Method for identifying fructus kochiae in Azhen blood-nourishing oral liquid
By using D101 macroporous adsorption resin and thin-layer chromatography combined with diferosin saponin Ic control in Azhen Yangxue oral solution, the eluting and expansion agent was optimized, and Azhen Yangxue oral treatment in the prior art was solved. The lack of specificity of the identification method for the liquid dermatitis has achieved higher identification accuracy and product quality monitoring.
Patent Information
- Application Number
- CN202410258885.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-06-14
- Filing Date
- 2024-03-07
- Publication Date
- 2024-12-17
AI Technical Summary
The current identification method of Azhen Yangxue Oral Liquid is not very specific, and it is difficult to effectively identify whether the Dermatitis comes from the Dermatitis itself or other medicinal materials.
The samples were pretreated and purified by D101 type macroporous adsorption resin column, and identified by thin-layer chromatography. The saponin Ic of the ferroferon was used as a reference product, combined with the development agent of n-butanol-ethanol-water-glacial acetic acid, and optimized. The ratio of elution and developer increases the specificity of identification.
A thin-layer identification method with higher specificity is established, which can identify the leukemia in Azhen Yangxue oral liquid without negative interference, effectively monitor product quality, and avoid background interference and misjudgment in the prior art.
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Figure CN119147686A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of traditional Chinese medicine preparation detection, and particularly relates to a method for identifying Kochia scoparia seeds in Azhen Yangxue oral liquid. Background Art
[0002] The current quality standard of Azhen Yangxue Oral Liquid is the national drug standard WS-5004 (B-0004)-2014Z. The identification method of item (3) of the current quality standard of this variety is as follows: take 50ml of this product, add 2ml of hydrochloric acid, heat and reflux in a water bath for 1 hour, cool, add 50ml of ethanol, shake for 10 minutes, shake and extract with petroleum ether (60-90℃) twice, 20ml each time, combine the petroleum ether extracts, evaporate to dryness, add 1ml of ethanol to the residue to dissolve it, and use it as the test solution. Take another oleanolic acid reference substance, add ethanol to make a solution containing 1mg per 1ml, and use it as the reference substance solution. According to the thin layer chromatography method (Appendix VI B of Part I of the 2010 edition of the Chinese Pharmacopoeia), 10 μl of the test solution and 5 μl of the reference solution were respectively spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-glacial acetic acid (12:4:0.5) was used as the developing agent. After development, the plate was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C for about 5 minutes, and examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatogram of the reference substance.
[0003] The main active ingredients of Kochia scoparia are saponin compounds (mainly triterpenoid saponins). Although oleanolic acid is one of the active ingredients of Kochia scoparia, many other medicinal materials such as jujube and papaya also contain oleanolic acid. If oleanolic acid is used as a control for the thin layer identification method of Kochia scoparia in Azhen Yangxue Oral Liquid, it is difficult to determine whether oleanolic acid comes from Kochia scoparia or other medicinal materials. The specificity is poor and it cannot effectively identify Kochia scoparia in Azhen Yangxue Oral Liquid. The identification method of Kochia scoparia in the current quality standard is not specific. Summary of the invention
[0004] The invention provides a method for identifying Kochia scoparia in Azhen Yangxue oral liquid, which solves the problem that the identification method for Kochia scoparia in Azhen Yangxue oral liquid has low specificity.
[0005] In view of the current quality standard problem, the present invention provides a method for identifying Kochia scoparia in Azhen Yangxue oral liquid, and the technical solution adopted is as follows:
[0006] A method for identifying Kochia scoparia in Azhen Yangxue oral liquid comprises the following steps:
[0007] (1) Preparation of test solution:
[0008] Take Azhen Yangxue oral liquid and add it to D101 macroporous adsorption resin column, elute with 150ml water, discard the water, elute with 60% methanol, discard the eluate, elute with methanol, collect the eluate, evaporate to dryness, add methanol to dissolve the residue, and use it as the test solution;
[0009] (2) Preparation of reference solution:
[0010] Take Kochia scoparia saponin Ic reference substance, add methanol to make a solution containing 0.5 mg per 1 ml, as the reference substance solution;
[0011] (3) Thin layer chromatography identification:
[0012] Pipette 10-20 μl of the test solution and 3-10 μl of the reference solution, spot them separately on the same silica gel thin layer plate, use n-butanol-ethanol-water-glacial acetic acid (8:2:3-5:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, bake at 105°C until the spots are clear, and examine under sunlight to see whether the spots of the same color appear in the corresponding positions of the test sample chromatogram and the reference chromatogram.
[0013] Furthermore, the step (1) is:
[0014] Take 10ml-20ml of Azhen Yangxue Oral Liquid, add it to a D101 macroporous adsorption resin column with an inner diameter of 1.0cm and a column height of 20cm, elute with 150ml of water, discard the water, elute with 150ml of 60% methanol, discard the eluate, elute with 150ml of methanol, collect the eluate, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution;
[0015] Preferably, the silica gel thin layer plate in step (3) is GF 254 Thin layer board.
[0016] Preferably, the developing agent is n-butanol-ethanol-water-glacial acetic acid in a ratio of 8:2:3:1.
[0017] Beneficial effects: By using the Kochia scoparia saponin Ic reference substance as a control, optimizing the sample pretreatment method of Azhen Yangxue oral liquid and investigating the durability of the method, a Kochia scoparia thin layer identification method with better specificity was established. Compared with the existing technology, this method has no negative interference and strong specificity. It can be used as a qualitative detection method for Azhen Yangxue oral liquid and can effectively monitor the quality of Azhen Yangxue oral liquid. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1Comparison chart of different eluents, where 1-negative control sample solution (30% methanol eluent), 2-test solution (30% methanol eluent), 3-negative control sample solution (60% methanol eluent), 4-test solution (60% methanol eluent), 5-negative control sample solution (80% methanol eluent), 6-test solution (80% methanol eluent), 7-negative control sample solution (methanol eluent), 8-test solution (methanol eluent), 9-control medicinal material solution
[0019] Figure 2 Comparison chart of different developing agents, where developing agent A is n-butanol-ethanol-glacial acetic acid (4:1:2:1), developing agent B is n-butanol-acetone-water (5:1:1), and developing agent C is n-butanol-ethanol-water (8:2:3), where 1-test solution (80% methanol eluent), 2-test solution (methanol eluent), 3-negative control sample solution (methanol eluent), 4-control medicinal material solution
[0020] Figure 3 Optimized developer chromatogram, where 1-test solution (80% methanol eluent), 2-test solution (methanol eluent), 3-negative control sample solution (methanol eluent), 4-control medicinal material solution
[0021] Figure 4 Chromatograms of methods 1-5 of Example 1, wherein 1-test solution 1, 2-test solution 2, 3-test solution 3, 4-test solution 4, 5-test solution 5, 6-test solution 6, 7-control medicinal material solution, 8-test solution 8, 9-test solution 9, 10-test solution 10, 11-test solution 11, 12-test solution 12.
[0022] Figure 5 Chromatogram of method 6-7 of Example 1, wherein 1-test solution 13, 2-test solution 14, 3-control medicinal material solution.
[0023] Figure 6 Example 1 Chromatograms of different thin layer plates, 1-negative sample lacking Kochia scoparia (20 μl), 2-kochia scoparia saponin Ic (5 μl), 3-control medicinal material solution (5 μl), 4-test solution 20221207 (10 μl), 5-test solution 20221207 (15 μl), 6-test solution 20221207 (20 μl), 7-test solution 20221208 (20 μl), 8-test solution 20221209 (20 μl)
[0024] Figure 7Example 1 Adaptability chromatogram, wherein 1-Azhen Yangxue Oral Liquid 20210503, 2-Azhen Yangxue Oral Liquid 20210504, 3-Azhen Yangxue Oral Liquid 20210505, 4-Azhen Yangxue Oral Liquid 20210601, 5-kochia scoparia saponin Ic, 6-control medicinal material solution, 7-Azhen Yangxue Oral Liquid 20210701, 8-Azhen Yangxue Oral Liquid 20210702, 9-Azhen Yangxue Oral Liquid 20211101, 10-Azhen Yangxue Oral Liquid 20211103, 11-Azhen Yangxue Oral Liquid 20211201.
[0025] Figure 8 Example 1 Adaptability chromatogram, wherein 1-Azhen Yangxue Oral Liquid 20220903, 2-Azhen Yangxue Oral Liquid 20220502, 3-Azhen Yangxue Oral Liquid 20220704, 4-Azhen Yangxue Oral Liquid 20211203, 5-negative control sample solution, 6-kochia scoparia saponin Ic, 7-control medicinal material solution, 8-Azhen Yangxue Oral Liquid 20220802, 9-Azhen Yangxue Oral Liquid 20211205, 10-Azhen Yangxue Oral Liquid 20221002, 11-Azhen Yangxue Oral Liquid 20220904.
[0026] Fig. 9 Example 2 chromatogram, wherein 1-test solution 20221207, 2-test solution 20221208, 3-control medicinal material solution 1, 4, control medicinal material solution 2, 5-reference solution, 6-test solution 20221209
[0027] Fig.10 Chromatogram of Comparative Example 1, wherein 1-method (1) test solution, 2-method (2) test solution, 3-method (3) test solution, 4-method (4) test solution, 5-method (5) test solution, 6-kochia scoparia control medicinal material solution DETAILED DESCRIPTION
[0028] The Azhen Yangxue oral liquid used in the following examples and comparative examples is the Azhen Yangxue oral liquid listed in the national drug standard WS-5004 (B-0004) -2014Z. In the examples or comparative examples, a negative control sample is set (according to the [prescription] under the Azhen Yangxue oral liquid, other medicinal materials lacking Kochia scoparia are weighed to prepare a negative control sample lacking Kochia scoparia, and a negative sample solution lacking Kochia scoparia is prepared in the same way as the test solution preparation method, and an equal volume of the sample solution is applied to a thin layer plate for thin layer identification).
[0029] Example 1 Method Investigation
[0030] 1. Experimental Materials
[0031] Samples: There are 20 batches in total, with batch numbers 20210503, 20210504, 20210505, 20210601, 20210701, 20210702, 20211101, 20211103, 20211201, 20211203, 20211205, 20220502, 20220704, 20220802, 20220903, 20220904, 20221002, 20221207, 20221208, and 20221209, all of which are produced by Jiuzhitang Co., Ltd.
[0032] Reference substances and reference medicinal materials: Kochia scoparia saponin Ic (batch number: 111723-201605, China Food and Drug Inspection Institutes), Kochia scoparia reference medicinal materials (batch number: 121148-200803, China Food and Drug Inspection Institutes).
[0033] High performance thin layer chromatography plate GF 254 , batch number: HX90224454; high-performance thin layer chromatography plate G, batch number: HX72407353. All are produced by Merck, Germany. They are dried at 105℃ for 1 hour before use, placed in a dryer to cool, and used for standby. The development cylinders are all double-slotted, the development method is upward development, and pre-saturation for 15 minutes.
[0034] The water was purified water, and the other reagents were of analytical grade.
[0035] 2. Experimental Methods
[0036] 2.1 Investigation of pre-treatment conditions
[0037] Since saponin compounds are the main chemical components of Kochia scoparia, based on the previous investigation, macroporous adsorption resin was used for sample pretreatment and purification, and chloroform-methanol-water development system was used for development. The specific method is as follows:
[0038] Take 10ml of Azhen Yangxue Oral Liquid (batch number: 20221207), add 40ml of methanol, ultrasonicate for 5 minutes, filter, evaporate the filtrate to dryness, add 30ml of water to dissolve, pass the aqueous solution through a D101 macroporous adsorption resin column (inner diameter 1.0cm, column length 20cm, macroporous adsorption resin weight about 15g) and elute with 150ml of water, and discard the aqueous solution. Sequentially use 30% methanol, 60% methanol, 80% methanol, and methanol 100ml each for elution, collect each eluate, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution. Take another 0.5g of Kochia scoparia control medicinal material, add 10ml of methanol, ultrasonicate for 30 minutes, filter, and use the filtrate as the control medicinal material solution. Take a negative control sample lacking Kochia scoparia again, and prepare a negative control solution according to the preparation method of the test solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 10 μl of each test solution and negative control solution and 5 μl of control medicinal material solution were taken and spotted on the same silica gel GF 254 On the thin layer plate, use the lower layer solution of chloroform-methanol-water (13:7:2) placed at 10℃ for 12 hours as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under sunlight. Figure 1 .
[0039] pass Figure 1 The results show that the 80% methanol eluent and methanol eluent of the test solution show spots of the same color at the corresponding positions of the chromatogram of the control medicinal material solution, without background interference, and the inspection effect is good. However, the spot separation is average. Considering that the target spots should be eluted as completely as possible, the elution steps will be simplified in the subsequent method research, that is, elution with 60% methanol and then elution with methanol again.
[0040] 2.2 Investigation of different developing agents
[0041] Take the test solution (80% methanol eluent and methanol eluent), negative control sample (methanol eluent) and control medicinal material solution under 2.1, and take 10μ1 of the test solution and negative control solution and 5μ1 of the control medicinal material solution, and apply them to the same silica gel GF 254 On the thin layer plate, n-butanol-ethanol-glacial acetic acid (4:1:2:1), n-butanol-acetone-water (5:1:1) and n-butanol-ethanol-water (8:2:3) were used as the developing agent, developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under sunlight. The results are shown in Figure 2 .
[0042] pass Figure 2The results show that n-butanol-ethanol-water (8:2:3) as a developing agent has a relatively good development effect, but the target spot formation is still poor. Therefore, on this basis, an appropriate amount of glacial acetic acid is added, that is, n-butanol-ethanol-water-glacial acetic acid (8:2:3:1) is used as a developing agent to improve the target spot formation, and the results are good. For details, see Figure 3 .
[0043] 2.3 Investigation of different elution solvents and volumes
[0044] On the basis of 2.1 and 2.2, the effects of different elution solvents and volumes of macroporous adsorption resin on the experimental results were further investigated. At the same time, the feasibility of removing impurities by macroporous adsorption resin after the pretreatment operation of adding water and diluting the sample without methanol ultrasonic extraction was investigated. Therefore, the sample with batch number 20221207 was taken and the test solution was prepared according to the following method:
[0045] Method 1: Take 10ml of Azhen Yangxue oral liquid, add 40ml of methanol, ultrasonicate for 5 minutes, filter, evaporate the filtrate, add 30ml of water to the residue to dissolve it, pass the aqueous solution through a D101 macroporous adsorption resin column (inner diameter 1.0cm, column length 20cm, macroporous adsorption resin weight about 15g), elute with 150ml of water, discard the aqueous solution, elute with 150ml of 30% methanol, collect the eluate, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, as the test solution 1; elute with 150ml of 80% methanol, collect the eluate, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, as the test solution 8.
[0046] Method 2: Take 10ml of Azhen Yangxue oral liquid, add 40ml of methanol, ultrasonicate for 5 minutes, filter, evaporate the filtrate, add 30ml of water to the residue to dissolve it, pass the aqueous solution through a D101 macroporous adsorption resin column (inner diameter 1.0cm, column length 20cm, macroporous adsorption resin weight about 15g), elute with 200ml of water, discard the aqueous solution, then elute with 200ml of 30% methanol, collect the eluate, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, as the test solution 2; then elute with 200ml of 80% methanol, collect the eluate, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, as the test solution 9.
[0047] Method 3: Take 10ml of Azhen Yangxue oral liquid, add 40ml of methanol, ultrasonicate for 5 minutes, filter, evaporate the filtrate, add 30ml of water to dissolve the residue, pass the aqueous solution through a D101 macroporous adsorption resin column (inner diameter 1.0cm, column length 20cm, macroporous adsorption resin weight about 15g), elute with 150ml of water, discard the aqueous solution, elute with 150ml of 60% methanol, and then add 50ml of 60% methanol for elution, collect the eluate, evaporate to dryness, add 1ml of methanol to dissolve the residue, as test solution 3 and 4; elute with 100ml of methanol, collect the eluate, evaporate to dryness, add 1ml of methanol to dissolve the residue, as test solution 10.
[0048] Method 4: Take 10ml of Azhen Yangxue oral liquid, add 40ml of methanol, ultrasonicate for 5 minutes, filter, evaporate the filtrate, add 30ml of water to the residue to dissolve it, pass the aqueous solution through a D101 macroporous adsorption resin column (inner diameter 1.0cm, column length 20cm, macroporous adsorption resin weight about 15g), elute with 150ml of water, discard the aqueous solution, then elute with 200ml of 60% methanol, collect the eluate, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, as the test solution 5; then elute with 150ml of methanol, collect the eluate, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, as the test solution 11.
[0049] Method 5: Take 10ml of Azhen Yangxue oral liquid, add 30ml of water, shake well, add to D101 macroporous adsorption resin column (inner diameter 1.0cm, column length 20cm, macroporous adsorption resin weight about 15g), elute with 150ml of water, discard the water, elute with 200ml of 60% methanol, collect the eluate, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, as the test solution 6; elute with 200ml of 80% methanol, collect the eluate, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, as the test solution 12.
[0050] Pipette 10μ1 of each test solution and 5μ1 of the control medicinal material solution under 2.1 and apply them to the same silica gel GF 254 On the thin layer plate, use n-butanol-ethanol-water-glacial acetic acid (8:2:3:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under sunlight. Figure 4 .
[0051] according to Figure 5 The results show that the sample was eluted with 150ml of water and then 150ml of 60% methanol after being adsorbed by D101 macroporous resin, which can effectively remove the background interference. After eluting with 150ml of methanol, the target spot can be well eluted and inspected.
[0052] 2.4 Investigation of sample column loading method
[0053] Based on Item 2.3, the method of sample column loading was further studied to investigate the feasibility of simplifying the pretreatment steps.
[0054] Therefore, take the sample with batch number 20221207 and prepare the test solution as follows:
[0055] Method 6: Take 10 ml of Azhen Yangxue oral liquid and add it directly to a D101 macroporous adsorption resin column (inner diameter 1.0 cm, column length 20 cm, macroporous adsorption resin weight about 15 g), elute with 150 ml of water, discard the water, elute with 150 ml of 60% methanol, discard the 60% methanol eluate, and elute with 150 ml of methanol, collect the methanol eluate, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, and use the test solution 13.
[0056] Method 7: Take 10ml of Azhen Yangxue oral liquid, add 40ml of methanol, ultrasonicate for 5 minutes, filter, evaporate the filtrate, add 30ml of water to dissolve the residue, pass the aqueous solution through a D101 macroporous adsorption resin column (inner diameter 1.0cm, column length 20cm, macroporous adsorption resin weight about 15g), elute with 150ml of water, discard the aqueous solution, then elute with 150ml of 60% methanol, discard the 60% methanol eluate, continue to elute with 150ml of methanol, collect the methanol eluate, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use the test solution 14.
[0057] Pipette 10μ1 of each test solution and 5μ1 of the control medicinal material solution under 2.1 and apply them to the same silica gel GF 254 On the thin layer plate, use n-butanol-ethanol-water-glacial acetic acid (8:2:3:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under sunlight. Figure 5 .
[0058] according to Figure 5 The results show that the target spots can be better viewed when the sample is directly loaded onto the column without being diluted with water, so method 6 is selected.
[0059] 2.5 Investigation of different thin layer plates and sample solution spotting amounts
[0060] Take the test sample solution of method 6 under item 2.4 and the reference medicinal material solution and reference substance solution under item 2.1 and apply them to silica gel G and silica gel GF respectively. 254 On the thin layer plate, the effect of different sample solution spotting volumes (10μl, 15μl, 20μl) on the results was investigated simultaneously. Figure 6 .
[0061] according to Figure 6The results show that when the sample solution is 20 μl, the silica gel GF 254 When the plate is thin, the inspection effect is better.
[0062] 2.5 Adaptability assessment
[0063] Using Method 6 under Item 2.4, a total of 17 batches of samples were tested, and the results all met the requirements. Figure 7 , 8 .
[0064] Considering that the Kochia seeds medicinal material is decocted in water in the production process of this variety, in order to improve the specificity of the method, the pretreatment method of the Kochia seeds control medicinal material is further optimized. Referring to the production process of this variety, the Kochia seeds control medicinal material is first decocted in water and then concentrated to an appropriate amount, and then prepared according to the preparation method of the test solution for investigation and research. The specific method is as follows:
[0065] Preparation of test solution: Take 10ml of the product and add it to a D101 macroporous adsorption resin column (inner diameter 1.0cm, column height 20cm), elute with 150ml of water, discard the water, then elute with 150ml of 60% methanol, discard the eluent, and continue to elute with 150ml of methanol, collect the eluent, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution.
[0066] Preparation of control medicinal material solution (tentative method under item 2.1): Take 0.5 g of Kochia scoparia reference medicinal material, add 10 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 5 ml of methanol to dissolve the residue, and use it as control medicinal material solution 1.
[0067] Preparation of control medicinal material solution (optimized method): Take 0.5 g of Kochia scoparia reference medicinal material, add 30 ml of water, heat and reflux for 30 minutes, filter, concentrate the filtrate to about 10 ml, and prepare control medicinal material solution 2 according to the preparation method of the test solution.
[0068] Preparation of reference solution: Take Kochia scoparia saponin Ic reference substance and add methanol to prepare a solution containing 0.5 mg per 1 ml as the reference solution.
[0069] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 20 μl of the test solution, 5 μl of the reference medicinal material solution and the reference substance solution were taken and spotted on the same silica gel GF 254 On the thin layer plate, use n-butanol-ethanol-water-glacial acetic acid (8:2:3:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under sunlight. Fig. 9 .
[0070] Depend on Fig. 9The results show that the Kochia scoparia reference medicinal material prepared by the optimized method shows the same color main spot (i.e., Kochia scoparia saponin Ic) at the corresponding position of the chromatogram of the test sample. Therefore, only the Kochia scoparia saponin Ic reference substance is retained in the detection method, the Kochia scoparia reference medicinal material is deleted, and the other detection conditions remain unchanged.
[0071] Example 2
[0072] A method for identifying Kochia scoparia in Azhen Yangxue oral liquid comprises the following steps:
[0073] (1) Preparation of test solution:
[0074] Take 10 ml of Azhen Yangxue oral liquid, add it to a D101 macroporous adsorption resin column with an inner diameter of 1.0 cm and a column height of 20 cm, elute with 150 ml of water, discard the water, elute with 150 ml of 60% methanol, discard the eluate, elute with 150 ml of methanol, collect the eluate, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as the test solution;
[0075] (2) Preparation of reference solution:
[0076] Take Kochia scoparia saponin Ic reference substance, add methanol to make a solution containing 0.5 mg per 1 ml, as the reference substance solution;
[0077] (3) Thin layer chromatography identification:
[0078] Pipette 10-20μ1 of the test solution and 3-10μ1 of the reference solution and apply them to the same silica gel GF. 254 On the thin layer plate, use n-butanol-ethanol-water-glacial acetic acid (8:2:3:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, bake at 105℃ until the spots are clear, and check under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the reference sample. Fig. 9 .
[0079] Comparative Example 1
[0080] Take the sample with batch number 20221207 and prepare the test solution as follows:
[0081] Method (1): Take 50 ml of Azhen Yangxue oral liquid, shake and extract it with water-saturated n-butanol for 3 times, 30 ml each time, combine the n-butanol liquid, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution 1.
[0082] Method (2): Take 10 ml of Azhen Yangxue oral liquid, add 30 ml of water-saturated n-butanol, ultrasonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution 2.
[0083] Method (3): Take 10 ml of Azhen Yangxue oral liquid, add 40 ml of methanol, ultrasonicate for 5 minutes, filter, evaporate the filtrate to dryness, add 10 ml of water to dissolve the residue, then shake and extract with water-saturated n-butanol for 3 times, 30 ml each time, combine the n-butanol solutions, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution 3.
[0084] Method (4): Take 10 ml of Azhen Yangxue oral liquid, add 40 ml of ethanol, ultrasonicate for 5 minutes, filter, evaporate the filtrate to dryness, add 10 ml of water to dissolve the residue, then shake and extract with water-saturated n-butanol for 3 times, 30 ml each time, combine the n-butanol solutions, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution 4.
[0085] Method (5): Take 10 ml of Azhen Yangxue oral liquid, add 40 ml of methanol, ultrasonicate for 5 minutes, filter, and extract the filtrate with ethyl acetate for 3 times, 30 ml each time, combine the ethyl acetate solutions, evaporate to dryness, and add 1 ml of methanol to dissolve the residue, which is used as the test solution 5.
[0086] Preparation of control medicinal material solution: Take 0.5 g of Kochia scoparia reference medicinal material, add 10 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 5 ml of methanol to the residue to dissolve it, and use it as the control medicinal material solution.
[0087] Pipette 10μ1 of the test solution and 5μ1 of the control drug solution and apply them to the same silica gel GF. 254 On the thin layer plate, use the upper layer solution of n-butanol-water-glacial acetic acid (4:5:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ for about 5 minutes, and examine under sunlight. The results are shown in Figure 2. Fig.10 .
[0088] pass Fig.10 The results show that the test solution of methods (1) to (4) does not show spots of the same color at the corresponding position of the chromatogram of the control medicinal material solution, and the separation effect is poor. The test solution of method (5) shows spots of the same color at the corresponding position of the chromatogram of the control medicinal material solution, and the spot separation is good, but the background interference is relatively serious, which affects the inspection effect, and the development time is relatively long (about 2 hours), which affects the detection efficiency.
Claims
1. A method for identifying Kochia scoparia in Azhen Yangxue oral liquid, characterized in that: The steps include: (1) Preparation of test solution: Take Azhen Yangxue oral liquid and add it to D101 macroporous adsorption resin column, elute with 150ml water, discard the water, elute with 60% methanol, discard the eluate, elute with methanol, collect the eluate, evaporate to dryness, add methanol to dissolve the residue, and use it as the test solution; (2) Preparation of reference solution: Take Kochia scoparia saponin Ic reference substance, add methanol to make a solution containing 0.5 mg per 1 ml, as the reference substance solution; (3) Thin layer chromatography identification: Pipette 10-20 μl of the test solution and 3-10 μl of the reference solution, spot them separately on the same silica gel thin layer plate, use 8:2:3-5:1 n-butanol-ethanol-water-glacial acetic acid as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, bake at 105°C until the spots are clear, and examine under sunlight to see if there are spots of the same color in the test sample chromatogram at the corresponding position in the reference chromatogram.
2. The method for identifying Kochia scoparia in Azhen Yangxue oral liquid according to claim 1, characterized in that: The step (1) is: Take 10ml~20ml of Azhen Yangxue oral liquid, add it to a D101 macroporous adsorption resin column with an inner diameter of 1.0cm and a column height of 20cm, elute with 150ml of water, discard the water, elute with 150ml of 60% methanol, discard the eluate, and elute with 150ml of methanol. Collect the eluate, evaporate it to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution.
3. The method for identifying Kochia scoparia in Azhen Yangxue oral liquid according to claim 1, characterized in that: The developing solvent is 8:2:3:1 n-butanol-ethanol-water-glacial acetic acid.
4. The method for identifying Kochia scoparia in Azhen Yangxue oral liquid according to claim 1, characterized in that: The silica gel thin layer plate in step (3) is GF 254 Thin layer board.