction (GT1706088), grind it into powder, take about 0.1g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh it, and ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0235] Chromatographic conditions: Agilent SB C18 (2.1 mm × 150 mm, 1.8 μm) column; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the specifications in Tables 24 to 28; flow rate: 0.3 ml / min; column temperature: 30°C; detection wavelength: 201 nm; injection volume: 2 μl.

[0236] The results are as follows Figures 10-14 When different gradients were optimized using acetonitrile-0.1% phosphoric acid solution as the mobile phase, the chromatographic peaks were too concentrated or the separation of some peaks was difficult to achieve ideal results.

[0237] Table 24 Gradient Optimization 1

[0238]

[0239] Table 25 Gradient Optimization 2

[0240]

[0241] Table 26 Gradient Optimization 3

[0242]

[0243] Table 27 Gradient Optimization 4

[0244]

[0245] Table 28 Gradient Optimization 5

[0246]

[0247] 2.2 In this experiment, acetonitrile:methanol (2:1)-0.1% phosphoric acid solution was selected as the mobile phase, and the gradient of the mobile phase was optimized:

[0248] Take an appropriate amount of Pulsatilla standard decoction (GT1706088), grind it into powder, take about 0.1g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh it, and ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0249] Chromatographic conditions: Agilent SB C18 (2.1 mm × 150 mm, 1.8 μm) column; acetonitrile:methanol (2:1) as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in Tables 29 to 30, respectively; flow rate, 0.30 ml / min; column temperature: 30°C; detection wavelength: 215 nm; injection volume: 2 μl.

[0250] The results are as follows Figures 15-16 The results showed that the effect of using acetonitrile:methanol (2:1)-0.1% phosphoric acid solution as the mobile phase was significantly better than that of acetonitrile-0.1% phosphoric acid solution. After gradient optimization, acetonitrile:methanol (2:1)-0.1% phosphoric acid solution was finally selected as the mobile phase, and gradient optimization 6 was selected as the elution gradient of the method.

[0251] Table 29 Gradient Optimization 6

[0252]

[0253] Table 30 Gradient Optimization 7

[0254]

[0255] 3. Investigation of wavelength

[0256] During the optimization of the gradient and mobile phase, three different detection wavelengths of 201 nm, 210 nm and 215 nm were investigated. The results are as follows: Figures 17-18 As shown in the figure, a comparison of three different wavelengths, 201nm, 210nm, and 215nm, revealed that the 201nm wavelength had a greater number of chromatographic peaks, but also greater baseline fluctuations than the 210nm wavelength. A comparison of the 210nm and 215nm wavelengths revealed the same number of chromatographic peaks, but the 215nm wavelength had a more even peak height ratio. Therefore, the 215nm wavelength was ultimately selected as the detection wavelength.

[0257] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0258] The embodiments described above only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all fall within the scope of protection of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing a characteristic spectrum of Pulsatilla chinensis medicinal materials, standard decoctions or Chinese medicine formula granules, characterized in that: The steps include: Prepare the test solution by taking Pulsatilla chinensis medicinal materials, standard decoction or Chinese medicine formula granules; The test solution is subjected to ultra-high performance liquid chromatography analysis; Among them, the conditions for ultra-high performance liquid chromatography analysis include: Mobile phase A is a mixture of acetonitrile and methanol in a volume ratio of 2:1, and mobile phase B is a phosphoric acid aqueous solution, wherein the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.2%. Gradient elution is adopted; the gradient elution program includes: from 0 min to 9 min, the volume percentage of mobile phase A changes from 8% to 10%; from 9 min to 18 min, the volume percentage of mobile phase A changes from 10% to 18%; from 18 min to 30 min, the volume percentage of mobile phase A changes from 18% to 26%; from 30 min to 38 min, the volume percentage of mobile phase A changes from 26% to 31%; from 38 min to 48 min, the volume percentage of mobile phase A changes from 31% to 45%. The chromatographic column was an octadecylsilane bonded silica column with a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm; The wavelength is 210nm~220nm; The characteristic map construction method further comprises the following steps: Take chicoric acid reference substance and pulsatilla saponin B4 reference substance to prepare a mixed reference solution; The mixed reference solution is subjected to ultra-high performance liquid chromatography analysis; In the steps of preparing the test solution: The extraction solvent of the Pulsatilla chinensis medicinal material is a methanol aqueous solution with a volume fraction of 40% to 60%; The extraction solvent of the Pulsatilla dasyphylla standard decoction is a methanol aqueous solution with a volume fraction of 40% to 60%; The extraction solvent of the Pulsatillae Chinese medicine formula granules is a methanol aqueous solution with a volume fraction of 40% to 60%; There are 9 characteristic peaks in the characteristic spectrum of the Pulsatilla medicinal material, standard decoction or Chinese medicine formula granules; among them, peak 1 is the chromatographic peak of monocaffeoyltartaric acid, peak 3 is the chromatographic peak of trans-monoferuloyl tartaric acid, peak 5 is the chromatographic peak of chicoric acid, peak 8 is the chromatographic peak of pulsatilla saponin B4, and peak 9 is the chromatographic peak of hedera saponin.

2. The method for constructing a characteristic spectrum of the Pulsatilla chinensis medicinal material, standard decoction or Chinese medicine formula granules according to claim 1, characterized in that: The conditions for ultra-high performance liquid chromatography analysis also include: column temperature of 28°C to 32°C; flow rate of 0.28 mL / min to 0.32 mL / min.

3. The method for constructing a characteristic spectrum of the Pulsatilla chinensis medicinal material, standard decoction or Chinese medicine formula granules according to claim 1, characterized in that: The following steps are also included: Take Pulsatilla chinensis control medicinal material and prepare reference solution; The reference solution was subjected to ultra-performance liquid chromatography analysis.

4. The method for constructing a characteristic spectrum of the Pulsatilla chinensis medicinal material, standard decoction or Chinese medicine formula granules according to claim 3, characterized in that: Preparation of the reference solution includes the following steps: The Pulsatilla chinensis control medicinal material was mixed with water, heated under reflux for extraction, and then filtered to obtain a filtrate.

5. The method for constructing a characteristic spectrum of the Pulsatilla chinensis medicinal material, standard decoction or Chinese medicine formula granules according to claim 1, characterized in that: The preparation of the mixed reference solution includes the following steps: The chicoric acid reference substance and pulsatilla saponin B4 reference substance were mixed with methanol.

6. The method for constructing a characteristic spectrum of the Pulsatilla chinensis medicinal material, standard decoction or Chinese medicine formula granules according to claim 1, characterized in that: The preparation of the test solution includes the following steps: Mixing the Pulsatilla chinensis medicinal material with a methanol aqueous solution having a volume fraction of 40% to 60%, performing ultrasonic extraction, and then filtering to obtain a filtrate; or Mixing the standard decoction of Pulsatillae with a methanol aqueous solution having a volume fraction of 40% to 60%, performing ultrasonic extraction, and then filtering to obtain a filtrate; or The Pulsatillae Chinese medicinal formula granules are mixed with a methanol aqueous solution with a volume fraction of 40% to 60%, subjected to ultrasonic extraction, and then filtered to obtain a filtrate.

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