CN106153664B - A kind of method, test solution and method for preparing test solution for detecting marine polysaccharide antioxidation - Google Patents
A kind of method, test solution and method for preparing test solution for detecting marine polysaccharide antioxidation Download PDFInfo
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 169
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 169
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 169
- 230000003064 anti-oxidating effect Effects 0.000 title claims abstract description 81
- 238000000034 method Methods 0.000 title claims abstract description 76
- 239000012085 test solution Substances 0.000 title claims abstract description 68
- -1 hydroxyl radical free radical Chemical class 0.000 claims abstract description 46
- 230000000694 effects Effects 0.000 claims abstract description 39
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 28
- 239000001301 oxygen Substances 0.000 claims abstract description 28
- 238000012360 testing method Methods 0.000 claims abstract description 20
- 239000000243 solution Substances 0.000 claims description 37
- 239000007788 liquid Substances 0.000 claims description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 24
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 13
- 229910052799 carbon Inorganic materials 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 7
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims 4
- 229960002163 hydrogen peroxide Drugs 0.000 claims 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims 2
- 239000004408 titanium dioxide Substances 0.000 claims 2
- 238000001514 detection method Methods 0.000 abstract description 76
- 230000003078 antioxidant effect Effects 0.000 abstract description 9
- 210000004027 cell Anatomy 0.000 description 58
- 238000001362 electron spin resonance spectrum Methods 0.000 description 38
- 239000000523 sample Substances 0.000 description 31
- 241000251511 Holothuroidea Species 0.000 description 17
- 241001261506 Undaria pinnatifida Species 0.000 description 17
- 238000010411 cooking Methods 0.000 description 17
- 241000237509 Patinopecten sp. Species 0.000 description 16
- 150000003254 radicals Chemical class 0.000 description 16
- 235000020637 scallop Nutrition 0.000 description 16
- 238000005516 engineering process Methods 0.000 description 13
- 210000002540 macrophage Anatomy 0.000 description 13
- 238000002360 preparation method Methods 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 238000012545 processing Methods 0.000 description 7
- VCUVETGKTILCLC-UHFFFAOYSA-N 5,5-dimethyl-1-pyrroline N-oxide Chemical compound CC1(C)CCC=[N+]1[O-] VCUVETGKTILCLC-UHFFFAOYSA-N 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 229940099259 vaseline Drugs 0.000 description 6
- 230000008901 benefit Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 4
- 230000007365 immunoregulation Effects 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000004969 inflammatory cell Anatomy 0.000 description 3
- 230000007794 irritation Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000002000 scavenging effect Effects 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 239000003636 conditioned culture medium Substances 0.000 description 2
- 231100001238 environmental toxicant Toxicity 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000005220 pharmaceutical analysis Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000004435 EPR spectroscopy Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 229920000869 Homopolysaccharide Polymers 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229920006280 packaging film Polymers 0.000 description 1
- 239000012785 packaging film Substances 0.000 description 1
- 150000005837 radical ions Chemical class 0.000 description 1
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- 210000001519 tissue Anatomy 0.000 description 1
- 238000012549 training Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- G01N24/10—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using electron paramagnetic resonance
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- High Energy & Nuclear Physics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of method, test solution and method for preparing test solution for detecting marine polysaccharide antioxidation.The method of the detection marine polysaccharide antioxidation includes the following steps：Step 101, prepare to be used for the test solution for detecting marine polysaccharide antioxidation；Step 102, hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O in the test solution are detected with ESR methods2 ‑) concentration；Step 103, the antioxidation activity height of detected polysaccharide sample is judged according to the testing result of step 102.The method accuracy of detection polysaccharide anti-oxidative effect proposed by the present invention is high, and can detect antioxidation of the marine polysaccharide to variety classes ROS.
Description
Technical field
The invention belongs to technical field of biological, more particularly to a kind of method for detecting marine polysaccharide antioxidation,
Test solution and the method for preparing test solution.
Background technology
All contain polysaccharide in plant, animal and microbial body in nature.Polysaccharide should in medicine and field of food health care
With extensive, quickly grow, there is good development prospect.Marine organisms cause marine organism due to special living environment
The building-up process of interior polysaccharide is different from terrestrial life, therefore produces the active material that many structures are novel, effect is special, wherein not
Few marine organisms have significant anti-tumor function and immunoregulation effect.
Marine polysaccharide can be divided into three categories according to source difference, algal polysaccharides, the micro- life of marine animal polysaccharide and ocean
Thing polysaccharide.Difference, the active just structure by polysaccharide, molecular weight etc. many factors are not influenced the bioactivity of homopolysaccharide,
There is very big difference in the effect of polysaccharide similar drug and health products, the bioactivity for detecting marine polysaccharide is to judge Polysaccharide medicine and guarantor
One of method of strong product effect.
There are animal individual difference, heavy workload, capital consumption are more for traditional Experiment of Zoology detection active polysaccharide method
The problems such as.The detection of antioxidation activity is usually detected by DCFH-DA in cell system at present, and fluorescence is utilized in detection process
Probe detects the active oxygen radical in test sample (ROS).In detection process, fluorescence microplate reader plays important work
With it judges cytoactive according to the light absorption value size of cell colony after addition fluorescence probe, and DCFH-DA is used for detecting carefully
Active oxygen radical in cell space system.The principle of DCFH-DA detection methods is as follows：Cell is through hydrogen peroxide (H2O2) start after processing
Inflammatory reaction is produced, easily triggers the generation of peroxidization, and again after polysaccharide sample is handled, can effectively it weaken this
Peroxidization, plays certain antioxidation, is added in cell system in DCFH-DA fluorescence probe mark systems
ROS, is respectively then that the fluorescence intensity in each hole is measured at 485nm and 530nm in excitation and launch wavelength, and then analyzes polysaccharide and resist
The effect of oxidation.The deficiency of this method is only to reflect the amount of ROS in test solution indirectly by fluorescent value, thus accuracy compared with
Difference, and fluorescent value only represents the total amount of ROS in test sample, and different types of ROS contents cannot be detected respectively.And
What the fluorescent value detected by DCFH-DA methods represented is the total amount of ROS in test sample, and this method can be examined accurately respectively
Go out different types of ROS contents.
Therefore, the method for existing detection polysaccharide anti-oxidative effect is there are poor accuracy, and cannot detect marine polysaccharide pair
The antioxidation of variety classes ROS.It is extremely necessary to establish a kind of method of new detection marine polysaccharide antioxidation activity.
Electron Spin Resonance Spectra (ESR) method is a kind of according to Electron Spin Resonance Spectra degree of absorption, checks tissue, thin
Free radical in born of the same parents or its extracting solution, and by it is bright up to the measure of (Lande) g factors come infer radical ion there are shape
The method of state.Electron Spin Resonance Spectra technology can relatively accurately detect the content of variety classes free radical, therefore have can
This technology can be applied to the antioxidation of detection marine polysaccharide.Figures 1 and 2 show that capture OH respectively with DMPO certainly
By base and O2 -The typical ESR spectrograms of free radical.
Electron Spin Resonance Spectra technology yet there are no in being related to external (cell system) detection related patents, on sea so far
The patent of foreign polysaccharide such as patent CN201210094611.8 is related to a kind of marine polysaccharide bi-component flocculant process sea-farming and gives up
The method of water, patent CN201010577744.1 are related to a kind of marine polysaccharide multilayer composite packaging film with antibacterial functions, specially
Sharp CN200610125844.4 is related to a kind of multi-functional antistaling agent for fruits and vegetables, and patent CN200510047285.5 is related to a kind of while surveys
Determine the detection method of cytoactive oxygen and reduced glutathione, patent CN200610049809.9 is related to a kind of cell of improvement
Interior active oxygen detection method, and on being not found in correlation using the research of the intracellular ROS of Electron Spin Resonance Spectra technology for detection
Patent, therefore utilize Electron Spin Resonance Spectra technology for detection marine polysaccharide antioxidation activity in cell system substantially to have and break through
Property meaning, therefore be hopeful, using on the basis of existing new and high technology, to realize more rapidly more accurately marine polysaccharide antioxygen
The Real_time quantitative detection of change effect.
The content of the invention
The object of the present invention is to provide a kind of method, test solution and side for preparing test solution for detecting marine polysaccharide antioxidation
Method, to solve the method for the detection polysaccharide anti-oxidative of prior art effect there are poor accuracy, and cannot detect marine polysaccharide pair
The technical problem of the antioxidation of variety classes ROS.
The present invention realizes that the technical solution of above-mentioned purpose is as follows：
A kind of method for detecting marine polysaccharide antioxidation, includes the following steps：
Step 101, prepare to be used for the test solution for detecting marine polysaccharide antioxidation；
Step 102, hydroxyl radical free radical (OH) and ultra-oxygen anion free radical in the test solution are detected with ESR methods
(O2 -) concentration；
Step 103, the antioxidation activity height of detected polysaccharide sample is judged according to the testing result of step 102.
The method of detection marine polysaccharide antioxidation as described above, further, wherein the step 101 is including as follows
Step：
Step A, takes polysaccharide sample to be configured to polysaccharide solution；
Step B, takes RAW264.7 cells, and adjusts cell density to 105~106A/mL；
Step C, the cell liquid for taking the step B to obtain, adds the hydrogen peroxide (H of 0.25mmol/L2O2) solution is to described
The polysaccharide solution in step A, obtains the test solution that several pieces concentration is between 0~200 μ g/mL and concentration is different, and to it
Cultivated, thus just obtained being used for the test solution for detecting marine polysaccharide antioxidation.
The method of detection marine polysaccharide antioxidation as described above, further, wherein the step A and the step
B is carried out at the same time, or the step B is carried out before the step A.
The method of detection marine polysaccharide antioxidation as described above, further, wherein being trained in the step A with DMEM
Support base or distilled water prepares the polysaccharide solution.
The method of detection marine polysaccharide antioxidation as described above, further, wherein the step B is included the use of
DMEM culture mediums adjust RAW264.7 cell densities.
The method of detection marine polysaccharide antioxidation as described above, further, wherein the step B includes adjusting
The RAW264.7 cells for crossing cell density are placed at 37 DEG C and carry out culture 24h.
The method of detection marine polysaccharide antioxidation as described above, further, wherein the step C will add H2O2
Cell liquid after solution is placed in 37 DEG C, CO2The CO2gas incubator culture 24h of concentration 5%, will be thin after addition polysaccharide solution
Cytosol is placed in 37 DEG C, CO2The CO2gas 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 of detection marine polysaccharide antioxidation as described above, further, wherein the step 102 is in room temperature bar
Carried out under part.
The present invention also proposes a kind of method prepared for detecting marine polysaccharide antioxidation test solution, including following step
Suddenly：
Step A, takes polysaccharide sample to be configured to polysaccharide solution；
Step B, takes RAW264.7 cells, and adjusts cell density to 105~106A/mL；
Step C, the cell liquid for taking the step B to obtain, adds the hydrogen peroxide (H of 0.25mmol/L2O2) solution is to described
The polysaccharide solution in step A, obtains the test solution that several pieces concentration is between 0~200 μ g/mL and concentration is different, and to it
Cultivated, thus just obtained being used for the test solution for detecting marine polysaccharide antioxidation.
The present invention also proposes a kind of test solution prepared for detecting marine polysaccharide antioxidation, it is used with preparation as above
Prepared in the method for detection marine polysaccharide antioxidation test solution.
The beneficial effects of the invention are as follows：
It is proposed by the present invention to prepare the method for being used for detecting marine polysaccharide antioxidation test solution and prepared with this method
Be used for detect the test solution of marine polysaccharide antioxidation, employ stable mononuclear macrophage strain RAW264.7 cells, should
Cell line had previously been widely used in molecular level to characterize the immunoregulation effect of various components, while was also common anti-
One of oxidation and inflammatory cell model.
Further, the present invention is proposed one kind according to above-mentioned preparation method and the test solution prepared and is total to based on electron spin
The method that vibration wave composes (ESR) technology for detection marine polysaccharide antioxidation, this method is by detecting mononuclear macrophage strain
ROS contents evaluate marine polysaccharide antioxidation activity in RAW264.7 cell supernatants, reduce detection difficulty, improve detection
As a result stability, enriches detection method.
Further it is proposed that detection method employ Electron Spin Resonance Spectra detection technique, its have detection model
Enclose the advantages that wide, detection object is extensive, testing conditions are simple, detection sensitivity is high.When the cell supernatant after medicine irritation
When middle ROS concentration is very low, it is likely to detect using common method, but sensitivity will be greatly improved using this method, and
Can in free radical aspect rational being explained as a result, providing one of detecting of analysis conventional method.
The present invention realizes fast and effective detection marine polysaccharide antioxidation activity, and detection process is succinct, and testing result is stablized
Reliably, and the present invention has widened thinking from the angle of free radical for the detection of this aspect.
In addition, method proposed by the present invention is expected to be applied to cell biology, Pharmaceutical Analysis, environmental toxicants detection etc.
Association area.
Brief description of the drawings
Fig. 1 is in the prior art with the ESR spectrograms of DMPO capture OH free radicals.
Fig. 2 is to capture O with DMPO in the prior art2 -The ESR spectrograms of free radical.
Fig. 3 is the flow chart of the detection marine polysaccharide antioxidation activity of one embodiment of the invention.
Fig. 4 (A) and Fig. 4 (B) is the ESR spectrums that detection of the embodiment of the present invention 1 to scallop polysaccharide antioxidation activity is obtained
Figure.
Fig. 5 (A) and Fig. 5 (B) is that detection of the embodiment of the present invention 2 to Undaria pinnatifida Suringar polysaccharide anti-oxidative activity is obtained
ESR spectrograms.
Fig. 6 (A) and Fig. 6 (B) is that detection of the embodiment of the present invention 3 to sea cucumber water cooking liquid polysaccharide anti-oxidative activity is obtained
ESR spectrograms.
Wherein,
Fig. 4 (A) show scallop polysaccharide to through H2O2The scavenging action of OH in the macrophage of processing；Shown in Fig. 4 (B)
It is scallop polysaccharide to through H2O2O in the macrophage of processing2 -Scavenging action.In Fig. 4：(a) blank control；(b)H2O2
0.25mmol/L；(c) 12.5 μ g/mL of scallop polysaccharide；(d) 25 μ g/mL of scallop polysaccharide；(e) 50 μ g/mL of scallop polysaccharide；(f) scallop
100 μ g/mL of polysaccharide；(g) 200 μ g/mL of scallop polysaccharide.
Fig. 5 (A) show Undaria pinnatifida Suringar polysaccharide to through H2O2The scavenging action of OH in the macrophage of processing；Fig. 5
(B) Undaria pinnatifida Suringar polysaccharide is shown to through H2O2O in the macrophage of processing2 -Scavenging action.In Fig. 5：(a) blank
Control；(b)H2O20.25mmol/L；(c) 12.5 μ g/mL of Undaria pinnatifida Suringar polysaccharide；(d) 25 μ g/ of Undaria pinnatifida Suringar polysaccharide
mL；(e) 50 μ g/mL of Undaria pinnatifida Suringar polysaccharide；(f) 100 μ g/mL of Undaria pinnatifida Suringar polysaccharide；(g) Undaria pinnatifida Suringar polysaccharide
200μg/mL。
Fig. 6 (A) show sea cucumber water cooking liquid polysaccharide to through H2O2The scavenging action of OH in the macrophage of processing；Fig. 6
(B) sea cucumber water cooking liquid polysaccharide is shown to through H2O2O in the macrophage of processing2 -Scavenging action.In Fig. 6：(a) blank pair
According to；(b)H2O20.25mmol/L；(c) 12.5 μ g/mL of sea cucumber water cooking liquid polysaccharide；(d) 25 μ g/mL of sea cucumber water cooking liquid polysaccharide；(e)
50 μ g/mL of sea cucumber water cooking liquid polysaccharide；(f) 100 μ g/mL of sea cucumber water cooking liquid polysaccharide；(g) 200 μ g/mL of sea cucumber water cooking liquid polysaccharide.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, actual conditions is not specified in embodiment
Person, the condition suggested according to normal condition or manufacturer carry out.Reagents or instruments used without specified manufacturer, being can be with
Conventional products that are commercially available.
The present invention is based on Electron Spin Resonance Spectra (ESR) technology, it is proposed that one kind detection marine polysaccharide antioxidation activity
Method, test solution and the method for preparing test solution.The method for elaborating this detection marine polysaccharide antioxidation activity in detail below, with
And prepare the test solution for being used for this detection method.
As shown in figure 3, the method for detection marine polysaccharide antioxidation proposed by the invention, includes the following steps：
Step 101, prepare to be used for the test solution for detecting marine polysaccharide antioxidation；
Step 102, hydroxyl radical free radical (OH) and ultra-oxygen anion free radical in the test solution are detected with ESR methods
(O2 -) concentration；
Step 103, the antioxidation activity height of detected polysaccharide sample is judged according to the testing result of step 102.
Detection method proposed by the present invention employs Electron Spin Resonance Spectra detection technique, its with detection range it is wide,
The advantages that detection object is extensive, testing conditions are simple, detection sensitivity is high.As ROS in the cell supernatant after medicine irritation
When concentration is very low, it is likely to detect using common method, but sensitivity will be greatly improved using this method, and can be certainly
Reasonably explained by what analysis conventional method on basic unit face detected as a result, providing one.
The present invention realizes fast and effective detection marine polysaccharide antioxidation activity, and detection process is succinct, and testing result is stablized
Reliably, and the present invention has widened thinking from the angle of free radical for the detection of this aspect.
RAW264.7 cell lines be previously widely used in molecular level to characterize the immunoregulation effect of various components,
It is also one of common anti-oxidant and inflammatory cell model at the same time.The cell strain growth density is larger, is typically larger than 106, but it is real
96 orifice plate nominal volumes are considered in the operation of border, and cell density is generally adjusted to 105~106It is more suitable.
Wherein, the step 101 includes the following steps：
Step A, takes polysaccharide sample to be configured to polysaccharide solution；
Specifically, the polysaccharide solution is prepared using DMEM culture mediums or distilled water.In usual cell experiment, sample uses
Culture medium is prepared, and to ensure that needed nutrient matter is sufficient in cell growth, but also be there is the example prepared with distilled water, is steamed
The benefit that distilled water is prepared is to avoid component complicated in culture medium, can investigate effect of the sample to cell merely.
Step B, takes RAW264.7 cells, and adjusts cell density to 105~106A/mL；
Specifically, RAW264.7 cell densities are adjusted using DMEM culture mediums, and will adjusted described in cell density
RAW264.7 cells, which are placed at 37 DEG C, carries out culture 24h.Usual mammalian cell carries out often in 37 DEG C of cell incubators
Rule culture, incubation time is depending on actual conditions.
Wherein, the step A can be also carried out at the same time with the step B, or the step B is in the advance of the step A
OK.In fact, the order of step A and step B can voluntarily be formulated without Compulsory Feature according to practical operation situation.
Step C, the cell liquid for taking the step B to obtain, adds the hydrogen peroxide (H of 0.25mmol/L2O2) solution is to described
The polysaccharide solution in step A, obtains the test solution that several pieces concentration is between 0~200 μ g/mL and concentration is different, and to it
Cultivated, thus just obtained being used for the test solution for detecting marine polysaccharide antioxidation.
Specifically, it will add H2O2Cell liquid after solution is placed in 37 DEG C, CO2The CO2gas incubator training of concentration 5%
24h is supported, the cell liquid after addition polysaccharide solution is placed in 37 DEG C, CO2The CO2gas 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
It is extremely short by the base service life, it is quenched in the short time, after being quenched, signal is no longer caught in, and is groped according to substantial amounts of experiment, sent out
It is existing, within 5~30min periods, it can completely detect the free radical of generation.
The step 102 carries out under normal temperature condition.In the present invention, step 102 suitably normal temperature condition with ESR equipment into
Row test, the results contrast tested out are accurate.
Specifically, ESR detection hydroxyl radical free radicals (OH) and ultra-oxygen anion free radical (O are being utilized2 -) during,
The design parameter difference 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℃.
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℃.
The step 103 judges the antioxidation activity of detected polysaccharide sample according to the testing result of the step 102
Just.
Specifically, it is cloudy according to the hydroxyl radical free radical (OH) in the marine polysaccharide sample to be tested for comparing various concentrations and super oxygen
Ion radical (O2 -) concentration, come judge the antioxidation activity of detected polysaccharide sample height.If test solution is more
Sugared concentration is higher, detected hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O2 -) concentration it is lower, and
Detected hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O2 -) concentration to the polysaccharide concentration of test solution
It is more sensitive, then illustrate that the antioxidation of detected polysaccharide sample is stronger, it is on the contrary then not strong.
The invention also provides a kind of method prepared for detecting marine polysaccharide antioxidation test solution, including following step
Suddenly：
Step A, takes polysaccharide sample to be configured to polysaccharide solution；
Step B, takes RAW264.7 cells, and adjusts cell density to 105~106A/mL；
Step C, the cell liquid for taking the step B to obtain, adds the hydrogen peroxide H of 0.25mM2O2Solution is to the step A
In the polysaccharide solution, obtain the test solution that several pieces concentration is between 0~200 μ g/mL and concentration is different, and it is trained
Support, thus just obtained being used for the test solution for detecting marine polysaccharide antioxidation.
The present invention also proposes a kind of test solution prepared for detecting marine polysaccharide antioxidation, it is used with preparation as above
Prepared in the method for detection marine polysaccharide antioxidation test solution.The test solution is used to detect marine polysaccharide antioxidation, energy
The difficulty of detection is reduced, improves the stability of testing result.
The detection of antioxidation activity of 1 scallop polysaccharide of example in macrophage
Step 101 ', prepare to be used for the test solution for detecting marine polysaccharide antioxidation；
Specifically, scallop polysaccharide sample is taken, the fan of 200 μ g/mL (micrograms per millilitre, similarly hereinafter) is configured to DMEM culture mediums
Pattra sugar juice；Take hydrogen peroxide (H2O2) sample, with the H of DMEM culture mediums preparation 0.25mmol/L2O2Solution；Trained with DMEM
Base is supported to adjust RAW264.7 cell densities to 3 × 105A/mL；The cell for adjusting density is placed in 96 porocyte culture plates
In, cultivate 24h at 37 DEG C；Appropriate H is added in 96 well culture plate2O2Solution, it is 0.25mmol/L to make its concentration；Will
It is 37 DEG C, CO that 96 porocyte culture plates, which are placed in temperature,2Concentration is to cultivate 24h in 5% CO2gas incubator；To 96 holes
Appropriate scallop polysaccharide solution is added in culture plate, it is respectively 0 μ g/mL, 12.5 μ g/mL, 25 μ to make scallop polysaccharide concentration in different holes
g/mL、50μg/mL、100μg/mL、200μg/mL；It is 37 DEG C, CO that 96 well culture plate is placed in temperature2Concentration is 5%
Continue to cultivate 30min and 5min respectively in CO2gas incubator, be respectively used to finally detect hydroxyl radical free radical (OH) and surpass
Oxygen anion free radical (O2 -) concentration；The 96 well culture plate difference polysaccharide concentration cell liquid (cell conditioned medium) is collected, this
It is the test solution for detecting scallop polysaccharide antioxidation.
Step 102 ', detect hydroxyl radical free radical (OH) and ultra-oxygen anion free radical in the test solution with ESR methods
(O2 -) concentration；
Specifically, test solution and ROS capturing agents (DMPO) are added into tube pipes and is fully mixed；Inhaled using capillary
Mixed liquor seals mouth under capillary to appropriate height, and with vaseline, then cleans the vaseline of tube wall remnants；Utilize ESR technologies
Detect hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O2 -) generation situation, obtain DMPO-OO under each polysaccharide concentration
(H) and DMPO-OH signal collection of illustrative plates is shown in Fig. 4.Utilizing ESR detection hydroxyl radical free radicals (OH) and ultra-oxygen anion free radical
(O2 -) during, the design parameter difference 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℃.
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℃.
The testing result of step 103 ', according to step 102 ' judges the antioxidation activity height of detected polysaccharide sample.
According to step 102 ' obtained in collection of illustrative plates judge the antioxidation activity of the scallop polysaccharide sample.As shown in Figure 4,
H2O2The peak height of control group occurs significantly to increase, in contrast, after the scallop polysaccharide for adding increasing concen-trations, OH and O2 -
ESR peak heights occur substantially to reduce, and show that scallop polysaccharide effectively prevents oxidation course, have preferable antioxidation.
The detection of antioxidation activity of the 2 Undaria pinnatifida Suringar polysaccharide of example in macrophage
Step 101 ", prepares to be used for the test solution for detecting marine polysaccharide antioxidation；
Specifically, RAW264.7 cells are taken, are adjusted RAW264.7 cell densities to 5 × 10 with DMEM culture mediums5A/
mL.Then, Undaria pinnatifida Suringar polysaccharide sample is taken, the Undaria pinnatifida Suringar polysaccharide solution of 200 μ g/mL is configured to distilled water；
Take hydrogen peroxide (H2O2) sample, with the H of distilled water preparation 0.25mmol/L2O2Solution；The cell for adjusting density is placed in 96
In porocyte culture plates, 24h is cultivated at 37 DEG C；Appropriate H is added in 96 well culture plate2O2Solution, makes its concentration be
0.25mmol/L；It is 37 DEG C, CO that 96 porocyte culture plates are placed in temperature2Concentration is in 5% CO2gas incubator
Cultivate 24h；Appropriate Undaria pinnatifida Suringar polysaccharide solution is added into 96 well culture plates, makes Undaria pinnatifida Suringar polysaccharide in different holes
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 96 well culture plate
It is 37 DEG C, CO to be placed in temperature2Concentration is to continue to cultivate 30min and 5min respectively in 5% CO2gas incubator, is respectively used to
Final detection hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O2 -) concentration；It is different to collect 96 well culture plate
The cell liquid (cell conditioned medium) of polysaccharide concentration, this is for detecting the to be tested of Undaria pinnatifida Suringar polysaccharide anti-oxidative effect
Liquid.
Step 102 ", hydroxyl radical free radical (OH) and ultra-oxygen anion free radical in the test solution are detected with ESR methods
(O2 -) concentration；
Specifically, test solution and ROS capturing agents (DMPO) are added into tube pipes and is fully mixed；Inhaled using capillary
Mixed liquor seals mouth under capillary to appropriate height, and with vaseline, then cleans the vaseline of tube wall remnants；Utilize ESR technologies
Detect hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O2 -) generation situation, obtain DMPO-OO under each polysaccharide concentration
(H) and DMPO-OH signal collection of illustrative plates is shown in Fig. 5.Utilizing ESR detection hydroxyl radical free radicals (OH) and ultra-oxygen anion free radical
(O2 -) during, the design parameter difference 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℃.
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℃.
The testing result of step 103 ", according to step 102 " judges the antioxidation activity height of detected polysaccharide sample.
According to step 102 " obtained in collection of illustrative plates judge the antioxidation activity of the Undaria pinnatifida Suringar polysaccharide sample.By
Knowable to Fig. 5, H2O2The peak height of control group occurs significantly to increase, and in contrast, adds the Undaria pinnatifida Suringar polysaccharide of increasing concen-trations
Afterwards, OH and O2 -ESR peak heights occur substantially to reduce, show Undaria pinnatifida Suringar polysaccharide effectively prevent aoxidize into
Journey, has preferable antioxidation.
The detection of antioxidation activity of the 3 sea cucumber water cooking liquid polysaccharide of example in macrophage
Step 101 " ', prepare to be used for the test solution for detecting marine polysaccharide antioxidation；
Specifically, sea cucumber water cooking liquid polysaccharide sample is taken, the sea cucumber water cooking liquid that 200 μ g/mL are configured to DMEM culture mediums is more
Sugar juice；Take hydrogen peroxide (H2O2) sample, with the H of DMEM culture mediums preparation 0.25mmol/L2O2Solution；With DMEM culture mediums
RAW264.7 cell densities are adjusted to 1 × 106A/mL；The cell for adjusting density is placed in 96 porocyte culture plates,
24h is cultivated at 37 DEG C；Appropriate H is added in 96 well culture plate2O2Solution, it is 0.25mmol/L to make its concentration；By described 96
It is 37 DEG C, CO that porocyte culture plates, which are placed in temperature,2Concentration is to cultivate 24h in 5% CO2gas incubator；To 96 well culture plates
Middle to add appropriate sea cucumber water cooking liquid polysaccharide solution, it is respectively 0 μ g/mL, 12.5 μ g/ to make sea cucumber water cooking liquid polysaccharide concentration in different holes
mL、25μg/mL、50μg/mL、100μg/mL、200μg/mL；It is 37 DEG C, CO that 96 well culture plate is placed in temperature2Concentration is
Continue to cultivate 30min and 5min respectively in 5% CO2gas incubator, be respectively used to finally detect hydroxyl radical free radical (OH)
And ultra-oxygen anion free radical (O2 -) concentration；The cell liquid of the 96 well culture plate difference polysaccharide concentration is collected (on cell
Clearly), this is the test solution for detecting the effect of sea cucumber water cooking liquid polysaccharide anti-oxidative.
Step 102 " ', detect hydroxyl radical free radical (OH) and ultra-oxygen anion free radical in the test solution with ESR methods
(O2 -) concentration；
Specifically, test solution and ROS capturing agents (DMPO) are added into tube pipes and is fully mixed；Inhaled using capillary
Mixed liquor seals mouth under capillary to appropriate height, and with vaseline, then cleans the vaseline of tube wall remnants；Utilize ESR technologies
Detect hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O2 -) generation situation, obtain DMPO-OO under each polysaccharide concentration
(H) and DMPO-OH signal collection of illustrative plates is shown in Fig. 6.Utilizing ESR detection hydroxyl radical free radicals (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℃.
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℃.
The testing result of step 103 " ', according to step 102 " ' judges that the antioxidation activity of detected polysaccharide sample is high
It is low.
According to step 102 " ' obtained in collection of illustrative plates judge the antioxidation activity of the sea cucumber water cooking liquid polysaccharide sample.By
Knowable to Fig. 6, H2O2The peak height of control group occurs significantly to increase, and in contrast, adds the sea cucumber water cooking liquid polysaccharide of increasing concen-trations
Afterwards, OH and O2 -ESR peak heights occur substantially to reduce, show that sea cucumber water cooking liquid polysaccharide effectively prevents oxidation course,
With preferable antioxidation.
It is proposed by the present invention to prepare the method for being used for detecting marine polysaccharide antioxidation test solution it was found from above-described embodiment
And the test solution for being used to detect marine polysaccharide antioxidation prepared with this method, employ stable mononuclear macrophage strain
RAW264.7 cells, the cell line be previously widely used in molecular level to characterize the immunoregulation effect of various components,
It is also one of common anti-oxidant and inflammatory cell model at the same time.
Further, the present invention according to above-mentioned preparation method and the test solution prepared propose based on electron spin resonance ripple
The method for composing (ESR) technology for detection marine polysaccharide antioxidation, by detecting on mononuclear macrophage strain RAW264.7 cells
ROS contents evaluate marine polysaccharide antioxidation activity in clear liquid, reduce detection difficulty, improve the stability of testing result,
Enrich detection method.
Further it is proposed that detection method employ Electron Spin Resonance Spectra detection technique, its have detection model
Enclose the advantages that wide, detection object is extensive, testing conditions are simple, detection sensitivity is high.When the cell supernatant after medicine irritation
When middle ROS concentration is very low, it is likely to detect using common method, but sensitivity will be greatly improved using this method, and
Can in free radical aspect rational being explained as a result, providing one of detecting of analysis conventional method.
The present invention realizes fast and effective detection marine polysaccharide antioxidation activity, and detection process is succinct, and testing result is stablized
Reliably, and the present invention has widened thinking from the angle of free radical for the detection of this aspect.
Therefore, method proposed by the present invention is expected to be applied to cell biology, Pharmaceutical Analysis, environmental toxicants detection etc.
Association area.
Above example is only the exemplary embodiment of the present invention, is not used in the limitation present invention, protection scope of the present invention
It is defined by the claims.Those skilled in the art can make the present invention respectively in the essence and protection domain of the present invention
Kind modification or equivalent substitution, this modification or equivalent substitution also should be regarded as being within the scope of the present invention.
Claims (8)
- A kind of 1. method for detecting marine polysaccharide antioxidation, it is characterised in that it includes the following steps：Step 101, prepare to be used for the test solution for detecting marine polysaccharide antioxidation；Step 102, hydroxyl radical free radical (OH) and ultra-oxygen anion free radical (O in the test solution are detected with ESR methods2 -·) Concentration；Step 103, the antioxidation activity height of detected polysaccharide sample is judged according to the testing result of step 102；Wherein, the step 101 includes the following steps：Step A, takes polysaccharide sample to be configured to polysaccharide solution；Step B, takes RAW264.7 cells, and adjusts cell density to 105~106A/mL；Step C, the cell liquid for taking the step B to obtain, using the hydrogen peroxide (H of 0.25mmol/L2O2) solution is by the step The concentration for the cell liquid that B is obtained is adjusted to 0.25mmol/L, will add the cell liquid after the hydrogenperoxide steam generator be placed in 37 DEG C, CO2The CO2gas incubator culture 24h of concentration 5%；Then the polysaccharide solution in the step A is added, is obtained some Part test solution that concentration is between 0~200 μ g/mL and concentration is different, 37 DEG C, CO are placed in by the test solution2The titanium dioxide of concentration 5% Carbon incubator cultivates 30min and 5min respectively, cultivates the test solution after 30min and is used to finally detect the dense of hydroxyl radical free radical (OH) Degree, cultivates the test solution after 5min and is used to finally detect ultra-oxygen anion free radical (O2 -) concentration, be thus just used for Detect the test solution of marine polysaccharide antioxidation.
- 2. the method as described in claim 1, it is characterised in that wherein described step A and the step B are carried out at the same time, or institute Step B is stated to carry out before the step A.
- 3. the method as described in claim 1, it is characterised in that matched somebody with somebody in wherein described step A with DMEM culture mediums or distilled water Make the polysaccharide solution.
- 4. the method as described in claim 1, it is characterised in that wherein described step B includes the use of the adjustment of DMEM culture mediums RAW264.7 cell densities.
- 5. the method as described in claim 1, it is characterised in that wherein described step B includes that the institute of cell density will be adjusted State RAW264.7 cells and be placed at 37 DEG C and carry out culture 24h.
- 6. the method as described in claim 1, it is characterised in that wherein described step 102 carries out under normal temperature condition.
- A kind of 7. method prepared for detecting marine polysaccharide antioxidation test solution, it is characterised in that it includes the following steps：Step A, takes polysaccharide sample to be configured to polysaccharide solution；Step B, takes RAW264.7 cells, and adjusts cell density to 105~106A/mL；Step C, the cell liquid for taking the step B to obtain, using the hydrogen peroxide (H of 0.25mmol/L2O2) solution is by the step The concentration for the cell liquid that B is obtained is adjusted to 0.25mmol/L, will add the cell liquid after the hydrogenperoxide steam generator be placed in 37 DEG C, CO2The CO2gas incubator culture 24h of concentration 5%；Then the polysaccharide solution in the step A is added, is obtained some Part test solution that concentration is between 0~200 μ g/mL and concentration is different, 37 DEG C, CO are placed in by the test solution2The titanium dioxide of concentration 5% Carbon incubator cultivates 30min and 5min respectively, cultivates the test solution after 30min and is used to finally detect the dense of hydroxyl radical free radical (OH) Degree, cultivates the test solution after 5min and is used to finally detect ultra-oxygen anion free radical (O2 -) concentration, thus just obtained described For detecting marine polysaccharide antioxidation test solution.
- 8. a kind of test solution for being used to detect marine polysaccharide antioxidation, it is characterised in that it uses side as claimed in claim 7 It is prepared by method.
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