Establishment of characteristic chromatogram of fried wax gourd seed dispensing granules and determination method and application thereof

Abstract

The present application relates to the field of traditional Chinese medicine identification, in particular to characteristic chromatogram construction, content determination method and application of fried winter melon seed formula granules.A characteristic chromatogram construction method of fried winter melon seed preparation includes the following steps: preparation of test sample solution; characteristic chromatogram is obtained by detecting the test sample solution by ultra performance liquid chromatography; the conditions of the ultra performance liquid chromatography detection include: a chromatographic column with octadecylsilane bonded silica gel as filler, detection wavelength of 258-262 nm, methanol as mobile phase A, water as mobile phase B, elution according to a specific gradient program.For the defects of few characteristic peaks and identified peaks in the existing characteristic chromatogram of fried winter melon seed preparation, the characteristic chromatogram construction method provided by the present application has more characteristic peaks and more identified characteristic peaks, which can more comprehensively control the product quality of fried winter melon seed preparation and strengthen the specific identification and overall quality control of fried winter melon seed preparation.

Description

Technical Field

[0001] This invention relates to the field of identification of traditional Chinese medicine, specifically to the construction of characteristic chromatograms and methods and applications for content determination of roasted winter melon seed formula granules. Background Technology

[0002] Winter melon seeds, originally called melon seeds or white melon seeds, are processed from the dried, mature seeds of the winter melon (Benincasa hispida (Thunb.) Cogn.), a plant in the Cucurbitaceae family. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), they are sweet and slightly cold in nature; they enter the lung, spleen, and small intestine meridians; and they have the effects of clearing heat and resolving phlegm, eliminating carbuncles and draining pus, and promoting diuresis. Pharmacological studies have shown that roasted winter melon seeds have anti-tumor, antioxidant, anti-inflammatory, analgesic, and anti-diabetic pharmacological effects.

[0003] Currently, there are relatively few studies on the characteristic spectral analysis and content determination of roasted winter melon seeds, as well as the identification of components. This also limits the quality control of roasted winter melon seed formulation granules. It is generally believed that the characteristic spectral analysis method can obtain more characteristic peaks and identify more characteristic peaks in order to more comprehensively control the quality of formulation granule products. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the shortcomings of the existing characteristic peaks and the lack of identified peaks in the characteristic spectrum of roasted winter melon seed formula granules, thereby providing a method and application for constructing characteristic spectrum of roasted winter melon seed formula granules and determining its content.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a method for constructing a characteristic spectrum of a roasted winter melon seed preparation, comprising the following steps:

[0007] Preparation of the test solution;

[0008] Characteristic chromatograms were obtained by ultra-high performance liquid chromatography (UHPLC) of the test sample solution.

[0009] The conditions for ultra-high performance liquid chromatography (UHPLC) detection include: a chromatographic column packed with octadecylsilane-bonded silica gel, a detection wavelength of 258–262 nm, methanol as mobile phase A, water as mobile phase B, and elution according to the following gradient program:

[0010]

[0011] Preferably, in the ultra-high performance liquid chromatography (UHPLC) detection conditions, the column length is 150 mm, the inner diameter is 2.1 mm, and the particle size of the packing material is 1.6–1.8 μm.

[0012] And / or, the chromatographic column is ACQUITY UPLC HSS T3 or CORTECSUPLCT3;

[0013] And / or, the column temperature is 28–32°C;

[0014] And / or, the flow rate is 0.19–0.21 ml / min;

[0015] And / or, the theoretical plate number, calculated based on the adenosine peak, should be no less than 10,000;

[0016] And / or, the detection wavelength is 260nm.

[0017] Preferably, the preparation process of the test sample solution is as follows: take the test sample, grind it finely, weigh it accurately, add solvent, weigh it, extract it, take it out, cool it, weigh it again, replenish the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate to obtain the test sample solution.

[0018] Preferably, the test sample is a roasted winter melon seed preparation; optionally, the roasted winter melon seed preparation includes roasted winter melon seed granules.

[0019] And / or, the extraction process is ultrasonic treatment and / or heat reflux treatment, preferably ultrasonic treatment;

[0020] And / or, the solvent in the test solution is a methanol aqueous solution or water with a volume percentage of 10% to 30%, preferably a methanol aqueous solution with a volume percentage of 10%;

[0021] And / or, relative to 0.2g of the test sample, the amount of solvent added is 20-25ml, preferably 20ml;

[0022] And / or, the extraction process takes 30 to 40 minutes, preferably 30 minutes.

[0023] Preferably, the method for constructing the characteristic spectrum further includes the steps of extracting the reference medicinal material of winter melon seed with an extractant, filtering the extract, taking the filtrate to prepare a reference solution of the reference medicinal material, and detecting the reference solution of the reference medicinal material by ultra-high performance liquid chromatography in the above-mentioned method for constructing the characteristic spectrum to obtain the reference spectrum of the reference medicinal material. Preferably, the extractant is a 10% (v / v) methanol aqueous solution.

[0024] Preferably, the method for constructing the characteristic chromatogram further includes the step of preparing a reference solution: accurately weighing uracil, hypoxanthine, uridine, guanosine, and adenosine respectively, adding solvents to prepare uracil solution, hypoxanthine solution, uridine solution, guanosine solution, and adenosine solution; and the step of detecting the reference solution using ultra-high performance liquid chromatography in the above construction method to obtain the reference chromatogram.

[0025] Preferably, each 1 ml of uracil solution contains 5-10 μg of uracil, each 1 ml of hypoxanthine solution contains 5-10 μg of hypoxanthine, each 1 ml of uridine solution contains 5-10 μg of uridine, each 1 ml of guanosine solution contains 5-10 μg of guanosine, and each 1 ml of adenosine solution contains 5-10 μg of adenosine.

[0026] And / or, the solvent in the reference solution is a 10% (v / v) aqueous methanol solution.

[0027] Preferably, the characteristic chromatogram of the test sample has 7 characteristic peaks, and the retention times of these peaks should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material; wherein, peaks 1, 3, 4, 6, and 7 should correspond to the retention times of the reference peaks of uracil, hypoxanthine, uridine, guanosine, and adenosine, respectively.

[0028] Peak 4 is the S peak. Calculate the relative retention times of characteristic peaks 2 and 5 with the S peak. The relative retention times of peaks 2 and 5 are within ±10% of the specified values. The specified values ​​are: 0.73 (peak 2) and 1.44 (peak 5).

[0029] Secondly, the present invention also provides a method for determining the content of roasted winter melon seeds in a preparation, comprising the following steps:

[0030] Prepare the test solution and the reference solution;

[0031] The test solution and reference solution were detected using ultra-high performance liquid chromatography (UHPLC) as described in the above-described characteristic chromatogram construction method; the reference standard included adenosine. The preparation methods for the test solution and reference solution in the content determination method are the same as those in the above-described characteristic chromatogram detection method.

[0032] Thirdly, the present invention also provides the application of the above-mentioned characteristic spectrum construction method and / or the above-mentioned content determination method in the quality detection of roasted winter melon seed preparations.

[0033] In this invention, unless otherwise specified, all percentages in the solution are volume percentages.

[0034] The technical solution of this invention has the following advantages:

[0035] 1. A method for constructing a characteristic chromatogram of a roasted winter melon seed preparation, comprising the following steps: preparation of a test solution; obtaining a characteristic chromatogram by ultra-high performance liquid chromatography (UHPLC) of the test solution; wherein the UHPLC detection conditions include: a chromatographic column packed with octadecylsilane-bonded silica gel, a detection wavelength of 258–262 nm, methanol as mobile phase A, water as mobile phase B, and elution according to a specific gradient program. Addressing the shortcomings of existing characteristic chromatograms of winter melon seed formulations, which have few characteristic peaks and identified peaks, the characteristic chromatogram construction method provided by this invention yields more characteristic peaks (7) and more identified characteristic peaks (5), enabling more comprehensive control over the quality of roasted winter melon seed formulation products and strengthening the specific identification and overall quality control of roasted winter melon seed preparations.

[0036] 2. In the method for determining the content of roasted winter melon seed preparations of the present invention, the content of trigonelline in roasted winter melon seed preparations is low (trigonelline is mainly found in the pulp of winter melon and has poor water solubility, making it particularly unsuitable for the content determination of roasted winter melon seed formula granules (formula granules are granules made by extraction, concentration, and drying using water as a solvent; they are only suitable for medicinal materials), and therefore unsuitable as an indicator component for content determination in preparations. Adenosine, on the other hand, is a relatively abundant component in the water extract of roasted winter melon seeds and has pharmacological effects such as immunomodulation, central nervous system sedation, antitumor, antibacterial, and antiviral activity. To quantitatively control the quality of roasted winter melon seed preparations, the determination of adenosine content is chosen as an important indicator for evaluating the quality of roasted winter melon seed preparations. It is more suitable than trigonelline for the quality control of roasted winter melon seed preparations, especially for roasted winter melon seed formula granules.

[0037] 3. In the quality testing application of the roasted winter melon seed preparation of the present invention, existing methods for characteristic chromatograms and content determination of roasted winter melon seed preparations are different, resulting in high testing costs and long testing times (even if the content determination index is the same substance, the testing method is different from the characteristic chromatogram method, and the testing time will be longer). Compared with the prior art, in the quality testing application of the present invention, the liquid chromatography detection method for characteristic chromatograms and content determination is the same, which can save testing costs and time and improve production efficiency. Attached Figure Description

[0038] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 This is a comparative characteristic spectrum of the roasted winter melon seed preparation in this invention;

[0040] Figure 2 These are the characteristic chromatograms of 15 batches of standard decoction of roasted winter melon seeds and 3 batches of formula granules in Example 1 of this invention;

[0041] Figure 3 These are the characteristic chromatograms of three batches of roasted winter melon seed granules in Example 1 of this invention;

[0042] Figure 4 These are characteristic comparison chromatograms of different chromatographic columns in Example 6 of the present invention;

[0043] Figure 5 This is the specific spectral diagram of the roasted winter melon seed particles in Embodiment 7 of the present invention;

[0044] Figure 6 This is the delayed spectrum of roasted winter melon seeds in Example 7 of the present invention;

[0045] Figure 7 This is the adenosine standard curve diagram in Example 7 of the present invention;

[0046] Figure 8 This is a measured characteristic spectrum of the roasted winter melon seed formula granules in Example 8 of the present invention;

[0047] Figure 9 These are characteristic chromatograms of the stir-fried winter melon seed formula granules and reference medicinal materials in Example 8 of this invention. Detailed Implementation

[0048] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0049] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0050] Instruments: ML204T electronic balance (Mettler-Toledo); JY2002 electronic balance (Mettler-Toledo); DZKW-4 electronic constant temperature water bath (Beijing Zhongxing Weiye Instrument Co., Ltd.); KQ-300DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); ACQUITY UPLC H-Class ultra-high performance liquid chromatograph, PDA Detector; TUV Detector, Empower 3 chromatography workstation;

[0051] Chromatographic column: ACQUITY UPLC HSS T3 (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm);

[0052] Test drug: Winter melon seeds (batch number: 2023-022701, Chengdu Glip Biotechnology Co., Ltd.);

[0053] Adenosine reference standard (batch number: 110879-202204, China National Institutes for Food and Drug Control);

[0054] Uric acid reference standard (batch number: 110887-202104, China National Institutes for Food and Drug Control, purity 99.6%);

[0055] Guanosine reference standard (batch number: 111977-202202, China National Institutes for Food and Drug Control, purity 93.6%);

[0056] Uracil reference standard (batch number: 100469-201302, China National Institutes for Food and Drug Control, 99.6%);

[0057] Hypoxanthine reference standard (batch number: 140661-202005, China National Institutes for Food and Drug Control, 99.4%);

[0058] Roasted winter melon seed granules: K437CP01, K437CP02, K437CP03;

[0059] Reagents: Methanol (Merck) was chromatographic grade, methanol and ethanol were analytical grade, and water was Watson distilled water.

[0060] Example 1

[0061] A method for constructing characteristic chromatograms and determining the content of a roasted winter melon seed preparation, using high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition), is described below:

[0062] (1) Solution preparation

[0063] Preparation of the test solution: Take an appropriate amount of roasted winter melon seed granules, grind them finely, and accurately weigh about 0.2g into a stoppered conical flask. Accurately add 20ml of 10vol% methanol aqueous solution, seal tightly, weigh, and sonicate (power 250W, frequency 40kHz) for 30 minutes. Remove, cool, and weigh again. Make up the weight loss with 10vol% methanol aqueous solution, shake well, filter, and collect the filtrate to obtain the test solution.

[0064] Preparation of reference solutions: Based on the availability of reference standards and the representativeness of the substances inferred from each characteristic peak in terms of pharmacological efficacy, uracil, adenosine, guanosine, uridine, and hypoxanthine were selected as reference standards. Simultaneously, considering the requirements for adenosine content determination, a method for preparing the reference solutions was determined. The specific process is as follows: Appropriate amounts of uracil, adenosine, guanosine, uridine, and hypoxanthine reference standards were accurately weighed and added to 10 vol% methanol aqueous solution to prepare solutions containing 8 μg of uracil, adenosine, guanosine, uridine, and hypoxanthine per 1 ml, respectively. These solutions were then used as reference solutions.

[0065] Preparation of reference solution for reference medicinal materials: Take 2g of winter melon seed reference material, add 20ml of 10vol% methanol aqueous solution, heat under reflux for 30 minutes, cool, filter, and take the filtrate to obtain the reference solution for reference medicinal materials.

[0066] (2) Detection by ultra-high performance liquid chromatography

[0067] The chromatographic column was an ACQUITY UPLC HSS T3 (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); the detection wavelength was 260 nm; methanol was used as mobile phase A and water as mobile phase B, with gradient elution performed according to the specifications in Table 1; the column temperature was 30 °C; and the flow rate was 0.2 mL per minute. The theoretical plate number, calculated based on the adenosine peak, should be no less than 10,000.

[0068] Accurately pipette 3 μl each of the reference solution, the reference medicinal material, and the test solution, inject them into the liquid chromatograph, and determine the result.

[0069] Table 1 Gradient Elution Table

[0070]

[0071] In this embodiment, 15 batches of standard decoction of roasted winter melon seeds (K437BJ01~K437BJ15) and three batches of formula granules of roasted winter melon seeds (K437CP01~K437CP03) were used as test samples. The preparation methods of the standard decoction and formula granules are as follows:

[0072] Standard decoction of roasted winter melon seeds: Take 10,000g of roasted winter melon seed slices, crush them, soak them for 30 minutes, and decoct twice. For the first decoction, add 7 times the amount of water as the slices and decoct for 20 minutes. For the second decoction, add 6 times the amount of water as the slices and decoct for 15 minutes. Filter the decoction while it is still hot through a 150-mesh filter cloth. Combine the two decoctions and concentrate them under low temperature and reduced pressure (65℃) until the material-to-liquid ratio is about 1:1 (relative density is 1.05-1.10). Freeze-dry and store in a sealed container to obtain the final product.

[0073] Formula for granulated roasted winter melon seeds: Take 10,000g of roasted winter melon seed slices, crush them, add water and decoct, filter, concentrate the filtrate into a clear extract (dry extract yield is 4.0wt%~7.0wt%), add appropriate amount of excipients, dry (or dry and pulverize), add appropriate amount of excipients again, mix well, granulate to make 1000g, which is the final product.

[0074] The liquid phase spectrum was obtained according to the aforementioned test method, and the test results are shown in Table 2. Figure 2 ( Figure 2 S1 to S18 correspond to items 1 to 18 in Table 2, respectively. Using the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" (version 2012.1), with the liquid chromatography spectrum of sample S1 as the reference spectrum, the full spectrum peaks were matched and fitted to generate a reference spectrum for the roasted winter melon seed formula granules, as shown below. Figure 1 As shown.

[0075] Table 2. List of standard decoction and granule samples of roasted winter melon seeds.

[0076]

[0077] Based on the common pattern diagrams of 15 batches of standard roasted winter melon seed decoction (K437BJ01~K437BJ15), seven common peaks were identified. These peaks yielded reference standards. The retention times of uracil, hypoxanthine, uridine, guanosine, and adenosine corresponded to peaks 1, 3, 4, 6, and 7, respectively. Peak 4 (uridine) was selected as the S peak for the following reasons: 1) Uric acid is a characteristic peak of roasted winter melon seed formulation granules and is an important representative component for quality control of roasted winter melon seeds; 2) Its elution position and peak area are moderate; 3) It has good stability.

[0078] Based on the above test results, it can be seen that by using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012.1 version), a reference spectrum was established for the standard decoction and formula granules of stir-fried winter melon seeds. A total of 7 common peaks were identified, and the correlation with the reference spectrum was good.

[0079] The characteristic chromatogram method and content detection method for roasted winter melon seed formula granules were verified according to the above method. Specifically, three batches of roasted winter melon seed formula granules (K437CP01, K437CP02, and K437CP03) were taken, and their characteristic chromatograms were obtained according to the method in Example 1. The results are as follows. Figure 3 As shown in Table 3, Figure 3 From top to bottom, they are K437CP01 ( Figure 3 (marked as S3), K437CP02 ( Figure 3 (marked as S2), K437CP03 ( Figure 3 (S1). Peak 1 is uracil; peak 3 is hypoxanthine; peak 4 (S) is uridine; peak 6 is guanosine; peak 7 is adenosine.

[0080] Table 3 Retention times and relative retention times for three batches of particles.

[0081]

[0082]

[0083] Based on the above test results, it can be seen that all seven characteristic peaks in the characteristic spectra of the three batches of particles are consistent with the control spectrum. Figure 1 Correspondingly, the relative retention times of each characteristic peak are all within ±10% of the standard decoction value, which meets the requirements. Therefore, the standard for the relative retention time of the particle characteristic peaks is consistent with the standard decoction standard, with the specified values ​​being 0.73 (peak 2) and 1.44 (peak 5).

[0084] Meanwhile, the adenosine content of roasted winter melon seeds was determined, and the results are shown in Table 4.

[0085] Table 4. Results of Particle Content in Roasted Winter Melon Seed Formula

[0086] batch number Adenosine content (mg / g) K437CP01 0.96 K437CP02 0.94 K437CP03 0.95

[0087] Using the above-mentioned content determination method, the adenosine content in the three batches of roasted winter melon seed formula granules was within the content limit range (0.20 mg / g to 1.00 mg / g).

[0088] Example 2: Investigation of the extraction method, extraction solvent, extraction time, and amount of extraction solvent used for the test sample.

[0089] (1) Examination of extraction methods

[0090] Accurately weigh 0.2g of roasted winter melon seed granules (K437CP01), add 20ml of 10vol% methanol aqueous solution, and perform ultrasonic treatment (250W power, 40kHz frequency) and reflux treatment for 30 minutes. Remove, cool, and weigh again. Make up the weight loss with 10vol% methanol aqueous solution, shake well, and filter to obtain the corresponding test solution. Accurately pipette 3μl of the filtrate and inject it into a high-performance liquid chromatograph. Determine the peak area according to the method in Example 1. The results are shown in Table 5 below. Determine the adenosine peak area and calculate the content. The results are shown in Table 6 below.

[0091] Table 5 Results of different extraction methods for roasted winter melon seeds

[0092] Extraction method Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 ultrasound 56093 49732 57336 146183 203286 345009 469365 reflux 55389 49268 58001 147569 213648 356247 462638

[0093] Table 6 Results of different extraction methods for roasted winter melon seeds

[0094]

[0095] The data results show that the peak areas of each characteristic peak are not significantly different under the two extraction methods. Taking all factors into consideration, ultrasound is the preferred extraction method for preparing the roasted winter melon seed granules.

[0096] (2) Investigation of extraction solvent

[0097] Take approximately 0.2 g of roasted winter melon seed granules (K437CP01), grind them finely, and accurately weigh them. Add water, 10 vol% methanol aqueous solution, 30 vol% methanol aqueous solution, 50 vol% methanol aqueous solution, 70 vol% methanol aqueous solution, methanol, 10 vol% ethanol aqueous solution, 30 vol% ethanol aqueous solution, 50 vol% ethanol aqueous solution, 70 vol% ethanol aqueous solution, and 20 ml of ethanol to the solution. Sonicate the solution (250 W, 40 kHz) for 30 min, remove, cool, and weigh again. Make up the weight with the appropriate solvent, shake well, and filter to obtain the corresponding test solution. Accurately pipette 3 μl of the filtrate and inject it into a high-performance liquid chromatograph. Determine the peak area according to the method in Example 1. The results are shown in Table 7 below. Determine the adenosine peak area and calculate the content. The results are shown in Table 8 below.

[0098] Table 7 Results of different extraction solvents for roasted winter melon seeds

[0099]

[0100] Table 8 Results of different extraction solvents for roasted winter melon seeds

[0101]

[0102] The test results show that when 50 vol% methanol aqueous solution, 70 vol% methanol aqueous solution, methanol, 10 vol% ethanol aqueous solution, 30 vol% ethanol aqueous solution, 50 vol% ethanol aqueous solution, 70 vol% ethanol aqueous solution, and ethanol are used as extraction solvents for roasted winter melon seed granules, peaks 1 to 4 have extremely poor peak shapes, and some characteristic peaks are lost. When water and 10 vol%–30 vol% methanol aqueous solutions are used as extraction solvents, the peak areas are larger when using 10 vol% methanol aqueous solution as the solvent. Taking all factors into consideration, 10 vol% methanol aqueous solution is preferred as the extraction solvent for preparing the roasted winter melon seed formula granules test sample.

[0103] (3) Investigation on the amount of extraction solvent used

[0104] Take roasted winter melon seed granules (K437CP01), grind them finely, and accurately weigh approximately 0.2g of each. Add 15ml, 20ml, and 25ml of 10vol% methanol aqueous solution to each, respectively, weigh, and sonicate (250W power, 40kHz frequency) for 30 minutes. Remove, cool, and weigh again. Make up the lost weight with 10vol% methanol aqueous solution, shake well, and filter to obtain the corresponding test solution. Accurately pipette 3μl of the filtrate and inject it into a high-performance liquid chromatograph. Determine the peak area according to the method in Example 1. The results are shown in Table 9 below. Determine the adenosine peak area and calculate the content. The results are shown in Table 10 below. To avoid the influence of the test sample concentration on the peak area, the peak area is converted to the test sample concentration by ratio.

[0105] Table 9. Test results of different extraction solvent usage for roasted winter melon seeds.

[0106]

[0107] Table 10 Test results of different extraction solvent usage for roasted winter melon seeds.

[0108]

[0109] The data results show that when the extraction solvent volume is 15 ml, the sample extraction is incomplete. When the extraction solvent volume is 20 ml and 25 ml, there is no significant difference in the characteristic peaks of the roasted winter melon seed granules. Considering the differences between batches and to ensure complete extraction of the test sample, the preferred solvent volume is 20 ml as the extraction solvent volume for the roasted winter melon seed granule test sample solution.

[0110] (4) Examination of extraction time

[0111] Take approximately 0.2 g of roasted winter melon seed granules (K4337CP01), grind them finely, and accurately weigh them. Add 20 ml of 10 vol% methanol aqueous solution, weigh again, and sonicate (250 W, 40 kHz) for 20 min, 30 min, and 40 min respectively. Remove, cool, and replenish the lost weight with 10 vol% methanol aqueous solution. Shake well, filter, and obtain the corresponding test solution. Accurately pipette 3 μl of the filtrate and inject it into a high-performance liquid chromatograph. Determine the peak area according to the method in Example 1. The results are shown in Table 11 below. Determine the adenosine peak area and calculate the content. The results are shown in Table 12 below.

[0112] Table 11 Results of different extraction times for roasted winter melon seeds

[0113]

[0114]

[0115] Table 12 Results of different extraction times for roasted winter melon seeds

[0116]

[0117] The data results show that there are almost no differences in the characteristic peaks of roasted winter melon seeds at different extraction times of 30 minutes and 40 minutes. Therefore, 30 minutes is preferred as the extraction time for the roasted winter melon seed sample solution.

[0118] Based on the above results, the preferred method for preparing the test solution of roasted winter melon seed granules is as follows: Take an appropriate amount of roasted winter melon seed granules, grind them finely, take approximately 0.2 g, accurately weigh it, place it in a stoppered conical flask, and accurately add 20 ml of 10 vol% methanol aqueous solution.

[0119] Seal the container, weigh it, sonicate it (power 250W, frequency 40kHz) for 30 minutes, remove it, let it cool, weigh it again, make up the weight loss with 10 vol% methanol aqueous solution, shake it well, filter it, and collect the filtrate to obtain the product.

[0120] Example 3: Investigation at different column temperatures

[0121] Roasted winter melon seed granules (batch number: K437CP01) were used to prepare a test solution according to the method described in Example 1. The solution was then tested at different column temperatures (28℃, 30℃, and 32℃) using the method described in Example 1 to examine the robustness of the experimental method to different column temperatures. The characteristic spectral test results are shown in Table 13 below, and the content determination test results are shown in Table 14 below.

[0122] Table 13 Retention Time and Relative Retention Time Results of Characteristic Spectra at Different Column Temperatures

[0123]

[0124] Table 14. Results of particle content determination in roasted winter melon seeds at different column temperatures.

[0125]

[0126] According to the above test results, under different column temperature conditions, the relative retention time of each characteristic peak is within ±10% of the specified value. Under different column temperature conditions, the RSD of the roasted winter melon seed formula granules is 1.17%, indicating that the column temperature durability of the present invention is good.

[0127] Example 4: Investigation of different flow rates

[0128] Roasted winter melon seed granules (batch number: K437CP01) were used to prepare a test solution according to the method described in the examples. The solution was then tested at different flow rates of 0.19 ml / min, 0.20 ml / min, and 0.21 ml / min, as described in Example 1, to examine the robustness of the experimental method to different flow rates. The characteristic spectral test results are shown in Table 15 below, and the content determination test results are shown in Table 16 below.

[0129] Table 15 Retention Time Results of Characteristic Maps for Different Flow Velocities

[0130]

[0131] Table 16 Results of Particle Content in Frying Winter Melon Seed Formulas with Different Flow Rates

[0132]

[0133] According to the above test results, under different flow rate conditions, the relative retention time of each characteristic peak is within ±10% of the specified value, and the RSD of the roasted winter melon seed formula granules is 1.74%; indicating that the flow rate durability of the present invention is good.

[0134] Example 5: Investigation at Different Wavelengths

[0135] Roasted winter melon seed granules (batch number: K437CP01) were used to prepare a test solution according to the method described in the examples. The solution was then measured at different wavelengths (258 nm, 260 nm, and 262 nm) as described in Example 1 to examine the robustness of the experimental method to different wavelengths. The characteristic spectral test results are shown in Table 17 below, and the content determination test results are shown in Table 18 below.

[0136] Table 17 Retention Time Results of Characteristic Spectra at Different Wavelengths

[0137]

[0138] Table 18 Results of Particle Content in Different Wavelength Roasted Winter Melon Seed Formulas

[0139]

[0140]

[0141] According to the above test results, under different wavelength conditions, the relative retention time of each characteristic peak is within ±10% of the specified value, and the RSD of the roasted winter melon seed formula granules is 0.68%, indicating that the wavelength durability of the present invention is good.

[0142] Example 6: Investigation of different chromatographic columns

[0143] Roasted winter melon seed granules (batch number: K437CP01) were used to prepare a test solution according to the preparation method of the test solution in Example 1. Different chromatographic columns were used, and the determination was performed according to the method in Example 1 to examine the robustness of the experimental method to the chromatographic column. The results are shown in Tables 19 and 20. Figure 4 As shown.

[0144] Column 1: ACQUITY UPLC BEH Shield RP18 column (column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm);

[0145] Column 2: CORTECSUPLCT3 column (150 mm in length, 2.1 mm in inner diameter, and 1.6 μm in particle size);

[0146] Column 3: ACQUITY UPLC HSS T3 column (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm);

[0147] Table 19 Retention times and relative retention times of characteristic chromatograms observed on different chromatographic columns

[0148]

[0149] Table 20: Results of Spectroscopy Analysis of Roasted Winter Melon Seed Granules Using Different Chromatographic Columns

[0150]

[0151] Based on the above test results, it can be seen that some chromatographic peaks were lost in column 1, and the retention time of peak 1 in column 2 was relatively early, making it susceptible to interference from the preceding peak. Taking all factors into consideration, it is recommended to select column ACQUITY UPLC HSS T3. The RSD of the content determination result of roasted winter melon seed formula granules was 1.19%. Different chromatographic columns have better durability than different methods for determining the content of roasted winter melon seed formula granules.

[0152] Example 7

[0153] This embodiment is used to perform methodological verification of the detection method in Embodiment 1:

[0154] (1) Specificity test

[0155] Take the test solution, reference solution, and blank solution of roasted winter melon seed granules, and inject them according to the chromatographic conditions of Example 1. Observe the peak positions of the test solution and reference solution, and the peak status of the blank solution. The results are as follows. Figure 5 As shown. According to Figure 5 The results show that the blank solution spectrum has no effect on the peak position of the test solution, indicating good specificity.

[0156] (2) Delayed testing

[0157] Take the sample solution of roasted winter melon seed granules, inject it according to the chromatographic conditions of Example 1, and record the characteristic retention chromatogram of the sample at twice the sampling time. The results are as follows. Figure 6 As shown. According to Figure 6 The results showed that no obvious hysteresis peak appeared after 30 minutes, and this chromatographic method met the analytical requirements of the Chinese Pharmacopoeia 2020 edition.

[0158] (3) Accuracy

[0159] Accurately weigh 2.459 mg of adenosine reference standard and place it in a 10 ml volumetric flask. Add an appropriate amount of 10 vol% methanol aqueous solution to dissolve it and dilute to the mark. Shake well to obtain the stock solution of the reference standard. Accurately transfer 1 ml, 2 ml, and 3 ml of the stock solution of the reference standard to 100 ml volumetric flasks, add 10% methanol to the mark, and shake well to obtain the mixed reference standard 1, mixed reference standard 2, and mixed reference standard 3 of roasted winter melon seeds.

[0160] Nine portions of roasted winter melon seed granules (K437CP01, adenosine content 0.96 mg / g) with known content were accurately weighed (0.1 g each) and numbered 1 to 9. For portions 1 to 3, 20 ml of the prepared mixed reference standard 1 solution was accurately added; for portions 4 to 6, 20 ml of the mixed reference standard 2 solution was accurately added; and for portions 7 to 9, 20 ml of the mixed reference standard 3 solution was accurately added. The mixture was weighed, sonicated (300 W, 40 kHz) for 30 minutes, cooled, and weighed again. The lost weight was replenished with 10 vol% methanol aqueous solution, shaken well, filtered, and the filtrate was collected. The test results are shown in Table 21 below. The recovery rate (%) was calculated as: (measured value - amount of analyte in the test sample) / amount of reference standard added × 100%.

[0161] Table 21 Results of Adenosine Recovery Rate Test

[0162]

[0163] Based on the above results, the recovery rate of adenosine ranged from 97.91% to 102.68%, with an average recovery rate of 100.2% and an RSD of 1.44%, which meets the method validation recovery rate requirements of the Chinese Pharmacopoeia 2020 edition, indicating that the results obtained using this method are accurate.

[0164] (4) Linearity and range

[0165] Take an appropriate amount of adenosine reference standard, accurately weigh 3.628 mg and place it in a 100 ml volumetric flask, add 10 vol% methanol aqueous solution and dilute to the mark to obtain the adenosine reference standard stock solution A;

[0166] 1) Accurately pipette 5 ml of solution A and dilute to 10 ml to prepare a mixed solution containing 0.01800 mg of adenosine per ml as reference standard 4.

[0167] 2) Accurately pipette 5 ml of solution A, dilute to 20 ml, and prepare a mixed solution containing 0.00902 mg of adenosine per ml as a reference standard.

[0168] 3) Accurately pipette 1 ml of solution A and dilute to 10 ml to prepare a mixed solution containing 0.00361 mg of adenosine per ml as reference standard 2.

[0169] 4) Accurately pipette 1 ml of solution A and dilute to 20 ml to prepare a mixed solution containing 0.00180 mg of adenosine per ml as reference standard 1.

[0170] Accurately pipette 3 μl of each reference solution and inject it into the ultra-high performance liquid chromatograph. Measure the peak area of ​​adenosine according to the conditions and methods described in the assay section. Plot the peak area as the ordinate (y) and the concentration (mg / ml) as the abscissa (x). Observe whether linearity is observed, and then perform linear regression using the least squares method to obtain the regression equation: adenosine y = 49790903.4187x + 4.0000, R0 2 =0.9999, with a linear range of 0.00180-0.03610 mg / ml. Test results are as follows... Figure 7 As shown in Table 22.

[0171] Table 22 Concentration and area of ​​adenosine standard curve

[0172] serial number 1 2 3 4 5 Adenosine concentration (μg / ml) 0.00180 0.00361 0.00902 0.01800 0.03610 Peak area 89365 182229 444343 904232 1794429

[0173] Linear range: Based on the results of precision, accuracy and linearity experiments, the linear range of adenosine is 0.00180 mg / g to 0.03610 mg / g.

[0174] (5) Stability test

[0175] Take 0.2g of roasted winter melon seed formula granules (batch number: K437CP01) as a sample, prepare the test solution according to the method of Example 1, and determine the characteristic spectrum at 0h, 2h, 4h, 6h, 8h, 10h, 12h and 24h according to the method of Example 1. Using peak 4 (uridine) as the reference peak, calculate the relative peak area and relative retention time of the other characteristic peaks, and calculate the RSD. The detection results of the characteristic spectrum are shown in Tables 23 and 24, and the detection results of the content determination (adenosine) are shown in Table 25.

[0176] Table 23 Stability Test (Characteristic Spectrum) Retention Time and Relative Retention Time Table

[0177]

[0178] Table 24 Stability Test (Characteristic Spectrum) Peak Area and Relative Peak Area Table

[0179]

[0180]

[0181] The stability test results showed that, after 24 hours of solution stability testing, the relative retention time (RSD) of each characteristic peak of the roasted winter melon seed granules was within the range of 0.47% to 1.25%, and the RSD of the relative peak area was within the range of 1.66% to 3.49%. The roasted winter melon seed granule test solution used in this experiment was stable within 24 hours.

[0182] Table 25 Results of the stability test of roasted winter melon seeds (including test results)

[0183]

[0184] According to the stability test results above, after examining the stability of the solution over 24 hours, the RSD of the peak area of ​​the roasted winter melon seed particles was 1.28%. The roasted winter melon seed particle test solution used in this experiment was stable within 24 hours.

[0185] (6) Repeatability test

[0186] Precision: Using a Waters UPLC H-Class with a TUV detector, 0.2g of roasted winter melon seed granules (batch number: K437CP01) was taken and divided into 6 portions. The chromatographic conditions of Example 1 were used to determine the characteristic chromatograms. Peak 4 (uridine) was used as the reference peak. The relative peak area and relative retention time of the remaining chromatographic peaks were calculated, and the RSD was calculated. The results of the characteristic chromatogram detection are shown in Tables 26 and 27, and the results of the content determination (adenosine) are shown in Table 28.

[0187] Table 26 Retention time and relative retention time of roasted winter melon seeds as a measure of repeatability (characteristic spectrum)

[0188]

[0189]

[0190] Table 27 Peak area and relative peak area of ​​the repeatability (characteristic spectrum) of roasted winter melon seeds.

[0191]

[0192] The results of the repeatability experiment show that the relative retention time RSD of the seven characteristic peaks is in the range of 0.04% to 0.33%, and the relative peak area RSD is in the range of 1.48% to 3.69%, indicating that the characteristic spectrum has good repeatability.

[0193] Table 28 Repeatability Tests (including measurements)

[0194]

[0195] Based on the above results, the RSD of the adenosine content in the sample was found to be 1.0%, indicating good repeatability. According to General Chapter 9101 of the 2020 edition of the Chinese Pharmacopoeia, the repeatability requirement is within 4% when the content of the analyte is 0.01%. Therefore, this method meets the analytical requirements.

[0196] Intermediate precision: Using an Agilent ultra-high performance liquid chromatograph with a TUV detector, 0.2g of roasted winter melon seed granules (batch number: K437CP01) was taken and divided into 6 portions. The chromatographic conditions of Example 1 were used to determine the characteristic chromatograms. Peak 4 (uridine) was used as the reference peak. The relative retention times of the remaining characteristic peaks were calculated and the RSD was calculated. The results of the characteristic chromatogram detection are shown in Table 29. The results of the content determination (adenosine) are shown in Table 30.

[0197] Table 29: Retention Time and Relative Retention Time for Intermediate Precision (Characteristic Spectrum) of Roasted Winter Melon Seeds

[0198]

[0199] The intermediate precision test results show that the relative retention time RSD of the seven identifier peaks of the six experimental samples is in the range of 0.02% to 1.04%; the relative retention time RSD between instruments is in the range of 0.25% to 1.36%, indicating that the intermediate precision of the characteristic spectrum is good.

[0200] Table 30 Intermediate Precision (including measurement) Results

[0201]

[0202] Based on the above results, the RSD of adenosine content was 0.60%, and the RSD of adenosine content in roasted winter melon seed granules determined by different instruments (Waters ultra-high performance liquid chromatograph and Agilent ultra-high performance liquid chromatograph) was 1.72%. According to General Chapter 9101 of the 2020 edition of the Chinese Pharmacopoeia, the precision requirement is within 4% when the content of the analyte is 0.01%. Therefore, the intermediate precision of this experiment meets the analytical requirements.

[0203] Example 8

[0204] Method validation was performed using roasted winter melon seed formula granules as the sample. The same test sample (roasted winter melon seed formula granules, batch number: K437CP01) was used for characteristic chromatogram and content testing. The characteristic chromatogram results are as follows: Figure 8 The results of the content determination are shown in Table 31 and Table 32. The characteristic chromatograms of the test sample and the reference medicinal material are shown in Table 32. Figure 9 As shown.

[0205] Table 31 System Applicability (Characteristic Map) Test Results

[0206] Retention time Peak area USP separation USP Theoretical Plates USP trail 4.035 56840 2.20 10236 1.10 6.130 49287 1.45 13993 1.00 6.552 61359 2.64 18534 1.11 8.271 155471 2.22 28804 1.43 11.815 212832 9.17 29625 1.44 13.578 349796 7.90 108756 1.37 18.391 467617 4.91 167515 1.33

[0207] The test results above show that the peak shape, resolution, tailing factor, and theoretical plate number of the roasted winter melon seed granules all meet the requirements of the Chinese Pharmacopoeia 2020 edition.

[0208] Table 32 System Suitability (Including Tests) Results (5 Repeated Injections of the Same Test Solution)

[0209] Sample injection Retention time Peak area USP trail USP Theoretical Plates 1 18.403 393978 1.24 218748 2 18.332 393696 1.24 200205 3 18.346 394245 1.24 196172 4 18.368 392801 1.24 196672 5 18.332 394965 1.24 191218 mean - 393937 - 200603 RSD% - 0.20 - -

[0210] The test results show that the average theoretical plate number of adenosine is 200603, the tailing factor is 1.24, and the repeatability RSD is 0.20%. The system suitability meets the analytical requirements of the Chinese Pharmacopoeia 2020 edition, and the instrument is in normal condition.

[0211] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic map of stir-fried wax gourd seed preparation, characterized by, It comprises the following steps: Preparation of the test sample solution, which is prepared by taking the test sample, grinding it, accurately weighing it, adding the solvent, weighing the weight, extracting and processing, taking it out, cooling it, re-weighing it, making up the weight loss with the corresponding solvent, shaking it, filtering it, and taking the subsequent filtrate to obtain the test sample solution; the test sample is fried wax gourd seed preparation, and the fried wax gourd seed preparation is fried wax gourd seed formula granules; the solvent in the test sample solution is a 10%-30% methanol aqueous solution or water by volume percentage; Preparation of the reference solution of the control sample: accurately weigh the uracil, hypoxanthine, uridine, guanosine, and adenosine, respectively, add the solvent to prepare the uracil solution, hypoxanthine solution, uridine solution, guanosine solution, and adenosine solution; Performing ultra-high performance liquid chromatography detection on the test sample solution and the reference solution of the control sample to obtain the characteristic spectrum and the control sample spectrum; The conditions for the ultra-high performance liquid chromatography detection include: a chromatographic column with octadecylsilane-bonded silica gel as the filler, the chromatographic column is an ACQUITY UPLC HSS T3, the column length of the chromatographic column is 150 mm, the inner diameter is 2.1 mm, the particle size of the filler is 1.8 μm, the detection wavelength is 258-262 nm, methanol is used as the mobile phase A, water is used as the mobile phase B, and elution is performed according to the following gradient program: 。 2. The feature map construction method of claim 1, wherein, In the conditions for the ultra-high performance liquid chromatography detection, the column temperature is 28-32 ℃; and / or, the flow rate is 0.19-0.21 ml / min; and / or, the theoretical plate number calculated based on the adenosine peak should be not less than 10,000; and / or, the detection wavelength is 260 nm.

3. The feature map construction method of claim 1, wherein, The extraction processing is ultrasonic treatment or / and hot reflux treatment; and / or, the solvent in the test sample solution is a 10% methanol aqueous solution by volume percentage; and / or, the amount of the solvent added is 20-25 ml relative to 0.2 g of the test sample; and / or, the duration of the extraction processing is 30-40 min.

4. The feature map construction method of claim 3, wherein, The extraction processing is ultrasonic treatment; and / or, the amount of the solvent added is 20 ml relative to 0.2 g of the test sample; and / or, the duration of the extraction processing is 30 min.

5. The feature map construction method according to any one of claims 1 to 4, characterized in that, The characteristic spectrum construction method further comprises the steps of preparing a reference solution of the control medicinal material by filtering and taking the subsequent filtrate after adding the extractant to the control medicinal material, and obtaining the reference spectrum of the control medicinal material by performing the ultra-high performance liquid chromatography detection on the reference solution of the control medicinal material according to the steps of the characteristic spectrum construction method in any one of claims 1-4.

6. The feature map construction method of claim 5, wherein, The extractant is a 10% methanol aqueous solution by volume percentage.

7. The feature map construction method of claim 1, wherein, Each 1 ml of the uracil solution contains 5-10 μg of uracil, each 1 ml of the hypoxanthine solution contains 5-10 μg of hypoxanthine, each 1 ml of the uridine solution contains 5-10 μg of uridine, each 1 ml of the guanosine solution contains 5-10 μg of guanosine, and each 1 ml of the adenosine solution contains 5-10 μg of adenosine; and / or, the solvent in the reference solution of the control sample is a 10% methanol aqueous solution by volume percentage.

8. The feature map construction method according to claim 1 or 2, characterized by, The characteristic spectrum of the test product has 7 characteristic peaks, and should correspond to the retention time of 7 characteristic peaks in the reference chromatogram of the control medicinal material; wherein, peak 1, peak 3, peak 4, peak 6 and peak 7 should correspond to the retention time of the reference peaks of uracil, hypoxanthine, uridine, guanosine and adenosine control respectively; Peak 4 is an S peak, and the relative retention time of peak 2 and peak 5 to the S peak is calculated, and the relative retention time of peak 2 and peak 5 is within ±10% of the specified value; the specified value is: peak 2 is 0.73, and peak 5 is 1.

44.

9. A method for the determination of the content of a roasted wax gourd seed preparation, characterized by The method comprises the following steps: The test product solution and the control reference solution are prepared; the preparation process of the test product solution is as follows: the test product is finely ground, precisely weighed, and then solvent is added and weighed, extracted and treated, taken out, cooled, weighed again, the lost weight is made up with corresponding solvent, shaken, filtered, and the filtered solution is taken as the test product solution; the solvent in the test product solution is 10%-30% methanol aqueous solution or water; the test product is fried wax gourd seed preparation, and the fried wax gourd seed preparation is fried wax gourd seed formula granules; The test product solution and the control reference solution are detected by the ultra-high performance liquid chromatography in the characteristic spectrum construction method of any one of claims 1-8; wherein, the control product comprises adenosine.

10. The application of the characteristic spectrum construction method of any one of claims 1-8 or / and the content determination method of claim 9 in the quality detection of fried wax gourd seed preparation.

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