CN117233313A - Thin layer chromatography method for distinguishing semen astragali Complanati medicinal materials and preparations thereof - Google Patents
Thin layer chromatography method for distinguishing semen astragali Complanati medicinal materials and preparations thereof Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 48
- 238000002360 preparation method Methods 0.000 title claims abstract description 32
- 238000004809 thin layer chromatography Methods 0.000 title claims abstract description 30
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- 239000000284 extract Substances 0.000 claims abstract description 16
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- 238000001514 detection method Methods 0.000 description 9
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- 238000005238 degreasing Methods 0.000 description 2
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- 150000002215 flavonoids Chemical class 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a thin-layer chromatography method, in particular to a thin-layer chromatography method for distinguishing semen astragali complanati medicinal materials and preparations thereof. Solves the technical problems of complex operation, high toxicity of reagents, long time consumption and weak distinguishing and identifying ability of semen astragali Complanati extract and formula particles in the existing thin-layer chromatography identification method of semen astragali Complanati. The method comprises the following steps: 1) Preparing a control liquid and a liquid to be identified; 2) Carrying out thin-layer sample application; 3) Ethyl acetate-methanol-formic acid with 14-16: 9 to 11: mixing uniformly in a volume ratio of 1-2 to obtain a developing agent; placing the developing agent into a developing cylinder, sealing the developing cylinder, and presaturating for 15-20 minutes; 4) Placing the spotted GF254 thin-layer plate into a spreading cylinder for spreading; 5) And developing and identifying. The method has good spot separation effect, prominent characteristic spots, no spot tailing and spot diffusion phenomenon, can rapidly identify and distinguish the semen astragali complanati medicinal materials and preparations thereof, and is suitable for commercial popularization and application.
Description
Technical Field
The invention relates to a thin-layer chromatography method, in particular to a thin-layer chromatography method for distinguishing semen astragali complanati medicinal materials and preparations thereof.
Background
Semen astragali Complanati is dry mature seed of Astragalus membranaceus Astragalus complanatus R.Br. Of Leguminosae, is a common traditional Chinese medicinal material, and has sweet nature and warm property, and can enter liver and kidney channels. Has effects of invigorating kidney, supporting yang, stopping nocturnal emission, reducing urination, nourishing liver, and improving eyesight. Semen astragali Complanati is mainly used for treating lumbago due to kidney deficiency, spermatorrhea, premature ejaculation, enuresis, frequent urination, leukorrhagia, dizziness, dim eyesight, etc. The present research shows that the semen astragali Complanati mainly contains flavonoid components such as amino acid, polypeptide, protein, phenols, tannins, sterols, triterpenes, alkaloids, semen astragali Complanati glycoside A, etc. Semen astragali Complanati can be used as both medicine and food within limited application range and dosage, and is favored by consumers. The traditional Chinese medicine formula particles, health products, solid beverages or functional foods often have semen astragali Complanati, such as YISHENLING particles (capsule), YINIUQI pill, SHENGLI Capsule, XIAOKEPING tablet, semen astragali Complanati particles, etc. According to the theory of traditional Chinese medicine, the semen astragali Complanati is in the form of water extraction in the preparations, so that the quality control of the product containing the semen astragali Complanati preparation can be well carried out by identifying the semen astragali Complanati extract.
Thin layer chromatography is one of the chromatographic methods and is an important experimental technique for rapid separation and qualitative analysis of small amounts of substances. Currently, there are few studies on thin-layer chromatography identification of semen astragali complanati, and the following brief summary of the existing methods for thin-layer chromatography identification of semen astragali complanati is provided:
the identification method of the semen astragali Complanati medicinal material in the 2020 edition of Chinese pharmacopoeia comprises the following steps: taking 0.2g of the product powder, adding 10ml of methanol, carrying out ultrasonic treatment for 30 minutes, cooling, filtering, evaporating filtrate to dryness, and adding 2ml of methanol into residues to dissolve, thereby obtaining a sample solution. And preparing a reference medicinal material solution of semen astragali Complanati 0.2g by the same method. And adding 60% ethanol into semen astragali Complanati glycoside A reference substance to obtain a solution containing 0.05mg per 1ml, and taking the solution as reference substance solution. According to a thin layer chromatography (general rule 0502) test, 2 μl of each of the above three solutions is sucked and respectively spotted on the same polyamide film, and the three solutions are developed by using ethanol-butanone-acetylacetone-water (3:3:1:13) as developing agents, taken out, dried, sprayed with aluminum trichloride test solution, dried by hot air, and inspected under an ultraviolet lamp (365 nm). In the sample chromatogram, fluorescent spots with the same color appear at the positions corresponding to the control chromatogram and the control chromatogram. However, the method adopts polyamide film detection, which is not suitable for the modern thin-layer automatic sample application technology, and the thin-layer automatic sample application technology uses a silica gel plate to apply sample, so that the old polyamide film detection brings a lot of inconveniences for the current detection, and therefore, the method for identifying the semen astragali complanati needs to be provided with simple operation and is suitable for the development of the current technology.
The thin layer identification method for the salted semen astragali complanati formula granule disclosed in Chinese patent CN110018269B is as follows: a. preparation of control medicinal material solution: extracting semen astragali Complanati control material powder with n-butanol, filtering, evaporating filtrate, and dissolving residue with n-butanol to obtain control material solution; b. preparation of a control solution: dissolving semen astragali Complanati glycoside A reference substance in 50% -70% ethanol to obtain reference substance solution; c. preparation of test solution: taking a sample to be detected, grinding, adding n-butanol for extraction, filtering, evaporating filtrate to dryness, and dissolving residues with n-butanol to obtain a sample solution; d. thin layer chromatography assay: taking a reference substance solution, a reference medicinal material solution and a test sample solution, respectively spotting on the same silica gel G plate, spreading with n-butanol-glacial acetic acid-water, air drying, spraying sulfuric acid ethanol solution, heating, and inspecting under an ultraviolet lamp; the volume ratio of the n-butanol to the glacial acetic acid to the water is (3-5) 1 (4-6). The unfolding system of the patent method belongs to an n-butanol system, and when an n-butanol reagent is used as the unfolding system, the unfolding time is too long, so that the operation is not facilitated.
The identification of semen astragali Complanati in the detection method of the capsule for strengthening body resistance of hepatitis B disclosed in Chinese patent CN104792917B is as follows: taking the product, taking the content of the product, placing the product into a conical flask, adding petroleum ether with the boiling range of 60-90 ℃, intermittently shaking, soaking overnight, filtering, volatilizing filtrate, and adding 70-80 v% ethanol into residues to dissolve the residues to obtain a sample solution; preparing semen astragali Complanati control medicinal material and herba Paederiae control medicinal material according to the preparation method of the sample solution, respectively; performing a test according to an annex VI B of the first edition of Chinese pharmacopoeia 2010, sucking the sample solution, the semen astragali complanati control medicinal material solution and the herba Paederiae control medicinal material solution, spotting on the same polyamide film, spreading with a methanol-water mixed solution with a volume ratio of methanol-water=7-9:3 as a developing agent, taking out, airing, spraying an acetic acid solution of lwt-2wt% of aluminum trichloride, heating for color development, and inspecting under a 365nm ultraviolet lamp; in the sample chromatogram, spots of the same color appear at the positions corresponding to the chromatogram of the semen astragali Complanati control, and spots of the same color do not appear at the positions corresponding to the chromatogram of the herba Paederiae control. The method is also a method of Chinese pharmacopoeia and is not suitable for distinguishing modern semen astragali Complanati.
The method for identifying semen astragali Complanati in the detection method of the Chinese patent CN104792916B of the Chinese medicinal preparation hepatitis B strengthening capsule is the method of Chinese pharmacopoeia at the core.
Chinese patent CN104792916B discloses a quality control method of semen astragali complanati preparation, which is characterized in that the identification of semen astragali complanati is as follows: preparing a sample solution by taking the sample as the sample solution; placing semen astragali Complanati reference material 1.0g in Soxhlet extractor, adding petroleum ether, refluxing under heating for 4 hr, volatilizing solvent from residue, adding 80% ethanol 20ml, refluxing under heating for 15 hr, cooling, filtering, and collecting filtrate as reference material solution; according to thin layer chromatography (appendix VIB of Chinese pharmacopoeia 2005 edition), sucking 2 μl of each of the above two solutions, respectively spotting on the same polyamide film, spreading with methanol-water (4:1) as developing agent, taking out, air drying, spraying 1% aluminum chloride ethanol solution, heating for developing color, and placing under ultraviolet lamp (365 nm) for inspection, wherein fluorescent spots of the same color appear on the positions corresponding to the control medicine chromatogram in the sample chromatogram. The method also adopts the method of Chinese pharmacopoeia.
Still other documents identify the following for thin layers of semen astragali Complanati: liu Jian et al were observed by paper chromatography at 365nm using an ultraviolet lamp. Chen Kun A thin layer plate of silica gel G was used as a developing agent with cyclohexane-ethyl acetate (8:2), and was observed under an ultraviolet lamp at 365 nm. Zhang Hua the sample was developed with an acidic dilute ethanol solution using n-butanol-methanol-water (13:3:2) using paper chromatography at a distance of 16cm and observed under UV light at 254 nm. Liu Chuanling under a chloroform-methanol-water (17:7:2) as a developing solvent, and was observed under an ultraviolet lamp at 365 nm. Zhang Yujie degreasing 22 commercially available semen astragali Complanati medicinal materials with petroleum ether, extracting with 75% ethanol under ultrasonic, evaporating filtrate, dissolving with methanol to obtain sample, adding semen astragali Complanati glycoside A as reference substance onto polyamide film, adding developing agent methanol-water (4:1), developing with 1% aluminum trichloride ethanol solution, and observing with ultraviolet lamp at 365 nm.
In summary, the existing thin-layer chromatography identification method of semen astragali Complanati mainly comprises a paper chromatography and a polyamide film method, but the paper chromatography and the polyamide film are not suitable for the modern thin-layer detection requirements, because the development system of n-butanol is long in time consumption, the toxicity of a chloroform development system is large, the damage to human bodies is large, petroleum ether degreasing is required for pretreatment, and the whole process is time-consuming and labor-consuming, the existing method is difficult to meet the requirements of semen astragali Complanati medicinal materials and preparations thereof in practical wide application and detection identification. In addition, the water extract and the formula granule of the semen astragali complanati are prepared from semen astragali complanati medicinal materials through a series of processes, wherein some processes damage the character characteristics of the raw medicinal materials, and in order to identify the traditional Chinese medicine water extract and the formula granule which have damaged characters, an identification method with strong distinguishing and identifying capabilities and simpler, more convenient and faster operation is needed to better control the quality.
Disclosure of Invention
The invention aims to solve the technical problems of complex operation, high toxicity of reagents used, long time consumption and weak distinguishing and distinguishing capability of semen astragali complanati extract and formula particles in the existing thin-layer chromatography distinguishing method of semen astragali complanati, and provides a thin-layer chromatography method for distinguishing semen astragali complanati medicinal materials and preparations thereof.
The technical scheme of the invention is as follows:
the invention discloses a thin-layer chromatography method for distinguishing semen astragali complanati medicinal materials and preparations thereof, which is characterized by comprising the following steps:
1) Preparing control liquid and liquid to be identified
Respectively placing a semen astragali complanati medicinal material control sample and a sample to be identified in a sealable test container, respectively adding 5-10 times of ethanol with the mass concentration of 50% -70% of the respective samples, respectively carrying out ultrasonic extraction for 30-60 minutes, standing to room temperature, filtering by adopting a microporous filter membrane, collecting filtrate of the semen astragali complanati medicinal material control sample as a first control solution, and collecting filtrate of the sample to be identified as a liquid to be identified; adding methanol into semen astragali Complanati glycoside A control sample to obtain second control solution;
the sample to be identified is commercially available semen astragali Complanati medicinal material, semen astragali Complanati extract or semen astragali Complanati formula granule;
2) Carrying out thin layer sample application
Sequentially spotting a first control liquid, a second control liquid and a liquid to be identified on different positions of the same silica gel GF254 thin layer plate at the same height to form spotting origins;
3) Preparation of the developing agent
3.1 Ethyl acetate-methanol-formic acid in 14-16: 9 to 11: mixing uniformly in a volume ratio of 1-2 to obtain a developing agent;
3.2 Placing the developing agent into a developing cylinder, sealing the developing cylinder, and presaturating for 15-20 minutes;
4) Unfolding
Opening the unfolding cylinder, and rapidly placing the spotted GF254 thin-layer plate into the unfolding cylinder to enable the GF254 thin-layer plate to lean against the unfolding cylinder; when the front edge of the developing agent moves up to 6 cm-10 cm away from the spotting origin, the GF254 thin-layer plate is taken out;
5) Color development and identification
5.1 Drying the GF254 thin-layer plate, spraying sulfuric acid ethanol color-developing agent with the mass ratio of 5% -10%, heating for 3-5 minutes at 103-107 ℃, cooling to room temperature, and then inspecting under 365nm ultraviolet light to obtain an inspection view;
5.2 Distinguishing and identifying the sample to be identified): if the spot formed by the liquid to be identified is the same in position and color as the spot formed by the first control liquid on the inspection view, and the spot same as the characteristic spot formed by the second control liquid appears in the spot of the liquid to be identified, the sample to be identified is a true product; otherwise, the sample to be identified is a counterfeit product.
Further, in step 3.1), the volume ratio of ethyl acetate-methanol-formic acid is 15:10:1.5.
Further, in the step 2), the distance between the spotting origin position and the bottom of the GF254 thin-layer plate is 1.0-1.5 cm.
Further, in step 4), the GF254 thin-layer plate is removed when the front of the developing agent is moved up to 8 to 10cm from the spotting origin.
Further, in step 3.2), the developing cylinder is a double-tank developing cylinder having a size of 10cm×10cm, 20cm×10cm, or 10cm×20cm, and the doses of developing agent in the double tanks are kept uniform.
Further, in step 5.1), the temperature of heating was 105 ℃.
Further, in the step 1), the addition amount of the ethanol is 8 times of the mass of each sample, and the mass concentration of the ethanol is 70%; ultrasonic extraction for 60 minutes; the concentration of the second control solution is 1mg/ml;
in the step 2), the distance between the point origin position and the bottom of the GF254 thin-layer plate is 1.0cm;
in step 3.2), presaturation is carried out for 20 minutes;
in the step 4), when the front edge of the developing agent ascends to 8cm away from the spotting origin, the GF254 thin-layer plate is taken out;
in the step 5.1), the mass ratio of the sulfuric acid ethanol color developing agent is 6%.
The invention has the beneficial effects that:
1. the invention provides a thin-layer chromatography method for distinguishing semen astragali complanati medicinal materials and preparations thereof, and provides a thin-layer distinguishing method which is quick, low in cost, simple, convenient, efficient, strong in specificity, visual in detection result and easy to identify, aiming at the distinguishing problem of the semen astragali complanati medicinal materials and preparations thereof.
2. The thin-layer chromatography method for distinguishing the semen astragali complanati medicinal material and the preparation thereof adopts the volume ratio of ethyl acetate-methanol-formic acid (14-16:9-11:1-2) to be mixed to obtain the developing agent, has good spot separation effect, prominent characteristic spots and no spot tailing and spot diffusion phenomenon.
3. The thin-layer chromatography method for distinguishing the semen astragali complanati medicinal materials and the preparations thereof selects 50% -100% ethanol to directly carry out ultrasonic treatment on the raw medicinal materials, filters the raw medicinal materials, takes filtrate as detection solution, has simple sample pretreatment and low cost, and is easy to operate.
4. The thin-layer chromatography method for distinguishing the semen astragali complanati medicinal material and the preparation thereof selects 5-10% of sulfuric acid ethanol for color development, and has the advantages of simple and quick operation, more thin-layer spots, obvious spot color and clear contrast.
Drawings
FIG. 1 is a chromatogram obtained in example 1 of a thin-layer chromatography method for discriminating a semen astragali Complanati medicinal material from its preparation according to the present invention (wherein, number s represents a semen astragali Complanati glycoside A control sample, number 1 represents a semen astragali Complanati medicinal material control sample, number 2 represents a commercially available semen astragali Complanati medicinal material, number 3 represents a laboratory-made semen astragali Complanati extract, and number 4 represents a commercially available semen astragali Complanati formula particle).
Detailed Description
The following describes in detail a thin layer chromatography method for discriminating a semen astragali Complanati medicinal material and its preparation by examples and drawings. In the following examples, the semen astragali Complanati medicinal material control sample and the semen astragali Complanati glycoside A control sample are both derived from Chinese medicine biological product laboratory, and the batch numbers are 121275-201503 and 111803-201704 respectively; in the sample to be identified, the semen astragali complanati medicinal material, semen astragali complanati formula granule and semen astragali complanati extract are all from commercial sources.
Example 1
Taking a proper amount of semen astragali Complanati medicinal material control sample and sample to be identified; in the embodiment, three samples to be identified are respectively commercially available semen astragali Complanati medicinal materials, semen astragali Complanati extract and semen astragali Complanati formula granules; respectively placing the semen astragali Complanati medicinal material control sample and three samples to be identified in four triangular flasks with plugs, respectively adding 8 times of ethanol with the mass concentration of 70% into the respective samples, respectively performing ultrasonic extraction for 60 minutes, standing to room temperature, filtering with a microporous filter membrane, collecting filtrate of the semen astragali Complanati medicinal material control sample as a first control solution, and collecting filtrate of the three samples to be identified as the liquids to be identified. And (3) taking a control sample of the semen astragali Complanati glycoside A, and adding methanol to prepare a second control solution with the concentration of 1 mg/ml. In other embodiments, the addition amount of the ethanol can be 5-10 times of the mass of each sample, and the mass concentration of the ethanol is 50% -70%; the ultrasonic extraction time is 30-60 minutes.
Sequentially spotting a first control liquid, a second control liquid and three liquids to be identified on different positions of the same silica gel GF254 thin layer plate at the same height to form spotting origins, wherein each liquid spot is performed twice; the distance of the spotting origin from the lower edge of the GF254 thin-layer plate was 1.0cm. In other embodiments, the spotting origin may be 1.0-1.5 cm from the lower edge of the GF254 lamina plate.
Mixing ethyl acetate-methanol-formic acid according to the volume ratio of 15:10:1.5 to obtain a developing agent, placing a proper amount of the developing agent into a double-tank developing cylinder, wherein the size of the developing cylinder can be 10 multiplied by 10cm, or 20 multiplied by 10cm, or 10 multiplied by 20cm, and the developing agent dosage in the double tanks is kept consistent. The deployment cylinder was sealed and pre-saturated for 20 minutes. In other embodiments, the volume ratio of ethyl acetate-methanol-formic acid may be 14 to 16:9 to 11: 1-2, the pre-saturation time period can be 15-20 minutes.
The unfolding cylinder is opened, the spotted GF254 thin-layer plate is quickly placed into the well-saturated unfolding cylinder, the GF254 thin-layer plate is leaned against the unfolding cylinder, and the developing agent in the unfolding cylinder cannot submerge the spotting origin. The developing agent was developed up the GF254 lamina plate until the front of the developing agent was up to 8cm from the spotting origin, and the GF254 lamina plate was removed. In other embodiments, the GF254 thin-layer plate is removed when the front of the developing agent is up to 6cm to 10cm from the point of origin.
Blowing the developing agent on the GF254 thin-layer plate by a blower, spraying the sulfuric acid ethanol color-developing agent with the mass ratio of 6%, and then placing the film on an electric heating plate and heating the film for 3-5 minutes at the temperature of 105 ℃. After being left to stand at room temperature, the glass was inspected under 365nm ultraviolet light to obtain an inspection view, as shown in FIG. 1. In other embodiments, the mass ratio of the sulfuric acid ethanol color reagent is 5% -10%.
As is evident from FIG. 1, the reference sample of semen astragali Complanati glycoside A, and the spots formed by the semen astragali Complanati, semen astragali Complanati extract and semen astragali Complanati granule to be identified are all bright blue. The positions and the colors of spots of the semen astragali complanati medicinal material, the semen astragali complanati extract and the semen astragali complanati formula particles are consistent with those of spots of semen astragali complanati medicinal material control samples, and spots consistent with characteristic spots of semen astragali complanati glycoside A control samples appear in the three samples to be identified, so that the semen astragali complanati medicinal material, the semen astragali complanati extract and the semen astragali complanati formula particles are genuine. The method has the advantages of clear contrast of each spot, good spot separation effect, prominent characteristic spots, no spot tailing and spot diffusion phenomenon, and the method can be used for identifying and distinguishing semen astragali complanati, has strong identifying and distinguishing capability, can quickly and efficiently obtain identifying results, and is suitable for commercial popularization and application.
Example 2
Taking a proper amount of semen astragali Complanati medicinal material control samples and samples to be identified, wherein two samples to be identified are semen astragali Complanati medicinal material and semen astragali Complanati extract respectively, placing semen astragali Complanati medicinal material control samples and two samples to be identified respectively in three triangular flasks with plugs, respectively adding 10 times of ethanol of the respective samples, respectively carrying out ultrasonic extraction for 60 minutes, standing at room temperature, filtering with a microporous filter membrane, collecting filtrate of the semen astragali Complanati medicinal material control samples as a first control solution, and collecting filtrate of the two samples to be identified as liquids to be identified. And (3) taking a control sample of the semen astragali Complanati glycoside A, and adding methanol to prepare a second control solution with the concentration of 1 mg/ml.
Sequentially spotting a first control liquid, a second control liquid and two liquids to be identified on different positions of the same silica gel GF254 thin-layer plate at the same height to form spotting origins; the distance of the spotting origin from the lower edge of the GF254 thin-layer plate was 1.0cm.
Mixing ethyl acetate-methanol-formic acid according to the volume ratio of 14:9:1 to obtain a developing agent, placing a proper amount of the developing agent into a double-tank developing cylinder, wherein the size of the developing cylinder can be 10 multiplied by 10cm, or 20 multiplied by 10cm, or 10 multiplied by 20cm, and the developing agent dosage in the double tanks is kept consistent. The deployment cylinder was sealed and pre-saturated for 20 minutes.
The unfolding cylinder is opened, the spotted GF254 thin-layer plate is quickly placed into the well-saturated unfolding cylinder, the GF254 thin-layer plate is leaned against the unfolding cylinder, and the developing agent in the unfolding cylinder cannot submerge the spotting origin. The developing agent is developed up the GF254 lamina plate until the front of the developing agent reaches 8cm from the origin of the spot, and the GF254 lamina plate is removed.
Blowing the developing agent on the thin layer plate by using a blower, spraying the sulfuric acid ethanol color developing agent with the mass ratio of 10%, and then placing the thin layer plate on an electric heating plate to heat the thin layer plate for 3-5 minutes at the temperature of 105 ℃. And (5) cooling to room temperature, and inspecting under 365nm ultraviolet light to obtain an inspected view.
Example 3
Taking a proper amount of semen astragali Complanati medicinal material control samples and samples to be identified, wherein two samples to be identified are semen astragali Complanati medicinal material and semen astragali Complanati extracts respectively, placing the semen astragali Complanati medicinal material control samples and the two samples to be identified in three triangular flasks with plugs respectively, adding 5 times of ethanol of the respective samples respectively, performing ultrasonic extraction for 50 minutes respectively, standing at room temperature, filtering by using a microporous filter membrane, collecting filtrate of the semen astragali Complanati control samples as a first control solution, and collecting filtrate of the two samples to be identified as liquids to be identified. And (3) taking a control sample of the semen astragali Complanati glycoside A, and adding methanol to prepare a second control solution with the concentration of 1 mg/ml.
Sequentially spotting a first control liquid, a second control liquid and two liquids to be identified on different positions of the same silica gel GF254 thin-layer plate at the same height to form spotting origins; the distance of the spotting origin from the lower edge of the GF254 thin-layer plate was 1.5cm.
Mixing ethyl acetate-methanol-formic acid according to the volume ratio of 16:11:2 to obtain a developing agent, placing a proper amount of the developing agent into a double-tank developing cylinder, wherein the size of the developing cylinder can be 10 multiplied by 10cm, or 20 multiplied by 10cm, or 10 multiplied by 20cm, and the developing agent dosage in the double tanks is kept consistent. The deployment cylinder was sealed and pre-saturated for 17 minutes.
The unfolding cylinder is opened, the spotted GF254 thin-layer plate is quickly placed into the well-saturated unfolding cylinder, the GF254 thin-layer plate is leaned against the unfolding cylinder, and the developing agent in the unfolding cylinder cannot submerge the spotting origin. The developing agent is developed up the GF254 lamina plate until the front of the developing agent reaches 10cm from the origin of the spot, and the GF254 lamina plate is removed.
Blowing the developing agent on the thin layer plate by a blower, spraying 5% sulfuric acid ethanol color developing agent, and heating on an electric heating plate at 103 ℃ for 3-5 minutes. And (5) cooling to room temperature, and inspecting under 365nm ultraviolet light to obtain an inspected view.
Example 4
Taking a proper amount of semen astragali Complanati medicinal material control samples and samples to be identified, wherein two samples to be identified are semen astragali Complanati medicinal material and semen astragali Complanati extracts respectively, placing the semen astragali Complanati medicinal material control samples and the two samples to be identified in three triangular flasks with plugs respectively, adding 10 times of ethanol of the respective samples respectively, respectively carrying out ultrasonic extraction for 30 minutes with the mass concentration of 50%, cooling to room temperature, filtering by adopting a microporous filter membrane, collecting filtrate of the semen astragali Complanati control samples as a first control liquid, and collecting filtrate of the two samples to be identified as liquids to be identified. And (3) taking a control sample of the semen astragali Complanati glycoside A, and adding methanol to prepare a second control solution with the concentration of 1 mg/ml.
Sequentially spotting a first control liquid, a second control liquid and two liquids to be identified on different positions of the same silica gel GF254 thin-layer plate at the same height to form spotting origins; the distance of the spotting origin from the lower edge of the GF254 thin-layer plate was 1.0cm.
Mixing ethyl acetate-methanol-formic acid according to the volume ratio of 15:10:1.5 to obtain a developing agent, placing a proper amount of the developing agent into a double-tank developing cylinder, wherein the size of the developing cylinder can be 10 multiplied by 10cm, or 20 multiplied by 10cm, or 10 multiplied by 20cm, and the developing agent dosage in the double tanks is kept consistent. The deployment cylinder was sealed and pre-saturated for 15 minutes.
The unfolding cylinder is opened, the spotted GF254 thin-layer plate is quickly placed into the well-saturated unfolding cylinder, the GF254 thin-layer plate is leaned against the unfolding cylinder, and the developing agent in the unfolding cylinder cannot submerge the spotting origin. The developing agent is developed up the GF254 lamina plate until the front of the developing agent reaches 6cm from the origin of the spot, and the GF254 lamina plate is removed.
Blowing the developing agent on the thin layer plate by a blower, spraying the sulfuric acid ethanol color developing agent with the mass ratio of 10%, and then placing the thin layer plate on an electric heating plate to heat the thin layer plate for 3 to 5 minutes at the temperature of 107 ℃. And (5) cooling to room temperature, and inspecting under 365nm ultraviolet light to obtain an inspected view.
The views obtained in examples 2-4 are similar to those obtained in example 1, and reference is made to FIG. 1.
Claims (7)
1. A thin layer chromatography method for identifying and distinguishing semen astragali complanati medicinal materials and preparations thereof, which is characterized by comprising the following steps:
1) Preparing control liquid and liquid to be identified
Respectively placing a semen astragali complanati medicinal material control sample and a sample to be identified in a sealable test container, respectively adding 5-10 times of ethanol with the mass concentration of 50% -70% of the respective samples, respectively carrying out ultrasonic extraction for 30-60 minutes, standing to room temperature, filtering by adopting a microporous filter membrane, collecting filtrate of the semen astragali complanati medicinal material control sample as a first control solution, and collecting filtrate of the sample to be identified as a liquid to be identified; adding methanol into semen astragali Complanati glycoside A control sample to obtain second control solution;
the sample to be identified is commercially available semen astragali Complanati medicinal material, semen astragali Complanati extract or semen astragali Complanati formula granule;
2) Carrying out thin layer sample application
Sequentially spotting a first control liquid, a second control liquid and a liquid to be identified on different positions of the same silica gel GF254 thin layer plate at the same height to form spotting origins;
3) Preparation of the developing agent
3.1 Ethyl acetate-methanol-formic acid in 14-16: 9 to 11: mixing uniformly in a volume ratio of 1-2 to obtain a developing agent;
3.2 Placing the developing agent into a developing cylinder, sealing the developing cylinder, and presaturating for 15-20 minutes;
4) Unfolding
Opening the unfolding cylinder, and rapidly placing the spotted GF254 thin-layer plate into the unfolding cylinder to enable the GF254 thin-layer plate to lean against the unfolding cylinder; when the front edge of the developing agent moves up to 6 cm-10 cm away from the spotting origin, the GF254 thin-layer plate is taken out;
5) Color development and identification
5.1 Drying the GF254 thin-layer plate, spraying sulfuric acid ethanol color-developing agent with the mass ratio of 5% -10%, heating for 3-5 minutes at 103-107 ℃, cooling to room temperature, and then inspecting under 365nm ultraviolet light to obtain an inspection view;
5.2 Distinguishing and identifying the sample to be identified): if the spot formed by the liquid to be identified is the same in position and color as the spot formed by the first control liquid on the inspection view, and the spot same as the characteristic spot formed by the second control liquid appears in the spot of the liquid to be identified, the sample to be identified is a true product; otherwise, the sample to be identified is a counterfeit product.
2. The thin layer chromatography method for distinguishing semen astragali Complanati medicinal materials from their preparations according to claim 1, wherein:
in step 3.1), the volume ratio of ethyl acetate-methanol-formic acid is 15:10:1.5.
3. A thin layer chromatography method for distinguishing semen astragali complanati medicinal materials from their preparations according to claim 1 or 2, characterized in that:
in the step 2), the distance between the point origin position and the bottom of the GF254 thin-layer plate is 1.0-1.5 cm.
4. A thin layer chromatography method for distinguishing semen astragali complanati medicinal materials from their preparations according to claim 3, wherein:
in step 4), the GF254 thin-layer plate is taken out when the front of the developing agent is moved up to 8-10 cm from the spotting origin.
5. The thin layer chromatography method for distinguishing semen astragali Complanati medicinal materials from their preparations according to claim 4, wherein:
in step 3.2), the deployment cylinder is a double-slot deployment cylinder, the size of the double-slot deployment cylinder is 10cm×10cm, 20cm×10cm or 10cm×20cm, and the doses of the deployment agent in the double slots are kept consistent.
6. The thin layer chromatography method for distinguishing semen astragali Complanati medicinal materials from their preparations according to claim 5, wherein:
in step 5.1), the temperature of heating was 105 ℃.
7. The thin layer chromatography method for distinguishing semen astragali Complanati medicinal materials from their preparations according to claim 6, wherein:
in the step 1), the addition amount of the ethanol is 8 times of the mass of each sample, and the mass concentration of the ethanol is 70%; ultrasonic extraction for 60 minutes; the concentration of the second control solution is 1mg/ml;
in the step 2), the distance between the point origin position and the bottom of the GF254 thin-layer plate is 1.0cm;
in step 3.2), presaturation is carried out for 20 minutes;
in the step 4), when the front edge of the developing agent ascends to 8cm away from the spotting origin, the GF254 thin-layer plate is taken out;
in the step 5.1), the mass ratio of the sulfuric acid ethanol color developing agent is 6%.
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