Fingerprint and content determination method of Jingqi huazhuo preparation

Abstract

The application provides a fingerprint spectrum and a content determination method of Jingqihuatong preparation, and belongs to the technical field of traditional Chinese medicines. The construction method comprises the following steps: detecting a test product solution containing the Jingqihuatong preparation by using high performance liquid chromatography, and generating a fingerprint spectrum according to the detection result; a mobile phase A is methanol, and a mobile phase B is 0.1% phosphoric acid aqueous solution; and gradient elution is carried out by using the mobile phases A and B. The fingerprint spectrum can simultaneously detect a plurality of chemical components in the Jingqihuatong preparation, has high separation degree, can systematically and stably fully reflect the quality of the product, and can effectively control the quality of the Jingqihuatong preparation. Meanwhile, the construction method is simple and efficient, can shorten the detection time, reduce the use of solvents, and greatly reduce the detection cost, has high specificity and selectivity for the analysis of unknown and known components, and is more beneficial to practical production and application.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and more specifically, to a fingerprint spectrum and content determination method for a preparation of astragalus extract for clearing turbidity. Background Technology

[0002] The formula for the Jingqi Huazhuo preparation is made from seven Chinese herbs: Astragalus membranaceus, Polygonatum sibiricum, Polygonatum odoratum, Curcuma zedoaria, Cynanchum paniculatum, Polygonum cuspidatum, and Centella asiatica. This formula works by promoting the upward movement of clear qi and the downward movement of turbid qi, thus invigorating qi, raising yang, and clearing turbidity. It is used to treat diabetic nephropathy of the spleen and kidney deficiency type with blood stasis obstructing the collaterals. Symptoms include foamy urine, lower back pain, frequent urination at night, and proteinuria.

[0003] In existing technologies, quality control studies mainly focus on determining the content of indicative components of the three medicinal ingredients in the prescription. This requires the combined use of an evaporative light scattering detector and an ultraviolet detector, which is cumbersome and greatly increases the detection time and cost. Furthermore, it only focuses on a few components in the prescription and cannot fully reflect the overall quality of the preparation.

[0004] The applicant has developed a fingerprinting method and content determination method for quality control using high performance liquid chromatography, enabling more comprehensive control of product quality.

[0005] The established chromatographic method can rapidly and accurately separate 12 characteristic peaks from the three medicinal materials Astragalus membranaceus, Polygonum cuspidatum, and Centella asiatica in the product, including three glycosides identified by known reference standards: polygalin, verbascoside, and emodin. The separation effect is good, and it can accurately quantify the active ingredients polygalin and emodin contained in Polygonum cuspidatum. The linear relationship of each component is good, and the recovery rate is good. It can meet the technical requirements for simultaneous determination of multiple contents and fingerprint chromatograms.

[0006] To address the shortcomings of existing technologies, this invention provides a fingerprint spectrum and content determination method for a strychnine-based turbidity-resolving preparation. Summary of the Invention

[0007] The purpose of this invention is to provide a fingerprint spectrum and content determination method for a purifying and turbidity-reducing preparation.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] In a first aspect, the present invention provides a method for constructing the fingerprint spectrum of a purifying and turbidity-reducing preparation, the method comprising the following steps:

[0010] The test solution containing the strychnine-based turbidity-reducing preparation was detected by high-performance liquid chromatography (HPLC), and the detection results were used to generate a fingerprint chromatogram. The HPLC detection used mobile phase A as methanol and mobile phase B as a 0.1% aqueous phosphoric acid solution. Gradient elution was performed using mobile phases A and B under the following conditions:

[0011]

[0012] The percentages in the table represent the percentage of each mobile phase in the total volume of the mobile phase.

[0013] Furthermore, in this high-performance liquid chromatography detection, the chromatographic column is an octadecylsilane-bonded silica column; the preferred specifications of the octadecylsilane-bonded silica column are 4.6 mm × 250 mm and 5 μm.

[0014] Furthermore, in this high-performance liquid chromatography detection, the column temperature is 30℃;

[0015] Furthermore, the detection wavelengths of this high-performance liquid chromatography are 210 nm and 260 nm;

[0016] Furthermore, the injection volume of this high-performance liquid chromatography is 5–20 μL, preferably 10 μL.

[0017] Furthermore, the flow rate of the mobile phase is 0.9–1.1 ml / min, preferably 1.0 ml / min.

[0018] Furthermore, it also includes preparing a reference solution and performing high-performance liquid chromatography (HPLC) on the reference solution to determine the peak assignment in the test solution based on the retention time of the reference solution; the retention time of the reference solution determines the peak assignment in the test solution; the reference solution includes polygalactosidin, verbascoside, and emodin.

[0019] Furthermore, the construction method includes the following steps:

[0020] (1) Preparation of the test solution: The strychnine-based turbidity-reducing preparation was dissolved in methanol;

[0021] (2) High performance liquid chromatography (HPLC) was performed using an octadecylsilane-bonded silica gel column; the mobile phase was methanol-0.1% phosphoric acid aqueous solution, the column temperature was 25℃, the flow rate was 1.0 mL / min, and the detection wavelength was 254 nm.

[0022] The gradient elution procedure is as follows:

[0023]

[0024] The percentages in the table represent the percentage of each mobile phase in the total volume of the mobile phase.

[0025] Furthermore, step (1) also includes sonicating for 30 minutes (power 100W, frequency 40kHz), cooling, shaking, and filtering.

[0026] Furthermore, step (1) also includes the use of an octadecylsilane-bonded silica gel column with dimensions of 4.6 mm × 250 mm and 5 μm.

[0027] Secondly, the present invention provides a fingerprint spectrum of a purifying and turbidity-reducing preparation, which includes 12 common characteristic peaks, with peak number 3 as the control peak. The relative retention times of the 12 common characteristic peaks are as follows:

[0028] The relative retention time of the common characteristic peak No. 1 is 0.41 ± 10%;

[0029] The relative retention time of the common characteristic peak No. 2 is 0.66 ± 10%;

[0030] The relative retention time of the characteristic peak No. 3 is 1.00.

[0031] The relative retention time of the common characteristic peak No. 4 is 1.04 ± 10%;

[0032] The relative retention time of the common characteristic peak No. 5 is 1.18 ± 10%;

[0033] The relative retention time of the common characteristic peak No. 6 is 1.20 ± 10%;

[0034] The relative retention time of the characteristic peak No. 7 is 1.28 ± 10%.

[0035] The relative retention time of the characteristic peak No. 8 is 1.38 ± 10%.

[0036] The relative retention time of the characteristic peak No. 9 is 1.61 ± 10%.

[0037] The relative retention time of the common characteristic peak at peak number 10 is 1.84 ± 10%.

[0038] The relative retention time of the characteristic peak No. 11 is 1.96 ± 10%.

[0039] The relative retention time of the characteristic peak at peak 12 is 2.34 ± 10%.

[0040] Furthermore, the fingerprint spectrum of this astragalus-based turbidity-resolving preparation, using peak 3 as the control peak, showed the following relative retention times for the 12 common characteristic peaks:

[0041] The relative retention time of the characteristic peak No. 1 is 0.41.

[0042] The relative retention time of the characteristic peak No. 2 is 0.66.

[0043] The relative retention time of the characteristic peak No. 3 is 1.00.

[0044] The relative retention time of the characteristic peak No. 4 is 1.04.

[0045] The relative retention time of the characteristic peak No. 5 is 1.18.

[0046] The relative retention time of the characteristic peak at peak 6 is 1.20.

[0047] The relative retention time of the characteristic peak No. 7 is 1.28.

[0048] The relative retention time of the characteristic peak at peak 8 is 1.38.

[0049] The relative retention time of the characteristic peak at peak 9 is 1.61.

[0050] The relative retention time of the characteristic peak at peak number 10 is 1.84.

[0051] The relative retention time of the characteristic peak at peak 11 is 1.96.

[0052] The relative retention time of the characteristic peak at peak 12 is 2.34.

[0053] Thirdly, this invention provides a fingerprint spectrum of the astragalus-based turbidity-resolving preparation as a standard fingerprint spectrum for the detection and quality control of the astragalus-based turbidity-resolving preparation.

[0054] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0055] This invention provides the establishment and application of a fingerprint spectrum detection method for astragalus-based turbidity-reducing preparations. This fingerprint spectrum can simultaneously detect multiple chemical components in the astragalus-based turbidity-reducing preparations, exhibiting high separation and systematically and stably reflecting the product quality, thereby effectively controlling the quality of the preparations. Furthermore, this construction method is simple and efficient, shortening detection time and reducing solvent usage, thus significantly lowering detection costs. It also demonstrates high specificity and selectivity for the analysis of both unknown and known components, making it more suitable for practical production applications. Attached Figure Description

[0056] Appendix Figure 1 Comparison of fingerprint patterns

[0057] Appendix Figure 2 Characteristic chromatograms of the first batch of formulations

[0058] Appendix Figure 3 Characteristic chromatograms of the second batch of formulations

[0059] Appendix Figure 4 Characteristic chromatograms of the third batch of formulations

[0060] Appendix Figure 5 Linear relationship between polygalactoside and emodin

[0061] Appendix Figure 6 Characteristic Peak Identification - Astragalus

[0062] Appendix Figure 7 Characteristic Peak Identification - Polygonatum

[0063] Appendix Figure 8 Characteristic Peak Identification - Polygonatum odoratum

[0064] Appendix Figure 9 Characteristic Peak Identification - Curcuma zedoaria

[0065] Appendix Figure 10 Characteristic Peak Identification - Xu Changqing

[0066] Appendix Figure 11 Characteristic Peak Identification - Polygonum cuspidatum

[0067] Appendix Figure 12 Characteristic Peak Identification - Centella Asiatica Detailed Implementation

[0068] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0069] The features and performance of the present invention will be further described in detail below with reference to embodiments:

[0070] The fingerprint pattern construction method of the Jingqi Huazhuo preparation includes the following steps:

[0071] (1) Types of mobile phases: Methanol is used as mobile phase A, and 0.1% phosphoric acid is used as mobile phase B.

[0072] (2) The chromatographic column is: C18, 4.6 mm × 250 mm, 5 μm.

[0073] (3) Flow rate: 1.0 ml / min

[0074] (4) Column temperature: 30℃

[0075] (5) Detection wavelengths: fingerprint spectrum 210nm; polygalactoside and emodin 260nm

[0076] (6) The gradient elution ratio is:

[0077]

[0078] (7) Preparation of test solution: Take 3g of the powder, add 25ml of methanol, sonicate for 30min, filter, and take the filtrate to obtain the test solution.

[0079] (8) Preparation method of reference solution: Take an appropriate amount of glutinin reference standard, accurately weigh it, add methanol to prepare a solution containing 0.1 mg per 1 ml, shake well, and the solution is obtained.

[0080] (9) Preparation of each negative test sample: Take 3g of each negative sample lacking Astragalus membranaceus, Polygonatum sibiricum, Polygonatum odoratum, Curcuma zedoaria, Cynanchum paniculatum, Polygonum cuspidatum, and Centella asiatica, and prepare them in the same way as the test sample solution.

[0081] (10) Preparation of each single herb test sample: Take 0.5-2g of Astragalus membranaceus, Polygonatum sibiricum, Polygonatum odoratum, Curcuma zedoaria, Cynanchum paniculatum, Polygonum cuspidatum and Centella asiatica and prepare them in the same way as the test sample solution.

[0082] (11) Preparation of mixed reference solution: Take appropriate amounts of verbenafil isoflavone glucoside reference standard, senna glycoside reference standard and emodin reference standard, accurately weigh them, add methanol to prepare a solution containing 40 μg, 0.1 mg and 20 μg per ml respectively.

[0083] (12) Determination method: Accurately pipette 10 μl of each of the reference solution, mixed reference solution and test solution into the liquid chromatograph, determine and record the chromatogram for 120 minutes.

[0084] The fingerprint chromatogram of the test sample should show 12 chromatographic peaks corresponding to the reference fingerprint chromatogram. Among them, the retention time of chromatographic peak No. 3 corresponds to that of the reference peak (S peak). The relative retention time of each characteristic peak and the S peak should be within 10% of the specified value. The specified values ​​are 0.41 (peak 1), 0.66 (peak 2), 1.00 (peak 3), 1.04 (peak 4), 1.18 (peak 5), 1.20 (peak 6), 1.28 (peak 7), 1.38 (peak 8), 1.61 (peak 9), 1.84 (peak 10), 1.96 (peak 11), and 2.34 (peak 12).

[0085] The contents of polygalactoside and emodin in the test sample were calculated using the external standard method.

[0086] According to the appendix Figure 1 The following table of chromatographic peak assignments was obtained by comparing the fingerprint spectrum.

[0087] Chromatographic Peak Attribution Table

[0088]

[0089]

[0090] Example 1: Fingerprint Spectrum Determination

[0091] Fingerprint spectroscopy was performed on three consecutive batches of samples according to the detection method determined in the above research. The results are attached. Figure 2-4 .

[0092] Table 13 Retention times of chromatographic peaks in formulations

[0093]

[0094] Example 2 Content Determination

[0095] The content of three consecutive batches of samples was determined according to the detection method established in the above research, and the results are shown in the table below.

[0096] Table 23 Results of Content Determination in Preparations

[0097]

[0098]

[0099] Example 3 Linear

[0100] Preparation of Polygonin stock solution: Take an appropriate amount of Polygonin reference standard, accurately weigh 19.13 mg (batch number: 111575-201603, content: 87.3%), dissolve and dilute in methanol to a 25 ml volumetric flask, shake well, and the concentration is 668.02 μg / ml. This solution is used as the linearity 7 reference solution for detection.

[0101] Preparation of emodin stock solution 1: Accurately weigh an appropriate amount of emodin reference standard: 22.72 mg (batch number: 110756-201913, content: 96.0%), dissolve and dilute in methanol to a 25 ml volumetric flask, and shake well to obtain a concentration of 872.45 μg / ml. Preparation of emodin stock solution 2: Accurately transfer 5 ml of emodin stock solution 1, dilute with methanol to a 25 ml volumetric flask, and shake well to obtain a concentration of 174.49 μg / ml.

[0102] Preparation of the linear 1 mixed reference solution: Accurately transfer 3 ml of styrax glycoside stock solution and 2 ml of emodin stock solution, dilute with methanol to a 10 ml volumetric flask, and shake well to obtain the solutions with concentrations of 200.41 μg / ml and 52.35 μg / ml, respectively.

[0103] Preparation of Linear 2 Mixed Reference Solution: Accurately transfer 3 ml of Linear 1 Mixed Reference Solution, dilute with methanol to a 5 ml volumetric flask, and shake well to obtain the solutions with concentrations of 120.24 μg / ml and 31.41 μg / ml, respectively.

[0104] Preparation of the linear 3 mixed reference solution: Accurately transfer 2 ml of the linear 1 mixed reference solution, dilute it with methanol to a 5 ml volumetric flask, and shake well to obtain the concentrations of 80.16 μg / ml and 20.94 μg / ml, respectively.

[0105] Preparation of Linear 4 Mixed Reference Solution: Accurately transfer 1 ml of Linear 1 Mixed Reference Solution into a 5 ml volumetric flask, dilute with methanol, and shake well to obtain the solutions with concentrations of 40.08 μg / ml and 10.47 μg / ml, respectively.

[0106] Preparation of linear 5 mixed reference solution: Accurately transfer 1 ml of linear 2 mixed reference solution, dilute with methanol to a 5 ml volumetric flask, and shake well to obtain the concentrations of 24.05 μg / ml and 6.28 μg / ml, respectively.

[0107] Preparation of linear 6 mixed reference solution: Accurately transfer 1 ml of linear 2 mixed reference solution, dilute with methanol to a 10 ml volumetric flask, and shake well to obtain the concentrations of 12.02 μg / ml and 3.14 μg / ml, respectively.

[0108] The mixed reference solutions of linearity 1–6 and linearity 7 of polydipsia were each injected in 10 μl for detection. Linearity was calculated with concentration on the x-axis and peak area on the y-axis. The results are shown in the figure and table below. Polydipsia showed a good linear relationship between concentration and peak area in the concentration range of 12.02–668.02 μg / ml; emodin showed a good linear relationship between concentration and peak area in the concentration range of 3.14–52.35 μg / ml. The linearity graphs for polydipsia and emodin are attached. Figure 5 .

[0109] Table 3. Results of linear relationship investigation of polygalactoside and emodin

[0110]

[0111] Example 4 Accuracy

[0112] The stock solution (concentration 668.02 μg / ml) was used to investigate the linear relationship between the stock solution and the polydipsia glycoside.

[0113] Preparation of emodin stock solution 2: Accurately transfer 5 ml of emodin stock solution 1 (see linearity study, concentration: 872.45 μg / ml), dilute with methanol to a 25 ml volumetric flask, and shake well to obtain a concentration of 174.49 μg / ml.

[0114] Take approximately 1g of the same Jingqi Huazhuo Granules sample (batch number: 20231001), accurately weigh it, and accurately add 2ml of the above-mentioned polydextrin stock solution and 21ml of emodin stock solution. Perform six parallel applications, accurately add 50ml of 70% ethanol, seal tightly, weigh, sonicate for 30min, cool, weigh again, replenish the lost weight with 70% ethanol, shake well, filter, accurately take 25ml of the filtrate, evaporate to dryness, dissolve the residue in 70% ethanol in a 10ml volumetric flask, shake well, and filter to obtain the final product. Inject and analyze the samples separately. The results are shown in the table below. The recovery rate of polydextrin ranged from 97.10% to 100.38%, with an average recovery rate of 100.49% and an RSD of 3.35%. The recovery rate of emodin ranged from 96.42% to 101.20%, with an average recovery rate of 99.70% and an RSD of 1.79%, indicating that the method has good accuracy.

[0115] Table 4 Results of the Recovery Rate Study

[0116]

[0117] Recovery rate calculation formula

[0118]

[0119] The above results show that the recovery rates of polygalactoside in each sample were between 90% and 108%, and the recovery rates of emodin were between 85% and 110%, which meet the requirements of the pharmacopoeia.

[0120] Based on the above research, this method can be used to determine the content of polygalactoside and emodin in Jingqi Huazhuo granules.

[0121] Example 5 Precision

[0122] A test solution was prepared according to the test sample preparation method. Five consecutive injections were performed for detection, and chromatograms were collected. Using polygalactosin as a reference peak, the relative retention times and relative peak areas of 12 characteristic peaks were calculated. The RSDs of the relative retention times were all less than 0.05%, and the RSDs of the relative peak areas were all less than 1%. Similarity analysis was performed on the chromatographic peaks from 5 to 110 min, with a time window width set at 0.5 min. The similarity results were all greater than 0.99, indicating that the instrument has good precision.

[0123] Table 5. Calculation results of relative retention time and relative peak area for precision.

[0124]

[0125] Example 6 Stability

[0126] A test solution was prepared according to the test solution preparation method. The solution was injected and analyzed at 0, 2, 4, 6, 8, 12, 16, 20, 24, 36, and 48 hours. Chromatographic images were collected, and the relative retention time and relative peak area of ​​the 12 characteristic peaks were calculated using polygalactosin as a reference peak. The RSD of the relative retention time was less than 0.1%, and the RSD of the relative peak area was less than 7%. Similarity analysis was performed on the chromatographic peaks from 5 to 110 minutes, with a time window width of 0.5 minutes. The similarity results were all greater than 0.99. This indicates that the test solution is basically stable within 48 hours.

[0127] Table 6. Calculation results of relative retention time and relative peak area for stability.

[0128]

[0129]

[0130] Example 7 Repeatability

[0131] Six parallel aliquots of the same sample were prepared according to the test sample preparation method. Each aliquot was injected and analyzed, and chromatograms were collected. Using polygalactosin as a reference peak, the relative retention times and relative peak areas of the 12 characteristic peaks were calculated. The results are shown in the table below. The RSD of the relative retention times was less than 0.2%, and the RSD of the relative peak areas was less than 8%. Similarity analysis was performed on the chromatographic peaks from 5 to 110 min, with a time window width of 0.5 min. The similarity results were all greater than 0.99, indicating that the method has good repeatability.

[0132] Table 7. Calculation results of relative retention time and relative peak area for repeatability.

[0133]

[0134]

[0135] Chromatogram is attached. Figures 6-12 .

[0136] All aspects not described in detail in this invention are common knowledge that can be readily understood by those skilled in the art. Although the invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which are obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of this invention fall within the scope of protection claimed by this invention.

Claims

1. A method for constructing a fingerprint of a Jingqi Huazhuo preparation, characterized in that, The formula for the Jingqi Huazhuo preparation is made from seven traditional Chinese medicines: Astragalus membranaceus, Polygonatum sibiricum, Polygonatum odoratum, Curcuma zedoaria, Cynanchum paniculatum, Polygonum cuspidatum, and Centella asiatica, through extraction and purification. The construction method includes the following steps: (1) The test solution containing the scutellaria baicalensis preparation was detected by high performance liquid chromatography, and the detection results were used to generate a fingerprint spectrum. The test solution was prepared by taking 3g of scutellaria baicalensis preparation powder, adding 25ml of methanol, sonicating for 30min, filtering, and taking the filtrate to obtain the test solution. (2) Prepare a reference solution and perform high performance liquid chromatography on the reference solution to determine the peak assignment in the test solution based on the retention time of the reference solution; the reference solution includes polysaccharide, verrucoside, and emodin; (3) The high-performance liquid chromatography (HPLC) detection uses an octadecylsilane-bonded silica column with dimensions of 4.6 mm × 250 mm and 5 μm; the detection wavelengths are 210 nm and 260 nm; the injection volume is 5–20 μL; the flow rate of the mobile phase is 0.9–1.1 ml / min; mobile phase A is methanol, and mobile phase B is a 0.1% aqueous solution of phosphoric acid; gradient elution is performed using mobile phase A and mobile phase B under the following conditions: The percentages in the table represent the percentage of each mobile phase in the total volume of the mobile phase.

2. The construction method according to claim 1, characterized in that, In the high-performance liquid chromatography detection, the column temperature is 30℃.

3. The construction method according to claim 2, characterized in that, The flow rate is 1.0 mL / min.

4. The construction method according to claim 3, characterized in that, The injection volume for the high-performance liquid chromatography is 10 μL.

5. The application of the fingerprint spectrum of the astragalus-based turbidity-resolving preparation according to claim 1 as a standard fingerprint spectrum in the detection and quality control of the astragalus-based turbidity-resolving preparation.

Images