Thin-layer chromatography identification method for simultaneously identifying various components of angelica sinensis potion

Abstract

The invention belongs to the technical field of quality control of traditional Chinese medicines, and particularly relates to a thin-layer chromatography identification method for simultaneously identifying various components of angelica sinensis potion. According to the thin-layer chromatography identification method, firstly, ethyl acetate-n-butyl alcohol is adopted for shaking extraction, ethyl acetate-n-butyl alcohol liquid is taken and evaporated to dryness, water-soluble components which have large interference on identification are removed, a methylbenzene-ethyl acetate-formic acid system is developed, and then angelica sinensis, ligusticum wallichii and ginger in the angelica sinensis potion can be identified at the same time; and further removing acidic tailing components which have great interference on identification from the test sample through alumina column chromatography, and developing by using an ethyl acetate-formic acid-glacial acetic acid-water system, so that white paeony root, divaricate saposhnikovia root, liquorice and polygonum multiflorum in the Chinese angelica potion can be identified at the same time. The seven medicinal materials in the angelica sinensis potion can be simply, rapidly and effectively identified only by two thin-layer plates, and the method has the advantages of clear color spots, no tailing, no crossing and good specificity.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine quality control, and specifically relates to a thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi. Background Art

[0002] Dangguiyinzi, also known as Dangguiyin, was first seen in the Song Dynasty's "Yanshi Jishengfang". It is one of the first batch of classic prescriptions published by the State Administration of Traditional Chinese Medicine. It belongs to the Chinese medicine compound preparation managed according to the ancient classic prescription catalog. This prescription is composed of angelica, white peony, Chuanxiong, raw rehmannia, tribulus, saposhnikovia, schizonepeta spicate, Polygonum multiflorum, astragalus, licorice, and ginger. In the prescription, angelica is used as the main medicine to nourish blood and harmonize blood, supplemented by astragalus to replenish qi and produce blood, Polygonum multiflorum nourishes blood and relieves constipation and detoxification, and ginger relieves the cold. In addition, white peony nourishes blood, Chuanxiong activates blood circulation, and raw rehmannia cools blood, which further enhances the effect of nourishing blood and activating blood circulation. Schizonepeta spicate and saposhnikovia are both emollients in wind medicine, and enter the blood, which are good at removing wind in the blood. Tribulus terrestris is used to dispel wind, relieve the exterior, and relieve itching, and is a total adjuvant. Roasted licorice harmonizes all the medicines, which is the guiding medicine. This prescription is mainly used to treat itching caused by dry blood, with the main manifestation being wind-itch. Therefore, the whole prescription nourishes blood to moisten dryness, and dispels wind to relieve itching, which is the characteristic of its composition.

[0003] The thin layer chromatography identification method is an important chromatographic technology, and it is also the main means of qualitative identification in the quality standards of Chinese herbal compound preparations. Danggui Yinzi is composed of eleven herbs. At present, there is no relevant research in the existing technology on the identification of Danggui Yinzi components using thin layer chromatography. Most of the identification methods under the [Identification] item of the quality standards for Chinese herbal compound preparations included in the 2020 edition of the "Chinese Pharmacopoeia" are classified according to the identification of medicinal flavors. That is, to establish one identification item, one test solution must be prepared, one thin layer plate must be used, and one thin layer plate must be developed once to identify one prescription medicinal flavor. If the thin layer chromatography identification method is used to achieve full coverage of the prescription medicinal flavors of Chinese herbal compound preparations, the number of tests required is often large, which is time-consuming and labor-intensive.

[0004] Therefore, how to establish a thin-layer chromatography detection method for Danggui Yinzi that can simultaneously identify multiple components with good separation and no interference has far-reaching significance for the quality control of Danggui Yinzi. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi. The identification method can use one extraction method to prepare two test sample solutions, realize the simultaneous identification of 7 of the 11 medicinal ingredients in the Danggui Yinzi formula, and has high stability and repeatability while ensuring the identification efficiency.

[0006] The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi of the present invention comprises the following steps: (1) Preparation of test solution Take Dangguiyinzi decoction, Dangguiyinzi fluid extract, Dangguiyinzi dry extract or Dangguiyinzi granules, first shake and extract with ethyl acetate-n-butanol mixed solution, then evaporate the extract to dryness, add methanol to dissolve the obtained residue, and use it as test solution 1; Take the test solution 1, add it to the alumina column, elute with methanol, collect the eluate, evaporate to dryness, and dissolve the residue with methanol as the test solution 2; (2) Preparation of reference solution Take ligustilide reference substance, 6-gingerol reference substance and liquiritin reference substance, and prepare ligustilide reference substance solution, 6-gingerol reference substance solution and liquiritin reference substance solution respectively; (3) Preparation of control medicinal material solution Take white peony root control medicinal material, Polygonum multiflorum control medicinal material and Saposhnikovia divaricata control medicinal material, add ethyl acetate-methanol to reflux respectively, filter, evaporate the filtrate to dryness, add methanol to dissolve the residue, add it to an alumina column, elute with methanol, collect the eluate, evaporate to dryness, and dissolve the residue in methanol to prepare white peony root control medicinal material solution, Polygonum multiflorum control medicinal material solution and Saposhnikovia divaricata control medicinal material solution respectively; (4) Thin layer chromatography detection The test solution 1 prepared in step (1), the ligustilide reference solution and the 6-gingerol reference solution prepared in step (2) are respectively spotted on the same silica gel G thin layer plate, developed with a toluene-ethyl acetate-formic acid system, taken out, air-dried, and examined under an ultraviolet lamp. In the chromatogram of the test sample, a fluorescent spot of the same color as that of the ligustilide reference is displayed at the position corresponding to that of the ligustilide reference. Then, the plate is sprayed with a vanillin sulfuric acid solution and heated until the spots are clearly colored. In the chromatogram of the test sample, a spot of the same color as that of the 6-gingerol reference is displayed at the position corresponding to that of the chromatogram of the 6-gingerol reference. The test solution 2 prepared in step (1), the liquiritin reference substance solution prepared in step (2), the white peony root reference medicinal material solution, the polygonum multiflorum reference medicinal material solution and the saposhnikovia divaricata reference medicinal material solution prepared in step (3) are respectively spotted on the same silica gel G thin layer plate, developed with an ethyl acetate-formic acid-glacial acetic acid-water system, taken out, air-dried, sprayed with a phosphomolybdic acid-sulfuric acid solution, heated until the spots are clearly colored, and examined under sunlight. In the chromatogram of the test sample, spots of the same color are shown at the corresponding positions of the chromatograms of the liquiritin reference substance, the white peony root reference medicinal material and the polygonum multiflorum reference medicinal material; in the chromatogram of the test sample, fluorescent spots of the same color are shown at the corresponding positions of the chromatograms of the liquiritin reference substance; and fluorescent main spots of the same color are shown at the corresponding positions of the chromatograms of the saposhnikovia divaricata control medicinal material.

[0007] in: The volume ratio of ethyl acetate to n-butanol in the ethyl acetate-n-butanol mixed solution in step (1) is 1-8:0.1-5.

[0008] The number of times of shaking extraction when preparing the test solution 1 in step (1) is 2 times.

[0009] In step (1), when the raw material is Dangguiyinzi dry extract or Dangguiyinzi granules, water should be added to dissolve it first, and then it should be shaken and extracted with an ethyl acetate-n-butanol mixed solution.

[0010] In step (1), when preparing the sample 2, eluting with methanol and collecting the eluate, specifically, first eluting with methanol, discarding the eluate, and then eluting with 50-70% methanol, and collecting the eluate.

[0011] In step (2), the ligustilide reference substance solution uses methanol as solvent, and each 1 mL of methanol contains 1 mg of ligustilide reference substance.

[0012] In step (2), the 6-gingerol reference substance solution is prepared using methanol as solvent, and each 1 mL of methanol contains 0.5 mg of 6-gingerol reference substance.

[0013] The liquiritin reference substance solution in step (2) uses methanol as solvent, and each 1 mL of methanol contains 1.0 mg of liquiritin reference substance.

[0014] The volume ratio of ethyl acetate to methanol in the ethyl acetate-methanol reflux system in step (3) is 1-19:0.1-5.

[0015] The mass volume ratio of the white peony root control medicinal material to ethyl acetate-methanol in step (3) is 0.5:30, expressed in g / mL.

[0016] The mass volume ratio of the Polygonum multiflorum reference medicinal material to ethyl acetate-methanol in step (3) is 0.1:30, in units of g / mL.

[0017] The mass volume ratio of the Fangfeng reference medicinal material to ethyl acetate-methanol in step (3) is 1.0:30, in units of g / mL.

[0018] In step (3), the method of eluting with methanol and collecting the eluate is as follows: first eluting with methanol, discarding the eluate, then eluting with 50-70% methanol, and collecting the eluate.

[0019] The alumina used in the alumina column in step (1) or the alumina column in step (3) is one of basic alumina, neutral alumina or acidic alumina.

[0020] In the toluene-ethyl acetate-formic acid system of step (4), the volume ratio of toluene, ethyl acetate and formic acid is 20-40: 2-8: 0.1-2.

[0021] In the ethyl acetate-formic acid-glacial acetic acid-water system of step (4), the volume ratio of ethyl acetate, formic acid, glacial acetic acid and water is 5-20: 0.1-2: 1-10: 1-10.

[0022] The wavelength of the ultraviolet lamp in step (4) is 365nm.

[0023] Most of the identification methods under the [Identification] item of the quality standard for compound Chinese medicine preparations included in the 2020 edition of the Chinese Pharmacopoeia are classified according to the identification of medicinal flavors, that is, to establish one identification item, one test solution must be prepared, one thin layer plate must be used, and one development must be carried out to identify one prescription medicinal flavor. The traditional model requires many tests and is time-consuming and laborious. The method described in the present invention has simple operation steps. First, ethyl acetate-n-butanol is used for shaking extraction, and the ethyl acetate-n-butanol solution is evaporated to remove the water-soluble components that interfere with the identification. The toluene-ethyl acetate-formic acid system is used for development, and the angelica, Chuanxiong and ginger in the angelica drink can be identified at the same time; then the test sample is subjected to alumina column chromatography to further remove the acidic tailing components that interfere with the identification, and the ethyl acetate-formic acid-glacial acetic acid-water system is used for development, and the white peony root, siler, licorice and polygonum multiflorum in the angelica drink can be identified at the same time. Only two thin layer plates are needed to simply, quickly and effectively identify the seven medicinal materials in Danggui Yinzi, and the color spots are clear, without tailing, without crossover and with good specificity. The identification method and effect described in the present invention have obvious technical progress, and the quality control of Danggui Yinzi has been further improved.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi described in the present invention can use one extraction method to prepare two test solutions, thereby achieving simultaneous identification of seven of the 11 medicinal ingredients in the Danggui Yinzi formula. While ensuring identification efficiency, it also has high stability and repeatability, solving the problems in the prior art of needing to prepare multiple test solutions and cumbersome detection procedures, and providing a reference basis for quality control of Danggui Yinzi, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the TLC spectrum of angelica, Chuanxiong and ginger thin layer identification under 365nm ultraviolet light (including the specificity investigation spectrum). The labels in the attached figure are as follows: 1. Ligustilide reference substance, 2. 6-gingerol reference substance; 3. Test solution 1, 4. Angelica and Chuanxiong negative test substance, 5. Ginger negative test substance; Figure 2This is the TLC spectrum of Angelica sinensis, Chuanxiong and ginger in Danggui Yinzi under sunlight (including the specificity investigation spectrum). The labels in the attached figure are as follows: 1. Ligustilide reference substance, 2. 6-gingerol reference substance, 3. Test solution 1, 4. Angelica sinensis and Chuanxiong negative test substance, 5. Ginger negative test substance; Figure 3 This is the TLC spectrum of licorice, fangfeng, heshouwu and white peony root in Danggui Yinzi under sunlight (including the specificity investigation spectrum), and the labels in the attached figure are as follows: 1. Licorice glycoside reference substance, 2. Fangfeng reference medicinal material, 3. Heshouwu reference medicinal material, 4. White peony reference medicinal material, 5. Test solution 2, 6. Licorice negative test sample, 7. Fangfeng negative test sample, 8. Heshouwu negative test sample, 9. White peony negative test sample; Figure 4 This is the TLC spectrum of licorice, fangfeng, polygonum multiflorum and white peony root in Danggui Yinzi under 365nm ultraviolet light (including specificity investigation spectrum). The marks in the attached figure are as follows: 1. liquiritin reference substance, 2. fangfeng reference medicinal material, 3. polygonum multiflorum reference medicinal material, 4. white peony root reference medicinal material, 5. test sample solution 2, 6. licorice negative test sample, 7. fangfeng negative test sample, 8. polygonum multiflorum negative test sample, 9. white peony negative test sample. DETAILED DESCRIPTION

[0026] The present invention is further described below with reference to the embodiments.

[0027] Negative samples were prepared in Examples 1-4. The purpose of preparing negative samples is to use negative samples to verify the specificity of the thin layer chromatography analysis method. By comparing the chromatograms of the negative test sample (a sample without the target medicinal material) solution with the reference solution and the test sample solution, it can be confirmed whether the target identification component is interfered by other components. If there is no spot interference of the same color at the corresponding position of the reference chromatogram and the test sample chromatogram in the negative test sample chromatogram, it indicates that the negative sample has no interference and the method specificity is good.

[0028] Example 1 Dangguiyinzi decoction was used as the sample to be tested, wherein the preparation methods of Dangguiyinzi decoction and negative samples were as follows: Angelica guiyinzi decoction: take 413g of angelica sinensis, 413g of white peony root, 413g of chuanxiong, 413g of raw rehmannia root, 413g of stir-fried tribulus terrestris, 413g of siler, 413g of schizonepeta spicate, 206.5g of polygonum multiflorum, 206.5g of astragalus, and 206.5g of stir-fried licorice, grind into coarse particles, add 96L of water, add 1062g of ginger, and boil for 1h to obtain angelica guiyinzi decoction.

[0029] Angelica and Chuanxiong negative sample: weigh the other 9 medicinal ingredients except Angelica and Chuanxiong. The composition, content and preparation method are the same as the preparation method of Angelica Decoction.

[0030] Ginger negative sample: weigh the other 10 medicinal ingredients except ginger. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi decoction.

[0031] White peony root negative sample: weigh the other 10 medicinal ingredients except white peony root. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi decoction.

[0032] Negative sample of Saposhnikovia divaricata: weigh the other 10 medicinal ingredients except Saposhnikovia divaricata. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi decoction.

[0033] Negative sample of Polygonum multiflorum: weigh the other 10 medicinal ingredients except Polygonum multiflorum. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi decoction.

[0034] Licorice negative sample: weigh the other 10 medicinal ingredients except licorice. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi decoction.

[0035] The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi described in Example 1, that is, the thin layer chromatography identification method for simultaneously identifying seven medicinal materials in Danggui Yinzi decoction, namely, Danggui, Chuanxiong, ginger, white peony root, fangfeng, He Shouwu, and liquorice, is composed of the following steps: (1) Preparation of test solution Take an appropriate amount of Danggui Yinzi decoction (equivalent to 21.6 g of the total amount of the decoction pieces), concentrate it to about 30 mL, and extract it twice with ethyl acetate-n-butanol (the volume ratio of ethyl acetate to n-butanol is 1:0.1), 30 mL each time, and combine the ethyl acetate-n-butanol solution, evaporate it to dryness, and dissolve the residue in 1 mL of methanol as the test solution 1.

[0036] Take the test solution 1, add it to a neutral alumina column (100-200 mesh, 5 g, inner diameter 1 cm, dry loading), elute with 20 mL of methanol, discard the eluent, elute with 20 mL of 50% methanol, collect the eluent, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution 2.

[0037] (2) Preparation of negative test solution of Angelica sinensis, Chuanxiong and ginger Weigh out an equal amount of Angelica sinensis and Chuanxiong negative sample and ginger negative sample in step (1), and prepare Angelica sinensis and Chuanxiong negative test solution and ginger negative test solution according to the method of test solution 1 in step (1).

[0038] (3) Preparation of negative test solutions of white peony root, siler, polygonum multiflorum and licorice Weigh out the white peony root negative sample, the fangfeng root negative sample, the polygonum multiflorum negative sample, and the licorice negative sample in the same amount as the decoction pieces in step (1), and prepare the white peony root negative test solution, the fangfeng root negative test solution, the polygonum multiflorum negative test solution, and the licorice negative test solution according to the method of the test solution 2 in step (1).

[0039] (4) Preparation of reference solution Take an appropriate amount of ligustilide reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the ligustilide reference substance solution; take an appropriate amount of 6-gingerol reference substance, add methanol to make a solution containing 0.5 mg per 1 mL, as the 6-gingerol reference substance solution; take an appropriate amount of glycyrrhizin reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the glycyrrhizin reference substance solution.

[0040] (5) Preparation of reference medicinal material solution Take 0.5 g of white peony root control medicinal material, 0.1 g of Polygonum multiflorum control medicinal material, and 1 g of Saposhnikovia divaricata control medicinal material, add 30 mL of ethyl acetate-methanol (the volume ratio of ethyl acetate to methanol is 1:0.1) respectively, reflux for 1 hour, filter, evaporate the filtrate, add 1 mL of methanol to the residue to dissolve it, add it to a neutral alumina column (100-200 mesh, 5 g, inner diameter of 1 cm, dry column), elute with 20 mL of methanol, discard the eluent, and then elute with 20 mL of 50% methanol, collect the eluent, evaporate to dryness, add 1 mL of methanol to dissolve the residue, and prepare white peony root control medicinal material solution, Polygonum multiflorum control medicinal material solution, and Saposhnikovia divaricata control medicinal material solution, respectively.

[0041] (6) Thin layer chromatography analysis 1 According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 8 μL of the test solution 1 prepared in step (1), the negative test solution of Angelica sinensis and Chuanxiong and the test solution of ginger prepared in step (2), 2 μL of the ligustilide reference solution and 5 μL of the 6-gingerol reference solution prepared in step (4) were taken and spotted on the same silica gel G thin layer plate, respectively, and developed with toluene-ethyl acetate-formic acid (the volume ratio of toluene, ethyl acetate and formic acid was 20:2:0.1), taken out, dried, and examined under ultraviolet light (365 nm). The results are shown in Table 1. Figure 1 .

[0042] from Figure 1 It can be seen that under 365nm inspection, in the chromatogram of the test sample, at the corresponding position with the ligustilide reference substance, a fluorescent spot of the same color appears, and there is no interference with the negative test sample of Angelica and Chuanxiong, with good specificity.

[0043] Then spray with 5% vanillin sulfuric acid solution and heat at 105℃ until the spots are clearly colored. Figure 2In the chromatogram of the test sample, a spot of the same color appears at the corresponding position of the chromatogram of the 6-gingerol reference sample. The ginger negative test sample has no interference and has good specificity.

[0044] (7) Thin layer chromatography analysis 2 According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 2 μL of the test solution 2 prepared in step (1) and the negative test solution of white peony, the negative test solution of saposhnikovia divaricata, the negative test solution of Polygonum multiflorum, and the negative test solution of Licorice prepared in step (3), 2 μL of the liquiritin reference solution prepared in step (4), 10 μL of the white peony reference medicinal material solution and the Polygonum multiflorum reference medicinal material solution prepared in step (5), and 1 μL of the saposhnikovia divaricata reference medicinal material solution were respectively spotted on the same silica gel G thin layer plate, developed with ethyl acetate-formic acid-glacial acetic acid-water (the volume ratio of ethyl acetate, formic acid, glacial acetic acid, and water is 5:0.1:1:10), taken out, dried, sprayed with phosphomolybdic acid-sulfuric acid solution (take 2 g of phosphomolybdic acid, add 20 mL of water to dissolve, then slowly add 30 mL of sulfuric acid and mix well), heated at 105°C until the spots are clearly colored, and inspected under sunlight. The results are shown in Table 1. Figure 3 .

[0045] from Figure 3 It can be seen that in the test sample chromatogram, spots of the same color appear at the corresponding positions with the chromatograms of the licorice glycoside reference sample, the chromatograms of the Polygonum multiflorum reference medicinal material, and the chromatograms of the white peony reference medicinal material. The licorice negative test sample, the Polygonum multiflorum negative test sample, and the white peony negative test sample have no interference and have good specificity.

[0046] Check under UV light (365nm), the results are shown in Figure 4 In the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the glycyrrhizin reference sample; in the chromatogram of the saposhnikovia divaricata reference medicinal material, a main fluorescent spot of the same color appears. There is no interference between the licorice-negative test sample and the saposhnikovia divaricata negative test sample, and the specificity is good.

[0047] Example 2 Dangguiyinzi fluid extract was used as the sample to be tested, wherein the preparation methods of Dangguiyinzi fluid extract and negative sample were as follows: Dangguiyinzi fluid extract: Dangguiyinzi decoction in Example 1 is concentrated to a relative density of 1.25 (70° C.) to obtain Dangguiyinzi fluid extract.

[0048] Angelica and Chuanxiong negative sample: weigh the other 9 medicinal ingredients except Angelica and Chuanxiong. The composition, content and preparation method are the same as the preparation method of Angelica Decoction Extract.

[0049] Ginger negative sample: weigh the other 10 medicinal ingredients except ginger. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi extract.

[0050] White peony root negative sample: weigh the other 10 medicinal ingredients except white peony root. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi extract.

[0051] Negative sample of Saposhnikovia divaricata: weigh the other 10 medicinal ingredients except Saposhnikovia divaricata. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi extract.

[0052] Negative sample of Polygonum multiflorum: weigh the other 10 medicinal ingredients except Polygonum multiflorum. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi extract.

[0053] Licorice negative sample: weigh the other 10 medicinal ingredients except licorice. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi decoction.

[0054] The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi described in Example 2, that is, the thin layer chromatography identification method for simultaneously identifying seven medicinal materials in Danggui Yinzi extract, namely, Danggui, Chuanxiong, ginger, white peony root, siler, Polygonum multiflorum, and liquorice, comprises the following steps: (1) Preparation of test solution Take an appropriate amount of Danggui Yinzi extract (equivalent to 21.6 g of the total amount of the slices), add 30 mL of water to dissolve, shake and extract twice with ethyl acetate-n-butanol (the volume ratio of ethyl acetate to n-butanol is 8:5), 30 mL each time, combine the ethyl acetate-n-butanol solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution 1; Take the test solution 1, add it to a basic alumina column (100-200 mesh, 5 g, inner diameter 1 cm, dry-packed), elute with 20 mL of methanol, discard the eluent, and then elute with 20 mL of 70% methanol, collect the eluent, evaporate it to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution 2.

[0055] (2) Preparation of negative test solution of Angelica sinensis, Chuanxiong and ginger Weigh out an equal amount of Angelica sinensis and Chuanxiong negative sample and ginger negative sample in step (1), and prepare Angelica sinensis and Chuanxiong negative test solution and ginger negative test solution according to the method of test solution 1 in step (1).

[0056] (3) Preparation of negative test solutions of white peony root, siler, polygonum multiflorum and licorice Weigh out the white peony root negative sample, the fangfeng root negative sample, the polygonum multiflorum negative sample, and the licorice negative sample in the same amount as the decoction pieces in step (1), and prepare the white peony root negative test solution, the fangfeng root negative test solution, the polygonum multiflorum negative test solution, and the licorice negative test solution according to the method of the test solution 2 in step (1).

[0057] (4) Preparation of reference solution Take an appropriate amount of ligustilide reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the ligustilide reference substance solution; take an appropriate amount of 6-gingerol reference substance, add methanol to make a solution containing 0.5 mg per 1 mL, as the 6-gingerol reference substance solution; take an appropriate amount of glycyrrhizin reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the glycyrrhizin reference substance solution.

[0058] (5) Preparation of reference medicinal material solution Take 0.5g of white peony root control medicinal material, 0.1g of Polygonum multiflorum control medicinal material, and 1g of Saposhnikovia divaricata control medicinal material, respectively, add 30mL of ethyl acetate-methanol (the volume ratio of ethyl acetate to methanol is 8:5), reflux for 1 hour, filter, evaporate the filtrate, add 1mL of methanol to the residue to dissolve it, add it to a neutral alumina column (100-200 mesh, 5g, inner diameter of 1cm, dry column), elute with 20mL of methanol, discard the eluent, and then elute with 20mL of 70% methanol, collect the eluent, evaporate to dryness, add 1mL of methanol to dissolve the residue, and prepare white peony root control medicinal material solution, Polygonum multiflorum control medicinal material solution, and Saposhnikovia divaricata control medicinal material solution, respectively.

[0059] (6) Thin layer chromatography analysis 1 According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 8 μL of the test solution 1 prepared in step (1), the negative test solution of Angelica sinensis and Chuanxiong and the test solution of ginger prepared in step (2), 2 μL of the ligustilide reference solution and 5 μL of the 6-gingerol reference solution prepared in step (4) were taken and spotted on the same silica gel G thin layer plate, developed with toluene-ethyl acetate-formic acid (the volume ratio of toluene, ethyl acetate and formic acid was 40:8:2), taken out, dried, and examined under ultraviolet light (365 nm).

[0060] Under 365nm inspection, in the chromatogram of the test sample, a fluorescent spot of the same color appeared at the corresponding position of the ligustilide reference sample. The Angelica and Chuanxiong negative test sample had no interference and had good specificity.

[0061] Then spray with 5% vanillin sulfuric acid solution and heat at 105℃ until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the 6-gingerol reference sample. The ginger-negative test sample has no interference and has good specificity.

[0062] (7) Thin layer chromatography analysis 2 According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 2 μL of the test solution 2 prepared in step (1) and the negative test solution of white peony, the negative test solution of saposhnikovia divaricata, the negative test solution of Polygonum multiflorum, and the negative test solution of Licorice prepared in step (3), 2 μL of the liquiritin reference solution prepared in step (4), 10 μL of the white peony reference medicinal material solution and the Polygonum multiflorum reference medicinal material solution prepared in step (5), and 1 μL of the saposhnikovia divaricata reference medicinal material solution are respectively spotted on the same silica gel G thin layer plate, developed with ethyl acetate-formic acid-glacial acetic acid-water (the volume ratio of ethyl acetate, formic acid, glacial acetic acid, and water is 20:2:10:10), taken out, dried, sprayed with phosphomolybdic acid-sulfuric acid solution (take 2 g of phosphomolybdic acid, add 20 mL of water to dissolve, then slowly add 30 mL of sulfuric acid and mix well), heated at 105°C until the spots are clearly colored, and inspected under sunlight. In the test sample chromatogram, spots of the same color appear at the corresponding positions with the chromatograms of the licorice glycoside reference sample, the chromatograms of the Polygonum multiflorum reference medicinal material, and the chromatograms of the white peony reference medicinal material. The licorice negative test sample, the Polygonum multiflorum negative test sample, and the white peony negative test sample have no interference and have good specificity.

[0063] When inspected under ultraviolet light (365nm), in the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the glycyrrhizin reference sample; in the chromatogram of the siler control medicinal material, a main fluorescent spot of the same color appears at the corresponding position. There is no interference between the licorice-negative test sample and the siler negative test sample, and the specificity is good.

[0064] Example 3 Dangguiyinzi dry extract was used as the sample to be tested, wherein the preparation methods of Dangguiyinzi dry extract and negative sample were as follows: Dangguiyinzi dry extract: Based on the preparation method of Dangguiyinzi extract in Example 2, further drying to a solid state can obtain Dangguiyinzi dry extract.

[0065] Angelica and Chuanxiong negative sample: weigh the other 9 medicinal ingredients except Angelica and Chuanxiong. The composition, content and preparation method are the same as the preparation method of Angelica Decoction dry extract.

[0066] Ginger negative sample: weigh the other 10 medicinal ingredients except ginger. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi dry extract.

[0067] White peony root negative sample: weigh the other 10 medicinal ingredients except white peony root. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi dry extract.

[0068] Negative sample of Saposhnikovia divaricata: weigh the other 10 medicinal ingredients except Saposhnikovia divaricata. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi dry extract.

[0069] Negative sample of Polygonum multiflorum: weigh the other 10 medicinal ingredients except Polygonum multiflorum. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi dry extract.

[0070] Licorice negative sample: weigh the other 10 medicinal ingredients except licorice. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi dry extract.

[0071] The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi described in Example 3, that is, the thin layer chromatography identification method for simultaneously identifying seven medicinal materials in Danggui Yinzi dry extract, namely, Danggui, Chuanxiong, ginger, white peony root, siler, Polygonum multiflorum, and liquorice, comprises the following steps: (1) Preparation of test solution Take an appropriate amount of Dangguiyinzi dry extract (equivalent to 21.6 g of the total amount of the slices), add 30 mL of water to dissolve it, shake and extract it twice with ethyl acetate-n-butanol (the volume ratio of ethyl acetate to n-butanol is 8:0.1), 30 mL each time, combine the ethyl acetate-n-butanol solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution 1; Take the test solution 1, add it to an acidic alumina column (100-200 mesh, 5 g, inner diameter 1 cm, dry-packed), elute with 20 mL of methanol, discard the eluent, and then elute with 20 mL of 50% methanol, collect the eluent, evaporate it to dryness, add 1 mL of methanol to dissolve the residue, and use it as the test solution 2.

[0072] (2) Preparation of negative test solution of Angelica sinensis, Chuanxiong and ginger Weigh out an equal amount of Angelica sinensis and Chuanxiong negative sample and ginger negative sample in step (1), and prepare Angelica sinensis and Chuanxiong negative test solution and ginger negative test solution according to the method of test solution 1 in step (1).

[0073] (3) Preparation of negative test solutions of white peony root, siler, polygonum multiflorum and licorice Weigh out the white peony root negative sample, the fangfeng root negative sample, the polygonum multiflorum negative sample, and the licorice negative sample in the same amount as the decoction pieces in step (1), and prepare the white peony root negative test solution, the fangfeng root negative test solution, the polygonum multiflorum negative test solution, and the licorice negative test solution according to the method of the test solution 2 in step (1).

[0074] (4) Preparation of reference solution Take an appropriate amount of ligustilide reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the ligustilide reference substance solution; take an appropriate amount of 6-gingerol reference substance, add methanol to make a solution containing 0.5 mg per 1 mL, as the 6-gingerol reference substance solution; take an appropriate amount of glycyrrhizin reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the glycyrrhizin reference substance solution.

[0075] (5) Preparation of reference medicinal material solution Take 0.5g of white peony root control medicinal material, 0.1g of Polygonum multiflorum control medicinal material, and 1g of Saposhnikovia divaricata control medicinal material, add 30mL of ethyl acetate-methanol (the volume ratio of ethyl acetate to methanol is 8:0.1) respectively, reflux for 1 hour, filter, evaporate the filtrate, add 1mL of methanol to the residue to dissolve it, add it to a neutral alumina column (100-200 mesh, 5g, inner diameter of 1cm, dry column), elute with 20mL of methanol, discard the eluent, and then elute with 20mL of 50% methanol, collect the eluent, evaporate to dryness, add 1mL of methanol to dissolve the residue, and prepare white peony root control medicinal material solution, Polygonum multiflorum control medicinal material solution, and Saposhnikovia divaricata control medicinal material solution, respectively.

[0076] (6) Thin layer chromatography analysis 1 According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 8 μL of the test solution 1 prepared in step (1), the negative test solution of Angelica sinensis and Chuanxiong and the test solution of ginger prepared in step (2), 2 μL of the ligustilide reference solution and 5 μL of the 6-gingerol reference solution prepared in step (4) were taken and spotted on the same silica gel G thin layer plate, developed with toluene-ethyl acetate-formic acid (the volume ratio of toluene, ethyl acetate and formic acid was 30:4:1), taken out, dried, and inspected under ultraviolet light (365 nm).

[0077] Under 365nm inspection, in the chromatogram of the test sample, a fluorescent spot of the same color appeared at the corresponding position of the ligustilide reference sample. The Angelica and Chuanxiong negative test sample had no interference and had good specificity.

[0078] Then spray with 5% vanillin sulfuric acid solution and heat at 105℃ until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the 6-gingerol reference sample. The ginger-negative test sample has no interference and has good specificity.

[0079] (7) Thin layer chromatography analysis 2 According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 2 μL of the test solution 2 prepared in step (1) and the negative test solution of white peony, the negative test solution of saposhnikovia divaricata, the negative test solution of Polygonum multiflorum, and the negative test solution of Licorice prepared in step (3), 2 μL of the liquiritin reference solution prepared in step (4), 10 μL of the white peony reference medicinal material solution and the Polygonum multiflorum reference medicinal material solution prepared in step (5), and 1 μL of the saposhnikovia divaricata reference medicinal material solution, are respectively spotted on the same silica gel G thin layer plate, developed with ethyl acetate-formic acid-glacial acetic acid-water (the volume ratio of ethyl acetate, formic acid, glacial acetic acid, and water is 15:1:5:5), taken out, dried, sprayed with phosphomolybdic acid-sulfuric acid solution (take 2 g of phosphomolybdic acid, add 20 mL of water to dissolve, then slowly add 30 mL of sulfuric acid and mix well), heated at 105°C until the spots are clearly colored, and inspected under sunlight. In the test sample chromatogram, spots of the same color appear at the corresponding positions with the chromatograms of the licorice glycoside reference sample, the chromatograms of the Polygonum multiflorum reference medicinal material, and the chromatograms of the white peony reference medicinal material. The licorice negative test sample, the Polygonum multiflorum negative test sample, and the white peony negative test sample have no interference and have good specificity.

[0080] When inspected under ultraviolet light (365nm), in the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the glycyrrhizin reference sample; in the chromatogram of the siler control medicinal material, a main fluorescent spot of the same color appears at the corresponding position. There is no interference between the licorice-negative test sample and the siler negative test sample, and the specificity is good.

[0081] Example 4 Dangguiyinzi granules were used as the sample to be tested, and the preparation methods of Dangguiyinzi granules and negative samples were as follows: Dangguiyinzi granules: Based on the preparation method of Dangguiyinzi dry extract in Example 3, dextrin is added and dry granulation is adopted to obtain Dangguiyinzi granule preparation.

[0082] Angelica and Chuanxiong negative sample: weigh the other 9 medicinal ingredients except Angelica and Chuanxiong. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi Granules.

[0083] Ginger negative sample: weigh the other 10 medicinal ingredients except ginger. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi Granules.

[0084] White peony root negative sample: weigh the other 10 medicinal ingredients except white peony root. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi granules.

[0085] Saposhnikovia negative sample: weigh the other 10 medicinal ingredients except Saposhnikovia. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi granules.

[0086] Negative sample of Polygonum multiflorum: weigh the other 10 medicinal ingredients except Polygonum multiflorum. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi granules.

[0087] Licorice negative sample: weigh the other 10 medicinal ingredients except licorice. The composition, content and preparation method are the same as the preparation method of Danggui Yinzi Granules.

[0088] The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi described in Example 4, that is, the thin layer chromatography identification method for simultaneously identifying seven medicinal materials in Danggui Yinzi granules, namely, Danggui, Chuanxiong, ginger, white peony root, siler, Polygonum multiflorum, and liquorice, comprises the following steps: (1) Preparation of test solution Take an appropriate amount of Danggui Yinzi granules (equivalent to 21.6 g of the total amount of the slices), add 30 mL of water to dissolve, shake and extract twice with ethyl acetate-n-butanol (the volume ratio of ethyl acetate to n-butanol is 2:1), 30 mL each time, combine the ethyl acetate-n-butanol solution, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution 1; Take the test solution 1, add it to a neutral alumina column (100-200 mesh, 5 g, inner diameter 1 cm, dry loading), elute with 20 mL of methanol, discard the eluent, elute with 20 mL of 50% methanol, collect the eluent, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution 2.

[0089] (2) Preparation of negative test solution of Angelica sinensis, Chuanxiong and ginger Weigh out an equal amount of Angelica sinensis and Chuanxiong negative sample and ginger negative sample in step (1), and prepare Angelica sinensis and Chuanxiong negative test solution and ginger negative test solution according to the method of test solution 1 in step (1).

[0090] (3) Preparation of negative test solutions of white peony root, siler, polygonum multiflorum and licorice Weigh out the white peony root negative sample, the fangfeng root negative sample, the polygonum multiflorum negative sample, and the licorice negative sample in the same amount as the decoction pieces in step (1), and prepare the white peony root negative test solution, the fangfeng root negative test solution, the polygonum multiflorum negative test solution, and the licorice negative test solution according to the method of the test solution 2 in step (1).

[0091] (4) Preparation of reference solution Take an appropriate amount of ligustilide reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the ligustilide reference substance solution; take an appropriate amount of 6-gingerol reference substance, add methanol to make a solution containing 0.5 mg per 1 mL, as the 6-gingerol reference substance solution; take an appropriate amount of glycyrrhizin reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the glycyrrhizin reference substance solution.

[0092] (5) Preparation of reference medicinal material solution Take 0.5g of white peony root control medicinal material, 0.1g of Polygonum multiflorum control medicinal material, and 1g of Saposhnikovia divaricata control medicinal material, respectively, add 30mL of ethyl acetate-methanol (the volume ratio of ethyl acetate to methanol is 19:1), reflux for 1 hour, filter, evaporate the filtrate, add 1mL of methanol to the residue to dissolve it, add it to a neutral alumina column (100-200 mesh, 5g, inner diameter of 1cm, dry column), elute with 20mL of methanol, discard the eluent, and then elute with 20mL of 50% methanol, collect the eluent, evaporate to dryness, add 1mL of methanol to dissolve the residue, and prepare white peony root control medicinal material solution, Polygonum multiflorum control medicinal material solution, and Saposhnikovia divaricata control medicinal material solution, respectively.

[0093] (6) Thin layer chromatography analysis 1 According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 8 μL of the test solution 1 prepared in step (1), the negative test solution of Angelica sinensis and Chuanxiong and the test solution of ginger prepared in step (2), 2 μL of the ligustilide reference solution and 5 μL of the 6-gingerol reference solution prepared in step (4) were taken and spotted on the same silica gel G thin layer plate, developed with toluene-ethyl acetate-formic acid (the volume ratio of toluene, ethyl acetate and formic acid was 30:4:1), taken out, dried, and inspected under ultraviolet light (365 nm).

[0094] Under 365nm inspection, in the chromatogram of the test sample, a fluorescent spot of the same color appeared at the corresponding position of the ligustilide reference sample. The Angelica and Chuanxiong negative test sample had no interference and had good specificity.

[0095] Then spray with 5% vanillin sulfuric acid solution and heat at 105℃ until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the 6-gingerol reference sample. The ginger-negative test sample has no interference and has good specificity.

[0096] (7) Thin layer chromatography analysis 2 According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 2 μL of the test solution 2 prepared in step (1) and the negative test solution of white peony, the negative test solution of saposhnikovia divaricata, the negative test solution of Polygonum multiflorum, and the negative test solution of Licorice prepared in step (3), 2 μL of the liquiritin reference solution prepared in step (4), 10 μL of the white peony reference medicinal material solution and the Polygonum multiflorum reference medicinal material solution prepared in step (5), and 1 μL of the saposhnikovia divaricata reference medicinal material solution are respectively spotted on the same silica gel G thin layer plate, developed with ethyl acetate-formic acid-glacial acetic acid-water (14:1:2:1), taken out, dried, sprayed with phosphomolybdic acid-sulfuric acid solution (take 2 g of phosphomolybdic acid, add 20 mL of water to dissolve, then slowly add 30 mL of sulfuric acid and mix well), heated at 105°C until the spots are clearly colored, and inspected under sunlight. In the test sample chromatogram, spots of the same color appear at the corresponding positions with the chromatograms of the licorice glycoside reference sample, the chromatograms of the Polygonum multiflorum reference medicinal material, and the chromatograms of the white peony reference medicinal material. The licorice negative test sample, the Polygonum multiflorum negative test sample, and the white peony negative test sample have no interference and have good specificity.

[0097] When inspected under ultraviolet light (365nm), in the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the glycyrrhizin reference sample; in the chromatogram of the siler control medicinal material, a main fluorescent spot of the same color appears at the corresponding position. There is no interference between the licorice-negative test sample and the siler negative test sample, and the specificity is good.

Claims

1. A thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi, characterized in that: It consists of the following steps: (1) Preparation of test solution Take Dangguiyinzi decoction, Dangguiyinzi fluid extract, Dangguiyinzi dry extract or Dangguiyinzi granules, first shake and extract with ethyl acetate-n-butanol mixed solution, then evaporate the extract to dryness, add methanol to dissolve the obtained residue, and use it as test solution 1; Take the test solution 1, add it to the alumina column, elute with methanol, collect the eluate, evaporate to dryness, and dissolve the residue with methanol as the test solution 2; (2) Preparation of reference solution Take ligustilide reference substance, 6-gingerol reference substance and liquiritin reference substance, and prepare ligustilide reference substance solution, 6-gingerol reference substance solution and liquiritin reference substance solution respectively; (3) Preparation of control medicinal material solution Take white peony root control medicinal material, Polygonum multiflorum control medicinal material and Saposhnikovia divaricata control medicinal material, add ethyl acetate-methanol to reflux respectively, filter, evaporate the filtrate to dryness, add methanol to dissolve the residue, add it to an alumina column, elute with methanol, collect the eluate, evaporate to dryness, and dissolve the residue in methanol to prepare white peony root control medicinal material solution, Polygonum multiflorum control medicinal material solution and Saposhnikovia divaricata control medicinal material solution respectively; (4) Thin layer chromatography detection The test solution 1 prepared in step (1), the ligustilide reference solution and the 6-gingerol reference solution prepared in step (2) are respectively spotted on the same silica gel G thin layer plate, developed with a toluene-ethyl acetate-formic acid system, taken out, air-dried, and examined under an ultraviolet lamp. In the chromatogram of the test sample, a fluorescent spot of the same color as that of the ligustilide reference is displayed at the position corresponding to that of the ligustilide reference. Then, the plate is sprayed with a vanillin sulfuric acid solution and heated until the spots are clearly colored. In the chromatogram of the test sample, a spot of the same color as that of the 6-gingerol reference is displayed at the position corresponding to that of the chromatogram of the 6-gingerol reference. The test solution 2 prepared in step (1), the liquiritin reference substance solution prepared in step (2), the white peony root reference medicinal material solution, the polygonum multiflorum reference medicinal material solution and the saposhnikovia divaricata reference medicinal material solution prepared in step (3) are respectively spotted on the same silica gel G thin layer plate, developed with an ethyl acetate-formic acid-glacial acetic acid-water system, taken out, air-dried, sprayed with a phosphomolybdic acid-sulfuric acid solution, heated until the spots are clearly colored, and examined under sunlight. In the chromatogram of the test sample, spots of the same color are shown at the corresponding positions of the chromatograms of the liquiritin reference substance, the white peony root reference medicinal material and the polygonum multiflorum reference medicinal material; in the chromatogram of the test sample, fluorescent spots of the same color are shown at the corresponding positions of the chromatograms of the liquiritin reference substance; and fluorescent main spots of the same color are shown at the corresponding positions of the chromatograms of the saposhnikovia divaricata control medicinal material.

2. The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi according to claim 1, characterized in that: The volume ratio of ethyl acetate to n-butanol in the ethyl acetate-n-butanol mixed solution in step (1) is 1-8:0.1-5.

3. The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi according to claim 1, characterized in that: In step (1), when preparing the sample 2, eluting with methanol and collecting the eluate, specifically, first eluting with methanol, discarding the eluate, and then eluting with 50-70% methanol, and collecting the eluate.

4. The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi according to claim 1, characterized in that: In step (2), the ligustilide reference substance solution is prepared using methanol as solvent, with each 1 mL of methanol containing 1 mg of ligustilide reference substance; In step (2), the 6-gingerol reference substance solution is prepared using methanol as solvent, and each 1 mL of methanol contains 0.5 mg of 6-gingerol reference substance; The liquiritin reference substance solution in step (2) uses methanol as solvent, and each 1 mL of methanol contains 1.0 mg of liquiritin reference substance.

5. The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi according to claim 1, characterized in that: The volume ratio of ethyl acetate to methanol in the ethyl acetate-methanol reflux system in step (3) is 1-19:0.1-5.

6. The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi according to claim 1, characterized in that: The mass volume ratio of the white peony root control medicinal material to ethyl acetate-methanol in step (3) is 0.5:30, in g / mL; The mass volume ratio of the Radix Polygoni Multiflori control medicinal material to ethyl acetate-methanol in step (3) is 0.1:30, in units of g / mL; The mass volume ratio of the Fangfeng reference medicinal material to ethyl acetate-methanol in step (3) is 1.0:30, in units of g / mL.

7. The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi according to claim 1, characterized in that: In step (3), the method of eluting with methanol and collecting the eluate is as follows: first eluting with methanol, discarding the eluate, then eluting with 50-70% methanol, and collecting the eluate.

8. The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi according to claim 1, characterized in that: The alumina used in the alumina column in step (1) or the alumina column in step (3) is one of basic alumina, neutral alumina or acidic alumina.

9. The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi according to claim 1, characterized in that: In the toluene-ethyl acetate-formic acid system of step (4), the volume ratio of toluene, ethyl acetate and formic acid is 20-40: 2-8: 0.1-2; In the ethyl acetate-formic acid-glacial acetic acid-water system of step (4), the volume ratio of ethyl acetate, formic acid, glacial acetic acid and water is 5-20: 0.1-2: 1-10: 1-10.

10. The thin layer chromatography identification method for simultaneously identifying multiple components of Danggui Yinzi according to claim 1, characterized in that: The wavelength of the ultraviolet lamp in step (4) is 365nm.

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