CN117405813A - Canavalia gladiata extract and Canavalia gladiata extract thin layer identification method - Google Patents
Canavalia gladiata extract and Canavalia gladiata extract thin layer identification method Download PDFInfo
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- 239000000284 extract Substances 0.000 title claims abstract description 113
- 240000003049 Canavalia gladiata Species 0.000 title claims abstract description 105
- 238000000034 method Methods 0.000 title claims abstract description 45
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 87
- 239000000463 material Substances 0.000 claims abstract description 64
- 239000013558 reference substance Substances 0.000 claims abstract description 47
- 239000000523 sample Substances 0.000 claims abstract description 37
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 210000000582 semen Anatomy 0.000 claims abstract description 22
- 238000004809 thin layer chromatography Methods 0.000 claims abstract description 15
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- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000010438 heat treatment Methods 0.000 claims abstract description 10
- 238000005507 spraying Methods 0.000 claims abstract description 8
- 238000001514 detection method Methods 0.000 claims abstract description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 42
- 235000010520 Canavalia ensiformis Nutrition 0.000 claims description 27
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 22
- 244000045232 Canavalia ensiformis Species 0.000 claims description 19
- 229940069765 bean extract Drugs 0.000 claims description 15
- 238000001704 evaporation Methods 0.000 claims description 14
- 241000220451 Canavalia Species 0.000 claims description 10
- 229960000583 acetic acid Drugs 0.000 claims description 9
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 9
- 239000012362 glacial acetic acid Substances 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
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- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 7
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- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 7
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 7
- 239000004473 Threonine Substances 0.000 claims description 7
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 7
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- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 6
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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)
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Abstract
The invention provides a thin-layer identification method of a Canavalia gladiata extract and a Canavalia gladiata extract, which comprises the following steps: pretreating Canavalia gladiata extract to obtain a sample solution; pretreating semen Canavaliae extract to obtain semen Canavaliae extract solution; decocting semen Canavaliae reference medicinal material in water, filtering, and adding methanol to obtain reference medicinal material solution; adding methanol solution into the reference substance to obtain reference substance solution; performing thin layer chromatography detection on the sample solution, the semen Canavaliae extract solution, the reference substance solution and the reference medicinal material solution, spraying ninhydrin solution, heating and inspecting, and if the sample solution and the reference medicinal material solution have fluorescent spots with the same sample color at the equal positions of Rf, obtaining semen Canavaliae extract. The thin-layer identification chromatographic method of the Canavalia gladiata extract is used for rapidly, accurately and effectively identifying the Canavalia gladiata extract. Meanwhile, whether a thin-layer chromatographic spectrum has fluorescent spots in a certain ratio shift range is used as a discrimination point for distinguishing the Canavalia gladiata extract from the Canavalia gladiata extract.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a thin-layer identification method for a Canavalia gladiata extract and a Canavalia gladiata extract.
Background
Canavalia gladiata is widely used, but currently there is less research on Canavalia gladiata. The content measurement and qualitative identification of the Canavalia gladiata are not regulated in Chinese pharmacopoeia and Chinese medicinal material standards and processing specifications of each province, and researches on the components of the Canavalia gladiata are only reported in literature (Konjac Ming, xie Jianfeng, li Yingchen, wang Ying, han Yongping. Optimization of ultrasonic extraction of Tibetan medicine Canavalia gladiata total flavonoids and antioxidative active Chinese patent medicines, 2016,38 (05): 1163-1167), but few in-depth researches on the Canavalia gladiata extracts are reported.
The semen Canavaliae belongs to leguminous plants, is a pseudo product of semen Canavaliae, has similar properties to semen Canavaliae, and can distinguish the two medicinal materials through property identification. However, the extract is extracted, the characteristics disappear, and the characteristics identification cannot be used for identifying the extract.
Therefore, a method with stronger specificity, stronger repeatability and effectiveness and controllability is required to be established, an effective means is provided for qualitative identification of the Canavalia gladiata extract, and the invention aims to establish a method capable of effectively identifying Canavalia gladiata and Canavalia gladiata extracts.
Disclosure of Invention
In view of the above, the present invention aims to provide a thin layer identification method for Canavalia gladiata extract and Canavalia gladiata extract.
The terms "comprising," "including," and "having" are used interchangeably herein to mean that the elements are included in an arrangement, meaning that the arrangement may exist in addition to the elements listed. It should also be understood that the use of "including," "comprising," and "having" descriptions herein also provides a "consisting of … …" scheme.
In the present application, the term "and/or" describes an association relationship of an association object, which means that three relationships may exist, for example, a and/or B may mean that a exists alone, a and B exist together, and B exists alone. Wherein A, B may be singular or plural.
In the present application, "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s).
It should be understood that, in various embodiments of the present application, the sequence number of each process described below does not mean that the execution sequence of some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
The invention provides construction and application of a thin-layer identification method of a Canavalia gladiata extract, wherein the chromatogram of a sample shows the same color spots compared with the chromatograms of a reference substance and a Canavalia gladiata reference medicinal material, and can be used for identifying the Canavalia gladiata extract. Compared with the thin-layer chromatograms of the reference substances and the sword bean reference medicinal materials, the sample has spots at the positions with the specific shift value of 0.225-0.257,0.495-0.605,0.599-0.732, and the mixed and fake sword bean extract has no obvious spots, and can be used as a discrimination point for distinguishing sword bean from the sword bean extract. The thin-layer method can rapidly and effectively identify the semen Canavaliae extract with lost decoction piece form and distinguish the semen Canavaliae extract from the semen Canavaliae extract, and has the advantages of simple operation, high precision and sensitivity, good stability and good application prospect.
The thin-layer identification chromatographic method for the Canavalia gladiata extract, which is established by the invention, can rapidly, accurately and effectively identify the Canavalia gladiata extract. Meanwhile, whether spots exist in a certain ratio shift range or not is observed through the thin-layer chromatographic spectrum, and the thin-layer chromatographic spectrum is used as an identification point for rapidly and effectively identifying the Canavalia gladiata and Canavalia gladiata extracts, so that guidance is provided for quality control of the Canavalia gladiata extracts and related preparations thereof.
The invention provides a thin-layer identification method of a Canavalia gladiata extract and a Canavalia gladiata extract, which comprises the following steps:
a) Pretreating Canavalia gladiata extract to obtain a sample solution;
pretreating semen Canavaliae extract to obtain semen Canavaliae extract solution;
b) Decocting semen Canavaliae reference medicinal material in water, filtering, and adding methanol to obtain reference medicinal material solution; adding methanol solution into the reference substance to obtain reference substance solution;
c) Carrying out thin layer chromatography detection on the sample solution, the jerusalem artichoke extract solution, the reference substance solution and the reference medicinal material solution, wherein the thin layer plate is a silica gel G thin layer plate; the developing agent is ethyl acetate-methanol-glacial acetic acid;
d) Spraying ninhydrin solution, heating and inspecting, wherein if the sample solution and the control medicinal material solution have fluorescence spots with the same color at the equal position of Rf, and have fluorescence spots at the positions of Rf=0.225-0.257,0.495-0.605,0.599-0.732, the sample solution is the Canavalia gladiata extract; when there is no fluorescent spot at rf=0.225 to 0.257,0.495 to 0.605,0.599 to 0.732, the extract is a jack bean extract.
The invention provides a Canavalia gladiata extract and a Canavalia gladiata extract, wherein the Canavalia gladiata extract is firstly taken and pretreated to obtain a sample solution.
The pretreatment of the Canavalia gladiata extract or Canavalia gladiata extract in the invention specifically comprises the following steps:
a) Adding methanol into Canavalia gladiata extract or Canavalia gladiata extract, performing ultrasonic treatment, filtering, volatilizing, dissolving residue in water, adding ethyl acetate, and shaking for extraction to obtain ethyl acetate layer extractive solution;
b) Evaporating the ethyl acetate layer extractive solution to dryness, adding water solvent, loading onto silica gel column, eluting with methanol, collecting eluate, evaporating to dryness, and dissolving the residue with methanol.
The pretreatment of the Canavalia gladiata extract provided by the invention comprises the steps of firstly adding methanol into Canavalia gladiata extract or Canavalia gladiata extract for ultrasonic treatment, filtering, volatilizing, adding ethyl acetate for shaking and extracting for 3 times to obtain ethyl acetate layer extract.
In some embodiments of the invention, the mass to volume ratio of the jack bean extract/jack bean extract, methanol, water, ethyl acetate is 0.5g:20mL:10mL:30mL;
in some embodiments of the invention, the ultrasonic time is 20-30 minutes;
evaporating the ethyl acetate layer extractive solution to dryness, adding water solvent, loading onto silica gel column, eluting with methanol, collecting eluate, evaporating to dryness, and dissolving the residue with methanol.
In some embodiments of the invention, the volume ratio of water to methanol is 5:20;
in some embodiments of the present invention, the silica gel column specification is: 160-200 meshes, 10g, 1.8cm inner diameter, and loading into a column by a dry method.
Decocting semen Canavaliae reference medicinal material in water, filtering, and adding methanol to obtain reference medicinal material solution;
the mass volume ratio of the sword bean control medicinal material to the water is 1g:50mL;
the time of the decoction is 20-30 min; preferably 30min.
Adding methanol solution into the reference substance to obtain reference substance solution;
the reference substances comprise threonine, valine and leucine; the concentration of each reference substance is 0.5mg/mL. The reference substance solution is a mixed reference substance solution.
Carrying out thin layer chromatography detection on the sample solution, the jerusalem artichoke extract solution, the reference substance solution and the reference medicinal material solution, wherein the thin layer plate is a silica gel G thin layer plate;
in some embodiments of the invention, the silica gel G thin layer panel is a azuril, merck or peninsula ocean prefabricated silica gel G panel. The result shows that the method has good durability and can meet the identification requirement.
In some embodiments of the invention, the developing agent is ethyl acetate-methanol-glacial acetic acid;
in some embodiments of the invention, the mass ratio of ethyl acetate-methanol-glacial acetic acid is 2:1:1.
Spraying ninhydrin solution, and heating for inspection.
The mass concentration of the ninhydrin solution is 1% -5%; the temperature for the heating inspection was 105 ℃.
In some embodiments of the invention, namely:
according to thin layer chromatography (rule 0502 of Chinese pharmacopoeia 2020 edition), sucking the above test sample solution and reference substance solution, respectively spotting on the same silica gel G thin layer plate, spreading with ethyl acetate-methanol-glacial acetic acid (2:1:1) as developing agent, taking out, air drying, spraying 1% -5% ninhydrin test solution, and heating at 105deg.C until the spot color is clear. Spots with the same color appear on the chromatogram of the sample at the positions corresponding to the chromatograms of the reference substance and the reference medicinal material.
In the preferred embodiment of the invention, the sample application amount of the thin layer chromatography sample is 2-8 mu L, the sample application amount of the control medicinal material solution is 2-8 mu L, and the sample application amount of the control sample is 1-4 mu L.
If the sample solution and the control medicinal material solution have fluorescence spots with the same color at the equal position of Rf, and have fluorescence spots at the positions of Rf=0.225-0.257,0.495-0.605,0.599-0.732, the sample solution is the Canavalia gladiata extract; when there is no fluorescent spot at rf=0.225 to 0.257,0.495 to 0.605,0.599 to 0.732, the extract is a jack bean extract.
In one embodiment, the Rf value is 0.25,0.55,0.666.
The inspection temperature of the invention is 105 ℃.
The method has better durability to different temperatures. Compared with the reference medicinal material map, the sample map and the reference medicinal material map show spots with the same color at the corresponding positions.
The inspection humidity is 32-75% rh.
The method has good durability to different humidity. Compared with the reference medicinal material map, the sample map and the reference medicinal material map show spots with the same color at the corresponding positions.
The invention provides a thin-layer identification method of a Canavalia gladiata extract and a Canavalia gladiata extract, which comprises the following steps: a thin layer identification method of Canavalia gladiata extract and Canavalia gladiata extract comprises: a) Pretreating Canavalia gladiata extract to obtain a sample solution; pretreating semen Canavaliae extract to obtain semen Canavaliae extract solution; b) Decocting semen Canavaliae reference medicinal material in water, filtering, and adding methanol to obtain reference medicinal material solution; adding methanol solution into the reference substance to obtain reference substance solution; c) Carrying out thin layer chromatography detection on the sample solution, the jerusalem artichoke extract solution, the reference substance solution and the reference medicinal material solution, wherein the thin layer plate is a silica gel G thin layer plate; the developing agent is ethyl acetate-methanol-glacial acetic acid; d) Spraying ninhydrin solution, heating and inspecting, wherein if the sample solution and the control medicinal material solution have fluorescence spots with the same color at the equal position of Rf, and have fluorescence spots at the positions of Rf=0.225-0.257,0.495-0.605,0.599-0.732, the sample solution is the Canavalia gladiata extract; when there is no fluorescent spot at rf=0.225 to 0.257,0.495 to 0.605,0.599 to 0.732, the extract is a jack bean extract. The thin-layer identification chromatographic method of the Canavalia gladiata extract is used for rapidly, accurately and effectively identifying the Canavalia gladiata extract. Meanwhile, whether a thin-layer chromatographic spectrum has fluorescent spots in a certain ratio shift range is used as a discrimination point for distinguishing the Canavalia gladiata extract from the Canavalia gladiata extract.
Drawings
FIG. 1 sample application amount investigation;
fig. 2 specificity investigation;
FIG. 3 is a different lamina plate study-Tianjin Si Li Da;
fig. 4 different lamina panels examined-merck;
FIG. 5 different lamellar plate surveys-Qingdao ocean;
FIG. 6 different temperatures-4 ℃;
FIG. 7 different temperatures-25 ℃;
FIG. 8 different humidity-32%;
FIG. 9 different humidity-75%;
FIG. 10 different lot verification;
FIG. 11 is a graph showing determination of Rf value at the identification point of Canavalia ectenes extract;
FIG. 12 is a graph of the results of the method of comparative example 1;
FIG. 13 is a graph showing the results of the method of comparative example 2.
Detailed Description
In order to further illustrate the present invention, a thin layer identification method of Canavalia gladiata extract, which is provided by the present invention, will be described in detail with reference to examples.
Ultrasonic machine, hot plate, water bath, mortar, thin layer imaging system: CAMAG TLC Visualizer A silica gel G thin layer plate (Qingdao ocean chemical Co., lot number: 20180527, tianjin Si Li Da technology Co., ltd., lot number: 191016, merck, lot number: HX 87183353)
Methanol, ethyl acetate, silica gel, glacial acetic acid, ninhydrin were all analytically pure, and water was ultrapure water (laboratory homemade).
Canavalia gladiata control medicinal material (Shanghai HongYongsheng biotechnology Co., ltd., lot number: 240072-202204), threonine (China food and drug assay institute, lot number: 140682-201302), valine (China food and drug assay institute, lot number: 140681-201703), leucine (China food and drug assay institute, lot number: 140687-201905), canavalia gladiata extract (Sichuan New green pharmaceutical technology development Co., lot number: 230401, 230402, 230403). Canavalia sonchifolia extract (Sichuan New green pharmaceutical technology development Co., ltd., lot Nos. 230301, 230302, 230303).
Example 1
Preparation of test solutions
Taking 0.5g of the product, grinding, adding 20ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, adding 10ml of water into residues to dissolve, adding ethyl acetate into the residues to shake and extract for 3 times, 10ml each time, combining ethyl acetate layers, evaporating to dryness, dissolving with 5ml of water, adding the mixture onto a silica gel column (160-200 meshes, 10g with the inner diameter of 1.8cm, and loading the column by a dry method), eluting with 20ml of methanol, collecting eluent, evaporating to dryness, adding 1ml of methanol into residues to dissolve, and taking the residue as a test sample solution.
Preparation of control medicinal material and control solution
Taking 1g of sword bean reference medicine, grinding, adding 50ml of water, decocting for 30 minutes, filtering, evaporating filtrate to dryness, adding 20ml of methanol into residues, and preparing the reference medicine solution by the same method. And taking threonine, valine and leucine as reference substances, and adding methanol to prepare a mixed solution containing 0.5mg of threonine, valine and leucine per 1ml as reference substance solution.
Assay
According to the thin layer chromatography (rule 0502 of Chinese pharmacopoeia 2020 edition), 6 μl of the sample solution and 3 μl of the reference solution are sucked and respectively spotted on the same silica gel G thin layer plate, ethyl acetate-methanol-glacial acetic acid (2:1:1) is used as developing agent, and the mixture is taken out, dried, sprayed with 1% -5% ninhydrin test solution, and heated at 105 ℃ until the spot color is clear. Spots with the same color appear on the chromatogram of the sample at the positions corresponding to the chromatograms of the reference substance and the reference medicinal material.
EXAMPLE 2 methodology investigation
2.1 sample application amount investigation
Under the experimental conditions, 1 μl, 2 μl, 3 μl and 4 μl of threonine, valine and leucine mixed control solution and 2 μl, 4 μl of Canavalia gladiata control solution and Canavalia gladiata extract solution are respectively taken and spotted on the same silica gel G thin layer plate. According to the established thin-layer chromatography conditions, the result is shown in figure 1, and when the mixed reference substance solution is 1-4 mu l, the sample application of the reference substance solution is 2-8 mu l, and the sample application of the sample solution is 2-8 ul, the thin-layer chromatography fluorescent spots are clear in color development, and the sample application amount of the mixed reference substance solution is 1-4 mu l, and the sample application amounts of the reference substance solution and the sample solution are 2-8 mu l. Comparing with the control and reference medicinal material patterns, the patterns of the sample and the control and reference medicinal material patterns show spots with the same color at the corresponding positions.
FIG. 1 sample application amount investigation. And (3) injection: 1. 4, 7 and 10 are threonine, valine and leucine mixed control sample application amounts of 1 μl, 2 μl, 3 μl and 4 μl; 2. 5, 8 and 11 are sample application amount of 2 μl, 4 μl, 6 μl and 8 μl of Canavalia gladiata control medicinal material; 3. the spotting amounts of the Canavalia gladiata extracts were 2. Mu.l, 4. Mu.l, 6. Mu.l and 8. Mu.l at 6, 9 and 12.
2.2 specificity investigation
According to the preparation method of the sample, the mixed reference substance solution, the sword bean reference medicinal material solution, the sword bean extract solution and the negative solution are prepared and respectively spotted on the same thin layer plate, developed according to the planned thin layer chromatography condition, and the result is shown in fig. 2, and the result shows that the negative sample has no interference to the sword bean extract sample, and the method has good specificity and can be used for identifying the sword bean extract solution.
Fig. 2 specificity study. 1, a negative solution is injected; 2 is a mixed reference substance; 3 is a sword bean control medicinal material; 4 is Canavalia gladiata extract
2.3 investigation of durability
2.3.1 comparison of different thin-layer plates
The test is carried out by respectively selecting Tianjin Si Li Da, merck and Qingdao ocean prefabricated silica gel G plates according to a planned test method, see figures 3-5, and the result shows that 3 brands of thin layer plates can all meet the identification requirement, and the method has good durability. Comparing with the control and reference medicinal material patterns, the patterns of the sample and reference medicinal material patterns show spots with the same color at the corresponding positions.
The results are shown in FIG. 3, and FIG. 3 shows a different lamina investigation-Tianjin Siderida. The injection comprises the following steps: 1 is a mixed reference substance; 2 is a Canavalia gladiata control medicinal material; 3 is Canavalia gladiata extract. FIG. 4 different lamellar plate surveys-merck notes 1 is a mixed control; 2 is a Canavalia gladiata control medicinal material; 3 is Canavalia gladiata extract. Fig. 5 different lamina plate surveys-Qingdao ocean, annotates: 1 is a mixed reference substance; 2 is a Canavalia gladiata control medicinal material; 3 is Canavalia gladiata extract.
2.3.2 comparison of different temperatures
And (5) taking the spotted thin-layer plates, and respectively unfolding the thin-layer plates at the low temperature of 4 ℃ and the normal temperature of 25 ℃. As can be seen from fig. 6 to 7, the method is excellent in durability against different temperatures. Comparing with the control and reference medicinal material patterns, the patterns of the sample and reference medicinal material patterns show spots with the same color at the corresponding positions.
FIG. 6 different temperatures of 4 ℃; and (3) injection: 1 is a mixed reference substance; 2 is a Canavalia gladiata control medicinal material; 3 is Canavalia gladiata extract. FIG. 7 different temperatures of 25 ℃; and (3) injection: and (3) injection: 1 is a mixed reference substance; 2 is a Canavalia gladiata control medicinal material; 3 is Canavalia gladiata extract.
2.3.3 comparison of different humidity
The spotted laminates were spread under 32% and 75% humidity conditions, respectively, as shown in fig. 8-9. As can be seen from the figure, the method has better durability to different humidity, and compared with the reference substance and the reference medicinal material patterns, the reference substance and the reference medicinal material patterns of the sample patterns display spots with the same color at the corresponding positions.
FIG. 8 shows different humidity levels of-32%, and 1 is a mixed reference substance; 2 is a Canavalia gladiata control medicinal material; 3 is Canavalia gladiata extract; FIG. 9 different humidity-75%, note: 1 is a mixed reference substance; 2 is a Canavalia gladiata control medicinal material; 3 is Canavalia gladiata extract.
2.3.4 verification
The thin-layer identification verification is carried out on 3 batches of Canavalia gladiata extracts, the experimental result is shown in figure 10, and the result shows that compared with the patterns of the reference substance and the reference medicinal material, the patterns of the reference substance and the reference medicinal material of the sample are in corresponding positions, and spots with the same color appear.
Fig. 10 is a different batch verification. 1, sword bean is used as a reference medicine; 2-4 is Canavalia gladiata extract SY2211001; SY2211002; SY2211003.
2.4 determination of the Rf value at the identification point of Canavalia gladiata extract and Canavalia gladiata extract
The Canavalia gladiata extract and Canavalia gladiata extract were examined by the same method as in example 1, and the results are shown in FIG. 11, and FIG. 11 shows the determination of the discrimination points Rf of the Canavalia gladiata extract and Canavalia gladiata extract; and (3) injection: 1 is a mixed reference substance; 2 is a Canavalia gladiata control medicinal material; 3-5 are Canavalia gladiata extracts 230401, 230402, 230403;6-8 is semen Canavaliae extract.
The results show that at the positions of the ratio shift values of 0.25,0.55 and 0.666, fluorescence spots appear in the Canavalia gladiata extract, while the mixed and fake Canavalia gladiata extract does not have obvious fluorescence spots, and can be used as a discrimination point for distinguishing Canavalia gladiata from the Canavalia gladiata extract.
Comparative example 1
Taking 0.5g of the product, grinding, adding 20ml of diluted ethanol, carrying out ultrasonic treatment for 20min, filtering, evaporating filtrate to dryness, and dissolving residues with 1ml of diluted ethanol to obtain a sample solution. Taking 1g of Canavalia gladiata reference medicinal material, grinding, adding 50ml of water, decocting for 30min, filtering, evaporating filtrate, dissolving residue in 1ml of diluted ethanol, and adding reference medicinal material solution. According to thin layer chromatography (rule 0502 of Chinese pharmacopoeia 2020 edition), sucking the above sample solution and medicinal solution 5ul each, respectively spotting on the same silica gel G thin layer plate, spreading with ethyl acetate-glacial acetic acid-methanol (9:3:1) as developing agent, taking out, air drying, spraying ninhydrin test solution, and heating at 105deg.C until the spot color is clear. Spots with the same color appear on the chromatogram of the sample at the positions corresponding to the chromatograms of the reference substance and the reference medicinal material. The experimental results are shown in FIG. 12.
FIG. 12 is a graph showing the results of the method of comparative example 1. And (3) injection: 1-3 is the sample application amount of the sword bean control medicine material of 2 μl, 5 μl and 8 μl; 4-5 is sample application amount of Canavalia gladiata extract 2 μl, 5 μl and 8 μl;
the result shows that the Canavalia gladiata extract has no obvious spots and cannot be identified by the thin layer identification method.
Comparative example 2
Taking 0.5g of the product, grinding, adding 20ml of diluted ethanol, carrying out ultrasonic treatment for 20min, filtering, evaporating filtrate to dryness, and dissolving residues with 1ml of diluted ethanol to obtain a sample solution. Taking 1g of Canavalia gladiata reference medicinal material, grinding, adding 50ml of water, decocting for 30min, filtering, evaporating filtrate, dissolving residue in 1ml of diluted ethanol, and adding reference medicinal material solution. According to thin layer chromatography (rule 0502 of Chinese pharmacopoeia 2020 edition), sucking the above sample solution and medicinal solution 5ul each, respectively, spotting on the same silica gel G thin layer plate, spreading with cyclohexane-formic acid-acetone (10:2:1) as developing agent, taking out, air drying, spraying ninhydrin test solution, and heating at 105deg.C until the spot color is clear. Spots with the same color appear on the chromatogram of the sample at the positions corresponding to the chromatograms of the reference substance and the reference medicinal material. The experimental results are shown in FIG. 13. FIG. 13 is a graph showing the results of the method of comparative example 2. And (3) injection: 1-2 is the sample application amount of the sword bean control medicine material 2 μl and 5 μl; 3-4 is the sample application amount of Canavalia gladiata extract 2. Mu.l and Canavalia gladiata extract 5. Mu.l.
The result shows that the sword bean extracts have no obvious difference by adopting the thin layer identification method, and the sword bean extracts cannot be identified.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A thin layer identification method of Canavalia gladiata extract and Canavalia gladiata extract comprises:
a) Pretreating Canavalia gladiata extract to obtain a sample solution;
pretreating semen Canavaliae extract to obtain semen Canavaliae extract solution;
b) Decocting semen Canavaliae reference medicinal material in water, filtering, and adding methanol to obtain reference medicinal material solution; adding methanol solution into the reference substance to obtain reference substance solution;
c) Carrying out thin layer chromatography detection on the sample solution, the jerusalem artichoke extract solution, the reference substance solution and the reference medicinal material solution, wherein the thin layer plate is a silica gel G thin layer plate; the developing agent is ethyl acetate-methanol-glacial acetic acid;
d) Spraying ninhydrin solution, heating and inspecting, wherein if the sample solution and the control medicinal material solution have fluorescence spots with the same color at the equal position of Rf, and have fluorescence spots at the positions of Rf=0.225-0.257,0.495-0.605,0.599-0.732, the sample solution is the Canavalia gladiata extract; when there is no fluorescent spot at rf=0.225 to 0.257,0.495 to 0.605,0.599 to 0.732, the extract is a jack bean extract.
2. The method according to claim 1, wherein the step a) of pretreatment of the jack bean extract or jack bean extract is specifically:
a) Adding methanol into Canavalia gladiata extract or Canavalia gladiata extract, performing ultrasonic treatment, filtering, volatilizing, dissolving residue in water, adding ethyl acetate, and shaking for extraction to obtain ethyl acetate layer extractive solution;
b) Evaporating the ethyl acetate layer extractive solution to dryness, adding water solvent, loading onto silica gel column, eluting with methanol, collecting eluate, evaporating to dryness, and dissolving the residue with methanol.
3. The method according to claim 1, wherein the mass to volume ratio of the sword bean extract/sword bean extract, methanol, water, ethyl acetate in step a) is 0.5g:20mL:10mL:30mL; the ultrasonic time is 20-30 min;
the volume ratio of the water to the methanol in the step b) is 5:20; the specification of the silica gel column is as follows: 160-200 meshes, 10g, 1.8cm inner diameter, and loading into a column by a dry method.
4. The method of claim 1, wherein the mass to volume ratio of the sword bean control drug and water in step B) is 1g:50mL;
the decoction time is 20-30 min;
the reference substances comprise threonine, valine and leucine; the concentration of the reference substance is 0.5mg/mL.
5. The method according to claim 1, wherein the sample application amount of the thin layer chromatography sample is 2-8 μl, the sample application amount of the control medicinal material solution is 2-8 μl, and the sample application amount of the control sample is 1-4 μl.
6. The process according to claim 1, wherein the mass ratio of ethyl acetate-methanol-glacial acetic acid in step C) is 2:1:1.
7. The method according to claim 1, wherein the ninhydrin solution in step D) has a mass concentration of 1% to 5%; the temperature for the heating inspection was 105 ℃.
8. The method of claim 1, wherein the silica gel G thin layer panel of step D) is a azuril, merck or peninsula ocean prefabricated silica gel G panel.
9. The method of claim 8, wherein step D) is performed at a viewing temperature of 105 ℃; the inspection humidity is 32-75% rh.
10. The method of claim 1, wherein the Rf value of step D) is 0.25,0.55,0.666.
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