A method for constructing a characteristic chromatogram of stir-fried foxtail millet and its preparation

Abstract

The application discloses a kind of fried millet sprout and the construction method of preparation characteristic map thereof, which utilizes ultra-high performance liquid chromatography to determine fried millet sprout and its preparation, so as to obtain the characteristic map of fried millet sprout.The present method uses ultrasonic-assisted ethanol solution extraction technology, greatly improves the preparation efficiency of test solution, is convenient to operate, significantly shortens the preparation time of test solution, and constructs the optimal mobile phase elution condition, reduces the detection time of fried millet sprout and its preparation characteristic map;The characteristic peak area obtained by the construction method of fried millet sprout and its preparation characteristic map disclosed in the application is moderate, the peak shape is clear, the peak separation degree is better, the repeatability is strong, and the sensitivity is better.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine ingredient detection, and particularly relates to a construction method of characteristic chromatogram of fried millet sprout and preparation thereof. BACKGROUND

[0002] Millet sprout is a processed product of mature fruit of Setaria italica (L.) Beauv. of Poaceae. The traditional Chinese medicine property proves that millet sprout has the functions of digesting and removing food retention, invigorating the spleen and stimulating appetite. The first edition of 2020 edition of Chinese Pharmacopoeia records that the surface of millet sprout is light yellow or powder white, and the fried millet sprout is prepared by frying the clean millet sprout, and the surface of the fried millet sprout is dark yellow, has aroma, tastes sweet, and has the effect of digesting and removing food retention.

[0003] At present, the medicinal forms of commercially available fried millet sprout mainly include fried millet sprout decoction pieces, fried millet sprout formula granules, fried millet sprout standard decoction and some compound preparations containing fried millet sprout. The fried millet sprout standard decoction is prepared by decocting the fried millet sprout decoction pieces with pure water, filtering while hot, combining the filtrates, then reducing pressure and concentrating, and low-temperature vacuum drying. The fried millet sprout formula granules are prepared by twice decocting the fried millet sprout decoction pieces, filtering while hot, combining the filtrates, low-temperature concentrating, spray drying, and dry granulating after adding excipients.

[0004] In recent years, the traditional Chinese medicine industry and researchers have carried out research on fried millet sprout and its preparations from various aspects. The 2020 edition of Chinese Pharmacopoeia does not regulate and limit the content of characteristic ingredients in fried millet sprout in the standard identification of fried millet sprout, which leads to uneven quality of fried millet sprout and its preparations in the market, and poor effect in actual clinical application. Therefore, it is necessary to find a method for describing and evaluating the overall quality of fried millet sprout. SUMMARY

[0005] The purpose of the present application is to provide a construction method of characteristic chromatogram of fried millet sprout and its preparation, so as to overcome the problems of long detection time, poor repeatability, low sensitivity and complex preparation method of test solution in the prior art. The present application uses ultra-high performance liquid chromatography to determine the characteristic ingredients of fried millet sprout and its preparation, and constructs a characteristic chromatogram, which has the characteristics of short detection time, strong stability, high sensitivity and the like, and has a strong promoting effect on the quality control and reasonable application of fried millet sprout and its preparation.

[0006] The technical solution adopted by the present application to solve its technical problems is:

[0007] A construction method of characteristic chromatogram of fried millet sprout and its preparation, comprising the following steps:

[0008] (1) Preparation of test solution: weigh the fried millet sprout and its preparation to be tested, add 70% ethanol solution, ultrasonic extraction, filtration, and collect the filtrate to obtain the test solution;

[0009] (2) taking 4-coumaric acid, 5-hydroxymethylfurfural and ferulic acid as the reference substance, using the ultra-high performance liquid chromatography to obtain the characteristic chromatogram of the roasted millet and its preparation;

[0010] The chromatographic conditions of the ultra-high performance liquid chromatography are as follows: one of YMC-Trait C18 (100*2.1mm, 1.9μm), ACQUITY UPLC HSS T3 (100*2.1mm, 1.8μm) or ZORBAX Extend C18 (2.1*100mm, 1.8μm) is used as the chromatographic column; acetonitrile is used as the mobile phase A, and 0.1-0.5% acetic acid aqueous solution is used as the mobile phase B for gradient elution, the flow rate is set to 0.1-0.3mL / min, the detection wavelength is set to 280-300nm, and the column temperature is 30-33℃;

[0011] The gradient elution conditions are as follows: 0-3.5min, the volume ratio of the mobile phase A to the mobile phase B is 5-10%:95-90%; 3.5-4.0min, the volume ratio of the mobile phase A to the mobile phase B is 10-14%:90-86%; 4.0-11.5min, the volume ratio of the mobile phase A to the mobile phase B is 14-20%:86-80%; 11.5-13.5min, the volume ratio of the mobile phase A to the mobile phase B is 20-22%:80-78%; 13.5-13.6min, the volume ratio of the mobile phase A to the mobile phase B is 22-5%:78-95%; 13.6-18.0min, the volume ratio of the mobile phase A to the mobile phase B is 5-5%:95-95%.

[0012] The preparation method of the reference substance is as follows: taking appropriate 4-coumaric acid reference substance, 5-hydroxymethylfurfural reference substance and ferulic acid reference substance, dissolving them in ethanol to prepare a mixed reference substance solution.

[0013] Further, the preparation method of the reference substance solution specifically includes: taking appropriate 4-coumaric acid reference substance, 5-hydroxymethylfurfural reference substance and ferulic acid reference substance, accurately weighing and determining, adding 70% ethanol to prepare a mixed solution, shaking, filtering, and taking the continuous filtrate to obtain the reference substance solution.

[0014] The preparation method of the reference substance solution of the medicinal material is as follows: taking appropriate roasted millet reference medicinal material in a conical flask with a plug, adding 70% ethanol, heating and refluxing for 1-1.5h, cooling, shaking, filtering, and taking the continuous filtrate as the reference medicinal material solution.

[0015] Further, the preparation method of the control medicinal material reference solution specifically comprises: taking about 2.5 g of fried millet seeds control medicinal material, accurately weighing, placing in a conical flask with a plug, adding 25 mL of 70% ethanol, heating refluxing for 1 h, cooling, shaking, filtering, taking the subsequent filtrate as the control medicinal material reference solution.

[0016] The preparation method of the test sample solution is as follows: taking a proper amount of fried millet seeds and its preparation sample in a conical flask with a plug, adding 70% ethanol, ultrasonic extraction for 20-40 min, taking out, cooling, shaking, filtering, and taking the subsequent filtrate as the control medicinal material reference solution.

[0017] Further, the preparation method of the test sample solution specifically comprises: taking a proper amount of fried millet seeds and its preparation sample to be tested, finely grinding, placing in a conical flask with a plug, adding 25 mL of 70% ethanol, ultrasonic extraction (power 250 W, frequency 53 kHz) for 40 min, taking out, cooling, shaking, filtering, and taking the subsequent filtrate.

[0018] The fried millet seeds and its preparation include fried millet seeds medicinal material, fried millet seeds decoction pieces, fried millet seeds formula granules or fried millet seeds standard decoction dry products.

[0019] The characteristic spectrum of the fried millet seeds and its preparation includes 6 characteristic peaks, with peak No. 5 as the reference peak, and the relative retention times of the remaining characteristic peaks should be within ±10% of the specified value, and the specified value is: 0.221 (peak 1), 0.310 (peak 2), 0.390 (peak 3), 0.951 (peak 4), 1.126 (peak 6); wherein peak No. 5 is the 4-coumaric acid peak.

[0020] The ultrasonic power is 200-250 W, and the concentration of the test sample solution is 15-30 mg / mL.

[0021] The concentration of the control reference solution is 4-coumaric acid 30 μg / mL, 5-hydroxymethylfurfural 10 μg / mL, and ferulic acid 5 μg / mL; and the concentration of the control medicinal material reference solution is 0.1 g / mL.

[0022] The chromatographic conditions are as follows: using YMC-Trait C18 (100*2.1 mm, 1.9 μm) column as the chromatographic column; gradient elution is performed with acetonitrile as the mobile phase A and 0.1% acetic acid aqueous solution as the mobile phase B, the flow rate is set to 0.3 mL / min, the detection wavelength is set to 300 nm, and the column temperature is 30°C.

[0023] The present application has the following beneficial effects:

[0024] 1. The application discloses a method for constructing a characteristic spectrum of fried millet sprouts and preparations thereof, which uses ultra-high performance liquid chromatography to determine the to-be-tested samples of the fried millet sprouts and the preparations thereof, and obtains the characteristic spectrum of the fried millet sprouts. The application adopts ultrasonic-assisted ethanol extraction of the test sample, which can effectively improve the dissolution rate of the effective components of the sample and greatly shorten the preparation time of the test sample solution.

[0025] 2. The method for constructing the characteristic spectrum of the fried millet sprouts and the preparations thereof has good peak shape and separation degree between peaks, strong repeatability, high separation efficiency, high sensitivity and the like, and can effectively detect the fried millet sprout medicinal materials, the fried millet sprout decoction pieces, the fried millet sprout standard decoction and the fried millet sprout formula granules.

[0026] 3. The characteristic spectrum constructed by the application takes the peak No. 5 as a reference peak, the peak No. 5 is identified as the 4-coumaric acid peak and is a representative peak of the effective components in the fried millet sprouts, the peak has good separation degree and moderate peak capacity, and the quality of the fried millet sprouts and the preparations thereof can be accurately controlled in a qualitative and quantitative manner by taking the peak as the reference peak. The six characteristic peaks determined in the characteristic spectrum are stable common peaks of 15 batches of fried millet sprouts from different origins and different batches, can accurately represent the characteristic components of the fried millet sprouts and the preparations thereof, thereby ensuring the effectiveness and accuracy of the quality control of the fried millet sprouts, and the six characteristic peaks have good peak shape and high separation degree, so that the retention time and the relative retention time of the characteristic peaks of the fried millet sprouts can be accurately controlled, thereby improving the reproducibility and the application range of the application.

[0027] 4. The application constructs the optimal elution condition of the fried millet sprouts and the preparations thereof, greatly shortens the detection time of the fried millet sprouts and the preparations thereof, and the characteristic spectrum has stable baseline, small external noise interference, good peak shape and separation degree and strong separation efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is the characteristic spectrum of the dry powder of the standard decoction of the 15 batches of fried millet sprouts in the embodiment 1 of the application;

[0029] Figure 2 is the characteristic spectrum of the mixed reference substance of the control in the embodiment 1 of the application;

[0030] Figure 3 is the control characteristic spectrum generated by the dry powder of the standard decoction of the 15 batches of fried millet sprouts in the embodiment 1 of the application;

[0031] Figure 4 is the three-dimensional isosbestic chromatogram of the ultra-high performance liquid chromatography in the experimental example 1 of the application;

[0032] Figure 5 is the characteristic spectrum of the experimental group 1 in the experimental example 2 of the application;

[0033] Figure 6 is the characteristic spectrum of the control group 1 in the experimental example 2 of the present application;

[0034] Figure 7 is the characteristic spectrum of the control group 2 in the experimental example 2 of the present application;

[0035] Figure 8 is the characteristic spectrum of the test solution of the experimental example 3 of the present application;

[0036] Figure 9 is the characteristic spectrum of the test solution of the experimental example 5 of the present application;

[0037] Figure 10 is the characteristic spectrum of the test solution of the experimental example 6 of the present application. DETAILED DESCRIPTION

[0038] Reagents and instruments used in the present application:

[0039] Reagent: Fried Millet Seed Control Drug Material (Batch No.: DSTYG001801, China Institute for Food and Drug Control), labeled No. R;

[0040] 4-coumaric acid control (Batch No.: 112037-201801, purity 99.3%, China Institute for Food and Drug Control), 5-hydroxymethylfurfural control (Batch No.: 111626-202013, purity 99.5%, China Institute for Food and Drug Control), and ferulic acid control (Batch No.: 110773-201915, purity 99.4%, China Institute for Food and Drug Control);

[0041] The preparation method of the fried millet seed standard decoction dry extract powder comprises the following steps: 100 g of fried millet seed slices are placed in a decoction pot, 1000 mL of pure water is added, soaked for 30 min, boiled with a strong fire, and then boiled for another 30 min with a weak fire. The extract is filtered through a 200-mesh filter screen, and the residue is added with 800 mL of pure water, boiled with a strong fire, and then boiled for 20 min with a weak fire. The two filtrates are combined, concentrated to a solid-liquid ratio of about 1:1 by a rotary evaporator, with a concentration temperature of 65°C. The concentrated solution is placed in a tray and dried under low temperature and vacuum, and then pulverized to obtain the dry extract powder.

[0042] The batch numbers and production places of the 15 batches of fried millet seed standard decoction dry extract powder used in the present application are as follows:

[0043] Table 1 Batch numbers and production places of 15 batches of fried millet seed standard decoction dry extract powder

[0044]

[0045] The preparation method of the fried millet bud formula granules is as follows: taking the fried millet bud decoction pieces, extracting twice, adding 10 times the amount of water for the first time, soaking for 30 minutes, decocting for 1 hour, adding 8 times the amount of water for the second time, decocting for 1 hour each time. After the decocting liquid is filtered through a 200-mesh double filter, the filtrate is concentrated to an extract with a solid content of 5% to 15% by a double-effect concentrator, and then the extract is transferred into a spray raw material tank. After spray drying, a proper amount of malt dextrin is added, mixed uniformly, and dry granulated, to obtain the fried millet bud formula granules.

[0046] The fried millet bud decoction pieces and the fried millet bud medicinal material used in the present application are produced in Dingyuan County, Chuzhou City, Anhui Province.

[0047] Reagents: ethanol (National Pharmaceutical Group Chemical Reagent Co., Ltd., batch number: 20210420), analytical pure; acetonitrile (National Pharmaceutical Group Chemical Reagent Co., Ltd., batch number: 20220521), chromatographically pure; pure water from ELGA option-Q ultra-pure water system.

[0048] Instruments: Waters series ultra-high performance liquid chromatograph (ACQUITY UPLC TM H-Class), American Alltech ELSD 6000 evaporative light scattering detector; Agilent 1290 series ultra-high performance liquid chromatograph (1290 binary gradient pump, 1290 automatic sampler, 1290 column oven), American Alltech ELSD 6100 evaporative light scattering detector; one-hundred-thousandth electronic balance (Sartorius); SK8200XS ultrasonic cleaner (Shanghai Keduo Ultrasonic Instrument Co., Ltd.).

[0049] Example 1

[0050] The present embodiment provides a method for constructing the characteristic chromatogram of fried millet bud and its preparation, and the method steps are specifically as follows:

[0051] (1) Preparation of reference solution of reference substance: take appropriate 4-coumaric acid reference substance, 5-hydroxymethylfurfural reference substance and ferulic acid reference substance, accurately weigh and determine, and prepare a mixed solution containing 4-coumaric acid 30 μg, 5-hydroxymethylfurfural 10 μg and ferulic acid 5 μg per 1 ml in 70% ethanol, to obtain the reference solution of reference substance;

[0052] (2) Preparation of reference solution of reference medicinal material: take about 2.5 g of millet bud reference medicinal material, accurately weigh and determine, place in a conical flask with a plug, add 25 mL of 70% ethanol, heat reflux for 1 hour, cool, shake uniformly, filter, and take the filtrate as the reference solution of reference medicinal material;

[0053] (3) Preparation of Test Solution: Accurately weigh about 0.5 g of the sample into a conical flask with a stopper, add 70% ethanol 25 ml, ultrasonic treatment (power 250 W, frequency 53 kHz) for 40 minutes, take out, cool, shake, filter, take the filtrate, and obtain the test solution;

[0054] (4) Chromatographic Conditions and System Suitability Test: octadecylsilane-bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.9 μm); acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution according to the following table; flow rate 0.3 mL per minute; column temperature 30 °C; detection wavelength 300 nm. The theoretical plate number should not be less than 5000 calculated according to the 4-coumaric acid peak;

[0055] (5) Determination: accurately pipette 1 μL of the reference solution and the test solution respectively into the liquid chromatograph, and determine, and obtain.

[0056] Table 2 Gradient Elution Program

[0057]

[0058] (6) Characteristic Peak Determination: prepare 15 batches of roasted wheat germ standard decoction dry extract powder test solution according to the method in step (3), and perform experiments according to the determination methods in steps (4) and (5), and obtain the characteristic chromatograms of 15 batches of roasted wheat germ standard decoction dry extract powder, which are shown in Figure 1 . Figure 1 The marker R is the characteristic chromatogram of the roasted wheat germ reference material, and the National Pharmacopoeia Committee promulgated the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System (2012 Edition)", taking the marker S1 liquid chromatogram as the reference chromatogram, the 15 batches of roasted wheat germ standard decoction UPLC chromatogram peaks were automatically matched to form a common peak mode chart, and the results showed that 6 common peaks were present in the 15 batches of roasted wheat germ standard decoction dry extract powder test solution, and the control characteristic chromatogram was established according to the median value, wherein the peak No. 5 was the 4-coumaric acid reference substance chromatographic peak, which was the index component specified in the Pharmacopoeia, and the peak area was moderate and the separation degree was good, so the peak was marked as S peak, and only 6 peaks corresponding to the control medicinal material were identified, and the standard control characteristic chromatogram was established, which is shown in Figure 2 , the relative retention time and RSD value were calculated, and the results are shown in Table 3.

[0059] Table 3 Actual Retention Time and Relative Retention Time of 15 Batches of Roasted Wheat Germ Standard Decoction Dry Extract Powder

[0060]

[0061]

[0062] From Table 3 and Figure 1 It can be seen that the relative retention times of the six characteristic peaks have no significant difference. Taking the average value of the relative retention time as the measured value, the relative retention times of the six characteristic peaks are 0.221 (peak 1), 0.310 (peak 2), 0.390 (peak 3), 0.951 (peak 4), 1.126 (peak 6), respectively, all of which are within ±10% of the specified value. The characteristic chromatograms of the 15 batches of roasted wheat germ standard decoction dry extract powder and the reference material of the roasted wheat germ control medicinal material were introduced into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System (2012 Edition)" issued by the National Pharmacopoeia Committee to obtain the similarity of the characteristic chromatograms of the 15 batches of roasted wheat germ standard decoction dry extract powder and the reference material of the roasted wheat germ control medicinal material, which were 0.988, 0.978, 0.992, 0.991, 1.000, 0.976, 1.000, 0.973, 0.995, 0.988, 0.987, 1.000, 0.995, 0.993, 1.000, respectively. This indicates that the 15 batches of roasted wheat germ standard decoction dry extract powder have a high degree of agreement with the reference material chromatogram of the roasted wheat germ control medicinal material. The characteristic chromatograms of the 15 batches of roasted wheat germ standard decoction dry extract powder are shown in Figure 1 , and the control characteristic chromatogram generated by the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System (2012 Edition)" from the chromatograms of the 15 batches of roasted wheat germ standard decoction dry extract powder is shown in Figure 3 . According to the similarity results and Figure 1 , it can be seen that the similarity of the 15 batches of roasted wheat germ standard decoction is high, the shapes of the six characteristic peaks are clear, and the separation degree is good. This construction method can better achieve precise control of the quality of roasted wheat germ.

[0063] Experimental Example 1: Investigation of Wavelength

[0064] The diode array detector was used to scan the test sample solution (S1) of the roasted wheat germ standard decoction dry extract powder at a wavelength of 190-400 nm, as shown in Figure 4 . The results showed that when the detection wavelength was around 300 nm, the response value of each chromatographic peak was moderate, the separation degree was good, and all aspects were better than those at other wavelengths. After comprehensive consideration, 300 nm was finally determined as the detection wavelength.

[0065] Experimental Example 2: Investigation of Elution Gradient Program

[0066] Three portions of the same batch of roasted wheat germ standard decoction dry extract powder to be tested (labeled S1) were taken, and test sample solutions were obtained according to the preparation method in step (3) of Example 1. Experimental group 1 was determined according to the chromatographic conditions in steps (4) and (5) of Example 1.

[0067] The difference between the control group 1 and the experimental group 1 is that the elution gradient program is different, and the elution gradient program of the control group 1 is shown in Table 4.

[0068] Table 4 Elution gradient program of the control group 1

[0069]

[0070] The difference between the control group 2 and the experimental group 1 is that the elution gradient program is different, and the elution gradient program of the control group 2 is shown in Table 5.

[0071] Table 5 Elution gradient program of the control group 2

[0072]

[0073] The characteristic spectrum of the experimental group 1, the control group 1 and the control group 2 is shown in Figures 5-7 ; it can be seen from the characteristic spectrum of the experimental group 1, the control group 1 and the control group 2 that the running baseline of the characteristic spectrum of the roasted millet standard decoction dry extract powder obtained by the experimental group 1 is more stable, the peak shape and separation degree of each peak are higher than those of the control group 1 and the control group 2, and the separation efficiency of the method of the experimental group 1 is higher, and the detection time is obviously less than that of the control group 1 and the control group 2.

[0074] Investigation of test sample solution preparation method in experimental example 3

[0075] Take the same batch of roasted millet and its preparation to be tested, a total of 4, and the mark numbers are S1, S2, S3 and S4 respectively. S1 is prepared according to the method in step (3) of example 1, and then the sample solution is determined according to the chromatographic method of step (4) and step (5) of example 1; the difference between S2 and S1 is that 70% ethanol is not used for ultrasonic assisted extraction, but directly used for heating reflux extraction for 1h, and the rest of the steps are the same; the difference between S3 and S1 is that 70% ethanol is not used for extraction and heating, but directly used for ultrasonic assisted water extraction for 1.5h, and the rest of the steps are the same; the difference between S4 and S1 is that 70% ethanol is not used for ultrasonic assisted extraction, but directly used for water heating reflux extraction for 1h, and the rest of the steps are the same. The characteristic spectrum obtained is shown in Figure 8 .

[0076] It can be seen from Figure 8 that the peak shape, peak separation degree and baseline level of the characteristic peak of the spectrum obtained by the experimental method used by mark number S1 have no obvious difference with S2, S3 and S4, but the preparation time of S1 is obviously lower than that of S2, S3 and S4, so as to shorten the time of constructing the characteristic spectrum of roasted millet and its preparation.

[0077] Investigation of test sample solution preparation time in experimental example 4

[0078] Three samples of the same batch of stir-fried barley sprouts to be tested were weighed and placed in conical flasks. The method in step (3) of Example 1 was followed, except that the ultrasonic time was set to 30 min, 40 min, and 50 min, respectively. The sample solutions were then measured by the chromatographic method in steps (4) and (5) of Example 1 to obtain the peak areas of each peak. The results are shown in Table 6.

[0079] Table 6 Preparation time of test solution

[0080]

[0081]

[0082] From the peak areas of the characteristic spectra obtained at different ultrasonic times, it can be seen that the preparation effect is best when the ultrasonic time is 40 minutes, so the preparation time is determined to be 40 minutes.

[0083] Experimental Example 5 Investigation of solvents for preparing test solution

[0084] Take the same batch of fried barley sprouts and their preparations to be tested, a total of 3 samples, marked as S1, S2, and S3, respectively. S1 is to prepare the fried barley sprout test solution according to the method in step (3) of Example 1, and then determine the sample solution according to the chromatographic method of steps (4) and (5) of Example 1; the difference between S2 and S1 is that when the sample is dissolved and prepared, the volume fraction of 70% ethanol is replaced by the volume fraction of 50% ethanol, and the other steps are the same; the difference between S3 and S1 is that when the sample is dissolved and prepared, the volume fraction of 70% ethanol is replaced by the volume fraction of 30% ethanol, and the other steps are the same. The characteristic spectrum obtained is shown in FIG. Figure 9 .

[0085] from Figure 9 As can be seen, the characteristic spectra obtained with 70% and 50% ethanol as the preparative solvents have moderate peak areas and clear peak shapes, while the characteristic spectra obtained with 30% ethanol as the preparative solvent have poor peak shapes, with some characteristic peaks missing. Considering that the low ethanol concentration and the high content of polysaccharides, proteins, and other components in the test solution would cause significant damage to the chromatographic column, 70% ethanol was selected as the preparative solvent.

[0086] Experimental Example 6 Investigation of the Test Sample of Stir-fried Barley Buds

[0087] Group S1: Take 0.5g of dry powder of standard decoction of fried barley sprouts, weigh it accurately, place it in a stoppered conical flask, add 25mL of 70% ethanol, and treat it ultrasonically (power 250W, frequency 53kHz) for 40min. Take it out, let it cool, shake it well, filter it, and take the filtrate to obtain the test solution.

[0088] S2 group: take 0.5g fried millet sprout formula granules, precision weighing, placed in a conical flask with a plug, add 70% ethanol 25ml, ultrasonic treatment (power 250w, frequency 53khz) 40min, take out, cool, shake, filter, take the filtrate, the test sample solution is obtained.

[0089] S3 group: take 0.5g fried millet sprout medicinal materials, precision weighing, placed in a conical flask with a plug, add 70% ethanol 25ml, ultrasonic treatment (power 250w, frequency 53khz) 40min, take out, cool, shake, filter, take the filtrate, the test sample solution is obtained.

[0090] S4 group: take 0.5g fried millet sprout decoction pieces, precision weighing, placed in a conical flask with a plug, add 70% ethanol 25ml, ultrasonic treatment (power 250w, frequency 53khz) 40min, take out, cool, shake, filter, take the filtrate, the test sample solution is obtained.

[0091] Then the test sample solution of S1 group, S2 group, S3 group and S4 group is determined according to the chromatographic method of step (4) and step (5) of example 1, and the characteristic spectrum obtained is as follows: Figure 10 .

[0092] Figure 10 It can be known that the characteristic spectrum of fried millet sprout constructed by the present application can be suitable for fried millet sprout standard decoction, fried millet sprout formula granules, fried millet sprout medicinal materials and fried millet sprout decoction pieces, and the application range is wide.

[0093] Experimental example 7 methodological investigation

[0094] (1) precision experiment

[0095] Repeatability investigation

[0096] Take the same batch of fried millet sprout to be tested, a total of 6, and the mark number is 1, 2, 3, 4, 5 and 6 respectively, prepare the test sample solution of fried millet sprout according to the method in step (3) of example 1, and then determine the sample solution according to the chromatographic method of step (4) and step (5) of example 1, obtain the characteristic spectrum, take the peak 5 as the reference peak, calculate the relative retention time and relative peak area respectively, and calculate the RSD value, the results are shown in table 7 and table 8.

[0097] Table 7 retention time table of fried millet sprout repeatability investigation

[0098]

[0099]

[0100] Table 8 peak area table of fried millet sprout repeatability investigation

[0101]

[0102] As can be seen from Table 7 and Table 8, the retention time RSD of each characteristic peak is in the range of 0.05-0.39%, and the RSD of relative peak area is in the range of 0.60-1.35%, indicating that the repeatability of the characteristic spectrum is good.

[0103] In conclusion, the roasted millet characteristic spectrum construction method has the advantages of high sample solution preparation efficiency, clear peak shape, high resolution and good repeatability; meanwhile, the construction method of the roasted millet characteristic spectrum can greatly improve the detection efficiency and reduce the detection time.

[0104] Obviously, the above embodiments only exemplify the preferred modes of the present application, and do not include all the inventive implementation ranges. Those of ordinary skill in the art can make various changes and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application is subject to the range defined by the claims.

Claims

1. A method for constructing a characteristic spectrum of stir-fried barley sprouts and their preparations, characterized in that: The following steps are involved: Preparation of test solution: Weigh the sample of fried barley sprouts and its preparations to be tested, add 70% ethanol solution, ultrasonically extract, filter, and collect the filtrate to obtain the test solution; Using 4-coumaric acid, 5-hydroxymethylfurfural and ferulic acid as reference substances, ultra-high performance liquid chromatography was used to obtain characteristic spectra of the stir-fried barley sprouts and their preparations; The stir-fried barley sprout and its preparation include stir-fried barley sprout medicinal materials, stir-fried barley sprout decoction pieces, stir-fried barley sprout formula granules or stir-fried barley sprout standard decoction dry products; The chromatographic conditions of the ultra-high performance liquid chromatography method are as follows: a YMC-Trait C18 column, an ACQUITY UPLC HSS T3 column, or a ZORBAX Extend C18 column is used as the chromatographic column; acetonitrile is used as the mobile phase A, and a 0.1-0.5% acetic acid aqueous solution is used as the mobile phase B for gradient elution, the flow rate is set to 0.1-0.3 mL / min, the detection wavelength is set to 280-300 nm, and the column temperature is 30-33°C; The gradient elution conditions are as follows: 0-3.5 min, the volume ratio of mobile phase A to mobile phase B is 5-10%:95-90%; 3.5-4.0 min, the volume ratio of mobile phase A to mobile phase B is 10-14%:90-86%; 4.0-11.5 min, the volume ratio of mobile phase A to mobile phase B is 14-20%:86-80%; 11.5-13.5 min, the volume ratio of mobile phase A to mobile phase B is 20-22%:80-78%; 13.5-13.6 min, the volume ratio of mobile phase A to mobile phase B is 22-5%:78-95%; 13.6-18.0 min, the volume ratio of mobile phase A to mobile phase B is 5-5%:95-95%.

2. The method for constructing a characteristic spectrum of a stir-fried barley sprout and its preparation according to claim 1, characterized in that: The preparation method of the reference substance is as follows: taking appropriate amounts of 4-coumaric acid reference substance, 5-hydroxymethylfurfural reference substance and ferulic acid reference substance, dissolving them in ethanol to prepare a mixed reference substance solution; The preparation method of the control medicinal material reference solution is as follows: take an appropriate amount of stir-fried barley sprout control medicinal material into a stoppered conical flask, add 70% ethanol, heat and reflux to extract for 1-1.5 hours, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution; The preparation method of the test solution is as follows: take an appropriate amount of fried barley sprouts and their preparations into a stoppered conical flask, add 70% ethanol, ultrasonically extract for 20-40 min, take out, cool, shake well, filter, and take the filtrate to obtain the test solution.

3. The method for constructing a characteristic spectrum of a stir-fried barley sprout and its preparation according to claim 2, characterized in that: The characteristic spectrum of the stir-fried barley sprouts and their preparations includes 6 characteristic peaks, with Peak 5 as the reference peak. The relative retention times of the remaining characteristic peaks should be within ±10% of the specified values, and the specified values ​​are: Peak 1 is 0.221, Peak 2 is 0.310, Peak 3 is 0.390, Peak 4 is 0.951, and Peak 6 is 1.126; among them, Peak 5 is the 4-coumaric acid peak.

4. The method for constructing a characteristic spectrum of a stir-fried barley sprout and its preparation according to claim 1, characterized in that: The ultrasonic power is 200-250 W, and the concentration of the test solution is 15-30 mg / mL.

5. The method for constructing a characteristic spectrum of a stir-fried barley sprout and its preparation according to claim 2, characterized in that: The preparation method of the test solution specifically includes taking an appropriate amount of the sample of the fried barley sprout and its preparation to be tested, grinding it, placing it in a stoppered conical flask, adding 25 ml of 70% ethanol, ultrasonically extracting it for 40 minutes at an ultrasonic power of 250 W and a frequency of 53 kHz, taking it out, cooling it, shaking it well, filtering it, and taking the filtrate to obtain the solution; The preparation method of the reference substance solution specifically comprises taking appropriate amounts of 4-coumaric acid reference substance, 5-hydroxymethylfurfural reference substance and ferulic acid reference substance, accurately weighing, adding 70% ethanol to prepare a mixed solution, shaking well, filtering, and taking the filtrate to obtain; The preparation method of the control medicinal material reference solution specifically includes taking 2.5 g of stir-fried barley sprout control medicinal material, accurately weighing, placing it in a stoppered conical flask, adding 25 mL of 70% ethanol, heating and reflux for 1 hour, cooling, shaking, filtering, and taking the filtrate as the control medicinal material reference solution.

6. The method for constructing a characteristic spectrum of a stir-fried barley sprout and its preparation according to claim 5, characterized in that: The concentrations of the reference substance solution are 30 μg / mL of 4-coumaric acid, 10 μg / mL of 5-hydroxymethylfurfural, and 5 μg / mL of ferulic acid; The concentration of the control medicinal material reference solution is 0.1 g / mL.

7. The method for constructing a characteristic spectrum of a stir-fried barley sprout and its preparation according to claim 1, characterized in that: The chromatographic conditions are as follows: a YMC-Trait C18 column with a specification of 100*2.1 mm, 1.9 µm was used as the chromatographic column; acetonitrile was used as the mobile phase A, 0.1% acetic acid aqueous solution was used as the mobile phase B for gradient elution, the flow rate was set to 0.3 mL / min, the detection wavelength was set to 300 nm, and the column temperature was 30 °C.

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