CN107271378A - The assay method of astaxanthin lipid antioxidation activity - Google Patents
The assay method of astaxanthin lipid antioxidation activity Download PDFInfo
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Abstract
The invention provides a kind of assay method of astaxanthin composition inhibition of lipid peroxidation, the assay method includes：Astaxanthin antioxidant composition, 10.0mg/mL egg yolk lecithins, 5.0mg/mL cholesterol and hexamethylene are mixed, ultrasound obtains film forming solution to solution clarification；By the film forming solution drop coating on carrier, astaxanthin composition double-layer quantum dots thin layer is obtained after drying；Carrier immersion free radical containing astaxanthin composition double-layer quantum dots thin layer is occurred to react in system solution, taken out afterwards；Occur to add 0.9 1.1mL1%TBA solution in system solution in above-mentioned free radical, it is subjected to absorbance measurement at 533nm after water-bath；This assay method can accurately, reliably review the anti-lipid peroxidation ability of astaxanthin, overcome the anti-system of aqueous free radical lipid peroxidation of the different astaxanthin composition of existing method dissolving characteristic, quantitative relation without determination, it is impossible to for it is accurate, it is reliable review astaxanthin composition anti-lipid peroxidation ability the problem of.
Description
Technical field
The present invention relates to the assay method of astaxanthin composition inhibition of lipid peroxidation.
Background technology
Astaxanthin (Astaxanthin, 3,3 '-dihydroxy -4,4 '-diketone-β, β '-carrotene), also known as astaxanthin, dragon
Shrimp shell pigment, is to be distributed widest fat soluble carotenoids in a kind of animal kingdom.Astaxanthin is water insoluble, is natural so far
The most strong oxidant found in boundary, its oxidation resistance is higher than bata-carotene more than 10 times, higher than vitamin E more than 550 times.
Astaxanthin also has very strong suppression tumour growth, strengthens immunologic function, resists a variety of physiological functions such as uv damage, thus
It is widely used in the fields such as food, medicine, cosmetics, health products, aquaculture.Astaxanthin and other antioxidants such as biological Huang
What the combination of ketone etc. can give full play to astaxanthin and other antioxidants cooperates with antioxidation, hence it is evident that the work of increase astaxanthin
Property, ten thousand emphasis researched and developed for astaxanthin product, but because astaxanthin is fat-soluble, and most antioxidants are water-soluble or alcohol
Dissolubility, it is difficult to find the appraisement system of the different anti-oxidant compositions of this kind of dissolving characteristic, constrains astaxanthin composition
Screening.
Lipid peroxidation refers to the oxidation deterioration of polyunsaturated fatty acid under oxidative conditions and lipid, this effect
To cell membrane, lipoprotein and other containing lipid conformation produce serious damage, including cell membrane fluidity and infiltrative
The damage of change, DNA and protein, and then influence cell normal function.Many major diseases, such as tumour occur, heart and brain blood
Pipe, diabetes and aging etc. are directed to the lipid peroxidation of free yl induction, therefore, find screening lipid peroxy and are turned into
It is significant with inhibitor.The method that the evaluation method of current lipid peroxidation inhibitor has in vitro and in vivo, it is square in vivo
Method needs to carry out a large amount of animal experiments, and the cost high cycle is long, and in vitro test Fang Lin is based on aqueous free radical and cell biological film
Or the peroxidatic reaction of lipid of lecithin vesicles, by detecting peroxidatic reaction of lipid product such as MDA (MBA) analog
Realize the evaluation of lipid peroxidation inhibitory activity.
Astaxanthin has obvious lipid peroxidation inhibitory activity, but current data are the result of animal experiment mostly, former
Because being that astaxanthin has obvious hydrophobicity, in existing aqueous free radical system, it is difficult to ginseng uniformly, quick and stable
With suppressing peroxidation of the free radical to Cell membrane lipids, consequently it is difficult to accurately, reliably review the suppression of astaxanthin composition
Lipid peroxidation capacity.
In summary, in the accurate aqueous free radical system of development stability, astaxanthin is different from other dissolving characteristics
The lipid peroxidation evaluation method of anti-oxidant compositions, is studied for astaxanthin and the exploitation of astaxanthin preparation has important valency
Value, also has important application prospect for the different antioxidant combination research of other dissolving characteristics.
The content of the invention
It is an object of the invention to provide a kind of assay method of astaxanthin composition inhibition of lipid peroxidation, this measure side
Method can accurately, reliably review the anti-lipid peroxidation ability of the different astaxanthin composition of dissolving characteristic, overcome foregoing
The existing anti-system of lipid peroxidation, the quantitative relation of no determination, it is impossible to the shrimp different for accurate, reliable review dissolving characteristic
The problem of blue or green promotor composition anti-lipid peroxidation ability.
In order to achieve the above object, the invention provides a kind of measure side of astaxanthin composition inhibition of lipid peroxidation
Method, the assay method includes：
(1) astaxanthin composition, 10.0mg/mL egg yolk lecithins, 5.0mg/mL cholesterol are mixed in and hexamethylene, surpassed
Sound to solution is clarified, and obtains film forming solution；
(2) it is 20~50 μ L film forming solutions drop coatings is thin in obtaining astaxanthin-double-layer quantum dots on carrier, after drying
Layer；
(3) the carrier immersion free radical containing astaxanthin composition-double-layer quantum dots thin layer is occurred anti-in solution system
Should, take out afterwards；Wherein, system solution occurs for the free radical by Fe of the 0.45-0.55mL concentration for 75mmol/L2+-
EDTA, 0.45-0.55mL concentration are 150mmol/L H2O2Solution and 1.45-1.55mL concentration buffer for 0.75mol/L PBS
Solution composition；
(4) 0.9-1.1mL1% thiobarbituricacidα-s (TBA) solution, water are added in system solution occurs for above-mentioned free radical
It is subjected to absorbance measurement at 533nm after bath；
(5) absorbance surveyed according to step (4) calculates lipid peroxidation inhibiting rate,
In formula, A0For blank sample absorbance, ASampleFor the absorbance of sample.
Preferably, the carrier is the long 6cm spectroscopically pure graphites rods of diameter 8mm, and using tetrafluoroethene pipe parcel, optical cement envelope
Dress.
Preferably, the carrier need to be polishing to light before with sand paper, and with ethanol and ultrasonic washing with clean water, blow
It is dry.
Preferably, the temperature of reaction is 36-38 DEG C in step (3), and the time of reaction is 10-30min.
Preferably, the temperature of water-bath is 85-88 DEG C in step (4), and the time of water-bath is 18-22min.
Preferably, the pH value of the PBS cushioning liquid is 7.3-7.5.
Beneficial effect：The invention provides a kind of assay method of astaxanthin composition lipid antioxidation activity, before overcoming
State the anti-system of aqueous free radical lipid peroxidation of the different astaxanthin composition of existing dissolving characteristic, the metering of no determination
Relation, it is impossible to for it is accurate, it is reliable review astaxanthin composition anti-lipid peroxidation ability the problem of；Meanwhile, utilize lecithin
Fat helps dissolution, and dissolving characteristic different astaxanthin and other antioxidants are dispersed in double-layer quantum dots and shape
Into good reactivity thin layer, with the free radical aqueous solution generation system reported and the detection of lipid peroxidation product light splitting light method
System has good suitability.Technical scheme of the present invention is easily achieved, and good reliability realizes dissolving characteristic different
The measure of astaxanthin composition anti-lipid peroxidation ability, anti-lipid peroxidation ability is that the most important activity of antioxidant refers to
One of mark, the present invention provides technical support for the astaxanthin combination antioxidant exploitation of high value.
Specifically apply mode
The following detailed description of the preferred embodiment of the present invention.
Embodiment 1
By 1：2 (w/w) astaxanthins-rutin composition, 10.0mg/mL egg yolk lecithins, 5.0mg/mL cholesterol are mixed in
And hexamethylene, it is ultrasonic to solution clarification, obtain film forming solution；By the film forming solution drop coating, in carrier, (carrier is spectroscopic pure stone
Inker, and using tetrafluoroethene pipe parcel, optical cement encapsulation, carrier need to be polishing to light before with sand paper, and with ethanol with
Ultrasonic washing with clean water, drying) on, obtain astaxanthin composition-double-layer quantum dots thin layer after drying；Astaxanthin combination will be contained
Thing-double-layer quantum dots thin layer carrier immersion free radical occur system solution in reaction (temperature of reaction be 36 DEG C, reaction when
Between be 10min), take out afterwards；Wherein, system solution occurs for the free radical by Fe of the 0.45mL concentration for 75mmol/L2+-
EDTA, 0.45mL concentration are 150mmol/L H2O2Solution and 1.45mL concentration constitute for 0.75mol/L PBS cushioning liquid；
Above-mentioned free radical occur system solution in add 0.9mL1%TBA solution, water-bath (temperature of water-bath be 85 DEG C, water-bath when
Between be 18min) after it is subjected to absorbance measurement at 533nm.
Embodiment 2
By 1：2 (w/w) astaxanthin-vitamin C compositions, 10.0mg/mL egg yolk lecithins, the mixing of 5.0mg/mL cholesterol
In and hexamethylene, ultrasound to solution clarification, obtain film forming solution；By the film forming solution drop coating, in carrier, (carrier is spectroscopic pure
Graphite rod, and using tetrafluoroethene pipe parcel, optical cement is encapsulated, and carrier need to be polishing to light before with sand paper, and use ethanol
With ultrasonic washing with clean water, drying) on, obtain astaxanthin composition-double-layer quantum dots thin layer after drying；Astaxanthin combination will be contained
Thing-double-layer quantum dots thin layer carrier immersion free radical occur system solution in reaction (temperature of reaction be 38 DEG C, reaction when
Between be 30min), take out afterwards；Wherein, system solution occurs for the free radical by Fe of the 0.55mL concentration for 75mmol/L2+-
EDTA, 0.55mL concentration are 150mmol/L H2O2Solution and 1.55mL concentration constitute for 0.75mol/L PBS cushioning liquid；
Above-mentioned free radical occur system solution in add 1.1mL1%TBA solution, water-bath (temperature of water-bath be 88 DEG C, water-bath when
Between be 22min) after it is subjected to absorbance measurement at 533nm.
Comparative example 1
By 1：2 (w/w) astaxanthins-rutin composition, 10.0mg/mL egg yolk lecithins, 5.0mg/mL cholesterol are mixed in
And water, ultrasound is to being completely dispersed, and adding reaction in free radical generation system solution, (temperature of reaction is 30 DEG C, the time of reaction
For 8min), take out afterwards；Wherein, system solution occurs for the free radical by Fe of the 0.4mL concentration for 75mmol/L2+-EDTA、
0.4mL concentration is 150mmol/L H2O2Solution and 1.4mL concentration constitute for 0.75mol/L PBS cushioning liquid；Above-mentioned
Free radical occurs to add 0.7mL1%TBA solution in system solution, and (temperature of water-bath is 80 DEG C, and the time of water-bath is for water-bath
Clear liquid is drawn with filtering head absorbance measurement is carried out at 533nm after 15min).
Comparative example 2
By 1：2 (w/w) astaxanthin-vitamin C compositions consolidate rutin, 10.0mg/mL egg yolk lecithins, 5.0mg/mL courages
Alcohol is mixed in and hexamethylene, and ultrasound is to being completely dispersed, and adding reaction in free radical generation system solution, (temperature of reaction is 30
DEG C, the time of reaction is 8min), take out afterwards；Wherein, the free radical generation system solution is by 0.4mL concentration
75mmol/L Fe2+- EDTA, 0.4mL concentration are 150mmol/L H2O2Solution and the PBS that 1.4mL concentration is 0.75mol/L
Cushioning liquid is constituted；0.7mL1%TBA solution is added in system solution occurs for above-mentioned free radical, (temperature of water-bath is for water-bath
80 DEG C, time of water-bath is 15min) after draw clear liquid with filtering head absorbance measurement carried out at 533nm.
Test case
The maximum lipid peroxidation inhibiting rate average value difference of above-described embodiment 1-2 astaxanthins five parallel determinations of composition
For 77.2% and 74.5%, during relative standard deviation is respectively 6.4% and 4.2%, comparative example 1,2, the shrimp of same amount is blue or green
The maximum lipid peroxidation inhibiting rate average value of five parallel determinations of promotor composition is 42.4% and 51.6%, relative standard deviation
Respectively 21.3% and 17.5%, the lipid peroxidation of embodiment 1-2 and comparative example 1,2 is calculated using following calculation formula
Inhibiting rate.
Calculation formula：
In formula, A0For blank sample absorbance, ASampleFor the absorbance of sample.
It can be obtained by calculating, the maximum lipid peroxidation inhibiting rate of embodiment 1 is 77.2%, relative standard deviation point
Not Wei 6.4%, the maximum lipid peroxidation inhibiting rate of embodiment 2 is 74.5%, and relative standard deviation is 4.2%, identical to use
Amount, the lipid peroxidation inhibiting rate of comparative example 1 is 42.4%, and relative standard deviation is 21.3%, the lipid peroxidation of comparative example 2
Inhibiting rate is 51.6%；Relative standard deviation is 17.5%.It can be seen that what is obtained within the scope of the present invention by above-mentioned data
Astaxanthin composition lipid peroxidation inhibiting rate has more preferable representativeness, and deviation is relatively low, and obtained outside the scope of the invention
Astaxanthin composition lipid peroxidation inhibiting rate representativeness is poor, and deviation is significantly higher.
Above-described is only the preferred embodiment of the present invention, it is noted that for one of ordinary skill in the art
For, without departing from the concept of the premise of the invention, various modifications and improvements can be made, these belong to the present invention
Protection domain.
Claims (6)
1. a kind of assay method of astaxanthin antioxidant composition inhibition of lipid peroxidation, it is characterised in that the measure side
Method includes：
(1) astaxanthin composition, 10.0mg/mL egg yolk lecithins, 5.0mg/mL cholesterol are mixed in and hexamethylene, ultrasound is extremely
Solution is clarified, and obtains film forming solution；
(2) 20~50 μ L film forming solutions drop coatings are obtained into astaxanthin antioxidant composition-bilayer phosphorus on carrier after drying
Adipose membrane thin layer；
(3) the carrier immersion free radical containing astaxanthin composition-double-layer quantum dots thin layer is occurred to react in solution system, it
After take out；Wherein, system solution occurs for the free radical by Fe of the 0.45-0.55mL concentration for 75mmol/L2+-EDTA、0.45-
0.55mL concentration is 150mmol/L H2O2Solution and 1.45-1.55mL concentration constitute for 0.75mol/L PBS cushioning liquid；
(4) 0.9-1.1mL1%TBA solution is added in system solution occurs for above-mentioned free radical, after water-bath by it at 533nm
Carry out absorbance measurement；
(5) absorbance surveyed according to step (4) calculates lipid peroxidation inhibiting rate,
In formula, A0For blank sample absorbance, ASampleFor the absorbance of sample.
2. assay method according to claim 1, wherein, the carrier is the long 6cm spectroscopically pure graphites rods of diameter 8mm, and
Wrapped up using tetrafluoroethene pipe, optical cement encapsulation.
3. assay method according to claim 1 or 2, wherein, the carrier need to be polishing to light before with sand paper,
And with ethanol and ultrasonic washing with clean water, drying.
4. assay method according to claim 1, wherein, the temperature of reaction is 36-38 DEG C in step (3), reaction when
Between be 10-30min.
5. assay method according to claim 1, wherein, the temperature of water-bath is 85-88 DEG C in step (4), water-bath when
Between be 18-22min.
6. assay method according to claim 1, wherein, the pH value of the PBS cushioning liquid is 7.3-7.5.
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| CN201710239952.2A CN107271378B (en) | 2017-04-13 | 2017-04-13 | Method for measuring anti-lipid peroxidation activity of astaxanthin |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109691672A (en) * | 2017-10-24 | 2019-04-30 | 国家海洋局第三海洋研究所 | A kind of liposome and preparation method thereof for encapsulating free astaxanthin |
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| CN1164387A (en) * | 1996-03-11 | 1997-11-12 | 巴斯福股份公司 | Stable carotenoid emulsions suitable for parenteral administration |
| US8349376B1 (en) * | 2011-03-08 | 2013-01-08 | Bezzek Mark S | Anti-dementia regimen |
| CN103767070A (en) * | 2014-01-15 | 2014-05-07 | 中国烟草总公司广东省公司 | Preparation method of squalene and astaxanthin composition liquid precursor lipidosome and radical reducing and harm reduction application of squalene and astaxanthin composition liquid precursor lipidosome |
| JP2016222612A (en) * | 2015-06-01 | 2016-12-28 | 昭和電工株式会社 | Cosmetic and skin external preparation |
-
2017
- 2017-04-13 CN CN201710239952.2A patent/CN107271378B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1164387A (en) * | 1996-03-11 | 1997-11-12 | 巴斯福股份公司 | Stable carotenoid emulsions suitable for parenteral administration |
| US8349376B1 (en) * | 2011-03-08 | 2013-01-08 | Bezzek Mark S | Anti-dementia regimen |
| CN103767070A (en) * | 2014-01-15 | 2014-05-07 | 中国烟草总公司广东省公司 | Preparation method of squalene and astaxanthin composition liquid precursor lipidosome and radical reducing and harm reduction application of squalene and astaxanthin composition liquid precursor lipidosome |
| JP2016222612A (en) * | 2015-06-01 | 2016-12-28 | 昭和電工株式会社 | Cosmetic and skin external preparation |
Non-Patent Citations (1)
| Title |
|---|
| CAITLIN S. BOON 等: "Factors Influencing the Chemical Stability of Carotenoids in Foods", 《CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109691672A (en) * | 2017-10-24 | 2019-04-30 | 国家海洋局第三海洋研究所 | A kind of liposome and preparation method thereof for encapsulating free astaxanthin |
| WO2019080193A1 (en) * | 2017-10-24 | 2019-05-02 | 国家海洋局第三海洋研究所 | Liposome encapsulating free astaxanthin and preparation method therefor |
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