CN117420253A - Thin layer identification method of angelica sinensis six-yellow decoction and compound preparation thereof - Google Patents

Thin layer identification method of angelica sinensis six-yellow decoction and compound preparation thereof Download PDF

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CN117420253A
CN117420253A CN202311189767.9A CN202311189767A CN117420253A CN 117420253 A CN117420253 A CN 117420253A CN 202311189767 A CN202311189767 A CN 202311189767A CN 117420253 A CN117420253 A CN 117420253A
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solution
butanol
water
medicinal material
mass
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张志强
韩红梅
刘利娟
周永康
吉艳慧
董晨虹
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Beijing Tcmages Pharmaceutical Co Ltd
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Beijing Tcmages Pharmaceutical Co Ltd
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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
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4055—Concentrating samples by solubility techniques
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • 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
    • G01N30/94—Development
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4055—Concentrating samples by solubility techniques
    • G01N2001/4061—Solvent extraction

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)

Abstract

The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a thin layer identification method of angelica six-yellow decoction and a compound preparation thereof. The preparation method of the test solution and the control medicinal material solution comprises at least one of a method 1 and a method 2; the developing agent comprises ethyl acetate, n-propanol and water. The thin layer identification method provided by the invention is simple in sample preparation method, easy to operate, less in loss in the sample preparation process, and capable of saving the test time and cost to a great extent; the method provided by the invention is negative, has no interference, has strong specificity, has good application prospect, and has important significance for identifying and researching the astragalus membranaceus thin layer in the compound preparation. The invention adopts the active ingredient reference substance and the reference medicinal material as the reference simultaneously, avoids the inconvenience of operation caused by the respective comparison of the reference substance and the reference medicinal material, and improves the accuracy rate at the same time.

Description

Thin layer identification method of angelica sinensis six-yellow decoction and compound preparation thereof
Technical Field
The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a thin layer identification method of angelica six-yellow decoction and a compound preparation thereof.
Background
The Liuhuang Shang Chuzi Liuyuan Lidong chongcang (Tibet of Liuyangxiang) is a classical prescription for treating night sweat, and is known as "holy medicine for treating night sweat". The Chinese angelica root, the six-yellow Shang You Chinese angelica, the dried rehmannia root, the prepared rehmannia root, the baical skullcap root, the golden thread, the amur corktree bark and the membranous milkvetch root have the main effects of nourishing yin, purging fire, consolidating exterior and arresting sweating, and are mainly used for treating night sweat syndromes of fire excess due to yin deficiency; the clinical symptoms are mainly fever and night sweat, facial barks and vexation, dry mouth and lips, dry stool, yellow urine, red tongue with yellow coating and rapid pulse.
Along with the enhancement of health care consciousness of people, the quality and the safety of Chinese patent medicine products are more required. In order to identify the astragalus membranaceus in the angelica six-yellow Shang Chufang, thin layer chromatography is disclosed in research on the reference of the angelica six-yellow Shang Wuzhi, and the steps of heating reflux, passing through neutral alumina column, extraction and the like are adopted in the preparation process of a sample, the sample preparation method is complex, the time consumption is long, and whether the astragalus membranaceus in the angelica six-yellow soup and a compound preparation thereof is fed or not cannot be rapidly confirmed. However, the formula achieves the effects of tonifying qi and strengthening exterior by using the astragalus root, and is one of important raw materials of the angelica six-yellow decoction.
Therefore, a method with high accuracy, time and cost saving and easy popularization is needed to control the qualitative identification of astragalus membranaceus in the angelica six-yellow Shang Fufang preparation and substance reference substance, so as to ensure the uniformity, stability and reliability of the quality of the angelica six-yellow Shang Fufang preparation.
Disclosure of Invention
Therefore, the invention aims to solve the technical problems of complex sample preparation method, long time consumption and low detection accuracy in the thin-layer identification of angelica hexa-yellow Shang Wu in the prior art. Therefore, a thin layer identification method of the angelica six-yellow decoction and the compound preparation thereof is provided.
For this purpose, the invention provides the following technical scheme:
a thin layer identification method of radix Angelicae sinensis six-yellow decoction and its compound preparation comprises the following steps:
taking a sample and astragalus control medicinal materials, and preparing a sample solution and a control medicinal material solution according to at least one of the following methods 1 and 2;
method 1: dissolving the sample and radix astragali reference materials in alkaline solution respectively, performing first solid-liquid separation, adding n-butanol into the absorbed liquid for mixing, performing second solid-liquid separation, absorbing n-butanol solution layer, adding water for mixing, performing third solid-liquid separation to obtain n-butanol solution layer, drying, dissolving in alcohol, and respectively preparing sample solution and reference medicinal material solution;
method 2: extracting the sample and radix astragali reference materials with alcohol respectively, separating solid and liquid, drying, dissolving in water, extracting with water saturated n-butanol, washing n-butanol extractive solution with alkaline solution, washing with n-butanol saturated water, collecting n-butanol solution, drying, dissolving in alcohol, and respectively preparing into sample solution and reference medicinal material solution;
and (3) identification: sample the sample solution, control medicinal material solution, and astragaloside IV control solution on the same thin layer plate, spreading with mixed solution containing ethyl acetate, n-propanol and water as spreading agent, taking out, air drying, spraying with color developing agent, heating, and inspecting under visible light.
Further, in the identification step, the adopted thin layer plate is a silica gel G thin layer plate; and/or heating at 95-105deg.C for 3-5min; and/or the sample application amount of the sample solution, the control medicinal material solution or the astragaloside IV control substance solution is 1-20 mu L.
In certain preferred embodiments, the sample application amount of the test solution, the control medicinal solution or the astragaloside IV control solution is 5-20 mu L.
In certain more preferred embodiments, the sample solution is spotted in an amount of 20. Mu.L, the control solution is spotted in an amount of 20. Mu.L, and the astragaloside IV control solution is spotted in an amount of 10. Mu.L.
Further, the color developing agent is sulfuric acid ethanol solution, and preferably, the concentration of the sulfuric acid ethanol solution is 5-15%.
Further, method 1 also satisfies one or more of the following A-H:
A. the volume ratio of the mass of the sample or the reference medicinal material to the alkaline solution is 0.5-6:15, preferably 2-4:15, wherein the ratio relationship of the mass to the volume is g/mL;
B. the first solid-liquid separation, the second solid-liquid separation or the third solid-liquid separation is centrifugal or filtering;
C. the volume ratio of the mass of the test sample or the reference medicinal material to the n-butanol is 0.5-6:5-30, preferably 2-4:10, wherein the ratio relationship of the mass to the volume is g/mL;
D. the volume ratio of the n-butanol to the water is 5-30:5-30, preferably 1:1;
E. in the dissolving step, the volume ratio of the mass of the test sample or the reference medicinal material to the alcohol is 0.5-6:0.5-3, preferably 2-4:1, wherein the proportioning relation of the mass and the volume is g/mL;
F. the mass volume concentration of the alkaline solution is 0.01-52g/100mL, preferably 0.01-4g/100mL;
G. the alcohol comprises one or more of methanol and ethanol; preferably methanol;
H. the alkaline solution comprises one or more of sodium hydroxide aqueous solution and potassium hydroxide aqueous solution.
Further, method 2 also satisfies one or more of the following a-j:
a. in the alcohol extraction step, the ratio of the mass of the test sample or the reference medicinal material to the volume of alcohol and the volume of water is 0.5-6:20-40:20-40, preferably 2-4:30:20, wherein the ratio relationship of the mass to the volume is g/mL;
b. the solid-liquid separation is centrifugation or filtration;
c. the ethanol extraction is carried out by ultrasonic extraction or thermal reflux extraction for at least 15min (e.g. 15-60 min)
d. The times of water-saturated n-butanol extraction are 1-5 times, and the volume ratio of the mass of the sample solution or the control medicinal material to the water-saturated n-butanol added in each extraction is 0.5-6:20, a step of; preferably 2-4:20, wherein the proportioning relation between the mass and the volume is g/mL;
e. the number of times of water washing of the alkaline solution or the n-butanol saturation is 1-5 times, and the volume ratio of the mass of the test sample or the control medicinal material to the alkaline solution or the n-butanol saturation water added during each washing is 0.5-6:20, a step of; preferably 2-4:20, wherein the ratio relationship of the mass to the volume is g/mL;
f. in the dissolving step, the volume ratio of the mass of the test sample or the reference medicinal material to the alcohol is 0.5-6:0.5-3, preferably 2-4:1, wherein the proportioning relation of the mass and the volume is g/mL;
g. the mass volume concentration of the alkaline solution is 0.01-52g/100mL, preferably 0.01-4g/100mL;
h. the alcohol comprises one or more of methanol and ethanol; preferably methanol;
j. the alkaline solution comprises one or more of sodium hydroxide aqueous solution and potassium hydroxide aqueous solution.
Further, the volume ratio of ethyl acetate, n-propanol and water in the developing agent is 5-10:1-8:1-6; preferably 6-8:6:4-5; more preferably 7:5:4.
further, the solvent of the reference substance solution is selected from methanol or methanol water solution with volume concentration of more than 80%; and/or, each 1mL of the reference substance solution contains 0.1-4 mg of astragaloside IV reference substance.
Further, the preparation method of the test solution comprises the steps of taking 0.5-6g of test powder, adding 15mL of alkaline solution with mass volume concentration of 0.01-4g/100mL, dissolving, centrifuging, absorbing supernatant, adding 5-30mL of n-butanol into the supernatant, shaking uniformly, centrifuging, absorbing an n-butanol solution layer, adding 5-30mL of water, shaking uniformly, centrifuging to obtain an n-butanol solution layer, evaporating to dryness, and adding 0.5-3mL of methanol for dissolving to obtain the test solution; or, taking 0.5-6g of test sample powder, adding 20-40mL of methanol, carrying out ultrasonic treatment for 15-60 minutes, filtering, evaporating filtrate to dryness, adding 10-30mL of water into residues for dissolution, extracting 1-5 times with water-saturated n-butanol, 20mL each time, combining n-butanol extract, washing 1-5 times with alkaline solution with the mass volume concentration of 0.01-4g/100mL, 20mL each time, washing 1-5 times with water-saturated n-butanol, 20mL each time, evaporating n-butanol solution to dryness, and adding 0.5-3mL of methanol into residues for dissolution to serve as a test sample solution;
the preparation method of the control medicinal material solution comprises the steps of taking 0.5-6g of astragalus mongholicus control medicinal material, adding 15mL of alkaline solution with mass volume concentration of 0.01-4g/100mL, dissolving, centrifuging, absorbing supernatant, adding 5-30mL of n-butanol into the supernatant, shaking up, centrifuging, absorbing an n-butanol solution layer, adding 5-30mL of water, shaking up, centrifuging to obtain an n-butanol solution layer, evaporating, and adding 0.5-3mL of methanol for dissolving to obtain the control medicinal material solution; or, taking 0.5-6g of astragalus control medicinal material, adding 20-40mL of methanol, carrying out ultrasonic treatment for 15-60 minutes, filtering, evaporating filtrate to dryness, adding 10-30mL of water into residues for dissolution, extracting with water-saturated n-butanol for 1-5 times, 20mL each time, combining n-butanol extract, washing with alkaline solution with the mass volume concentration of 0.01-4g/100mL for 1-5 times, 20mL each time, washing with water-saturated n-butanol for 1-5 times, 20mL each time, evaporating n-butanol liquid for dryness, and adding 0.5-3mL of methanol into residues for dissolution to serve as control medicinal material solution.
Further, the preparation method of the test sample solution comprises the steps of taking 2-4g of test sample powder, adding 15mL of sodium hydroxide aqueous solution with mass volume concentration of 0.5-2g/100mL, dissolving, centrifuging, absorbing supernatant, adding 10mL of n-butanol into the supernatant, shaking up, centrifuging, absorbing an n-butanol solution layer, adding 10mL of water, shaking up, centrifuging to obtain an n-butanol solution layer, evaporating to dryness, and adding 1mL of methanol for dissolving to obtain the test sample solution; or, taking 2-4g of test sample powder, adding 30mL of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, adding 20mL of water into residues for dissolution, extracting 2 times with water-saturated n-butanol, 20mL each time, combining n-butanol extract solutions, washing 2 times with sodium hydroxide aqueous solution with the mass volume concentration of 0.5-2g/100mL each time, 20mL each time, washing 2 times with water-saturated n-butanol, 20mL each time, evaporating n-butanol solution to dryness, and adding 1mL of methanol into residues for dissolution to serve as a test sample solution;
the preparation method of the control medicinal material solution comprises the steps of taking 2-4g of astragalus mongholicus control medicinal material, adding 15mL of sodium hydroxide aqueous solution with mass volume concentration of 0.5-2g/100mL, dissolving, centrifuging, absorbing supernatant, adding 10mL of n-butanol into the supernatant, shaking uniformly, centrifuging, absorbing an n-butanol solution layer, adding 10mL of water, shaking uniformly, centrifuging to obtain an n-butanol solution layer, evaporating to dryness, and adding 1mL of methanol for dissolving to obtain the control medicinal material solution; or, taking 2-4g of astragalus control medicinal material, adding 30mL of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, dissolving residues in 20mL of water, extracting with water-saturated n-butanol for 2 times, 20mL each time, combining n-butanol extract solutions, washing with sodium hydroxide aqueous solution with the mass volume concentration of 0.5-2g/100mL for 2 times, 20mL each time, washing with water saturated with n-butanol for 2 times, 20mL each time, evaporating n-butanol solution to dryness, dissolving residues in methanol l mL, and taking the residues as control medicinal material solution.
Further, the test sample is a solid preparation, a liquid preparation or a semisolid preparation of the angelica sinensis six-yellow decoction, preferably a powder, a granule, a tablet, a capsule, an oral liquid, an injection, an emulsion, a suspension or an ointment.
The Chinese angelica six-yellow decoction and the compound preparation thereof are prepared by taking Chinese angelica, dried rehmannia root, prepared rehmannia root, baikal skullcap root, coptis root, amur corktree bark and astragalus root as raw material medicines according to the conventional process in the field. Wherein the mass ratio of the Chinese angelica, the radix rehmanniae, the prepared rehmannia root, the baical skullcap root, the golden thread, the amur corktree bark and the membranous milkvetch root is 2-3:2-3:2-3:2-3:2-3:2-3:4-6; preferably 2.5:2.5:2.5:2.5:2.5:2.5:5.
the technical scheme of the invention has the following advantages:
1. the invention provides a thin layer identification method of angelica sinensis six-yellow decoction and a compound preparation thereof, and the preparation method of a test solution and a control medicinal material solution comprises at least one of a method 1 and a method 2; the developing agent comprises ethyl acetate, n-propanol and water. The thin layer identification method provided by the invention is simple in sample preparation method, easy to operate, less in loss in the sample preparation process, and capable of saving the test time and cost to a great extent; the method provided by the invention is negative, has no interference, has strong specificity, has good application prospect, and has important significance for identifying and researching the astragalus membranaceus thin layer in the compound preparation. The invention adopts the active ingredient reference substance and the reference medicinal material as the reference, avoids the inconvenience of operation caused by the reference substance and the reference medicinal material respectively, improves the accuracy rate of the reference substance and the reference medicinal material, and particularly can obtain more characteristic spots when the method 1 is adopted for preparing the sample solution and the reference medicinal material solution, thereby obviously improving the accuracy.
2. According to the thin-layer identification method of the angelica sinensis six-yellow decoction and the compound preparation thereof, the common low-toxicity reagent is adopted for experiments, so that the safety of experimental operation is greatly improved, and the harm to the environment and experimental personnel is reduced. The identification method provided by the invention has no special requirements on temperature and humidity, the temperature and the humidity have no influence on the identification result, and the identification method has better durability and reproducibility. Has good specificity, and discharges the possibility that other components cause interference to generate the same spots.
3. The thin layer identification method provided by the invention comprises the following steps of: 1-8:1-6 (especially ethyl acetate, n-propanol and water with the volume ratio of 6-8:6:4-5) are used as developing agents, the obtained thin layer has clearer spots and higher separation degree, and the optimal volume ratio is 7:5:4. compared with the prior art, the preparation method of the sample solution provided by the invention is simple and easy to operate, does not comprise complex operation processes such as heating reflux, over-neutral alumina column and the like, saves the use amount of organic solvents, and shortens the time for preparing the sample solution.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a sheet profile of developing agent 1 in example 2 of the present invention; wherein, from left to right, the method comprises the following steps: 1. a vagina 1;2 is supplied with 1;3. pair 1;4. astragaloside IV reference solution; 5. calycosin glucoside control solution; 6. pair 2;7. 2, supplying; 8. a vagina 2;9. a vagina 3;10. 3, supplying; 11. pair 3;12. astragaloside IV reference solution; 13. calycosin glucoside control solution; 14. pair 4;15. 4, supplying; 16. a vagina 4;17. astragaloside IV reference solution; 18. calycosin glucoside control solution; 19. pair 5;20. 5, supplying; 21. a vagina 5;
FIG. 2 is a thin layer pattern of developing agent 2 in example 2 of the present invention; the steps are as follows from left to right: 1. a vagina 1;2 is supplied with 1;3. pair 1;4. a vagina 2; 5.2, supplying; 6. pair 2;7. a vagina 3;8. 3, supplying; 9. pair 3;10. astragaloside IV reference solution; 11. calycosin glucoside control solution; 12. a vagina 4;13. 4, supplying; 14. pair 4;15. a vagina 5;16. 5, supplying; 17. pair 5;18. astragaloside IV reference solution;
FIG. 3 is a thin layer pattern of developing agent 3 in example 2 of the present invention; the steps are as follows from left to right: 1. a vagina 1;2 is supplied with 1;3. pair 1;4. a vagina 2; 5.2, supplying; 6. pair 2;7. a vagina 3;8. 3, supplying; 9. pair 3;10. astragaloside IV reference solution; 11. calycosin glucoside control solution; 12. a vagina 4;13. 4, supplying; 14. pair 4;15. a vagina 5;16. 5, supplying; 17. pair 5;18. astragaloside IV reference solution;
FIG. 4 is a thin layer pattern of developing agent 4 in example 2 of the present invention; the steps are as follows from left to right: 1. a vagina 1;2 is supplied with 1;3. pair 1;4. a vagina 2; 5.2, supplying; 6. pair 2;7. a vagina 3;8. 3, supplying; 9. pair 3;10. astragaloside IV reference solution; 11. calycosin glucoside control solution; 12. a vagina 4;13. 4, supplying; 14. pair 4;15. a vagina 5;16. 5, supplying; 17. pair 5;18. astragaloside IV reference solution;
FIG. 5 is a thin layer pattern of developing agent 5 in example 2 of the present invention; the steps are as follows from left to right: 1. a vagina 1;2 is supplied with 1;3. pair 1;4. a vagina 2; 5.2, supplying; 6. pair 2;7. a vagina 3;8. 3, supplying; 9. pair 3;10. astragaloside IV reference solution; 11. calycosin glucoside control solution; 12. a vagina 4;13. 4, supplying; 14. pair 4;15. a vagina 5;16. 5, supplying; 17. pair 5;18. astragaloside IV reference solution;
FIG. 6 is a thin layer pattern of developing agent 6 in example 2 of the present invention; the steps are as follows from left to right: 1. a vagina 1;2 is supplied with 1;3. pair 1;4. a vagina 2; 5.2, supplying; 6. pair 2;7. a vagina 3;8. 3, supplying; 9. pair 3;10. astragaloside IV reference solution; 11. calycosin glucoside control solution; 12. a vagina 4;13. 4, supplying; 14. pair 4;15. a vagina 5;16. 5, supplying; 17. pair 5;18. astragaloside IV reference solution;
FIG. 7 is a thin layer chromatography of different spotting amounts in example 3 of the present invention; 1. 5 μl of test solution; 2. 10 μl of test solution; 3. 15 μl of test solution; 4. 20 μl of test solution; 5. 10 mu L of astragaloside IV reference substance solution; 6. 10 mu L of control medicinal material solution; 7. 15 mu L of control medicinal material solution; 8. 20 μl of control medicinal solution;
FIG. 8 is a thin layer chromatography of a specificity study of the present invention; 1. the test solution JF-DGLH-DG2020019;2. radix astragali control medicinal material solution; 3. astragaloside IV reference solution; 4. angelica sinensis Liuhuang Shang Huangqi single negative control solution; 5. angelica sinensis Liuhuang Shang Huangqi single positive control solution;
FIG. 9 is a thin layer chromatography at ambient temperature under investigation of the development temperature of the present invention; wherein, 1, a sample solution, 2, an astragaloside IV reference substance solution and 3, an astragalus reference medicinal material solution;
FIG. 10 is a thin layer chromatography under low and medium temperature conditions under development temperature investigation of the present invention; wherein, 1, a sample solution, 2, an astragaloside IV reference substance solution and 3, an astragalus reference medicinal material solution;
FIG. 11 is a thin layer chromatography under low humidity conditions in a developed humidity study of the present invention; wherein, 1, a sample solution, 2, an astragaloside IV reference substance solution and 3, an astragalus reference medicinal material solution;
FIG. 12 is a thin layer chromatography under high humidity conditions in a developed humidity study of the present invention; wherein, 1, a sample solution, 2, an astragaloside IV reference substance solution and 3, an astragalus reference medicinal material solution;
FIG. 13 is a thin layer chromatography using Qingdao ocean plates (Qingdao ocean chemical Co., ltd., lot number: 20200707) in a thin layer plate study of the present invention; wherein, 1, astragaloside IV reference substance solution, 2, test substance solution and 3, astragalus reference medicinal material solution;
FIG. 14 shows a thin layer plate (Chii 326005; huang Wu silica gel development test plant; batch No. 20161007; wherein, 1. Astragaloside IV reference solution, 2. Sample solution, 3. Astragalus reference solution) using smoke bench Chii 32600in the thin layer plate investigation of the present invention;
FIG. 15 is a thin layer chromatogram of a silver dragon thin layer plate (institute of chemical industry, ministry of tobacco, lot number 20190808) used in a thin layer plate study of the present invention; wherein, 1, astragaloside IV reference substance solution, 2, test substance solution and 3, astragalus reference medicinal material solution;
FIG. 16 is a graph of the use of ethyl acetate in the development of the invention: n-propanol: thin layer chromatography with water (6:6:5) as developing agent;
FIG. 17 is a graph of the use of ethyl acetate in the development of the invention: n-propanol: thin layer chromatography with water (8:6:4) as developing agent;
fig. 18 shows thin layer chromatography according to example 4 of the present invention, in order from left to right: 1-4,7-8 sample solutions (batch numbers: JF-DGLH-DG2020001, JF-DGLH-DG2020002, JF-DGLH-DG2020003, JF-DGLH-DG2020004, JF-DGLH-DG2020005, JF-DGLH-DG2020006, respectively) 5. Astragaloside IV reference solution; 6. radix astragali control medicinal material solution; 9. astragalus negative control solution.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge. The proportion of each solvent in the developing agent is volume ratio.
Instrument, reagent and reagent
Instrument: an electronic balance: JJ500, a double jetty test instrumentation factory in the well-established market; an electronic balance: MSA6.6S-OCE-DM available from Saidolekulare instruments Co., ltd; thin layer imaging system: goodLook-1000, shanghai family philosophy Biochemical technology Co., ltd; ultrasonic cleaner: KQ-100DE, kunshan ultrasonic instruments Inc. (500W, 40 KHz.); electric heating constant temperature water bath kettle: DK-S26, a science and technology Co., ltd; PH meter: FE28, metrele-Tolyduo instruments Co., ltd; silica gel G plate: the institute of chemical industry in the tobacco stand market.
Reagent: the chemical reagents such as methanol, ethanol, n-butanol, water, ethyl acetate, sulfuric acid, sodium hydroxide, hydrochloric acid, anhydrous sodium sulfate, chloroform and the like are all analytically pure.
Reagent: astragaloside IV (Chinese food and drug verification institute, lot number: 111781-201717), calycosin glucoside (Chinese food and drug verification institute, lot number: 111920-201606), radix astragali control medicine (Chinese food and drug verification institute, lot number: 120904-201620).
The preparation method of the angelica hexa-yellow Shang Dong dry powder comprises the following steps: taking decoction pieces of radix Angelicae sinensis and six yellow decoction, respectively pulverizing decoction pieces, weighing 2.5g of decoction pieces of radix Angelicae sinensis, radix rehmanniae Preparata, cortex Phellodendri, scutellariae radix and Coptidis rhizoma respectively, placing into a decoction pot, adding 400ml of purified water, boiling with strong fire 500w, adjusting to slow fire 300w, and decocting with cover for 30min. Filtering with 200 mesh gauze, cooling the filtrate to room temperature, and drying. Preparing Angelica sinensis Liuhuang Shang Dong dry powder with different batches according to the method by adopting the raw materials of different batches, wherein the batches are: JF-DGLH-DG2020019, JF-DGLH-DG2020001, JF-DGLH-DG2020002, JF-DGLH-DG2020003, JF-DGLH-DG2020004, JF-DGLH-DG2020005, JF-DGLH-DG2020006.
The preparation method of the angelica hexa-yellow Shang Huangqi single-negative freeze-dried powder comprises the following steps: 2.5g of each of the crushed Chinese angelica, dried rehmannia root, prepared rehmannia root, amur corktree bark, baical skullcap root and golden thread decoction pieces are put into a decoction pot, 400ml of purified water is added, the mixture is boiled by strong fire 500w and then is adjusted to slow fire 300w, and the mixture is decocted for 30min. Filtering with 200 mesh gauze, cooling the filtrate to room temperature, and drying. Lot number: JF-DGLH-DG2020026.
The preparation method of the angelica hexa-yellow Shang Huangqi single positive sample freeze-dried powder comprises the following steps: weighing 5g of crushed astragalus decoction pieces, placing into a decoction kettle, adding 400ml of purified water, boiling with strong fire 500w, adjusting to slow fire 300w, and decocting with a cover for 30min. Filtering with 200 mesh gauze, cooling the filtrate to room temperature, and drying. Lot number: JF-DGLH-DG2020035.
Example 1
The embodiment provides a method for identifying angelica hexa-yellow Shang Ji preparation thereof, which comprises the following steps:
preparation of test solution: taking Angelica sinensis Liuhuang Shang Dong dry powder as a test article, taking 4g of the test article, adding 15mL of 1% (w/v, g/100 mL) sodium hydroxide aqueous solution, dissolving, centrifuging, absorbing supernatant, adding 10mL of n-butanol, shaking uniformly, centrifuging, absorbing an n-butanol solution layer, adding 10mL of water, shaking uniformly, centrifuging to obtain an n-butanol solution layer, evaporating to dryness, and adding 1mL of methanol for dissolving to obtain the Chinese medicinal composition.
Preparation of control medicinal material solution: taking 4g of astragalus control medicinal material, adding 15mL of 1% (w/v, g/100 mL) sodium hydroxide aqueous solution, dissolving, centrifuging, absorbing supernatant, adding 10mL of n-butanol into the supernatant, shaking uniformly, centrifuging, absorbing an n-butanol solution layer, adding 10mL of water, shaking uniformly, centrifuging to obtain an n-butanol solution layer, evaporating to dryness, and adding 1mL of methanol for dissolving.
Preparation of a control solution: adding methanol into astragaloside IV reference substance to obtain solution containing 0.5mg per L of astragaloside IV reference substance.
Sucking 20 μl of the sample solution, 20 μl of the control medicinal material solution and 10 μl of the control medicinal material solution, respectively spotting on the same thin layer plate, and adding ethyl acetate: n-propanol: spreading with water (7:5:4) as spreading agent, air drying, spraying 10% sulfuric acid ethanol solution, heating to color spots, and inspecting under visible light.
Example 2 sample preparation method and examination of developing Agents
The same batch of angelica six-yellow Shang Dong dry powder, astragalus control medicinal material and astragalus single-negative freeze-dried powder are respectively taken and divided into five parts, and sample solutions are respectively prepared according to the following five methods.
Method 1: about 4g of angelica hexa-yellow Shang Dong dry powder (sieving with a fourth sieve) is taken, placed in a conical flask with a plug, 80% methanol solution containing 4% of concentrated ammonia test solution (4 ml of concentrated ammonia test solution is taken, 80% methanol is added to 100ml, shaking is carried out uniformly) is added, 50ml of the mixture is sealed, heating reflux is carried out for 1 hour, filtering and evaporating is carried out, and 10ml of 80% methanol is added to residues for dissolution, thus obtaining a sample solution. Designated as supply 1. And (3) preparing a radix astragali control medicinal material, and preparing a control medicinal material solution according to the same method as the sample solution, wherein the control medicinal material solution is denoted as a pair 1. Taking Angelica sinensis Liuhuang Shang Huangqi single negative powder, preparing a negative control solution according to the same method as the test sample solution, and marking as yin 1.
Method 2: about 4g of angelica hexa-yellow Shang Dong dry powder (sieved by a fourth sieve) is taken, put into a conical flask with a plug, 50ml of methanol is added, the mixture is sealed, heated and refluxed for 4 hours, filtered, evaporated to dryness, and 10ml of methanol is added into residues to be dissolved to be used as a test solution. Designated as supply 2. And preparing a control medicinal material of astragalus according to the same method as the sample solution. Designated as pair 2. Taking angelica hexa-yellow Shang Huangqi single negative powder, and preparing a negative control solution according to the same method as that of the sample solution. The result is denoted as yin 2.
Method 3: adding ethanol 30mL into 4g of angelica hexa-yellow Shang Dong dry powder, heating and refluxing for 20 minutes, filtering, evaporating the filtrate to dryness, adding 0.3% (w/v, g/100 mL) sodium hydroxide aqueous solution 15mL into the residue to dissolve, filtering, adjusting the pH value of the filtrate to 5-6 by dilute hydrochloric acid, shaking and extracting by using ethyl acetate 15mL, separating ethyl acetate liquid, filtering by using filter paper paved with a proper amount of anhydrous sodium sulfate, and evaporating the filtrate to dryness. The residue was dissolved in 1ml of ethyl acetate to prepare a sample solution. Designated as supply 3. And preparing a control medicinal material of astragalus according to the same method as the sample solution. Designated as pair 3. Taking angelica hexa-yellow Shang Huangqi single negative powder, and preparing a negative control solution according to the same method as that of the sample solution. The result is denoted as yin 3.
Method 4: 2g of angelica hexa-yellow Shang Dong dry powder is taken, 30mL of methanol is added, ultrasonic treatment is carried out for 30 minutes, filtering is carried out, filtrate is evaporated to dryness, residues are added with 20mL of water to dissolve, the mixture is extracted for 2 times by using water saturated n-butanol, 20mL of each time, n-butanol extract is combined, the mixture is washed for 2 times by using 1% (w/v, g/100 mL) sodium hydroxide aqueous solution, 20mL of each time is washed for 2 times by using water saturated n-butanol, 20mL of each time is carried out, n-butanol solution is evaporated to dryness, and residues are added with l mL of methanol to dissolve to be used as a test solution. Designated as supply 4. And preparing a control medicinal material of astragalus according to the same method as the sample solution. Designated as pair 4. Taking angelica hexa-yellow Shang Huangqi single negative powder, and preparing a negative control solution according to the same method as that of the sample solution. The result is denoted as yin 4.
Method 5: taking 4g of angelica hexa-yellow Shang Dong dry powder, adding 15mL of 1% (w/v, g/100 mL) sodium hydroxide aqueous solution, dissolving, centrifuging, absorbing supernatant, adding 10mL of n-butanol into the supernatant, shaking uniformly, centrifuging, absorbing an n-butanol solution layer, adding 10mL of water, shaking uniformly, centrifuging to obtain an n-butanol solution layer, evaporating to dryness, and adding 1mL of methanol for dissolving to obtain a sample solution. Designated as supply 5. And preparing a control medicinal material of astragalus according to the same method as the sample solution. Designated as pair 5. Taking angelica hexa-yellow Shang Huangqi single negative powder, and preparing a negative control solution according to the same method as that of the sample solution. Marked as yin 5.
Preparing a reference substance solution: adding methanol into astragaloside IV reference substance to obtain solution containing 0.5mg per L/L, and obtaining astragaloside IV reference substance solution; adding methanol into calycosin glucoside reference substance to obtain solution containing 0.5mg per l mL, and obtaining calycosin glucoside reference substance solution.
Sucking 20 μl of the sample solution, 20 μl of the control medicinal solution, 20 μl of the negative control solution, 10 μl of the astragaloside IV control solution and 10 μl of the calycosin glucoside control solution, respectively spotting on the same silica gel G thin layer plate, respectively developing with the following 6 solutions as developing agents, air drying, spraying 10% sulfuric acid ethanol solution, heating to spot color development, and inspecting under visible light.
Developing agent 1: trichloromethane-methanol (10:1);
developing agent 2: a lower solution of chloroform-methanol-water (13:7:2);
developing agent 3: an upper solution of n-butanol-ethyl acetate-water (4:1:5);
developing agent 4: a lower layer solution prepared by standing chloroform-ethyl acetate-methanol-water (10:20:11:5) below 10deg.C for 12 hr;
developing agent 5: ethyl acetate-butanone-formic acid-water (5:3:3:1);
developing agent 6: ethyl acetate: n-propanol: water (7:5:4). The results are shown in FIGS. 1-6 and the tables below.
TABLE 1 identification results of test samples in each sample preparation method and developing agent
The small knot: as is clear from the results, 1) the calycosin glucoside was not developed, so that the control was astragaloside IV. 2) The spot information after the spray color developing agent is heated is more, and the spot information is easier to observe, so the results listed in the experiment are all observed under a white light source after the spray color developing agent is heated. 3) When different sample preparation methods are examined, the corresponding spots of the astragaloside IV reference substances in the sample preparation solutions obtained by the sample preparation methods 1, 2 and 3 cannot be observed, and other negative non-interference spots cannot be found, and the corresponding spots of the astragaloside IV reference substances in the sample preparation solutions obtained by the sample preparation methods 4 and five can be observed; 4) When different developing agents are investigated, the astragaloside control substances in the developing agent 1 are not developed, the astragaloside Rf value in the developing agent 4 is too small, the astragaloside Rf values in the developing agents 2, 3 and 5 are moderate, negative and non-interference, but other negative and non-interference spots cannot be found in the control medicinal materials; 5) The accuracy of thin layer identification of the combination of sample preparation methods 4 or 5 in combination with developer 6 is significantly improved over other combination methods. Spots with the same color are displayed on the positions corresponding to the chromatograms of the reference substance and the reference medicinal material in the chromatogram of the test substance, and the spot Rf values are moderate and negative without interference; 6) The sample preparation method 5 combines the developing agent 6 as the optimal combination, spots with the same color are arranged on the positions corresponding to the reference substances in the chromatogram of the test sample, the positions and the colors of the spots in the solution of the reference substances correspond to each other, and the spot Rf values are moderate and negative without interference.
EXAMPLE 3 methodology investigation
(1) Investigation of sample application amount
Sucking 10 μl of control solution, and sample solutions with different sample application amounts (5 μl, 10 μl, 15 μl, 20 μl), and respectively applying radix astragali control solution with different sample application amounts (10 μl, 15 μl, 20 μl) on the same thin layer plate with ethyl acetate: n-propanol: spreading with water (volume ratio of 7:5:4) as spreading agent, air drying, spraying 10% sulfuric acid ethanol solution, heating to color, and inspecting under visible light. Wherein each solution was prepared in the same manner as in example 1. The results are shown in FIG. 7.
As can be seen from fig. 7, at each sample amount, the thin layer chromatography of the test sample solution showed spots of the same color at the same position as the control sample solution, and also corresponded to two spots in the control medicinal material solution, showing a total of 3 characteristic spots. The sample application amounts of 10 mu l of the reference substance solution, 20 mu l of the reference medicinal material solution and 20 mu l of the test sample solution can identify the astragalus membranaceus in the angelica six-yellow Shang Wuzhi standard more clearly and accurately, so the reference substance solution is preferable, and the sample application amounts of the reference medicinal material solution and the test sample solution are respectively 10 mu l, 20 mu l and 20 mu l.
(2) Investigation of specificity
Test solution, control solution and control solution were prepared in the same manner as in example 1.
Angelica sinensis Liuhuang Shang Huangqi single positive control solution: 4g of Danggui Liuhuang Shang Huangqi single positive lyophilized powder is used to replace the test sample, and Danggui Liuhuang Shang Huangqi single positive control solution is prepared according to the preparation method of the test sample solution of example 1.
Angelica sinensis Liuhuang Shang Huangqi single negative control solution: taking 4g of Danggui Liuhuang Shang Huangqi single negative lyophilized powder to replace a test sample, and preparing an Danggui Liuhuang Shang Huangqi single negative control solution according to the preparation method of the test sample solution of the embodiment 1.
Sucking the sample solution, the reference substance solution, the astragalus control medicinal solution, the angelica six yellow Shang Huangqi single negative control solution and the angelica six yellow Shang Huangqi single positive control solution, respectively spotting on the same thin layer plate, and using ethyl acetate: n-propanol: spreading with water (7:5:4) as spreading agent, air drying, spraying 10% sulfuric acid ethanol solution, heating to color spots, and inspecting under visible light. Wherein, the preparation method of the test solution, the reference solution and the astragalus root reference medicinal material solution is the same as that of the embodiment 1. The results are shown in FIG. 8.
The result shows that the thin layer chromatography of the test solution and the reference solution show spots with the same color at the same position, and the spots also correspond to the two spots in the reference solution. The spot Rf values are moderate and negative without interference. The method has good specificity.
(3) Investigation of the development temperature and humidity
A. Sample solutions, control medicinal material solutions and control solutions are prepared and spotted according to the method in the embodiment 1, and are developed under the conditions of 15.2 ℃ and 20% humidity and 5.0 ℃ and 82% humidity, and the rest process conditions are the same as the embodiment 1, so that the method has good adaptability to different temperatures, and the results are shown in figures 9 and 10.
B. The sample solution, the control medicinal material solution and the control solution are prepared and spotted according to the method in the embodiment 1, and the rest process conditions are the same as the embodiment 1 under the humidity environments of 15-30% and 75-90%, respectively, and the result shows that the method has good adaptability to different humidity, and the results are shown in figures 11 and 12.
(4) Investigation of different lamina plates
Preparing and spotting a sample solution, a control medicinal material solution and a control solution according to the method in the embodiment 1, respectively, adopting different thin-layer plates, and adopting the rest process conditions as in the embodiment 1; the results show that the method has better adaptability to different thin-layer plates, and the results are shown in figures 13-15.
(5) Investigation of different developing Agents
The same batch of 20. Mu.l of the sample solution, 20. Mu.l of the reference substance solution and 10. Mu.l of the astragalus control medicinal solution in example 1 were pipetted onto the same thin layer plate, respectively, with ethyl acetate: n-propanol: spreading with water (6:6:5) as spreading agent, air drying, spraying 10% sulfuric acid ethanol solution, heating to color spots, and inspecting under visible light. Wherein, batch number of test article: JF-DGLH-DG2020001. The results are shown in FIG. 16, 1. Sample solution, 2. Astragaloside IV control solution, 3. Astragalus control medicinal solution.
20 μl of the sample solution prepared in the same batch of example 1, 20 μl of the control solution and 10 μl of the astragalus control solution are sucked, and the sample solution, the control solution and the astragalus control solution are respectively spotted on the same thin layer plate, and ethyl acetate is used for preparing the sample solution: n-propanol: spreading with water (8:6:4) as spreading agent, air drying, spraying 10% sulfuric acid ethanol solution, heating to color spots, and inspecting under visible light. Wherein, batch number of test article: JF-DGLH-DG2020001. The results are shown in FIG. 17, 1. Astragaloside IV reference solution, 2. Test solution, 3. Astragalus control solution.
As can be seen from fig. 9, 16 and 17, the chromatogram of the sample solution with the 3 developing agent ratios shows spots of the same color at the same positions as the chromatogram of the control medicinal solution and the chromatogram of the control solution.
Example 4
This example provides verification of a thin layer authentication method, specifically including,
taking 6 batches of angelica six-yellow Shang Dong dry powder as a test product, preparing a test product solution according to the method of the embodiment 1, respectively preparing a control medicinal material solution and a control product solution according to the method of the embodiment 1, taking astragalus negative control medicinal material, preparing astragalus negative control solutions according to the same method of the embodiment 1 of the test product solution, respectively taking 20 mu L of each of the test product solutions of different batches and 20 mu L of the control medicinal material solution, 10 mu L of the control product solution and 10 mu L of the astragalus negative control solution, respectively spotting the control medicinal material solution and the control solution on the same thin-layer plate, and using ethyl acetate: n-propanol: spreading with water (7:5:4) as spreading agent, air drying, spraying 10% sulfuric acid ethanol solution, heating to color spots, and inspecting under visible light.
According to the display of fig. 18, the chromatogram of the test solution shows spots with the same color at the same position as the chromatogram of the control solution, and the color spectrum of the control solution shows spots with the same color, the span is moderate, and 3 characteristic spots are provided, so that no negative interference exists, which indicates that the identification method provided by the invention has good stability.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (10)

1. A thin layer identification method of Chinese angelica six-yellow decoction and a compound preparation thereof is characterized by comprising the following steps,
taking a sample and astragalus control medicinal materials, and preparing a sample solution and a control medicinal material solution according to at least one of the following methods 1 and 2 respectively:
method 1: dissolving the sample and radix astragali reference materials in alkaline solution respectively, performing first solid-liquid separation, adding n-butanol into the absorbed liquid for mixing, performing second solid-liquid separation, absorbing n-butanol solution layer, adding water for mixing, performing third solid-liquid separation to obtain n-butanol solution layer, drying, dissolving in alcohol, and respectively preparing sample solution and reference medicinal material solution;
method 2: extracting the sample and radix astragali reference materials with alcohol respectively, separating solid and liquid, drying, dissolving in water, extracting with water saturated n-butanol, washing n-butanol extractive solution with alkaline solution, washing with n-butanol saturated water, collecting n-butanol solution, drying, dissolving in alcohol, and respectively preparing into sample solution and reference medicinal material solution;
and (3) identification: sample the sample solution, control medicinal material solution, and astragaloside IV control solution on the same thin layer plate, spreading with mixed solution containing ethyl acetate, n-propanol and water as spreading agent, taking out, air drying, spraying with color developing agent, heating, and inspecting under visible light.
2. The method of claim 1, wherein the thin layer plate used in the step of identifying is a silica gel G thin layer plate; and/or heating at 95-105deg.C for 3-5min; and/or the sample application amount of the sample solution, the control medicinal material solution or the astragaloside IV control substance solution is 1-20 mu L.
3. The thin layer identification method according to claim 1 or 2, wherein the color developing agent is sulfuric acid ethanol solution, preferably, the concentration of the sulfuric acid ethanol solution is 5-15%.
4. A thin layer authentication method according to any one of claims 1-3, wherein method 1 further satisfies one or more of the following a-H:
A. the volume ratio of the mass of the sample or the reference medicinal material to the alkaline solution is 0.5-6:15, preferably 2-4:15, wherein the ratio relationship of the mass to the volume is g/mL;
B. the first solid-liquid separation, the second solid-liquid separation or the third solid-liquid separation is centrifugal or filtering;
C. the volume ratio of the mass of the test sample or the reference medicinal material to the n-butanol is 0.5-6:5-30, preferably 2-4:10, wherein the ratio relationship of the mass to the volume is g/mL;
D. the volume ratio of the n-butanol to the water is 5-30:5-30, preferably 1:1;
E. in the dissolving step, the volume ratio of the mass of the test sample or the reference medicinal material to the alcohol is 0.5-6:0.5-3, preferably 2-4:1, wherein the proportioning relation of the mass and the volume is g/mL;
F. the mass volume concentration of the alkaline solution is 0.01-52g/100mL, preferably 0.01-4g/100mL;
G. the alcohol comprises one or more of methanol and ethanol; preferably methanol;
H. the alkaline solution comprises one or more of sodium hydroxide aqueous solution and potassium hydroxide aqueous solution.
5. The thin layer authentication method according to any one of claims 1 to 4, wherein method 2 further satisfies one or more of the following a-j:
a. in the alcohol extraction step, the ratio of the mass of the test sample or the reference medicinal material to the volume of alcohol and the volume of water is 0.5-6:20-40:10-30, preferably 2-4:30:20, wherein the ratio relationship of the mass to the volume is g/mL;
b. the solid-liquid separation is centrifugation or filtration;
c. extracting with alcohol for at least 15min by ultrasonic extraction or thermal reflux extraction;
d. the times of water saturated n-butanol extraction are 1-5 times, and the volume ratio of the mass of the test or control medicinal material to the water saturated n-butanol added in each extraction is 0.5-6:20, a step of; preferably 2-4:20, wherein the proportioning relation between the mass and the volume is g/mL;
e. the number of times of water washing of the alkaline solution or the n-butanol saturation is 1-5 times, and the volume ratio of the mass of the test sample or the control medicinal material to the alkaline solution or the n-butanol saturation water added during each washing is 0.5-6:20, a step of; preferably 2-4:20, wherein the ratio relationship of the mass to the volume is g/mL;
f. in the dissolving step, the volume ratio of the mass of the test sample or the reference medicinal material to the alcohol is 0.5-6:0.5-3, preferably 2-4:1, wherein the proportioning relation of the mass and the volume is g/mL;
g. the mass volume concentration of the alkaline solution is 0.01-52g/100mL, preferably 0.01-4g/100mL;
h. the alcohol comprises one or more of methanol and ethanol; preferably methanol;
j. the alkaline solution comprises one or more of sodium hydroxide aqueous solution and potassium hydroxide aqueous solution.
6. The thin layer identification method according to any one of claims 1 to 4, wherein the volume ratio of ethyl acetate, n-propanol and water in the developing agent is 5 to 10:1-8:1-6; preferably 6-8:6:4-5; more preferably 7:5:4.
7. the method for identifying thin layers according to any one of claims 1 to 4, wherein the solvent of the reference solution is selected from methanol or an aqueous methanol solution having a volume concentration of 80% or more; and/or, each 1mL of the reference substance solution contains 0.1-4 mg of astragaloside IV reference substance.
8. The thin layer identification method according to any one of claims 1 to 6, wherein the preparation method of the test solution comprises the steps of taking 0.5 to 6g of test powder, adding 15mL of alkaline solution with mass volume concentration of 0.01 to 4g/100mL, dissolving, centrifuging, absorbing supernatant, adding 5 to 30mL of n-butanol into the supernatant, shaking up, centrifuging, absorbing an n-butanol solution layer, adding 5 to 30mL of water, shaking up, centrifuging to obtain an n-butanol solution layer, evaporating to dryness, and adding 0.5 to 3mL of methanol for dissolving to obtain the test solution; or, taking 0.5-6g of test sample powder, adding 20-40mL of methanol, carrying out ultrasonic treatment for 15-60 minutes, filtering, evaporating filtrate to dryness, adding 10-30mL of water into residues for dissolution, extracting 1-5 times with water-saturated n-butanol, 20mL each time, combining n-butanol extract, washing 1-5 times with alkaline solution with the mass volume concentration of 0.01-4g/100mL, 20mL each time, washing 1-5 times with water-saturated n-butanol, 20mL each time, evaporating n-butanol solution to dryness, and adding 0.5-3mL of methanol into residues for dissolution to serve as a test sample solution;
the preparation method of the control medicinal material solution comprises the steps of taking 0.5-6g of astragalus mongholicus control medicinal material, adding 15mL of alkaline solution with mass volume concentration of 0.01-4g/100mL, dissolving, centrifuging, absorbing supernatant, adding 5-30mL of n-butanol into the supernatant, shaking up, centrifuging, absorbing an n-butanol solution layer, adding 5-30mL of water, shaking up, centrifuging to obtain an n-butanol solution layer, evaporating, and adding 0.5-3mL of methanol for dissolving to obtain the control medicinal material solution; or, taking 0.5-6g of astragalus control medicinal material, adding 20-40mL of methanol, carrying out ultrasonic treatment for 15-60 minutes, filtering, evaporating filtrate to dryness, adding 10-30mL of water into residues for dissolution, extracting with water-saturated n-butanol for 1-5 times, 20mL each time, combining n-butanol extract, washing with alkaline solution with the mass volume concentration of 0.01-4g/100mL for 1-5 times, 20mL each time, washing with water-saturated n-butanol for 1-5 times, 20mL each time, evaporating n-butanol liquid for dryness, and adding 0.5-3mL of methanol into residues for dissolution to serve as control medicinal material solution.
9. The method for identifying thin layers according to any one of claims 1 to 7, wherein the preparation method of the test solution comprises the steps of taking 2 to 4g of the test powder, adding 15mL of sodium hydroxide aqueous solution with mass volume concentration of 0.5 to 2g/100mL, dissolving, centrifuging, absorbing supernatant, adding 10mL of n-butanol into the supernatant, shaking up, centrifuging, absorbing an n-butanol solution layer, adding 10mL of water, shaking up, centrifuging to obtain an n-butanol solution layer, evaporating to dryness, and adding 1mL of methanol for dissolving to obtain the test solution; or, taking 2-4g of test sample powder, adding 30mL of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, adding 20mL of water into residues for dissolution, extracting 2 times with water-saturated n-butanol, 20mL each time, combining n-butanol extract solutions, washing 2 times with sodium hydroxide aqueous solution with the mass volume concentration of 0.5-2g/100mL each time, 20mL each time, washing 2 times with water-saturated n-butanol, 20mL each time, evaporating n-butanol solution to dryness, and adding 1mL of methanol into residues for dissolution to serve as a test sample solution;
the preparation method of the control medicinal material solution comprises the steps of taking 2-4g of astragalus mongholicus control medicinal material, adding 15mL of sodium hydroxide aqueous solution with mass volume concentration of 0.5-2g/100mL, dissolving, centrifuging, absorbing supernatant, adding 10mL of n-butanol into the supernatant, shaking uniformly, centrifuging, absorbing an n-butanol solution layer, adding 10mL of water, shaking uniformly, centrifuging to obtain an n-butanol solution layer, evaporating to dryness, and adding 1mL of methanol for dissolving to obtain the control medicinal material solution; or, taking 2-4g of astragalus control medicinal material, adding 30mL of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, dissolving residues in 20mL of water, extracting with water-saturated n-butanol for 2 times, 20mL each time, combining n-butanol extract solutions, washing with sodium hydroxide aqueous solution with the mass volume concentration of 0.5-2g/100mL for 2 times, 20mL each time, washing with water saturated with n-butanol for 2 times, 20mL each time, evaporating n-butanol solution to dryness, dissolving residues in methanol l mL, and taking the residues as control medicinal material solution.
10. The method for thin layer identification according to any one of claims 1 to 9, wherein the test substance is a solid preparation, a liquid preparation or a semisolid preparation of angelica sinensis, preferably a powder, a granule, a tablet, a capsule, an oral liquid, an injection, an emulsion, a suspension or an ointment.
CN202311189767.9A 2023-09-14 2023-09-14 Thin layer identification method of angelica sinensis six-yellow decoction and compound preparation thereof Pending CN117420253A (en)

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