Method for constructing characteristic spectrum of Qi-invigorating and blood-maintaining preparation, and its characteristic spectrum and application

Abstract

The present invention relates to a method for constructing a characteristic spectrum of a Qi-invigorating and blood-reinvigorating preparation, as well as its characteristic spectrum and application. By adopting specific high-performance liquid chromatography (HPLC) detection and analysis conditions for the Qi-invigorating and blood-reinvigorating preparation, a characteristic spectrum of the Qi-invigorating and blood-reinvigorating preparation containing multiple characteristic peaks is constructed. The method for constructing the characteristic spectrum of the Qi-invigorating and blood-reinvigorating preparation has the advantages of good stability, high precision, good repeatability, etc. after methodological verification. It can comprehensively reflect the effective ingredients contained in the Qi-invigorating and blood-reinvigorating preparation, and further objectively, comprehensively and accurately evaluate the quality of the Qi-invigorating and blood-reinvigorating preparation, which is of great significance for controlling the quality of the Qi-invigorating and blood-reinvigorating preparation and ensuring the clinical efficacy.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and in particular to a method for constructing a characteristic spectrum of a Qi-invigorating and blood-maintaining preparation, as well as the characteristic spectrum and application thereof. Background Art

[0002] Yiqiweixue preparations are pure traditional Chinese medicines made from pig blood extract, astragalus root, and jujube. They tonify blood and replenish qi. They are primarily used to treat symptoms such as sallow or pale complexion, dizziness, fatigue, shortness of breath, laziness, spontaneous sweating, pale lips and tongue, and a weak pulse caused by qi and blood deficiency. These preparations include Yiqiweixue Capsules, Yiqiweixue Tablets, Yiqiweixue Granules, and Yiqiweixue Granules (without added sucrose).

[0003] Qi-invigorating and blood-replenishing preparations are made from three herbs and have a relatively complex chemical composition. Quality control of these preparations is essential for ensuring their clinical effectiveness. Traditionally, there are few reports on these preparations, and a lack of comprehensive characteristic spectra that reflect the active ingredients in these preparations. The characteristic spectra of traditional Chinese medicines (TCMs) are a comprehensive, quantifiable identification method that can comprehensively reflect the types and quantities of chemical components in TCMs and their preparations, thereby providing a holistic description and evaluation of drug quality. Therefore, there is an urgent need to provide a method for detecting the characteristic spectra of TCMs to better control product quality. Summary of the Invention

[0004] Based on this, it is necessary to provide a method for constructing a characteristic spectrum of Qi-invigorating and blood-reinforcing preparations. The characteristic spectrum of Qi-invigorating and blood-reinforcing preparations constructed by this method has multiple characteristic peaks, which can comprehensively reflect the effective ingredients contained in the Qi-invigorating and blood-reinforcing preparations, and realize objective, comprehensive and accurate quality control of the Qi-invigorating and blood-reinforcing preparations.

[0005] One aspect of the present invention provides a method for constructing a characteristic spectrum of a Qi-invigorating and blood-reinforcing preparation, comprising the following steps:

[0006] Extract the Yiqiweixue preparation using an extraction solvent to prepare a test solution;

[0007] Performing high performance liquid chromatography on the test solution to obtain a chromatogram of the corresponding test sample;

[0008] The conditions for the HPLC detection include: mobile phase A is methanol, mobile phase B is phosphoric acid aqueous solution, and gradient elution is used;

[0009] The gradient elution procedure includes:

[0010] From 0 to 8 minutes, the volume percentage of the mobile phase A is maintained at 4%;

[0011] From 8 to 20 minutes, the volume percentage of the mobile phase A increases from 4% to 15%;

[0012] From 20 min to 55 min, the volume percentage of the mobile phase A increases from 15% to 80%;

[0013] 55min~65min, the volume percentage of the mobile phase A is maintained at 80%;

[0014] The chromatograms of the test samples were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system for processing, and the main chromatographic peaks existing in the chromatograms of different batches of test samples were selected as characteristic peaks to construct the characteristic spectrum of the Qi-invigorating and blood-maintaining preparation.

[0015] In one embodiment, the Qi-invigorating and blood-replenishing preparation includes one or more of Qi-invigorating and blood-replenishing capsules, Qi-invigorating and blood-replenishing tablets, Qi-invigorating and blood-replenishing granules, and Qi-invigorating and blood-replenishing granules (without added sucrose).

[0016] In one embodiment, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.02%-0.1%.

[0017] In one embodiment, the high performance liquid chromatography detection further satisfies at least one of the following conditions:

[0018] (1) The injection volume is 5 μL-50 μL;

[0019] (2) The detection wavelength is 190nm-400nm;

[0020] (3) Flow rate: 0.8 mL / min-1.2 mL / min;

[0021] (4) The column temperature is 25℃-35℃.

[0022] In one embodiment, the step of extracting the Qi-Yinqi-Weixue preparation using an extraction solvent satisfies at least one of the following conditions:

[0023] (1) The extraction method is one or more of ultrasonic extraction, reflux extraction, maceration extraction, decoction extraction, or oscillation extraction;

[0024] (2) The extraction time is 20-60 minutes;

[0025] (3) The extraction solvent includes methanol aqueous solution or methanol; optionally, the volume percentage of methanol in the methanol aqueous solution is 20%-95%.

[0026] In one embodiment, the weight-to-volume ratio of the Qi-invigorating and blood-maintaining preparation to the extraction solvent is (2-20) g: (15-100) mL.

[0027] In one embodiment, the method for constructing the characteristic spectrum of the Qi-invigorating and blood-reinforcing preparation further comprises the following steps:

[0028] Dissolving the reference substance using a dissolving reagent to prepare a reference substance solution; the reference substance solution includes a mixed reference substance reference solution and a mixed reference medicinal material reference solution;

[0029] Performing high performance liquid chromatography on the reference substance solution to obtain a chromatogram of the corresponding reference substance;

[0030] performing common peak identification on the chromatographic peaks in the characteristic spectrum of the Yiqiweixue preparation according to the chromatogram of the reference substance;

[0031] Wherein, the reference substances include one or more of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, calycosin, astragalus and jujube.

[0032] In one embodiment, the dissolving agent is a methanol aqueous solution or water; optionally, the volume percentage of methanol in the methanol aqueous solution is 5%-50%.

[0033] In one embodiment, the characteristic peaks of the characteristic spectrum of the Qi-invigorating and blood-retaining preparation include 7 characteristic peaks, of which 5 identified characteristic peaks include: characteristic peak No. 3 is guanosine, characteristic peak No. 4 is 5-hydroxymethylfurfural, characteristic peak No. 5 is calycosin glucoside, characteristic peak No. 6 is formononetin, and characteristic peak No. 7 is calycosin.

[0034] Another aspect of the present invention provides the use of the characteristic spectrum of the Qi-invigorating and blood-reinvigorating preparation constructed by the method for constructing the characteristic spectrum of the Qi-invigorating and blood-reinvigorating preparation as described above in the quality control of the Qi-invigorating and blood-reinvigorating preparation.

[0035] Compared with the prior art, this application has the following beneficial effects:

[0036] This application constructs a characteristic spectrum of the Qi-invigorating and blood-retaining preparation containing multiple characteristic peaks by adopting specific high-performance liquid chromatography detection and analysis conditions for the Qi-invigorating and blood-retaining preparation. The method for constructing the characteristic spectrum of the Qi-invigorating and blood-retaining preparation has the advantages of good stability, high precision, and good repeatability after methodological verification. It can comprehensively reflect the effective ingredients contained in the Qi-invigorating and blood-retaining preparation, and then objectively, comprehensively and accurately evaluate the quality of the Qi-invigorating and blood-retaining preparation, which is of great significance for controlling the quality of the Qi-invigorating and blood-retaining preparation and ensuring clinical efficacy.

[0037] Furthermore, this method has the advantages of high separation efficiency, good reproducibility, and high sensitivity. It can detect the complex multi-source material system of Qi-invigorating and blood-replenishing preparations, comprehensively obtain the characteristic information of the material basis of Qi-invigorating and blood-replenishing preparations, and evaluate the uniformity and stability of the quality of Qi-invigorating and blood-replenishing preparations, which is of great significance to improving the overall quality control level of Qi-invigorating and blood-replenishing preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] To more clearly illustrate the specific embodiments of this application or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without inventive effort. The drawings are only for further understanding of the exemplary embodiments and descriptions of the present invention, constitute a part of the present invention, and do not constitute an undue limitation of the present invention.

[0039] Figure 1 : is the reference solution spectrum, where S1 is the mixed reference solution 1, S2 is the mixed reference solution 2, S3 is the jujube reference medicinal material solution, S4 is the astragalus reference medicinal material solution, and S5 is the mixed reference medicinal material reference solution.

[0040] Figure 2 It is a peak identification spectrum, where S1 is the mixed reference substance solution 1, S2 is the mixed reference substance solution 2, S3 is the jujube reference medicinal material solution, S4 is the astragalus reference medicinal material solution, S5 is the Yiqi Weixue tablet test solution, S6 is the Yiqi Weixue granule (no sucrose added) test solution, S7 is the Yiqi Weixue granule test solution, and S8 is the Yiqi Weixue capsule test solution.

[0041] Figure 3 The chromatograms of 11 batches of Yiqiweixue tablets in one embodiment are shown.

[0042] Figure 4 This is a comparative characteristic spectrum of Yiqiweixue tablets in one embodiment.

[0043] Figure 5 The chromatograms are of 15 batches of Yiqiweixue Capsules in one embodiment.

[0044] Figure 6 This is a comparative characteristic spectrum of Yiqiweixue Capsule in one embodiment.

[0045] Figure 7 This is a chromatogram of 13 batches of Yiqiweixue granules (no sucrose added) in one embodiment.

[0046] Figure 8 This is a comparative characteristic spectrum of the Yiqi Weixue Granules (no sucrose added) in one embodiment.

[0047] Figure 9 The chromatograms of 15 batches of Yiqiweixue granules in one embodiment are shown.

[0048] Figure 10 This is a comparative characteristic spectrum of Yiqi Weixue Granules in one embodiment. DETAILED DESCRIPTION

[0049] The following is a further detailed description of the detection method of the characteristic spectrum of the Qi-Yinjiang and Blood-Weiwei preparations of the present application, as well as their characteristic spectrum and applications, with reference to specific examples. This application can 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.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0051] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0052] As used herein, "one or more" refers to any one, any two, or any two or more of the listed items.

[0053] Herein, the optional scope of "and / or", "or / and", and "and / or" includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, and the said any and all combinations include any two relevant listed items, any more relevant listed items, or a combination of all relevant listed items.

[0054] Herein, “further”, “further”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present invention.

[0055] In the present invention, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Furthermore, 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 specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0056] Unless otherwise specified, the percentage contents mentioned in the present invention refer to mass percentage for solid-liquid mixture and solid-solid mixture, and refer to volume percentage for liquid-liquid mixture.

[0057] The percentage concentrations mentioned in the present invention, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0058] The temperature parameters in the present invention, unless otherwise specified, allow both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range of instrument control. Fluctuations within the range of ±0.8°C, ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, and ±0.1°C are allowed. Normal temperature or room temperature in the present invention refers to no temperature control operation, generally refers to 4°C to 35°C, preferably refers to 20±5°C.

[0059] In recent years, with the rapid development of chromatography and mass spectrometry, TCM characteristic chromatographic techniques have been widely applied and improved. Spectroscopy (including ultraviolet, visible, and infrared spectroscopy), spectroscopy (including mass spectrometry and nuclear magnetic resonance spectroscopy), chromatography (including thin-layer chromatography, high-performance liquid chromatography, gas chromatography, etc.), and various instrument-coupled techniques can all be used to study TCM characteristic chromatographic techniques. These techniques enable comprehensive analysis of Chinese medicinal materials, decoction pieces, and Chinese patent medicines, providing a reliable basis for quality control.

[0060] Among the existing detection methods for Qi-invigorating and blood-retaining preparations, thin-layer chromatography has low separation efficiency and sensitivity, making it difficult to detect trace components; while the content determination method of high-performance liquid chromatography has a single indicator, only determining astragaloside IV, which cannot cover the entire substance group of Qi-invigorating and blood-retaining preparations.

[0061] Based on this, it is necessary to provide a method for constructing a characteristic spectrum of Qi-invigorating and blood-retaining preparations. During the research process, the technical personnel of this application found through chromatographic conditions and methodological investigations that by adopting specific HPLC conditions to detect the characteristic spectrum of Qi-invigorating and blood-retaining preparations, a characteristic spectrum of Qi-invigorating and blood-retaining preparations containing multiple characteristic peaks can be constructed. This method is simple, reliable, objective, and has good precision, repeatability and stability.

[0062] One aspect of the present invention provides a method for constructing a characteristic spectrum of a Qi-invigorating and blood-reinforcing preparation, comprising the following steps:

[0063] Extract the Yiqiweixue preparation using an extraction solvent to prepare a test solution;

[0064] Performing high performance liquid chromatography on the test solution to obtain a chromatogram of the corresponding test sample;

[0065] The conditions for the HPLC detection include: mobile phase A is methanol, mobile phase B is phosphoric acid aqueous solution, and gradient elution is used;

[0066] The gradient elution procedure includes:

[0067] From 0 to 8 minutes, the volume percentage of the mobile phase A is maintained at 4%;

[0068] From 8 to 20 minutes, the volume percentage of the mobile phase A increases from 4% to 15%;

[0069] From 20 min to 55 min, the volume percentage of the mobile phase A increases from 15% to 80%;

[0070] 55min~65min, the volume percentage of the mobile phase A is maintained at 80%;

[0071] The chromatograms of the test samples were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system for processing, and the main chromatographic peaks existing in the chromatograms of different batches of test samples were selected as characteristic peaks to construct the characteristic spectrum of the Qi-invigorating and blood-maintaining preparation.

[0072] Specifically, the formulation of reference maps and judgment standards for Qi-invigorating and blood-maintaining preparations usually involves testing no less than 10 batches, and generally 10 to 30 batches.

[0073] In some examples, the Qi-invigorating and blood-replenishing preparations include one or more of Qi-invigorating and blood-replenishing capsules, Qi-invigorating and blood-replenishing tablets, Qi-invigorating and blood-replenishing granules, and Qi-invigorating and blood-replenishing granules (without added sucrose).

[0074] In some examples, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.02%-0.1%. It is understood that the volume percentage of phosphoric acid in the phosphoric acid aqueous solution includes but is not limited to 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% and 0.1%.

[0075] In some examples, the conditions for the HPLC detection include: an injection volume of 5 μL-50 μL. It is understood that the injection volume for the HPLC detection includes but is not limited to 5 μL, 10 μL, 20 μL, 30 μL, 40 μL and 50 μL.

[0076] In some examples, the HPLC detection conditions include: a detection wavelength of 190 nm to 400 nm. It is understood that the detection wavelengths of the HPLC detection include but are not limited to 250 nm, 255 nm, 260 nm, 265 nm, 270 nm, and 275 nm.

[0077] In some examples, the HPLC detection conditions include: a flow rate of 0.8 mL / min-1.2 mL / min. It is understood that the HPLC detection flow rate includes but is not limited to 0.8 mL / min, 0.85 mL / min, 0.9 mL / min, 0.94 mL / min, 0.98 mL / min, 1.02 mL / min, 1.06 mL / min, 1.1 mL / min, and 1.2 mL / min.

[0078] In some examples, the HPLC detection conditions include: a column temperature of 25° C.-35° C. It is understood that the column temperature of the HPLC detection includes but is not limited to 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., and 35° C.

[0079] In some of these examples, the conditions for the HPLC detection include: the chromatographic column is filled with octadecylsilane bonded silica gel.

[0080] In some examples, the conditions for the step of extracting the Qi-invigorating and blood-weixing preparation using an extraction solvent include: the extraction method is one or more of ultrasonic extraction, reflux extraction, maceration extraction, decoction extraction or oscillation extraction.

[0081] In some examples, the conditions for extracting the Qi-Yiweixue preparation using an extraction solvent include: an extraction time of 20 minutes to 60 minutes. It is understood that the extraction time includes but is not limited to 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, and 60 minutes.

[0082] In some examples, the conditions for extracting the Qi-Yiweixue preparation using an extraction solvent include: the extraction solvent includes a methanol-water solution or methanol; optionally, the volume percentage of methanol in the methanol-water solution is 20%-95%. Further, the volume percentage of methanol in the methanol-water solution is 70%-95%.

[0083] In some examples, the weight-to-volume ratio of the Qi-invigorating and blood-maintaining preparation to the extraction solvent is (2-20) g: (15-100) mL. It is understood that the weight-to-volume ratio of the Qi-invigorating and blood-maintaining preparation to the extraction solvent includes but is not limited to 3 g: 25 mL, 5 g: 25 mL, 3 g: 35 mL, 5 g: 35 mL, 3 g: 50 mL, and 5 g: 50 mL. Furthermore, the weight-to-volume ratio of the Qi-invigorating and blood-maintaining preparation to the extraction solvent is (3-15) g: (25-50) mL.

[0084] In some examples, the method for constructing the characteristic spectrum of the Qi-invigorating and blood-reinforcing preparation further includes the following steps:

[0085] Dissolving the reference substance using a dissolving reagent to prepare a reference substance solution; the reference substance solution includes a mixed reference substance reference solution and a mixed reference medicinal material reference solution;

[0086] Performing high performance liquid chromatography on the reference substance solution to obtain a chromatogram of the corresponding reference substance;

[0087] performing common peak identification on the chromatographic peaks in the characteristic spectrum of the Yiqiweixue preparation according to the chromatogram of the reference substance;

[0088] Wherein, the reference substances include one or more of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, calycosin, astragalus and jujube.

[0089] In some examples, the concentrations of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin and calycosin in the mixed control reference solution are 10 μg / mL-30 μg / mL.

[0090] In some examples, the preparation steps of the mixed control medicinal material reference solution include: taking 1g of astragalus medicinal material and 1g of jujube medicinal material, adding 10% methanol aqueous solution to dilute the solution 50 times, reflux extraction, centrifugal speed at 4000r / min, and taking the supernatant after centrifugation for 10 minutes; the amount of the supernatant taken is 25mL.

[0091] In some examples, the conditions for preparing the test solution include: ultrasonic conditions of 250W and 40kHz, and the filtrate obtained after evaporation is 10mL-30mL.

[0092] In some examples, the dissolving agent is a methanol-water solution or water; optionally, the volume percentage of methanol in the methanol-water solution is 5%-50%.

[0093] In some of the examples, the characteristic peaks of the characteristic spectrum of the Qi-invigorating and blood-retaining preparation include 7 characteristic peaks, of which 5 identified characteristic peaks include: characteristic peak No. 3 is guanosine, characteristic peak No. 4 is 5-hydroxymethylfurfural, characteristic peak No. 5 is calycosin glucoside, characteristic peak No. 6 is formononetin, and characteristic peak No. 7 is calycosin.

[0094] In some of the examples, the characteristic spectrum of the Qi-invigorating and blood-retaining preparation stipulates that the relative retention time of characteristic peak No. 1 is 0.18, and the relative retention time of characteristic peak No. 2 is 0.28. The values ​​are determined by comprehensive analysis of the sampling results of multiple batches of different dosage forms and different instruments. It is required that in the subsequent detection process using this method, the relative retention time of characteristic peak No. 1 and characteristic peak No. 2 shall not exceed ±10%, and the RSD% of multiple injection results shall not exceed 3%.

[0095] In one specific example, the method for constructing the characteristic spectrum of the Qi-invigorating and blood-reinforcing preparation includes the following steps:

[0096] (1) Preparation of reference solution:

[0097] Mixed reference substance solution 1: Weigh appropriate amount of guanosine and 5-hydroxymethylfurfural reference substances, dissolve them in 10% methanol, dilute to the mark, shake well, filter, and obtain the filtrate.

[0098] Mixed reference substance solution 2: Weigh appropriate amounts of calycosin glucoside, formononetin, and calycosin reference substance, dissolve them in 50% methanol, dilute to the mark, shake well, filter, and obtain the filtrate.

[0099] Mixed control medicinal material reference solution: weigh appropriate amount of astragalus control medicinal material and jujube control medicinal material, add 10% methanol for reflux extraction, centrifuge, take appropriate amount of supernatant and evaporate to dryness, add 10% methanol to dissolve the residue, transfer to a volumetric flask, add 10% methanol to dilute to the scale line, shake well, filter, and take the filtrate to obtain.

[0100] (2) Preparation of test solution:

[0101] Yiqiweixue tablets: Take an appropriate amount of sample, grind it into powder, take an appropriate amount of fine powder, accurately weigh it, put it into a conical flask, accurately add an appropriate amount of methanol, weigh it, ultrasonicate it, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, accurately measure an appropriate amount of filtrate, evaporate it to dryness, dissolve the residue in water, transfer it to a volumetric flask, dilute it to the scale line with water, shake it well, filter it, and take the filtrate.

[0102] Yiqiweixue Capsules: Take an appropriate amount of sample, grind it into powder, take an appropriate amount of fine powder, accurately weigh it, put it into a conical flask, accurately add an appropriate amount of methanol, weigh it, ultrasonicate it, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, accurately measure an appropriate amount of filtrate, evaporate it to dryness, dissolve the residue in water, transfer it to a volumetric flask, dilute it to the scale line with water, shake it well, filter it, and take the filtrate.

[0103] Yiqiweixue Granules: Take an appropriate amount of sample, grind it into powder, take an appropriate amount of fine powder, weigh it accurately, put it into a conical flask, add an appropriate amount of 90% methanol accurately, weigh it, ultrasonicate it, let it cool, weigh it again, make up the lost weight with 90% methanol, shake it well, filter it, accurately measure an appropriate amount of filtrate, evaporate it to dryness, dissolve the residue in water, transfer it to a volumetric flask, dilute it to the scale line with water, shake it well, filter it, and take the filtrate.

[0104] Yiqiweixue Granules (no sucrose added): Take an appropriate amount of sample, grind it into powder, take an appropriate amount of fine powder, accurately weigh it, place it in a conical flask, accurately add an appropriate amount of 90% methanol, weigh the weight, ultrasonicate it, let it cool, weigh it again, make up the lost weight with 90% methanol, shake it well, filter it, accurately measure an appropriate amount of filtrate, evaporate it to dryness, dissolve the residue in water, transfer it to a volumetric flask, dilute it to the scale line with water, shake it well, filter it, and take the filtrate.

[0105] (3) Chromatographic conditions

[0106] The results were detected by high performance liquid chromatography, with octadecylsilane bonded silica gel as the filler, methanol as the mobile phase A, and phosphoric acid aqueous solution as the mobile phase B, using gradient elution;

[0107] The gradient elution procedure includes:

[0108] From 0 to 8 minutes, the volume percentage of the mobile phase A is maintained at 4%;

[0109] From 8 to 20 minutes, the volume percentage of the mobile phase A increases from 4% to 15%;

[0110] From 20 min to 55 min, the volume percentage of the mobile phase A increases from 15% to 80%;

[0111] From 55 min to 65 min, the volume percentage of the mobile phase A was maintained at 80%.

[0112] (4) Characteristic spectrum determination

[0113] The reference solution of step (1) and the test solution of step (2) were respectively injected into the high performance liquid chromatograph, and the chromatographic conditions of step (3) were used for detection to obtain the corresponding chromatograms of the reference and test samples, and the relative retention time of each characteristic peak was calculated.

[0114] (5) Establishment of control characteristic map

[0115] The chromatograms of the test samples were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system for processing, and the main chromatographic peaks existing in the chromatograms of different batches of test samples were selected as characteristic peaks to construct a control characteristic spectrum of the Qi-invigorating and blood-maintaining preparation.

[0116] Another aspect of the present invention provides the use of the characteristic spectrum of the Qi-invigorating and blood-reinvigorating preparation constructed by the method for constructing the characteristic spectrum of the Qi-invigorating and blood-reinvigorating preparation as described above in the quality control of the Qi-invigorating and blood-reinvigorating preparation.

[0117] The present invention will be further described in detail below with reference to specific examples. For experimental parameters not specified in the following specific examples, reference should be made to the guidance provided in this application document. Reference may also be made to experimental manuals in the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturer. It will be understood that the instruments and raw materials used in the following examples are relatively specific and are not limited thereto in other specific examples.

[0118] Some of the instruments and materials used in the examples are as follows:

[0119] Instruments: LC-2050 high-performance liquid chromatograph (Shimadzu Malaysia Factory); Agilent 1260II high-performance liquid chromatograph (Agilent China Technology Co., Ltd.); direct-active atomic force detector (DAD); SQP electronic analytical balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); JA5003 electronic analytical balance (Shanghai Sunny Optical Hengping Scientific Instruments Co., Ltd.); KQ5200DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0120] Reference substances: guanosine (batch number 111977-202202), 5-hydroxymethylfurfural (batch number 111626-202316), calycosin glucoside (batch number: 111920-201907), calycosin isoflavone (batch number: 112117-202301), astragalus reference medicinal material (batch number: 120974-201813), and jujube reference medicinal material (121093-201809) were all purchased from the China Food and Drug Inspection Institutes; formononetoin (batch number 15943) and calycosin isoflavone (batch number: 15747) were purchased from Shanghai Shidande Standard Technology Service Co., Ltd.

[0121] Reagents: methanol (chromatographic grade); acetonitrile (chromatographic grade); methanol (analytical grade); phosphoric acid (analytical grade); ultrapure water.

[0122] Test samples: Yiqiweixue tablets (batch numbers: 20190718, 20190816, 20190916, 20210309, 20220401S, 20220601, 20220701, 20220702, 20221201S, 20221202S, 20221203S; specification: 0.57 g / tablet; source: Guangdong Hongshanhu Pharmaceutical Co., Ltd.); Yiqiweixue capsules (batch number: 2022101 2. 20221104, 20221113, 20231005, 20230203, 20230521, 20231010, 20231001, 20231104, 20231106, 20240304, 20240306, 20240514, 20240515; Specification: 0.45g / pill; Source: Guangdong Red Coral Pharmaceutical Co., Ltd.; Yiqi Weixue Granules (Sugar-Free) (Batch No.: 20220801, 20220802, 20220803, 20230401, 20230402, 20230403, 20230404, 20231201S, 20231202S, 20240101S, 20240102S, 20240301S, 20240302S; Specification: 5g / bag; Source: Guangdong Hongshanhu Pharmaceutical Co., Ltd.); Yiqiweixue Granules (Batch No. : 20220801, 20220905, 20221001, 20221106, 20221202, 20230808, 20231010, 20231109, 10231209, 20231214, 20240110, 20240207, 20240412, 20240502, 20240503; Specification: 10g / bag; Source: Guangdong Red Coral Pharmaceutical Co., Ltd.).

[0123] Investigation Example 1

[0124] In the research scheme for the characteristic spectrum of the Qi-Yinweixue preparation of this application, the inventors of this application, through extensive experimental exploration, have optimized the reference solution preparation steps, the test sample preparation steps, the chromatographic conditions steps, the characteristic spectrum determination steps, and the control characteristic spectrum establishment steps, and have obtained a stable, reliable, and easy-to-operate detection method. The preferred screening schemes of the research technology of this application are provided below:

[0125] 1. Preparation of reference solution

[0126] In the reference solution preparation step of the present application, the final solvents of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, and calycosin reference substances were investigated, and 10%, 20%, 50%, 80%, and 100% methanol aqueous solutions were investigated, respectively. The results showed that guanosine and 5-hydroxymethylfurfural reference substances were stable in 10% methanol and had no solvent effect; calycosin glucoside, formononetin, and calycosin were stable in 50% methanol solution and had no solvent effect.

[0127] In the preparation steps of the mixed control medicinal material reference solution, water, 10% methanol, and 40% methanol extraction solvents were investigated, heating reflux and ultrasonic extraction methods were investigated, and extraction time was investigated. The optimal preparation method was 10% methanol reflux extraction for 2 hours, centrifugation, and 25 mL of the supernatant was taken, evaporated to dryness, and the residue was dissolved in 10% methanol, transferred to a 2 mL volumetric flask, fixed to volume, filtered, and the filtrate was obtained.

[0128] 2. Preparation of test solution

[0129] In the preparation step of the test solution, the present application investigated the mass volume ratio of the sample to the extraction solvent, the range of which includes but is not limited to (3g~20g): (20mL~60mL), the extraction solvent, the range of which includes but is not limited to methanol, ethanol and methanol aqueous solution and ethanol aqueous solution in different volume ratios, the extraction method (reflux, ultrasound), and the extraction time (20min~60min).

[0130] The results showed that the most preferred ratio of Yiqiweixue tablets was 5 g: 25 mL (methanol), which was treated with ultrasound (250 W, 40 kHz) for 60 min; the most preferred ratio of Yiqiweixue capsules was 3.5 g: 25 mL (methanol), which was treated with ultrasound (250 W, 40 kHz) for 60 min; the most preferred ratio of Yiqiweixue granules was 15 g: 50 mL (90% methanol), which was treated with ultrasound (250 W, 40 kHz) for 40 min; the most preferred ratio of Yiqiweixue granules (no sucrose added) was 5 g: 50 mL (90% methanol), which was treated with ultrasound (250 W, 40 kHz) for 40 min.

[0131] 3. Screening of HPLC Conditions

[0132] 3.1 Elution gradient screening and detection wavelength selection

[0133] By consulting the literature to determine the properties of the ingredients contained in the prescription medicine, methanol-0.05% phosphoric acid was preliminarily selected as the mobile phase. This application uses a diode array detector for detection, and when conducting the mobile phase elution gradient investigation, the full wavelength (190nm-400nm scanning) is turned on; after adjustment and screening, the tentative chromatographic conditions are obtained, and the detection wavelength is 272nm. At this wavelength, the chromatographic peak response value is good and the baseline is flat; the gradient elution program is 0-8min, the methanol volume percentage is maintained at 4%; 8min-20min, the methanol volume percentage is 4%→15%; 20min-55min, the methanol volume percentage is 15%→80%; 55min-65min, the methanol volume percentage is maintained at 80%.

[0134] 3.2 Investigation of mobile phase components

[0135] The present invention compared the elution effects of several different elution systems, including acetonitrile-0.1% formic acid, acetonitrile-0.02% formic acid, acetonitrile-water, methanol-water, methanol-0.1% phosphoric acid, methanol-0.05% phosphoric acid, and methanol-0.02% phosphoric acid. The results showed that methanol and 0.05% phosphoric acid solution were used as the mobile phase, achieving excellent separation of the components in the Yiqiweixue preparation. Therefore, methanol and 0.05% phosphoric acid solution were ultimately selected as the mobile phase.

[0136] 3.3 Flow rate, column temperature, chromatographic column, injection volume investigation and screening

[0137] This application compared flow rates of 0.9 mL / min, 1.0 mL / min, and 1.1 mL / min, with the results showing that 1.0 mL / min was optimal. The column temperature was investigated at 28°C to 32°C, with 30°C being optimal. Chromatographic columns filled with octadecylsilane-based bonded silica from different manufacturers and models were investigated, with ZORBAX SB-C18 ultimately being the optimal choice. Injection volumes, including but not limited to 20 μL to 60 μL, were investigated, with 30 μL being the optimal choice.

[0138] 3.4 Characteristic Peak Selection and Identification Results in This Application

[0139] In the process of establishing the characteristic spectrum of the Yiqiweixue preparation, 7 characteristic peaks were selected by consulting relevant literature and regulatory requirements, and Peak 5 was the reference peak S. Figure 1 At the same time, the seven characteristic peaks were attributed and identified. Among them, the reference substance identified five characteristic peaks, corresponding to five different components. Characteristic peak No. 3 was guanosine, characteristic peak No. 4 was 5-hydroxymethylfurfural, characteristic peak No. 5 (reference peak S) was calycosin glucoside, characteristic peak No. 6 was formononetin, characteristic peak No. 7 was calycosin isoflavone, characteristic peak No. 1 that was not identified by the reference substance was a common peak of jujube and astragalus, characteristic peak No. 2 was a unique peak of jujube. For details of the identification chromatogram, see Figure 2The identification results are shown in Table 1:

[0140] Table 1 Characteristic peak identification results

[0141]

[0142] Example 1

[0143] This embodiment provides the detection of the characteristic spectrum of Yiqiweixue tablets, which is as follows:

[0144] Step 1: Preparation of reference solution:

[0145] Mixed control medicinal material reference solution: Take 1g of Astragalus membranaceus control medicinal material and 1g of jujube control medicinal material respectively, place them in a stoppered conical flask, accurately add 50mL of 10% methanol, weigh the weight, heat and reflux for 2h, cool, weigh again, make up the lost weight with 10% methanol, shake well, centrifuge (4000r / min) for 10min, accurately measure 25mL of supernatant, evaporate to dryness, dissolve the residue in 10% methanol, transfer to a 2mL volumetric flask, add 10% methanol to dilute to the scale, shake well, filter, and take the filtrate as the mixed control medicinal material reference solution.

[0146] Mixed reference substance solution 1: Accurately weigh appropriate amounts of guanosine and 5-hydroxymethylfurfural standards, add 10% methanol to make a solution containing approximately 20 μg of guanosine and 5-hydroxymethylfurfural per 1 mL, filter, and use the filtrate as reference substance solution 1.

[0147] Mixed reference substance solution 2: Accurately weigh appropriate amounts of calycosin glucoside, formononetin, and calycosin standard products, add 50% methanol to prepare a solution containing approximately 20 μg of calycosin glucoside, formononetin, and calycosin per 1 mL. Filter and take the filtrate as reference substance solution 2.

[0148] Step 2. Preparation of test solution: Take an appropriate amount of sample, grind it into powder, take about 5g of fine powder, accurately weigh it, put it into a conical flask, accurately add 25mL of methanol, weigh it, ultrasonically treat it (power 200w, frequency 40KHz) for 60 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, accurately measure 15mL of the filtrate, evaporate it to dryness, dissolve the residue in water, transfer it to a 2mL volumetric flask, dilute it to the scale with water, shake it well, filter it, and take the filtrate.

[0149] Step 3, liquid chromatography conditions: A ZORBAX SB-C18 (250×4.6 mm, 5 μm) column was used, with methanol as mobile phase A and 0.05% phosphoric acid solution as mobile phase B, gradient elution was performed, the detection wavelength was 272 nm, the column temperature was 30°C, and the flow rate was 1 mL / min. The gradient elution program was as follows: from 0 to 8 minutes, mobile phase A was maintained at 4%, and mobile phase B was maintained at 96%; from 8 minutes to 20 minutes, mobile phase A increased from 4% to 15%, and mobile phase B decreased from 96% to 85%; from 20 minutes to 55 minutes, mobile phase A increased from 15% to 80%, and mobile phase B decreased from 85% to 20%; from 55 minutes to 65 minutes, mobile phase A was maintained at 80%, and mobile phase B was maintained at 20%.

[0150] Step 4, determination: accurately aspirate the reference solution and take 30 μL of each of the 11 batches of test solution prepared according to the preparation method of the test solution in step 2, inject them into the high performance liquid chromatography instrument for detection, and record the chromatogram, such as Figure 3 The chromatograms of the test solutions of 11 batches of Yiqiweixue tablets were respectively introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system; the 7 main chromatographic peaks present in the chromatograms of each batch of Yiqiweixue tablets were selected as characteristic peaks; the control characteristic spectrum of Yiqiweixue tablets was generated by the average value calculation method, see Figure 4 In the characteristic spectra of 11 batches of Yiqiweixue tablets, characteristic peak No. 3 is guanosine with an average retention time of 11.903 min, characteristic peak No. 4 is 5-hydroxymethylfurfural with an average retention time of 15.356 min, characteristic peak No. 5 is calycosin glucoside with an average retention time of 36.932 min, characteristic peak No. 6 is formononetin with an average retention time of 42.637 min, and characteristic peak No. 7 is calycosin with an average retention time of 45.807 min. The peak corresponding to the calycosin glucoside reference peak is peak S (characteristic peak No. 5), and the relative retention times of the unidentified characteristic peaks No. 1 and No. 2 are calculated. The average relative retention times of the 11 batches are: 0.181 (characteristic peak No. 1) and 0.282 (characteristic peak No. 2).

[0151] The methodological research on the characteristic spectrum detection method of Yiqiweixue tablets is as follows:

[0152] (1) Precision study

[0153] Prepare a test solution of Yiqiweixue Tablets (Batch No. 20221201S) using the test solution method described in Step 2. Analyze the solution using the detection method described in Step 3, with six replicate injections. The test solution's characteristic chromatogram should show seven characteristic peaks corresponding to the retention times of the seven characteristic peaks in the reference herbal material chromatogram. Characteristic peaks 3, 4, 5, 6, and 7 should correspond to the retention times of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, and the calycosin reference material. The peak corresponding to the calycosin glucoside reference material peak is designated as peak S (characteristic peak 5). Calculate the relative retention times of the unidentified characteristic peaks 1 and 2 relative to peak S. The relative retention times should be within ±10% and the RSD values ​​within ±3%. The specified values ​​are: 0.18 (characteristic peak 1) and 0.28 (characteristic peak 2). The retention time / relative retention time of each characteristic peak is summarized in Table 2 below. The results show that the parallel injection precision of this method is good.

[0154] Table 2 Precision study results of Yiqiweixue tablets

[0155]

[0156] (2) Reproducible studies

[0157] Six samples of Yiqiweixue Tablets (Batch No. 20221201S) were precisely weighed in parallel. Six test solutions were prepared according to the method in Step 2. Analyze according to the detection method in Step 3. The test sample's characteristic spectrum should show seven characteristic peaks, corresponding to the retention times of the seven characteristic peaks in the chromatogram of the reference medicinal material. Characteristic Peaks 3, 4, 5, 6, and 7 should correspond to the retention times of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, and the chromatographic peaks of the calycosin glucoside reference material. The peak corresponding to the calycosin glucoside reference material peak was designated as Peak S (Peak S5). The relative retention times of the unidentified Peaks 1 and 2 relative to Peak S were calculated, and the relative retention times were within ±10% and the RSD values ​​within ±3%. The specified values ​​were: 0.18 (Peak S1) and 0.28 (Peak S2). The retention time / relative retention time of each characteristic peak is summarized in Table 3 below. The results show that the method has good repeatability.

[0158] Table 3 Results of the reproducibility study of Yiqiweixue tablets

[0159]

[0160] (3) Stability study

[0161] Yiqiweixue tablets (batch number: 20221201S) were prepared as a test solution according to the test solution method in step 2. The test solution was analyzed according to the detection method in step 3. Samples were injected at different times of 0, 5, 10, 15, 20, and 25 hours. Seven characteristic peaks appeared in the characteristic spectrum of the test sample and corresponded to the retention times of the seven characteristic peaks in the chromatogram of the reference medicinal material. Among them, characteristic peaks 3, 4, 5, 6, and 7 should correspond to the retention times of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, and calycosin reference substance chromatographic peaks. The peak corresponding to the calycosin glucoside reference peak was designated as peak S (characteristic peak 5). The relative retention times of the unidentified characteristic peaks 1 and 2 and peak S were calculated. The relative retention times were within ±10% and the RSD values ​​were within ±3%. The specified values ​​are: 0.18 (characteristic peak No. 1) and 0.28 (characteristic peak No. 2). The retention time / relative retention time of each characteristic peak is summarized in Table 4 below. The results show that the chromatographic peaks of the Yiqiweixue tablets test solution hardly change within 25 hours, indicating that the method has good stability.

[0162] Table 4 Stability study results of Yiqiweixue tablets

[0163]

[0164] Example 2

[0165] This embodiment provides the detection of the characteristic spectrum of Yiqiweixue Capsule, which is as follows:

[0166] Step 1: Preparation of reference solution: same as in Example 1.

[0167] Step 2. Preparation of test solution: Take an appropriate amount of sample, grind it into powder, take about 3.5g of fine powder, accurately weigh it, put it into a conical flask, accurately add 25mL of methanol, weigh it, ultrasonically treat it (power 200w, frequency 40KHz) for 60 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, accurately measure 10mL of the filtrate, evaporate it to dryness, dissolve the residue in water, transfer it to a 2mL volumetric flask, dilute it to the scale with water, shake it well, filter it, and take the filtrate.

[0168] Step 3, liquid chromatography conditions: same as in Example 1.

[0169] Step 4, determination: accurately aspirate the reference solution and take 30 μL of each of the 15 batches of Yiqiweixue Capsules prepared according to the preparation method of the test solution in step 2, inject them into the high performance liquid chromatography instrument for detection, and record the chromatogram. Figure 5The chromatograms of 15 batches of Yiqiweixue Capsule test solution were respectively imported into the Chinese medicine chromatographic fingerprint similarity evaluation system, and the 7 main chromatographic peaks present in the chromatograms of each batch of Yiqiweixue Capsule were selected as characteristic peaks; the control characteristic spectrum of Yiqiweixue Capsule was generated by the average value calculation method, see Figure 6 In the characteristic spectra of 15 batches of Yiqiweixue Capsules, characteristic peak No. 3 is guanosine with an average retention time of 11.917 min, characteristic peak No. 4 is 5-hydroxymethylfurfural with an average retention time of 15.384 min, characteristic peak No. 5 is calycosin glucoside with an average retention time of 36.936 min, characteristic peak No. 6 is formononetin with an average retention time of 42.627 min, and characteristic peak No. 7 is calycosin with an average retention time of 45.802 min. The peak corresponding to the calycosin glucoside reference peak is the S peak (characteristic peak No. 5), and the relative retention times of the unidentified characteristic peaks No. 1 and No. 2 are calculated. The average relative retention times of the 15 batches are: 0.181 (characteristic peak No. 1) and 0.282 (characteristic peak No. 2).

[0170] The methodological research on the characteristic spectrum detection method of Yiqiweixue Capsules is as follows:

[0171] (1) Precision study

[0172] Prepare a test solution using Yiqiweixue Capsules (Batch No. 20221012) according to the test solution method in Step 2. Analyze using the detection method in Step 3, with six replicate injections. The test sample's characteristic spectrum should exhibit seven characteristic peaks corresponding to the retention times of the seven characteristic peaks in the chromatogram of the control medicinal material reference. Characteristic peaks 3, 4, 5, 6, and 7 should correspond to the retention times of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, and the calycosin glucoside reference peak. The peak corresponding to the calycosin glucoside reference peak is designated as peak S (characteristic peak 5). Calculate the relative retention times of the unidentified characteristic peaks 1 and 2 relative to peak S. The relative retention times should be within ±10% and the RSD values ​​within ±3%. The specified values ​​are: 0.18 (characteristic peak 1) and 0.28 (characteristic peak 2). The retention time / relative retention time of each characteristic peak is summarized in Table 5 below. The results show that the parallel injection precision of this method is good.

[0173] Table 5 Precision study results of Yiqiweixue Capsules

[0174]

[0175] (2) Reproducible studies

[0176] Precision weigh six portions of Yiqiweixue Capsules (Batch No. 20221012) in parallel. Prepare six test sample solutions according to the method in Step 2. Analyze according to the detection method in Step 3. The test sample's characteristic spectrum should exhibit seven characteristic peaks corresponding to the retention times of the seven characteristic peaks in the chromatogram of the reference medicinal material. Characteristic peaks 3, 4, 5, 6, and 7 should correspond to the retention times of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, and the chromatographic peaks of the calycosin glucoside reference. The peak corresponding to the calycosin glucoside reference peak is designated as peak S (characteristic peak 5). Calculate the relative retention times of the unidentified characteristic peaks 1 and 2 relative to peak S. The relative retention times should be within ±10% and the RSD values ​​within ±3%. The specified values ​​are: 0.18 (characteristic peak 1) and 0.28 (characteristic peak 2). The retention time / relative retention time of each characteristic peak is summarized in Table 6 below. The results show that the method has good repeatability.

[0177] Table 6 Results of the reproducibility study of Yiqiweixue Capsules

[0178]

[0179] (3) Stability study

[0180] Yiqiweixue Capsules (batch number: 20221012) were prepared as a test solution according to the test solution method in step 2. The test solution was analyzed according to the detection method in step 3. Samples were injected at different times of 0, 5, 10, 15, 20, and 25 hours. Seven characteristic peaks appeared in the characteristic spectrum of the test sample, corresponding to the retention times of the seven characteristic peaks in the chromatogram of the reference medicinal material. Among them, characteristic peaks 3, 4, 5, 6, and 7 corresponded to the retention times of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, and calycosin reference substance chromatographic peaks. The peak corresponding to the calycosin glucoside reference peak was designated as peak S (characteristic peak 5). The relative retention times of the unidentified characteristic peaks 1 and 2 and peak S were calculated, and the relative retention times were within ±10% and the RSD values ​​were within ±3%. The specified values ​​are: 0.18 (characteristic peak No. 1) and 0.28 (characteristic peak No. 2). The retention time / relative retention time of each characteristic peak is summarized in Table 7 below. The results show that the chromatographic peaks of the Yiqiweixue Capsule test solution hardly change within 25 hours, indicating that the method has good stability.

[0181] Table 7 Stability study results of Yiqiweixue capsule

[0182]

[0183] Example 3

[0184] This example provides the detection of the characteristic spectrum of Yiqiweixue granules (no sucrose added), as follows:

[0185] Step 1: Preparation of reference solution: same as in Example 1.

[0186] Step 2. Preparation of test solution: Take an appropriate amount of sample, grind it into powder, take about 5g of fine powder, accurately weigh it, put it into a conical flask, accurately add 50mL of 90% methanol, weigh the weight, ultrasonically treat (power 200w, frequency 40KHz) for 40 minutes, let it cool, weigh it again, make up the lost weight with 90% methanol, shake it well, filter it, accurately measure 25mL of the filtrate, evaporate it to dryness, dissolve the residue in water, transfer it to a 2mL volumetric flask, dilute it to the scale with water, shake it well, filter it, and take the filtrate.

[0187] Step 3, liquid chromatography conditions: same as in Example 1.

[0188] Step 4: Accurately aspirate the reference solution and 30 μL of each of the 13 batches of Yiqiweixue granules (no sucrose added) prepared according to the preparation method of the test solution in step 2, inject them into the high performance liquid chromatography instrument for detection, and record the chromatogram. Figure 7 The chromatograms of the test solutions of 13 batches of Yiqiweixue granules were respectively introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system; the 7 main chromatographic peaks present in the chromatograms of each batch of Yiqiweixue granules (no sucrose added) were selected as characteristic peaks; the control characteristic spectrum of Yiqiweixue capsule was generated by the average value calculation method, see Figure 8 In the characteristic spectra of 13 batches of Yiqiweixue Granules (no sucrose added), characteristic peak No. 3 is guanosine with an average retention time of 11.874 min, characteristic peak No. 4 is 5-hydroxymethylfurfural with an average retention time of 15.333 min, characteristic peak No. 5 is calycosin glucoside with an average retention time of 36.946 min, characteristic peak No. 6 is formononetin with an average retention time of 42.654 min, and characteristic peak No. 7 is calycosin with an average retention time of 45.712 min. The peak corresponding to the calycosin glucoside reference peak is the S peak (characteristic peak No. 5), and the relative retention times of the unidentified characteristic peaks No. 1 and No. 2 are calculated. The average relative retention times of the 13 batches are: 0.181 (characteristic peak No. 1) and 0.281 (characteristic peak No. 2).

[0189] The methodological study of the characteristic spectrum detection method of Yiqiweixue Granules (no sucrose added) is as follows:

[0190] (1) Precision study

[0191] Prepare a test solution using Yiqiweixue Granules (no sucrose added) (Batch No. 20230403) according to the test solution method in Step 2. Analyze using the detection method in Step 3, with six replicate injections. The test solution's characteristic spectrum should exhibit seven characteristic peaks corresponding to the retention times of the seven characteristic peaks in the chromatogram of the control medicinal material reference. Characteristic peaks 3, 4, 5, 6, and 7 should correspond to the retention times of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, and the calycosin glucoside reference peak. The peak corresponding to the calycosin glucoside reference peak is designated as peak S (characteristic peak 5). Calculate the relative retention times of unidentified characteristic peaks 1 and 2 relative to peak S. The relative retention times should be within ±10% and the RSD values ​​within ±3%. The specified values ​​are: 0.18 (characteristic peak 1) and 0.28 (characteristic peak 2). The retention time / relative retention time of each characteristic peak is summarized in Table 8 below. The results show that the parallel injection precision of this method is good.

[0192] Table 8 Precision study results of Yiqiweixue granules (no sucrose added)

[0193]

[0194] (2) Reproducible studies

[0195] Precision weigh six portions of Yiqiweixue Granules (no sucrose added) (Batch No. 20230403) in parallel. Prepare six test sample solutions according to the method in Step 2. Analyze according to the detection method in Step 3. The test sample's characteristic spectrum should exhibit seven characteristic peaks, corresponding in retention time to the seven characteristic peaks in the chromatogram of the reference medicinal material. Characteristic Peaks 3, 4, 5, 6, and 7 should correspond to the retention times of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, and the chromatographic peaks of the calycosin glucoside reference. The peak corresponding to the calycosin glucoside reference peak is designated as Peak S (Peak S5). Calculate the relative retention times of the unidentified Peaks 1 and 2 relative to Peak S. The relative retention times should be within ±10% and the RSDs within ±3%. The specified values ​​are: 0.18 (Peak S1) and 0.28 (Peak S2). The retention time / relative retention time of each characteristic peak is summarized in Table 9 below. The results show that the method has good repeatability.

[0196] Table 9 Repeatability study results of Yiqiweixue granules (no sucrose added)

[0197]

[0198] (3) Stability study

[0199] Yiqiweixue granules (no sucrose added) (batch number: 20230403) were used to prepare a test solution according to the test solution method in step 2. The test solution was analyzed according to the detection method in step 3. Samples were injected at different times of 0, 5, 10, 15, 20, and 25 hours. Seven characteristic peaks appeared in the characteristic spectrum of the test sample and corresponded to the retention times of the seven characteristic peaks in the chromatogram of the reference medicinal material. Among them, characteristic peaks 3, 4, 5, 6, and 7 should correspond to the retention times of guanosine, 5-hydroxymethylfurfural, guanosine glucoside, formononetin, and guanosine reference material chromatogram peaks. The peak corresponding to the guanosine glucoside reference material peak was designated as peak S (characteristic peak 5). The relative retention times of the unidentified characteristic peaks 1 and 2 and peak S were calculated. The relative retention times were within ±10% and the RSD values ​​were within ±3%. The specified values ​​are: 0.18 (characteristic peak No. 1) and 0.28 (characteristic peak No. 2). The retention time / relative retention time of each characteristic peak is summarized in Table 10 below. The results show that the chromatographic peaks of the Yiqiweixue granules (no sucrose added) test solution show almost no change within 25 hours, indicating that the method has good stability.

[0200] Table 10 Stability study results of Yiqiweixue granules (no sucrose added)

[0201]

[0202] Example 4

[0203] This embodiment provides the detection of the characteristic spectrum of Yiqi Weixue granules, which is as follows:

[0204] Step 1: Preparation of reference solution: same as in Example 1.

[0205] Step 2. Preparation of test solution: Take an appropriate amount of sample, grind it into powder, take about 15g of fine powder, accurately weigh it, put it into a conical flask, accurately add 50mL of 90% methanol, weigh the weight, ultrasonically treat (power 200w, frequency 40KHz) for 40 minutes, let it cool, weigh it again, make up the lost weight with 90% methanol, shake it well, filter it, accurately measure 30mL of the filtrate, evaporate it to dryness, dissolve the residue in water, transfer it to a 5mL volumetric flask, dilute it to the scale with water, shake it well, filter it, and take the filtrate.

[0206] Step 3, liquid chromatography conditions: same as in Example 1.

[0207] Step 4: Accurately pipette the reference solution and 30 μL of each of the 15 batches of Yiqiweixue granules prepared according to the preparation method of the test solution in step 2, inject them into the high performance liquid chromatography instrument for detection, and record the chromatogram. Figure 9The chromatograms of 15 batches of Yiqiweixue granules test solution were respectively introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system; the 7 main chromatographic peaks present in the chromatograms of each batch of Yiqiweixue tablets were selected as characteristic peaks; the control characteristic spectrum of Yiqiweixue tablets was generated by the average value calculation method, see Figure 10 In the characteristic spectra of 15 batches of Yiqiweixue Capsules, characteristic peak No. 3 is guanosine with an average retention time of 12.048 min, characteristic peak No. 4 is 5-hydroxymethylfurfural with an average retention time of 15.532 min, characteristic peak No. 5 is calycosin glucoside with an average retention time of 36.988 min, characteristic peak No. 6 is formononetin with an average retention time of 42.765 min, and characteristic peak No. 7 is calycosin with an average retention time of 45.884 min. The peak corresponding to the calycosin glucoside reference peak is the S peak (characteristic peak No. 5), and the relative retention times of the unidentified characteristic peaks No. 1 and No. 2 are calculated. The average relative retention times of the 15 batches are: 0.183 (characteristic peak No. 1) and 0.286 (characteristic peak No. 2).

[0208] The methodological research on the characteristic spectrum detection method of Yiqi Weixue Granules is as follows:

[0209] (1) Precision study

[0210] Prepare a test solution using Yiqi Weixue Granules (Batch No. 20231209) according to the test solution method in Step 2. Analyze using the detection method in Step 3, with six replicate injections. The test solution's characteristic chromatogram should exhibit seven characteristic peaks corresponding in retention time to the seven characteristic peaks in the chromatogram of the mixed control medicinal material reference solution. Characteristic peaks 3, 4, 5, 6, and 7 should correspond to the retention times of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, and the calycosin glucoside reference substance. The peak corresponding to the calycosin glucoside reference substance peak is designated as peak S (characteristic peak 5). Calculate the relative retention times of unidentified characteristic peaks 1 and 2 relative to peak S. The relative retention times should be within ±10% and the RSD within ±3%. The specified values ​​are: 0.18 (characteristic peak 1) and 0.28 (characteristic peak 2). The retention time / relative retention time of each characteristic peak is summarized in Table 11 below. The results show that the parallel injection precision of this method is good.

[0211] Table 11 Precision study results of Yiqiweixue granules

[0212]

[0213] (2) Reproducible studies

[0214] Precision weigh six portions of Yiqiweixue Granules (Batch No. 20231209) in parallel. Prepare six test sample solutions according to the method in Step 2. Analyze according to the detection method in Step 3. The test sample's characteristic spectrum should exhibit seven characteristic peaks, corresponding in retention time to the seven characteristic peaks in the chromatogram of the reference medicinal material. Characteristic Peaks 3, 4, 5, 6, and 7 should correspond to the retention times of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, and the calycosin reference material. The peak corresponding to the calycosin glucoside reference material peak is designated as Peak S (Peak S5). Calculate the relative retention times of the unidentified Peaks 1 and 2 relative to Peak S. The relative retention times should be within ±10% and the RSDs within ±3%. The specified values ​​are: 0.18 (Peak S1) and 0.28 (Peak S2). The retention time / relative retention time of each characteristic peak is summarized in Table 12 below. The results show that the method has good repeatability.

[0215] Table 12 Results of the repeatability study of Yiqiweixue granules

[0216]

[0217] (3) Stability study

[0218] Yiqiweixue granules (batch number: 20231209) were prepared according to the test solution method in step 2. The test solution was analyzed according to the detection method in step 3. Samples were injected at different times of 0, 5, 10, 15, 20, and 25 hours. Seven characteristic peaks appeared in the characteristic spectrum of the test sample and corresponded to the retention times of the seven characteristic peaks in the chromatogram of the reference medicinal material. Among them, characteristic peaks 3, 4, 5, 6, and 7 should correspond to the retention times of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, and calycosin reference substance chromatogram peaks. The peak corresponding to the calycosin glucoside reference peak was designated as peak S (characteristic peak 5). The relative retention times of the unidentified characteristic peaks 1 and 2 and peak S were calculated. The relative retention times were within ±10% and the RSD values ​​were within ±3%. The specified values ​​are: 0.18 (characteristic peak 1) and 0.28 (characteristic peak 2). The retention time / relative retention time of each characteristic peak is summarized in Table 13 below. The results show that the chromatographic peaks of the Yiqiweixue granule test solution hardly change within 25 hours, indicating that the method has good stability.

[0219] Table 13 Yiqiweixue granules stability study results

[0220]

[0221] The control characteristic spectra obtained in Examples 1, 2, 3, and 4 were similar, with all four dosage forms having seven characteristic peaks. The relative retention times of the unlabeled characteristic peaks were within ±10% of the specified value, and the RSD% range was between 0.00% and 0.45%, which was much less than the specified ±3%. The above experimental results demonstrate that the characteristic spectrum detection method for the Qi-Yiweixue preparations (Yiqiweixue Tablets, Yiqiweixue Capsules, Yiqiweixue Granules, and Yiqiweixue Granules (No Sucrose Added)) provided in this application has good stability, high precision, and good repeatability. It can comprehensively and objectively evaluate the quality of Qi-Yiweixue preparations, which is of great significance for ensuring clinical efficacy.

[0222] 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.

[0223] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the spirit of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description may be used to interpret the content of the claims.

Claims

1. A method for constructing a characteristic spectrum of a Qi-invigorating and blood-replenishing preparation, characterized in that: The following steps are involved: Extracting the Qi-Yinweixue preparation using an extraction solvent to prepare a test solution; the extraction solvent includes a methanol-water solution or methanol; the Qi-Yinweixue preparation includes one or more of Qi-Yinweixue capsules, Qi-Yinweixue tablets, Qi-Yinweixue granules, and sucrose-free Qi-Yinweixue granules; and the extraction method is ultrasonic extraction; Performing high performance liquid chromatography on the test solution to obtain a chromatogram of the corresponding test sample; The conditions for the HPLC detection include: mobile phase A is methanol, mobile phase B is phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.05%; gradient elution is used; the detection wavelength is 272 nm; the column temperature is 30°C; the injection volume is 30 μL; The gradient elution procedure includes: From 0 to 8 minutes, the volume percentage of the mobile phase A is maintained at 4%; From 8 to 20 minutes, the volume percentage of the mobile phase A increased from 4% to 15%; From 20 min to 55 min, the volume percentage of the mobile phase A increased from 15% to 80%; From 55 to 65 minutes, the volume percentage of the mobile phase A is maintained at 80%; Flow rate: 0.8 mL / min-1.2 mL / min; The chromatogram of the test sample is introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system for processing, and the main chromatographic peaks present in the chromatograms of different batches of test samples are selected as characteristic peaks to construct the characteristic spectrum of the Qi-invigorating and blood-maintaining preparation; The characteristic peaks of the characteristic spectrum of the Qi-invigorating and blood-replenishing preparation include 7 characteristic peaks, of which 5 identified characteristic peaks include: characteristic peak No. 3 is guanosine, characteristic peak No. 4 is 5-hydroxymethylfurfural, characteristic peak No. 5 is calycosin glucoside, characteristic peak No. 6 is formononetin, and characteristic peak No. 7 is calycosin.

2. The method for constructing the characteristic spectrum of the Qi-invigorating and blood-reinforcing preparation according to claim 1, wherein: The steps of extracting the Yiqiweixue preparation using an extraction solvent meet the following conditions: (1) The extraction time is 20-60 minutes; (2) In the methanol aqueous solution, the volume percentage of methanol is 20%-95%.

3. The method for constructing the characteristic spectrum of the Qi-invigorating and blood-reinforcing preparation according to claim 1, wherein: The weight-to-volume ratio of the Qi-invigorating and blood-maintaining preparation to the extraction solvent is (2-20) g: (15-100) mL.

4. The method for constructing a characteristic spectrum of a Qi-invigorating and blood-replenishing preparation according to any one of claims 1 to 3, wherein: The method for constructing the characteristic spectrum of the Qi-invigorating and blood-reinforcing preparation further comprises the following steps: Dissolving the reference substance using a dissolving reagent to prepare a reference substance solution; the reference substance solution includes a mixed reference substance reference solution and a mixed reference medicinal material reference solution; Performing high performance liquid chromatography on the reference substance solution to obtain a chromatogram of the corresponding reference substance; performing common peak identification on the chromatographic peaks in the characteristic spectrum of the Yiqiweixue preparation according to the chromatogram of the reference substance; Wherein, the reference substances include one or more of guanosine, 5-hydroxymethylfurfural, calycosin glucoside, formononetin, calycosin, astragalus and jujube.

5. The method for constructing the characteristic spectrum of the Qi-invigorating and blood-reinforcing preparation according to claim 4, wherein: The dissolving agent is a methanol aqueous solution or water.

6. The method for constructing the characteristic spectrum of the Qi-invigorating and blood-reinforcing preparation according to claim 5, wherein: In the methanol aqueous solution, the volume percentage of methanol is 5%-50%.

7. Use of the characteristic spectrum of the Qi-invigorating and blood-reinforcing preparation constructed by the method for constructing the characteristic spectrum of the Qi-invigorating and blood-reinforcing preparation according to any one of claims 1 to 6 in the detection of the Qi-invigorating and blood-reinforcing preparation.

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