Construction method and application of fingerprint spectrum of motherwort preparation

Abstract

The invention relates to a construction method and application of a fingerprint spectrum of a herba leonuri preparation, and the method comprises the following steps: extracting the herba leonuri preparation by using an extraction solvent to prepare a test solution; performing high performance liquid chromatography detection on the test solution, and constructing a fingerprint spectrum of the motherwort preparation; the conditions of the high performance liquid chromatography detection are as follows: a mobile phase A is a formic acid aqueous solution, a mobile phase B is acetonitrile, and gradient elution is adopted. According to the characteristics of the components contained in the motherwort preparation, the fingerprint spectrum of the motherwort preparation containing a plurality of characteristic peaks is constructed, and the construction method of the fingerprint spectrum of the motherwort preparation is simple, convenient, reliable, high in objectivity and good in precision, repeatability, specificity and stability. Furthermore, the separation degree of each characteristic peak of the fingerprint of the motherwort preparation is relatively good, the characteristics of main chemical components of the motherwort preparation can be relatively comprehensively reflected, and an overall quality control basis can be provided for the quality standard of the motherwort preparation.

Description

Technical Field

[0001] The present application relates to the technical field of detection of traditional Chinese medicine preparations, and in particular to a method for constructing and applying a fingerprint spectrum of a Leonurus japonicus preparation. Background Art

[0002] Motherwort is the fresh or dried aerial part of Leonurus japonicas Houtt., a plant of the Labiatae family. Fresh products are harvested from the seedling stage in spring to the pre-flowering stage in early summer; dried products are harvested in summer when the stems and leaves are lush and the flowers are not or just beginning to bloom, and then dried in the sun, or cut and dried in the sun; they are widely distributed throughout the country. Motherwort contains a variety of trace elements such as selenium and manganese. Selenium has the effects of enhancing cellular immunity, improving the body's defense function, and relieving arteriosclerosis. Manganese can prevent aging, fatigue, and oxidation, and can also inhibit the growth of cancer cells. At the same time, motherwort has the effects of diuresis, swelling, and uterine contraction. It is an important medicine used by doctors in all dynasties to treat gynecological diseases. At present, the preparations made of motherwort as raw materials mainly include motherwort injection, motherwort granules, motherwort capsules, motherwort oral liquid, motherwort tablets, motherwort paste, and fresh motherwort capsules. Among them, motherwort granules are common preparations in clinical practice. Their effectiveness and safety have been recognized in long-term clinical practice, and they are convenient to carry and take, so they are very popular.

[0003] The whole herb of Leonurus can be used as medicine, and the active ingredient is leonuri. Leonurus contains a variety of alkaloids such as leonurine, stachydrine, leonuridine, leonurin, benzoic acid, potassium chloride, etc., and there are many ingredients. The fingerprint of the existing Leonurus preparations cannot fully reflect the intrinsic quality of Leonurus preparations. Therefore, it is necessary to establish a fingerprint of Leonurus preparations that can characterize its main chemical components, which can provide a more comprehensive and effective evaluation for the quality control of Leonurus preparations, thereby ensuring its safety. Summary of the invention

[0004] Based on this, the purpose of this application includes providing a method for constructing and applying a fingerprint spectrum of a Leonurus herb preparation.

[0005] The technical solution of this application is as follows:

[0006] One aspect of the present invention provides a method for constructing a fingerprint of a Leonurus japonicus preparation, comprising the following steps:

[0007] Extracting the Leonurus japonicus preparation using an extraction solvent to prepare a test solution;

[0008] Performing high performance liquid chromatography on the test solution to construct a fingerprint of the motherwort preparation;

[0009] The conditions for the high performance liquid chromatography detection include: mobile phase A is formic acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is adopted.

[0010] In one embodiment, the gradient elution procedure comprises:

[0011] From 0 to 20 min, the volume percentage of the mobile phase B increased from 2% to 5%;

[0012] From 20min to 30min, the volume percentage of the mobile phase B increased from 5% to 12%;

[0013] From 30min to 56min, the volume percentage of the mobile phase B increased from 12% to 21%;

[0014] From 56 min to 65 min, the volume percentage of the mobile phase B increased from 21% to 26%.

[0015] In one embodiment, the volume percentage of formic acid in the formic acid aqueous solution is 0.1%~0.3%.

[0016] In one embodiment, the high performance liquid chromatography detection satisfies at least one of the following conditions:

[0017] (1) The detection wavelength is 250nm~350nm;

[0018] (2) Flow rate: 0.5 mL / min~2 mL / min;

[0019] (3) The injection volume is 0.5 μL~5 μL;

[0020] (4) The column temperature is 25℃~35℃.

[0021] In one embodiment, the extracting step satisfies at least one of the following conditions:

[0022] (1) The extraction method is ultrasonic extraction or reflux extraction;

[0023] (2) The extraction time is 15min~60min;

[0024] (3) The extraction solvent includes methanol or a methanol-water solution.

[0025] In one embodiment, the volume percentage of methanol in the methanol-water solution is 25% to 75%.

[0026] In one embodiment, the method for constructing the fingerprint of the motherwort preparation further comprises the following steps:

[0027] Dissolve the reference substance using a dissolving reagent to prepare a reference substance solution;

[0028] Performing high performance liquid chromatography on the reference solution to obtain a reference spectrum;

[0029] According to the reference spectrum, the chromatographic peaks of the fingerprint spectrum of the motherwort preparation are identified as common peaks;

[0030] Wherein, the reference substance includes one or more of ferulic acid, leonurine hydrochloride, stachydrine hydrochloride, rutin, quercetin and hyperoside.

[0031] In one embodiment, the fingerprint of the motherwort preparation contains 15 common peaks, among which two characteristic peaks are identified: Peak 9 is leonurine hydrochloride, and Peak 13 is ferulic acid.

[0032] In one embodiment, the motherwort preparation includes motherwort granules, motherwort capsules or motherwort tablets.

[0033] Another aspect of the present invention provides the application of the method for constructing the fingerprint of the motherwort preparation as described above in the quality control of the motherwort preparation.

[0034] Compared with the prior art, this application has the following beneficial effects:

[0035] According to the characteristics of the ingredients contained in the motherwort preparation, the present application constructs a fingerprint spectrum of the motherwort preparation containing multiple characteristic peaks by detecting and analyzing the motherwort preparation under specific high performance liquid chromatography conditions. The method for constructing the fingerprint spectrum of the motherwort preparation is simple, reliable, and objective, and has good precision, repeatability, specificity and stability.

[0036] Furthermore, the fingerprint of the motherwort preparation has good separation of the characteristic peaks, contains 15 common peaks, can more comprehensively reflect the main chemical composition characteristics of the motherwort preparation, and identify two characteristic peaks such as ferulic acid and leonurine hydrochloride; and the detection results of different batches of motherwort preparations have high similarity, and the generated control fingerprint can be used as an overall similarity evaluation of the material basis of the motherwort preparation, which can provide an overall quality control basis for the quality standards of the motherwort preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 It is the high performance liquid chromatography superimposed fingerprint of the Leonurus granules in Example 1 and the reference fingerprint R graph.

[0039] Figure 2The HPLC chromatogram of the mixed standard solution in Example 1 is shown in FIG.

[0040] Figure 3 The HPLC chromatograms of the Motherwort Granules, Motherwort and brown sugar in Example 1 are shown; wherein A: Motherwort Granules; B: Motherwort; C: brown sugar.

[0041] Figure 4 It is the HPLC chromatogram of the Leonurus granules, mixed reference substance and blank test solution in Example 1; wherein, A: Leonurus granules; B: mixed reference substance; C: blank test solution.

[0042] Figure 5 This is the HPLC chromatogram of the intermediate precision of the fingerprint instrument of Leonurus granules in Example 1, wherein A: equipment J-20-C-008; B: equipment J-20-C-093.

[0043] Figure 6 This is the HPLC chromatogram of the intermediate precision of the chromatographic column of the fingerprint of Leonurus granules in Example 1, wherein A: chromatographic column JZ-SY-C002; B: chromatographic column QC-C18-24-0401; C: chromatographic column QC-C18-24-0402. DETAILED DESCRIPTION

[0044] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. 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 this application.

[0046] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0047] As used herein, "one or more" refers to any one, any two, or any two or more of the listed items.

[0048] Herein, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0049] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0050] Only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form an undefined range; and any lower limit can be combined with other lower limits to form an undefined range, and any upper limit can be combined with any other upper limit to form an undefined range. In addition, each separately disclosed point or single value itself can be combined with any other point or single value as a lower limit or upper limit or with other lower limits or upper limits to form an undefined range. The use of numerical ranges represented by endpoints includes all numbers within the range and any range within the range, for example, 1 to 5 includes 1, 1.1, 1.3, 1.5, 2, 2.75, 3, 3.80, 4 and 5, etc.

[0051] The percentage contents involved in this application, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

[0052] The percentage concentrations mentioned in this application, 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.

[0053] The temperature parameters in this application, 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 controlled by the instrument. Fluctuations within the range of ±5°C, ±2°C, ±1°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 this application refers to no temperature control operation, generally 4°C~35°C, preferably 20±5°C.

[0054] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0055] Traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method based on the systematic study of chemical components of traditional Chinese medicine. It is mainly used to evaluate the authenticity, quality and stability of Chinese medicinal materials and semi-finished products of traditional Chinese medicine preparations. Traditional Chinese medicines are multi-component complex systems, so the evaluation of their quality should adopt a detection method that is suitable for them and can provide rich identification information. Traditional Chinese medicine fingerprint can more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine, and can provide an overall description and evaluation of the quality of the medicine. It further reflects the quality of traditional Chinese medicine and the overall efficacy of traditional Chinese medicine. As a traditional Chinese medicine preparation, the fingerprint of motherwort preparation can reflect its unique chemical composition and pharmacodynamic characteristics, and has a wide range of application value in the quality control of traditional Chinese medicine.

[0056] Based on this, one aspect of the present invention provides a method for constructing a fingerprint of a Leonurus japonicus preparation, comprising the following steps:

[0057] Extracting the Leonurus japonicus preparation using an extraction solvent to prepare a test solution;

[0058] Performing high performance liquid chromatography on the test solution to construct a fingerprint of the motherwort preparation;

[0059] The conditions for the high performance liquid chromatography detection include: mobile phase A is formic acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is adopted.

[0060] In some examples, the gradient elution procedure includes:

[0061] From 0 to 20 min, the volume percentage of the mobile phase B increased from 2% to 5%;

[0062] From 20min to 30min, the volume percentage of the mobile phase B increased from 5% to 12%;

[0063] From 30min to 56min, the volume percentage of the mobile phase B increased from 12% to 21%;

[0064] From 56 min to 65 min, the volume percentage of the mobile phase B increased from 21% to 26%.

[0065] In some examples, the volume percentage of formic acid in the formic acid aqueous solution is 0.1% to 0.3%. It is understandable that the volume percentage of formic acid in the formic acid aqueous solution includes but is not limited to 0.1%, 0.15%, 0.2%, 0.25%, and 0.3%.

[0066] In some examples, the conditions for the HPLC detection include: a detection wavelength of 250 nm to 350 nm. It is understandable that the detection wavelength of the HPLC includes but is not limited to 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, and 350 nm.

[0067] In some of the examples, the conditions for the HPLC detection include: a flow rate of 0.5 mL / min to 2 mL / min. It is understandable that the flow rate for the HPLC detection includes but is not limited to 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, 1.6 mL / min, 1.7 mL / min, 1.8 mL / min, 1.9 mL / min, and 2 mL / min.

[0068] In some examples, the conditions for the HPLC detection include: the injection volume is 0.5 μL to 5 μL. It is understandable that the injection volume for the HPLC detection includes but is not limited to 0.5 μL, 1 μL, 1.5 μL, 2 μL, 2.5 μL, 3 μL, 3.5 μL, 4 μL, 4.5 μL, and 5 μL.

[0069] In some examples, the conditions for the HPLC detection include: a column temperature of 25° C. to 35° C. It is understandable that the column temperature for 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.

[0070] In some of the examples, the conditions of the extraction step include: the extraction method is ultrasonic extraction or reflux extraction.

[0071] In some examples, the conditions of the extraction step include: the extraction time is 15 min to 60 min. It can be understood that the extraction time includes but is not limited to 15 min, 20 min, 30 min, 40 min, 45 min, 50 min, and 60 min.

[0072] In some examples, the conditions of the extracting step include: the extraction solvent includes methanol or a methanol-water solution.

[0073] In some of the examples, the volume percentage of methanol in the methanol-water solution is 25% to 75%. It is understandable that the volume percentage of methanol in the methanol-water solution includes but is not limited to 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75%.

[0074] In some examples, the method for constructing the fingerprint of the motherwort preparation further includes the following steps:

[0075] Dissolve the reference substance using a dissolving reagent to prepare a reference substance solution;

[0076] Performing high performance liquid chromatography on the reference solution to obtain a reference spectrum;

[0077] According to the reference spectrum, the chromatographic peaks of the fingerprint spectrum of the motherwort preparation are identified as common peaks;

[0078] Wherein, the reference substance includes one or more of ferulic acid, leonurine hydrochloride, stachydrine hydrochloride, rutin, quercetin and hyperoside.

[0079] In some of the examples, the dissolving agent includes methanol, methanol-water solution or ethanol; in the methanol-water solution, the percentage of methanol is 50% to 100%.

[0080] In some of the examples, the fingerprint of the motherwort preparation contained 15 common peaks, among which two characteristic peaks were identified: Peak 9 was leonurine hydrochloride, and Peak 13 was ferulic acid.

[0081] In some examples, the motherwort preparation includes motherwort granules, motherwort capsules or motherwort tablets. Further, the motherwort preparation is motherwort granules.

[0082] Another aspect of the present invention provides the application of the method for constructing the fingerprint of the motherwort preparation as described above in the quality control of the motherwort preparation.

[0083] The following is further described in conjunction with specific embodiments. The raw materials involved in the following specific embodiments, unless otherwise specified, can all be commercially available; the instruments used, unless otherwise specified, can all be commercially available; the processes involved, unless otherwise specified, are all routinely selected by those skilled in the art.

[0084] The present invention is further described in detail below in conjunction with specific examples. For experimental parameters not specified in the following specific examples, reference is made to the instructions given in the present application document, and reference may also be made to the experimental manuals in the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturer. It is understood that the instruments and raw materials used in the following examples are relatively specific, and may not be limited thereto in other specific examples.

[0085] The following are specific embodiments.

[0086] Example 1

[0087] 1. Materials and Methods

[0088] Some of the instruments and materials used in the examples are as follows:

[0089] Testing instruments: electronic analytical balance, high performance liquid chromatograph, ultrasonic cleaner.

[0090] Test sample: The 7 batches of brown sugar motherwort granules used in the example were all from Guangzhou Nuojin Pharmaceutical Co., Ltd., with batch numbers 3373002, 4371001, 3376004, 4371002, 4371003, 4371004, and 4371005, respectively.

[0091] The preparation method of the test solution is as follows: take 4.0 g of Leonurus granules, accurately weigh, place in a 150 mL stoppered conical flask, accurately add 25 mL of 50% methanol, seal, weigh and then ultrasonically treat for 45 minutes, cool to room temperature, weigh again, make up the lost weight with 50% methanol, shake well, filter with a 0.45 μm microporous filter membrane, collect the filtrate, and obtain.

[0092] The reference substances include: ferulic acid reference substance, leonurine hydrochloride reference substance, stachydrine hydrochloride rutin reference substance, quercetin reference substance, and hyperoside reference substance.

[0093] The preparation method of the mixed reference solution is as follows: take appropriate amounts of ferulic acid reference and leonurine hydrochloride reference, accurately weigh them, and add methanol to make a mixed solution containing 100 μg of ferulic acid and 100 μg of leonurine hydrochloride per 1 mL.

[0094] The preparation method of the auxiliary material solution such as brown sugar is as follows: take 2.6g of brown sugar, 0.6g of dextrin and 0.6g of starch respectively, weigh them accurately, put them in a 150mL stoppered conical flask, accurately add 25mL of 50% methanol, seal it, weigh it and then ultrasonically treat it for 45min, cool it to room temperature, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it with a 0.45μm microporous filter membrane, collect the filtrate, and obtain it.

[0095] The preparation method of motherwort solution is as follows: take 5.8 g of motherwort, accurately weigh it, put it in a 150 mL stoppered conical flask, accurately add 25 mL of 50% methanol, seal it, weigh it and then ultrasonically treat it for 45 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it with a 0.45 μm microporous filter membrane, collect the filtrate, and obtain it.

[0096] Blank solution: Take an appropriate amount of 50% methanol, filter it through a 0.45μm microporous filter membrane, and collect the filtrate to obtain.

[0097] The chromatographic conditions are shown in Table 1 below:

[0098] Table 1

[0099]

[0100] 2. Establishment of fingerprint and identification of common peaks

[0101] Seven batches of brown sugar motherwort granules were used to prepare test solution according to the test solution preparation method described above. The test solution, blank solution, mixed reference solution, brown sugar and other auxiliary material solutions and motherwort solution were injected and analyzed according to the above chromatographic conditions. The results are as follows: Figures 1 to 3 As shown. The fingerprint spectrum of the 7 batches of Yi Mu Cao Granules was analyzed to find their common characteristic peaks. The chromatograms of the 7 batches of Yi Mu Cao Granules were imported into the "Chinese Medicine Fingerprint Similarity Evaluation System (2012 Edition)", and the chromatogram of sample S2 was used as the reference spectrum. The superimposed fingerprint spectrum was automatically matched in the form of full spectrum peaks, and the reference fingerprint spectrum R was generated by the average method. The common peaks were identified by comparing with the spectrum of the mixed reference solution.

[0102] The results showed that through fingerprint analysis of Yi Mu Cao Granules and their negative samples, 15 characteristic peaks were matched in the chromatograms of 7 batches of Yi Mu Cao Granules. By comparing with the spectrum of the mixed reference solution, 2 common peaks were identified, namely, Leonurine Hydrochloride (Peak 9) and Ferulic Acid (Peak 13).

[0103] 3. Similarity analysis of fingerprints

[0104] By comparing the chromatograms, 15 characteristic peaks with good separation and stable peaks in each batch of fingerprints were determined. The 2012 version of the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" was used to generate a reference fingerprint based on the 15 peaks and perform similarity analysis. The results are shown in Table 2.

[0105] Table 2 Similarity evaluation results of 7 batches of Leonurus granules

[0106]

[0107] The results showed that the similarity of the lowest similarity batch was 0.878, and the others were basically above 0.99. This shows that the similarity of each batch of Motherwort Granules is high, and the generated control fingerprint can be used as an overall similarity evaluation of the material basis of Motherwort Granules.

[0108] IV. Methodological Validation

[0109] 4.1 Specificity Investigation

[0110] Accurately pipette 20 μL of each of the test solution, mixed reference solution and blank solvent of Leonurus Granules, inject into the liquid chromatograph, and measure according to the chromatographic conditions as described above to check whether there is interference with the extraction solvent. The results are as follows: Figure 4 shown.

[0111] The results showed that the blank test solution had almost no absorption under the chromatographic conditions and had no interference with the chromatographic analysis of the test sample, indicating that the established method had good specificity.

[0112] 4.2 Repeatability study

[0113] Weigh 6 portions of the same batch of Yi Mu Cao granules to prepare the test solution, and inject and analyze under the optimal chromatographic conditions. Record the peak area, use peak 11 as the reference peak, and calculate the RSD value of the peak area. The results are shown in Tables 3 and 4.

[0114] Table 3 Relative peak areas of repeatability of fingerprint spectrum of Leonurus granules

[0115]

[0116] Table 4 Relative retention time of repeatability study of fingerprint spectrum of Leonurus granules

[0117]

[0118] The results showed that the RSD of the relative peak areas of the common peaks were all less than 3.97% (n=6), and the RSD of the relative retention time were all less than 0.70% (n=6), indicating that the method had good repeatability.

[0119] 4.3 Precision investigation

[0120] Take the motherwort granules as the test sample, inject 6 times continuously, measure under the optimal chromatographic conditions, take peak 11 as the reference peak, calculate the RSD value of the relative peak area of ​​each common peak, and the results are shown in Tables 5 and 6.

[0121] Table 5 Relative peak areas of the fingerprint precision of Leonurus granules

[0122]

[0123] Table 6 Relative retention time of the precision study of fingerprint spectrum of Leonurus granules

[0124]

[0125] As a result, the RSD of the relative peak area of ​​each common peak was less than 3.59% (n=6), and the RSD of the relative retention time was less than 0.83% (n=6), indicating that the precision of the instrument was good.

[0126] 4.4 Stability investigation

[0127] The test samples of Leonurus granules were measured under the optimal chromatographic conditions at 0, 6h, 12h, 18h, 24h, 30h and 36h after preparation. Peak 11 was used as the reference peak to calculate the RSD value of the peak area. The results are shown in Tables 7 and 8.

[0128] The results showed that the RSD of the relative peak areas of the common peaks were less than 3.97% (n=7), and the RSD of the relative retention time were less than 0.75% (n=7), indicating that the method had good stability.

[0129] Table 7 Relative peak areas of stability test of fingerprint of Leonurus granules

[0130]

[0131] Table 8 Relative retention time of stability study of fingerprint of Leonurus granules

[0132]

[0133] The results showed that the RSD of the relative peak areas of the common peaks were all less than 3.97% (n=7), and the RSD of the relative retention time were all less than 0.75% (n=7), indicating that the method had good stability.

[0134] 4.5 Intermediate precision study

[0135] 4.5.1 Liquid Phase Intermediate Precision Study

[0136] At present, the laboratory liquid chromatography is mainly Thermo Fisher U3000 (equipment number: J-20-C-093, J-20-C-008). One sample of Leonurus granules was prepared for fingerprint determination on different liquid chromatography, and similarity analysis was performed using the 2012 version of the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System". The results are as follows Figure 5 and as shown in Table 9.

[0137] Table 9 Evaluation of the similarity of the instrument intermediate precision of the fingerprint spectrum of Leonurus granules

[0138]

[0139] The results showed that the similarity of the fingerprint spectra of Leonurus granules measured in different numbered liquid phases reached 0.999, indicating that the intermediate precision of the liquid phase was good.

[0140] 4.5.2 Column intermediate precision

[0141] One sample of Leonurus granules was prepared and its fingerprint was determined using three ODS C18 (4.6×250mm, 5μm) columns (department numbers: JZ-SY-C002, QC-C18-24-0401, QC-C18-24-0402). The 2012 edition of the “Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System” was used for similarity analysis. The results are as follows: Figure 6 As shown in Table 10.

[0142] Table 10 Evaluation of the similarity of the intermediate precision of the chromatographic column of the fingerprint of Leonurus granules

[0143]

[0144] The results showed that the similarity of the fingerprint spectra of Leonurus granules measured in different numbered liquid phases reached 0.994, indicating that the intermediate precision of the liquid phase was good.

[0145] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

[0146] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A method for constructing a fingerprint of a motherwort preparation, characterized in that: The following steps are involved: Extracting the Leonurus japonicus preparation using an extraction solvent to prepare a test solution; Performing high performance liquid chromatography on the test solution to construct a fingerprint of the motherwort preparation; The conditions for the high performance liquid chromatography detection include: mobile phase A is formic acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is adopted.

2. The method for constructing the fingerprint of the motherwort preparation according to claim 1, characterized in that: The procedure of the gradient elution includes: From 0 to 20 min, the volume percentage of the mobile phase B increased from 2% to 5%; From 20min to 30min, the volume percentage of the mobile phase B increased from 5% to 12%; From 30min to 56min, the volume percentage of the mobile phase B increased from 12% to 21%; From 56 min to 65 min, the volume percentage of the mobile phase B increased from 21% to 26%.

3. The method for constructing the fingerprint of the motherwort preparation according to claim 1, characterized in that: In the formic acid aqueous solution, the volume percentage of formic acid is 0.1% to 0.3%.

4. The method for constructing the fingerprint of the motherwort preparation according to claim 1, characterized in that: The high performance liquid chromatography detection satisfies at least one of the following conditions: (1) The detection wavelength is 250nm~350nm; (2) Flow rate: 0.5 mL / min~2 mL / min; (3) The injection volume is 0.5 μL~5 μL; (4) The column temperature is 25℃~35℃.

5. The method for constructing the fingerprint of the motherwort preparation according to claim 1, characterized in that: The extraction step satisfies at least one of the following conditions: (1) The extraction method is ultrasonic extraction or reflux extraction; (2) The extraction time is 15min~60min; (3) The extraction solvent includes methanol or a methanol-water solution.

6. The method for constructing the fingerprint of the motherwort preparation according to claim 5, characterized in that: In the methanol aqueous solution, the volume percentage of methanol is 25% to 75%.

7. The method for constructing the fingerprint of the motherwort preparation according to claim 1, characterized in that: The method for constructing the fingerprint of the motherwort preparation also includes the following steps: Dissolve the reference substance using a dissolving reagent to prepare a reference substance solution; Performing high performance liquid chromatography on the reference solution to obtain a reference spectrum; According to the reference spectrum, the chromatographic peaks of the fingerprint spectrum of the motherwort preparation are identified as common peaks; Wherein, the reference substance includes one or more of ferulic acid, leonurine hydrochloride, stachydrine hydrochloride, rutin, quercetin and hyperoside.

8. The method for constructing the fingerprint of the Leonurus japonicus preparation according to any one of claims 1 to 7, characterized in that: The fingerprint of the motherwort preparation contains 15 common peaks, among which two characteristic peaks are identified: peak 9 is leonurine hydrochloride, and peak 13 is ferulic acid.

9. The method for constructing the fingerprint of the Leonurus japonicus preparation according to any one of claims 1 to 7, characterized in that: The motherwort preparation includes motherwort granules, motherwort capsules or motherwort tablets.

10. Application of the method for constructing the fingerprint of the Leonurus herb preparation according to any one of claims 1 to 9 in the quality control of the Leonurus herb preparation.

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