Method for simultaneously identifying stachydrine hydrochloride and leonurine

Abstract

The invention discloses a method for simultaneously identifying stachydrine hydrochloride and leonurine. The method comprises the following steps: preparing a test solution, preparing a reference solution and identifying according to a thin-layer chromatography. The preparation of the test sample solution comprises the following steps: dissolving a test sample in methanol, heating and refluxing, evaporating the filtrate to dryness, adding hydrochloric acid into the obtained residue for dissolving, loading on a cation exchange resin column, eluting with a mixed solution, collecting the eluent, and evaporating to dryness to obtain the test sample solution. A preparation method of the mixed solution comprises the following steps: mixing 15-30 ml of stronger ammonia water, 2-4 g of sodium chloride and 70 ml of absolute ethyl alcohol, and adding water to 100 ml. According to the method, the test sample is prepared through elution of the mixed solution, the stachydrine hydrochloride and the leonurine can be separated and identified at the same time by combining elution of the cation exchange column and regulation and control of all parameters, the color development is clear, the operation is simple and convenient, the separation effect is good, the sample treatment time can be shortened to 4.5 h, the detection efficiency is improved, and the detection cost is reduced. The method can be effectively applied to rapid and efficient identification of motherwort drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of identification of active ingredients in traditional Chinese patent medicines, and particularly to a method for simultaneously identifying stachydrine hydrochloride and leonurine. Background Art

[0002] Yixue'an Granules are used for treating gynecological diseases and are traditional Chinese patent medicines for stopping bleeding and regulating menstruation, so they can play a role in stopping bleeding. The main active ingredients include motherwort, etc., which can promote blood circulation to stop bleeding and regulate qi to relieve pain. It can be applied to the situation of women with lochia not stopping after childbirth, uterine bleeding not stopping after induced abortion, or excessive menstrual bleeding with blood stasis syndrome, and can also play a role in shortening the bleeding time and relieving abdominal pain. Motherwort (Leonurus japonicus) is a commonly used traditional Chinese medicine for promoting blood circulation to remove blood stasis and promoting diuresis to reduce swelling. Its main active ingredients mainly include stachydrine hydrochloride and leonurine. In the field of traditional Chinese medicine research and quality control, accurately identifying the active ingredients in traditional Chinese medicines is crucial. The accurate and efficient identification of these two ingredients, stachydrine hydrochloride and leonurine, is of great significance in ensuring the quality, efficacy, safety, pharmacological research and clinical application of motherwort and its preparations.

[0003] Currently, the detection methods for stachydrine hydrochloride and leonurine mainly include high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). Although HPLC has high precision, the equipment is expensive and the operation is complex, which is not suitable for grass-roots laboratories or rapid detection requirements; while in the traditional TLC method for separating stachydrine hydrochloride and leonurine, problems such as spot tailing, poor resolution and long detection time are often caused by improper selection of the developing system or insensitive color development method. In addition, some identification methods can only detect a single component and cannot effectively identify stachydrine hydrochloride and leonurine simultaneously, with poor separation effect, increasing the detection workload and cost. Summary of the Invention

[0004] Aiming at the above deficiencies, the present invention provides a method for simultaneously identifying stachydrine hydrochloride and leonurine, which is simple to operate, short in detection time, good in separation effect, stable and efficient, and can simultaneously identify stachydrine hydrochloride and leonurine in motherwort. The specific technical solution is as follows:

[0005] A method for simultaneously identifying stachydrine hydrochloride and leonurine includes: preparation of a test solution, preparation of a reference solution, and identification by thin layer chromatography.

[0006] The preparation of the test solution comprises the following steps: taking a test sample, dissolving it in methanol, heating and refluxing, cooling, filtering, collecting the filtrate, evaporating to dryness in a water bath, adding hydrochloric acid to obtain a residue to dissolve it, filtering, applying the filtrate to a cation exchange resin column, washing with water until colorless, eluting with a mixed solution, collecting the eluate, evaporating to dryness, and dissolving the residue in methanol to obtain a test solution;

[0007] The preparation of the reference solution comprises the following steps: taking Leonurus japonicus, adding hydrochloric acid solution or methanol, heating to reflux, filtering, applying cation exchange resin to the filtrate, washing with water until colorless, eluting with a mixed solution, collecting the eluate, evaporating to dryness, dissolving the residue in methanol to obtain the reference solution;

[0008] The preparation method of the 100 ml mixed solution comprises: mixing 15-30 ml of concentrated ammonia water, 2-5 g of sodium chloride and 70 ml of anhydrous ethanol, and adding water to 100 ml; the volume concentration of the concentrated ammonia water is 25%-28%.

[0009] Preferably, the developing agent used for identification by thin layer chromatography consists of acetone, anhydrous ethanol and hydrochloric acid in a volume ratio of 9-11:5-7:1; the test is carried out according to the thin layer chromatography "Chinese Pharmacopoeia" 2020 edition Part IV General Rules 0502, 8-12ul of the test solution and the reference solution are respectively spotted on the same silica gel G thin layer plate with sodium carboxymethyl cellulose as an adhesive, developed with a developing agent, taken out to dry, sprayed with a color developer, baked at 100-110°C until the spots are clearly colored, and then inspected under a light source.

[0010] Preferably, the preparation method of the 100 ml mixed solution comprises: mixing 20 ml of concentrated aqueous ammonia, 3 g of sodium chloride, and 70 ml of anhydrous ethanol, and adding water to 100 ml.

[0011] Preferably, the developing solvent used for identification by thin layer chromatography consists of acetone, anhydrous ethanol and hydrochloric acid in a volume ratio of 10:6:1.

[0012] Preferably, the developer is prepared by mixing a dilute potassium bismuth iodide test solution and a ferric chloride test solution in a volume ratio of 10:1, and the spraying amount of the developer is 0.2 ml-1.0 ml.

[0013] Preferably, the model of the cation exchange resin column is 732 sodium type.

[0014] Preferably, the heating reflux temperature is 60-80°C and the time is 50-70 minutes.

[0015] Preferably, the test sample is Yixuean granules.

[0016] Preferably, the elution volume of the mixed solution is 20-70 ml.

[0017] Preferably, the preparation of the test solution comprises the following steps: taking 7.8-8.3 g of the test sample, dissolving it in 28-32 ml of methanol, heating and refluxing for 50-70 min, cooling, filtering, collecting the filtrate, evaporating to dryness in a water bath, adding 18-22 ml of 1% hydrochloric acid to obtain the residue and dissolving it, filtering, applying the filtrate to a cation exchange resin column, washing with water until colorless, eluting with 20-70 ml of the mixed solution, collecting the eluate, evaporating to dryness, and dissolving the residue in 0.8-1.2 ml of methanol to obtain the test solution.

[0018] Preferably, the preparation of the reference solution comprises the following steps: taking 4.8-5.2 g of Leonurus herb, dissolving it in 28-32 ml of 1% hydrochloric acid solution or 28-32 ml of methanol, heating and refluxing for 50-70 min, filtering, applying a cation exchange resin to the filtrate, washing with water until colorless, eluting with 20-70 ml of a mixed solution, collecting the eluate, evaporating to dryness, and dissolving the residue in 0.8-1.2 ml of methanol to obtain a reference solution.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The invention provides a method for simultaneously identifying stachydrine hydrochloride and leonurine. The method adopts a mixed solution of concentrated ammonia water, sodium chloride solution and anhydrous ethanol for elution to prepare a test sample, and combines the elution of a cation exchange column, the regulation of various parameters and the use of an improved color developer, so that stachydrine hydrochloride and leonurine can be separated and identified on the same thin layer plate at the same time. The method has clear color development, simple operation and good separation effect, and the sample processing time can be shortened to 4.5 hours, thereby improving the detection efficiency. The method can be effectively applied to the rapid and efficient identification of motherwort medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 It is a silica gel G thin layer plate diagram of Example 1 of the present invention; wherein 1 is the Yixuean granule test solution, 2 is the motherwort control medicinal material solution, 3 is the motherwortine reference substance, and 4 is the stachydrine hydrochloride reference substance;

[0023] Figure 2 It is a silica gel G thin layer plate diagram of Example 2 of the present invention; wherein 1 is the Yixuean granule test solution, 2 is the motherwort control medicinal material solution, 3 is the motherwortine reference substance, and 4 is the stachydrine hydrochloride reference substance;

[0024] Figure 3Silica gel G thin layer plate diagram of Comparative Example 1 of the present invention; wherein, 1 is the test solution A of Yixue'an granules, 1 is the test solution B of Yixue'an granules, 3 is the solution of the control medicinal material of Leonurus japonicus, 4 is the negative control solution without Leonurus japonicus; 5 is the reference solution of stachydrine hydrochloride;

[0025] Figure 4 Silica gel G thin layer plate diagram of Comparative Example 2 of the present invention; wherein, 1 is the sugar-free test solution of Yixue'an granules, 2 is the sugar-containing test solution of Yixue'an granules, 3 is the solution of the control medicinal material of Leonurus japonicus, 4 is the reference solution of stachydrine hydrochloride, 5 is the negative control solution without Leonurus japonicus;

[0026] Figure 5 Silica gel G thin layer plate diagram of Comparative Example 3 of the present invention; wherein, 1 is the test solution of Yixue'an granules eluted with 3% sodium chloride solution by mass concentration, 2 is the test solution of Yixue'an granules eluted with 5% ammonia water solution by volume concentration, 3 is the test solution of Yixue'an granules eluted with a mixed solution of 20 ml of 25% ammonia water solution by volume concentration, 70 ml of absolute ethanol and water added to 100 ml, 4 is the test solution of Yixue'an granules eluted with a mixed solution of 20 ml of 25% ammonia water solution by volume concentration, 3 g of sodium chloride, 70 ml of absolute ethanol and water added to 100 ml, 5 is the solution of the control medicinal material of Leonurus japonicus, 6 is the reference solution of stachydrine hydrochloride, 7 is the negative control solution without Leonurus japonicus;

[0027] Figure 6 Silica gel G thin layer plate diagram of Comparative Example 4 of the present invention; wherein, 1 is the test solution of Yixue'an granules, 2 is the solution of the control medicinal material of Leonurus japonicus, 3 is the reference substance of leonurine, 4 is the reference solution of stachydrine hydrochloride. Detailed Description of the Invention

[0028] The following is a detailed description of the specific embodiments of the present invention, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Unless otherwise defined, all the technical terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention. Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0029] Example 1

[0030] The identification method for simultaneously identifying stachydrine hydrochloride and leonurine in this example specifically includes the following steps:

[0031] 1. Preparation of the test solution

[0032] Take 8.0 g of sugar-free Yixue'an Granules, dissolve it in 30 ml of methanol, heat under reflux for 60 min, cool, filter, collect the filtrate, evaporate to dryness in a water bath, add 20 ml of 1% hydrochloric acid to the residue to dissolve, filter, load the filtrate onto a 732 sodium-type cation exchange resin column (5 g, 1 cm column), wash with water until colorless, elute with 30 ml of a mixed solution (26 ml of concentrated ammonia water, 2 g of sodium chloride, 70 ml of absolute ethanol, add water to 100 ml), collect the eluate, evaporate to dryness, add 1.0 ml of methanol to dissolve the residue to obtain the test solution of Yixue'an Granules; the concentration of concentrated ammonia water is 25%.

[0033] 2. Preparation of reference solution

[0034] Take 5.0 g of the control crude drug of Leonurus japonicus (batch number: 120912 - 202110; National Institutes for Food and Drug Control), dissolve it in 30 ml of methanol, heat under reflux for 60 min, filter, load the filtrate onto a cation exchange resin (5 g, 1 cm column), wash with water until colorless, elute with 30 ml of a mixed solution (26 ml of concentrated ammonia water, 2 g of sodium chloride, 70 ml of absolute ethanol, add water to 100 ml), collect the eluate, evaporate to dryness, add 1.0 ml of methanol to dissolve the residue to obtain the solution of the control crude drug of Leonurus japonicus.

[0035] 3. Preparation of stachydrine hydrochloride reference substance

[0036] Take the reference substance of stachydrine hydrochloride (batch number: 111823 - 202206; National Institutes for Food and Drug Control), add ethanol to make a solution containing 3 mg per 1 mL as the reference solution.

[0037] 4. Preparation of stachydrine hydrochloride reference substance

[0038] Take the reference substance of stachydrine hydrochloride (batch number: 110712 - 202111; National Institutes for Food and Drug Control), add ethanol to make a solution containing 3 mg per 1 mL as the reference solution.

[0039] 5. Identification by thin layer chromatography

[0040] The developing solvent used for identification by thin layer chromatography is composed of acetone, absolute ethanol and hydrochloric acid in a volume ratio of 10:6:1; conduct the test according to the general rules 0502 in Part IV of the Chinese Pharmacopoeia 2020 Edition for thin layer chromatography. Pipette 10 μl each of the test solution and the reference solution, respectively spot them on the same silica gel G thin layer plate with sodium carboxymethyl cellulose as the binder, develop with the developing solvent, take out and dry in the air, spray with the color developing agent, the spraying amount is 0.5 ml, bake at 105 °C until the spots are clearly developed, and then examine under the light source. In the chromatogram of the test sample, at the corresponding position to the chromatogram of the reference substance, there are spots showing the same color, as Figure 1 shown. The method in this example can simultaneously identify stachydrine hydrochloride and leonurine, and it takes about 5.5 h.

[0041] Example 2

[0042] The method for simultaneously identifying stachydrine hydrochloride and leonurine in this example specifically includes the following steps:

[0043] 1. Preparation of test solution

[0044] Take 8.0 g of sugar-free Yixue'an granules, dissolve them in 30 ml of methanol, heat under reflux for 60 min, cool, filter, collect the filtrate, evaporate to dryness in a water bath, add 20 ml of 1% hydrochloric acid to the residue to dissolve, filter, load the filtrate onto a 732 sodium-type cation exchange resin column (5 g, 1 cm column), wash with water until colorless, elute with 70 ml of a mixed solution (20 ml of concentrated ammonia water, 3 g of sodium chloride, 70 ml of absolute ethanol, add water to 100 ml), collect the eluate, evaporate to dryness, and dissolve the residue in 1.0 ml of methanol to obtain the test solution of Yixue'an granules; the concentration of concentrated ammonia water is 25%.

[0045] 2. Preparation of control solution

[0046] Take 5.0 g of the control medicinal material of Leonurus japonicus (batch number: 120912 - 202110; National Institutes for Food and Drug Control), dissolve it in 30 ml of 1% hydrochloric acid solution, heat under reflux for 60 min, filter, load the filtrate onto a cation exchange resin (5 g, 1 cm column), wash with water until colorless, elute with 70 ml of a mixed solution (20 ml of concentrated ammonia water, 3 g of sodium chloride, 70 ml of absolute ethanol, add water to 100 ml), collect the eluate, evaporate to dryness, and dissolve the residue in 1.0 ml of methanol to obtain the solution of the control medicinal material of Leonurus japonicus.

[0047] 3. Preparation of leonurine reference substance

[0048] Take the reference substance of stachydrine hydrochloride (batch number: 111823 - 202206; National Institutes for Food and Drug Control), add ethanol to make a solution containing 3 mg per 1 mL as the reference substance solution.

[0049] 4. Preparation of stachydrine hydrochloride reference substance

[0050] Take the reference substance of stachydrine hydrochloride (batch number: 110712 - 202111; National Institutes for Food and Drug Control), add ethanol to make a solution containing 3 mg per 1 mL as the reference substance solution.

[0051] 5. Identification by thin layer chromatography

[0052] The developing solvent used for identification by thin-layer chromatography consists of acetone, absolute ethanol, and hydrochloric acid in a volume ratio of 10:6:1; the test is carried out according to the thin-layer chromatography method in General Principles 0502, Volume IV of Chinese Pharmacopoeia 2020 Edition. 10 μl of the test solution and the reference solution are respectively spotted on the same silica gel G thin-layer plate using sodium carboxymethylcellulose as the binder. The plate is developed with the developing solvent, taken out and dried in air, sprayed with the coloring agent in a spraying amount of 0.5 ml, baked at 105 °C until the spots are clearly developed, and then examined under a light source. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference substance, such as Figure 2 shown. The method of this example can simultaneously identify stachydrine hydrochloride and leonurine, and it takes about 4.5 h.

[0053] Comparative Example 1

[0054] 1. Preparation of the test solution

[0055] Take 5.0 g of sugar-free Yixue'an granules, grind them finely, add 50 ml of methanol, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, add 20 ml of water to dissolve, adjust the pH value to 1.5 with dilute hydrochloric acid, filter, add the filtrate to a 001X7 type 732 Na-type strong acidic cation exchange resin column, soak for 5 minutes, release at a flow rate of 1 ml / minute, wash with water until the eluate is nearly colorless, discard the aqueous solution, then elute with 50 ml of 2 mol / L ammonia solution, collect the eluate, evaporate to dryness in a water bath, and dissolve the residue in 1 ml of methanol to obtain the test solution A of Yixue'an granules; prepare the test solution B of Yixue'an granules in the same way.

[0056] 2. Preparation of the reference solution

[0057] Take 1 g of the reference medicinal material of Leonurus japonicus Houtt., and prepare the reference medicinal material solution of Leonurus japonicus Houtt. according to the method described in step 1; then take an appropriate amount of stachydrine hydrochloride reference substance, add methanol to make a solution containing 1 mg per 1 ml as the stachydrine hydrochloride reference solution; take 5.0 g of Yixue'an granules without Leonurus japonicus Houtt., grind them finely, and prepare the negative reference solution without Leonurus japonicus Houtt. in the same way as in "1. Preparation of the test solution".

[0058] 3. Identification by thin-layer chromatography

[0059] According to the thin-layer chromatography test published in Appendix VIB of Part I of Chinese Pharmacopoeia 2005 Edition, 10 μl of the above-mentioned test solution A and B of Yixue'an Granules and the reference medicinal material solution of Leonurus japonicus were each taken, 10 μl of the negative control solution without Leonurus japonicus, and 5 μl of the reference solution of stachydrine hydrochloride were spotted on the same silica gel G thin-layer plate using sodium carboxymethylcellulose solution as the binder. Using n-butanol:hydrochloric acid:ethyl acetate = 8:3:1 as the developing solvent, develop, take out, air-dry until there is no smell of the developing solvent, and spray with dilute bismuth potassium iodide test solution; in the chromatogram of the test sample, at the positions corresponding to the chromatogram of the reference medicinal material and the reference solution, spots of the same color appear, and it can be examined under light source. For example, Figure 3 as shown. Only the single component stachydrine hydrochloride was detected in this comparative example.

[0060] Comparative Example 2

[0061] 1. Preparation of the test solution

[0062] Take 8 g of sugar-free Yixue'an Granules, dissolve in 30 ml of methanol, reflux and extract at 90 °C for 60 min, cool, filter, collect the filtrate, evaporate to dryness in a water bath. Dissolve the obtained residue in 8 ml of water, centrifuge at 4000 revolutions per minute for 10 min, take the supernatant, evaporate to dryness in a water bath, dissolve in 5 ml of absolute ethanol, and load onto an activated carbon-aluminum oxide column wetted with absolute ethanol. The inner diameter of the activated carbon-aluminum oxide column is 20 mm, and its packing is composed of 0.5 g of activated carbon and 2 g of 300-mesh aluminum oxide evenly mixed; elute with 30 ml of absolute ethanol, collect the eluate, evaporate to dryness, dissolve the obtained residue in 1 ml of absolute ethanol, take the supernatant, and use it as the sugar-free test solution of Yixue'an Granules; take 25 g of sugar-containing Yixue'an Granules and prepare the sugar-containing test solution of Yixue'an Granules in the same way.

[0063] 2. Preparation of the reference solution

[0064] Take 3 g of Leonurus japonicus medicinal material and prepare the reference medicinal material solution of Leonurus japonicus using the method described in step 1; Preparation of the reference solution of stachydrine hydrochloride: Take the reference substance of stachydrine hydrochloride and dissolve it in absolute ethanol to a concentration of 5 mg / ml; Take 5.0 g of Yixue'an Granules without Leonurus japonicus, grind them finely, and prepare the negative control solution without Leonurus japonicus in the same way as "1. Preparation of the test solution";

[0065] 3. Identification by thin-layer chromatography

[0066] Take 10 μl each of the test solution, Leonurus japonicus control solution, stachydrine hydrochloride reference substance solution, and negative control, and spot them in parallel on the same silica gel G plate. Develop with an eluent of acetone - absolute ethanol - hydrochloric acid in a volume ratio of 10:6:1. After air drying, heat at 105 °C for 15 min, let it cool, spray with a 10% sulfuric acid absolute ethanol solution, dry at 105 °C for 3 min, then spray with a mixed solution of dilute bismuth potassium iodide test solution - 1% ferric chloride absolute ethanol solution in a volume ratio of 10:1, and examine under a light source. If Figure 4 as shown. The sample treatment time for this comparative example is about 9 h.

[0067] Comparative Example 3

[0068] 1. Preparation of the test solution

[0069] Take 8.0 g of sugar - free Yixue'an granules, add 30 ml of methanol, heat under reflux for 1 h, filter, evaporate to dryness, dissolve with 20 ml of 1% hydrochloric acid, filter, pass the filtrate through the pre - treated cation exchange resin (5 g, 1 cm column), wash with water until colorless, elute with 50 ml of 3% sodium chloride solution by mass concentration, collect the eluate, evaporate to dryness, and dissolve the residue with 1 ml of methanol to obtain the test solution of Yixue'an granules eluted with 3% sodium chloride solution by mass concentration; prepare the test solution of Yixue'an granules eluted with 5% ammonia water by volume concentration and the test solution of Yixue'an granules eluted with a mixed solution of 20 ml of 25% ammonia water and 70 ml of absolute ethanol diluted to 100 ml with water in the same method

[0070] the test solution of Yixue'an granules eluted with the solution

[0071] 2. Preparation of the reference substance solution

[0072] Take 5.0 g of Leonurus japonicus medicinal materials, add 30 ml of methanol, heat under reflux for 1 h, filter, evaporate the filtrate to dryness, and dissolve the residue with 1 ml of methanol to obtain the reference medicinal material solution of Leonurus japonicus; Preparation of stachydrine hydrochloride reference substance solution: Take stachydrine hydrochloride reference substance, dissolve it with absolute ethanol to a concentration of 5 mg / ml; Take 8.0 g of Yixue'an granules without Leonurus japonicus, grind them finely, and prepare the negative reference substance solution without Leonurus japonicus in the same method as in "1. Preparation of the test solution".

[0073] 3. Identification by thin - layer chromatography

[0074] Test according to the thin-layer chromatography method (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2020 Edition). Pipette 10 μl of the test solution and the reference crude drug solution respectively, and spot them on the same silica gel G thin-layer plate using sodium carboxymethyl cellulose as the binder. Use acetone-anhydrous ethanol-hydrochloric acid (10:6:1) as the developing solvent, develop, take out, air dry, spray with the color-developing agent, the spraying amount is 0.5 ml, and dry at 105 °C until the spots are clearly developed. In the chromatogram of the test solution, at the positions corresponding to the chromatogram of the reference crude drug, spots of the same color appear. Visual inspection can be carried out under a light source. As Figure 5 shown, the test sample prepared by eluting with the mixed solution of ammonia water, sodium chloride and anhydrous ethanol in this comparative example can simultaneously identify stachydrine hydrochloride and leonurine, and the effect is better.

[0075] Comparative Example 4

[0076] The method for simultaneously identifying stachydrine hydrochloride and leonurine in this comparative example specifically includes the following steps:

[0077] 1. Preparation of the test solution

[0078] Take 8.0 g of sugar-free Yixue'an granules, dissolve them in 30 ml of methanol, heat under reflux for 60 min, cool, filter, collect the filtrate, evaporate to dryness in a water bath, add 20 ml of 1% hydrochloric acid to the residue to dissolve, filter, pass the filtrate through a 732 sodium-type cation exchange resin column (5 g, 1 cm column), wash with water until colorless, elute with 70 ml of a mixed solution (12 ml of concentrated ammonia water, 5 g of sodium chloride, 70 ml of anhydrous ethanol, add water to 100 ml), collect the eluate, evaporate to dryness, and dissolve the residue in 1.0 ml of methanol to obtain the test solution of Yixue'an granules; the concentration of concentrated ammonia water is 25%.

[0079] 2. Preparation of the reference solution

[0080] Take 5.0 g of the reference crude drug of Leonurus japonicus (batch number: 120912-202110; National Institutes for Food and Drug Control), dissolve it in 30 ml of 1% hydrochloric acid solution, heat under reflux for 60 min, filter, pass the filtrate through a cation exchange resin (5 g, 1 cm column), wash with water until colorless, elute with 70 ml of a mixed solution (12 ml of concentrated ammonia water, 5 g of sodium chloride, 70 ml of anhydrous ethanol, add water to 100 ml), collect the eluate, evaporate to dryness, and dissolve the residue in 1.0 ml of methanol to obtain the reference solution of the reference crude drug of Leonurus japonicus.

[0081] 3. Preparation of the reference substance of leonurine

[0082] Take the reference substance of stachydrine hydrochloride (batch number: 111823-202206; National Institutes for Food and Drug Control), and make a solution containing 3 mg per 1 mL with ethanol as the reference solution.

[0083] 4. Preparation of the reference substance of stachydrine hydrochloride

[0084] Take the stachydrine hydrochloride reference substance (batch number: 110712 - 202111; National Institutes for Food and Drug Control), and add ethanol to prepare a solution containing 3 mg per 1 mL as the reference substance solution.

[0085] 5. Identification by thin - layer chromatography

[0086] The developing agent used for identification by thin - layer chromatography is composed of acetone, absolute ethanol and hydrochloric acid in a volume ratio of 10:6:1; conduct the test according to the general rules 0502 in Part IV of the Chinese Pharmacopoeia 2020 Edition for thin - layer chromatography. Pipette 10 μL each of the test solution and the reference substance solution, and spot them on the same silica gel G thin - layer plate with sodium carboxymethylcellulose as the binder respectively. Develop with the developing agent, take out and air - dry, spray with the color - developing agent, the spraying amount is 0.5 mL, bake at 105 °C until the spots are clearly developed, and then examine under the light source. In the chromatogram of the test sample, at the position corresponding to the chromatogram of the reference substance, there are spots showing the same color, as Figure 6 shown. The method of this comparative example can simultaneously identify stachydrine hydrochloride and leonurine, which takes about 4.5 h, and the separation effect of the method of this comparative example is poor.

[0087] In summary, the present invention provides a method for simultaneously identifying stachydrine hydrochloride and leonurine. By using a mixed solution of concentrated ammonia water, sodium chloride solution and absolute ethanol for elution to prepare the test sample, and combining the elution of the cation - exchange column, the regulation of each parameter and the use of the improved color - developing agent, stachydrine hydrochloride and leonurine can be simultaneously separated and identified on the same thin - layer plate, with clear color development, simple operation and good separation effect. The time consumed for sample treatment can be shortened to 4.5 h, improving the detection efficiency, and it can be effectively applied to the rapid and efficient identification of leonurus drugs.

[0088] The foregoing description of the specific exemplary embodiments of the present invention is for the purposes of illustration and exemplification. These descriptions are not intended to limit the present invention to the precise forms disclosed, and obviously, many changes and variations are possible in light of the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the present invention, as well as various different selections and changes. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for simultaneously identifying stachydrine hydrochloride and leonurine, characterized in that: include: Preparation of test solution, preparation of reference solution and identification by thin layer chromatography; The preparation of the test solution comprises the following steps: taking a test sample, dissolving it in methanol, heating and refluxing, cooling, filtering, collecting the filtrate, evaporating to dryness in a water bath, adding hydrochloric acid to obtain a residue to dissolve it, filtering, applying the filtrate to a cation exchange resin column, washing with water until colorless, eluting with a mixed solution, collecting the eluate, evaporating to dryness, and dissolving the residue in methanol to obtain a test solution; The preparation of the reference solution comprises the following steps: taking Leonurus japonicus, adding hydrochloric acid solution or methanol, heating to reflux, filtering, applying cation exchange resin to the filtrate, washing with water until colorless, eluting with a mixed solution, collecting the eluate, evaporating to dryness, dissolving the residue in methanol to obtain the reference solution; The preparation method of the 100 ml mixed solution comprises: mixing 15-30 ml of concentrated ammonia water, 2-4 g of sodium chloride and 70 ml of anhydrous ethanol, and adding water to 100 ml; the volume concentration of the concentrated ammonia water is 25%-28%.

2. The method for simultaneously identifying stachydrine hydrochloride and leonurine according to claim 1, characterized in that: The developing agent used for thin layer chromatography identification is composed of acetone, anhydrous ethanol and hydrochloric acid in a volume ratio of 9-11:5-7:1; the test is carried out according to the thin layer chromatography "Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502", 8-12ul of the test solution and the reference solution are respectively taken and spotted on the same silica gel G thin layer plate with sodium carboxymethyl cellulose as an adhesive, developed with a developing agent, taken out and dried, sprayed with a color developer, baked at 100-110°C until the spots are clearly colored, and then inspected under a light source.

3. The method for simultaneously identifying stachydrine hydrochloride and leonurine according to claim 1, characterized in that: The preparation method of the 100 ml mixed solution comprises: mixing 20 ml of concentrated ammonia water, 3 g of sodium chloride, and 70 ml of anhydrous ethanol, and adding water to 100 ml.

4. The method for simultaneously identifying stachydrine hydrochloride and leonurine according to claim 2, characterized in that: The developing solvent used in the thin layer chromatography identification is composed of acetone, anhydrous ethanol and hydrochloric acid in a volume ratio of 10:6:

1.

5. The method for simultaneously identifying stachydrine hydrochloride and leonurine according to claim 1, characterized in that: The color developer is prepared by mixing a dilute potassium bismuth iodide test solution and a ferric chloride test solution in a volume ratio of 10:1, and the spraying amount is 0.2ml-1.0ml.

6. The method for simultaneously identifying stachydrine hydrochloride and leonurine according to claim 1, characterized in that: The model of the cation exchange resin column is 732 sodium type.

7. The method for simultaneously identifying stachydrine hydrochloride and leonurine according to claim 1, characterized in that: The heating reflux temperature is 60-80°C and the time is 50-70 minutes.

8. The method for simultaneously identifying stachydrine hydrochloride and leonurine according to claim 1, characterized in that: The test sample is Yixuean granules.

9. The method for simultaneously identifying stachydrine hydrochloride and leonurine according to claim 1, characterized in that: The preparation of the test solution comprises the following steps: taking 7.8-8.3 g of the test sample, dissolving it in 28-32 ml of methanol, heating and refluxing for 50-70 min, cooling, filtering, collecting the filtrate, evaporating to dryness in a water bath, adding 18-22 ml of 1% hydrochloric acid to obtain the residue and dissolving it, filtering, applying the filtrate to a cation exchange resin column, washing with water until colorless, eluting with 20-70 ml of a mixed solution, collecting the eluate, evaporating to dryness, adding 0.8-1.2 ml of methanol to obtain the residue and dissolving it to obtain the test solution.

10. The method for simultaneously identifying stachydrine hydrochloride and leonurine according to claim 1, characterized in that: The preparation of the reference solution comprises the following steps: taking 4.8-5.2 g of Leonurus herb, dissolving it in 28-32 ml of 1% hydrochloric acid solution or 28-32 ml of methanol, heating and refluxing for 50-70 min, filtering, applying a cation exchange resin to the filtrate, washing with water until colorless, eluting with 20-70 ml of a mixed solution, collecting the eluate, evaporating to dryness, and dissolving the residue in 0.8-1.2 ml of methanol to obtain a reference solution.

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