CN101339174B - Method for determining carthamus tinctorius yellow color content - Google Patents
Method for determining carthamus tinctorius yellow color content Download PDFInfo
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Abstract
The invention relates to a method for measuring the content of safflower yellow pigment; the method comprises the following detailed steps: by the steps of identifying the safflower yellow pigment of a safflower yellow pigment product, detecting the content of total flavone and detecting the content of hydroxysafflor yellow A, the safflower yellow pigment of effective parts of the safflower can be evaluated effectively, the mass of intermediate of the safflower yellow pigment or pharmaceutical preparations of the safflower yellow pigment can be controlled, and the mass of the pharmaceutical preparations of the safflower yellow pigment can be further effectively controlled, thus ensuring the safe, effective and controllable preparation. The method of the invention also has the advantages of ease, shortcut and effectiveness and high accuracy and specificity.
Description
Technical field
The present invention relates to a kind of carthamin yellow detection method, specifically, relate to a kind of method that detects carthamin yellow and content.Belong to drug world.
Background technology
Safflower belongs to the dried floral of feverfew safflower Carthamus tinctorius L., has the effect that activates blood circulation and disperses blood clots, stimulates the menstrual flow.Safflower is world today research and uses one of " focus " plant, to its The Chemical Constituents, and existing lot of documents report, carthamin yellow proves the main pharmacodynamics position of safflower through pharmacological research, and wherein main active is a carthamin yellow.
The bibliographical information of the existing a large amount of carthamin yellows of prior art or carthamin yellow active component and preparation thereof.About the assay method of carthamin yellow or carthamin yellow, some reports are arranged also.In order to control carthamin yellow or carthamin yellow pharmaceutical preparation quality effectively, guarantee raw material or intermediate quality, a kind of convenient, carthamin yellow product quality product valency method, particularly detection method effectively need be provided, thereby can guarantee that the medicine preparation quality effectively and controlled.
Summary of the invention
For this reason, fundamental purpose of the present invention provide a kind of effective, controlled, detect the method for carthamin yellow easily, and then can assess carthamin yellow product quality and quality effectively.
For achieving the above object, technical solution of the present invention is as follows:
The invention provides a kind of method that detects carthamin yellow, it comprises the steps:
(1) detection of carthamin yellow is with carthamin yellow in the test sample of thin-layered chromatography or ultraviolet spectrophotometry detection carthamin yellow;
(2) content of total flavone is measured, and uses ultraviolet spectrophotometry, is reference substance with the hydroxyl radical carthamin yellow carthamus A, and the mensuration wavelength is 360nm, general flavone content in the test sample of mensuration carthamin yellow;
(3) assay of hydroxyl radical carthamin yellow carthamus A, use high performance liquid chromatography, with the hydroxyl radical carthamin yellow carthamus A is reference substance, with octadecylsilane chemically bonded silica is stationary phase, mixed solvent with methyl alcohol, acetonitrile, water and phosphoric acid is a moving phase, the mensuration wavelength is 360nm, and theoretical cam curve is calculated by the hydroxyl radical carthamin yellow carthamus A peak should be not less than 3000, measures the content of hydroxyl radical carthamin yellow carthamus A in the carthamin yellow test sample.
Carthamin yellow of the present invention all is the general flavone that extraction from safflower, separation, purifying obtain, and can obtain carthamin yellow by the extraction from the safflower crude drug of prior art known method, separation, purifying.For example carthamin yellow that extraction in the disclosed safflower, separation, purifying obtain in the patented claims such as CN1600817A, CN1935176A, CN101007828A or carthamin yellow or safflower extract product etc.The aforementioned patent applications disclosure is introduced the application as a reference.
The above-mentioned described method of the present invention, wherein the detection of step (1) carthamin yellow can detect by thin-layered chromatography.For example can carthamin yellow product in contrast, adopt methods such as polyamide thin-layer chromatography, silica gel thin-layer chromatography to detect carthamin yellow in the test samples.Also can adopt ultraviolet spectrophotometry, test sample is measured its maximum absorption wavelength, the carthamin yellow in the working sample in certain wavelength coverage.
Wherein, as preferred embodiment, the present invention adopts thin-layered chromatography to detect carthamin yellow in the test sample of carthamin yellow.And the described thin-layer chromatography of preferred steps (1), be to be adsorbent with the polyamide, the mixed solvent of water, ethanol, formic acid and diacetone is a developping agent, is reference substance with the hydroxyl radical carthamin yellow carthamus A, after the Development of Thin-Layer Chromatography, test sample still manifests identical point with the corresponding position of reference substance chromatogram.
Wherein, be more preferably the mixed solvent that described developping agent is water, ethanol, formic acid and diacetone, compatibility by volume, water: ethanol: formic acid: diacetone is 5: 1.5: 1: 0.5 mixed solvent.
As another embodiment of the present invention, wherein step (1) ultraviolet spectrophotometry detects carthamin yellow in the test sample of carthamin yellow, and there is maximum absorption band at the place at 221～227nm wavelength.
Wherein, the above-mentioned described detection method of the present invention, the moving phase described in the step (3) wherein, preferred compatibility by volume, methyl alcohol: acetonitrile: water: phosphoric acid is 32: 3: 64.5: 0.5 mixed solvent.
Another purpose of the present invention provides a kind of method that detects the injection carthamin yellow, and wherein said carthamin yellow is the general flavone that extraction separates from safflower, purifying obtains, and it comprises the steps:
(1) detection of carthamin yellow detects carthamin yellow in the injection carthamin yellow with thin-layered chromatography or ultraviolet spectrophotometry;
(2) content of total flavone is measured, and uses ultraviolet spectrophotometry, is reference substance with the hydroxyl radical carthamin yellow carthamus A, and the detection wavelength is 360nm, determines the general flavone content in the injection carthamin yellow;
(3) assay of hydroxyl radical carthamin yellow carthamus A, use high performance liquid chromatography, with the hydroxyl radical carthamin yellow carthamus A is reference substance, with octadecylsilane chemically bonded silica is stationary phase, use methyl alcohol: acetonitrile: water: the volume ratio of phosphoric acid is 32: 3: 64.5: 0.5 mixed solvent is a moving phase, the detection wavelength is 360nm, and theoretical cam curve is calculated by the hydroxyl radical carthamin yellow carthamus A peak should be not less than 3000, determines the hydroxyl radical carthamin yellow carthamus A content in the injection carthamin yellow.
Wherein, above-mentioned described method, preferred steps (1) adopts the carthamin yellow in the thin-layered chromatography detection injection carthamin yellow, thin-layer chromatography is to be adsorbent with the polyamide, water: ethanol: formic acid: the volume ratio of diacetone is 5: 1.5: 1: 0.5 mixed solvent is a developping agent, and hydroxyl radical carthamin yellow carthamus A is a reference substance.
Above-mentioned described method, wherein step (1) adopts the carthamin yellow in the ultraviolet spectrophotometry detection injection carthamin yellow, and there is maximum absorption band at the place at 221～227nm wavelength.
The detection method of above-mentioned described injection carthamin yellow, it detects the carthamin yellow freeze drying powder injection, specification is every bottle of 50mg/, and wherein general flavone content must not be less than 38.25mg in the step (2), and hydroxyl radical carthamin yellow carthamus A content should be 31.5～38.5mg in the step (3).
Injection carthamin yellow of the present invention also is the higher general flavone of purity that extraction from safflower, separation, purifying obtain, and can obtain carthamin yellow by the extraction from the safflower crude drug of prior art known method, separation, purifying.For example carthamin yellow that extraction in the disclosed safflower, separation, purifying obtain in the patented claims such as CN1600817A, CN1935176A, CN101007828A or carthamin yellow or safflower extract product etc.The aforementioned patent applications disclosure is introduced the application as a reference.
Injection carthamin yellow for example of the present invention, can prepare by the following method: take by weighing safflower, the deionized water that adds 10-13 times of medicinal material weight, extracted 20-25 minute in 100 ℃, filter, the deionized water that filter residue adds 10 times of medicinal material weight repeats to extract once by above-mentioned condition again, filters.Merge secondary raffinate, be cooled to room temperature, after the usefulness hydro-extractor was centrifugal, extracting centrifugal liquid was standby.
Above-mentioned centrifugate slowly is added to handled in the good HZ801 macroporous adsorptive resins of balance, the post blade diameter length ratio is 1: 12, and last sample flow velocity is per minute 10ml.Behind the end of the sample, use the flow velocity wash-out of the deionized water of normal temperature with per minute 20ml.The eluent of carthamin yellow is rich in collection, and eluent gets carthamin yellow crude product concentrate in 80～90 ℃ of concentrating under reduced pressure.
Gel LH-20 post on the carthamin yellow crude product concentrate, the blade diameter length ratio of post is 1: 5, and applied sample amount is 10% of a bed volume, and water for injection wash-out, flow velocity are per minute 5ml, collect to contain the carthamin yellow part.Collect liquid behind 80～90 ℃ of concentrating under reduced pressure, get carthamin yellow elaboration concentrate,, get injection carthamin yellow (perhaps being called the carthamin yellow freeze-dried powder) through freeze drying.
As preferably, injection carthamin yellow of the present invention, wherein general flavone content is not less than 75%, and hydroxyl radical carthamin yellow carthamus A content is 60%～80%.
As preferably, injection carthamin yellow of the present invention is the carthamin yellow freeze-dried powder, and the preparation specification is the 50mg/ bottle, and wherein general flavone content must not be less than 38.25mg, and carthamus tinctorius yellow color content should be 31.5～38.5mg.
The present invention finds, by step to carthamin yellow discriminating in the carthamin yellow product, general flavone content detection and hydroxyl radical carthamin yellow carthamus A content detection, can estimate the active component carthamin yellow of safflower effectively, can control the intermediate quality of carthamin yellow or carthamin yellow pharmaceutical formulation, and then can control carthamin yellow medicine preparation quality effectively, guarantee preparation safety, effective and controlled.Particularly by adopting specific mixed solvent proportioning, this simple, fast method of utilization thin-layered chromatography can identify carthamin yellow in the carthamin yellow product very effectively, and specificity and accuracy height are obtained amazing invention effect.The inventive method not only can be assessed the quality and the quality of carthamin yellow and pharmaceutical preparation thereof effectively, be the Quality Control effective measures, and method is easy, quick, effective, the advantage of accuracy and high-specificity.
Embodiment
Following examples are in order further to explain and explanation content of the present invention, should not to be understood that purport of the present invention is limited or limits.
Embodiment 1: the carthamin yellow test sample detects
Test sample: injection carthamin yellow (specification: 50mg, manufacturer: Zhejiang Yongning Pharmaceutical Co., Ltd, lot number: 080303)
Step 1: carthamin yellow detects:
Adopt the polyamide thin layer plate, reference substance is a hydroxyl radical carthamin yellow carthamus A, and developping agent is water-ethanol-formic acid-diacetone (5: 1.5: 1: 0.5).It is an amount of to get test sample, adds water and makes the solution that contains 2mg among every 1ml, as test liquid.Other gets reference substance and adds water and make the solution liquid in contrast that contains 2mg among every 1ml.Get the polyamide thin layer plate, specification 10 * 20cm draws each 2 μ l of above-mentioned two kinds of solution, puts respectively on same thin layer plate, takes out, dries after launching with developping agent.Showing a spot with the reference substance same position in the test sample as a result.Development system is better, the R of hydroxyl radical carthamin yellow carthamus A
fBe about 0.7, degree of separation is better.
Step 2: determination of total flavonoids
The preparation of reference substance solution: precision takes by weighing through the hydroxyl radical carthamin yellow carthamus A reference substance of phosphorus pentoxide vacuum drying after 24 hours an amount of, adds water and makes solution that every 1ml contains 45 μ g product solution in contrast.
The preparation of typical curve: precision is measured above-mentioned reference substance solution 0,1.0,2.0,4.0,6.0,8.0ml places the 10ml volumetric flask respectively, adds water to scale and shakes up, and does blank simultaneously with water.Adopting day island proper Tianjin UV-260 ultraviolet scene photometer to measure, measure absorbance log at the wavelength place of 360nm, is ordinate with the absorbance log, and concentration is horizontal ordinate, makes typical curve.
Measure: get this product 25mg, accurately claim surely, place the 100ml measuring bottle, be dissolved in water and be diluted to scale.Precision is measured 2ml, places the 25ml measuring bottle, and thin up shakes up to scale, adopts day island proper Tianjin UV-260 ultraviolet scene photometer to measure, and measures absorbance log at the wavelength place of 360nm, reads general flavone amount the need testing solution from typical curve.
The result records that general flavone content is 43.66mg in the test sample.
Step 3: hydroxyl radical carthamin yellow carthamus A assay
The reference substance solution preparation: precision is measured through the hydroxyl radical carthamin yellow carthamus A reference substance of phosphorus pentoxide vacuum drying after 24 hours an amount of, adds water and makes the solution that every 1ml contains 0.20mg, shakes up promptly.
Need testing solution preparation: get this product 25mg, accurately claim surely, place the 100ml measuring bottle, be dissolved in water and be diluted to scale, shake up promptly.
Chromatographic condition: adopt high performance liquid chromatography, with octadecylsilane chemically bonded silica is stationary phase, use methyl alcohol: acetonitrile: water: the volume ratio of phosphoric acid is 32: 3: 64.5: 0.5 mixed solvent is a moving phase, the detection wavelength is 360nm, and theoretical cam curve is calculated by the hydroxyl radical carthamin yellow carthamus A peak should be not less than 3000.
Measure: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject liquid chromatograph, measure peak area, calculate promptly.
It is 36.7mg by dry product calculating hydroxyl carthamin yellow A-containing that the result records this product.
Embodiment 2: thin-layer chromatography detects carthamin yellow
Test sample: self-control injection carthamin yellow
Reference substance: hydroxyl radical carthamin yellow carthamus A
Thin layer version: polyamide thin layer plate
Developping agent:
1, methanol-water (8: 2)
2, water-ethanol-diacetone (4: 2: 1.5)
3, alcohol-water (3: 2)
4, alcohol-water-butanone-diacetone is (3: 13: 3: 1)
5, benzene-methyl alcohol-butanone (6: 2: 2)
6, water-ethanol-formic acid-diacetone is (5: 1.5: 1: 0.5)
It is an amount of to get test sample, adds water and makes the solution that contains 2mg among every 1ml, as test liquid.Other gets reference substance and adds water and make the solution liquid in contrast that contains 2mg among every 1ml.Get the polyamide thin layer plate, specification 10 * 20cm draws each 2 μ l of above-mentioned two kinds of solution, puts respectively on same thin layer plate, takes out, dries after launching with developping agent.Result such as following table 1.
Table 1: carthamin yellow thin layer result
| The developping agent sequence number | The result |
| 1 | Two spots of forward position and initial point |
| 2 | Banded no point |
| 3 | Initial point does not launch |
| 4 | Initial point |
| 5 | Initial point does not launch |
| 6 | 1 point, R fAbout 0.7 |
The result shows, developping agent 6 is carthamin yellow in the test sample effectively, and development system is better, the R of hydroxyl radical carthamin yellow carthamus A
fBe about 0.7, degree of separation is better.
The present invention is described according to preferred embodiment.Should be understood that the description of front and embodiment are just to illustrating the present invention.Under prerequisite without departing from the spirit and scope of the present invention, those skilled in the art can design multiple alternative of the present invention and improvement project, and it all should be understood to be within protection scope of the present invention.
Claims (4)
1. method that detects carthamin yellow, the general flavone that the extraction from safflower of wherein said carthamin yellow system, separation, purifying obtain, it comprises the steps:
(1) detection of carthamin yellow, with the carthamin yellow in thin-layered chromatography or the ultraviolet spectrophotometry detection carthamin yellow test sample, wherein, described thin-layer chromatography is to be adsorbent with the polyamide, water: ethanol: formic acid: the volume ratio of diacetone is 5: 1.5: 1: 0.5 mixed solvent is a developping agent, hydroxyl radical carthamin yellow carthamus A is a reference substance, and described ultraviolet spectrophotometry detects has maximum absorption band at 221～227nm wavelength place;
(2) measuring content of total flavone, use ultraviolet spectrophotometry, is reference substance with the hydroxyl radical carthamin yellow carthamus A, and the mensuration wavelength is 360nm, measures the general flavone content in the carthamin yellow test sample;
(3) content of mensuration hydroxyl radical carthamin yellow carthamus A, use high performance liquid chromatography, with the hydroxyl radical carthamin yellow carthamus A is reference substance, with octadecylsilane chemically bonded silica is stationary phase, use methyl alcohol: acetonitrile: water: the volume ratio of phosphoric acid is 32: 3: 64.5: 0.5 mixed solvent is a moving phase, the mensuration wavelength is 360nm, and theoretical cam curve is calculated by the hydroxyl radical carthamin yellow carthamus A peak should be not less than 3000, measures the content of hydroxyl radical carthamin yellow carthamus A in the carthamin yellow test sample.
2. method according to claim 1, wherein step (1) adopts the carthamin yellow in the thin-layered chromatography detection carthamin yellow test sample.
3. method according to claim 1, wherein step (1) adopts the carthamin yellow in the ultraviolet spectrophotometry detection carthamin yellow test sample.
4. according to each described method of claim 1-3, wherein general flavone content must not be less than 38.25mg in the step (2), and hydroxyl radical carthamin yellow carthamus A content should be 31.5～38.5mg in the step (3).
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| CN102692461B (en) * | 2012-05-22 | 2013-12-18 | 辽宁中医药大学 | Whole-time three-wavelength fusion method for simultaneously determining contents of four ingredients in Flos Carthami |
| CN102879389B (en) * | 2012-09-27 | 2015-01-07 | 山东阿如拉药物研究开发有限公司 | Liver tonifying, blood activating and eyesight improving pill of Tibetan medicine compound and quality detection method of preparation of pill |
| CN104198414A (en) * | 2014-09-05 | 2014-12-10 | 北京智云达科技有限公司 | Method for detecting artificially synthesized edible pigments |
| CN105115965A (en) * | 2015-07-27 | 2015-12-02 | 南昌大学 | Soft drink synthetic pigment fast detection method and kit |
| CN107607669B (en) * | 2017-08-30 | 2020-01-31 | 新疆奇沐医药研究院（有限公司） | Quality detection method for safflower and safflower formula granules |
| CN110376142B (en) * | 2019-08-20 | 2022-07-01 | 陕西中医药大学 | Detection method for quality grade of traditional Chinese medicine safflower |
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