A HPLC detection method for Dashanzhao pills, fingerprint spectrum of Dashanzhao pills and application thereof

Abstract

The present invention relates to the field of drug analysis technology, and specifically to a Da Shanzhao Pill HPLC detection method, Da Shanzhao Pill fingerprint and application; the detection method includes Da Shanzhao Pill fingerprint and main index component quantitative method. A systematic investigation was conducted on different extraction methods, extraction time, extraction solvent, and extraction volume of samples to screen out the optimal extraction conditions; and the measurement data was systematically analyzed using chemometric tools. The results of the method are reliable and highly accurate, and can reflect the overall quality of commercially available Da Shanzhao Pills; the method can effectively monitor the quality status of Da Shanzhao Pills, and provide a scientific evaluation strategy for the control of the quality consistency and effectiveness of the preparation.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a HPLC detection method for Da Shanzhao Pills, a Da Shanzhao Pills fingerprint spectrum and applications. Background Art

[0002] The "Requirements for Submission of Material Standards for Traditional Chinese Medicine Compound Preparations of Ancient Classic Prescriptions (Draft for Comments)" requires that "in terms of the material standards for classic prescriptions and the quality requirements for classic prescription preparations, in principle, the information on each ingredient in the prescription should be reflected in items such as content determination or fingerprint spectrum." This provides a reference guideline and quality standard for the development and research of traditional Chinese medicine compound preparations of ancient classic prescriptions. As a classic Chinese medicine prescription, Da Shanzha Pills are a large honey pill made by mixing fine powders of hawthorn, bran-fried Liushenqu, and fried malt with refined honey and water. It has the functions of digesting food, removing stagnation, and harmonizing the spleen and stomach. [1] . "Nature and flavor" is a unique attribute of traditional Chinese medicine, reflecting the essential characteristics of traditional Chinese medicine and the core of the theory of traditional Chinese medicine properties. Da Shanzha Pills are sour, and the sour taste can be astringent. The material basis of this type of medicine is mainly organic acids. Da Shanzha Pills and compound digestive enzymes both promote the recovery of gastrointestinal digestive function, and compared with compound digestive enzymes, Da Shanzha Pills are low-priced, high-quality and safe. [2] Hawthorn, as the main medicine, is rich in flavonoids, phenolic acids and other ingredients, and has a variety of modern pharmacological effects. [3-4] Based on the Chinese Pharmacopoeia, the quality control of Da Shanzha Wan is currently mainly based on the content determination of a single ingredient, and the formulation time is relatively long. [5-6] , lack of systematic analysis. Da Shanzhao Pills are safe, widely used, in high demand, and produced by many companies. There is no literature report on whether there are differences in quality between different manufacturers and batches, and on the overall quality. Moreover, in recent years, the drugs sold on the market [7] , or food [8] , there are differences in quality, and it is difficult to control their quality.

[0003] In order to better control the quality of Da Shanzha Pills, the present invention is based on the concept of overall quality control of Chinese medicine preparations, uses fingerprint technology to establish a characteristic fingerprint of Da Shanzha Pills, and quantitatively determines the main indicator components chlorogenic acid, vanillic acid, hyperoside, isochlorogenic acid B, and isochlorogenic acid A. With the help of chemometric statistical analysis software, comprehensive quality characteristic analysis of multiple batches of Da Shanzha Pills is carried out to ensure the safety, stability and effectiveness of Da Shanzha Pills, and provide a reference for the quality control of Da Shanzha Pills preparations.

[0004] References

[0005] [1] Chinese Pharmacopoeia [S]. Volume 1. 2020: 519-520.

[0006] [2] Luo Shaoju, Wu Jiaming, Zhang Xutao, et al. Comparative study of Da Shanzha Wan and compound digestive enzymes in promoting gastrointestinal digestive function [J]. Guangming Chinese Medicine, 2015, 30(12): 2552-2554.

[0007] [3] Ren Weiguang, Zhang Cuiying. Research progress of hawthorn and prediction of quality markers[J]. Northwest Pharmaceutical Journal, 2023, 38(04): 214-220.

[0008] [4] Shi Guoyu, Wu Weihong, Shang Qingjie, et al. Research progress on the pharmacological effects and health care applications of hawthorn as both medicine and food[J]. Modern Food, 2020(15):126-128.

[0009] [5] Song Jie. Determination of ursolic acid in Dashanzhao pills by HPLC[J]. Medical Theory and Practice, 2011, 24(21): 2636-2637.

[0010] [6] Luo Lan, Guo Libing, Zeng Changqing. Determination of total flavonoids in Dashanzhao Pills[J]. Lishizhen Traditional Chinese Medicine, 2007(09):2207-2208.

[0011] [7] Liu Xianlei, Li Kun, Zhang Hui, et al. Study on UPLC fingerprint and chemical pattern recognition of commercially available Danshen formula granules[J]. Journal of Pharmaceutical Analysis, 2022, 42(11): 2000-2009.

[0012] [8] Hui Ju, Chu Baijun, Chen Shaocong, et al. Quality analysis of eleven commercially available sunflower seed oil products [J / OL]. China Oils and Fats: 1-10 [2023-12-24]. Summary of the invention

[0013] The primary purpose of the present invention is to provide a HPLC detection method for Da Shanzha Wan, wherein Da Shanzha Wan is extracted to obtain a test solution, which is then tested by HPLC; the HPLC test conditions are:

[0014] Column: Eclipse Plus C 18 Chromatographic column, 250 mm × 4.6 mm, 5 μm;

[0015] Mobile phase: acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution;

[0016] Flow rate: 1.0 mL / min;

[0017] Detection wavelength: 250nm;

[0018] Column temperature: 30°C;

[0019] Injection volume: 10 μL;

[0020] The gradient elution program was as follows: 0-5 min, 5%-6% A; 5-12 min, 6%-12% A; 12-20 min, 12%-15% A; 20-26 min, 15%-19% A; 26-40 min, 19%-33% A; 40-55 min, 33%-55% A; 55-60 min, 55%-75% A; 60-65 min, 75%-90% A; 65-70 min, 90%-5% A; 70-72 min, 5% A.

[0021] The second object of the present invention is to provide a method for detecting the fingerprint of Da Shanzha Wan, comprising the following steps:

[0022] S1. Preparation of reference substance solution: accurately weigh each reference substance, place it in a volumetric flask, dissolve it with methanol, and make up to volume to obtain a reference substance solution containing a predetermined concentration of a standard component;

[0023] S2. Preparation of test solution: accurately weigh the sample and mix it with diatomaceous earth in a ratio of 1:1, add extraction solvent for extraction, let it stand and cool, centrifuge to obtain the supernatant, concentrate under reduced pressure, make up to volume, filter, and obtain the filtrate;

[0024] S3. Establishment of HPLC characteristic fingerprint: Using the detection method, obtain the chromatogram of the reference solution and the test sample fingerprint of the test solution under the same chromatographic conditions.

[0025] Preferably, the standard ingredients described in step S1 are chlorogenic acid, vanillic acid, hyperoside, isochlorogenic acid B and isochlorogenic acid A.

[0026] Preferably, the mass concentrations of chlorogenic acid, vanillic acid, hyperoside, isochlorogenic acid B and isochlorogenic acid A in step S1 are 1.0251, 1.0747, 0.9186, 0.8900, 1.0104 and 0.9731 mg / mL, respectively.

[0027] Preferably, the method for extracting the sample solution in step S2 is ultrasonic extraction or thermal reflux extraction.

[0028] Preferably, the method for extracting the sample solution in step S2 is hot reflux extraction.

[0029] Preferably, the extraction volume of the test solution in step S2 is 10 to 50 ml.

[0030] Preferably, the extraction volume of the test solution described in step S2 is 25 ml.

[0031] Preferably, the extraction solvent of the test solution described in step S2 is 30% methanol, 50% methanol, 70% methanol, methanol or 95% ethanol.

[0032] Preferably, the extraction solvent of the test solution described in step S2 is methanol.

[0033] Preferably, the filtration in step S2 uses a microporous filter membrane.

[0034] Preferably, the microporous filter membrane described in step S2 is a 0.45 μm organic microporous filter membrane.

[0035] The third object of the present invention is to provide the application of the detection method in detecting commercially available Da Shanzha Wan.

[0036] The beneficial effects of the present invention are:

[0037] (1) The present invention provides a HPLC detection method for Da Shanzha Wan, and systematically investigates different sample extraction methods (ultrasound, hot reflux), extraction time (30 min, 1 h), extraction solvents of methanol (30, 50, 70, 100%), 95% ethanol, and extraction volumes (10, 25, 50 mL). The results show that when 25 mL of methanol is added to 2 g of sample and heated under reflux for extraction for 1 h, the extraction rate of the sample is the best and the response value is the highest, so this method is determined to be the optimal extraction condition;

[0038] (2) The characteristic fingerprint of Da Shanzha Wan was established using the optimal extraction conditions, and the common peaks were determined and the main index components were identified based on the rich chromatographic information;

[0039] (3) The present invention utilizes the HPLC detection method to detect and evaluate the Da Shanzha Pills that have been marketed in China, so as to control the quality of the Da Shanzha Pills on the market and ensure the clinical efficacy of the Da Shanzha Pills. The HPLC detection method can realize the quality monitoring of the Da Shanzha Pills, provide a reference basis for ensuring the safety, stability and quality control of the Da Shanzha Pills, and promote the inheritance and development of traditional Chinese medicine.

[0040] (4) The results of the method described are reliable and accurate, and can reflect the overall quality of the commercially available Da Shanzhao Pills. This method can effectively monitor the quality of Da Shanzhao Pills and provide a scientific evaluation strategy for controlling the quality consistency and effectiveness of the preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Chromatograms of different gradient elution conditions

[0042] Note: The gradient elution methods from top to bottom are (1), (2), (3) and (4);

[0043] Figure 2 HPLC chromatogram of Dashanzhao Pills

[0044] Note: A. Mixed reference; B. Sample; C. Negative sample; 1-chlorogenic acid; 2-vanillic acid; 3-hyperoside; 4-isochlorogenic acid B; 5-isochlorogenic acid A;

[0045] Figure 3 HPLC fingerprint of Dashanzhao Pills

[0046] Note: Peaks identified: 5. Chlorogenic acid 6. Vanillic acid 7. Hyperoside 9. Isochlorogenic acid B 10. Isochlorogenic acid A

[0047] Figure 4 HPLC comparative fingerprint of Dashanzhao Pills

[0048] Figure 5 Chromatogram of mixed reference solution for fingerprint peak identification of Dashanzhao Pills

[0049] Figure 6 Radar chart of the content of 5 ingredients

[0050] Figure 7 Cluster analysis diagram

[0051] Figure 8 Principal component score plot

[0052] Fig. 9 OPLS-DA score plot

[0053] Fig.10 Permutation test plot of OPLS-DA model 200 times

[0054] Fig.11 VIP score chart DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with test examples and specific implementation methods. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0056] Example 1: A HPLC detection method for Da Shanzha Wan

[0057] 1. Instruments and Reagents

[0058] 1.1 Instrument

[0059] Waters e2695 liquid chromatograph (Waters, USA); temperature-adjustable electric heating mantle (Beijing Kewei Yongxing Instrument Co., Ltd.); XSE205DU electronic balance (Mettler-Toledo, Switzerland); energy-saving intelligent constant temperature bath (Ningbo Xinzhi Biotechnology Co., Ltd.); Centrifuge 5810R centrifuge (Eppendorf); rotary evaporator (IKA).

[0060] 1.2 Reagents

[0061] Chlorogenic acid (batch number RDD-L00701908029, purity 98%) was from Chengdu Ruifensidedan Biotechnology Co., Ltd.; vanillic acid (batch number: 110722-201815, purity: 91.7%), hyperoside (batch number: 111521-201809, purity: 94.9%) were from China Food and Drug Inspection Institute; isochlorogenic acid A (batch number: 7867, purity: 98%), isochlorogenic acid B (batch number: 3089, purity: 98%) were from Shanghai Shidande Standard Technology Service Co., Ltd. Acetonitrile and methanol were chromatographically pure, purified water was from Watsons, and other reagents were analytically pure; sample source information is shown in Table 1.

[0062] Table 1 Da Shanzhao Wan sample information

[0063]

[0064]

[0065] 2 Preparation of test solution

[0066] Take an appropriate amount of sample, cut it into pieces, weigh it, mix it with diatomaceous earth in equal proportions and grind it into powder. Weigh 2g of powder accurately (accurate to 0.001g), place it in a 150mL conical flask, accurately pipette 10-50mL of extraction solvent, weigh it, extract it for 1h, let it stand and cool, weigh it again, make up with extraction solvent, centrifuge (8000r / min, 5min), take out the supernatant, concentrate it under reduced pressure at 60℃, transfer the concentrate to a 5mL volumetric flask, rinse it with a small amount of extraction solvent several times, transfer the washing liquid to the volumetric flask, make up the volume of extraction solvent to the scale, shake it well and filter it through a 0.45μm organic microporous filter membrane, and take the filtrate.

[0067] 3. Investigation of chromatographic conditions and test sample preparation methods

[0068] 3.1 Optimization of different gradient elution conditions

[0069] (1) Chromatographic conditions: Eclipse Plus C 18Chromatographic column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A)-0.1% formic acid solution (B), gradient elution (0-12 min, 10%-15% A; 12-26 min, 15%-27% A; 26-42 min, 27%-40% A; 42-43 min, 40%-52% A; 43-62 min, 52%-52% A; 62-63 min, 52%-70% A; 63-72 min, 70%-90% A; 72-74 min, 90%-10% A; 74-75 min, 10% A); flow rate: 1.0 mL min -1 ; Column temperature 30℃; Detection wavelength 250nm; Injection volume 10μL.

[0070] (2) Chromatographic conditions: Eclipse Plus C 18 Chromatographic column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A)-0.1% formic acid solution (B), gradient elution (0-5 min, 5%-8% A; 5-12 min, 8%-15% A; 12-26 min, 15%-27% A; 26-42 min, 27%-40% A; 42-43 min, 40%-52% A; 43-50 min, 52%-70% A; 50-55 min, 70%-90% A; 55-58 min, 90%-5% A; 58-60 min, 5% A); flow rate: 1.0 mL min -1 ; Column temperature 30℃; Detection wavelength 250nm; Injection volume 10μL.

[0071] (3) Chromatographic conditions: Eclipse Plus C 18 Chromatographic column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A)-formic acid solution (B), gradient elution (0-5 min, 5%-6% A; 5-12 min, 6%-12% A; 12-26 min, 12%-24% A; 26-42 min, 24%-35% A; 42-43 min, 35%-48% A; 43-50 min, 48%-59% A; 50-55 min, 59%-75% A; 55-60 min, 75%-90% A; 60-63 min, 90%-5% A; 63-65 min, 5% A); flow rate: 1.0 mL min -1 ; Column temperature 30℃; Detection wavelength 250nm; Injection volume 10μL.

[0072] (4) Chromatographic conditions: Eclipse Plus C 18Chromatographic column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A)-formic acid solution (B), gradient elution (0-5 min, 5%-6% A; 5-12 min, 6%-12% A; 12-20 min, 12%-15% A; 20-26 min, 15%-19% A; 26-40 min, 19%-33% A; 40-55 min, 33%-55% A; 55-60 min, 55%-75% A; 60-65 min, 75%-90% A; 65-70 min, 90%-5% A; 70-72 min, 5% A); flow rate: 1.0 mL min -1 ; Column temperature 30℃; Detection wavelength 250nm; Injection volume 10μL.

[0073] Figure 1 Method A failed to effectively separate the components in the test sample, and the overall chromatographic peak response value was too low; Figure 1 In the time period of 0 to 40 minutes under method B, the proportion of organic phase was too large, there was accumulation with the chromatographic peak, and the baseline was not stable; Figure 1 Under method C, the organic phase ratio was still large during the period of 15 to 40 minutes, and the chromatographic peaks did not completely achieve baseline separation; and a bifurcation peak appeared at 46 minutes; Figure 1 Under the method of D, the overall chromatographic information is rich, the separation of each chromatographic peak is good, and the response is high. Therefore, the mobile phase gradient elution method (4) is determined to be the best.

[0074] 3.2 Investigation of sample preparation and extraction methods

[0075] Ultrasonic extraction: Weigh 2 parts of the sample and diatomaceous earth in equal proportions and grind them into fine powder, 2g / part, accurately add 25mL methanol, and weigh. Extract by ultrasound (power 300W, frequency 40kHz) for 30min and 1h, respectively, let it stand and cool, weigh again, make up the lost weight with methanol, centrifuge (8000r / min, 5min), take out the supernatant, concentrate under reduced pressure at 60℃, transfer the concentrate to a 5mL volumetric flask, make up to the scale with methanol, shake well, filter with 0.45μm organic microporous filter membrane, measure on the machine, and determine the extraction method based on the content of the index components.

[0076] Hot reflux extraction: Weigh 2 parts of the sample and diatomaceous earth in equal proportions and grind them into fine powder, 2g / part, accurately add 25mL methanol, and weigh. Perform hot reflux extraction for 30min and 1h, respectively, let it stand and cool, weigh it again, make up the lost weight with methanol, centrifuge (8000r / min, 5min), take out the supernatant, concentrate under reduced pressure at 60℃, transfer the concentrate to a 5mL volumetric flask, make up to the scale with methanol, shake well, filter with 0.45μm organic microporous filter membrane, and measure it on the machine. Determine the extraction method based on the content of the index component.

[0077] Table 2 Content of index components under different extraction methods (mg / g)

[0078]

[0079] As shown in Table 2, the contents of the index components of the test samples extracted by ultrasonic extraction or hot reflux extraction for 1 hour were significantly higher than those of ultrasonic extraction or hot reflux extraction for 30 minutes; compared with the test samples extracted by hot reflux extraction for 1 hour, the contents of the index components in the test samples extracted by hot reflux extraction for 1 hour were higher than those in the test samples extracted by ultrasonic extraction for 1 hour. Therefore, it was finally determined that the test samples were extracted by hot reflux extraction for 1 hour.

[0080] 3.3 Investigation of extraction volume for sample preparation

[0081] Accurately weigh the sample and mix it with an equal proportion of diatomaceous earth and grind it into fine powder. 3 portions and 2g / portion, accurately add 10ml, 25ml, and 50ml of methanol respectively, weigh the weight, extract under hot reflux for 1h, let it stand and cool, then weigh the weight again, make up the lost weight with methanol, centrifuge (8000r / min, 5min) and take out the supernatant, concentrate under reduced pressure at 60℃, transfer the concentrate to a 5mL volumetric flask, make up to the scale with methanol, shake well and filter with 0.45μm organic microporous filter membrane, and measure on the machine.

[0082] Table 3 Content of index components under different extraction volumes (mg / g)

[0083]

[0084] As shown in Table 3, when the extraction volume is 25ml and 50ml, the content of each index component is significantly higher than that of the extraction solvent of 10ml; the content of each index component is similar when the extraction volume is 25ml compared with 50ml, indicating that 25ml as the extraction solvent can meet the extraction needs. Considering environmental friendliness and cost saving, 25ml is determined as the final extraction volume.

[0085] 3.4 Investigation of extraction solvents for sample preparation

[0086] Accurately weigh the sample and mix it with diatomaceous earth in equal proportion and grind it into fine powder, 5 portions, 2g / portion, accurately add 30% methanol, 50% methanol, 70% methanol, methanol, 25ml of 95% ethanol respectively, weigh the weight, extract under hot reflux for 1h, let it stand and cool, weigh again, make up the lost weight with methanol, take out the supernatant after centrifugation (8000r / min, 5min), concentrate under reduced pressure at 60℃, transfer the concentrate to a 5mL volumetric flask, wash with the corresponding solvent and make up to the scale, shake well and filter with 0.45μm organic microporous filter membrane, and measure on the machine.

[0087] Table 4 Content of index components under different extraction solvents (mg / g)

[0088]

[0089] After the samples were extracted with the above-mentioned different solvents, it was found that the samples extracted with 30% methanol and 50% methanol had a large resistance to passing through the membrane and were not easy to filter during the filtration process with a 0.45μm organic microporous filter membrane. The 95% ethanol extract was not easy to transfer to the volumetric flask after concentration, and lumps appeared, which were not easy to be washed off the bottle wall and had a large loss. Combined with the results of the determination of the index components in Table 4, the extraction effect of methanol was better than other solvents as a whole, so methanol was selected as the final extraction solvent.

[0090] The above systematic investigation conducted on different sample extraction methods (ultrasound, hot reflux), extraction time (30min, 1h), extraction solvents of methanol (30, 50, 70, 100%), 95% ethanol, and extraction volume (10, 25, 50mL). The results showed that when 25mL of methanol was added to 2g of sample and heated to reflux for extraction for 1h, the sample extraction rate was the best and the response value was the highest. Therefore, this method was determined to be the optimal extraction condition.

[0091] The following examples use the optimal extraction conditions to establish fingerprints.

[0092] Example 2: A method for detecting the fingerprint of Da Shanzhao Wan

[0093] 1. Solution Preparation

[0094] 1.1 Reference solution

[0095] Accurately weigh appropriate amounts of chlorogenic acid, vanillic acid, hyperoside, isochlorogenic acid B, isochlorogenic acid A and other standard substances, respectively, in 10 mL volumetric flasks, dissolve and dilute with methanol to obtain single reference substance solutions with mass concentrations of 1.0251, 1.0747, 0.9186, 0.8900, 1.0104, and 0.9731 mg / mL for chlorogenic acid, vanillic acid, hyperoside, isochlorogenic acid B, and isochlorogenic acid A, respectively. Accurately pipette appropriate amounts of the above single reference substance solutions, dilute with methanol to 5 mL volumetric flasks, obtain mixed reference substance solutions with mass concentrations of 82.000, 10.7470, 36.7440, 17.8000, 16.1664, and 27.2468 μg / mL for chlorogenic acid, vanillic acid, hyperoside, isochlorogenic acid B, and isochlorogenic acid A, respectively, and store at 4 ° C for use.

[0096] 1.2 Preparation of test solution

[0097] Take an appropriate amount of sample, cut it into pieces, weigh 2g, mix it with diatomaceous earth in equal proportions and grind it into powder. Weigh 2g of powder accurately (accurate to 0.001g), place it in a 150mL conical flask, accurately transfer 25mL of methanol solution, weigh it, heat it under reflux and extract it for 1h, let it stand and cool it, weigh it again, make up with methanol solution, centrifuge it (8000r / min, 5min), take out the supernatant, concentrate it under reduced pressure at 60℃, transfer the concentrate to a 5mL volumetric flask, rinse it with methanol in small amounts several times, transfer the washing liquid to the volumetric flask, make up to the mark with methanol, shake it well and filter it with a 0.45μm organic microporous filter membrane, and take the filtrate.

[0098] 1.3 Preparation of negative sample solution

[0099] Da Shanzha Pills are composed of three herbs: hawthorn, malt, and fried glutinous rice with six medicinal herbs. Because the 2020 edition of the "Chinese Pharmacopoeia" states that the three powders are directly mixed with refined honey, etc., the preparation method of Da Shanzha Pills is consistent with that of the test sample solution in the negative test, and a single sample solution of hawthorn, malt, and fried glutinous rice with six medicinal herbs is obtained.

[0100] 2. Chromatographic conditions

[0101] Combined with the above chromatographic conditions optimization, the selected chromatographic conditions are: Eclipse Plus C 18 Chromatographic column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution; flow rate: 1.0 mL / min, detection wavelength: 250 nm, column temperature: 30 ° C, injection volume: 10 μL, gradient elution program see Table 5.

[0102] Table 5 Gradient elution program

[0103]

[0104] Take the above three solutions, inject under the chromatographic condition of "2", record the chromatogram, and the target components all reach baseline separation. In the negative sample chromatogram, chlorogenic acid and vanillic acid can be detected in the three herbs. In addition, hyperoside and isochlorogenic acid B can be detected in hawthorn, isochlorogenic acid A and isochlorogenic acid B can be detected in Liushenqu, and isochlorogenic acid A can be detected in malt. The overall chromatographic peak response values ​​in malt and Liushenqu are relatively low. For details, see Figure 2 .

[0105] 3 Methodological investigation

[0106] 3.1 Precision test

[0107] The prepared test solution (No.: H4) was accurately drawn and continuously sampled for 6 times according to the chromatographic conditions under item "4". Chlorogenic acid was used as the reference peak to calculate the RSD values ​​of the relative retention time and relative peak area of ​​each target component. The results showed that the RSD values ​​were all less than 3%, indicating that the instrument had good precision.

[0108] 3.2 Repeatability test

[0109] Take 6 portions of sample (No.: H4), weigh accurately, prepare the test solution according to the method under "1.2", continuously inject and measure under the chromatographic conditions under "2", and record its chromatogram. Taking chlorogenic acid as the reference peak, calculate the RSD value of the relative retention time and relative peak area of ​​each target component. The results show that the RSD values ​​are all less than 3%, indicating that the method has good repeatability.

[0110] 3.3 Stability test

[0111] Accurately pipette the prepared test solution (No.: H4), prepare the test solution according to the method under "1.2", and measure at 0, 2, 4, 8, 12, and 24 hours under the chromatographic conditions of "2", and record the chromatogram. Taking chlorogenic acid as the reference peak, calculate the RSD value of the relative retention time and relative peak area of ​​each target component. The results show that the RSD values ​​are all less than 3%, indicating that the test solution has good stability in the measurement results within 24 hours.

[0112] 3.4 Establishment of fingerprint

[0113] Take appropriate amount of Da Shanzha Wan samples from 12 batches produced by representative manufacturers, prepare them into test solutions according to the method under "1.2" conditions, and inject 10 μL of samples respectively under "2" chromatographic conditions, and record the chromatograms. The results were analyzed and processed by the Chinese medicine chromatographic fingerprint similarity evaluation system, set H4 as the reference spectrum, and automatically match the chromatographic peaks of other samples with the reference spectrum to generate the Da Shanzha Wan superimposed spectrum and reference spectrum, see Figure 3 , Figure 4; The chromatogram of the mixed reference solution used for chromatographic peak identification in the characteristic fingerprint is as follows Figure 5 As shown, peak 5 was used as the reference peak (S), and the relative retention time and relative peak area of ​​each common peak were calculated.

[0114] 3.5 Similarity Evaluation

[0115] The fingerprints of each batch of samples were compared with the control fingerprints, and the similarity results were 0.941, 0.992, 0.994, 1.000, 0.991, 0.966, 0.919, 0.959, 0.963, 0.949, 0.987 and 0.941, respectively. The similarities were all greater than 0.9, indicating that the similarity between the batches was good.

[0116] 4. Content Determination

[0117] 4.1 Linear Relationship

[0118] Pipette 0.1, 0.2, 0.4, 0.8, 1.2, 1.6 mL of the mixed reference solution under "1.1" respectively, place them in 2 mL volumetric flasks, dilute to the mark with methanol, shake well, prepare mixed reference solutions with gradient concentrations, and measure according to the chromatographic conditions under "2". Draw the standard curve with the mass concentration of the reference solution (X, μg / mL) as the abscissa and the peak area (Y) as the ordinate, see Table 6.

[0119] Table 6 Regression equations and correlation coefficients of each component

[0120]

[0121] 4.2 Methodological Review

[0122] The test solution was prepared according to the method under "1.2". According to the chromatographic conditions under "2", the sample was injected 6 times continuously. The RSD of the peak area of ​​chlorogenic acid, vanillic acid, hyperoside, isochlorogenic acid B, and isochlorogenic acid A was <3.0%, indicating that the instrument had good precision. The sample (No.: H4) was accurately weighed, and 6 solutions were prepared in parallel according to the method under "1.2". The samples were injected and measured according to the chromatographic conditions under "2". The average contents of chlorogenic acid, vanillic acid, hyperoside, isochlorogenic acid B, and isochlorogenic acid A were 0.1630, 0.0197, 0.1163, 0.0186, and 0.0357 mg / g, respectively, and the RSD was <3.0%. The results showed that the method had good repeatability. Accurately weigh the sample (No.: H4), prepare the test solution according to the method under "2.2", and measure it at 0, 2, 4, 8, 12h, and 24h according to the chromatographic conditions under "4". The RSDs of the peak areas of chlorogenic acid, vanillic acid, hyperoside, isochlorogenic acid B, and isochlorogenic acid A were all <3.0%. The results showed that this method had good stability within 24h.

[0123] 4.3 Sample recovery experiment

[0124] Accurately weigh 0.5 g of sample (No.: H4) and add 6 replicates to the reference solution at a ratio of 1:1. Prepare the test solution according to the method under "1.2" and calculate the recovery rate. The average recoveries of chlorogenic acid, vanillic acid, hyperoside, isochlorogenic acid B and isochlorogenic acid A (n=6) were 98.09%, 107.15%, 109.35%, 96.27% and 105.27%, respectively. The RSD values ​​were all less than 4.5%. The specific results are shown in Table 7.

[0125] Table 7 Sample recovery test results

[0126]

[0127]

[0128] 4.4 Sample determination

[0129] For 51 batches of Da Shanzha Wan, the test solution was prepared according to the method under "1.2", and three replicates were injected and analyzed under the chromatographic conditions under "2". See Table 8 for details.

[0130] The results showed that the mean of chlorogenic acid in all samples was 0.1330 mg / g, the mean of vanillic acid was 0.0211 mg / g, the mean of hyperoside was 0.1184 mg / g, the mean of isochlorogenic acid B was 0.0233 mg / g, and the mean of isochlorogenic acid A was 0.0410 mg / g. The target compounds of many companies obviously exceeded the mean of all samples by ±30%, indicating that there were large differences in the preparation processes among companies, and the quality of single medicinal materials needed further in-depth research; the contents of all target components in Q2 companies exceeded the mean of all samples by ±30% to varying degrees. In order to further demonstrate the content differences of the indicator components in different samples, the content radar chart was drawn using Origin 21 software, see Figure 6 .

[0131] Table 8 Assay results (n=3, mg / g)

[0132]

[0133]

[0134] 5. Chemometric Analysis

[0135] 5.1 Cluster analysis

[0136] The common peaks of 51 samples were identified, and 16 common peaks were obtained. The relative retention time of the common peaks was obtained by taking chlorogenic acid as the reference peak, as shown in Table 9. The peak area of ​​the common peak was used as the original data, and cluster analysis was performed using SPSS25. The specific results are shown in Figure 7 It can be seen that when the inter-group distance is 20, all samples can be divided into two categories, Q1, Q3-Q13 are clustered into one category, and Q2 is clustered into another category alone. This shows that the preparation process within the enterprise is relatively stable.

[0137] Table 9 Relative retention time of common peaks

[0138]

[0139]

[0140] 5.2 Principal Component Analysis (PCA)

[0141] The most basic dimensionality reduction analysis (PCA) was performed using the 16 common peak areas of 51 batches of samples as variables, imported into SPSS25.0 software, and extracted with eigenvalue > 1 as the criterion. Four principal component factors were obtained, and the cumulative variance contribution rate reached 75.709%, indicating that these four principal component factors can basically represent the 16 common components to reflect and evaluate the sample quality. The principal components and variance contribution rates are shown in Table 11. The common component peak areas were imported into SIMCA 14.1 software, and a score chart was generated after analysis, see Figure 8 , which is basically consistent with the results of cluster analysis.

[0142] Table 10 Principal component eigenvalues ​​and variance contribution rates

[0143]

[0144] 5.3 Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA)

[0145] To further verify the accuracy of the principal component analysis results, the peak areas of the common components were imported into SIMCA 14.1 to establish the OPLS-DA model. 2 X, R 2 Y was 0.940 and 0.603 respectively, the prediction ability parameter Q2 was 0.536, and the OPLS-DA score graph was shown in Fig. 9 , which is consistent with the results of principal component analysis. To prevent overfitting, 200 permutation tests were performed. Fig.10 , indicating that the model is stable and has good predictive ability. Variable importance projection (VIP) is an important indicator for screening different compounds between different samples. The larger the VIP value, the greater the impact on the difference. After analysis, the VIP values ​​of 16 common components were obtained. The results are shown in Fig.11Taking the VIP value greater than 1 as the standard, 8 variables were obtained, among which peak 5 was chlorogenic acid, peak 7 was hyperoside, and peak 9 was isochlorogenic acid B, which were the main markers causing sample differences.

[0146] The results of the batch content test of 51 batches of Da Shanzha Wan showed that the content of each manufacturer's samples was basically maintained at ±30% of their respective mean values, indicating that the uniformity of samples of different batches within the enterprise was good and the production process environment was stable and reliable. On this basis, the contents of the five components of manufacturer Q2 were mostly higher than ±30% of the mean of all samples, indicating that there were large differences in the preparation processes between enterprises and the quality of medicinal materials needed further in-depth research. Chemometric analysis was performed on the 16 common components of 51 batches of samples. The cluster analysis results showed that when the Euclidean distance was 15, all samples could be roughly divided into three categories; when the Euclidean distance was 20, the samples could be clustered into two categories; among them, Q2 was always clustered into one category alone. It can be seen from the principal component analysis score diagram that the analysis results are consistent with the clustering results. OPLS-DA screened out 8 variable factors that affect the differences between samples, including chlorogenic acid, hyperoside and isochlorogenic acid B for quantitative analysis. These three target ingredients are mainly found in hawthorn. Da Shanzha Wan stipulates that hawthorn should be used as raw product, but it does not rule out that some companies use other processed products instead. Luo Qiaomei et al. [8] The chemical composition of the Da Shanzha Pills was analyzed before and after the hawthorn was fried and charred, and it was found that the raw and charred products had some common ingredients, including chlorogenic acid. Therefore, companies should strictly control the medicinal materials and quality before production to ensure accurate feeding.

[0147] In this study, a multi-component content determination method for Da Shanzha Pills was established based on HPLC, and the quality of 51 batches of samples was evaluated as a whole by combining chemometric statistical analysis methods. The results of this method are reliable and accurate, and can reflect the quality of Da Shanzha Pills and single medicinal materials on the market; the obvious difference in appearance color can also reflect the overall quality of Da Shanzha Pills samples, and the results can provide a reference for the quality control of Da Shanzha Pills on the market.

[0148] In summary, the present invention provides a HPLC detection method for Da Shanzha Pills, and systematically investigates different sample extraction methods (ultrasound, hot reflux), extraction time (30min, 1h), extraction solvents such as methanol (30, 50, 70, 100%), 95% ethanol, and extraction volumes (10, 25, 50mL). The results show that when 25mL of methanol is added to 2g of sample and heated under reflux for extraction for 1h, the sample extraction rate is the best and the response value is the highest, so the method is determined to be the optimal extraction condition; the characteristic fingerprint of Da Shanzha Pills is established using the optimal extraction condition, and based on the rich chromatographic information, the common peaks are determined and the main index components are identified; the present invention uses the HPLC detection method to detect and evaluate the Da Shanzha Pills that have been marketed in China, so as to control the quality of the commercially available Da Shanzha Pills and ensure the clinical efficacy of the Da Shanzha Pills; the HPLC detection method can realize the quality monitoring of the Da Shanzha Pills, provide a reference basis for ensuring the safety, stability and quality control of the Da Shanzha Pills, and promote the inheritance and development of traditional Chinese medicine. The results of the method described are reliable and accurate, and can reflect the overall quality of the commercially available Da Shanzhao Pills; this method can effectively monitor the quality status of Da Shanzhao Pills and provide a scientific evaluation strategy for controlling the quality consistency and effectiveness of the preparation.

Claims

1. A HPLC detection method for Da Shanzhao Pills, characterized in that: The test solution was prepared by extracting Da Shan Zha Wan, and then the solution was determined by HPLC. Preparation of reference substance solution: accurately weigh each reference substance, place it in a volumetric flask, dissolve it with methanol, and make up to volume to obtain a reference substance solution containing a predetermined concentration of a standard component; the standard components are chlorogenic acid, vanillic acid, hyperoside, isochlorogenic acid B and isochlorogenic acid A; Preparation of test solution: accurately weigh the sample and mix it with diatomaceous earth in a ratio of 1:1, add extraction solvent for extraction, let it stand and cool, centrifuge to take the supernatant, reduce pressure and concentrate, fix the volume, filter, and take the filtrate; the extraction method is hot reflux extraction, and the extraction solvent is methanol; HPLC assay conditions are: Column: Eclipse Plus C 18 Chromatographic column, 250 mm × 4.6 mm, 5 μm; Mobile phase: acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution; Flow rate: 1.0 mL / min; Detection wavelength: 250nm; Column temperature: 30°C; Injection volume: 10 μL; The gradient elution program was as follows: 0-5 min, 5%-6% A; 5-12 min, 6%-12% A; 12-20 min, 12%-15% A; 20-26 min, 15%-19% A; 26-40 min, 19%-33% A; 40-55 min, 33%-55% A; 55-60 min, 55%-75% A; 60-65 min, 75%-90% A; 65-70 min, 90%-5% A; 70-72 min, 5% A.

2. A method for detecting the fingerprint of Da Shanzhao Wan, characterized in that: The steps include: S1. Preparation of reference substance solution: accurately weigh each reference substance, place it in a volumetric flask, dissolve it with methanol, and make up to volume to obtain a reference substance solution containing a predetermined concentration of standard components; S2. Preparation of test solution: accurately weigh the sample and mix it with diatomaceous earth in a ratio of 1:1, add extraction solvent for extraction, let it stand and cool, centrifuge to obtain the supernatant, concentrate under reduced pressure, make up to volume, filter, and obtain the filtrate; S3. Establishment of HPLC characteristic fingerprint: using the detection method described in claim 1, obtaining the chromatogram of the reference solution and the test sample fingerprint of the test solution under the same chromatographic conditions; The extraction method of the test solution described in step S2 is hot reflux extraction, and the extraction solvent is methanol; The standard components described in step S1 are chlorogenic acid, vanillic acid, hyperoside, isochlorogenic acid B and isochlorogenic acid A.

3. The detection method according to claim 2, characterized in that The mass concentrations of chlorogenic acid, vanillic acid, hyperoside, isochlorogenic acid B and isochlorogenic acid A in step S1 are 1.0251, 1.0747, 0.9186, 1.0104 and 0.9731 mg / mL, respectively.

4. The detection method according to claim 2, characterized in that The extraction volume of the test solution described in step S2 is 10 to 50 ml.

5. The detection method according to claim 2, characterized in that: The filtration described in step S2 uses a microporous filter membrane.

6. The detection method according to claim 5, characterized in that The microporous filter membrane described in step S2 is a 0.45 μm organic microporous filter membrane.

7. Use of the detection method according to any one of claims 2 to 6 in detecting commercially available Da Shanzha Wan.

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