CN115508494A - Thin-layer identification method for rhizoma sparganii formula granules and standard decoction thereof - Google Patents

Thin-layer identification method for rhizoma sparganii formula granules and standard decoction thereof Download PDF

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CN115508494A
CN115508494A CN202211118806.1A CN202211118806A CN115508494A CN 115508494 A CN115508494 A CN 115508494A CN 202211118806 A CN202211118806 A CN 202211118806A CN 115508494 A CN115508494 A CN 115508494A
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rhizoma sparganii
thin
solution
standard decoction
identification method
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张宇静
周敏
周海琴
张云天
翟燕娟
顾超
何雪霞
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Jiangyin Tianjiang Pharmaceutical Co Ltd
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Jiangyin Tianjiang Pharmaceutical Co Ltd
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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
    • 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
    • G01N30/95—Detectors specially adapted therefor; Signal analysis
    • 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
    • G01N2030/945—Application of reagents to undeveloped plate
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Abstract

The invention discloses a thin-layer identification method for trigone formula granules and standard decoction thereof. The method improves the quality controllability of the rhizoma sparganii medicine, is beneficial to quality supervision and management, and is simple, convenient, rapid, high in efficiency, low in cost and environment-friendly.

Description

Thin-layer identification method for rhizoma sparganii formula granules and standard decoction thereof
Technical Field
The invention relates to the field of quality control of traditional Chinese medicines, in particular to a thin-layer identification method for rhizoma sparganii formula granules and standard decoction thereof.
Background
Rhizoma Sparganii is loaded in "materia Medica" of Tang Dynasty, and is a dried tuber of Sparganii stoloniferum Buch. It is pungent, bitter and neutral in flavor, enters liver and spleen meridians, has the actions of breaking blood and promoting qi circulation, resolving food stagnation and relieving pain, and is clinically combined with E.zedoary to treat abdominal mass, dysmenorrhea, amenorrhea due to blood stasis, thoracic obstruction, heartache due to stagnation of food, distending pain due to food stagnation, etc.
The traditional Chinese medicine prescription granule is a novel prescription medication with uniform specification, uniform dosage and uniform quality standard, which is processed by taking traditional Chinese medicine decoction pieces as raw materials and water as a solvent and carrying out production processes of extraction, concentration, drying, granulation and the like. The traditional Chinese medicine decoction is used under the guidance of the traditional Chinese medicine theory based on the traditional Chinese medicine standard decoction, and compared with the traditional Chinese medicine decoction, the traditional Chinese medicine decoction has the characteristics of convenience in taking, easiness in storage, portability, controllable quality and the like.
The rhizoma sparganii formula granules and the standard decoction are prepared from rhizoma sparganii medicinal materials through a series of processes, and some processes can damage the character characteristics of the raw medicinal materials. Therefore, the identification of the aqueous extract and the formula granules which lose the original appearance character has important significance on the quality control of the rhizoma sparganii. Under the thin-layer chromatography identification condition of the rhizoma sparganii medicinal material item in the 2020 edition of Chinese pharmacopoeia, a rhizoma sparganii test sample is extracted by ethanol, and the characteristic is that the rhizoma sparganii test sample is an alcohol-soluble component and is not suitable for rhizoma sparganii formula granules and standard decoction taking water as a solvent. The thin-layer identification method of the rhizoma sparganii decocted in water and the prepared granules and standard decoction is not referred to in literature reports.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the defects and shortcomings of the prior art, the invention provides a thin-layer identification method for rhizoma sparganii formula particles and standard decoction thereof, which improves the quality controllability of rhizoma sparganii drugs, is beneficial to quality supervision and management, and is simple, convenient, rapid, high in efficiency, low in cost and environment-friendly.
The technical scheme is as follows: the invention relates to a thin-layer identification method for a rhizoma sparganii formula particle and a standard decoction thereof, which is characterized by comprising the following steps of: the method comprises the following steps:
1) Dissolving rhizoma Sparganii formula granule and standard decoction sample in water respectively, extracting with ethyl acetate solution under shaking, standing for layering, collecting supernatant, evaporating to dry, and dissolving residue with alcohol solvent to obtain formula granule and standard decoction sample solution;
2) Decocting rhizoma Sparganii with water or heating under reflux, filtering, extracting with ethyl acetate solution under shaking, standing for layering, collecting supernatant, evaporating to dryness, and dissolving the residue with alcohol solvent to obtain control solution;
3) Respectively spotting the sample solution and the reference medicinal material solution on the same thin layer plate, spreading with cyclohexane-ethyl acetate-formic acid as developing agent, air drying, and obtaining thin layer chromatogram under ultraviolet lamp to complete identification operation.
When the rhizoma sparganii formula particles and the standard decoction sample are dissolved by adding water in the step 1), the mass-to-volume ratio of the rhizoma sparganii formula particles or the rhizoma sparganii standard decoction to the water is 1.
Wherein, when the rhizoma sparganii reference medicinal material is decocted by adding water in the step 2), the mass volume of the reference medicinal material and water is 1-2 g/mL, the treatment time of decoction or heating reflux is 15-60 min, the shaking extraction frequency of the ethyl acetate solution is 1-4 times, and the volume ratio of the ethyl acetate solution to the reference medicinal material aqueous solution is 1.
Wherein the volume ratio of cyclohexane to ethyl acetate to formic acid in the developing solvent in the step 3) is 3:1-3.
Wherein, the sample amount of the test solution in the step 3) is 2 to 10 mul, and the sample amount of the reference medicinal material solution is 5 to 20 mul.
Wherein, the inspection is carried out under 365nm ultraviolet lamp in the step 3).
Wherein, the thin layer plate in the step 3) is a silica gel G thin layer plate.
Wherein, fluorescent spots with the same color are displayed on the thin-layer chromatogram in the step 3) at the positions corresponding to the chromatogram of the reference medicinal material solution in the chromatogram of the test solution.
Principle analysis: according to the chemical structure and properties of each effective component of the traditional Chinese medicine, a test sample and a reference medicinal solution are simply, conveniently and quickly prepared by adopting a proper extraction solvent according to a similar compatible extraction principle. And then proper developing agent is adopted for developing, and various chemical components can be well separated on the thin-layer plate along with the selected developing agent according to different adsorption, desorption, re-adsorption and re-desorption capabilities. And then, with the help of effective components with similar polarities, different color spots are shown on the same thin-layer plate under different inspection conditions, and a multi-information thin-layer chromatogram is obtained.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: the invention adopts a simple and quick pretreatment method to obtain a test solution and a reference medicinal solution, which are respectively spotted on the same thin-layer plate, are unfolded by using a proper developing agent and are inspected under ultraviolet light to obtain a multi-information triangular thin-layer chromatogram. At present, thin-layer identification reports about rhizoma sparganii water extracts and preparations thereof are not found. The invention not only provides a method for identifying the common burreed rhizome formula granules and the standard decoction, improves the quality controllability of the common burreed rhizome formula granules and the standard decoction, but also is more beneficial to quality supervision and management. The method is simple, convenient, rapid, high in efficiency, low in cost and environment-friendly.
Drawings
FIG. 1 is a thin layer chromatogram of trigone formula granules in example 1 of the present invention;
FIG. 2 is a thin layer chromatogram of the preparation method of various test samples in example 1 of the present invention;
FIG. 3 is a thin layer chromatogram of different control preparation methods in example 1 of the present invention;
FIG. 4 is a thin layer chromatogram of a trigone standard decoction in example 2 of the present invention;
FIG. 5 is a thin layer chromatogram of the sample solution of the test sample and the reference solution in different spot amounts in example 3 of the present invention;
FIG. 6 is a thin layer chromatogram of the trigonal formula granules of example 3 of the present invention at different temperatures;
FIG. 7 is a thin layer chromatogram of the triangular prism-shaped formula particles with different humidity in example 3 of the present invention.
Detailed Description
The technical solution of the present invention is further described with reference to the accompanying drawings and the detailed description.
The invention adopts the following instruments and reagents:
the instrument comprises the following steps: a thin-layer automatic imager (CAMAG TLC VIUALIZER), a thousandth balance (AR 223CN, aohaus), a silica gel G thin-layer plate (Qingdao oceanic factory), a GKC114 temperature-controlled water bath (Nantong Huatai laboratory instruments Co., ltd.), and an AS165W type centrifuge (Su Wang (Shanghai) commercial Co., ltd.).
Cyclohexane (national medicine group chemical reagent, inc.), ethyl acetate (national medicine group chemical reagent, inc.) and formic acid (national medicine group chemical reagent, inc.) are analytically pure and water is ultrapure water;
the rhizoma sparganii reference medicinal material (121521-201504) is purchased from China pharmaceutical biologicals assay research institute;
rhizoma Sparganii formula pellets (batch Nos.: 19050199, 19050209, 19050219) were provided by Jiangyin Tianjiang pharmaceutical Co., ltd.
The present invention can be carried out according to the procedures or conditions of the conventional experimental procedures described in the literature in the field, without specifying the specific experimental procedures or conditions. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1:
preparation of a test solution: taking 1g of three different batches of rhizoma sparganii formula particles (batch number: 19050199, 19050209, 19050219, manufactured by Jiangyin Tianjiang pharmaceutical industry Co., ltd.), adding 20mL of water, slightly heating to dissolve, cooling, extracting twice with ethyl acetate by shaking and shaking, 20mL each time, combining ethyl acetate solutions, evaporating to dryness, and adding 1mL of methanol to dissolve residues to obtain a sample solution.
Preparation of reference drug solution: taking another rhizoma Sparganii 5g as reference material, adding 50mL of water, decocting for 30min, boiling slightly, filtering, evaporating the filtrate to dryness, concentrating the filtrate to about 20mL, cooling, extracting twice with ethyl acetate under shaking, each time 20mL, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 1mL of methanol to obtain the reference material solution.
Thin layer identification: the test solution 5 μ l and the control solution 15 μ l were pipetted on the same silica gel G thin layer plate, developed with cyclohexane-ethyl acetate-formic acid (3.
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. The results are shown in FIG. 1, where 1-3 in FIG. 1 correspond to the chromatogram of the test sample of the rhizoma sparganii formula particles with the lot numbers 19050199, 19050209 and 19050219 respectively, and S is the chromatogram of the rhizoma sparganii reference drug.
1. Examination of the preparation method of the test solution:
the method comprises the following steps: collecting rhizoma Sparganii formula granule (batch No. 19050199) lg, adding methanol 20ml, ultrasonic treating for 20 min, filtering, evaporating filtrate to dryness, and dissolving residue with methanol 1ml to obtain sample solution.
The second method comprises the following steps: taking 1g of the product powder, adding 20ml of ethanol, performing ultrasonic treatment for 20 minutes, filtering, evaporating the filtrate to dryness, and dissolving the residue with 1ml of ethanol to obtain a test solution.
The third method comprises the following steps: taking 1g of the product, adding 20ml of water, slightly heating to dissolve, cooling, extracting with ethyl acetate twice with shaking, 20ml each time, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 1ml of methanol to obtain a sample solution.
Taking 5g of rhizoma sparganii reference medicinal material, and preparing a reference medicinal material solution by adopting the conditions of the example 1.
Respectively sucking 5 mu L of the three test solution samples and 15 mu L of the reference solution samples, dropping the three test solution samples and the reference solution samples on the same silica gel G thin-layer plate, developing the three test solution samples by taking cyclohexane-ethyl acetate-formic acid (3. As can be seen from FIG. 2, the sample chromatogram spots of the rhizoma sparganii formula particle prepared by the method (i.e., method III) of the present invention are clear and rich, the separation effect is good, and the chromatogram of the test sample corresponds to the chromatogram spots of the reference drug.
2. Examination of control solution preparation method:
the method comprises the following steps: collecting rhizoma Sparganii control material 5g, adding water 50ml, boiling for 30min, filtering, concentrating the filtrate to about 20ml, cooling, extracting with ethyl acetate twice (20 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 1ml to obtain control solution.
The second method comprises the following steps: taking 5g of rhizoma Sparganii as reference material, adding 20ml of water, soaking for 30min, extracting with ethyl acetate twice with shaking, each time extracting with 20ml, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 1ml of methanol to obtain reference material solution.
Taking 5g of rhizoma sparganii reference medicinal material, and preparing a reference medicinal material solution by adopting the conditions of the example 1.
Respectively sucking 15 μ L of the above three control drug solutions, spotting on the same silica gel G thin layer plate, developing with cyclohexane-ethyl acetate-formic acid (3. As can be seen from fig. 3, the rhizoma sparganii reference drug adopts two different extraction methods, and the chromatographic spot separation effect is similar; spots corresponding to the reference medicinal materials can be detected in the sample, but the extraction efficiency of the first method is higher, and the obtained fluorescent spots are clearer.
Example 2:
this example is different from example 1 in that the trigonal standard decoction (aqueous extract) of the sample solution used in the present invention, lot numbers DG221811112, DG221811113 and DG221811114, was prepared in the same manner as in example 1, and the results are shown in fig. 4. In fig. 4, 1-3 correspond to the chromatogram of the sample of the standard decoction of rhizoma sparganii with the lot numbers of DG221811112, DG221811113 and DG221811114 respectively, and S is the chromatogram of the rhizoma sparganii reference drug.
Example 3:
this example performed durability verification of the thin layer authentication in example 1, specifically including:
(1) Investigation of different spot sizes:
taking rhizoma sparganii formula particles (lot number: 19050199) and a rhizoma sparganii reference drug, preparing a test solution and a reference drug solution according to the method described in embodiment 1, respectively taking 2 muL, 5 muL, 8 muL, 10 muL of the rhizoma sparganii formula particle test solution and 5 muL, 10 muL, 15 muL, 20 muL of the rhizoma sparganii reference drug solution, spotting on the same silica gel G thin-layer plate, taking cyclohexane-ethyl acetate-formic acid (3: 1.5).
(2) Investigation of different temperatures:
taking the trigone formula particles (batch numbers: 19050199, 19050209 and 19050219) and the trigone reference drug, preparing a test solution and a reference drug solution respectively according to the method described in example 1, taking 5 microliter of the trigone formula particle test solution and 15 microliter of the trigone reference drug solution, respectively, spotting on the same silica gel G thin layer plate, taking cyclohexane-ethyl acetate-formic acid (3. The experimental result shows that the temperature has small influence on the thin layer identification of the rhizoma sparganii formula particles, and the thin layer identification method has good durability to different temperatures.
(3) Investigation of different humidities:
taking the trigone formula particles (batch numbers: 19050199, 19050209 and 19050219) and the trigone reference drug, preparing a test solution and a reference drug solution respectively according to the method described in example 1, taking 5 microliter of the trigone formula particle test solution and 15 microliter of the trigone reference drug solution, respectively, spotting on the same silica gel G thin layer plate, taking cyclohexane-ethyl acetate-formic acid (3. The experimental result shows that the influence of humidity on the thin layer identification of the rhizoma sparganii formula particles is small, and the durability of the thin layer identification method to different humidity is good.
Comprehensive analysis: the thin-layer identification method of the invention obtains the test sample and the reference medicinal solution by a simple and quick pretreatment method, respectively dots the test sample and the reference medicinal solution on the same thin-layer plate, and after the thin-layer plate is unfolded, corresponding fluorescent spots are detected under ultraviolet light, thereby greatly improving the quality controllability of the identified object, being beneficial to quality supervision, and having the advantages of simple and quick method, high efficiency, low cost, no environmental pollution, rich chromatographic spot information, good separation degree and good reproducibility. The method of the invention does not use color developing agent, and the clear spots can be displayed by directly observing under ultraviolet light (365 nm). Compared with the existing identification method, the inspection process is simple, convenient and quick, and the damage to the health of operators and the pollution to the environment are reduced.
The traditional clinical medication of rhizoma sparganii usually adopts a water decoction mode, which shows the treatment effect of water-soluble components, and most of the functional components are water-soluble. The thin-layer identification method provided by the invention presents the components with higher water solubility in the rhizoma sparganii, so that various information of the water-soluble components in the rhizoma sparganii can be better kept, and further more thin-layer information can be provided in the rhizoma sparganii standard decoction (water extract) or the rhizoma sparganii formula particles. The thin-layer chromatogram of the rhizoma sparganii reference medicinal material, the standard decoction (water extract) and the formula granules can be used for quality control in process engineering, so that the clinical medication effect is ensured; meanwhile, a thin-layer identification reference picture and a method are provided for various preparations of the rhizoma sparganii which is extracted with water and used as a medicine.

Claims (8)

1. A thin-layer identification method for rhizoma sparganii formula granules and standard decoction thereof is characterized by comprising the following steps: the method comprises the following steps:
1) Dissolving rhizoma Sparganii formula granule and standard decoction sample in water respectively, extracting with ethyl acetate solution under shaking, standing for layering, collecting supernatant, evaporating to dry, and dissolving residue with alcohol solvent to obtain formula granule and standard decoction sample solution;
2) Decocting rhizoma Sparganii with water or heating under reflux, filtering, extracting with ethyl acetate solution under shaking, standing for layering, collecting supernatant, evaporating to dryness, and dissolving the residue with alcohol solvent to obtain control solution;
3) Respectively spotting the sample solution and the reference medicinal material solution on the same thin layer plate, spreading with cyclohexane-ethyl acetate-formic acid as developing agent, air drying, and obtaining thin layer chromatogram under ultraviolet lamp to complete identification operation.
2. The thin-layer identification method for rhizoma sparganii formula particles and standard decoction thereof according to claim 1, characterized by comprising the following steps: when the rhizoma sparganii formula particles and the standard decoction sample are dissolved by adding water in the step 1), the mass-to-volume ratio of the rhizoma sparganii formula particles or the rhizoma sparganii standard decoction to the water is 1.
3. The thin-layer identification method for rhizoma sparganii formula granules and standard decoction thereof according to claim 1, which is characterized in that: when the rhizoma sparganii reference medicinal material in the step 2) is decocted by adding water, the mass volume of the reference medicinal material and water is 1-2 g/mL, the treatment time of decoction or heating reflux is 15-60 min, the number of times of shaking extraction of the ethyl acetate solution is 1-4, and the volume ratio of the ethyl acetate solution to the reference medicinal material aqueous solution is 1.5-1.
4. The thin-layer identification method for rhizoma sparganii formula granules and standard decoction thereof according to claim 1, which is characterized in that: the volume ratio of cyclohexane to ethyl acetate to formic acid in the developing agent in the step 3) is 3:1-3.
5. The thin-layer identification method for rhizoma sparganii formula granules and standard decoction thereof according to claim 1, which is characterized in that: in the step 3), the sample amount of the test solution is 2-10 mul, and the sample amount of the reference medicinal material solution is 5-20 mul.
6. The thin-layer identification method for rhizoma sparganii formula granules and standard decoction thereof according to claim 1, which is characterized in that: and in the step 3), the inspection is carried out under a 365nm ultraviolet lamp.
7. The thin-layer identification method for rhizoma sparganii formula granules and standard decoction thereof according to claim 1, which is characterized in that: the thin layer plate in the step 3) is a silica gel G thin layer plate.
8. The thin-layer identification method for rhizoma sparganii formula granules and standard decoction thereof according to claim 1, which is characterized in that: and in the thin-layer chromatogram in the step 3), fluorescent spots with the same color are displayed in the positions, corresponding to the positions of the chromatogram of the solution of the reference medicinal material, in the chromatogram of the solution of the test solution.
CN202211118806.1A 2022-09-13 2022-09-13 Thin-layer identification method for rhizoma sparganii formula granules and standard decoction thereof Pending CN115508494A (en)

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