Characteristic chromatogram construction and content determination method and application of fried Chinese waxgourd seed formula granules

Abstract

The invention relates to the field of traditional Chinese medicine identification, in particular to a specific chromatogram construction and content determination method and application of fried Chinese waxgourd seed formula granules. The specific chromatogram construction method of the fried Chinese waxgourd seed preparation comprises the following steps: preparing a test solution; performing ultra-high performance liquid chromatography detection on the test solution to obtain a characteristic spectrum; the detection conditions of the ultra-high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is taken as a chromatographic column of a filler, the detection wavelength is 258-262 nm, methanol is taken as a mobile phase A, water is taken as a mobile phase B, and elution is performed according to a specific gradient procedure. Aiming at the defect that the existing characteristic spectrum of the fried Chinese waxgourd seed preparation has few characteristic peaks and identification peaks, the characteristic spectrum construction method provided by the invention can obtain more characteristic peaks and more identified characteristic peaks, so that the product quality of the fried Chinese waxgourd seed preparation can be more comprehensively controlled, and the specific identification and the overall quality control of the fried Chinese waxgourd seed preparation are enhanced.

Description

Technical Field

[0001] The present invention relates to the field of identification of traditional Chinese medicines, and particularly to the construction of characteristic chromatograms, content determination methods and applications of stir-fried winter melon seed formula granules. Background Art

[0002] The original name of winter melon seeds was melon seeds, also known as white melon seeds. Stir-fried winter melon seeds are the processed products of the dried and mature seeds of the winter melon (Benincasa hispida (Thunb.) Cogn.) of the Cucurbitaceae family. It was first recorded in "Shennong Ben Cao Jing", with a sweet taste and slightly cold nature; it belongs to the lung, spleen, and small intestine meridians; it has the effects of clearing heat and resolving phlegm, eliminating carbuncles and discharging pus, and promoting diuresis. Pharmacological studies have shown that stir-fried winter melon seeds have pharmacological effects such as anti-tumor, antioxidant, anti-inflammatory, analgesic, and anti-diabetic effects.

[0003] At present, there are relatively few studies on the characteristic chromatograms and content determination of stir-fried winter melon seeds, and there are also few component identifications. There are also relatively large limitations in the quality control of stir-fried winter melon seed formula granules. Generally, it is considered that the more characteristic peaks obtained by the characteristic chromatogram method and the more identified characteristic peaks, the more comprehensive the quality control of the formula granule products can be. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the small number of characteristic peaks and identified peaks in the characteristic chromatogram of the existing stir-fried winter melon seed formula granules, so as to provide a method for constructing a characteristic chromatogram, a content determination method and an application of stir-fried winter melon seed formula granules to solve the above problems.

[0005] To achieve the above object, the present invention provides the following technical solutions:

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

[0007] Preparation of the test solution;

[0008] Performing ultra-high performance liquid chromatography detection on the test solution to obtain a characteristic chromatogram;

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

[0010]

[0011] Preferably, in the conditions for the ultra-high performance liquid chromatography detection, the column length of the chromatographic column is 150 mm, the inner diameter is 2.1 mm, and the particle size of the filler is 1.6-1.8 μm;

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

[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 number of theoretical plates calculated by the adenosine peak should be not less than 10,000;

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

[0017] Preferably, the preparation process of the test solution is as follows: Take the test sample, grind it finely, weigh it accurately, add the solvent, weigh the weight, perform extraction treatment, take it out, let it cool, weigh the weight again, make up the lost weight with the corresponding solvent, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0018] Preferably, the test sample is the stir-fried winter melon seed preparation. Optionally, the stir-fried winter melon seed preparation includes stir-fried winter melon seed formula granules;

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

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

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

[0022] And / or, the duration of the extraction treatment is 30 - 40 min, preferably 30 min.

[0023] Preferably, the characteristic fingerprint construction method further includes the steps of preparing a reference solution of the control medicinal material by extracting the control medicinal material of winter melon seed with an extractant, filtering, and taking the subsequent filtrate, and detecting the reference solution of the control medicinal material by the ultra-high performance liquid chromatography method in the above characteristic fingerprint construction method to obtain the reference fingerprint of the control medicinal material. Preferably, the extractant is an aqueous methanol solution with a volume percentage of 10%.

[0024] Preferably, the characteristic fingerprint construction method further includes the preparation step of the reference solution of the reference substance: Weigh uracil, hypoxanthine, uridine, guanosine, and adenosine accurately, and add the solvent to make uracil solution, hypoxanthine solution, uridine solution, guanosine solution, and adenosine solution; and the step of detecting the reference solution of the reference substance by the ultra-high performance liquid chromatography method in the above construction method to obtain the reference fingerprint of the reference substance.

[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 substance solution is an aqueous methanol solution with a volume percentage of 10%.

[0027] Preferably, the characteristic chromatogram of the test sample has 7 characteristic peaks, and should correspond to the retention times of the 7 characteristic peaks in the chromatogram of the reference crude drug; among them, peak 1, peak 3, peak 4, peak 6, and peak 7 should respectively correspond to the retention times of the reference peaks of uracil, hypoxanthine, uridine, guanosine, and adenosine reference substances;

[0028] Peak 4 is the S peak, calculate the relative retention times of peak 2 and peak 5 characteristic peaks with respect to the S peak, and the relative retention times of peak 2 and peak 5 are within the range of ±10% of the specified values; the specified values are: 0.73 (peak 2), 1.44 (peak 5).

[0029] In the second aspect, the present invention also provides a method for determining the content of stir-fried winter melon seeds preparation, including the following steps:

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

[0031] Respectively use the ultra-high performance liquid chromatography method in the above-mentioned characteristic chromatogram construction method to detect the test sample solution and the reference substance solution; among them, the reference substance includes adenosine. The preparation methods of the test sample solution and the reference substance solution in the content determination method are the same as those of the test sample solution and the reference substance solution in the above-mentioned characteristic chromatogram detection method.

[0032] In the third aspect, the present invention also provides the application of the above-mentioned characteristic chromatogram construction method or / and the above-mentioned content determination method in the quality detection of stir-fried winter melon seeds preparation.

[0033] In the present invention, unless otherwise specified, the percentages of the solutions are all volume percentages.

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

[0035] 1. A method for constructing a characteristic fingerprint of a stir-fried winter melon seed preparation, comprising the following steps: preparation of a test solution; performing ultra-high performance liquid chromatography (UHPLC) detection on the test solution to obtain a characteristic fingerprint; the conditions for the UHPLC detection include: a chromatographic column filled with octadecylsilyl-bonded silica gel, a detection wavelength of 258 - 262 nm, methanol as mobile phase A, water as mobile phase B, and elution carried out according to a specific gradient program. Aiming at the defects of few characteristic peaks and identified peaks in the characteristic fingerprint of the existing winter melon seed formula granules, the characteristic fingerprint construction method provided by the present invention has more characteristic peaks (7) and more identified characteristic peaks (5), can more comprehensively control the product quality of the stir-fried winter melon seed formula granules, and strengthens the specific identification and overall quality control of the stir-fried winter melon seed preparation.

[0036] 2. In the content determination method of the stir-fried winter melon seed preparation of the present invention, since the content of trigonelline in the stir-fried winter melon seed medicinal material is relatively low in the stir-fried winter melon seed preparation (trigonelline mainly exists in the winter melon pulp and has poor water solubility, especially not suitable for the content determination of stir-fried winter melon seed formula granules (formula granules are granule preparations made by taking water as a solvent, through extraction, concentration, and drying), and it is only applicable to the medicinal material), it is not suitable as an index component for the content determination of the preparation. Adenosine, as a component with a relatively high content in the water extract of stir-fried winter melon seeds, has pharmacological effects such as immunomodulation, sedation of the central nervous system, anti-tumor, antibacterial, and antiviral. In order to quantitatively control the quality of the stir-fried winter melon seed preparation, the content of adenosine is selected as an important index for evaluating the quality of the stir-fried winter melon seed preparation, which is more suitable for the quality control of the stir-fried winter melon seed preparation than trigonelline, especially suitable for stir-fried winter melon seed formula granules.

[0037] 3. In the quality detection application of the stir-fried winter melon seed preparation of the present invention, the characteristic fingerprint and content determination of the existing stir-fried winter melon seed preparation are different methods, with problems such as high detection cost and long time consumption (even if the content determination index is the same substance, the detection time will be longer because the determination method is different from that of the characteristic fingerprint). Compared with the prior art, in the quality detection application of the present invention, the liquid phase detection methods of the characteristic fingerprint and content determination are the same, which can save detection cost and time and improve production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0039] Figure 1 It is the control characteristic fingerprint of the stir-fried winter melon seed preparation in the present invention;

[0040] Figure 2 They are the characteristic chromatograms of 15 batches of standard decoctions of stir-fried winter melon seeds and 3 batches of formula granules in Example 1 of the present invention;

[0041] Figure 3 They are the three-batch verification characteristic chromatograms of stir-fried winter melon seed granules in Example 1 of the present invention;

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

[0043] Figure 5 They are the specificity chromatograms of stir-fried winter melon seed granules in Example 7 of the present invention;

[0044] Figure 6 They are the delay chromatograms of stir-fried winter melon seed granules in Example 7 of the present invention;

[0045] Figure 7 They are the standard curve graphs of adenosine in Example 7 of the present invention;

[0046] Figure 8 They are the measured characteristic chromatograms of stir-fried winter melon seed formula granules in Example 8 of the present invention;

[0047] Figure 9 They are the characteristic chromatograms of stir-fried winter melon seed formula granules and reference substances of control medicinal materials in Example 8 of the present invention. Detailed implementation manners

[0048] The following embodiments are provided to better further understand the present invention, which are not limited to the described optimal implementation manners, and do not constitute limitations to the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other existing technologies falls within the protection scope of the present invention.

[0049] For those not specifying specific experimental steps or conditions in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not specifying the manufacturers, they are all conventional reagent products that can be obtained through commercial purchase.

[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 type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); ACQUITY UPLC H-Class ultra-high performance liquid chromatograph, PDA Detector detector; TUV Detector ultraviolet detector, Empower 3 chromatographic workstation;

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

[0052] Test drugs: Control crude drug of Benincasa hispida var. chieh-qua seeds (batch number: 2023-022701, Chengdu Gelipu Biotechnology Co., Ltd.);

[0053] Reference substance of adenosine (batch number: 110879-202204, National Institutes for Food and Drug Control);

[0054] Reference substance of uridine (batch number: 110887-202104, National Institutes for Food and Drug Control, purity 99.6%);

[0055] Reference substance of guanosine (batch number: 111977-202202, National Institutes for Food and Drug Control, purity 93.6%);

[0056] Reference substance of uracil (batch number: 100469-201302, National Institutes for Food and Drug Control, 99.6%);

[0057] Reference substance of hypoxanthine (batch number: 140661-202005, National Institutes for Food and Drug Control, 99.4%);

[0058] Fried Benincasa hispida var. chieh-qua seed formula granules: K437CP01, K437CP02, K437CP03;

[0059] Reagents: Methanol (Merck) is chromatographically pure, methanol and ethanol are both analytically pure, and water is Watsons distilled water.

[0060] Example 1

[0061] A method for constructing the characteristic chromatogram and determining the content of a fried Benincasa hispida var. chieh-qua seed preparation is determined by high performance liquid chromatography (General Principles 0512 of the Chinese Pharmacopoeia 2020 Edition). The specific process is as follows:

[0062] (1) Solution preparation

[0063] Preparation of the test solution: Take an appropriate amount of fried Benincasa hispida var. chieh-qua seed formula granules, grind them finely, take about 0.2 g, weigh accurately, and place them in a stoppered conical flask. Accurately add 20 ml of 10 vol% methanol aqueous solution, stopper tightly, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, take out, let cool, weigh again, make up the lost weight with 10 vol% methanol aqueous solution, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0064] Preparation of reference substance solution: According to the availability of reference substances and the representativeness of the substances inferred from each characteristic peak in terms of pharmacodynamic effects, uracil, adenosine, guanosine, uridine, and hypoxanthine were determined as reference substances for the reference solution. At the same time, taking into account the requirements for the determination of adenosine content, the preparation method of the reference solution was determined. The specific process was as follows: Appropriate amounts of uracil, adenosine, guanosine, uridine, and hypoxanthine reference substances were accurately weighed and dissolved in 10 vol% methanol aqueous solution to prepare a uracil solution containing 8 μg per 1 ml, an adenosine solution containing 8 μg per 1 ml, a guanosine solution containing 8 μg per 1 ml, a uridine solution containing 8 μg per 1 ml, and a hypoxanthine solution containing 8 μg per 1 ml, respectively, as the reference substance solution.

[0065] Preparation of reference solution of control crude drug: Take 2 g of control crude drug of Benincasa hispida (Thunb.) Cogn. var. chieh-qua How seeds, add 20 ml of 10 vol% methanol aqueous solution, heat under reflux for 30 minutes, cool, filter, and take the subsequent filtrate to obtain the reference solution of control crude drug.

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

[0067] The chromatographic column was 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 was used as mobile phase B, and gradient elution was carried out according to the regulations in Table 1 below; the column temperature was 30 °C; the flow rate was 0.2 ml per minute. The number of theoretical plates calculated based on the adenosine peak should not be less than 10000.

[0068] Accurately pipette 3 μl of each of the reference substance solution, reference solution of control crude drug, and test solution, inject them into the liquid chromatograph, and determine, then the results can be obtained.

[0069] Table 1 Gradient elution table

[0070]

[0071] In this example, 15 batches of standard decoctions of stir-fried Benincasa hispida (Thunb.) Cogn. var. chieh-qua How seeds (K437BJ01 - K437BJ15) and three batches of formula granules of stir-fried Benincasa hispida (Thunb.) Cogn. var. chieh-qua How seeds (K437CP01 - K437CP03) were taken as test samples, and the preparation methods of the standard decoctions and formula granules were as follows:

[0072] Standard decoction of stir-fried Benincasa hispida (Thunb.) Cogn. var. chieh-qua How seeds: Take 10000 g of stir-fried Benincasa hispida (Thunb.) Cogn. var. chieh-qua How seeds, crush them, soak for 30 minutes, decoct twice. For the first decoction, add 7 times the amount of water of the medicinal materials and decoct for 20 minutes; for the second decoction, add 6 times the amount of water of the medicinal materials and decoct for 15 minutes. Filter the medicinal liquid while it is hot with a 150-mesh filter cloth, combine the two decoction liquids, concentrate under reduced pressure at low temperature (65 °C) until the ratio of material to liquid is about 1:1 (relative density 1.05 - 1.10), and then freeze-dry and store in a sealed manner to obtain the product.

[0073] Stir-fried winter melon seed formula granules: Take 10,000 g of stir-fried winter melon seed decoction pieces, crush them, add water for decoction, filter, concentrate the filtrate into a clear paste (the yield of dry extract is 4.0 wt% - 7.0 wt%), add appropriate amount of excipients, dry (or dry and pulverize), then add appropriate amount of excipients again, mix evenly, granulate, and make 1,000 g, thus obtaining.

[0074] Obtain its liquid chromatogram according to the aforementioned test method, and the test results are shown in Table 2. Figure 2 ( Figure 2 Among them, S1 - S18 respectively correspond to No. 1 - 18 in Table 2), use the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" (version 2012.1), take the liquid chromatogram of sample S1 as the reference chromatogram, calculate by median, match all peaks, and fit to generate the control chromatogram of stir-fried winter melon seed formula granules, as Figure 1 shown.

[0075] Table 2 List of standard decoctions and formula granules of stir-fried winter melon seeds

[0076]

[0077] According to the common pattern diagram of 15 batches of standard decoctions of stir-fried winter melon seeds (K437BJ01 - K437BJ15), 7 common peaks are confirmed, and reference substances can be obtained. Among them, uracil, hypoxanthine, uridine, guanosine, and adenosine respectively correspond to the retention times of peak 1, peak 3, peak 4, peak 6, and peak 7. Select peak 4 (uridine) as the S peak for the following reasons: 1) Uridine is used as the characteristic identification peak of stir-fried winter melon seed formula granules and is an important representative component for the quality control of stir-fried winter melon seeds; 2) The peak position and peak area are moderate; 3) It has good stability.

[0078] According to the above test results, it can be known that using the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" (version 2012.1) to establish the control chromatogram for the standard decoction and formula granules of stir-fried winter melon seeds, a total of 7 common peaks are identified, and the similarity correlation with the control chromatogram is good.

[0079] Verify the characteristic chromatogram method and content detection method for stir-fried winter melon seed formula granules according to the above method. Specifically: Take 3 batches of stir-fried winter melon seed formula granules (K437CP01, K437CP02, K437CP03), and obtain their characteristic chromatograms according to the method of Example 1. The results are as Figure 3 shown in Table 3. Figure 3 From top to bottom are K437CP01 ( Figure 3 marked as S3 in Figure 3 ), K437CP02 ( Figure 3 marked as S2 in

[0080] Table 3 Retention Time and Relative Retention Time of Three Batches of Granules

[0081]

[0082]

[0083] According to the above test results, it can be seen that the 7 characteristic peaks in the characteristic chromatograms of the three batches of granules can all correspond to the control chromatogram ( Figure 1 ), and the relative retention time of each characteristic peak is within the range of ±10% of the specified value of the standard decoction, meeting the requirements. Therefore, the relative retention time standard of the granule characteristic peaks is consistent with the standard of the standard decoction, and the specified values are: 0.73 (peak 2), 1.44 (peak 5).

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

[0085] Table 4 Content Results of Stir-Fried Winter Melon Seed Formula Granules

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

[0087] Using the above content determination method, the adenosine component contents in the three batches of stir-fried winter melon seed formula granules were measured, and all were within the content limit range (0.20 mg / g - 1.00 mg / g).

[0088] Example 2 Investigation of the Extraction Method, Extraction Solvent, Extraction Time and Extraction Solvent Usage of the Test Sample

[0089] (1) Investigation of the extraction method

[0090] Take 0.2 g of stir-fried winter melon seed formula granules (K437CP01), accurately weigh, accurately add 20 ml of 10 vol% methanol aqueous solution, ultrasonically treat (power 250 W, frequency 40 kHz) and heat reflux for 30 minutes respectively, take out, let cool, weigh, make up the lost weight with 10 vol% methanol aqueous solution, shake well, filter, and obtain the corresponding test sample solution. Accurately pipette 3 μl of the continued filtrate, inject it into the high performance liquid chromatograph, and determine according to the method in Example 1, determine the peak area of each peak, and the results are shown in Table 5 below; determine the peak area of the adenosine peak and calculate the content, and the results are shown in Table 6 below.

[0091] Table 5 Results of Different Extraction Methods for Stir-Fried Winter Melon Seed Granules

[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 Stir-Fried Winter Melon Seed Granules

[0094]

[0095] According to the above data results, under the two extraction methods, the differences in the peak areas of each characteristic peak are not significant. Considering comprehensively, ultrasound is preferentially selected as the extraction method for preparing the test sample of semen benincasae formula granules.

[0096] (2) Investigation of extraction solvents

[0097] Take the semen benincasae formula granules (K437CP01), grind them finely, take about 0.2 g, weigh accurately, accurately add 20 ml of 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 ethanol respectively, perform ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 min, take out, let it cool, weigh again, make up the weight with the corresponding solvent, shake well, filter, and obtain the corresponding test sample solutions. Accurately pipette 3 μl of the consecutive filtrate, inject it into the high performance liquid chromatograph, and determine according to the method of Example 1. Determine the peak areas of each peak, and the results are shown in Table 7 below; determine the peak area of adenosine, calculate the content, and the results are shown in Table 8 below.

[0098] Table 7 Results of different extraction solvents for semen benincasae granules

[0099]

[0100] Table 8 Results of different extraction solvents for semen benincasae granules

[0101]

[0102] According to the above test results, 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 semen benincasae granules, the peak shapes of peaks 1 - 4 are extremely poor, and some characteristic peaks are lost. When water and 10 vol% - 30 vol% methanol aqueous solutions are used as extraction solvents, the peak areas of each characteristic peak are larger when using 10 vol% methanol aqueous solution as the solvent. Considering comprehensively, 10 vol% methanol aqueous solution is preferentially selected as the extraction solvent for preparing the test sample of semen benincasae formula granules.

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

[0104] Take the prepared granules of stir-fried winter melon seeds (K437CP01), grind them into fine powder. Weigh approximately 0.2 g respectively and accurately. Precisely add 15 ml, 20 ml, and 25 ml of 10 vol% methanol aqueous solution respectively, weigh, perform ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 min, take out, let it cool, weigh again, make up the lost weight with 10 vol% methanol aqueous solution, shake well, filter, and obtain the corresponding test solution. Precisely pipette 3 μl of the consecutive filtrate, inject it into the high performance liquid chromatograph, and determine according to the method in Example 1. Determine the peak areas of each peak, and the results are shown in Table 9 below; determine the peak area of adenosine, calculate the content, and the results are shown in Table 10 below; to avoid the influence of the test sample concentration on the peak area, calculate the ratio of each peak area to the test sample concentration for conversion.

[0105] Table 9 Test Results of Different Dosages of Extraction Solvents for Stir-fried Winter Melon Seeds Granules

[0106]

[0107] Table 10 Test Results of Different Dosages of Extraction Solvents for Stir-fried Winter Melon Seeds Granules

[0108]

[0109] According to the above data results, when the dosage of the extraction solvent is 15 ml, the sample extraction is incomplete. When the dosage of the extraction solvent is 20 ml and 25 ml, there are no obvious differences in the characteristic peaks of the stir-fried winter melon seeds granules. Considering the differences between batches and ensuring complete extraction of the test sample, the preferred dosage of the test sample solvent added is 20 ml, which is used as the dosage of the extraction solvent for the test solution of the stir-fried winter melon seeds granules.

[0110] (4) Investigation of Extraction Time

[0111] Take the prepared granules of stir-fried winter melon seeds (K4337CP01), grind them into fine powder. Weigh approximately 0.2 g and accurately. Precisely add 20 ml of 10 vol% methanol aqueous solution, weigh, perform ultrasonic treatment (power 250 W, frequency 40 kHz) for 20 min, 30 min, and 40 min respectively, take out, let it cool, make up the lost weight with 10 vol% methanol aqueous solution, shake well, filter, and obtain the corresponding test solution. Precisely pipette 3 μl of the consecutive filtrate, inject it into the high performance liquid chromatograph, and determine according to the method in Example 1. Determine the peak areas of each peak, and the results are shown in Table 11 below; determine the peak area of adenosine, calculate the content, and the results are shown in Table 12 below.

[0112] Table 11 Results of Different Extraction Times for Stir-fried Winter Melon Seeds Granules

[0113]

[0114]

[0115] Table 12 Results of Stir-fried Benincasa hispida Seeds Granules at Different Extraction Times

[0116]

[0117] According to the above data results, there are almost no differences in the characteristic peaks of stir-fried Benincasa hispida seeds granules at extraction times of 30 minutes and 40 minutes. It is preferred to use 30 minutes as the extraction time for the test solution of stir-fried Benincasa hispida seeds granules.

[0118] From the above results, it can be seen that the preferred treatment method for the test solution of stir-fried Benincasa hispida seeds granules is as follows: Take an appropriate amount of stir-fried Benincasa hispida seeds granules, grind them finely, take about 0.2 g, weigh accurately, place them in a stoppered conical flask, accurately add 20 ml of 10 vol% methanol aqueous solution,

[0119] Stopper tightly, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, take out, let it cool, weigh again, make up the lost weight with 10 vol% methanol aqueous solution, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0120] Example 3 Investigation of Different Column Temperatures

[0121] Take stir-fried Benincasa hispida seeds granules (batch number: K437CP01), prepare the test solution according to the preparation method of the test solution in Example 1, and measure them at different column temperatures of 28 °C, 30 °C, and 32 °C according to the method in Example 1 to investigate the durability of the experimental method for different column temperatures. The test results of the characteristic spectrogram are shown in Table 13 below, and the test results of the content determination are shown in Table 14 below.

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

[0123]

[0124] Table 14 Results of Content Determination of Stir-fried Benincasa hispida Seeds Granules at Different Column Temperatures

[0125]

[0126] According to the above test results, it can be known that under different column temperature conditions, the relative retention times of each characteristic peak are within ±10% of the specified value. Under different column temperature conditions, the RSD of the content determination results of stir-fried Benincasa hispida seeds formula granules is 1.17%; it shows that the column temperature durability of the present invention is good.

[0127] Example 4 Investigation of Different Flow Rates

[0128] Take the stir-fried winter melon seed granules (batch number: K437CP01), prepare the test solution according to the preparation method of the test solution in the example, and measure according to the method of Example 1 at different flow rates of 0.19 ml / min, 0.20 ml / min, and 0.21 ml / min respectively to investigate the durability of the experimental method for different flow rates. The test results of the characteristic chromatogram are shown in Table 15 below, and the test results of the content determination are shown in Table 16 below.

[0129] Table 15 Results of retention time of characteristic chromatograms at different flow rates

[0130]

[0131] Table 16 Results of the content of stir-fried winter melon seed formula granules at different flow rates

[0132]

[0133] According to the above test results, it can be seen that under different flow rate conditions, the relative retention times of each characteristic peak are all within ±10% of the specified value, and the RSD of the content determination result of the stir-fried winter melon seed formula granules is 1.74%; it shows that the flow rate durability of the present invention is good.

[0134] Example 5 Investigation at different wavelengths

[0135] Take the stir-fried winter melon seed granules (batch number: K437CP01), prepare the test solution according to the preparation method of the test solution in the example, and measure according to the method of Example 1 at different wavelengths of 258 nm, 260 nm, and 262 nm respectively to investigate the durability of the experimental method for different wavelengths. The test results of the characteristic chromatogram are shown in Table 17 below, and the test results of the content determination are shown in Table 18 below.

[0136] Table 17 Results of retention time of characteristic chromatograms at different wavelengths

[0137]

[0138] Table 18 Results of the content of stir-fried winter melon seed formula granules at different wavelengths

[0139]

[0140]

[0141] According to the above test results, it can be seen that under different wavelength conditions, the relative retention times of each characteristic peak are all within ±10% of the specified value, and the RSD of the content determination result of the stir-fried winter melon seed formula granules is 0.68%; it shows that the wavelength durability of the present invention is good.

[0142] Example 6 Investigation with different chromatographic columns

[0143] Take the stir-fried winter melon seed granules (batch number: K437CP01), prepare the test solution according to the preparation method of the test solution in Example 1, use different chromatographic columns respectively, and determine according to the method in Example 1 to investigate the durability of the experimental method for the chromatographic column. The results are shown in Table 19, Table 20 and Figure 4 as shown below.

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

[0145] Chromatographic column 2: CORTECS UPLC T3 chromatographic column (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm);

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

[0147] Table 19 Retention time and relative retention time of characteristic chromatograms investigated with different chromatographic columns

[0148]

[0149] Table 20 Content determination results of stir-fried winter melon seed formula granules with different chromatographic columns

[0150]

[0151] According to the above test results, it can be seen that there are individual chromatographic peaks missing in chromatographic column 1, and the retention time of peak 1 in chromatographic column 2 is relatively forward and is easily interfered by the previous peak. Considering comprehensively, it is recommended to preferably use the ACQUITY UPLC HSS T3 chromatographic column. The RSD of the content determination result of the stir-fried winter melon seed formula granules is 1.19%, and the durability of the content determination method of the stir-fried winter melon seed formula granules with different chromatographic columns is good.

[0152] Example 7

[0153] This example is used to conduct a methodological verification on the detection method in Example 1:

[0154] (1) Specificity test

[0155] Take the test solution of the stir-fried winter melon seed formula granules, the reference substance solution of the reference substance, and the blank solution, inject samples according to the chromatographic conditions in Example 1, check the peak positions of the test solution and the reference substance solution of the reference substance, and the peak situation of the blank solution. The results are as Figure 5 shown below. According to Figure 5 the display, in the chromatogram of the blank solution, there is no influence on the peak position of the test solution, and the specificity is good.

[0156] (2) Delay test

[0157] Take the prepared granules of stir-fried Benincasa hispida seeds as the test solution, inject according to the chromatographic conditions in Example 1, and record the characteristic retention chromatogram of the test solution for 2 times the acquisition time. The results are as Figure 6 shown. According to Figure 6 the results, no obvious lag peak appears after 30 minutes, and this chromatographic method meets the analysis requirements of (Chinese Pharmacopoeia 2020 Edition).

[0158] (3) Accuracy

[0159] Accurately weigh 2.459 mg of adenosine reference substance and place it in a 10 ml volumetric flask. Add an appropriate amount of 10 vol% methanol aqueous solution to dissolve and dilute to the mark, shake well, and use it as the mother solution of the reference solution. Accurately pipette 1 ml, 2 ml, and 3 ml of the reference mother solution into 100 ml volumetric flasks respectively, add 10% methanol to dilute to the mark, shake well, and use them as the mixed reference substances 1, 2, and 3 of stir-fried Benincasa hispida seeds.

[0160] Take 9 portions of stir-fried Benincasa hispida seeds granules with known content (K437CP01, adenosine content 0.96 mg / g), each portion is 0.1 g, accurately weigh and number them from 1 to 9. Among them, for No. 1 - 3, accurately add 20 ml of the above-prepared mixed reference substance 1 solution; for No. 4 - 6, accurately add 20 ml of the mixed reference substance 2 solution; for No. 7 - 9, accurately add 20 ml of the mixed reference substance 3 solution. Weigh, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, let it cool, weigh again, make up the lost weight with 10 vol% methanol aqueous solution, shake well, filter, and take the continuous filtrate to obtain the solution. The test results are shown in Table 21 below. Among them, recovery rate (%) = (measured value - amount of the tested component contained in the test sample) / amount of added reference substance × 100%.

[0161] Table 21 Results of adenosine recovery test

[0162]

[0163] According to the above measurement results, the recovery rate of adenosine is between 97.91% and 102.68%, the average recovery rate is 100.2%, and the RSD value is 1.44%, meeting the requirements of the recovery rate for methodology verification in (Chinese Pharmacopoeia 2020 Edition), indicating that the results measured by this method are accurate.

[0164] (4) Linearity and range

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

[0166] 1) Pipette 5 ml of Solution A precisely, make up the volume to 10 ml to prepare a mixed solution containing 0.01800 mg of adenosine per 1 ml as Reference Substance 4.

[0167] 2) Pipette 5 ml of Solution A precisely, make up the volume to 20 ml to prepare a mixed solution containing 0.00902 mg of adenosine per 1 ml as Reference Substance 3.

[0168] 3) Pipette 1 ml of Solution A precisely, make up the volume to 10 ml to prepare a mixed solution containing 0.00361 mg of adenosine per 1 ml as Reference Substance 2.

[0169] 4) Pipette 1 ml of Solution A precisely, make up the volume to 20 ml to prepare a mixed solution containing 0.00180 mg of adenosine per 1 ml as Reference Substance 1.

[0170] Precisely pipette 3 μl of each reference substance solution, inject it into an ultra-high performance liquid chromatograph, and determine the peak area of the adenosine chromatographic peak according to the conditions and method under the content determination item. Take the peak area of the chromatographic peak as the ordinate y, and the concentration (unit: mg / ml) as the abscissa x, observe whether it is linear, and then perform linear regression by the least squares method to obtain the regression equation: for adenosine, y = 49790903.4187x + 4.0000, R 2 = 0.9999, and its linear range is 0.00180 - 0.03610 mg / ml. The test results are as shown in Figure 7 and Table 22.

[0171] Table 22 Concentrations and Areas of Adenosine Standard Curve

[0172] 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: According to 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 0.2 g of the prepared stir-fried winter melon seed formula granule (batch number: K437CP01) sample, prepare the test solution according to the method in Example 1, and perform the determination according to the method in Example 1 at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h respectively to obtain its characteristic chromatogram. Take the 4th peak (uridine) as the reference peak, calculate the relative peak areas and relative retention times of the remaining characteristic peaks, and calculate the RSD. The detection results of the characteristic chromatogram are shown in Table 23 and Table 24, and the detection results of the content determination (adenosine) are shown in Table 25.

[0176] Table 23 Retention Times and Relative Retention Times in the Stability Test (Characteristic Chromatogram)

[0177]

[0178] Table 24 Peak area and relative peak area table of stability test (characteristic chromatogram)

[0179]

[0180]

[0181] According to the above stability test results, after investigating the solution stability for 24 hours, the relative retention time RSD of each characteristic peak of Stir-fried Winter Melon Seeds Granules is in the range of 0.47% - 1.25%, and the RSD of the relative peak area of each characteristic peak is in the range of 1.66% - 3.49%. The test solution of Stir-fried Winter Melon Seeds Granules used in this experiment is stable within 24 hours.

[0182] Table 25 Investigation results of the stability (content determination) of Stir-fried Winter Melon Seeds Granules

[0183]

[0184] According to the above stability test results, after investigating the solution stability for 24 hours, the RSD of the peak area of Stir-fried Winter Melon Seeds Granules is 1.28%. The test solution of Stir-fried Winter Melon Seeds Granules used in this experiment is stable within 24 hours.

[0185] (6) Repeatability test

[0186] Precision: Using Waters UPLC H-Class, TUV detector, take 0.2 g of Stir-fried Winter Melon Seeds Granules (batch number: K437CP01), take 6 portions, determine according to the chromatographic conditions of Example 1 to obtain its characteristic chromatogram. Using the 4th peak (uridine) as the reference peak, calculate the relative peak area and relative retention time of the remaining chromatographic peaks, and calculate the RSD. The test results of the characteristic chromatogram are shown in Tables 26 and 27, and the test results of the content determination (adenosine) are shown in Table 28.

[0187] Table 26 Retention time and relative retention time table for the repeatability (characteristic chromatogram) investigation of Stir-fried Winter Melon Seeds Granules

[0188]

[0189]

[0190] Table 27 Peak area and relative peak area table for the repeatability (characteristic chromatogram) investigation of Stir-fried Winter Melon Seeds Granules

[0191]

[0192] According to the results of the above reproducibility experiments, the RSD of the relative retention times of the 7 characteristic peaks is in the range of 0.04% - 0.33%, and the RSD of the relative peak areas is in the range of 1.48% - 3.69%, indicating that the reproducibility of this characteristic chromatogram is good.

[0193] Table 28 Reproducibility Test (Including Measurement)

[0194]

[0195] Based on the above measurement results, the RSD of the adenosine content in the sample was found to be 1.0%, with good reproducibility. According to General Rule 9101 in the 2020 Edition of the Chinese Pharmacopoeia, when the content of the component to be determined is 0.01%, the reproducibility requirement is within 4%. Therefore, this method meets the analysis requirements.

[0196] Intermediate precision: Using an Agilent ultra-high performance liquid chromatograph with a TUV detector, take 0.2 g of stir-fried winter melon seed granules (batch number: K437CP01), take 6 portions, and determine according to the chromatographic conditions in Example 1 to obtain its characteristic chromatogram. Using the 4th peak (uridine) as the reference peak, calculate the relative retention times of the remaining characteristic peaks, and calculate the RSD. The detection results of the characteristic chromatogram are shown in Table 29, and the detection results of the content determination (adenosine) are shown in Table 30.

[0197] Table 29 Investigation of Retention Time and Relative Retention Time for Intermediate Precision (Characteristic Chromatogram) of Stir-Fried Winter Melon Seed Granules

[0198]

[0199] According to the results of the above intermediate precision experiments, the RSD of the relative retention times of the 7 labeled peaks of the six experimental samples is in the range of 0.02% - 1.04%; the RSD of the relative retention times between instruments is in the range of 0.25% - 1.36%. The results indicate that the intermediate precision of this characteristic chromatogram is good.

[0200] Table 30 Results Table of Intermediate Precision (Including Measurement)

[0201]

[0202] Based on the above measurement results, the RSD of the adenosine content is 0.60%, and the RSD of the adenosine content in the stir-fried winter melon seed formula granules determined by different instruments (Waters ultra-high performance liquid chromatograph, Agilent ultra-high performance liquid chromatograph) is 1.72%; according to General Rule 9101 in the 2020 Edition of the Chinese Pharmacopoeia, when the content of the component to be determined is 0.01%, the precision requirement is within 4%. Therefore, the intermediate precision of this experiment meets the analysis requirements.

[0203] Example 8

[0204] Taking the stir-fried winter melon seed formula granule as a sample for method validation, the same test sample (stir-fried winter melon seed formula granule, batch number: K437CP01) was used for characteristic fingerprint and content determination. The test results of the characteristic fingerprint are as shown in Figure 8 and Table 31, and the test results of the content determination are shown in Table 32. The characteristic fingerprints of the test sample and the reference crude drug reference substance are as shown in Figure 9 shown.

[0205] Table 31 Results of system suitability (characteristic fingerprint) test

[0206] Retention time Peak area USP resolution USP theoretical plate number USP tailing 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] It can be seen from the above test results that for the detection of stir-fried winter melon seed formula granule, the peak shapes, resolution, tailing factors, and theoretical plate numbers of each characteristic peak all meet the requirements of the Chinese Pharmacopoeia (2020 Edition).

[0208] Table 32 Results of system suitability (content determination) test (the same test sample solution was injected 5 times repeatedly)

[0209] Injection Retention time Peak area USP tailing USP theoretical plate number 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 value - 393937 - 200603 RSD% - 0.20 - -

[0210] According to the above test results, the average value of the 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 analysis requirements of the Chinese Pharmacopoeia (2020 Edition), and the instrument status is normal.

[0211] Obviously, the above embodiments are only examples clearly described and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or alterations derived therefrom are still within the protection scope of this invention.

Claims

1. A method for constructing a characteristic spectrum of a stir-fried wax gourd seed preparation, characterized in that: The following steps are involved: Preparation of test solution; The test solution is subjected to ultra-high performance liquid chromatography to obtain a characteristic spectrum; The conditions for the ultra-high performance liquid chromatography detection include: a chromatographic column with octadecylsilane bonded silica as a filler, a detection wavelength of 258 to 262 nm, methanol as mobile phase A, water as mobile phase B, and elution according to the following gradient program:

2. The method for constructing a characteristic map according to claim 1, characterized in that: In the conditions of the ultra-high performance liquid chromatography detection, the column length of the chromatographic column is 150 mm, the inner diameter is 2.1 mm, and the particle size of the filler is 1.6 to 1.8 μm; and / or, the chromatographic column is ACQUITY UPLC HSS T3 or CORTEC UPLC T3; and / or, the column temperature is 28-32°C; and / or, the flow rate is 0.19 to 0.21 ml / min; and / or, the number of theoretical plates calculated based on the adenosine peak should be no less than 10,000; And / or, the detection wavelength is 260nm.

3. The method for constructing a characteristic map according to claim 1, characterized in that: The preparation process of the test solution is as follows: take the test sample, grind it, accurately weigh it, add a solvent, weigh it, extract it, take it out, cool it, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate to obtain the test solution.

4. The method for constructing a characteristic map according to claim 3, characterized in that: The test sample is a stir-fried wax gourd seed preparation, and optionally, the stir-fried wax gourd seed preparation includes stir-fried wax gourd seed formula granules; And / or, the extraction treatment is ultrasonic treatment and / or heat reflux treatment, preferably ultrasonic treatment; 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%; and / or, relative to 0.2 g of the test sample, the amount of the solvent added is 20 to 25 ml, preferably 20 ml; And / or, the extraction treatment lasts for 30 to 40 minutes, preferably 30 minutes.

5. The method for constructing a characteristic map according to any one of claims 1 to 4, characterized in that: The characteristic spectrum construction method also includes the steps of extracting wax gourd seeds with an extractant, filtering the extracted wax gourd seeds, and preparing a reference medicinal material solution, and detecting the reference medicinal material solution with ultra-high performance liquid chromatography in the characteristic spectrum construction method according to any one of claims 1 to 4 to obtain a reference medicinal material spectrum. Preferably, the extractant is a 10% by volume methanol aqueous solution.

6. The method for constructing a characteristic map according to any one of claims 1 to 5, characterized in that: The characteristic spectrum construction method also includes the steps of preparing a reference substance solution: accurately weighing uracil, hypoxanthine, uridine, guanosine, and adenosine respectively, and adding a solvent to prepare a uracil solution, a hypoxanthine solution, a uridine solution, a guanosine solution, and an adenosine solution; And the step of detecting the reference substance solution by ultra-high performance liquid chromatography to obtain the reference substance spectrum in the construction method according to any one of claims 1 to 5.

7. The method for constructing a characteristic map according to claim 6, characterized in that: 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; And / or, the solvent in the reference substance solution is a methanol aqueous solution with a volume percentage of 10%.

8. The method for constructing a characteristic spectrum according to any one of claims 1 to 7, characterized in that: The characteristic spectrum of the test sample has 7 characteristic peaks, and the retention times of the 7 characteristic peaks in the chromatogram of the reference medicinal material reference material should correspond to each other; wherein, peak 1, peak 3, peak 4, peak 6, and peak 7 should correspond to the retention times of the reference peaks of uracil, hypoxanthine, uridine, guanosine, and adenosine, respectively; Peak 4 is S peak. The relative retention time of peak 2, peak 5 characteristic peaks and S peak is calculated. The relative retention time of peak 2 and peak 5 is within ±10% of the specified value; the specified value is: 0.73 (peak 2), 1.44 (peak 5).

9. A method for determining the content of a stir-fried wax gourd seed preparation, characterized in that: The following steps are involved: Prepare test sample solutions and reference sample solutions; The test solution and the reference solution are detected by the ultra-high performance liquid chromatography method in any one of claims 1 to 8, respectively; wherein the reference solution includes adenosine.

10. Application of the characteristic spectrum construction method according to any one of claims 1 to 8 and / or the content determination method according to claim 9 in the quality inspection of stir-fried wax gourd seed preparations.

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