CN104568928A - Method for screening antioxidant active component - Google Patents
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Abstract
The invention discloses a method for screening an antioxidant active component. The method comprises the following steps: a) applying the sample of a test sample solution on a thin-layer plate, evaporating a sample solvent for further use or further developing the sample solvent with an appropriate developing solvent for further use; b) dipping the obtained product in a linoleic acid solution, taking the product out from an evaporating solvent, and placing the obtained product under an ultraviolet lamp of 254nm or 366nm for illumination; c) dipping the obtained product in a developer solution, taking the product out and heating the product for color development; d) inspecting the obtained product, wherein a blue spot in a green background under the ultraviolet lamp of 366nm and a white spot in a pink background under visible light are the antioxidant active component. According to the method, complex instrument and equipment are eliminated, the antioxidant active component can be screened out quickly in a high throughput, the screening result is vivid, and easy to identify and remember, and the sensitivity and specificity are higher than those obtained by an ordinary screening method, so that a convenient and practical way is provided for screening the antioxidant active component.
Description
Technical field
The present invention relates to a kind of screening technique of oxidation-resistant active ingredient, specifically, relate to a kind of method utilizing TLC-bioautography technology screening to have the oxidation-resistant active ingredient of anti-linoleic acid peroxidation lipid reactant.
Background technology
Oxygen free radical reaction and peroxidatic reaction of lipid play an important role in the metabolic processes of body, both are in coordination and dynamic balance state under normal circumstances, maintain many biochemical reactions and immune response in body, once this coordination and mobile equilibrium produce disorderly and imbalance, not normal and the immunologic function of a series of metabolism will be caused to reduce, form oxygen radical chain reaction, infringement biological membrane and function thereof, so that form cell transparency pathology, fiberization, large area cellular damage causes neural further, tissue, organ equivalent damage, this reaction is just lipid peroxidation.Process (the PerOxide of the ROS oxidative biological film occurred in lipid peroxidation process, LPO), as MDA (Malonaldehyde, and 4-hydroxyl nonenoic acid (4-hydroxynonenal MDA), HNE), thus the mobility of cell membrane and permeability are changed, finally cause the change of eucaryotic cell structure and function.That studies free radical and natural along with people deepens continuously, and finds the growing interest that natural causes people.
Adopt linoleic acid for the antioxidant of screening and evaluation anti-peroxidation lipid reactant and its power in prior art more, antioxidant or measured object is added in the system that linoleic acid exists, make linoleic acid generation peroxidatic reaction of lipid through excess temperature bath, then detect linoleic Peroxidation Product (measuring the generation of MDA or 4-hydroxyl nonenoic acid) by ultraviolet spectrophotometry.The method consumes time is long, during screening complex component, easily be subject to the restriction of solvent, test sample concentration, test sample dissolubility and test sample composition diversity, therefore the scope of tested test sample is little, Influence on test result factor is many, and be a total analysis result, and can not obtaining specifically what composition, to have anti-peroxidation lipid reactant active; And traditional ultraviolet spectrophotometry once can only analyze a test sample, cannot realize under a platform, analyze the high flux screening pattern of multiple test sample.In addition, also has the thin-layered chromatography being Testing index with linoleic fluorescence duration time (Fluorescence Persistence Time), linoleic acid is under ultraviolet irradiation, easy generation peroxidatic reaction of lipid, fluorescer on lipid peroxide and thin layer plate reacts, generate not fluorescent material, form slightly dark background colour.When there being the spot of polyphenoils to exist, linoleic acid peroxidation can be stoped to react, the fluorescence duration time at this spot place is extended, until polyphenoils is exhausted, linoleic acid just starts oxidation, and fluorescence spot starts to disappear.The method must adopt the thin layer plate of 254nm fluorescer, and detection sensitivity is very low, and great majority analysis test sample has fluorescent quenching spot (blackening), just severe jamming is had, there will be false negative result, and fluorescence disappearance required time is long, therefore detection time is very long.
TLC-bioautography technology is a kind of a kind of fast activity screening technique TLC separation technology combined with bio-assay technique.Be applied to the multiplex DPPH method of TLC-bioautography technology of antioxidation activity screening at present, because DPPH is a kind of stable free radical, active spot (yellow) and background color (purple) significant difference after colour developing, the good method of a kind of antioxidation activity of therefore can yet be regarded as screening; But DPPH free radical is a kind of Inorganic radicals, and in esse free radical in inorganic matter; Therefore, so far there are no adopts TLC-bioautography technology to carry out the screening of the oxidation-resistant active ingredient with anti-linoleic acid peroxidation lipid reactant.
Summary of the invention
For the problems referred to above that prior art exists, the object of this invention is to provide a kind of screening technique of oxidation-resistant active ingredient, to utilize TLC-bioautography technology, from natural products or monomeric compound, screening has the oxidation-resistant active ingredient of anti-linoleic acid peroxidation lipid reactant fast.
For achieving the above object, the technical solution used in the present invention is as follows:
Screen a method for oxidation-resistant active ingredient, comprise the steps:
A) by need testing solution point sample on thin layer plate, fling to sample solvent for subsequent use or re-use after suitable developping agent launches take out, dry for subsequent use;
B) thin layer plate after a) processing through step be impregnated in Linoleic Acid solution, then takes out and volatilize solvent, be placed in the uviol lamp of 254nm or 366nm under irradiate;
C) by through step b) thin layer plate after process impregnated in chromogenic reagent solution, and then take out, carry out heating and develop the color;
D) to through step c) thin layer plate after colour developing inspects: the white dot under the blue spot under the green background occurred under the uviol lamp of 366nm and the pink background that occurs under visible light is oxidation-resistant active ingredient.
Preferably, described thin layer plate selects silica gel plate or reversed phase thin layer plate.
Preferably, described Linoleic Acid solution select volume fraction be 1 ~ 5% linoleic acid hexane solution.
Preferably, step b) be irradiate 10 ~ 60 minutes under the uviol lamp being placed in 254nm or 366nm.
Preferably, described chromogenic reagent solution is the mixed solution formed by thiobarbituricacidα-, trichloroacetic acid and ethanol water, wherein: the mass concentration of thiobarbituricacidα-is 0.7 ~ 10.7mg/mL, the mass concentration of trichloroacetic acid is 0 ~ 45mg/mL, and the volume fraction of ethanol is 30 ~ 70%.
As optimal case, in described chromogenic reagent solution, the mass concentration of thiobarbituricacidα-is 4.7 ~ 8.7mg/mL, the mass concentration of trichloroacetic acid is 25 ~ 30mg/mL, and the volume fraction of ethanol is 50%.
Preferably, step c) be heat 6 ~ 25 minutes at 40 ~ 100 DEG C.
Compared with prior art, the present invention has following beneficial effect:
1) the inventive method does not need complicated instrument and equipment, simple to operate, and experiment expends low, is applicable to common laboratory operation, and sensitivity and high-specificity are in conventional screening assays;
2) the selection result is visual in image, is easy to identification and memory;
3) screening process take thin layer plate as medium, is not subject to the restriction of solvent and test sample concentration, makes the kind of tested test sample more extensive;
4) can fast, high flux screening goes out to have the oxidation-resistant active ingredient of anti-linoleic acid lipid peroxidation;
5) in conjunction with thin layer scanning, the quantitative test of antagonism linoleic acid lipid peroxidation capacity can be realized.
Accompanying drawing explanation
Fig. 1 be embodiment 1 inspect result figure;
Fig. 2 be embodiment 2 inspect result figure;
Fig. 3 be embodiment 3 inspect result figure;
Fig. 4 be embodiment 4 inspect result figure;
Fig. 5 be embodiment 5 inspect result figure;
Fig. 6 be embodiment 6 inspect result figure;
Fig. 7 be embodiment 7 inspect result figure;
Fig. 8 be embodiment 8 inspect result figure;
Fig. 9 be embodiment 9 inspect result figure;
Figure 10 be embodiment 10 inspect result figure.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is further elaborated:
Embodiment 1
Thin layer plate A: get perilla seed powder 1g, accurately weighed, add methyl alcohol 5mL, after ultrasonic 25 minutes, get supernatant liquid filtering, filtrate is as need testing solution; Separately get Rosmarinic acid and cyanidenon reference substance, add methyl alcohol and make the solution of every 1mL containing 1mg respectively, product solution in contrast; Respectively draw reference substance solution 1 μ L and need testing solution 10 μ L, point sample on same silica GF254 thin layer plate, after flinging to methyl alcohol with normal hexane-dichloromethane-ethyl acetate-formic acid (volume ratio is for 2:5:2.5:0.5) for developping agent launches; Take out, dry for subsequent use.
Thin layer plate B: get saline cistanche powder 0.5g, accurately weighed, add methyl alcohol 10mL, get supernatant after ultrasonic 25 minutes, filter, filtrate is as need testing solution; Separately get acteoside reference substance, add methyl alcohol and make the solution of every 1mL containing 1mg, product solution in contrast; Respectively draw reference substance solution 4 μ L and need testing solution 4 μ L, point sample on same silica GF254 thin layer plate, after flinging to methyl alcohol with acetate-methanol-formic acid-water (volume ratio is for 7.5:0.5:1:0.5) for developping agent launches; Take out, dry for subsequent use.
The preparation of Linoleic Acid solution: accurately measure 0.6mL linoleic acid, is dissolved in the normal hexane of 20mL, is mixed with linoleic acid hexane solution.
The preparation of chromogenic reagent solution: it is in the ethanol water of 50% that 0.87g thiobarbituricacidα-and 2.5g trichloroacetic acid are added 100mL volume fraction, is stirred to dissolve and mixes.
Thin layer plate A and B all be impregnated in Linoleic Acid solution, take out and volatilize normal hexane, to be then placed under the uviol lamp of 254nm the irradiation of 3cm place 40 minutes; Take out thin layer plate A and B, then impregnated in chromogenic reagent solution; Take out, then within 6 minutes, develop the color in 100 DEG C of heating; Under the uviol lamp of 366nm and under visible ray, the thin layer plate after colour developing is inspected respectively, inspect result as shown in Figure 1.
As seen from Figure 1: the blue spot under point has the thin layer plate A of purple perilla increment and point to have the thin layer plate B of saline cistanche all to occur green background under the uviol lamp of 366nm, and all there is the white dot under pink background under visible light, illustrate that perilla seed and saline cistanche are the oxidation-resistant active ingredient with anti-linoleic acid peroxidation lipid reactant, consistent with existing result of study, further illustrate the inventive method and there is feasibility.
Embodiment 2
Thin layer plate A: accurately take 1mg watermiscible vitamin E (Trolox), be dissolved in 1mL absolute ethyl alcohol, is mixed with the solution that concentration is 1mg/mL; Draw 2 μ L solution point samples in silica G F
254on thin layer plate A, for subsequent use after flinging to ethanol.
Thin layer plate B: accurately take 1mg cupreol, is dissolved in 1mL absolute ethyl alcohol, is mixed with the solution that concentration is 1mg/mL; Draw 2 μ L solution point samples on silica GF254 thin layer plate B, for subsequent use after flinging to ethanol.
Thin layer plate C: accurately take 1mg Nuciferine, is dissolved in 1mL absolute ethyl alcohol, is mixed with the solution that concentration is 1mg/mL; Draw 2 μ L solution point samples on silica GF254 thin layer plate C, for subsequent use after flinging to ethanol.
The preparation of Linoleic Acid solution: accurately measure 0.6mL linoleic acid, is dissolved in the normal hexane of 20mL, is mixed with linoleic acid hexane solution.
The preparation of chromogenic reagent solution: it is in the ethanol water of 50% that 0.87g thiobarbituricacidα-and 2.5g trichloroacetic acid are added 100mL volume fraction, is stirred to dissolve and mixes.
Thin layer plate A, B, C all be impregnated in Linoleic Acid solution, take out and volatilize normal hexane, to be then placed under the uviol lamp of 254nm the irradiation of 3cm place 40 minutes; Take out thin layer plate A, B, C, then impregnated in chromogenic reagent solution; Take out, then within 6 minutes, develop the color in 100 DEG C of heating; Under the uviol lamp of 366nm and under visible ray, the thin layer plate after colour developing is inspected respectively, inspect result as shown in Figure 2.
As seen from Figure 2: put the blue spot under having the thin layer plate A of watermiscible vitamin E to occur green background under the uviol lamp of 366nm, and occurred the white dot under pink background under visible light; And point has the thin layer plate B of cupreol and put the blue spot under having the thin layer plate C of Nuciferine all not occur green background under the uviol lamp of 366nm, and all there is not the white dot under pink background under visible light yet; Illustrate that watermiscible vitamin E is the oxidation-resistant active ingredient with anti-linoleic acid peroxidation lipid reactant, and cupreol and Nuciferine all do not have anti-linoleic acid lipid peroxidation, consistent with existing result of study, further illustrate the inventive method and there is feasibility.
Embodiment 3
The preparation of need testing solution: accurately take 1mg watermiscible vitamin E (Trolox), be dissolved in 1mL absolute ethyl alcohol, is mixed with the solution that concentration is 1mg/mL.
The preparation of Linoleic Acid solution: accurately measure 0.2,0.4,0.6,0.8 and 1.0mL linoleic acid, be dissolved in respectively in the normal hexane of 20mL, is mixed with the linoleic acid hexane solution (being designated as Linoleic Acid solution 1,2,3,4,5 successively) of variable concentrations.
The preparation of chromogenic reagent solution: it is in the ethanol water of 50% that 0.87g thiobarbituricacidα-and 2.5g trichloroacetic acid are added 100mL volume fraction, is stirred to dissolve and mixes.
Get the parallel point sample of need testing solution on 5 silica GF254 thin layer plates, point sample amount is 2 μ L, then flings to ethanol, for subsequent use.
Above-mentioned 5 thin layer plates be impregnated in respectively (corresponding thin layer plate is designated as thin layer plate 1,2,3,4,5 successively) in Linoleic Acid solution 1,2,3,4,5, take out and volatilize normal hexane, to be then placed under the uviol lamp of 254nm the irradiation of 3cm place 40 minutes; Take out thin layer plate, then impregnated in chromogenic reagent solution; Take out, then within 6 minutes, develop the color in 100 DEG C of heating; Under the uviol lamp of 366nm and under visible ray, the thin layer plate after colour developing is inspected respectively, inspect result as shown in Figure 3.
As seen from Figure 3: select volume fraction be 1 ~ 5% linoleic acid hexane solution all can be inspected result clearly.
Embodiment 4
The preparation of need testing solution: accurately take 1mg watermiscible vitamin E (Trolox), be dissolved in 1mL absolute ethyl alcohol, is mixed with the solution that concentration is 1mg/mL.
The preparation of Linoleic Acid solution: accurately measure 0.6mL linoleic acid, is dissolved in the normal hexane of 20mL, is mixed with linoleic acid hexane solution.
The preparation of chromogenic reagent solution: it is in the ethanol water of 50% that 0.87g thiobarbituricacidα-and 2.5g trichloroacetic acid are added 100mL volume fraction, is stirred to dissolve and mixes.
Get the parallel point sample of need testing solution on cellulose thin-layer chromatography plate (being designated as A), polyam ide TLC chromatosheet (being designated as B), ODS reversed phase thin layer plate (being designated as C), point sample amount is 2 μ L, then flings to ethanol, for subsequent use.
Above-mentioned 3 thin layer plates be impregnated in Linoleic Acid solution respectively, then take out and volatilize normal hexane, to be placed under the uviol lamp of 254nm the irradiation of 3cm place 40 minutes; Take out thin layer plate, then impregnated in chromogenic reagent solution; Take out, then within 6 minutes, develop the color in 100 DEG C of heating; Under the uviol lamp of 366nm and under visible ray, the thin layer plate after colour developing is inspected respectively, inspect result as shown in Figure 4.
As seen from Figure 4: select cellulose thin-layer chromatography plate (being designated as A) and polyam ide TLC chromatosheet, all can only inspect out result clearly under the uviol lamp of 366nm, result of inspecting under visible light is not very clear; But select ODS reversed phase thin layer plate (being designated as C), then under the uviol lamp of 366nm He under visible ray, all can inspect out result clearly.
Embodiment 5
The preparation of need testing solution: accurately take 1mg watermiscible vitamin E (Trolox), be dissolved in 1mL absolute ethyl alcohol, is mixed with the solution that concentration is 1mg/mL.
The preparation of Linoleic Acid solution: accurately measure 0.6mL linoleic acid, is dissolved in the normal hexane of 20mL, is mixed with linoleic acid hexane solution.
The preparation of chromogenic reagent solution: it is in the ethanol water of 50% that 0.87g thiobarbituricacidα-and 2.5g trichloroacetic acid are added 100mL volume fraction, is stirred to dissolve and mixes.
Get the parallel point sample of need testing solution on 6 silica GF254 thin layer plates, point sample amount is 2 μ L, then flings to ethanol, for subsequent use.
Above-mentioned 6 thin layer plates be impregnated in Linoleic Acid solution respectively, taking-up volatilizes normal hexane, is then placed in the treatment with irradiation (corresponding thin layer plate is designated as thin layer plate 1,2,3,4,5,6 successively) that 3cm place under the uviol lamp of 254nm carries out 10min, 20min, 30min, 40min, 50min and 60min respectively; Take out thin layer plate, then impregnated in chromogenic reagent solution; Take out, then within 6 minutes, develop the color in 100 DEG C of heating; Under the uviol lamp of 366nm and under visible ray, the thin layer plate after colour developing is inspected respectively, inspect result as shown in Figure 5.
As seen from Figure 5: the irradiation time of uviol lamp is selected and all can be inspected result clearly in 10 ~ 60 minutes.
Embodiment 6
The preparation of need testing solution: accurately take 1mg watermiscible vitamin E (Trolox), be dissolved in 1mL absolute ethyl alcohol, is mixed with the solution that concentration is 1mg/mL.
The preparation of Linoleic Acid solution: accurately measure 0.6mL linoleic acid, is dissolved in the normal hexane of 20mL, is mixed with linoleic acid hexane solution.
The preparation of chromogenic reagent solution: respectively by 0.87g thiobarbituricacidα-and 0,0.05,0.1,0.2,0.3,0.4,0.5,1.5,2.5,3.5, to add 100mL volume fraction be in the ethanol water of 50% for the trichloroacetic acid of 4.5g, be stirred to dissolve and mix, the mass concentration being mixed with trichloroacetic acid is followed successively by 0,0.5,1.0,2.0,3.0,4.0,5.0,15,25,35, the chromogenic reagent solution (being designated as chromogenic reagent solution 1-11 successively) of 45mg/mL.
Get the parallel point sample of need testing solution on 11 silica GF254 thin layer plates, point sample amount is 2 μ L, then flings to ethanol, for subsequent use.
Above-mentioned 11 thin layer plates be impregnated in Linoleic Acid solution respectively, take out and volatilize normal hexane, be then placed in the treatment with irradiation that 3cm place under the uviol lamp of 254nm carries out 40min; Take out thin layer plate again, impregnated in chromogenic reagent solution 1-11 respectively; Take out, then within 6 minutes, develop the color in 100 DEG C of heating; Under the uviol lamp of 366nm and under visible ray, the thin layer plate after colour developing is inspected respectively, inspect result as shown in Figure 6.
As seen from Figure 6: select the mass concentration of trichloroacetic acid to be that the chromogenic reagent solution of 0 ~ 45mg/mL all can be inspected result clearly.
Embodiment 7
The preparation of need testing solution: accurately take 1mg watermiscible vitamin E (Trolox), be dissolved in 1mL absolute ethyl alcohol, is mixed with the solution that concentration is 1mg/mL.
The preparation of Linoleic Acid solution: accurately measure 0.6mL linoleic acid, is dissolved in the normal hexane of 20mL, is mixed with linoleic acid hexane solution.
The preparation of chromogenic reagent solution: respectively 0.07,0.17,0.27,0.47,0.67,0.87 and the thiobarbituricacidα-of 1.07g and 2.5g trichloroacetic acid being added 100mL volume fraction is in the ethanol water of 50%, be stirred to dissolve and mix, the mass concentration being mixed with trichloroacetic acid is followed successively by 0.7,1.7,2.7,4.7,6.7,8.7, the chromogenic reagent solution (being designated as chromogenic reagent solution 1-7 successively) of 10.7mg/mL.
Get the parallel point sample of need testing solution on 7 silica GF254 thin layer plates, point sample amount is 2 μ L, then flings to ethanol, for subsequent use.
Above-mentioned 7 thin layer plates be impregnated in Linoleic Acid solution respectively, take out and volatilize normal hexane, be then placed in the treatment with irradiation that 3cm place under the uviol lamp of 254nm carries out 40min; Take out thin layer plate again, impregnated in chromogenic reagent solution 1-7 respectively; Take out, then within 6 minutes, develop the color in 100 DEG C of heating; Under the uviol lamp of 366nm and under visible ray, the thin layer plate after colour developing is inspected respectively, inspect result as shown in Figure 7.
As seen from Figure 7: select the mass concentration of thiobarbituricacidα-to be that the chromogenic reagent solution of 0.7 ~ 10.7mg/mL all can be inspected result clearly.
Embodiment 8
The preparation of need testing solution: respectively 0.01,0.02,0.04,0.06 and 0.08mg watermiscible vitamin E (Trolox) are dissolved in 1mL absolute ethyl alcohol, be mixed with concentration be followed successively by 0.01,0.02,0.04,0.06, the need testing solution (being designated as need testing solution 1-5 successively) of 0.08mg/mL.
The preparation of Linoleic Acid solution: accurately measure 0.6mL linoleic acid, is dissolved in the normal hexane of 20mL, is mixed with linoleic acid hexane solution.
The preparation of chromogenic reagent solution: it is in the ethanol water of 50% that 0.87g thiobarbituricacidα-and 2.5g trichloroacetic acid are added 100mL volume fraction, is stirred to dissolve and mixes.
By above-mentioned need testing solution 1-5, point sample is on 5 silica GF254 thin layer plates respectively, and point sample amount is 2 μ L, then flings to ethanol, for subsequent use.
Above-mentioned 5 thin layer plates be impregnated in Linoleic Acid solution respectively, take out and volatilize normal hexane, be then placed in the treatment with irradiation that 3cm place under the uviol lamp of 254nm carries out 40min; Take out thin layer plate again, impregnated in chromogenic reagent solution respectively; Take out, then within 6 minutes, develop the color in 100 DEG C of heating; Under the uviol lamp of 366nm and under visible ray, the thin layer plate after colour developing is inspected respectively, inspect result as shown in Figure 8.
As seen from Figure 8: the minimal detectable concentration of the inventive method is 0.02mg/mL.
Embodiment 9
The preparation of need testing solution: accurately take 1mg watermiscible vitamin E (Trolox), be dissolved in 1mL absolute ethyl alcohol, is mixed with the solution that concentration is 1mg/mL.
The preparation of Linoleic Acid solution: accurately measure 0.6mL linoleic acid, is dissolved in the normal hexane of 20mL, is mixed with linoleic acid hexane solution.
The preparation of chromogenic reagent solution: it is in the ethanol water of 50% that 0.87g thiobarbituricacidα-and 2.5g trichloroacetic acid are added 100mL volume fraction, is stirred to dissolve and mixes.
Get the parallel point sample of need testing solution on 4 silica GF254 thin layer plates, point sample amount is 2 μ L, then flings to ethanol, for subsequent use.
Above-mentioned 4 thin layer plates be impregnated in Linoleic Acid solution respectively, take out and volatilize normal hexane, be then placed in the treatment with irradiation that 3cm place under the uviol lamp of 254nm carries out 40min; Take out thin layer plate again, impregnated in chromogenic reagent solution respectively; Take out, respectively 40 DEG C of heating 23 minutes (being designated as A), 60 DEG C of heating 20 minutes (being designated as B), heat 6 minutes (being designated as D) at heating 13 minutes (being designated as C) and 100 DEG C at 80 DEG C; Finally under the uviol lamp of 366nm and under visible ray, the thin layer plate after colour developing is inspected respectively, inspect result as shown in Figure 9.
As seen from Figure 9: at 40 ~ 100 DEG C, carry out heating colour developing, all under uviol lamp, result can be inspected clearly; But only 100 DEG C of heating colour developings, both under the uviol lamp of 366nm He under visible ray, all result can be inspected clearly.
Embodiment 10
The preparation of need testing solution: accurately take n-propyl gallate (PG, Propyl gallate, be designated as 1), butylhydroxy anisole (BHA, Butylated hydroxyanisole, be designated as 2), 2,6-BHT (BHT, Butylatedhydroxytoluene, be designated as 3), ditert-butylhydro quinone (TBHQ, Tertiary butylhydroquinone, is designated as 4), watermiscible vitamin E (Trolox is designated as 5) each 1mg, be dissolved in 1mL absolute ethyl alcohol respectively, be all mixed with the solution that concentration is 1mg/mL.
The preparation of Linoleic Acid solution: accurately measure 0.6mL linoleic acid, is dissolved in the normal hexane of 20mL, is mixed with linoleic acid hexane solution.
The preparation of chromogenic reagent solution: it is in the ethanol water of 50% that 0.87g thiobarbituricacidα-and 2.5g trichloroacetic acid are added 100mL volume fraction, is stirred to dissolve and mixes.
By above-mentioned need testing solution point sample on same silica GF254 thin layer plate, point sample amount is 2 μ L, then flings to ethanol, for subsequent use.
Above-mentioned thin layer plate be impregnated in Linoleic Acid solution, take out and volatilize normal hexane, be then placed in the treatment with irradiation that 3cm place under the uviol lamp of 254nm carries out 40min; Take out thin layer plate again, impregnated in chromogenic reagent solution; Take out, 100 DEG C of heating 6 minutes; Finally under the uviol lamp of 366nm and under visible ray, the thin layer plate after colour developing is inspected, inspect result as shown in Figure 10.
As seen from Figure 10: above-mentioned test sample all demonstrates and inspects result clearly under the uviol lamp of 366nm and under visible ray, illustrates that the inventive method can realize fast, high flux screening goes out oxidation-resistant active ingredient, and inspects result accurately and reliably.
Visible in sum: the inventive method does not need complicated instrument and equipment, can fast, high flux screening goes out to have the oxidation-resistant active ingredient of anti-linoleic acid lipid peroxidation, not only the selection result visual in image, be easy to identification and memory, and sensitivity and high-specificity are in conventional screening assays, and be not subject to the restriction of solvent and test sample concentration, make the kind of tested test sample more extensive, being applicable to common laboratory operation, providing a kind of approach of convenient practicality for filtering out the oxidation-resistant active ingredient with anti-linoleic acid lipid peroxidation.
Finally be necessary described herein: above embodiment, only for being described in more detail technical scheme of the present invention, can not be interpreted as limiting the scope of the invention; Some nonessential improvement that those skilled in the art's foregoing according to the present invention is made and adjustment all belong to protection scope of the present invention.
Claims (6)
1. screen a method for oxidation-resistant active ingredient, it is characterized in that, comprise the steps:
A) by need testing solution point sample on thin layer plate, fling to sample solvent for subsequent use or re-use after suitable developping agent launches take out, dry for subsequent use;
B) thin layer plate after a) processing through step be impregnated in Linoleic Acid solution, then takes out and volatilize solvent, be placed in the uviol lamp of 254nm or 366nm under irradiate;
C) by through step b) thin layer plate after process impregnated in chromogenic reagent solution, and then take out, carry out heating and develop the color;
D) to through step c) thin layer plate after colour developing inspects: the white dot under the blue spot under the green background occurred under the uviol lamp of 366nm and the pink background that occurs under visible light is oxidation-resistant active ingredient.
2. the method for claim 1, is characterized in that: described thin layer plate selects silica gel plate or reversed phase thin layer plate.
3. the method for claim 1, is characterized in that: described Linoleic Acid solution select volume fraction be 1 ~ 5% linoleic acid hexane solution.
4. the method for claim 1, is characterized in that: step b) be irradiate 10 ~ 60 minutes under the uviol lamp being placed in 254nm or 366nm.
5. the method for claim 1, it is characterized in that: described chromogenic reagent solution is the mixed solution formed by thiobarbituricacidα-, trichloroacetic acid and ethanol water, wherein: the mass concentration of thiobarbituricacidα-is 0.7 ~ 10.7mg/mL, the mass concentration of trichloroacetic acid is 0 ~ 45mg/mL, and the volume fraction of ethanol is 30 ~ 70%.
6. the method for claim 1, is characterized in that: step c) be heat 6 ~ 25 minutes at 40 ~ 100 DEG C.
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| CN108776121A (en) * | 2018-04-08 | 2018-11-09 | 广州卡马生物科技有限公司 | It is a kind of based on Fluorescence Quenching Principle to the high-throughput screening method of antioxidant in medicinal material |
| CN109061027A (en) * | 2018-05-07 | 2018-12-21 | 江南大学 | The method of HPTLC- biological developing screening oil synthesis phenol antioxidant |
| CN109804249A (en) * | 2017-06-14 | 2019-05-24 | 清滔制药株式会社 | Method for measuring human body fluid oxidative stress |
| CN113670875A (en) * | 2021-08-19 | 2021-11-19 | 宁德师范学院 | Method for screening antioxidant by using phascolosoma esculenta |
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| CN109061027A (en) * | 2018-05-07 | 2018-12-21 | 江南大学 | The method of HPTLC- biological developing screening oil synthesis phenol antioxidant |
| CN109061027B (en) * | 2018-05-07 | 2020-10-09 | 江南大学 | Method for synthesizing phenolic antioxidant by HPTLC-biological development screening of oil |
| CN113670875A (en) * | 2021-08-19 | 2021-11-19 | 宁德师范学院 | Method for screening antioxidant by using phascolosoma esculenta |
| CN113670875B (en) * | 2021-08-19 | 2023-04-28 | 宁德师范学院 | Method for screening antioxidant by using delicious euglena |
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