TLC Identification Method for Dan'e Fukang Decoction Paste

Abstract

The present invention belongs to the technical field of identification of traditional Chinese medicinal materials, and particularly relates to a thin-layer chromatography identification method for Dan'e Fukang decoction extract. The method of the present invention can identify multiple specific characteristic components contained in two kinds of medicinal material slices by using the same thin-layer plate and the same developing system. The method is as follows: a. Take Dan'e Fukang decoction extract, and prepare test solution 1 and test solution 2 with petroleum ether as the solvent; b. Prepare reference substance solutions of tanshinone IIA, tetrahydropalmatine, germacrone, and ligustilide; c. Spot test solution 1, the reference substance solution of tanshinone IIA, and the reference substance solution of tetrahydropalmatine on the same thin-layer plate, and develop with developing agent 1; spot test solution 2, the reference substance solution of germacrone, and the reference substance solution of ligustilide on the same thin-layer plate, and develop with developing agent 2. After development, color, examine, and compare; the color spots of the related characteristic components identified by the method of the present invention are clear, the spots are well separated, and the recognition rate is relatively high, and it has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of identification of traditional Chinese medicinal materials, and particularly relates to a thin-layer chromatography identification method for Dan'e Fukang Decoction Paste. Background Art

[0002] Dan'e Fukang Decoction Paste is composed of Salvia miltiorrhiza, Curcuma zedoaria, Bupleurum yunnanense, Panax notoginseng, Paeonia lactiflora, Angelica sinensis, Sparganium stoloniferum, Cyperus rotundus, Corydalis yanhusuo, Glycyrrhiza uralensis, etc., and has the effects of promoting blood circulation to remove blood stasis, soothing the liver and regulating qi, regulating menstruation and relieving pain, softening and resolving hard masses, etc., and is a commonly used drug for treating endometriosis clinically.

[0003] The State Food and Drug Administration's national drug standard has included the identification method for Dan'e Fukang Decoction Paste. The method is as follows: Take Dan'e Fukang Decoction Paste, and prepare a test solution with ether as the solvent; take tanshinone II A reference substance, add anhydrous ethanol to make a solution containing 0.5 mg per 1 mL as the tanshinone II A reference substance solution; according to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2015 Edition), take 5 - 10 μL of each of the above two solutions, and spot them on the same silica gel G thin-layer plate respectively. Use toluene-ethyl acetate (9.5:0.5) as the developing agent, develop, take out, dry, in the test solution chromatogram, at the position corresponding to the reference substance chromatogram, there are spots showing the same color.

[0004] Take tetrahydropalmatine reference substance, add anhydrous ethanol to make a solution containing 0.5 mg per 1 mL as the tetrahydropalmatine reference substance solution. According to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2015 Edition), take 5 - 10 μL of each of the above test solution and the tetrahydropalmatine reference substance solution respectively, and spot them on the same silica gel G thin-layer plate respectively. Use n-hexane-chloroform-methanol (6:3.2:0.8) as the developing agent, develop, take out, dry, fume with iodine vapor for several minutes, take out, place in the air to volatilize the iodine adsorbed on the plate completely, and then examine under ultraviolet light (365 nm). In the test solution chromatogram, at the position corresponding to the reference substance chromatogram, there are fluorescent spots showing the same color.

[0005] Take Dan'e Fukang Decoction Paste, and prepare a test solution with ether as the solvent; take germacrone reference substance, add anhydrous ethanol to make a solution containing 0.4 mg per 1 mL as the reference substance solution. According to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2015 Edition), take 10 - 15 μL of each of the test solution and the germacrone reference substance solution, and spot them on the same silica gel G thin-layer plate respectively. Use petroleum ether (30 - 60 °C)-acetone-ethyl acetate (94:5:1) as the developing agent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat at 105 °C until the spots are clearly developed. In the test solution chromatogram, at the position corresponding to the reference substance chromatogram, there are spots showing the same color.

[0006] The thin-layer chromatography identification method of the above-mentioned Dan'e Fukang decoction extract uses solvents with high toxicity and great harm to the human body. Moreover, the existing identification methods can only identify one characteristic component using one thin-layer plate, with cumbersome steps, low efficiency, waste of manpower and material resources, and high costs. Summary of the Invention

[0007] The purpose of the present invention is to provide a thin-layer chromatography identification method for Dan'e Fukang decoction extract.

[0008] The present invention provides a thin-layer chromatography identification method for Dan'e Fukang decoction extract, which includes the following steps:

[0009] a. Take Dan'e Fukang decoction extract, use petroleum ether as a solvent to prepare test solution 1 and test solution 2;

[0010] b. Prepare reference substance solutions of tanshinone IIA, tetrahydropalmatine, germacrone, and ligustilide;

[0011] c. Spot test solution 1, the reference substance solution of tanshinone IIA, and the reference substance solution of tetrahydropalmatine on the same thin-layer plate, and develop using developer 1; spot test solution 2, the reference substance solution of germacrone, and the reference substance solution of ligustilide on the same thin-layer plate, and develop using developer 2. After development, color, inspect, and compare. The developer 1 is composed of the following components in volume ratio: toluene - ethyl acetate - methanol 17 - 20:0.5 - 1.5:0.1 - 1; the developer 2 is composed of the following components in volume ratio: petroleum ether - ethyl acetate 90 - 97:9 - 2.

[0012] Further, in step c, the developer 1 is: the volume ratio of toluene - ethyl acetate - methanol is 19:1:0.5.

[0013] Further, in step c, the developer 2 is: the volume ratio of petroleum ether - ethyl acetate is 94:5.

[0014] Further, the boiling point of the petroleum ether is 60 - 90 °C.

[0015] Further, in step a, the preparation method of the test solution 1 is: take Dan'e Fukang decoction extract, add water, shake well, adjust the pH value to 9 - 10 with ammonia test solution, extract with petroleum ether with a boiling point of 60 - 90 °C by shaking, combine the petroleum ether solutions at 60 - 90 °C, dehydrate with anhydrous sodium sulfate, filter, evaporate the filtrate to dryness, and dissolve the residue in absolute ethanol to obtain test solution 1.

[0016] Preferably, the preparation method of the test sample solution 1 is as follows: Take 5 g of Dan'e Fukang Extract, add 20 mL of water, shake well, adjust the pH value to 9 - 10 with ammonia test solution, extract 3 times with petroleum ether having a boiling point of 60 - 90°C, 20 mL each time, combine the petroleum ether solution at 60 - 90°C, add an appropriate amount of anhydrous sodium sulfate for dehydration, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of absolute ethanol to obtain the test sample solution 1.

[0017] Further, in step a, the preparation method of the test sample solution 2 is as follows: Take Dan'e Fukang Extract, add water, shake well, extract with petroleum ether having a boiling point of 60 - 90°C by shaking, combine the petroleum ether solution at 60 - 90°C, wash with water, add anhydrous sodium sulfate for dehydration, filter, evaporate the filtrate to dryness, dissolve the residue in absolute ethanol to obtain the test sample solution 2.

[0018] Preferably, the preparation method of the test sample solution 2 is as follows: Take 5 g of Dan'e Fukang Extract, add 40 mL of water, shake well, extract 3 times with petroleum ether having a boiling point of 60 - 90°C, 40 mL each time, combine the petroleum ether solution at 60 - 90°C, wash 3 times with water, 40 mL each time, add an appropriate amount of anhydrous sodium sulfate for dehydration, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of absolute ethanol to obtain the test sample solution 2.

[0019] Further, in step c, the test sample solution 1, tanshinone IIA reference substance solution and tetrahydropalmatine reference substance solution are spotted on the same thin layer plate, developed with developing agent 1, taken out, dried, examined under a daylight lamp to obtain the position of tanshinone IIA in the test sample on the thin layer plate; the dried thin layer plate is fumigated with iodine vapor for several minutes, taken out, and after the iodine adsorbed on the plate is volatilized in the air, examined under a 365 nm ultraviolet lamp to obtain the position of tetrahydropalmatine in the test sample on the thin layer plate.

[0020] Further, in step c, the test sample solution 2, germacrone reference substance solution and ligustilide reference substance solution are spotted on the same thin layer plate, developed with developing agent 2, taken out, dried, examined under a 365 nm ultraviolet lamp to obtain the position of ligustilide in the test sample on the thin layer plate; spray the dried thin layer plate with 10% sulfuric acid ethanol solution, heat at 105°C for 10 s and then examine under a 365 nm ultraviolet lamp to obtain the position of germacrone in the test sample on the thin layer plate.

[0021] The beneficial effects of the present invention are:

[0022] The thin layer chromatography identification method of Dan'e Fukang decoction of the present invention solves the defect that only one characteristic component is identified by one thin layer plate by optimizing the conditions such as the preparation of the test solution and the development system. The method of the present invention can identify multiple exclusive characteristic components contained in two kinds of medicinal material slices by using the same thin layer plate and the same development system, saving manpower, time and cost. The reagents used in the method of the present invention are less toxic and stable, avoiding the use of toxic reagents such as ether, chloroform, acetone, etc., reducing the use of toluene and reducing the harm to the operator. The relevant characteristic components identified by the method of the present invention have clear color spots, good spot separation, high recognition, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a thin layer chromatogram of Example 1 before color development; wherein 1 is tanshinone IIA reference substance, 2 is test sample 1-1, 3 is test sample 1-2, 4 is test sample 1-3, 5 is test sample 1-4, 6 is test sample 1-5, and 7 is tetrahydropalmatine reference substance.

[0024] Figure 2 It is a thin layer chromatogram after color development of Example 1; wherein 1 is tanshinone IIA reference substance, 2 is test sample 1-1, 3 is test sample 1-2, 4 is test sample 1-3, 5 is test sample 1-4, 6 is test sample 1-5, and 7 is corydaline reference substance.

[0025] Figure 3 This is the thin layer chromatogram of the extraction solvent of Experimental Example 1 before color development; 1 is the tanshinone IIA reference substance, 2 is the corydalis ethylamine reference substance, 3 is the test sample with ether as the extraction solvent, 4 is the test sample with 60-90°C petroleum ether as the extraction solvent, 5 is the test sample with ethyl acetate as the extraction solvent; 6 is the corydalis ethylamine reference substance, and 7 is the tanshinone IIA reference substance.

[0026] Figure 4 This is the thin layer chromatogram after color development of the extraction solvent of Experimental Example 1; wherein 1 is the tanshinone IIA reference substance, 2 is the corydalis ethylamine reference substance, 3 is the test substance with ether as the extraction solvent, 4 is the test substance with 60-90°C petroleum ether as the extraction solvent, 5 is the test substance with ethyl acetate as the extraction solvent; 6 is the corydalis ethylamine reference substance, and 7 is the tanshinone IIA reference substance.

[0027] Figure 5 The thin layer chromatogram of n-hexane-chloroform-methanol (6:3.2:0.8) before color development was systematically investigated for Experimental Example 1; 1 is the tanshinone IIA reference substance, 3 is the corydalis butylene glycol reference substance, and 2 and 4 are both test substances.

[0028] Figure 6For Test Example 1, a TLC chromatogram after development with n-hexane-chloroform-methanol (6:3.2:0.8) and color development was examined; among them, 1 is the reference substance of tanshinone IIA, 3 is the reference substance of tetrahydropalmatine, and 2 and 4 are both test samples.

[0029] Figure 7 For Test Example 1, a TLC chromatogram before development with benzene-ethyl acetate-methanol (19:1:0.3) and color development was examined; among them, 1 is the reference substance of tanshinone IIA, 3 is the reference substance of tetrahydropalmatine, and 2 and 4 are both test samples.

[0030] Figure 8 For Test Example 1, a TLC chromatogram after development with benzene-ethyl acetate-methanol (19:1:0.3) and color development was examined; among them, 1 is the reference substance of tanshinone IIA, 3 is the reference substance of tetrahydropalmatine, and 2 and 4 are both test samples.

[0031] Figure 9 For Test Example 1, a TLC chromatogram before development with dichloromethane-ethyl acetate-methanol (19:1:1) and color development was examined; among them, 1 is the reference substance of tanshinone IIA, 3 is the reference substance of tetrahydropalmatine, and 2 and 4 are both test samples.

[0032] Figure 10 For Test Example 1, a TLC chromatogram after development with dichloromethane-ethyl acetate-methanol (19:1:1) and color development was examined; among them, 1 is the reference substance of tanshinone IIA, 3 is the reference substance of tetrahydropalmatine, and 2 and 4 are both test samples.

[0033] Figure 11 For Test Example 1, a TLC chromatogram before development with toluene-ethyl acetate-methanol (19:1:0.5) and color development was examined; among them, 1 is the reference substance of tanshinone IIA, 3 is the reference substance of tetrahydropalmatine, and 2 and 4 are both test samples.

[0034] Figure 12 For Test Example 1, a TLC chromatogram after development with toluene-ethyl acetate-methanol (19:1:0.5) and color development was examined; among them, 1 is the reference substance of tanshinone IIA, 3 is the reference substance of tetrahydropalmatine, and 2 and 4 are both test samples.

[0035] Figure 13 A TLC chromatogram for the specificity study of Salvia miltiorrhiza f. mandarinensis in Test Example 1; among them, 1 is the reference substance of tanshinone IIA, 2 is the negative of Salvia miltiorrhiza f. mandarinensis, 3 is the reference substance of tetrahydropalmatine, and 4 is the negative of Corydalis yanhusuo.

[0036] Figure 14 A TLC chromatogram for the specificity study of Corydalis yanhusuo in Test Example 1; among them, 1 is the reference substance of tanshinone IIA, 2 is the negative of Salvia miltiorrhiza f. mandarinensis, 3 is the reference substance of tetrahydropalmatine, and 4 is the negative of Corydalis yanhusuo.

[0037] Figure 15TLC chromatogram of Example 2 without using a color developer; where 1 is the reference substance of germacrone, 2 is the test sample 2-1, 3 is the test sample 2-2, 4 is the test sample 2-3, 5 is the test sample 2-4, 6 is the test sample 2-5, and 7 is the reference substance of ligustilide.

[0038] Figure 16 TLC chromatogram of Example 2 using a color developer; where 1 is the reference substance of germacrone, 2 is the test sample 2-1, 3 is the test sample 2-2, 4 is the test sample 2-3, 5 is the test sample 2-4, 6 is the test sample 2-5, and 7 is the reference substance of ligustilide.

[0039] Figure 17 TLC chromatogram of Test Example 2 for investigating extraction solvents using a color developer; where 1 is the reference substance of germacrone, 2 is the test sample with ether as the extraction solvent, 3 is the test sample with petroleum ether at 60-90°C as the extraction solvent, and 4 is the test sample with ethyl acetate as the extraction solvent.

[0040] Figure 18 TLC chromatogram of Test Example 2 for investigating extraction solvents without using a color developer; where 1 is the reference substance of ligustilide, 2 is the test sample with petroleum ether at 60-90°C as the extraction solvent, 3 is the test sample with ether as the extraction solvent, 4 is the test sample with ethyl acetate as the extraction solvent, and 5 is the reference substance of ligustilide.

[0041] Figure 19 TLC chromatogram of Test Example 2 for investigating the developing system of petroleum ether-acetone:ethyl acetate (94:5:1) at 30-60°C; where 1 is the reference substance of germacrone, 2 and 3 are both test samples, and 4 is the reference substance of germacrone.

[0042] Figure 20 TLC chromatogram of Test Example 2 for investigating the developing system of petroleum ether-ethyl acetate (94:5) at 60-90°C with a color developer; where 1 is the reference substance of germacrone, 2 and 3 are both test samples, and 4 is the reference substance of ligustilide.

[0043] Figure 21 TLC chromatogram of Test Example 2 for investigating the developing system of cyclohexane-ethyl acetate (8:5); where 1 is the reference substance of germacrone, 2 and 3 are both test samples, and 4 is the reference substance of germacrone.

[0044] Figure 22 TLC chromatogram of Test Example 2 for investigating the developing system of n-hexane-ethyl acetate (4:1); where 1 and 4 are both reference substances of ligustilide, and 2 and 3 are both test samples.

[0045] Figure 23 TLC chromatogram of Test Example 2 for investigating the developing system of n-hexane-ether (7:3); where 1 and 4 are both reference substances of ligustilide, and 2 and 3 are both test samples.

[0046] Figure 24 For Test Example 2's developing system, the thin-layer chromatogram of cyclohexane-dichloromethane-ethyl acetate-formic acid (4:1:1:0.1) was examined; among them, 1 and 4 are both reference substances of ligustilide, and 2 and 3 are both test samples.

[0047] Figure 25 For Test Example 2's developing system, the thin-layer chromatogram of n-hexane-ethyl acetate (9:1) was examined; among them, 1 and 4 are both reference substances of ligustilide, and 2 and 3 are both test samples.

[0048] Figure 26 For Test Example 2's developing system, the thin-layer chromatogram of cyclohexane-ethyl acetate-formic acid (4:1:0.1) was examined; among them, 1 and 4 are both reference substances of ligustilide, and 2 and 3 are both test samples.

[0049] Figure 27 For Test Example 2's developing system, the thin-layer chromatogram of petroleum ether-ethyl acetate (94:5) at 60 - 90 °C without a developer was examined; among them, 1 is the reference substance of germacrone, 2 and 3 are both test samples, and 4 is the reference substance of ligustilide.

[0050] Figure 28 For Test Example 2's developer examination, the thin-layer chromatogram of 5% vanillin sulfuric acid ethanol solution was examined; among them, 1 and 4 are both reference substances of germacrone, and 2 and 3 are both test samples.

[0051] Figure 29 For Test Example 2's developer examination, the thin-layer chromatogram of 5% vanillin sulfuric acid solution was examined; among them, 1 and 4 are both reference substances of germacrone, and 2 and 3 are both test samples.

[0052] Figure 30 For Test Example 2's developer examination, the thin-layer chromatogram of 10% sulfuric acid ethanol solution was examined; among them, 1 and 4 are both reference substances of germacrone, and 2 and 3 are both test samples.

[0053] Figure 31 For the thin-layer chromatogram of the zedoary specificity examination in Test Example 2; among them, 1 and 4 are both reference substances of germacrone, 2 is the test sample, and 3 is the zedoary negative.

[0054] Figure 32 For the thin-layer chromatogram of the angelica specificity examination in Test Example 2; among them, 1 and 4 are both reference substances of ligustilide, 2 is the test sample, and 3 is the angelica negative. Detailed implementation mode

[0055] The present invention will be further elaborated in detail through specific embodiments below.

[0056] Example 1 Identification of tanshinone IIA and tetrahydropalmatine in Dan'e Fukang Ointment

[0057] Preparation of test solution: Take 5 batches of Dan'e Fukang decoction extract, weigh 5 g each, add 20 mL of water, shake well, adjust the pH value to 9 - 10 with ammonia test solution, extract with petroleum ether with a boiling point of 60 - 90 °C by shaking for 3 times, 20 mL each time. Combine the petroleum ether solution at 60 - 90 °C, dehydrate with an appropriate amount of anhydrous sodium sulfate, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of anhydrous ethanol to obtain test solutions 1-1, 1-2, 1-3, 1-4, and 1-5.

[0058] Preparation of reference solution: Take tanshinone IIA reference substance, and make a solution containing 0.5 mg per 1 mL with anhydrous ethanol as the tanshinone IIA reference solution; take tetrahydropalmatine reference substance, and make a solution containing 0.2 mg per 1 mL with anhydrous ethanol as the tetrahydropalmatine reference solution.

[0059] Identification: According to the thin-layer chromatography method (General Principles 0502 of Chinese Pharmacopoeia 2020 Edition) for testing, absorb 5 - 10 μL of test solutions 1-1 - 1-5, tanshinone IIA reference solution, and tetrahydropalmatine reference solution respectively, and spot them on the same silica gel G thin-layer plate. Use toluene - ethyl acetate - methanol as the developing solvent with a volume ratio of 19:1:0.5. Develop, take out, dry in air, and examine under daylight. At the position corresponding to the chromatogram of tanshinone IIA reference substance, spots of the same color appear. Then, expose the dried thin-layer plate to iodine vapor for several minutes, take out, let the adsorbed iodine on the plate evaporate completely in air, and then examine under ultraviolet light at 365 nm. In the chromatogram of the test sample, at the position corresponding to the chromatogram of tetrahydropalmatine reference substance, fluorescent spots of the same color appear. See Figure 1 and Figure 2 。

[0060] Test Example 1

[0061] Preparation of reference solution: Take tanshinone IIA reference substance, and make a solution containing 0.5 mg per 1 mL with anhydrous ethanol as the tanshinone IIA reference solution. Take tetrahydropalmatine reference substance, and make a solution containing 0.5 mg per 1 mL with anhydrous ethanol as the tetrahydropalmatine reference solution.

[0062] 1.1 Investigation on extraction solvents for Radix Salviae Miltiorrhizae and Rhizoma Corydalis

[0063] Preparation of test solution: Take 5 g of Dan'e Fukang decoction extract, add 20 mL of water, shake well, adjust the pH value to 9 - 10 with ammonia test solution, extract with ether by shaking for 3 times, 20 mL each time. Combine the ether solution, dehydrate with an appropriate amount of anhydrous sodium sulfate, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of anhydrous ethanol to obtain the ether solution of Dan'e Fukang decoction extract as the test solution.

[0064] Take 5 g of Dan'e Fukang Decoction Extract, add 20 mL of water, shake well, adjust the pH value to 9 - 10 with ammonia test solution, extract with petroleum ether with a boiling point of 60 - 90 °C by shaking 3 times, 20 mL each time. Combine the petroleum ether solutions at 60 - 90 °C, add an appropriate amount of anhydrous sodium sulfate for dehydration, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of anhydrous ethanol to obtain the petroleum ether solution at 60 - 90 °C of the test sample of Dan'e Fukang Decoction Extract.

[0065] Take 5 g of Dan'e Fukang Decoction Extract, add 20 mL of water, shake well, adjust the pH value to 9 - 10 with ammonia test solution, extract with ethyl acetate by shaking 3 times, 20 mL each time. Combine the ethyl acetate solutions, add an appropriate amount of anhydrous sodium sulfate for dehydration, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of anhydrous ethanol to obtain the ethyl acetate solution of the test sample of Dan'e Fukang Decoction Extract.

[0066] According to the test of thin - layer chromatography (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2015 Edition), absorb 5 μL each of the reference substance solution of tanshinone IIA, the reference substance solution of tetrahydropalmatine, the ether solution of the test sample of Dan'e Fukang Decoction Extract, the petroleum ether solution at 60 - 90 °C of the test sample of Dan'e Fukang Decoction Extract, and the ethyl acetate solution of the test sample of Dan'e Fukang Decoction Extract, and spot them respectively on the same high - efficiency silica gel G thin - layer plate. Use toluene - ethyl acetate - methanol (volume ratio is 19:1:0.5) as the developing agent, develop, take out, dry in air, and examine for tanshinone IIA under daylight; then fume the dried thin - layer plate with iodine vapor for several minutes, take out, let the iodine adsorbed on the plate volatilize completely in air, and then examine for tetrahydropalmatine under ultraviolet light at 365 nm. See Figure 3 and Figure 4 。

[0067] From Figure 3 and Figure 4 it can be seen that there is no obvious difference in the spots of tanshinone IIA and tetrahydropalmatine developed after extraction of the Dan'e Fukang Decoction Extract sample with petroleum ether at 60 - 90 °C and with ether. However, since the harmfulness of ether is greater than that of petroleum ether at 60 - 90 °C, petroleum ether at 60 - 90 °C is selected as the extraction solvent for the test sample of Dan'e Fukang Decoction Extract.

[0068] 1.2 Investigation on the developing system for Salvia miltiorrhiza f. alba and Corydalis yanhusuo

[0069] Test according to the thin-layer chromatography method (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2015 Edition). Absorb 5 μL each of the reference substance solutions of tanshinone IIA, tetrahydropalmatine, and the 60 - 90 °C petroleum ether solution of the test sample of Dan'e Fukang Decoction, spot them on the same silica gel G thin-layer plate, and use hexane-chloroform-methanol (volume ratio 6:3.2:0.8), benzene-ethyl acetate-methanol (volume ratio 19:1:0.3), dichloromethane-ethyl acetate-methanol (volume ratio 19:1:1), and toluene-ethyl acetate-methanol (volume ratio 19:1:0.5) as the developing agents respectively. Develop, take out, dry in air, and examine tanshinone IIA under daylight; then, after drying the thin-layer plate, fume it with iodine vapor for several minutes, take out, let the iodine adsorbed on the plate volatilize completely in air, and examine tetrahydropalmatine under ultraviolet light at 365 nm. See Figures 5 to 12 。

[0070] From Figures 5 to 12 It can be seen that both the toluene-ethyl acetate-methanol (19:1:0.5) and benzene-ethyl acetate-methanol (19:1:0.3) developing systems have good separation effects. Since toluene has lower toxicity than benzene, toluene-ethyl acetate-methanol (19:1:0.5) is selected as the developing agent.

[0071] 1.3 Specificity investigation of Salvia miltiorrhiza f. alba and Corydalis yanhusuo

[0072] Take 5 g of the Dan'e Fukang Decoction-Salvia miltiorrhiza f. alba negative sample, add 20 mL of water, shake well, adjust the pH value to 9 - 10 with ammonia test solution, extract with 60 - 90 °C petroleum ether by shaking 3 times, 20 mL each time. Combine the 60 - 90 °C petroleum ether solutions, add an appropriate amount of anhydrous sodium sulfate for dehydration, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of anhydrous ethanol to obtain the Dan'e Fukang Decoction-Salvia miltiorrhiza f. alba negative sample solution.

[0073] Take 5 g of the Dan'e Fukang Decoction-Corydalis yanhusuo negative sample, add 20 mL of water, shake well, adjust the pH value to 9 - 10 with ammonia test solution, extract with 60 - 90 °C petroleum ether by shaking 3 times, 20 mL each time. Combine the 60 - 90 °C petroleum ether solutions, add an appropriate amount of anhydrous sodium sulfate for dehydration, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of anhydrous ethanol to obtain the Dan'e Fukang Decoction-Corydalis yanhusuo negative sample solution.

[0074] Test according to the thin-layer chromatography method (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2015 Edition). Pipette 5 μL each of the tanshinone IIA reference substance solution, tetrahydropalmatine reference substance solution, the 60 - 90 °C petroleum ether test solution of Dan'e Fukang medicinal extract, the Dan'e Fukang medicinal extract - Salvia miltiorrhiza Bunge f. alba negative sample solution, and the Dan'e Fukang medicinal extract - Corydalis yanhusuo W. T. Wang negative sample solution, and spot them respectively on the same silica gel G thin-layer plate. Using toluene - ethyl acetate - methanol (19:1:0.5) as the developing solvent, develop, take out, dry in air, and examine under daylight; then expose the dried thin-layer plate to iodine vapor for several minutes, take out, allow the iodine adsorbed on the plate to evaporate completely in air, and then examine under ultraviolet light at 365 nm. See attachment Figure 13 and Figure 14 .

[0075] It can be seen from Figure 13 and Figure 14 that in the chromatogram of the test solution, at the positions corresponding to the chromatogram of the reference substance, spots of the same color appear; there are no spots in the negative test solution, indicating good specificity.

[0076] It can be seen from Figure 14 that in the chromatogram of the Salvia miltiorrhiza Bunge f. alba negative test solution, in addition to the spot of tetrahydropalmatine, there are also 5 obvious specific characteristic spots of Corydalis yanhusuo W. T. Wang, realizing the simultaneous identification of multiple characteristic components.

[0077] Example 2 Identification of germacrone and ligustilide in Dan'e Fukang medicinal extract

[0078] Preparation of the test solution: Take 5 batches of Dan'e Fukang medicinal extract, weigh 5 g each, add 40 mL of water, shake well, extract with 60 - 90 °C petroleum ether by shaking 3 times, 40 mL each time. Combine the 60 - 90 °C petroleum ether solutions, wash with water 3 times, 40 mL each time, dehydrate with an appropriate amount of anhydrous sodium sulfate, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of anhydrous ethanol to obtain the test solutions 2 - 1, 2 - 2, 2 - 3, 2 - 4, and 2 - 5.

[0079] Preparation of the reference substance solution: Take germacrone reference substance, and make a solution containing 0.4 mg per 1 mL with anhydrous ethanol as the germacrone reference substance solution; take ligustilide reference substance, and make a solution containing 1 mg per 1 mL with anhydrous ethanol as the ligustilide reference substance solution.

[0080] Test according to the thin-layer chromatography method (General Principle 0502 of Chinese Pharmacopoeia 2020 Edition). Pipette 1 - 5 μL of the test solution, the reference solution of germacrone, and the reference solution of ligustilide respectively, and spot them on the same silica gel G thin-layer plate. Use petroleum ether - ethyl acetate (94:5) at 60 - 90 °C as the developing solvent, develop, take out, air dry, and examine under ultraviolet light at 365 nm. In the chromatogram of the test solution, at the position corresponding to the chromatogram of the ligustilide reference substance, there are fluorescent spots of the same color. Then, spray the air-dried thin-layer plate with 10% sulfuric acid ethanol solution, heat at 105 °C for dozens of seconds, and examine under ultraviolet light at 365 nm. In the chromatogram of the test solution, at the position corresponding to the chromatogram of the germacrone reference substance, there are fluorescent spots of the same color. See Figure 15 、 Figure 16 。

[0081] Test Example 2

[0082] Preparation of reference solutions: Take germacrone reference substance, add anhydrous ethanol to make a solution containing 0.4 mg per 1 mL as the germacrone reference solution. Take ligustilide reference substance, add anhydrous ethanol to make a solution containing 1 mg per 1 mL as the ligustilide reference solution.

[0083] 2.1 Investigation of extraction solvents for zedoary turmeric and angelica

[0084] Take 5 g of Dan'e Fukang Extract, add 40 mL of water, shake well, extract with ether by shaking 3 times, 40 mL each time, combine the ether layers, wash with water 3 times, 40 mL each time, dehydrate the ether layer with an appropriate amount of anhydrous sodium sulfate, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of anhydrous ethanol to obtain the ether solution of Dan'e Fukang Extract as the test solution.

[0085] Take 5 g of Dan'e Fukang Extract, add 40 mL of water, shake well, extract with petroleum ether at 60 - 90 °C by shaking 3 times, 40 mL each time, combine the petroleum ether layers at 60 - 90 °C, wash with water 3 times, 40 mL each time, dehydrate the petroleum ether layer at 60 - 90 °C with an appropriate amount of anhydrous sodium sulfate, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of anhydrous ethanol to obtain the petroleum ether solution of Dan'e Fukang Extract at 60 - 90 °C as the test solution.

[0086] Take 5 g of Dan'e Fukang Extract, add 40 mL of water, shake well, extract with ethyl acetate by shaking 3 times, 40 mL each time, combine the ethyl acetate layers, wash with water 3 times, 40 mL each time, dehydrate the ethyl acetate layer with an appropriate amount of anhydrous sodium sulfate, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of anhydrous ethanol to obtain the ethyl acetate solution of Dan'e Fukang Extract as the test solution.

[0087] Test according to the thin-layer chromatography method (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2015 Edition). Pipette 5 μL each of the zingiberone reference substance solution, the ethyl ether solution of the test sample of Dan'e Fukang Extract, the petroleum ether solution (60 - 90°C) of the test sample of Dan'e Fukang Extract, and the ethyl acetate solution of the test sample of Dan'e Fukang Extract, and spot them respectively on the same high-performance silica gel G thin-layer plate. Use petroleum ether - ethyl acetate (94:5) at 60 - 90°C as the developing solvent, develop, take out, air dry, spray with 5% vanillin sulfuric acid ethanol solution, heat at 105°C for several tens of seconds, and examine under daylight. See Figure 17 。

[0088] Test according to the thin-layer chromatography method (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2015 Edition). Pipette 5 μL each of the ligustilide reference substance solution, the ethyl ether solution of the test sample of Dan'e Fukang Extract, the petroleum ether solution (60 - 90°C) of the test sample of Dan'e Fukang Extract, and the ethyl acetate solution of the test sample of Dan'e Fukang Extract, and spot them respectively on the same high-performance silica gel G thin-layer plate. Use n-hexane - ethyl acetate (4:1) as the developing solvent, develop, take out, air dry, and examine under ultraviolet light at 365 nm. See Figure 18 。

[0089] As can be seen from the above figure, there is no obvious difference in the zingiberone spots developed from the test sample of Dan'e Fukang Extract after extraction with petroleum ether at 60 - 90°C and after extraction with ethyl ether. The difference in the ligustilide spots is relatively large. The resolution and discriminability of the ligustilide spots after extraction with petroleum ether at 60 - 90°C are higher. Therefore, petroleum ether at 60 - 90°C is selected as the extraction solvent for the test sample of Dan'e Fukang Extract.

[0090] 2.2 Investigation of the developing system

[0091] Test according to the thin-layer chromatography method (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2015 Edition). Pipette 5 μL each of the zingiberone reference substance solution and the petroleum ether solution (60 - 90°C) of the test sample of Dan'e Fukang Extract, and spot them respectively on the same high-performance silica gel G thin-layer plate. Use petroleum ether - acetone - ethyl acetate (94:5:1) at 30 - 60°C, petroleum ether - ethyl acetate (94:5) at 60 - 90°C, and cyclohexane - ethyl acetate (8:5) as the developing solvents respectively, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C for several tens of seconds, and examine under ultraviolet light at 365 nm. See Figure 19 、 Figure 20 、 Figure 21 。

[0092] Test according to the thin-layer chromatography method (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2015 Edition). Pipette 5 μL each of the reference substance solution of ligustilide and the 60 - 90°C petroleum ether solution of the test sample of Dan'e Fukang Extract onto the same high-efficiency silica gel G thin-layer plate. Using n-hexane - ethyl acetate (4:1), n-hexane - diethyl ether (7:3), cyclohexane - dichloromethane - ethyl acetate - formic acid (4:1:1:0.1), n-hexane - ethyl acetate (9:1), cyclohexane - ethyl acetate - formic acid (4:1:0.1), 60 - 90°C petroleum ether - ethyl acetate (94:5) as the developing solvents respectively, develop, take out, air-dry, and examine under ultraviolet light at 365 nm. Observe Figure 22 、 Figure 23 、 Figure 24 、 Figure 25 、 Figure 26 、 Figure 27 。

[0093] 2.3 Investigation of Color Reagents

[0094] Test according to the thin-layer chromatography method (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2015 Edition). Pipette 5 μL each of the reference substance solution of germacrone and the 60 - 90°C petroleum ether solution of the test sample of Dan'e Fukang Extract onto the same high-efficiency silica gel G thin-layer plate. Using 60 - 90°C petroleum ether - ethyl acetate (94:5) as the developing solvent, develop, take out, air-dry, spray with 5% vanillin sulfuric acid ethanol solution, heat at 105°C until the spots are clearly developed. Observe Figure 28 。

[0095] Test according to the thin-layer chromatography method (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2015 Edition). Pipette 5 μL each of the reference substance solution of germacrone and the 60 - 90°C petroleum ether solution of the test sample of Dan'e Fukang Extract onto the same high-efficiency silica gel G thin-layer plate. Using 60 - 90°C petroleum ether - ethyl acetate (94:5) as the developing solvent, develop, take out, air-dry, spray with 5% vanillin sulfuric acid solution, heat at 105°C until the spots are clearly developed. Observe Figure 29 。

[0096] Test according to the thin-layer chromatography method (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2015 Edition). Pipette 5 μL each of the reference substance solution of germacrone and the 60 - 90°C petroleum ether solution of the test sample of Dan'e Fukang Extract onto the same high-efficiency silica gel G thin-layer plate. Using 60 - 90°C petroleum ether - ethyl acetate (94:5) as the developing solvent, develop, take out, air-dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C for dozens of seconds, and examine under ultraviolet light at 365 nm. Observe Figure 30 。

[0097] From Figures 28 to 30It can be seen that both the 10% sulfuric acid ethanol solution with 5% vanillin and the 10% sulfuric acid ethanol solution have good color development effects, and the 10% sulfuric acid ethanol solution is easier to operate. Therefore, the 10% sulfuric acid ethanol solution was selected as the color developer.

[0098] 2.4 Specificity investigation

[0099] Take 5 g of the negative sample of Dan'e Fukang Decoction - Zedoary, add 40 mL of water, shake well, extract with petroleum ether at 60 - 90 °C by shaking 3 times, 40 mL each time. Combine the petroleum ether solution at 60 - 90 °C, wash with water 3 times, add an appropriate amount of anhydrous sodium sulfate to dehydrate the petroleum ether solution at 60 - 90 °C, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of anhydrous ethanol to obtain the negative sample solution of Dan'e Fukang Decoction - Zedoary.

[0100] Take 5 g of the negative sample of Dan'e Fukang Decoction - Angelica, add 40 mL of water, shake well, extract with petroleum ether at 60 - 90 °C by shaking 3 times, 40 mL each time. Combine the petroleum ether solution at 60 - 90 °C, wash with water 3 times, add an appropriate amount of anhydrous sodium sulfate to dehydrate the petroleum ether solution at 60 - 90 °C, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of anhydrous ethanol to obtain the negative sample solution of Dan'e Fukang Decoction - Angelica.

[0101] According to the thin-layer chromatography method (General Principles 0502, Volume IV of the Chinese Pharmacopoeia 2015 Edition), 5 μL of the germacrone reference substance solution, the 60 - 90 °C petroleum ether solution of the test sample of Dan'e Fukang Decoction, and the negative sample solution of Dan'e Fukang Decoction - Zedoary were respectively absorbed and spotted on the same high-performance silica gel G thin-layer plate. Using petroleum ether - ethyl acetate (94:5) at 60 - 90 °C as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C for dozens of seconds, and examine under ultraviolet light (365 nm). It can be seen that Figure 31 In the chromatogram of the test sample of Dan'e Fukang Decoction, in addition to the germacrone spot, there is also a specific characteristic spot of the zedoary medicinal material, realizing the simultaneous identification of multiple characteristic components.

[0102] According to the thin-layer chromatography method (General Principles 0502, Volume IV of the Chinese Pharmacopoeia 2015 Edition), 5 μL of the ligustilide reference substance solution, the 60 - 90 °C solution of the test sample of Dan'e Fukang Decoction, and the negative sample solution of Dan'e Fukang Decoction - Angelica were respectively absorbed and spotted on the same high-performance silica gel G thin-layer plate. Using petroleum ether - ethyl acetate (94:5) at 60 - 90 °C as the developing agent, develop, take out, dry, and examine under ultraviolet light at 365 nm. It can be seen that Figure 32 .

Claims

1. TLC identification method for Dan'e Fukang Decoction Extract, characterized in that, It includes the following steps: a. Take Dan'e Fukang decoction extract, and prepare test solution 1 and test solution 2 with petroleum ether as the solvent. b. Prepare reference substance solutions of tanshinone IIA, tetrahydropalmatine, germacrone or ligustilide respectively. c. Spot test solution 1, the reference substance solution of tanshinone IIA and the reference substance solution of tetrahydropalmatine on the same thin layer plate, and develop with developer 1; spot test solution 2, the reference substance solution of germacrone and the reference substance solution of ligustilide on the same thin layer plate, and develop with developer 2. After development, color it, examine it, and make a comparison. The preparation method of the said test solution 1 is: Take Dan'e Fukang decoction extract, add water, shake well, adjust the pH value to 9 - 10 with ammonia test solution, extract with petroleum ether with a boiling point of 60 - 90 °C by shaking, combine the petroleum ether solution at 60 - 90 °C, dehydrate with anhydrous sodium sulfate, filter, evaporate the filtrate to dryness, dissolve the residue in absolute ethanol to obtain test solution 1. The preparation method of the said test solution 2 is: Take Dan'e Fukang decoction extract, add water, shake well, extract with petroleum ether with a boiling point of 60 - 90 °C by shaking, combine the petroleum ether solution at 60 - 90 °C, wash with water, dehydrate with anhydrous sodium sulfate, filter, evaporate the filtrate to dryness, dissolve the residue in absolute ethanol to obtain test solution 2. The said developer 1 is composed of the following components in volume ratio: toluene - ethyl acetate - methanol 17 - 20:0.5 - 1.5:0.1 - 1. The said developer 2 is composed of the following components in volume ratio: petroleum ether - ethyl acetate 90 - 97:9 - 2.

2. The TLC identification method of Dan'e Fukang decoction extract according to claim 1, characterized in that: The boiling point of the said petroleum ether is 60 - 90 °C.

3. The TLC identification method of the Dan'e Fukang decoction extract according to claim 1, characterized in that: In step c, the said developer 1 is: the volume ratio of toluene - ethyl acetate - methanol is 19:1:0.

5.

4. The TLC identification method of Dan'e Fukang Extract according to claim 1, characterized in that: In step c, the said developer 2 is: the volume ratio of petroleum ether - ethyl acetate is 94:

5.

5. The TLC identification method of Dan'e Fukang Decoction Extract according to claim 1, characterized in that: In step c, for the thin layer plate developed with developer 1, dry it in the air, examine it under a daylight lamp to obtain the position of tanshinone IIA in the test sample on the thin layer plate; iodine color the dried thin layer plate and examine it under a 365 nm ultraviolet lamp to obtain the position of tetrahydropalmatine in the test sample on the thin layer plate.

6. The TLC identification method of the Dan'e Fukang decoction extract according to claim 1, characterized in that: In step c, for the thin layer plate developed with developer 2, dry it in the air, examine it under a 365 nm ultraviolet lamp to obtain the position of ligustilide in the test sample on the thin layer plate; spray the dried thin layer plate with sulfuric acid - ethanol solution, heat it and then examine it under a 365 nm ultraviolet lamp to obtain the position of germacrone in the test sample on the thin layer plate.

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