阅读说明：本技术 一种利用薄层色谱快速鉴别杜仲叶和皮三种活性成分的方法 (Method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by thin-layer chromatography ) 是由 王志宏 彭密军 王翔 杨秋玲 于 2019-09-16 设计创作，主要内容包括：本发明公开了一种利用薄层色谱快速鉴别杜仲叶和皮三种活性成分的方法。该方法包括以下步骤：1)分别配制桃叶珊瑚苷、京尼平苷酸和绿原酸对照品溶液；2)分别配制杜仲叶和皮的待测样品溶液；3)使用定量毛细管分别吸取对照品溶液和待测样品溶液适量,点样于GF 254硅胶板,以乙酸丁酯：甲醇：甲酸为混合溶剂作为展开剂,通过254nm紫外灯观察和5.0％硫酸-乙醇溶液为显色剂进行薄层色谱鉴别,观察样品溶液的薄层色谱硅胶板中出现斑点的位置和颜色。结果显示,所得的色谱图中杜仲叶和皮的三种活性成分呈现不同的斑点。本发明鉴别方法专属性强,操作方便,可用于同时快速准确鉴别杜仲叶和皮中桃叶珊瑚苷、京尼平苷酸和绿原酸。(The invention discloses a method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by using thin-layer chromatography. The method comprises the following steps: 1) respectively preparing aucubin, geniposide and chlorogenic acid reference substance solutions; 2) respectively preparing to-be-detected sample solutions of eucommia ulmoides leaves and cortex; 3) respectively sucking appropriate amounts of a reference substance solution and a sample solution to be detected by using a quantitative capillary, spotting on a GF 254 silica gel plate, and adding butyl acetate: methanol: formic acid is used as a mixed solvent as a developing agent, thin-layer chromatography identification is carried out by using a 254nm ultraviolet lamp for observation and using a 5.0% sulfuric acid-ethanol solution as a color developing agent, and the position and the color of spots appearing in a thin-layer chromatography silica gel plate of a sample solution are observed. The results show that the obtained chromatogram shows different spots of the three active ingredients of the leaves and the bark of eucommia ulmoides. The identification method has strong specificity and convenient operation, and can be used for simultaneously, rapidly and accurately identifying aucubin, geniposide and chlorogenic acid in eucommia ulmoides leaves and barks.)

1. A method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by using thin-layer chromatography is characterized by comprising the following steps:

(1) accurately weighing aucubin, geniposide and chlorogenic acid reference substances, respectively, dissolving with methanol or ethanol solution, and preparing each reference substance solution;

(2) accurately weighing eucommia leaf and bark sample powder to be detected, respectively placing the eucommia leaf and bark sample powder into a conical flask with a plug, dissolving the eucommia leaf and bark sample powder by using a methanol or ethanol solution, carrying out ultrasonic or heating auxiliary treatment, cooling at room temperature, and filtering to obtain filtrate, namely the sample solution to be detected;

(3) respectively sucking 5-15 microlitres of different reference substance solutions prepared in the step (1) and different sample solutions to be detected prepared in the step (2) by using a quantitative capillary tube, and sequentially spotting different positions of the same horizontal line in the same GF 254 thin-layer silica gel plate;

(4) carrying out thin-layer chromatography by using butyl acetate-methanol-formic acid as a developing agent according to a certain volume ratio, taking out a thin-layer silica gel plate after the chromatography is finished, and airing;

(5) and detecting spots on the thin-layer silica gel plate, and identifying aucubin, geniposide and chlorogenic acid.

2. The method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by thin layer chromatography according to claim 1, wherein the step (5) is to detect spots on a thin layer silica gel plate, and specifically comprises the following steps:

inspecting the thin-layer silica gel plate obtained in the step (4) at a wavelength of 254nm by using an ultraviolet lamp analyzer, and comparing characteristic spots in the chromatogram;

when the ratio shift value Rf of the chlorogenic acid in the reference solution and the sample solution to be detected is 0.48-0.71, obvious spots appear;

the specific shift value Rf of the geniposide in the reference solution and the sample solution to be detected is 0.37-0.61, and obvious spots also appear;

uniformly spraying 5.0% sulfuric acid-ethanol color developing agent on the same thin-layer silica gel plate inspected under the ultraviolet lamp, heating at 105 deg.C for 5min, and observing spot change of the thin-layer chromatography silica gel plate;

and (3) generating corresponding spots on the thin-layer silica gel plate after color development and heating at the position where the specific migration value Rf is 0.25-0.50 between the reference solution and the aucubin in the sample solution to be detected.

3. The method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by thin layer chromatography according to claim 1, wherein the methanol or ethanol solution in the step (1) is an aqueous solution containing 60-100% of methanol or ethanol by volume fraction, and the concentration of the reference solution is 1.0 mg/mL.

4. The method for rapidly identifying the three active ingredients of the eucommia ulmoides leaves and the eucommia ulmoides bark by using the thin layer chromatography as claimed in claim 1, wherein the sample powder to be tested in the step (2) is sieved by a sieve with 20-100 meshes, and the weighing amount is 0.1-1.0 g.

5. The method for rapidly identifying the three active ingredients of the leaves and the barks of eucommia ulmoides oliv by using the thin layer chromatography as claimed in claim 1, wherein the methanol or ethanol solution in the step (2) is an aqueous solution containing 50-100% by volume of methanol or ethanol.

6. The method for rapidly identifying the three active ingredients of the leaves and the barks of the eucommia ulmoides oliv by using the thin layer chromatography as claimed in claim 1, wherein the ultrasonic or heat-assisted treatment of the step (2) is specifically as follows: ultrasonic-assisted dissolution is carried out for 10-30 min, or water bath heating at 70 ℃ is carried out for 10-30 min.

7. The method for rapidly identifying the three active ingredients of the eucommia ulmoides leaves and the eucommia ulmoides bark by using the thin layer chromatography as claimed in claim 1, wherein the developing agent in the step (4) is butyl acetate-methanol-formic acid which consists of (6.0-8.5): (1.5-4.5): 0.5-2.5) by volume ratio.

8. The method for rapidly identifying the three active ingredients of folium cortex eucommiae and the cortex eucommiae by thin layer chromatography according to claim 1, wherein the thin layer chromatography in the step (4) is performed by one-time development, and the solvent in the chromatographic cylinder is pre-saturated for 20-30min before the development.

9. The method for rapidly identifying the three active ingredients of the leaves and the barks of eucommia ulmoides oliv by using the thin layer chromatography as claimed in claim 1, wherein the sample powder to be tested in the step (2) is prepared by the following method: microwave deactivating enzyme of fresh folium Eucommiae and cortex Eucommiae, oven drying or oven drying at 45-60 deg.C, removing impurities, and keeping in dark place; respectively crushing the dried eucommia leaves and skins by a high-speed universal crusher, and sieving by a sieve of 20-100 meshes to obtain sample powder; carrying out ultrasonic assisted extraction on the sample powder twice by using 50-80% v/v ethanol solution for 0.5h each time, wherein the material-liquid ratio is 1: 1-20, after the extraction is finished, carrying out reduced pressure concentration on the extract at 60 ℃, when the solid content reaches 20%, carrying out spray drying to obtain a brown yellow uniform powder sample, sieving with an 80-mesh sieve, uniformly mixing, and carrying out sealed preservation to obtain the sample powder to be detected.

10. The method for rapidly identifying the three active ingredients of folium cortex eucommiae and cortex eucommiae by thin layer chromatography as claimed in claim 1, wherein the sample solution to be tested in step (2) is prepared according to the following method: taking 0.1g of sample powder sample to be detected, placing the sample powder sample into a color comparison tube with a plug, adding 10mL of methanol or ethanol solution with the volume fraction of 50-100%, fully shaking up, dissolving for 10min with the assistance of ultrasound or heating, cooling at room temperature, and filtering to obtain filtrate, namely the sample solution to be detected.

Technical Field

The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by using thin-layer chromatography.

Background

Eucommia ulmoides leaves (Eucommia ulmoides Oliv. leaves) are dry leaves of Eucommia ulmoides belonging to genus Eucommia of family Eucommiaceae, mainly collected in the season of blooming branches and leaves in summer and autumn, and are dried in the sun or at low temperature after microwave enzyme deactivation. Modern researches show that the chemical components of folium Eucommiae are similar to those of cortex Eucommiae, and part of folium Eucommiae can replace cortex Eucommiae. The eucommia ulmoides leaves are rich in active ingredients such as polyphenol, iridoid and flavonoid, have the effects of reducing blood pressure and blood fat, enhancing immunity, resisting inflammation, resisting oxidation, inhibiting bacteria and the like, and have high development value. Folium Eucommiae is collected in Chinese pharmacopoeia from 2005, wherein the identification method of folium Eucommiae is mainly based on thin layer chromatography, and by using chlorogenic acid as observation index through silica gel H thin layer plate under 365nm ultraviolet lamp, and observing fluorescence spot. Although the chlorogenic acid compound can be obviously identified by the method, the eucommia ulmoides leaf resource is identified, and the method still has room for improvement because the target compound is single. In addition to chlorogenic acid inspection, other methods for identifying eucommia ulmoides leaves have been reported to use aucubin as a characteristic compound, but due to differences in polarity of different compounds, a color development method and an inspection method, a single compound and a single inspection method are usually used in many cases. Therefore, a rapid and more accurate method for identifying the eucommia ulmoides leaves and identifying the characteristic compounds in the eucommia ulmoides leaves can be established, and the method has a very wide application prospect. In 2018, in 4 months, the national Weijian committee plans to manage the eucommia leaves as the substances of both food and Chinese medicinal materials, so that the method ensures the land based nature of the eucommia resources and has important significance for the development of the eucommia leaf industry by strictly controlling the quality of the eucommia resources from the source. The chemical component analysis result of the eucommia ulmoides leaves shows that the content of chlorogenic acid in the eucommia ulmoides leaves is higher, and in addition, compared with other compounds, the content of aucubin and geniposide is considerable, and the compounds belong to representative characteristic compounds in the eucommia ulmoides leaves. The eucommia bark is a traditional precious Chinese medicinal material in China, has the effects of reducing three, resisting six, increasing one and the like, has high medicinal value, and simultaneously chlorogenic acid, aucubin and geniposide are also main characteristic components in the eucommia bark. Therefore, the method for qualitatively analyzing aucubin, geniposide and chlorogenic acid at the same time with high efficiency has important research value for rapidly and more accurately identifying eucommia ulmoides leaves and barks and related compounds.

Although the identification and differentiation of the three compounds in the leaves and the barks of eucommia ulmoides can be realized by precise instrument analysis (such as high performance liquid chromatography and the like) from the technical aspect, a method which is simple, convenient, easy, efficient and accurate to realize the simultaneous identification and differentiation of the three characteristic components in the leaves and the barks of eucommia ulmoides is not seen at present. This real problem is also urgently solved by those skilled in the art.

Disclosure of Invention

The invention aims to provide a method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by using thin-layer chromatography.

The technical scheme adopted by the invention is as follows:

a method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by thin-layer chromatography comprises the following steps:

(1) accurately weighing aucubin, geniposide and chlorogenic acid reference substances, respectively, dissolving with methanol or ethanol solution, and preparing each reference substance solution;

(2) accurately weighing eucommia leaf and bark sample powder to be detected, respectively placing the eucommia leaf and bark sample powder into a conical flask with a plug, dissolving the eucommia leaf and bark sample powder by using a methanol or ethanol solution, carrying out ultrasonic or heating auxiliary treatment, cooling at room temperature, and filtering to obtain filtrate, namely the sample solution to be detected;

(3) respectively sucking 5-15 microlitres of different reference substance solutions prepared in the step (1) and different sample solutions to be detected prepared in the step (2) by using a quantitative capillary tube, and sequentially spotting different positions of the same horizontal line in the same GF 254 thin-layer silica gel plate;

(4) carrying out thin-layer chromatography by using butyl acetate-methanol-formic acid as a developing agent according to a certain volume ratio, taking out a thin-layer silica gel plate after the chromatography is finished, and airing;

(5) and detecting spots on the thin-layer silica gel plate, and identifying aucubin, geniposide and chlorogenic acid.

The step (5) of detecting the spots of the thin-layer silica gel plate is carried out by the following method:

inspecting the thin-layer silica gel plate obtained in the step (4) at a wavelength of 254nm by using an ultraviolet lamp analyzer, and comparing characteristic spots in the chromatogram;

obvious spots appear in the ratio shift value Rf of the chlorogenic acid in the reference solution and the sample solution to be detected within the range of 0.48-0.71;

obvious spots also appear in the range of the specific shift value Rf of 0.37-0.61 of the geniposide in the reference solution and the sample solution to be detected;

uniformly spraying 5.0% sulfuric acid-ethanol color developing agent on the same thin-layer silica gel plate inspected under the ultraviolet lamp, heating at 105 deg.C for 5min, and observing spot change of the thin-layer chromatography silica gel plate;

and (3) generating corresponding spots on the thin-layer silica gel plate after color development and heating, wherein the corresponding spots are generated in the range of a specific transfer value Rf of 0.25-0.50 between the reference substance solution and the aucubin in the sample solution to be detected. According to different proportions of the developing solvent, corresponding spots appear in three different active ingredients in the eucommia ulmoides in corresponding ranges, and a good separation effect is achieved. In addition, the volume ratio of the used amount of the developing solvent is beyond the range, and the separation effect of the sample is not obvious.

Preferably, the methanol or ethanol solution in the step (1) is an aqueous solution containing 60-100% of methanol or ethanol by volume fraction, and the concentration of the reference solution is 1.0 mg/mL.

Preferably, the powder of the sample to be tested in the step (2) is sieved by a sieve with 20-100 meshes, and the weighing amount is 0.1-1.0 g.

Preferably, the methanol or ethanol solution in the step (2) is an aqueous solution containing 50-100% of methanol or ethanol by volume fraction.

Preferably, the ultrasonic or heat-assisted treatment in the step (2) is specifically: ultrasonic-assisted dissolution is carried out for 10-30 min, or water bath heating at 70 ℃ is carried out for 10-30 min.

Preferably, the developing agent in the step (4) is composed of butyl acetate-methanol-formic acid according to the volume ratio of (6.0-8.5): (1.5-4.5): 0.5-2.5).

Preferably, the thin layer chromatography in the step (4) is performed once, and before the thin layer chromatography is performed, the solvent in the chromatographic cylinder should be pre-saturated for 20-30 min.

Preferably, the powder of the sample to be tested in the step (2) is prepared according to the following method: microwave deactivating enzyme of fresh folium Eucommiae and cortex Eucommiae, oven drying or oven drying at 45-60 deg.C, removing impurities, and keeping in dark place; respectively crushing the dried eucommia leaves and skins by a high-speed universal crusher, and sieving by a sieve of 20-100 meshes to obtain sample powder; carrying out ultrasonic assisted extraction on the sample powder twice by using 50-80% v/v ethanol solution for 0.5h each time, wherein the material-liquid ratio is 1: 1-20, after the extraction is finished, carrying out reduced pressure concentration on the extract at 60 ℃, when the solid content reaches 20%, carrying out spray drying to obtain a brown yellow uniform powder sample, sieving with an 80-mesh sieve, uniformly mixing, and carrying out sealed preservation to obtain the sample powder to be detected.

Preferably, the sample solution to be tested in step (2) is prepared according to the following method: taking 0.1g of sample powder sample to be detected, placing the sample powder sample into a color comparison tube with a plug, adding 10mL of methanol or ethanol solution with the volume fraction of 50-100%, fully shaking up, dissolving for 10min with the assistance of ultrasound or heating, cooling at room temperature, and filtering to obtain filtrate, namely the sample solution to be detected.

The thin layer chromatography of the above step (4) is developed in a two-tank thin layer chromatography tank according to the following steps (a) and (b):

(a) accurately transferring a proper amount of butyl acetate, methanol and formic acid solution, and uniformly mixing the butyl acetate, the methanol and the formic acid solution according to the volume ratio of (6.0-8.5) to (1.5-4.5) to (0.5-2.5) to obtain a developing agent; placing the developing solvent in the first tank of a dry and clean double-tank thin-layer chromatography cylinder;

(b) placing the spotted thin-layer plate in the second groove of the double-groove thin-layer chromatography cylinder in the step (a), sealing a cylinder cover, pre-saturating for 5-30min (preferably 20-30min), transferring the developing agent in the first groove of the double-groove thin-layer chromatography cylinder to a second operation with a thin-layer chromatography plate for development, wherein the liquid level of the developing agent is not higher than the horizontal line of the spotted sample, the chromatography distance is 9.0cm, after finishing chromatography, taking out the thin-layer silica gel plate, and airing.

The GF 254 thin-layer silica gel plate can be a self-made thin-layer silica gel plate or can be obtained from a market.

The invention has the beneficial effects that:

the thin-layer chromatography identification method provided by the invention is strong in specificity and convenient to operate, three different characteristic components (aucubin, geniposide and chlorogenic acid) of eucommia leaves and eucommia bark in the obtained thin-layer chromatogram show different spots, and the method can be used for quickly and accurately identifying aucubin, geniposide and chlorogenic acid in eucommia leaves and eucommia bark at the same time.

Drawings

FIG. 1 is a thin layer chromatogram of example 1. Wherein, A and B are the same thin silica gel plate, FIG. 1(A) shows the thin chromatographic spots under the condition of ultraviolet lamp, and FIG. 1(B) shows the corresponding spot change condition under the heating condition after spraying color developing agent.

FIG. 2 is a thin layer chromatogram of example 2. Wherein, A and B are the same thin silica gel plate, FIG. 2(A) is the appearance of thin chromatographic spots under the condition of an ultraviolet lamp, and FIG. 2(B) is the change condition of the corresponding spots under the heating condition after spraying the color developing agent.

FIG. 3 is a thin layer chromatogram of example 3. Wherein, A and B are the same thin silica gel plate, FIG. 3(A) is the appearance of thin chromatographic spots under the condition of an ultraviolet lamp, and FIG. 3(B) is the change condition of the corresponding spots under the heating condition after spraying the color developing agent.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.