CN110632236A - Method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by thin-layer chromatography - Google Patents
Method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by thin-layer chromatography Download PDFInfo
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Abstract
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.
Description
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.
Example 1
A method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by thin-layer chromatography comprises the following steps:
(1) preparation of control solutions
10.1mg of aucubin reference substance, 10.0mg of geniposide reference substance and 10.3mg of chlorogenic acid reference substance are accurately weighed in sequence, fully dissolved by 60% methanol solution in volume fraction respectively, and the volume is determined to be 10mL, so that standard reference substance solution with 1.01mg/mL of aucubin, 1.00mg/mL of geniposide and 1.03mg/mL of chlorogenic acid is prepared.
(2) Preparation of eucommia ulmoides leaf and bark sample solution to be tested
Collecting fresh folium Eucommiae and cortex Eucommiae in 7-8 months, deactivating enzyme with microwave, oven drying at 45 deg.C, removing impurities, and keeping in dark place; respectively crushing dried eucommia leaves and skins by a high-speed universal crusher, sieving by a 100-mesh sieve to obtain sample powder, performing ultrasonic-assisted extraction twice by using 50% v/v ethanol solution for 0.5h each time, wherein the material-liquid ratio is 1:15, after the extraction is finished, performing reduced pressure concentration on an extracting solution at 60 ℃, obtaining a brown-yellow uniform powder sample when the solid content reaches 20%, sieving by an 80-mesh sieve, uniformly mixing, and performing sealed preservation to obtain the to-be-detected sample powder of the eucommia leaves or skins;
weighing about 0.1g of eucommia ulmoides leaf or bark sample powder to be detected, respectively placing the powder into color comparison tubes with plugs, adding 10mL of 50% methanol solution by volume fraction, fully shaking up, dissolving for 10min under the assistance of ultrasound, cooling at room temperature, and filtering to obtain filtrate, namely the eucommia ulmoides leaf or bark sample solution to be detected.
(3) Spotting is carried out
And (3) respectively sucking 10 mu L 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, sequentially spotting the solutions at different positions of the same horizontal line of the same GF 254 thin-layer silica gel plate, wherein the samples are spaced at proper intervals (about 1.0cm), and the spotting origin points at two ends are more than 0.5cm away from the edge of the thin-layer silica gel plate, so that the edge effect is avoided.
(4) Is unfolded
Spreading the spotted thin-layer silica gel plate in a double-groove thin-layer chromatography cylinder, wherein the spreading is carried out 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 according to the volume ratio of 6.3:1.5:1.0 to obtain a developing solvent; 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 silica gel plate in the second groove of the double-groove thin-layer chromatography cylinder in the step (a), sealing a cylinder cover, sealing and storing at normal temperature, presaturating for 30min, transferring the developing agent in the first groove of the double-groove thin-layer chromatography cylinder to the second groove in which the thin-layer chromatography plate is placed for development, wherein in the process of transferring the developing agent, the volume of the developing agent needs to be proper, the liquid level of the developing agent is not higher than the horizontal line of the spotting, performing thin-layer chromatography in the normal-temperature environment, the chromatography distance is 9.0cm, after the chromatography is finished, taking out the thin-layer silica gel plate, and airing to finish the thin-layer chromatography.
(5) Detection of spots
And (5) placing the thin-layer silica gel plate in the step (4) under an ultraviolet lamp with the wavelength of 254nm for inspection, 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 0.53, obvious spots appear;
the specific shift value Rf of the geniposide in the reference solution and the sample solution to be detected is 0.41, and obvious spots appear;
5.0% sulfuric acid-ethanol solution color developing agent (5 mL sulfuric acid is diluted to 100mL by adding ethanol) is prepared and mixed evenly. Uniformly spraying 5.0% sulfuric acid-ethanol color developing agent on the same thin-layer silica gel plate inspected under the ultraviolet lamp by using a thin-layer manual sprayer, heating at 105 ℃ for 5min, and observing spot change of the thin-layer chromatography silica gel plate;
after the thin-layer silica gel plate is subjected to color development and heating, corresponding spots appear on the reference substance solution and the aucubin in the sample solution to be detected at the position with the specific shift value Rf of 0.30, and no obvious spots corresponding to geniposide and chlorogenic acid are observed at the positions with the Rf of 0.41 and 0.53. The specific analysis result is shown in fig. 1, wherein a and B are the same thin-layer silica gel plate, fig. 1(a) shows the thin-layer chromatography spots under the condition of an ultraviolet lamp, and fig. 1(B) shows the change of the corresponding spots under the heating condition after the color developing agent is sprayed.
Example 2
A method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by thin-layer chromatography comprises the following steps:
(1) preparation of control solutions
10.1mg of aucubin reference substance, 10.0mg of geniposide reference substance and 10.3mg of chlorogenic acid reference substance are accurately weighed in sequence, fully dissolved by 100% methanol solution in volume fraction respectively, and the volume is determined to be 10mL, so that standard reference substance solution with 1.01mg/mL of aucubin, 1.00mg/mL of geniposide and 1.03mg/mL of chlorogenic acid is prepared.
(2) Preparation of eucommia ulmoides leaf and bark sample solution to be tested
Collecting fresh folium Eucommiae and cortex Eucommiae in 7-8 months, microwave deactivating enzyme, oven drying at 60 deg.C, removing impurities, and keeping in dark place; respectively crushing dried eucommia leaves and skins by a high-speed universal crusher, sieving by a 100-mesh sieve to obtain sample powder, performing ultrasonic-assisted extraction twice by using 80% v/v ethanol solution for 0.5h each time, wherein the material-liquid ratio is 1:20, after the extraction is finished, performing reduced pressure concentration on an extracting solution at 60 ℃, obtaining a brown-yellow uniform powder sample when the solid content reaches 20%, sieving by an 80-mesh sieve, uniformly mixing, and performing sealed preservation to obtain the to-be-detected sample powder of the eucommia leaves or skins;
weighing about 0.1g of eucommia ulmoides leaf or bark sample powder to be detected, respectively placing the powder into colorimetric tubes with plugs, adding 10mL of methanol solution with volume fraction of 100%, fully shaking up, heating in water bath at 70 ℃ for auxiliary dissolution for 10min, cooling at room temperature, and filtering to obtain filtrate, namely the eucommia ulmoides leaf or bark sample solution to be detected.
(3) Spotting is carried out
And (3) respectively sucking 15 mu L 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, sequentially spotting the solutions at different positions of the same horizontal line of the same GF 254 thin-layer silica gel plate, wherein the samples are spaced at proper intervals (about 1.0cm), and the origin points of spotting at two ends are more than 0.5cm away from the edge of the thin-layer silica gel plate, so that the edge effect is avoided.
(4) Is unfolded
Spreading the spotted thin-layer silica gel plate in a double-groove thin-layer chromatography cylinder, wherein the spreading is carried out 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 according to the volume ratio of 6.5:2.0:1.5 to obtain a developing solvent; 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 silica gel plate in the second groove of the double-groove thin-layer chromatography cylinder in the step (a), sealing a cylinder cover, sealing and storing at normal temperature, presaturating for 20min, transferring the developing agent in the first groove of the double-groove thin-layer chromatography cylinder to the second groove in which the thin-layer chromatography plate is placed for development, wherein in the process of transferring the developing agent, the volume of the developing agent needs to be proper, the liquid level of the developing agent is not higher than the horizontal line of the spotting, performing thin-layer chromatography in the normal-temperature environment, the chromatography distance is 9.0cm, after the chromatography is finished, taking out the thin-layer silica gel plate, and airing to finish the thin-layer chromatography.
(5) Detection of spots
And (5) placing the thin-layer silica gel plate in the step (4) under an ultraviolet lamp with the wavelength of 254nm for inspection, 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 0.60, obvious spots appear;
the specific shift value Rf of the geniposide in the reference solution and the sample solution to be detected is 0.48, and obvious spots appear;
5.0% sulfuric acid-ethanol solution color developing agent (5 mL sulfuric acid is diluted to 100mL by adding ethanol) is prepared and mixed evenly. Uniformly spraying 5.0% sulfuric acid-ethanol color developing agent on the same thin-layer silica gel plate inspected under the ultraviolet lamp by using a thin-layer manual sprayer, heating at 105 ℃ for 5min, and observing spot change of the thin-layer chromatography silica gel plate;
after the thin-layer silica gel plate is subjected to color development and heating, corresponding spots appear in the positions of the specific shift value Rf of 0.36 of the aucubin in the reference solution and the sample solution to be detected, and spots corresponding to geniposide also appear near Rf of 0.48, but no spots corresponding to chlorogenic acid are observed at Rf of 0.60. The specific analysis result is shown in fig. 2, wherein a and B are the same thin layer silica gel plate, fig. 2(a) shows the thin layer chromatography spots under the condition of an ultraviolet lamp, and fig. 2(B) shows the change of the corresponding spots under the heating condition after the color developing agent is sprayed.
Example 3
A method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by thin-layer chromatography comprises the following steps:
(1) preparation of control solutions
Accurately weighing 10.1mg of aucubin reference substance, 10.0mg of geniposide reference substance and 10.3mg of chlorogenic acid reference substance in turn, fully dissolving the two substances respectively by using ethanol solution with volume fraction of 60%, and fixing the volume to 10mL to prepare standard reference substance solution with aucubin concentration of 1.01mg/mL, geniposide concentration of 1.00mg/mL and chlorogenic acid concentration of 1.03 mg/mL.
(2) Preparation of eucommia ulmoides leaf and bark sample solution to be tested
Collecting fresh folium Eucommiae and cortex Eucommiae in 7-8 months, deactivating enzyme with microwave, oven drying at 50 deg.C, removing impurities, and keeping in dark place; respectively crushing dried eucommia leaves and skins by a high-speed universal crusher, sieving by a 20-mesh sieve to obtain sample powder, performing ultrasonic-assisted extraction twice by using 70% v/v ethanol solution for 0.5h each time, wherein the material-liquid ratio is 1:10, after the extraction is finished, performing reduced pressure concentration on an extracting solution at 60 ℃, obtaining a brown-yellow uniform powder sample when the solid content reaches 20%, sieving by a 80-mesh sieve, uniformly mixing, and performing sealed preservation to obtain the to-be-detected sample powder of the eucommia leaves or skins;
weighing about 0.1g of eucommia ulmoides leaf or bark sample powder to be detected, respectively placing the powder into colorimetric tubes with plugs, adding 10mL of ethanol solution with volume fraction of 50%, fully shaking up, heating in water bath at 70 ℃ for auxiliary dissolution for 30min, cooling at room temperature, and filtering to obtain filtrate, namely the eucommia ulmoides leaf or bark sample solution to be detected.
(3) Spotting is carried out
And (3) respectively sucking 5 mu L 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, sequentially spotting the solutions at different positions of the same horizontal line of the same GF 254 thin-layer silica gel plate, wherein the samples are spaced at proper intervals (about 1.0cm), and the origin points of spotting at two ends are more than 0.5cm away from the edge of the thin-layer silica gel plate, so that the edge effect is avoided.
(4) Is unfolded
Spreading the spotted thin-layer silica gel plate in a double-groove thin-layer chromatography cylinder, wherein the spreading is carried out 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 according to the volume ratio of 6.7:2.5:2 to obtain a developing solvent; 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 silica gel plate in the second groove of the double-groove thin-layer chromatography cylinder in the step (a), sealing a cylinder cover, sealing and storing at normal temperature, presaturating for 20min, transferring the developing agent in the first groove of the double-groove thin-layer chromatography cylinder to the second groove in which the thin-layer chromatography plate is placed for development, wherein in the process of transferring the developing agent, the volume of the developing agent needs to be proper, the liquid level of the developing agent is not higher than the horizontal line of the spotting, performing thin-layer chromatography in the normal-temperature environment, the chromatography distance is 9.0cm, after the chromatography is finished, taking out the thin-layer silica gel plate, and airing to finish the thin-layer chromatography.
(5) Detection of spots
And (5) placing the thin-layer silica gel plate in the step (4) under an ultraviolet lamp with the wavelength of 254nm for inspection, 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 0.64, obvious spots appear;
the specific shift value Rf of the geniposide in the reference solution and the sample solution to be detected is 0.54, and obvious spots appear;
5.0% sulfuric acid-ethanol solution color developing agent (5 mL sulfuric acid is diluted to 100mL by adding ethanol) is prepared and mixed evenly. Uniformly spraying 5.0% sulfuric acid-ethanol color developing agent on the same thin-layer silica gel plate inspected under the ultraviolet lamp by using a thin-layer manual sprayer, heating at 105 ℃ for 5min, and observing spot change of the thin-layer chromatography silica gel plate;
after the thin-layer silica gel plate is subjected to color development and heating, corresponding spots appear on the reference substance solution and the aucubin in the sample solution to be detected at the position with the specific shift value Rf of 0.41, and no spots corresponding to geniposide and chlorogenic acid are observed at the positions with Rf of 0.54 and 0.64. The specific analysis result is shown in fig. 3, wherein a and B are the same thin layer silica gel plate, fig. 3(a) shows the thin layer chromatography spots under the condition of an ultraviolet lamp, and fig. 3(B) shows the change of the corresponding spots under the heating condition after the color developing agent is sprayed.
Example 4
A method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by thin-layer chromatography is compared with example 2, and the method is characterized in that a developing agent is formed by mixing butyl acetate-methanol-formic acid according to a volume ratio of 8.5:4.5:2.5, the rest steps are the same as example 2, and the results are as follows:
and (4) placing the thin-layer silica gel plate under an ultraviolet lamp with the wavelength of 254nm for inspection, 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 0.71, obvious spots appear;
the specific shift value Rf of the geniposide in the reference solution and the sample solution to be detected is 0.61, and obvious spots appear;
5.0% sulfuric acid-ethanol solution color developing agent (5 mL sulfuric acid is diluted to 100mL by adding ethanol) is prepared and mixed evenly. Uniformly spraying 5.0% sulfuric acid-ethanol color developing agent on the same thin-layer silica gel plate inspected under the ultraviolet lamp by using a thin-layer manual sprayer, heating at 105 ℃ for 5min, and observing spot change of the thin-layer chromatography silica gel plate;
and (3) forming corresponding spots on the reference solution and aucubin in the sample solution to be detected at a position with a specific shift value Rf of 0.50 by using a thin-layer silica gel plate after color development and heating, and simultaneously, no spots corresponding to geniposide and chlorogenic acid are observed at positions with Rf of 0.61 and 0.71.
Example 5
A method for rapidly identifying three active ingredients of folium cortex eucommiae and cortex eucommiae by thin-layer chromatography comprises the following steps compared with example 2, wherein the developing agent is butyl acetate: methanol: formic acid was mixed in a volume ratio of 6.0:1.5:0.5, and the rest of the procedure was the same as in example 2, with the following results:
and (4) placing the thin-layer silica gel plate under an ultraviolet lamp with the wavelength of 254nm for inspection, 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, 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, and obvious spots appear;
5.0% sulfuric acid-ethanol solution color developing agent (5 mL sulfuric acid is diluted to 100mL by adding ethanol) is prepared and mixed evenly. Uniformly spraying 5.0% sulfuric acid-ethanol color developing agent on the same thin-layer silica gel plate inspected under the ultraviolet lamp by using a thin-layer manual sprayer, heating at 105 ℃ for 5min, and observing spot change of the thin-layer chromatography silica gel plate;
and (3) forming corresponding spots on the reference solution and aucubin in the sample solution to be detected at a position with a specific shift value Rf of 0.26 on a thin-layer silica gel plate after color development and heating, and simultaneously, no spots corresponding to geniposide and chlorogenic acid are observed at positions with Rf of 0.37 and 0.48.
According to a large number of experiments, the invention discovers that three different active ingredients in eucommia ulmoides can generate corresponding spots in corresponding ranges according to different proportions of the developing solvents, and a better separation effect is achieved. However, when the volume ratio of the used amount of the developing solvent is beyond the range of the invention, the volume ratio of butyl acetate-methanol-formic acid is (6.0-8.5): (1.5-4.5): 0.5-2.5), the separation effect of the sample is not obvious. Meanwhile, chlorogenic acid, geniposide and aucubin cannot be effectively separated by changing the composition of the developing solvent.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (10)
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.
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