Characteristic chromatogram construction method and application of polygonum and folium isatidis formula granules

Abstract

The invention provides a specific chromatogram construction method and application of polygonum-folium isatidis formula granules. The specific chromatogram construction method comprises the following steps: acquiring a specific chromatogram of a to-be-detected substance by adopting a high performance liquid chromatography; the specific chromatographic conditions of the high performance liquid chromatography are provided, octadecylsilane chemically bonded silica is used as a chromatographic column of a filler, the detection wavelength is 305-315 nm, acetonitrile is used as a mobile phase A, a 0.04%-0.06% phosphoric acid aqueous solution is used as a mobile phase B, and elution is carried out according to a specific gradient program. The specificity of the material information corresponding to the characteristic peak in the characteristic spectrum obtained by the construction method is stronger, the related information in the characteristic spectrum represented by the method is more comprehensive, and the overall quality control of the polygonum-folium isatidis can be better realized.

Description

Technical Field

[0001] The present invention relates to the field of identification of traditional Chinese medicines, and particularly to a method for constructing a characteristic fingerprint of Polygonum tinctorium Ait. formula granules and its application. Background Art

[0002] Polygonum tinctorium Ait. is the dried leaf of the plant Polygonum tinctorium Ait. of the Polygonaceae family. It is harvested twice when the branches and leaves are lush in summer and autumn, the stems and branches and impurities are removed, and then dried. It is distributed in Liaoning, Hebei, Shaanxi, Shandong and other places. It has the effects of clearing heat and detoxifying, cooling blood and removing spots, and is mainly used for symptoms such as febrile disease, maculae, cough due to lung heat, sore throat, mumps, erysipelas, and carbuncles.

[0003] Relatively few studies have been conducted on the characteristic fingerprint of Polygonum tinctorium Ait., and the identified components are few. The currently disclosed characteristic components are all nucleoside components, such as guanosine, uridine, adenosine, etc.; however, there is currently no literature reporting that the nucleoside components in Polygonum tinctorium Ait. are its active ingredients, and using nucleoside components as the characteristic peak components of the characteristic fingerprint for quality control lacks specificity. Summary of the Invention

[0004] The characteristic fingerprint of the water extract of Polygonum tinctorium Ait. and its preparations obtained by the currently disclosed characteristic fingerprint construction method has fewer specific characteristic peaks and is not conducive to the overall quality control. The present invention provides a method for constructing a characteristic fingerprint with more specific characteristic peaks, which can better realize the overall quality control of the water extract of Polygonum tinctorium Ait. and its preparations.

[0005] A method for constructing a characteristic fingerprint of a water extract of Polygonum tinctorium Ait. and its preparations, which uses high performance liquid chromatography to obtain the characteristic fingerprint of the analyte; the chromatographic conditions of the high performance liquid chromatography are as follows:

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

[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] and / or, the column temperature is 28 - 32 °C;

[0010] and / or, the flow rate is 0.9 - 1.1 ml / min;

[0011] and / or, the theoretical plate number calculated based on the 4-coumaric acid peak should be not less than 5000.

[0012] In the described high performance liquid chromatography method, the chromatographic column is SHIMADZU Shim-pack C18-AQ or YMC-Triart C18.

[0013] When the analyte is the test sample, the preparation process of the test sample solution is as follows: Take the water extract of Polygonum tinctorium Ait. or the preparation of the water extract of Polygonum tinctorium Ait., add a solvent, perform extraction treatment, shake well, filter, and take the subsequent filtrate to obtain it.

[0014] When the analyte is the control medicinal material, the preparation process of the reference substance solution of the control medicinal material is as follows: Take the control medicinal material, add water, perform heat reflux treatment, let it cool, centrifuge, take the supernatant and concentrate it under reduced pressure until nearly dry, add a solvent and perform ultrasonic treatment, take it out, let it cool, shake well and filter, and obtain the subsequent filtrate.

[0015] When the analyte is the reference substance, the preparation process of the reference substance solution of the reference substance is as follows: Take the reference substance, accurately weigh it, and prepare a reference substance solution of the reference substance with a solvent.

[0016] The reference substances include the reference substance of 4-coumaric acid and the reference substance of ferulic acid.

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

[0018] The preparations of the water extract of Polygonum tinctorium Ait. include the water extract paste of Polygonum tinctorium Ait., the dried powder of the water extract of Polygonum tinctorium Ait., the formula granules of Polygonum tinctorium Ait. or the water extract preparation of Polygonum tinctorium Ait.

[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 reference substance solution of the reference substance is a methanol solution.

[0021] And / or, 15 - 50 ml of solvent is added to every 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 time is 20 min or more.

[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 time is 20 - 30 min.

[0025] The characteristic chromatogram includes characteristic peaks from peak 1 to peak 14.

[0026] Taking the characteristic peak corresponding to 4-coumaric acid as peak 5, the characteristic peak corresponding to ferulic acid as peak 6, and peak 5 as the S peak, calculate the relative retention times of other characteristic peaks with respect to the S peak, and the relative retention times should be within the range of ±10% of the specified values. The specified values are as follows: peak 1: 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.

[0027] Application of the characteristic chromatogram constructed by the above construction method in the overall quality control of water extracts and preparations of Polygonum tinctorium Lour.

[0028] The technical solution of the present invention has the following advantages:

[0029] 1. The present invention provides a method for constructing a characteristic chromatogram of water extracts and preparations of Polygonum tinctorium Lour. The characteristic peaks in the characteristic chromatogram obtained by this construction method include at least 14 characteristic peaks, and the substance information of the characteristic chromatogram is more comprehensive. At the same time, the characteristic peaks include the characteristic peaks corresponding to 4-coumaric acid and ferulic acid. Among them, the pharmacological effects of 4-coumaric acid are anti-inflammatory, antioxidant, immunomodulatory, anti-tumor, antiplatelet aggregation, protecting cardiovascular, preventing and improving diabetes, protecting nerves, antibacterial, etc., and the pharmacological effects of ferulic acid are antibacterial and anti-inflammatory, preventing and treating coronary heart disease, protecting women's ovaries and inhibiting liver injury, etc. This has a certain correlation with the efficacy of Polygonum tinctorium Lour., and its specificity is stronger, more fully characterizing the efficacy chemical components in the water extracts and preparations of Polygonum tinctorium Lour., and can better realize the overall quality control of the water extracts and preparations of Polygonum tinctorium Lour.

[0030] 2. The construction method of the present invention can not only reflect the content of active ingredients in the formula granules of Polygonum tinctorium Lour. from the overall chromatographic appearance, effectively characterize its quality, but also is conducive to better controlling the quality of the product as a whole, providing an effective guarantee for the production and clinical application of drugs.

[0031] 3. The construction method of the present invention has the advantages of simple method, stability, high precision, good reproducibility, etc., can quickly and accurately identify the quality of the product, and provides a scientific basis for comprehensively establishing the quality control standard of the formula granules of Polygonum tinctorium Lour. Description of the Drawings

[0032] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0033] Figure 1 It is the characteristic chromatogram of Polygonum tinctorium Lour. formula granules in Example 1 of the present invention.

[0034] Figure 2 It is the comparison chromatogram of the test solution and the reference substance solution of the reference substance in Example 1 of the present invention.

[0035] Figure 3 It is the characteristic chromatogram of the test solution and the reference medicinal material reference substance solution in Example 1 of the present invention.

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

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

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

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

[0040] Figure 8 It is the characteristic chromatogram obtained by detecting Polygonum tinctorium Lour. formula granules with the chromatographic column of the same serial number in Comparative Example 1 of the present invention.

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

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

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

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

[0045] Figure 13 It is the characteristic chromatogram obtained with the chromatographic column of serial number USAC011511 in Comparative Example 1 of the present invention. Detailed implementation manners

[0046] For those not specifying specific experimental steps or conditions in the examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments not indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

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

[0048] Test drugs: 4-coumaric acid reference substance (batch number: 112037-202102, National Institutes for Food and Drug Control, purity 99.7%);

[0049] Ferulic acid reference substance (batch number: 110773-202316, National Institutes for Food and Drug Control, purity 99.3%);

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

[0051] Polygonum tinctorium Lour. herb: K478YC16; Polygonum tinctorium Lour. cut crude drug: K478YP16;

[0052] Polygonum tinctorium Lour. freeze-dried powder: K478BJ01, K478BJ02, K478BJ03, K478BJ04, K478BJ05, K478BJ06, K478BJ07, K478BJ08, K478BJ09, K478BJ010, K478BJ011, K478BJ012, K478BJ013, K478BJ014, K478BJ015, K478BJ016, K478BJ017, K478BJ018, K478BJ019, K478BJ020, K478BJ021, K478BJ022, K478BJ023, K478BJ024;

[0053] Polygonum tinctorium Lour. formula granules: K478CP16, K478CP17, K478CP18.

[0054] Reagents: Methanol is of chromatographic grade, ethanol is of analytical grade, water is Watsons distilled water, acetonitrile (MERCK) is of chromatographic grade; phosphoric acid (MERCK) is of chromatographic grade; methanol (MERCK) is of chromatographic grade; water is distilled water (Watsons); other reagents are all of analytical grade.

[0055] Example 1

[0056] A method for constructing the characteristic fingerprint of Polygonum tinctorium Lour. preparation, the specific process is as follows:

[0057] 1. Preparation of the solution of the analyte

[0058] Preparation of the test solution: Take an appropriate amount of the test sample (Polygonum tinctorium Lour. formula granules K478CP16), grind it finely, take about 0.1 g, place it in a stoppered conical flask, accurately add 15 ml of 30% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 20 minutes, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0059] Preparation of the reference solution of the control medicinal material: Take 0.5 g of Polygonum tinctorium Lour. medicinal material, place it in a stoppered conical flask, add 50 ml of water, heat under reflux for 30 minutes, take it out, let it cool, centrifuge, take the supernatant, concentrate it under reduced pressure to nearly dry, add 15 ml of 30% methanol to the residue, ultrasonically treat (power 250 W, frequency 40 kHz) for 20 minutes, take it out, let it cool, shake well, filter, and take the subsequent filtrate to obtain the reference solution of the control medicinal material.

[0060] Reference solution of the reference substance: Take appropriate amounts of 4-coumaric acid reference substance and ferulic acid reference substance, accurately weigh them, dissolve them in methanol to prepare a mixed reference solution containing 10 μg of each per 1 ml as the reference solution of the reference substance.

[0061] 2. Chromatographic method

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

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

[0064] Table 1

[0065]

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

[0067] Take 24 batches of freeze-dried Polygonum tinctorium Lour. powder and three batches of Polygonum tinctorium Lour. formula granules respectively, obtain their liquid chromatograms according to the method in Example 1, use the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" (version 2012.1), take the S1 liquid chromatogram as the reference chromatogram, calculate by the median, and obtain the characteristic chromatogram of Polygonum tinctorium Lour. formula granules, as Figure 1 shown, and identify the common peaks.

[0068] Table 2 List of Standard Decoction Freeze-dried Powder and Granules of Polygonum tinctorium Lour. Leaves

[0069]

[0070] A total of 14 common peaks were identified from 24 batches of standard decoctions and 3 batches of granules of Polygonum tinctorium Lour. Leaves, and the similarity range was between 0.988 and 0.999.

[0071] According to their retention times and peak areas in the characteristic chromatogram, peak 5 was determined to be 4-coumaric acid and peak 6 was determined to be ferulic acid. Using 4-coumaric acid in peak 5 as the reference peak, the relative retention times and relative peak areas of characteristic peaks 1-4 and 7-14 with respect to peak 5 were calculated. The relative retention time and relative peak area data of the 24 batches of freeze-dried powder of the standard decoction of Polygonum tinctorium Lour. Leaves were processed, and the results are as follows.

[0072] Table 3 Relative Retention Time Table of the Characteristic Chromatogram of 24 Batches of Standard Decoctions of Polygonum tinctorium Lour. Leaves

[0073]

[0074]

[0075] Table 4 Relative Retention Time Table of the Characteristic Chromatogram of 24 Batches of Standard Decoctions of Polygonum tinctorium Lour. Leaves

[0076]

[0077]

[0078] Table 5 Relative Peak Area Table of the Characteristic Chromatogram of 24 Batches of Standard Decoctions of Polygonum tinctorium Lour. Leaves

[0079]

[0080] Table 6 Relative Peak Area Table of the Characteristic Chromatogram of 24 Batches of Standard Decoctions of Polygonum tinctorium Lour. Leaves

[0081]

[0082]

[0083] After processing the data of the liquid chromatograms of 24 batches of standard decoctions of Polygonum tinctorium Lour. leaves, the similarity between the characteristic chromatograms of the 24 batches of standard decoctions of Polygonum tinctorium Lour. leaves and the control chromatogram was greater than 0.9. The relative retention times of the 14 common peaks in the characteristic chromatogram of the standard decoction of Polygonum tinctorium Lour. leaves had small differences, all within the range of ±10%, and the average value of the relative retention times was selected as the specified value; the specified values were: 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%. However, the differences in the relative peak areas were relatively large, and their RSD values were 44.87% - 93.52%, which did not meet the quality control requirements. Therefore, the relative peak areas were not included in the standard.

[0084] Take the test solution of Polygonum tinctorium Lour. granules, the reference solution of kaempferol - 3 - O - β - D - glucuronide, and the mixed reference solution of 4 - coumaric acid and ferulic acid reference substances, and perform detection and comparison according to the above chromatographic conditions to infer the characteristic peaks in Polygonum tinctorium Lour. leaves. The results are shown in Figure 2 . The characteristic chromatogram shows 14 peaks, among which peak 5 is 4 - coumaric acid, peak 6 is ferulic acid, and peak 7 is kaempferol - 3 - O - β - D - glucuronide, and there is no interference from negative excipients and blank solutions. In the characteristic chromatogram obtained in the present invention, the characteristic peak components are mainly organic acid components. Among them, 4 - coumaric acid and ferulic acid, as representative components of organic acids in Polygonum tinctorium Lour. leaves, mainly have pharmacological effects such as antibacterial, anti - inflammatory, antiviral, antioxidant, and anti - tumor.

[0085] Take about 0.5 g of Polygonum tinctorium Lour. cut - pieces, accurately weigh, add 50 ml of water, heat under reflux for 30 minutes, take out, cool, centrifuge, take the supernatant, concentrate it under reduced pressure until nearly dry, add 15 ml of 30% methanol to the residue, ultrasonically treat (power 250 W, frequency 40 kHz) for 20 minutes, shake well, filter, and take the subsequent filtrate to obtain the accompanying cut - piece solution. Take about 0.1 g of Polygonum tinctorium Lour. granules and prepare the test solution of granules in the same method. Compare the characteristic chromatogram of Polygonum tinctorium Lour. granules with the characteristic chromatogram of Polygonum tinctorium Lour. cut - pieces. The results are shown in Figure 3 .

[0086] Through Figure 3 comparison of the chromatograms, it can be seen that the 14 characteristic peaks of Polygonum tinctorium Lour. granules and Polygonum tinctorium Lour. cut - pieces are consistent. It shows that from Polygonum tinctorium Lour. cut - pieces to formula granules, the substances corresponding to the characteristic peaks are completely transferred, but the transfer rates are different, and the correlation between the two is good.

[0087] Example 2

[0088] This example is used to perform method validation on the chromatographic conditions in Example 1.

[0089] 1. Specificity study

[0090] Approximately 1.0 g of Polygonum tinctorium Lour. formula granules and maltodextrin were taken respectively, and the test solution of Polygonum tinctorium Lour. formula granules and the negative control solution of excipients were prepared respectively according to the method of Example 1. The results showed that the negative solution had no interference on the characteristic peaks, indicating that the method had good specificity.

[0091] 2. Repeatability investigation

[0092] Six portions of Polygonum tinctorium Lour. formula granules were taken, ground fine, and determined according to the method of Example 1 to obtain their characteristic chromatograms. Using the 5th peak as the reference peak S, the relative retention times and relative peak areas of the 1st - 4th characteristic peaks and the 7th - 14th characteristic peaks were calculated, and the RSD was calculated.

[0093] The results of the repeatability experiment showed that the RSD of the relative retention times of the six repeatability experiment samples was in the range of 0.0% - 0.03%, and the RSD of the relative peak areas was in the range of 0.25% - 2.70%, indicating that the repeatability of the characteristic chromatogram was good.

[0094] 3. Intermediate precision investigation

[0095] Polygonum tinctorium Lour. formula granules were taken, ground fine, and sample preparation was carried out according to the method of Example 1. Different analysts carried out intermediate precision tests at different times using another high - performance liquid chromatograph (TUV detector), determined, and obtained their characteristic chromatograms. Using the 5th peak as the reference peak S, the relative retention times of the 1st - 4th characteristic peaks and the 7th - 14th characteristic peaks with respect to the 5th peak were calculated, and the RSD value was calculated.

[0096] The results of intermediate precision showed that the RSD of the relative retention times of the six intermediate precision experiment samples was in the range of 0.00% - 0.490%; compared with the repeatability samples, the RSD of the relative retention times of the intermediate precision was in the range of 0.55% - 1.59%. From the data, it can be seen that the intermediate precision of this method was good.

[0097] 4. Stability investigation

[0098] The same test solution of granules was taken and determined at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h respectively. The relative retention times of the 1st - 4th characteristic peaks and the 7th - 14th characteristic peaks with respect to the 5th peak were calculated, and the RSD was calculated.

[0099] The results of stability showed that after investigating the solution stability for 24 hours, the RSD% of the relative retention times of the samples was in the range of 0.00% - 0.03%, and the RSD% of the relative peak areas was in the range of 0.37% - 3.30%. From the data, it can be seen that the test solution was relatively stable within 24 hours.

[0100] Example 3

[0101] A method for constructing a characteristic chromatogram of a Polygonum tinctorium Lour. leaf preparation, which is different from Example 1 in that the preparation of the test solution is different.

[0102] (1) Selection of extraction solvent

[0103] Weigh 0.1 g of Polygonum tinctorium Lour. leaf granules powder accurately, place it in a stoppered conical flask, accurately add 15 ml of the corresponding solvents (water, 30% methanol, 50% methanol, 70% methanol, methanol, 30% ethanol, 50% ethanol, 70% ethanol, ethanol) respectively, perform ultrasonic treatment (power 250 W, frequency 40 kHz) for 20 minutes, take it out, let it cool, shake well, filter, take the continuous filtrate, inject it into a high performance liquid chromatograph, and determine according to the chromatographic conditions in Standard Example 1. The results are shown in Table 7-8 and Figure 4 as shown.

[0104] Table 7 Investigation table of extraction solvents

[0105]

[0106] Table 8 Investigation table of extraction solvents

[0107]

[0108] It can be seen from the data results that when methanol is used as the extraction solvent, the response values of each characteristic peak are low; when ethanol is used as the extraction solvent, characteristic peaks 1-4 are missing; when 70% ethanol aqueous solution is used as the extraction solvent, the peak shape is not good, and there is no obvious difference in the characteristic peak information when the other 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 solution. For reasons such as taking into account the peak area and peak shape, the present invention preferably uses an alcohol aqueous solution or water below 50% as the solvent. Among them, after extraction with 30% methanol aqueous solution, the detectable peaks are relatively rich, the peak areas of each characteristic peak are relatively high, and the peak shape is better. Therefore, 30% methanol aqueous solution is selected as the extraction solvent for the characteristic chromatogram of Polygonum tinctorium Lour. leaf granules.

[0109] (2) Selection of solvent dosage

[0110] Accurately weigh 0.1 g of the granules powder of this product, place it in a stoppered conical flask, accurately add 10 ml, 15 ml, 25 ml, 50 ml of water respectively, tightly stopper, weigh, perform ultrasonic treatment for 20 minutes (power 250 W, frequency 40 kHz), take it out, let it cool, shake well, filter, take the continuous filtrate, and you will get it. Accurately absorb 10 μl of the continuous filtrate, inject it into a high performance liquid chromatograph, and detect according to the chromatographic conditions in Example 1. The results are as follows.

[0111] Table 9 Investigation of extraction solvent dosage

[0112]

[0113] Table 10 Investigation of the amount of extraction solvent

[0114]

[0115]

[0116] The analysis results show that when the amount of the extraction solvent is 10 ml, the sample is not completely extracted at different amounts of the extraction solvent. When the amounts of the extraction solvent are 15 ml, 25 ml, and 50 ml, the differences in the number of characteristic peaks and the response values are small. Considering the durability of the method and the principle of economy, the amount of the solvent is selected as 15 ml.

[0117] (3) Selection of extraction time

[0118] Accurately weigh 0.1 g of the granule powder of this product, place it in a stoppered conical flask, accurately add 15 ml of 30% methanol, stopper it tightly, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 10, 20, and 30 minutes respectively, take it out, let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution. Accurately pipette 10 μl of the subsequent filtrate, inject it into a high performance liquid chromatograph, and determine it according to the chromatographic conditions in Example 1. The results are as follows.

[0119] Table 11 Investigation table of extraction time

[0120]

[0121] Table 12 Investigation table of extraction time

[0122]

[0123] It can be seen from the above results that the sample is not completely extracted after ultrasonication for 10 minutes, and the peak areas of each characteristic peak are not much different when ultrasonication is carried out for 20 and 30 minutes. To ensure its complete extraction, the extraction time is selected as 20 minutes.

[0124] (4) Selection of extraction method

[0125] Accurately weigh 0.1 g of the granule powder of this product, place it in a stoppered conical flask, accurately add 15 ml of 30% methanol, stopper it tightly, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) and reflux it by heating for 20 minutes respectively, take it out, let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution. Accurately pipette 10 μl of the subsequent filtrate, inject it into a high performance liquid chromatograph, and determine it according to the chromatographic conditions in Example 1. The results are as follows.

[0126] Table 13 Investigation table of extraction method

[0127]

[0128] Table 14 Investigation table of extraction method

[0129]

[0130] As can be seen from the above results, the peak areas of each characteristic peak are not much different when the samples are treated by ultrasound and reflux. Considering the convenience of the experiment, ultrasound is therefore selected as the extraction method.

[0131] Example 4

[0132] A method for constructing a characteristic chromatogram of Polygonum tinctorium Lour. preparation is different from that of Example 1 in that the chromatographic conditions are as follows:

[0133] (1) Investigation of different column temperatures

[0134] Take Polygonum tinctorium Lour. granules, prepare a test solution according to the preparation method of the test solution in Example 1, and measure it according to the method in Example 1 at different column temperatures (28 °C, 30 °C, and 32 °C) respectively to investigate the durability of the experimental method for column temperature. The results are as follows:

[0135] Table 15 Retention time and relative retention time of Polygonum tinctorium Lour. at different column temperatures

[0136]

[0137]

[0138] Table 16 Retention time and relative retention time of Polygonum tinctorium Lour. at different column temperatures

[0139]

[0140] The results of the investigation of different column temperatures show that the relative retention times of each characteristic peak in the sample are all within the range of ±10% of the specified value, indicating that the durability of this method for different column temperatures is good.

[0141] (2) Investigation of different flow rates

[0142] Take Polygonum tinctorium Lour. formula granules, prepare a test solution according to the preparation method of the test solution in Example 1, and measure it according to the method in Example 1 at different flow rates (0.9 ml / min, 1.0 ml / min, and 1.1 ml / min) respectively to investigate the durability of the experimental method for flow rate. The results are as follows.

[0143] Table 17 Retention time and relative retention time of Polygonum tinctorium Lour. at different flow rates

[0144]

[0145] Table 18 Retention time and relative retention time of Polygonum tinctorium Lour. at different flow rates

[0146]

[0147] The results of the investigation at different flow rates showed that the RSD of the relative retention time of the labeled peaks in the sample was in the range of 0.19% to 2.89%, and the relative retention times of each characteristic were within ±10% of the specified value. This method has good durability for different flow rates.

[0148] (3) Investigation at different acid concentrations

[0149] Take Polygonum tinctorium Lour. granules, prepare the test solution according to the preparation method of the test solution in Example 1, and determine it according to the method in Example 1 using different acid concentrations (0.04% phosphoric acid, 0.05% phosphoric acid, and 0.06% phosphoric acid) respectively to investigate the durability of the experimental method for the flow rate. The results are as follows:

[0150] Table 19 Retention time and relative retention time of Polygonum tinctorium Lour. at different acid concentrations

[0151]

[0152] Table 20 Retention time and relative retention time of Polygonum tinctorium Lour. at different acid concentrations

[0153]

[0154]

[0155] The results of the investigation at different acid concentrations showed that the RSD of the relative retention time of the labeled peaks in the sample was in the range of 0.02% to 0.56%, and the relative retention times of each characteristic were within ±10% of the specified value. This method has good durability for different acid concentrations.

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

[0157] Take Polygonum tinctorium Lour. granules, prepare the test solution according to the method in Example 1, and determine it according to the method in Example 1 using different types of chromatographic columns

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

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

[0160] Investigate the durability of the experimental method for different chromatographic columns. The results are as follows:

[0161] Table 21 Retention time and relative retention time of Polygonum tinctorium Lour. for different types of chromatographic columns

[0162]

[0163] Table 22 Investigation of retention time and relative retention time of Polygonum tinctorium leaves on different types of chromatographic columns

[0164]

[0165]

[0166] The investigation results of different types of chromatographic columns show that: from the data and chromatogram results, it can be seen that the peak 1 and peak 7 of chromatographic column 1 are missing, and compared with chromatographic column 2, the peak shapes of each characteristic peak of chromatographic column 3 are not good. Therefore, it is recommended 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] To ensure that the fixed chromatographic column can stably reproduce its characteristic chromatogram, different serial numbers of the same type of chromatographic column were used to conduct the determination according to the method of Example 1, and the durability of the sample for different serial numbers of the same type 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 different serial numbers of the same type are as follows:

[0173] Table 23 Investigation of retention time and relative retention time of Polygonum tinctorium leaves with different serial numbers of the same type

[0174]

[0175] Table 24 Retention time and relative retention time of Polygonum tinctorium leaves with different serial numbers of the same type

[0176]

[0177]

[0178] The investigation results of chromatographic columns with the same model but different serial numbers show that: from the data and chromatogram results, it can be seen that the relative retention time RSD of the labeled peaks in the samples measured by the three chromatographic columns is in the range of 0.00% to 0.10%. This method is feasible for using this model of chromatographic column.

[0179] Example 5

[0180] A method for constructing the characteristic chromatogram of Polygonum tinctorium Lour. preparation, which is different from Example 1 in that the detection wavelength is different.

[0181] Take Polygonum tinctorium Lour. formula granules, prepare them into a test solution according to the preparation method of the test solution in Example 1, and measure them according to the method in Example 1 at different detection wavelengths (305 nm, 310 nm, and 315 nm) respectively to investigate the durability of the experimental method for the wavelength. The detection results are as Figures 5 - 7 and the following table shows.

[0182] Table 25 Retention time and relative retention time of Polygonum tinctorium Lour. at different wavelengths

[0183]

[0184] Table 26 Retention time and relative retention time of Polygonum tinctorium Lour. at different wavelengths

[0185]

[0186]

[0187] The investigation results of different flow rates show that: the relative retention time RSD of the labeled peaks in the sample is in the range of 0.00 to 0.00%, and the relative retention time of each characteristic is within the range of ±10% of the specified value. This method has good durability for different wavelengths.

[0188] Comparative Example 1

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

[0190] Chromatographic column: Agilent ZORBAX BONUS-RP (4.6 mm × 200 mm); flow rate 1 ml / min; column temperature 30 °C; detection wavelength 260 nm; mobile phase is methanol-water, and elution is carried out according to the gradient conditions shown in Table 27.

[0191] Table 27

[0192]

[0193] The obtained characteristic chromatogram is as Figure 8 shown.

[0194] Meanwhile, detections were carried out using chromatographic columns with the following different serial numbers. Among them, the chromatographic column is Agilent ZORBAX BONUS-RP, serial number USAC008396, L.N. B19042, as Figure 9 shown, the positions of peaks 5 and 6 are normal; the chromatographic column is Agilent ZORBAX BONUS-RP, serial number USAC008638, L.N. B19186, as Figure 10 shown, the positions of peaks 5 and 6 are normal; the chromatographic column is Agilent ZORBAX BONUS-RP, serial number USAC009539, L.N. B20199, as Figure 11 shown, peaks 5 and 6 are not baseline separated; the chromatographic column is Agilent ZORBAX BONUS-RP, serial number USAC010218, L.N. B21380, as Figure 12 shown, peaks 5 and 6 almost overlap; the chromatographic column is Agilent ZORBAX BONUS-RP, serial number USAC011511, L.N. B23222, as Figure 13 shown, peaks 5 and 6 are reversed.

[0195] From Figures 8 - 13 the results, it can be seen that the characteristic chromatogram obtained by the method of this comparative example has fewer characteristic peaks of Polygonum tinctorium Lour.; the baseline of the characteristic chromatogram is not stable and the peak shape is not good; the identified peaks are nucleoside components and lack specificity; the later reproducibility of this method is unstable and the same model chromatographic column cannot stably reproduce.

[0196] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or alterations derived therefrom are still within the protection scope of this invention.

Claims

1. A method for constructing a characteristic fingerprint of water extract of Polygonum tinctorium Lour. and its preparation, characterized in that, Obtain the characteristic chromatogram of the analyte by high performance liquid chromatography; the chromatographic conditions of the high performance liquid chromatography are as follows: A chromatographic column filled with octadecylsilyl-bonded silica gel; the detection wavelength is 305 - 315 nm; acetonitrile is used as mobile phase A, and an aqueous solution of 0.04% - 0.06% phosphoric acid is used as mobile phase B, and elution is carried out according to the following gradient program:

2. The construction method according to claim 1, wherein 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; and / or, the column temperature is 28 - 32 °C; and / or, the flow rate is 0.9 - 1.1 ml / min; and / or, the theoretical plate number calculated based on the 4-coumaric acid peak should be not less than 5000.

3. The construction method according to claim 1, characterized in that, In the high performance liquid chromatography, the chromatographic column is SHIMADZU Shim-pack C18-AQ or YMC-Triart C18.

4. The construction method according to any one of claims 1 to 3, characterized in that When the analyte is the test sample, the preparation process of the test sample solution is as follows: Take the water extract of Polygonum tinctorium Ait. or the preparation of the water extract of Polygonum tinctorium Ait., add a solvent, perform extraction treatment, shake well, filter, and take the subsequent filtrate to obtain it; When the analyte is the control crude drug, the preparation process of the reference substance solution of the control crude drug is as follows: Take the control crude drug, add water, perform heat reflux treatment, cool, centrifuge, take the supernatant and concentrate it under reduced pressure to near dryness, add a solvent for ultrasonic treatment, take it out, cool, shake well and filter, and obtain the subsequent filtrate; When the analyte is the reference substance, the preparation process of the reference substance solution of the reference substance is as follows: Take the reference substance, accurately weigh it, and prepare a reference substance solution of the reference substance with a solvent.

5. The construction method according to claim 4, characterized in that, The reference substances include 4-coumaric acid reference substance and ferulic acid reference substance.

6. The construction method according to claim 4, characterized in that, In the preparation of the test sample solution, the extraction treatment method is heating reflux or ultrasonic treatment.

7. The construction method according to any one of claims 4-6, characterized in that The preparations of the water extract of Polygonum tinctorium Ait. include the water extract paste of Polygonum tinctorium Ait., the dried powder of the water extract of Polygonum tinctorium Ait., the formula granules of Polygonum tinctorium Ait. or the water extract preparation of Polygonum tinctorium Ait.; and / or, the solvent used in the preparation process of the test sample solution is an aqueous alcohol solution with a mass concentration of less than 50% or water; and / or, the solvent used in the preparation process of the reference substance solution of the reference substance is a methanol solution; and / or, 15 - 50 ml of solvent is added to every 0.1 g of the test sample in the test sample solution; and / or, in the preparation of the test sample solution, the ultrasonic time is more than 20 min.

8. The construction method according to any one of claims 1 to 7, characterized in that, The solvent in the test sample solution is an aqueous alcohol solution with a mass concentration of 30% - 50% or water; and / or, in the preparation of the test sample solution, the ultrasonic time is 20 - 30 min.

9. The construction method according to any one of claims 1-8, characterized in that, The characteristic chromatogram includes characteristic peaks from peak 1 to peak 14; Taking the characteristic peak corresponding to 4-coumaric acid as peak 5, the characteristic peak corresponding to ferulic acid as peak 6, and peak 5 as the S peak, calculate the relative retention time of other characteristic peaks with respect to the S peak, and the relative retention time should be within the range of ±10% of the specified value. The specified values are successively peak 1: 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.

10. Use of the characteristic spectrum constructed by the construction method according to any one of claims 1-9 in the overall quality control of the water extract and preparation of Polygonum tinctorium Lour.

Images