阅读说明：本技术 一种三叶青药材中山柰酚-3-o-芸香糖苷含量的测定方法 (Method for determining content of kaempferol-3-O-rutinoside in radix tetrastigme medicinal material ) 是由 李洪玉 张红心 李礼斌 王娜妮 许平翠 寿旦 于 2020-04-24 设计创作，主要内容包括：本发明提供了一种三叶青药材中山柰酚-3-O-芸香糖苷含量的测定方法,属于化学检测技术领域。本发明使用高效液相色谱法对三叶青药材中山柰酚-3-O-芸香糖苷的含量进行测定,取样量小、提取溶剂用量少、提取溶液不需要浓缩,测定方法简便快速且重复性好,能够对三叶青药材的质量进行准确评价。实施例结果表明,本发明方法所得谱图山柰酚-3-O-芸香糖苷峰与其他峰的分离度大于1.5,说明其分离度好；在线性关系试验中,本发明测定方法进样量在0.004～0.12μg范围内线性关系良好；在重复性试验中,本发明测定方法山柰酚-3-O-芸香糖苷含量的相对标准偏差为2.76％,说明本发明方法重复性良好。(The invention provides a method for determining the content of kaempferol-3-O-rutinoside in a radix tetrastigme medicinal material, and belongs to the technical field of chemical detection. The method uses the high performance liquid chromatography to determine the content of kaempferol-3-O-rutinoside in the radix tetrastigme medicinal material, has the advantages of small sample amount, small using amount of extraction solvent, no need of concentrating the extraction solution, simple, convenient and quick determination method and good repeatability, and can accurately evaluate the quality of the radix tetrastigme medicinal material. The example results show that the separation degree of the kaempferol-3-O-rutinoside peak and other peaks obtained by the method is more than 1.5, which indicates that the separation degree is good; in a linear relation test, the linear relation of the sample introduction amount of the measuring method is good within the range of 0.004-0.12 mu g; in a repeatability test, the relative standard deviation of the content of the kaempferol-3-O-rutinoside in the determination method is 2.76%, which shows that the repeatability of the method is good.)

1. A method for determining the content of kaempferol-3-O-rutinoside in radix tetrastigme medicinal materials is characterized by comprising the following steps:

(1) mixing radix tetrastigme powder with methanol, and sequentially performing ultrasonic extraction and microfiltration to obtain a filtrate as a test solution, wherein the mass of the radix tetrastigme powder is 0.11-0.22 g, and the volume of the methanol is 5-10 m L;

(2) sampling the test solution to perform high performance liquid chromatography test to obtain a test result of the test solution;

the conditions of the high performance liquid chromatography test comprise:

the chromatographic column is CAPCE LL PAK C18, the length of the chromatographic column is 250mm, the diameter of the chromatographic column is 4.6mm, and the particle size of the filler is 5 mu m;

the column temperature of the high performance liquid chromatography is 30 ℃;

the mobile phase A of the high performance liquid chromatography is acetonitrile, the mobile phase B is pure water, and the flow rate of the mobile phases A and B is 1.0m L/min;

the elution mode of the mobile phase is gradient elution, and the procedure of the gradient elution is as follows:

at 0min, the volume ratio of the mobile phase A to the mobile phase B is 15: 85 parts by weight;

at 30min, the volume ratio of the mobile phase A to the mobile phase B is 30: 70;

(3) obtaining the theoretical sample amount of the test sample solution according to a preset standard curve and the test result of the liquid chromatogram of the test sample obtained in the step (2), wherein the standard curve is a linear relation curve between the sample amount of the kaempferol-3-O-rutinoside reference sample and the peak area of the liquid chromatogram;

(4) and (3) obtaining the content of kaempferol-3-O-rutinoside in the radix tetrastigme medicinal material according to the theoretical sample amount obtained in the step (3) and the actual sample amount obtained in the step (2).

2. The method according to claim 1, wherein the radix tetrastigme powder has a particle size of 40 mesh or more.

3. The method according to claim 1, wherein the power of the ultrasonic extraction is 300 to 400W, the frequency is 40kHz, and the time is 50 to 60 min.

4. An assay method as claimed in claim 1, wherein the microfiltration process is microporous membrane filtration.

5. The method according to claim 4, wherein the microporous membrane for microfiltration is an alcohol-soluble microporous membrane having a pore size of 0.45 μm.

6. The method according to claim 1, wherein the sample size of the HPLC test is 5 to 10 μ L.

7. The assay method according to claim 1, wherein the detector of the high performance liquid chromatography test is an ultraviolet-visible light detector; the detection wavelength of the detector is 350 nm.

Technical Field

The invention relates to the technical field of production of medicine intermediates, and particularly relates to a method for determining the content of kaempferol-3-O-rutinoside in a radix tetrastigme medicinal material.

Background

Radix tetrastigme (Tetrastigma hemsleyanum Diels et Gilg) is a perennial evergreen vine of Tetrastigma hemsleyanum in Vitaceae and is a unique rare medicinal plant in China. Radix Apioris Fortunei has effects of clearing heat and detoxicating, relieving swelling and pain, eliminating phlegm and resolving hard mass, and can be used for treating febrile convulsion, pneumonia, hepatitis, venomous snake bite, etc. Radix Apioris Fortunei is mainly distributed in Zhejiang, Jiangxi, Fujian, Hunan, Guangxi, Guizhou, etc.

At present, the quality control component of the radix tetrastigme medicinal material is the flavonoid substance in the radix tetrastigme, when the high performance liquid chromatography is used for measuring the flavonoid substance in the radix tetrastigme, the variety of the flavonoid substance in the radix tetrastigme is more, the required sample amount is 3.0-4.0 g, the dosage of an extraction solvent is more, 40-50 m L, the solution needs to be concentrated after extraction, and the measuring method is more complicated.

Disclosure of Invention

In view of the above, the invention aims to provide a method for determining the content of kaempferol-3-O-rutinoside in a radix tetrastigme medicinal material. The method has the advantages that the kaempferol-3-O-rutinoside in the radix tetrastigme medicinal material is subjected to high performance liquid chromatography test, the sampling amount is small, the using amount of an extraction solvent is small, the method is simple, and the quality of the radix tetrastigme medicinal material can be accurately evaluated.

In order to achieve the purpose of the invention, the invention provides the following technical scheme:

the invention provides a method for determining the content of kaempferol-3-O-rutinoside in a radix tetrastigme medicinal material, which comprises the following steps:

(1) mixing radix tetrastigme powder with methanol, and sequentially performing ultrasonic extraction and microfiltration to obtain a filtrate as a test solution, wherein the mass of the radix tetrastigme powder is 0.11-0.22 g, and the volume of the methanol is 5-10 m L;

(2) sampling the test solution to perform high performance liquid chromatography test to obtain a test result of the test solution;

the conditions of the high performance liquid chromatography test comprise:

the chromatographic column is CAPCE LL PAK C18, the length of the chromatographic column is 250mm, the diameter of the chromatographic column is 4.6mm, and the particle size of the filler is 5 mu m;

the column temperature of the high performance liquid chromatography is 30 ℃;

the mobile phase A of the high performance liquid chromatography is acetonitrile, the mobile phase B is pure water, and the flow rate of the mobile phases A and B is 1.0m L/min;

the elution mode of the mobile phase is gradient elution, and the procedure of the gradient elution is as follows:

at 0min, the volume ratio of the mobile phase A to the mobile phase B is 15: 85 parts by weight;

at 30min, the volume ratio of the mobile phase A to the mobile phase B is 30: 70;

(3) obtaining the theoretical sample amount of the test sample solution according to a preset standard curve and the test result of the liquid chromatogram of the test sample obtained in the step (2), wherein the standard curve is a linear relation curve between the sample amount of the kaempferol-3-O-rutinoside reference sample and the peak area of the liquid chromatogram;

(4) and (3) obtaining the content of kaempferol-3-O-rutinoside in the radix tetrastigme medicinal material according to the theoretical sample amount obtained in the step (3) and the actual sample amount obtained in the step (2).

Preferably, the particle size of the radix tetrastigme powder is more than or equal to 40 meshes.

Preferably, the power of ultrasonic extraction is 300-400W, the frequency is 40kHz, and the time is 50-60 min.

Preferably, the microfiltration method is microporous membrane filtration.

Preferably, the microporous membrane for microfiltration is an alcohol-soluble microporous membrane, and the pore diameter is 0.45 μm.

Preferably, the sample injection amount of the high performance liquid chromatography test is 5-10 mu L.

Preferably, the detector for the high performance liquid chromatography test is an ultraviolet visible light detector; the detection wavelength of the detector is 350 nm.

The invention provides a method for measuring the content of kaempferol-3-O-rutinoside in a radix tetrastigme medicinal material, wherein the kaempferol-3-O-rutinoside has higher content in flavonoids of the radix tetrastigme medicinal material and is an active component with antioxidant, anti-inflammatory and anti-tumor effects of the radix tetrastigme. The method uses the kaempferol-3-O-rutinoside in the radix tetrastigme medicinal material as a quality control component, and measures the content of the kaempferol-3-O-rutinoside in the radix tetrastigme medicinal material by high performance liquid chromatography, so that the method has the advantages of small sampling amount, small using amount of an extraction solvent, no need of concentrating the extraction solution, simple, convenient and quick measurement method, good repeatability and capability of accurately evaluating the quality of the radix tetrastigme medicinal material. The example results show that the separation degree of the kaempferol-3-O-rutinoside peak and other peaks obtained by the method is more than 1.5, which indicates that the separation degree is good; in a linear relation test, the linear relation of the sample introduction amount of the measuring method is good within the range of 0.004-0.12 mu g; in a repeatability test, the relative standard deviation of the content of the kaempferol-3-O-rutinoside in the determination method is 2.76%, which shows that the repeatability of the method is good.

Drawings

FIG. 1 is an HP L C profile of a 1 μ L (0.004mg/m L) control solution;

FIG. 2 is an HP L C profile of a 3 μ L (0.004mg/m L) control solution;

FIG. 3 is an HP L C profile of a 5 μ L (0.004mg/m L) control solution;

FIG. 4 is an HP L C profile of a 7 μ L (0.004mg/m L) control solution;

FIG. 5 is an HP L C profile of a 10 μ L (0.004mg/m L) control solution;

FIG. 6 is an HP L C profile of a 3 μ L (0.04mg/m L) control solution;

FIG. 7 is a standard curve of a control solution;

FIG. 8 is an HP L C spectrum of the test solution of example 1;

FIG. 9 is an HP L C spectrum of the test solution of example 2;

FIG. 10 is an HP L C spectrum of the test solution of example 3;

FIG. 11 is a HP L C spectrum of the test solution of example 4;

FIG. 12 is a HP L C profile of the test solution of example 5.

Detailed Description

The invention provides a method for determining the content of kaempferol-3-O-rutinoside in a radix tetrastigme medicinal material, which comprises the following steps:

(1) mixing radix tetrastigme powder with methanol, and sequentially performing ultrasonic extraction and microfiltration to obtain a filtrate as a test solution, wherein the mass of the radix tetrastigme powder is 0.11-0.22 g, and the volume of the methanol is 5-10 m L;

(2) sampling the test solution to perform high performance liquid chromatography test to obtain a test result of the test solution;

the conditions of the high performance liquid chromatography test comprise:

the chromatographic column is CAPCE LL PAK C18, the length of the chromatographic column is 250mm, the diameter of the chromatographic column is 4.6mm, and the particle size of the filler is 5 mu m;

the column temperature of the high performance liquid chromatography is 30 ℃;

the mobile phase A of the high performance liquid chromatography is acetonitrile, the mobile phase B is pure water, and the flow rate of the mobile phase is 1.0m L/min;

the elution mode of the mobile phase is gradient elution, and the procedure of the gradient elution is as follows:

at 0min, the volume ratio of the mobile phase A to the mobile phase B is 15: 85 parts by weight;

at 30min, the volume ratio of the mobile phase A to the mobile phase B is 30: 70;

(3) obtaining the theoretical sample amount of the test sample solution according to a preset standard curve and the test result of the liquid chromatogram of the test sample obtained in the step (2), wherein the standard curve is a linear relation curve between the sample amount of the kaempferol-3-O-rutinoside reference sample and the peak area of the liquid chromatogram;

(4) and (3) obtaining the content of kaempferol-3-O-rutinoside in the radix tetrastigme medicinal material according to the theoretical sample amount obtained in the step (3) and the actual sample amount obtained in the step (2).

The radix tetrastigme powder is mixed with methanol, ultrasonic extraction and microfiltration are sequentially carried out, and filtrate obtained by microfiltration is test solution. In the present invention, the preparation method of the radix tetrastigme powder preferably comprises the following steps:

sequentially pulverizing and sieving radix Apioris Fortunei to obtain radix Apioris Fortunei powder.

The present invention does not require any particular way of comminution, as is well known to those skilled in the art. In the invention, the screening is preferably carried out by a 40-60-mesh screen, and the particle size of the radix tetrastigme powder is preferably not less than 40 meshes, and more preferably not less than 60 meshes.

In the invention, the mass of the radix tetrastigme powder is 0.11-0.22 g, preferably 0.2g, the volume of the methanol is 5-10 m L, preferably 10m L, the invention has no special requirement on the mixing mode of the radix tetrastigme powder and the methanol, and the mixing mode known by a person skilled in the art can be used, specifically stirring and mixing are adopted.

In the present invention, the filtration mode is preferably microporous membrane filtration, the microporous membrane is preferably alcohol-soluble microporous membrane, and the pore diameter of the microporous membrane is preferably 0.45 μm.

The filtrate obtained by the method does not need to be concentrated, and the filtrate is directly used as a sample solution for sample injection.

After a test solution is obtained, the test solution is sampled and subjected to high performance liquid chromatography test, and a test result of the test solution is obtained, wherein the sampling amount of the high performance liquid chromatography test is preferably 5-10 mu L, and the conditions of the high performance liquid chromatography test comprise:

the chromatographic column is CAPCE LL PAK C18, the length of the chromatographic column is 250mm, the diameter of the chromatographic column is 4.6mm, and the particle size of the filler is 5 mu m;

the column temperature of the high performance liquid chromatography is 30 ℃;

the mobile phase A of the high performance liquid chromatography is acetonitrile, the mobile phase B is pure water, and the flow rate of the mobile phase is 1.0m L/min;

the elution mode of the mobile phase is gradient elution, and the procedure of the gradient elution is as follows:

at 0min, the volume ratio of the mobile phase A to the mobile phase B is 15: 85 parts by weight;

at 30min, the volume ratio of the mobile phase A to the mobile phase B is 30: 70.

in the present invention, the detector for high performance liquid chromatography is preferably an ultraviolet-visible light detector; the detection wavelength of the detector is preferably 350 nm. According to the invention, through screening the detection wavelength and setting the elution program, the kaempferol-3-O-rutinoside component can be well separated without adding acid in the elution phase during high performance liquid chromatography separation.

After a test result of the liquid chromatogram of the test sample is obtained, the theoretical sample amount of the test sample solution is obtained according to a preset standard curve and the test result of the liquid chromatogram of the test sample obtained in the step (2), and the standard curve is a linear relation curve between the sample amount of the kaempferol-3-O-rutinoside reference sample and the peak area of the liquid chromatogram.

In the present invention, the standard curve is preferably obtained by:

providing a kaempferol-3-O-rutinoside reference solution;

performing high performance liquid chromatography analysis on the reference substance solutions with different sample volumes to obtain a reference substance liquid chromatogram and a kaempferol-3-O-rutinoside peak area;

and drawing a standard curve by taking the peak area as a vertical coordinate and the sample amount of the kaempferol-3-O-rutinoside as a horizontal coordinate.

In the present invention, the concentration of the control solution is preferably 0.004mg/m L. in the present invention, the providing of the kaempferol-3-O-rutinoside control solution preferably comprises the steps of:

weighing 1.6mg of kaempferol-3-O-rutinoside reference substance, adding methanol to dissolve to a constant volume of 1m L, putting 0.25m L into a 10m L volumetric flask, adding methanol to a constant volume, taking 1m L solution to a 10m L volumetric flask, adding methanol to a constant volume, and obtaining the kaempferol-3-O-rutinoside reference substance solution with the concentration of 0.004mg/m L.

In the present invention, when performing high performance liquid chromatography analysis on different sample volumes of control solutions, the sample volumes of the selected control solutions are preferably 1 μ L (concentration of 0.004mg/m L), 3 μ L0 (concentration of 0.004mg/m L1), 5 μ L (concentration of 0.004mg/m L), 7 μ L (concentration of 0.004mg/m L), 10 μ L (concentration of 0.004mg/m L) and 3 μ L (concentration of 0.04mg/m L), and the conditions, types and detection wavelengths of the high performance liquid chromatography test on the control solutions are the same as above, and are not described herein again.

After a reference substance liquid chromatogram and a kaempferol-3-O-rutinoside peak area are obtained, the method takes the peak area as a vertical coordinate and the kaempferol-3-O-rutinoside sample injection amount (mu g) as a horizontal coordinate to draw a standard curve. The method for drawing the standard curve has no special requirement, and the method for drawing the standard curve, which is well known to those skilled in the art, can be used. In the invention, the sampling quantity is positively correlated with the peak area of kaempferol-3-O-rutinoside. As a specific example of the present invention, the standard curve is Y-175.74 x + 2047.3.

After a standard curve is obtained, according to the liquid chromatogram of the test sample, the peak area of the kaempferol-3-O-rutinoside and the standard curve, the sample injection quality (mu g) of the kaempferol-3-O-rutinoside in the test sample solution is obtained; after the quality sample amount of the kaempferol-3-O-rutinoside in the test solution is obtained, calculating according to a formula I to obtain the mass concentration of the kaempferol-3-O-rutinoside in the test solution;

in the formula I, C_{Test article}The mass concentration of kaempferol-3-O-rutinoside in the test solution is mg/m L;

m_{sample introduction}kaempferol-3-O-rutinoside as test solutionThe sample injection mass of (1) mg;

V_{sample introduction}M L, the sample injection volume of the test solution;

after the mass concentration of the test solution is obtained, the mass content of kaempferol-3-O-rutinoside in the radix tetrastigme powder is calculated according to the formula II:

in the formula II, A is the mass percentage of kaempferol-3-O-rutinoside in the radix tetrastigme powder;

C_{test article}The mass concentration of kaempferol-3-O-rutinoside in the test solution is mg/m L;

V_{test article}M L, the volume of the test solution;

m_{radix tetrastigme}Is the mass of radix tetrastigme powder, mg.

The method uses the high performance liquid chromatography to determine the content of kaempferol-3-O-rutinoside in the radix tetrastigme medicinal material, has the advantages of small sample amount, small using amount of extraction solvent, no need of concentrating the extraction solution, simple, convenient and quick determination method and good repeatability, and can accurately evaluate the quality of the radix tetrastigme medicinal material.

The method for determining the kaempferol-3-O-rutinoside content in Hevea triloba medicinal materials provided by the present invention is described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.