Construction method of hplc characteristic chromatogram of standard decoction of vinegar wistaria

Abstract

The present application relates to a kind of construction methods of vinegar jinyinhua standard decoction HPLC characteristic map, the present application adopts high performance liquid chromatography, Kromasil 100-5C18 chromatographic column, with methanol and 0.05% phosphoric acid aqueous solution mixture as mobile phase gradient elution is carried out, under the condition that flow rate is 1.0mL / min, column temperature is 35 ℃, detection wavelength is 238nm, establish vinegar jinyinhua standard decoction HPLC characteristic map, chromatogram is matched by "Chinese medicine chromatographic fingerprint similarity evaluation system" (2004A edition) peak, 14 common peaks are defined, 6 characteristic peaks are identified as jinyinhua-5-O-madder glycoside, luteolin, tiliroside, apigenin, hydroxyl jinyinhua and jinyinhua, and the similarity of 21 batches of vinegar jinyinhua standard decoction characteristic map is evaluated, sample similarity result is above 0.95;The HPLC characteristic map analysis method of vinegar jinyinhua standard decoction constructed by the present application is simple, accurate, reproducible, provides methodological basis for the quality evaluation and control of vinegar jinyinhua formula granules system.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis and detection technology, specifically relating to a method for constructing an HPLC characteristic spectrum of a standard decoction of Daphne genkwa in vinegar. Background Technology

[0002] Daphne genkwa Sieb. et Zucc., a plant in the Thymelaeaceae family, is a dried flower bud. Also known as Du Yuan or Mouse Flower, it has a bitter and pungent taste, is warm in nature, and is poisonous. It was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). The 2010 edition of the *Pharmacopoeia of the People's Republic of China* records its functions and indications as purging water retention, detoxifying, and killing parasites. It is used for edema, abdominal distension, ascites, phlegm accumulation, shortness of breath and cough, difficulty in urination and defecation, and externally for scabies, tinea capitis, and frostbite. It enters the lung, spleen, and kidney meridians. Due to its poisonous nature, its application is limited; its processed form has traditionally been processed with vinegar to reduce toxicity before being used medicinally. Daphne genkwa is rich in chemical components, including diterpenoid esters, flavonoids, coumarins, lignans, chlorogenic acids, and phenolic glycosides, among which diterpenoids and flavonoids are the main bioactive components. The toxicity of Daphne genkwa can be reduced after being processed with vinegar. There are early literature reports that the content of Daphne genkwa ester A decreases and the content of Daphne genkwa serotonin increases slightly after processing with vinegar.

[0003] Traditional Chinese medicine (TCM) granules, as a new form of TCM, are gaining increasing acceptance among patients due to their small size, portability, and preservation of the original components. Standard decoctions, serving as a crucial bridge to assess the consistency in quality between single-herb granules and their corresponding single-herb clinical decoctions, are of significant importance for quality control. Characteristic chromatograms of TCM are an effective means of evaluating the overall quality of TCM, offering the advantage of comprehensively characterizing its components and being widely used in qualitative and quantitative analysis. Currently, there are no literature reports on the characteristic chromatogram studies of vinegar-processed Daphne genkwa standard decoctions. Pang Nannan et al. studied the components of Daphne genkwa using RP-HPLC with a Kromasil C18 column (250mm × 4.6mm). Gradient elution was performed using methanol-0.05% phosphoric acid aqueous solution as the mobile phase (5 μm), with a flow rate of 1.0 mL / min, a column temperature of 35℃, and a detection wavelength of 238 nm. Twenty-one common peaks in the components of *Daphne genkwa* were identified, establishing a common pattern for *Daphne genkwa* medicinal materials. The fingerprint similarity of 17 out of 19 batches of *Daphne genkwa* medicinal materials was above 0.90. (Pang Nannan, Bi Kaishun, Yan Baoqing, et al. Study on high performance liquid chromatography fingerprint of *Daphne genkwa* [J]. Chinese Traditional and Herbal Drugs, 2010(5)) :4), while this study focuses on Daphne genkwa, which is a raw material of traditional Chinese medicine. As we all know, the raw materials of traditional Chinese medicine and the finished products cannot be generalized. Since vinegar Daphne genkwa is a processed product of Daphne genkwa, it is a sample made by boiling vinegar Daphne genkwa with water according to the standard decoction preparation process. Its components and efficacy are different from those of Daphne genkwa. The method used in this literature only focuses on the quality control of Daphne genkwa medicinal material and does not involve the preparations made from it, and therefore does not involve the construction of the components and characteristic spectra of the standard decoction of vinegar Daphne genkwa.

[0004] In summary, there are currently no reports on the standard decoction of Daphne genkwa in vinegar. Therefore, an HPLC characteristic spectrum of the standard decoction of Daphne genkwa in vinegar was established, which can serve as a material benchmark for quality comparison with the single-ingredient preparation of Daphne genkwa in vinegar granules. This is of great significance for the quality control of Daphne genkwa in vinegar granule preparations. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention proposes a method for constructing HPLC characteristic chromatograms of vinegar-processed Daphne genkwa standard decoction, which comprehensively characterizes the characteristic chromatograms of the components in the vinegar-processed Daphne genkwa standard decoction. The construction method of this invention is simple, accurate, and has good reproducibility. By establishing an HPLC characteristic chromatogram analysis method, it provides a methodological basis for the quality evaluation and control of vinegar-processed Daphne genkwa formula granule system.

[0006] The method for constructing the HPLC characteristic chromatogram of vinegar-processed Daphne genkwa standard decoction provided by this invention includes the following steps:

[0007] S1. Preparation of reference solution

[0008] Accurately weigh appropriate amounts of six reference standards: genkwa-5-O-rubiain, luteolin, titanin, apigenin, hydroxygenkwa-5-O-rubiain, and genkwa-5-O-rubiain. Dissolve them in methanol to prepare a mixed solution, shake well, and use it as the reference solution.

[0009] S2. Preparation of the test solution

[0010] Accurately weigh the freeze-dried powder of vinegar-processed Daphne genkwa standard decoction, place it in a stoppered conical flask, accurately add methanol, seal tightly, weigh, sonicate, remove and cool, weigh again, replenish the lost weight with methanol, shake well, filter and collect the filtrate to obtain the vinegar-processed Daphne genkwa standard decoction test solution.

[0011] S3, Feature Map Construction

[0012] The reference solution and the test solution were injected under the following high-performance liquid chromatography conditions to construct HPLC characteristic chromatograms.

[0013] Furthermore, the high-performance liquid chromatography conditions for step S3 are as follows:

[0014] The reference solution and the test solution were injected under the following high-performance liquid chromatography conditions to construct the characteristic HPLC chromatograms.

[0015] Column: Kromasil 100-5C18 (4.6mm × 250mm, 5μm);

[0016] Column temperature: 35℃;

[0017] Injection volume: 20 μL;

[0018] Detection wavelength: 238nm;

[0019] Flow rate: 1.0 mL / min;

[0020] Mobile phase: methanol (A), 0.05% phosphoric acid-water (B), gradient elution according to Table 3 below;

[0021] Table 1 Gradient Elution Table

[0022]

[0023]

[0024] Secondly, the method for constructing the HPLC characteristic chromatogram of the vinegar-processed Daphne genkwa standard decoction provided by this invention is applied in vinegar-processed Daphne genkwa compound preparations.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) Selection of chromatographic column: Two chromatographic columns, Kromasil Cl8 (250mm×4.6mm, 5um) and Nanospectral Analysis Chorm C18 (250mm×4.6mm, 5um), were selected for comparative experiments. The results showed that the column efficiency of the two chromatographic columns was not much different and the separation effect was good. Both can be used as experimental columns.

[0027] (2) Selection of mobile phase: In the experiment, methanol-water, methanol-0.05% phosphoric acid aqueous solution, acetonitrile-0.8% acetic acid aqueous solution and 0.05% formic acid acetonitrile-0.05% formic acid aqueous solution were selected. Finally, methanol-0.05% phosphoric acid aqueous solution was determined as the mobile phase. The elution procedure was determined through repeated experiments.

[0028] (3) Selection of detection wavelength: A full-wavelength scan was performed using a PDA detector to obtain 3D spectra. The results showed that the baseline of the chromatogram at 238 nm was stable, with a large number of chromatographic peaks and rich information. Therefore, 238 nm was selected as the optimal detection wavelength.

[0029] (4) Spectra at 210nm, 220nm, 230nm, 238nm, 240nm, 250nm, 260nm, and 270nm were extracted from the above 3D chromatograms and compared. The chromatogram results showed that the baselines of the 210nm, 220nm, and 230nm chromatograms drifted. Comparing the number of peaks at 240nm, 250nm, 260nm, and 270nm, the number of peaks at 240nm was higher. In addition, comparing the chromatograms at 238nm and 240nm, it was found that the peak areas of each chromatographic peak in the 238nm chromatogram were larger. Therefore, the optimal absorption wavelength for the characteristic chromatogram was finally selected as 238nm.

[0030] (5) This study established a high-performance liquid chromatography (HPLC) characteristic chromatographic analysis method for vinegar-processed Daphne genkwa standard decoction, comprehensively, systematically, and characteristically reflecting its chemical composition. Fourteen common peaks were identified, and six of them were pinpointed. This method exhibits good precision, repeatability, intermediate precision, and stability. Corresponding characteristic peaks were found in the characteristic chromatograms of different batches, comprehensively reflecting the overall characteristics of different batches of vinegar-processed Daphne genkwa standard decoction samples. The established HPLC characteristic chromatographic method can provide a methodological basis for the quality evaluation and control of vinegar-processed Daphne genkwa granule formulations. Attached Figure Description

[0031] Figure 1 The effect of different chromatographic columns (Kromasil C18 column) on the high performance liquid chromatography of vinegar-processed Daphne genkwa standard decoction.

[0032] Figure 2 The effect of different chromatographic columns (ChromCore C18 column) on the high performance liquid chromatography of vinegar-processed Daphne genkwa standard decoction.

[0033] Figure 3 The effect of different mobile phases (methanol-water) on the high performance liquid chromatography of vinegar-processed Daphne genkwa standard decoction.

[0034] Figure 4 The effect of different mobile phases (methanol-0.05% phosphoric acid) on the high performance liquid chromatography of vinegar-processed Daphne genkwa standard decoction.

[0035] Figure 5 The effect of different mobile phases (acetonitrile-0.8% acetic acid) on the high performance liquid chromatography of vinegar-processed Daphne genkwa standard decoction.

[0036] Figure 6 The effect of different mobile phases (0.05% formic acid acetonitrile - 0.05% formic acid) on the high performance liquid chromatography of vinegar-processed Daphne genkwa standard decoction.

[0037] Figure 7 This is a 3D spectrum obtained from full-wavelength scanning during the high-performance liquid chromatography (HPLC) detection of the standard decoction of Daphne genkwa in vinegar.

[0038] Figure 8 The effect of different detection wavelengths (210 nm) on the high performance liquid chromatography of vinegar-processed Daphne genkwa standard decoction.

[0039] Figure 9 The effect of different detection wavelengths (220 nm) on the high performance liquid chromatography of vinegar-processed Daphne genkwa standard decoction.

[0040] Figure 10 The effect of different detection wavelengths (230 nm) on the high performance liquid chromatography of vinegar-processed Daphne genkwa standard decoction.

[0041] Figure 11 The effect of different detection wavelengths (238 nm) on the high performance liquid chromatography of vinegar-processed Daphne genkwa standard decoction.

[0042] Figure 12 The effect of different detection wavelengths (240 nm) on the high performance liquid chromatography of vinegar-processed Daphne genkwa standard decoction was investigated.

[0043] Figure 13 The effect of different detection wavelengths (250 nm) on the high performance liquid chromatography of vinegar-processed Daphne genkwa standard decoction was investigated.

[0044] Figure 14 The effect of different detection wavelengths (260 nm) on the high performance liquid chromatography of vinegar-processed Daphne genkwa standard decoction.

[0045] Figure 15 The effect of different detection wavelengths (270 nm) on the high performance liquid chromatography of vinegar-processed Daphne genkwa standard decoction.

[0046] Figure 16 This is a high-performance liquid chromatography (HPLC) peak matching diagram of 21 batches of vinegar-processed Daphne genkwa standard decoction.

[0047] Figure 17 A comparative characteristic spectrum of the common pattern of vinegar-processed Daphne genkwa standard decoction. Detailed Implementation

[0048] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0049] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0050] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of the invention.

[0052] The following embodiments further describe the present invention, but these embodiments are not intended to limit the scope of protection of the present invention.

[0053] The sources and models of the instruments and materials used in the embodiments of this invention are shown in Table 1;

[0054] Table 1. Source and Model of Instruments and Materials

[0055]

[0056] The medicinal material of Daphne genkwa is the dried flower bud of Daphne genkwa, a plant of the Thymelaeaceae family. After being processed with vinegar, Daphne genkwa is obtained by vinegar-processing. The preparation process of vinegar-processed Daphne genkwa according to the standard decoction is as follows:

[0057] Each batch of Daphne genkwa was processed according to the vinegar-processing method in the pharmacopoeia to obtain vinegar-processed Daphne genkwa slices. 100g of each batch of vinegar-processed Daphne genkwa slices were taken and decocted twice with water. For the first decoction, 15 times the amount of water was added, and the mixture was soaked for 30 minutes. After boiling over high heat (500W), it was simmered over low heat (300W) for 30 minutes. The mixture was then filtered while hot through a 350-mesh sieve, and the filtrate was cooled to room temperature. For the second decoction, 12 times the amount of water was added, and the mixture was boiled over high heat (500W) and then simmered over low heat (300W) for 30 minutes. The mixture was then filtered while hot through a 350-mesh sieve, and the filtrate was cooled to room temperature. The two filtrates were combined and concentrated under reduced pressure to a volume of approximately 150g of clear extract. The extract was dispensed into 10ml vials, with 5ml dispensed into each vial. The vials were then freeze-dried under vacuum, and the extract was capped with aluminum.

[0058] Twenty-one batches of freeze-dried powder of vinegar-processed Daphne genkwa standard decoction were obtained according to the above preparation method. The sample numbers and sources are shown in Table 2.

[0059] Table 2. Sample numbers and sources of 21 batches of freeze-dried powder of vinegar-processed Daphne genkwa standard decoction.

[0060]

[0061]

[0062] Example 1: Screening of chromatographic conditions for constructing HPLC characteristic chromatograms of vinegar-processed Daphne genkwa standard decoction

[0063] (1) Selection of chromatographic columns: Two chromatographic columns, Kromasil Cl8 (250mm×4.6mm, 5um) and Nanospectral Analysis Chorm C18 (250mm×4.6mm, 5um), were used for comparative experiments. (See attached...) Figure 1 and attached Figure 2 The results showed that the column efficiencies of the two types of chromatographic columns were similar, and both had good separation effects, so both could be used as experimental columns.

[0064] (2) Selection of mobile phase: In the experiment, methanol-water, methanol-0.05% phosphoric acid aqueous solution, acetonitrile-0.8% acetic acid aqueous solution, and 0.05% formic acid-acetonitrile-0.05% formic acid aqueous solution were selected. Figure 3 ~Attached Figure 6 Ultimately, methanol-0.05% phosphoric acid aqueous solution was selected as the mobile phase, and the elution procedure was determined through repeated experiments.

[0065] (3) Selection of detection wavelength: A PDA detector is used for full-wavelength scanning to obtain a 3D spectrum (attached). Figure 7 ), and the spectra at 210nm, 220nm, 230nm, 238nm, 240nm, 250nm, 260nm, and 270nm were extracted from the above 3D chromatograms for comparison (see appendix). Figure 8 ~Attached Figure 15The results showed that the baselines of the chromatograms at 210nm, 220nm, and 230nm were drifted. Comparing the number of peaks at 240nm, 250nm, 260nm, and 270nm, the number of peaks at 240nm was higher. The baseline of the chromatogram at 238nm was stable, with a large number of peaks and rich information. In addition, comparing the chromatograms at 238nm and 240nm, it was found that the peak areas of each chromatogram at 238nm were larger. Therefore, the optimal absorption wavelength for the characteristic chromatogram was finally selected as 238nm.

[0066] Example 2: Construction of HPLC characteristic chromatogram of vinegar-processed Daphne genkwa standard decoction

[0067] Part 1: Construction Method

[0068] The specific steps are as follows:

[0069] S1. Preparation of reference solution

[0070] Accurately weigh 10 mg each of the six reference standards: genkwa-5-O-rubiain, luteolin, titanin, apigenin, hydroxygenkwa-5-O-rubiain, and genkwa-5-O-rubiain. Dissolve them in methanol to prepare a mixed solution, shake well, and use it as the reference solution.

[0071] S2. Preparation of the test solution

[0072] Accurately weigh 0.1 g of the freeze-dried powder of vinegar-processed Daphne genkwa standard decoction, place it in a 100 ml stoppered conical flask, accurately add 25 ml of 70% methanol, seal tightly, weigh, sonicate for 1.0 h, remove, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter through a 0.22 μm microporous membrane, and collect the filtrate to obtain the vinegar-processed Daphne genkwa standard decoction test solution;

[0073] S3, Feature Map Construction

[0074] The reference solution and the test solution were injected under the following high-performance liquid chromatography conditions to construct the characteristic HPLC chromatograms.

[0075] Column: Kromasil 100-5C18 (4.6mm × 250mm, 5μm);

[0076] Column temperature: 35℃;

[0077] Injection volume: 20 μL;

[0078] Detection wavelength: 238nm;

[0079] Flow rate: 1.0 mL / min;

[0080] Mobile phase: methanol (A), 0.05% phosphoric acid-water (B), gradient elution according to Table 3 below;

[0081] Table 3 Gradient Elution Table

[0082]

[0083] Part Two: Methodological Examination

[0084] (1) Precision test

[0085] Following the construction method in Part 1, 20 μL of the vinegar-processed Daphne genkwa standard decoction test solution from batch S5 was injected six times consecutively. The relative retention time and relative peak area of ​​the common peaks were calculated using peak 14 as a reference. The results showed that the RSD of the relative retention time of each common peak ranged from 0.00% to 0.11%, and the RSD of the relative peak area ranged from 0.21% to 1.72%, indicating that the instrument was stable and the method had good precision. The results are shown in Tables 4 and 5.

[0086] Table 4. Relative retention times of all peaks in the precision test.

[0087]

[0088] Table 5. Relative peak areas of common peaks in precision tests.

[0089]

[0090] (2) Repeatability test

[0091] Following the construction method described in Part I, six parallel standard decoction solutions of Daphne genkwa (vinegar-coated daphne) were prepared and analyzed. The relative retention time and relative peak area of ​​the common peaks were calculated using peak 14 as a reference. The results showed that the RSD of the relative retention time of each common peak ranged from 0.00% to 0.27%, and the RSD of the relative peak area ranged from 0.48% to 3.71%, indicating good repeatability of the method. The results are shown in Tables 6 and 7.

[0092] Table 6. Relative retention times of common peaks in repeatability tests

[0093]

[0094] Table 7 Relative peak areas of common peaks in repeatability tests

[0095]

[0096] (3) Intermediate precision test

[0097] Different personnel conducted experiments on different dates using different numbered liquid chromatography instruments. Six parallel standard decoction solutions of Daphne genkwa (vinegar-coated daphne) were prepared according to the construction method described in Part I, and analyzed. The relative retention time and relative peak area of ​​the common peaks were calculated using peak 14 as a reference. Tables 8 and 9 show that the RSD of the relative retention time of each common peak was 0.00%–0.10%, and when summarized with the relative retention time data of each characteristic peak in the repeatability test of the six samples, the RSD was 0.00%–0.27%. The RSD of the relative peak area of ​​each characteristic peak was 0.59%–4.32%, and when summarized with the relative peak area data of each characteristic peak in the repeatability test of the six samples, the RSD was 0.86%–3.47%, indicating good intermediate precision of the method.

[0098] Table 8. Relative retention times of all peaks in the intermediate precision test.

[0099]

[0100] Table 9 shows the relative peak areas of the intermediate precision tests.

[0101]

[0102] (4) Stability test

[0103] Following the construction method described in Part I, the test solution of vinegar-processed Daphne genkwa standard decoction (batch number S5) was injected and analyzed under the chromatographic conditions specified in Part I at 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h. Chromatograms were recorded, and the relative retention time and relative peak area of ​​the common peaks were calculated using peak 14 as a reference. The results showed that the RSD of the relative retention time of each common peak ranged from 0.00% to 1.77%, and the RSD of the relative peak area ranged from 0.12% to 4.37%, indicating that the test solution exhibited good stability within 24 hours. The results are shown in Tables 10 and 11.

[0104] Table 10: Relative retention times of common peaks in stability tests

[0105]

[0106] Table 11 Relative peak areas of common peaks in stability tests

[0107]

[0108] Part Three: Feature Map Construction and Feature Peak Identification

[0109] (1) Establishment of feature maps

[0110] Twenty-one batches of vinegar-processed Daphne genkwa standard decoction test solutions were injected and analyzed according to the chromatographic conditions described in Part I of the construction method. Chromatograms of the 21 batches of samples were obtained and imported into the "Traditional Chinese Medicine Chromatographic Characteristic Chromatogram / Characteristic Chromatogram Similarity Evaluation System" (Version 2004A). Common peak matching was performed using the median method. The results are shown in [Figure 1]. Figure 16 The control characteristic spectrum of the common pattern of the generated vinegar-processed Daphne genkwa standard decoction is shown in [reference needed]. Figure 17 Fourteen common chromatographic peaks were identified. The 14th common peak (genistein) with suitable peak area and retention time was selected as the reference peak (S peak). With the retention time and peak area of ​​genistein as 1, the relative retention time and relative peak area of ​​the remaining common peaks were calculated. The results are shown in Tables 12 and 13.

[0111] Table 1221 shows the characteristic chromatograms of the standard decoction samples containing vinegar-processed Daphne genkwa, including the relative retention times of the common peaks.

[0112]

[0113] Table 1321 shows the characteristic chromatograms of the standard decoction samples containing vinegar-processed Daphne genkwa, including the common peaks and their relative peak areas.

[0114]

[0115]

[0116] (2) Identification of the components of characteristic peaks

[0117] Take the reference solution from the construction method in Part 1, and inject it for detection according to the chromatographic conditions in the construction method in Part 1. Compare the characteristic chromatogram of the obtained reference solution with the characteristic chromatogram of the vinegar-processed Daphne genkwa standard decoction sample. The retention times of the chromatographic peaks show that peak 8 is genkwa-5-O-rubiain, peak 10 is luteolin, peak 11 is titannin, peak 12 is apigenin, peak 13 is hydroxygenkwa, and peak 14 is genkwa.

[0118] (3) Similarity evaluation

[0119] The similarity evaluation system for the chromatographic fingerprint / characteristic chromatogram of traditional Chinese medicine (version 2004A) issued by the National Pharmacopoeia Commission was used to evaluate the similarity of the characteristic chromatograms of 21 batches of vinegar-processed Daphne genkwa standard decoction. The results are shown in Table 14. The results show that the similarity of the characteristic chromatograms of the 21 batches of vinegar-processed Daphne genkwa standard decoction is relatively high, all above 0.95.

[0120] Table 14. Comparison of similarity among 21 batches of vinegar-processed Daphne genkwa standard decoctions

[0121]

[0122] It should be noted that the specific features, structures, materials or characteristics described in this specification can be combined in any way. For the sake of brevity, not all possible combinations of the various technical features in the above embodiments have been described. Without contradiction, those skilled in the art can combine and integrate the different embodiments and features described in this specification.

[0123] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for constructing the HPLC characteristic chromatogram of a standard decoction of Daphne genkwa in vinegar, characterized in that, The construction method includes the following steps: S1. Preparation of reference solution Accurately weigh appropriate amounts of six reference standards: genkwa-5-O-rubiain, luteolin, titanin, apigenin, hydroxygenkwa-5-O-rubiain, and genkwa-5-O-rubiain. Dissolve them in methanol to prepare a mixed solution, shake well, and use it as the reference solution. S2. Preparation of the test solution Accurately weigh the freeze-dried powder of vinegar-processed Daphne genkwa standard decoction, place it in a stoppered conical flask, accurately add methanol, seal tightly, weigh, sonicate, remove and cool, weigh again, replenish the lost weight with methanol, shake well, filter and collect the filtrate to obtain the vinegar-processed Daphne genkwa standard decoction test solution. S3, Feature Map Construction The reference solution and the test solution were injected under the following high-performance liquid chromatography conditions to construct the characteristic HPLC chromatograms. The high-performance liquid chromatography (HPLC) conditions in step S3 were achieved using a Kromasil 100-5C18 column. In the high performance liquid chromatography conditions of step S3, mobile phase A is methanol and mobile phase B is 0.05% phosphoric acid-water. The elution procedure for the mobile phase is as follows: At 0 min, the proportion of mobile phase B is 85% and the proportion of mobile phase A is 15%. At 5 minutes, the proportion of mobile phase B was 78%, and the proportion of mobile phase A was 22%. At 9 minutes, the proportion of mobile phase B was 76%, and the proportion of mobile phase A was 24%. At 12 minutes, the proportion of mobile phase B was 70%, and the proportion of mobile phase A was 30%. At 22 min, the proportion of mobile phase B was 59%, and the proportion of mobile phase A was 41%. At 29 min, the proportion of mobile phase B was 55%, and the proportion of mobile phase A was 45%. At 41 min, the proportion of mobile phase B was 55%, and the proportion of mobile phase A was 45%. At 57 min, the proportion of mobile phase B was 40%, and the proportion of mobile phase A was 60%. At 66 min, the proportion of mobile phase B was 17%, and the proportion of mobile phase A was 83%. At 76 min, the proportion of mobile phase B was 7%, and the proportion of mobile phase A was 93%. At 78 minutes, the proportion of mobile phase B was 7%, and the proportion of mobile phase A was 93%.

2. The method for constructing the HPLC characteristic chromatogram of the vinegar-processed Daphne genkwa standard decoction according to claim 1, characterized in that, The column temperature in the high-performance liquid chromatography (HPLC) conditions of step S3 is 35°C.

3. The method for constructing the HPLC characteristic chromatogram of the vinegar-processed Daphne genkwa standard decoction according to claim 1, characterized in that, In step S3, the injection volume in the high-performance liquid chromatography conditions is 20 μL.

4. The method for constructing the HPLC characteristic chromatogram of the vinegar-processed Daphne genkwa standard decoction according to claim 1, characterized in that, The detection wavelength in the high-performance liquid chromatography (HPLC) conditions of step S3 is 238 nm.

5. The method for constructing the HPLC characteristic chromatogram of the vinegar-processed Daphne genkwa standard decoction according to claim 1, characterized in that, The flow rate in the high-performance liquid chromatography (HPLC) conditions of step S3 is 1.0 mL / min.

6. The application of the method for constructing the HPLC characteristic chromatogram of the vinegar-processed Daphne genkwa standard decoction according to any one of claims 1 to 5 in the preparation of vinegar-processed Daphne genkwa compound preparations.

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