CN113759067A - Thin-layer chromatography construction method, identification method and application thereof - Google Patents
Thin-layer chromatography construction method, identification method and application thereof Download PDFInfo
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- CN113759067A CN113759067A CN202110901598.1A CN202110901598A CN113759067A CN 113759067 A CN113759067 A CN 113759067A CN 202110901598 A CN202110901598 A CN 202110901598A CN 113759067 A CN113759067 A CN 113759067A
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Abstract
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly provides a thin-layer chromatography construction method, an identification method and application thereof, wherein the thin-layer chromatography construction method comprises the following steps: preparing a radix ranunculi ternati test solution: mixing a radix ranunculi ternati test sample, water and inorganic acid, heating for hydrolysis, extracting with an organic solvent to obtain an extracting solution, drying, and dissolving to obtain a test sample solution; the thin-layer chromatography detection is carried out on the test solution to obtain the thin-layer chromatography of the test, and the construction method has the advantages of simple and convenient operation steps, short time consumption, small solvent types and dosage, cost saving and easy popularization and application; and the multiple components in the test solution are well separated, so that the spot separation effect in the thin-layer detection is good and clear, a plurality of spots can be displayed, the identification process is quicker and more convenient, and the identification result is more accurate and reliable.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a thin-layer chromatography construction method, an identification method and application thereof.
Background
The radix Ranunculi Ternati is derived from dried root tuber of Ranunculus tenuis Thunb of Ranunculaceae, and has effects of clearing away heat and toxic materials, softening hard masses, eliminating phlegm, resolving hard mass, relieving swelling, and preventing malaria. The radix ranunculi ternati mainly contains saccharides, lipids, amino acids, flavones, glycosides and other components, and has clinical applications of resisting tubercle bacillus, resisting tumors, relieving cough, eliminating phlegm, diminishing inflammation and the like.
At present, thin-layer identification in pharmacopeia quality standard of radix ranunculi ternati mainly aims at amino acid ingredients in the radix ranunculi ternati, but the thin-layer identification is difficult to detect in radix ranunculi ternati decoction-labeled freeze-dried powder, and the transferability of the radix ranunculi ternati ingredients in medicinal materials, decoction pieces and water extracts cannot be qualitatively investigated; in the prior art, a thin-layer identification method for formula particles is not searched for by the ternate buttercup root, so that whether the technological process of the ternate buttercup root from medicinal materials, decoction pieces to ternate buttercup root standard decoction freeze-dried powder and ternate buttercup root formula particles is stable or not can not be evaluated.
For example, yangxianfen discloses an identification method of ternate buttercup root, and chenge and the like disclose an analysis comparison method of wild and wild ternate buttercup root medicinal materials.
Therefore, a qualitative identification method which can be simultaneously applied to the components of the radix ranunculi ternati in the radix ranunculi ternati medicinal materials, decoction pieces, the radix ranunculi ternati standard decoction freeze-dried powder and the radix ranunculi ternati formula particles is needed to be established, so as to serve as one of qualitative evaluation methods for the stability of the technical processes of the radix ranunculi ternati from the medicinal materials, the decoction pieces to the radix ranunculi ternati standard decoction freeze-dried powder and the radix ranunculi ternati formula particles.
Disclosure of Invention
Therefore, the invention aims to solve the problem that the methods in the prior art cannot be simultaneously applied to identifying the ranunculus ternatus component in the ranunculus ternatus medicinal materials, the decoction pieces and the ranunculus ternatus medicinal preparation.
Therefore, the invention provides a thin-layer chromatography construction method, which comprises the following steps:
preparing a radix ranunculi ternati test solution: mixing a radix ranunculi ternati test sample, water and inorganic acid, heating for hydrolysis, extracting with an organic solvent to obtain an extracting solution, drying, and dissolving to obtain a test sample solution;
and (4) carrying out thin-layer chromatography detection on the test solution to obtain the thin-layer chromatography of the test.
The thin-layer chromatography detection comprises the following steps:
(1) spotting the ternate buttercup root sample solution on a thin-layer plate;
(2) developing with mixed solution of cyclohexane-ethyl acetate-formic acid as developing agent, taking out, volatilizing solvent, and developing to obtain the final product.
Further, the adopted thin layer plate is a silica gel G thin layer plate;
preferably, the length of the thin-layer plate is more than or equal to 10cm, and more preferably 10-20 cm;
preferably, the spotting volume is 1-30 μ l; more preferably 1-4. mu.l.
Further, in the thin-layer chromatography detection, ultraviolet color development or sulfuric acid ethanol solution is adopted as a color developing agent for color development. Specifically, an ethanol solution containing 10% by volume of sulfuric acid may be used.
In certain preferred embodiments, the color is developed using 365nm UV light.
Further, in the preparation of the ternate buttercup root test solution, any one or more of the following a-g is satisfied:
a, the ratio of the mass of the ternate buttercup root test sample to the volume of water and inorganic acid is 0.3-0.7: 20-50: 1-3, wherein, if the unit of mass is g, the unit of corresponding volume is ml.
b, the volume ratio of the mass of the ternate buttercup root test sample to the organic solvent is 0.3-0.7: 30-60, wherein if the unit of mass is g, the unit of corresponding volume is ml.
c, heating the hydrolysis to 90-100 ℃ for 0.5-2 h.
And d, extracting with the organic solvent for 1-5 times, adding 20-60 times of the organic solvent for each extraction, and combining the extractive solutions for each time.
e, the inorganic acid is hydrochloric acid and/or sulfuric acid;
f, the organic solvent is selected from at least one of ethyl acetate, n-butyl alcohol and diethyl ether;
g, after the extraction step, a step of concentrating the extracting solution is also included; or, the method further comprises the step of drying and dissolving the extract, preferably, the solvent adopted in the dissolving step is at least one of methanol, ethanol and ethyl acetate. More preferably, the ratio of the mass of the ternate buttercup root test sample to the volume of the solvent is 0.3-0.7: 1-3, wherein, if the unit of mass is g, the unit of corresponding volume is ml.
Further, in the preparation process of the ternate buttercup root test sample solution, water and the inorganic acid can be mixed with the ternate buttercup root test sample in sequence, or can be mixed into the aqueous solution containing the inorganic acid and then mixed into the ternate buttercup root test sample.
The radix ranunculi ternati test sample is selected from radix ranunculi ternati medicinal materials, decoction pieces or radix ranunculi ternati herbal preparations, wherein the radix ranunculi ternati herbal preparations are prepared by taking radix ranunculi ternati as raw material medicines according to the conventional technical means in the field and adding or not adding pharmaceutically conventional auxiliary materials.
Preferably, the ungula herbal formulation is selected from a solid, semi-solid or liquid formulation of ungula ternata.
Wherein, the solid preparation can be but not limited to tablets, powders, granules, capsules and the like;
semisolid formulations can be, but are not limited to, ointments, pastes, creams, and the like;
the liquid preparation can be, but is not limited to, decoction, mixture, oral liquid, injection, emulsion, suspension, nano liquid preparation and the like.
The decoction of radix Ranunculi Ternati can be water extract of radix Ranunculi Ternati or decoction pieces.
The powder of the radix Ranunculi Ternati herbal medicine can be selected from lyophilized powder of standard decoction of radix Ranunculi Ternati.
The invention also provides thin-layer chromatography obtained by the construction method.
The invention also provides a method for identifying the ternate buttercup root, the decoction pieces or the ternate buttercup root medicinal preparation, which comprises the step of comparing the thin-layer chromatography of the ternate buttercup root to be detected to the thin-layer chromatography of the reference medicinal material;
the thin-layer chromatography of the radix ranunculi ternati test sample to be tested is obtained by using the radix ranunculi ternati test sample to be tested according to any one of the construction methods.
Preferably, the thin-layer chromatography of the control medicinal material is prepared by using the ternate buttercup root control medicinal material according to the same preparation method of the test solution, and in order to make the color development of the control medicinal material more obvious, in a preferred scheme, the thin-layer chromatography of the control medicinal material is prepared by mixing the ternate buttercup root medicinal material with water, decocting, carrying out solid-liquid separation and drying to obtain a ternate buttercup root medicinal material extract, and then taking the ternate buttercup root medicinal material extract to replace the ternate buttercup root test solution and obtaining the ternate buttercup root medicinal material extract according to any one of the construction methods. The experimental result obtained without the steps of decoction and solid-liquid separation has an unclear map, and the definition can be improved by increasing the concentration or the sample amount of the reference medicinal material solution.
In the thin-layer chromatography construction of the reference medicinal material, the decocting temperature is 90-150 ℃, and the time is 0.5-2 h; and/or the mass ratio of the ternate buttercup root medicinal material to the volume of water is 0.5-3: 20-100 parts of; and/or, the solid-liquid separation is filtration or centrifugation.
The invention also provides application of the method for identifying the radix ranunculi ternati medicinal material, the decoction pieces or the radix ranunculi ternati medicinal preparation in quality detection of the radix ranunculi ternati medicinal material, the decoction pieces or the radix ranunculi ternati medicinal preparation.
The invention also provides a quality detection method of the traditional Chinese medicine product,
comprises the step of identifying the radix ranunculi ternati medicinal material, the decoction pieces or the radix ranunculi ternati medicinal preparation according to the method.
The technical scheme of the invention has the following advantages:
1. the thin-layer chromatography construction method provided by the invention comprises the steps of preparing a radix ranunculi ternati test sample solution, and carrying out thin-layer chromatography detection on the test sample solution to obtain the thin-layer chromatography of the test sample. The preparation method using the specific test solution has the advantages of simple and convenient operation steps, short time consumption, small solvent types and dosage and easy popularization and application; and various components in the test solution are well separated, so that the spot separation effect in the thin-layer detection is good and clear, a plurality of spots can be shown, the identification process is quicker and more convenient, and the identification result is more accurate and reliable.
2. The thin-layer chromatography construction method provided by the invention combines the cyclohexane-ethyl acetate-formic acid as the developing agent, so that the spot separation effect is better and clearer, and particularly, the thin-layer chromatography construction method adopts the following steps that the volume ratio is 0.5-1.5: 1: the developing agent under the specific volume ratio can be applied to qualitative identification of the radix ranunculi ternati ingredients in radix ranunculi ternati medicinal materials, decoction pieces, radix ranunculi ternati standard decoction freeze-dried powder and radix ranunculi ternati formula particles, and has the advantages of high separation degree, many spots, clearness, no aggregation, comprehensive chromatographic information and no negative interference.
3. According to the thin-layer chromatography construction method provided by the invention, in the thin-layer chromatography detection, a sample solution is spotted on a thin-layer plate, the adopted thin-layer plate is a silica gel G thin-layer plate, the price is low, the performance is good, preferably, the length of the thin-layer plate is more than or equal to 10cm, and the separation degree of a sample can be improved.
4. According to the construction method of the thin-layer chromatography, the developed thin-layer plate is developed under 365nm ultraviolet, so that the color of the characteristic spot is more obvious, the component to be detected is easily distinguished from other components, and the specificity is strong.
5. The construction method of the thin-layer chromatography provided by the invention particularly controls the ratio of the mass of the radix ranunculi ternati test sample to the volume of water and inorganic acid to be 0.3-0.7: 20-50: 1-3; or the volume ratio of the mass of the ternate buttercup root test sample to the organic solvent is 0.3-0.7: 30-60, or heating and hydrolyzing at 90-100 deg.C for 0.5-2 h; the identification process is faster and more convenient, and the identification result is more accurate and reliable.
6. According to the identification method provided by the invention, 5 spots with the same color as the corresponding positions of the chromatogram of the radix ranunculi ternati reference medicinal material exist in the chromatogram of the test sample, and the identification result is accurate and reliable.
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 example 1;
FIG. 2 is a thin layer chromatogram of example 2;
FIG. 3 is a thin layer chromatogram of example 3;
FIG. 4 is a thin layer chromatogram of example 4;
FIG. 5 is a thin layer chromatogram for sample application amount optimization in Experimental example 1;
FIG. 6 is a thin layer chromatogram at 28 ℃ and 15% humidity in Experimental example 1;
FIG. 7 is a thin layer chromatogram at a temperature of 3 ℃ and a humidity of 48% in Experimental example 1;
FIG. 8 is a thin layer chromatogram at 28 ℃ and 79% humidity in Experimental example 1;
FIG. 9 is a thin layer chromatogram of a silica gel G thin layer plate produced by Merck KGaA in Experimental example 1;
FIG. 10 is a thin layer chromatogram of a silica gel G thin layer plate of Nicoti chemical industry research institute in Experimental example 1;
FIG. 11 is a thin layer chromatogram of silica gel G thin layer plate of the yellow Business silica gel development test plant, cheese 32600, Takeda, example 1;
FIG. 12 is a thin layer chromatogram of standard decoction lyophilized powder of radix Ranunculi Ternati of different lot numbers;
FIG. 13 is a thin layer chromatogram of various lot numbers of Ranunculus ternatus thumb formula granules;
FIG. 14 is a thin layer chromatogram of different lot numbers of radix Ranunculi Ternati;
FIG. 15 is a thin layer chromatogram of Experimental example 2;
FIG. 16 is a thin layer chromatogram of comparative example 1;
FIG. 17 is a thin layer chromatogram of comparative example 2;
FIG. 18 is a thin layer chromatogram of comparative example 3;
fig. 19 is a thin layer chromatogram of comparative example 4.
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.
The instrument comprises the following steps: JY20002 electronic balance (Mettler Tulipuo), DZKW-4 constant temperature water bath (Beijing Zhongxing Weiji instruments Co., Ltd.) silica gel G plate (Merck KGaA, tobacco platform chemical industry research institute, tobacco platform Chi 32600yellow silica gel development test factory).
Reagent testing: cat's claw control medicinal material (batch No. 121593-
The standard decoction of radix ranunculi ternati lyophilized powder: 200602-containing materials 464400-01, 200610-containing materials 453400-02, 200618-containing materials 461670-03, 200703-containing materials 458000-04, 200703-containing materials 458000-05, 200703-containing materials 456250-06, 200703-containing materials 473200-07, 200703-containing materials 474250-08, 200703-containing materials 473000-09, 200703-containing materials 475500-10, 200831-containing materials 458000-11, 200831-containing materials 458000-12, 200831-containing materials 456250-13, 200831-containing materials 473200-14, 200831-containing materials 474250-15, 200831-containing materials 473000-16, 200831-containing materials 475500-17 and 200831-containing materials 454150-18.
Ranunculi ternata formulation granules (batch numbers: KL200602-464400-01, KL200610-453400-02 and KL 200618-461670-03).
Reagent: cyclohexane, ethyl acetate, n-butanol, toluene, methanol, ethanol, acetic acid, formic acid and hydrochloric acid are analytically pure.
The standard decoction freeze-dried powder of radix ranunculi ternati and the formula granules of the radix ranunculi ternati can be prepared by adopting the conventional method, for example, the preparation method of the standard decoction freeze-dried powder of the radix ranunculi ternati and the formula granules of the radix ranunculi ternati comprises the following steps:
(1) preparing standard radix ranunculi ternati decoction freeze-dried powder: decocting appropriate amount of radix Ranunculi Ternati for 2 times, adding 8 times of water for the first decoction (adding 8ml of water for each gram of radix Ranunculi Ternati), boiling, extracting for 30min, adding 6 times of water for the second decoction (adding 6ml of water for each gram of radix Ranunculi Ternati), boiling, extracting for 25min, filtering, concentrating the filtrate at 65 deg.C under reduced pressure to obtain pure extract with relative density of 1.05 + -0.03 (60 deg.C), and spray drying.
(2) Preparation of the ternate buttercup root formula particle: taking a proper amount of the radix ranunculi ternati medicinal materials, decocting for 2 times, adding 12 times of water for one decoction (adding 12ml of water for each gram of the radix ranunculi ternati medicinal materials), extracting for 30min after boiling, adding 6 times of water for the second decoction (adding 6ml of water for each gram of the radix ranunculi ternati medicinal materials), extracting for 25min after boiling, filtering, concentrating the filtrate at 65 ℃ under reduced pressure to obtain a pure extract with the relative density of 1.05 +/-0.03 (60 ℃), spray-drying, adding maltodextrin with the amount of 10% of the decoction pieces (adding 0.1g of auxiliary materials for each gram of the radix ranunculi ternati medicinal materials), and granulating to obtain the traditional Chinese medicine.
Example 1
This embodiment provides a method for identifying Ranunculus ternatus, comprising the following steps:
(1) preparation of a test solution: collecting radix Ranunculi Ternati standard decoction lyophilized powder 0.5g, grinding, adding water 30ml and hydrochloric acid 2ml, heating and hydrolyzing at 100 deg.C for 1 hr, cooling, extracting with ethyl acetate under shaking for 2 times, each time 20ml, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 2ml to obtain sample solution.
(2) Preparation of reference drug solution: collecting herba Ranunculi Ternati control medicinal material 1g, adding water 50ml, boiling for 30min, filtering, spin-drying the filtrate, adding water 30ml and hydrochloric acid 2ml, heating at 100 deg.C for hydrolysis for 1 hr, cooling, extracting with ethyl acetate shaking for 2 times (20 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 2ml to obtain control medicinal material solution.
(3) And (3) thin-layer chromatography detection: sucking 2 μ l of the sample solution and 3 μ l of the control solution, respectively dropping on the same silica gel G thin layer plate, presaturating with cyclohexane-ethyl acetate-formic acid (1: 1: 0.1) as developing agent for 30min, developing, taking out, air drying, and inspecting under ultraviolet (365 nm).
The results are shown in FIG. 1.
Wherein the number 1 is the chromatogram of the test sample; and 2, reference medicine chromatogram.
As can be seen by comparison, under the condition of the developing solvent, spots of the test sample and corresponding positions of the reference drug chromatogram show the same color, and the separation effect is good.
Example 2
This embodiment provides a method for identifying Ranunculus ternatus, comprising the following steps:
(1) preparation of a test solution: collecting radix Ranunculi Ternati standard decoction lyophilized powder 0.5g, grinding, adding water 30ml and hydrochloric acid 2ml, heating and hydrolyzing at 100 deg.C for 1 hr, cooling, extracting with ethyl acetate under shaking for 2 times, each time 20ml, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 2ml to obtain sample solution.
(2) Preparation of reference drug solution: collecting herba Ranunculi Ternati control medicinal material 1g, adding water 50ml, boiling for 30min, filtering, spin-drying the filtrate, adding water 30ml and hydrochloric acid 2ml, heating at 100 deg.C for hydrolysis for 1 hr, cooling, extracting with ethyl acetate shaking for 2 times (20 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 2ml to obtain control medicinal material solution.
(3) And (3) thin-layer chromatography detection: sucking 2 μ l of the sample solution and 3 μ l of the reference solution, respectively dropping on the same silica gel G thin layer plate, pre-saturating with cyclohexane-ethyl acetate-formic acid (0.5: 1: 0.1) as developing agent for 30min, developing, taking out, air drying, and inspecting under ultraviolet (365 nm).
The results are shown in FIG. 2.
Wherein the number 1 is the chromatogram of the test sample; and 2, reference medicine chromatogram.
As can be seen by comparison, under the condition of the developing solvent, spots of the test sample and corresponding positions of the reference drug chromatogram show the same color, and the separation effect is good.
Example 3
This embodiment provides a method for identifying Ranunculus ternatus, comprising the following steps:
(1) preparation of a test solution: collecting radix Ranunculi Ternati standard decoction lyophilized powder 0.5g, grinding, adding water 30ml and hydrochloric acid 2ml, heating and hydrolyzing at 100 deg.C for 1 hr, cooling, extracting with ethyl acetate under shaking for 2 times, each time 20ml, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 2ml to obtain sample solution.
(2) Preparation of reference drug solution: collecting herba Ranunculi Ternati control medicinal material 1g, adding water 50ml, boiling for 30min, filtering, spin-drying the filtrate, adding water 30ml and hydrochloric acid 2ml, heating at 100 deg.C for hydrolysis for 1 hr, cooling, extracting with ethyl acetate shaking for 2 times (20 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 2ml to obtain control medicinal material solution.
(3) And (3) thin-layer chromatography detection: sucking 2 μ l of the sample solution and 3 μ l of the reference solution, respectively dropping on the same silica gel G thin layer plate, pre-saturating with cyclohexane-ethyl acetate-formic acid (1.5: 1: 0.1) as developing agent for 30min, developing, taking out, air drying, and inspecting under ultraviolet (365 nm).
The results are shown in FIG. 3.
Wherein the number 1 is the chromatogram of the test sample; and 2, reference medicine chromatogram.
As can be seen by comparison, under the condition of the developing solvent, spots of the test sample and corresponding positions of the reference drug chromatogram show the same color, and the separation effect is good.
Example 4
This embodiment provides a method for identifying Ranunculus ternatus, comprising the following steps:
(1) preparation of a test solution: taking 0.5g of radix Ranunculi Ternati standard decoction lyophilized powder, grinding, adding 6% hydrochloric acid water solution 30ml, heating and hydrolyzing at 100 deg.C for 1 hr, cooling, extracting with ethyl acetate for 2 times with shaking, each time 20ml, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 2ml to obtain sample solution.
(2) Preparation of reference drug solution: collecting herba Ranunculi Ternati control material 1g, adding water 50ml, boiling for 30min, filtering, spin-drying the filtrate, adding 6% hydrochloric acid water solution 30ml, heating and hydrolyzing at 100 deg.C for 1 hr, cooling, shaking with ethyl acetate for 2 times, extracting 20ml each time, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 2ml to obtain control solution.
(3) And (3) thin-layer chromatography detection: sucking 2 μ l of the sample solution and 3 μ l of the control solution, respectively dropping on the same silica gel G thin layer plate, presaturating with cyclohexane-ethyl acetate-formic acid (1: 1: 0.1) as developing agent for 30min, developing, taking out, air drying, and inspecting under ultraviolet (365 nm).
The results are shown in FIG. 4.
Wherein the number 1 is the chromatogram of the test sample; and 2, reference medicine chromatogram.
As can be seen by comparison, under the condition of the developing solvent, spots of the test sample and corresponding positions of the reference drug chromatogram show the same color, and the separation effect is good.
Example 5
This embodiment provides a method for identifying Ranunculus ternatus, comprising the following steps:
(1) preparation of a test solution: collecting radix Ranunculi Ternati standard decoction lyophilized powder 0.5g, grinding, adding water 30ml and hydrochloric acid 2ml, heating and hydrolyzing at 100 deg.C for 1 hr, cooling, extracting with ethyl acetate for 20ml each time for 2 times, and mixing ethyl acetate solutions to obtain test solution.
(2) Preparation of reference drug solution: collecting herba Ranunculi Ternati control medicinal material 1g, adding water 50ml, boiling for 30min, filtering, spin-drying the filtrate, adding water 30ml and hydrochloric acid 2ml, heating at 100 deg.C for hydrolysis for 1 hr, cooling, extracting with ethyl acetate shaking for 2 times (20 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 2ml to obtain control medicinal material solution.
(3) And (3) thin-layer chromatography detection: sucking 2 μ l of the sample solution and 3 μ l of the control solution, respectively dropping on the same silica gel G thin layer plate, presaturating with cyclohexane-ethyl acetate-formic acid (1: 1: 0.1) as developing agent for 30min, developing, taking out, air drying, and inspecting under ultraviolet (365 nm).
The spot of the sample chromatogram and the spot of the reference medicinal material chromatogram have the same color, and the separation effect is good.
Experimental example 1 methodological examination
1. Spot quantity optimization
In the research, the sample application amount of the test solution is inspected by adopting the preparation methods and sample application amount requirements of the test solution and the ternate buttercup root control medicinal material in the thin-layer identification of example 1 based on the content difference of the chemical components of the ternate buttercup root medicinal material and the ternate buttercup root formula granules.
The preparation of the test solution and the control solution was carried out by taking the standard decoction lyophilized powder of radix Ranunculi Ternati (200602-. Wherein the numbers 1-3 are sequentially 1 mul, 2 mul and 3 mul of standard decoction lyophilized powder of radix Ranunculi Ternati (200602-464400-01); the numbers 4-6 are sequentially 1 mul, 2 mul and 3 mul of the sample application amount of the standard ternate buttercup root decoction freeze-dried powder (200610-; the numbers 7-9 are 1 mul, 2 mul and 3 mul of the standard decoction lyophilized powder of radix Ranunculi Ternati (200618-; the serial numbers 10-12 are 2 mul, 3 mul and 4 mul of the reference medicinal material of the ternate buttercup root in sequence.
As can be seen from the figure, when the spotting amount of the standard decoction lyophilized powder of radix ranunculi ternati is 2 μ l and the spotting amount of the control medicine of radix ranunculi ternati is 3 μ l, the TLC spots are most clear and the resolution is the best at the spotting amount.
2. Durability examination
Preparing a ternate buttercup root standard decoction freeze-dried powder test solution and a ternate buttercup root reference medicinal material solution respectively according to the method of example 1, dropping the solutions on the same silica gel G thin layer plate (Taiwan cheese 32600), and respectively carrying out thin layer preparation according to the conditions of example 1 under the conditions of the temperature of 28 ℃ and the humidity of 15%; developing at 3 deg.C and humidity of 48% and at 28 deg.C and humidity of 79%, taking out, air drying, developing, inspecting under ultraviolet lamp (365nm), and obtaining thin layer chromatogram shown in FIGS. 6-8.
According to the maps, the separation effect of the standard ternate buttercup root decoction freeze-dried powder is better under different temperature and humidity conditions. Experimental results show that the thin layer identification method has good durability on temperature and humidity.
3. Investigation of different lamella plates
Trade name of thin layer plate: thin layer chromatography silica gel plate, manufacturer: sesame, 32600, yellow silica gel development test plant, tabacco; specification: 10X 20cm, model: HSG, lot number: 2016, month 10, 07;
trade name of thin layer plate: thin layer chromatography silica gel plate, manufacturer: merck KGaA, specification: 20X 20cm, model: TLC Silica gel 60, batch number: HX 87183353;
trade name of thin layer plate: thin layer chromatography silica gel plate, manufacturer: the chemical industry institute of cigarette tai city, specification: 200X 100mm, type: HSG, lot number: 20161028.
a test solution of standard decoction lyophilized powder of radix Ranunculi Ternati and a control solution of radix Ranunculi Ternati are prepared according to the method in example 1, and are spotted on the silica gel G thin layer plate produced by different manufacturers, and developed according to the thin layer condition in example 1, and the results are shown in FIGS. 9-11.
According to the atlas, the thin-layer identification is carried out on the cat's claw grass standard decoction freeze-dried powder by using silica gel G thin-layer plates produced by different manufacturers (Merck KGaA, Qingdao ocean factory, Taiwan Chinesis silica gel development and test factory 32600).
4. Sample assay
Identifying standard decoction lyophilized powder of radix Ranunculi Ternati, radix Ranunculi Ternati formula granule and radix Ranunculi Ternati medicinal material of different batches according to the method of example 1 to obtain a thin layer diagram, wherein the thin layer board is silica gel G board, and the manufacturer: sesame, 32600, yellow silica gel development test plant, tabacco; specification: 10X 20cm, model: HSG, lot number: 2016, month 10, and day 07, see FIGS. 12-14.
In FIG. 12, the number 1 is the standard decoction lyophilized powder of radix Ranunculi Ternati 200602-; number 2 is radix Ranunculi Ternati standard decoction lyophilized powder 200610-; the number 3 is the standard decoction freeze-dried powder of the radix ranunculi ternati 200618-; the number 4 is the standard decoction of radix ranunculi ternati lyophilized powder 200703-; the number 5 is the standard decoction freeze-dried powder 200703-; the number 6 is the standard decoction of radix ranunculi ternati 200703 and 456250-06; the number 7 is the standard decoction freeze-dried powder 200703 and 473200-07 of radix ranunculi ternati; the number 8 is the standard decoction freeze-dried powder 200703 and 474250-08 of radix ranunculi ternati; the number 9 is the standard decoction of radix ranunculi ternati 200703-; the number 10 is the standard decoction of radix ranunculi ternati 200703 and 475500-10; the number 11 is the standard decoction freeze-dried powder of radix ranunculi ternati 200831-; the number 12 is the standard decoction freeze-dried powder of radix ranunculi ternati 200831-; number 13 is radix Ranunculi Ternati standard decoction lyophilized powder 200831-; the number 14 is the standard decoction freeze-dried powder of radix ranunculi ternati 200831-; the number 15 is the standard decoction freeze-dried powder of radix ranunculi ternati 200831-; the number 16 is the standard decoction freeze-dried powder 200831-473000-16 of radix ranunculi ternati; the number 17 is the standard decoction freeze-dried powder of radix ranunculi ternati 200831-; number 18 is radix Ranunculi Ternati standard decoction lyophilized powder 200831-; the number 19 is the control drug 121593 and 201202 of radix ranunculi ternati.
In FIG. 13, the number 1 is the Uncaria tomentosa formula particle 200602-464400-01; the number 2 is the ternate buttercup root formula particle 200610-453400-02; the number 3 is the ranunculus ternatus thumb formula particle 200618-461670-03; the number 4 is the control medicinal material 121593 and 201202 of radix ranunculi ternati.
In FIG. 14, the number 1 is radix ranunculi ternati 200602-464400-01; the number 2 is the control medicinal material 121593 and 201202 of radix ranunculi ternati.
Experimental example 2
The influence of different inorganic acids, extracting agents and solvents on thin-layer chromatography is examined,
(1) respectively adopting sulfuric acid and nitric acid as inorganic acids, and preparing a test solution by the following method: taking 0.5g of radix Ranunculi Ternati standard decoction lyophilized powder, grinding, adding 30ml of water and 2ml of inorganic acid, heating and hydrolyzing at 100 deg.C for 1 hr, cooling, extracting with ethyl acetate under shaking for 2 times, each time 20ml, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 2ml of methanol to obtain a sample solution;
(2) respectively adopting ethyl ether and n-butanol as extracting agents, and preparing a test solution by adopting the following method: taking 0.5g of radix Ranunculi Ternati standard decoction lyophilized powder, grinding, adding 30ml of water and 2ml of inorganic acid, heating and hydrolyzing at 100 deg.C for 1 hr, cooling, extracting with 20ml each time for 2 times by shaking with extractant, mixing extractive solutions, evaporating to dryness, and dissolving residue with 2ml of methanol to obtain sample solution;
(3) respectively adopting ethanol and ethyl acetate as dissolving solvents, and preparing a test solution by the following method: taking 0.5g of radix Ranunculi Ternati standard decoction lyophilized powder, grinding, adding 30ml of water and 2ml of hydrochloric acid, heating and hydrolyzing at 100 deg.C for 1 hr, cooling, extracting with ethyl acetate under shaking for 2 times, each time 20ml, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 2ml of solvent to obtain sample solution;
(4) preparation of reference drug solution: collecting herba Ranunculi Ternati control material 1g, adding water 50ml, boiling for 30min, filtering, spin-drying the filtrate, adding 6% hydrochloric acid water solution 30ml, heating and hydrolyzing at 100 deg.C for 1 hr, cooling, shaking with ethyl acetate for 2 times, extracting 20ml each time, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 2ml to obtain control solution.
(5) And (3) thin-layer chromatography detection: sucking 2 μ l of the sample solution and 3 μ l of the control solution, respectively dropping on the same silica gel G thin layer plate, presaturating with cyclohexane-ethyl acetate-formic acid (1: 1: 0.1) as developing agent for 30min, developing, taking out, air drying, and inspecting under ultraviolet (365 nm). In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution.
In fig. 15, reference numeral 1 denotes a sample solution obtained by extraction with sulfuric acid; 2 is a test solution extracted by nitric acid; 3 is a test solution extracted by diethyl ether; 4 is a sample solution extracted by n-butyl alcohol; 5 is a test solution dissolved by ethanol; 6 is a test sample solution dissolved by ethyl acetate; 7 is the reference solution.
As can be seen from the figure, the chromatographic spots of the sample obtained by the solution of the sample extracted by sulfuric acid, the solution of the sample extracted by ethyl ether and the solution of the sample dissolved by ethanol and ethyl acetate have the same color as the corresponding positions of the chromatogram of the reference medicinal material, and the separation effect is good, the chromatographic spots of the sample obtained by the solution of the sample extracted by n-butanol have slightly poor color, and the chromatographic spots of the sample extracted by nitric acid cannot develop color.
Comparative example 1
The comparative example provides a method of identifying catclaw buttercup and its formulation, comprising the steps of:
(1) preparation of a test solution: the method comprises the following steps of preparing a test sample solution by taking a radix ranunculi ternati medicinal material, radix ranunculi ternati standard decoction freeze-dried powder and radix ranunculi ternati formula particles as test samples according to the following operation, taking 0.5g of the test sample, grinding, adding 30ml of water and 2ml of hydrochloric acid, heating and hydrolyzing at 100 ℃ for 1 hour, cooling, shaking and extracting with ethyl acetate for 2 times, 20ml each time, combining ethyl acetate solutions, evaporating to dryness, and dissolving residues with 2ml of methanol to obtain the test sample solution.
(2) Preparation of reference drug solution: collecting herba Ranunculi Ternati control medicinal material 1g, adding water 50ml, boiling for 30min, filtering, spin-drying the filtrate, adding water 30ml and hydrochloric acid 2ml, heating at 100 deg.C for hydrolysis for 1 hr, cooling, extracting with ethyl acetate shaking for 2 times (20 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 2ml to obtain control medicinal material solution.
(3) And (3) thin-layer chromatography detection: sucking 2 μ l of the sample solution and 3 μ l of the reference solution, respectively dropping on the same silica gel G thin layer plate, pre-saturating with toluene-ethyl acetate-formic acid (20: 4: 0.5) as developing agent for 30min, developing, taking out, air drying, and inspecting under ultraviolet (365 nm).
The results are shown in FIG. 16.
Wherein the number 1 is the radix ranunculi ternati medicinal material 200602-464400-01; the number 2 is the standard decoction freeze-dried powder of radix ranunculi ternati 200602-464400-01; the number 3 is the ternate buttercup root formula particle 200602-464400-01; the number 4 is the control medicinal material 121593 and 201202 of radix ranunculi ternati.
As can be seen, under the condition of the developing agent, the separation effect of each fluorescent spot of the thin-layer chromatography is poor, and the tailing is serious.
Comparative example 2
The comparative example provides a method of identifying catclaw buttercup and its formulation, comprising the steps of:
(1) preparation of a test solution: the method comprises the following steps of preparing a test sample solution by taking a radix ranunculi ternati medicinal material, radix ranunculi ternati standard decoction freeze-dried powder and radix ranunculi ternati formula particles as test samples according to the following operation, taking 0.5g of the test sample, grinding, adding 30ml of water and 2ml of hydrochloric acid, heating and hydrolyzing at 100 ℃ for 1 hour, cooling, shaking and extracting with ethyl acetate for 2 times, 20ml each time, combining ethyl acetate solutions, evaporating to dryness, and dissolving residues with 2ml of methanol to obtain the test sample solution.
(2) Preparation of reference drug solution: collecting herba Ranunculi Ternati control medicinal material 1g, adding water 50ml, boiling for 30min, filtering, spin-drying the filtrate, adding water 30ml and hydrochloric acid 2ml, heating at 100 deg.C for hydrolysis for 1 hr, cooling, extracting with ethyl acetate shaking for 2 times (20 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 2ml to obtain control medicinal material solution.
(3) And (3) thin-layer chromatography detection: sucking 2 μ l of the sample solution and 3 μ l of the control solution, respectively dropping on the same silica gel G thin layer plate, presaturating with cyclohexane-acetone-ethyl acetate (5: 2: 1) as developing agent for 30min, developing, taking out, air drying, and inspecting under ultraviolet (365 nm).
The results are shown in FIG. 17.
Wherein the number 1 is the radix ranunculi ternati medicinal material 200602-464400-01; the number 2 is the standard decoction freeze-dried powder of radix ranunculi ternati 200602-464400-01; the number 3 is the ternate buttercup root formula particle 200602-464400-01; the number 4 is the control medicinal material 121593 and 201202 of radix ranunculi ternati.
As can be seen, under the condition of the developing agent, the definition of each fluorescent spot in the thin-layer chromatography is poor, the number of spots is small, and the presented information is less.
Comparative example 3
The comparative example provides a method of identifying a catclaw buttercup formula particle, comprising the steps of:
(1) preparation of a test solution: taking 0.5g of radix Ranunculi Ternati formula granule, adding 10ml of ethyl acetate, heating and refluxing for 30min, cooling, filtering, evaporating filtrate to dryness, and dissolving residues with 2ml of ethyl acetate respectively to obtain a sample solution as a sample solution.
(2) Preparation of reference drug solution: collecting herba Ranunculi Ternati control medicinal material 1g, adding water 50ml, boiling for 30min, filtering, spin-drying the filtrate, adding water 30ml and hydrochloric acid 2ml, heating at 100 deg.C for hydrolysis for 1 hr, cooling, extracting with ethyl acetate shaking for 2 times (20 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 2ml to obtain control medicinal material solution.
(3) And (3) thin-layer chromatography detection: sucking 2 μ l of the sample solution and 3 μ l of the control solution, respectively dropping on the same silica gel G thin layer plate, presaturating with cyclohexane-ethyl acetate-formic acid (1: 1: 0.1) as developing agent for 30min, developing, taking out, air drying, and inspecting under ultraviolet (365 nm).
The result is shown in FIG. 18, wherein the number 1 is the control medicinal material 121593-; no. 2 is the test solution of the ranunculus ternatus formulation particle 200602-464400-01 and the comparative example 3.
As can be seen from the figure, the test sample obtained by this preparation method has no separation spots in the chromatogram.
Comparative example 4
The comparative example provides a method of identifying a catclaw buttercup formula particle, comprising the steps of:
(1) preparation of a test solution: taking 0.5g of radix Ranunculi Ternati formula granule, adding 10ml of methanol, heating and refluxing for 30min, cooling, filtering, evaporating filtrate, dissolving residue with 2m of ethanol respectively to obtain test solution as test solution.
(2) Preparation of reference drug solution: collecting herba Ranunculi Ternati control medicinal material 1g, adding water 50ml, boiling for 30min, filtering, spin-drying the filtrate, adding water 30ml and hydrochloric acid 2ml, heating at 100 deg.C for hydrolysis for 1 hr, cooling, extracting with ethyl acetate shaking for 2 times (20 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 2ml to obtain control medicinal material solution.
(3) And (3) thin-layer chromatography detection: sucking 2 μ l of the sample solution and 3 μ l of the control solution, respectively dropping on the same silica gel G thin layer plate, presaturating with cyclohexane-ethyl acetate-formic acid (1: 1: 0.1) as developing agent for 30min, developing, taking out, air drying, and inspecting under ultraviolet (365 nm).
The result is shown in FIG. 19, wherein the number 1 is the control medicinal materials 121593 and 201202 of radix ranunculi ternati; no. 2 is the test solution of the ternate buttercup root formula particle 200602-464400-01 and the comparative example 4
As can be seen from the figure, the test sample obtained by this preparation method has no separation spots in the chromatogram.
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 therefrom are within the scope of the invention.
Claims (10)
1. A thin layer chromatography construction method is characterized by comprising the following steps:
preparing a radix ranunculi ternati test solution: mixing a radix ranunculi ternati test sample, water and inorganic acid, heating for hydrolysis, and extracting with an organic solvent to obtain a test sample solution;
and (4) carrying out thin-layer chromatography detection on the test solution to obtain the thin-layer chromatography of the test.
2. The construction method according to claim 1, wherein the thin layer chromatography detection comprises the following steps:
(1) spotting the ternate buttercup root sample solution on a thin-layer plate;
(2) developing with mixed solution of cyclohexane-ethyl acetate-formic acid as developing agent, taking out, volatilizing solvent, and developing to obtain the final product.
3. The construction method according to claim 2, wherein the thin layer chromatography detection further satisfies any one or more of the following A-C:
a, adopting a silica gel G thin layer plate as a thin layer plate; preferably, the spotting volume is 1-4 μ l;
b, the volume ratio of cyclohexane-ethyl acetate-formic acid is 0.5-1.5: 1: 0.1;
c, developing color under ultraviolet or by adopting a sulfuric acid ethanol solution as a color developing agent; preferably, the color is developed by 365nm ultraviolet light.
4. The construction method according to any one of claims 1 to 3, wherein the preparation of the ternate buttercup root test solution satisfies any one or more of the following a-g:
a, the ratio of the mass of the ternate buttercup root test sample to the volume of water and inorganic acid is 0.3-0.7: 20-50: 1-3;
b, the volume ratio of the mass of the ternate buttercup root test sample to the organic solvent is 0.3-0.7: 30-60 parts of;
c, heating and hydrolyzing at 90-100 ℃ for 0.5-2 h;
d, extracting with organic solvent for 1-5 times, adding 20-60 times of organic solvent for each extraction, and mixing extractive solutions;
e, the inorganic acid is hydrochloric acid and/or sulfuric acid;
f, the organic solvent is selected from at least one of ethyl acetate, n-butyl alcohol and diethyl ether;
g, after the extraction step, a step of concentrating the extracting solution is also included; or, the method further comprises the step of drying and dissolving the extract, preferably, the solvent adopted in the dissolving step is at least one of methanol, ethanol and ethyl acetate.
5. The constructing method according to any one of claims 1 to 4, wherein the radix ranunculi ternati test sample is selected from radix ranunculi ternati medicinal materials, decoction pieces or radix ranunculi ternati medicinal preparations, preferably, the radix ranunculi ternati medicinal preparations are selected from solid preparations, semi-solid preparations or liquid preparations of radix ranunculi ternati.
6. A thin layer chromatography obtained by the method of construction according to any one of claims 1 to 5.
7. A method for identifying radix Ranunculi Ternati medicinal material, decoction pieces or radix Ranunculi Ternati medicinal preparation is characterized by comprising the step of comparing the thin-layer chromatography of a to-be-detected radix Ranunculi Ternati sample with that of a control medicinal material;
wherein the thin-layer chromatography of the radix ranunculi ternati test sample to be tested is obtained by using the radix ranunculi ternati test sample to be tested according to the construction method of any one of claims 1 to 5;
the thin layer chromatography of the control medicinal material is obtained by using the ternate buttercup root control medicinal material according to the construction method of any one of claims 1 to 5, and preferably, before the ternate buttercup root control medicinal material is used according to the construction method of any one of claims 1 to 5, the thin layer chromatography further comprises the steps of mixing the ternate buttercup root medicinal material with water, decocting, carrying out solid-liquid separation, and drying to obtain the ternate buttercup root medicinal material extract.
8. The method according to claim 7, wherein in the thin layer chromatography construction of the reference drug, the decoction temperature is 90-150 ℃ and the time is 0.5-2 h; and/or the mass ratio of the ternate buttercup root medicinal material to the volume of water is 0.5-3: 20-100 parts of; and/or, the solid-liquid separation is filtration or centrifugation.
9. The use of the method of identifying a radix ranunculi ternati medicinal material, decoction piece or radix ranunculi ternati medicinal preparation of claim 7 or 8 in the quality detection of the radix ranunculi ternati medicinal material, decoction piece or radix ranunculi ternati medicinal preparation.
10. A quality detection method of a traditional Chinese medicine product is characterized in that,
comprising the step of identifying a medicinal material, decoction pieces or a medicinal preparation of radix ranunculi ternati according to the method of claim 6 or 7.
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