CN111308007A - Quality control method of artemisia apiacea granules - Google Patents
Quality control method of artemisia apiacea granules Download PDFInfo
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- CN111308007A CN111308007A CN202010181667.1A CN202010181667A CN111308007A CN 111308007 A CN111308007 A CN 111308007A CN 202010181667 A CN202010181667 A CN 202010181667A CN 111308007 A CN111308007 A CN 111308007A
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- 238000003908 quality control method Methods 0.000 title claims abstract description 30
- 235000011570 Artemisia caruifolia var apiacea Nutrition 0.000 title claims abstract description 28
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- 239000008187 granular material Substances 0.000 title claims abstract description 26
- ZFHSKBJBODQVBX-AXTRIDKLSA-N Pechueloic acid Chemical compound C[C@H]1CC[C@@H](C(=C)C(O)=O)CC2=C(C)C(=O)C[C@@H]12 ZFHSKBJBODQVBX-AXTRIDKLSA-N 0.000 claims abstract description 118
- CRDNMYFJWFXOCH-YPKPFQOOSA-N (3z)-3-(3-oxo-1h-indol-2-ylidene)-1h-indol-2-one Chemical compound N/1C2=CC=CC=C2C(=O)C\1=C1/C2=CC=CC=C2NC1=O CRDNMYFJWFXOCH-YPKPFQOOSA-N 0.000 claims abstract description 50
- 238000004809 thin layer chromatography Methods 0.000 claims abstract description 30
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- CRDNMYFJWFXOCH-UHFFFAOYSA-N isoindigotin Natural products N1C2=CC=CC=C2C(=O)C1=C1C2=CC=CC=C2NC1=O CRDNMYFJWFXOCH-UHFFFAOYSA-N 0.000 claims abstract description 25
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- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 claims description 9
- 229960004191 artemisinin Drugs 0.000 claims description 9
- CFZLNSBZTUMWNS-UHFFFAOYSA-N benzene;chloroform;propan-2-one Chemical compound CC(C)=O.ClC(Cl)Cl.C1=CC=CC=C1 CFZLNSBZTUMWNS-UHFFFAOYSA-N 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 5
- 239000000377 silicon dioxide Substances 0.000 claims description 5
- 239000013074 reference sample Substances 0.000 claims description 3
- 235000003092 Artemisia dracunculus Nutrition 0.000 claims description 2
- 240000001851 Artemisia dracunculus Species 0.000 claims description 2
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 57
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
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- INCWELKXTZCRSA-UHFFFAOYSA-N ethyl acetate;methanol;hydrate Chemical compound O.OC.CCOC(C)=O INCWELKXTZCRSA-UHFFFAOYSA-N 0.000 description 1
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- 229940126680 traditional chinese medicines Drugs 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/74—Optical detectors
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- G01N30/94—Development
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- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/95—Detectors specially adapted therefor; Signal analysis
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a quality control method of Artemisia apiacea granules, which revises a thin-layer chromatography identification method of rupestonic acid, (R, S) -goitrin and indirubin, adds a high performance liquid chromatography determination method of the content of rupestonic acid which is a characteristic active ingredient of rupestonic acid, can more scientifically and effectively control the product quality and ensure the curative effect. The characteristic active ingredients of the three medicinal materials in the medicament are identified by adopting thin-layer chromatography, the sample treatment method is simple and convenient, the separation degree is good, and the spots are clear; the high performance liquid chromatography is used for measuring the content of the rupestonic acid in the medicine, and has the advantages of simple operation, strong specificity, good repeatability and high accuracy. The quality control method of the artemisia apiacea granules is a quality control method which has more comprehensive and complete detection items, more scientific and reasonable quality control indexes and simpler and more convenient operation, can further improve the quality level of products and ensure the curative effect.
Description
Technical Field
The invention belongs to the technical field of quality control of veterinary Chinese medicinal preparations, and particularly relates to a quality control method of Artemisia apiacea Hance granules.
Background
The prescription of the artemisia apiacea granules is derived from 'Compound Achillea alpina granules' of the Lung system of the national Chinese patent drug standards compilation department of the State drug administration, and the standard number is WS-11137(ZD-1137) -2002 ]. The formula of the product comprises: 240g of alpine yarrow herb, 240g of isatis root and 240g of dyers woad leaf; the preparation process comprises the following steps: decocting the above three medicinal materials in water for 2 times, each time for 2 hr, filtering, mixing filtrates, concentrating the filtrate to obtain fluid extract, adding equal volume of ethanol, standing for 24 hr, collecting supernatant, recovering ethanol and concentrating to obtain soft extract, adding appropriate amount of sucrose and dextrin, mixing, wet granulating, drying, and making into 1000 g.
In the prescription composition, Artemisia rupestris L is a plant of Artemisia of Compositae, and the herb is used as a medicine, and is currently collected in the first book of drug standards and traditional Chinese medicines of Ministry of health. The medicinal material is recorded in Bencao gang mu Shi Yi of Zhao Zhimin in Qing Dynasty of China at first, is produced in Xinjiang alone in China, and has a long history of folk medication. Isatis root (Radix Isatidis) is the dried root of Isatis Indigotica Fort of Cruciferae, and is recorded in Shen nong Ben Cao Jing. Folium Isatidis (Folium Isatidis) is dried leaf of Isatis Indigotica Fort of Cruciferae. Both ban lan Gen and Da Qing Ye are recorded in 2015 edition of Chinese veterinary pharmacopoeia.
The quality standard with the original number of WS-11137(ZD-1137) -2002 is used for identifying the components of isatis root and dyers woad leaf in the prescription, namely indigo and indirubin, by thin-layer chromatography, but spots in a chromatogram are unclear, and even no spots are displayed at corresponding positions of the indigo identification chromatogram; the raw quality standard also identifies the artemisia rupestris in the prescription by thin-layer chromatography, but the identification reference substance selected by the method is an artemisia rupestris reference medicinal material, the comparison medicinal material is influenced by the production place, the harvesting time and the like compared with the reference substance, the uniformity of batches cannot be ensured, the extraction and treatment procedures of the reference medicinal material are complicated, the interference of identification maps is large, and the identification effect is poor; the original quality standard has no content determination item, and the characteristic active ingredients of the isatis root are not identified according to spring, which is not beneficial to the quality control. At present, a plurality of documents disclose identification of artemisia rupestris and content determination methods of an artemisia rupestris characteristic active ingredient, namely artemisia rupestris keto acid, in the artemisia rupestris granule prescription, but the disclosed methods have insufficient extraction and purification degree of a test solution, and have the defects of many chromatographic impurity peaks, strong interference, poor separation degree and unstable peak output time during determination. The identification and content measurement of rupestonic acid in alpine yarrow are respectively disclosed in the documents "research on quality standard of alpine yarrow in Xinjiang (Laiyu Guli, Abudura et al, medicine guide, volume 30, No. 9, 2011) and" determination of content of rupestonic acid in alpine yarrow in Xinjiang by high performance liquid chromatography "(Macheng et al, Shizhen national medicine, volume 19, No. 4, 2008), but the method is only suitable for determination of medicinal material rupestonic acid and is not suitable for traditional Chinese medicine preparation with complex components; the document, "research on quality standards of compound herba Achillea Wilsonianae granule" (Limon et al, journal of traditional Chinese medicine, Vol. 21, No. 11, 2003) discloses a quality control method of compound herba Achillea Wilsonianae granule, but the identification method has no significant difference from the original standard, only the content determination of auxiliary drugs folium Isatidis and radix Isatidis in the prescription is increased, and the content determination of main drug herba Achillea Wilsonianae in the prescription is not increased; the document "content of rupestonic acid in compound rupestris capsule" by high performance liquid chromatography (zhangxuefeng et al, military science, vol. 34, No. 4, 2012) discloses a high performance liquid chromatography method for measuring rupestonic acid in compound rupestris capsule, but it is found that temperature has a great influence on the measurement, the retention time is extremely unstable, and the repeatability is poor.
In conclusion, the existing preparation quality control method only establishes a partial identification method for medicinal materials in a prescription, does not establish an identification method for characteristic active ingredients of a main medicament, particularly has no content measurement method, and has the defects of lacking comprehensiveness of test items and unsatisfactory method effects. Therefore, in order to ensure the safe and effective clinical use and stable quality of the artemisia apiacea granules, a more comprehensive and more complete quality control method needs to be established.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a quality control method of Artemisia apiacea granules, which is an improvement on the basis of the original quality standard, revises a thin-layer chromatography identification method of rupestonic acid, (R, S) -goitrin and indirubin, and adds a high performance liquid chromatography determination method of the content of rupestonic acid which is a characteristic active ingredient of rupestonic acid, so that the product quality can be more scientifically and effectively controlled, and the curative effect is ensured.
The appendices in the invention refer to corresponding appendices in the second part of the Chinese animal pharmacopoeia 2015 year edition. .
The specific technical scheme is as follows:
a quality control method of Artemisia apiacea granules comprises identifying rupestonic acid, R, S-goitrin and indirubin; also comprises the content measurement of rupestonic acid;
wherein, the identification of rupestonic acid, R, S-goitrin and indirubin adopts thin layer chromatography; the content of rupestonic acid is determined by high performance liquid chromatography.
Further, in the identification of rupestonic acid: using methanol or ethanol as solvent, respectively dropping the sample solution and the reference solution on the same silica gel GF254On the thin layer plate, petroleum ether-ethyl acetate-glacial acetic acid is used as a developing agent;
further, in the identification of R, S-goitrin: the test sample takes dichloromethane or trichloromethane as a solvent, and the reference sample takes methanol or ethanol as a solvent; respectively dropping the test solution and the reference solution on the same silica gel GF254Petroleum ether-ethyl acetate is used as a developing agent on the thin-layer plate;
further, in the identification of indirubin: using trichloromethane as solvent, respectively dropping the sample solution and the reference solution on the same silica gel G thin layer plate, and using benzene-trichloromethane-acetone as developing agent.
Developing the three test samples and the reference sample, taking out the silica gel GF254 thin-layer plate, air drying, and inspecting under an ultraviolet lamp; taking out the silica gel G thin-layer plate, drying in the air and inspecting. If the inspection result is: in the chromatogram of the test sample, at the position corresponding to the chromatogram of the reference substance, the silica gel GF254 thin-layer plate shows fluorescent spots, and the silica gel G thin-layer plate shows spots with the same color as the reference substance, so that the sample to be tested of the Artemisia apiacea particles respectively contains rupestonic acid, R, S-goitrin or indirubin.
Wherein, in the identification of rupestonic acid: the volume ratio of the developing solvent oil ether to the ethyl acetate to the glacial acetic acid is (10-30): (5-15): 1, the temperature of petroleum ether is 30-90 ℃;
wherein, in the identification of R, S-goitrin: the volume ratio of the developing solvent petroleum ether to the ethyl acetate is (1-5): 1, the temperature of petroleum ether is 30-90 ℃;
wherein, in the identification of indirubin: the volume ratio of the developing agent benzene-trichloromethane-acetone is (2-9): (1-8): 1.
wherein, in the identification of rupestonic acid, the preparation method of the test solution is as follows: grinding the artemisia isatis leaf particles into powder, adding a solvent, wherein the dosage ratio of the artemisia isatis leaf particle powder to the solvent is 1 g: (4-16) mL; performing ultrasonic treatment for 10-60 min, filtering, taking filtrate, evaporating to dryness, adding a solvent again to dissolve evaporated substances, wherein the ratio of the arteannuin particle powder to the solvent added again is 1 g: (0.2-1) mL to obtain a test solution;
wherein, in the identification of R, S-goitrin, the preparation method of the test solution is as follows: grinding the artemisia isatis leaf particles into powder, adding water to dissolve the powder, adding a solvent, shaking and extracting for 1-4 times, wherein the dosage ratio of the artemisia isatis leaf particle powder to the water is 1 g: (2-10) mL, wherein the dosage ratio of the arteannuin particle powder to the solvent used in each extraction is 1 g: (2-10) mL; centrifuging the solvent layer, combining the solvent supernatant, and concentrating until the ratio of arteannuin particle powder to concentrated solution is 1 g: (0.1-0.5) mL to obtain a test solution;
in the identification of indirubin, the preparation method of the test solution comprises the following steps: grinding the artemisia isatis leaf particles into powder, adding water to dissolve the powder, adding trichloromethane, shaking and extracting for 1-4 times, wherein the dosage ratio of the artemisia isatis leaf particle powder to the water is 2 g: (3-10) mL, wherein the dosage ratio of the arteannuin particle powder to the chloroform used for each extraction is 1 g: (2-10) mL; centrifuging the chloroform layer, combining the chloroform clarified liquid, evaporating to dryness, adding chloroform again to the evaporated matter for dissolving, wherein the ratio of tarragon green particle powder to chloroform added again is 1 g: (0.05-0.25) mL to obtain a test solution.
Wherein, in the identification of rupestonic acid, the concentration of the reference solution is 0.05-0.15 mg/mL-1The sample application amount is 5 mul;
wherein, in the identification of R, S-goitrin, the concentration of the reference substance solution is 0.3-0.9 mg/mL-1The sample amount is 10 mul;
wherein, in the identification of the indirubin, the concentration of the reference substance solution is 0.05-0.15 mg/mL-1The amount of the spot was 10. mu.l.
Further, taking rupestonic acid as a reference substance in the determination of the content of rupestonic acid; octadecylsilane chemically bonded silica is used as a filling agent; methanol-acetonitrile-0.4% phosphoric acid solution is used as a mobile phase; the detection wavelength is 221-265 nm.
Wherein in the mobile phase, the volume ratio of methanol-acetonitrile-0.4% phosphoric acid solution is (11-68): (5-45): (16-92).
Wherein, in the content measurement of rupestonic acid, the preparation method of the test sample comprises the following steps:
grinding the artemisia apiacea particles into powder, adding methanol or ethanol as a solvent, and carrying out ultrasonic treatment for 10-50 min, wherein the dosage ratio of the artemisia apiacea particle powder to the solvent is 6 g: (20-80) mL; supplementing with methanol or ethanol, reducing weight, shaking, filtering, and collecting filtrate to obtain test solution.
Preferably, after the ultrasonic treatment, the reaction system is cooled to below 25 ℃, and then methanol or ethanol is used for complementing the weight loss.
Wherein, in the content measurement of rupestonic acid, the reference substance is methanol or ethanol solution with the concentration of 13-74 mug.mL < -1 >.
In the case where the concentration is not specified, the concentration of methanol is 100% (anhydrous methanol) and the concentration of ethanol is 95% (volume concentration) in the present invention.
The invention has the following beneficial effects:
the method adopts thin-layer chromatography to identify the characteristic active ingredients of the three medicinal materials in the medicament, and has the advantages of simple and convenient sample processing method, good separation degree and clear spots; the high performance liquid chromatography is used for measuring the content of the rupestonic acid in the medicine, and has the advantages of simple operation, strong specificity, good repeatability and high accuracy. The quality control method of the artemisia apiacea granules is a quality control method which has more comprehensive and complete detection items, more scientific and reasonable quality control indexes and simpler and more convenient operation, can further improve the quality level of products and ensure the curative effect.
Drawings
FIG. 1 is a thin layer chromatogram for identifying rupestonic acid in example 1;
FIG. 2 is a thin-layer chromatogram for identification of R, S-goitrin in example 1;
FIG. 3 is a thin layer chromatogram for indirubin identification in example 1
FIG. 4 is a liquid chromatogram of a control in example 1;
FIG. 5 is a liquid chromatogram of a negative sample in example 1;
FIG. 6 is a liquid chromatogram of the sample to be tested in example 1.
Detailed Description
The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
The reagents and materials in the examples are commercially available, and in the case where concentrations are not specified, the concentration of methanol is 100% (anhydrous methanol) and the concentration of ethanol is 95% (volume concentration).
Example 1
A quality control method of Artemisia apiacea granules comprises the following steps:
(1) qualitatively identifying characteristic active ingredient of herba Achilleae in the medicine, namely herba Achilleae acid, by thin layer chromatography: taking 5g of the fine powder of the Artemisia apiacea particles, adding 40mL of ethanol, performing ultrasonic treatment for 20min, filtering, taking the filtrate, evaporating to dryness, adding 3mL of ethanol again to dissolve the evaporated substance to obtain a sample solution. Adding ethanol into another rupestonic acid control to obtain a solution containing 0.08mg per 1mL as control solution.
Performing thin-layer chromatography (appendix 0502) test, sucking 5 μ l of each of the two solutions, respectively dropping on the same silica gel GF254 thin-layer plate, developing with petroleum ether (30-60 deg.C) -ethyl acetate-glacial acetic acid (volume ratio 15:8:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp. The chromatogram of the test sample showed fluorescent spots at positions corresponding to those of the control chromatogram, and the results are shown in FIG. 1.
(2) Qualitatively identifying characteristic active ingredient of radix Isatidis- (R, S) -goitrin by thin layer chromatography: taking 10g of the fine powder of the Artemisia apiacea particles, adding 80mL of water for dissolving, adding dichloromethane, and shaking for extraction for 3 times, wherein 80mL of dichloromethane is used each time; the dichloromethane layer was centrifuged, and the dichloromethane supernatant was combined and concentrated to 3mL as a sample solution. And adding ethanol into (R, S) -goitrin reference substance to obtain a solution containing 0.6mg per 1mL as reference substance solution.
Performing thin-layer chromatography (appendix 0502) test, sucking 10 μ l of each of the two solutions, respectively dropping on the same silica gel GF254 thin-layer plate, developing with petroleum ether (30-60 deg.C) -ethyl acetate (volume ratio of 2:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp. The chromatogram of the test sample showed fluorescent spots at positions corresponding to those of the control chromatogram, and the results are shown in FIG. 2.
(3) Qualitatively identifying characteristic active component of folium Isatidis in the medicine, indirubin, by thin layer chromatography: collecting 20g of the fine powder, adding 50mL of water to dissolve, adding chloroform, shaking and extracting for 3 times, using 50mL of chloroform each time, centrifuging chloroform layer, mixing chloroform clear solution, evaporating, adding 3mL of chloroform to dissolve the evaporated substance to obtain sample solution. Additionally, taking indirubin reference substance, and adding chloroform to make into solution containing 0.08mg per 1mL as reference substance solution.
Performing thin layer chromatography (appendix 0502) test, sucking 10 μ l of each of the two solutions, dropping on the same silica gel G thin layer plate, developing with benzene-chloroform-acetone (volume ratio 6: 3: 1) as developing agent, taking out, and air drying. Spots of the same color as the control appear in the chromatogram of the test solution at positions corresponding to the chromatograms of the control solution.
As shown in FIG. 3, the test sample and the control sample showed pink spots with the same color at the corresponding positions of the chromatogram. It should be noted that since the patent application document cannot submit a color drawing and a specific color cannot be displayed, if necessary, the applicant can provide color artwork as evidence.
(4) And (4) checking: according to the requirements of annex 0106 granule, its properties, granularity, water content, dissolubility and loading amount are in accordance with the regulations.
(5) Determining the content of rupestonic acid by high performance liquid chromatography (appendix 0512):
chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol-acetonitrile-0.4% phosphoric acid solution (volume ratio is 47: 15: 30) is used as a mobile phase; the detection wavelength was 254 nm.
Preparation of control solutions: precisely weighing a rupestonic acid reference substance, and adding ethanol to obtain a solution containing 39 μ g of rupestonic acid per 1 mL.
Preparation of a test solution: taking a proper amount of artemisia apiacea granules, grinding, taking 6g of fine powder, precisely weighing, placing in a conical flask with a plug, precisely adding 60mL of ethanol, sealing the plug, weighing, carrying out ultrasonic treatment for 20min, cooling to below 25 ℃, weighing again, supplementing the weight loss by ethanol, shaking uniformly, filtering, and taking filtrate to obtain a test solution. .
The determination method comprises the following steps: precisely sucking 20 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring. The product contains herba Achilleae ketonic acid (C) per 1g15H20O3) Calculated, not less than 0.14 mg. The reference liquid chromatogram is shown in FIG. 4, the negative sample liquid chromatogram is shown in FIG. 5, and the sample liquid chromatogram to be tested is shown in FIG. 6.
Example 2
A quality control method of Artemisia apiacea granules comprises the following steps:
(1) qualitatively identifying characteristic active ingredient of herba Achilleae in the medicine, namely herba Achilleae acid, by thin layer chromatography: taking 5g of the fine powder of the Artemisia apiacea particles, adding 20mL of methanol, performing ultrasonic treatment for 60min, filtering, taking the filtrate, evaporating to dryness, adding 1mL of methanol again to dissolve the evaporated substance to obtain a sample solution. Adding methanol into another rupestonic acid control to obtain a solution containing 0.05mg per 1mL as control solution.
Performing thin-layer chromatography (appendix 0502) test, sucking 5 μ l of each of the two solutions, respectively dropping on the same silica gel GF254 thin-layer plate, developing with petroleum ether (60-90 deg.C) -ethyl acetate-glacial acetic acid (volume ratio 30: 5:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp. The test chromatogram shows fluorescent spots at positions corresponding to those of the control chromatogram.
(2) Qualitatively identifying characteristic active ingredient of radix Isatidis- (R, S) -goitrin by thin layer chromatography: taking 10g of the fine powder of the Artemisia apiacea particles, adding 20mL of water for dissolving, adding dichloromethane, and shaking for extraction for 4 times, wherein 20mL of dichloromethane is used each time; the dichloromethane layer was centrifuged, and the dichloromethane supernatant was combined and concentrated to 1mL as a sample solution. Methanol was added to the (R, S) -goitrin control solution to prepare a solution containing 0.3mg per 1mL of the control solution.
Performing thin-layer chromatography (appendix 0502) test, sucking 10 μ l of each of the two solutions, respectively dropping on the same silica gel GF254 thin-layer plate, developing with petroleum ether (30-60 deg.C) -ethyl acetate (volume ratio 1: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp. The test chromatogram shows fluorescent spots at positions corresponding to those of the control chromatogram.
(3) Qualitatively identifying characteristic active component of folium Isatidis in the medicine, indirubin, by thin layer chromatography: collecting 20g of the fine powder, adding 30mL of water to dissolve, adding chloroform, shaking and extracting for 4 times with 30mL of chloroform each time, centrifuging chloroform layer, mixing chloroform clear solution, evaporating, adding 1mL of chloroform to dissolve the evaporated substance to obtain sample solution. Additionally, taking indirubin reference substance, and adding chloroform to make into solution containing 0.05mg per 1mL as reference substance solution.
Performing thin layer chromatography (appendix 0502) test, sucking 10 μ l of each of the two solutions, dropping on the same silica gel G thin layer plate, developing with benzene-chloroform-acetone (volume ratio of 2: 8:1) as developing agent, taking out, and air drying. Spots of the same color as the control appear in the chromatogram of the test solution at positions corresponding to the chromatograms of the control solution.
(4) And (4) checking: according to the requirements of annex 0106 granule, its properties, granularity, water content, dissolubility and loading amount are in accordance with the regulations.
(5) Determining the content of rupestonic acid by high performance liquid chromatography (appendix 0512):
chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol-acetonitrile-0.4% phosphoric acid solution (volume ratio is 11: 45: 16) is used as a mobile phase; the detection wavelength was 221 nm. Preparation of control solutions: taking a rupestonic acid reference substance, precisely weighing, and adding methanol to prepare a solution containing 13 μ g per 1 mL.
Preparation of a test solution: taking a proper amount of artemisia apiacea granules, grinding, taking 6g of fine powder, precisely weighing, placing in a conical flask with a plug, precisely adding 80mL of methanol, sealing the plug, weighing, carrying out ultrasonic treatment for 10min, cooling to below 25 ℃, weighing again, supplementing the weight loss by using methanol, shaking uniformly, filtering, and taking filtrate to obtain a test solution.
The determination method comprises the following steps: precisely sucking 20 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring. Every 1g of the product contains Achillea alpinaKeto acid (C)15H20O3) Not less than 0.05 mg.
Example 3
(1) Qualitatively identifying characteristic active ingredient of herba Achilleae in the medicine, namely herba Achilleae acid, by thin layer chromatography: taking 5g of the fine powder of the Artemisia apiacea particles, adding 80mL of ethanol, performing ultrasonic treatment for 10min, filtering, taking the filtrate, evaporating to dryness, adding 5mL of ethanol again to dissolve the evaporated substance to obtain a sample solution. Adding ethanol into another rupestonic acid control to obtain a solution containing 0.15mg per 1mL as control solution.
Performing thin-layer chromatography (appendix 0502) test, sucking 5 μ l of each of the two solutions, respectively dropping on the same silica gel GF254 thin-layer plate, developing with petroleum ether (30-60 deg.C) -ethyl acetate-glacial acetic acid (volume ratio 10: 15: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp. The test chromatogram shows fluorescent spots at positions corresponding to those of the control chromatogram.
(2) Qualitatively identifying characteristic active ingredient of radix Isatidis- (R, S) -goitrin by thin layer chromatography: taking 10g of the fine powder of the Artemisia apiacea particles, adding 100mL of water for dissolving, adding dichloromethane, and shaking for extraction for 4 times, wherein 100mL of dichloromethane is used each time; the dichloromethane layer was centrifuged, and the dichloromethane supernatant was combined and concentrated to 5mL as a test solution. And adding ethanol into (R, S) -goitrin control to obtain a solution containing 0.9mg per 1mL as control solution.
Performing thin-layer chromatography (appendix 0502) test, sucking 10 μ l of each of the two solutions, respectively dropping on the same silica gel GF254 thin-layer plate, developing with petroleum ether (60-90 deg.C) -ethyl acetate (volume ratio 5:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp. The test chromatogram shows fluorescent spots at positions corresponding to those of the control chromatogram.
(3) Qualitatively identifying characteristic active component of folium Isatidis in the medicine, indirubin, by thin layer chromatography: collecting 20g of the pulverized powder, adding 100mL of water to dissolve, adding chloroform, shaking and extracting for 1 time, using 100mL of chloroform, centrifuging chloroform layer, mixing chloroform clear solution, evaporating, adding 5mL of chloroform to dissolve the evaporated substance to obtain sample solution. Additionally, taking indirubin reference substance, and adding chloroform to make into solution containing 0.15mg per 1mL as reference substance solution.
Performing thin layer chromatography (appendix 0502) test, sucking 10 μ l of each of the two solutions, dropping on the same silica gel G thin layer plate, developing with benzene-chloroform-acetone (volume ratio 9: 1: 1) as developing agent, taking out, and air drying. Spots of the same color as the control appear in the chromatogram of the test solution at positions corresponding to the chromatograms of the control solution.
(4) And (4) checking: according to the requirements of annex 0106 granule, its properties, granularity, water content, dissolubility and loading amount are in accordance with the regulations.
(5) Determining the content of rupestonic acid by high performance liquid chromatography (appendix 0512):
chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol-acetonitrile-0.4% phosphoric acid solution (volume ratio is 68: 5: 92) is used as a mobile phase; the detection wavelength was 265 nm.
Preparation of control solutions: precisely weighing a rupestonic acid reference substance, and adding ethanol to obtain a solution containing 74 μ g of rupestonic acid per 1 mL.
Preparation of a test solution: taking a proper amount of artemisia apiacea granules, grinding, taking 6g of fine powder, precisely weighing, placing in a conical flask with a plug, precisely adding 20mL of ethanol, sealing the plug, weighing, carrying out ultrasonic treatment for 50min, cooling to below 25 ℃, weighing again, supplementing the weight loss by ethanol, shaking uniformly, filtering, and taking filtrate to obtain a test solution.
The determination method comprises the following steps: precisely sucking 20 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring. The product contains herba Achilleae ketonic acid (C) per 1g15H20O3) Calculated, not less than 0.25 mg.
Experiment of
The quality control method of the invention is an optimal scheme obtained by screening a large number of experimental studies, and the experimental study process of the invention is as follows.
Experiment 1 thin-layer chromatography identification method for characteristic active ingredient of artemisia rupestris-ketoacid
1.1 preparation method of test solution screening:
the method comprises the following steps: taking 5g of the ground powder, adding 80mL of methanol, carrying out ultrasonic treatment for 10 minutes, filtering, taking the filtrate, evaporating to dryness, adding 5mL of methanol again to dissolve the evaporated substance to be used as a test solution.
The second method comprises the following steps: taking 5g of the ground powder, adding 40mL of ethanol, carrying out ultrasonic treatment for 20 minutes, filtering, taking the filtrate, evaporating to dryness, adding 3mL of ethanol again to dissolve the evaporated substance to be used as a test solution.
The third method comprises the following steps: taking 5g of the ground powder of the product, adding 20mL of methanol, carrying out ultrasonic treatment for 60 minutes, filtering, taking the filtrate, evaporating to dryness, adding 1mL of methanol again to dissolve the evaporated substance to be used as a test solution.
The method four comprises the following steps: taking 5g of the ground powder, adding 80mL of ethanol, heating and refluxing for 40 minutes, filtering, taking the filtrate, evaporating to dryness, adding 2mL of ethanol again to dissolve the evaporated substance to be used as a test solution.
The method five comprises the following steps: taking 5g of the ground powder of the product, adding 50mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, taking the filtrate, evaporating to dryness, adding 2mL of ethanol again to dissolve the evaporated substance to be used as a test solution.
The method six: taking 5g of the ground powder of the product, adding 20mL of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, taking the filtrate, evaporating to dryness, adding 2mL of ethanol again to dissolve the evaporated substance to be used as a test solution.
As a result: the first, third and sixth spots are fuzzy and have more impurities, the fourth spot is not present, the second and fifth spots are clear, but the second spot is not trailing and is clearest, so the best choice is the second spot.
1.2 unfolding the system screening:
1) ethyl acetate-methanol-concentrated ammonia test solution (volume ratio 17: 2:1) (ii) a
2) Ethyl acetate-methanol-water (volume ratio 40: 5:1) (ii) a
3) Toluene-methanol-formic acid (volume ratio 18: 3: 1) (ii) a
4) Petroleum ether (30-60 ℃), ethyl acetate-glacial acetic acid (volume ratio 20: 10: 1) (ii) a
5) Petroleum ether (30-60 ℃), ethyl acetate and glacial acetic acid (the volume ratio is 15:8: 1);
6) petroleum ether (60-90 ℃), ethyl acetate-glacial acetic acid (volume ratio 10: 5: 1).
As a result: the Rf values of systems 1), 2), 3) are high, the Rf values of systems 4), 6) are low, the spot roundness is low, the Rf value of system 5) is moderate, the spot is round, and therefore the optimal choice is system 5).
Experiment 2 thin-layer chromatography identification method screening of characteristic active ingredient of radix Isatidis- (R, S) -goitrin
2.1 preparation method screening of test solution:
the method comprises the following steps: dissolving 10g of the fine powder in 100mL of water, extracting with chloroform 100mL for 1 time, centrifuging chloroform layer, mixing chloroform supernatant, and concentrating to 5mL to obtain test solution.
The second method comprises the following steps: dissolving the pulverized powder 10g in water 20mL, extracting with dichloromethane for 4 times (each time using dichloromethane 20 mL), mixing dichloromethane solutions, evaporating to dryness, and dissolving the residue with dichloromethane 2mL to obtain sample solution.
The third method comprises the following steps: dissolving the pulverized powder 10g in water 80mL, extracting with dichloromethane 80mL for 3 times, centrifuging dichloromethane layer, mixing dichloromethane supernatant, and concentrating to 3mL to obtain test solution.
As a result: the first method has serious spot tailing, the second method has serious emulsification, cannot be separated and purified, and the third method has high separation efficiency and clear spots, so the third method is the best selection.
2.2 unfolding system screening:
1) petroleum ether (30-60 ℃) -ethyl acetate (volume ratio 2:1) (ii) a
2) Benzene-chloroform-acetone (volume ratio 5: 4: 1) (ii) a
3) Petroleum ether (60-90 ℃) -ethyl acetate (volume ratio 5: 1).
As a result: system 2) smear severe, system 3) spot rounding low, system 1) best, and therefore the best choice is system 1).
Experiment 3 thin-layer chromatography identification method screening of indigowoad leaf characteristic active component indirubin
3.1 preparation method screening of test solution:
the method comprises the following steps: dissolving 10g of the product in 30mL of water, extracting with 30mL of chloroform under shaking for 1 time, mixing chloroform solutions, evaporating to dryness, and dissolving the evaporated material with 1mL of chloroform to obtain a sample solution.
The second method comprises the following steps: collecting 20g of the fine powder, adding 50mL of water to dissolve, adding chloroform, shaking and extracting for 3 times, using 50mL of chloroform each time, centrifuging chloroform layer, mixing chloroform clear solution, evaporating, adding 3mL of chloroform to dissolve the evaporated substance to obtain sample solution.
The third method comprises the following steps: dissolving 10g of the product in 100mL of water, adding chloroform, shaking for 4 times, using 100mL of chloroform each time, mixing the chloroform solutions, evaporating, and adding 5mL of chloroform to dissolve the evaporated product to obtain a sample solution.
As a result: the extract obtained by the first method and the third method is seriously emulsified, cannot be separated and purified, and the second method has high separation efficiency and clear spots, so the second method is the best method.
3.2 unfolding the system for screening:
1) petroleum ether (60-90 ℃) -ethyl acetate (volume ratio 1: 1) (ii) a
2) Benzene-chloroform-acetone (volume ratio 6: 3: 1) (ii) a
3) Petroleum ether (30-60 ℃) and ethyl acetate (volume ratio is 2: 1).
As a result: system 1), 3) smear severe, system 2) best, and therefore system 2) is the best choice.
Experiment 4 content determination method screening of characteristic active ingredient of artemisia rupestris-ketonic acid
4.1 instruments and reagents
The instrument comprises the following steps: an Agilent 1100 high performance liquid chromatography system; SUPELCO ODS C18 column (4.6 mm. times.250 mm, 5 μm); shimadzu UV-2550 ultraviolet-visible spectrophotometer.
Reagent testing: a rupestonic acid control; methanol, ethanol, acetonitrile (chromatographically pure); other reagents were analytically pure.
Sample preparation: artemisia apiacea granules (lot 190202) and negative control of Achillea alpina.
4.2 screening of test solution preparation method
The method comprises the following steps: taking a proper amount of the product, grinding, taking 6g of fine powder, precisely weighing, placing in a 20mL measuring flask, adding a proper amount of methanol, carrying out ultrasonic treatment for 10 minutes, cooling to below 25 ℃, adding methanol to the scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the product.
The second method comprises the following steps: taking a proper amount of the product, grinding, taking 3g of fine powder, precisely weighing, placing into a 80mL measuring flask, adding a proper amount of 50% ethanol, carrying out ultrasonic treatment for 40 minutes, cooling to below 25 ℃, adding 50% ethanol to the scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the product.
The third method comprises the following steps: taking a proper amount of the product, grinding, taking 6g of fine powder, precisely weighing, placing in a conical flask with a plug, precisely adding 60mL of ethanol, sealing the plug, weighing, carrying out ultrasonic treatment for 20 minutes, cooling to below 25 ℃, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, and taking a subsequent filtrate.
Tests show that the first and second methods have unstable peak-producing time, more impurity peaks and poor resolution, so the third method is selected as the preparation method of the test solution.
4.3 screening of chromatographic conditions
Preparing a rupestonic acid reference solution with a proper concentration by using methanol or ethanol as a solvent, placing the solution in an ultraviolet spectrophotometer for scanning, wherein the set interval is 200-400 nm, and the scanning result shows that the absorbance value of rupestonic acid at the wavelength of 254nm is the maximum. Therefore, 254nm was chosen as the detection wavelength.
And (3) flow comparison:
1) methanol-0.4% phosphoric acid solution (volume ratio 50: 50) under the condition, the retention time of a rupestonic acid peak is proper, but the separation condition of a main peak and front and rear impurity peaks in a chromatogram map is poor, accurate measurement cannot be carried out, the proportion of a mobile phase is adjusted before and after trying, and the main peak and the impurity peaks are difficult to separate all the time after trying the proportion of the mobile phase of a plurality of different methanol-0.4 percent phosphoric acid solutions;
2) methanol-acetonitrile-0.4% phosphoric acid solution (volume ratio 47: 15: 30) when the condition is used for measurement, the retention time is increased, and the separation effect of the main peak and the impurity peak of the sample is better, so that a methanol-acetonitrile-0.4% phosphoric acid solution (volume ratio 47: 15: 30) as the mobile phase.
4.4 System suitability and specificity testing
And respectively taking a blank solution, a reference substance solution, a test solution and a negative reference substance solution, and injecting samples, wherein the separation degree of the rupestonic acid reaches more than 1.5 under the chromatographic condition, and the negative sample has no absorption peak near the corresponding position, which indicates that the method can meet the detection requirement.
4.5 Linear survey
Precisely measuring a rupestonic acid reference substance, diluting with methanol, respectively making into rupestonic acid reference substance solutions of 7.5, 15, 30, 45, 60, and 75 μ g/mL, filtering, collecting subsequent filtrates, injecting sample according to the above chromatographic conditions, measuring, and performing linear regression with peak area (y) as ordinate and control concentration (x) as abscissa to obtain regression equation: the results are shown in table 1, wherein y is 96.558x-136.322, and R2 is 0.9997, and the linear relation is good, and the sampling amount of the rupestonic acid is in the range of 0.15-1.5 mu g.
TABLE 1 Linear relationship test data
4.6 precision test
And (3) taking the reference substance solution, injecting the reference substance solution into a liquid chromatograph according to a method, repeatedly injecting samples for 6 times, calculating RSD according to the peak area, and showing that the precision is good as shown in table 2.
TABLE 2 precision test data
4.7 stability test
A solution of the same sample (lot 190202) was prepared and injected at 0, 2, 4, 6, 8, 10, and 12h, respectively, and the RSD was calculated by measuring the peak area. The results are shown in table 3, which shows that the sample is stable over 12 h.
Table 3 stability test data
4.8 repeatability test
6 parts of the same sample solution (lot 190202) were prepared, and the sample was injected, and the content was measured to calculate RSD. The results are shown in Table 4, indicating that the process is very reproducible.
TABLE 4 repeatability test data
4.9 sample recovery test
6 parts of a sample (lot No. 190202) having a known content were precisely weighed, and each of the 6 parts was placed in a volumetric flask, and a control (. mu.g.mL) was precisely added thereto-1) And (5) preparing 10mL of solution, preparing a sample solution according to the method, carrying out sample injection measurement, and calculating the sample injection recovery rate. The results are shown in Table 5, indicating that the process yields are good.
TABLE 5 sample recovery test data
4.10 sample measurement
Three samples (190201, 190202, 190203) were tested under the conditions and methods described above, and the amount of rupestonic acid in the samples was calculated from the peak area, and the results are shown in Table 6.
Table 6 results of sample measurement (n ═ 3)
The test process does not need to be carried out according to the sequence of steps, and meanwhile, the conventional item detection can be carried out, for example, the sample is brown or brownish particles; sweet and slightly bitter. And the sample also conforms to the regulations of the granules in the appendix 0106 of the second part of the 2015 edition of Chinese veterinary pharmacopoeia.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A quality control method of Artemisia apiacea granules is characterized by comprising the identification of rupestonic acid, R, S-goivochen and indirubin; also comprises the content measurement of rupestonic acid;
wherein, the identification of rupestonic acid, R, S-goitrin and indirubin adopts thin layer chromatography; the content of rupestonic acid is determined by high performance liquid chromatography.
2. The quality control method according to claim 1,
in the identification of rupestonic acid: using methanol or ethanol as solvent, respectively dropping the sample solution and the reference solution on the same silica gel GF254On the thin layer plate, petroleum ether-ethyl acetate-glacial acetic acid is used as a developing agent;
in the identification of R, S-goitrin: the test sample takes dichloromethane or trichloromethane as a solvent, and the reference sample takes methanol or ethanol as a solvent; respectively dropping the test solution and the reference solution on the same silica gel GF254Petroleum ether-ethyl acetate is used as a developing agent on the thin-layer plate;
in the identification of indirubin: using trichloromethane as solvent, respectively dropping the sample solution and the reference solution on the same silica gel G thin layer plate, and using benzene-trichloromethane-acetone as developing agent.
3. The quality control method according to claim 2,
in the identification of rupestonic acid: the volume ratio of the developing solvent oil ether to the ethyl acetate to the glacial acetic acid is (10-30): (5-15): 1, the temperature of petroleum ether is 30-90 ℃;
in the identification of R, S-goitrin: the volume ratio of the developing solvent petroleum ether to the ethyl acetate is (1-5): 1, the temperature of petroleum ether is 30-90 ℃;
in the identification of indirubin: the volume ratio of the developing agent benzene-trichloromethane-acetone is (2-9): (1-8): 1.
4. the quality control method according to claim 2 or 3,
in the identification of rupestonic acid, the preparation method of the test solution is as follows: grinding the artemisia isatis leaf particles into powder, adding a solvent, wherein the dosage ratio of the artemisia isatis leaf particle powder to the solvent is 1 g: (4-16) mL; performing ultrasonic treatment for 10-60 min, filtering, taking filtrate, evaporating to dryness, adding a solvent again to dissolve evaporated substances, wherein the ratio of the arteannuin particle powder to the solvent added again is 1 g: (0.2-1) mL to obtain a test solution;
in the identification of R, S-goitrin, the preparation method of the test solution is as follows: grinding the artemisia isatis leaf particles into powder, adding water to dissolve the powder, adding a solvent, shaking and extracting for 1-4 times, wherein the dosage ratio of the artemisia isatis leaf particle powder to the water is 1 g: (2-10) mL, wherein the dosage ratio of the arteannuin particle powder to the solvent used each time is 1 g: (2-10) mL; centrifuging the solvent layer, combining the solvent supernatant, and concentrating until the ratio of arteannuin particle powder to concentrated solution is 1 g: (0.1-0.5) mL to obtain a test solution;
in the identification of indirubin, the preparation method of the test solution is as follows: grinding the artemisia isatis leaf particles into powder, adding water to dissolve the powder, adding trichloromethane, shaking and extracting for 1-4 times, wherein the dosage ratio of the artemisia isatis leaf particle powder to the water is 2 g: (3-10) mL, wherein the dosage ratio of the arteannuin particle powder to the chloroform used each time is 2 g: (3-10) mL; centrifuging the chloroform layer, combining the chloroform clarified liquid, evaporating to dryness, adding chloroform again to the evaporated matter for dissolving, wherein the ratio of tarragon green particle powder to chloroform added again is 1 g: (0.05-0.25) mL to obtain a test solution.
5. The quality control method according to claim 2 or 3,
in the identification of rupestonic acid, the concentration of the reference solution is 0.05-0.15 mg/mL-1The sample application amount is 5 mul;
in the identification of R, S-goitrin, the concentration of the reference solution is 0.3-0.9 mg/mL-1The sample amount is 10 mul;
in the identification of indirubin, the concentration of the reference substance solution is 0.05-0.15 mg/mL-1The amount of the spot was 10. mu.l.
6. The quality control method according to claim 1, wherein in the thin layer chromatography detection, under an ultraviolet lamp, a sample chromatogram shows a fluorescent spot or a spot having the same color as a reference substance at a position corresponding to the reference substance chromatogram, and it is determined that the arteannuin granules contain rupestonic acid, R, S-goitrin or indirubin.
7. The quality control method according to claim 1, wherein in the measurement of the content of rupestonic acid, rupestonic acid is used as a control; octadecylsilane chemically bonded silica is used as a filling agent; methanol-acetonitrile-0.4% phosphoric acid solution is used as a mobile phase; the detection wavelength is 221-265 nm.
8. The quality control method according to claim 7, wherein the mobile phase contains methanol-acetonitrile-0.4% phosphoric acid solution in a volume ratio of (11-68): (5-45): (16-92).
9. The quality control method according to claim 7, wherein the test sample is prepared by measuring the content of rupestonic acid as follows:
grinding the artemisia apiacea particles into powder, adding methanol or ethanol as a solvent, and carrying out ultrasonic treatment for 10-50 min, wherein the dosage ratio of the artemisia apiacea particle powder to the solvent is 6 g: (20-80) mL; supplementing with methanol or ethanol, reducing weight, shaking, filtering, and collecting filtrate to obtain test solution.
10. The quality control method according to claim 7, wherein the control substance has a concentration of 13 to 74 μ g/mL in the measurement of the content of rupestonic acid-1Methanol (b) ofOr an ethanol solution.
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