CN109187841B - Thin-layer identification method of coptidis flos - Google Patents
Thin-layer identification method of coptidis flos Download PDFInfo
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- CN109187841B CN109187841B CN201811009945.4A CN201811009945A CN109187841B CN 109187841 B CN109187841 B CN 109187841B CN 201811009945 A CN201811009945 A CN 201811009945A CN 109187841 B CN109187841 B CN 109187841B
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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
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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
- G01N30/94—Development
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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
- G01N30/95—Detectors specially adapted therefor; Signal analysis
Abstract
The invention discloses a thin-layer identification method of coptis chinensis flowers, which comprises the following steps: (1) taking the coptidis flos to be identified, drying and crushing, taking 0.25g of the coptidis flos powder in a conical flask, adding 25ml of anhydrous methanol, carrying out ultrasonic treatment for 30min, filtering, and taking a subsequent filtrate as a test solution; (2) taking berberine hydrochloride, and preparing into 800ug/ml solution as reference solution; (3) sucking the test solution and the reference solution, respectively dropping on the same silica gel G thin layer plate, developing with 60-90 deg.C petroleum ether-chloroform-methanol as developing agent at volume ratio of 5:3:2, taking out, air drying, placing under 365nm ultraviolet lamp for inspection, wherein in the chromatogram of the test solution, spots of the same color appear at corresponding positions with Rf value of 0.93 + -0.02, and spots appear at positions with Rf values of 0.35 + -0.05 and 0.77 + -0.05 compared with the chromatogram of the reference solution. The method is beneficial to the quality control of the coptidis flos and provides scientific basis for formulating the quality standard of the coptidis flos.
Description
Technical Field
The invention relates to a thin-layer identification method of coptis chinensis flowers, belonging to the technical field of traditional Chinese medicine analysis.
Background
The flower of coptis (lysmachia davurica Ledeb.) is a flower of coptis (coptischinensis franch.) belonging to the family ranunculaceae, coptis deltoidea c.y.chengthsiao or coptis (c.teteawal.) and is reported in the literature to contain abundant amino acids and mineral elements useful for the human body. The coptis as a traditional Chinese medicinal material has strong antibacterial and fire-purging capabilities, and modern medical research shows that the coptis also has more than twenty functions of reducing blood sugar, reducing blood fat, resisting platelet aggregation and the like. The flower is used as a part rich in plant nutrient components, and the development and utilization of the coptis flower have important economic and social values. The thin-layer identification is helpful for the quality control of the coptidis flos. At present, no thin-layer identification method of the coptis chinensis flowers is recorded in pharmacopoeia and Chinese medicinal material standards of various provinces.
Disclosure of Invention
The invention aims to provide a thin-layer identification method of coptis chinensis flowers.
The thin-layer identification method of the coptis chinensis flower provided by the invention comprises the following steps:
(1) taking the coptidis flos to be identified, drying and crushing, taking 0.25g of the coptidis flos powder in a conical flask, adding 25ml of anhydrous methanol, carrying out ultrasonic treatment for 30min, filtering, and taking a subsequent filtrate as a test solution;
(2) taking berberine hydrochloride, and preparing into 800ug/ml solution as reference solution;
(3) sucking the test solution and the reference solution, respectively dropping on the same thin layer plate, developing with 60-90 deg.C petroleum ether-chloroform-methanol as developing agent at volume ratio of 5:3:2, taking out, air drying, placing under 365nm ultraviolet lamp for inspection, wherein in the chromatogram of the test solution, spots with the same color appear at the corresponding positions with Rf value of 0.93 + -0.02, and spots appear at the positions with Rf values of 0.35 + -0.05 and 0.77 + -0.05 compared with the chromatogram of the reference solution.
Further, in the step (3), the development temperature was 29 ℃, the development distance was 9.0cm, and the amount of the spot was 10. mu.l.
The identification method of the invention is beneficial to the quality control of the coptidis flos, provides scientific basis for establishing the quality standard of the coptidis flos and lays a foundation for the sustainable utilization and development of the coptidis flos.
Drawings
FIG. 1 is a thin-layer chromatogram for identification of a test sample and a reference sample, wherein A is berberine hydrochloride reference sample, and 1-6 are 20180310-1-1, 20180310-2-1, 20180310-3-1, 20180310-4-1, 20180310-5-1,20180316-6-1 flos Coptidis test sample in sequence.
FIG. 2 is a sample thin-layer chromatogram obtained by using different thin-layer plates, wherein the left plate is a silica gel H thin-layer plate, the right plate is a silica gel G thin-layer plate, and 1-6 are 20180310-1-1, 20180310-2-1, 20180310-3-1, 20180310-4-1, 20180310-5-1,20180316-6-1 golden flower samples in sequence.
FIG. 3 is a thin-layer chromatogram of a test sample developed under different temperature conditions, wherein the left side is 6 ℃, the right side is 29 ℃, and the 1-6 are 20180310-1-1, 20180310-2-1, 20180310-3-1, 20180310-4-1, 20180310-5-1,20180316-6-1 golden flower test samples in sequence.
FIG. 4 is a sample thin-layer chromatogram under different spot sizes, wherein the left side is 10 μ l spot size, the middle is 20 μ l spot size, the right side is 27 μ l spot size, and 1-6 are 20180310-1-1, 20180310-2-1, 20180310-3-1, 20180310-4-1, 20180310-5-1,20180316-6-1, respectively.
FIG. 5 is a thin layer chromatogram of a sample under different spreading distances, wherein the spreading distance on the left is 7.7cm, the middle is 8.3cm, the right is 9cm, and 1-6 are 20180310-1-1, 20180310-2-1, 20180310-3-1, 20180310-4-1, 20180310-5-1,20180316-6-1 of a flower of Coptis chinensis Franch in sequence.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments, but the scope of the present invention is not limited thereto.
A. The instrument comprises the following steps: BJ-150 type high-speed multifunctional pulverizer, No. 2 pharmacopoeia sieve, thin-layer glass plate (100mX200m), HS-3120 ultrasonic cleaner, FA2004 analysis electronic balance (Shanghai Liangping Instrument and meters Co., Ltd.), 101-1-S type electrothermal blowing drying oven (Chengdou Yayuan science and technology Co., Ltd.), UV-8 three-purpose ultraviolet analyzer (Wuxi Kodao Instrument plant), silica gel G thin-layer plate (Qingdao Kaihua), silica gel H thin-layer plate (Qingdao Kaihua), micro-injector, conical flask, capillary glass tube (produced by Waixi medical university Instrument plant) with inner diameter of 0.5mm and length of 100mm, 20cm × 10cm chromatography cylinder (vertical type), thermometer, funnel and filter paper.
B. Reagent: anhydrous methanol, petroleum ether (60-90 ℃), chloroform, ammonia water, sodium carboxymethylcellulose (experimental reagent, Chengduo chemical reagent field), berberine hydrochloride (Sichuan Vickqi Biotech Co., Ltd.), and other reagents are analytically pure.
C. Sample Source number
Example 1
(1) Preparation of a test solution: drying the coptidis flos to be identified at a constant temperature of 60 ℃, crushing, putting 0.25g of the coptidis flos powder into a conical flask, adding 25ml of anhydrous methanol, carrying out ultrasonic treatment for 30min, filtering, and taking a subsequent filtrate as a test solution;
(2) preparation of control solutions: taking berberine hydrochloride, and preparing into 800ug/ml solution as reference solution;
(3) thin layer chromatography conditions, development and inspection
Thin-layer plate: silica gel G thin layer plate;
developing agent: petroleum ether (60-90 ℃) -trichloromethane-methanol (5:3: 2);
sample amount of spotting: 10 mu l of the mixture;
the unfolding distance is as follows: 9.0 cm;
development temperature: 29 ℃;
the unfolding mode is as follows: a double-groove unfolding cylinder in a vertical mode (20cm multiplied by 10 mm); carrying out upward development, namely presaturating with ammonia water for 30min, discarding the ammonia water, developing, taking out and airing;
and (6) inspection: viewing under 365nm ultraviolet lamp;
as a result: in the chromatogram of the test solution, spots of the same color appear at positions corresponding to Rf values of 0.91 to 0.95, and spots of the same color appear at positions corresponding to Rf values of 0.30 to 0.37 and 0.72 to 0.82, as compared with the chromatogram of the control solution.
Example 2 screening of thin layer conditions
The sample solution used in this example was the same as in example 1.
(1) Investigation of different thin layer panels
Respectively absorbing the sample solution to be tested, spotting the sample solution on a silica gel H thin-layer plate and a silica gel G thin-layer plate, developing according to the thin-layer chromatography conditions and the development mode of the embodiment 1, and observing under a 365nm ultraviolet lamp, wherein the thin-layer chromatogram is shown in figure 2, and the result shows that the development effect of the sample on the silica gel H thin-layer plate is poor, the spot separation effect is poor, and in comparison, the development effect on the silica gel G thin-layer plate is more ideal, and the spots are clearer.
(2) Unfolding temperature investigation
The sample solution was spotted on two identical silica gel G thin layer plates (same manufacturer and production lot) respectively, developed according to the thin layer chromatography conditions and development method of example 1, the development temperature was set to 6 ℃ and 29 ℃ respectively, and examined under a 365nm ultraviolet lamp, and the thin layer chromatogram was as shown in FIG. 3, and the results showed that the spots were more rounded and clear at the development temperature of 29 ℃.
(3) Examination of sample application amount
The sample solution is sucked and respectively spotted on three same silica gel G thin-layer plates (same manufacturer and production batch), the three same silica gel G thin-layer plates are developed according to the thin-layer chromatography condition and the development mode of the embodiment 1, only the spotting amount is respectively set to be 10 mu l, 20 mu l and 27 mu l, the spotting amount is detected under a 365nm ultraviolet lamp, the thin-layer chromatogram is shown in figure 4, and the result shows that when the spotting amount is 10 mu l, the spots are clear and do not trail, and the more the spotting amount is, the more the trail is obvious.
(4) Spread distance survey
The sample solution is sucked and respectively spotted on three same silica gel G thin-layer plates (same manufacturers and production batches), the three same silica gel G thin-layer plates are unfolded according to the thin-layer chromatography conditions and the unfolding mode of the embodiment 1, only the unfolding distances are respectively set to be 7.7cm, 8.3cm and 9.0cm, the thin-layer chromatograms are viewed under a 365nm ultraviolet lamp, as shown in figure 5, the result shows that 4 spots appear when the unfolding distance is about 7cm, spots with unstable components are not obvious along with the increase of the unfolding distance, and three spots are clearer when the unfolding distance is 9 cm.
Claims (2)
1. The thin-layer identification method of the coptidis flos is characterized by comprising the following steps:
(1) taking the coptidis flos to be identified, drying and crushing, taking 0.25g of the coptidis flos powder in a conical flask, adding 25ml of anhydrous methanol, carrying out ultrasonic treatment for 30min, filtering, and taking a subsequent filtrate as a test solution;
(2) taking berberine hydrochloride, preparing solution with concentration of 800 mug/mL as reference solution;
(3) sucking the test solution and the reference solution, respectively dropping on the same silica gel G thin layer plate, developing with 60-90 deg.C petroleum ether-chloroform-methanol as developing agent at volume ratio of 5:3:2, taking out, air drying, placing under 365nm ultraviolet lamp for inspection, wherein in the chromatogram of the test solution, spots of the same color appear at corresponding positions with Rf value of 0.93 + -0.02, and spots appear at positions with Rf values of 0.35 + -0.05 and 0.77 + -0.05 compared with the chromatogram of the reference solution.
2. The thin-layer identification method of coptidis flos according to claim 1, characterized in that: in the step (3), the development temperature is 29 ℃, the development distance is 9cm, and the sample amount is 10 mu l.
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| JP2001302525A (en) * | 2000-02-16 | 2001-10-31 | Shiseido Co Ltd | External preparation for skin care |
| KR100353262B1 (en) * | 2000-03-15 | 2002-09-18 | 주식회사 한국야쿠르트 | Perilla frutescence extract Method which effects to prevention and treatment of a stomach ulcer and its use and process of obtaining for berberine therefrom |
| EP1454993A1 (en) * | 2003-03-05 | 2004-09-08 | Max-Planck-Gesellschaft Zur Förderung Der Wissenschaften E.V. | Method for identifying the function of a gene |
| CN1733105B (en) * | 2005-08-18 | 2011-04-20 | 贵阳云岩西创药物科技开发有限公司 | Detection method of Golden Preparation for treating gynecological disease |
| CN101928285A (en) * | 2010-08-05 | 2010-12-29 | 重庆市中药研究院 | Method for extracting major alkaloids from rhizoma coptidis |
| CN104478878B (en) * | 2014-11-08 | 2017-05-31 | 吕梁学院 | A kind of method that separation from green peel of walnut prepares alkaloid |
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