CN118091008A - Thin-layer identification method of Chinese chive seed medicinal material - Google Patents
Thin-layer identification method of Chinese chive seed medicinal material Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/94—Development
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides a thin-layer identification method of Chinese chive seed medicinal materials, which is characterized by comprising the steps of preparing a sample solution, preparing a reference medicinal material solution and identifying the thin layer. The method is simple, convenient, rapid, accurate, high in efficiency and low in cost, can be well used for quality control of the Chinese chive seed medicinal materials, and can also effectively distinguish common mixed and fake products in the market, such as the Chinese onion seed medicinal materials and the stramonium seed medicinal materials.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to a thin-layer identification method of Chinese chive seed medicinal materials.
Background
Semen Allii Tuberosi is dry mature seed of Allium tuberosum Rottl.ex Spreng. Of Liliaceae, has warm nature, pungent and sweet taste, and can enter liver and kidney channels, has effects of warming liver and kidney, supporting yang and stopping nocturnal emission, and can be used for treating liver and kidney deficiency, soreness of waist and knees, sexual impotence and spermatorrhea, enuresis and frequent urination, and leukorrhagia. All the regions of the country are mainly produced in Shandong, henan, hebei, shanxi and Anhui provinces. Modern pharmacological researches show that semen Allii Tuberosi has effects of improving sexual function, improving immunity, resisting oxidation and aging. It mainly contains alkaloid, steroid saponin, nucleic acid and nucleoside and fatty acid components. The market of Chinese chive seed medicines is investigated, and it is found that, except for genuine products, chinese chives (dried mature seeds of Allium fistulosum Allium distulosum L. In Liliaceae) and Datura seed (dried mature seeds of Datura stramonium Datura stramonium L. In Solanaceae) with similar characters are often used as or mixed with Chinese chive seeds for sale, but the two have great difference with Chinese chive seed efficacy, especially the toxicity of Datura stramonium seed is great, poisoning can be caused by improper use, and the Chinese chive seed is not mixed. Therefore, it is needed to establish a thin-layer chromatography identification method for Chinese chive seed medicinal materials, which can fill the gap of the thin-layer identification item of Chinese chive seed in the 2020 edition of Chinese pharmacopoeia, and can be used as a specific identification method for distinguishing Chinese chive seed medicinal materials and mixed and counterfeit products thereof, and has important practical significance for improving the standard and quality control of Chinese chive seed medicinal materials and ensuring clinical accuracy and safe medication.
At present, the thin-layer chromatography identification method of the Chinese chive seed medicinal material and the identification research of the Chinese chive seed medicinal material and the common mixed products in the market are insufficient, zhu et al research the identification of the Chinese chive seed and the two mixed products thereof, and the Chinese chive seed and the mixed products thereof are distinguished through the properties, the powder microscopic characteristics and the thin-layer chromatography of the medicinal material, but the spots of the thin-layer chromatography in the research are beyond the Rf value range (0.2-0.8) specified by Chinese pharmacopoeia and can not be used as the identification basis in principle (Zhu, zheng Jiming. The identification of the Chinese chive seed and the two mixed products thereof [ J ]. Chinese pharmaceutical industry, 2012, 21 (2): 69-70.). Zhou Yunmei et al studied the identification of leek seeds and mixes, and identified true or false by microscopic identification by observing both cross sections and powders under a microscope, but microscopic identification of powders was low in resolution, time-consuming, labor-consuming, and highly subjective (Zhou Yunmei, xu Yulin. Identification of leek seeds and mixes [ J ]. Modern medical sanitation, 2005 (4): 458.). The above has some defects for the thin-layer chromatography of Chinese chives and the distinguishing research of common mixed and fake products in the market thereof, the property identification of the medicinal materials is excessively dependent on experience, is more limited by experience, knowledge and the like of an identifier, and especially has great difficulty in identification and popularization for varieties with very similar appearance properties, and the property identification cannot be carried out after the preparation; the existing thin-layer chromatography method has the defects of less extracted component information, single spot, incapability of better representing the quality of the Chinese chives and incapability of better distinguishing the Chinese chives from common mixed and counterfeited products in the market.
Disclosure of Invention
Problems to be solved by the invention
In order to solve the problems of the prior art. The invention aims to create a thin-layer identification method of the Chinese chive seed medicinal material, which is efficient, quick and accurate, is simple and easy to operate, has obvious identification characteristics, is easy to judge in result, has good specificity, can accurately identify the Chinese chive seed medicinal material, and can effectively distinguish the Chinese chive seed medicinal material from market common approximations, mixed imitations such as Chinese chive seed medicinal material and mandolin seed medicinal material.
Solution for solving the problem
The invention provides the following technical scheme:
[1] The invention provides a thin-layer identification method of Chinese chive seed medicinal materials, wherein the method comprises the steps of preparing a sample solution, preparing a control medicinal material solution and identifying the thin layer,
The preparation method of the sample solution comprises decocting semen Allii Tuberosi powder in water, filtering, concentrating, cooling, adding ethyl acetate or ethyl formate, shaking, extracting, evaporating to dryness, and dissolving the residue with alcohol solvent;
preparing a control medicinal material solution by taking a semen allii tuberosi control medicinal material according to the preparation step of the test sample solution;
The thin layer identification step comprises the steps of taking a sample solution and a reference medicinal material solution, respectively spotting the sample solution and the reference medicinal material solution on the same thin layer chromatographic plate, taking chloroform-ethyl formate-glacial acetic acid or toluene-ethyl acetate-formic acid as developing agents, and further taking the volume ratio of the chloroform: ethyl formate: glacial acetic acid= (3-8): (2-5): 1, a step of; toluene-ethyl acetate-formic acid= (5-9): (1.5-4): 1.
[2] The thin layer identification method according to [1], wherein the mass volume ratio of the medicinal material powder to the water used for decoction is 1g (30-65 mL).
[3] The thin layer identification method according to [1] or [2], wherein the decoction time is 20 to 45 minutes.
[4] The thin layer identification method according to any one of [1] to [3], wherein the number of times of shaking extraction by adding ethyl acetate or ethyl formate is at least 2, and further the amount of ethyl acetate or ethyl formate added is 10 to 30 ml/time.
[5] The thin layer identification method according to any one of the above [1] to [4], wherein the alcohol solvent is methanol or a methanol aqueous solution.
[6] The thin layer identification method according to any one of the technical schemes [1] to [5], wherein the thin layer identification step further comprises the steps of unfolding, airing, spraying a thin layer color developing agent, heating until spots develop clearly, and inspecting under an ultraviolet lamp.
[7] The thin layer identification method according to [6], wherein the thin layer developer is a 10% sulfuric acid ethanol solution.
[8] The thin layer identification method according to any one of the above [1] to [7], wherein the amount of spotting on the same silica gel G thin layer plate in the thin layer identification step is 5 to 10. Mu.L.
[9] The thin layer identification method according to any one of [1] to [8], wherein the method further comprises comparing the chromatogram of the sample with the chromatogram of the reference medicinal material, and judging whether fluorescent spots of the same color are displayed at the corresponding positions.
[10] The invention also provides an application of the thin layer identification method in any one of the technical schemes [1] to [9] in identification of the Chinese chive seed medicinal material and the mixed and fake products thereof.
ADVANTAGEOUS EFFECTS OF INVENTION
The thin-layer identification method of the Chinese chive seed medicinal material can better develop and separate the compound with larger polarity in the Chinese chive seed medicinal material, has rich and clear spots after color development, better separation degree, obvious identification characteristics, easy judgment of results and better specificity, reportedly the Chinese chive seed contains various components such as alkaloids, steroid saponins, nucleic acid, nucleosides, fatty acid components and the like, can better represent the quality of the Chinese chive seed medicinal material, improves the quality controllability of the identified product, has higher practical value, and has the advantages of simplicity, rapidness, high efficiency and low cost.
In addition, the thin-layer identification method of the Chinese chive seed medicinal material can effectively distinguish Chinese chive seeds from common mixed and fake products in the market, is convenient to popularize and apply, and provides basis for clinical accurate medication.
The above description does not disclose all embodiments of the present invention and all advantages of the present invention.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. The drawings in the following description are illustrative of certain embodiments of the invention and other drawings may be made by those skilled in the art without undue burden.
Fig. 1 shows the medicinal material properties of leek seed, onion seed and stramonium seed medicinal materials adopted in the embodiment of the invention.
FIG. 2 shows thin layer analysis (TLC) patterns (1-3: sample solution preparation method I, 4-6: sample solution preparation method II, 7-9: sample solution preparation method III, S1: control medicinal material solution preparation method I, S2: control medicinal material solution preparation method II, S3: control medicinal material solution preparation method III, and 10: negative control) of the preparation methods of different sample solutions and control solutions used in example 1 of the present invention.
FIG. 3 shows thin layer analysis (TLC) patterns of different color developers (3-1 is 10% sulfuric acid ethanol solution as color developer, 3-2 is 5% phosphomolybdic acid ethanol solution as color developer, 3-3 is 2% vanillin sulfuric acid solution as color developer, 1-3 are three parallel test sample solutions, and S is semen Allii Tuberosi control medicinal material).
FIG. 4 shows thin layer analysis (TLC) patterns of different developing agents (4-1 is chloroform-ethyl formate-glacial acetic acid as developing agent, 4-2 is toluene-ethyl acetate-formic acid as developing agent, 4-3 is chloroform-toluene-ethyl formate-glacial acetic acid as developing agent, 1-3 are three parallel test sample solutions, and S is semen Allii Tuberosi control medicinal material).
FIG. 5 shows thin layer analysis (TLC) patterns of different spotting amounts (leek seed medicinal material: 1 (1. Mu.L), 2 (3. Mu.L), 3 (5. Mu.L), 4 (10. Mu.L), leek seed control medicinal material: S1 (1. Mu.L), S2 (3. Mu.L), S3 (5. Mu.L), S4 (10. Mu.L)).
Fig. 6 shows a thin layer analysis (TLC) spectrum of a specificity test of a thin layer chromatography identification method of leek seed medicinal material (1 to 3 are 3 parallels of test solution, S is leek seed control medicinal material, and 4 is negative control).
FIG. 7 shows thin layer analysis (TLC) patterns of thin layer chromatography identification tests of different batches of leek seed medicinal material (1-9: leek seed medicinal material batches S1-S9, S: leek seed control medicinal material).
FIG. 8 shows a thin layer analysis (TLC) chart of an identification test of the Chinese chive seed medicinal material, the Chinese onion seed medicinal material and the Datura seed medicinal material (1-3: the Datura seed medicinal material (S13-S15), 4-6: the Chinese onion seed medicinal material (S10-S12), 7-9: the Chinese chive seed medicinal material (S1-S3), and S: the Chinese chive seed reference medicinal material).
Detailed Description
Numerous specific details are set forth in the following description in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, well known methods, procedures, means, equipment and steps have not been described in detail so as not to obscure the present invention.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In the present specification, the numerical range indicated by the term "numerical value a to numerical value B" means a range including the end point numerical value A, B.
In the present specification, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.
It should be understood that, as used in the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.
Reference in the specification to "one or more particular/preferred embodiments/aspects," "another or other particular/preferred embodiments/aspects," "one or another embodiment/aspect," "one or another technical aspect," etc., means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.
The term "comprising" in the description of the invention and the claims and in the above figures and any variants thereof is intended to cover a non-exclusive inclusion. For example, a process, method, or system, article, or apparatus that comprises a list of steps or elements is not limited to only those listed but may optionally include additional steps or elements not listed or inherent to such process, method, article, or apparatus.
The term "control" (or "reference") as used herein refers to a standard drug that has been identified as being of a species, for use in identifying a sample to be tested, unless otherwise indicated.
The term "test sample" as used herein refers to an experimental sample used for detection or identification, unless otherwise indicated.
The term "thin layer chromatography" as used herein is a micro, rapid, simple method of separation analysis, unless otherwise indicated. A suitable stationary phase is coated onto a glass plate, plastic or aluminum substrate in a uniform thin layer. And after spotting and developing, comparing the obtained sample with a chromatogram obtained by the same method according to a comparison shift value (Rf) and a proper control substance, and performing drug identification, impurity inspection or content determination. The method is an important experimental technique for rapidly separating and qualitatively analyzing a small amount of substances, and is also used for tracking the reaction progress. Has the characteristics of short unfolding time, strong separation capability, high sensitivity and the like. Thin layer chromatography is one of important inspection items of Chinese medicinal materials, and can judge the authenticity and quality of medicinal materials by comparing the chromatographic behaviors of the test sample and the control medicinal materials.
The term "spotting" as used herein refers to the process of dropping droplets of a sample solution to be separated, identified onto a thin-layer plate, unless otherwise specified.
The term "developing" as used herein, unless otherwise indicated, refers to the process by which the developing agent carries the sample components to migrate a distance on the lamina plate by capillary action of the stationary phase on the lamina plate.
The invention provides a thin-layer identification method of Chinese chive seed medicinal materials, which comprises the steps of obtaining a sample solution and a reference medicinal material solution by a simple and quick preparation method, respectively dotting on the same thin-layer plate, unfolding, and inspecting under an ultraviolet lamp. The method is simple, convenient, quick, high in efficiency and low in cost, can accurately identify the Chinese chive seed medicinal materials, can effectively distinguish the common approximate products, the mixed pseudo Chinese onion seed medicinal materials and the Datura seed medicinal materials in the market, and can be well applied to quality control of the Chinese chive seed medicinal materials.
A thin-layer identification method of Chinese chive seed medicinal materials is characterized by comprising the steps of preparing a sample solution, preparing a control medicinal material solution and identifying the thin layer.
< Preparation of sample solution >
The inventor examines the preparation method of the sample solution, compares the final extraction results, and finally determines: the preparation method of the test solution comprises the steps of taking semen allii tuberosi medicinal material powder, adding water for decoction and filtration, concentrating, cooling, adding ethyl acetate or ethyl formate for shaking extraction, evaporating to dryness, and adding alcohol solvent into residues for dissolution to obtain the test solution.
In some embodiments of the invention, the preparation of the test solution comprises taking a proper amount of semen Allii Tuberosi, wherein the "medicinal material" refers to raw materials of Chinese medicine which are not processed or prepared into finished products; pulverizing, sieving with second sieve, preferably decocting with water (such as distilled water, ultrapure water, etc.), wherein water extraction is closer to traditional decoction decocting manner of traditional Chinese medicine than ethanol extraction (such as methanol, ethanol, etc.), and can better characterize clinical quality of medicinal materials; in order to ensure the extraction efficiency, the mass volume ratio of the medicinal powder to the water used for decoction is 1g (30-65 mL), further can be 1g (35-60 mL), and is more preferably 1g (40-55 mL); the time for the water to decoct may be 20 to 45 minutes, preferably 25 to 40 minutes, and in some embodiments of the invention, the time for the decoct is 30 minutes. In the preparation of the sample solution, the filtration is not particularly limited, and may be any conventional filtration means in the art, such as filter paper, cotton filtration, etc. The filtrate obtained after filtration is subjected to concentration treatment, and can be evaporated to dryness in a water bath, heated and concentrated by a rotary evaporator or concentrated by a reduced pressure evaporation method, and in some specific embodiments of the invention, the filtrate is concentrated to 20mL.
The preparation of the sample solution also comprises the steps of adding the extraction solvent after cooling, and the cooling mode is not particularly limited in the invention, and the sample solution can be placed at normal temperature; for the extraction solvent, ethyl acetate or ethyl formate may be selected for extraction, in some embodiments of the invention, ethyl acetate is selected as the extraction solvent; the extraction mode is preferably shaking extraction to facilitate the operation, and in order to make the thin layer chromatography spots clearer and the separation effect better, it is preferable to add ethyl acetate or ethyl formate at least twice, wherein the amount of ethyl acetate or ethyl formate can be 10-30 mL/time, preferably 15-25 mL/time, more preferably 20 mL/time. The ethyl acetate or ethyl formate solutions are then combined, evaporated to dryness, and an alcoholic solvent is added to the residue, in some embodiments of the invention, the alcoholic solvent is selected from methanol, ethanol, and the like, with methanol being preferred from the standpoint of solubility. In some specific embodiments of the present invention, the alcohol solvent is added in an amount of preferably 0.3 mL-1.0 mL, more preferably 0.4 mL-0.8 mL, and still more preferably 0.5mL, in terms of dissolution efficiency.
The preparation method of the sample solution provided by the invention is simple, convenient, quick, high in efficiency and good in extraction effect, thin-layer chromatographic spots of the sample prepared by the method are clear and rich, the separation effect is good, and the sample chromatogram corresponds to the chromatographic spots of the reference medicinal material.
< Preparation of control medicinal solution >
The preparation of the control medicinal material solution comprises taking the leek seed control medicinal material, and the rest preparation methods refer to the preparation of the sample solution.
In addition, in some preferred embodiments of the present invention, the thin layer identification method of the present invention may further comprise preparation of a negative control solution, wherein the preparation of the negative control solution of the present invention comprises taking a negative sample not containing semen Allii Tuberosi, and the remaining preparation methods refer to < preparation of sample solution > described above.
< Thin layer authentication step >
The invention has conducted intensive researches on developing agents in the thin layer identification step, and the inventor discovers that chloroform-ethyl formate-glacial acetic acid and toluene-ethyl acetate-formic acid are used as developing agents, and compared with other developing agents such as chloroform-toluene-ethyl formate-glacial acetic acid, the developing agents are more beneficial to obtaining thin layer chromatograms of chives with multiple information and clear and rich spots.
In some embodiments of the present invention, the thin layer identification step includes taking a test solution and a control solution, respectively spotting the test solution and the control solution on the same thin layer chromatography plate, in some embodiments of the present invention, preferably spotting the thin layer chromatography plate on a silica gel thin layer plate, using chloroform-ethyl formate-glacial acetic acid or toluene-ethyl acetate-formic acid as developing agent, developing, taking out, airing, spraying a thin layer color developing agent, heating until spots develop clearly, and inspecting under an ultraviolet lamp. In some embodiments of the present invention, the sample application amount of the test solution or the control solution is 5. Mu.L to 10. Mu.L, and further 7. Mu.L to 10. Mu.L, and if the sample application amount is too low, the spot separation degree is relatively poor and the spots cannot be clearly displayed. The silica gel thin layer plate is selected from silica gel G thin layer plates, because other types of thin layer plates, such as silica gel GF254 thin layer plates, contain fluorescent substances, are mainly used for observing fluorescence under the ultraviolet light with the wavelength of 254nm, and have requirements on the wavelength; the high-efficiency silica gel G thin layer plate is mainly used for varieties with poor separation effect of common plates, has small development time difference and is high in cost. The invention uses chloroform-ethyl formate-glacial acetic acid or toluene-ethyl acetate-formic acid as developing agent to develop, which is favorable for separating various chemical components of semen allii tuberosi well on the thin layer plate. In some preferred embodiments of the present invention, to obtain better separation, the developing agent of the present invention comprises, by volume, chloroform: ethyl formate: glacial acetic acid= (3-8): (2-5): 1, further (4 to 7): (3-5): 1, further 6:3:1; toluene-ethyl acetate-formic acid= (5-9): (1.5-4): 1, further (6 to 8.5): (2-3): 1, further 7:2:1. in some embodiments of the invention, the thin layer of color reagent is selected from 10% sulfuric acid in ethanol, which can obtain better color effect relative to 5% phosphomolybdic acid in ethanol and 2% vanillin sulfuric acid; for the preparation method of the color developing agent, a conventional method can be adopted for preparation, for example, 10mL of concentrated sulfuric acid is added into 90mL of absolute ethyl alcohol, and the mass fraction of the adopted concentrated sulfuric acid is 95% -98%. And then heated at a temperature of 100 to 110 c, preferably 105 c. The invention can find that the chromatogram of the sample and the chromatogram of the reference medicine show clear spots with different colors when being inspected under an ultraviolet lamp (365 nm), and compared with the inspection under sunlight, the spots have clear color development and rich colors under ultraviolet light. In some embodiments of the invention, the test sample chromatogram shows fluorescent spots of the same color at positions corresponding to the control chromatogram.
In some specific embodiments of the present invention, the thin layer identification step of the present invention may further comprise taking a sample solution, a control medicinal solution and a negative control solution, respectively spotting on the same silica gel G thin layer plate, spreading with chloroform-ethyl formate-glacial acetic acid as a developing agent, taking out, drying, spraying a thin layer of color-developing agent, heating until spots develop clearly, and inspecting under an ultraviolet lamp. The result shows that the fluorescence spots with the same color appear on the positions corresponding to the control medicine chromatogram in the test sample chromatogram, and the negative control is not interfered.
By adopting the thin-layer identification method of the Chinese chive seed medicinal material, the chromatogram of the sample which is consistent with the spots of the reference medicinal material can be obtained, the separation degree and the spotting property in the chromatogram are better, the unfolding time is shorter, the detection is clear, and the quality of the Chinese chive seed medicinal material can be better controlled.
Examples
The technical scheme of the invention will be further described below in conjunction with specific examples. It should be understood that the following examples are illustrative and not intended to limit the scope of the present invention.
< Instrument and reagent >
A thin-layer automatic imager (CAMAH TLC VISUALIZER), a full-automatic thin-layer spotter (CAMAG TLC VISUALIZER), an ME204E one ten thousandth balance (Metrer-Tolyo), a KQ-250E ultrasonic cleaner (Kunshan ultrasonic electronics Co., ltd.), a silica gel G thin-layer plate (Qingdao marine chemical plant, qingdao Kang Yexin medical silica gel desiccant Co., ningpo solar desiccant Co., ltd.).
Methanol (national chemical reagent Co., ltd.), ethanol (national chemical reagent Co., ltd.), toluene (Shanghai Lingfeng chemical reagent Co., ltd.), ethyl acetate (national chemical reagent Co., ltd.), and formic acid (national chemical reagent Co., ltd.) all of which are analytically pure.
Semen Allii Tuberosi reference material (batch number 121358-200401) is purchased from China medicine biological product testing institute.
The 9 batches of Chinese chives and the 3 batches of shallot and the 3 batches of stramonium seeds are all from main production areas and area of land of China, wherein the Chinese chives meet the requirements of the 2020 edition of Chinese pharmacopoeia, the information of medicinal materials is shown in table 1, and the properties of the medicinal materials are shown in figure 1.
Table 1: chinese chive seed, shallot seed and stramonium seed medicinal material information table
Embodiment one: construction of thin-layer identification method of Chinese chive seed medicinal material
Investigation of preparation methods of sample solution and reference solution
< Preparation of sample solution >
The method comprises the following steps: taking 1g of the product powder, adding 50ml of water, decocting for 30 minutes, filtering, concentrating the filtrate to 20ml, cooling, shaking and extracting twice with 20ml of ethyl acetate each time, combining ethyl acetate solutions, evaporating to dryness, and adding 0.5ml of methanol into residues to dissolve the residues to obtain a sample solution.
The second method is as follows: taking 1g of the product powder, adding 20ml of methanol, carrying out ultrasonic treatment for 30 minutes, concentrating the filtrate to dryness, adding 20ml of water into the residue to dissolve, adding 20ml of ethyl acetate into the residue, shaking and extracting twice, merging the ethyl acetate solutions each time, evaporating to dryness, and adding 0.5ml of methanol into the residue to dissolve the residue to obtain a sample solution.
And a third method: taking 1g of the product powder, adding 20ml of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 0.5ml of methanol into residues to dissolve the residues to obtain a sample solution.
< Preparation of control medicinal solution >
The method comprises the following steps: taking 1g of semen Allii Tuberosi reference medicinal material, adding 50ml of water, decocting for 30min, filtering, concentrating the filtrate to 20ml, cooling, shaking and extracting twice with ethyl acetate, 20ml each time, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 0.5ml of methanol to obtain reference medicinal material solution.
The second method is as follows: taking 1g of semen Allii Tuberosi reference medicine, adding 20ml of methanol, carrying out ultrasonic treatment for 30 minutes, concentrating the filtrate to dryness, adding 20ml of water into the residue to dissolve, adding 20ml of ethyl acetate each time, shaking and extracting twice, combining ethyl acetate solutions, evaporating to dryness, and adding 0.5ml of methanol into the residue to dissolve to obtain reference medicine solution.
And a third method: taking semen Allii Tuberosi reference medicine 1g, adding ethanol 20ml, ultrasonic treating for 30 min, filtering, evaporating filtrate, and dissolving residue with methanol 0.5ml to obtain test solution.
< Preparation of negative control solution >
Taking 1g of negative control medicinal material sample of the semen allii tuberosi, and preparing a negative control solution according to the method one in the preparation of the test solution.
< Thin layer authentication >
Sample (lot number: S1) was prepared into test solution according to the above three preparation methods (3 parts in parallel for each method, 9 parts in total) respectively, and control medicinal solution was prepared according to the above three preparation methods, meanwhile negative control solution was prepared according to method one for the negative control sample, which was spotted on the same silica gel G thin layer plate respectively, and developed with chloroform-ethyl formate-glacial acetic acid (6:3:1) as developing agent, taken out, dried in the air, sprayed with 10% sulfuric acid ethanol solution, heated at 105℃until the spot color development was clear, and examined under ultraviolet light (365 nm). The results are shown in FIG. 2 (wherein 1-3 is the first preparation method of the sample solution, 4-6 is the second preparation method of the sample solution, 7-9 is the third preparation method of the sample solution, S1 is the first preparation method of the control medicinal material solution, S2 is the second preparation method of the control medicinal material solution, S3 is the third preparation method of the control medicinal material solution, and 10 is the negative control).
The experimental results show that: after the water is added for decoction, the spots are extracted by shaking with ethyl acetate, the separation degree is good, and the water extraction is closer to the traditional decoction mode, so that the clinical quality of the medicinal materials can be better represented. Therefore, the first method is selected as a preparation method of the test solution of the Chinese chive seed medicinal material and the reference medicinal material solution.
Investigation of thin layer authentication method
< Investigation of color developer >
Taking 1g of semen Allii Tuberosi powder (batch number: S1), adding 50ml of water, decocting for 30min, filtering, concentrating the filtrate to 20ml, cooling, extracting twice with ethyl acetate with shaking, 20ml each time, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 0.5ml of methanol to obtain test solution (parallel 3 parts). And 1g of semen allii tuberosi reference medicine is prepared to obtain a reference medicine solution. According to thin layer chromatography (rule 0502 of Chinese pharmacopoeia 2020 edition), sucking 5 μl of each of the above two solutions, respectively spotting on the same silica gel G thin layer plate, spreading with chloroform-ethyl formate-glacial acetic acid (6:3:1) as developing agent, taking out, air drying, respectively spraying 10% sulfuric acid ethanol solution, 5% phosphomolybdic acid ethanol solution, and 2% vanillin sulfuric acid solution, heating at 105deg.C until the spots develop clearly, and placing under ultraviolet light (365 nm) for inspection. The thin layer analysis (TLC) patterns sprayed with different color developers are respectively shown in figure 3, wherein 3-1 is a thin layer analysis (TLC) pattern with 10% sulfuric acid ethanol solution as the color developer, 3-2 is a thin layer analysis (TLC) pattern with 5% phosphomolybdic acid ethanol solution as the color developer, 3-3 is a thin layer analysis (TLC) pattern with 2% vanillin sulfuric acid solution as the color developer, 1-3 are 3 parallel test sample solutions, and S is a Chinese chive seed control medicinal material.
The experimental results show that: the color developing agent sprayed with 10% sulfuric acid ethanol solution has clear and rich spots under ultraviolet light (365 nm) than the spots of other two color developing agents. The color development conditions were therefore selected as follows: spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, and inspecting under ultraviolet lamp (365 nm).
< Examination of developing Agents >
Taking 1g of semen allii tuberosi powder, adding 50ml of water, decocting for 30 min, filtering, concentrating the filtrate to 20ml, cooling, shaking and extracting twice with 20ml of ethyl acetate each time, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 0.5ml of methanol to obtain a sample solution. And 1g of semen allii tuberosi reference medicine is prepared to obtain a reference medicine solution. According to thin layer chromatography (rule 0502 of Chinese pharmacopoeia 2020 edition), sucking 5 μl of each of the above two solutions, respectively spotting on the same silica gel G thin layer plate, respectively using chloroform-ethyl formate-glacial acetic acid (6:3:1), toluene-ethyl acetate-formic acid (7:2:1), and chloroform-toluene-ethyl formate-glacial acetic acid (4:2:3:1) as developing agents, developing, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until spot color is clear, and placing under ultraviolet lamp (365 nm) for inspection.
The experimental results show that: the chloroform-ethyl formate-glacial acetic acid and toluene-ethyl acetate-formic acid are used as developing agents, have good separation effects and are rich in spots, and a developing system composed of the chloroform, the ethyl formate and the glacial acetic acid which are relatively safe is selected as the developing agent through comparison. The thin layer analysis (TLC) patterns of different developing agents are shown in FIG. 4, wherein 4-1 is a thin layer analysis (TLC) pattern of chloroform-ethyl formate-glacial acetic acid as a developing agent, 4-2 is a thin layer analysis (TLC) pattern of toluene-ethyl acetate-formic acid as a developing agent, 4-3 is a thin layer analysis (TLC) pattern of chloroform-toluene-ethyl formate-glacial acetic acid as a developing agent, 1-3 are 3 parallel sample solutions, and S is a leek seed reference medicinal material.
< Investigation of sample application amount >
Taking semen Allii Tuberosi (batch number: S1), preparing into sample solution and control solution according to the above sample preparation method, respectively dispensing 1 μl, 3 μl,5 μl and 10 μl on the same silica gel G thin layer plate, spreading with chloroform-ethyl formate-glacial acetic acid (6:3:1) as developing agent, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, and inspecting under ultraviolet lamp (365 nm). The results are shown in FIG. 5 (leek seed drug: 1 (1. Mu.L), 2 (3. Mu.L), 3 (5. Mu.L), 4 (10. Mu.L), leek seed control drug: S1 (1. Mu.L), S2 (3. Mu.L), S3 (5. Mu.L), S4 (10. Mu.L)).
The results show that: when the sample application amount of the sample solution and the control medicinal material solution is 5 mu L, the spots in the corresponding positions of the sample chromatogram and the control medicinal material chromatogram clearly correspond to each other, and no other interference exists, so that the sample application amounts of the sample solution and the control medicinal material solution are 5 mu L.
Methodological verification of thin-layer identification method of semen allii tuberosi medicinal material
< Specificity test >
Sample application is carried out on a sample solution (batch number: S1, 3 parts in parallel), a control medicinal material solution (S) and a chives-deficiency seed negative control solution (4) on the same silica gel G thin layer plate, the sample application is carried out by taking chloroform-ethyl formate-glacial acetic acid (6:3:1) as a developing agent, the developing agent is taken out, the sample application is dried in the air, 10% sulfuric acid ethanol solution is sprayed, the sample application is heated at 105 ℃ until the spots are clear, and the sample application is carried out under an ultraviolet lamp (365 nm) for detection, and the results are shown in fig. 6 (1-3 are 3 parallels of the sample solution, S is a chives seed control medicinal material, and 4 is a negative control).
The result shows that the chromatogram of the test sample of the Chinese chive seed medicinal material and the chromatogram of the reference medicinal material show fluorescent spots with the same color at the corresponding positions, and the negative control has no interference. The specificity of the thin layer identification method is better.
< Specificity test >
1. Investigation of different temperatures
Taking 5 mu L of sample solution (batch number: S1) and 5 mu L of control medicinal solution, respectively spotting on the same silica gel G thin layer plate, spreading with chloroform-ethyl formate-glacial acetic acid (6:3:1) as spreading agent under different temperature conditions (room temperature, 4 ℃), taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the spot color is clear, and inspecting under ultraviolet lamp (365 nm).
The result shows that under different temperature conditions, the chromatogram of the tested sample of the Chinese chive seed medicinal material and the chromatogram of the reference medicinal material show fluorescent spots with the same color at the corresponding positions, and the separation effect is good. Experimental results show that the temperature has no great influence on thin-layer identification of the Chinese chive seed medicinal material, and the thin-layer identification method is good in durability for different temperatures.
2. Investigation of different humidity
Taking 5 mu L of sample solution (batch number: S1) and 5 mu L of control medicinal solution, respectively spotting on the same silica gel G thin layer plate, spreading with chloroform-ethyl formate-glacial acetic acid (6:3:1) as developing agent under different humidity conditions, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the spots develop clearly, and inspecting under ultraviolet lamp (365 nm).
The result shows that under the condition of different humidity, the chromatogram of the tested sample of the Chinese chive seed medicinal material and the chromatogram of the reference medicinal material show fluorescent spots with the same color at the corresponding positions, and the separation effect is good. Experimental results show that the humidity has no great influence on thin-layer identification of the Chinese chive seed medicinal materials, and the thin-layer identification method is good in durability to different humidity.
3. Investigation of different lamina plates
Taking 5 mu L of sample solution (batch number: S1) and 5 mu L of control medicinal solution, respectively spotting on silica gel G thin layer plates (Qingdao marine chemical plant, qingdao Kang Yexin medical silica gel desiccant Co., ningshan solar desiccant Co., ltd.) of different brands, spreading with chloroform-ethyl formate-glacial acetic acid (6:3:1) as developing agent, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the spot color is clear, and inspecting under ultraviolet lamp (365 nm).
The result shows that the chromatogram of the tested sample of the Chinese chive seed medicinal material and the chromatogram of the reference medicinal material show fluorescent spots with the same color at the corresponding positions, and the separation effect is good. The results show that the thin layer patterns of the silica gel G thin layer plates of different brands are not obviously different, and the thin layer identification method is better in durability on the thin layer plates of different brands.
Embodiment two: verification of thin-layer identification method of chives seed medicinal materials in different batches
Taking different batches of semen allii tuberosi medicinal materials (S1-S9), preparing a sample solution according to the preparation method of the determined sample solution, preparing a reference substance solution by taking semen allii tuberosi reference medicinal materials according to the preparation method of the determined reference medicinal material solution, respectively spotting on the same silica gel G thin layer plate, spreading with chloroform-ethyl formate-glacial acetic acid (6:3:1) as a developing agent, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until the spot color is clear, and placing an ultraviolet lamp (365 nm) for inspection. The results are shown in FIG. 7 (1-9: leek seed lot S1-S9, S: leek seed control).
The result shows that the test solution of the leek seed medicinal material in different batches shows fluorescent spots with the same color at the positions corresponding to the chromatogram of the control medicinal material. The thin-layer chromatography identification method can be used for better developing and separating the compounds with larger polarity in the Chinese chive seed medicinal materials, and has the advantages of rich and clear spots after color development, better separation degree and easy identification result judgment.
Embodiment III: identification of Chinese chive seed medicinal material and Chinese onion seed medicinal material and Datura seed medicinal material
Taking 3 batches of semen Allii Tuberosi medicinal materials (S1-S3), semen allii fistulosi medicinal materials (S10-S12) and semen Daturae medicinal materials (S13-S15), preparing a sample solution according to a preparation method of a determined sample solution, preparing a reference substance solution of semen allii tuberosi reference medicinal materials according to the preparation method of the determined reference medicinal material solution, respectively spotting on the same silica gel G thin layer plate, using chloroform-ethyl formate-glacial acetic acid (6:3:1) as developing agent, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until the spot color is clear, and placing an ultraviolet lamp (365 nm) for inspection. The results are shown in FIG. 8 (wherein 1-3 are Datura seed medicinal materials (S13-S15), 4-6 are Allium seed medicinal materials (S10-S12), 7-9 are semen Allii Tuberosi medicinal materials (S1-S3), and S is semen Allii Tuberosi reference medicinal material).
The result shows that under the condition of the thin-layer chromatography, the chromatographic spots of the semen allii tuberosi, the semen allii fistulosi and the semen stramonium are obviously different, the position a1 in the thin-layer chromatogram of the semen stramonium is obviously the spot, and the semen allii tuberosi and the semen allii fistulosi have no spot at the corresponding positions, so that the semen stramonium and other two can be distinguished; compared with the thin-layer chromatogram of the allium tuberosum medicinal material, the thin-layer chromatogram of the allium tuberosum medicinal material has one more spot at the position a2, so that the allium tuberosum and the allium tuberosum can be distinguished, and in addition, the sample solution of the allium tuberosum medicinal material shows fluorescent spots with the same color at the positions corresponding to the chromatogram of the reference medicinal material.
The above examples are only intended to illustrate several embodiments of the invention, which are described in more detail and are not to be construed as limiting the scope of the invention in any way. It should be clear to a person skilled in the art that several variations and modifications are possible without departing from the inventive concept, which fall within the scope of the present invention.
Claims (10)
1. A thin-layer identification method of Chinese chive seed medicinal materials is characterized by comprising the steps of preparing a sample solution, preparing a control medicinal material solution and identifying the thin layer,
The preparation method of the sample solution comprises decocting semen Allii Tuberosi powder in water, filtering, concentrating, cooling, adding ethyl acetate or ethyl formate, shaking, extracting, evaporating to dryness, and dissolving the residue with alcohol solvent;
preparing a control medicinal material solution by taking a semen allii tuberosi control medicinal material according to the preparation step of the test sample solution;
The thin layer identification step comprises the steps of taking a sample solution and a reference medicinal material solution, respectively spotting the sample solution and the reference medicinal material solution on the same thin layer chromatographic plate, taking chloroform-ethyl formate-glacial acetic acid or toluene-ethyl acetate-formic acid as developing agents, and further taking the volume ratio of the chloroform: ethyl formate: glacial acetic acid= (3-8): (2-5): 1, a step of; toluene-ethyl acetate-formic acid= (5-9): (1.5-4): 1.
2. The thin layer identification method according to claim 1, wherein the mass-to-volume ratio of the medicinal material powder to the water used for decoction is 1g (30-65 mL).
3. The thin layer identification method according to claim 1 or 2, wherein the decoction time is 20 to 45 minutes.
4. A thin layer authentication method according to any one of claims 1 to 3, wherein the number of shaking extractions performed by adding ethyl acetate or ethyl formate is at least 2, further wherein the amount of ethyl acetate or ethyl formate added is 10 to 30 ml/time.
5. The thin layer authentication method according to any one of claims 1 to 4, wherein the alcohol solvent is methanol or an aqueous methanol solution.
6. The method according to any one of claims 1 to 5, wherein the thin layer identification step further comprises spreading, air drying, spraying a thin layer of color developing agent, heating until the spots develop clearly, and inspecting under an ultraviolet lamp.
7. The method of claim 6, wherein the thin layer developer is 10% sulfuric acid ethanol solution.
8. The thin layer identification method according to any one of claims 1 to 7, wherein the amount of spotting of spots on the same silica gel G thin layer plate in the thin layer identification step is 5 to 10 μl.
9. The thin layer identification method according to any one of claims 1 to 8, further comprising comparing the chromatogram of the sample with the chromatogram of the reference drug to determine whether fluorescent spots of the same color are displayed at the corresponding positions.
10. Use of the thin layer identification method according to any one of claims 1-9 for identifying leek seed medicinal material and its mixed counterfeit products.
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