Thin-layer Identification Method for Dipsacus asperoides, Paeonia veitchii, and Notopterygium incisum in Huoxue Antong Tincture

Abstract

The present invention discloses a thin-layer identification method for Dipsaci Radix, Paeoniae Radix Rubra, and Notopterygii Rhizoma et Radix in Huoxue Antong Tincture. With the same test sample solution, three medicinal herbs, namely Dipsaci Radix, Paeoniae Radix Rubra, and Notopterygii Rhizoma et Radix in Huoxue Antong Tincture, can be simultaneously identified on one thin-layer plate. This method is simple, rapid, with a large amount of information, effectively improving the detection efficiency, reducing the test cost, having a short detection cycle, being accurate, reliable, with good reproducibility and high accuracy, and being able to effectively and comprehensively control the quality of Huoxue Antong Tincture.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection of traditional Chinese medicine components, and particularly relates to a thin-layer identification method for Dipsaci Radix, Paeoniae Radix Rubra, and Notopterygii Rhizoma et Radix in Huoxue Antong Tincture. Background Art

[0002] Huoxue Antong Tincture is a traditional Chinese medicine compound preparation developed by the First Affiliated Hospital of Hunan University of Chinese Medicine. It has the effects of expelling wind and removing dampness, promoting blood circulation and relieving pain, and is used for lumbocrural pain caused by wind-dampness obstruction. Huoxue Antong Tincture is made from 19 traditional Chinese medicines such as Taxilli Herba, Notopterygii Rhizoma et Radix, Dipsaci Radix, Paeoniae Radix Rubra, Achyranthis Bidentatae Radix, Salviae Miltiorrhizae Radix, Rehmanniae Radix Preparata, etc.

[0003] As is well known, thin-layer identification is basically for one variety. If there are N identifications, N test solution samples need to be prepared, N thin-layer plates, developed N times, and the traditional identification mode for N kinds of medicinal materials is adopted. To exclude interference, the sample pretreatment procedure is complex and cumbersome, requiring a large amount of organic reagents for repeated purification treatment, which is time-consuming, laborious, reagent-consuming, polluting the environment, harmful to health, and has a long detection cycle. In Huoxue Antong Tincture, Rehmanniae Radix Preparata, Dipsaci Radix, Salviae Miltiorrhizae Radix, Paeoniae Radix Rubra, and Notopterygii Rhizoma et Radix are all characteristic index components. To identify 5 characteristic index components in Huoxue Antong Tincture, 5 test solution samples need to be prepared, developed 5 times on 5 thin-layer plates; this identification method is cumbersome to operate, has a high inspection cost, and low efficiency. In addition, Huoxue Antong Tincture is obtained by soaking 19 kinds of medicinal materials in ethanol at a specified concentration, so it contains water-soluble components and alcohol-soluble components of 19 kinds of medicinal materials, and the total components are complex. It is difficult to avoid the interference of other medicinal material components by conventional thin-layer test sample treatment methods. Therefore, there is an urgent need to find a simple, fast, more stable, and specific identification method to improve the detection efficiency, reduce the detection cost, and effectively control the quality of Huoxue Antong Tincture. Summary of the Invention

[0004] To solve the above problems, the purpose of the present invention is to provide a thin-layer identification method for Dipsaci Radix, Paeoniae Radix Rubra, and Notopterygii Rhizoma et Radix in Huoxue Antong Tincture.

[0005] To achieve the above invention purpose, the present invention adopts the following technical solutions:

[0006] A thin-layer identification method for Dipsaci Radix, Paeoniae Radix Rubra, and Notopterygii Rhizoma et Radix in Huoxue Antong Tincture, which comprises the following steps:

[0007] (1) Preparation of test solution: Take 100 - 200 ml of Huoxue Antong Tincture, evaporate it on a water bath until there is no alcohol smell. Add 20 - 40 ml of water to the residue to dissolve it, and filter. Add 20 - 40 ml of ether to the filtrate, shake and extract. Discard the ether layer. Add water - saturated n - butanol solution to the aqueous layer and shake and extract 2 - 4 times, 30 - 40 ml each time. Combine the n - butanol layers. Wash the n - butanol layer with 25 - 50 ml of ammonia test solution or 1 - 3% sodium hydroxide solution 1 - 2 times, discard the washing solution, then wash it with water saturated with n - butanol 1 - 2 times, 30 - 40 ml each time. Discard the aqueous layer. Evaporate the n - butanol layer to dryness. Dissolve the residue in 1 - 3 ml of methanol or ethanol to obtain the test solution;

[0008] (2) Preparation of reference solution: Separately take reference substance of asperosaponin VI, add methanol to make a solution containing 1 mg per 1 ml as the first reference solution;

[0009] Separately take reference substance of paeoniflorin, add ethanol to make a solution containing 1 mg per 1 ml as the second reference solution;

[0010] Separately take 0.5 g of the reference crude drug of Notopterygium incisum, add 10 - 50 ml of methanol, ultrasonically treat for 30 - 40 minutes, filter, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the third reference crude drug solution;

[0011] (3) Thin - layer identification: According to the thin - layer chromatography test, absorb 3 - 8 μl of the above - mentioned first reference solution, 3 - 8 μl of the second reference solution, 3 - 8 μl of the third reference crude drug solution, and 3 - 8 μl of the test solution, and spot them on the same thin - layer plate respectively. Use the lower layer solution of chloroform - methanol - water placed at 8 - 10 °C for 12 hours as the developing agent, and the volume ratio of chloroform, methanol, and water is 24 - 28∶12 - 16∶3 - 6. Develop under the conditions of temperature 20 - 25 °C and humidity 50 - 60% RH. Take out, dry in air, and examine under ultraviolet light at 365 nm. In the test solution chromatogram, at the position corresponding to the chromatogram of the reference crude drug of Notopterygium incisum, there are fluorescent spots of the same color. Then, spray with a vanillin - sulfuric acid solution with a mass percentage concentration of 4 - 8% or a sulfuric acid - ethanol solution with a mass percentage concentration of 5 - 15%, heat at 100 - 110 °C until the spots are clearly colored, and examine under daylight. In the test solution chromatogram, at the positions corresponding to the chromatograms of the reference substances of asperosaponin VI and paeoniflorin, there are daylight spots of the same color respectively.

[0012] A thin - layer identification method for Dipsacus asperoides, Paeonia lactiflora, and Notopterygium incisum in Huoxue Antong Tincture, which comprises the following steps:

[0013] (1) Preparation of the test solution: Take 100 - 200 ml of Huoxue Antong Tincture, evaporate it on a water bath until the alcohol smell disappears. Add 20 - 40 ml of water to the residue to dissolve it, and filter. Add 20 - 60 ml of ether to the filtrate, shake and extract. Discard the ether layer. Add water-saturated n-butanol solution to the aqueous layer and shake and extract 2 - 4 times, 20 - 30 ml each time. Combine the n-butanol layers. Wash with water saturated with n-butanol 2 - 3 times, 20 ml each time, and discard the aqueous washings. Evaporate the n-butanol layer to dryness. Add 10 - 15 ml of water to the residue to dissolve it, load it onto a D101 macroporous adsorption resin column, and elute successively with water, 30% ethanol, and 70% ethanol. Collect the 70% ethanol eluate, evaporate it to dryness. Add 1 - 3 ml of methanol to the residue to dissolve it, and use it as the test solution.

[0014] (2) Preparation of the reference solution: Separately take the reference substance of Dipsacoside VI, add methanol to make a solution containing 1 mg per 1 ml, and use it as the first reference solution.

[0015] Separately take the reference substance of Paeoniflorin, add ethanol to make a solution containing 1 mg per 1 ml, and use it as the second reference solution.

[0016] Separately take 0.5 g of the reference crude drug of Notopterygium incisum, add 10 - 50 ml of methanol, ultrasonically treat for 30 - 40 minutes, filter, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as the third reference crude drug solution.

[0017] (3) TLC identification: According to the TLC test method, respectively pipette 3 - 8 μl of the above-mentioned first reference solution, second reference solution, third reference crude drug solution, and test solution, and spot them on the same TLC plate. Use the lower layer solution of chloroform - methanol - water placed at 8 - 10 °C for 12 hours as the developing agent, and the volume ratio of chloroform, methanol, and water is 24 - 28∶12 - 16∶3 - 6. Develop at a temperature of 20 - 25 °C and a humidity of 50 - 60% RH. Take out, air dry, and examine under ultraviolet light at 365 nm. In the test solution chromatogram, at the position corresponding to the chromatogram of the reference crude drug of Notopterygium incisum, there are fluorescent spots of the same color. Then, spray with a vanillin sulfuric acid solution with a mass percentage concentration of 4 - 8% or a sulfuric acid ethanol solution with a mass percentage concentration of 5 - 15%, heat at 100 - 110 °C until the spots develop clearly, and examine under daylight. In the test solution chromatogram, at the positions corresponding to the chromatograms of the reference substances of Dipsacoside VI and Paeoniflorin, there are daylight spots of the same color respectively.

[0018] Furthermore, the above-mentioned TLC plate is a silica gel G TLC plate, silica gel GF 254 TLC plate or high-performance silica gel G TLC plate.

[0019] Preferably, the above-mentioned TLC plate uses a silica gel G plate from Merck, Germany.

[0020] Furthermore, in the above Huoxue Antong Tincture, the mass ratio of Dipsacus asperoides, Paeonia lactiflora, and Notopterygium incisum is 1:1:1.53 - 1.86.

[0021] Application of the above thin-layer identification method in the quality control of Huoxue Antong Tincture.

[0022] Due to the adoption of the above technical solution, the present invention has the following advantages:

[0023] The thin-layer identification method for Dipsacus asperoides, Paeonia lactiflora, and Notopterygium incisum in the Huoxue Antong Tincture has simple operation, appropriate selection of developing agent, short detection period, accurate and reliable results, good reproducibility, and high accuracy; with the same test solution, using an exclusive developing agent to simultaneously identify the three medicinal flavors of Dipsacus asperoides, Paeonia lactiflora, and Notopterygium incisum in the Huoxue Antong Tincture on one thin-layer plate, it is simple, fast, has a large amount of information, effectively improves the detection efficiency, and reduces the test cost; it can effectively and comprehensively control the quality of Huoxue Antong Tincture, ensure the safety of medication, guarantee its clinical efficacy, and provide a theoretical basis for the in-depth study of the effective substances of Huoxue Antong Tincture. Description of the Drawings

[0024] Figure 1 is the thin-layer chromatogram of each sample solution in Example 1 of the thin-layer identification method for Dipsacus asperoides, Paeonia lactiflora, and Notopterygium incisum in the Huoxue Antong Tincture of the present invention;

[0025] Figure 2 is the thin-layer chromatogram of each sample solution in Example 2 of the thin-layer identification method for Dipsacus asperoides, Paeonia lactiflora, and Notopterygium incisum in the Huoxue Antong Tincture of the present invention;

[0026] Figure 3 is the thin-layer chromatogram of each sample solution in Example 3 of the thin-layer identification method for Dipsacus asperoides, Paeonia lactiflora, and Notopterygium incisum in the Huoxue Antong Tincture of the present invention;

[0027] Figure 4 is the thin-layer chromatogram of each sample solution in Example 4 of the thin-layer identification method for Dipsacus asperoides, Paeonia lactiflora, and Notopterygium incisum in the Huoxue Antong Tincture of the present invention;

[0028] Figure 5 is the thin-layer chromatogram of each sample solution in Example 5 of the thin-layer identification method for Dipsacus asperoides, Paeonia lactiflora, and Notopterygium incisum in the Huoxue Antong Tincture of the present invention;

[0029] Figure 6 is the thin-layer chromatogram of each sample solution in Example 6 of the thin-layer identification method for Dipsacus asperoides, Paeonia lactiflora, and Notopterygium incisum in the Huoxue Antong Tincture of the present invention. Detailed Embodiments

[0030] The technical solution of the present invention will be further described in detail below with reference to the drawings and embodiments.

[0031] Example 1

[0032] A thin-layer identification method for Dipsacus asperoides, Paeonia lactiflora, and Notopterygium incisum in Huoxue Antong Tincture includes the following specific steps:

[0033] (1) Preparation of the test solution: Take 100 ml of Huoxue Antong Tincture, evaporate it on a water bath until the alcohol smell disappears, dissolve the residue in 20 ml of water, and filter for 20 min; add 20 ml of ether to the filtrate, shake and extract once, discard the ether layer, add water-saturated n-butanol to the aqueous layer and shake and extract twice, 30 ml each time, combine the n-butanol layers; wash the n-butanol layer with 25 ml of ammonia test solution once, discard the ammonia test solution, then wash with 30 ml of water saturated with n-butanol once, discard the aqueous layer, evaporate the n-butanol layer to dryness, dissolve the residue in 1 ml of ethanol to obtain the test solution;

[0034] (2) Preparation of the reference solution: Separately take the reference substance of asperosaponin VI, and make a solution containing 1 mg per 1 ml with methanol as the first reference solution;

[0035] Separately take the reference substance of paeoniflorin, and make a solution containing 1 mg per 1 ml with ethanol as the second reference solution;

[0036] Separately take 0.5 g of the control crude drug of Notopterygium incisum, add 30 ml of methanol, ultrasonically treat for 30 min, filter, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the third control crude drug solution;

[0037] (3) Thin-layer identification: According to the test of thin-layer chromatography (General Principles 0502, Volume IV of the Chinese Pharmacopoeia 2020 Edition), absorb 5 μl of the above-mentioned first reference solution, 5 μl of the second reference solution, 5 μl of the third control crude drug solution, and 5 μl of each of the 3 batches of test solutions, and spot them on the same silica gel G thin-layer plate respectively. Use the lower layer solution of chloroform-methanol-water placed at 8°C or below for 12 hours as the developing agent, and the volume ratio between chloroform, methanol, and water is 26∶14∶4. Develop at a temperature of 20°C and a humidity of 50% RH, take out, dry in air, and examine under ultraviolet light at 365 nm; in the test solution chromatogram, at the position corresponding to the chromatogram of the control crude drug of Notopterygium incisum, there are fluorescent spots showing the same color; then, spray with 5% vanillin sulfuric acid solution, heat at 100°C until the spots develop clearly, and examine under daylight. In the test solution chromatogram, at the positions corresponding to the chromatograms of the reference substances of asperosaponin VI and paeoniflorin, there are daylight spots showing the same color respectively, to achieve the identification of Dipsaci Radix, Paeoniae Radix Rubra, and Notopterygii Rhizoma et Radix.

[0038] Figure 1 is the thin-layer chromatogram of each sample solution in Example 1, Figure 1a is the thin-layer chromatogram of each sample solution when examined under ultraviolet light, Figure 1bIt is the thin-layer chromatogram of each sample solution when inspected under daylight; among them, 1 is 5 μl of the solution of the control crude drug of Notopterygium incisum, 2 is 5 μl of the solution of the reference substance of paeoniflorin, 3 is 5 μl of the solution of the reference substance of asperosaponin VI, 4 is 5 μl of the first test solution, 5 is 5 μl of the second test solution, and 6 is 5 μl of the third test solution. It can be known from Figure 1 that at the positions corresponding to the chromatograms of the reference substance of asperosaponin VI, the reference substance of paeoniflorin, and the control crude drug of Notopterygium incisum, spots of the same color appear respectively.

[0039] Example 2

[0040] A thin-layer identification method for Dipsacus asper Wall., Paeonia lactiflora Pall., and Notopterygium incisum in Huoxue Antong Tincture, which includes the following specific steps:

[0041] (1) Preparation of the test solution: Take 150 ml of Huoxue Antong Tincture, evaporate it on a water bath until there is no alcohol smell, add 35 ml of water to the residue to dissolve it, and filter for 25 min; add 30 ml of ether to the filtrate, shake and extract once, discard the ether layer, add water-saturated n-butanol to the aqueous layer and shake and extract 3 times, 35 ml each time, combine the n-butanol layers; wash the n-butanol layer with 35 ml of ammonia test solution once, discard the ammonia test solution, and then wash it with 35 ml of water saturated with n-butanol once, discard the aqueous layer, evaporate the n-butanol layer to dryness, and dissolve the residue in 2 ml of ethanol to obtain the test solution;

[0042] (2) Preparation of the reference substance solution: Take another reference substance of asperosaponin VI, add methanol to make a solution containing 1 mg per 1 ml as the first reference substance solution;

[0043] Take another reference substance of paeoniflorin, add ethanol to make a solution containing 1 mg per 1 ml as the second reference substance solution;

[0044] Take another 0.5 g of the control crude drug of Notopterygium incisum, add 30 ml of methanol, ultrasonically treat for 35 min, filter, evaporate to dryness, and dissolve the residue in 1 ml of methanol as the third control crude drug solution;

[0045] (3) Thin-layer identification: Test according to the thin-layer chromatography method (General Principles 0502, Volume IV of the Chinese Pharmacopoeia 2020 Edition). Pipette 5 μl of the above-mentioned first reference substance solution, 5 μl of the second reference substance solution, 5 μl of the third reference medicinal material solution, and 5 μl of each of the 3 batches of test sample solutions, and spot them on the same silica gel G thin-layer plate respectively. Use the lower layer solution of chloroform-methanol-water placed at 10°C or below for 12 hours as the developing agent, and the volume ratio of chloroform, methanol, and water is 28:16:6. Develop at a temperature of 22°C and a humidity of 55% RH. Take out, dry in the air, and examine under ultraviolet light at 365 nm. In the chromatogram of the test sample, at the position corresponding to the chromatogram of the Notopterygii Rhizoma et Radix reference medicinal material, fluorescent spots of the same color appear. Then, spray with 6% vanillin sulfuric acid solution, heat at 102°C until the spots are clearly developed, and examine under daylight. In the chromatogram of the test sample, at the positions corresponding to the chromatograms of the asperosaponin VI reference substance and the paeoniflorin reference substance, daylight spots of the same color appear respectively, realizing the identification of Dipsaci Radix, Paeoniae Radix Rubra, and Notopterygii Rhizoma et Radix.

[0046] Figure 2 is the thin-layer chromatogram of each sample solution in Example 2, Figure 2a which is the thin-layer chromatogram of each sample solution when examined under ultraviolet light, Figure 2b and which is the thin-layer chromatogram of each sample solution when examined under daylight; among them, 1 is 5 μl of the Notopterygii Rhizoma et Radix reference medicinal material solution, 2 is 5 μl of the paeoniflorin reference substance solution, 3 is 5 μl of the asperosaponin VI reference substance solution, 4 is 5 μl of the first test sample solution, 5 is 5 μl of the second test sample solution, and 6 is 5 μl of the third test sample solution. It can be seen from Figure 2 that at the positions corresponding to the chromatograms of the asperosaponin VI reference substance, the paeoniflorin reference substance, and the Notopterygii Rhizoma et Radix reference medicinal material, spots of the same color appear respectively.

[0047] Example 3

[0048] A thin-layer identification method for Dipsaci Radix, Paeoniae Radix Rubra, and Notopterygii Rhizoma et Radix in Huoxue Antong Tincture, which comprises the following specific steps:

[0049] (1) Preparation of the test sample solution: Take 200 ml of Huoxue Antong Tincture, evaporate it on a water bath until there is no alcohol smell, add 40 ml of water to the residue to dissolve it, and filter for 30 minutes; add 40 ml of ether to the filtrate, shake and extract 2 times, discard the ether layer, add water-saturated n-butanol to the aqueous layer and shake and extract 4 times, 40 ml each time, combine the n-butanol layers; wash the n-butanol layer with 50 ml of 1% sodium hydroxide solution 2 times, discard the ammonia test solution, then wash with water saturated with n-butanol 2 times, 40 ml each time, discard the aqueous layer, evaporate the n-butanol layer to dryness, dissolve the residue in 3 ml of ethanol to obtain the test sample solution;

[0050] (2) Preparation of the reference substance solution: Separately take the asperosaponin VI reference substance, add methanol to make a solution containing 1 mg per 1 ml as the first reference substance solution;

[0051] Separately take paeoniflorin reference substance, add ethanol to make a solution containing 1 mg per 1 ml, as the second reference substance solution;

[0052] Separately take 0.5 g of the reference medicinal material of Notopterygium incisum, add 30 ml of methanol, ultrasonically treat for 35 minutes, filter, evaporate to dryness, dissolve the residue in 1 ml of methanol, as the third reference medicinal material solution;

[0053] (3) Thin-layer identification: Conduct the test according to the thin-layer chromatography method (General Principles 0502, Volume IV of the Chinese Pharmacopoeia 2020 Edition). Pipette 5 μl of the above-mentioned first reference substance solution, 5 μl of the second reference substance solution, 5 μl of the third reference medicinal material solution, and 5 μl of each of the 3 batches of test sample solutions, and spot them on the same silica gel GF 254 thin-layer plate. Use the lower-layer solution of chloroform-methanol-water placed at 10°C or below for 12 hours as the developing agent. The volume ratio among chloroform, methanol, and water is 24:12:3. Develop at a temperature of 25°C and a humidity of 60% RH. Take out, dry in the air, and examine under ultraviolet light at 365 nm; in the chromatogram of the test sample, at the position corresponding to the chromatogram of the reference medicinal material of Notopterygium incisum, there are fluorescent spots showing the same color; then, spray with 15% sulfuric acid ethanol solution, heat at 105°C until the spots develop clearly, and examine under daylight. In the chromatogram of the test sample, at the positions corresponding to the chromatograms of asperosaponin VI reference substance and paeoniflorin reference substance, there are daylight spots showing the same color respectively, realizing the identification of Dipsaci Radix, Paeoniae Radix Rubra, and Notopterygium incisum.

[0054] Figure 3 is the thin-layer chromatogram of each sample solution in Example 3, Figure 3a is the thin-layer chromatogram of each sample solution when examined under ultraviolet light, Figure 3b is the thin-layer chromatogram of each sample solution when examined under daylight; among them, 1 is 5 μl of the reference medicinal material solution of Notopterygium incisum, 2 is 5 μl of the paeoniflorin reference substance solution, 3 is 5 μl of the asperosaponin VI reference substance solution, 4 is 5 μl of the first test sample solution, 5 is 5 μl of the second test sample solution, and 6 is 5 μl of the third test sample solution. As can be seen from Figure 2, at the positions corresponding to the chromatograms of asperosaponin VI reference substance, paeoniflorin reference substance, and the reference medicinal material of Notopterygium incisum, there are spots showing the same color respectively.

[0055] Example 4

[0056] A method for thin-layer identification of Dipsaci Radix, Paeoniae Radix Rubra, and Notopterygium incisum in Huoxue Antong Tincture, which includes the following specific steps:

[0057] (1) Preparation of the test solution: Take 100 ml of Huoxue Antong Tincture, evaporate it on a water bath until the alcohol smell disappears, dissolve the residue in 20 ml of water, and filter; extract the filtrate with ether by shaking 2 times, 20 ml each time, discard the ether layer, extract the aqueous layer with n-butanol saturated with water by shaking 3 times, 20 ml each time, combine the n-butanol layers; wash with water saturated with n-butanol 2 times, 20 ml each time, discard the washing liquid, evaporate the n-butanol layer to dryness, dissolve the residue in 10 ml of water, pass through a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column height 12 cm), elute successively with 50 ml each of water, 30% ethanol, and 70% ethanol, collect the 70% ethanol eluate, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution;

[0058] (2) Preparation of the reference solution: Separately take the reference substance of Dipsacoside VI, add methanol to make a solution containing 1 mg per 1 ml as the first reference solution;

[0059] Separately take the reference substance of Paeoniflorin, add ethanol to make a solution containing 1 mg per 1 ml as the second reference solution;

[0060] Separately take 0.5 g of the reference crude drug of Notopterygium incisum, add 30 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the third reference crude drug solution;

[0061] (3) Thin-layer identification: Conduct the test according to the thin-layer chromatography method (General Rules 0502, Volume IV, Chinese Pharmacopoeia 2020 Edition). Pipette 5 μl of the above-mentioned first reference solution, 5 μl of the second reference solution, 5 μl of the third reference crude drug solution, and 5 μl of each of the 3 batches of test solutions, and spot them on the same silica gel G thin-layer plate respectively. Use the lower layer solution of chloroform-methanol-water placed at 9°C for 12 hours as the developing solvent, with the volume ratio of chloroform, methanol, and water being 24:12:3. Develop at a temperature of 20°C and a humidity of 53% RH, take out, air dry, and examine under ultraviolet light at 365 nm; in the chromatogram of the test sample, at the position corresponding to the chromatogram of the reference crude drug of Notopterygium incisum, there are fluorescent spots showing the same color; then, spray with 10% sulfuric acid ethanol solution, heat at 100°C until the spots develop clearly, and examine under daylight. In the chromatogram of the test sample, at the positions corresponding to the chromatograms of the reference substances of Dipsacoside VI and Paeoniflorin, there are daylight spots showing the same color respectively, to achieve the identification of Dipsacus asperoides, Paeonia lactiflora, and Notopterygium incisum.

[0062] Figure 4 is the thin-layer chromatogram of each sample solution in Example 4, Figure 4a which is the thin-layer chromatogram of each sample solution when examined under ultraviolet light, Figure 4bIt is the thin-layer chromatogram of each sample solution when examined under daylight; among them, 1 is 5 μl of the solution of the control crude drug of Notopterygium incisum, 2 is 5 μl of the solution of the reference substance of paeoniflorin, 3 is 5 μl of the solution of the reference substance of asperosaponin VI, 4 is 5 μl of the first test solution, 5 is 5 μl of the second test solution, and 6 is 5 μl of the third test solution. It can be seen from Figure 4 that at the positions corresponding to the chromatograms of the reference substance of asperosaponin VI, the reference substance of paeoniflorin, and the control crude drug of Notopterygium incisum, spots of the same color appear respectively.

[0063] Example 5

[0064] A thin-layer identification method for Dipsacus asperoides, Paeonia lactiflora, and Notopterygium incisum in Huoxue Antong Tincture, which comprises the following specific steps:

[0065] (1) Preparation of the test solution: Take 150 ml of Huoxue Antong Tincture, evaporate it to alcohol-free on a water bath, add 30 ml of water to the residue to dissolve it, and filter; extract the filtrate with ether by shaking for 2 times, 25 ml each time, discard the ether layer, extract the aqueous layer with n-butanol saturated with water by shaking for 3 times, 20 ml each time, combine the n-butanol layers; wash with water saturated with n-butanol for 2 times, 20 ml each time, discard the water washing layer, evaporate the n-butanol layer to dryness, add 10 ml of water to the residue to dissolve it, pass through a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column height 12 cm), elute successively with 50 ml each of water, 30% ethanol, and 70% ethanol, collect the 70% ethanol eluate, evaporate it to dryness, and add 1 ml of methanol to the residue to dissolve it as the test solution;

[0066] (2) Preparation of the reference substance solution: Take another reference substance of asperosaponin VI, add methanol to make a solution containing 1 mg per 1 ml as the first reference substance solution;

[0067] Take another reference substance of paeoniflorin, add ethanol to make a solution containing 1 mg per 1 ml as the second reference substance solution;

[0068] Take another 0.5 g of the control crude drug of Notopterygium incisum, add 40 ml of methanol, ultrasonically treat for 35 minutes, filter, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it as the third control crude drug solution;

[0069] (3) Thin-layer identification: Test according to the thin-layer chromatography method (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2020 Edition). Pipette 5 μl of the above-mentioned first reference substance solution, 5 μl of the second reference substance solution, 5 μl of the third reference crude drug solution, and 5 μl of each of the 3 batches of test sample solutions, and spot them on the same silica gel G thin-layer plate respectively. Use the lower layer solution of chloroform-methanol-water placed at 9°C for 12 hours as the developing solvent, and the volume ratio of chloroform, methanol, and water is 24:14:4. Develop at a temperature of 22°C and a humidity of 55% RH. Take out, dry in air, and examine under ultraviolet light at 365 nm; in the chromatogram of the test sample, at the position corresponding to the chromatogram of the Notopterygii Rhizoma et Radix reference crude drug, fluorescent spots of the same color appear; then, spray with a 6% vanillin sulfuric acid solution by mass percentage concentration, heat at 100°C until the spots develop clearly, and examine under daylight. In the chromatogram of the test sample, at the positions corresponding to the chromatograms of the asperosaponin VI reference substance and the paeoniflorin reference substance, daylight spots of the same color appear respectively, realizing the identification of Dipsaci Radix, Paeoniae Radix Rubra, and Notopterygii Rhizoma et Radix.

[0070] Figure 5 is the thin-layer chromatogram of each sample solution in Example 5, Figure 5a which is the thin-layer chromatogram of each sample solution when examined under ultraviolet light, Figure 5b and which is the thin-layer chromatogram of each sample solution when examined under daylight; among them, 1 is 5 μl of the Notopterygii Rhizoma et Radix reference crude drug solution, 2 is 5 μl of the paeoniflorin reference substance solution, 3 is 5 μl of the asperosaponin VI reference substance solution, 4 is 5 μl of the first test sample solution, 5 is 5 μl of the second test sample solution, and 6 is 5 μl of the third test sample solution. It can be seen from Figure 5 that at the positions corresponding to the chromatograms of the asperosaponin VI reference substance, the paeoniflorin reference substance, and the Notopterygii Rhizoma et Radix reference crude drug, spots of the same color appear respectively.

[0071] Example 6

[0072] A thin-layer identification method for Dipsaci Radix, Paeoniae Radix Rubra, and Notopterygii Rhizoma et Radix in Huoxue Antong Tincture, which comprises the following specific steps:

[0073] (1) Preparation of the test sample solution: Take 200 ml of Huoxue Antong Tincture, evaporate it on a water bath until there is no alcohol smell, add 40 ml of water to the residue to dissolve it, and filter; extract the filtrate with ether by shaking 2 times, 30 ml each time, discard the ether layer, and extract the aqueous layer with water-saturated n-butanol by shaking 3 times, 30 ml each time, and combine the n-butanol layers; wash with n-butanol-saturated water 3 times, 20 ml each time, discard the washing solution, evaporate the n-butanol layer to dryness, add 15 ml of water to the residue to dissolve it, pass through a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column height 12 cm), elute successively with 50 ml of water, 30% ethanol, and 70% ethanol each, collect the 70% ethanol eluate, evaporate to dryness, and add 3 ml of methanol to the residue to dissolve it as the test sample solution;

[0074] (2) Preparation of reference substance solution: Separately take asperosaponin VI reference substance, add methanol to prepare a solution containing 1 mg per 1 ml as the first reference substance solution;

[0075] Separately take paeoniflorin reference substance, add ethanol to prepare a solution containing 1 mg per 1 ml as the second reference substance solution;

[0076] Separately take 0.5 g of the control medicinal material of Notopterygium incisum, add 45 ml of methanol, ultrasonically treat for 40 minutes, filter, evaporate to dryness, dissolve the residue in 1 ml of methanol to prepare the third control medicinal material solution;

[0077] (3) Thin layer identification: Conduct the test according to the thin layer chromatography method (General Principles 0502, Volume IV of the Chinese Pharmacopoeia 2020 Edition). Pipette 5 μl of the above-mentioned first reference substance solution, 5 μl of the second reference substance solution, 5 μl of the third control medicinal material solution, and 5 μl of the test solution respectively, and spot them on the same silica gel G thin layer plate. Use the lower layer solution of chloroform - methanol - water placed at 10°C or below for 12 hours as the developing agent, and the volume ratio of chloroform, methanol, and water is 28∶16∶6. Develop at a temperature of 25°C and a humidity of 60% RH, take out, air dry, and examine under ultraviolet light at 365 nm. In the test solution chromatogram, at the position corresponding to the chromatogram of the control medicinal material of Notopterygium incisum, fluorescent spots of the same color appear; then, spray with a vanillin sulfuric acid solution with a mass percentage concentration of 8%, heat at 106°C until the spots are clearly developed, and examine under daylight. In the test solution chromatogram, at the positions corresponding to the chromatograms of asperosaponin VI reference substance and paeoniflorin reference substance, daylight spots of the same color appear respectively.

[0078] Figure 6 is the thin layer chromatogram of each sample solution in Example 6, Figure 6a is the thin layer chromatogram of each sample solution when examined under ultraviolet light, Figure 6b is the thin layer chromatogram of each sample solution when examined under daylight; among them, 1 is 5 μl of the control medicinal material solution of Notopterygium incisum, 2 is 5 μl of the paeoniflorin reference substance solution, 3 is 5 μl of the asperosaponin VI reference substance solution, 4 is 5 μl of the first test solution, 5 is 5 μl of the second test solution, and 6 is 5 μl of the third test solution. It can be seen from Figure 5 that at the positions corresponding to the chromatograms of asperosaponin VI reference substance, paeoniflorin reference substance, and the control medicinal material of Notopterygium incisum, spots of the same color appear respectively.

[0079] The above are only the preferred embodiments of the present invention, rather than limitations on the present invention. Without departing from the spirit and scope of the present invention, all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of patent protection of the present invention.

Claims

1. A thin layer chromatography identification method for Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in the tincture for promoting blood circulation and relieving pain, wherein the mass ratio of Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in the tincture for promoting blood circulation and relieving pain is 1:1:1.53-1.86, and the thin layer chromatography identification method comprises the following steps: (1) Preparation of test solution; (2) Preparation of reference solution: Take Dipsacus asper saponin VI reference substance, add methanol to prepare a solution containing 1 mg per 1 ml, as the first reference solution; take Paeoniflorin reference substance, add ethanol to prepare a solution containing 1 mg per 1 ml, as the second reference solution; take 0.5 g of Notopterygium root reference medicinal material, add 10 ml to 50 ml of methanol, ultrasonically treat for 30 to 40 minutes, filter, evaporate to dryness, and add 1 ml of methanol to the residue to dissolve it, as the third reference medicinal material solution; (3) Thin layer identification; characterized by: (1) Preparation of test solution: Take 100ml-200ml of Huoxue Antong Tincture, steam it on a water bath until there is no alcohol smell, add 20ml-40ml of water to dissolve the residue, and filter; add 20-40ml of ether to the filtrate, shake and extract, discard the ether solution, add water-saturated n-butanol solution to the water layer, shake and extract 2-4 times, 30ml-40ml each time, and combine the n-butanol solution; wash the n-butanol solution with 25ml-50ml of 1-3% sodium hydroxide solution 1-2 times, discard the washing solution, and then wash it with water saturated with n-butanol 1-2 times, 30ml-40ml each time, discard the water solution, evaporate the n-butanol solution to dryness, add 1ml-3ml of methanol or ethanol to dissolve the residue, and obtain the test solution; (3) Thin layer chromatography: According to the thin layer chromatography test, take 3-8 μl of the first reference solution, 3-8 μl of the second reference solution, 3-8 μl of the third reference medicinal material solution, and 3-8 μl of the test solution, and apply them to the same silica gel GF 254 On the thin layer plate, a lower layer solution of chloroform-methanol-water placed below 8-10°C for 12 hours is used as a developing agent, and the volume ratio of chloroform, methanol and water is 24-28:12-16:3-6. The plate is developed at a temperature of 20-25°C and a humidity of 50-60%RH, taken out, air-dried, and inspected under 365nm ultraviolet light. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the Notopterygium root reference medicinal material; then, it is sprayed with a 4-8% mass percentage concentration of vanillin sulfuric acid solution or a 5-15% sulfuric acid ethanol solution, heated at 100-110°C until the spots are clearly colored, and inspected under sunlight. In the chromatogram of the test sample, sunlight spots of the same color appear at the corresponding positions of the chromatograms of the Dipsacus asper saponin VI reference substance and the paeoniflorin reference substance, respectively.

2. The thin layer identification method of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii in the blood-activating and pain-relieving tincture according to claim 1 is characterized by: The volume ratio of the developing solvent, chloroform, methanol and water in the step (3) is 24-26:12-14:3-5.

3. The thin layer identification method of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii in the blood-activating and pain-relieving tincture according to claim 2 is characterized in that: The volume ratio of the developing solvent in step (3) is 26:14:4 among chloroform, methanol and water.

4. A thin layer chromatography identification method for Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in the tincture for promoting blood circulation and relieving pain, wherein the mass ratio of Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in the tincture for promoting blood circulation and relieving pain is 1:1:1.53-1.86, and the thin layer chromatography identification method comprises the following steps: (1) Preparation of test solution; (2) Preparation of reference solution: Take the reference substance of Dipsacus asper saponin VI, add methanol to prepare a solution containing 1 mg per 1 ml, as the first reference solution; take the reference substance of Paeoniflorin, add ethanol to prepare a solution containing 1 mg per 1 ml, as the second reference solution; take 0.5 g of Notopterygium root reference medicinal material, add 10 ml to 50 ml of methanol, ultrasonically treat for 30 to 40 minutes, filter, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, as the third reference medicinal material solution; (3) Thin layer identification; characterized in that it comprises the following steps: (1) Preparation of test solution: Take 100ml-200ml of Huoxue Antong Tincture, steam it on a water bath until there is no alcohol smell, add 20ml-40ml of water to the residue to dissolve it, and filter it; add 20-60ml of ether to the filtrate, shake and extract, discard the ether solution, add saturated n-butanol solution with water to extract 2-4 times, 20ml-30ml each time, and combine the n-butanol solution; wash with n-butanol saturated water 2-3 times, 20ml each time, discard the water washing solution, evaporate the n-butanol solution to dryness, add 10-15ml of water to dissolve the residue, apply it to D101 macroporous adsorption resin column, elute with water, 30% ethanol, and 70% ethanol in turn, collect the 70% ethanol eluate, evaporate to dryness, and add 1ml-3ml of methanol to dissolve the residue as the test solution; (3) Thin layer chromatography: According to the thin layer chromatography test, take 3-8 μl of the first reference solution, 3-8 μl of the second reference solution, 3-8 μl of the third reference medicinal material solution, and 3-8 μl of the test solution, and apply them to the same silica gel GF 254 On the thin layer plate, a lower layer solution of chloroform-methanol-water placed below 8-10°C for 12 hours is used as a developing agent, the volume ratio of chloroform, methanol and water is 24:12:4 or 28:16:6, and the plate is developed at a temperature of 20-25°C and a humidity of 50-60%RH, taken out, air-dried, and inspected under 365nm ultraviolet light. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the Notopterygium root reference medicinal material; then, it is sprayed with a 4-8% mass percentage concentration of vanillin sulfuric acid solution or a 5-15% sulfuric acid ethanol solution, heated at 100-110°C until the spots are clearly colored, and inspected under sunlight. In the chromatogram of the test sample, sunlight spots of the same color appear at the corresponding positions of the chromatograms of the Dipsacus asper saponin VI reference substance and the paeoniflorin reference substance, respectively.

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