Establishment of characteristic chromatogram of fructus armeniacae, fried fructus armeniacae and its preparation and application

Abstract

The application provides a kind of Fuling drink piece, fried Fuling drink piece and the construction method and application of its preparation characteristic map, the construction method of fried Fuling and its preparation characteristic map, comprising: preparing test sample solution;Using high performance liquid chromatography to detect test sample solution, chromatographic conditions include with octadecylsilane bonded silica as filler, with methanol as mobile phase A, with 0.08%-0.4% formic acid as mobile phase B, and using specific elution procedure, while shortening the detection time also significantly improves the separation effect of multiple effective components, so that the characteristic map contains more characteristic peaks, greatly enriches the atlas information, realizes the effective separation of characteristic peaks such as uridine, thymine, gallic acid and guanosine, the characteristic of the obtained characteristic map is obvious, fully reflects the integrity and characteristic of the drug preparation of fried Fuling, and the baseline is stable, the peak shape is good, which provides basis for comprehensively detecting the quality of Fuling drink piece, fried Fuling and its pharmaceutical preparations.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection, specifically to a method and application for constructing characteristic chromatograms of Liu Shen Qu (a type of medicinal fermentation), stir-fried Liu Shen Qu, and their preparations. Background Technology

[0002] Liu Shen Qu (Six-Ingredient Dioscorea Flavouring) slices are a fermented starter made from Polygonum hydropiper, Artemisia annua, Xanthium sibiricum, red adzuki bean, bitter almond, wheat bran, and flour; stir-fried Liu Shen Qu slices are made by stir-frying Liu Shen Qu slices.

[0003] According to literature reports, Liu Shen Qu (a type of fermented medicinal herb) contains a relatively large number of chemical components, mainly amylase, yeast, volatile oils, glycosides, and B vitamins. The chemical components in stir-fried Liu Shen Qu slices are even more complex and diverse. Furthermore, traditional Chinese medicine is a system where complex components work together in a coordinated manner to exert therapeutic effects, requiring comprehensive and multi-faceted accurate quality control of medicinal materials. Although stir-fried Liu Shen Qu slices are a traditional fermentation preparation, a national pharmacopoeia standard has not yet been established. Currently, Liu Shen Qu varies significantly across the country in terms of formulation, compatibility, and fermentation processes, resulting in substantial quality differences. Quality control, primarily based on morphological identification, lacks quantitative indicators, and quality standards need improvement. Moreover, due to the complexity of the compound composition and natural fermentation, research on its pharmacodynamic material basis is relatively weak. Summary of the Invention

[0004] The problem this invention aims to solve is that there is no publicly available method for effectively detecting stir-fried Shenqu (a type of medicinal fermentation) slices or its preparations in the prior art; therefore, this invention provides a method for constructing and applying characteristic chromatograms of Shenqu slices, stir-fried Shenqu slices and their preparations.

[0005] In a first aspect, the present invention provides a method for constructing characteristic chromatograms of Shenqu (a type of medicinal fermentation), stir-fried Shenqu, and their preparations, comprising the following steps:

[0006] Preparation of the test solution;

[0007] The test solution was analyzed by high performance liquid chromatography (HPLC). The chromatographic conditions included using octadecylsilane-bonded silica gel as the stationary phase, methanol as mobile phase A, and 0.08%–0.4% formic acid as mobile phase B, with gradient elution as specified in the table below:

[0008]

[0009] In some embodiments, a chromatographic column with dimensions of 250 mm × 4.6 mm and a particle size of 5 μm is used;

[0010] And / or, the injection volume of the test solution is 5-20 μL;

[0011] And / or, the detection wavelength is 220-280nm, the column temperature is 18-22℃, and the flow rate is 0.8-1.2mL / min.

[0012] In some embodiments, the preparation method of the test solution is as follows: weigh the test sample, add solvent to extract, obtain the extract, separate the solid and liquid, and take the liquid, which is the test solution;

[0013] Preferably, the method for preparing the test solution further satisfies any one or more of the following A:

[0014] A. The ratio of the mass of the test sample to the volume of the solvent is 0.1-0.5:5-20; the relationship between mass and volume is g / mL.

[0015] B. The extraction method is either reflux extraction or ultrasonic extraction;

[0016] C. Extraction time is ≥20 min;

[0017] D. The solvent is selected from at least one of water, methanol and ethanol, preferably an aqueous methanol solution;

[0018] E. The solid-liquid separation is selected from centrifugation or membrane filtration;

[0019] More preferably, the solid-liquid separation specifically involves: filtering the extract through a filter membrane and evaporating it to dryness; adding the solvent to dissolve it, filtering it again through a filter membrane, and taking the filtrate as the test solution;

[0020] Alternatively, the solid-liquid separation specifically involves: adding a solvent to the extract to replenish the weight lost during the extraction process, shaking well, evaporating to dryness, adding the solvent to dissolve, filtering through a filter membrane, and taking the filtrate as the test solution;

[0021] Alternatively, the solid-liquid separation specifically involves: adding solvent to the extract to replenish the weight lost during extraction, shaking well, and evaporating to dryness; adding the solvent to dissolve, filtering through a filter membrane, and evaporating to dryness again; adding the solvent again to dissolve, filtering through a filter membrane again, and collecting the filtrate as the test solution. In some embodiments, the method for constructing the Liushenqu herbal slices further includes the step of preparing a reference solution by adding solvent to at least one of uridine, thymine, and guanosine as a reference standard, and the step of detecting the reference solution using high-performance liquid chromatography in the construction method to obtain a reference standard chromatogram;

[0022] Alternatively, the method for constructing the stir-fried Shenqu slices and its preparations may further include the step of preparing a reference solution by adding solvent to a reference standard using at least one of uridine, thymine, gallic acid, and guanosine, and the step of obtaining a reference standard chromatogram by detecting the reference solution according to the high performance liquid chromatography method in the construction method described above.

[0023] Preferably, the solvents are selected from at least one of methanol, ethanol, and water;

[0024] More preferably, 1 mL of the reference solution contains 0.005 to 50 μg of the respective reference standard.

[0025] In some embodiments, the characteristic spectrum of the Liu Shen Qu herbal slices has 6 common characteristic peaks, including peak 1 being uridine, peak 2 being thymine, and peak 6 being the characteristic peak corresponding to guanosine.

[0026] Preferably, peak 1 is designated as peak S, and the relative retention times of other characteristic peaks relative to peak 1 are within ±10% of a specified value. The specified values ​​for each characteristic peak are as follows:

[0027] Peak 2: 1.09, Peak 3: 1.21, Peak 4: 1.81, Peak 5: 2.13, Peak 6: 2.55.

[0028] In some embodiments, the characteristic chromatogram of the stir-fried Shenqu slices and its preparations has 6 common characteristic peaks, including peak 1 being uridine, peak 2 being thymine, peak 4 being gallic acid, and peak 6 being the characteristic peak corresponding to guanosine.

[0029] Preferably, peak 1 is designated as peak S, and the relative retention times of other characteristic peaks relative to peak 1 are within ±10% of a specified value. The specified values ​​for each characteristic peak are as follows:

[0030] Peak 2: 1.09, Peak 3: 1.21, Peak 4: 1.35, Peak 5: 1.83, Peak 6: 2.56.

[0031] Secondly, the present invention provides an application of the method for constructing characteristic chromatograms of the aforementioned Shenqu decoction pieces, stir-fried Shenqu decoction pieces and their preparations in the quality detection and identification of Shenqu decoction pieces, stir-fried Shenqu decoction pieces and their preparations.

[0032] Thirdly, the present invention also provides a method for quality testing of Shenqu (a type of medicinal fermentation) slices, including the step of comparing the characteristic spectrum of the Shenqu product to be tested with the reference characteristic spectrum of Shenqu slices.

[0033] The characteristic spectrum of the tested Liushenqu product was constructed using the tested Liushenqu product according to the construction method described above, and the control characteristic spectrum of the Liushenqu slices was selected from any one of the following (1)-(3):

[0034] (1) It has 6 common characteristic peaks, including peak 1 being uridine, peak 2 being thymine, and peak 6 being the characteristic peak corresponding to guanosine;

[0035] Taking peak 1 as peak S, the relative retention times of other characteristic peaks relative to peak 1 are within ±10% of the specified value. The specified values ​​for each characteristic peak are:

[0036] Peak 2: 1.09, Peak 3: 1.21, Peak 4: 1.81, Peak 5: 2.13, Peak 6: 2.55.

[0037] (2) Using single or multiple batches of Liushenqu (a type of medicinal herb) to obtain a comparative feature map of Liushenqu slices according to the construction method described above;

[0038] (3) Using multiple batches of Liushenqu obtained according to the construction method described above, a control feature map was prepared by means of the average or median method.

[0039] Fourthly, the present invention also provides a quality testing method for stir-fried Shenqu (medicated leaven) slices and its pharmaceutical preparations, including the step of comparing the characteristic chromatogram of the stir-fried Shenqu slices product to be tested with the control characteristic chromatogram of stir-fried Shenqu slices or its pharmaceutical preparations.

[0040] The characteristic chromatogram of the tested stir-fried Shenqu herbal slices is constructed using the tested stir-fried Shenqu herbal slices according to the construction method described above, and the control characteristic chromatogram of the stir-fried Shenqu herbal slices or its pharmaceutical preparation is selected from any one of the following (1)-(3):

[0041] (1) It has 6 common characteristic peaks, including peak 1 for uridine, peak 2 for thymine, peak 4 for gallic acid, and peak 6 for guanosine; with peak 1 as the S peak, the relative retention times of the other characteristic peaks relative to peak 1 are within ±10% of the specified value, and the specified values ​​for each characteristic peak are:

[0042] Peak 2: 1.09, Peak 3: 1.21, Peak 4: 1.35, Peak 5: 1.83, Peak 6: 2.56.

[0043] (2) The characteristic spectrum of the pharmaceutical preparation of stir-fried Shenqu decoction pieces obtained by using single or multiple batches of the pharmaceutical preparation of stir-fried Shenqu decoction pieces according to the construction method described above;

[0044] (3) Using multiple batches of stir-fried Shenqu decoction pieces, the characteristic chromatograms obtained according to the construction method described above are used to prepare a control characteristic chromatogram by means of average value or median.

[0045] Fifthly, the present invention also provides a method for identifying stir-fried Shenqu (a type of medicinal fermentation) slices or its preparations with Shenqu slices, comprising: using the product to be identified to construct a characteristic spectrum of the product to be identified according to the construction method described above;

[0046] If the characteristic spectrum of the product to be identified has 6 common characteristic peaks, including peak 1 being uridine, peak 2 being thymine, peak 4 being gallic acid, and peak 6 being the characteristic peak corresponding to guanosine; then the product to be identified is stir-fried Shenqu (medicated leaven) slices or its preparations.

[0047] If the characteristic spectrum of the product to be identified has 6 common characteristic peaks, including peak 1 corresponding to uridine, peak 2 to thymine, and peak 6 to guanosine, then the product to be identified is Shenqu (medicated leaven) slices.

[0048] And / or, if the relative peak area of ​​peak 3 and peak 1 is greater than 1.3, the product to be identified is stir-fried Shenqu (a type of medicinal fermented vegetable) slices or its preparation; if the relative peak area of ​​peak 3 and peak 1 is less than 1.2, the product to be identified is Shenqu (another type of medicinal fermented vegetable) slices.

[0049] The technical solution of this invention has the following advantages:

[0050] 1. The method for constructing characteristic chromatograms of Liu Shen Qu (a type of medicinal fermented vegetable), fried Liu Shen Qu, and their pharmaceutical preparations provided by this invention uses octadecylsilane-bonded silica gel as a filler, methanol as mobile phase A, and 0.08%-0.4% formic acid as mobile phase B, and employs a specific elution procedure. This method significantly improves the separation effect of multiple active ingredients while shortening the detection time, resulting in more characteristic peaks in the characteristic chromatograms, greatly enriching the chromatographic information. It achieves effective separation of characteristic peaks including uridine, thymine, gallic acid, and guanosine. The obtained characteristic chromatograms are highly distinctive, fully reflecting the integrity and characteristics of the pharmaceutical preparations made from fried Liu Shen Qu, with a stable baseline and good peak shape, providing a basis for comprehensive quality testing of fried Liu Shen Qu and its pharmaceutical preparations.

[0051] 2. The method for constructing the characteristic spectrum of the pharmaceutical preparation of stir-fried Shenqu (a type of medicinal herb) provided by the present invention can select the S-peak uridine as the internal reference peak in the characteristic spectrum, and can determine the 6 common characteristic peaks of stir-fried Shenqu, and calculate the relative retention time of each common characteristic peak based on the S-peak uridine. This is beneficial for the comprehensive quality detection and overall quality control of stir-fried Shenqu, thereby helping to improve the safety and stability of the drug.

[0052] 3. The quality detection method for Liushenqu slices or stir-fried Liushenqu slices or their pharmaceutical preparations provided by the present invention can simultaneously construct a characteristic spectrum of the product to be tested and compare it with the corresponding control characteristic spectrum. The method is simple and facilitates comprehensive quality control of Liushenqu or stir-fried Liushenqu slices and their pharmaceutical preparations.

[0053] 4. The method for identifying stir-fried Shenqu slices and its pharmaceutical preparations from Shenqu slices provided by the present invention involves comparing the characteristic chromatogram of the product to be identified with the reference characteristic chromatogram of Shenqu slices, stir-fried Shenqu slices or their preparations, thereby achieving the identification of stir-fried Shenqu slices and its preparations from Shenqu slices. Attached Figure Description

[0054] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0055] Figure 1 The fingerprint spectrum of the freeze-dried powder of the standard decoction of stir-fried Shenqu (a type of medicinal herb) in Example 1; wherein peak 1 (S): uridine; peak 2: thymine; peak 4: gallic acid; peak 6: guanosine;

[0056] Figure 2 This is a localization map of the characteristic spectra of uridine reference standard, guanosine reference standard, gallic acid reference standard and thymine reference standard and the freeze-dried powder of stir-fried Shenqu decoction in Example 1;

[0057] Figure 3 The chromatogram for gradient condition 1 in Experimental Example 2;

[0058] Figure 4 The chromatogram for gradient condition 2 in Experimental Example 2;

[0059] Figure 5 The chromatogram for gradient condition 3 in Experimental Example 2;

[0060] Figure 6 The chromatogram for gradient condition 4 in Experimental Example 2;

[0061] Figure 7 The chromatogram for gradient condition 5 in Experimental Example 2;

[0062] Figure 8 The chromatogram for gradient condition 6 in Experimental Example 2;

[0063] Figure 9 The chromatogram for gradient condition 7 in Experimental Example 2;

[0064] Figure 10 This is the full-wavelength scan diagram from Experiment Example 2;

[0065] Figure 11 The chromatogram of 0.4% acetic acid in Experimental Example 2;

[0066] Figure 12 The chromatogram of 0.4% phosphate in Experimental Example 2;

[0067] Figure 13 The chromatogram of the 0.4% formic acid solution in Experiment Example 2 is shown below.

[0068] Figure 14 The chromatogram of the Waters Xselect HSS T3 column in Experiment Example 2;

[0069] Figure 15 The chromatogram of the Diamonsil Plus 5μm C18-A column in Experiment Example 2;

[0070] Figure 16 The Morphling AQ-C18 column used in Experimental Example 2;

[0071] Figure 17 The chromatogram of 100% methanol in Experimental Example 2;

[0072] Figure 18 The chromatogram for 50% methanol in Experimental Example 2 is shown below.

[0073] Figure 19 The chromatogram of 20% methanol in Experimental Example 2 is shown.

[0074] Figure 20 The characteristic chromatograms of the freeze-dried powder of the standard decoction of 15 batches of stir-fried Shenqu (a type of medicinal herb) in Experimental Example 5 are shown.

[0075] Figure 21 Characteristic chromatograms of 15 batches of stir-fried Shenqu (a type of medicinal herb) from Experimental Example 5;

[0076] Figure 22 The characteristic chromatogram of the stir-fried Shenqu (medicated leaven) slices in Experiment Example 5 is shown below;

[0077] Figure 23 The characteristic chromatograms of 15 batches of Shenqu (a traditional Chinese medicine) slices in Experimental Example 5;

[0078] Figure 24 The characteristic chromatogram of the six Shenqu decoction pieces in Experiment Example 5 is shown.

[0079] Figure 25 This is a comparison of the characteristic spectra of Liu Shen Qu slices, stir-fried Liu Shen Qu slices, and standard decoction of slices in Example 5. Detailed Implementation

[0080] The following embodiments are provided to better understand the present invention and are not intended to limit the preferred embodiments. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention. Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in the art. Reagents or instruments used that do not specify the manufacturer are all commercially available conventional reagent products. Percentages not specified in the present invention are volume percentages.

[0081] The instruments and reagents used in the following examples or test cases are as follows:

[0082] 1. Instruments and reagents

[0083] 1.1 Instruments

[0084] The chromatographic systems are shown in Table 1 below:

[0085] Table 1 Chromatograph

[0086]

[0087] The chromatographic columns are shown in Table 2 below:

[0088] Table 2 Chromatographic Columns

[0089]

[0090] Other instruments are listed in Table 3 below:

[0091] Table 3 Other Instruments

[0092]

[0093]

[0094] 1.2 The test drugs are shown in Table 4 below:

[0095] Table 4. Test Drugs

[0096]

[0097] Information on multiple batches of Liu Shen Qu (a traditional Chinese medicine), stir-fried Liu Shen Qu, and freeze-dried powder of standard decoction of stir-fried Liu Shen Qu is shown in Table 5 below:

[0098] Table 5. Batch numbers of Liushenqu and its preparations

[0099]

[0100] The following method can be used to process 6 Shenqu (a type of medicinal fermented herb): Take 6 Shenqu slices, place them in a stir-frying machine, and stir-fry at 150℃ for 15 minutes until the surface is yellow, occasionally with scorched spots, hard and brittle, and has a fragrant aroma. Remove and let cool.

[0101] The specific preparation process of the standard decoction of stir-fried Shenqu (a type of medicinal herb) is as follows: Take 100g of stir-fried Shenqu, place it in a clay pot, add 10 times the amount of water and soak for 30 minutes. First, bring it to a boil over high heat, then simmer over low heat for 30 minutes. Filter it while hot through a 200-mesh filter cloth. Add 8 times the amount of water to the dregs, bring it to a boil over high heat, then simmer over low heat for 25 minutes. Filter it while hot through a 200-mesh filter cloth. Combine the two filtrates, cool the filtrate to room temperature, and concentrate it under reduced pressure and low temperature (60℃) to a concentrated extract with a material-to-liquid ratio close to 2:1. Place it in a freeze dryer and freeze dry (-42℃, 40pa) until dry. Pulverize it into powder to obtain the "standard decoction" (freeze-dried powder).

[0102] 1.3 Reagents

[0103] Methanol and formic acid were of chromatographic grade (TEDIA), and water was ultrapure water; all other reagents were of analytical grade.

[0104] There are many types of prepared Liu Shen Qu (a type of fermented medicinal herb), and this invention uses a standard decoction of Liu Shen Qu as a reference. Since the standard decoction of Liu Shen Qu is extracted through boiling in water, the water solubility of its chemical components must be considered, as well as their content. Substances with low content may not be readily apparent after water dissolution. Therefore, among amylase, yeast, volatile oils, glycosides, and B vitamins, nucleosides and polyphenols have higher content and better water solubility. Nucleosides play a role in regulating the intestines and aiding in nutrient digestion and absorption. Other components, due to poor water solubility or low content, are not easily apparent in the standard decoction. Therefore, nucleosides are used as the main chemical components in the characteristic spectrum of the standard decoction of Liu Shen Qu.

[0105] Example 1

[0106] This embodiment provides a method for constructing the characteristic spectrum of freeze-dried powder of stir-fried Shenqu standard decoction, including the following steps:

[0107] Preparation of the test solution: Accurately weigh 0.2 g of the freeze-dried powder of the standard decoction of stir-fried Shenqu (batch number 2103001Y), place it in a stoppered conical flask, add 10 ml of 50% methanol, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with 50% methanol, shake well, evaporate to dryness in a water bath, add 2 ml of 50% methanol to dissolve it, filter it, evaporate to dryness in a water bath, accurately add 2 ml of 50% methanol to dissolve it, filter it, and take the filtrate to obtain the test solution.

[0108] Preparation of the reference solution for stir-fried Shenqu (a type of fermented medicinal herb): Accurately weigh 2g of stir-fried Shenqu reference slices (batch number DSTYC009801), add 50ml of water, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of 50% methanol, filter, and use the filtrate as the reference solution. Preparation of the reference solution: Accurately weigh appropriate amounts of uridine, guanosine, and gallic acid reference standards, add 50% methanol to prepare a solution containing 50μg of each reference standard per 1ml.

[0109] High-performance liquid chromatography (HPLC) detection: Inject 10 μl each of the reference solution and the test solution into the HPLC system (Waters chromatograph). Chromatographic conditions are as follows: Waters Xselect HSS T3 column, packed with octadecylsilane-bonded silica gel (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A, and 0.4% formic acid as mobile phase B, eluted according to the specifications in Table 6; detection wavelength 270 nm; column temperature 20℃; flow rate 1.0 mL / min. The theoretical plate number, calculated based on the uridine peak, should not be less than 3000.

[0110] Table 6 Chromatographic conditions of Example 1

[0111]

[0112] The fingerprint spectrum of the freeze-dried powder of the standard decoction of stir-fried Shenqu was established according to the above method. The results are shown in Table 7 below. Figure 1 As shown.

[0113] Table 7. Comparison of Relative Retention Times of Standard Decoction Powder of Stir-fried Six-Ingredient Dioscorea Berry and Freeze-dried Powder

[0114]

[0115] Table 8. Comparison of relative peak areas in standard decoctions and freeze-dried powders of stir-fried Liu Shen Qu (a traditional Chinese medicine).

[0116]

[0117] The results showed that the characteristic chromatogram of the freeze-dried powder of the standard decoction of stir-fried Shenqu (a type of medicinal fermentation) had a stable baseline, good peak shape, good separation effect, and significant characteristic components, with 6 characteristic peaks. Through localization and identification with reference standards, peak 1 was identified as uridine, peak 2 as thymine, peak 4 as gallic acid, and peak 6 as guanosine. The localization chromatogram is shown in [reference needed]. Figure 2 With peak 1 as the S-peak, the relative retention times of other characteristic peaks relative to peak 1 are as follows: peak 2: 1.09, peak 3: 1.20, peak 4: 1.35, peak 5: 1.82, peak 6: 2.56.

[0118] Experimental Example 1: System Adaptability Assessment

[0119] Chromatographic conditions: Waters chromatograph, Waters Xselect HSS T3 column, octadecylsilane-bonded silica gel as packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A, 0.4% formic acid as mobile phase B, gradient elution according to the chromatographic conditions specified in Table 6 of Example 1; detection wavelength 270 nm. Column temperature 20 °C, flow rate 1.0 mL / min. The theoretical plate number, calculated based on the uridine peak, should be no less than 3000.

[0120] The batch number of the test sample (freeze-dried powder of standard decoction of stir-fried Shenqu) was 2103001Y. The preparation of the reference solution of stir-fried Shenqu and the test sample solution was the same as in Example 1.

[0121] The assay method involves precisely pipetting 10 μl each of the reference solution and the test solution of stir-fried Shenqu (a type of medicinal fermentation), injecting them into the liquid chromatograph, and determining the content.

[0122] The system compatibility of the reference solution and the test solution of the stir-fried Shenqu (a type of medicinal herb) was determined by HPLC, and the results are shown in Table 9.

[0123] Table 9 System Adaptability

[0124]

[0125] The results showed that the characteristic spectrum of the freeze-dried powder of the standard decoction of stir-fried Shenqu was stable with good baseline, good peak shape, good separation effect, and good system adaptability, making it suitable for the determination of the characteristic spectrum of the freeze-dried powder of the standard decoction of stir-fried Shenqu.

[0126] Experimental Example 2: Investigation of chromatographic conditions for freeze-dried powder of stir-fried Shenqu (a traditional Chinese medicine) decoction.

[0127] The batch number of the test sample (freeze-dried powder of standard decoction of stir-fried Shenqu) was 2103001Y. The preparation of the reference solution, control solution and test sample solution of stir-fried Shenqu were the same as in Example 1.

[0128] HPLC detection was performed using a Waters Xselect HSS T3 column on a Waters chromatograph.

[0129] 1.1 Gradient Selection

[0130] An octadecylsilane-bonded silica gel column (250 mm column length, 4.6 mm inner diameter, 5 μm particle size) was used as the packing material. 0.4% formic acid solution was used as mobile phase A, and methanol as mobile phase B. The detection wavelength was 270 nm, the column temperature was 20 °C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL. Different elution gradients (Table 10-16 below) were used to determine the same sample solution.

[0131] Table 10 Gradient Condition 1

[0132]

[0133] Table 11 Gradient Condition 2

[0134]

[0135] Table 12 Gradient Condition 3

[0136]

[0137] Table 13 Gradient Condition 4

[0138]

[0139] Table 14 Gradient Condition 5

[0140]

[0141] Table 15 Gradient Condition 6

[0142]

[0143] Table 16 Gradient Conditions 7

[0144]

[0145] Test results as follows Figure 3-9 By comparing the chromatograms of samples measured by different elution programs, the gradient with richer chromatographic information, better resolution of the main chromatographic peaks, more stable baseline, and more reasonable analysis time is selected.

[0146] The suitability parameters for each gradient chromatographic peak system are shown in Table 17:

[0147] Table 17 System Suitability Parameters for Each Gradient Chromatographic Peak

[0148]

[0149]

[0150]

[0151] The results showed that the chromatographic peak separation improved with increasing elution time. Gradient 5 exhibited better separation and richer chromatographic information compared to the previous gradients. While gradient 6 improved separation by extending the elution time, some tail peaks remained unseparated. Referring to gradient 5 and combining it with gradient 6, gradient 7 was obtained. The resulting chromatogram showed richer chromatographic information, more major peaks, better resolution, and a more stable baseline. Therefore, gradient 7 was selected as the preferred mobile phase for subsequent condition screening. The chromatograms of gradients 1-7 showed that peaks after 60 minutes of elution were not relevant. Therefore, the preferred elution time range was 0-60 minutes, as shown in the table below.

[0152] Table 18 shows the final determined elution gradient.

[0153]

[0154] 1.2 Selection of detection wavelength

[0155] An octadecylsilane-bonded silica gel column (250 mm column length, 4.6 mm inner diameter, 5 μm particle size) was used as the packing material. 0.4% formic acid solution was used as mobile phase A, and methanol as mobile phase B. Elution was performed according to the elution gradient determined in Table 18. The column temperature was 20℃, the flow rate was 1.0 mL / min, and the injection volume was 10 μL. The absorption of the standard decoction (lyophilized powder) of stir-fried Liu Shen Qu (a type of Chinese herbal medicine) at different wavelengths was determined using full-wavelength scanning. Chromatograms at 220 nm, 270 nm, 280 nm, and 300 nm were recorded within the range of 210–300 nm. The wavelength with the best peak shape and the most peaks was selected as the optimal wavelength. The full-wavelength scan chromatogram is shown below. Figure 10 The HPLC chromatograms were compared, and the system adaptability parameters for different absorption wavelengths and chromatographic peaks are shown in Table 19.

[0156] Table 19 System adaptability parameters for chromatographic peaks at different absorption wavelengths

[0157]

[0158]

[0159] The composition of stir-fried Shenqu (a type of medicinal herb) is relatively complex. After fermentation, the compounds in the original medicinal material decompose, reducing their original content and generating new compounds. Referring to the full-wavelength scan and HPLC chromatogram, when the detection wavelength range is 220-300 nm, the chromatogram at a wavelength of 270 nm has a large number of chromatographic peaks, moderate intensity, and stable baseline. Therefore, the absorption wavelength of 270 nm, which has relatively rich chromatographic peak information and relatively better system adaptability parameters, is selected as the detection wavelength for the characteristic chromatogram of the standard decoction (lyophilized powder) of stir-fried Shenqu for further investigation.

[0160] 1.3 Investigation of mobile phase composition

[0161] An octadecylsilane-bonded silica gel column (250 mm column length, 4.6 mm inner diameter, 5 μm particle size) was used as the packing material. Elution was performed according to the elution gradient determined in Table 18. The detection wavelength was 270 nm, the column temperature was 20 °C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL. Methanol was used as mobile phase B, and different acid types (0.4% acetic acid solution, 0.4% phosphoric acid solution, and 0.4% formic acid solution) were used as mobile phase A to investigate and obtain a suitable mobile phase system. Specific results are shown in […]. Figures 11-13 See Tables 20-22.

[0162] Table 20 System adaptability parameters for 0.4% acetic acid chromatographic peak

[0163]

[0164]

[0165] Table 21 System adaptability parameters for 0.4% phosphate chromatographic peaks

[0166]

[0167] Table 22 System adaptability parameters for chromatographic peaks of 0.4% formic acid solution

[0168]

[0169] The results showed that different acids had a significant impact on the chromatograms. The chromatograms eluted with the mobile phases of methanol-0.4% phosphoric acid and methanol-0.4% acetic acid had unstable baselines, few chromatographic peaks, or poor resolution. The chromatograms eluted with the mobile phase of methanol-0.4% formic acid had richer chromatographic information, more major chromatographic peaks, better resolution, and more stable baselines. Therefore, the methanol-0.4% formic acid solution mobile phase system with relatively better separation performance was selected for subsequent condition screening and investigation in order to obtain better separation results.

[0170] 1.4 Investigation of different acid concentrations

[0171] An octadecylsilane-bonded silica gel column (250 mm column length, 4.6 mm inner diameter, 5 μm particle size) was used as the packing material. Formic acid solutions of different concentrations were used as mobile phase A, and methanol as mobile phase B. Elution was performed according to the elution gradient determined in Table 18. The detection wavelength was 270 nm, the column temperature was 20 °C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL. Different concentrations of acid solutions (0.3% formic acid, 0.4% formic acid, and 0.5% formic acid) were investigated to obtain a suitable mobile phase system. The results are shown in Table 23.

[0172] Table 23 Parameters for testing the applicability of the system at different formic acid concentrations

[0173]

[0174] The results showed that different formic acid concentrations had little effect on each chromatographic peak, but the chromatogram eluted with 0.4% formic acid solution showed better peak shape and resolution of the main chromatographic peaks. Therefore, 0.4% formic acid solution was selected for subsequent condition screening.

[0175] 1.5 Investigation of different flow velocities

[0176] An octadecylsilane-bonded silica gel column (250 mm column length, 4.6 mm inner diameter, 5 μm particle size) was used as the packing material. 0.4% formic acid solution was used as mobile phase A, and methanol as mobile phase B. Elution was performed according to the elution gradient determined in Table 18. The detection wavelength was 270 nm, the column temperature was 20 °C, and the injection volume was 10 μL. Measurements were performed at flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min to investigate the effect of different flow rates on the separation of characteristic chromatograms of the lyophilized powder of *Chao Liu Shen Qu* (a traditional Chinese medicine). The results are shown in Tables 24-26.

[0177] Table 24 Parameters for assessing system suitability at a flow rate of 0.8 ml / min

[0178]

[0179] Table 25 Parameters for assessing system suitability at a flow rate of 1.0 ml / min

[0180]

[0181] Table 26 Parameters for assessing system suitability at a flow rate of 1.2 ml / min

[0182]

[0183] The results showed that different flow rates had a significant impact on the retention time of some chromatographic peaks, as well as on peak resolution and asymmetry. At a flow rate of 0.8 ml / min, the elution time was generally delayed, while at 1.2 ml / min, the elution time was generally advanced, resulting in some peaks not being baseline separated. A flow rate of 1.0 ml / min showed relatively good peak separation; therefore, 1.0 ml / min was selected as the preferred flow rate for subsequent screening.

[0184] 1.6 Investigation at different column temperatures

[0185] An octadecylsilane-bonded silica gel column (250 mm column length, 4.6 mm inner diameter, 5 μm particle size) was used as the packing material. 0.4% formic acid solution was used as mobile phase A, and methanol as mobile phase B. Elution was performed according to the elution gradient determined in Table 18. The detection wavelength was 270 nm, the flow rate was 1.0 mL / min, and the injection volume was 10 μL. The effect of different column temperatures on the separation of characteristic chromatograms of the lyophilized powder of the stir-fried Liushenqu standard decoction was investigated at column temperatures of 18℃, 20℃, and 22℃. The results are shown in Tables 27-29.

[0186] Table 27 Parameters for System Suitability Testing at Column Temperature of 18℃

[0187]

[0188] Table 28 Parameters for System Suitability Testing at a Column Temperature of 20℃

[0189]

[0190]

[0191] Table 29 Parameters for System Suitability Testing at Column Temperature of 22℃

[0192]

[0193] The results showed that different column temperatures had a significant impact on the retention time of chromatographic peaks. At a column temperature of 18℃, the elution time was prolonged, and peak 5 was not completely eluted. At a column temperature of 22℃, the elution time was shortened, and the separation of each peak was poor. At a column temperature of 20℃, the system adaptability parameters of each peak were relatively good. Taking all factors into consideration, a column temperature of 20℃ was selected as the optimal temperature for subsequent condition screening and investigation.

[0194] 1.7 Investigation of different brands of chromatographic columns

[0195] The column was packed with octadecylsilane-bonded silica gel (250 mm length, 4.6 mm inner diameter, 5 μm particle size), with 0.4% formic acid solution as mobile phase A and methanol as mobile phase B. Elution was performed according to the elution gradient determined in Table 18. The detection wavelength was 270 nm, the column temperature was 20 °C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL. The effect of different brands of chromatographic columns on the characteristic chromatogram of the lyophilized powder of the standard decoction of stir-fried Liushenqu was investigated. The different brands of chromatographic columns investigated were: Waters Xselect HSST3, 250 mm × 4.6 mm, 5 μm (column 1), Diamonsil Plus 5 μm C18-A, 250 mm × 4.6 mm, 5 μm (column 2), and Morphling AQ-C18, 250 mm × 4.6 mm, 5 μm (column 3). Specific results are shown in [Table 18]. Figures 14-16 See Tables 30-32.

[0196] Table 30 Waters Xselect HSS T3 column system suitability parameters

[0197]

[0198] Table 31. Suitability parameters for Diamonsil Plus 5μm C18-A Column system evaluation

[0199]

[0200] Table 32 Morphling AQ-C18 Column Testing System Suitability Parameters

[0201]

[0202] The results showed that different brands of chromatographic columns had a significant impact on the retention time of each chromatographic peak. The Waters Xselect HSST3 column showed the best separation effect for each chromatographic peak, while the other two brands performed poorly in terms of resolution, symmetry factor, and other parameters. Therefore, the preferred column brand is the Waters Xselect HSS T3 (250mm × 4.6mm, 5μm) column.

[0203] 1.8 Determination of chromatographic conditions

[0204] Chromatographic conditions and system suitability: Waters chromatograph, Waters Xselect HSS T3 column, octadecylsilane-bonded silica gel as packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); mobile phase A was 0.4% formic acid, and mobile phase B was pure methanol, with gradient elution as specified in Table 18; detection wavelength was 270 nm, column temperature was 20 °C, and flow rate was 1.0 ml per minute.

[0205] Example 3: Determination of the preparation method of the test solution

[0206] The purpose of this experiment is to investigate the effect of the preparation of the test solution on the detection of the test solution of the freeze-dried powder of the standard decoction of stir-fried Shenqu (a type of Chinese herbal medicine).

[0207] The preparation of the reference solution and the reference decoction piece solution shall be in accordance with Experimental Example 1. The chromatographic conditions shall be in accordance with the chromatographic conditions determined in 1.8.

[0208] 3.1 Investigation of methanol concentration as extraction solvent

[0209] Preparation of the test solution: Accurately weigh 0.2 g of the freeze-dried powder of the standard decoction of stir-fried Shenqu (batch number 2103001Y), place it in a stoppered conical flask, and accurately add 10 ml of extraction solvents of different concentrations (methanol, 50% methanol, and 20% methanol), weigh, stopper tightly, and sonicate (250 W, 40 kHz) for 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the test solution. HPLC analysis is performed; see [link to HPLC results]. Figures 17-19 And Table 33.

[0210] Table 33. Parameters for Adaptability of Chromatographic Peaks Based on Methanol Concentration as Extraction Solvent

[0211]

[0212] HPLC analysis, combined with the chromatographic parameters of each peak, showed that the total peak area of ​​the characteristic chromatographic peaks obtained by extraction with different methanol concentrations varied. The peak area of ​​the chromatographic peaks obtained by extraction with 50% methanol was significantly larger than that obtained by extraction with other methanol concentrations. Considering all factors, 50% methanol was selected as the preferred extraction solvent.

[0213] 3.2 Examination of extraction time

[0214] The effects of different extraction times (20, 30, and 40 minutes) on the extraction of the main characteristic peaks in the characteristic chromatogram of the standard decoction of stir-fried Shenqu were investigated.

[0215] Preparation of the test solution: Accurately weigh 0.2 g of the freeze-dried powder of the standard decoction of stir-fried Shenqu (batch number 2103001Y), place it in a stoppered conical flask, accurately add 10 ml of 50% methanol, weigh, stopper tightly, and sonicate (power 250W, frequency 40kHz) for 20, 30, and 40 minutes respectively. Cool, shake well, filter, evaporate to dryness in a water bath, accurately add 2 ml of 50% methanol to dissolve, filter, and collect the filtrate. The results of HPLC analysis are shown in Table 34.

[0216] Table 34 Extraction time parameters for chromatographic peak system adaptability.

[0217]

[0218]

[0219] Results analysis: A comprehensive comparison of the results of three different extraction times showed that different extraction times did not affect the retention time of each peak; there was no significant difference in peak area after ultrasonic treatment for 20 minutes, 30 minutes and 40 minutes, indicating that extraction was complete after ultrasonic treatment for 30 minutes, and there were no significant differences in the separation degree, asymmetry and theoretical plate number of each peak. The optimal extraction time is 30 minutes.

[0220] 3.3 Sampling Quantity Examination

[0221] The effect of different sampling amounts on the characteristic chromatogram of the standard decoction of stir-fried Shenqu (a type of medicinal herb) was investigated.

[0222] Preparation of the test solution: Accurately weigh 0.1g, 0.2g, and 0.5g of the freeze-dried powder of the standard decoction of stir-fried Shenqu (batch number 2103001Y), place them in stoppered conical flasks, accurately add 10ml of 50% methanol to each flask, weigh, stopper tightly, and sonicate (power 250W, frequency 40kHz) for 30 minutes respectively. After cooling, shake well, filter, evaporate to dryness in a water bath, accurately add 2ml of 50% methanol to dissolve, filter, and collect the filtrate. The results of HPLC analysis are shown in Table 35.

[0223] Table 35 Sampling quantity parameters for chromatographic peak system adaptability assessment

[0224]

[0225] Experimental results show that different sample amounts have no significant impact on the retention time, resolution, symmetry factor, and theoretical plate number of each characteristic peak. Furthermore, at a sample amount of 0.2 g, the peak areas of the characteristic peaks in the lyophilized powder of the standard decoction of stir-fried Shenqu (a type of Chinese herbal medicine) are relatively moderate. Therefore, 0.2 g is the preferred sample amount for this method.

[0226] 3.4 Investigation of different solvent volumes

[0227] The effect of different solvent volumes (5 ml, 10 ml, 20 ml) on the characteristic chromatogram of the standard decoction of stir-fried Shenqu (a type of Chinese herbal medicine) was investigated.

[0228] Preparation of the test solution: Accurately weigh 0.2 g of the freeze-dried powder of the standard decoction of stir-fried Shenqu (batch number 2103001Y), place it in a stoppered conical flask, and accurately add 5 ml, 10 ml, and 20 ml of 50% methanol respectively. Weigh the flasks, stopper them tightly, and sonicate them (power 250 W, frequency 40 kHz) for 30 minutes respectively. Cool, shake well, filter, evaporate to dryness in a water bath, accurately add 2 ml of 50% methanol to dissolve, filter, and collect the filtrate. The results of HPLC analysis are shown in Table 36.

[0229] Table 36 Solvent Volume Assessment: Chromatographic Peak System Adaptability Parameters

[0230]

[0231] HPLC analysis was conducted, with the information content and separation effect of the chromatographic peaks as the main evaluation indicators. The results showed that different solvent volumes mainly affected the peak area of ​​the chromatogram. When the solvent volume was 20 ml, the solution concentration was low, the peak area was too small, and integration error was easily caused. When the solvent volume was 5 ml, the peak area was large, and the system adaptability parameters were relatively poor. Therefore, 10 ml was selected as the extraction solvent volume.

[0232] 3.5 Validation of the test sample preparation method

[0233] Based on the above results, the preparation method for the test solution of the freeze-dried powder of the standard decoction of stir-fried Shenqu (a type of medicinal herb) is determined as follows:

[0234] Accurately weigh 0.2g of the freeze-dried powder of the standard decoction of stir-fried Shenqu (a type of medicinal preparation), place it in a stoppered conical flask, accurately add 10ml of 50% methanol, weigh, seal tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, weigh again, replenish the lost weight with 50% methanol, shake well, evaporate to dryness in a water bath, add 2ml of 50% methanol to dissolve, filter, evaporate to dryness in a water bath, accurately add 2ml of 50% methanol to dissolve, filter, and collect the filtrate to obtain the final product.

[0235] Experimental Example 4: Analytical Methodology Validation

[0236] Preparation of the test solution: Accurately weigh 0.2 g of the freeze-dried powder of the standard decoction of stir-fried Shenqu (batch number 2103001Y), place it in a stoppered conical flask, add 10 ml of 50% methanol, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with 50% methanol, shake well, evaporate to dryness in a water bath, add 2 ml of 50% methanol to dissolve it, filter it, evaporate to dryness in a water bath, accurately add 2 ml of 50% methanol to dissolve it, filter it, and take the filtrate to obtain the test solution.

[0237] Preparation of reference solution for stir-fried Shenqu (a type of Chinese herbal medicine): Take 2g of stir-fried Shenqu reference slices (batch number DSTYC009801), accurately weigh it, add 50ml of water, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 20ml of 50% methanol to dissolve the residue, filter, and take the filtrate as the reference solution for the reference slices.

[0238] Preparation of reference solutions: Take appropriate amounts of uridine, guanosine, and gallic acid reference standards, accurately weigh them, and add 50% methanol to prepare a solution containing 50 μg of each reference standard per 1 ml.

[0239] Detection by high performance liquid chromatography: The chromatographic conditions are the same as those determined in 1.8 of Experimental Example 2.

[0240] 4.1 Instrument precision test

[0241] The same sample solution was injected six times under chromatographic conditions. Chromatograms were recorded, and the relative retention times and relative peak areas of the six characteristic peaks were determined and analyzed. The results showed that the RSD of the relative retention times of each characteristic peak and the reference peak S (peak 1) was less than 2%, and the RSD of the relative peak areas of each characteristic peak and the reference peak S (peak 1) was less than 14%. This indicates good instrument precision.

[0242] 4.2 Repeatability Experiment

[0243] Six samples from the same batch of test samples (batch number: 21030010Y) were taken, and the relative retention times and relative peak areas of the six common peaks were measured and analyzed. The results showed that the RSD of the relative retention times of each characteristic peak and the reference peak S (peak 1) was less than 2%, and the RSD of the relative peak areas of each characteristic peak and the reference peak S (peak 1) was less than 12%, indicating that the method has good repeatability.

[0244] 4.3 Stability Test

[0245] The same sample was injected at 0, 2, 4, 6, 8, 10, 12, and 24 hours after preparation. The relative retention times and relative peak areas of the six common peaks were measured and analyzed to determine the stability of the sample solution. The results showed that the RSD of the relative retention times of each characteristic peak and the reference S peak (peak 1) was less than 2%, and the RSD of the relative peak areas of each characteristic peak and the reference S peak (peak 4) was less than 10%. This indicates that the sample solution was relatively stable within 24 hours.

[0246] 4.5 Durability

[0247] 4.5.1 Investigation of different flow velocities

[0248] Take the same batch of test sample solution (batch number: 21030010Y), and change the chromatographic conditions only by the flow rate. Measure the sample at flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min. Examine the relative retention time and relative peak area of ​​each characteristic peak and the reference peak S (peak 1) when the flow rate changes slightly, and analyze the results. The results show that the relative retention time RSD% is less than 2% for all peaks, and the relative peak area RSD for peak 6 is 60%. Flow rate has a significant impact on the relative peak area. Therefore, a flow rate of 1.0 ml / min is preferred.

[0249] 4.5.2 Investigation at different column temperatures

[0250] Using the same sample, the chromatographic conditions were varied except for the column temperature. The column temperatures were set to 18℃, 20℃, and 22℃, and the sample was injected and analyzed. The chromatograms were recorded, and the relative retention times and relative peak areas of the characteristic peaks were analyzed. The relative retention times and relative peak areas of each characteristic peak and the reference peak S (peak 1) were examined when the column temperature changed. The results showed that the relative retention time RSD% was less than 4%, while the RSD of the relative peak area of ​​peak 4 was relatively large, approximately 36.2%. This indicates that different column temperatures have a significant impact on the relative peak area of ​​the characteristic chromatograms. Therefore, the optimal column temperature for this method is 20℃.

[0251] 4.5.3 Examination of different brands of chromatographs

[0252] A sample solution of the same lyophilized powder of stir-fried Shenqu (a traditional Chinese medicine) decoction was taken. The chromatographic conditions were changed only by the chromatograph, and the determination was performed using Shimadzu, Waters, and Agilent chromatographs. The chromatograms were recorded, and the retention times of the characteristic peaks were analyzed. The relative retention times of each characteristic peak and the reference peak S (peak 1) were examined when the liquid phase changed. The results showed that the relative retention time RSD% was less than 2%. This indicates that different chromatographs have little effect on the relative retention times of characteristic peaks 1, 2, 3, 5, and 6 (varying within ±10%), and that different instruments have good robustness to the characteristic chromatogram construction method of this invention.

[0253] The above experiments show that the method for constructing the characteristic chromatograms of the fried six-ingredient herbal decoction and its pharmaceutical preparations provided by the present invention, using octadecylsilane-bonded silica gel as the filler, methanol as mobile phase A, and 0.08%-0.4% formic acid as mobile phase B, and employing a specific elution procedure, not only shortens the detection time but also significantly improves the separation effect of multiple active ingredients. This results in the characteristic chromatograms containing more characteristic peaks, greatly enriching the chromatographic information and achieving effective separation of characteristic peaks including uridine, thymine, gallic acid, and guanosine. The obtained characteristic chromatograms are highly distinctive, fully reflecting the integrity and characteristics of the fried six-ingredient herbal decoction pharmaceutical preparation. The baseline is stable, and the peak shape is good. The S-peak uridine is selected as the internal reference peak in the characteristic chromatogram, and six common characteristic peaks of the fried six-ingredient herbal decoction can be identified. The relative retention time of each common characteristic peak can be calculated based on the S-peak uridine, which is beneficial for the comprehensive quality detection and overall quality control of the fried six-ingredient herbal decoction, thereby helping to improve the safety and stability of the drug.

[0254] Example 5: Establishment of Feature Map

[0255] The "Traditional Chinese Medicine Chromatographic Characteristic Chromatographic Similarity Evaluation System 2012 Edition" software, compiled by the Pharmacopoeia Commission, was used to generate a reference characteristic chromatogram of the freeze-dried powder of the standard decoction of stir-fried Shenqu (a type of fermented medicinal herb) using multiple batches of representative samples. A reference characteristic chromatogram of stir-fried Shenqu was also generated using multiple batches of representative samples.

[0256] Preparation of the test solution: Take the test sample (a batch of stir-fried Shenqu decoction standard decoction freeze-dried powder or stir-fried Shenqu decoction pieces or Shenqu decoction pieces).

[0257] Accurately weigh 0.2g and place it in a stoppered conical flask. Add 10ml of 50% methanol, weigh the flask, and sonicate (250W power, 40kHz frequency) for 30 minutes. Let it cool, weigh it again, and replenish the lost weight with 50% methanol. Shake well, evaporate to dryness in a water bath, add 2ml of 50% methanol to dissolve the solution, filter, evaporate to dryness in a water bath, accurately add 2ml of 50% methanol to dissolve the solution, filter, and collect the filtrate.

[0258] Preparation of the reference solution for stir-fried Shenqu (a type of fermented medicinal herb): Accurately weigh 2g of stir-fried Shenqu reference slices (batch number DSTYC009801), add 50ml of water, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of 50% methanol, filter, and use the filtrate as the reference solution. Preparation of the reference solution: Accurately weigh appropriate amounts of uridine, guanosine, and gallic acid reference standards, add 50% methanol to prepare a solution containing 50μg of each reference standard per 1ml.

[0259] Detection by high performance liquid chromatography: The chromatographic conditions are the same as those determined in 1.8 of Experimental Example 2.

[0260] 5.1 Characteristic chromatogram determination of 15 batches of stir-fried Shenqu (a traditional Chinese medicine) decoction standard preparations

[0261] Take 15 batches of freeze-dried powder of standard decoction of stir-fried Shenqu (a type of medicinal herb), and perform the above-mentioned test methods. The test results are as follows: Figure 20 And Table 37.

[0262] Table 37. Results of relative retention times of standard decoctions from 15 batches of stir-fried Shenqu (a type of medicinal herb).

[0263]

[0264] The test results showed that the relative retention times of each characteristic peak in the characteristic chromatogram of the lyophilized powder of the 15 batches of stir-fried Shenqu (a type of Chinese herbal medicine) decoction were all within ±10% of the values ​​specified in the reference chromatogram of the stir-fried Shenqu decoction. The generated reference characteristic chromatogram of the lyophilized powder of the stir-fried Shenqu decoction is shown below. Figure 1 As shown, there are 6 chromatographic peaks with good resolution. Taking peak 1 as the S peak, the relative retention times of the other characteristic peaks relative to peak 1 are as follows: Peak 2:

[0265] 1.09, Peak 3: 1.20, Peak 4: 1.35, Peak 5: 1.82, Peak 6: 2.56.

[0266] 5.2 Characteristic Spectrum Determination of 15 Batches of Fried Shenqu Herbal Slices

[0267] Take 15 batches of stir-fried Shenqu (a type of medicinal herb) slices, and perform the above-mentioned testing method. The test results are as follows: Figure 21 See Tables 38 and 39.

[0268] Table 38. Results of relative retention time determination of characteristic chromatograms of 15 batches of stir-fried Shenqu (a type of Chinese herbal medicine).

[0269]

[0270]

[0271] Table 39. Results of relative peak area determination of characteristic chromatograms of 15 batches of stir-fried Shenqu (a type of Chinese herbal medicine).

[0272]

[0273] The test results showed that the relative retention times of each characteristic peak in the characteristic chromatograms of 15 batches of stir-fried Shenqu medicinal slices were all within ±10% of the values ​​specified in the reference chromatograms of stir-fried Shenqu medicinal slices.

[0274] Generate a comparative characteristic spectrum of stir-fried Shenqu (a traditional Chinese medicine) slices, such as... Figure 22 As shown, there are 6 chromatographic peaks with good separation. Taking peak 1 as the S peak, the relative retention times of the other characteristic peaks relative to peak 1 are as follows: peak 2: 1.09, peak 3: 1.21, peak 4: 1.35, peak 5: 1.83, and peak 6: 2.56.

[0275] 5.3 Characteristic Spectrum Determination of 15 Batches of Liu Shen Qu Herbal Pieces

[0276] Fifteen batches of Shenqu (a traditional Chinese medicine) slices were taken and tested according to the above-mentioned method. The test results are shown in Tables 40 and 41. Figure 23 .

[0277] Table 40: Relative Retention Time Results of Characteristic Pictorial Data of 15 Batches of Liu Shen Qu Herbal Pieces

[0278]

[0279] Table 41. Relative peak area results of characteristic chromatograms of 15 batches of Liu Shen Qu (a traditional Chinese medicine) slices.

[0280]

[0281]

[0282] The test results showed that the relative retention times of each characteristic peak in the characteristic chromatograms of 15 batches of Liushenqu (a traditional Chinese medicine) were all within ±10% of the values ​​specified in the Liushenqu reference chromatogram. The generated Liushenqu reference characteristic chromatogram is shown below. Figure 24 As shown, the six chromatographic peaks with good separation include peak 1 (S): uridine; peak 2: thymine; peak 6: guanosine; taking peak 1 as the S peak, the relative retention times of the other characteristic peaks relative to peak 1 are as follows: peak 2: 1.09, peak 3: 1.21, peak 4: 1.81, peak 5: 2.13, peak 6: 2.55.

[0283] 5.4 Characteristic Spectral Transmission Relationship between Six-Shenqu Slices, Fried Six-Shenqu Slices, and Fried Six-Shenqu Standard Decoction

[0284] A comparison of the characteristic spectra of the standard decoction of stir-fried Shenqu, stir-fried Shenqu slices, and Shenqu slices was conducted, and the results are as follows: Figure 25 S1(5): Comparison chart of Liu Shen Qu (a type of medicinal herb) slices; S2(5): Comparison chart of stir-fried Liu Shen Qu (a type of medicinal herb) slices; S3(5): Comparison chart of standard decoction of stir-fried Liu Shen Qu (a type of medicinal herb).

[0285] The chromatograms show changes in the characteristic peaks obtained from the characteristic chromatograms of Liushenqu (a type of fermented medicinal herb), stir-fried Liushenqu, and the standard decoction of stir-fried Liushenqu. A total of five characteristic peaks were observed. After stir-frying, the peak area of ​​peak 3 significantly increased, and a new gallic acid characteristic peak (peak a) was added. Peak b showed significant degradation and completely disappeared in some batches. Therefore, the chemical composition of Liushenqu changed significantly before and after stir-frying. After the stir-fried Liushenqu was prepared into a standard decoction, the six characteristic peaks appearing in the characteristic chromatogram were stably transferred to the standard decoction, indicating good value transfer of each characteristic peak during the process from stir-fried Liushenqu to the standard decoction.

[0286] Example 2

[0287] This embodiment provides a quality testing method for stir-fried Shenqu (medicated leaven) slices and its pharmaceutical preparations, including comparing the characteristic chromatogram of the stir-fried Shenqu slices to be tested with the control characteristic chromatogram of stir-fried Shenqu slices or its pharmaceutical preparations.

[0288] Among them, the feature spectrum of the stir-fried Shenqu herbal slices to be tested was constructed using the stir-fried Shenqu herbal slices to be tested according to the construction method described above;

[0289] The comparative characteristic chromatogram of stir-fried Shenqu slices or its pharmaceutical preparations is directly adopted from the comparative characteristic chromatogram of stir-fried Shenqu slices or standard decoction obtained in Experimental Example 5; (or, the comparative characteristic chromatogram of stir-fried Shenqu slices or its pharmaceutical preparations is adopted from the characteristic chromatogram of the pharmaceutical preparations of stir-fried Shenqu slices obtained by the construction method of Example 1 from single batches or multiple batches of the pharmaceutical preparations of stir-fried Shenqu slices; the comparative characteristic chromatogram of stir-fried Shenqu slices or its pharmaceutical preparations can also be prepared by using the characteristic chromatogram of the pharmaceutical preparations of stir-fried Shenqu slices obtained by the construction method of Example 1 from multiple batches of the pharmaceutical preparations of stir-fried Shenqu slices through the average value or median method to prepare the comparative characteristic chromatogram).

[0290] Example 3

[0291] This embodiment provides a method for identifying stir-fried Shenqu (a type of medicinal fermentation) slices or its preparations with Shenqu slices. The characteristic spectrum of the product to be identified is constructed using the construction method of Example 1.

[0292] If the characteristic spectrum of the product to be identified has 6 common characteristic peaks, including peak 1 being uridine, peak 2 being thymine, peak 4 being gallic acid, and peak 6 being the characteristic peak corresponding to guanosine; then the product to be identified is stir-fried Shenqu (medicated leaven) slices or its preparations.

[0293] If the characteristic spectrum of the product to be identified has 6 common characteristic peaks, including peak 1 being uridine, peak 2 being thymine, and peak 6 being the characteristic peak corresponding to guanosine, then the product to be identified is Liu Shen Qu (a traditional Chinese medicine preparation); or, if the relative peak area of ​​peak 3 and peak 1 is greater than 1.3, then the product to be identified is fried Liu Shen Qu (another traditional Chinese medicine preparation) or its preparation; if the relative peak area of ​​peak 3 and peak 1 is less than 1.2, then the product to be identified is Liu Shen Qu (another traditional Chinese medicine preparation).

[0294] The above experiments demonstrate that the method for distinguishing between the pharmaceutical preparation of stir-fried Liu Shen Qu and its counterfeit products provided by this invention involves comparing the characteristic spectrum of the product to be identified with the control characteristic spectrum of stir-fried Liu Shen Qu or its preparation. This allows for the identification of stir-fried Liu Shen Qu and its preparation from counterfeit products made of flour and wheat bran, or counterfeit products made without the addition of red beans or bitter almonds.

[0295] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing the preparation characteristic chromatogram of Fructus Crataegi, which is characterized in that, Includes the following steps: Preparation of the test solution: The test solution is prepared by weighing the test sample, extracting it with a solvent to obtain the extract, separating the solid and liquid, and taking the liquid, which is the test solution; the solvent is selected from methanol or methanol-water solution; the method for constructing the Liushenqu decoction pieces also includes the step of preparing a reference solution by adding a solvent with uridine, thymine, and guanosine as reference standards; the method for constructing the fried Liushenqu decoction pieces and its preparations also includes the step of preparing a reference solution by adding a solvent with uridine, thymine, gallic acid, and guanosine as reference standards; High-performance liquid chromatography (HPLC) was used to detect the test solution and reference solution. The chromatographic conditions included: octadecylsilane-bonded silica gel as the stationary phase; detection wavelength of 270-280 nm; column temperature of 20℃; flow rate of 1 mL / min; mobile phase A of methanol; and mobile phase B of 0.08%-0.4% formic acid. Gradient elution was performed according to the specifications in the table below. 。 2. The construction method according to claim 1, characterized in that, A chromatographic column with dimensions of 250 mm × 4.6 mm and a particle size of 5 μm was used. And / or, the injection volume of the test solution is 5-20 µL.

3. The construction method according to claim 1, characterized in that, The method for preparing the test solution also satisfies any one or more of the following A: A. The ratio of the mass of the test sample to the volume of the solvent is 0.1-0.5:5-20; the relationship between mass and volume is g / mL. B. The extraction method is either reflux extraction or ultrasonic extraction; C. Extraction time is ≥20 min; D. The solid-liquid separation is selected from centrifugation or membrane filtration.

4. The construction method according to claim 1, characterized in that, The solid-liquid separation is specifically as follows: the extract is filtered through a filter membrane and evaporated to dryness; the solvent is added to dissolve it, and the extract is filtered through a filter membrane again, and the filtrate is the test solution. Alternatively, the solid-liquid separation specifically involves: adding a solvent to the extract to replenish the weight lost during the extraction process, shaking well, evaporating to dryness, adding the solvent to dissolve, filtering through a filter membrane, and taking the filtrate as the test solution; Alternatively, the solid-liquid separation may specifically involve: adding solvent to the extract to replenish the weight lost during extraction, shaking well, and evaporating to dryness; adding the solvent to dissolve the extract, filtering through a filter membrane, and evaporating to dryness again; adding the solvent again to dissolve the extract, filtering through a filter membrane again, and taking the filtrate as the test solution.

5. The construction method according to claim 1, characterized in that, In the preparation of the reference solution, the solvents are selected from at least one of methanol, ethanol and water.

6. The construction method according to claim 5, characterized in that, Each 1 mL of the reference solution contains 0.005–50 µg of the respective reference standard.

7. The construction method according to any one of claims 1-6, characterized in that, The characteristic spectrum of the Liushenqu herbal slices has 6 common characteristic peaks, including peak 1 which is uridine, peak 2 which is thymine, and peak 6 which is the characteristic peak corresponding to guanosine.

8. The construction method according to claim 7, characterized in that, In the characteristic spectrum of the Six-Ingredient Dioscorea Medication, peak 1 is designated as peak S. The relative retention times of other characteristic peaks relative to peak 1 are within ±10% of a specified value. The specified values ​​for each characteristic peak are as follows: Peak 2: 1.09, Peak 3: 1.21, Peak 4: 1.81, Peak 5: 2.13, Peak 6: 2.

55.

9. The construction method according to any one of claims 1-6, characterized in that, The characteristic chromatogram of the stir-fried Shenqu slices and its preparations has 6 common characteristic peaks, including peak 1 being uridine, peak 2 being thymine, peak 4 being gallic acid, and peak 6 being the characteristic peak corresponding to guanosine.

10. The construction method according to claim 9, characterized in that, In the characteristic chromatogram of the stir-fried Shenqu slices and its preparations, peak 1 is designated as peak S. The relative retention times of other characteristic peaks relative to peak 1 are within ±10% of a specified value. The specified values ​​for each characteristic peak are as follows: Peak 2: 1.09, Peak 3: 1.21, Peak 4: 1.35, Peak 5: 1.83, Peak 6: 2.

56.

11. The application of the method for constructing the characteristic chromatograms of Liushenqu slices, stir-fried Liushenqu slices and their preparations as described in any one of claims 1-10 in the quality detection of Liushenqu slices, stir-fried Liushenqu slices and their preparations.

12. A method for quality testing of Shenqu (a traditional Chinese medicine) slices, characterized in that, This includes the step of comparing the characteristic chromatogram of the product to be tested, Liushenqu, with the control characteristic chromatogram of Liushenqu decoction pieces. The characteristic spectrum of the tested Liushenqu product is constructed using the tested Liushenqu product according to any one of the construction methods described in claims 1-6, and the control characteristic spectrum of the Liushenqu slices is selected from any one of the following (1)-(3): (1) It has 6 common characteristic peaks, including peak 1 being uridine, peak 2 being thymine, and peak 6 being the characteristic peak corresponding to guanosine; Taking peak 1 as peak S, the relative retention times of other characteristic peaks relative to peak 1 are within ±10% of the specified value. The specified values ​​for each characteristic peak are: Peak 2: 1.09, Peak 3: 1.21, Peak 4: 1.81, Peak 5: 2.13, Peak 6: 2.55; (2) A comparative feature spectrum of Liushenqu decoction pieces obtained by using a single batch or multiple batches of Liushenqu decoction pieces according to any one of the construction methods described in claims 1-6; (3) Using multiple batches of Liushenqu medicinal slices, the feature maps obtained by any of the construction methods described in claims 1-6 are used to prepare a comparative feature map of Liushenqu medicinal slices by means of the average value or median.

13. A method for quality testing of stir-fried Shenqu (a type of medicinal fermentation) slices and its pharmaceutical preparations, characterized in that, The process includes comparing the characteristic chromatogram of the tested stir-fried Shenqu (a type of medicinal fermented wheat) slices with the control characteristic chromatogram of stir-fried Shenqu slices or its pharmaceutical preparations. The characteristic chromatogram of the tested stir-fried Shenqu herbal slices is constructed using the tested stir-fried Shenqu herbal slices according to any one of the construction methods described in claims 1-6, and the control characteristic chromatogram of the stir-fried Shenqu herbal slices or its pharmaceutical preparation is selected from any one of the following (1)-(3): (1) It has 6 common characteristic peaks, including peak 1 is uridine, peak 2 is thymine, peak 4 is gallic acid, and peak 6 is the characteristic peak corresponding to guanosine. Taking peak 1 as peak S, the relative retention times of other characteristic peaks relative to peak 1 are within ±10% of the specified value. The specified values ​​for each characteristic peak are: Peak 2: 1.09, Peak 3: 1.21, Peak 4: 1.35, Peak 5: 1.83, Peak 6: 2.56; (2) The characteristic spectrum of the pharmaceutical preparation of fried shenqu decoction pieces obtained by using single or multiple batches of fried shenqu decoction pieces or their pharmaceutical preparations according to any one of the construction methods described in claims 1-6; (3) The characteristic spectrum obtained by the construction method described in any one of claims 1-6 using multiple batches of stir-fried Shenqu decoction pieces is used to make a control characteristic spectrum by the average value or median method.

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