阅读说明：本技术 姜竹茹中药配方颗粒中生姜的鉴别方法 (Method for identifying ginger in ginger bamboo shavings traditional Chinese medicine formula granules ) 是由 吕渭升 潘晓君 杨文惠 侯栩轩 叶梅霞 李振雨 何民友 陈向东 孙冬梅 魏梅 于 2021-09-01 设计创作，主要内容包括：本发明涉及一种姜竹茹中药配方颗粒中生姜的鉴别方法,所述鉴别方法包括提供对照品溶液、制备供试品溶液并采用薄层色谱法对所述对照品溶液和所述供试品溶液进行检测的步骤；所述对照品溶液中的对照品包含姜酮和6-姜辣素；制备所述供试品溶液的步骤包括：取姜竹茹中药配方颗粒,制备含有姜酮和6-姜辣素的提取液；采用聚酰胺柱对所述含有姜酮和6-姜辣素的提取液进行柱层析,柱层析的条件包括：层析柱采用聚酰胺柱,洗脱液为甲醇体积百分比为25％～55％的甲醇溶液,洗脱方式采用等度洗脱。该方法实现了基于薄层色谱法鉴别姜竹茹中药配方颗粒中生姜,为姜竹茹中药配方颗粒产品的质量控制提供科学的、快速的、低成本的新方法。(The invention relates to a method for identifying ginger in ginger bamboo shavings traditional Chinese medicine formula particles, which comprises the steps of providing a reference substance solution, preparing a test solution, and detecting the reference substance solution and the test solution by adopting a thin-layer chromatography; the reference substance in the reference substance solution comprises zingerone and 6-gingerol; the step of preparing the test solution comprises: taking the ginger-bamboo shavings traditional Chinese medicine formula granules, and preparing an extracting solution containing zingerone and 6-gingerol; performing column chromatography on the extract containing the zingerone and the 6-gingerol by adopting a polyamide column, wherein the conditions of the column chromatography comprise that: the chromatographic column adopts a polyamide column, the eluent is methanol solution with the volume percentage of methanol of 25-55 percent, and the elution mode adopts isocratic elution. The method realizes identification of rhizoma Zingiberis recens in the granule based on thin layer chromatography, and provides a scientific, rapid and low-cost new method for quality control of granule product.)

1. The identification method of ginger in the ginger-bamboo shavings traditional Chinese medicine formula particles is characterized by comprising the steps of providing a reference substance solution, preparing a test solution, and detecting the reference substance solution and the test solution by adopting a thin-layer chromatography;

the reference substance in the reference substance solution comprises zingerone and 6-gingerol;

the step of preparing the test solution comprises:

taking the ginger-bamboo shavings traditional Chinese medicine formula granules, and preparing an extracting solution containing zingerone and 6-gingerol;

performing column chromatography on the extracting solution containing the zingerone and the 6-gingerol, wherein the conditions of the column chromatography comprise that: the chromatographic column adopts a polyamide column, and the eluent adopts methanol water solution with the methanol volume percentage of 25-55 percent.

2. The method for identifying ginger in the ginger-bamboo shavings traditional Chinese medicine formula granules according to claim 1, wherein the step of preparing the extracting solution containing zingerone and 6-gingerol comprises:

taking the ginger bamboo shavings traditional Chinese medicine formula particles, extracting by taking a methanol water solution as an extraction solvent, and collecting an alcohol extract;

removing the solvent in the alcohol extract, dissolving the obtained residue a in water, extracting with ethyl acetate, and collecting an organic phase;

the solvent was removed from the organic phase and the residue b was dissolved in methanol.

3. The method for identifying ginger in the ginger bamboo shavings traditional Chinese medicine formula particles as claimed in claim 2, wherein the number of times of extracting with the ethyl acetate is 1-3, and the amount of the ethyl acetate extracted from 1g of the ginger bamboo shavings traditional Chinese medicine formula particles is 6-10 mL per 1 time; or/and the ethyl acetate is used for extracting by adopting a shaking way.

4. The method for identifying ginger in the ginger-bamboo shavings traditional Chinese medicine formula granules according to claim 2 or 3, wherein the volume percentage of the methanol-containing extraction solvent is 78-85%.

5. The method for identifying ginger in the ginger bamboo shavings traditional Chinese medicine formula particles as claimed in claim 2 or 3, wherein the dosage of the extraction solvent is 18 mL-22 mL for every 1g of the ginger bamboo shavings traditional Chinese medicine formula particles; or/and ultrasonic extraction is adopted in the extraction mode of the extraction solvent.

6. The method for identifying ginger in ginger-bamboo shavings traditional Chinese medicine formula granules according to any one of claims 1 to 3, wherein the parameter conditions of the polyamide column comprise: the grain diameter of the filler is 100-200 meshes, the mass of the filler is 1.5-2.5 g, the inner diameter is 1.5-2.5 cm, and the column is packed by a dry method.

7. The method for identifying ginger in ginger-bamboo shavings-traditional Chinese medicine formula granules according to any one of claims 1 to 3, wherein a solvent in the control solution comprises methanol.

8. The method for identifying ginger in the ginger-bamboo shavings traditional Chinese medicine formula particle as claimed in claim 7, wherein each 1mL of the control solution contains 0.18mg to 0.22mg of the zingiberone and 0.18mg to 0.22mg of the 6-gingerol.

9. The method for identifying ginger in the ginger-bamboo shavings traditional Chinese medicine formula granules according to any one of claims 1 to 3 and 8, wherein the method for identifying ginger further comprises the following steps: preparing a negative sample solution from the ginger-lacking negative sample, and detecting the negative sample solution by adopting thin-layer chromatography in the detection process.

10. The method for identifying ginger in ginger-bamboo shavings traditional Chinese medicine formula granules according to claim 9, wherein the negative sample lacking ginger is selected from bamboo shavings traditional Chinese medicine formula granules or maltodextrin.

11. The method for identifying ginger in the ginger-bamboo shavings traditional Chinese medicine formula granules according to any one of claims 1 to 3, 8 and 10, wherein a thin layer chromatography plate adopted by the thin layer chromatography is a silica gel G plate.

12. The method for identifying ginger in the ginger-bamboo shavings traditional Chinese medicine formula granules according to any one of claims 1 to 3, 8 and 10, wherein a developing agent adopted by the thin layer chromatography is (1.8-2.2): (0.8-1.2) 1 of a mixture of petroleum ether, chloroform and ethyl acetate, wherein the boiling point of the petroleum ether is 60-90 ℃.

13. The method for identifying ginger in the ginger-bamboo shavings traditional Chinese medicine formula granules according to any one of claims 1 to 3, 8 and 10, wherein the color developing agent adopted by the thin layer chromatography is a vanillin sulfuric acid test solution.

Technical Field

The invention relates to the technical field of traditional Chinese medicine detection, and particularly relates to a method for identifying ginger in ginger bamboo shavings traditional Chinese medicine formula particles.

Background

The ginger-bamboo shavings traditional Chinese medicine formula particle is obtained by extracting ginger-bamboo shavings decoction pieces with water, concentrating and drying. The ginger and bamboo shavings decoction pieces are prepared by processing bamboo shavings, are roasted or fried with ginger juice to be yellow, are similar to the bamboo shavings, have yellow surface and slightly have ginger fragrance. Zhu Ru with ginger is good at resolving phlegm and stopping vomiting.

The identification of the ginger bamboo shavings traditional Chinese medicine formula granules faces the problems that: the traditional Chinese medicine formula particle is a particle prepared by carrying out water extraction, separation, concentration, drying and granulation on single traditional Chinese medicine decoction pieces. The traditional Chinese medicine formula granules are obtained from traditional Chinese medicine decoction pieces, and the drug effect substances are basically consistent with the decoction pieces of the traditional Chinese medicine decoction pieces. Traditionally, people can identify the types of medicinal materials by eye observation, nose smell, mouth taste and hand touch, and the traditional Chinese medicine formula granules are changed into finished products without the appearance shape of the medicinal pieces from shaped medicinal pieces through a series of processing, and are difficult to identify by traditional visual observation, nose smell and the like. Because the traditional Chinese medicine formula particle loses the characteristics of appearance, smell and the like which are recognized by the traditional decoction pieces, and plays an important role in the prescription as solid decoction preparation, the identification of the types of medicinal materials contained in the traditional Chinese medicine formula particle is particularly important by adopting a proper technical means.

At present, the identification of ginger in the ginger bamboo shavings traditional Chinese medicine formula particle is blank, so a method for identifying ginger in the ginger bamboo shavings traditional Chinese medicine formula particle is urgently needed to be provided.

Disclosure of Invention

Based on the background technology, the invention mainly aims to provide a method for identifying ginger in ginger bamboo shavings traditional Chinese medicine formula particles.

The purpose of the invention can be realized by the following technical scheme:

a method for identifying ginger in ginger-bamboo shavings traditional Chinese medicine formula particles comprises the steps of providing a reference substance solution, preparing a test substance solution, and detecting the reference substance solution and the test substance solution by adopting a thin-layer chromatography;

the reference substance in the reference substance solution comprises zingerone and 6-gingerol;

the step of preparing the test solution comprises:

taking the ginger-bamboo shavings traditional Chinese medicine formula granules, and preparing an extracting solution containing zingerone and 6-gingerol;

performing column chromatography on the extracting solution containing the zingerone and the 6-gingerol, wherein the conditions of the column chromatography comprise that: the chromatographic column adopts a polyamide column, and the eluent is methanol water solution with the methanol volume percentage of 25-55%.

In one embodiment, the step of preparing the extract solution containing zingerone and 6-gingerol comprises:

taking the ginger bamboo shavings traditional Chinese medicine formula particles, extracting by taking a methanol water solution as an extraction solvent, and collecting an alcohol extract;

removing the solvent in the alcohol extract, dissolving the obtained residue a in water, extracting with ethyl acetate, and collecting an organic phase;

the solvent was removed from the organic phase and the residue b was dissolved in methanol.

In one embodiment, the number of times of extraction with the ethyl acetate is 1 to 3, and the dosage of the ethyl acetate is 6 to 10mL for each 1g of the ginger bamboo shavings traditional Chinese medicine formula particles.

In one embodiment, the ethyl acetate extraction mode adopts oscillation extraction.

In one embodiment, the extraction solvent comprises from 78% to 85% by volume methanol.

In one embodiment, the dosage of the extraction solvent is 18 mL-22 mL for each 1g of the ginger bamboo shavings traditional Chinese medicine formula particles.

In one embodiment, the extraction with the extraction solvent is performed by ultrasonic extraction.

In one embodiment, the parameter conditions of the polyamide column include: the grain diameter of the filler is 100-200 meshes, the mass of the filler is 1.5-2.5 g, the inner diameter is 1.5-2.5 cm, and the column is packed by a dry method.

In one embodiment, the solvent in the control solution comprises methanol.

In one embodiment, each 1mL of the control solution comprises 0.18mg to 0.22mg of the zingerone and 0.18mg to 0.22mg of the 6-gingerol.

In one embodiment, the authentication method further comprises the steps of: preparing a negative sample solution from the ginger-lacking negative sample, and detecting the negative sample solution by adopting thin-layer chromatography in the detection process.

In one embodiment, the negative sample of ginger is selected from bamboo shavings traditional Chinese medicine formula granules or maltodextrin.

In one embodiment, the thin layer chromatography plate used in the thin layer chromatography is a silica gel G plate.

In one embodiment, the developing agent adopted by the thin layer chromatography is (1.8-2.2): (0.8-1.2) 1 of a mixture of petroleum ether, chloroform and ethyl acetate, wherein the boiling point of the petroleum ether is 60-90 ℃.

In one embodiment, the color reagent used in the thin layer chromatography is vanillin sulfuric acid test solution.

Compared with the prior art, the invention has the following beneficial effects:

the ginger-bamboo shavings traditional Chinese medicine formula particle is obtained by extracting ginger-bamboo shavings decoction pieces with water, concentrating and drying, relates to two traditional Chinese medicines of bamboo shavings and ginger, generates complex chemical components in the processing process of the ginger-bamboo shavings decoction pieces and the preparation process of the traditional Chinese medicine formula particle, and the components in the bamboo shavings possibly interfere with the components in the ginger in the identification process. Therefore, it is difficult to identify ginger in the ginger-bamboo shavings traditional Chinese medicine formula granules simply by referring to the traditional ginger component thin layer chromatography identification method (including the ginger component thin layer chromatography recorded in pharmacopoeia).

Therefore, in the process of preparing a test solution, the prepared extracting solution containing the zingerone and the 6-gingerol is purified by adopting proper column chromatography conditions, so that interference components (including components in the bamboo shavings) can be effectively removed, the effective purification of the zingerone and the 6-gingerol which are characteristic components of the ginger is realized, the thin-layer chromatography is successfully applied to the detection of the ginger in the ginger bamboo shavings traditional Chinese medicine formula particles, the Rf value of each spot of a chromatogram obtained by the detection is moderate, the integral separation effect is good, and the spots are clear. The method is rapid, stable, special, and durable, and can accurately display the characteristics of rhizoma Zingiberis recens component, and provide a scientific, rapid, and low-cost new method for quality control of granule product of rhizoma Zingiberis recens-caulis Bambusae in Taenia.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a chromatogram obtained from the thin-layer chromatography of different sample amounts of a traditional Chinese medicine granule in ginger and bamboo shavings; silica gel G is adopted to prefabricate a thin-layer plate (SG, batch number: 20210309, institute of chemical industry of cigarette Taiwan); in the figure: 1.5 mul of ginger bamboo shavings traditional Chinese medicine formula granules (CG 20060002); 2. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 20060002); 3. ginger bamboo shavings traditional Chinese medicine formula granules; (CG20060002) 20. mu.L; 4. mixing with 5 μ L of reference substance (upper: zingerone; lower: 6-gingerol); 5. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol); 6. mixing with 15 μ L of reference substance (upper: zingerone; lower: 6-gingerol);

FIG. 2 is a thin layer chromatography specificity verification chromatogram of a granule of a traditional Chinese medicine formula of rhizoma Zingiberis recens, caulis Bambusae in Taenia; silica gel G is adopted to prefabricate a thin-layer plate (SG, batch number: 20210309, institute of chemical industry of cigarette Taiwan); in the figure: 1. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006002); 2. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006003); 3. 10 mul of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006004); 4. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol); 5. negative sample of bamboo shavings Chinese medicinal prescription granule (CG2006011) 10 μ L;

FIG. 3 is a thin-layer chromatogram for identification of granule prepared from rhizoma Zingiberis recens, caulis Bambusae in Taenia, and Chinese medicinal materials at room temperature; silica gel G is adopted to prefabricate a thin-layer plate (SG, batch number: 20210309, institute of chemical industry of cigarette Taiwan); in the figure: 1. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006002); 2. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006003); 3. 10 mul of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006004); 4. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol);

FIG. 4 is a thin-layer chromatogram for identification of granule of rhizoma Zingiberis recens, caulis Bambusae in Taenia, and Chinese medicinal materials at low temperature; silica gel G is adopted to prefabricate a thin-layer plate (SG, batch number: 20210309, institute of chemical industry of cigarette Taiwan); in the figure: 1. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006002); 2. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006003); 3. 10 mul of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006004); 4. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol);

FIG. 5 is a thin-layer chromatogram for identification of granule prepared from rhizoma Zingiberis recens, caulis Bambusae in Taenia, and Chinese medicinal materials under high humidity; silica gel G is adopted to prefabricate a thin-layer plate (SG, batch number: 20210309, institute of chemical industry of cigarette Taiwan); in the figure: 1. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006002); 2. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006003); 3. 10 mul of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006004); 4. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol);

FIG. 6 is a thin-layer chromatogram for identification of granule of rhizoma Zingiberis recens, caulis Bambusae in Taenia, and Chinese medicinal materials under low humidity; silica gel G is adopted to prefabricate a thin-layer plate (SG, batch number: 20210309, institute of chemical industry of cigarette Taiwan); in the figure: 1. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006002); 2. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006003); 3. 10 mul of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006004); 4. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol);

FIG. 7 is a chromatogram for investigation of silica gel G plates from different manufacturers; silica gel G is adopted to prefabricate a thin-layer plate (G, batch number: 20191018, Qingdao ocean chemical Co., Ltd.); in the figure: 1. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006002); 2. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006003); 3. 10 mul of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006004); 4. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol);

FIG. 8 is a silica gel G plate inspection chromatogram of different manufacturers (Spectraceae); silica gel G is adopted to prefabricate a thin-layer plate (G, batch number: 20210326, Qingdao spectral separation materials Co., Ltd.); in the figure: 1. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006002); 2. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006003); 3. 10 mul of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006004); 4. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol);

FIG. 9 is a silica gel G plate investigation chromatogram from different manufacturers; silica gel G was used to prepare thin-layer plates (TLC Silica gel 60, batch: HXD4333226, Merck GmbH); in the figure: 1. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006002); 2. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006003); 3. 10 mul of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006004); 4. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol);

FIG. 10 is a silica gel G plate investigation chromatogram of different manufacturers (Yinlong Nicotiana); silica gel G prefabricated thin layer plate (SG, batch number: 20210309, institute of chemical industry, cigarette Tai city); 1. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006002); 2. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006003); 3. 10 mul of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006004); 4. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol);

FIG. 11 is a thin layer chromatography of ginger and bamboo shavings traditional Chinese medicine formula granules; silica gel G prefabricated thin layer plate (SG, batch number: 20210309, institute of chemical industry, cigarette Tai city); in the figure: 1. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006002); 2. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006003); 3. 10 mul of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006004); 4. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol);

fig. 12 is a thin layer chromatography of the ginger bamboo shavings traditional Chinese medicine formula granules of example 3; in the figure: 1. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006002); 2. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006003); 3. 10 mul of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006004); 4. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol);

fig. 13 is a thin layer chromatography of the ginger bamboo shavings traditional Chinese medicine formula granules of example 4; in the figure: 1. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006002); 2. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006003); 3. 10 mul of ginger bamboo shavings traditional Chinese medicine formula granules (CG 2006004); 4. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol);

FIG. 14 is a thin layer chromatography of the ginger-bamboo shavings traditional Chinese medicine formula granules of example 5; in the figure: 1.3 mul of ginger bamboo shavings traditional Chinese medicine formula particles (extracted by methanol); 2.3 mul of ginger bamboo shavings traditional Chinese medicine formula particles (extracted by 80% methanol); 3. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula particles (extracted by 50% methanol); 4. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol);

FIG. 15 is a thin layer chromatography of the ginger and bamboo shavings traditional Chinese medicine formula granules of example 6; in the figure: 1. 10 mu L of ginger bamboo shavings traditional Chinese medicine formula particles (ultrasonic and extraction); 2. 10 mul of ginger bamboo shavings traditional Chinese medicine formula particles (extraction); 3. mixing with 10 μ L of reference substance (upper: zingerone; lower: 6-gingerol);

FIG. 16 is a thin layer chromatography of the ginger, bamboo shavings and traditional Chinese medicine formula granules obtained from different eluents; in the figure: 1. washing the part with water 10 μ L; 2. washing with water, and eluting 10 μ L of 10% methanol; 3. washing with water, and eluting with 30% methanol to obtain 10 μ L eluate; 4. directly eluting 10 μ L of the fraction with 30% methanol; 5. mixing reference substances (upper: zingerone; lower: 6-gingerol);

FIG. 17 is a thin layer chromatography of Zhuru traditional Chinese medicine formula granules obtained from different eluents; in the figure: 10 μ L of 1.30% methanol; 2.60% methanol elution 10 μ L; 3. 10 μ L of zingerone control; 4.6-gingerol control 10 μ L;

FIG. 18 is a thin-layer chromatography of the ginger-bamboo shavings Chinese medicinal granule obtained from different chromatographic columns; in the figure: 1. petroleum ether (60-90 ℃) elutes 10 mu L; 2. petroleum ether (60-90 deg.C) and ethyl acetate (1:1) eluting 10 μ L; 3. eluting with ethyl acetate to obtain 10 μ L solution; 4. methanol elution 10. mu.L; elution 10. mu.L from a 5.30% methanol-polyamide column; 6. 10 μ L of zingerone control; 7.6-gingerol control 10. mu.L.

Detailed Description

In order to facilitate an understanding of the present invention, the present invention will be described in more detail below. It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as being limited to the embodiments or examples set forth herein. Rather, these embodiments or examples are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments or examples only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of two or more of the associated listed items, including any and all combinations of two or more of the associated listed items, or all of the associated listed items.

In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.

In the present invention, the numerical range is defined to include both end points of the numerical range unless otherwise specified.

The percentage contents referred to in the present invention mean, unless otherwise specified, mass percentages for solid-liquid mixing and solid-solid phase mixing, and volume percentages for liquid-liquid phase mixing.

The percentage concentrations referred to in the present invention refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system to which the component is added.

The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a treatment within a certain temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.

At present, the qualitative method of traditional Chinese medicine components mainly comprises physicochemical tests, thin-layer chromatography, high performance liquid chromatography, gas-mass spectrometry, ultra-high performance liquid chromatography-mass spectrometry and the like. High performance liquid chromatography or liquid chromatography-mass spectrometry, etc., the required instruments are more precise, leading to greatly increased inspection cost. Thin-layer chromatography, also known as thin-layer chromatography, is a method in which a sample solution is spotted on a thin-layer plate, developed with a developing agent in a developing container, and the components contained in the sample are separated and used for identification, examination, and content measurement. The thin layer chromatography is one of the most widely applied methods in the plane chromatography, and has the advantages of easy mastering, cheap equipment and simple and flexible operation. However, the application of the thin-layer chromatography in the ginger bamboo shavings traditional Chinese medicine formula granules is still blank, and the invention aims to apply the thin-layer chromatography to the ginger bamboo shavings traditional Chinese medicine formula granules and fill the blank of quality control.

The invention provides a method for identifying ginger in ginger bamboo shavings traditional Chinese medicine formula particles, which comprises the steps of providing a reference substance solution, preparing a test solution, and detecting the reference substance solution and the test solution by adopting a thin-layer chromatography;

the reference substance in the reference substance solution comprises zingerone and 6-gingerol;

the step of preparing the test solution comprises:

taking the ginger-bamboo shavings traditional Chinese medicine formula granules, and preparing an extracting solution containing zingerone and 6-gingerol;

performing column chromatography on the extract containing the zingerone and the 6-gingerol by adopting a polyamide column, wherein the conditions of the column chromatography comprise that: the chromatographic column adopts a polyamide column, and the eluent is methanol water solution with the methanol volume percentage of 25-55%.

The volume percentage of methanol in the eluent applicable to the invention can be 25%, 30%, 35%, 40%, 45%, 50%, 55%, and more preferably, 25% to 35%.

In one example, the step of preparing the extract solution containing zingerone and 6-gingerol comprises:

taking the ginger bamboo shavings traditional Chinese medicine formula particles, extracting by taking a methanol solution as an extraction solvent, and collecting an alcohol extract;

removing the solvent in the alcohol extract, dissolving the obtained residue a in water, extracting with ethyl acetate, and collecting an organic phase;

the solvent was removed from the organic phase and the residue b was dissolved in methanol.

In one example, the number of times of extraction with the ethyl acetate is 1 to 3 (e.g., 1, 2, 3), and the amount of the ethyl acetate used for each 1g of the bambusae caulis traditional Chinese medicine formula particles is 6mL to 10mL (e.g., 6mL, 8mL, 10mL) for each 1 extraction.

In one example, the ethyl acetate extraction mode is shaking extraction.

In one example, the extraction solvent contains methanol in a volume percentage of 78% to 85%. For example 78%, 80%, 82%, 85%.

In one example, the amount of the extraction solvent used per 1g of the ginger-bamboo shavings traditional Chinese medicine formula particle is 18mL to 22mL (e.g. 18mL, 20mL, 22 mL).

In one example, the extraction with the extraction solvent is performed by ultrasonic extraction.

In one example, the parameter conditions of the polyamide column include: the grain diameter of the filler is 100 meshes-200 meshes, the mass of the filler is 1.5 g-2.5 g, and the inner diameter is 1.5 cm-2.5 cm. For example, a polyamide column (100 mesh to 200 mesh, 2g, 2 cm).

In one example, the step of preparing the test solution comprises:

taking the ginger bamboo shavings traditional Chinese medicine formula particles, performing ultrasonic extraction by using a methanol solution with the methanol percentage of 78-85% as an extraction solvent, and collecting an extracting solution a;

removing the solvent in the extracting solution a, dissolving the obtained residue a' with water, extracting with ethyl acetate by shaking, and collecting the extracting solution b;

the solvent in the extract b was removed and the residue b' was dissolved in methanol.

Performing column chromatography on the extract containing the zingerone and the 6-gingerol by adopting a polyamide column, wherein the conditions of the column chromatography comprise that: the chromatographic column adopts a polyamide column, and the eluent is methanol solution with the methanol volume percentage of 25-55%.

In the process of extracting the characteristic components of the zingerone and the 6-gingerol, the invention combines a methanol solution ultrasonic extraction method, an ethyl acetate liquid-liquid extraction method and a column chromatography method to purify and enrich ginger components, has better effect than the common ultrasonic method or the liquid-liquid extraction method which is simply used, and can remove interfering components.

In one example, the solvent in the control solution comprises methanol.

In one example, each 1mL of the control solution comprises 0.18mg to 0.22mg of the zingiberone and 0.18mg to 0.22mg of the 6-gingerol, e.g., 0.18mg of the zingiberone and 0.18mg of the 6-gingerol, 0.22mg of the zingiberone and 0.22mg of the 6-gingerol, 0.2mg of the zingiberone and 0.2mg of the 6-gingerol.

In one example, the authentication method further includes the steps of: preparing a negative sample solution from the ginger-lacking negative sample, and detecting the negative sample solution by adopting thin-layer chromatography in the detection process.

In one example, the negative sample of ginger is selected from bamboo shavings traditional Chinese medicine formula granules or maltodextrin.

In one example, the thin layer chromatography plate used in the thin layer chromatography is a silica gel G plate.

In one example, the thin layer chromatography adopts a developing solvent with a volume ratio of (1.8-2.2): (0.8-1.2) 1 of a mixture of petroleum ether, chloroform and ethyl acetate, wherein the boiling point of the petroleum ether is 60-90 ℃. Examples thereof include petroleum ether (60 ℃ C. -90 ℃ C.) -chloroform-ethyl acetate (2:1:1), petroleum ether (60 ℃ C. -90 ℃ C.) -chloroform-ethyl acetate (1.8:1.2:1), and petroleum ether (60 ℃ C. -90 ℃ C.) -chloroform-ethyl acetate (2.2:0.8: 1).

In one example, the color reagent used in the thin layer chromatography is vanillin sulfuric acid test solution.

The test methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biomaterials, if not specifically mentioned, are commercially available.

Example 1

The embodiment provides a method for identifying ginger in ginger bamboo shavings traditional Chinese medicine formula granules, which mainly adopts thin-layer chromatography for identification and comprises the following steps:

1. instruments, reagents and reagents

The instrument comprises the following steps: a thin layer auto imager (CAMAG REPROSTAR 3, Switzerland Camma), a full-AUTOMATIC thin layer sample applicator (AUTOMATIC TLC SAMPLER4, Switzerland Camma), a ten-thousandth balance (ME204E, Switzerland Mettler), an ultrasonic cleaner (KQ-500DE, Kunshan ultrasonic Instrument Co., Ltd.), an ultra-pure water system (Milli-Q Direct, Merck Co., Ltd.), and a water bath (HWS-28, Shanghai-constant technology Co., Ltd.); a double-tank deployment cylinder; silica gel G thin-layer plate (G, lot # 20191008, Qingdao ocean chemical Co., Ltd.), Silica gel G thin-layer plate (G, lot # 20210326, Qingdao Sekugaku Seiko Co., Ltd.), Silica gel G thin-layer plate (TLC Silica gel 60, lot # HXD4333226, Merck Co., Ltd.), Silica gel G thin-layer plate (SG, lot # 20210309, Nicoti City chemical industry research institute).

Reagent: methanol (batch No. SY22009009, Sdn science, Inc.); petroleum ether (60-90 ℃ C.) (batch No. SY22007012, Fochen chemical Co., Ltd.); trichloromethane (batch number: SY22011016, Guangzhou chemical agent plant); ethyl acetate (batch No. SY22009001, Szegaku K.K.); polyamide powder (batch No. SY21901016, chemical reagents of national drug group, Ltd.); the water was ultrapure water (self-made in the laboratory).

Reagent testing: ginger bamboo shavings traditional Chinese medicine formula granules (batch numbers CG2006002, CG2006003 and CG 2006004; source: Guangdong party pharmaceutical Co., Ltd.); bamboo shavings Chinese medicinal formula granules (batch number CG 2006011; source: Guangdong one-party pharmacy Co., Ltd.); 6-gingerol (batch No. 111833-202007, China institute for testing food and drug); zingerone (batch No. 111807-201802, China institute for testing food and drug).

2. Preparation of the solution

2.1 preparation of test solutions

Taking about 2.5g of the ginger-bamboo shavings traditional Chinese medicine formula particles, grinding, adding 50mL of 80% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, drying filtrate by distillation, adding 20mL of water into residues for dissolving, shaking and extracting by ethyl acetate for 2 times, 20mL each time, combining ethyl acetate solutions, drying by distillation, adding 2mL of methanol into residues for dissolving, adding the residues onto a polyamide column (polyamide 100-200 meshes, 2g, 2cm in inner diameter, and loading into the column by a dry method), eluting by 50mL of 30% methanol, collecting eluent, drying by distillation, and adding 1mL of methanol into residues for dissolving to obtain a sample solution.

2.2 preparation of control solutions

Accurately weighing appropriate amount of 6-gingerol and zingerone, and adding methanol to obtain mixed reference solutions each containing 0.2mg per 1 mL.

2.3 preparation of negative sample solution

Taking 2.5g of a bamboo shaving traditional Chinese medicine formula particle sample, grinding, adding 50mL of 80% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, evaporating filtrate to dryness, adding 20mL of water to the residue to dissolve, shaking and extracting for 2 times by using ethyl acetate, extracting for 20mL each time, combining ethyl acetate solutions, evaporating to dryness, adding 2mL of methanol to the residue to dissolve, adding the residue to a polyamide column (100-200 meshes of polyamide, 2g, 2cm in inner diameter, and filling the column by a dry method), eluting by using 50mL of 30% methanol, collecting eluent, evaporating to dryness, and adding 1mL of methanol to the residue to dissolve to serve as a negative control solution.

2.4 preparation of color-developing agent

Dissolving vanillin 0.1g in 10mL of sulfuric acid.

3 conditions of thin layer chromatography

Thin-layer plate: silica gel G plate.

Developing agent: petroleum ether (60-90 deg.C) -trichloromethane-ethyl acetate (2:1: 1).

Sample application mode: spraying stripe-shaped sample application.

The unfolding mode is as follows: and (3) adopting a double-groove unfolding cylinder, wherein the thin-layer plate is pre-saturated for 15 minutes, and the unfolding distance is about 8 cm.

And (6) inspection: spraying vanillin sulfuric acid solution, and heating at 105 deg.C until the spots are clearly developed.

4 investigation of different sample quantities of ginger-bamboo shavings traditional Chinese medicine formula particles

Respectively sucking the sample solution of rhizoma Zingiberis recens-caulis Bambusae in Taenia Chinese medicinal formula granule (CG2006002) and the mixed reference solution of 6-gingerol and zingiberone on the same silica gel G thin layer plate, spreading with petroleum ether (60-90 deg.C) -chloroform-ethyl acetate (2:1:1) as developing agent, taking out, air drying, spraying vanillin sulfuric acid solution, heating at 105 deg.C until the spots are clear, and inspecting in sunlight.

The results are shown in FIG. 1: as can be seen from FIG. 1, when the sample amount of the sample solution is 10 μ L and the sample amount of the mixed reference solution is 10 μ L, the color development of the sample and reference chromatograms is clear, the resolution is good, no tailing phenomenon occurs, and the background is free of interference. Spots of the same color appear in the chromatogram of the test solution at positions corresponding to those in the chromatogram of the control solution. Therefore, the sample solution was selected to be dispensed in an amount of 10. mu.L, and the control solution was selected to be dispensed in an amount of 10. mu.L.

5 investigation of specificity of ginger-bamboo shavings traditional Chinese medicine formula granules

Respectively sampling the test solution of the rhizoma zingiberis bambusae caulis et folium caulis in taeniis traditional Chinese medicine formula granule, the mixed reference solution of 6-gingerol and zingiberone, and the negative reference solution on the same silica gel G thin layer plate, developing with petroleum ether (60-90 ℃) -trichloromethane-ethyl acetate (2:1:1) as a developing agent, taking out, air drying, spraying vanillin sulfuric acid test solution, heating at 105 ℃ until the spots are clearly developed, and inspecting under sunlight.

The results are shown in FIG. 2: as can be seen from FIG. 2, the test sample chromatogram shows spots of the same color at the positions corresponding to those of the control chromatogram, and the negative sample is not interfered. The thin layer method is proved to have good specificity.

6 investigation of different temperatures

Respectively sampling the test solution of the rhizoma zingiberis bambusae caulis in taeniam traditional Chinese medicine formula granule, the 6-gingerol and zingiberone mixed reference solution on the same silica gel G thin-layer plate, respectively developing at normal temperature and low temperature by using petroleum ether (60-90 ℃) -trichloromethane-ethyl acetate (2:1:1) as a developing agent, taking out, drying in the air, spraying vanillin sulfuric acid test solution, heating at 105 ℃ until the spots are clear in color, and inspecting in the sunlight.

The results of the experiment are shown in FIGS. 3 and 4: as can be seen from the figures 3 and 4, the color spots of the color spectrums of the ginger-bamboo shavings traditional Chinese medicine formula granules and the 6-gingerol and zingiberone reference substance are clear, the separation degree is good, and no tailing phenomenon exists. Spots with the same color appear on the chromatogram of the test sample at the positions corresponding to the chromatogram of the reference substance, which shows that the temperature reduction has no obvious influence on the thin-layer identification of the traditional Chinese medicine formula granules of the ginger bamboo shavings, and shows that the thin-layer identification method has good durability under the conditions of normal temperature and low temperature.

7 different humidity investigation

Respectively sampling the test solution of the rhizoma Zingiberis recens-caulis Bambusae in Taenia Chinese medicinal granule, the 6-gingerol and zingiberone mixed reference solution on the same silica gel G thin layer plate, developing under high humidity and low humidity conditions with petroleum ether (60-90 deg.C) -chloroform-ethyl acetate (2:1:1) as developing agent, taking out, air drying, spraying vanillin sulfuric acid solution, heating at 105 deg.C until the spots are clear, and inspecting in sunlight.

The results of the experiment are shown in FIGS. 5 and 6: as can be seen from figures 5 and 6, under high humidity and low humidity conditions, the spots of the chromatogram of the test sample of the caulis bambusae in taeniam Chinese medicinal formula granule and the chromatogram of the 6-gingerol and zingiberone reference sample are clear in color, better in separation degree, free of trailing phenomenon and free of interference on background. Spots of the same color appear in the chromatogram of the test solution at positions corresponding to those in the chromatogram of the control solution. The thin layer identification method has no obvious influence on the thin layer identification of the traditional Chinese medicine formula granules of the ginger bamboo shavings, and the thin layer identification method has good durability to different humidity.

8 thin layer plate investigation of different manufacturers

Respectively taking a test solution of a traditional Chinese medicine formula particle of caulis bambusae in taeniam, 6-gingerol and zingerone mixed reference solution, spotting the solution on silica gel G thin-layer plates (marine silica gel G plate, Nicoti silver dragon silica gel G plate and Merck silica gel G plate) of different manufacturers, respectively developing under the same temperature and humidity conditions by using petroleum ether (60-90 ℃) -trichloromethane-ethyl acetate (2:1:1) as a developing agent, taking out, drying in the air, spraying a vanillin sulfuric acid test solution, heating at 105 ℃ until spots are clearly developed, and inspecting under sunlight.

The experimental results are shown in fig. 7, 8, 9 and 10: as can be seen from FIGS. 7, 8, 9 and 10, the spots of the chromatogram of the test sample of the Zhuru traditional Chinese medicine formula particle and the chromatograms of the 6-gingerol and the zingerone reference substance of the Zhuru traditional Chinese medicine formula particle of ginger are clear in color and better in separation degree by adopting silica gel G thin layer plates (marine silica gel G plate, Spermacology silica gel G plate, Yinlong silica gel G plate and Merck silica gel G plate) of different manufacturers. Spots of the same color appear in the chromatogram of the test solution at positions corresponding to those in the chromatogram of the control solution. The durability of the thin layer identification method on silica gel G thin layer plates of different manufacturers is good.

9 small knot

The Rf value of each spot of the chromatogram obtained by the embodiment is moderate, the integral separation effect is good, the spots are clear, the negative is free from interference, and the method is proved to have good durability through different temperature and humidity investigation. Spots of the same color appear in the chromatogram of the test solution at positions corresponding to those in the chromatogram of the control solution. The method is proved to be well used for identifying ginger components in the ginger bamboo shavings traditional Chinese medicine formula particles by thin-layer chromatography with 6-gingerol and zingerone as reference.

Example 2

The embodiment provides a method for identifying ginger in ginger bamboo shavings traditional Chinese medicine formula granules, which comprises the following steps:

1 preparation of test solutions

Taking about 2.5g of bamboo shavings traditional Chinese medicine formula granules (CG2006002, CG2006003 and CG2006004) respectively, grinding, adding 50mL of 80% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, drying filtrate by distillation, adding 20mL of water into residues to dissolve, shaking and extracting for 2 times by using ethyl acetate, wherein 20mL of ethyl acetate is used for each time, combining ethyl acetate solution, drying by distillation, adding 2mL of methanol into residues to dissolve, adding the residues onto a polyamide column (polyamide is 100-200 meshes, 2g, the inner diameter is 2cm, and the column is filled by a dry method), eluting by using 50mL of 30% methanol, collecting eluent, drying by distillation, adding 1mL of methanol into residues to dissolve, and using the obtained solution as a sample solution.

2 preparation of control solutions

Accurately weighing appropriate amount of 6-gingerol and zingerone, and adding methanol to obtain mixed reference solutions each containing 0.2mg per 1 mL.

2.3 unfolding

According to thin layer chromatography (general rule 0502 of 2020 version of Chinese pharmacopoeia), 10 μ L of each of the two solutions is absorbed, and the two solutions are respectively spotted on the same silica gel G thin layer plate, and petroleum ether (60-90 ℃) and trichloromethane-ethyl acetate (2:1:1) are used as developing agents, and the developing distance is about 8 cm.

2.4 color development

Spraying vanillin sulfuric acid solution, and heating at 105 deg.C until the spots are clearly developed.

2.5 determination of results

The test results are shown in FIG. 11: in the chromatograms of the three test samples, spots with the same color are displayed at the corresponding positions of the chromatograms of the reference sample, which indicates that the three batches of ginger-bamboo shavings formula granules contain ginger components and meet the regulations.

Example 3

This example is a modification of example 2, and the modification to example 2 mainly includes a step of "1 preparing a sample solution", and specifically includes:

taking about 2.5g of bamboo shavings traditional Chinese medicine formula granules (CG2006002, CG2006003 and CG2006004) respectively, grinding, adding 50mL of 78% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, evaporating filtrate, adding 20mL of water into residue to dissolve, shaking and extracting for 1 time by using ethyl acetate, 15mL each time, combining ethyl acetate solution, evaporating to dryness, adding 2mL of methanol into residue to dissolve, adding the residue onto a polyamide column (polyamide is 100-200 meshes, 1.5g, the inner diameter is 1.5cm, and the column is filled by a dry method), eluting by using 50mL of 25% methanol, collecting eluent, evaporating to dryness, adding 1mL of methanol into residue to dissolve, and taking the residue as a sample solution.

The test results are shown in FIG. 12: in the chromatograms of the three test samples, spots with the same color are displayed at the corresponding positions of the chromatograms of the reference sample, which indicates that the three batches of ginger-bamboo shavings traditional Chinese medicine formula granules contain ginger components and meet the regulations.

Example 4

This example is a modification of example 2, and the modification to example 2 mainly includes a step of "1 preparing a sample solution", and specifically includes:

taking about 2.5g of bamboo shavings traditional Chinese medicine formula granules (CG2006002, CG2006003 and CG2006004) respectively, grinding, adding 50mL of 85% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, evaporating filtrate, adding 20mL of water into residues to dissolve, shaking and extracting with ethyl acetate for 3 times, 25mL each time, combining ethyl acetate solution, evaporating to dryness, adding 2mL of methanol into residues to dissolve, adding the residues onto a polyamide column (polyamide 100-200 meshes, 2.5g, the inner diameter is 2.5cm, loading into the column by a dry method), eluting with 50mL of 55% methanol, collecting eluent, evaporating to dryness, adding 1mL of methanol into residues to dissolve, and taking the residues as a sample solution.

The test results are shown in FIG. 13: in the chromatograms of the three test samples, spots with the same color are displayed at the corresponding positions of the chromatograms of the reference sample, which indicates that the three batches of ginger-bamboo shavings traditional Chinese medicine formula granules contain ginger components and meet the regulations.

Example 5

This example is a modification of example 2, and the modification to example 2 mainly includes a step of "1 preparing a sample solution", and specifically includes:

taking about 2.5g of ginger-bamboo shavings traditional Chinese medicine formula granules (CG2006002) respectively, grinding, adding 3 parts of methanol, 80% methanol and 50% methanol respectively, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, evaporating filtrate to dryness, dissolving residue methanol 2mL to obtain a sample solution.

The test results are shown in FIG. 14: in the chromatograms of the three samples, spots are relatively blurred because of only pure ultrasonic extraction and no impurity removal and purification steps. However, it can still be seen from the figure that the 80% methanol extraction of the sample spot is much more than the methanol extraction and the 50% methanol extraction, because both zingerone and 6-gingerol are slightly soluble in water and insoluble in the 50% methanol solution, so the spot is not observed in the chromatogram. Whereas, the extraction with 80% methanol is more intense than the extraction with methanol, and the extraction efficiency is higher than that with methanol. Therefore, 80% methanol is selected as the extraction solvent of the first step, which provides a basis for the subsequent purification experiment to extract the zingerone and 6-gingerol to the maximum extent.

Example 6

This example is a modification of example 2, and the modification to example 2 mainly includes a step of "1 preparing a sample solution", and specifically includes:

taking about 2.5g of the ginger-bamboo shavings traditional Chinese medicine formula particles, grinding, respectively adding 50mL of 80% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, drying the filtrate by distillation, adding 20mL of water into the residue to dissolve, shaking and extracting for 2 times with ethyl acetate, 20mL each time, combining ethyl acetate solutions, drying by distillation, adding 1mL of methanol into the residue to dissolve to obtain a sample solution 1.

Taking about 2.5g of rhizoma Zingiberis recens caulis Bambusae in Taenia Chinese medicinal granule, grinding, adding 20mL of water for dissolving, extracting with ethyl acetate under shaking for 2 times, each time 20mL, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 1mL of methanol to obtain sample solution 2.

The test results are shown in FIG. 15: the liquid-liquid extraction is carried out by simply using ethyl acetate, the permeability of the ethyl acetate to particles is poor, so that the extraction efficiency is low, 6-gingerol is hardly extracted, and the characteristic components of the ginger, namely, zingerone and 6-gingerol cannot be simultaneously detected. While the ultrasonic treatment with 80% methanol and the extraction with ethyl acetate can extract zingerone and 6-gingerol simultaneously, but the chromatographic background is deep, the main spots are not obvious enough, other spots near the zingerone spots interfere with each other, the separation degree is not good, and further purification is needed.

Comparative example 1

The comparison example provides a method for identifying ginger in ginger bamboo shavings traditional Chinese medicine formula particles, which comprises the following steps:

1 preparation of test solutions

Taking about 2.5g of the ginger-bamboo shavings traditional Chinese medicine formula granules, paralleling 2 parts, grinding, respectively adding 50mL of 80% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, drying the filtrate by distillation, adding 20mL of water into the residue for dissolving, shaking and extracting for 2 times by using ethyl acetate, 20mL each time, combining ethyl acetate solutions, drying by distillation, adding 2mL of methanol into the residue for dissolving, and adding the mixture onto a polyamide column (polyamide is 100-200 meshes, 2g, the inner diameter is 2cm, and the column is filled by a dry method).

The 1 st sample is eluted by 50mL of water, then eluted by 50mL of 10% methanol and 50mL of 30% methanol, the eluates are respectively collected and evaporated to dryness, and 1mL of methanol is added into the residue to dissolve the residue to obtain test solution 1, 2 and 3.

The 2 nd sample was directly eluted with 50mL of 30% methanol, the eluate was collected and evaporated to dryness, and the residue was dissolved in 1mL of methanol to give a sample solution 4.

2 preparation of control solutions

Accurately weighing appropriate amount of 6-gingerol and zingerone, and adding methanol to obtain control solutions containing 0.2mg per 1 mL.

3 unfolding

According to thin layer chromatography (general 0502 of the 2020 version of Chinese pharmacopoeia), 10 μ L of the above solution was drawn up and spotted on the same silica gel G thin layer plate (SG, lot number: 20210309, Nicoti, chemical industry research institute), and developed with petroleum ether (60-90 ℃) -chloroform-ethyl acetate (2:1:1) as developing agent at a development distance of about 8 cm.

4 color development

Spraying vanillin sulfuric acid solution, and heating at 105 deg.C until the spots are clearly developed.

5 determination of results

As a result: the polyamide adsorption belongs to hydrogen bond adsorption, and the elution capacity of a solvent is from weak to strong, wherein water is methanol or ethanol, acetone is diluted sodium hydroxide or ammonia water is formamide, dimethylformamide is dimethylformamide and urea aqueous solution. Generally, when the elution solvent is selected, water or methanol-water/ethanol-water solution with different proportions is firstly used for elution. As can be seen from fig. 16:

in the 1 st sample, water is used as an elution solution to elute part of the zingerone component, the rest of the zingerone and the 6-gingerol are both in the 30% methanol part, and the two main components are not in the same part.

The sample No. 2 was eluted directly with 30% methanol, and both zingerone and 6-gingerol were in the same location. And the water washing does not remove too many impurities except the two main components, so the impurity removal effect is not obvious. Therefore, 30% methanol is selected to directly elute better.

Comparative example 2

The comparison example provides a method for identifying ginger in ginger bamboo shavings traditional Chinese medicine formula particles, which comprises the following steps:

1 preparation of test solutions

Taking about 2.5g of the ginger-bamboo shavings traditional Chinese medicine formula granules, paralleling 2 parts, grinding, respectively adding 50mL of 80% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, drying the filtrate by distillation, adding 20mL of water into the residue for dissolving, shaking and extracting for 2 times by using ethyl acetate, 20mL each time, combining ethyl acetate solutions, drying by distillation, adding 2mL of methanol into the residue for dissolving, and adding the mixture onto a polyamide column (polyamide is 100-200 meshes, 2g, the inner diameter is 2cm, and the column is filled by a dry method).

The 1 st sample was eluted with 50mL of 30% methanol, the 2 nd sample was eluted with 50mL of 60% methanol, and the eluates were collected and evaporated to dryness, and the residue was dissolved in 1mL of methanol to give test solutions 1 and 2.

2 preparation of control solutions

Accurately weighing appropriate amount of 6-gingerol and zingerone, and adding methanol to obtain control solutions each containing 0.2mg per 1 mL.

3 unfolding

According to thin layer chromatography (general 0502 of the 2020 version of Chinese pharmacopoeia), 10 μ L of the above solution was drawn up and spotted on the same silica gel G thin layer plate (SG, lot number: 20210309, Nicoti, chemical industry research institute), and developed with petroleum ether (60-90 ℃) -chloroform-ethyl acetate (2:1:1) as developing agent at a development distance of about 8 cm.

4 color development

Spraying vanillin sulfuric acid solution, and heating at 105 deg.C until the spots are clearly developed.

5 determination of results

As a result: as can be seen from fig. 17, both major components were eluted with 30% methanol or 60% methanol, but 30% methanol eluted less impurities and the background of the chromatographic band was lighter than that eluted with 60% methanol. And 30% methanol is preferred as an elution solvent for environmental protection and reduced use of organic solvents.

Comparative example 3

The comparison example provides a method for identifying ginger in ginger bamboo shavings traditional Chinese medicine formula particles, which comprises the following steps:

1 preparation of test solutions

Taking about 2.5g of the bamboo shavings traditional Chinese medicine formula particles, paralleling 4 parts, grinding, respectively adding 50mL of 80% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, drying filtrate by distillation, adding 20mL of water into residues for dissolving, shaking and extracting for 2 times by using ethyl acetate, 20mL each time, combining ethyl acetate solutions, drying by distillation, adding 2mL of methanol into residues for dissolving, adding the residues onto a silica gel column (column chromatography silica gel 100-200 meshes, 2g, the inner diameter is 2cm, loading the column by a dry method), respectively eluting by using petroleum ether (60-90 ℃), ethyl acetate (1:1), ethyl acetate and 50mL of methanol, collecting eluent, drying by distillation, and adding 1mL of methanol into residues for dissolving respectively to obtain sample solutions 1, 2, 3 and 4.

Taking about 2.5g of the ginger and bamboo shavings traditional Chinese medicine formula particles, grinding, respectively adding 50mL of 80% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, drying filtrate by distillation, adding 20mL of water into residues for dissolving, shaking and extracting for 2 times by ethyl acetate, 20mL each time, combining ethyl acetate solutions, drying by distillation, adding 2mL of methanol into residues for dissolving, adding the residues onto a polyamide column (polyamide 100-200 meshes, 2g, the inner diameter of the column is 2cm, and the column is filled by a dry method), eluting by 50mL of 30% methanol, collecting eluent, drying by distillation, and adding 1mL of methanol into residues for dissolving to obtain a sample solution 5.

2 preparation of control solutions

Accurately weighing appropriate amount of 6-gingerol and zingerone, and adding methanol to obtain control solutions containing 0.2mg per 1 mL.

3 unfolding

According to thin layer chromatography (general 0502 of the 2020 version of Chinese pharmacopoeia), 10 μ L of the above solution was drawn up and spotted on the same silica gel G thin layer plate (SG, lot number: 20210309, Nicoti, chemical industry research institute), and developed with petroleum ether (60-90 ℃) -chloroform-ethyl acetate (2:1:1) as developing agent at a development distance of about 8 cm.

4 color development

Spraying vanillin sulfuric acid solution, and heating at 105 deg.C until the spots are clearly developed.

5 determination of results

As a result: as can be seen from FIG. 18, the 6-gingerol and zingerone components of ginger were purified by silica gel column, and eluted with petroleum ether (60 ℃ C. to 90 ℃ C.), and no component was eluted. The chromatogram obtained by elution with petroleum ether (60 ℃ C. -90 ℃ C.): ethyl acetate (1:1), ethyl acetate, methanol is not very different, indicating that it is sufficient to elute the desired components with petroleum ether (60 ℃ C. -90 ℃ C.): ethyl acetate (1: 1). However, compared with the polyamide column, the background is darker, other impurities are dried near the 6-gingerol spot, the separation degree and the definition of the spot are poor, and the impurity removal effect is inferior to that of the polyamide column. Thus, polyamide was chosen as the column chromatography packing.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments, are within the scope of the present invention as set forth in the appended claims. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.