1. A method for identifying chicoric acid, liquiritin and curcumin in a natural plant antibacterial growth-promoting feed additive is characterized in that thin-layer chromatography is used for identification, a developing solvent is a lower-layer solution of chloroform-ethyl acetate-formic acid-water, and the span length is 14-17 cm; the test solution is obtained by subjecting the test to ultrasonic treatment in 80% methanol solution at 27-35 deg.C; the ratio of the trichloromethane to the ethyl acetate to the formic acid to the water is 12:24:5:4 or 12:24:7: 4.

2. The method of claim 1 wherein the chicoric acid control solution is 0.1-0.3mg chicoric acid per 1mL of 80% methanol; the liquiritin reference solution contains 0.4-0.6mg liquiritin per 1mL 70% ethanol; the curcumin control solution contains 0.4-0.6mg curcumin per 1mL absolute ethanol.

3. The method of claim 2, wherein the color development is performed by ultraviolet 365nm inspection or spraying a color developing agent, heating and drying until the color development of the spot is clear, and inspecting in the sun.

4. The method of claim 3, comprising:

(1) dissolving a sample to be detected by using 80% methanol at 30 ℃ to obtain a test sample solution;

(2) the solvent of chicoric acid reference solution is 80% ethanol; the solvent of liquiritin reference substance solution is 70% ethanol; the solvent of curcumin reference solution is absolute ethyl alcohol;

(3) sequentially spotting the prepared test solution and reference solution on different positions of the same horizontal line of the same thin-layer plate, then placing the thin-layer plate in a developing agent for upward development, wherein the developing agent is chloroform-ethyl acetate-formic acid-water (12:24:7:4), and the development distance is 17 cm;

(4) after the development is finished, taking out the thin layer plate, inspecting under a 365nm ultraviolet lamp or spraying 10% sulfuric acid ethanol solution, heating and drying in the air, and inspecting under sunlight.

5. The method according to claim 4, wherein in step (2) the concentration of the chicoric acid control solution is 0.2 mg/mL; the concentration of the solvent of the liquiritin reference substance solution is 0.5 mg/mL; the concentration of curcumin control solution was 0.5 mg/mL.

6. The method according to any one of claims 1-5, wherein the natural plant antibacterial, growth-promoting feed additive is prepared from the following raw materials in parts by weight: 120 parts of astragalus extract, 100 parts of eucommia bark extract, 120 parts of honeysuckle extract, 110 parts of coptis extract, 100 parts of cinnamon extract, 70 parts of turmeric extract, 80 parts of quassia extract, 90 parts of dandelion extract, 60 parts of aloe extract, 60 parts of nutmeg extract, 90 parts of liquorice extract, 10 parts of adhesive and 10 parts of water.

7. A thin-layer chromatography method for identifying liquiritin is characterized in that liquiritin reference solution is prepared by using 70% ethanol, the concentration of the liquiritin reference solution is 0.5mg/mL, a developing agent is trichloromethane-ethyl acetate-formic acid-water (12:24:7:4), and a color developing agent is a 10% sulfuric acid ethanol solution, and after the color developing agent is sprayed, heating and drying are carried out until spots are clearly developed, and the spots are inspected under sunlight.

8. A thin layer chromatography method for identifying chicoric acid is characterized in that a chicoric acid reference solution is prepared by using absolute ethyl alcohol, the concentration of the chicoric acid reference solution is 0.5mg/mL, a developing agent is chloroform-ethyl acetate-formic acid-water (12:24:7:4), and the detection is carried out under a 365nm ultraviolet lamp.

9. A thin-layer chromatography method for identifying curcumin is characterized in that a curcumin reference solution is prepared by using 80% ethanol, the concentration of the curcumin reference solution is 0.2mg/mL, a developing agent is trichloromethane-ethyl acetate-formic acid-water (12:24:7:4), and after the curcumin reference solution is inspected under a 365nm ultraviolet lamp or sprayed by a 10% ethanol sulfate solution, the curcumin reference solution is heated and dried until spots are clearly developed and inspected under sunlight.

10. Use of the method according to any one of claims 1 to 6 or the thin layer chromatography method according to any one of claims 7 to 9 in the anti-counterfeiting detection of an antibacterial, growth-promoting feed additive in a natural plant.

Background

The natural plant antibacterial growth-promoting feed additive (ZL201410834689.8) has good killing effect on gram-positive bacteria (such as staphylococcus aureus) and gram-negative bacteria (such as escherichia coli and salmonella), integrates the effects of bacteriostasis, sterilization and antivirus, and improvement of immunity and disease resistance, can completely replace feed antibiotics, is green and environment-friendly, has moderate cost, and is simple and convenient in production method and easy to operate.

In order to standardize the market sale and facilitate the rapid identification of products by consumers, a method for simply, conveniently and rapidly identifying the natural plant antibacterial growth-promoting feed additive (ZL201410834689.8) is needed.

Disclosure of Invention

The invention aims to provide a method for rapidly and efficiently identifying the authenticity of a natural plant antibacterial growth-promoting feed additive.

The granular formulation of the natural plant antibacterial growth-promoting feed additive is prepared from the following raw materials in parts by weight: 120 parts of astragalus extract, 100 parts of eucommia bark extract, 120 parts of honeysuckle extract, 110 parts of coptis extract, 100 parts of cinnamon extract, 70 parts of turmeric extract, 80 parts of quassia extract, 90 parts of dandelion extract, 60 parts of aloe extract, 60 parts of nutmeg extract, 90 parts of liquorice extract, 10 parts of adhesive and 10 parts of water.

When thin-layer chromatography identification is carried out on chicoric acid, liquiritin and curcumin in the natural plant antibacterial growth-promoting feed additive, as the components of the natural plant antibacterial growth-promoting feed additive are complex and the properties of the chicoric acid, the liquiritin and the curcumin are different, in the identification process, the difficulties to be overcome are as follows: no color development of the control, incomplete development of the test or control solution, insufficient stretching of the spot, and the like.

The invention firstly determines a method for identifying chicoric acid, liquiritin and curcumin in the natural plant antibacterial growth-promoting feed additive by thin-layer chromatography, which comprises the following steps: the developing agent is a lower layer solution of chloroform-ethyl acetate-formic acid-water, and the span distance is 14-17 cm; the test solution is obtained by subjecting the test to ultrasonic treatment in 80% methanol solution at 27-35 deg.C; the ratio of the trichloromethane to the ethyl acetate to the formic acid to the water is 12:24:5:4 or 12:24:7: 4.

In the method provided by the invention, the chicoric acid control solution is prepared by adding 0.1-0.3mg of chicoric acid in every 1mL of 80% methanol; the liquiritin reference solution contains 0.4-0.6mg liquiritin per 1mL 70% ethanol; the curcumin control solution contains 0.4-0.6mg curcumin per 1mL absolute ethanol.

In the method provided by the invention, the color development mode is that the color is inspected under ultraviolet 365nm or 10% sulfuric acid ethanol solution is sprayed, and the color development of spots is clear after heating and drying, and the color is inspected under sunlight.

Specifically, the method for identifying chicoric acid, liquiritin and curcumin in the natural plant antibacterial growth-promoting feed additive by thin-layer chromatography comprises the following steps:

(1) dissolving a sample to be detected by using 80% methanol at 30 ℃ to obtain a test sample solution;

(2) the solvent of chicoric acid reference solution is 80% ethanol; the solvent of liquiritin reference substance solution is 70% ethanol; the solvent of curcumin reference solution is absolute ethyl alcohol;

(3) sequentially spotting the prepared test solution and reference solution on different positions of the same horizontal line of the same thin-layer plate, then placing the thin-layer plate in a developing agent, and developing upwards, wherein the developing agent is chloroform-ethyl acetate-formic acid-water (12:24:7:4), and the developing distance is 17 cm;

(4) after the development is finished, taking out the thin layer plate, inspecting under a 365nm ultraviolet lamp or spraying a color developing agent, heating and drying in the air, and inspecting under sunlight.

Wherein, in the step (2), the concentration of the chicoric acid reference solution is 0.2 mg/mL; the concentration of the solvent of the liquiritin reference substance solution is 0.5 mg/mL; the concentration of curcumin control solution was 0.5 mg/mL.

The invention also claims a thin-layer chromatography method for identifying liquiritin, wherein a liquiritin reference substance solution is prepared by using 70% ethanol, the concentration of the liquiritin reference substance solution is 0.5mg/mL, a developing agent is trichloromethane-ethyl acetate-formic acid-water (12:24:7:4), and a color developing agent is a 10% ethanol sulfate solution, and after the color developing agent is sprayed, the liquiritin reference substance solution is heated and dried until spots are clearly developed and is inspected under sunlight.

And a thin-layer chromatography method for identifying chicoric acid, wherein a chicoric acid reference solution is prepared by using absolute ethyl alcohol, the concentration of the chicoric acid reference solution is 0.5mg/mL, a developing agent is chloroform-ethyl acetate-formic acid-water (12:24:7:4), and the detection is carried out under a 365nm ultraviolet lamp.

And a thin-layer chromatography method for identifying curcumin, wherein a curcumin reference solution is prepared by using 80% ethanol, the concentration of the curcumin reference solution is 0.2mg/mL, a developing agent is chloroform-ethyl acetate-formic acid-water (12:24:7:4), and the curcumin reference solution is heated and dried until spots are clearly developed after being inspected under a 365nm ultraviolet lamp or sprayed by a 10% ethanol sulfate solution, and is inspected under sunlight.

According to the understanding of the technical personnel in the field, the invention also claims the application of the method or the thin layer chromatography method in the anti-counterfeiting detection of the natural plant antibacterial growth-promoting feed additive.

The invention has the beneficial effects that:

(1) the method provided by the invention has the advantages that the developing effect of the developing agent of the test sample on the thin-layer plate is better, the chromatogram is clear, the separation effect is good, and the impurity band is less when the test sample solution is prepared in a water bath at 30 ℃;

(2) the invention determines the optimal developing solvent and the solution concentration of a reference substance for identifying the chicoric acid, the liquiritin and the curcumin in the antibacterial growth-promoting feed additive of the natural plant by thin-layer chromatography;

(3) by adopting the method provided by the invention, the chicoric acid, the liquiritin and the curcumin in the natural plant antibacterial growth-promoting feed additive can be efficiently and conveniently detected, the standard market sale is realized, and the rapid identification of products by consumers is facilitated.

Drawings

FIG. 1 is a thin layer chromatography color development of chicoric acid and curcumin under a 365nm ultraviolet lamp in example 1 of the present invention.

FIG. 2 is a color development of glycyrrhizin and curcumin by thin layer chromatography under sunlight in example 1 of the present invention.

FIG. 3 is a thin layer chromatography color development of chicoric acid and curcumin under a 365nm ultraviolet lamp in example 2 of the present invention.

FIG. 4 is a color development of glycyrrhizin and curcumin by thin layer chromatography under sunlight in example 2 of the present invention.

FIG. 5 is a thin layer chromatogram of comparative example 1 of the present invention.

FIG. 6 is a thin layer chromatogram of comparative example 2 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.

Unless otherwise specified, test materials, reagents, instruments and the like used in the examples of the present invention are commercially available; all technical measures in the examples of the present invention are conventional measures well known to those skilled in the art, unless otherwise specified.

Example 1

The detection object of the embodiment: ZL201410834689.8 feed additive prepared as in example 1. The control substances used in this example are chicoric acid, liquiritin and curcumin, and the thin-layer chromatography identification method in this example comprises the following steps:

1. preparation of a test solution: dissolving 1g of the Elephantopi product in 20mL of 80% methanol at 30 deg.C for 20min by ultrasonic treatment (60W, 80Hz), and filtering to obtain a filtrate as a sample solution.

2. Preparation of control solutions:

adding 80% methanol into chicoric acid control to obtain solution containing 0.2mg per 1 mL;

adding 70% ethanol into liquiritin reference substance to obtain solution containing 0.5mg per 1 ml;

adding anhydrous ethanol into curcumin control, and making into solutions each containing 0.5mg per 1 ml.

3. Sample application: thin-layer plate: a silica gel G plate;

the sample application sequence is that the sample, chicoric acid, liquiritin and curcumin are tested;

sample amount of spotting: 10. mu.L, 10. mu.L.

4. Unfolding: the mode is upward expansion;

developing agent: chloroform-ethyl acetate-formic acid-water (12:24:7:4) lower layer solution;

and (3) span extension: 17 cm.

5. Color development: taking out, air drying, and inspecting under ultraviolet 365 nm; spraying 10% ethanol sulfate solution, drying at 105 deg.C until the spots are clear, and inspecting in sunlight.

The experimental result is shown in fig. 1 and fig. 2, the test sample chromatogram band is clear, and the test sample chromatogram shows the same color spot on the position corresponding to the reference chromatogram.

Example 2

The detection object of the embodiment: ZL201410834689.8 feed additive prepared as in example 1. The control substances used in this example are chicoric acid, liquiritin and curcumin, and the thin-layer chromatography identification method in this example comprises the following steps:

1. preparation of a test solution: dissolving 1g of the Elephantopi product in 10mL of 80% methanol at 30 deg.C for 20min by ultrasonic treatment (60W, 80Hz), and filtering to obtain a filtrate as a sample solution.

2. Preparation of control solutions:

adding 80% methanol into chicoric acid control to obtain solution containing 0.2mg per 1 mL;

adding 70% ethanol into liquiritin reference substance to obtain solution containing 0.5mg per 1 ml;

adding anhydrous ethanol into curcumin control, and making into solution containing 0.5mg per 1 ml.

3. Sample application: thin-layer plate: a silica gel G plate;

the sample application sequence is that the sample, chicoric acid, liquiritin and curcumin are tested;

sample amount of spotting: 10. mu.L, 10. mu.L.

4. Unfolding: the mode is upward expansion;

and (3) span extension: 14 cm;

developing agent: chloroform-ethyl acetate-formic acid-water (12:24: 5: 4) as the lower layer.

5. Color development: taking out, air drying, and inspecting under ultraviolet 365 nm; spraying 10% ethanol sulfate solution, drying at 105 deg.C until the spots are clear, and inspecting in sunlight. See fig. 3, 4.

The results in fig. 3 and fig. 4 confirm that the chicoric acid and the curcumin are inspected under the ultraviolet of 365nm, the color is clear, and the chicoric acid and the curcumin display the same color spots at the same position as the Ellison sample; the liquiritin and the curcumin are sprayed by 10 percent of sulfuric acid ethanol solution, and are placed in an oven at 105 ℃ until spots are clear, and the liquiritin and the curcumin are inspected under sunlight and have clear color development, and the spots with the same color as the Elephantopi sample are displayed at the same position.

Comparative example 1

The test object of this comparative example: ZL201410834689.8 feed additive prepared as in example 1. The control substances used in this example are chicoric acid, liquiritin and curcumin, and the thin-layer chromatography identification method in this example comprises the following steps:

1. preparation of a test solution: dissolving 1g of the Elephantopi product in 20mL of methanol at 60 deg.C for 20min by ultrasonic treatment (45W, 80Hz), and filtering to obtain a filtrate as a sample solution.

2. Preparation of control solutions:

adding 80% methanol into chicoric acid control to obtain solution containing 0.2mg per 1 mL;

adding 70% ethanol into liquiritin reference substance to obtain solution containing 20 μ g per 1 ml;

adding anhydrous ethanol into curcumin control, and making into solution containing 0.5mg per 1 ml.

3. Sample application: thin-layer plate: a silica gel G plate;

the sample application sequence is test sample, chicoric acid, liquiritin and curcumin;

sample amount of spotting: 10. mu.L, 20. mu.L, 10. mu.L.

4. Unfolding: the mode is upward expansion;

and (3) span extension: 17 cm;

developing agent: chloroform-methanol-formic acid-water (40: 20: 0.9: 8) as the lower layer.

5. Color development: taking out, air drying, and inspecting under ultraviolet 365 nm. See fig. 5.

The experimental result of the comparative example shows that the spots of chicory acid are inconsistent with the spots at the corresponding positions of the Elephantopus sample; ② no obvious point of liquiritin.

The reason is presumed to be that when (1) the sample is dissolved, the sample is not fully dissolved due to the fact that the water temperature is too high at 60 ℃ and the methanol concentration is not proper, and the sample is not fully dispersed on the thin-layer plate; (2) the developer system is not suitable; (3) the color development method is not suitable; (4) the concentration of the liquiritin reference substance solution is not appropriate.

Comparative example 2

The test object of this comparative example: ZL201410834689.8 feed additive prepared as in example 1. The control substances used in this example are chicoric acid, liquiritin and curcumin, and the thin-layer chromatography identification method in this example comprises the following steps:

1. preparation of a test solution: dissolving 1g of the Elephantopi product in 10mL of 80% methanol at 30 deg.C for 20min by ultrasonic treatment (60W, 80Hz), and filtering to obtain a filtrate as a sample solution.

2. Preparation of control solutions:

adding 80% methanol into chicoric acid control to obtain solution containing 0.2mg per 1 mL;

adding 70% ethanol into liquiritin reference substance to obtain solution containing 20 μ g per 1 ml;

adding anhydrous ethanol into curcumin control, and making into solution containing 0.5mg per 1 ml.

3. Sample application:

thin-layer plate: a silica gel G plate;

the sample application sequence is that the sample, chicoric acid, liquiritin and curcumin are tested;

sample amount of spotting: 10. mu.L, 20. mu.L, 10. mu.L.

4. Unfolding: the mode is upward expansion;

and (3) span extension: 16 cm;

developing agent: chloroform-ethyl acetate-formic acid-water (12:24: 5: 4) as the lower layer.

5. Color development: taking out, air drying, and inspecting under ultraviolet 365 nm. And (3) chromatographic identification: spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.

Relative to comparative example 1, the solvent for the sample preparation was adjusted from 20ml of pure methanol to 10ml of 80% methanol; the ultrasonic power is increased from 45W to 60W; the water temperature was reduced from 60 ℃ to 30 ℃. FIG. 6 shows that the sample developed clear spectrum, but liquiritin did not develop color, and the detection method or concentration of liquiritin control was changed.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.