Thin-layer identification method for teasel root, red paeony root and notopterygium root in blood-activating pain-relieving tincture

Abstract

The invention discloses a thin-layer identification method for teasel root, red paeony root and notopterygium root in blood-activating and pain-relieving tincture, which uses the same test solution to simultaneously identify three medicines of teasel root, red paeony root and notopterygium root in blood-activating and pain-relieving tincture on a thin-layer plate, is simple, convenient and rapid, has large information amount, effectively improves the detection efficiency, reduces the detection cost, and has the advantages of short detection period, accuracy, reliability and good reproducibility. The accuracy is high, and the quality of the blood-activating and pain-relieving tincture can be effectively and comprehensively controlled.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine component detection, and particularly relates to a thin layer identification method of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii in a blood-activating and pain-relieving tincture. Background Art

[0002] Huoxue Antong Tincture is a Chinese medicine compound preparation developed by the First Affiliated Hospital of Hunan University of Chinese Medicine, which has the effects of dispelling wind and dampness, promoting blood circulation and relieving pain. It is used for waist and leg pain caused by rheumatic obstruction. Huoxue Antong Tincture is made of 19 Chinese medicines, including mistletoe, notopterygium, radix dipsaci, red peony root, rhizoma achyranthis Bidentatae, salvia miltiorrhizae, and rehmannia glutinosa.

[0003] As we all know, thin layer identification is basically one variety. If there are N identifications, N test sample solutions, N thin layer plates, N times of development, and N traditional identification modes of medicinal materials need to be identified. In order to eliminate interference, the sample pretreatment procedure is complicated and cumbersome, and a large amount of organic reagents are required for repeated purification, which is time-consuming, labor-intensive, reagent-consuming, polluting the environment, harmful to health, and has a long detection cycle. In Huoxue Antong Tincture, Radix Rehmanniae, Radix Dipsaci, Radix Salviae Miltiorrhizae, Radix Paeoniae Rubrae, and Radix Notopterygii are all characteristic indicator components. In order to identify the five characteristic indicator components in Huoxue Antong Tincture, five test sample solutions need to be prepared, and five times of development are completed on five thin layer plates; this identification method is cumbersome to operate, with high inspection costs and low efficiency. In addition, Huoxue Antong Tincture is obtained by soaking 19 medicinal materials in ethanol at a specified concentration, so it contains water-soluble components and alcohol-soluble components of 19 medicinal materials, and the total composition is complex. Conventional thin layer sample processing methods are difficult to avoid interference from other medicinal materials. Therefore, there is an urgent need to find a simple, fast, more stable and specific identification method to improve detection efficiency, reduce detection costs, and effectively control the quality of Huoxue Antong Tincture. Summary of the invention

[0004] To solve the above problems, the present invention aims to provide a thin layer identification method of Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in a blood-activating and analgesic tincture.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme:

[0006] A thin layer identification method of Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in a blood-activating and pain-relieving tincture, comprising the following steps:

[0007] (1) Preparation of the 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 20-40ml of ether to the filtrate, shake and extract, discard the ether solution, add water-saturated n-butanol solution to the water layer and 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 ammonia test solution or 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 the residue to dissolve it, and obtain the test solution;

[0008] (2) Preparation of reference solution: Take another reference substance of Dipsacoside VI and add methanol to prepare a solution containing 1 mg per 1 ml as the first reference solution;

[0009] Take another reference substance of paeoniflorin and add ethanol to make a solution containing 1 mg per 1 ml as the second reference substance solution;

[0010] Take another 0.5 g of Notopterygium root control 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, and use it as the third control medicinal material solution;

[0011] (3) Thin layer identification: According to the thin layer chromatography test, 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 are respectively applied to the same thin layer plate, and the lower layer solution of chloroform-methanol-water placed below 8-10°C for 12 hours is used as the developing solvent, and the volume ratio of chloroform, methanol, and water is 24-28:12-16:3-6. The plate is placed at a temperature of 20-25°C and a humidity of 50-60%R H conditions, taken out, dried, and examined under 365nm ultraviolet light. In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the Notopterygium root reference medicinal material. Then, spray it with 4-8% vanillin sulfuric acid solution or 5-15% sulfuric acid ethanol solution in mass percentage concentration, heat at 100-110℃ until the spots are clearly colored, and examine it under sunlight. In the chromatogram of the test sample, sunlight spots of the same color appeared at the corresponding positions of the chromatograms of the Dipsacus asper saponin VI reference substance and the Paeoniflorin reference substance, respectively.

[0012] A thin layer identification method of Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in a blood-activating and pain-relieving tincture, comprising the following steps:

[0013] (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;

[0014] (2) Preparation of reference solution: Take another reference substance of Dipsacoside VI and add methanol to prepare a solution containing 1 mg per 1 ml as the first reference solution;

[0015] Take another reference substance of paeoniflorin and add ethanol to make a solution containing 1 mg per 1 ml as the second reference substance solution;

[0016] Take another 0.5 g of Notopterygium root control 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, and use it as the third control medicinal material solution;

[0017] (3) Thin layer identification: According to the thin layer chromatography test, 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 are respectively applied to the same thin layer plate, and the lower layer solution of chloroform-methanol-water placed below 8-10°C for 12 hours is used as the developing solvent, and the volume ratio of chloroform, methanol, and water is 24-28:12-16:3-6. The plate is placed at a temperature of 20-25°C and a humidity of 50-60%R H conditions, taken out, dried, and examined under 365nm ultraviolet light. In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the Notopterygium root reference medicinal material. Then, spray it with 4-8% vanillin sulfuric acid solution or 5-15% sulfuric acid ethanol solution in mass percentage concentration, heat at 100-110℃ until the spots are clearly colored, and examine it under sunlight. In the chromatogram of the test sample, sunlight spots of the same color appeared at the corresponding positions of the chromatograms of the Dipsacus asper saponin VI reference substance and the Paeoniflorin reference substance, respectively.

[0018] Furthermore, the above-mentioned thin layer plate is a silica gel G thin layer plate, a silica gel GF 254 Thin layer plate or high-efficiency silica gel G thin layer plate.

[0019] Preferably, the thin layer plate is a German Merck silica gel G plate.

[0020] Furthermore, in the above-mentioned blood-activating and pain-relieving tincture, the mass ratio of Dipsacus asper, Red Peony Root and Notopterygium wilfordii is 1:1:1.53-1.86.

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

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

[0023] The thin layer identification method of Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in Huoxue Antong Tincture is simple to operate, has appropriate developing agent selection, short detection cycle, is accurate and reliable, has good reproducibility and high accuracy; the same test solution is used and a dedicated developing agent is adopted to simultaneously identify the three medicinal flavors of Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in Huoxue Antong Tincture on one thin layer plate, which is simple and fast, has a large amount of information, effectively improves the detection efficiency and reduces the inspection cost; the quality of Huoxue Antong Tincture can be effectively and comprehensively controlled, the safety of medication is guaranteed, the clinical efficacy is ensured, and a theoretical basis is provided for in-depth research on the pharmacological material basis of Huoxue Antong Tincture. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Fig. 1 is a thin layer chromatogram of each sample solution in Example 1 of the thin layer identification method of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii in the tincture for promoting blood circulation and relieving pain of the present invention;

[0025] 2 is a thin layer chromatogram of each sample solution in Example 2 of the thin layer identification method of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii in the tincture for promoting blood circulation and relieving pain of the present invention;

[0026] 3 is a thin layer chromatogram of each sample solution in Example 3 of the thin layer identification method of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii in the tincture for promoting blood circulation and relieving pain of the present invention;

[0027] 4 is a thin layer chromatogram of each sample solution in Example 4 of the thin layer identification method of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii in the tincture for promoting blood circulation and analgesia of the present invention;

[0028] 5 is a thin layer chromatogram of each sample solution in Example 5 of the thin layer identification method of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii in the tincture for promoting blood circulation and analgesia of the present invention;

[0029] 6 is a thin layer chromatogram of each sample solution in Example 6 of the thin layer identification method of Dipsacus asper, Red Peony Root and Notopterygium in the blood-activating and pain-relieving tincture of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0031] Example 1

[0032] A thin layer chromatography identification method of Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in a blood-activating and pain-relieving tincture, comprising the following specific steps:

[0033] (1) Preparation of the test solution: Take 100 ml of Huoxue Antong Tincture, steam it on a water bath until there is no alcohol smell, add 20 ml of water to the residue to dissolve it, and filter it for 20 minutes; add 20 ml of ether to the filtrate, shake and extract it once, discard the ether solution, add water-saturated n-butanol to the water layer and shake and extract it twice, 30 ml each time, and combine the n-butanol solutions; wash the n-butanol solution once with 25 ml of ammonia test solution, discard the ammonia test solution, and then wash it once with 30 ml of water saturated with n-butanol, discard the water solution, evaporate the n-butanol solution to dryness, and add 1 ml of ethanol to the residue to dissolve it to obtain the test solution;

[0034] (2) Preparation of reference solution: Take another reference substance of Dipsacoside VI and add methanol to prepare a solution containing 1 mg per 1 ml as the first reference solution;

[0035] Take another reference substance of paeoniflorin and add ethanol to make a solution containing 1 mg per 1 ml as the second reference substance solution;

[0036] Take another 0.5 g of Notopterygium root control medicinal material, add 30 ml of methanol, ultrasonically treat for 30 min, filter, evaporate to dryness, add 1 ml of methanol to the residue to dissolve, and use it as the third control medicinal material solution;

[0037] (3) Thin layer identification: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 5 μl of the 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 three batches of test sample solutions were respectively spotted on the same silica gel G thin layer plate, and the lower layer solution of chloroform-methanol-water placed below 8°C for 12 hours was used as the developing solvent. The volume ratio of chloroform, methanol, and water was 26:14:4. The plate was heated at 20°C and 40°C. Unfold under 50% RH conditions, take out, dry, and examine 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 reference medicinal material Notopterygium wilfordii; then, spray with 5% vanillin sulfuric acid solution, heat at 100°C until the spots are clearly colored, and examine 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 reference material Dipsacus asper saponin VI and the reference material Paeoniflorin, respectively, to achieve the identification of Dipsacus asper, Paeonia lactiflora, and Notopterygium wilfordii.

[0038] FIG. 1 is a 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 a thin layer chromatogram of each sample solution when examined under sunlight; among them, 1 is 5μl of Notopterygium root control medicinal material solution, 2 is 5μl of peony glycoside reference solution, 3 is 5μl of Dipsacus root saponin VI reference solution, 4 is 5μl of the first test solution, 5 is 5μl of the second test solution, and 6 is 5μl of the third test solution. As shown in Figure 1, spots of the same color appear at the corresponding positions of the Dipsacus root saponin VI reference, peony glycoside reference, and Notopterygium root control medicinal material chromatograms.

[0039] Example 2

[0040] A thin layer chromatography identification method of Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in a blood-activating and pain-relieving tincture, comprising the following specific steps:

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

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

[0043] Take another reference substance of paeoniflorin and 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 Notopterygium root control medicinal material, add 30 ml of methanol, ultrasonically treat for 35 min, filter, evaporate to dryness, add 1 ml of methanol to the residue to dissolve, and use it as the third control medicinal material solution;

[0045] (3) Thin layer identification: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 5 μl of the first reference solution, 5 μl of the second reference solution, 5 μl of the third reference medicinal material solution, and 5 μl of each of the three batches of test sample solutions were respectively spotted on the same silica gel G thin layer plate, and the lower layer solution of chloroform-methanol-water placed below 10°C for 12 hours was used as the developing solvent. The volume ratio of chloroform, methanol, and water was 28:16:6. The temperature was 22°C, Unfold under 55% RH conditions, take out, dry, and examine 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 wilfordii reference medicinal material; then, spray with 6% vanillin sulfuric acid solution, heat at 102°C until the spots are clearly colored, and examine 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, to achieve the identification of Dipsacus asper, Paeonia lactiflora, and Notopterygium wilfordii.

[0046] FIG. 2 is a thin layer chromatogram of each sample solution in Example 2, Figure 2a is the thin layer chromatogram of each sample solution when examined under ultraviolet light, Figure 2b It is a thin layer chromatogram of each sample solution when examined under sunlight; among them, 1 is 5μl of Notopterygium root control medicinal material solution, 2 is 5μl of peony glycoside reference solution, 3 is 5μl of Dipsacus root saponin VI reference solution, 4 is 5μl of the first test solution, 5 is 5μl of the second test solution, and 6 is 5μl of the third test solution. As shown in Figure 2, spots of the same color appear at the corresponding positions of the Dipsacus root saponin VI reference, peony glycoside reference, and Notopterygium root control medicinal material chromatograms.

[0047] Example 3

[0048] A thin layer chromatography identification method of Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in a blood-activating and pain-relieving tincture, comprising the following specific steps:

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

[0050] (2) Preparation of reference solution: Take another reference substance of Dipsacoside VI and add methanol to prepare a solution containing 1 mg per 1 ml as the first reference solution;

[0051] Take another reference substance of paeoniflorin and add ethanol to make a solution containing 1 mg per 1 ml as the second reference substance solution;

[0052] Take another 0.5 g of Notopterygium root control medicinal material, add 30 ml of methanol, ultrasonically treat for 35 min, filter, evaporate to dryness, add 1 ml of methanol to the residue to dissolve, and use it as the third control medicinal material solution;

[0053] (3) Thin layer identification: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 5 μl of the first reference solution, 5 μl of the second reference solution, 5 μl of the third reference medicinal material solution, and 5 μl of each of the three batches of test solution were taken and spotted on the same silica gel GF 254 On the thin layer plate, a lower layer solution of chloroform-methanol-water placed below 10°C for 12 hours is used as a developing agent, the volume ratio of chloroform, methanol and water is 24:12:3, and the plate is developed at a temperature of 25°C and a humidity of 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 15% sulfuric acid ethanol solution, heated at 105°C until the spots are clearly colored, and inspected under sunlight, and 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, to achieve the identification of Dipsacus asper, Paeonia lactiflora and Notopterygium root.

[0054] FIG3 is a 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 It is a thin layer chromatogram of each sample solution when examined under sunlight; among them, 1 is 5μl of Notopterygium root control medicinal material solution, 2 is 5μl of peony glycoside reference solution, 3 is 5μl of Dipsacus root saponin VI reference solution, 4 is 5μl of the first test solution, 5 is 5μl of the second test solution, and 6 is 5μl of the third test solution. As shown in Figure 2, spots of the same color appear at the corresponding positions of the Dipsacus root saponin VI reference, peony glycoside reference, and Notopterygium root control medicinal material chromatograms.

[0055] Example 4

[0056] A thin layer chromatography identification method of Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in a blood-activating and pain-relieving tincture, comprising the following specific steps:

[0057] (1) Preparation of test solution: Take 100 ml of Huoxue Antong Tincture, steam it on a water bath until there is no alcohol smell, dissolve the residue in 20 ml of water and filter; extract the filtrate with ether and shake it twice, 20 ml each time, discard the ether solution, extract the water solution with water-saturated n-butanol 3 times, 20 ml each time, and combine the n-butanol solution; wash with n-butanol saturated water twice, 20 ml each time, discard the water washing solution, evaporate the n-butanol solution to dryness, dissolve the residue in 10 ml of water, pass it through a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column height 12 cm), and elute with water, 30% ethanol, and 70% ethanol 50 ml each in sequence, collect the 70% ethanol eluate, evaporate to dryness, dissolve the residue in 1 ml of methanol, and use it as the test solution;

[0058] (2) Preparation of reference solution: Take another reference substance of Dipsacoside VI and add methanol to prepare a solution containing 1 mg per 1 ml as the first reference solution;

[0059] Take another reference substance of paeoniflorin and add ethanol to make a solution containing 1 mg per 1 ml as the second reference substance solution;

[0060] Take another 0.5 g of Notopterygium root control medicinal material, add 30 ml of methanol, ultrasonically treat for 30 min, filter, evaporate to dryness, add 1 ml of methanol to the residue to dissolve, and use it as the third control medicinal material solution;

[0061] (3) Thin layer identification: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 5 μl of the first reference solution, 5 μl of the second reference solution, 5 μl of the third reference medicinal material solution, and 5 μl of each of the three batches of test sample solutions were respectively spotted on the same silica gel G thin layer plate, and the lower layer solution of chloroform-methanol-water placed below 9°C for 12 hours was used as the developing solvent. The volume ratio of chloroform, methanol, and water was 24:12:3. The plate was heated at 20°C and 40°C. Unfold under humidity of 53% RH, take out, dry, and examine 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 reference medicinal material Notopterygium wilfordii; then, spray with 10% sulfuric acid ethanol solution, heat at 100°C until the spots are clearly colored, and examine 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 reference material Dipsacus asper saponin VI and the reference material Paeoniflorin, respectively, to achieve the identification of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii.

[0062] FIG4 is a thin layer chromatogram of each sample solution in Example 4, Figure 4a is the thin layer chromatogram of each sample solution when examined under ultraviolet light, Figure 4bIt is a thin layer chromatogram of each sample solution when examined under sunlight; among them, 1 is 5μl of Notopterygium root control medicinal material solution, 2 is 5μl of peony glycoside reference solution, 3 is 5μl of Dipsacus root saponin VI reference solution, 4 is 5μl of the first test solution, 5 is 5μl of the second test solution, and 6 is 5μl of the third test solution. As shown in Figure 4, spots of the same color appear at the corresponding positions of the Dipsacus root saponin VI reference, peony glycoside reference, and Notopterygium root control medicinal material chromatograms.

[0063] Example 5

[0064] A thin layer chromatography identification method of Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in a blood-activating and pain-relieving tincture, comprising the following specific steps:

[0065] (1) Preparation of test solution: Take 150 ml of Huoxue Antong Tincture, steam it on a water bath until there is no alcohol smell, dissolve the residue in 30 ml of water and filter; extract the filtrate with ether and shake it twice, 25 ml each time, discard the ether solution, extract the water solution with water-saturated n-butanol and shake it three times, 20 ml each time, and combine the n-butanol solution; wash it with n-butanol-saturated water twice, 20 ml each time, discard the water washing solution, evaporate the n-butanol solution to dryness, dissolve the residue in 10 ml of water, pass it through a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column height 12 cm), and elute it with 50 ml each of water, 30% ethanol, and 70% ethanol in sequence, collect the 70% ethanol eluate, evaporate it to dryness, dissolve the residue in 1 ml of methanol, and use it as the test solution;

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

[0067] Take another reference substance of paeoniflorin and 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 Notopterygium root control medicinal material, add 40 ml of methanol, ultrasonically treat for 35 min, filter, evaporate to dryness, add 1 ml of methanol to the residue to dissolve, and use it as the third control medicinal material solution;

[0069] (3) Thin layer identification: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 5 μl of the 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 three batches of test sample solutions were respectively spotted on the same silica gel G thin layer plate, and the lower layer solution of chloroform-methanol-water placed below 9°C for 12 hours was used as the developing solvent. The volume ratio of chloroform, methanol, and water was 24:14:4. The temperature was 22°C and the humidity was 55 Unfold under %RH conditions, take out, dry, and examine under 365nm ultraviolet light; in the chromatogram of the test sample, at the corresponding position of the chromatogram of the Notopterygium wilfordii reference medicinal material, a fluorescent spot of the same color appears; then, spray with 6% by mass concentration of vanillin sulfuric acid solution, heat at 100°C until the spots are clearly colored, and examine under sunlight, in the chromatogram of the test sample, at the corresponding positions of the chromatograms of the Dipsacus asper saponin VI reference substance and the Paeoniflorin reference substance, sunlight spots of the same color appear respectively, thereby achieving the identification of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii.

[0070] FIG5 is a thin layer chromatogram of each sample solution in Example 5, Figure 5a is the thin layer chromatogram of each sample solution when examined under ultraviolet light, Figure 5b It is a thin layer chromatogram of each sample solution when examined under sunlight; among them, 1 is 5μl of Notopterygium root control medicinal material solution, 2 is 5μl of peony glycoside reference solution, 3 is 5μl of Dipsacus root saponin VI reference solution, 4 is 5μl of the first test solution, 5 is 5μl of the second test solution, and 6 is 5μl of the third test solution. As shown in Figure 5, spots of the same color appear at the corresponding positions of the Dipsacus root saponin VI reference, peony glycoside reference, and Notopterygium root control medicinal material chromatograms.

[0071] Example 6

[0072] A thin layer chromatography identification method of Radix Dipsaci, Radix Paeoniae Rubra and Radix Notopterygii in a blood-activating and pain-relieving tincture, comprising the following specific steps:

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

[0074] (2) Preparation of reference solution: Take another reference substance of Dipsacoside VI and add methanol to prepare a solution containing 1 mg per 1 ml as the first reference solution;

[0075] Take another reference substance of paeoniflorin and add ethanol to make a solution containing 1 mg per 1 ml as the second reference substance solution;

[0076] Take another 0.5 g of Notopterygium root control medicinal material, add 45 ml of methanol, ultrasonically treat for 40 minutes, filter, evaporate to dryness, add 1 ml of methanol to the residue to dissolve, and use it as the third control medicinal material solution;

[0077] (3) Thin layer identification: According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 5 μl of the first reference solution, 5 μl of the second reference solution, 5 μl of the third reference medicinal material solution, and 5 μl of the test solution were respectively spotted on the same silica gel G thin layer plate, and the lower layer solution of chloroform-methanol-water placed below 10°C for 12 hours was used as the developing solvent. The volume ratio of chloroform, methanol, and water was 28:16:6. The plate was heated at 25 ℃ and humidity 60% RH, taken out, dried, and examined under 365nm ultraviolet light. In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the Notopterygium root reference medicinal material. Then, it was sprayed with 8% by mass percentage concentration of vanillin sulfuric acid solution, heated at 106℃ until the spots were clearly colored, and examined under sunlight. In the chromatogram of the test sample, sunlight spots of the same color appeared at the corresponding positions of the chromatograms of the Dipsacus asper saponin VI reference substance and the Paeoniflorin reference substance, respectively.

[0078] FIG6 is a 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 It is a thin layer chromatogram of each sample solution when examined under sunlight; among them, 1 is 5μl of Notopterygium root control medicinal material solution, 2 is 5μl of peony glycoside reference solution, 3 is 5μl of Dipsacus root saponin VI reference solution, 4 is 5μl of the first test solution, 5 is 5μl of the second test solution, and 6 is 5μl of the third test solution. As shown in Figure 5, spots of the same color appear at the corresponding positions of the Dipsacus root saponin VI reference, peony glycoside reference, and Notopterygium root control medicinal material chromatograms.

[0079] The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Without departing from the spirit and scope of the present invention, all equivalent changes and modifications made within 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 identification method of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii in a blood-activating and pain-relieving tincture, characterized in that: It includes the following steps: (1) Preparation of the 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 20-40ml of ether to the filtrate, shake and extract, discard the ether solution, add water-saturated n-butanol solution to the water layer and 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 ammonia test solution or 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 the residue to dissolve it, and obtain the test solution; (2) Preparation of reference solution: Take another reference substance of Dipsacoside VI and add methanol to prepare a solution containing 1 mg per 1 ml as the first reference solution; Take another reference substance of paeoniflorin and add ethanol to make a solution containing 1 mg per 1 ml as the second reference substance solution; Take another 0.5 g of Notopterygium root control 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, and use it as the third control medicinal material solution; (3) Thin layer identification: According to the thin layer chromatography test, 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 are respectively applied to the same thin layer plate, and the lower layer solution of chloroform-methanol-water placed below 8-10°C for 12 hours is used as the developing solvent, and the volume ratio of chloroform, methanol, and water is 24-28:12-16:3-6. The plate is placed at a temperature of 20-25°C and a humidity of 50-60%R H conditions, taken out, dried, and examined under 365nm ultraviolet light. In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the Notopterygium root reference medicinal material. Then, spray it with 4-8% vanillin sulfuric acid solution or 5-15% sulfuric acid ethanol solution in mass percentage concentration, heat at 100-110℃ until the spots are clearly colored, and examine it under sunlight. In the chromatogram of the test sample, sunlight spots of the same color appeared 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, chloroform, methanol and water in step (3) is 26:14:

4.

4. A thin layer identification method of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii in a blood-activating and pain-relieving tincture, characterized in that: It includes 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; (2) Preparation of reference solution: Take another reference substance of Dipsacoside VI and add methanol to prepare a solution containing 1 mg per 1 ml as the first reference solution; Take another reference substance of paeoniflorin and add ethanol to make a solution containing 1 mg per 1 ml as the second reference substance solution; Take another 0.5 g of Notopterygium root control 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, and use it as the third control medicinal material solution; (3) Thin layer identification: According to the thin layer chromatography test, 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 are respectively applied to the same thin layer plate, and the lower layer solution of chloroform-methanol-water placed below 8-10°C for 12 hours is used as the developing solvent, and the volume ratio of chloroform, methanol, and water is 24-28:12-16:3-6. The plate is placed at a temperature of 20-25°C and a humidity of 50-60%R H conditions, taken out, dried, and examined under 365nm ultraviolet light. In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the Notopterygium root reference medicinal material. Then, spray it with 4-8% vanillin sulfuric acid solution or 5-15% sulfuric acid ethanol solution in mass percentage concentration, heat at 100-110℃ until the spots are clearly colored, and examine it under sunlight. In the chromatogram of the test sample, sunlight spots of the same color appeared at the corresponding positions of the chromatograms of the Dipsacus asper saponin VI reference substance and the Paeoniflorin reference substance, respectively.

5. The thin layer identification method of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii in the blood-activating and pain-relieving tincture according to claim 4 is characterized by: The volume ratio of the developing solvent, chloroform, methanol and water in step (3) is 24:12:

4.

6. The thin layer identification method of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii in the blood-activating and pain-relieving tincture according to claim 4 is characterized by: The volume ratio of the developing solvent, chloroform, methanol and water in step (3) is 28:16:

6.

7. 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 or 2 is characterized by: The thin layer plate is a silica gel G thin layer plate, a silica gel GF 254 Thin layer plate or high-efficiency silica gel G thin layer plate.

8. 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 or 2 is characterized by: In the blood-activating and pain-relieving tincture, the mass ratio of Dipsacus asper, Paeonia lactiflora rubra and Notopterygium wilfordii is 1:1:1.53-1.

86.

9. Use of the thin layer identification method of Dipsacus asper, Paeonia lactiflora and Notopterygium wilfordii in Huoxue Antong tincture as claimed in any one of claims 1 to 8 in the quality control of Huoxue Antong tincture.

Images