CN110702834A - Non-interference rapid thin-layer identification method for kidney-tonifying toxin-expelling granules - Google Patents
Non-interference rapid thin-layer identification method for kidney-tonifying toxin-expelling granules Download PDFInfo
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Abstract
The invention relates to an interference-free rapid thin-layer identification method for kidney-tonifying toxin-expelling granules. The method is characterized in that: although the granules are compound preparations consisting of 11 medicinal materials, 5 medicinal materials are not extracted and purified, 70% methanol ultrasonic supernatant solution is directly adopted, the same test solution is adopted, and the interference of other components can be eliminated through the polarity, the acidity and alkalinity of a developing agent, the selectivity of a color developing agent and the limitation of inspection conditions, so that the information spots of a reference substance and the reference medicinal materials are only presented or the spot definition is improved; removing impurities from the other 3 medicinal materials by secondary chromatographic column, eluting with nontoxic ethanol to obtain test solution, and detecting on the same thin layer plate; the method is simple, convenient, fast, environment-friendly, clear in spot and free of interference, and the identification subscription-increasing rate is 72.7%. Wherein, the same plate thin layer identification of the giant knotweed and the dandelion is carried out; spraying 10% sulfuric acid ethanol solution to excite only epimedium component to show high selectivity of bright blue fluorescence; is the first report.
Description
Technical Field
The invention relates to an interference-free rapid thin-layer identification method for kidney-tonifying toxin-expelling granules. Although the granule is a compound preparation consisting of 11 medicinal materials, 5 medicinal materials do not need extraction and impurity removal, 70% methanol ultrasonic supernatant solution is directly adopted, and the mutual matching is proper through the polarity of developing agent, acidity and alkalinity, the selectivity of color developing agent and the limitation of inspection condition, so that the interference of other components can be eliminated, and only the information spots of a reference substance and the reference medicinal material are presented or the spot definition is improved; and removing impurities from the other 3 medicinal materials by secondary chromatographic column, eluting with nontoxic ethanol to obtain test solution, and detecting on the same thin layer plate. The method is simple, convenient, fast, environment-friendly, clear in spot and free of interference.
Background
The so-called interference-free rapid thin layer identification method is characterized in that the interference of a plurality of other components in the compound preparation can be eliminated through the polarity and the composition proportion of the developing agent, the acid-base property of the developing agent, the selectivity of the color developing agent and the limitation of detection conditions, and the interference is properly matched, so that the test solution only presents information spots of a reference substance or a reference medicinal material or the spot definition is improved; and the method is simple, convenient and fast, environment-friendly and clear in spots.
As is known, in the case of a compound preparation consisting of a plurality of medicinal materials, due to the fact that a plurality of chemical components of the medicinal materials are subjected to thin-layer identification, components with similar properties are present in the same developing agent, interference on components to be detected is caused, even a plurality of spots cannot be separated, and the thin-layer identification cannot be carried out. The more the medicines composing the prescription are, the more the thin-layer identification is increased, the more the difficulty is, and the more the interference components to be judged need to be eliminated according to the chemical properties of the components to be detected. For example, an alkaline component such as an alkaloid is salified in the presence of an acid, dissolved in an aqueous acid solution, extracted with an organic solvent to remove fat-soluble impurities, dissociated in the presence of a base, adjusted to be alkaline in the aqueous acid solution, and then the dissociated alkaloid is extracted with an organic solvent to eliminate interference of water-soluble components. The thin-layer identification of acid components, such as cholic acid in infantile ZHIBAO pill, comprises salifying cholic acid with alkali, dissolving in aqueous alkali solution, extracting with organic solvent to remove fat-soluble impurities, dissociating with acid, adjusting the aqueous alkali solution to acidity, extracting with organic solvent to free cholic acid, and removing interference of water-soluble components; the operation is carried out twice, and interference components are eliminated. Neutral components, such as astragaloside IV thin layer identification in TIANDAN TONGLUO Capsule, are extracted by methanol under reflux, the extractive solution is evaporated to dryness, dissolved in water, chloroform is used for extracting liposoluble impurities, n-butanol is used for extracting astragaloside IV, the extractive solution is washed with alkaline water solution for 3 times, and acid components and water soluble components interference in the extractive solution are further removed. Etc. in the preparation of the test article, a clear thin layer of spots is obtained by removing numerous interfering components through repeated solvent transfer using a similar compatible extraction principle.
Therefore, a finished preparation, particularly more than ten prescriptions, which is collected in the first edition of the Chinese pharmacopoeia 2015, appears, the collection rate of thin-layer identification is not up to 40% on average, even if the individual variety reaches more than 40%, the thin-layer identification is also a variety, and if N identifications exist, N sample solutions are prepared, N thin-layer plates are developed for N times, and the traditional identification mode for identifying N medicinal materials is realized. In order to eliminate interference, the pretreatment procedure of the sample is complex and troublesome, a large amount of organic reagents are needed for repeated purification treatment, the labor and time are wasted, the reagents are wasted, the environment is polluted, the health is harmed, and the detection period is long. The method for identifying the Dalitong particle thin layer recorded in the first edition of the Chinese pharmacopoeia 2015 is taken as an example and is clear at a glance. The Dalitong granules are prepared from 12 traditional Chinese medicines, 7 thin-layer identifications are collected under the standard item, the collection rate is 58%, and the specific method comprises the following steps:
[ IDENTIFICATION ] collecting 10g of this product, grinding, adding 20ml of methanol, subjecting to ultrasonic treatment for 30 min, filtering, evaporating the filtrate to dryness, dissolving the residue with 60ml of n-butanol saturated with water, shaking and extracting with 1% sodium hydroxide solution for 3 times (20 ml each time), discarding the sodium hydroxide solution, washing the n-butanol solution with 50ml of water saturated with n-butanol, discarding the water solution, evaporating the n-butanol solution to dryness, and dissolving the residue with 1ml of methanol to obtain a sample solution. Taking 2g of bupleurum root as a reference medicinal material, adding 100ml of water, decocting for 1 hour, filtering, evaporating filtrate to dryness, adding 10ml of ethanol into residues for dissolving, filtering, evaporating filtrate to dryness, adding 20ml of methanol into residues, and preparing a reference medicinal solution by the same method from ultrasonic treatment for 30 minutes. Performing thin layer chromatography (general rule 502) test, sucking 10 μ l of each of the 2 solutions, respectively dropping on the same silica gel G thin layer plate, developing with ethyl acetate-ethanol-water (8: 2: 1) as developing agent, taking out, air drying, spraying 2% p-dimethylaminobenzaldehyde 40% sulfuric acid solution, heating at 60 deg.C until the spots are clearly developed, and inspecting under sunlight. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the reference solution.
(2) Grinding 12g of the product, adding 50ml of methanol, performing ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, dissolving residue in 20ml of water, extracting with ethyl acetate for 2 times, each time extracting for 20ml, mixing ethyl acetate solutions, evaporating to dryness, and dissolving residue in 1ml of methanol to obtain a sample solution. Adding methanol into dehydrocostus lactone reference substance to obtain solution containing 0.5mg per 1ml as reference substance solution. Performing thin layer chromatography (general rule 502) test, sucking 10 μ l of sample solution and 5 μ l of reference solution, respectively dropping on the same silica gel G thin layer plate, developing with upper layer solution of cyclohexane-ethyl formate-formic acid (15: 5: 1) as developing agent, taking out, air drying, spraying 1% vanillin sulfuric acid solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under sunlight. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.
(3) Collecting 12g of the product, grinding, adding 40ml of ethanol, heating and refluxing for 30 min, filtering, evaporating filtrate, dissolving residue in 10ml of water, shaking and extracting with diethyl ether for 2 times (15ml, 10ml), discarding ethyl ether solution, shaking and extracting with water saturated n-butanol for 3 times (15ml, 10ml, 10ml), mixing n-butanol solutions, evaporating to dryness, dissolving residue in 2ml of methanol to obtain sample solution. Adding methanol into hesperidin control to obtain solution containing 0.2mg per 1ml as control solution. Performing thin layer chromatography (general rule 502) test, sucking 1 μ l of sample solution and 4 μ l of reference solution, respectively dropping on the same polyamide film to form strips, developing with chloroform-acetone-methanol (5: 1) as developing agent, taking out, air drying, spraying 2% aluminum trichloride ethanol solution, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test solution, fluorescent stripes with the same color appear at the corresponding positions of the chromatogram of the reference solution.
(4) Taking 12g of the product, grinding, adding 50ml of methanol, carrying out ultrasonic treatment for 20 minutes, filtering, evaporating filtrate to dryness, adding 20ml of water into residue to dissolve the residue, adjusting the pH value to 2-3 by using dilute hydrochloric acid, shaking and extracting by using ethyl acetate for 2 times, wherein 20ml of water is added for each time, combining ethyl acetate solutions, evaporating to dryness, and adding 2ml of methanol into residue to dissolve the residue to obtain a sample solution. The caffeic acid control solution is prepared by adding methanol to obtain a solution containing 0.5mg per 1 ml. Performing thin layer chromatography (general rule 502) test, sucking sample solution 5 μ l and reference solution 2 μ l, respectively dropping on the same silica gel G thin layer plate, developing with butyl acetate-formic acid-water (7: 2.5) upper layer solution as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). The test chromatogram shows fluorescent spots of the same color at the positions corresponding to those of the control chromatogram.
(5) Taking 2g of hawthorn control medicinal material, adding 100ml of water, decocting for 1 hour, filtering, concentrating the filtrate to 20ml, adjusting the pH value to 1-2 by using dilute hydrochloric acid, shaking and extracting for 2 times by using ethyl acetate, wherein 20ml of ethyl acetate is used for each time, combining ethyl acetate solutions, evaporating to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain the control medicinal material solution. Performing thin layer chromatography (general rule 502) test, collecting 10 μ l of test solution and 30 μ l of control solution under [ identification ] item (2), respectively dropping on the same silica gel G thin layer plate, developing with cyclohexane-ethyl acetate-formic acid (20: 1) as developing agent, taking out, air drying, spraying with 2% ferric trichloride ethanol solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under sunlight. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the reference solution.
(6) 30g of the product is taken, ground, added with 7.5ml of concentrated ammonia test solution and 100ml of trichloromethane, ultrasonically treated for 30 minutes, filtered, added with 10ml of diluted hydrochloric acid and 20ml of water into the filtrate, shaken, separated into acid water solution, added with concentrated ammonia test solution to adjust the pH value to about 9, shaken and extracted for 2 times with 20ml of trichloromethane each time, the trichloromethane solution is combined, volatilized to be dry, and 0.5ml of methanol is added into the residue to be dissolved to be used as test solution. Taking 1g of Arecae semen as reference material, adding concentrated ammonia solution 3ml and chloroform 20ml, and preparing the reference material solution by the same method from "ultrasonic treatment for 30 minutes". Performing thin-layer chromatography (general rule 502) test, collecting 40 μ l sample solution and 20 μ l reference material solution, respectively dropping on the same silica gel G thin-layer plate, developing with toluene-chloroform-methanol-concentrated ammonia solution (10: 4: 1.5: 0.1) as developing agent, taking out, air drying, spraying diluted bismuth potassium iodide solution, and inspecting under sunlight. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.
(7) Taking 20g of the product, grinding, adding 5ml of concentrated ammonia test solution and 60ml of chloroform, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a test solution. Adding methanol into tetrahydropalmatine control to obtain 1mg solution per 1ml, and making into control solution. Performing thin layer chromatography (general rule 502) test, sucking sample solution 40 μ l and reference solution 2 μ l, respectively dropping on the same silica gel G thin layer plate prepared with 1% sodium hydroxide solution, developing with cyclohexane-ethyl acetate (3: 2) as developing agent, taking out, air drying, fumigating with iodine vapor until the color of spot is clear, volatilizing iodine adsorbed on the plate, and inspecting under ultraviolet lamp (365 nm). The test chromatogram shows fluorescent spots of the same color at the positions corresponding to those of the control chromatogram.
In the thin-layer identification of 7 medicinal materials in the above example, 7 sample solutions are prepared, 7 thin-layer plates are unfolded 7 times, and according to the specified operation protocol, only the sample pretreatment time is 20 hours, 96g of sample is needed, 760ml of organic extraction solvent is needed, 70ml of developing agent and 3 hours of unfolding time are added, and the thin-layer identification of 1 batch of samples takes 3 days and 830ml of organic solvent. The premise is that the identification of the four medicinal materials is carried out by taking a reference substance as a reference, if the reference medicinal materials are added, the sample processing time and the cost of the solvent are more, and the reference substance is used as a reference, so that the information quantity is single, and the quality supervision is not facilitated.
If the method meets the retest, the detection time and the organic reagent are doubled, and the detection speed cannot be matched with the mechanized mass production. Therefore, a simple and convenient and rapid detection method is found, the detection efficiency is improved, the detection cost is reduced, the environmental pollution is reduced, the spot definition is improved, the goal of detection personnel to struggle is achieved, and under the background condition, the research on an interference-free rapid thin-layer identification method is carried out by taking kidney-tonifying toxin-expelling particles as research objects, and the success is achieved.
The weight ratio of the kidney-tonifying toxin-expelling granules is as follows:
the preparation process comprises the following steps: sterilizing Ginseng radix and Notoginseng radix with flowing steam, drying, and pulverizing into fine powder; extracting herba Epimedii, herba Taraxaci, rhizoma Polygoni Cuspidati, and radix astragali with 70% ethanol for 2 times, each for 2.5 hr, filtering, mixing filtrates, and recovering ethanol; fructus Psoraleae (parched), semen Cuscutae, fructus Lycii, rehmanniae radix, and radix Puerariae by extracting with water for 2 times, each for 2.5 hr, filtering, mixing filtrates, concentrating at 70 deg.C under reduced pressure to obtain fluid extract with relative density of 1.10-1.15, mixing with ethanol extract, concentrating at 70 deg.C under reduced pressure to obtain soft extract with relative density of 1.25-1.30, adding the fine powder, and drying under reduced pressure. Mixing the dry extract, a proper amount of dextrin and steviosin, pulverizing, granulating with appropriate amount of ethanol, drying, and making into 1000g granule.
Disclosure of Invention
An interference-free rapid thin-layer identification method of the kidney-tonifying toxin-expelling granules is established. Namely, although the granules are compound preparations consisting of 11 medicinal materials, 5 medicinal materials do not need extraction and impurity removal, 70% methanol ultrasonic supernatant solution and the same test solution are directly adopted, and are matched appropriately through the polarity of developing agents, the acidity and alkalinity, the selectivity of color developing agents and the limitation of inspection conditions, so that the interference of other components can be eliminated, and only information spots of reference products and reference medicinal materials are presented or the definition of the spots is improved; and removing impurities from the other 3 medicinal materials by secondary chromatographic column, eluting with nontoxic ethanol to obtain test solution, and detecting on the same thin layer plate. The method is simple, convenient, fast, environment-friendly, clear in spot and free of interference. Wherein, the same plate thin layer identification of the giant knotweed and the dandelion is carried out; spraying 10% sulfuric acid ethanol solution to excite only epimedium component to show high selectivity of bright blue fluorescence; is the first report.
The technical scheme adopted by the invention for solving the technical problems is as follows:
(1) taking 1g of sample, grinding, adding 2ml of 70% methanol, carrying out ultrasonic treatment for 10 minutes, centrifuging, and taking supernate as a test sample solution; taking appropriate amount of psoralen and isopsoralen as reference substances, and adding methanol to obtain solutions containing 3mg of psoralen and isopsoralen per 1ml as reference substance solutions; taking 0.2g of fructus psoraleae as a reference medicinal material, adding 2ml of 70% methanol, carrying out ultrasonic treatment for 15 minutes, centrifuging, and taking supernate as a reference medicinal material solution; respectively taking 5-8 mul of a reference solution and a reference medicinal material solution and 8-10 mul of a test solution, respectively dropping the solutions on the same silica gel G thin-layer plate, developing by using n-hexane-ethyl acetate as a developing agent in a volume ratio of 4: 2, taking out, airing, spraying a 2% potassium hydroxide ethanol solution, drying by hot air, placing under an ultraviolet lamp 365nm for inspection, and respectively displaying fluorescent spots with the same color on positions corresponding to the chromatograms of the reference and reference medicinal materials in the chromatogram of the test solution;
(2) taking appropriate amount of emodin reference substance, adding methanol to obtain solution containing 0.3mg per 1ml as reference substance solution; taking 0.5g of dandelion as a reference medicinal material and 0.2g of polygonum cuspidatum as a reference medicinal material, respectively adding 2ml of 70% methanol, carrying out ultrasonic treatment for 20 minutes, centrifuging, and taking supernate as respective reference medicinal material solutions; sucking 3-5 mul of a reference solution, 10 mul of a dandelion reference solution, 5 mul of a polygonum cuspidatum reference solution and 5-6 mul of a test solution under the item (1), respectively dropping the solutions on the same silica gel G thin-layer plate, developing by using cyclohexane-ethyl acetate-acetone-formic acid as a developing agent in a volume ratio of 10: 3: 2: 0.5, taking out, drying in the air, placing the developed developing agent under an ultraviolet lamp of 365nm, and displaying the same yellow fluorescent spots in the chromatogram of the test solution at positions corresponding to the chromatograms of the reference solution and the polygonum cuspidatum reference solution; spraying 3% aluminum trichloride ethanol solution, and inspecting under 365nm ultraviolet lamp to obtain bright blue fluorescent main spot in the chromatogram of the test sample at the position corresponding to the chromatogram of the herba Taraxaci control material;
(3) taking appropriate amount of puerarin as reference substance, adding methanol to obtain solution containing 0.1mg per 1ml as reference substance solution; taking 0.1g of radix Puerariae as a reference medicinal material, adding 2ml of methanol, performing ultrasonic treatment for 20 minutes, centrifuging, and taking supernate as a reference medicinal material solution; sucking 4 μ l of a reference medicinal material solution, 5-6 μ l of a reference substance solution, and 3-5 μ l of a test solution under the item (1), respectively dropping on the same silica gel G thin layer plate, developing by using chloroform-ethyl acetate-methanol-concentrated ammonia with the volume ratio of 4.1: 1: 4.2: 0.8 as a developing agent, taking out, drying in the air, and placing under an ultraviolet lamp 365nm for inspection, wherein in the chromatogram of the test solution, fluorescent main spots with the same color are respectively displayed at positions corresponding to the chromatograms of the reference substance and the reference medicinal material;
(4) taking appropriate amount of icariin as control, adding methanol to obtain solution containing 2mg per 1ml as control solution; taking 0.2g of herba epimedii control medicinal material, adding 2ml of methanol, carrying out ultrasonic treatment for 20 minutes, centrifuging, and taking supernate as a control medicinal material solution; respectively taking 3-5 mul of each of a reference solution and a reference medicinal material solution, respectively dropping 2-3 mul of the test solution under the item (1) on the same silica gel G thin-layer plate, taking ethyl acetate-butanone-formic acid-water with the volume ratio of 10: 1 as a developing agent, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until spots are clearly developed, viewing under 365nm ultraviolet light, and respectively displaying main fluorescent spots with the same color on positions corresponding to the chromatograms of the reference and the reference medicinal materials in the chromatogram of the test solution;
(5) taking 2.5g of the product, grinding, adding 20ml of 70% methanol, carrying out ultrasonic treatment for 20 minutes, filtering, evaporating the methanol from the filtrate in a water bath, passing the residual aqueous solution through a D101 type macroporous resin (the inner diameter is 1.5cm, the column height is 12cm), eluting with 50ml of water and then 50ml of 20% ethanol, discarding the eluent, eluting with 80ml of 70% ethanol, collecting the eluent, evaporating to dryness, transferring the residue with 2ml of ethanol to an alkaline alumina column (the inner diameter is 1cm, the alumina is 1.5g), eluting with 50ml of 80% ethanol, evaporating to dryness the eluent, and dissolving the residue with 1ml of methanol to obtain a sample solution; taking appropriate amount of ginsenoside Rg1, Re and astragaloside IV as reference substances, adding methanol to obtain solutions each containing 1mg per 1ml as reference substance solution; sucking 3-4 μ l of each reference solution and 3-5 μ l of each test solution, respectively spotting the reference solutions and the test solutions on the same silica gel G thin-layer plate in strips, placing layered dichloromethane-methanol-water (17: 7: 2) lower layer solution at 10 ℃ as a developing agent, developing, taking out, drying, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until the spots are clearly developed, and respectively developing strip spots with the same color on positions corresponding to the reference chromatogram in the test solution chromatogram.
The principle of the invention is as follows:
firstly, according to the difference of chemical structures and polarities of effective components of the traditional Chinese medicine, along with the movement of a developing agent, the adsorption and desorption capacities on a thin layer plate are different, the interference of components with different polarities is eliminated, and component spots with close polarities are separated; then, by means of the characteristics of the developing agent that the acid adsorbs alkaline substances and the alkali adsorbs acidic substances, the adsorbed components are retained at the bottom of the thin-layer plate and are not developed along with the developing agent, so that the interference of the acid and alkaline components is eliminated; finally, the color developing selectivity of the color developing agent to the compound and the limitation of the inspection condition are utilized to eliminate the interference of components which are not selected and inspected; through the combined interference elimination measures, the traditional operation steps of extracting and removing impurities by using an organic solvent, evaporating the extract to dryness and polluting the environment are avoided, the resolution of information spots or spots only showing a reference substance or a reference medicinal material can be improved on the developed thin-layer plate by directly adopting a 70% methanol ultrasonic supernatant solution, and the interference of a plurality of other medicinal components in the compound preparation is eliminated. The method is simple, convenient, rapid, environment-friendly and clear in spots.
The invention has the following innovation points and beneficial effects:
(1) the method breaks through the problems of repeated extraction and impurity removal of the solvent which is always adopted in the thin-layer identification, and elimination of interference of other components, and is tedious, time-consuming and environment-polluting. The new measure of thin layer identification and impurity removal is that the interference of various components which are not detected is eliminated by the polarity of the developing agent, the acidity and alkalinity, the selectivity of the color developing agent and the limitation of the inspection condition, and the sample application of the supernatant is realized only by using proper solvent ultrasound. Although the composition has a plurality of medicinal materials, the non-inspection components do not interfere the inspection components, and only the spots or the spot definition of the reference substance to be detected and the reference medicinal material is improved.
(2) Firstly, thin layer identification of psoralea fruit in a sample is carried out, n-hexane-ethyl acetate with volume ratio of 4: 2 is used as developing agent, polar components are controlled at the original point according to partial fat solubility of the developing agent, so that the polar components are not developed, then only the high selectivity of fluorescence enhancement of psoralen and isopsoralen and the limitation of fluorescence detection under the ultraviolet lamp 365nm are detected by means of 2% potassium hydroxide ethanol solution, a developed and developed thin layer plate is detected under the ultraviolet lamp 365nm, a test sample and a reference medicinal material only show fluorescence spots of psoralen and isopsoralen, and the components of other 10 medicinal materials are concealed or eliminated (figure 1). In addition, the non-toxic 2% potassium hydroxide ethanol solution replaces the 10% potassium hydroxide methanol solution with high toxicity, so that the strong base corrosivity of the color developing agent is reduced, and the safety is improved.
(3) Then thin-layer identification of giant knotweed and dandelion in a sample is carried out, wherein after cyclohexane-ethyl acetate-acetone-formic acid with the volume ratio of 10: 3: 2: 0.5 is unfolded, the medium polarity and the acidity of the unfolding agent are used for dragging the alkaline components, the acidic components in the giant knotweed and the dandelion are pushed, the unfolding agent is used for carrying out adsorption and desorption, so that the emodin in the giant knotweed is positioned in front of each spot, and the test sample shows 1 clear bright yellow fluorescent spot at the position corresponding to the chromatography of the giant knotweed control medicinal material and the emodin control by the inherent fluorescence and inspection under the ultraviolet lamp of 365 nm; the test sample presents 1 red fluorescent spot at the position corresponding to the chromatogram of the dandelion control drug, and can not be used as an identification characteristic point due to no exclusive use (figure 2); however, by means of the selectivity of 3% aluminum trichloride ethanol solution to flavonoids and the limitation of ultraviolet light 365nm inspection, a developed color thin-layer plate is inspected under the ultraviolet light 365nm, a test sample presents 1 clear bright blue fluorescent spot on the position corresponding to the color spectrum of the dandelion control drug, the original red spot of the dandelion is faded (figure 3), and the definition and specificity of the spot are improved. The simultaneous identification of giant knotweed rhizome and dandelion and the high selectivity of the aluminum trichloride ethanol solution to dandelion are first reported.
(4) The thin-layer identification of the kudzuvine root in a sample is realized by taking trichloromethane-ethyl acetate-methanol-concentrated ammonia with the volume ratio of 4.1: 1: 4.2: 0.8 as developing agent, adsorbing some components with high acidity by virtue of partial water solubility and alkalinity of the developing agent, keeping the components at the original point, not developing with the developing agent, only some water-soluble components with weak acidity and neutrality, and developing upwards with the developing agent, so that the developed thin-layer plate mainly presents main fluorescence spots of puerarin and kudzuvine root medicinal materials, and the components of other 10 medicinal materials are basically concealed or eliminated (figure 4).
(5) Thin-layer identification of herba Epimedii in sample is carried out by developing with ethyl acetate-butanone-formic acid-water at volume ratio of 10: 1, promoting only flavonoid components such as icariin in herba Epimedii with the aid of water solubility and acidity of developing agent, adsorbing and desorbing with developing agent, separating well, generating fluorescence effect on herba Epimedii with the aid of 10% sulfuric acid ethanol solution, allowing developed and developed thin-layer plate to show 6 bright blue fluorescence spots of icariin and herba Epimedii, and concealing or eliminating other 10 medicinal materials (FIG. 5). Compared with the map reported in the literature, the thin layer spots have greatly improved information content and definition (fig. 7 and 8). Spraying 10% ethanol sulfate solution to excite fluorescence of herba Epimedii component, and eliminating interference of other medicinal components in the prescription.
(6) The thin-layer identification of ginseng, pseudo-ginseng and astragalus root in the sample is characterized by avoiding the extraction and impurity removal of organic solvents polluting the environment, removing water-soluble components only by 2 chromatographic columns, namely D101 type macroporous resin columns, removing a large amount of acidic components by alkaline alumina columns, eluting by nontoxic aqueous ethanol, and evaporating the eluent to dryness to obtain a test solution. The layered lower layer solution of dichloromethane-methanol-water (17: 7: 2) is placed at 10 ℃ as a developing agent and is developed. The 10% sulfuric acid ethanol solution is used for developing color, simultaneously, the ginsenoside Rg1, Re and astragaloside IV in the sample are identified, the spots are clear, and no interference exists (figure 6). The method for preparing the test solution by using the ginseng, the pseudo-ginseng and the astragalus root to remove impurities only through a chromatographic column and eluting the impurities with nontoxic ethanol is reported for the first time.
(7) Except for ginseng, pseudo-ginseng and astragalus, the thin-layer identification of other 5 medicinal materials adopts the identification forms of a reference substance and a reference medicinal material, the reference substance determines the spot component, and the reference medicinal material improves the information content of detection. The identification rate of increase of order reaches 72.7 percent, which is higher than that of compound preparations with the same formula.
Drawings
FIG. 1 shows TLC pattern of fluorescent spots observed under 365nm UV light after spraying 2% KOH solution on the sample, psoralea fruit medicinal material, psoralen and isopsoralen.
FIG. 2 is a fluorescent TLC chart of samples, giant knotweed rhizome and emodin under 365nm ultraviolet lamp.
FIG. 3 is a TLC image of bright blue fluorescent spots of samples and dandelion herbs after being sprayed with 3% alchlor ethanol solution for color development and being inspected under an ultraviolet lamp of 365 nm.
FIG. 4 is a TLC image of bright blue fluorescence spots of samples, radix Puerariae and puerarin under 365nm UV light.
FIG. 5 is a TLC image of bright blue fluorescent spots of samples, epimedium herb materials and icariin, which are observed under an ultraviolet lamp of 365nm after being sprayed with 10% sulfuric acid ethanol solution for color development.
FIG. 6 is a spot TLC pattern of the sample and the control inspected in the sun after developing color by spraying 10% ethanol sulfate solution.
FIG. 7 is a TLC chart of UV 365nm inspection of Epimedium herb reported in literature [ 1 ].
FIG. 8 is a TLC image of 365nm ultraviolet light inspection after color development of epimedium vein relaxing pills by spraying aluminum trichloride.
In FIG. 1, 2 and 3 are samples; 4. fructus Psoraleae; 5. psoralen; 6. isopsoralen; 7. blank.
FIGS. 2 and 3 are chromatograms of the same thin-layer plate under different inspection conditions, wherein 1 is blank of dandelion; 2. dandelion; 3. 4, 5, sampling; 6. giant knotweed rhizome; 7. emodin; 8. rhizoma Polygoni Cuspidati blank
Figure 5, 1 icariin; 2. herba Epimedii crude drug; 3. 4, 5 samples; 6. blank space
In fig. 6, 1, ginseng and pseudo-ginseng are blank; 2. radix astragali blank; 3. 4, 5, sampling; 6. ginsenoside Re; 7. astragaloside IV; 8. ginsenoside Rg 1.
1 in fig. 7, epimedium 161101; 2. epimedium 161102; 3. icariin 110737-
1, blank in fig. 8; 2. 3, 4, sampling; 5. icariin
The specific implementation mode of the invention is as follows:
(1) taking 1g of sample, grinding, adding 2ml of 70% methanol, carrying out ultrasonic treatment for 10 minutes, centrifuging, and taking supernate as a test sample solution; taking appropriate amount of psoralen and isopsoralen as reference substances, and adding methanol to obtain solutions containing 3mg of psoralen and isopsoralen per 1ml as reference substance solutions; taking 0.2g of fructus psoraleae as a reference medicinal material, adding 2ml of 70% methanol, carrying out ultrasonic treatment for 15 minutes, centrifuging, and taking supernate as a reference medicinal material solution; respectively taking 5-8 mul of a reference solution and a reference medicinal material solution and 8-10 mul of a test solution, respectively dropping the solutions on the same silica gel G thin-layer plate, developing by using n-hexane-ethyl acetate as a developing agent in a volume ratio of 4: 2, taking out, airing, spraying a 2% potassium hydroxide ethanol solution, drying by hot air, placing under an ultraviolet lamp 365nm for inspection, and respectively displaying fluorescent spots with the same color on positions corresponding to the chromatograms of the reference and reference medicinal materials in the chromatogram of the test solution;
(2) taking appropriate amount of emodin reference substance, adding methanol to obtain solution containing 0.3mg per 1ml as reference substance solution; taking 0.5g of dandelion as a reference medicinal material and 0.2g of polygonum cuspidatum as a reference medicinal material, respectively adding 2ml of 70% methanol, carrying out ultrasonic treatment for 20 minutes, centrifuging, and taking supernate as respective reference medicinal material solutions; sucking 3-5 mul of a reference solution, 10 mul of a dandelion reference solution, 5 mul of a polygonum cuspidatum reference solution and 5-6 mul of a test solution under the item (1), respectively dropping the solutions on the same silica gel G thin-layer plate, developing by using cyclohexane-ethyl acetate-acetone-formic acid as a developing agent in a volume ratio of 10: 3: 2: 0.5, taking out, drying in the air, placing the developed developing agent under an ultraviolet lamp of 365nm, and displaying the same yellow fluorescent spots in the chromatogram of the test solution at positions corresponding to the chromatograms of the reference solution and the polygonum cuspidatum reference solution; spraying 3% aluminum trichloride ethanol solution, and inspecting under 365nm ultraviolet lamp to obtain bright blue fluorescent main spot in the chromatogram of the test sample at the position corresponding to the chromatogram of the herba Taraxaci control material;
(3) taking appropriate amount of puerarin as reference substance, adding methanol to obtain solution containing 0.1mg per 1ml as reference substance solution; taking 0.1g of radix Puerariae as a reference medicinal material, adding 2ml of methanol, performing ultrasonic treatment for 20 minutes, centrifuging, and taking supernate as a reference medicinal material solution; sucking 4 μ l of a reference medicinal material solution, 5-6 μ l of a reference substance solution, and 3-5 μ l of a test solution under the item (1), respectively dropping on the same silica gel G thin layer plate, developing by using chloroform-ethyl acetate-methanol-concentrated ammonia with the volume ratio of 4.1: 1: 4.2: 0.8 as a developing agent, taking out, drying in the air, and placing under an ultraviolet lamp 365nm for inspection, wherein in the chromatogram of the test solution, fluorescent main spots with the same color are respectively displayed at positions corresponding to the chromatograms of the reference substance and the reference medicinal material;
(4) taking appropriate amount of icariin as control, adding methanol to obtain solution containing 2mg per 1ml as control solution; taking 0.2g of herba epimedii control medicinal material, adding 2ml of methanol, carrying out ultrasonic treatment for 20 minutes, centrifuging, and taking supernate as a control medicinal material solution; respectively taking 3-5 mul of each of a reference solution and a reference medicinal material solution, respectively dropping 2-3 mul of the test solution under the item (1) on the same silica gel G thin-layer plate, taking ethyl acetate-butanone-formic acid-water with the volume ratio of 10: 1 as a developing agent, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until spots are clearly developed, viewing under 365nm ultraviolet light, and respectively displaying main fluorescent spots with the same color on positions corresponding to the chromatograms of the reference and the reference medicinal materials in the chromatogram of the test solution;
(5) taking 2.5g of the product, grinding, adding 20ml of 70% methanol, carrying out ultrasonic treatment for 20 minutes, filtering, evaporating the methanol from the filtrate in a water bath, passing the residual aqueous solution through a D101 type macroporous resin (the inner diameter is 1.5cm, the column height is 12cm), eluting with 50ml of water and then 50ml of 20% ethanol, discarding the eluent, eluting with 80ml of 70% ethanol, collecting the eluent, evaporating to dryness, transferring the residue with 2ml of ethanol to an alkaline alumina column (the inner diameter is 1cm, the alumina is 1.5g), eluting with 50ml of 80% ethanol, evaporating to dryness the eluent, and dissolving the residue with 1ml of methanol to obtain a sample solution; taking appropriate amount of ginsenoside Rg1, Re and astragaloside IV as reference substances, adding methanol to obtain solutions each containing 1mg per 1ml as reference substance solution; sucking 3-4 μ l of each reference solution and 3-5 μ l of each test solution, respectively spotting the reference solutions and the test solutions on the same silica gel G thin-layer plate in strips, placing layered dichloromethane-methanol-water (17: 7: 2) lower layer solution at 10 ℃ as a developing agent, developing, taking out, drying, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until the spots are clearly developed, and respectively developing strip spots with the same color in the positions corresponding to the reference chromatogram in the test solution chromatogram.
Reference to the literature
【1】 Chinese medicine epimedium thin-layer chromatography identification in literature uploaded to Baidu library by sun-handed flower 2016.12.27 "
【2】 Wushifu, Wanhong, Yingxingtuowan pill thin-layer chromatography identification method research, pharmaceutical research, 33(6)328-331 in 2014.
Claims (3)
1. An interference-free rapid thin-layer identification method of kidney-tonifying toxin-expelling granules is characterized in that:
(1) taking 1g of sample, grinding, adding 2ml of 70% methanol, carrying out ultrasonic treatment for 10 minutes, centrifuging, and taking supernate as a test sample solution; taking appropriate amount of psoralen and isopsoralen as reference substances, and adding methanol to obtain solutions containing 3mg of psoralen and isopsoralen per 1ml as reference substance solutions; taking 0.2g of fructus psoraleae as a reference medicinal material, adding 2ml of 70% methanol, carrying out ultrasonic treatment for 15 minutes, centrifuging, and taking supernate as a reference medicinal material solution; respectively taking 5-8 mul of a reference solution and a reference medicinal material solution and 8-10 mul of a test solution, respectively dropping the solutions on the same silica gel G thin-layer plate, developing by using n-hexane-ethyl acetate as a developing agent in a volume ratio of 4: 2, taking out, airing, spraying a 2% potassium hydroxide ethanol solution, drying by hot air, placing under an ultraviolet lamp 365nm for inspection, and respectively displaying fluorescent spots with the same color on positions corresponding to the chromatograms of the reference and reference medicinal materials in the chromatogram of the test solution;
(2) taking appropriate amount of emodin reference substance, adding methanol to obtain solution containing 0.3mg per 1ml as reference substance solution; taking 0.5g of dandelion as a reference medicinal material and 0.2g of polygonum cuspidatum as a reference medicinal material, respectively adding 2ml of 70% methanol, carrying out ultrasonic treatment for 20 minutes, centrifuging, and taking supernate as respective reference medicinal material solutions; sucking 3-5 mul of a reference solution, 10 mul of a dandelion reference solution, 5 mul of a polygonum cuspidatum reference solution and 5-6 mul of a test solution under the item (1), respectively dropping the solutions on the same silica gel G thin-layer plate, developing by using cyclohexane-ethyl acetate-acetone-formic acid as a developing agent in a volume ratio of 10: 3: 2: 0.5, taking out, drying in the air, placing the developed developing agent under an ultraviolet lamp of 365nm, and displaying the same yellow fluorescent spots in the chromatogram of the test solution at positions corresponding to the chromatograms of the reference solution and the polygonum cuspidatum reference solution; spraying 3% aluminum trichloride ethanol solution, and inspecting under 365nm ultraviolet lamp to obtain bright blue fluorescent main spot in the chromatogram of the test sample at the position corresponding to the chromatogram of the herba Taraxaci control material;
(3) taking appropriate amount of puerarin as reference substance, adding methanol to obtain solution containing 0.1mg per 1ml as reference substance solution; taking 0.1g of radix Puerariae as a reference medicinal material, adding 2ml of methanol, performing ultrasonic treatment for 20 minutes, centrifuging, and taking supernate as a reference medicinal material solution; sucking 4 μ l of a reference medicinal material solution, 5-6 μ l of a reference substance solution, and 3-5 μ l of a test solution under the item (1), respectively dropping on the same silica gel G thin layer plate, developing by using chloroform-ethyl acetate-methanol-concentrated ammonia with the volume ratio of 4.1: 1: 4.2: 0.8 as a developing agent, taking out, drying in the air, and placing under an ultraviolet lamp 365nm for inspection, wherein in the chromatogram of the test solution, fluorescent main spots with the same color are respectively displayed at positions corresponding to the chromatograms of the reference substance and the reference medicinal material;
(4) taking appropriate amount of icariin as control, adding methanol to obtain solution containing 2mg per 1ml as control solution; taking 0.2g of herba epimedii control medicinal material, adding 2ml of methanol, carrying out ultrasonic treatment for 20 minutes, centrifuging, and taking supernate as a control medicinal material solution; respectively taking 3-5 mul of each of a reference solution and a reference medicinal material solution, respectively dropping 2-3 mul of the test solution under the item (1) on the same silica gel G thin-layer plate, taking ethyl acetate-butanone-formic acid-water with the volume ratio of 10: 1 as a developing agent, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until spots are clearly developed, viewing under 365nm ultraviolet light, and respectively displaying main fluorescent spots with the same color on positions corresponding to the chromatograms of the reference and the reference medicinal materials in the chromatogram of the test solution;
(5) taking 2.5g of the product, grinding, adding 20ml of 70% methanol, carrying out ultrasonic treatment for 20 minutes, filtering, evaporating the methanol from the filtrate in a water bath, passing the residual aqueous solution through a D101 type macroporous resin (the inner diameter is 1.5cm, the column height is 12cm), eluting with 50ml of water and then 50ml of 20% ethanol, discarding the eluent, eluting with 80ml of 70% ethanol, collecting the eluent, evaporating to dryness, transferring the residue with 2ml of ethanol to an alkaline alumina column (the inner diameter is 1cm, the alumina is 1.5g), eluting with 50ml of 80% ethanol, evaporating to dryness the eluent, and dissolving the residue with 1ml of methanol to obtain a sample solution; taking appropriate amount of ginsenoside Rg1, Re and astragaloside IV as reference substances, adding methanol to obtain solutions each containing 1mg per 1ml as reference substance solution; sucking 3-4 μ l of each reference solution and 3-5 μ l of each test solution, respectively spotting the reference solutions and the test solutions on the same silica gel G thin-layer plate in strips, placing layered dichloromethane-methanol-water (17: 7: 2) lower layer solution at 10 ℃ as a developing agent, developing, taking out, drying, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until the spots are clearly developed, and respectively developing strip spots with the same color in the positions corresponding to the reference chromatogram in the test solution chromatogram.
2. The method for identifying the non-interfering rapid thin layer of the kidney-tonifying and toxin-expelling granules according to claim 1, wherein the granules are a compound preparation consisting of 11 medicinal materials, but 5 medicinal materials are directly treated by using a 70% methanol ultrasonic supernatant solution without extraction and impurity removal, and interference of other components can be eliminated by the polarity of a developing agent, the acidity and alkalinity, the selectivity of a color developing agent and the limitation of inspection conditions, and only information spots of a reference substance and the reference medicinal materials are presented or the spot definition is improved.
3. The interference-free rapid thin-layer identification method of kidney-tonifying and toxin-expelling granules according to claim 1, further characterized in that the thin-layer identification of 5 medicinal materials is completed by adopting the same test solution on 4 thin-layer plates, and only 15ml of preparation solvents of the test solution, the reference medicinal materials and the reference substance are needed, and the time is 30 minutes.
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