CN113759030B - Thin-layer identification method for glabrous sarcandra herb and glabrous sarcandra herb formula preparation - Google Patents
Thin-layer identification method for glabrous sarcandra herb and glabrous sarcandra herb formula preparation Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a thin-layer identification method of glabrous sarcandra herb and a glabrous sarcandra herb formula preparation. The method comprises respectively preparing a sample solution, a reference medicinal material solution and a reference solution, spotting the sample solution, the reference medicinal material solution and the reference solution on a same silica gel thin layer plate, developing with a developing agent, drying, and inspecting under an ultraviolet lamp; wherein the developing solvent comprises ammonia water, ethyl acetate and isopropanol. The thin-layer identification method provided by the invention can be used for identifying a single medicinal material of glabrous sarcandra herb and identifying a prescription preparation of the glabrous sarcandra herb. The identification method provided by the invention does not need to use a color developing agent in the identification process, and the identification method can be directly inspected under a fluorescent lamp, so that the fluorescent spots are clear and the separation degree is high.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a thin-layer identification method of glabrous sarcandra herb and a glabrous sarcandra herb formula preparation.
Background
Herba Pileae Scriptae is derived from dried whole herb of Sarcandra glabra (Thunb.) Nakai of Chloranthaceae. Has effects of clearing heat and cooling blood, promoting blood circulation and removing macula, dispelling pathogenic wind and dredging collaterals, and is mainly used for treating blood heat induced macula and rash, rheumatalgia, and traumatic injury. At present, in the market, the prescription preparation taking the glabrous sarcandra herb as the raw medicinal material mainly comprises an anticancer pill, a heat clearing and inflammation diminishing capsule, a glabrous sarcandra herb tablet and the like. In the prior art, in the quality standard of an anticancer pill product, in the identification result obtained by the identification item aiming at the glabrous sarcandra herb thin-layer chromatography identification method, the chromatogram of a test sample only shows one spot, no reference is made to a reference substance, and the developed system is benzene-ethyl acetate-methanol-isopropanol-concentrated ammonia water which is relatively complex.
In addition, in the thin layer chromatography identification and content determination of sarcandra glabra sanqing granules, which is published by Deng Ruihan et al, ethyl acetate needs to be added for extraction for many times when a test solution is prepared, the preparation of the test solution is complex and tedious, and a development system of the thin layer identification is toluene-ethyl acetate-formic acid, so that the toxicity of chemical reagents is high. Aiming at the problems of the prior art in identifying the glabrous sarcandra herb and the glabrous sarcandra herb formula preparation by a thin layer, the invention provides a thin layer identification method of the glabrous sarcandra herb and the glabrous sarcandra herb formula preparation.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defects that the test solution is complex, the development system is complex and toxic chemical reagents exist in the process of identifying the glabrous sarcandra herb thin layer in the prior art. Therefore, a thin-layer identification method of the glabrous sarcandra herb and the glabrous sarcandra herb formula preparation is provided.
Therefore, the invention provides the following technical scheme.
The invention provides a thin-layer identification method of glabrous sarcandra herb and a glabrous sarcandra herb prescription preparation, which comprises the following steps,
preparation of a test solution: taking a proper amount of a sample to be detected, and adding an organic solvent to prepare a sample solution;
preparation of reference drug solution: taking a proper amount of glabrous sarcandra herb reference medicinal material, and adding an organic solvent to prepare a reference medicinal material solution;
preparation of control solutions: taking a proper amount of isofraxidin, adding a solvent, and preparing a reference substance solution;
spotting the sample solution, the reference solution and the reference solution on the same silica gel thin layer plate, developing with ammonia water, ethyl acetate and isopropanol as developing agent, taking out, air drying, and inspecting under ultraviolet lamp.
The developing solvent comprises ammonia water, ethyl acetate and isopropanol in a volume ratio of (2.8-3.2) to (0.8-1.2) to (4.8-5.2).
The developing agent comprises ammonia water, ethyl acetate and isopropanol in a volume ratio of 3.
The preparation method of the test solution comprises grinding a sample to be tested into fine powder, collecting the fine powder, adding methanol, performing ultrasonic treatment, filtering, collecting filtrate, evaporating to dryness, adding methanol into the residue, and dissolving the residue to obtain a test solution;
the preparation method of the reference medicinal solution comprises collecting herba Pileae Scriptae reference medicinal material, adding methanol, ultrasonic processing, filtering, collecting filtrate, evaporating, and adding methanol into residue to dissolve the residue to obtain reference medicinal solution.
The preparation method of the reference solution comprises the steps of taking isofraxidin, adding methanol and preparing the isofraxidin into the reference solution;
each 1ml of the control solution contains 0.4-0.6mg of isofraxidin.
The preparation method of the test solution comprises grinding a sample to be tested into fine powder, collecting 0.8-1.1g of the fine powder, adding 23-26ml of 60% methanol, performing ultrasonic treatment for 25-35min, filtering, collecting filtrate, evaporating to dryness, adding 0.8-1.2ml of methanol into residues, and dissolving the residues to obtain the test solution;
the preparation method of the reference medicinal solution comprises collecting 0.8-1.1g herba Pileae Scriptae reference medicinal material, adding 23-26ml 60% methanol for 25-35min, performing ultrasonic treatment for 25-35min, filtering, collecting filtrate, evaporating, adding 0.8-1.2ml methanol into residue, and dissolving the residue to obtain reference medicinal solution.
The wavelength of the ultraviolet lamp is 365nm.
The sample to be detected is a glabrous sarcandra herb medicinal material and/or a glabrous sarcandra herb prescription preparation.
Further, the amount of each solution to be spotted was 0.5 to 3. Mu.L.
The technical scheme of the invention has the following advantages:
1. the invention provides a thin-layer identification method of glabrous sarcandra herb and glabrous sarcandra herb prescription preparation, which comprises the steps of respectively preparing a test solution, a reference medicinal material solution and a reference solution, spotting the test solution, the reference medicinal material solution and the reference solution on the same silica gel thin-layer plate, developing by using a developing agent, drying and then placing under an ultraviolet lamp for inspection; wherein the developing solvent comprises ammonia water, ethyl acetate and isopropanol. The thin-layer identification method provided by the invention can be used for identifying a single medicinal material of glabrous sarcandra herb and identifying a prescription preparation of glabrous sarcandra herb.
The identification method provided by the invention does not need to use a color developing agent in the identification process, and the identification method can be directly inspected under a fluorescent lamp, so that the fluorescent spots are clear and the separation degree is high. The invention takes ammonia water, ethyl acetate and isopropanol as developing agents which are determined by factors such as the dissolubility of analyzed substances in a test sample, the structure of the analyzed substances and the like, the ethyl acetate and the isopropanol have good separation effect on the test sample, and the ammonia water is added to adjust the degree of separation of the development and reduce the phenomenon of spot tailing. The developing solvent does not contain high-toxicity and explosive solvents, reduces the experiment cost and environmental pollution, and protects the body health of operators. The identification method provided by the invention has no special requirements on temperature and humidity, the temperature and humidity have no influence on the identification result, and the identification method has better durability and reproducibility.
The invention takes the glabrous sarcandra herb as the reference medicinal material solution, the spots of the test sample solution correspond to the spots of the reference medicinal material one by one, and the invention has good specificity and eliminates the possibility of generating the same spots due to interference caused by other components.
2. According to the thin layer identification method provided by the invention, ammonia water, ethyl acetate and isopropanol in a specific ratio are used as developing agents, so that the obtained thin layer has clearer spots and higher separation degree. Compared with the prior art, the preparation method of the test solution provided by the invention is simple and easy to operate, does not contain complicated operation processes such as reflux and extraction, saves the use amount of organic solvent, and shortens the time for preparing the test solution.
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, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a thin-layer chromatogram of a test solution, a blank solution and a reference solution in examples 1 to 4 of the present invention;
FIG. 2 is a thin layer chromatogram of a blank solution, a reference drug solution, a reference solution and a test solution of comparative example 1 and comparative example 2 according to the present invention;
FIG. 3 is a thin layer chromatogram for spot size examination in example 5 of the present invention;
FIGS. 4 to 7 are thin layer chromatograms for temperature and humidity investigation in example 5 of the present invention;
FIGS. 8 to 10 are thin layer chromatograms of different thin layer plate studies according to example 5 of the present invention;
FIG. 11 is a thin layer chromatogram for different extraction solvent studies of example 5 of the present invention;
FIG. 12 is a thin layer chromatogram verified by the thin layer identification method of example 6 of the present invention.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Experimental materials and instruments
Experimental materials:
anti-cancer pill, hainan longshengtang pharmaceutical limited, lot number: 180802; carrying out thin-layer identification by taking the anticancer plain pills as a test sample of a glabrous sarcandra herb formula preparation;
glabrous sarcandra herb as a reference medicinal material, china institute for food and drug assay, lot number: 121293-201404;
ammonia, national drug group chemical reagents ltd, batch No.: 20190522;
isopropyl alcohol, national pharmaceutical group chemical agents ltd, batch No.: 20190116;
formic acid, national pharmaceutical group chemical reagents ltd, batch No.: 20190301;
ethyl acetate, national pharmaceutical group chemical agents limited, lot number: 20191108;
isofraxidin, chinese food & drug institute, batch number: 110837-201206; purity: 99.5 percent;
methanol, national pharmaceutical group chemical reagents ltd, batch No.: 20190618.
an experimental instrument:
one-ten-thousandth balance, type: ML204T; the manufacturer: mertler-deliduous instruments (shanghai) ltd;
one-tenth-ten-thousandth balance, type: 125P-CE; the manufacturer: sidorist scientific instruments (Beijing) Inc.;
ultrasonic cleaner, model: KQ-100DE; the manufacturer: kunshan ultrasonic instruments, inc.;
thin layer imager, model: goodLook-1000; the manufacturer: shanghai science and technology, inc.;
the silica gel G thin-layer plate is manufactured by Qingdao ocean chemical industry Co., ltd (batch number: 20190307); tintario silica gel development Co., ltd. (batch No.: 20161007); the institute for chemical industry of cigarette Tai (batch number: 20190808).
Example 1
This example provides a thin-layer identification method for a glabrous sarcandra herb formulation, which comprises the following steps,
preparation of a test solution: grinding into fine powder, collecting 0.9180g fine powder, adding 25ml60% methanol, ultrasonic treating for 30min, filtering, collecting filtrate, evaporating, adding 1ml methanol into residue, and dissolving the residue to obtain test solution.
Preparation of reference drug solution: collecting 1.0000g herba Pileae Scriptae control, adding 25ml60% methanol, ultrasonic treating for 30min, filtering, collecting filtrate, evaporating, and adding 1ml methanol into residue to dissolve the residue to obtain herba Pileae Scriptae control solution.
Preparation of control solutions: adding methanol into isofraxidin reference substance to obtain reference substance solution, wherein each 1ml of reference substance solution contains 0.5mg of isofraxidin.
Preparation of a blank solution: 1ml of methanol was used as a blank solution.
The method comprises the steps of sucking 1 mu L of blank solution, 1 mu L of test solution, 0.5 mu L of glabrous sarcandra herb contrast medicinal material solution and 0.5 mu L of isofraxidin contrast solution, respectively dropping the solutions on the same silica gel G thin-layer plate (Qingdao ocean chemical Co., ltd.), taking ammonia water-ethyl acetate-isopropanol (volume ratio is 3. The developing agent of the present example was developed at a temperature of 20. + -. 1 ℃ and a humidity of 21. + -. 1%. Wherein, the identification result of the blank solution is shown as 1 in figure 1; the test solution is shown as 2 in figure 1; the isofraxidin control solution is shown in figure 1 at 4; the herba Pileae Scriptae reference medicinal solution is shown in 5 of figure 1.
As can be seen from FIG. 1, the TLC of the test solution shows the same color of fluorescent spots as the control solution and the reference solution at the same positions, and has moderate Rf value.
Example 2
This example provides a thin layer assay method of a glabrous sarcandra herb formulation, which is different from example 1 in that the amount of spotting is different when thin layer assay is performed, the amount of spotting of the sample solution is 1.5. Mu.L, and the thin layer assay result is shown in 3 in FIG. 1.
Example 3
This example provides a thin layer assay method of a glabrous sarcandra herb formulation, which is different from example 1 in that the amount of spotting is different when thin layer assay is performed, the amount of spotting of the sample solution is 2 μ L, and the thin layer assay result is shown in fig. 1, 6.
Example 4
This example provides a thin layer assay method of a glabrous sarcandra herb formulation, which is different from example 1 in that the amount of spotting is different when thin layer assay is performed, the amount of spotting of the sample solution is 2.5. Mu.L, and the thin layer assay result is shown in 7 in FIG. 1.
Comparative example 1
The comparative example provides a thin-layer identification method of a glabrous sarcandra herb prescription preparation, which comprises the following steps,
preparing a test solution: grinding into fine powder, collecting 0.9180g fine powder, adding 25ml60% methanol, ultrasonic treating for 30min, filtering, collecting filtrate, evaporating, adding 1ml methanol into residue, and dissolving the residue to obtain test solution.
Preparation of reference drug solution: taking 1.0000g herba Pileae Scriptae control medicinal material, adding 25ml60% methanol, ultrasonic treating for 30min, filtering, taking filtrate, evaporating to dryness, adding 1ml methanol into residue, and dissolving the residue to obtain herba Pileae Scriptae control medicinal material solution;
taking 1.0002g of herba Scutellariae Barbatae reference medicinal material, adding 25ml of 60% methanol, performing ultrasonic treatment for 30min, filtering, taking filtrate, evaporating to dryness, adding 1ml of methanol into residue, and dissolving the residue to obtain herba Scutellariae Barbatae reference medicinal material solution;
collecting 1.0001g herba Hedyotidis Diffusae control, adding 25ml60% methanol, ultrasonic processing for 30min, filtering, collecting filtrate, evaporating to dryness, adding 1ml methanol into residue, and dissolving the residue to obtain herba Hedyotidis Diffusae control solution.
Preparation of control solutions: adding methanol into isofraxidin reference substance to obtain reference substance solution, wherein each 1ml of reference substance solution contains 0.5mg of isofraxidin;
adding methanol into scutellarin control to obtain control solution, wherein each 1ml of control solution contains 0.5mg of scutellarin;
preparation of a blank solution: 1ml of methanol was used as a blank solution.
The method comprises the following steps of sucking 1 mu L of blank solution, 3 mu L of test solution, 2 mu L of glabrous sarcandra herb contrast solution, 1.5 mu L of isofraxidin contrast solution, 4 mu L of spreading hedyotis herb contrast solution, 2 mu L of scutellarin contrast solution and 5 mu L of sculellaria barbata contrast solution, respectively spotting the solution on the same silica gel G thin layer plate (Qingdao ocean chemical Co., ltd.), developing by taking ammonia water-absolute ethyl alcohol-n-butyl alcohol (volume ratio is 3. The developing of the developing solvent of this comparative example was carried out at a temperature of 23. + -. 5 ℃ and a humidity of 40. + -. 5%. Wherein, the identification result of the blank solution is shown as 1 in figure 2; herba Hedyotidis Diffusae reference medicinal material solution is shown in figure 2; the herba Pileae Scriptae reference medicinal solution is shown in figure 2 at 3; the isofraxidin control solution is shown in figure 2 at 4; the test solution is shown as 5 in FIG. 2; the wild Scutellariae radix reference solution is shown as 7 in figure 2, and herba Scutellariae Barbatae reference solution is shown as 8 in figure 2.
Comparative example 2
The comparison example provides a thin layer identification method of the glabrous sarcandra herb formula preparation, which is different from the comparison example 1 in that the sample application amount is different, the sample application amount of the sample solution of the comparison example is 4 mu L, and the thin layer chromatography is shown as 6 in figure 2.
Through the identification chromatogram in fig. 2, the test solution, the reference solution and the reference solution show fluorescence spots with the same color at the same position, but have trailing phenomenon and poor unfolding effect, and the unfolding agent system using ammonia water, absolute ethyl alcohol and n-butyl alcohol as unfolding agents is not suitable for identifying the glabrous sarcandra herb formula preparation.
In addition, as can be seen from the chromatogram in fig. 2, the spots of the herba Hedyotidis Diffusae reference medicinal material solution and the herba Scutellariae Barbatae reference medicinal material solution are not clear, and the development effect is not good. The wild scutellaria baicalensis contrast solution has poor unfolding effect. Therefore, the invention takes the glabrous sarcandra herb as a reference medicinal material and isofraxidin as a reference substance.
Example 5
This example provides a methodological review of the thin layer identification method
(1) Investigation of amount of dots
A test solution, a control solution and a reference solution are prepared according to the method in example 1, 1 μ L of blank solution is taken, 0.5 μ L, 1 μ L, 1.5 μ L and 2 μ L of test solution are taken for the test solution, 0.5 μ L of reference solution and 0.5 μ L of control solution are taken for the control solution, the blank solution and the reference solution are respectively spotted on the same silica gel G thin-layer plate (Qingdao ocean chemical Co., ltd.), ammonia water-ethyl acetate-isopropanol (volume ratio 3. FIG. 3 shows that when the sample amount of the sample solution is 1 μ L, the sample amount of the control solution is 0.5 μ L, and the sample amount of the control solution is 0.5 μ L, the thin layer chromatography has clear spots, good resolution, and moderate Rf. The blank solution corresponds to 1 in fig. 3; the test solution of 0.5. Mu.L, 1. Mu.L, 1.5. Mu.L and 2. Mu.L respectively correspond to 2, 3, 6 and 7 in FIG. 3; the isofraxidin control solution corresponds to 4 in fig. 3; the herba Pileae Scriptae control solution corresponds to 5 in FIG. 3.
(2) Examination of development temperature and humidity
A. A test solution, a control solution and a reference solution are prepared according to the method in example 1, 1 μ L of the test solution, 0.5 μ L of the glabrous sarcandra herb control solution, 0.5 μ L of the isofraxidin control solution and 1 μ L of the blank solution are respectively spotted on the same silica gel G thin-layer plate (Qingdao ocean chemical Co., ltd.), developed under the conditions of the temperature of 28 ± 5 ℃ and the humidity of 44 ± 5%, taken out, dried, placed under an ultraviolet lamp (365 nm) for inspection, and a thin-layer chromatogram is shown in fig. 4 by taking ammonia water-ethyl acetate-isopropanol (volume ratio of 3. The blank solution corresponds to 1 in fig. 4; the test solution corresponds to 3 in fig. 4; herba Pileae Scriptae reference medicinal solution corresponds to 4 in FIG. 4; the isofraxidin control solution corresponds to 2 in figure 4.
B. A test solution, a control solution and a reference solution are prepared according to the method in example 1, 1 μ L of the test solution, 0.5 μ L of the glabrous sarcandra herb control solution, 0.5 μ L of the isofraxidin control solution and 1 μ L of the blank solution are respectively spotted on the same silica gel G thin-layer plate (Qingdao ocean chemical Co., ltd.), developed under the conditions of 6 ± 5 ℃ and 45 ± 5% humidity, taken out, dried, placed under an ultraviolet lamp (365 nm) for inspection, and a thin-layer chromatogram is shown in fig. 5, wherein the developing agent is ammonia water-ethyl acetate-isopropanol (volume ratio is 3. The blank solution corresponds to 1 in fig. 5; the test solution corresponds to 3 in fig. 5; herba Pileae Scriptae reference solution corresponds to 4 in FIG. 5; the isofraxidin control solution corresponds to 2 in figure 5.
C. A test solution, a control solution and a reference solution are prepared according to the method in example 1, 1 μ L of the test solution, 0.5 μ L of the glabrous sarcandra herb control solution, 0.5 μ L of the isofraxidin control solution and 1 μ L of the blank solution are respectively spotted on the same silica gel G thin-layer plate (Qingdao ocean chemical Co., ltd.), developed under the conditions that the temperature is 27 ± 5 ℃ and the humidity is 9 ± 5%, taken out, dried, placed under an ultraviolet lamp (365 nm) for inspection, and a thin-layer chromatogram is shown in fig. 6, wherein ammonia water-ethyl acetate-isopropanol (volume ratio is 3. The blank solution corresponds to 1 in fig. 6; the test solution corresponds to 3 in fig. 6; herba Pileae Scriptae reference medicinal solution corresponds to 4 in FIG. 6; the isofraxidin control solution corresponds to 2 in figure 6.
D. A test solution, a control solution and a reference solution are prepared according to the method in example 1, 1 μ L of the test solution, 0.5 μ L of the glabrous sarcandra herb control solution, 0.5 μ L of the isofraxidin control solution and 1 μ L of the blank solution are respectively spotted on the same silica gel G thin-layer plate (Qingdao ocean chemical Co., ltd.), developed under the conditions of 27 ± 5 ℃ and 68 ± 5% of humidity, taken out, dried, placed under an ultraviolet lamp (365 nm) for inspection, and a thin-layer chromatogram is shown in fig. 7 by taking ammonia water-ethyl acetate-isopropanol (volume ratio of 3. The blank solution corresponds to 1 in fig. 7; the test solution corresponds to 3 in fig. 7; herba Pileae Scriptae reference medicinal solution corresponds to 4 in FIG. 7; the isofraxidin control solution corresponds to 2 in figure 7.
Fig. 4-fig. 7 are thin layer chromatography charts showing that the test solution, the reference solution and the reference solution show spots of the same color at the same positions, and the spot separation effect is good. The thin layer identification method provided by the invention has good durability to temperature and humidity, can be used for identification under various temperatures and humidities, and has small influence on the identification result by the temperature and the humidity.
(3) Investigation of different lamella plates
A. Preparing a test solution, a reference medicinal material solution and a reference solution according to the method in example 1, taking 1 μ L of the test solution, 0.5 μ L of the reference medicinal material solution, 0.5 μ L of the reference solution and 1 μ L of the blank solution, respectively spotting on the same silica gel G thin-layer plate (Qingdao ocean chemical Co., ltd.), developing with ammonia water-ethyl acetate-isopropanol (volume ratio: 3: 1). The blank solution corresponds to 1 in fig. 8; the test solution corresponds to 3 in fig. 8; herba Pileae Scriptae reference solution corresponds to 4 in FIG. 8; the isofraxidin control solution corresponds to 2 in figure 8.
B. A test solution, a reference solution and a reference solution are prepared according to the method in example 1, and 1 μ L of the test solution, 0.5 μ L of the reference solution and 1 μ L of the blank solution are respectively spotted on the same silica gel G thin layer plate (fumitory Jiang You silica gel development ltd), ammonia water-ethyl acetate-isopropanol (volume ratio 3. The blank solution corresponds to 1 in fig. 9; the test solution corresponds to 3 in fig. 9; herba Pileae Scriptae reference solution corresponds to 4 in FIG. 9; the isofraxidin control solution corresponds to 2 in figure 9.
C. A test solution, a reference drug solution and a reference solution are prepared according to the method in example 1, and 1 μ L of the test solution, 0.5 μ L of the reference drug solution, 0.5 μ L of the reference solution and 1 μ L of the blank solution are respectively spotted on the same silica gel G thin layer plate (tai chemical industry research institute), ammonia water-ethyl acetate-isopropanol (volume ratio 3. The blank solution corresponds to 1 in fig. 10; the test solution corresponds to 3 in fig. 10; herba Pileae Scriptae reference medicinal solution corresponds to 4 in FIG. 10; the isofraxidin control solution corresponds to 2 in figure 10.
The thin-layer chromatography in fig. 8-10 can show that the thin-layer plates produced by tiaojiang friend silica gel development ltd and Qingdao ocean chemical ltd have better thin-layer chromatography effect, can display fluorescent spots with the same color, and have clear spots and good correspondence.
(4) Investigation of different extraction solvents
Preparation of a test solution: grinding into fine powder to obtain fine powder, adding ethanol (50 ml) into 1.0001g of the fine powder, ultrasonic treating for 30min, cooling, filtering, evaporating the filtrate, and dissolving the residue with methanol (1 ml) to obtain test solution.
The preparation method of herba Pileae Scriptae reference medicinal solution is the same as the above test solution.
Preparation of control solutions: adding methanol into isofraxidin reference substance to obtain reference substance solution, wherein each 1ml of reference substance solution contains 0.5mg of isofraxidin.
Taking 1 μ L of a test solution, 0.5 μ L of a reference drug solution, 0.5 μ L of a reference solution and 1 μ L of a blank solution, respectively spotting on the same silica gel G thin-layer plate (Qingdao ocean chemical Co., ltd.), taking ammonia water-ethyl acetate-isopropanol (volume ratio is 3.
From the identification results of fig. 11, the spots of the control drug solution and the spots of the test solution are not clear, and ethanol is not suitable as an extraction solvent.
Example 6
This example provides validation of a thin layer authentication method, including,
taking 1 μ L of blank solution, 1 μ L of each of test solution of different batches, 0.5 μ L of contrast solution and 0.5 μ L of contrast solution, respectively spotting on the same silica gel G thin-layer plate (Qingdao ocean chemical Co., ltd.), taking ammonia water-ethyl acetate-isopropanol (volume ratio is 3. In fig. 12, 1 is a blank solution, 8 is a glabrous sarcandra herb reference solution, and 9 is an isofraxidin reference solution; the test solution batches are 181201, 180901, 180803, 190301, 190601, 180802, 190201, 181101, 190101, 181001, 190501, respectively, which correspond to 2, 3, 4, 5, 6, 7, 10, 11, 12, 13 and 14 in fig. 12.
According to the display of fig. 12, the chromatogram of the test solution, the chromatogram of the control solution and the chromatogram of the control solution display spots of the same color at the same positions, and the spread distance is moderate, which indicates that the identification method provided by the invention has good stability.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.
Claims (7)
1. A thin-layer identification method of glabrous sarcandra herb and glabrous sarcandra herb formula preparation is characterized by comprising the following steps,
preparation of a test solution: taking a proper amount of a sample to be detected, and adding a methanol solution to prepare a sample solution;
preparation of reference drug solution: taking a proper amount of glabrous sarcandra herb reference medicinal material, and adding a methanol solution to prepare a reference medicinal material solution;
preparation of control solutions: taking a proper amount of isofraxidin, and adding a solvent to prepare a reference substance solution;
spotting the sample solution, the reference solution and the reference solution on the same silica gel thin layer plate, developing with ammonia water, ethyl acetate and isopropanol as developing agent, taking out, air drying, and inspecting under ultraviolet lamp;
the glabrous sarcandra herb formula preparation is an anti-cancer pill;
the volume ratio of ammonia water, ethyl acetate and isopropanol in the developing solvent is (2.8-3.2): (0.8-1.2): 4.8-5.2.
2. The thin layer identification method of claim 1, wherein the developing solvent comprises ammonia, ethyl acetate and isopropanol in a volume ratio of 3.
3. The thin-layer identification method according to claim 1 or 2, wherein the sample solution is prepared by grinding a sample to be tested into fine powder, collecting the fine powder, adding a methanol solution, performing ultrasonic treatment, filtering, collecting the filtrate, evaporating to dryness, adding methanol to the residue, and dissolving the residue to obtain a sample solution;
the preparation method of the reference medicinal material solution comprises collecting herba Pileae Scriptae reference medicinal material, adding methanol solution, ultrasonic processing, filtering, collecting filtrate, evaporating, and adding methanol into residue to dissolve the residue to obtain reference medicinal material solution.
4. The thin layer identification method as claimed in claim 1 or 2, wherein the control solution is prepared by preparing isofraxidin and adding methanol to obtain a control solution;
each 1ml of the control solution contains 0.4-0.6mg of isofraxidin.
5. The thin layer identification method of claim 3, wherein the sample solution is prepared by grinding a sample to be tested into fine powder, collecting 0.8-1.1g of the fine powder, adding 23-26ml of 60% methanol, performing ultrasonic treatment for 25-35min, filtering, collecting the filtrate, evaporating, adding 0.8-1.2ml of methanol into the residue, and dissolving the residue to obtain a sample solution;
the preparation method of the reference medicinal material solution comprises collecting 0.8-1.1g herba Pileae Scriptae reference medicinal material, adding 23-26ml 60% methanol, ultrasonic treating for 25-35min, filtering, collecting filtrate, evaporating, and adding 0.8-1.2ml methanol into residue to dissolve the residue to obtain reference medicinal material solution.
6. The method of claim 1 or 2, wherein the uv lamp has a wavelength of 365nm.
7. The method of claim 1 or 2, wherein the amount of spots is 0.5-3 μ L.
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