A method for constructing and applying the characteristic spectrum of Polygonum multiflorum leaf formula granules

Abstract

This invention provides a method for constructing and applying characteristic chromatograms of Polygonum multiflorum (Isatis tinctoria) formulation granules. High-performance liquid chromatography (HPLC) is used to obtain the characteristic chromatograms of the analytes. Specific HPLC conditions are provided, including an octadecylsilane-bonded silica column as the packing material, a detection wavelength of 305–315 nm, acetonitrile as mobile phase A, and 0.04%–0.06% phosphoric acid aqueous solution as mobile phase B, eluted according to a specific gradient program. The characteristic chromatograms obtained by this method exhibit stronger specificity of the substance information corresponding to the characteristic peaks, and the relevant information in the characteristic chromatograms characterized by this method is more comprehensive, enabling better overall quality control of Polygonum multiflorum (Isatis tinctoria).

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine identification, and particularly relates to a characteristic spectrum construction method and application of Polygoni Orientalis Leaf Granules. BACKGROUND

[0002] Polygoni Orientalis Leaf is the dry leaves of Polygonum tinctorium Ait. of Polygonaceae. It is collected twice in summer and autumn when the branches and leaves are lush, and then impurities are removed and dried. It is distributed in Liaoning, Hebei, Shaanxi, Shandong and other places. It has the effects of clearing heat and resolving toxicity, cooling blood and removing spots, and is mainly used for treating warm disease fever, rash and macule, lung heat and cough, laryngeal obstruction, mumps, erysipelas and carbuncle.

[0003] There are relatively few characteristic spectrum studies on Polygoni Orientalis Leaf, and the identified components are few. The characteristic components disclosed at present are all nucleosides, such as guanosine, uridine and adenosine. However, there is no literature report that the nucleosides in Polygoni Orientalis Leaf are the effective components, and the nucleosides are not specific enough as the characteristic peak components for quality control. SUMMARY

[0004] The characteristic spectrum construction method disclosed in the prior art has few specific characteristic peaks in the characteristic spectrum of the water extract of Polygoni Orientalis Leaf and its preparation, which is not conducive to the overall quality control. The present application provides a characteristic spectrum construction method which has more specific characteristic peaks and can better achieve the overall quality control of the water extract of Polygoni Orientalis Leaf and its preparation.

[0005] A characteristic spectrum construction method of a water extract of Polygoni Orientalis Leaf and its preparation, which adopts high performance liquid chromatography to obtain the characteristic spectrum of the test object.

[0006] The chromatographic column is filled with octadecylsilane-bonded silica gel, the detection wavelength is 305-315 nm, acetonitrile is used as the mobile phase A, and 0.04%-0.06% phosphoric acid aqueous solution is used as the mobile phase B, and the elution is performed according to the following gradient program:

[0007]

[0008] In the chromatographic conditions of the high performance liquid chromatography, the column length of the chromatographic column is 250 mm, the column inner diameter is 4.6 mm, and the particle size is 5 μm.

[0009] The column temperature is 28-32 ℃.

[0010] The flow rate is 0.9-1.1 ml / min.

[0011] The theoretical plate number calculated according to 4-coumaric acid peak should be not less than 5000.

[0012] The high performance liquid chromatography is SHIMADZU Shim-pack C18-AQ or YMC-Triart C18.

[0013] When the test substance is the test sample, the preparation process of the test sample solution is as follows: taking the water extract of Polygoni Orientalis or the preparation of the water extract of Polygoni Orientalis, adding a solvent, extracting and treating, shaking, filtering, and taking the filtrate, thereby obtaining the test sample solution.

[0014] When the test substance is the control medicinal material, the preparation process of the control medicinal material reference solution is as follows: taking the control medicinal material, adding water, hot refluxing, cooling, centrifuging, taking the supernatant, concentrating to near dryness under reduced pressure, adding a solvent for ultrasonic treatment, taking out, cooling, shaking, filtering, and taking the filtrate, thereby obtaining the control medicinal material reference solution.

[0015] When the test substance is the control sample, the preparation process of the control sample reference solution is as follows: taking the control sample, accurately weighing, and adding a solvent to prepare the control sample reference solution.

[0016] The control sample includes 4-coumaric acid control sample and ferulic acid control sample.

[0017] In the preparation of the test sample solution, the extraction and treatment method is heating refluxing or ultrasonic treatment.

[0018] The preparation of the water extract of Polygoni Orientalis includes water extract paste, water extract dried powder, formula granules, and water extract preparation of Polygoni Orientalis.

[0019] And / or, the solvent used in the preparation process of the test sample solution is an alcohol aqueous solution with a mass concentration of 50% or less or water.

[0020] And / or, the solvent used in the preparation process of the control sample reference solution is a methanol solution.

[0021] And / or, 15-50 ml of the solvent is added to 0.1 g of the test sample in the test sample solution.

[0022] And / or, in the preparation of the test sample solution, the ultrasonic treatment time is more than 20 min.

[0023] Preferably, the solvent used in the preparation process of the test sample solution is an alcohol aqueous solution with a mass concentration of 30%-50% or water.

[0024] And / or, in the preparation of the test sample solution, the ultrasonic treatment time is 20-30 min.

[0025] The characteristic spectrum includes characteristic peaks of peaks 1-14.

[0026] The relative retention time of other characteristic peaks to the S peak should be within the range of ±10% of the specified value, and the specified value is 0.55 for peak 1, 0.62 for peak 2, 0.71 for peak 3, 0.77 for peak 4, 1.17 for peak 7, 1.22 for peak 8, 1.34 for peak 9, 1.42 for peak 10, 1.45 for peak 11, 1.54 for peak 12, 1.58 for peak 13, and 1.60 for peak 14.

[0027] The characteristic spectrum constructed by the construction method is applied in the overall quality control of the water extract of Polygonum orientale and the preparation.

[0028] The technical scheme of the present application has the following advantages:

[0029] 1. The construction method of the characteristic spectrum of the water extract of Polygonum orientale and the preparation provided by the present application has at least 14 characteristic peaks in the characteristic spectrum obtained by the construction method, and the material information of the characteristic spectrum is more comprehensive. Meanwhile, the characteristic peaks include the characteristic peaks corresponding to 4-coumaric acid and ferulic acid. The pharmacological action of 4-coumaric acid is anti-inflammatory, antioxidant, immune regulation, anti-tumor, anti-platelet aggregation, cardiovascular protection, prevention and improvement of diabetes, nerve protection, and antibacterial action, etc. The pharmacological action of ferulic acid is antibacterial and anti-inflammatory, prevention and treatment of coronary heart disease, protection of women's ovaries, and inhibition of liver damage, etc. This has certain correlation with the efficacy of Polygonum orientale, and the specificity is stronger, and the efficacy chemical components in the water extract of Polygonum orientale and the preparation are more fully represented, so that the overall quality control of the water extract of Polygonum orientale and the preparation can be better realized.

[0030] 2. The construction method of the present application can not only reflect the content of the effective components of the Polygonum orientale formula granules from the overall appearance of the chromatogram, but also effectively represent the quality, and is beneficial to better control the quality of the product from the whole, so as to provide effective protection for the production and clinical application of the drug.

[0031] 3. The construction method of the present application has the advantages of simple method, stability, high precision, and good reproducibility, and can quickly and accurately identify the quality of the product, so as to provide a scientific basis for the overall establishment of the quality control standard of the Polygonum orientale formula granules. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0033] Figure 1 is the characteristic chromatogram of the Hedy sariptae Formula Granules in Example 1 of the present application.

[0034] Figure 2 is the comparison chromatogram of the test sample solution and the reference solution of the control sample in Example 1 of the present application.

[0035] Figure 3 is the characteristic chromatogram of the test sample solution and the reference solution of the control sample in Example 1 of the present application.

[0036] Figure 4 is the characteristic chromatogram of different extraction solvents in Example 3 of the present application.

[0037] Figure 5 is the characteristic chromatogram of different detection wavelengths (305 nm) in Example 5 of the present application.

[0038] Figure 6 is the characteristic chromatogram of different detection wavelengths (310 nm) in Example 5 of the present application.

[0039] Figure 7 is the characteristic chromatogram of different detection wavelengths (315 nm) in Example 5 of the present application.

[0040] Figure 8 is the characteristic chromatogram of the same serial number of the chromatographic column in Comparative Example 1 of the present application for detecting Hedy sariptae Formula Granules.

[0041] Figure 9 is the characteristic chromatogram obtained by the chromatographic column with serial number USAC008396 in Comparative Example 1 of the present application.

[0042] Figure 10 is the characteristic chromatogram obtained by the chromatographic column with serial number USAC008638 in Comparative Example 1 of the present application.

[0043] Figure 11 is the characteristic chromatogram obtained by the chromatographic column with serial number USAC009539 in Comparative Example 1 of the present application.

[0044] Figure 12 is the characteristic chromatogram obtained by the chromatographic column with serial number USAC010218 in Comparative Example 1 of the present application.

[0045] Figure 13 is the characteristic chromatogram obtained by the chromatographic column with serial number USAC011511 in Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0046] The specific experimental steps or conditions not mentioned in the examples can be carried out according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments used are not marked by the manufacturer, which are conventional reagent products that can be obtained by market purchase.

[0047] Instrument: Waters e2695 high performance liquid chromatograph (TUV Detector detector); ML204T electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); JA1002 electronic balance (Shanghai Puchun Metrology Instrument Co., Ltd.); MSA6·6S-0CE-DM electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); KQ-100DE digital ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0048] Reagent: 4-coumaric acid reference substance (batch number: 112037-202102, China Institute for Drug Control, purity 99.7%);

[0049] Ferulic acid reference substance (batch number: 110773-202316, China Institute for Drug Control, purity 99.3%);

[0050] Kaempferol-3-O-β-D-glucuronide (batch number: STC12401, Shanghai Shidand Standard Technology Service Co., Ltd., purity 98%);

[0051] Rehmannia root: K478YC16; Rehmannia root slices: K478YP16;

[0052] Rehmannia root: K478YC16; Rehmannia root slices: K478YP16;

[0053] Rehmannia root: K478YC16; Rehmannia root slices: K478YP16;

[0054] Reagents: methanol for chromatography, ethanol for analytical purity, water for distilled water, acetonitrile (MERCK) for chromatography, phosphoric acid (MERCK) for chromatography, methanol (MERCK) for chromatography, water for distilled water (Aesop), and other reagents for analytical purity.

[0055] Example 1

[0056] A method for constructing a characteristic spectrum of a Rehmannia root preparation, the specific process being as follows:

[0057] 1. Preparation of the test solution

[0058] Preparation of the test solution: Take the test product (K478CP16) and grind it finely. Take about 0.1 g and place it in a conical flask with a stopper. Precisely add 15 ml of 30% methanol and ultrasonically treat it (power 250 W, frequency 40 kHz) for 20 minutes. Shake it well and filter it. Take the filtrate to obtain the test solution.

[0059] Preparation of the reference solution of the control medicinal material: Take 0.5 g of the medicinal material of Kiangi leaf and place it in a conical flask with a stopper. Add 50 ml of water and heat it under reflux for 30 minutes. Take it out and cool it. Centrifuge it and take the supernatant. Concentrate it under reduced pressure to near dryness. Add 15 ml of 30% methanol to the residue and ultrasonically treat it (power 250 W, frequency 40 kHz) for 20 minutes. Take it out and cool it. Shake it well and filter it. Take the filtrate to obtain the reference solution of the control medicinal material.

[0060] Preparation of the reference solution of the control: Take the appropriate amount of 4-coumaric acid and ferulic acid control and precisely weigh it. Add methanol to prepare a mixed reference solution of the control containing 10 μg per 1 ml as the reference solution of the control.

[0061] 2. Chromatographic method

[0062] The following chromatographic conditions are used for detection:

[0063] The chromatographic column filled with octadecylsilane-bonded silica gel (SHIMMADZU Shim-pack GIST C18-AQ, column length 250 mm, inner diameter 4.6 mm, particle size 5 μm) is used. Acetonitrile is used as the mobile phase A and 0.05% phosphoric acid solution is used as the mobile phase B. Gradient elution is performed according to the following Table 1. The flow rate is 1.0 ml per minute. The column temperature is 30°C. The detection wavelength is 310 nm. The theoretical plate number calculated according to the peak of 4-coumaric acid should not be less than 5000.

[0064] Table 1

[0065]

[0066] Precisely take 3 μl of the reference solution of the control medicinal material, the reference solution of the control, and the test solution, respectively, and inject them into the liquid chromatograph. Measure them to obtain the results.

[0067] Take 24 batches of Kiangi leaf lyophilized powder and three batches of Kiangi leaf formula granules, respectively, and obtain their liquid chromatograms according to the method in Example 1. Use the S1 liquid chromatogram as the reference chromatogram. Calculate the median value using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprint (2012.1 version) to obtain the characteristic chromatogram of the Kiangi leaf formula granules as shown in Table 2, and identify the common peaks. Figure 1 ​

[0068] Table 2 List of Polygoni Orientalis Radix Decoction and Polygoni Orientalis Radix Granules

[0069]

[0070] The 24 batches of Polygoni Orientalis Radix standard decoction and 3 batches of granules were identified by common peaks, and 14 common peaks were identified, with a similarity range of 0.988-0.999.

[0071] According to the retention time and peak area in the characteristic spectrum, peak No. 5 was determined to be 4-coumaric acid, and peak No. 6 was determined to be ferulic acid. With peak No. 5, 4-coumaric acid, as the reference peak, the relative retention time and relative peak area of peaks No. 1-4 and No. 7-14 were calculated. The relative retention time and relative peak area data of the 24 batches of Polygoni Orientalis Radix standard decoction were processed, and the results were as follows.

[0072] Table 3 Relative retention time table of 24 batches of Polygoni Orientalis Radix standard decoction characteristic spectrum

[0073]

[0074]

[0075] Table 4 Relative retention time table of 24 batches of Polygoni Orientalis Radix standard decoction characteristic spectrum

[0076]

[0077]

[0078] Table 5 Relative peak area table of 24 batches of Polygoni Orientalis Radix standard decoction characteristic spectrum

[0079]

[0080] Table 6 Relative peak area table of 24 batches of Polygoni Orientalis Radix standard decoction characteristic spectrum

[0081]

[0082]

[0083] After data processing of 24 batches of Hedyseri standard decoction liquid chromatogram, the similarity of the characteristic chromatogram of 24 batches of Hedyseri standard decoction and the control chromatogram is greater than 0.9. The relative retention time difference of 14 common peaks of Hedyseri standard decoction characteristic chromatogram is small, all within ±10%, and the average value of the relative retention time is selected as the specified value; the specified value is: 0.55 (peak 1), 0.62 (peak 2), 0.71 (peak 3), 0.78 (peak 4), 1.17 (peak 7), 1.22 (peak 8), 1.34 (peak 9), 1.41 (peak 10), 1.45 (peak 11), 1.54 (peak 12), 1.58 (peak 13), 1.59 (peak 14). Allowable error: ±10%. The difference of relative peak area is large, and the RSD value is 44.87%-93.52%, which does not meet the quality control requirements, so the relative peak area is not included in the standard.

[0084] Take Hedyseri granules test solution, kaempferol-3-O-beta-D glucuronic acid reference solution, and 4-coumaric acid control and ferulic acid mixed control reference solution, and detect and compare according to the above chromatographic conditions, and deduce the characteristic peaks in Hedyseri, and the results are shown in Figure 2 . The characteristic chromatogram shows 14 peaks, of which peak 5 is 4-coumaric acid, peak 6 is ferulic acid, and peak 7 is kaempferol-3-O-beta-D glucuronic acid, and the negative accessories and blank solution have no interference. In the characteristic chromatogram obtained by the present application, the characteristic peak components are mainly organic acid components. Among them, 4-coumaric acid and ferulic acid are representative components of organic acids in Hedyseri, which have antibacterial, anti-inflammatory, antiviral, antioxidant, antitumor and other pharmacological effects.

[0085] Take about 0.5g of Hedyseri decoction pieces, accurately weigh, add water 50ml, heat reflux for 30 minutes, take out, cool, centrifuge, take the supernatant, reduce pressure and concentrate to nearly dry, add 30% methanol 15ml to the residue, ultrasonic treatment (power 250W, frequency 40kHz) for 20 minutes, shake evenly, filter, and take the filtrate, which is the accompanying decoction piece solution. Another Hedyseri granules about 0.1g is prepared into granule test solution by the same method. Compare the characteristic chromatogram of Hedyseri granules with the characteristic chromatogram of Hedyseri decoction pieces, and the results are shown in Figure 3 .

[0086] Through the comparison of the chromatogram Figure 3 , it can be known that the 14 characteristic peaks of Hedyseri granules and Hedyseri decoction pieces are consistent. It shows that the characteristic peaks corresponding to the substances are all transferred from Hedyseri decoction pieces to formula granules, but the transfer rate is different, and the correlation between the two is good.

[0087] Example 2

[0088] This example is used to verify the chromatographic conditions in example 1.

[0089] 1. Specificity study

[0090] About 1.0 g of Houttuynia cordata Thunb. Dispensing Granules and malt dextrin were taken respectively, and the Houttuynia cordata Thunb. Dispensing Granules test sample solution and excipient negative control solution were prepared according to the method of Example 1. The results showed that the negative solution had no interference with the characteristic peaks, and the method had good specificity.

[0091] 2. Reproducibility study

[0092] Six portions of Houttuynia cordata Thunb. Dispensing Granules were taken, finely ground, and determined according to the method of Example 1. The relative retention time and relative peak area of the characteristic peaks 1-4 and 7-14 were calculated with the peak No. 5 as the reference peak S, and the RSD was calculated.

[0093] The reproducibility experiment results showed that the relative retention time RSD of the six reproducibility experiment samples was in the range of 0.00%-0.03%, and the relative peak area RSD was in the range of 0.25%-2.70%, indicating that the reproducibility of the characteristic spectrum was good.

[0094] 3. Intermediate precision study

[0095] Houttuynia cordata Thunb. Dispensing Granules were taken, finely ground, and sample preparation was performed according to the method of Example 1. The intermediate precision test was performed by different analysts at different times using another high performance liquid chromatograph (TUV detector), and the characteristic spectrum was obtained. The relative retention time of the characteristic peaks 1-4 and 7-14 to the peak No. 5 was calculated, and the RSD value was calculated.

[0096] The intermediate precision results showed that the relative retention time RSD of the six intermediate precision experiment samples was in the range of 0.00%-0.490%, and the relative retention time RSD of the intermediate precision compared with the reproducibility samples was in the range of 0.55%-1.59%. The data showed that the intermediate precision of the method was good.

[0097] 4. Stability study

[0098] The same granule test sample solution was taken, and the determination was performed at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h. The relative retention time of the characteristic peaks 1-4 and 7-14 to the peak No. 5 was calculated, and the RSD was calculated.

[0099] The stability results showed that the solution stability was investigated for 24 hours, and the relative retention time RSD% of the sample was in the range of 0.00%-0.03%, and the relative peak area RSD% was in the range of 0.37%-3.30%. The data showed that the test sample solution was stable within 24 hours.

[0100] Example 3

[0101] A construction method of a Polygonum multiflorum Thunb. preparation characteristic spectrum, which is different from example 1 in the preparation of a test sample solution.

[0102] (1) Selection of extraction solvent

[0103] Take 0.1 g of Polygonum multiflorum Thunb. granule powder, accurately weigh, and place in a conical flask with a plug. Respectively, accurately add 15 ml of corresponding solvent (water, 30% methanol, 50% methanol, 70% methanol, methanol, 30% ethanol, 50% ethanol, 70% ethanol, ethanol), ultrasonic treatment (power 250 W, frequency 40 kHz) for 20 minutes, take out, cool, shake, filter, take the filtrate, and inject into a high performance liquid chromatograph. According to the chromatographic conditions of standard example 1, the results are shown in Tables 7-8 and Figure 4 .

[0104] Table 7 Extraction solvent investigation table

[0105]

[0106] Table 8 Extraction solvent investigation table

[0107]

[0108] From the data results, it can be seen that when methanol is used as the extraction solvent, the response value of each characteristic peak is low; when ethanol is used as the extraction solvent, peaks 1-4 characteristic peaks are missing; when 70% ethanol aqueous solution is used as the extraction solvent, the peak type is poor, and there is no obvious difference in the characteristic peak information when the rest of the solvents are used as the extraction solvent. Therefore, an alcohol aqueous solution or water can be selected as the solvent used in the preparation process of the test sample solution. In order to take into account the peak area and peak type, etc., the present application preferably uses an alcohol aqueous solution of 50% or less or water as the solvent. Among them, 30% methanol aqueous solution can detect more abundant peaks after extraction, each characteristic peak has a higher peak area, and the peak type is better. Therefore, the Polygonum multiflorum Thunb. granule characteristic spectrum selects 30% methanol aqueous solution as the extraction solvent.

[0109] (2) Selection of solvent amount

[0110] Accurately weigh 0.1 g of the granule powder, place in a conical flask with a plug, respectively accurately add water 10 ml, 15 ml, 25 ml, 50 ml, seal, weigh, ultrasonic treatment for 20 minutes (power 250 W, frequency 40 kHz), take out, cool, shake, filter, take the filtrate, and inject into a high performance liquid chromatograph. According to the chromatographic conditions of standard example 1, the results are shown in the following table.

[0111] Table 9 Extraction solvent amount investigation

[0112]

[0113] Table 10 Extraction solvent amount investigation

[0114]

[0115]

[0116] The analysis results show that under different solvent amounts, when the extraction solvent amount is 10 ml, the sample is not completely extracted. When the extraction solvent amount is 15 ml, 25 ml, or 50 ml, the number of characteristic peaks and response values are relatively small. Therefore, the solvent amount is selected as 15 ml in consideration of the method robustness and the principle of saving.

[0117] (3) Selection of extraction time

[0118] Precisely weigh 0.1 g of the granular powder of the product into a conical flask with a stopper, precisely add 30% methanol 15 ml, tightly seal, weigh, and ultrasonically treat (power 250 W, frequency 40 kHz) for 10, 20, and 30 minutes, respectively, take out, cool, shake, filter, and take the filtrate, which is obtained. Precisely take 10 μl of the filtrate and inject it into a high-performance liquid chromatograph for determination according to the chromatographic conditions of Example 1, and the results are as follows.

[0119] Table 11 Extraction time investigation table

[0120]

[0121] Table 12 Extraction time investigation table

[0122]

[0123] From the above results, it can be seen that the sample is not completely extracted for 10 minutes of ultrasonic treatment, and the peak areas of the characteristic peaks are not significantly different for 20 and 30 minutes of ultrasonic treatment. Therefore, the extraction time is selected as 20 minutes to ensure complete extraction.

[0124] (4) Selection of extraction method

[0125] Precisely weigh 0.1 g of the granular powder of the product into a conical flask with a stopper, precisely add 30% methanol 15 ml, tightly seal, weigh, and ultrasonically treat (power 250 W, frequency 40 kHz) and heat reflux for 20 minutes, respectively, take out, cool, shake, filter, and take the filtrate, which is obtained. Precisely take 10 μl of the filtrate and inject it into a high-performance liquid chromatograph for determination according to the chromatographic conditions of Example 1, and the results are as follows.

[0126] Table 13 Extraction method investigation table

[0127]

[0128] Table 14 Extraction method investigation table

[0129]

[0130] From the above results, it can be seen that the peak areas of each characteristic peak are not much different when the sample is treated by ultrasonic and reflux. Considering the convenience of the experiment, the extraction method is selected as ultrasonic.

[0131] Example 4

[0132] A method for constructing a characteristic spectrum of a Hedy s rhizoma et Radix preparation, which differs from example 1 in that the chromatographic conditions are different, and specifically as follows:

[0133] (1) Investigation of different column temperatures

[0134] Take Hedy s rhizoma et Radix granules, prepare the test solution according to the preparation method of the test solution in example 1, and determine according to the method in example 1 using different column temperatures (28℃, 30℃ and 32℃) respectively, to investigate the robustness of the experimental method to column temperature. The results are as follows:

[0135] Table 15 Retention time and relative retention time of Hedy s rhizoma et Radix at different column temperatures

[0136]

[0137]

[0138] Table 16 Retention time and relative retention time of Hedy s rhizoma et Radix at different column temperatures

[0139]

[0140] The results of the investigation of different column temperatures show that the relative retention times of the characteristic peaks in the sample are within ±10% of the specified values, indicating that the method has good robustness to different column temperatures.

[0141] (2) Investigation of different flow rates

[0142] Take Hedy s rhizoma et Radix granules, prepare the test solution according to the preparation method of the test solution in example 1, and determine according to the method in example 1 using different flow rates (0.9ml / min, 1.0ml / min and 1.1ml / min) respectively, to investigate the robustness of the experimental method to flow rate. The results are as follows.

[0143] Table 17 Retention time and relative retention time of Hedy s rhizoma et Radix at different flow rates

[0144]

[0145] Table 18 Retention time and relative retention time of Hedy s rhizoma et Radix at different flow rates

[0146]

[0147] The results of the different flow rate investigation show that the RSD of the relative retention time of the marker peaks in the sample is in the range of 0.19% to 2.89%, and the relative retention time of each characteristic is within the range of ±10% of the specified value, and the method has good robustness to different flow rates.

[0148] (3) Investigation of different acid concentrations

[0149] The Hedyotiis diffusae leaves granules were prepared into a test sample solution according to the preparation method of the test sample solution in Example 1, and different acid concentrations (0.04% phosphoric acid, 0.05% phosphoric acid, and 0.06% phosphoric acid) were used to determine the retention time and relative retention time of each characteristic according to the method in Example 1, and the results are as follows:

[0150] Table 19 Investigation of the retention time and relative retention time of Hedyotiis diffusae leaves at different acid concentrations

[0151]

[0152] Table 20 Investigation of the retention time and relative retention time of Hedyotiis diffusae leaves at different acid concentrations

[0153]

[0154]

[0155] The results of the different acid concentration investigation show that the RSD of the relative retention time of the marker peaks in the sample is in the range of 0.02% to 0.56%, and the relative retention time of each characteristic is within the range of ±10% of the specified value, and the method has good robustness to different acid concentrations.

[0156] (4) Investigation of different types of chromatographic columns

[0157] The Hedyotiis diffusae leaves granules were prepared into a test sample solution according to the method in Example 1, and different types of chromatographic columns were used to determine the retention time and relative retention time of each characteristic according to the method in Example 1,

[0158] Chromatographic column 1: Waters XSelect HSS T3 (4.6*250mm, 5μm)

[0159] Chromatographic column 2: SHIMMADZU Shim-pack GIST C18-AQ (4.6*250mm, 5μm) Chromatographic column 3: YMC-Triart C18 (4.6*250mm, 5μm);

[0160] The results of the investigation of the robustness of the experimental method to different chromatographic columns are as follows:

[0161] Table 21 Investigation of the retention time and relative retention time of Hedyotiis diffusae leaves at different types of chromatographic columns

[0162]

[0163] Table 22 Investigation of retention time and relative retention time of different types of chromatographic column of Polygoni Orientalis Herba

[0164]

[0165]

[0166] The investigation results of different types of chromatographic column showed that: from the data and spectrum results, it could be known that the peaks No. 1 and No. 7 of the chromatographic column 1 were lost, the peak type of each characteristic peak of the chromatographic column 3 was not good compared with the chromatographic column 2, therefore, it was suggested to fix the chromatographic column SHIMADZU Shim-pack GIST C18-AQ (4.6*250mm, 5μm).

[0167] (5) Investigation of different serial numbers of the same chromatographic column

[0168] In order to ensure that the fixed chromatographic column could be stable and reproducible, the same type of chromatographic column with different serial numbers was determined by the method of Example 1, and the durability of the sample to the same type with different serial numbers was investigated.

[0169] Chromatographic column 1: SHIMMADZU Shim-pack GIST C18-AQ (4.6*250mm, 5μm), SN number: 21G27316;

[0170] Chromatographic column 2: SHIMMADZU Shim-pack GIST C18-AQ (4.6*250mm, 5μm), SN number: 22C52948;

[0171] Chromatographic column 3: SHIMMADZU Shim-pack GIST C18-AQ (4.6*250mm, 5μm), SN number: 22C52937;

[0172] The durability results of the same type with different serial numbers were as follows:

[0173] Table 23 Investigation of retention time and relative retention time of the same type with different serial numbers of Polygoni Orientalis Herba

[0174]

[0175] Table 24 Retention time and relative retention time of the same type with different serial numbers of Polygoni Orientalis Herba

[0176]

[0177]

[0178] The same type and different serial number chromatographic column investigation results show that: from the data and atlas results, the relative retention time RSD of the identified peaks in the sample measured by three chromatographic columns is in the range of 0.00% to 0.10%, and this type of chromatographic column can be used in this method.

[0179] Example 5

[0180] A construction method of a Polygonum multiflorum Thunb. preparation characteristic chromatogram, which is different from example 1 in that the detection wavelength is different.

[0181] Take Polygonum multiflorum Thunb. formula granules, prepare a test solution according to the preparation method of the test solution in example 1, and use different detection wavelengths (305 nm, 310 nm and 315 nm) to determine according to the method in example 1, to investigate the robustness of the experimental method to wavelength. The detection results are shown in Figures 5-7 and the following table.

[0182] Table 25 Investigation of retention time and relative retention time of Polygonum multiflorum Thunb. at different wavelengths

[0183]

[0184] Table 26 Investigation of retention time and relative retention time of Polygonum multiflorum Thunb. at different wavelengths

[0185]

[0186]

[0187] Different flow rate investigation results show that: the relative retention time RSD of the identified peaks in the sample is in the range of 0.00 to 0.00%, and the relative retention time of each characteristic is within ±10% of the specified value, and the method has good robustness to different wavelengths.

[0188] Comparative example 1

[0189] The difference between this comparative example and example 1 is that the chromatographic conditions are different, as follows:

[0190] Chromatographic column Agilent ZORBAX BONUS-RP (4.6 mm x 200 mm); flow rate 1 ml / min; column temperature 30℃; detection wavelength 260 nm; mobile phase is methanol-water, eluted according to the gradient conditions shown in table 27.

[0191] Table 27

[0192]

[0193] The characteristic chromatogram obtained by detection is shown in Figure 8 .

[0194] Simultaneously, the following chromatographic columns with different serial numbers were used for detection, including the Agilent ZORBAX BONUS-RP column, serial number USAC008396, LNB19042, as shown below. Figure 9 As shown, peaks 5 and 6 are in normal positions; the chromatographic column is Agilent ZORBAX BONUS-RP, serial number USAC008638, LNB19186. Figure 10 As shown, peaks 5 and 6 are in normal positions; the chromatographic column is Agilent ZORBAX BONUS-RP, serial number USAC009539, LNB20199. Figure 11 As shown, peaks 5 and 6 were not baseline separated; the chromatographic column was Agilent ZORBAX BONUS-RP, serial number USAC010218, LNB21380. Figure 12 As shown, peaks 5 and 6 almost overlap; the chromatographic column is Agilent ZORBAX BONUS-RP, serial number USAC011511, LNB23222. Figure 13 As shown, peaks 5 and 6 are reversed.

[0195] pass Figures 8-13 The results show that the characteristic chromatograms obtained by this comparative method have few characteristic peaks of Polygonum tinctoria; the baseline of the characteristic chromatograms is unstable and the peak shape is poor; the identified peaks are nucleoside components and lack specificity; the reproducibility of this method is unstable in the later stages and cannot be stably reproduced with the same type of chromatographic column.

[0196] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic chromatogram of a water extract of Polygonum multiflorum Thunb and a preparation thereof, characterized in that, The characteristic spectrum of the test substance is obtained by high performance liquid chromatography; the chromatographic conditions of the high performance liquid chromatography are: The chromatographic column is SHIMADZU Shim-pack C18-AQ; the detection wavelength is 305-315 nm; acetonitrile is used as mobile phase A, and 0.04-0.06% phosphoric acid aqueous solution is used as mobile phase B, and elution is carried out according to the following gradient program: The preparation process of the test sample solution is as follows: taking the water extract of Hedyseri Herba or the preparation of the water extract of Hedyseri Herba, adding a solvent, extracting and treating, shaking, filtering, and taking the continuous filtrate, thereby obtaining the test sample solution; The preparation process of the reference solution of the control substance is as follows: taking the control substance, accurately weighing, adding a solvent to prepare the reference solution of the control substance; The control substance includes the control substance of 4-coumaric acid and the control substance of ferulic acid; The preparation of the water extract of Hedyseri Herba is the water extract of Hedyseri Herba, the dried powder of the water extract of Hedyseri Herba or the formula granules of Hedyseri Herba; The solvent used in the preparation process of the test sample solution is an alcohol aqueous solution with a mass concentration of 50% or less or water.

2. The construction method of claim 1, wherein, In the chromatographic conditions of the high performance liquid chromatography, the column length of the chromatographic column is 250 mm, the inner diameter of the column is 4.6 mm, and the particle size is 5 μm; And / or, the column temperature is 28-32 ℃; And / or, the flow rate is 0.9-1.1 ml / min; And / or, the theoretical plate number calculated according to the peak of 4-coumaric acid should be not less than 5000.

3. The construction method according to claim 1, characterized in that, The preparation process of the reference solution of the control medicinal material is as follows: taking the control medicinal material, adding water, heat refluxing, cooling, centrifuging, taking the supernatant, concentrating to near dryness under reduced pressure, adding a solvent for ultrasonic treatment, taking out, cooling, shaking, filtering, and taking the continuous filtrate, thereby obtaining the reference solution of the control medicinal material.

4. The construction method of claim 1, wherein, In the preparation of the test sample solution, the extraction and treatment method is heating refluxing or ultrasonic treatment.

5. The construction method according to any one of claims 1-4, characterized in that, The solvent used in the preparation process of the reference solution of the control substance is a methanol solution; And / or, 15-50 ml of the solvent is added to 0.1 g of the test sample in the test sample solution; And / or, in the preparation of the test sample solution, the ultrasonic treatment time is more than 20 min.

6. The construction method according to any one of claims 1 to 4, characterized in that, The solvent in the test sample solution is an alcohol aqueous solution with a mass concentration of 30-50% or water; And / or, the ultrasonic treatment time in the preparation of the test sample solution is 20-30 min.

7. The construction method according to any one of claims 1 to 4, characterized in that, The characteristic peaks in the characteristic spectrum include peak 1-peak 14; The characteristic peak corresponding to 4-coumaric acid is peak 5, the characteristic peak corresponding to ferulic acid is peak 6, peak 5 is S peak, and the relative retention time of other characteristic peaks to S peak should be within ±10% of the specified value, and the specified value is peak 1 in turn: 0.55, peak 2: 0.62, peak 3: 0.71, peak 4: 0.77, peak 7: 1.17, peak 8: 1.22, peak 9: 1.34, peak 10: 1.42, peak 11: 1.45, peak 12: 1.54, peak 13: 1.58, peak 14: 1.

60.

8. The application of the characteristic spectrum constructed by the construction method according to any one of claims 1-7 in the overall quality control of the water extract of Hedyseri Herba and the preparation thereof.

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