CN102507922B - Method for rapidly determining total antioxidant capacity of marine bivalves - Google Patents
Method for rapidly determining total antioxidant capacity of marine bivalves Download PDFInfo
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Abstract
The invention relates to a method for determining antioxidant capacity of ocean shellfishes, in particular to a method for rapidly determining total antioxidant capacity of marine bivalves, which replaces a conventional spectrophotometer with a full wave frequency scanning type microplate reader a 96 ferment plate for determining total antioxidant capacity of processed samples, can determine batches of samples rapidly and accurately in a short time and accordingly can detect interspecies resistance study of the marine bivalves rapidly. The method rapidly determining total antioxidant capacity of marine bivalves can determine the total antioxidant capacity of the marine bivalves with different varieties and different textures in batch mode, and determination speed is improved by about 10 times compared with a conventional method.
Description
Technical field
The present invention relates to a kind of method of measuring seashells oxidation resistance, is the method for a kind of Fast Measurement ocean bivalve shellfish TAC specifically.
Background technology
TAC (Total antioxidant capability, TAC) is the ability of weighing active oxygen radical (Reactive oxygen radical species, ROS) excessive in animal removing body, is a composite target.Under disease and stress situation, animal body oxygen demand can sharply increase, metabolic disorder, causes oxidative stress, in cell, can produce and accumulate a large amount of ROS.ROS causes by having an effect with protein, nucleic acid and lipid that protein inactivation is even degraded, DNA chain ruptures, lipid peroxidation etc., thereby cause eucaryotic cell structure and function to be destroyed, oxidative stress in primosome simultaneously, if can not remove in time, finally cause body oxidative damage, serious harm body health, even causes death.Under normal circumstances, the generation of the interior ROS of cell and removing are always in dynamic balance state.In biosystem evolutionary process; zooblast has also formed a set of anti-oxidative defense system by antioxidase system (as superoxide dismutase SOD, cat catalase, Glutathione peroxidase GPx etc.) and anti-oxidant non-enzyme system (as carotenoid, ubiquinone, selenium, vitamin, compounds containing thiol groups etc.) inherence in order to protect self to escape injury, thereby has certain oxidation resistance.Therefore, research body removes that the ability of ROS maintains homeostasis to it and degeneration-resistant resistance against diseases is significant.
Marine products bivalve is a most important guiding principle in Mollusca, wherein has many types, as blood clam, clam, scallop, razor clam etc. not only have important economic worth, is also important sea-farming object simultaneously.But, up to now, research to these species oxidation resistances is but known little about it, only in indivedual kinds such as mussel, razor clam, there is a small amount of report, and its assay method is all to adopt ferric ion reduction resistance to oxidation analytic approach (Ferric reducing antioxidant power, FRAP), after processing sample, utilize spectrophotometer to carry out.The method of this routine has several shortcomings: 1, length consuming time.After sample preparation completes, each sample still needs 1 minute; 2, many by sample amount.Need at least 2ml of sample size of use at every turn; 3, poor repeatability.Because sample open-assembly time in air is long, the variation between sample repeated measures is larger; 4, a little less than practicality.Be merely able to measure single species or minority species at every turn, can not measure in batches, a little less than practicality.The method of this conventional determining TAC can not meet the needs of miscellaneous marine products bivalve shellfish scientific research far away, therefore, must set up the method for Fast Measurement TAC a kind of.Up to now, there is not yet any report about TAC method in Fast Measurement marine products bivalve shellfish body.
Summary of the invention
Object of the present invention is in order to provide a kind of few, reproducible, practical and measure fast the method for marine products bivalve shellfish TAC by sample amount.
The TAC of sample after the present invention utilizes the all-wave sweep type microplate reader replacement frequently spectrophotometric determination of 96 hole ELISA Plate to process, can be within the extremely short time, batch sample is measured fast and accurately, thereby provided a method for quick for resistance research between the kind of marine products bivalve shellfish.
Technical scheme of the present invention is: utilize FRAP method to process the different tissues of different marine products bivalve shellfish, obtain after the supernatant of testing sample, utilize all-wave frequency sweep type microplate reader to carry out Fast Measurement to its TAC, its operation steps is:
1, set up Trolox typical curve: 1. accurately take Trolox [(±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid], with 95% ethanol preparation mother liquor, and be diluted to gradient concentration, then get equivalent and add the distilled water and FRAP working fluid (the 0.3M acetate buffer: 10 mM TPTZ solution: 20 mM FeCl that in different test tubes, add respectively again 3 times and 30 times
3solution=10:1:1 ratio is mixed, with prepare front half an hour temporarily) build up Trolox reaction system, replace Trolox solution in contrast with 95% ethanol simultaneously; 2. after 37 ℃ of water-bath 30 min, immediately with ice-water bath cessation reaction.3. then draw every part of potpourri 200 μ l, with all-wave frequently sweep type microplate reader at 593 nm places its light absorption values of mensuration and calculate typical curve.
2, set up quantification of protein typical curve: 1. accurately take standard bovine serum albumin, prepare mother liquor with distilled water, and be diluted to gradient concentration, then get equivalent and add the 0.1g/L Coomassie brilliant blue-G250 dye liquor that adds again 5 times of volumes in different test tubes; 2. mix and standing 5min, then draw every duplicate samples 200 μ l, with all-wave frequently sweep type microplate reader at 595 nm places its light absorption values of mensuration and calculate typical curve.
3, sample of tissue: the tissue such as the closed shell flesh, the gill, outer embrane to the bivalve marine shellfish living samples respectively, and with physiological saline (0.86%) the cleaning tissue sample of being got of precooling, whole process is carried out on ice.
4, sample preparation: each tissue sample takes respectively a certain amount of tissue sample in centrifuge tube after tentatively pulverizing and stirring, and adding volume is the PBS damping fluid of 9 times of sample qualities, carries out homogenate under ice-water bath condition with 10000-15000 r/min rotating speed.With 12000 g centrifugal 10 min at 4 ℃, for subsequent use after getting supernatant and being diluted to required concentration afterwards.
5, the mensuration of testing sample Trolox concentration: replace the Trolox solution in step 1 with testing sample supernatant, all the other operate with step 1, then light absorption value are converted into Trolox concentration (μ M, C
1).
6, the mensuration of testing sample protein concentration: replace the standard protein solution in step 2 with testing sample supernatant, all the other operate with step 2, then calculate its protein concentration (μ g/ml, C according to quantification of protein typical curve
2).
7, the calculating of sample TAC (TAC): TAC=C
1/ C
2(μ mol/mg), micromole's number of the Trolox that every milligram of histone is suitable.
Advantage of the present invention and good effect are as follows:
1, take short.After sample preparation completes, each sample only needed for 10 seconds, less than 1/10 of conventional method;
2, few by sample amount.At every turn need to sample size be only 200 μ l, less than 1/10 of conventional method;
3, reproducible.Because sample measurement is quick, in air open-assembly time short, the variation between sample repeated measures is less, reproducible;
4, practical.Multiple tissues of multiple species are in bulk practically applicable to the research of miscellaneous marine products bivalve shellfish different tissues inoxidizability very much.
The present invention is a kind of method of Fast Measurement marine products bivalve shellfish TAC, can study the oxidation resistance of different marine products bivalve shellfish, different tissues fast and efficiently by this technology, to marine products bivalve shellfish resistance, research has great importance.
Embodiment
1, set up Trolox typical curve: (1) accurately takes 25.029mg Trolox, the mother liquor that is 1mM with 100mL95% ethanol compound concentration, and be diluted to 0.2mM, 0.4mM, 0.6mM, 0.8mM isoconcentration, then all gets 60 μ l and adds and in different test tubes, add respectively the distilled water of 180 μ l and the FRAP working fluid of 18000 μ L (0.3M acetate buffer: 10mM TPTZ solution: 20mM FeCl again
3solution=10:1:1 ratio is mixed, with prepare front half an hour temporarily) build up Trolox reaction system, replace Trolox solution in contrast with 95% ethanol simultaneously; After (2) 37 ℃ of water-bath 30min, immediately with ice-water bath cessation reaction.(3) then draw every part of potpourri 200 μ l, with all-wave frequently sweep type microplate reader 593nm place its light absorption value of mensuration and calculate typical curve (
y=0.0007
x-0.0107,
r 2=0.9983, wherein
yfor light absorption value,
xfor the concentration (μ M) of Trolox).
2, set up quantification of protein typical curve: (1) accurately takes standard bovine serum albumin 100mg, with 100mL distilled water compound concentration be the mother liquor of 1000 μ g/ml, and be diluted to 100 μ g/ml, 200 μ g/ml, 300 μ g/ml, 400 μ g/ml, 500 μ g/ml isoconcentrations, then get equivalent and add the 0.1g/l Coomassie brilliant blue-G250 dye liquor that adds again 5 times of volumes in different test tubes; (2) mix and standing 5min, then draw every duplicate samples 200 μ l, with all-wave frequently sweep type microplate reader 595 nm places its light absorption values of mensuration and calculate typical curve (
y=0.0031
x+ 0.0086,
r 2=0.9933, wherein
yfor light absorption value,
xfor protein content (μ g/ml)).
3, animal to be detected and sampling: comb pen shell to be determined (
atrinapectinata), blood clam (
scapharcasubcrenata), Taiwan life shellfish (
amusiumjaponicum taiwanicum), Ruditapes philippinarum (
ruditapesphilippinarum) and Perna viridis (
perna viridis) etc. five kinds of marine products bivalve shellfish all come from the wild species of In The Eastern Guangdong Sea Area.The tissue such as the closed shell flesh to its live body, the gill, outer embrane samples respectively, and cleans with the physiological saline (0.86%) of precooling the tissue sample of being got, and whole process is carried out on ice.
4, sample preparation: each tissue sample takes respectively the tissue sample of 0.1mg in centrifuge tube after tentatively pulverizing and stirring, and adding volume is the PBS damping fluid of 9 times of sample qualities, carries out homogenate under ice-water bath condition with 10000-15000 r/min rotating speed.With 12000 g centrifugal 10 min at 4 ℃, for subsequent use after getting supernatant and being diluted to required concentration afterwards.
5, the mensuration of testing sample Trolox concentration: replace the Trolox solution in step 1 with testing sample supernatant, all the other operate with step 1, then light absorption value are converted into Trolox concentration (μ M, C
1).Totally 45 of institute's test sample product (5 kinds marine products bivalve shellfish X 3 kinds organize 3 of X to repeat=45 samples) 3min consuming time altogether.
6, the mensuration of testing sample protein concentration: replace the standard protein solution in step 2 with testing sample supernatant, all the other operate with step 2, then calculate its protein concentration (μ g/ml, C according to quantification of protein typical curve
2).Institute's test sample product are totally 45 common 3min consuming time.
7, according to formula TAC=C
1/ C
2the TAC of (μ mol/mg) calculation sample.The TAC (TAC) (μ mol/mg) of 5 kinds of each tissues of marine products bivalve shellfish is listed in table 1:
The TAC (μ mol/mg) of several marine products bivalve shellfish of table 1. different tissues
Trolox typical curve of the present invention and quantification of protein typical curve both can be applicable to later each sample determination after setting up, and the TAC that can measure in batches marine products bivalve shellfish variety classes, different tissues within the extremely short time, finding speed improves 10 times of left and right than conventional method.The present invention obviously has the advantages such as quick, efficient, accurate, thereby for resistance research between the kind of marine products bivalve shellfish provides a method for quick, to marine products bivalve shellfish resistance, research has great importance.
Claims (1)
1. a method for Fast Measurement ocean bivalve shellfish TAC, comprises the following steps:
(1) set up Trolox typical curve:
1. accurately take Trolox[(±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid], with 95% ethanol preparation mother liquor, and be diluted to gradient concentration, and then get equivalent volumes solution and add and in different test tubes, add respectively again the distilled water of 3 times and the FRAP working fluid of 30 times, wherein FRAP working fluid is by 0.3M acetate buffer: 10mM TPTZ solution: 20mMFeCl
3solution=10:1:1 volume ratio mixes, and with prepare front half an hour temporarily, builds up Trolox reaction system, replaces Trolox solution in contrast with 95% ethanol simultaneously;
2. after 37 ℃ of water-bath 30min, immediately with ice-water bath cessation reaction;
3. then draw every part of potpourri 200 μ l, with all-wave frequently sweep type microplate reader at 593nm place its light absorption value of mensuration and calculate typical curve;
(2) set up quantification of protein typical curve:
1. accurately take standard bovine serum albumin, prepare mother liquor with distilled water, and be diluted to gradient concentration, then get equivalent and add the 0.1g/L Coomassie brilliant blue-G250 dye liquor that adds again 5 times of volumes in different test tubes;
2. mix and standing 5min, then draw every duplicate samples 200 μ l, with all-wave frequently sweep type microplate reader at 595nm place its light absorption value of mensuration and calculate typical curve;
(3) sample of tissue: the tissue such as the closed shell flesh, the gill, outer embrane to the bivalve marine shellfish living samples respectively, and with the physiological saline of precooling, concentration is 0.86%, the cleaning tissue sample of getting, whole process is carried out on ice;
(4) sample preparation: after each tissue sample is tentatively pulverized and stirred, take respectively a certain amount of tissue sample in centrifuge tube, adding volume is the PBS damping fluid of 9 times of sample qualities, under ice-water bath condition, carry out homogenate with 10000-15000r/min rotating speed, with 12000g centrifugal 10min at 4 ℃, for subsequent use after getting supernatant and being diluted to required concentration afterwards; Described PBS damping fluid is ml/mg with the unit of the volume mass ratio of tissue sample;
(5) mensuration of testing sample Trolox concentration: replace the Trolox solution in step (1) with testing sample supernatant, all the other operate same step (1), then light absorption value is converted into Trolox concentration, unit is μ M and is defined as C
1;
(6) mensuration of testing sample protein concentration: replace the standard protein solution in step (2) with testing sample supernatant, all the other operate same step (2), then calculate its protein concentration according to quantification of protein typical curve, unit is μ g/ml and is defined as C2;
(7) calculating of sample TAC (TAC): TAC=C
1/ C
2, unit is μ mol/mg, micromole's number of the Trolox that every milligram of histone is suitable.
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| CN110069745B (en) * | 2019-04-12 | 2023-03-03 | 山东省科学院海洋仪器仪表研究所 | Method for calculating biological life activity of bivalve shellfish |
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Non-Patent Citations (8)
| Title |
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| Integration of biochemical,histochemical and toxicogenomic indices for the assessment of health status of mussels from the Tamar Estuary,U.K;J.P.Shaw;《Marine Environmental Research》;20110730;第72卷;第13-24页 * |
| IrisF.F.The Ferric Reducing Ability of Plasma(FRAP) as a Measure of"Antioxidant Power":The FRAP Assay.《ANALYTICAL BIOCHEMISTRY》.1996,第239卷第70-76页. |
| J.P.Shaw.Integration of biochemical,histochemical and toxicogenomic indices for the assessment of health status of mussels from the Tamar Estuary,U.K.《Marine Environmental Research》.2011,第72卷第13-24页. |
| The Ferric Reducing Ability of Plasma(FRAP) as a Measure of"Antioxidant Power":The FRAP Assay;IrisF.F;《ANALYTICAL BIOCHEMISTRY》;19960715;第239卷;第70-76页 * |
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| 抗氧化能力指数(ORAC)测定原理及应用;续洁琨;《中国药理学通报》;20060831;第22卷(第8期);第1015-1021页 * |
| 拳参抗氧化活性研究;常星;《精细化工中间体》;20090430;第39卷(第2期);第28-33页 * |
| 续洁琨.抗氧化能力指数(ORAC)测定原理及应用.《中国药理学通报》.2006,第22卷(第8期),第1015-1021页. |
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