CN104459012A - Thin-layer identification method of effective part of flavones of fruit of rosa davurica pall - Google Patents
Thin-layer identification method of effective part of flavones of fruit of rosa davurica pall Download PDFInfo
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- CN104459012A CN104459012A CN201410823341.9A CN201410823341A CN104459012A CN 104459012 A CN104459012 A CN 104459012A CN 201410823341 A CN201410823341 A CN 201410823341A CN 104459012 A CN104459012 A CN 104459012A
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Abstract
The invention discloses a thin-layer identification method of an effective part of flavones of fruit of rosa davurica pall, belongs to the technical field of traditional Chinese medicine identification, and relates to a thin-layer identification method and the optimal thin-layer identification condition of main components in the effective part of a traditional Chinese medicine. The thin-layer identification method comprises the following steps of carrying out sample application on a test solution of the effective part of flavones of the fruit of rosa davurica pall and a reference solution of rutin, hyperin or quercetin on chromatography silica gel (G)-thin plate (aluminum foil sheet base) by a microsyringe; identifying rutin and hyperin thin layers by adopting an acetic ether-formic acid-water developing system; and identifying a quercetin thin layer by adopting a benzene-acetic ether-formic acid developing system, and qualitatively judging the types of main components contained in the test solution according to the positions and colors of spots of the test solution and the reference solution by adopting AlCl3 ethanol solution as a developing agent, so that a thin-layer identification method which is relatively high in specificity, and is convenient and simple to operate is provided for the quality control of the effective part of the flavones of the fruit of rosa davurica pall.
Description
Technical field
The present invention relates to the thin-layer identification method of rose hip flavone effective part, belong to TCD identificafion technical field.
Background technology
Rosa bella (
rosa davurica Pall.) be rose family Rosa defoliation bush plant, its fruit is rose hip.Modern medicine study finds, rose hip has anti-ageing, antifatigue, resist oxygen lack, and strengthen immune function of human body effect, long-term taking can significantly improve SOD content in human body.Rose hip is the precious resources of integration of drinking and medicinal herbs, be rich in organic acid, the various active compositions such as flavones, cumarin, sterol, triterpene, alkene, saponin(e, volatile oil, mineral matter, trace element, flavone compound wherein have anti-oxidant, reduce peroxidatic reaction of lipid, reduce myocardial consumption of oxygen, treatment of vascular sclerosis, step-down, the effect such as anti-ageing, Green Tea Extract and anticancer, anti-cancer.It is a kind of rare green resource having Development volue and potentiality.
At present, many researchists expand fundamental research to rose hip, the extraction of its active component, purifying process and biologically active are studied, Zhong Fangli etc. are studied under the TLC distinguish condition of rose hip crude extract in the rose sheet of mountain, compare the expansion effect of benzene-ethyl acetate-glacial acetic acid (5:2:0.1) and chloroform-methanol-water (28:10:1) two systems, result determination benzene-ethyl acetate-glacial acetic acid system slightly well.The people such as Guo Shuhua have also carried out rose hip composition and have differentiated and quantitative test, but the TLC distinguish research of relevant rose hip crude extract is deep.
Active component refers to that the content when the class in a herb or compound Chinese medicine extract or a few class chemical composition reaches more than 50% of total extract, and this class or the known chemical composition of a few class are considered to effective constituent, namely the mixture of this class or a few constituents is considered to active component.Compared with traditional Chinese medicine preparation, the drug substance of active component Chinese medicine is basic or drug effect is basic obtains clearly substantially, and drug effect increases than traditional Chinese medicine, determined curative effect, and take safe and reliable, toxic and side effect is low.At present, the research and development of active component Chinese medicine day by day obtain the attention of people.
At present, Zhong Fangli etc. have been studied the extraction of rose hip general flavone, purification process and medical usage and have applied for patent (patent name: rose hip extractive of general flavone and extracting method thereof and its medical usage, the patent No.: ZL201110091577.4).In order in the comprehensive utilization of rose hip active component and exploitation, its quality can be controlled preferably, the thin-layer identification method of rose hip flavone effective part is studied.TLC distinguish has easy and simple to handle, rapid, and specificity is comparatively strong, has no the research report that relevant thin-layer method differentiates rose hip flavone effective part at present.
Summary of the invention
An object of the present invention be to provide a kind of simply, the thin-layer identification method of rose hip flavone effective part easily and efficiently.
Two of object of the present invention is to provide the thin-layer identification method can differentiating three kinds of major components in rose hip flavone effective part.
Three of object of the present invention there is provided the top condition can differentiating three kinds of major components in rose hip flavone effective part.
The concrete research contents of TLC distinguish of rose hip flavone effective part is as follows:
1. the preparation of rose hip flavone effective part need testing solution
Precision take be dried to constant weight rose hip flavone effective part medicinal extract in right amount in volumetric flask, add ethanol dissolve, constant volume is for subsequent use.
2. the preparation of reference substance solution
It is in right amount each that precision takes the rutin, Quercetin, the Hyperoside reference substance that are dried to constant weight, is placed in volumetric flask respectively, dissolves constant volume with methyl alcohol, for subsequent use.
3. the preparation of thin layer plate
Take a certain amount of silica G, add distilled water in right amount, after swelling a period of time, in mortar after grinding, its uniform spreading is being cleaned up in advance and on dry glass plate, naturally drying, before using, put 105 DEG C of activation 30min-1h.
4. remove the preparation of the negative controls of general flavone
Take rose hip flavone effective part extract appropriate, use water-soluble solution, the solution obtained crosses D-101 resin column, efflux repeats upper prop, to efflux without flavones reaction (doing color discrimination test to not reacting by hydrochloric acid magnesium powder method), collecting efflux, water-bath volatilizing and dissolves with appropriate 60% ethanol, the negative controls of general flavone must be removed, for subsequent use.
5. the configuration of developping agent
Configure developping agent 1 respectively: ethyl acetate-formic acid-water (8:1:1); Developping agent 2: ethyl acetate-formic acid-water (9:1:1); Developping agent 3: ethyl acetate-formic acid-water (7:1:1); Developping agent 4: ethyl acetate-formic acid-water (10:1:1); Developping agent 5: ethyl acetate-formic acid-water (12:1:1); Developping agent 6: ethyl acetate-formic acid-water (20:1:1); Developping agent 7: methyl alcohol-glacial acetic acid-water (4:1:5); Developping agent 8: benzene-ethyl acetate-formic acid (5:4:1); Developping agent 9: benzene-ethyl acetate-formic acid (6:4:1); Developping agent 10: chloroform-methanol-water (28:10:1), and development system is compared.
6. the configuration of developer
Developer 1:FeCl
3the configuration of solution: take FeCl
3.6H
2o is appropriate, dissolves with distilled water, and in volumetric flask constant volume, for subsequent use.
Developer 2:AlCl
3the configuration of ethanolic solution: take anhydrous AlCl
3in right amount, be slowly repeatedly dissolved in absolute ethyl alcohol on a small quantity, and with absolute ethyl alcohol constant volume in volumetric flask, for subsequent use.
7. thin-layer chromatography
(1) before point sample point sample, removed by the adsorbent apart from plate 1-2mm, expose glass plate, to prevent edge effect, two edge points are 1.5cm apart from two panel edges, and use micro syringe point sample, spot diameter is 3-5mm.
(2) launch that the excellent thin layer plate of point is put into configured in advance to get well and the chromatography cylinder reached capacity, the degree of depth immersing developping agent is apart from initial point 2-2.5mm, when to be deployed dose edge to top is 1-1.5cm onboard, is taken out by plate, naturally dries.
(3) thin layer plate dried evenly is sprayed upper developer by colour developing, as 105 DEG C of baking ovens or with heat gun colour developing, observes and calculates R after colour developing in dark box type uv analyzer (365nm)
fvalue.
8. the optimization of rose hip flavone effective part TLC distinguish condition
(1) the selection rose hip extractive of general flavone of developping agent is after macroporous resin purification, and its general flavone content >=50%, its principal ingredient is respectively rutin, Hyperoside and Quercetin.Hyperoside, has another name called Quercetin-3-O-
β-D-galactopyranoside.Belong to flavonol glycosides compounds.Aglycon is Quercetin, and glycosyl is galactopyranose.Rutin, also known as rutin sophorin, also flavonol glycosides compounds is belonged to, aglycon is Quercetin, glycosyl is 1 molecule glucose and 1 molecule rhamnose, the polarity of Hyperoside and rutin is close, and Quercetin polarity compared with both is slightly little, and when developping agent is selected, the molecular structure of three and polarity all produce larger impact to the selection of developping agent system.Evaluation, selection and optimization have been carried out to the expansion effect of 10 kinds of developping agents.As accompanying drawing 1, accompanying drawing 2, accompanying drawing 3.
(2) selection evaluation self-control silica G plate (glass chip), self-control silica gel H plate (glass chip) of silica gel plate,--thin plate (aluminium foil base), purchase chromatographic silica gel (G)--rose hip flavone effective part thin-layer chromatography effect of thin plate (glass chip) of purchasing chromatographic silica gel (G).As accompanying drawing 4, accompanying drawing 5.
(3) selection of developer
Use 5%AlCl respectively
3and 9%FeCl
3solution chromogenic reagent, contrast color developing effect, determines best developer.
(4) selection of test sample TLC distinguish concentration
Respectively test sample original liquid concentration, stoste are diluted to 2 times, 5 times, 10 times and carried out thin-layer chromatography discriminating, determine the test sample TLC distinguish concentration be comparatively suitable for.
(5) selection of point sample volume
Draw 2 μ L, 3 μ L, 5 μ L test samples and reference substance point sample on silica gel plate with micro syringe respectively, contrast thin-layer chromatography effect, determines best point sample volume.
(6) best thin-layer chromatography condition is determined
According to the optimum results of above each factor, determine the TLC distinguish condition of the best of dahurian rosa leaf fruit flavone effective part, as accompanying drawing 6, accompanying drawing 7.
Accompanying drawing illustrates:
Accompanying drawing 1 is for developping agent with ethyl acetate-formic acid-water (8:1:1).
Accompanying drawing 2 is for developping agent with methyl alcohol-glacial acetic acid-water (4:1:5).
Accompanying drawing 3 is rutins, Hyperoside reference substance launches in ethyl acetate formic acid water 8:1:1, and Quercetin reference substance launches in benzene-acetic acid formic acid 6:4:1.
Accompanying drawing 4 uses self-control silica G plate (glass chip) thin-layer chromatography effect.
Accompanying drawing 5 uses commercially available chromatographic silica gel (G)--thin plate (aluminium foil base) thin-layer chromatography effect.
Accompanying drawing 6 is thin-layer chromatography effects under top condition.
Accompanying drawing 7 is thin-layer chromatography effects under top condition.
The invention has the beneficial effects as follows:
(1) at present there are no the thin-layer identification method closing rose hip flavone effective part, the invention provides a kind of thin-layer identification method of rose hip flavone effective part, for the quality monitoring in its application development process provides simple and efficient method;
(2) the invention provides a kind of thin-layer identification method differentiating three kinds of major components in rose hip flavone effective part, make its TLC distinguish more accurately, selectivity is stronger;
(3) the invention provides a kind of best TLC distinguish condition differentiating three kinds of major components in rose hip flavone effective part.
embodiment
embodiment 1
1. the preparation of rose hip flavone effective part need testing solution
Precision take be dried to constant weight rose hip medicinal extract 0.1607g in 25mL volumetric flask, add 60% ethanol dissolve, constant volume; This solution is filtered, gets subsequent filtrate 5mL in 10mL volumetric flask, add 60% ethanol constant volume; Get subsequent filtrate 2mL in 10mL volumetric flask, add 60% ethanol constant volume, get subsequent filtrate 1mL and in 10mL volumetric flask, add 60% ethanol, constant volume, for subsequent use.
2. the preparation of reference substance solution
Precision takes the control substance of Rutin 5.02mg being dried to constant weight, Quercetin reference substance 5.00mg, and Hyperoside reference substance 5.05mg, is placed in 10mL volumetric flask respectively, dissolves constant volume with methyl alcohol, for subsequent use.
3. remove the preparation of the negative controls of general flavone
Take rose hip flavone effective part extract appropriate, use water-soluble solution, the solution obtained crosses D-101 resin column, efflux repeats upper prop, to efflux without flavones reaction (doing color discrimination test to not reacting by hydrochloric acid magnesium powder method), collecting efflux, water-bath volatilizing and dissolves with appropriate 60% ethanol, the negative controls of general flavone must be removed, for subsequent use.
4. the configuration of developping agent
The best developping agent of configuration rutin and Hyperoside: ethyl acetate-formic acid-water (8:1:1); The best developping agent of configuration Quercetin: benzene-ethyl acetate-formic acid (6:4:1).
5. the configuration of developer
Configure better developer AlCl
3ethanolic solution: take anhydrous AlCl
34.1705g, is slowly repeatedly dissolved in absolute ethyl alcohol on a small quantity, and with absolute ethyl alcohol constant volume in 100mL volumetric flask and get final product, for subsequent use.
6. thin-layer chromatography
(1) point sample
Select chromatographic silica gel (G
)-thin plate (aluminium foil base), uses micro syringe point sample, and spot diameter is 3-5mm, and the point sample amount of need testing solution is 5 μ L; The point sample amount of Hyperoside reference substance solution is 3 μ L, and the point sample amount of control substance of Rutin solution is 5 μ L; When the point sample amount of Quercetin reference substance solution is 2 μ L, fluorescent effect is obvious and clear.
(2) launch
The excellent thin layer plate of point is put into configured in advance get well and the chromatography cylinder reached capacity, the degree of depth immersing developping agent is apart from initial point 2-2.5mm, when to be deployed dose edge to top is 1-1.5cm onboard, is taken out by plate, naturally dries.
(3) develop the color
The thin layer plate dried evenly is sprayed upper AlCl
3ethanolic solution developer, as 105 DEG C of baking ovens or with heat gun colour developing, observes and calculates R after colour developing in dark box type uv analyzer (365nm)
fvalue, is shown in accompanying drawing 6, accompanying drawing 7.
Claims (7)
1. a thin-layer identification method for rose hip flavone effective part, is characterized in that comprising the steps:
(1) rose hip flavone effective part need testing solution is prepared;
(2) rutin of variable concentrations, Quercetin, Hyperoside reference substance solution is prepared;
(3) negative controls of general flavone is removed in preparation;
(4) get rose hip flavone effective part need testing solution, rutin, Quercetin, Hyperoside reference substance solution, remove the negative controls of general flavone and do TLC distinguish analysis, be primarily characterized in that need testing solution and the spot of reference substance solution at identical position display same color, the non-display dot of negative controls, then show containing this composition in need testing solution, and there is not the interference of other factors.
2. the thin-layer identification method of rose hip flavone effective part according to claim 1, is characterized in that, can differentiate the 1-2 kind principal ingredient in rose hip flavone effective part simultaneously.
3. the thin-layer identification method of rose hip flavone effective part according to claim 1, it is characterized in that, its concrete operation step is: get need testing solution, rutin, Hyperoside or Quercetin reference substance solution, with micro syringe in the upper point sample of chromatographic silica gel (G)-thin plate (aluminium foil base), after launching with developping agent, dry, colour developing, dry, need testing solution and the spot of reference substance solution at identical position display same color.
4. the thin-layer identification method of the rose hip flavone effective part described in claim 3, is characterized in that, developping agent system used is respectively: rutin and Hyperoside TLC distinguish adopt ethyl acetate-formic acid-water to launch system; Quercetin TLC distinguish adopts benzene-ethyl acetate-formic acid to launch system.
5. the thin-layer identification method of the rose hip flavone effective part described in claim 3, is characterized in that, the concrete volume ratio of developping agent system used is: ethyl acetate-formic acid-water=8:1:1; Benzene-ethyl acetate-formic acid=6:4:1.
6. the thin-layer identification method of the rose hip flavone effective part described in claim 3, is characterized in that, developer used is AlCl
3ethanolic solution.
7. the thin-layer identification method of the rose hip flavone effective part described in claim 3, is characterized in that, developer used is AlCl
3ethanol solution concentration is about 0.04g/mL.
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Cited By (1)
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| CN114252549A (en) * | 2020-09-25 | 2022-03-29 | 广州白云山中一药业有限公司 | Identification method of salt-steamed dodder |
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| CN114252549A (en) * | 2020-09-25 | 2022-03-29 | 广州白云山中一药业有限公司 | Identification method of salt-steamed dodder |
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Application publication date: 20150325 |