CN113295816A - Thin layer chromatography detection method for Yihechun preparation - Google Patents
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Abstract
The invention belongs to the technical field of medicine quality control, and particularly relates to a thin-layer chromatography detection method of Yihe spring preparation, which comprises the following steps: (1) preparing a test solution; (2) preparing a reference solution; (3) detecting with thin layer, spraying color developing agent, heating until the color development of spots is clear, and respectively inspecting under sunlight and ultraviolet lamp; and obtaining a chromatogram inspection view. Compared with the existing quality standard, the R of the ginsenoside Rg1 obtained by the inventionfThe value reaches 0.2-0.8, and the identification result is more comprehensive after the ginseng reference medicinal materials are added.
Description
Technical Field
The invention belongs to the technical field of medicine quality control, and particularly relates to a thin-layer chromatography detection method of Yihechun preparation.
Background
The Yihe Chun oral liquid is prepared with 15 kinds of Chinese medicinal materials, including ginseng, cyathula root, dog kidney, prepared rehmannia root, pilose antler, epimedium, Chinese chive seed, cnidium fruit, cynomorium, deer penis, glehnia root, etc. and through extraction, and has the functions of invigorating kidney and strengthening Yang, and is used in treating impotence, spermatorrhea, soreness of waist and knees, etc. caused by kidney Yang deficiency. Wherein ginseng is the monarch drug, and in order to ensure the product quality, it is particularly important to research a method for identifying ginseng in Yichun oral liquid.
The traditional high performance liquid chromatography has the advantages of high sensitivity, high accuracy and the like, and becomes one of important means for controlling the quality of the traditional Chinese medicine, the basic mode is isocratic and gradient elution, and the components can be well separated by combining and applying the two modes. In the prior art, the content of the ginsenoside Rg1, Re and Rb1 in the traditional Chinese medicine preparation containing ginseng is usually determined by adopting a high performance liquid chromatography.
Chinese patent application CN102435697A discloses a method for measuring the content of ginsenoside Rg1 and Rb1 in a Xinshu capsule, which is a gradient elution method and simultaneously detects Rb1 and Rg 1.
Chinese patent application CN104807905A discloses a method for measuring the content of ginsenoside Rg1 and Re in Xinnaoning tablets. The method adopts a gradient elution method to simultaneously detect Re and Rg1, and has the advantages of good linear relation, high precision, good stability, high recovery rate, simplicity, convenience, rapidness, accuracy and feasibility.
The prior art discloses that the content detection of the ginsenosides Rg1, Re and Rb1 in the Chinese medicinal preparation is carried out by adopting an HPLC isocratic and gradient combination mode. However, since the Yihechun preparation is composed of 15 kinds of Chinese herbs, its components are more complex than other Chinese herbal preparations; meanwhile, the combination mode of isocratic elution and gradient elution is difficult to count, the separation mechanism of gradient elution is complex, the experimental scheme disclosed by the existing literature cannot meet the separation and content detection of the ginsenosides Rg1, Re and Rb1 in the Yihe spring preparation, and the HPLC method needs expensive corresponding instruments and has long separation time.
The thin layer chromatography is also called thin layer chromatography, can rapidly carry out the qualitative determination of the medicine components, and is a chromatographic separation technology which takes a support coated on a support plate as a stationary phase and takes a proper solvent as a mobile phase to separate, verify and quantify a mixed sample. However, the prescription of Yihe and Chun oral liquid has more traditional Chinese medicines and complex components, and the Rf values of the ginsenoside Rg1 and Re reference product spots in the existing standard are both less than 0.1, so the effect is not good.
Therefore, the thin-layer chromatography identification method suitable for the ginseng in Yihe and Chun oral liquid has important significance.
Disclosure of Invention
In order to overcome the technical problems, the invention provides a thin-layer chromatography detection method for ginseng in Yihe spring preparation, which is simple and feasible and can obtain accurate identification results.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
a thin layer chromatography detection method for radix Ginseng in Yihechun preparation comprises the following steps:
respectively dripping test solution, control solution and control solution of Yichun preparation on the same silica gel G thin layer plate, developing with n-butanol-ethyl acetate-water as developing agent, taking out, air drying, spraying color developing agent, heating until the spots are clearly developed, and respectively inspecting under sunlight and ultraviolet lamp (365 nm); and obtaining a chromatogram inspection view.
A thin layer chromatography detection method for radix Ginseng in YIHECHUN preparation comprises the following steps:
(1) preparing a test solution:
a) preparing the Yihe-Chun preparation into aqueous solution, extracting the water phase after ether extraction with n-butanol, washing the n-butanol extract with ammonia test solution, concentrating and drying to obtain n-butanol extract;
b) eluting n-butanol extract by macroporous resin column chromatography, collecting eluate, concentrating, drying, and preparing sample solution with dried extract;
(2) preparation of control solutions:
preparing a reference substance solution by using a ginsenoside Rg1 reference substance; and/or preparing a reference solution from the ginseng medicinal material;
(3) thin layer detection:
respectively spotting the sample solution and the reference solution on a thin layer plate, developing with n-butanol-ethyl acetate-water system as developing agent, developing, inspecting, and comparing.
Preferably, the elution condition of the column chromatography is that water, 30-50% ethanol and 60-80% ethanol are used for elution in sequence, and 60-80% ethanol eluent is collected; preferably eluting with water, 40% ethanol, and 70% ethanol, and collecting 70% ethanol eluate.
Preferably, the eluent for column chromatography is 1.7-3.1 column volumes of water, 1.0-2.0 column volumes of 40% ethanol, 1.7-2.5 column volumes of 70% ethanol.
Preferably, the elution solvent for column chromatography is 2.4 column volumes of water, 1.4 column volumes of 40% ethanol, 2.4 column volumes of 70% ethanol.
Preferably, the times of ether extraction are 1-3 times, the times of n-butanol extraction are 1-4 times, and the ammonia test solution is washed for 1-3 times; the concentration and drying are drying by distillation.
Preferably, the thin layer plate is a silica gel G thin layer plate.
Preferably, the method for preparing the test solution of the Yihechun preparation is as follows:
(1) extracting wet Yihechun preparation with diethyl ether 30ml for 2 times (20 ml each time) to obtain diethyl ether solution and water phase, discarding diethyl ether solution, extracting water phase with water saturated n-butanol for 3 times (30ml, 20ml), and mixing n-butanol extractive solutions;
(2) washing n-butanol extract with ammonia solution for 2 times (40 ml each time), placing on water bath, evaporating to dryness, and dissolving residue in water 30ml to obtain water solution;
(3) passing the water solution through D101 macroporous resin column (inner diameter of 1.5cm, column height of 12cm), sequentially eluting with water 50ml, 40% ethanol 30ml and 70% ethanol 50ml, collecting 70% ethanol eluate, and dissolving the residue with methanol 1 ml.
Preferably, the sample amount of the test solution is 3-7 μ l, the sample amount of the ginsenoside Rg1 reference solution is 1-4 μ l, and the sample amount of the ginseng medicinal material reference solution is 1-3 μ l.
Preferably, the sample amount of the test solution is 3 μ l, the sample amount of the ginsenoside Rg1 reference solution is 2 μ l, and the sample amount of the ginseng material reference solution is 2 μ l.
Preferably, the developing solvent is an upper layer solution of n-butanol-ethyl acetate-water in a volume ratio of 3-5:1-2: 4-7.
Preferably, the developing solvent is an upper layer solution of n-butanol-ethyl acetate-water in a volume ratio of 4:1: 5.
Preferably, the color developing agent is 5-20% sulfuric acid ethanol solution.
Preferably, the color developing agent is a 10% ethanol sulfate solution.
Preferably, the condition of color development is heating;
preferably, the heating temperature for the color development is 90-110 ℃.
Preferably, the heating temperature for color development is 105 ℃.
Preferably, the inspection is performed under sunlight and/or ultraviolet light.
Preferably, the dried substance is dissolved by methanol to prepare a test solution; dissolving the ginsenoside Rg1 reference substance with methanol to obtain reference solution.
Preferably, the step of preparing the reference solution by using the ginseng medicinal material comprises the following steps:
heating and refluxing Ginseng radix reference material and chloroform, adding water saturated n-butanol into the residue, ultrasonic treating, collecting supernatant, adding 2-5 times of ammonia solution, shaking, standing for layering, collecting supernatant, evaporating to dry, and adding methanol into the residue.
Preferably, the step of preparing the reference solution by using the ginseng medicinal material comprises the following steps:
collecting Ginseng radix control material 1g, adding chloroform 40ml, heating and refluxing for 1 hr, discarding chloroform solution, volatilizing solvent from residue, adding water 0.5ml, stirring for moistening, adding saturated n-butanol 10ml, ultrasonic treating for 30 min, collecting supernatant, adding 3 times of ammonia solution, shaking, standing for layering, collecting supernatant, evaporating to dry, and dissolving residue with methanol 1 ml.
The Yihechun preparation of the present invention is selected from any one of granules, tablets, capsules, oral liquids and other pharmaceutically acceptable dosage forms.
The Yihechun preparation is prepared with fifteen kinds of Chinese medicinal materials, including ginseng, cyathula root, dog's kidney, cynomorium, pilose antler, epimedium, deer's penis, glehnia root, borneol, cnidium fruit, prepared rehmannia root, fried Chinese chive seed, raspberry, prepared aconite root and sweetgum fruit.
Preferably, the Yihe spring preparation is prepared from fifteen raw materials of 60-90g of ginseng, 40-60g of medicinal cyathula root, 80-120g of dog kidney (prepared), 80-120g of cynomorium songaricum, 10-15g of pilose antler (unhaired), 240g of epimedium 160-.
Preferably, the Yihe spring preparation composition is prepared from 75g of ginseng, 50g of medicinal cyathula root, 100g of dog kidney (prepared), 100g of cynomorium songaricum, 12.5g of pilose antler (unhaired), 200g of epimedium herb, 3g of penis cervi (prepared), 100g of straight ladybell root, 2.5g of borneol, 200g of common cnidium fruit, 100g of prepared rehmannia root, 125g of fried Chinese chive seed, 75g of raspberry, 50g of prepared common monkshood daughter root, 75g of beautiful sweetgum fruit and fifteen raw materials.
The preparation method of the Yihechun preparation of the present invention refers to nineteenth volume of the Chinese medicine prescription preparation of the ministry of health in accordance with the standards of the medicine. The method comprises the following steps:
taking the fifteen raw materials, cutting off prepared rehmannia root, raspberry, monkshood, medicinal cyathula root, sweetgum fruit, cynomorium songaricum, epimedium herb, straight ladybell root, tuber onion seed and common cnidium fruit, putting the cut-off raw materials into an extractor, adding a proper amount of water, soaking for 8-12 hours, decocting for three times, filtering for several times, combining filtrates, concentrating to a proper amount, and performing spray drying to obtain an extract 1 for later use;
grinding Ginseng radix, testis et Pentis Canis, penis et testis Cervi, and cornu Cervi Pantotrichum into fine powder;
grinding Borneolum, mixing with the above fine powder and extract 1 to obtain extract, and making into capsule or solution. The Yihechun extract can also be used directly as Yihechun preparation.
Compared with the prior art, the invention has the following beneficial effects:
compared with the existing quality standard, the Rf value of the ginsenoside Rg1 obtained by the invention reaches more than 0.2, and the identification result is more comprehensive after ginseng control medicinal materials are added.
Drawings
FIG. 1: in the sunlight lower inspection view of the embodiment 1, the chromatographic points from left to right are respectively: ginsenoside Re reference substance, ginsenoside Rg1 reference substance, and test solution 3 μ L, 5 μ L, and 7 μ L;
FIG. 2: in the view of the ultraviolet lamp in example 1, the color spectrum points from left to right are respectively: ginsenoside Re reference substance, ginsenoside Rg1 reference substance, and test solution 3 μ L, 5 μ L, and 7 μ L;
FIG. 3: in the sunlight lower inspection view of the development system in the embodiment 2, the color spectrum points from left to right are respectively: ginsenoside Re reference substance, ginsenoside Rg1 reference substance, and test solution;
FIG. 4: in example 2, the ultraviolet underinspection view of the system is developed, and the color spectrum points from left to right are respectively: ginsenoside Re reference substance, ginsenoside Rg1 reference substance, and test solution;
FIG. 5: in the sunlight lower inspection chart of the second system in the embodiment 2, the color spectrum points from left to right are respectively: ginsenoside Re reference substance, ginsenoside Rg1 reference substance, and test solution;
FIG. 6: in the ultraviolet light lower inspection view of the second system developed in the embodiment 2, the color spectrum points from left to right are respectively: ginsenoside Re reference substance, ginsenoside Rg1 reference substance, and test solution;
FIG. 7: in the medium daylight underinspection view of the third system developed in example 2, the color spectrum points from left to right are respectively: ginsenoside Re reference substance, ginsenoside Rg1 reference substance, and test solution;
FIG. 8: in the ultraviolet light lower inspection view of the system three in the embodiment 2, the color spectrum points from left to right are respectively: ginsenoside Re reference substance, ginsenoside Rg1 reference substance, and test solution;
FIG. 9: in the sunlight lower inspection view of the embodiment 3, the chromatographic points from left to right are respectively: ginsenoside Re reference substance, ginsenoside Rg1 reference substance, 70% ethanol sample solution, and 40% ethanol sample solution;
FIG. 10: in the view of the ultraviolet lamp lower inspection in example 3, the color spectrum points from left to right are respectively: ginsenoside Re reference substance, ginsenoside Rg1 reference substance, 70% ethanol sample solution, and 40% ethanol sample solution;
FIG. 11: in example 3, day-down viewing (manual tracing), the chromatographic points from left to right are: ginsenoside Re reference substance, ginsenoside Rg1 reference substance, 70% ethanol sample solution, and 40% ethanol sample solution;
FIG. 12: in the ultraviolet lamp lower inspection view (manual tracing) in example 3, the chromatographic points from left to right are respectively: ginsenoside Re reference substance, ginsenoside Rg1 reference substance, 70% ethanol sample solution, and 40% ethanol sample solution;
FIG. 13: in the sunlight lower inspection view of the embodiment 4, the chromatographic points from left to right are respectively: ginsenoside Rg1 reference substance, 70% ethanol sample solution, and reference medicinal material solution;
FIG. 14: in the view of the ultraviolet lamp in example 4, the color spectrum points from left to right are respectively: ginsenoside Rg1 reference substance, 70% ethanol sample solution, and reference medicinal material solution;
FIG. 15: in the day-light screening view (manual tracing) of example 4, the chromatographic points from left to right are: ginsenoside Rg1 reference substance, 70% ethanol sample solution, and reference medicinal material solution;
FIG. 16: in the ultraviolet lamp lower inspection view (manual tracing) in example 4, the chromatographic points from left to right are respectively: ginsenoside Rg1 reference substance, 70% ethanol sample solution, and reference medicinal material solution;
FIG. 17: in the sunlight lower inspection view of the embodiment 5, the chromatographic points from left to right are respectively: ginsenoside Rg1 reference substance, reference medicinal solution, negative sample, 17080810 test sample, 18072301 test sample, and 18122904 test sample;
FIG. 18: in the view of the ultraviolet lamp lower inspection in example 5, the color spectrum points from left to right are respectively: ginsenoside Rg1 reference substance, reference medicinal solution, negative sample, 17080810 test sample, 18072301 test sample, and 18122904 test sample;
FIG. 19: in the day-light screening view (manual tracing) of example 5, the chromatographic points from left to right are: ginsenoside Rg1 reference substance, reference medicinal solution, negative sample, 17080810 test sample, 18072301 test sample, and 18122904 test sample;
FIG. 20: in the ultraviolet lamp lower inspection view (manual tracing) in example 5, the chromatographic points from left to right are respectively: ginsenoside Rg1 reference substance, reference medicinal solution, negative sample, 17080810 test sample, 18072301 test sample, and 18122904 test sample;
the invention will now be further illustrated with reference to the accompanying drawings and examples:
Detailed Description
Example 1 comparison of dot amounts
1. Preparation of test solution
Extracting Yihe spring oral liquid 30mL with diethyl ether for 2 times (20 mL each time), discarding the diethyl ether solution, extracting the water solution with saturated n-butanol for 3 times (30mL, 20mL), mixing the n-butanol extractive solutions, washing with ammonia solution for 2 times (40 mL each time), evaporating the n-butanol extractive solution on water bath, and dissolving the residue with methanol 1mL to obtain sample solution.
2. Preparation of control solutions
Accurately weighing ginsenoside Rg1, and adding methanol to obtain control solution with concentration of 2 mg/mL; accurately weighing ginsenoside Re, and adding methanol to obtain control solution with concentration of 2 mg/mL.
3. Spotting and developing
Sucking 3 μ L, 5 μ L, 7 μ L and 2 μ L of 1.2 reference solution, respectively dropping on the same silica gel G thin layer plate, spreading with lower layer solution of chloroform-ethyl acetate-methanol-water (15:40:22:10) at temperature below 10 deg.C as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, heating at 105 deg.C until color development is clear, and placing in sunlight and ultraviolet lamp (365nm) respectively to observe the result as shown in figure 1 and figure 2, but Rf value of ginsenoside Rg1 is less than 0.1, and 3 μ L of the test solution is selected as the dropping amount. The experimental results are shown in fig. 1 and fig. 2.
Example 2 comparison of unfolding systems
1. Preparation of test solution
Extracting Yihe spring oral liquid 30mL with diethyl ether for 2 times (20 mL each time), discarding the diethyl ether solution, extracting the water solution with saturated n-butanol for 3 times (30mL, 20mL), mixing the n-butanol extractive solutions, washing with ammonia solution for 2 times (40 mL each time), evaporating the n-butanol extractive solution on water bath, and dissolving the residue with methanol 1mL to obtain sample solution.
2. Preparation of control solutions
Accurately weighing ginsenoside Rg1, and adding methanol to obtain control solution with concentration of 2 mg/mL; accurately weighing ginsenoside Re, and adding methanol to obtain control solution with concentration of 2 mg/mL.
3. Spotting is carried out
4. Is unfolded
Deployment system
Developing with chloroform-ethyl acetate-methanol-water (15:40:22:10) at lower layer solution below 10 deg.C as developing agent, taking out, air drying, developing again, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, heating at 105 deg.C until spots develop clearly, and viewing under sunlight and ultraviolet lamp (365nm) respectively to obtain results shown in figures 3 and 4, wherein Rf values of ginsenoside Re and Rg1 are both greater than 0.2, but in the chromatogram of the sample, the spot shapes at corresponding positions with the chromatogram of ginsenoside Re and Rg1 reference substance are less than round, and the spot corresponding to ginsenoside Rg1 has interference of yellow spot. The experimental results are shown in fig. 3 and 4.
Deployment system two
Developing with n-butanol-ethyl acetate-methanol-water (15:40:22:10) as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol solution, heating at 105 deg.C until the color of spots is clear, and respectively placing under sunlight and ultraviolet lamp (365nm) to observe the results as shown in FIGS. 5 and 6, wherein in the chromatogram of the sample, the spots can not be separated and identified at the corresponding position of the chromatogram of the reference substance. The experimental results are shown in fig. 5 and 6.
Deployment system three
Taking an upper layer solution of n-butanol-ethyl acetate-water (4:1:5) as a developing agent, developing, taking out, drying in the air, spraying a 10% sulfuric acid ethanol solution, heating at 105 ℃ until spots are clearly developed, and respectively placing under sunlight and an ultraviolet lamp (365nm) to observe results as shown in figures 7 and 8, wherein in a sample chromatogram, spots and fluorescent spots with the same color are respectively displayed on corresponding positions of a reference chromatogram, the Rf values of ginsenoside Re and Rg1 are both more than 0.2, but the ginsenoside Re position in the sample chromatogram has interference of yellow fluorescent spots and the separation effect of the sample spots is poor. The experimental results are shown in fig. 7 and 8.
EXAMPLE 3 improvement of test solution treatment method
1. Preparation of test solution
Extracting Yihe spring oral liquid 30mL with diethyl ether for 2 times (20 mL each time), discarding the diethyl ether, extracting the water solution with saturated n-butanol for 3 times (30mL, 20mL), mixing the n-butanol extractive solutions, washing with ammonia test solution for 2 times (40 mL each time), evaporating the n-butanol extractive solution in water bath, dissolving the residue with 30mL of water, passing through D101 type macroporous resin (inner diameter 1.5cm, column height 12cm), eluting with 50mL of water, 30mL of 40% ethanol and 50mL of 70% ethanol, collecting eluates of each part, evaporating, and dissolving the residue with 1mL of methanol to obtain sample solution.
2. Preparation of control solutions
Accurately weighing ginsenoside Rg1, and adding methanol to obtain control solution with concentration of 2 mg/mL; accurately weighing ginsenoside Re, and adding methanol to obtain control solution with concentration of 2 mg/mL.
3. Spotting and developing
Absorbing 40% of a test solution, 3 μ L of 70% elution part and 2 μ L of a reference solution, respectively dropping on the same silica gel G thin layer plate, taking an upper layer solution of n-butyl alcohol-ethyl acetate-water (4:1:5) as a developing agent, developing, taking out, drying in the air, spraying a 10% sulfuric acid ethanol solution, heating at 105 ℃ until the color development of spots is clear, respectively placing the spots under sunlight and an ultraviolet lamp (365nm) to detect the results as shown in figures 9 and 10, respectively, in the chromatogram of the test solution at the 70% ethanol part, spots with the same color and fluorescent spots are respectively displayed on the corresponding positions of the reference chromatogram, the separation degree is good, the Rf values of ginsenoside Re and Rg1 are both greater than 0.2, but the position of ginsenoside Re has yellow fluorescent interference; in the chromatogram of the test sample with 40% ethanol part, the total spots are light and difficult to identify, and 70% ethanol part is selected as the test sample solution. In this embodiment, a TLC photograph and a manual tracing drawing drawn according to the TLC photograph are provided respectively to more clearly express the effect of this embodiment, which are shown in fig. 9, fig. 10, fig. 11, and fig. 12, respectively.
Example 4 increasing Ginseng control
1. Preparation of test solution
Extracting Yihe spring oral liquid 30mL with diethyl ether for 2 times (20 mL each time), discarding the diethyl ether, extracting the water solution with saturated n-butanol for 3 times (30mL, 20mL), mixing the n-butanol extractive solutions, washing with ammonia solution for 2 times (40 mL each time), evaporating the n-butanol solution in water bath, dissolving the residue with 30mL of water, passing through D101 type macroporous resin (inner diameter 1.5cm, column height 12cm), eluting with 50mL of water, 30mL of 40% ethanol and 50mL of 70% ethanol, collecting eluates of each part, wherein 70% is the main experimental part, evaporating, and dissolving the residue with 1mL of methanol to obtain the sample solution.
2. Preparation of control solutions
Accurately weighing ginsenoside Rg1, and adding methanol to obtain control solution with concentration of 2 mg/mL.
3. Preparation of reference drug solution
Collecting Ginseng radix control material 1g, adding chloroform 40ml, heating and refluxing for 1 hr, discarding chloroform solution, volatilizing solvent from residue, adding water 0.5ml, stirring for moistening, adding saturated n-butanol 10ml, ultrasonic treating for 30 min, collecting supernatant, adding 3 times of ammonia solution, shaking, standing for layering, collecting supernatant, evaporating to dry, and dissolving residue with methanol 1 ml.
4. Spotting and developing
Sucking 3 μ L of test solution, 2 μ L of control solution, and 2 μ L of control solution, respectively dropping on the same silica gel G thin layer plate, developing with upper solution of n-butanol-ethyl acetate-water (4:1:5) as developing agent, taking out, air drying, spraying 10% ethanol sulfate solution, heating at 105 deg.C until the color development of spots is clear, and respectively placing in sunlight and ultraviolet lamp (365nm) to observe the results as shown in FIGS. 13 and 14. In this embodiment, a TLC photograph and a manual tracing drawing drawn according to the TLC photograph are respectively provided to more clearly express the effect of this embodiment, which are respectively shown in fig. 13, fig. 14, fig. 15, and fig. 16.
EXAMPLE 5 three batches of samples
1. Preparation of test solution
Extracting Yihe spring oral liquid (batch numbers 17080810, 18072301, 18122904)30mL with diethyl ether for 2 times, each time 20mL, discarding the diethyl ether, extracting the water solution with saturated n-butanol for 3 times (30mL, 20mL), mixing the n-butanol extractive solutions, washing with ammonia test solution for 2 times, each time 40mL, evaporating the n-butanol extractive solution on water bath, dissolving the residue with purified water 30mL, passing through D101 type macroporous resin (inner diameter 1.5cm, column height 12cm), eluting with water 50mL, 40% ethanol 30mL and 70% ethanol 50mL, collecting 70% ethanol part, evaporating, dissolving the residue with methanol 1mL, and collecting the sample solution;
2. preparation of control solutions
Accurately weighing ginsenoside Rg1, and adding methanol to obtain control solution with concentration of 2 mg/mL.
3. Preparation of reference drug solution
Collecting Ginseng radix control material 1g, adding chloroform 40ml, heating and refluxing for 1 hr, discarding chloroform solution, volatilizing solvent from residue, adding water 0.5ml, stirring for moistening, adding saturated n-butanol 10ml, ultrasonic treating for 30 min, collecting supernatant, adding 3 times of ammonia solution, shaking, standing for layering, collecting supernatant, evaporating to dry, and dissolving residue with methanol 1 ml.
4. Negative sample solution preparation
Preparing a negative sample without ginseng, taking 30mL of the negative sample, adding diethyl ether for extraction for 2 times, 20mL each time, removing the diethyl ether solution, extracting the water solution for 3 times by using saturated n-butanol (30mL, 30mL and 20mL), merging n-butanol extract, washing by using an ammonia test solution for 2 times, 40mL each time, placing the n-butanol solution on a water bath for evaporation, adding 30mL of pure water to dissolve residues, passing through D101 type macroporous resin (the inner diameter is 1.5cm, the column height is 12cm), eluting by using 50mL of water, 30mL of 40% ethanol and 50mL of 70% ethanol in sequence, collecting 70% ethanol eluent, evaporating to dryness, adding 1mL of methanol to dissolve the residues to obtain a negative sample solution.
5. Spotting and developing
Taking 3 μ L of test solution, 3 μ L of negative sample solution, 2 μ L of control solution, and 2 μ L of control solution, respectively spreading on the same silica gel G thin layer plate, spreading with n-butanol-ethyl acetate-water (4:1:5) upper layer solution as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed, and respectively inspecting under sunlight and ultraviolet lamp (365 nm). In the chromatogram of the test solution, spots or fluorescent spots of the same color are respectively displayed at the corresponding positions of the chromatogram of the control solution. The method has comprehensive and accurate detection results. In this embodiment, a TLC photograph and a manual tracing drawing drawn according to the TLC photograph are provided respectively to more clearly express the effect of this embodiment, which are shown in fig. 17, fig. 18, fig. 19, and fig. 20 respectively.
The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.
Claims (10)
1. A thin layer chromatography detection method for radix Ginseng in Yihechun preparation comprises the following steps:
(1) preparing a test solution:
a) preparing the Yihe-Chun preparation into aqueous solution, extracting the water phase after ether extraction with n-butanol, washing the n-butanol extract with ammonia test solution, concentrating and drying to obtain n-butanol extract;
b) eluting n-butanol extract by macroporous resin column chromatography, collecting eluate, concentrating, drying, and preparing sample solution with dried extract;
(2) preparation of control solutions:
preparing a reference substance solution by using a ginsenoside Rg1 reference substance; and/or preparing a reference solution from the ginseng medicinal material;
(3) thin layer detection:
respectively spotting the sample solution and the reference solution on a thin layer plate, developing with n-butanol-ethyl acetate-water system as developing agent, developing, inspecting, and comparing.
2. The detection method as claimed in claim 1, wherein the elution conditions of the column chromatography are sequentially water, 30-50% ethanol, 60-80% ethanol, collecting 60-80% ethanol eluate; preferably eluting with water, 40% ethanol, and 70% ethanol, and collecting 70% ethanol eluate.
3. The detection method as claimed in claim 1, wherein the developing solvent is an upper solution of n-butanol-ethyl acetate-water in a volume ratio of 3-5:1-2: 4-7; preferably an n-butanol-ethyl acetate-water supernatant in a volume ratio of 4:1: 5.
4. The detection method according to claim 1, wherein the color developing reagent is a 5-20% ethanol sulfate solution, preferably a 10% ethanol sulfate solution, and the color developing condition is heating.
5. The detection method according to claim 1, wherein the dried product is dissolved in methanol to prepare a test solution; dissolving the ginsenoside Rg1 reference substance with methanol to obtain reference solution.
6. The detection method according to claim 1, wherein the step of preparing the reference solution from the ginseng material comprises: heating and refluxing Ginseng radix reference material and chloroform, adding water saturated n-butanol into the residue, ultrasonic treating, collecting supernatant, adding 2-5 times of ammonia solution, shaking, standing for layering, collecting supernatant, evaporating to dry, and adding methanol into the residue.
7. The detection method according to claim 1, wherein the sample amount of the test solution is 3-7 μ L, the sample amount of the ginsenoside Rg1 control solution is 1-4 μ L, and the sample amount of the ginseng material control solution is 1-3 μ L.
8. The assay of claim 1, wherein the test solution of the Yihechun preparation is prepared by:
extracting wet Yihechun preparation with diethyl ether 30ml for 2 times (20 ml each time) to obtain diethyl ether solution and water phase, discarding diethyl ether solution, extracting water phase with water saturated n-butanol for 3 times, and mixing n-butanol extractive solutions;
washing n-butanol extract with ammonia solution for 2 times (40 ml each time), placing on water bath, evaporating to dryness, and dissolving residue in water 30ml to obtain water solution;
passing the water solution through D101 macroporous resin column, eluting with water 50ml, 40% ethanol 30ml and 70% ethanol 50ml sequentially, collecting 70% ethanol eluate, and dissolving the residue after evaporation with 1ml methanol.
9. The assay method of any one of claims 1 to 8, wherein the Yihechun preparation is prepared from fifteen materials of ginseng, cyathula root, dog kidney, cynomorium songaricum, hairy antler, epimedium, deer penis, adenophora stricta, borneol, cnidium fruit, prepared rehmannia root, leek seed, raspberry, aconite root, and sweetgum fruit.
10. The assay of claim 9, wherein the Yihechun formulation is selected from any one of granules, tablets, capsules, oral liquids and other pharmaceutically acceptable dosage forms.
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