阅读说明：本技术 一种蛇泡筋的表儿茶素的含量测定方法 (Method for measuring content of epicatechin in snake-soaked tendon ) 是由 李永辉 吴娇霞 吴毓皇 徐晗 李立言 于 2021-08-25 设计创作，主要内容包括：本发明公开了一种蛇泡筋的表儿茶素的含量测定方法,采用高效液相色谱法测定蛇泡筋中表儿茶素的含量,所述高效液相色谱条件为：色谱柱：Hanbonlichrospher C18；流动相：乙酸水-乙腈；流速：0.8-1.2ml/min；时间：10-15min；检测波长：280nm；柱温：35-42℃；进样量：0.002-0.012ml。本发明所述方法精密、快速,专属性强,对建立蛇泡筋的质量标准,奠定了理论基础和实验依据；对扩大蛇泡筋的药用资源,促进蛇泡筋产业化利用具有重要的意义。(The invention discloses a method for measuring the content of epicatechin in a snake-soaked tendon, which adopts a high performance liquid chromatography to measure the content of epicatechin in the snake-soaked tendon, wherein the conditions of the high performance liquid chromatography are as follows: a chromatographic column: hanbonlichospher C18; mobile phase: acetic acid water-acetonitrile; flow rate: 0.8-1.2 ml/min; time: 10-15 min; detection wavelength: 280 nm; column temperature: 35-42 ℃; sample introduction amount: 0.002-0.012 ml. The method is precise, rapid and high in specificity, and lays theoretical foundation and experimental basis for establishing the quality standard of the snake-soaked tendon; has important significance for expanding the medicinal resources of the snake-soaked tendon and promoting the industrialized utilization of the snake-soaked tendon.)

1. A method for measuring the content of epicatechin in the snake-soaking tendon is characterized by comprising the following steps: the method for measuring the epicatechin content in the snake-soaked tendon by adopting the high performance liquid chromatography specifically comprises the following steps:

(1) preparation of control solutions: dissolving epicatechin control in methanol to obtain control solution;

(2) preparation of a test solution: dissolving the powder of the root of Chinese goosebeery herb in ethanol to obtain a medicinal solution, weighing, carrying out ultrasonic treatment and cooling, and filtering to obtain a filtrate as a test solution;

(3) injecting the reference solution and the sample solution into a high performance liquid chromatograph, and detecting the content of epicatechin; the high performance liquid chromatography conditions are as follows:

a chromatographic column: hanbon lichrospher C18;

mobile phase: acetic acid water-acetonitrile;

flow rate: 0.8-1.2 ml/min;

time: 10-15 min;

detection wavelength: 280 nm;

column temperature: 35-42 ℃;

sample introduction amount: 0.002-0.012 ml.

2. The method for measuring the epicatechin content of the tendon Serpentis as set forth in claim 1, wherein: the epicatechin concentration in the reference solution in the step (1) is 0.064 mg/ml.

3. The method for measuring the epicatechin content of the tendon Serpentis as set forth in claim 1, wherein: and (3) crushing the root of the Chinese gooseberry twig in the step (2) and screening the crushed root of the Chinese gooseberry twig with a No. 2-4 sieve to prepare the powder of the root of the Chinese gooseberry twig, wherein the mass-volume ratio of the powder of the root of the Chinese gooseberry twig to the ethanol is 1g to 20-80 ml.

4. The method for measuring the epicatechin content of the tendon Serpentis as set forth in claim 1, wherein: the mass concentration of the ethanol is 30-70%.

5. The method for measuring the epicatechin content of the tendon Serpentis as set forth in claim 1, wherein: the ultrasonic treatment conditions are as follows: the ultrasonic power is 200-300W, the frequency is 30-50kHz, and the ultrasonic time is 10-30 min.

6. The method for measuring the epicatechin content of the tendon Serpentis as set forth in claim 1, wherein: and the weight determination is that after cooling, the ethanol is added to ensure that the weight of the medicinal material solution is equal to the weighed weight.

7. The method for measuring the epicatechin content of the tendon Serpentis as set forth in claim 1, wherein: and (3) the volume ratio of the acetic acid water to the acetonitrile in the mobile phase in the step (3) is 21: 4.

8. The method for measuring the epicatechin content of the tendon Serpentis as set forth in claim 1, wherein: the acetic acid water solution is 2% acetic acid water solution.

9. The method for measuring the epicatechin content of the tendon Serpentis as set forth in claim 1, wherein: the length of the chromatographic column is 250mm, and the inner diameter of the chromatographic column is 4.6 mm.

10. The method for measuring the epicatechin content of the tendon Serpentis as set forth in claim 1, wherein: in the step (3), the flow rate is 1.0ml/min, the time is 15min, the column temperature is 40 ℃, and the sample injection amount is 0.01 ml.

Technical Field

The application relates to the technical field of traditional Chinese medicines, in particular to a method for measuring the content of epicatechin of a snake tendon.

Background

The root of the snake-soaked tendon is the medicinal part of the snake-soaked tendon Rubus cochinchinensis, and is mainly distributed in low-altitude mountainous regions, hills and roadside forests in Hainan province and Guangdong province. It is slightly pungent, sweet, astringent and warm in taste. Has the functions of dispelling wind, relieving pain, dispersing pathogen accumulation and removing toxic material. Can be used for treating rheumatic lumbago, skelalgia, diabetes, intestinal gas, hemorrhoid, and eczema. The chemical components of the snake-soaked tendon are less researched, and the Rubus parvifoliusL of the same genus mainly contains tanning, flavonoid glycoside, saccharides, phenols and amino acid components. At present, no research report on a method for measuring the content of the snake-soaking tendon exists.

The content determination of epicatechin in other plants or medicinal materials is shown in CN110567984A, which discloses a detection method for evaluating the quality of a medicinal material of the root of a Ficus Nipponica, and discloses a content determination method for epicatechin in the medicinal material of the root of the Ficus Nipponica; CN103336069B discloses a high performance liquid phase determination method for phenolic compounds in peach fruits and also discloses a determination method for epicatechin in peach fruits. However, since different plants or herbs contain different components and contents, even the content measurement method of the same substance may be different depending on the influence of the interfering component and the content.

Disclosure of Invention

Therefore, the application provides a method for measuring the content of epicatechin in the snake-soaked tendon, so as to establish the quality standard of the snake-soaked tendon, expand the medicinal resources of the snake-soaked tendon and promote the industrialized utilization of the snake-soaked tendon.

The technical scheme of the application is realized as follows:

a method for measuring the content of epicatechin in the tendon of Snake, which adopts high performance liquid chromatography to measure the content of epicatechin in the tendon of Snake, comprises the following steps:

(1) preparation of control solutions: dissolving epicatechin control in methanol to obtain control solution; (2) preparation of a test solution: dissolving the powder of the root of Chinese goosebeery herb in ethanol to obtain a medicinal solution, weighing, carrying out ultrasonic treatment, cooling, weighing, and filtering, wherein the filtered filtrate is a test solution; effective components in the root medicinal material of the Chinese wampee rhizome can be fully extracted through ultrasonic treatment, and experimental errors are reduced; the application uses ethanol as the solvent of the test sample, and the main reasons are that the ethanol has better safety than methanol, high boiling point and less solvent volatilization in the extraction process.

(3) Injecting the reference solution and the sample solution into a high performance liquid chromatograph, and detecting the content of epicatechin; the high performance liquid chromatography conditions are as follows:

a chromatographic column: hanbon lichrospher C18250X 4.6 mm;

mobile phase: acetic acid water-acetonitrile;

flow rate: 0.8-1.2 ml/min;

time: 10-15 min;

detection wavelength: 280 nm;

column temperature: 35-42 ℃;

sample introduction amount: 0.002-0.012 ml.

The further technical proposal is that the epicatechin concentration in the reference substance solution in the step (1) is 0.064 mg/ml.

The further technical scheme is that the medicinal material of the root of the tendon dipped in the step (2) is crushed and sieved by a No. 2-4 sieve to prepare medicinal material powder of the root of the tendon dipped in the snake, and the mass volume ratio of the medicinal material powder of the root of the tendon dipped in the snake to the ethanol is 1g:20-80 ml.

The further technical scheme is that the mass concentration of the ethanol is 30-70%.

The further technical scheme is that the ultrasonic treatment conditions are as follows: the ultrasonic power is 200-300W, the frequency is 30-50kHz, and the ultrasonic time is 10-30 min.

The further technical scheme is that the weight determination is that after cooling, the ethanol is added to ensure that the weight of the medicinal material solution is equal to the weighed weight.

The further technical scheme is that the volume ratio of the acetic acid water to the acetonitrile in the flowing phase in the step (3) is 21: 4.

The further technical scheme is that the acetic acid water solution is an acetic acid water solution with the mass concentration of 2%.

The further technical scheme is that the length of the chromatographic column is 250mm, and the inner diameter of the chromatographic column is 4.6 mm.

The further technical scheme is that in the step (3), the flow rate is 1.0ml/min, the time is 15min, the column temperature is 40 ℃, and the sample injection amount is 0.01 ml.

Methanol and acetonitrile used in this application are both chromatographically pure, available from Fisher, usa.

Compared with the prior art, the beneficial effects of this application are:

(1) the method for measuring the content of epicatechin in the snake-soaked tendon lays a theoretical foundation and an experimental basis for establishing the quality standard of the snake-soaked tendon; has important significance for expanding the medicinal resources of the snake-soaked tendon and promoting the industrialized utilization of the snake-soaked tendon.

(2) The method for measuring the content of epicatechin in the soaking sinew is suitable for measuring the content of epicatechin in the soaking sinew in different producing areas, and the content range of epicatechin in the soaking sinew medicinal materials is 1.76-9.17 mg/g through measurement.

(3) According to the method for measuring the content of the epicatechin of the tendon immersed in the snake, the epicatechin is in a good linear relation within a range of 128-768ng, the average recovery rate of the method is 101.11%, the RSD is 2.95%, and the RSD of the method precision is less than 3%.

(4) According to the method for measuring the content of the epicatechin in the snake-soaking tendon, the epicatechin is well separated from a sample, and a solvent does not interfere with the sample.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only preferred embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise.

FIG. 1 is an HPLC chromatogram of a control of example 1 of the present application.

FIG. 2 is an HPLC chromatogram of the test article (lot No. 201906001) of example 1 of the present application.

FIG. 3 is a line graph of the linear range and detection limit of epicatechin control solutions of example 1 of the present application.

Detailed Description

For a better understanding of the present technology, specific examples are provided below, which are further described in conjunction with the accompanying drawings.

The No. 2, 3 or 4 sieve mentioned in the application is in R40/3 series according to the standard drug sieve specification of Chinese pharmacopoeia in 2015, the inner diameter of the sieve pore of the No. 2 sieve is 850 mu m +/-29 mu m, and the sieve pore is 25 meshes; the inner diameter of the mesh of the No. 3 sieve is 355 mu m +/-13 mu m, and the mesh number is 50 meshes; the screen hole inner diameter of the No. 4 screen is 250 mu m +/-9.9 mu m, and the mesh number is 65 meshes.

Example 1

A method for measuring the content of epicatechin in the tendon of Snake, which adopts high performance liquid chromatography to measure the content of epicatechin in the tendon of Snake, comprises the following steps:

(1) preparation of control solutions: dissolving epicatechin control in methanol to obtain control solution;

(2) preparation of a test solution: dissolving the powder of the root of Chinese goosebeery herb in ethanol to obtain a medicinal solution, weighing, carrying out ultrasonic treatment, cooling, weighing, and filtering, wherein the filtered filtrate is a test solution;

(3) injecting the reference solution and the sample solution into a high performance liquid chromatograph, and detecting the content of epicatechin; the high performance liquid chromatography conditions are as follows:

a chromatographic column: hanbon lichrospher C18250X 4.6 mm;

mobile phase: acetic acid water-acetonitrile;

flow rate: 1.0 ml/min;

time: 15 min;

detection wavelength: 280 nm;

column temperature: 40 deg.C

Sample introduction amount: 0.010ml

The epicatechin concentration in the reference solution in the step (1) is 0.064 mg/ml. And (3) crushing the root of the Chinese gooseberry twig in the step (2) and screening the crushed root of the Chinese gooseberry twig by a No. 2 sieve to prepare powder of the root of the Chinese gooseberry twig, wherein the mass-to-volume ratio of the powder of the root of the Chinese gooseberry twig to the ethanol is 1g to 50 ml. The mass concentration of the ethanol is 50%. The ultrasonic treatment conditions are as follows: the ultrasonic power is 250W, the frequency is 40kHz, and the ultrasonic time is 20 min. And the weight determination is that after cooling, the ethanol is added to ensure that the weight of the medicinal material solution is equal to the weighed weight. And (3) the volume ratio of the acetic acid water to the acetonitrile in the mobile phase in the step (3) is 21: 4. The acetic acid water solution is 2% acetic acid water solution.

Example 2

A method for measuring the content of epicatechin in the tendon of Snake, which adopts high performance liquid chromatography to measure the content of epicatechin in the tendon of Snake, comprises the following steps:

(1) preparation of control solutions: dissolving epicatechin control in methanol to obtain control solution;

(2) preparation of a test solution: dissolving the powder of the root of Chinese goosebeery herb in ethanol to obtain a medicinal solution, weighing, carrying out ultrasonic treatment, cooling, weighing, and filtering, wherein the filtered filtrate is a test solution;

(3) injecting the reference solution and the sample solution into a high performance liquid chromatograph, and detecting the content of epicatechin; the high performance liquid chromatography conditions are as follows:

a chromatographic column: hanbon lichrospher C18250X 4.6 mm;

mobile phase: acetic acid water-acetonitrile;

flow rate: 0.8 ml/min;

time: 10 min;

detection wavelength: 280 nm;

column temperature: 35 deg.C

Sample introduction amount: 0.002ml

The epicatechin concentration in the reference solution in the step (1) is 0.064 mg/ml. And (3) crushing the root of the Chinese gooseberry twig in the step (2) and screening the crushed root of the Chinese gooseberry twig by a No. 3 sieve to prepare powder of the root of the Chinese gooseberry twig, wherein the mass-to-volume ratio of the powder of the root of the Chinese gooseberry twig to the ethanol is 1g to 20 ml. The mass concentration of the ethanol is 30%. The ultrasonic treatment conditions are as follows: the ultrasonic power is 200W, the frequency is 30kHz, and the ultrasonic time is 10 min. And the weight determination is that after cooling, the ethanol is added to ensure that the weight of the medicinal material solution is equal to the weighed weight. And (3) the volume ratio of the acetic acid water to the acetonitrile in the mobile phase in the step (3) is 21: 4. The acetic acid water solution is 2% acetic acid water solution.

Example 3

A method for measuring the content of epicatechin in the tendon of Snake, which adopts high performance liquid chromatography to measure the content of epicatechin in the tendon of Snake, comprises the following steps:

(1) preparation of control solutions: dissolving epicatechin control in methanol to obtain control solution;

(2) preparation of a test solution: dissolving the powder of the root of Chinese goosebeery herb in ethanol to obtain a medicinal solution, weighing, carrying out ultrasonic treatment, cooling, weighing, and filtering, wherein the filtered filtrate is a test solution;

(3) injecting the reference solution and the sample solution into a high performance liquid chromatograph, and detecting the content of epicatechin; the high performance liquid chromatography conditions are as follows:

a chromatographic column: hanbon lichrospher C18250X 4.6 mm;

mobile phase: acetic acid water-acetonitrile;

flow rate: 1.2 ml/min;

time: 12 min;

detection wavelength: 280 nm;

column temperature: 42 deg.C

Sample introduction amount: 0.012ml

The epicatechin concentration in the reference solution in the step (1) is 0.064 mg/ml. And (3) crushing the root of the Chinese gooseberry twig in the step (2) and screening the crushed root of the Chinese gooseberry twig by a No. 4 sieve to prepare powder of the root of the Chinese gooseberry twig, wherein the mass-to-volume ratio of the powder of the root of the Chinese gooseberry twig to the ethanol is 1g to 80 ml. The mass concentration of the ethanol is 70%. The ultrasonic treatment conditions are as follows: the ultrasonic power is 300W, the frequency is 50kHz, and the ultrasonic time is 30 min. And the weight determination is that after cooling, the ethanol is added to ensure that the weight of the medicinal material solution is equal to the weighed weight. And (3) the volume ratio of the acetic acid water to the acetonitrile in the mobile phase in the step (3) is 21: 4. The acetic acid water solution is 2% acetic acid water solution.

Comparative example 1

The mobile phase was acetonitrile-0.1% aqueous phosphoric acid and the other steps were the same as in example 1.

Comparative example 2

The methanol in the control solution was replaced with a methanol solution containing 0.1% phosphoric acid, and the ethanol with a mass concentration of 30% in the test solution was replaced with a methanol solution containing 0.1% phosphoric acid, and the other steps were the same as in example 1.

The results of the measurement of the epicatechin content in examples 1 to 3 and comparative examples 1 to 2 show that the epicatechin content in examples 1 to 3 according to the present invention is separated from the adjacent peaks at the baseline, the retention time of the rosmarinic acid peak, the number of theoretical plates, and both meet the requirements, and the number of theoretical plates in the measurement method in comparative examples 1 to 2 does not meet the requirements, and the degree of separation is low. The results are shown in Table 1.

TABLE 1 System suitability results

Name (R)	Retention time (min)	Peak area	Number of theoretical plate	Degree of separation
					Example 1	8.548	342786.2	9465.0	1.97
Example 2	8.484	347563.8	9398.5	1.92
					Example 3	8.647	345538.1	9422.7	1.95
Comparative example 1	10.468	156368.4	5487.3	1.42
					Comparative example 2	9.156	27389.5	5259.4	1.28

Verification experiment of example 1

1. Investigation of linear relationships

Taking a reference substance solution (0.064mg/ml), sequentially injecting 2 muL, 4 muL, 6 muL, 8 muL, 10 muL and 12 muL by adopting a sequential injection method, and drawing a standard curve by taking the injection amount (ng) as a horizontal coordinate and the peak area as a vertical coordinate. The results show that epicatechin is well correlated linearly in the range of 128-768ng (see fig. 3), and the linear regression equation is 832.1X +6083, and R is 0.9998.

2. Precision survey

Precisely weighing 1.0g of root medicinal powder (201906001, sieved by a No. two sieve) of the root of the Chinese string snake, putting the powder into a conical flask with a plug, adding 50ml of 50% ethanol, weighing, carrying out ultrasonic extraction for 20 minutes, taking out, cooling, complementing the loss weight by 50% ethanol, filtering, continuously injecting samples for 7 times from the subsequent filtrate, recording the peak area of epicatechin, and calculating the RSD value to examine the precision, wherein the result is shown in Table 2. The results show that: the RSD of the epicatechin peak area was 3.17%, indicating that the instrument precision was good.

TABLE 2 results of precision investigation

3. Stability survey

Precisely weighing 1.0g of root medicinal powder of the root of the sinkiang snakegourd (201906001, sieved by a No. two sieve), placing the powder in a conical flask with a plug, adding 50ml of 50% ethanol, weighing, carrying out ultrasonic extraction for 20 minutes, taking out, cooling, complementing the lost weight with 50% ethanol, filtering, taking subsequent filtrates, injecting samples with 0h, 2h, 3.5h, 5.5h, 7.5h, 11.5h, 15.5h and 19.5h respectively, recording the peak area of epicatechin, and calculating the RSD value to test the stability of the sample, wherein the result is shown in Table 3. The results show that: epicatechin was stable over 19.5h with a peak area RSD of 2.58%.

Table 3 stability test results

4. Repeatability survey

Precisely weighing 6 parts of the root medicinal powder (201906001, sieved by a No. two sieve) of the root of the sinew flexuosus, wherein each part is about 1.0g, placing the powder into a conical flask with a plug, adding 50ml of 50% ethanol, weighing the weight, carrying out ultrasonic extraction for 20 minutes, taking out, cooling, complementing the loss weight by 50% ethanol, filtering, taking a subsequent filtrate for sample injection, recording the peak area of epicatechin, calculating the content and RSD of epicatechin in the medicinal material, and observing the repeatability, wherein the result shows that the RSD of the repeatability of the extraction method is 4.42%. The results are shown in Table 4.

TABLE 4 results of repeated investigation

5. Investigation of sample recovery

Precisely weighing 6 parts of the root medicinal powder (201906001, sieved by a No. two sieve) of the root of the common burreed rhizome, wherein each part is about 0.5g, placing the powder into a conical flask with a plug, respectively adding 15ml of a reference substance solution (0.064mg/ml), then adding 35ml of 50% ethanol, weighing, ultrasonically extracting for 20 minutes, taking out, cooling, complementing the weight loss by 50% ethanol, filtering, taking a subsequent filtrate for sample injection, recording the peak area of epicatechin, and calculating the sample injection recovery of the epicatechin, wherein the result is shown in Table 5.

TABLE 5 sample recovery results

Examples of the experiments

The results of measuring the epicatechin content in the soaking tendon medicinal materials of different batches by using the method for measuring the epicatechin content of the soaking tendon in the embodiment 1 of the application are shown in table 6, which shows that the difference of the epicatechin content in the soaking tendon root is large and ranges from 1.76 mg/g to 9.17 mg/g.

TABLE 6 content of epicatechin in different batches of the herb of Redbeton Serpentis

The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.