Detection method of selfheal granules

Abstract

The invention relates to a detection method of traditional Chinese medicine components, in particular to a detection method of selfheal granules. Measuring the content of rosmarinic acid serving as an effective component in the selfheal granules by adopting an ultra-high performance liquid chromatography; the method comprises the following steps: (1) preparing a to-be-detected sample solution, namely precisely weighing a selfheal particle sample, dissolving the selfheal particle sample into an organic solvent A, weighing, performing ultrasonic treatment, uniformly shaking and filtering the solution subjected to ultrasonic treatment for later use; (2) preparation of a reference substance solution: precisely weighing a rosmarinic acid reference substance, and dissolving the rosmarinic acid reference substance with an organic solvent B to prepare the rosmarinic acid reference substance solution; (4) liquid chromatographic analysis: detecting and analyzing the reference substance solution and the to-be-detected sample solution by using an ultra-performance liquid chromatograph; and (5) result analysis: establishing a standard curve and verifying the method. According to the detection method provided by the invention, a better separation degree can be obtained, the analysis time is greatly shortened, and the analysis efficiency is improved.

Description

Technical Field

[0001] The invention relates to a method for detecting components of traditional Chinese medicine, and in particular to a method for detecting herba prunellae granules. Background Art

[0002] Prunella vulgaris L. is a perennial herbaceous plant of the Lamiaceae family and the genus Prunella, mainly produced in Jiangsu, Zhejiang, Anhui, Henan and other places. Prunella vulgaris has a wide range of medical applications. The Chinese medicine granules prepared by Prunella vulgaris can be directly used in the prescription preparation of clinical Chinese medicine. Patients can directly take it according to the prescribed dosage. It is convenient to take, the dosage is small, the quality is controllable, it is easy to store and easy to carry, which makes its content determination method technology particularly important.

[0003] Modern pharmacological research shows that rosmarinic acid has multiple pharmacological activities such as antioxidant, antibacterial and anti-inflammatory, anti-tumor, anti-depression and anti-anxiety, improvement of kidney disease, and liver protection.

[0004] With the widespread use of Prunella vulgaris, its safety has also received widespread attention, and reports on its adverse reactions have gradually increased, such as skin itching, papules, and even fainting due to anaphylactic shock caused by taking Prunella vulgaris. Existing studies have shown that the content of rosmarinic acid in phenolic acids in Prunella vulgaris is relatively high, which affects the quality of Prunella vulgaris. Therefore, the study and construction of a new method for analyzing rosmarinic acid in Prunella vulgaris has important practical significance for ensuring the quality and safety of Prunella vulgaris. Chinese patent CN101862373B discloses a quality control method for Xiasangju granules, which uses high performance liquid chromatography to determine the content of rosmarinic acid, a characteristic component of Prunella vulgaris. However, its analysis time is long, which is not conducive to efficient operation. Summary of the invention

[0005] In order to solve the above technical problems, the first aspect of the present invention provides a method for detecting Prunella vulgaris granules, which uses ultra high performance liquid chromatography to determine the content of rosmarinic acid, an effective ingredient in Prunella vulgaris granules; the steps include:

[0006] (1) Preparation of sample solution: accurately weigh a sample of Prunella vulgaris granules, dissolve it in organic solvent A, weigh it, and then perform ultrasonic treatment. Shake the ultrasonically treated solution evenly, filter it, and set aside.

[0007] (2) Preparation of reference solution: accurately weigh rosmarinic acid reference substance and dissolve it in organic solvent B to prepare rosmarinic acid reference solution;

[0008] (4) Liquid chromatography analysis: using ultra-high performance liquid chromatography to detect and analyze the reference solution and the sample solution;

[0009] (5) Result analysis: Establish standard curve and verify the method.

[0010] In one embodiment, the organic solvent A and the organic solvent B both include any one of methanol and ethanol.

[0011] In a preferred embodiment, the organic solvent A and the organic solvent B are both ethanol.

[0012] In a more preferred embodiment, the ethanol is an ethanol aqueous solution with a mass concentration of 49.5-50.5%.

[0013] In one embodiment, the concentration of the rosmarinic acid reference solution is 0.1 mg / mL.

[0014] In one embodiment, the ultrasonic treatment is performed under the following conditions: ultrasonic power is 200-300 W, and ultrasonic frequency is 40-60 kHz.

[0015] In one embodiment, the ultrasonic treatment is carried out under the following conditions: the ultrasonic power is 250 W and the ultrasonic frequency is 40 kHz.

[0016] In one embodiment, the ultrasonic treatment time is 30-40 min, which can be 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, and 40 min.

[0017] In one embodiment, in step (1), the solution is weighed again after ultrasonic treatment, and the weight is supplemented with organic solvent A to ensure that the quality of the solution before and after ultrasonic treatment remains consistent.

[0018] In a preferred embodiment, the step (1) is specifically as follows: accurately weigh 0.2 g of a sample of Prunella vulgaris granules, dissolve it in 25 mL of organic solvent A, weigh it, and then perform ultrasonic treatment, weigh the ultrasonically treated solution again, and make up the weight with organic solvent A, ensure that the quality of the solution before and after ultrasonic treatment is consistent, then shake and filter it for later use;

[0019] In one embodiment, the liquid chromatography column has a size of 100 mm×2.1 mm×1.8 μm.

[0020] The small particle size chromatographic column used in the present invention, on the one hand, helps to increase the contact area between the sample and the stationary phase, thereby improving the separation efficiency; on the other hand, the surface area to volume ratio of the column increases, so that the number of theoretical plates per unit length (theoretical plate height) increases, which helps to improve the separation ability of the column.

[0021] In a preferred embodiment, the chromatographic column of the liquid chromatography is waters ACQUITY HSS T3.

[0022] In one embodiment, the mobile phase of the liquid chromatography comprises at least one of phosphoric acid and methanol.

[0023] In a preferred embodiment, the mobile phase of the liquid chromatography is methanol-0.1% phosphoric acid aqueous solution.

[0024] In one embodiment, the mass ratio of the 0.1% phosphoric acid aqueous solution to methanol is 58:42.

[0025] In one embodiment, the filler of the liquid chromatography is octadecylsilane bonded silica gel.

[0026] In one embodiment, the flow rate of the liquid chromatography is 0.3-1 mL / min, which can be 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, and 1 mL / min.

[0027] In one embodiment, the wavelength column temperature of the liquid chromatography is 330°C.

[0028] In one embodiment, the calculation formula of the test result of the sample solution to be tested is: i =C×25mL / 0.2g; where

[0029] Xi——the concentration of rosmarinic acid in the sample solution to be tested, in mg / g;

[0030] C——Concentration of rosmarinic acid obtained according to the standard curve, in mg / L.

[0031] Beneficial effects:

[0032] 1. The present invention adopts ultra high performance liquid chromatography to measure the content of rosmarinic acid, an effective ingredient in the herbaceous herb granules, and uses the measured rosmarinic acid content as the herbaceous herb index cost to evaluate the quality of the herbaceous herb, thereby replacing the existing high performance liquid chromatography detection method and improving the detection sensitivity and precision.

[0033] 2. The ultra-high performance liquid chromatograph of the present invention adopts a small-particle chromatographic column, specifically 100 mm×2.1 mm×1.8 μm, which can obtain better separation, greatly shorten the analysis time, and improve the analysis efficiency.

[0034] 3. The detection method provided by the present invention has selectivity, precision and accuracy that meet the requirements.

[0035] 4. The recovery rate of the detection method of Prunella Vulgaris granules established by the present invention is 99.6%-100.8%, and the RSD is 0.6%, which has good repeatability.

[0036] 5. The detection method of Prunella Vulgaris granules established in the present invention is simple, rapid and sensitive. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 It is the liquid chromatogram of Example 1.

[0039] Figure 2 It is the liquid chromatogram of Comparative Example 1. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the examples and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The experimental methods for which specific conditions are not specified in the examples are carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be purchased commercially.

[0041] Example 1

[0042] The first aspect of this embodiment provides a method for detecting Prunella vulgaris granules, which uses ultra-high performance liquid chromatography to determine the content of rosmarinic acid, an effective ingredient in Prunella vulgaris granules; the steps include:

[0043] (1) Preparation of the sample solution to be tested: accurately weigh 0.2 g of Prunella vulgaris granules, dissolve it in 25 mL of organic solvent A, weigh it, and then perform ultrasonic treatment. Weigh the ultrasonically treated solution again, and make up the weight with organic solvent A. Ensure that the quality of the solution before and after ultrasonic treatment is consistent, then shake well and filter it for later use;

[0044] (2) Preparation of reference solution: accurately weigh rosmarinic acid reference substance and dissolve it in organic solvent B to prepare rosmarinic acid reference solution;

[0045] (4) Liquid chromatography analysis: using ultra-high performance liquid chromatography to detect and analyze the reference solution and the sample solution;

[0046] (5) Result analysis: Establish standard curve and verify the method.

[0047] The organic solvent A and the organic solvent B are both ethanol aqueous solutions with a mass concentration of 50%.

[0048] The conditions of the ultrasonic treatment are: ultrasonic power is 250W, and ultrasonic frequency is 40kHz.

[0049] The ultrasonic treatment time is 30 min.

[0050] The concentration of the rosmarinic acid reference substance solution is 0.1 mg / mL.

[0051] The specification of the chromatographic column of the liquid chromatography is waters ACQUITY HSS T3 (100 mm×2.1 mm×1.8 μm).

[0052] The filler of the liquid chromatography is octadecylsilane bonded silica gel.

[0053] The mobile phase of the liquid chromatography is methanol-0.1% phosphoric acid aqueous solution. The mass ratio of the 0.1% phosphoric acid aqueous solution to methanol is 58:42.

[0054] The flow rate of the liquid chromatography was 0.3 mL / min.

[0055] The wavelength column temperature of the liquid chromatography is 330°C.

[0056] The calculation formula of the test result of the sample solution to be tested is: i =C×25mL / 0.2g; where

[0057] X i ——The concentration of rosmarinic acid in the sample solution to be tested, in mg / g;

[0058] C——Concentration of rosmarinic acid obtained according to the standard curve, in mg / L.

[0059] Figure 1 It is the liquid chromatogram of Example 1. It can be seen from the figure that the time used for analysis is 8 minutes.

[0060] The method selectivity, precision and accuracy of Example 1 were also tested:

[0061] Method selectivity: The sample solution and the reference solution of this example show the same chromatographic peak at the same retention time.

[0062] Method precision: Six samples were measured in parallel using the test method of Example 1, and the rsd of the test results was 0.6%, meeting the requirement of rsd≤3%.

[0063] Method accuracy: The test method of Example 1 was used to perform a spiked test, and the recovery rate of the test results was 99.6%-100.8%, meeting the requirement of a recovery rate of 90%-108%.

[0064] Comparative Example 1

[0065] The specific implementation of this comparative example 1 is the same as that of Example 1, except that the specification of the chromatographic column of the liquid chromatography is waters ACQUITY HSS T3 (250 mm×4.6 mm×5 μm).

[0066] Figure 2 It is the liquid chromatogram of comparative example 1. It can be seen from the figure that the time used for analysis is 18 minutes.

Claims

1. A method for detecting Prunella Vulgaris granules, characterized in that: The content of rosmarinic acid, an active ingredient in Prunella vulgaris granules, is determined by ultra-high performance liquid chromatography; the steps include: (1) Preparation of sample solution: accurately weigh a sample of Prunella vulgaris granules, dissolve it in organic solvent A, weigh it, and then perform ultrasonic treatment. Shake the ultrasonically treated solution evenly, filter it, and set aside. (2) Preparation of reference solution: accurately weigh rosmarinic acid reference substance and dissolve it in organic solvent B to prepare rosmarinic acid reference solution; (4) Liquid chromatography analysis: using ultra-high performance liquid chromatography to detect and analyze the reference solution and the sample solution; (5) Result analysis: Establish standard curve and verify the method.

2. The method for detecting Prunella Vulgaris granules according to claim 1, characterized in that: The organic solvent A and the organic solvent B both include any one of methanol and ethanol.

3. The method for detecting Prunella Vulgaris granules according to claim 1, characterized in that: The concentration of the rosmarinic acid reference substance solution is 0.1 mg / mL.

4. The method for detecting Prunella Vulgaris granules according to claim 1, characterized in that: The conditions of the ultrasonic treatment are: ultrasonic power is 200-300W, and ultrasonic frequency is 40-60kHz.

5. The method for detecting Prunella Vulgaris granules according to claim 4, characterized in that: The conditions of the ultrasonic treatment are: ultrasonic power is 250W, and ultrasonic frequency is 40kHz.

6. The method for detecting Prunella Vulgaris granules according to claim 5, characterized in that: The ultrasonic treatment time is 30-40 min.

7. The method for detecting Prunella Vulgaris granules according to claim 5, characterized in that: In the step (1), the solution is weighed again after the ultrasonic treatment, and the weight is supplemented with organic solvent A to ensure that the quality of the solution before and after the ultrasonic treatment remains consistent.

8. The method for detecting Prunella Vulgaris granules according to claim 1, characterized in that: The specifications of the chromatographic column of the liquid chromatography are 100 mm×2.1 mm×1.8 μm.

9. The method for detecting Prunella Vulgaris granules according to claim 8, characterized in that: The mobile phase of the liquid chromatography comprises at least one of phosphoric acid and methanol.

10. The method for detecting Prunella Vulgaris granules according to claim 9, characterized in that: The mass ratio of the phosphoric acid to methanol is 58:42.

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