A thin-layer chromatography identification method for multi-testing of a single plate of Shengmaiyin

Abstract

The present invention belongs to a detection method for traditional Chinese medicine preparations, and relates to a thin-layer chromatography identification method for multi-testing of a single plate of Shengmaiyin; the thin-layer chromatography identification method includes: (1) preparation of a test solution; (2) preparation of a reference substance solution; (3) preparation of a reference crude drug solution: (4) thin-layer chromatography identification: respectively spotting the test solution, the reference substance solution and the reference crude drug solution on the same reversed-phase thin-layer plate, using acetonitrile-methanol-water with a volume ratio of 3:3:2 as the developing agent, developing, taking out, drying, spraying with sulfuric acid ethanol solution, heating, and inspecting. The present invention uses a single thin-layer plate to simultaneously identify the three herbs in the whole formula of Shengmaiyin, and has the advantages of simplicity, rapidity, high efficiency, etc. At the same time, the chromatographic spots are clear, and the specificity and reproducibility are good. Compared with the prior art, the pretreatment method of the present invention is simple, and the reagents used have low toxicity; at the same time, it makes up for the technical defect that there is no quality control of ginsenoside Rg3 in Shengmaiyin by thin-layer identification in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of detection of traditional Chinese medicine preparations. Specifically, it relates to a thin-layer chromatography identification method for multi-testing of a single plate of Shengmaiyin. Background Art

[0002] Shengmaiyin is composed of three herbs: red ginseng, ophiopogon root, and schisandra fruit, and has the effects of replenishing qi and restoring pulse, nourishing yin and promoting fluid production. It can be used for qi and yin deficiency, palpitations and shortness of breath, and spontaneous sweating with a weak pulse.

[0003] Currently, the current quality standard of Shengmaiyin is the first part of the Chinese Pharmacopoeia 2020 edition, including character, identification, inspection, and content determination. Under the identification item, red ginseng uses ginsenoside diol and ginsenoside triol as references, ophiopogon root uses the reference medicinal material of ophiopogon root as a reference, and schisandra fruit uses schisandrol A and the reference medicinal material of schisandra fruit as references. The preparation methods of the test solutions used for identifying each herb are different, and the developing agents are also different. The specific information is shown in Table 1 below.

[0004] Table 1 Thin-layer identification method of Shengmaiyin included in the Chinese Pharmacopoeia

[0005]

[0006] For the thin-layer identification method of Shengmaiyin included in the Chinese Pharmacopoeia, ginsenoside diol and ginsenoside triol are used as identification indicators for red ginseng. Ginsenoside diol is the degradation product of ginsenoside diol type-A (ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rd, etc.), and ginsenoside triol type-B (ginsenoside Rg1, ginsenoside Re, and ginsenoside Rf) hydrolyzes to form ginsenoside triol under the conditions of adding acid and heating. The above two components are the acid-heated degradation products of Shengmaiyin, not the original components in the preparation, and can be produced by the hydrolysis of many ginsenoside components. Using these as identification indicators, the specificity is not strong. Existing public information shows that Shengmaiyin contains ginsenoside Rg3, which has significant pharmacological effects and has the effect of anti-arrhythmia. Its mechanism of action is to block the occurrence of ion overload and protect myocardial cells from hypoxia. In addition, the application of ginsenoside Rg3 in the treatment or prevention of enterovirus infection. Therefore, it is of great significance to control the quality of ginsenoside Rg3 in Shengmaiyin to ensure the safety and effectiveness of clinical medication, but there is no method for identifying ginsenoside Rg3 in Shengmaiyin by thin-layer chromatography in the existing technology. e , 1 , 1 and ginsenoside Rd, etc.) of ginsenoside diol type-A, and ginsenoside triol type-B (ginsenoside Rg1 1 , ginsenoside Re e and ginsenoside Rf) hydrolyzes to form ginsenoside triol under the conditions of adding acid and heating. The above two components are the acid-heated degradation products of Shengmaiyin, not the original components in the preparation, and can be produced by the hydrolysis of many ginsenoside components. Using these as identification indicators, the specificity is not strong. Existing public information shows that Shengmaiyin contains ginsenoside Rg3, which has significant pharmacological effects and has the effect of anti-arrhythmia. Its mechanism of action is to block the occurrence of ion overload and protect myocardial cells from hypoxia. In addition, the application of ginsenoside Rg3 in the treatment or prevention of enterovirus infection. Therefore, it is of great significance to control the quality of ginsenoside Rg3 in Shengmaiyin to ensure the safety and effectiveness of clinical medication, but there is no method for identifying ginsenoside Rg3 in Shengmaiyin by thin-layer chromatography in the existing technology.

[0007] Currently, there is no thin-layer chromatography identification method for multi-testing of a single plate of Shengmaiyin in the existing technology; for the thin-layer identification method of Shengmaiyin included in the Chinese Pharmacopoeia, three test solutions are prepared separately, and moreover, the pretreatment methods of the test samples and the reference medicinal material solutions are complex, and the reagents used are highly toxic; the single herbs are detected separately, resulting in problems such as large workload, high detection cost, time-consuming, laborious, and reagent-consuming. Summary of the Invention

[0008] In view of the problems existing in the above-mentioned prior art, the present invention provides a thin-layer chromatography identification method for multi-testing of a single plate of Shengmaiyin. The present invention is a thin-layer chromatography identification method for simultaneously determining the three herbs in the whole formula of Shengmaiyin with a single thin-layer plate and a single test solution, which can simultaneously identify ginseng rubra, ophiopogon japonicus, and schisandra chinensis, ensuring the product quality of Shengmaiyin and the safety of clinical medication.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions.

[0010] A thin-layer chromatography identification method for multi-testing of a single plate of Shengmaiyin includes the following steps:

[0011] S1. Preparation of the test solution: Take a sample of Shengmaiyin, extract it with water-saturated n-butanol, wash it, evaporate to dryness, dissolve the residue in methanol, separate the solid and liquid, and take the supernatant to obtain the test solution;

[0012] S2. Preparation of the reference solution: Take reference substances of ginsenoside Rg3 and schisandrol A, and add methanol to them respectively to obtain the reference solutions;

[0013] S3. Preparation of the reference medicinal material solution: Take reference medicinal materials of ginseng rubra, ophiopogon japonicus, and schisandra chinensis, and prepare the reference medicinal material solutions respectively;

[0014] S4. Thin-layer chromatography identification: Absorb the test solution, reference solution, and reference medicinal material solution and spot them on the same reversed-phase thin-layer plate respectively, use acetonitrile-methanol-water with a volume ratio of 3:3:2 as the developing agent, develop, take out, dry in the air, spray with sulfuric acid-ethanol reagent, heat, and examine. Then make a comparison.

[0015] Preferably, the ratio of Shengmaiyin to water-saturated n-butanol in step S1 is 20 mL: 15 - 25 mL; more preferably 20 mL: 20 mL.

[0016] In some embodiments, the preparation method of the test solution is: Take 20 mL of Shengmaiyin sample, extract it with 15 - 25 mL of water-saturated n-butanol, wash it, evaporate the water-saturated n-butanol solution to dryness, dissolve the residue in 1 mL of methanol, separate the solid and liquid, and take the supernatant to obtain the test solution.

[0017] Preferably, the preparation method of the reference solution in step S2 is: Take reference substances of ginsenoside Rg3 and schisandrol A, and add methanol to them respectively to obtain the reference solution of ginsenoside Rg3 and the reference solution of schisandrol A.

[0018] Further preferably, the concentration of the ginsenoside Rg3 reference substance solution is 0.3 - 0.8 mg / mL, and the concentration of the schisandrol A reference substance solution is 0.8 - 1.2 mg / mL.

[0019] In some embodiments, the preparation method of the reference substance solution is as follows: Take the ginsenoside Rg3 reference substance and add methanol thereto to prepare a ginsenoside Rg3 reference substance solution with a concentration of 0.3 - 0.8 mg / mL; take the schisandrol A reference substance and add methanol thereto to prepare a schisandrol A reference substance solution with a concentration of 0.8 - 1.2 mg / mL.

[0020] Preferably, the preparation method of the reference crude drug solution in step S3 is as follows:

[0021] Take the red ginseng reference crude drug, add methanol and citric acid thereto, reflux, filter, evaporate the filtrate to dryness, dissolve the residue in water, then extract with water-saturated n-butanol, wash and evaporate to dryness, dissolve the residue in methanol, perform solid-liquid separation, and take the supernatant to prepare the red ginseng reference crude drug solution;

[0022] Take the ophiopogon japonicus reference crude drug, decoct with water, filter, add hydrochloric acid and water to the filtrate, boil, cool, extract with chloroform, evaporate the organic phase to dryness, dissolve the residue in methanol, perform solid-liquid separation, and take the supernatant to prepare the ophiopogon japonicus reference crude drug solution;

[0023] Take the schisandra chinensis reference crude drug, ultrasonically treat with dichloromethane, filter, evaporate the filtrate to dryness, dissolve the residue in ethyl acetate, perform solid-liquid separation, and take the supernatant to prepare the schisandra chinensis reference crude drug solution.

[0024] Further preferably, in the preparation process of the red ginseng reference crude drug solution of the present invention, the volume fraction of the methanol is 50 - 70%, more preferably 60%; the ratio of the red ginseng reference crude drug, methanol and citric acid is 2 - 6 g : 40 - 80 mL : 1.5 - 2.0 g, more preferably 4 g : 60 mL : 1.84 g; the reflux is carried out at 55 - 75°C for 1 - 6 h, more preferably at 65°C for 3 h;

[0025] In the preparation process of the ophiopogon japonicus reference crude drug solution of the present invention, the ratio of the ophiopogon japonicus reference crude drug and water is 1 g : 15 - 25 mL, more preferably 1 g : 20 mL; the decocting time is 5 - 20 min, more preferably 10 min; the ratio of the hydrochloric acid and water is 0.3 - 0.8 mL : 1 mL, more preferably 0.5 mL : 1 mL; the boiling time is 3 - 8 min, more preferably 5 min;

[0026] In the preparation process of the Ophiopogon japonicus reference crude drug solution of the present invention, the ratio of the Schisandra chinensis reference crude drug to dichloromethane is 1 g: 30 - 50 mL, and more preferably 1 g: 40 mL; the time of ultrasonic treatment is 10 - 50 min, and more preferably 30 min.

[0027] In some embodiments, the method for preparing the reference crude drug solution is as follows:

[0028] Take 2 - 6 g of the Ginseng rubra reference crude drug, add 40 - 80 mL of 50 - 70% methanol and 1.5 - 2.0 g of citric acid, reflux at 55 - 75 °C for 1 - 6 h, filter, evaporate the filtrate to dryness, dissolve the residue in water and then extract with water-saturated n-butanol, wash and evaporate to dryness, dissolve the residue in methanol, perform solid-liquid separation, take the supernatant to obtain the Ginseng rubra reference crude drug solution;

[0029] Take 1 g of the Ophiopogon japonicus reference crude drug, add 15 - 25 mL of water and decoct for 5 - 20 min, filter, add 0.3 - 0.8 mL of hydrochloric acid and 1 mL of water to the filtrate, boil for 3 - 8 min, cool, extract with 15 - 25 mL of chloroform, evaporate the organic phase to dryness, dissolve the residue in methanol, perform solid-liquid separation, take the supernatant to obtain the Ophiopogon japonicus reference crude drug solution;

[0030] Take 1 g of the Schisandra chinensis reference crude drug, add 30 - 50 mL of dichloromethane and perform ultrasonic treatment for 10 - 50 min, filter, evaporate the filtrate to dryness, dissolve the residue in ethyl acetate, perform solid-liquid separation, take the supernatant to obtain the Schisandra chinensis reference crude drug solution.

[0031] Preferably, the sulfuric acid ethanol reagent in step S4 is a 8 - 12 wt% sulfuric acid ethanol solution, and more preferably 10 wt%; the heating conditions are: heating temperature 102 - 105 °C, heating time 60 - 120 s; the inspection conditions are: visible light and 254 nm.

[0032] In some embodiments, the identification method of thin-layer chromatography in step S4 is as follows:

[0033] Absorb 3 - 7 μL of the test solution, reference solution and reference crude drug solution, respectively spot them on the same reverse-phase thin-layer plate, use acetonitrile - methanol - water with a volume ratio of 3:3:2 as the developing agent, develop, take out, dry, spray with an 8 - 12 wt% sulfuric acid ethanol solution, heat at 102 - 105 °C for 60 - 120 s, place it under daylight or 254 nm for inspection, compare, in the test solution chromatogram, at the positions corresponding to the reference solution chromatogram and the reference crude drug chromatogram, spots of the same color should appear.

[0034] It should be noted that:

[0035] The "solid-liquid separation" involved in the present invention is a conventional technical means for separating solid and liquid in the art, such as filtration, centrifugation, etc.

[0036] In the present invention, the "washing" involved is liquid-liquid extraction. First, 15 - 25 mL of ammonia test solution is added for washing, a total of 1 - 5 times, and then 15 - 25 mL of water saturated with n-butanol is added for washing, a total of 1 - 5 times.

[0037] The thin-layer chromatography identification method of the present invention is not limited to Shengmaiyin, and is also applicable to the quality control of products containing red ginseng, ophiopogon root, and schisandra fruit.

[0038] The beneficial effects of the present invention are as follows:

[0039] 1. The present invention provides a method for simultaneously identifying the three herbs in the whole formula of Shengmaiyin on a single thin-layer plate. The thin-layer identification method of Shengmaiyin included in the Chinese Pharmacopoeia uses three developing systems, namely cyclohexane - acetone (2:1), chloroform - acetone (4:1), and petroleum ether - ethyl formate - formic acid (15:5:1), to identify the three herbs. The present invention only needs to use one developing system of acetonitrile - methanol - water (3:3:2) to complete the thin-layer identification of all the herbs in the formula. The proportion of the developing agent used is appropriate, the toxicity of the reagent is small, and the preparation method is simple.

[0040] 2. The present invention uses one test solution to simultaneously identify the three herbs of red ginseng, ophiopogon root, and schisandra fruit in the whole formula. Compared with the thin-layer identification method included in the Chinese Pharmacopoeia, which requires the preparation of three test solutions respectively, this method only needs to prepare one test solution and uses the liquid-liquid extraction method, so the operation is simple. Moreover, the pretreatment of the thin-layer identification method included in the Chinese Pharmacopoeia uses toxic reagents such as hydrochloric acid, sulfuric acid, chloroform, and ether, while the pretreatment method of the present invention only uses reagents such as n-butanol, ammonia test solution, and water, and the toxicity is relatively small.

[0041] 3. The present invention simultaneously uses two reference substance solutions and three reference medicinal material solutions as references. After color development, it is inspected under daylight and 254 nm. The color chromatogram is more fully expressed, giving more room for analysts to think and judge.

[0042] 4. The Chinese Pharmacopoeia uses ginsenoside diol and ginsenoside triol as identification indicators. The present invention uses ginsenoside Rg3 reference substance and red ginseng reference medicinal material as references to identify the ginsenoside Rg3 component in the preparation, and has good specificity.

[0043] 5. When the present invention detects Shengmaiyin, the chromatographic spots of the test solution are clear, and there is no interference from each negative sample without the corresponding herb. The method has good reproducibility and can be used for the quality control of Shengmaiyin. Description of the Drawings

[0044] Figure 1Thin layer chromatogram developed for the sample detection of Example 1 (from top to bottom: visual inspection under daylight, visual inspection at 254 nm; in the figure, No. 1 is the reference solution of ginsenoside Rg3, No. 2 is the reference medicinal material solution of red ginseng, No. 3 is the reference medicinal material solution of Ophiopogon japonicus, No. 4 is the test solution of Shengmaiyin of batch 2408002, No. 5 is the test solution of Shengmaiyin of batch 2408004, No. 6 is the reference medicinal material solution of Schisandra chinensis, No. 7 is the reference solution of schisandrol A, and the sample application amounts of No. 1-7 are all 5 μL, the temperature is 23 °C, and the humidity is 53%);

[0045] Figure 2 Thin layer chromatogram developed for the investigation of the sample application amounts of the red ginseng-free negative sample and the Shengmaiyin test solution in Example 2 (from top to bottom: visual inspection under daylight, visual inspection at 254 nm; in the figure, No. 1 is the reference solution of ginsenoside Rg3, No. 2 is the reference medicinal material solution of red ginseng, No. 3 is the red ginseng-free negative test solution, No. 4 is the test solution of Shengmaiyin of batch 2408002, No. 5 is the reference solution of ginsenoside Rg3, No. 6 is the reference medicinal material solution of red ginseng, No. 7 is the red ginseng-free negative test solution, No. 8 is the test solution of Shengmaiyin of batch 2408002, No. 9 is the reference solution of ginsenoside Rg3, No. 10 is the reference medicinal material solution of red ginseng, No. 11 is the red ginseng-free negative test solution, No. 12 is the test solution of Shengmaiyin of batch 2408002, the sample application amount of No. 1-4 is 3 μL, the sample application amount of No. 5-8 is 5 μL, the sample application amount of No. 9-12 is 7 μL, the temperature is 22 °C, and the humidity is 80%);

[0046] Figure 3 Thin layer chromatogram developed for the investigation of the sample application amounts of the Ophiopogon japonicus-free negative sample and the Shengmaiyin test solution in Example 3 (from top to bottom: visual inspection under daylight, visual inspection at 254 nm; in the figure, No. 1 is the reference medicinal material solution of Ophiopogon japonicus, No. 2 is the Ophiopogon japonicus-free negative test solution, No. 3 is the test solution of Shengmaiyin of batch 2408002, No. 4 is the reference medicinal material solution of Ophiopogon japonicus, No. 5 is the Ophiopogon japonicus-free negative test solution, No. 6 is the test solution of Shengmaiyin of batch 2408002, No. 7 is the reference medicinal material solution of Ophiopogon japonicus, No. 8 is the Ophiopogon japonicus-free negative test solution, No. 9 is the test solution of Shengmaiyin of batch 2408002, the sample application amount of No. 1-3 is 3 μL, the sample application amount of No. 4-6 is 5 μL, the sample application amount of No. 7-9 is 7 μL, the temperature is 22 °C, and the humidity is 80%);

[0047] Figure 4TLC chromatograms for investigating the sample application volumes of the negative sample without Schisandra chinensis and the test sample of Shengmaiyin in Example 4 (from top to bottom: visual inspection under daylight, visual inspection at 254 nm; in the figure, No. 1 is the solution of schisandrol A reference, No. 2 is the solution of the medicinal material of Schisandra chinensis for reference, No. 3 is the negative test sample solution without Schisandra chinensis, No. 4 is the test sample solution of Shengmaiyin of batch 2408002, No. 5 is the solution of schisandrol A reference, No. 6 is the solution of the medicinal material of Schisandra chinensis for reference, No. 7 is the negative test sample solution without Schisandra chinensis, No. 8 is the test sample solution of Shengmaiyin of batch 2408002, No. 9 is the solution of schisandrol A reference, No. 10 is the solution of the medicinal material of Schisandra chinensis for reference, No. 11 is the negative test sample solution without Schisandra chinensis, No. 12 is the test sample solution of Shengmaiyin of batch 2408002, the sample application volume of No. 1-4 is 3 μL, the sample application volume of No. 5-8 is 5 μL, the sample application volume of No. 9-12 is 7 μL, the temperature is 23 °C, and the humidity is 53%);

[0048] Figure 5 TLC chromatograms for investigating the specificity of each sample without a certain herb in Example 5 (from top to bottom: visual inspection under daylight, visual inspection at 254 nm; in the figure, No. 1 is the solution of ginsenoside Rg3 reference, No. 2 is the solution of the medicinal material of red ginseng for reference, No. 3 is the negative test sample solution without red ginseng, No. 4 is the test sample solution of Shengmaiyin of batch 2408002, No. 5 is the negative test sample solution without Ophiopogon japonicus, No. 6 is the solution of the medicinal material of Ophiopogon japonicus for reference, No. 7 is the test sample solution of Shengmaiyin of batch 2408004, No. 8 is the negative test sample solution without Schisandra chinensis, No. 9 is the solution of the medicinal material of Schisandra chinensis for reference, No. 10 is the solution of schisandrol A reference, the sample application volume of No. 1-10 is 5 μL, the temperature is 23 °C, and the humidity is 53%);

[0049] Figure 6 TLC chromatogram of red ginseng in Shengmaiyin included in the Pharmacopoeia for Comparative Example 1 (in the figure, No. 1 is the test sample solution of Shengmaiyin of batch 2408002, No. 2 is the mixed reference solution of ginsendiol and ginsenoside R1, the sample application volume of No. 1 and 2 is 10 μL, the temperature is 20 °C, and the humidity is 50%);

[0050] Figure 7 TLC chromatogram of Ophiopogon japonicus in Shengmaiyin included in the Pharmacopoeia for Comparative Example 2 (in the figure, No. 1 is the test sample solution of Shengmaiyin of batch 2408002, No. 2 is the solution of the medicinal material of Ophiopogon japonicus for reference, the sample application volume of No. 1 and 2 is 5 μL, the temperature is 20 °C, and the humidity is 50%);

[0051] Figure 8 TLC chromatogram of Schisandra chinensis in Shengmaiyin included in the Pharmacopoeia for Comparative Example 3 (in the figure, No. 1 is the test sample solution of Shengmaiyin of batch 2408002, No. 2 is the solution of the medicinal material of Schisandra chinensis for reference, No. 3 is the solution of schisandrol A reference, the sample application volume of No. 1-3 is 5 μL, the temperature is 20 °C, and the humidity is 50%). Detailed implementation manners

[0052] The following description of the embodiments is only used to help understand the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art of this technology, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. The following description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but can be applied to a wider range that conforms to the principles and novel features disclosed herein. Although any methods and materials similar or equivalent to those described in the present invention can be used in the implementation or testing of the present invention, preferred methods and materials are listed herein.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0054] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0055] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available. It is worth noting that in this example, the concentration of hydrochloric acid is 36 - 38% (g / g), and the ammonia test solution is 40% aqueous ammonia solution (v / v).

[0056] Example 1

[0057] The Shengmaiyin samples were detected using reference substances and solutions of various reference crude drugs.

[0058] (1) Preparation of the test solution: Take 20 mL of Shengmaiyin, extract it 3 times with water-saturated n-butanol, 20 mL each time. Combine the water-saturated n-butanol layers, wash them 3 times with ammonia test solution, 20 mL each time, and then wash them 3 times with water saturated with n-butanol, 20 mL each time. Evaporate the water-saturated n-butanol solution on a water bath to dryness, dissolve the residue in 1 mL of methanol, centrifuge, and take the supernatant to obtain the test solution.

[0059] (2) Preparation of the reference substance solution: Take reference substance ginsenoside Rg3, and make a reference substance solution containing 0.5 mg per 1 mL with methanol; then take reference substance schisandrol A, and make a reference substance solution containing 1.0 mg per 1 mL with methanol.

[0060] (3) Preparation of each reference crude drug solution: Take 4 g of the reference crude drug of red ginseng, add 60 mL of 60% methanol, then add 1.84 g of citric acid, reflux in a water bath at 65°C for 3 h, filter, evaporate to dryness, dissolve the residue in 15 mL of water, extract with water-saturated n-butanol three times, 20 mL each time, combine the water-saturated n-butanol layers, wash with ammonia test solution three times, 20 mL each time, then wash with water saturated with n-butanol three times, 20 mL each time, evaporate the water-saturated n-butanol solution on a water bath to dryness, dissolve the residue in 2 mL of methanol, centrifuge, and take the supernatant to obtain the reference crude drug solution of red ginseng;

[0061] Take 1 g of the reference crude drug of Ophiopogon japonicus, add 20 mL of water, decoct for 10 minutes, filter, add 0.5 mL of hydrochloric acid and 1 mL of water to the filtrate, heat to boiling for 5 minutes, cool, extract with 20 mL of chloroform by shaking, separate the chloroform layer, evaporate to dryness, dissolve the residue in 1 mL of methanol, centrifuge, and take the supernatant to obtain the reference crude drug solution of Ophiopogon japonicus;

[0062] Take 1 g of the reference crude drug of Schisandra chinensis, add 40 mL of dichloromethane, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethyl acetate, centrifuge, and take the supernatant to obtain the reference crude drug solution of Schisandra chinensis.

[0063] (4) Pipette 5 μL of the test solution, reference solution and each reference crude drug solution respectively, spot them on the same reverse-phase thin-layer plate, use acetonitrile - methanol - water (volume ratio 3:3:2) as the developing solvent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clear, and examine under daylight and 254 nm.

[0064] The results showed that: in the chromatogram of the test solution, at the positions corresponding to the chromatograms of the reference substance and the reference crude drug, spots of the same color were shown. When detecting two batches of Shengmaiyin samples, the reproducibility was good and the resolution of each identification spot was good, as Figure 1 shown.

[0065] Example 2

[0066] Examine the sample application amounts of the negative sample without red ginseng and the test solution of Shengmaiyin.

[0067] (1) Preparation of the test solution: Take 20 mL of Shengmaiyin, extract with water-saturated n-butanol three times, 20 mL each time, combine the water-saturated n-butanol layers, wash with ammonia test solution three times, 20 mL each time, then wash with water saturated with n-butanol three times, 20 mL each time, evaporate the water-saturated n-butanol solution on a water bath to dryness, dissolve the residue in 1 mL of methanol, centrifuge, and take the supernatant to obtain the test solution.

[0068] (2) Preparation of the reference solution: Take the reference substance of ginsenoside Rg3, add methanol to make a reference solution containing 0.5 mg per 1 mL.

[0069] (3) Preparation of the control crude drug solution: Take 4 g of the control crude drug of red ginseng, add 60 mL of 60% methanol, then add 1.84 g of citric acid, reflux in a water bath at 65 °C for 3 h, filter, evaporate to dryness, dissolve the residue in 15 mL of water, extract with water-saturated n-butanol three times, 20 mL each time, combine the water-saturated n-butanol layers, wash with ammonia test solution three times, 20 mL each time, then wash with water saturated with n-butanol three times, 20 mL each time, evaporate the water-saturated n-butanol solution on a water bath to dryness, dissolve the residue in 2 mL of methanol, centrifuge, and take the supernatant to obtain the control crude drug solution of red ginseng.

[0070] (4) Preparation of the negative test solution without red ginseng: Prepare a negative sample without red ginseng according to the prescription process of Shengmaiyin. Measure 20 mL of the negative sample without red ginseng, extract with water-saturated n-butanol three times, 20 mL each time, combine the water-saturated n-butanol layers, wash with ammonia test solution three times, 20 mL each time, then wash with water saturated with n-butanol three times, 20 mL each time, evaporate the water-saturated n-butanol solution on a water bath to dryness, dissolve the residue in 1 mL of methanol, centrifuge, and take the supernatant to obtain the negative test solution without red ginseng.

[0071] (5) Pipette 3 μL, 5 μL, and 7 μL of the reference substance solution, the control crude drug solution, the test solution, and the negative test solution without red ginseng respectively, and spot them on the same reverse-phase thin-layer plate. Use acetonitrile - methanol - water (volume ratio 3:3:2) as the developing solvent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clear, and examine under daylight and at 254 nm.

[0072] The results showed that: when the sample volumes of the negative sample without red ginseng and the test solution of Shengmaiyin were between 3 μL and 7 μL, the specificity of ginsenoside Rg3 was good, as Figure 2 shown.

[0073] Example 3

[0074] Examine the sample volumes of the negative sample without Ophiopogon japonicus and the test solution of Shengmaiyin.

[0075] (1) Preparation of the test solution: Take 20 mL of Shengmaiyin, extract with water-saturated n-butanol three times, 20 mL each time, combine the water-saturated n-butanol layers, wash with ammonia test solution three times, 20 mL each time, then wash with water saturated with n-butanol three times, 20 mL each time, evaporate the water-saturated n-butanol solution on a water bath to dryness, dissolve the residue in 1 mL of methanol, centrifuge, and take the supernatant to obtain the test solution.

[0076] (2) Preparation of the control crude drug solution: Take 1 g of the control crude drug of Ophiopogon japonicus, add 20 mL of water, decoct for 10 minutes, filter, add 0.5 mL of hydrochloric acid and 1 mL of water to the filtrate, heat to boiling for 5 minutes, cool, extract with 20 mL of chloroform by shaking, separate the chloroform layer, evaporate to dryness, dissolve the residue in 1 mL of methanol, centrifuge, and take the supernatant to obtain the control crude drug solution of Ophiopogon japonicus.

[0077] (3) Preparation of the negative reference solution without Ophiopogon japonicus: Prepare a negative sample without Ophiopogon japonicus according to the prescription process of Shengmaiyin. Measure 20 mL of the negative sample without Ophiopogon japonicus, extract it with water-saturated n-butanol three times, 20 mL each time. Combine the water-saturated n-butanol layers, wash them with ammonia test solution three times, 20 mL each time, then wash them with water saturated with n-butanol three times, 20 mL each time. Evaporate the water-saturated n-butanol solution on a water bath until dry. Dissolve the residue in 1 mL of methanol, centrifuge, and take the supernatant to obtain the negative reference solution without Ophiopogon japonicus.

[0078] (4) Pipette 3 μL, 5 μL, and 7 μL of the reference medicinal material solution, the test solution, and the negative reference solution without Ophiopogon japonicus respectively, and spot them on the same reverse-phase thin-layer plate. Use acetonitrile - methanol - water (volume ratio 3:3:2) as the developing solvent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clear, and examine under daylight and 254 nm.

[0079] The results showed that: when the spotting amounts of the negative sample without Ophiopogon japonicus and the test solution of Shengmaiyin were between 3 - 7 μL, the specificity was good at the corresponding positions of the main spots of the reference medicinal material of Ophiopogon japonicus, as Figure 3 shown.

[0080] Example 4

[0081] Examine the spotting amounts of the negative sample without Schisandra chinensis and the test solution of Shengmaiyin.

[0082] (1) Preparation of the test solution: Take 20 mL of Shengmaiyin, extract it with water-saturated n-butanol three times, 20 mL each time. Combine the water-saturated n-butanol layers, wash them with ammonia test solution three times, 20 mL each time, then wash them with water saturated with n-butanol three times, 20 mL each time. Evaporate the water-saturated n-butanol solution on a water bath until dry. Dissolve the residue in 1 mL of methanol, centrifuge, and take the supernatant to obtain the test solution.

[0083] (2) Preparation of the reference solution: Take schisandrol A reference substance, and make a reference solution containing 1 mg per 1 mL with methanol.

[0084] (3) Preparation of the reference medicinal material solution: Take 1 g of Schisandra chinensis reference medicinal material, add 40 mL of dichloromethane, ultrasonically treat for 30 minutes, filter, evaporate the filtrate until dry, dissolve the residue in 1 mL of ethyl acetate, centrifuge, and take the supernatant to obtain the Schisandra chinensis reference medicinal material solution.

[0085] (4) Preparation of the negative reference solution without Schisandra chinensis: Prepare a negative sample without Schisandra chinensis according to the prescription process of Shengmaiyin. Measure 20 mL of the negative sample without Schisandra chinensis, extract it with water-saturated n-butanol three times, 20 mL each time. Combine the water-saturated n-butanol layers, wash them with ammonia test solution three times, 20 mL each time, then wash them with water saturated with n-butanol three times, 20 mL each time. Evaporate the water-saturated n-butanol solution on a water bath until dry. Dissolve the residue in 1 mL of methanol, centrifuge, and take the supernatant to obtain the negative reference solution without Schisandra chinensis.

[0086] (5) Pipette 3 μL, 5 μL, and 7 μL of the reference solution, reference crude drug solution, test solution, and negative reference solution without Schisandra chinensis respectively, and spot them on the same reverse-phase thin-layer plate. Use acetonitrile - methanol - water (volume ratio 3:3:2) as the developing solvent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clear, and examine under daylight and 254 nm.

[0087] The results showed that: when the spotting volume of the negative sample without Schisandra chinensis and the test solution of Shengmaiyin was 3 μL, the specificity of schisandrol A was good; when the spotting volume was 5 μL and 7 μL, there were spot tails near schisandrol A in the chromatogram of the negative reference solution without Schisandra chinensis, which affected the judgment of its specificity; because its specificity was good at 3 μL, it was determined that schisandrol A had specificity, as Figure 4 shown.

[0088] Example 5

[0089] Examine the specificity of each negative sample without certain ingredients.

[0090] (1) Preparation of the test solution: Take 20 mL of Shengmaiyin, extract it with water-saturated n-butanol three times, 20 mL each time. Combine the water-saturated n-butanol layers, wash them with ammonia test solution three times, 20 mL each time, then wash them with water saturated with n-butanol three times, 20 mL each time. Evaporate the water-saturated n-butanol solution on a water bath until dry. Dissolve the residue in 1 mL of methanol, centrifuge, and take the supernatant to obtain the test solution.

[0091] (2) Preparation of the reference solution: Take reference substance ginsenoside Rg3, dissolve it in methanol to prepare a reference solution containing 0.5 mg per 1 mL; then take reference substance schisandrol A, dissolve it in methanol to prepare a reference solution containing 1 mg per 1 mL.

[0092] (3) Preparation of the reference crude drug solution: Take 4 g of red ginseng reference crude drug, add 60 mL of 60% methanol, then add 1.84 g of citric acid, reflux in a water bath at 65 °C for 3 h, filter, evaporate to dryness, dissolve the residue in 15 mL of water, extract with water-saturated n-butanol three times, 20 mL each time. Combine the water-saturated n-butanol layers, wash with ammonia test solution three times, 20 mL each time, then wash with water saturated with n-butanol three times, 20 mL each time. Evaporate the water-saturated n-butanol solution on a water bath to dryness, dissolve the residue in 2 mL of methanol, centrifuge, and take the supernatant to obtain the red ginseng reference crude drug solution;

[0093] Take 1 g of ophiopogon japonicus reference crude drug, add 20 mL of water, decoct for 10 minutes, filter, add 0.5 mL of hydrochloric acid and 1 mL of water to the filtrate, heat to boiling for 5 minutes, cool, extract with 20 mL of chloroform by shaking, separate the chloroform layer, evaporate to dryness, dissolve the residue in 1 mL of methanol, centrifuge, and take the supernatant to obtain the ophiopogon japonicus reference crude drug solution;

[0094] Take 1 g of schisandra chinensis reference crude drug, add 40 mL of dichloromethane, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethyl acetate, centrifuge, and take the supernatant to obtain the schisandra chinensis reference crude drug solution.

[0095] (4) Preparation of the negative test solutions without each flavor: Prepare negative samples without red ginseng, ophiopogon japonicus, and schisandra chinensis respectively according to the prescription process of Shengmaiyin. Measure 20 mL of each negative sample respectively, extract with water-saturated n-butanol three times, 20 mL each time. Combine the water-saturated n-butanol layers, wash with ammonia test solution three times, 20 mL each time, then wash with water saturated with n-butanol three times, 20 mL each time. Evaporate the water-saturated n-butanol solution on a water bath to dryness, dissolve the residue in 1 mL of methanol, centrifuge, and take the supernatant to obtain the negative test solutions without each flavor.

[0096] (5) Pipette 5 μL of the reference solution, reference crude drug solution, test solution, and each negative test solution without flavor respectively, spot them on the same reversed-phase thin-layer plate, use acetonitrile - methanol - water (volume ratio 3:3:2) as the developing solvent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clear, and examine under daylight and 254 nm.

[0097] The results showed that: The specificity of each negative test solution without flavor was good, as Figure 5 shown.

[0098] Comparative Example 1

[0099] The identification method of red ginseng in Shengmaiyin included in the first part of Chinese Pharmacopoeia (2020 edition).

[0100] (1) Preparation of the reference solution: Take reference substances of ginsenoside Rb1, ginsenoside Re, and ginsenoside Rg1, and make a mixed solution containing 1 mg of each in 1 mL with anhydrous ethanol.

[0101] (2) Preparation of the test solution: Take 20 mL of Shengmaiyin, shake and extract with 20 mL of n-butanol. Evaporate the n-butanol solution to dryness. Dissolve the residue in 15 mL of 45% ethanol solution of sulfuric acid (7→100), heat under reflux for 1 hour, evaporate the ethanol, shake and extract with 10 mL of chloroform. Separate the chloroform layer, wash it with water until neutral, dehydrate it with an appropriate amount of anhydrous sodium sulfate, filter, and concentrate the filtrate to 1 mL to obtain the test solution.

[0102] (3) Pipette 10 μL each of the reference solution and the test solution and spot them on the same silica gel G thin-layer plate. Use cyclohexane-acetone (volume ratio 2:1) as the developing solvent, develop, take out, air-dry, spray with sulfuric acid-methanol solution (1→2), heat at 105 °C for about 10 minutes, and examine under an ultraviolet lamp (365 nm). As Figure 6 shown.

[0103] Comparative Example 2

[0104] The identification method of Ophiopogonis Radix in Shengmaiyin included in Part I of Chinese Pharmacopoeia (2020 Edition).

[0105] (1) Preparation of the reference medicinal material solution: Take 1 g of the reference medicinal material of Ophiopogonis Radix, add 20 mL of water, decoct for 10 minutes, filter. Add 0.5 mL of hydrochloric acid and 1 mL of water to the filtrate, heat and boil for 5 minutes, cool, shake and extract with 20 mL of chloroform. Separate the chloroform layer and concentrate it to 1 mL.

[0106] (2) Preparation of the test solution: Take 10 mL of this product, add 0.5 mL of hydrochloric acid and 1 mL of water, heat and boil for 5 minutes, cool, shake and extract with 20 mL of chloroform. Separate the chloroform layer and concentrate it to 1 mL.

[0107] (3) Pipette 5 μL each of the reference solution and the test solution and spot them on the same silica gel G thin-layer plate. Use chloroform-acetone (volume ratio 4:1) as the developing solvent, develop, take out, air-dry, spray with 10% sulfuric acid-ethanol solution, heat at 100 °C until the spots are clearly developed, and examine under daylight. As Figure 7 shown.

[0108] Comparative Example 3

[0109] The identification method of Schisandrae Chinensis Fructus in Shengmaiyin included in Part I of Chinese Pharmacopoeia (2020 Edition).

[0110] (1) Preparation of the reference solution: Take schisandrol A reference substance, dissolve it in chloroform to make a solution containing 1 mg per 1 mL as the reference solution.

[0111] (2) Preparation of control crude drug solution: Weigh 1 g of Schisandra chinensis control crude drug, add 20 mL of chloroform, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of ethanol to obtain the control crude drug solution.

[0112] (3) Preparation of test solution: Take 10 mL of this product, add 20 mL of water, shake well, extract with ether by shaking 3 times, 30 mL each time, combine the ether layers, evaporate to dryness, dissolve the residue in 1 mL of ethanol to obtain the test solution.

[0113] (4) Pipette 5 - 10 μL of the above test solution, 2 - 5 μL of the control crude drug solution and the reference substance solution respectively onto the same silica gel GF254 thin layer plate, use the upper layer solution of petroleum ether (30 - 60 °C) - ethyl formate - formic acid (volume ratio 15:5:1) as the developing solvent, develop, take out, air dry, and examine under an ultraviolet lamp (254 nm), as Figure 8 shown.

[0114] The above is a further description of the present invention in combination with specific embodiments, but these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention.

Claims

1. A thin layer chromatography identification method for Shengmai Yin with one plate and multiple tests, characterized in that: The steps include: S1. Preparation of test solution: Take a sample of Shengmai Drink, extract it with water-saturated n-butanol, wash it, evaporate it to dryness, dissolve the residue with methanol, separate the solid and liquid, take the supernatant, and prepare the test solution; S2. Preparation of reference solution: Take ginsenoside Rg3 reference substance and schisandra alcohol A reference substance, add methanol respectively, and prepare reference solution; S3, preparation of control medicinal material solution: taking red ginseng control medicinal material, ophiopogon control medicinal material and schisandra chinensis control medicinal material, and preparing control medicinal material solutions respectively; The preparation method of the control medicinal material solution in step S3 is: Take a red ginseng reference medicinal material, add a methanol solution and citric acid to reflux, filter, evaporate the filtrate to dryness, dissolve the residue in water, then extract with saturated n-butanol, wash, evaporate to dryness, dissolve the residue in methanol, separate the solid and liquid, take the supernatant, and prepare a red ginseng reference medicinal material solution; Take Ophiopogon japonicus reference medicinal material, add water to decoct, filter, add hydrochloric acid and water to the filtrate, boil, cool, extract with chloroform, evaporate the organic phase to dryness, add methanol to dissolve the residue, separate the solid and liquid, take the supernatant, and prepare the Ophiopogon japonicus reference medicinal material solution; Take Schisandra chinensis reference medicinal material, add dichloromethane for ultrasonic treatment, filter, evaporate the filtrate to dryness, add ethyl acetate to dissolve the residue, separate the solid and liquid, take the supernatant, and prepare Schisandra chinensis reference medicinal material solution; S4. Thin layer chromatography identification: Pipette the test sample solution, reference sample solution and reference medicinal material solution and spot them on the same reverse phase thin layer plate respectively, use acetonitrile-methanol-water with a volume ratio of 3:3:2 as the developing solvent, develop, take out, dry, spray with sulfuric acid ethanol solution, heat, inspect and compare.

2. The thin layer chromatography identification method according to claim 1, characterized in that: The ratio of Shengmai Yin to water-saturated n-butanol in step S1 is 20 mL:15-25 mL.

3. The thin layer chromatography identification method according to claim 2, characterized in that: The preparation method of the test solution described in step S1 is: take 20mL of Shengmai Drink sample, add 15-25mL of water-saturated n-butanol to extract, wash, evaporate the water-saturated n-butanol solution to dryness, add 1mL of methanol to dissolve the residue, separate the solid and liquid, take the supernatant, and prepare the test solution.

4. The thin layer chromatography identification method according to claim 1, characterized in that: The preparation method of the reference solution described in step S2 is: taking ginsenoside Rg3 reference substance and adding methanol to prepare a ginsenoside Rg3 reference substance solution with a concentration of 0.3-0.8 mg / mL; taking schisandra alcohol A reference substance and adding methanol to prepare a schisandra alcohol A reference substance solution with a concentration of 0.8-1.2 mg / mL.

5. The thin layer chromatography identification method according to claim 1, characterized in that: In the preparation process of the red ginseng reference medicinal material solution, the volume fraction of the methanol is 50-70%; the ratio of the red ginseng reference medicinal material, the methanol solution and the citric acid is 2-6g:40-80mL:1.5-2.0g; the reflux is refluxed at 55-75°C for 1-6h; And / or in the process of preparing the Ophiopogon japonicus reference medicinal material solution, the ratio of the Ophiopogon japonicus reference medicinal material to water is 1 g: 15-25 mL; the decoction time is 5-20 min; the ratio of hydrochloric acid to water is 0.3-0.8 mL: 1 mL; the boiling time is 3-8 min; And / or in the process of preparing the Schisandra chinensis reference medicinal material solution, the ratio of the Schisandra chinensis reference medicinal material to dichloromethane is 1 g: 30-50 mL; and the time of the ultrasonic treatment is 10-50 min.

6. The thin layer chromatography identification method according to claim 5, characterized in that: The preparation method of the control medicinal material solution is: Take 2-6 g of red ginseng reference medicinal material, add 40-80 mL of 50-70% methanol solution and 1.5-2.0 g of citric acid, reflux at 55-75° C. for 1-6 h, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with saturated n-butanol, wash, evaporate to dryness, dissolve the residue in methanol, separate the solid and liquid, and take the supernatant to prepare a red ginseng reference medicinal material solution; Take 1 g of Radix Ophiopogonis reference medicinal material, add 15-25 mL of water and boil for 5-20 min, filter, add 0.3-0.8 mL of hydrochloric acid and 1 mL of water to the filtrate, boil for 3-8 min, cool, add 15-25 mL of chloroform to extract, evaporate the organic phase to dryness, add methanol to dissolve the residue, separate the solid and liquid, take the supernatant, and prepare the Radix Ophiopogonis reference medicinal material solution; Take 1 g of Schisandra chinensis reference medicinal material, add 30-50 mL of dichloromethane, and perform ultrasonic treatment for 10-50 min. Filter, evaporate the filtrate to dryness, add ethyl acetate to dissolve the residue, separate the solid and liquid, and take the supernatant to prepare Schisandra chinensis reference medicinal material solution.

7. The thin layer chromatography identification method according to claim 1, characterized in that: The concentration of the sulfuric acid ethanol solution in step S4 is 8-12wt%; And / or the inspection conditions are: visible light and 254nm.

8. The thin layer chromatography identification method according to claim 7, characterized in that: The identification method of the thin layer chromatography in step S4 is: 3-7 μL of the test solution, the reference solution and the reference medicinal material solution are respectively spotted on the same reversed thin layer plate, and acetonitrile-methanol-water with a volume ratio of 3:3:2 is used as the developing solvent, developed, taken out, dried, sprayed with 8-12wt% sulfuric acid ethanol solution, heated at 102-105°C for 60-120s, and inspected under sunlight or 254nm. For comparison, in the test sample chromatogram, spots of the same color appear at the corresponding positions of the reference sample chromatogram and the reference medicinal material chromatogram.

9. Application of the thin layer chromatography identification method according to any one of claims 1 to 8 in quality control of products containing red ginseng, ophiopogon japonicus and schisandra chinensis.

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