CN106153664A - A kind of detect the method for marine polysaccharide antioxidation, test solution and the method preparing test solution - Google Patents
A kind of detect the method for marine polysaccharide antioxidation, test solution and the method preparing test solution Download PDFInfo
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Abstract
The invention discloses and a kind of detect the method for marine polysaccharide antioxidation, test solution and the method preparing test solution.The method of described detection marine polysaccharide antioxidation comprises the steps: step 101, prepares the test solution to be measured for detecting marine polysaccharide antioxidation；Step 102, detects hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O in described test solution to be measured by ESR method2 ‑) concentration；Step 103, judges the antioxidant activity height of the polysaccharide sample detected according to the testing result of step 102.The method accuracy of the detection polysaccharide anti-oxidative effect that the present invention proposes is high, and can detect the marine polysaccharide antioxidation to variety classes ROS.
Description
Technical field
The invention belongs to technical field of biological, particularly relate to a kind of detect marine polysaccharide antioxidation method,
Test solution and the method preparing test solution.
Background technology
Polysaccharide is all contained in plant, animal and microbial body in nature.Polysaccharide should at medicine and field of food health care
With extensively, quickly grow, there is good DEVELOPMENT PROSPECT.Marine organisms, due to special living environment, cause marine organism
The building-up process of interior polysaccharide is different from terrestrial life, therefore produces many novel structures, acts on special active substance, the most not
Few marine organisms have significant anti-tumor function and immunoregulation effect.
Marine polysaccharide can be divided into three major types, Sargassum polysaccharides, the micro-life of marine animal polysaccharide and ocean according to source difference
Thing polysaccharide.The not biological activity of homopolysaccharide difference, activity height is affected by many factors such as the structure of polysaccharide, molecular weight,
There is the biggest difference in effect of polysaccharide medicine and health product, the biological activity of detection marine polysaccharide is to judge Polysaccharide medicine and guarantor
One of method of strong product effect.
There is animal individual difference in active polysaccharide method, workload is big, capital consumption is many in traditional Experiment of Zoology detection
Etc. problem.In cell system, the detection of antioxidant activity is generally detected by DCFH-DA at present, utilizes fluorescence during detection
Probe detects the reactive oxygen free radical (ROS) in test sample.During detection, fluorescence microplate reader plays important work
With, it judges cytoactive according to the light absorption value size of cell colony after adding fluorescent probe, and DCFH-DA is used for detecting carefully
Reactive oxygen free radical in cell space system.The principle of DCFH-DA detection method is as follows: cell is through hydrogen peroxide (H2O2) start after process
Produce inflammatory reaction, easily trigger the generation of peroxidization, and again after polysaccharide sample treatment, can effectively weaken this
Peroxidization, plays certain antioxidation, adds in DCFH-DA fluorescent probe labelling system in cell system
ROS, then excite and launch wavelength be respectively at 485nm and 530nm measure each hole fluorescence intensity, and then analyze polysaccharide resist
The effect of Oxidation.The deficiency of the method is the amount of ROS in test solution that only indirectly reflected by fluorescent value, and therefore accuracy is relatively
Difference, and fluorescent value only represents the total amount of ROS in test sample, and different types of ROS content can not be detected respectively.And
What the fluorescent value detected by DCFH-DA method represented is the total amount of ROS in test sample, and this method can be examined the most respectively
Go out different types of ROS content.
Therefore, there is poor accuracy in the method for existing detection polysaccharide anti-oxidative effect, and can not detect marine polysaccharide pair
The antioxidation of variety classes ROS.A kind of method setting up new detection marine polysaccharide antioxidant activity is the most necessary.
Electron Spin Resonance Spectra (ESR) method is a kind of according to Electron Spin Resonance Spectra degree of absorption, checks and organizes, carefully
Free radical in born of the same parents or its extracting solution, and the existence shape of radical ion is inferred by the bright mensuration reaching (Lande) g-factor
The method of state.Electron Spin Resonance Spectra technology can relatively accurately detect the content of variety classes free radical, and therefore having can
This technology can be applied to detect the antioxidation of marine polysaccharide.Figures 1 and 2 show that and capture OH respectively with DMPO certainly
By base and O2 -Free radical typical ESR spectrogram.
Electron Spin Resonance Spectra technology yet there are no in relating to external (cell system) detection Patents so far, about sea
Patent such as patent CN201210094611.8 of ocean polysaccharide relates to a kind of marine polysaccharide bi-component flocculant process sea-farming and gives up
The method of water, patent CN201010577744.1 relates to a kind of marine polysaccharide multilayer composite packaging film with antibacterial functions, specially
Profit CN200610125844.4 relates to a kind of multi-functional antistaling agent for fruits and vegetables, and patent CN200510047285.5 relates to one and surveys simultaneously
Determining the detection method of cytoactive oxygen and reduced glutathion, patent CN200610049809.9 relates to the cell of a kind of improvement
Interior active oxygen detection method, and be not found in being correlated with about the research utilizing the intracellular ROS of Electron Spin Resonance Spectra technology for detection
Patent, therefore utilize Electron Spin Resonance Spectra technology for detection marine polysaccharide antioxidant activity in cell system substantially to have breakthrough
Property meaning, is therefore hopeful on the basis of utilizing existing new and high technology, it is achieved more rapid marine polysaccharide antioxygen more accurately
The Real_time quantitative detection of change effect.
Summary of the invention
It is an object of the invention to provide a kind of side detected the method for marine polysaccharide antioxidation, test solution and prepare test solution
, there is poor accuracy solving the method for the detection polysaccharide anti-oxidative effect of prior art, and marine polysaccharide pair can not be detected in method
The technical problem of the antioxidation of variety classes ROS.
The technical scheme that the present invention realizes above-mentioned purpose is as follows:
A kind of method detecting marine polysaccharide antioxidation, comprises the steps:
Step 101, prepares the test solution to be measured for detecting marine polysaccharide antioxidation；
Step 102, detects hydroxyl radical free radical (OH) and ultra-oxygen anion free radical in described test solution to be measured by ESR method
(O2 -) concentration；
Step 103, judges the antioxidant activity height of the polysaccharide sample detected according to the testing result of step 102.
The method detecting marine polysaccharide antioxidation as above, further, wherein said step 101 includes as follows
Step:
Step A, takes polysaccharide sample preparation and becomes polysaccharide solution；
Step B, takes RAW264.7 cell, and adjusts cell density to 105～106Individual/mL；
Step C, takes the Cell sap that described step B obtains, and adds the hydrogen peroxide (H of 0.25mmol/L2O2) solution is to described
Described polysaccharide solution in step A, obtains the test solution that some parts of concentration are between 0～200 μ g/mL and concentration is different, and to it
Cultivate, the most just obtained the test solution described to be measured for detecting marine polysaccharide antioxidation.
The method detecting marine polysaccharide antioxidation as above, further, wherein said step A and described step
B is carried out simultaneously, or described step B was carried out before described step A.
The method detecting marine polysaccharide antioxidation as above, further, trains with DMEM in wherein said step A
Support base or described polysaccharide solution prepared by distilled water.
The method detecting marine polysaccharide antioxidation as above, further, wherein said step B includes using
DMEM culture medium adjusts RAW264.7 cell density.
The method detecting marine polysaccharide antioxidation as above, further, wherein said step B includes adjusting
Cross the described RAW264.7 cell of cell density to be placed at 37 DEG C and carry out cultivating 24h.
The method detecting marine polysaccharide antioxidation as above, further, wherein said step C will add H2O2
Cell sap after solution is placed in 37 DEG C, CO2The CO2 gas incubator of concentration 5% cultivates 24h, thin by add after polysaccharide solution
Cytosol is placed in 37 DEG C, CO2The CO2 gas incubator of concentration 5% cultivates 30min and 5min respectively, is respectively used to finally detect hydroxyl
Base free radical (OH) and ultra-oxygen anion free radical (O2 -) concentration.
The method detecting marine polysaccharide antioxidation as above, further, wherein said step 102 is at room temperature bar
Carry out under part.
The present invention also proposes a kind of method prepared for detecting marine polysaccharide antioxidation test solution, including walking as follows
Rapid:
Step A, takes polysaccharide sample preparation and becomes polysaccharide solution；
Step B, takes RAW264.7 cell, and adjusts cell density to 105～106Individual/mL；
Step C, takes the Cell sap that described step B obtains, and adds the hydrogen peroxide (H of 0.25mmol/L2O2) solution is to described
Described polysaccharide solution in step A, obtains the test solution that some parts of concentration are between 0～200 μ g/mL and concentration is different, and to it
Cultivate, the most just obtained the test solution described to be measured for detecting marine polysaccharide antioxidation.
The present invention also proposes a kind of test solution prepared for detecting marine polysaccharide antioxidation, and it is used with preparation as above
Prepared by the method in detection marine polysaccharide antioxidation test solution.
The invention has the beneficial effects as follows:
The preparation that the present invention proposes is for detecting the method for marine polysaccharide antioxidation test solution and preparing by the method
For detecting the test solution of marine polysaccharide antioxidation, have employed stable mononuclear phagocyte strain RAW264.7 cell, should
Cell strain had previously been widely used in molecular level to characterize the immunoregulation effect of various component, was also conventional resisting simultaneously
One of oxidation and inflammatory cell model.
Further, the present invention proposes one based on electron spin altogether according to above-mentioned preparation method and the test solution prepared
The method of wave spectrum (ESR) technology for detection marine polysaccharide antioxidation of shaking, the method is by detection mononuclear phagocyte strain
In RAW264.7 cell supernatant, ROS content evaluates marine polysaccharide antioxidant activity, reduces detection difficulty, improves detection
The stability of result, enriches detection method.
Further it is proposed that detection method have employed Electron Spin Resonance Spectra detection technique, it has detection model
Enclose wide, detection object is extensive, testing conditions simple, detection sensitivity advantages of higher.When the cell supernatant after medicine irritation
When middle ROS concentration is the lowest, utilize common method to be likely to detect, but utilize this method will be greatly improved sensitivity, and
Can in free radical aspect analysis conventional method detection result, provide a rational explanation.
Present invention achieves the most effectively detection marine polysaccharide antioxidant activity, detection process is succinct, and testing result is stable
Reliably, and the present invention has widened thinking from the detection that the angle of free radical is this aspect.
It addition, the method that the present invention proposes is expected to be applied to cytobiology, pharmaceutical analysis, environmental toxicants detection etc.
Association area.
Accompanying drawing explanation
Fig. 1 is the ESR spectrogram in prior art with DMPO capture OH free radical.
Fig. 2 is to capture O with DMPO in prior art2 -The ESR spectrogram of free radical.
Fig. 3 is the flow chart of the detection marine polysaccharide antioxidant activity of one embodiment of the invention.
Fig. 4 (A) and Fig. 4 (B) is the ESR spectrum that the detection of scallop polysaccharide antioxidant activity is obtained by the embodiment of the present invention 1
Figure.
Fig. 5 (A) and Fig. 5 (B) is that the detection of Undaria pinnatifida Suringar polysaccharide anti-oxidative activity is obtained by the embodiment of the present invention 2
ESR spectrogram.
Fig. 6 (A) and Fig. 6 (B) is that the detection of Stichopus japonicus water cooking liquid polysaccharide anti-oxidative activity is obtained by the embodiment of the present invention 3
ESR spectrogram.
Wherein,
Fig. 4 (A) show scallop polysaccharide to through H2O2The scavenging action of OH in the macrophage processed；Shown in Fig. 4 (B)
For scallop polysaccharide to through H2O2O in the macrophage processed2 -Scavenging action.In Fig. 4: (a) blank；(b)H2O2
0.25mmol/L；(c) scallop polysaccharide 12.5 μ g/mL；(d) scallop polysaccharide 25 μ g/mL；(e) scallop polysaccharide 50 μ g/mL；(f) scallop
Polysaccharide 100 μ g/mL；(g) scallop polysaccharide 200 μ g/mL.
Fig. 5 (A) show Undaria pinnatifida Suringar polysaccharide to through H2O2The scavenging action of OH in the macrophage processed；Fig. 5
(B) Undaria pinnatifida Suringar polysaccharide it is shown to through H2O2O in the macrophage processed2 -Scavenging action.In Fig. 5: (a) is blank
Comparison；(b)H2O20.25mmol/L；(c) Undaria pinnatifida Suringar polysaccharide 12.5 μ g/mL；(d) Undaria pinnatifida Suringar polysaccharide 25 μ g/
mL；(e) Undaria pinnatifida Suringar polysaccharide 50 μ g/mL；(f) Undaria pinnatifida Suringar polysaccharide 100 μ g/mL；(g) Undaria pinnatifida Suringar polysaccharide
200μg/mL。
Fig. 6 (A) show Stichopus japonicus water cooking liquid polysaccharide to through H2O2The scavenging action of OH in the macrophage processed；Fig. 6
(B) Stichopus japonicus water cooking liquid polysaccharide it is shown to through H2O2O in the macrophage processed2 -Scavenging action.In Fig. 6: (a) is blank right
According to；(b)H2O20.25mmol/L；(c) Stichopus japonicus water cooking liquid polysaccharide 12.5 μ g/mL；(d) Stichopus japonicus water cooking liquid polysaccharide 25 μ g/mL；(e)
Stichopus japonicus water cooking liquid polysaccharide 50 μ g/mL；(f) Stichopus japonicus water cooking liquid polysaccharide 100 μ g/mL；(g) Stichopus japonicus water cooking liquid polysaccharide 200 μ g/mL.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, unreceipted actual conditions in embodiment
Person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or instrument unreceipted production firm person, be permissible
By city available from conventional products.
The present invention is based on Electron Spin Resonance Spectra (ESR) technology, it is proposed that a kind of detection marine polysaccharide antioxidant activity
Method, test solution and the method for preparing test solution.The method elaborating this detection marine polysaccharide antioxidant activity in detail below, with
And preparation is for the test solution of this detection method.
As it is shown on figure 3, the method for detection marine polysaccharide antioxidation proposed by the invention, comprise the steps:
Step 101, prepares the test solution to be measured for detecting marine polysaccharide antioxidation；
Step 102, detects hydroxyl radical free radical (OH) and ultra-oxygen anion free radical in described test solution to be measured by ESR method
(O2 -) concentration；
Step 103, judges the antioxidant activity height of the polysaccharide sample detected according to the testing result of step 102.
The detection method that the present invention proposes have employed Electron Spin Resonance Spectra detection technique, its have detection range wide,
Detection object is extensive, testing conditions simple, detection sensitivity advantages of higher.As ROS in the cell supernatant after medicine irritation
When concentration is the lowest, utilize common method to be likely to detect, but utilize this method will be greatly improved sensitivity, and can be certainly
By the result of analysis conventional method detection on basic unit face, provide a rational explanation.
Present invention achieves the most effectively detection marine polysaccharide antioxidant activity, detection process is succinct, and testing result is stable
Reliably, and the present invention has widened thinking from the detection that the angle of free radical is this aspect.
RAW264.7 cell strain had previously been widely used in molecular level to characterize the immunoregulation effect of various component,
Also it is one of conventional antioxidation and inflammatory cell model simultaneously.This cell strain growth density is relatively big, and typically larger than 106, but real
In view of 96 orifice plate nominal volume in the operation of border, typically cell density is adjusted to 105～106The most suitable.
Wherein, described step 101 comprises the steps:
Step A, takes polysaccharide sample preparation and becomes polysaccharide solution；
Specifically, DMEM culture medium or distilled water is used to prepare described polysaccharide solution.Generally in cell experiment, sample is multiplex
Culture medium is prepared, to ensure that in cell growth, desired nutritional material is sufficient, but the example that also existence is prepared with distilled water, steam
The benefit of distilled water preparation is to avoid component complicated in culture medium, it is possible to the simple investigation sample effect to cell.
Step B, takes RAW264.7 cell, and adjusts cell density to 105～106Individual/mL；
Specifically, use DMEM culture medium to adjust RAW264.7 cell density, and will adjust described in cell density
RAW264.7 cell is placed at 37 DEG C and carries out cultivating 24h.Generally mammalian cell is all carried out often in 37 DEG C of cell culture incubators
Rule are cultivated, and incubation time is depending on practical situation.
Wherein, described step A also is able to carry out with described step B simultaneously, or described step B is in the advance of described step A
OK.It practice, the order of step A and step B is without Compulsory Feature, can formulate voluntarily according to practical operation situation.
Step C, takes the Cell sap that described step B obtains, and adds the hydrogen peroxide (H of 0.25mmol/L2O2) solution is to described
Described polysaccharide solution in step A, obtains the test solution that some parts of concentration are between 0～200 μ g/mL and concentration is different, and to it
Cultivate, the most just obtained the test solution described to be measured for detecting marine polysaccharide antioxidation.
Specifically, it will add H2O2Cell sap after solution is placed in 37 DEG C, CO2The CO2 gas incubator training of concentration 5%
Supporting 24h, the Cell sap after adding polysaccharide solution is placed in 37 DEG C, CO2The CO2 gas incubator of concentration 5% is cultivated respectively
30min and 5min, is respectively used to finally detect hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O2 -) concentration.From
Extremely short by the base life-span, i.e. there is cancellation in the short time, after cancellation, signal is no longer caught in, and gropes according to substantial amounts of experiment, sends out
Existing, within 5～30min time periods, the free radical of generation can completely be detected.
Described step 102 is carried out under normal temperature condition.In the present invention, step 102 is suitably entered at normal temperature condition ESR equipment
Row test, the results contrast tested out is accurate.
Specifically, ESR detection hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O is being utilized2 -During),
The design parameter of ESR is the most as follows:
OH: X-band；Modulating frequency: 100kHz；Resonant frequency: 9.8GHz；Microwave power: 20mW；Modulation amplitude:
0.2mT；Central magnetic field: 336mT；Sweep time: 30s；Sweep length: 10mT；Time constant: 0.1s；Temperature: 26 DEG C.
O2 -: X-band；Modulating frequency: 100kHz；Resonant frequency: 9.44GHz；Microwave power: 1.16mW；Modulated amplitude
Degree: 0.1mT；Central magnetic field: 336mT；Sweep time: 30s；Sweep length: 10mT；Time constant: 3s；Temperature: 26 DEG C.
Described step 103 judges the antioxidant activity of the polysaccharide sample detected according to the testing result of described step 102
Just.
Specifically, cloudy according to the hydroxyl radical free radical (OH) in the marine polysaccharide sample to be tested comparing variable concentrations and super oxygen
Ion radical (O2 -) concentration, judge detected polysaccharide sample antioxidant activity height.If test solution to be measured is many
Sugar concentration is the highest, detected hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O2 -) concentration the lowest, and
Detected hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O2 -) concentration treat the polysaccharide concentration of test fluid
The most sensitive, then the antioxidation of the polysaccharide sample that explanation is detected is the strongest, otherwise the strongest.
The invention allows for a kind of method prepared for detecting marine polysaccharide antioxidation test solution, including walking as follows
Rapid:
Step A, takes polysaccharide sample preparation and becomes polysaccharide solution；
Step B, takes RAW264.7 cell, and adjusts cell density to 105～106Individual/mL；
Step C, takes the Cell sap that described step B obtains, and adds the hydrogen peroxide H of 0.25mM2O2Solution is to described step A
In described polysaccharide solution, obtain the test solution that some parts of concentration are between 0～200 μ g/mL and concentration is different, and it trained
Support, the most just obtained the test solution described to be measured for detecting marine polysaccharide antioxidation.
The present invention also proposes a kind of test solution prepared for detecting marine polysaccharide antioxidation, and it is used with preparation as above
Prepared by the method in detection marine polysaccharide antioxidation test solution.It is used for detecting marine polysaccharide antioxidation, energy by this test solution
Reduce the difficulty of detection, improve the stability of testing result.
The detection of example 1 scallop polysaccharide antioxidant activity in macrophage
Step 101 ', prepare the test solution to be measured for detecting marine polysaccharide antioxidation；
Specifically, take scallop polysaccharide sample, be configured to the fan of 200 μ g/mL (micrograms per millilitre, lower same) by DMEM culture medium
Pattra sugar juice；Take hydrogen peroxide (H2O2) sample, with the H of DMEM culture medium preparation 0.25mmol/L2O2Solution；Train with DMEM
Support base to adjust RAW264.7 cell density to 3 × 105Individual/mL；The cell adjusting density is placed in 96 porocyte culture plates
In, at 37 DEG C, cultivate 24h；Appropriate H is added in described 96 well culture plates2O2Solution so that it is concentration is 0.25mmol/L；Will
It is 37 DEG C, CO that described 96 porocyte culture plates are placed in temperature2Concentration be 5% CO2 gas incubator in cultivate 24h；To 96 holes
Culture plate adds appropriate scallop polysaccharide solution, makes scallop polysaccharide concentration in different hole be respectively 0 μ g/mL, 12.5 μ g/mL, 25 μ
g/mL、50μg/mL、100μg/mL、200μg/mL；It is 37 DEG C, CO that described 96 well culture plates are placed in temperature2Concentration is 5%
CO2 gas incubator continues respectively cultivate 30min and 5min, be respectively used to finally detect hydroxyl radical free radical (OH) and surpass
Oxygen anion free radical (O2 -) concentration；Collect described 96 well culture plates difference polysaccharide concentration Cell sap (cell conditioned medium), this
It it is i.e. the test solution to be measured for detecting scallop polysaccharide antioxidation.
Step 102 ', detect hydroxyl radical free radical (OH) and ultra-oxygen anion free radical in described test solution to be measured by ESR method
(O2 -) concentration；
Specifically, in tube pipe, add test solution to be measured and ROS trapping agent (DMPO) and fully mix；Capillary tube is utilized to inhale
Mixed liquor is to suitably height, and seals capillary tube end opening with vaseline, then cleans the vaseline that tube wall is remaining；Utilize ESR technology
Detection hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O2 -) generate situation, obtain DMPO-OO under each polysaccharide concentration
And Fig. 4 is shown in by DMPO-OH signal collection of illustrative plates (H).Utilizing ESR detection hydroxyl radical free radical (OH) and ultra-oxygen anion free radical
(O2 -During), the design parameter of ESR is the most as follows:
OH: X-band；Modulating frequency: 100kHz；Resonant frequency: 9.8GHz；Microwave power: 20mW；Modulation amplitude:
0.2mT；Central magnetic field: 336mT；Sweep time: 30s；Sweep length: 10mT；Time constant: 0.1s；Temperature: 26 DEG C.
O2 -: X-band；Modulating frequency: 100kHz；Resonant frequency: 9.44GHz；Microwave power: 1.16mW；Modulated amplitude
Degree: 0.1mT；Central magnetic field: 336mT；Sweep time: 30s；Sweep length: 10mT；Time constant: 3s；Temperature: 26 DEG C.
The testing result of step 103 ', according to step 102 ' judges the antioxidant activity height of the polysaccharide sample detected.
According to step 102 ' obtained in collection of illustrative plates judge described scallop polysaccharide sample antioxidant activity.As shown in Figure 4,
H2O2The peak height of matched group occurs significantly to increase, in contrast, after adding the scallop polysaccharide of increasing concen-trations, OH and O2 -'s
ESR peak height all occurs substantially to reduce, and shows that scallop polysaccharide effectively prevents oxidation course, has preferable antioxidation.
The detection of example 2 Undaria pinnatifida Suringar polysaccharide antioxidant activity in macrophage
Step 101 ", prepare the test solution to be measured for detecting marine polysaccharide antioxidation；
Specifically, take RAW264.7 cell, by DMEM culture medium, RAW264.7 cell density is adjusted to 5 × 105Individual/
mL.Then, take Undaria pinnatifida Suringar polysaccharide sample, be configured to the Undaria pinnatifida Suringar polysaccharide solution of 200 μ g/mL with distilled water；
Take hydrogen peroxide (H2O2) sample, with the H of distilled water preparation 0.25mmol/L2O2Solution；The cell adjusting density is placed in 96
In porocyte culture plate, at 37 DEG C, cultivate 24h；Appropriate H is added in described 96 well culture plates2O2Solution so that it is concentration is
0.25mmol/L；Described 96 porocyte culture plates are placed in temperature is 37 DEG C, CO2Concentration is in the CO2 gas incubator of 5%
Cultivate 24h；In 96 well culture plates, add appropriate Undaria pinnatifida Suringar polysaccharide solution, make Undaria pinnatifida Suringar polysaccharide in different hole
Concentration is respectively 0 μ g/mL, 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL；By described 96 well culture plates
Being placed in temperature is 37 DEG C, CO2Concentration be 5% CO2 gas incubator in respectively continue cultivate 30min and 5min, be respectively used to
Finally detect hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O2 -) concentration；Collect described 96 well culture plates different
The Cell sap (cell conditioned medium) of polysaccharide concentration, this is i.e. for detecting the to be tested of Undaria pinnatifida Suringar polysaccharide anti-oxidative effect
Liquid.
Step 102 ", detect hydroxyl radical free radical (OH) and ultra-oxygen anion free radical in described test solution to be measured by ESR method
(O2 -) concentration；
Specifically, in tube pipe, add test solution to be measured and ROS trapping agent (DMPO) and fully mix；Capillary tube is utilized to inhale
Mixed liquor is to suitably height, and seals capillary tube end opening with vaseline, then cleans the vaseline that tube wall is remaining；Utilize ESR technology
Detection hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O2 -) generate situation, obtain DMPO-OO under each polysaccharide concentration
And Fig. 5 is shown in by DMPO-OH signal collection of illustrative plates (H).Utilizing ESR detection hydroxyl radical free radical (OH) and ultra-oxygen anion free radical
(O2 -During), the design parameter of ESR is the most as follows:
OH: X-band；Modulating frequency: 100kHz；Resonant frequency: 9.8GHz；Microwave power: 20mW；Modulation amplitude:
0.2mT；Central magnetic field: 336mT；Sweep time: 30s；Sweep length: 10mT；Time constant: 0.1s；Temperature: 26 DEG C.
O2 -: X-band；Modulating frequency: 100kHz；Resonant frequency: 9.44GHz；Microwave power: 1.16mW；Modulated amplitude
Degree: 0.1mT；Central magnetic field: 336mT；Sweep time: 30s；Sweep length: 10mT；Time constant: 3s；Temperature: 26 DEG C.
Step 103 ", according to step 102 " testing result judge detected polysaccharide sample antioxidant activity height.
According to step 102 " obtained in collection of illustrative plates judge described Undaria pinnatifida Suringar polysaccharide sample antioxidant activity.By
Fig. 5 understands, H2O2The peak height of matched group occurs significantly to increase, and in contrast, adds the Undaria pinnatifida Suringar polysaccharide of increasing concen-trations
After, OH and O2 -ESR peak height all occur substantially to reduce, show Undaria pinnatifida Suringar polysaccharide effectively prevent aoxidize into
Journey, has preferable antioxidation.
The detection of example 3 Stichopus japonicus water cooking liquid polysaccharide antioxidant activity in macrophage
Step 101 " ', prepare the test solution to be measured for detecting marine polysaccharide antioxidation；
Specifically, taking Stichopus japonicus water cooking liquid polysaccharide sample, the Stichopus japonicus water cooking liquid being configured to 200 μ g/mL by DMEM culture medium is many
Sugar juice；Take hydrogen peroxide (H2O2) sample, with the H of DMEM culture medium preparation 0.25mmol/L2O2Solution；Use DMEM culture medium
RAW264.7 cell density is adjusted to 1 × 106Individual/mL；The cell adjusting density is placed in 96 porocyte culture plates,
24h is cultivated at 37 DEG C；Appropriate H is added in described 96 well culture plates2O2Solution so that it is concentration is 0.25mmol/L；By described 96
It is 37 DEG C, CO that porocyte culture plate is placed in temperature2Concentration be 5% CO2 gas incubator in cultivate 24h；To 96 well culture plates
Middle addition appropriate Stichopus japonicus water cooking liquid polysaccharide solution, makes Stichopus japonicus water cooking liquid polysaccharide concentration in different hole be respectively 0 μ g/mL, 12.5 μ g/
mL、25μg/mL、50μg/mL、100μg/mL、200μg/mL；It is 37 DEG C, CO that described 96 well culture plates are placed in temperature2Concentration is
The CO2 gas incubator of 5% continues respectively cultivate 30min and 5min, be respectively used to finally detect hydroxyl radical free radical (OH)
And ultra-oxygen anion free radical (O2 -) concentration；Collect the Cell sap of described 96 well culture plate difference polysaccharide concentrations (on cell
Clearly), this is i.e. the test solution to be measured for detecting Stichopus japonicus water cooking liquid polysaccharide anti-oxidative effect.
Step 102 " ', detect hydroxyl radical free radical (OH) and ultra-oxygen anion free radical in described test solution to be measured by ESR method
(O2 -) concentration；
Specifically, in tube pipe, add test solution to be measured and ROS trapping agent (DMPO) and fully mix；Capillary tube is utilized to inhale
Mixed liquor is to suitably height, and seals capillary tube end opening with vaseline, then cleans the vaseline that tube wall is remaining；Utilize ESR technology
Detection hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O2 -) generate situation, obtain DMPO-OO under each polysaccharide concentration
And Fig. 6 is shown in by DMPO-OH signal collection of illustrative plates (H).Utilizing ESR detection hydroxyl radical free radical (OH) and ultra-oxygen anion free radical
(O2 -During), the design parameter of ESR is as follows:
OH: X-band；Modulating frequency: 100kHz；Resonant frequency: 9.8GHz；Microwave power: 20mW；Modulation amplitude:
0.2mT；Central magnetic field: 336mT；Sweep time: 30s；Sweep length: 10mT；Time constant: 0.1s；Temperature: 26 DEG C.
O2 -: X-band；Modulating frequency: 100kHz；Resonant frequency: 9.44GHz；Microwave power: 1.16mW；Modulated amplitude
Degree: 0.1mT；Central magnetic field: 336mT；Sweep time: 30s；Sweep length: 10mT；Time constant: 3s；Temperature: 26 DEG C.
Step 103 " testing result of ', according to step 102 " ' judges that the antioxidant activity of polysaccharide sample detected is high
Low.
According to step 102 " ' obtained in collection of illustrative plates judge described Stichopus japonicus water cooking liquid polysaccharide sample antioxidant activity.By
Fig. 6 understands, H2O2The peak height of matched group occurs significantly to increase, and in contrast, adds the Stichopus japonicus water cooking liquid polysaccharide of increasing concen-trations
After, OH and O2 -ESR peak height all occur substantially to reduce, show that Stichopus japonicus water cooking liquid polysaccharide effectively prevents oxidation course,
There is preferable antioxidation.
Knowable to above-described embodiment, the preparation that the present invention proposes is for the method detecting marine polysaccharide antioxidation test solution
And with the test solution for detecting marine polysaccharide antioxidation that the method is prepared, have employed stable mononuclear phagocyte strain
RAW264.7 cell, this cell strain had previously been widely used in molecular level to characterize the immunoregulation effect of various component,
Also it is one of conventional antioxidation and inflammatory cell model simultaneously.
Further, the present invention according to above-mentioned preparation method and the test solution prepared propose based on electron spin resonance ripple
The method of spectrum (ESR) technology for detection marine polysaccharide antioxidation, by detection mononuclear phagocyte strain RAW264.7 cell
In clear liquid, ROS content evaluates marine polysaccharide antioxidant activity, reduces detection difficulty, improves the stability of testing result,
Enrich detection method.
Further it is proposed that detection method have employed Electron Spin Resonance Spectra detection technique, it has detection model
Enclose wide, detection object is extensive, testing conditions simple, detection sensitivity advantages of higher.When the cell supernatant after medicine irritation
When middle ROS concentration is the lowest, utilize common method to be likely to detect, but utilize this method will be greatly improved sensitivity, and
Can in free radical aspect analysis conventional method detection result, provide a rational explanation.
Present invention achieves the most effectively detection marine polysaccharide antioxidant activity, detection process is succinct, and testing result is stable
Reliably, and the present invention has widened thinking from the detection that the angle of free radical is this aspect.
Therefore, the method that the present invention proposes is expected to be applied to cytobiology, pharmaceutical analysis, environmental toxicants detection etc.
Association area.
Above example is only the exemplary embodiment of the present invention, is not used in the restriction present invention, protection scope of the present invention
It is defined by the claims.The present invention can be made respectively in the essence of the present invention and protection domain by those skilled in the art
Planting amendment or equivalent, this amendment or equivalent also should be regarded as being within the scope of the present invention.
Claims (10)
1. the method detecting marine polysaccharide antioxidation, it is characterised in that it comprises the steps:
Step 101, prepares the test solution to be measured for detecting marine polysaccharide antioxidation；
Step 102, detects hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O in described test solution to be measured by ESR method2 -·)
Concentration；
Step 103, judges the antioxidant activity height of the polysaccharide sample detected according to the testing result of step 102.
2. the method for claim 1, it is characterised in that wherein said step 101 comprises the steps:
Step A, takes polysaccharide sample preparation and becomes polysaccharide solution；
Step B, takes RAW264.7 cell, and adjusts cell density to 105～106Individual/mL；
Step C, takes the Cell sap that described step B obtains, and adds the hydrogen peroxide (H of 0.25mmol/L2O2) solution is to described step
Described polysaccharide solution in A, obtains the test solution that some parts of concentration are between 0～200 μ g/mL and concentration is different, and carries out it
Cultivate, the most just obtained the test solution described to be measured for detecting marine polysaccharide antioxidation.
3. method as claimed in claim 2, it is characterised in that wherein said step A and described step B are carried out simultaneously, or institute
State step B to carry out before described step A.
4. method as claimed in claim 2, it is characterised in that join by DMEM culture medium or distilled water in wherein said step A
Make described polysaccharide solution.
5. method as claimed in claim 2, it is characterised in that wherein said step B includes using DMEM culture medium to adjust
RAW264.7 cell density.
6. method as claimed in claim 2, it is characterised in that wherein said step B includes adjusting the institute of cell density
State RAW264.7 cell be placed at 37 DEG C carry out cultivate 24h.
7. method as claimed in claim 2, it is characterised in that wherein said step C will add H2O2Cell sap after solution is put
In 37 DEG C, CO2The CO2 gas incubator of concentration 5% cultivates 24h, and the Cell sap after adding polysaccharide solution is placed in 37 DEG C, CO2
The CO2 gas incubator of concentration 5% cultivates 30min and 5min respectively, be respectively used to finally to detect hydroxyl radical free radical (OH) and
Ultra-oxygen anion free radical (O2 -) concentration.
8. the method for claim 1, it is characterised in that wherein said step 102 is carried out under normal temperature condition.
9. the method prepared for detecting marine polysaccharide antioxidation test solution, it is characterised in that it comprises the steps:
Step A, takes polysaccharide sample preparation and becomes polysaccharide solution；
Step B, takes RAW264.7 cell, and adjusts cell density to 105～106Individual/mL；
Step C, takes the Cell sap that described step B obtains, and adds the hydrogen peroxide (H of 0.25mmol/L2O2) solution is to described step
Described polysaccharide solution in A, obtains the test solution that some parts of concentration are between 0～200 μ g/mL and concentration is different, and carries out it
Cultivate, the most just obtained the test solution described to be measured for detecting marine polysaccharide antioxidation.
10. the test solution prepared for detecting marine polysaccharide antioxidation, it is characterised in that it is with such as claim 9 institute
Prepared by the method stated.
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