Thin-layer chromatography identification method for one-plate multi-detection of pulse-activating decoction

Abstract

The invention belongs to a detection method of a traditional Chinese medicine preparation, and relates to a one-plate multi-detection thin-layer chromatography identification method of pulse-activating decoction. The thin-layer chromatography identification method comprises the following steps: (1) preparing a test solution; (2) preparing a reference substance solution; (4) thin-layer chromatography identification: sucking the test sample solution, the reference substance solution and the reference medicinal material solution, respectively dripping on the same reversed-phase thin-layer plate, developing by taking acetonitrile-methanol-water with the volume ratio of 3: 3: 2 as a developing solvent, taking out, airing, spraying a sulfuric acid ethanol solution, heating and inspecting. The method adopts one thin-layer plate to simultaneously identify the whole-formula three medicines in the pulse-activating decoction, has the advantages of simplicity, rapidness, high efficiency and the like, and meanwhile, has clear chromatographic spots and good specificity and reproducibility. Compared with the prior art, the pretreatment method is simple, and used reagents are low in toxicity; meanwhile, the technology for controlling the quality of the ginsenoside Rg3 in the pulse-activating decoction through thin-layer identification does not exist in the prior art is made up.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine preparation detection, and in particular to a thin layer chromatography identification method for Shengmai Yin with one plate and multiple tests. Background Art

[0002] Shengmai Yin is composed of three herbs: red ginseng, ophiopogon and schisandra chinensis. It has the effects of replenishing qi and pulse, nourishing yin and promoting body fluid. It can be used for qi and yin deficiency, palpitations, shortness of breath, weak pulse and spontaneous sweating.

[0003] At present, the current quality standard of Shengmai Yin is the 2020 edition of the Chinese Pharmacopoeia, which includes properties, identification, inspection and content determination. Under the identification item, red ginseng is referenced to ginsenodiol and ginsenotriol, Ophiopogon japonicus is referenced to Ophiopogon japonicus control medicinal materials, and Schisandra chinensis is referenced to Schisandrin A and Schisandra chinensis control medicinal materials. The preparation method of the test solution used for identification of each medicine is different, and the developing agent is also different. The specific information is shown in Table 1 below.

[0004] Table 1 Thin layer chromatography identification method of Shengmai Yin included in the Chinese Pharmacopoeia

[0005]

[0006] The thin-layer identification method of Shengmai Yin included in the Chinese Pharmacopoeia uses ginsenoside diol and ginsenotriol as identification indicators for red ginseng. Ginsenoside diol is the degradation product of ginsenoside diol type-A (ginsenoside Rb, ginsenoside Rb1 and ginsenoside Rd, etc.), and ginsenoside triol type-B (ginsenoside Rg1, ginsenoside R e and ginsenoside Rf) are hydrolyzed under acid-adding and heating conditions to generate ginsenoside triol. The above two components are degradation products of Shengmai Drink after acid heating, and are not original components in the preparation. They can be produced by hydrolysis of many ginsenoside components. Using this as an identification index has low specificity. Existing public information shows that Shengmai Drink contains ginsenoside Rg3, which has significant pharmacological effects and has anti-arrhythmic effects. Its mechanism of action is to protect myocardial cells from hypoxia by blocking the occurrence of ion overload. In addition, the application of ginsenoside Rg3 in the treatment or prevention of enterovirus infection. Therefore, quality control of ginsenoside Rg3 in Shengmai Drink is of great significance to ensure the safety and effectiveness of clinical medication, but there is no method for identifying ginsenoside Rg3 in Shengmai Drink by thin layer chromatography in the prior art.

[0007] At present, there is no thin layer chromatography identification method for Shengmai Drink that can perform multiple tests on one plate in the existing technology; the thin layer chromatography identification method for Shengmai Drink included in the "Chinese Pharmacopoeia" prepares three test sample solutions separately, and the pretreatment methods of the test sample and control medicinal material solutions are complicated, and the reagents used are highly toxic; testing single herbs separately results in a large workload, high testing costs, time-consuming, labor-intensive, and wasteful testing reagents. 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 Shengmai Drink with one plate and multiple tests. The present invention is a thin layer chromatography identification method for simultaneously measuring the three herbs in the whole formula of Shengmai Drink with one thin layer plate and one test solution, and can simultaneously identify red ginseng, Ophiopogon japonicus and Schisandra chinensis, thereby ensuring the product quality of Shengmai Drink and the safety of clinical use of the drug.

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

[0010] A thin layer chromatography identification method for Shengmai Yin with one plate and multiple tests, comprising the following steps:

[0011] 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;

[0012] S2. Preparation of reference solution: Take ginsenoside Rg3 reference substance and schisandra alcohol A reference substance, add methanol respectively, and prepare reference solution;

[0013] 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;

[0014] 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 reagent, heat, inspect and compare.

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

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

[0017] Preferably, the preparation method of the reference solution in step S2 is: take ginsenoside Rg3 reference substance and schisandrae alcohol A reference substance, add methanol respectively, to prepare ginsenoside Rg3 reference substance solution and schisandrae alcohol A reference substance solution.

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

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

[0020] Preferably, the preparation method of the control medicinal material solution in step S3 is:

[0021] Take a red ginseng reference medicinal material, add methanol 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;

[0022] 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;

[0023] 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.

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

[0025] In the preparation process of the Ophiopogon japonicus control medicinal material solution of the present invention, the ratio of the Ophiopogon japonicus control medicinal material to water is 1g:15-25mL, and more preferably 1g:20mL; the decoction time is 5-20min, and more preferably 10min; the ratio of hydrochloric acid to water is 0.3-0.8mL:1mL, and more preferably 0.5mL:1mL; the boiling time is 3-8min, and more preferably 5min;

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

[0027] In some embodiments, the preparation method of the control medicinal material solution is:

[0028] Take 2-6 g of red ginseng reference medicinal material, 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, then 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;

[0029] 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;

[0030] 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.

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

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

[0033] 3-7 μL of the test solution, reference solution and reference medicinal material solution were taken and spotted on the same reverse phase thin layer plate respectively, and acetonitrile-methanol-water with a volume ratio of 3:3:2 was used as the developing solvent. The plate was developed, taken out, dried, sprayed with 8-12 wt% 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 appeared at the corresponding positions of the reference sample chromatogram and the reference medicinal material chromatogram.

[0034] It is worth mentioning that:

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

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

[0037] The thin layer chromatography identification method of the present invention is not limited to Shengmai Yin, but is also applicable to the quality control of products containing red ginseng, ophiopogon japonicus and schisandra chinensis.

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

[0039] 1. The present invention provides a method for simultaneously identifying the three herbs of the whole prescription of Shengmai Yin on a thin layer plate. The thin layer identification method of Shengmai Yin recorded 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, namely acetonitrile-methanol-water (3:3:2), to complete the thin layer identification of the herbs of the whole prescription. The developing agent reagent used has a moderate ratio, the reagent toxicity is low, and the preparation method is simple.

[0040] 2. The present invention uses one test solution to simultaneously identify three herbs, namely, red ginseng, ophiopogon japonicus and schisandra chinensis. Compared with the thin-layer identification method included in the Chinese Pharmacopoeia, which requires the preparation of three test solutions respectively, the present method only requires the preparation of one test solution and uses liquid-liquid extraction, and the method is simple to operate. In addition, the thin-layer identification method included in the Chinese Pharmacopoeia uses toxic reagents such as hydrochloric acid, sulfuric acid, chloroform and ether for pretreatment, while the pretreatment method of the present invention only uses reagents such as n-butanol, ammonia test solution and water, and is relatively less toxic.

[0041] 3. The present invention uses two reference substance solutions and three reference medicinal material solutions as references at the same time. After color development, the samples are inspected under sunlight and 254 nm. The color spectrum is more fully expressed, giving the analyst more room for thinking and judgment.

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

[0043] 5. When the present invention detects Shengmai Drink, the chromatographic spots of the test sample are clear, and there is no interference with the negative samples lacking flavor. The method has good reproducibility and can be used for quality control of Shengmai Drink. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a thin layer chromatogram developed for the sample test in Example 1 (from top to bottom: daylight inspection, 254nm inspection; in the figure, No. 1 is ginsenoside Rg3 control solution, No. 2 is red ginseng control medicinal material solution, No. 3 is Ophiopogon japonicus control medicinal material solution, No. 4 is 2408002 batch Shengmai Yin test solution, No. 5 is 2408004 batch Shengmai Yin test solution, No. 6 is Schisandra chinensis control medicinal material solution, No. 7 is Schisandrae Chinensis Alcohol control solution, the sample volume of No. 1-7 is 5μL, the temperature is 23°C, and the humidity is 53%);

[0045] Figure 2 The thin layer chromatograms of the negative sample lacking red ginseng and the sample spotting amount of Shengmai Drink test sample in Example 2 (from top to bottom: daylight inspection, 254nm inspection; No. 1 in the figure is the ginsenoside Rg3 control solution, No. 2 is the red ginseng control medicinal material solution, No. 3 is the negative test sample solution lacking red ginseng, No. 4 is the 2408002 batch of Shengmai Drink test sample solution, No. 5 is the ginsenoside Rg3 control solution, No. 6 is the red ginseng control medicinal material solution, No. 7 is the negative test sample solution lacking red ginseng Ginseng negative test solution, No. 8 is 2408002 batch Shengmai Drink test solution, No. 9 is ginsenoside Rg3 control solution, No. 10 is red ginseng control medicinal material solution, No. 11 is red ginseng negative test solution, No. 12 is 2408002 batch Shengmai Drink test solution, No. 1-4 spotting volume is 3μL, No. 5-8 spotting volume is 5μL, No. 9-12 spotting volume is 7μL, temperature is 22°C, humidity is 80%);

[0046] Figure 3 This is a thin layer chromatogram of the sample spotting amount of the negative sample lacking Ophiopogon japonicus and the Shengmai Drink test sample in Example 3 (from top to bottom: daylight inspection, 254nm inspection; in the figure, No. 1 is the Ophiopogon japonicus control medicinal material solution, No. 2 is the negative test sample solution lacking Ophiopogon japonicus, No. 3 is the 2408002 batch of Shengmai Drink test sample solution, No. 4 is the Ophiopogon japonicus control medicinal material solution, No. 5 is the negative test sample solution lacking Ophiopogon japonicus, No. 6 is the 2408002 batch of Shengmai Drink test sample solution, No. 7 is the Ophiopogon japonicus control medicinal material solution, No. 8 is the negative test sample solution lacking Ophiopogon japonicus, No. 9 is the 2408002 batch of Shengmai Drink test sample solution, the sample spotting amount of No. 1-3 is 3μL, the sample spotting amount of No. 4-6 is 5μL, the sample spotting amount of No. 7-9 is 7μL, the temperature is 22°C, and the humidity is 80%);

[0047] Figure 4 The thin layer chromatograms of the negative samples lacking Schisandra chinensis and the sample spotting amount of Shengmai Drink test sample in Example 4 (from top to bottom: daylight inspection, 254nm inspection; No. 1 in the figure is the Schisandra alcohol A control solution, No. 2 is the Schisandra chinensis control medicinal material solution, No. 3 is the negative test sample solution lacking Schisandra chinensis, No. 4 is the 2408002 batch of Shengmai Drink test sample solution, No. 5 is the Schisandra alcohol A control solution, No. 6 is the Schisandra chinensis control medicinal material solution, No. 7 is the negative test sample solution lacking Schisandra chinensis The sample solution is negative for Schisandra chinensis, the ...

[0048] Figure 5This is a thin layer chromatogram of the specificity investigation of each taste-deficient sample in Example 5 (from top to bottom: daylight inspection, 254nm inspection; in the figure, No. 1 is the ginsenoside Rg3 control solution, No. 2 is the red ginseng control medicinal material solution, No. 3 is the negative test solution lacking red ginseng, No. 4 is the 2408002 batch of Shengmai Drink test solution, No. 5 is the negative test solution lacking Ophiopogon japonicus, No. 6 is the Ophiopogon japonicus control medicinal material solution, No. 7 is the 2408004 batch of Shengmai Drink test solution, No. 8 is the negative test solution lacking Schisandra chinensis, No. 9 is the Schisandra chinensis control medicinal material solution, No. 10 is the Schisandrin A control solution, the sample volume of No. 1-10 is 5 μL, the temperature is 23°C, and the humidity is 53%);

[0049] Figure 6 This is the thin layer chromatogram of Shengmai Drink red ginseng included in the Pharmacopoeia of Comparative Example 1 (No. 1 in the figure is the test solution of Shengmai Drink batch 2408002, No. 2 is the mixed reference solution of ginsenodiol and ginsenotriol, the sample volume of No. 1 and 2 is 10 μL, the temperature is 20°C, and the humidity is 50%);

[0050] Figure 7 This is the thin layer chromatogram of Shengmai Drink Ophiopogon japonicus included in the Pharmacopoeia of Comparative Example 2 (No. 1 in the figure is the test solution of Shengmai Drink batch 2408002, No. 2 is the Ophiopogon japonicus control medicinal material solution, the sample volume of No. 1 and No. 2 is 5μL, the temperature is 20℃, and the humidity is 50%);

[0051] Figure 8 This is the thin layer chromatogram of Schisandra chinensis of Shengmai Drink included in the Pharmacopoeia of Comparative Example 3 (No. 1 in the figure is the test solution of Shengmai Drink batch 2408002, No. 2 is the Schisandra chinensis reference medicinal material solution, No. 3 is the Schisandra alcohol A reference solution, the sample volume of No. 1-3 is 5 μL, the temperature is 20°C, and the humidity is 50%). DETAILED DESCRIPTION

[0052] The following examples are 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, without departing from the principles of the present invention, the present invention may also be subjected to several improvements and modifications, which also fall within the scope of protection of the claims of the present invention. The following description of the disclosed embodiments enables professionals and technicians in this field to implement or use the present invention. It will be apparent to professionals and technicians in this field that various modifications to these embodiments, and the general principles defined herein may 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 the embodiments shown herein, but may be applied to a wider range consistent with the principles and novel features disclosed herein. Although any methods and materials similar or equivalent to those described in the present invention may be used in the implementation or testing of the present invention, preferred methods and materials are listed herein.

[0053] 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.

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

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

[0056] Example 1

[0057] The Shengmai Drink samples were tested using reference substances and various reference medicinal material solutions.

[0058] (1) Preparation of test solution: Take 20 mL of Shengmai Yin, extract it with water-saturated n-butanol for 3 times, 20 mL each time, combine the water-saturated n-butanol layers, wash it with ammonia test solution for 3 times, 20 mL each time, and then wash it with water saturated with n-butanol for 3 times, 20 mL each time. Evaporate the water-saturated n-butanol solution on a water bath, add 1 mL of methanol to dissolve the residue, centrifuge, and take the supernatant to prepare the test solution.

[0059] (2) Preparation of reference solution: Take ginsenoside Rg3 reference substance and add methanol to prepare a reference solution containing 0.5 mg per 1 mL; then take schisandra alcohol A reference substance and add methanol to prepare a reference solution containing 1.0 mg per 1 mL.

[0060] (3) Preparation of each control medicinal material solution: Take 4 g of red ginseng control medicinal material, add 60 mL of 60% methanol, and 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 3 times, 20 mL each time, combine the water-saturated n-butanol layers, wash with ammonia test solution 3 times, 20 mL each time, and then wash with water saturated with n-butanol 3 times, 20 mL each time, evaporate the water-saturated n-butanol solution in a water bath, dissolve the residue in 2 mL of methanol, centrifuge, and take the supernatant to prepare the red ginseng control medicinal material solution;

[0061] Take 1 g of Radix Ophiopogonis as a reference medicinal material, 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, extract with 20 mL of chloroform by shaking, take the chloroform solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve, centrifuge, take the supernatant, and prepare the Radix Ophiopogonis as a reference medicinal material solution;

[0062] Take 1 g of Schisandra chinensis reference medicinal material, add 40 mL of dichloromethane, and ultrasonically treat for 30 minutes. Filter, evaporate the filtrate to dryness, add 1 mL of ethyl acetate to the residue to dissolve, centrifuge, and take the supernatant to prepare Schisandra chinensis reference medicinal material solution.

[0063] (4) Take 5 μL of the test solution, reference solution and each reference medicinal material solution 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, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clear, and examine under sunlight and 254 nm.

[0064] The results showed that in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions in the chromatogram of the reference sample and the reference medicinal material. The reproducibility of the two batches of Shengmaiyin samples was good, and the separation of each identification spot was good. Figure 1 shown.

[0065] Example 2

[0066] The spotting amount of negative samples lacking red ginseng and the test solution of Shengmai Drink was investigated.

[0067] (1) Preparation of test solution: Take 20 mL of Shengmai Yin, extract it with water-saturated n-butanol for 3 times, 20 mL each time, combine the water-saturated n-butanol layers, wash it with ammonia test solution for 3 times, 20 mL each time, and then wash it with water saturated with n-butanol for 3 times, 20 mL each time. Evaporate the water-saturated n-butanol solution on a water bath, add 1 mL of methanol to dissolve the residue, centrifuge, and take the supernatant to prepare the test solution.

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

[0069] (3) Preparation of reference medicinal material solution: Take 4 g of red ginseng reference medicinal material, add 60 mL of 60% methanol, and 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 3 times, 20 mL each time, combine the water-saturated n-butanol layers, wash with ammonia test solution 3 times, 20 mL each time, and then wash with water saturated with n-butanol 3 times, 20 mL each time, evaporate the water-saturated n-butanol solution in a water bath, dissolve the residue in 2 mL of methanol, centrifuge, and take the supernatant to prepare the red ginseng reference medicinal material solution.

[0070] (4) Preparation of negative test solution lacking red ginseng: prepare a negative sample without red ginseng according to the prescription process of Shengmai Yin, measure 20 mL of the negative sample lacking red ginseng, extract it with water-saturated n-butanol for 3 times, 20 mL each time, combine the water-saturated n-butanol layers, wash it with ammonia test solution for 3 times, 20 mL each time, and then wash it with water saturated with n-butanol for 3 times, 20 mL each time, evaporate the water-saturated n-butanol solution on a water bath, add 1 mL of methanol to dissolve the residue, centrifuge, and take the supernatant to prepare the negative test solution lacking red ginseng.

[0071] (5) 3 μL, 5 μL, and 7 μL of the reference solution, reference medicinal material solution, test solution, and red ginseng-deficient negative test solution were respectively spotted on the same reverse phase thin layer plate, and acetonitrile-methanol-water (volume ratio 3:3:2) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C until the spots were clear, and examined under sunlight and 254 nm.

[0072] The results showed that the amount of the negative samples lacking red ginseng and the test solution of Shengmai Drink was between 3μL and 7μL, and the ginsenoside Rg3 had good specificity, such as Figure 2 shown.

[0073] Example 3

[0074] The sample spot amount of negative samples lacking Ophiopogon japonicus and Shengmai Drink test solution was investigated.

[0075] (1) Preparation of test solution: Take 20 mL of Shengmai Yin, extract it with water-saturated n-butanol for 3 times, 20 mL each time, combine the water-saturated n-butanol layers, wash it with ammonia test solution for 3 times, 20 mL each time, and then wash it with water saturated with n-butanol for 3 times, 20 mL each time. Evaporate the water-saturated n-butanol solution on a water bath, add 1 mL of methanol to dissolve the residue, centrifuge, and take the supernatant to prepare the test solution.

[0076] (2) Preparation of reference medicinal material solution: Take 1 g of Ophiopogon japonicus reference medicinal material, 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, extract with 20 mL of chloroform by shaking, take the chloroform solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, centrifuge, take the supernatant, and prepare the Ophiopogon japonicus reference medicinal material solution.

[0077] (3) Preparation of negative test solution lacking Ophiopogon japonicus: prepare negative sample without Ophiopogon japonicus according to the prescription process of Shengmai Yin, measure 20 mL of negative sample lacking Ophiopogon japonicus, extract it with water-saturated n-butanol for 3 times, 20 mL each time, combine the water-saturated n-butanol layers, wash it with ammonia test solution for 3 times, 20 mL each time, and then wash it with water saturated with n-butanol for 3 times, 20 mL each time, evaporate the water-saturated n-butanol solution on a water bath, add 1 mL of methanol to dissolve the residue, centrifuge, take the supernatant, and prepare the negative test solution lacking Ophiopogon japonicus.

[0078] (4) 3 μL, 5 μL, and 7 μL of the control medicinal material solution, test solution, and Ophiopogon japonicus-deficient negative test solution were respectively spotted on the same reverse phase thin layer plate, and acetonitrile-methanol-water (volume ratio 3:3:2) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C until the spots were clear, and examined under sunlight and 254 nm.

[0079] The results showed that the spotting volume of the negative samples lacking Ophiopogon japonicus and the test solution of Shengmai Yin was between 3-7 μL, and the specificity was good at the corresponding position of the main spot of the Ophiopogon japonicus control medicinal material, such as Figure 3 shown.

[0080] Example 4

[0081] The spotting amount of the negative samples lacking Schisandra chinensis and the test solution of Shengmai Drink was investigated.

[0082] (1) Preparation of test solution: Take 20 mL of Shengmai Yin, extract it with water-saturated n-butanol for 3 times, 20 mL each time, combine the water-saturated n-butanol layers, wash it with ammonia test solution for 3 times, 20 mL each time, and then wash it with water saturated with n-butanol for 3 times, 20 mL each time. Evaporate the water-saturated n-butanol solution on a water bath, add 1 mL of methanol to dissolve the residue, centrifuge, and take the supernatant to prepare the test solution.

[0083] (2) Preparation of reference solution: Take Schisandrin A reference substance and add methanol to prepare a reference solution containing 1 mg per 1 mL.

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

[0085] (4) Preparation of negative test solution lacking Schisandra chinensis: According to the prescription process of Shengmai Yin, a negative sample without Schisandra chinensis was prepared. 20 mL of negative sample lacking Schisandra chinensis was measured and extracted with water-saturated n-butanol for 3 times, 20 mL each time. The water-saturated n-butanol layers were combined and washed with ammonia test solution for 3 times, 20 mL each time. Then, the water-saturated n-butanol solution was washed with water saturated with n-butanol for 3 times, 20 mL each time. The water-saturated n-butanol solution was evaporated on a water bath. 1 mL of methanol was added to the residue to dissolve it. The mixture was centrifuged and the supernatant was collected to prepare the negative test solution lacking Schisandra chinensis.

[0086] (5) 3 μL, 5 μL, and 7 μL of the reference solution, reference medicinal material solution, test solution, and Schisandrae Chinensis negative test solution were respectively spotted on the same reverse phase thin layer plate, and acetonitrile-methanol-water (volume ratio 3:3:2) was used as the developing solvent. The plate was developed, taken out, dried, sprayed with 10% ethanolic sulfuric acid solution, heated at 105°C until the spots were clear, and examined under sunlight and 254 nm.

[0087] The results showed that when the sample volume of the negative sample lacking Schisandra chinensis and the test solution of Shengmai Drink was 3μL, the specificity of Schisandrae Chinensis A was good; when the sample volume was 5μL and 7μL, there was a spot tail near Schisandrae Chinensis A in the chromatogram of the negative sample lacking Schisandra chinensis, which affected its specificity judgment; because its specificity was good at 3μL, it was judged that Schisandrae Chinensis A was specific, such as Figure 4 shown.

[0088] Example 5

[0089] The specificity of each taste-deficient negative sample was investigated.

[0090] (1) Preparation of test solution: Take 20 mL of Shengmai Yin, extract it with water-saturated n-butanol for 3 times, 20 mL each time, combine the water-saturated n-butanol layers, wash it with ammonia test solution for 3 times, 20 mL each time, and then wash it with water saturated with n-butanol for 3 times, 20 mL each time. Evaporate the water-saturated n-butanol solution on a water bath, add 1 mL of methanol to dissolve the residue, centrifuge, and take the supernatant to prepare the test solution.

[0091] (2) Preparation of reference solution: Take ginsenoside Rg3 reference standard and add methanol to prepare a reference solution containing 0.5 mg per 1 mL; then take schisandra alcohol A reference standard and add methanol to prepare a reference solution containing 1 mg per 1 mL.

[0092] (3) Preparation of reference medicinal material solution: Take 4 g of red ginseng reference medicinal material, add 60 mL of 60% methanol, and 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 3 times, 20 mL each time, combine the water-saturated n-butanol layers, wash three times with ammonia test solution, 20 mL each time, and then wash three times with water saturated with n-butanol, 20 mL each time, evaporate the water-saturated n-butanol solution in a water bath, dissolve the residue in 2 mL of methanol, centrifuge, and take the supernatant to prepare the red ginseng reference medicinal material solution;

[0093] Take 1 g of Radix Ophiopogonis as a reference medicinal material, 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, extract with 20 mL of chloroform by shaking, take the chloroform solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve, centrifuge, take the supernatant, and prepare the Radix Ophiopogonis as a reference medicinal material solution;

[0094] Take 1 g of Schisandra chinensis reference medicinal material, add 40 mL of dichloromethane, and ultrasonically treat for 30 minutes. Filter, evaporate the filtrate to dryness, add 1 mL of ethyl acetate to the residue to dissolve, centrifuge, and take the supernatant to prepare Schisandra chinensis reference medicinal material solution.

[0095] (4) Preparation of negative test solutions of each flavor deficiency: According to the prescription process of Shengmai Yin, negative samples without red ginseng, Ophiopogon japonicus, and Schisandra chinensis were prepared respectively. 20 mL of each negative sample was measured and extracted with water-saturated n-butanol for 3 times, 20 mL each time. The water-saturated n-butanol layers were combined, washed with ammonia test solution for 3 times, 20 mL each time, and then washed with water saturated with n-butanol for 3 times, 20 mL each time. The water-saturated n-butanol solution was evaporated on a water bath, 1 mL of methanol was added to the residue to dissolve it, centrifuged, and the supernatant was taken to prepare the negative test solutions of each flavor deficiency.

[0096] (5) Take 5 μL of the reference solution, reference medicinal material solution, test solution and each taste-deficient negative test solution 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, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clear, and examine under sunlight and 254 nm.

[0097] The results showed that the specificity of each taste-deficient negative test product was good, such as Figure 5 shown.

[0098] Comparative Example 1

[0099] The identification method of red ginseng in Shengmai Drink is included in the 2020 edition of the "Chinese Pharmacopoeia".

[0100] (1) Preparation of reference solution: Take ginsenodiol reference substance and ginsenotriol reference substance and add anhydrous ethanol to prepare a mixed solution containing 1 mg of each substance per 1 mL.

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

[0102] (3) Take 10 μL 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, dry, spray with sulfuric acid methanol solution (1→2), heat at 105℃ for about 10 minutes, and examine under ultraviolet light (365nm). Figure 6 shown.

[0103] Comparative Example 2

[0104] The identification method of Ophiopogon japonicus in Shengmai Drink is included in the 2020 edition of the Chinese Pharmacopoeia.

[0105] (1) Preparation of reference medicinal material solution: Take 1 g of Radix Ophiopogonis and add 20 mL of water. Boil for 10 minutes and filter. Add 0.5 mL of hydrochloric acid and 1 mL of water to the filtrate. Boil for 5 minutes. Cool. Shake and extract with 20 mL of chloroform. Take the chloroform solution and concentrate it to 1 mL.

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

[0107] (3) Pipette 5 μL of the reference solution and the test solution onto the same silica gel G thin layer plate, use chloroform-acetone (volume ratio 4:1) as the developing solvent, develop, remove, dry, spray with 10% sulfuric acid ethanol solution, heat at 100°C until the spots are clearly colored, and examine under sunlight. Figure 7 shown.

[0108] Comparative Example 3

[0109] The identification method of Schisandra chinensis in Shengmai Yin is included in the 2020 edition of the Chinese Pharmacopoeia.

[0110] (1) Preparation of reference solution: Take Schisandrin A reference substance and add chloroform to prepare a solution containing 1 mg per 1 mL as the reference solution.

[0111] (2) Preparation of control medicinal material solution: 1 g of Schisandra chinensis control medicinal material was added with 20 mL of chloroform, and ultrasonically treated for 30 minutes. The mixture was filtered, and the filtrate was evaporated to dryness. 1 mL of ethanol was added to the residue to dissolve it, and the solution was used as the control medicinal material solution.

[0112] (3) Preparation of test solution: Take 10 mL of the product, add 20 mL of water, shake well, extract with ether three times, 30 mL each time, combine the ether solutions, evaporate to dryness, add 1 mL of ethanol to dissolve the residue, and use this as the test solution.

[0113] (4) 5-10 μL of the test solution, 2-5 μL of the reference drug solution and the reference substance solution were respectively spotted on the same silica gel GF254 thin layer plate, and the upper layer solution of petroleum ether (30-60°C)-ethyl formate-formic acid (volume ratio of 15:5:1) was used as the developing agent. The plate was developed, taken out, dried, and examined under ultraviolet light (254 nm). Figure 8 shown.

[0114] The above is a further description of the present invention in conjunction with specific embodiments, but these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection 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; 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: The preparation method of the control medicinal material solution in step S3 is: Take a red ginseng reference medicinal material, add methanol 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.

6. The thin layer chromatography identification method according to claim 5, 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, methanol and 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.

7. The thin layer chromatography identification method according to claim 6, 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 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, then 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.

8. 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.

9. The thin layer chromatography identification method according to claim 8, 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.

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

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