A method for evaluating the anti-photooxidative ability of astaxanthin

Abstract

The present invention relates to the technical field of astaxanthin antioxidant capacity detection, specifically, an evaluation method for evaluating astaxanthin's resistance to light oxidation. By selecting a suitable photosensitizer, active oxygen probe, illumination time, reaction reagent concentration, and spectral measurement mode and method for astaxanthin, the evaluation of astaxanthin's resistance to light oxidation can be achieved. This is the first time to establish an experimental scheme and detection method for astaxanthin's resistance to light oxidation. The patent has low requirements for experimental equipment and has the advantages of being simple and convenient, easy to operate, accurate and reliable in measurement, and quantitatively analyzeable.

Description

Technical Field

[0001] The invention relates to the technical field of astaxanthin antioxidant capacity detection, and in particular to a detection and analysis method for evaluating astaxanthin's ability to resist light oxidation. Background Art

[0002] Astaxanthin is a non-water-soluble antioxidant with extremely strong antioxidant properties. Its structure consists of a polyolefin in the middle of the molecule connected to two saturated ionone rings at the ends. It contains hydroxyl and ketone groups, so it is a type of lutein. In recent years, astaxanthin, as an extraordinary natural antioxidant, has received increasing attention in the fields of food, cosmetics, etc.

[0003] Radiation is everywhere in our lives. Light radiation can induce the production of a variety of reactive oxygen species and free radicals, which lead to negative consequences such as photoaging and photodamage. Among them, singlet oxygen is a type of reactive oxygen species with high reactivity. It is easy to react with macromolecules in the body such as DNA, proteins and lipid molecules, causing cell damage. The oxidation of singlet oxygen on biological molecules is related to a variety of diseases such as arthritis, cataracts and skin cancer. It is very important to choose suitable natural antioxidants to protect biological antioxidants.

[0004] Astaxanthin is a natural antioxidant with good antioxidant properties, but there are few studies on astaxanthin against photooxidation, especially the removal of singlet oxygen produced by photooxidation, which requires the development of effective test methods. This requires solving two problems: one is to consider how to conveniently establish a photooxidation experiment, and the other is how to effectively detect astaxanthin to remove the reactive oxygen species produced by light, mainly singlet oxygen. In the past, there were methods for measuring singlet oxygen, including fluorescence, chromatography, chemiluminescence, and electron spin resonance. For example, the Chinese invention patent with publication number CN111233905A discloses a fluorescent probe and application for selectively detecting singlet oxygen in a solution phase. The patent detects singlet oxygen by a prepared fluorescent probe, and the fluorescence method is used. However, the absorption band of the probe is 518 / 545nm, and astaxanthin has a large absorption here, so it cannot be used for the detection of astaxanthin anti-oxidation. Therefore, there is an urgent need for a detection method for astaxanthin to produce reactive oxygen against photooxidation. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to provide a method for evaluating the photo-oxidation resistance of astaxanthin.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] A method for evaluating the photo-oxidation resistance of astaxanthin, and establishing an experimental scheme and evaluation method for astaxanthin to resist photo-oxidation: using methylene blue as a photosensitizer, using xenon lamp irradiation, adding a 660nm filter, and generating singlet oxygen under light conditions; using DPBF as a probe to detect singlet oxygen, adding different concentrations of astaxanthin, and using ultraviolet-visible absorption spectroscopy to detect changes in the absorption peak of DPBF at 380-440nm;

[0008] The sample without astaxanthin was used as the control, and the quenching constant k of astaxanthin for singlet oxygen was calculated according to the following formula: r :

[0009] S0 / S=1+k r [A] / (k d +k Q [P])

[0010] Among them, S0 represents the initial reduction rate of the DPBF probe of the sample without astaxanthin;

[0011] S represents the initial reduction rate of the DPBF probe in the sample containing astaxanthin;

[0012] [A] represents the initial concentration of astaxanthin, and [P] represents the initial concentration of DPBF;

[0013] k d represents the decay rate constant of singlet oxygen itself;

[0014] k Q represents the rate constant of the reaction between singlet oxygen and DPBF;

[0015] By k r Determine the singlet oxygen scavenging ability of astaxanthin.

[0016] Beneficial effects: First, the present application establishes a light oxidation experimental scheme. In this patent, methylene blue (MB) is used as a photosensitizer, and xenon lamp irradiation is used. A 660nm filter is added. Under light conditions, methylene blue can produce highly oxidizing reactive oxygen - singlet oxygen; secondly, a suitable method for detecting astaxanthin to remove reactive oxygen generated by light is established. Here, it is mainly for singlet oxygen. 1,3-diphenylisobenzofuran (DPBF) is used to detect singlet oxygen. The ultraviolet-visible absorption spectroscopy is used to detect the change of the absorption peak of DPBF at 380-440nm. By processing the spectral data, and according to the initial concentration of astaxanthin, the initial concentration of DPBF, the initial reduction rate of the DPBF probe in the sample, the decay rate constant of singlet oxygen itself, and the rate constant of the reaction between singlet oxygen and DPBF, the quenching constant k of astaxanthin for singlet oxygen is calculated. r , to determine the ability of astaxanthin to scavenge singlet oxygen.

[0017] The method for evaluating the photo-oxidation resistance of astaxanthin established in the present application has low requirements for experimental equipment and has the advantages of being simple, convenient and highly operational. It uses DPBF and methylene blue as the main detection reagents, which are cheap, easy to obtain and highly recognized, effectively reducing the detection cost.

[0018] Preferably, the method for evaluating astaxanthin against light oxidation comprises the following steps:

[0019] (1) dissolving astaxanthin in an organic solvent to prepare a mother solution, and then diluting it with anhydrous ethanol to prepare an astaxanthin reserve solution; dissolving DPBF in anhydrous ethanol to prepare a DPBF-ethanol solution, and dissolving methylene blue in deionized water to prepare a methylene blue-water solution;

[0020] (2) Add astaxanthin reserve solution, DPBF-ethanol solution, and methylene blue-water solution into a cuvette, fill it up with ethanol-water mixed solution, and seal the cuvette; use anhydrous ethanol as a control;

[0021] (3) After the samples containing astaxanthin and the control samples were exposed to light, the samples were scanned using a UV-visible spectrophotometer to observe changes in the absorption peak of DPBF at 380-440 nm;

[0022] (4) According to the change of DPBF absorption peak, the initial reduction rate of DPBF probe in astaxanthin-containing samples and control samples was calculated, and the quenching constant k of astaxanthin for singlet oxygen was calculated by substituting it into the formula: r .

[0023] Preferably, the organic solvent in step (1) is selected from one or more combinations of dimethyl sulfoxide, chloroform and acetone.

[0024] Preferably, in step (2), the final concentrations of DPBF and methylene blue are 6 μmol / L and 6 μmol / L, respectively.

[0025] Preferably, the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 67%-100%.

[0026] Beneficial effects: The present invention adds 67%-100% ethanol to the reaction system, which can fully dissolve the fat-soluble antioxidant astaxanthin and prevent the system from becoming turbid.

[0027] Preferably, the concentration of astaxanthin in the cuvette of step (2) is 0.5-2.0 μmol / L.

[0028] Beneficial effect: When the concentration of astaxanthin is 0.5-2.0 μmol / L, the detection method of the present application can quickly determine the changes in the components in the solution, thereby realizing the evaluation of the ability of low-concentration astaxanthin to scavenge active oxygen (singlet oxygen) generated by photo-oxidation, and low-concentration detection can also save sample costs.

[0029] Preferably, the concentration of DPBF in the cuvette of step (2) is 6-10 μmol / L, and the concentration of methylene blue is 6-9 μmol / L.

[0030] Preferably, in step (3), a xenon lamp is used as the light source for inducing methylene blue to produce singlet oxygen, and the light intensity is adjustable.

[0031] Beneficial effect: The present invention obtains light of a wavelength suitable for exciting the photosensitizer methylene blue through the light emitted by the xenon lamp, thereby increasing the yield of singlet oxygen.

[0032] Preferably, in step (3), a filter is used to remove stray light in the light emitted by the xenon lamp, and the wavelength of the transmitted light is 660nm.

[0033] Beneficial effects: The present application utilizes a filter to remove stray light from the light emitted by a xenon lamp, and concentrates on utilizing monochromatic light in the 660nm band, thereby further improving the excitation effect of methylene blue and thereby increasing the yield of active singlet oxygen.

[0034] Preferably, the illumination time of the sample in step (3) is 0 to 120 seconds.

[0035] Beneficial effects: The present application measures the generation of singlet oxygen in the reaction solution by observing the change in DPBF absorption over time under different illumination times, and takes the DPBF absorption peak at 120s of illumination for statistical analysis.

[0036] Preferably, the step (4) calculates the quenching constant k of astaxanthin for singlet oxygen r k in the formula d 3×10 in water 5 s -1 , in ethanol it is 6.7×10 4 s -1 ;k Q 8x10 8 mol -1 s -1 .

[0037] The advantages of the present invention are:

[0038] 1. This patent establishes for the first time an experimental scheme and evaluation method for evaluating astaxanthin's resistance to photo-oxidation. In the experiment, methylene blue is used as a photosensitizer, and 660nm light can effectively generate active singlet oxygen with strong oxidizing ability, thereby establishing a photo-oxidation experimental scheme; DPBF is used as a probe to detect the reaction between astaxanthin and singlet oxygen, and by observing the changes in the absorption spectrum at 380-440nm, a method for detecting astaxanthin's removal of active oxygen generated by light is established, thereby inferring the quenching constant of astaxanthin for singlet oxygen, thereby achieving a quantitative evaluation of astaxanthin's resistance to photo-oxidation;

[0039] 2. The detection method of astaxanthin against light oxidation established by this patent has low requirements for experimental equipment and has the advantages of being simple, convenient and highly operable. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is an operation flow chart of Example 1 of the present application;

[0041] Figure 2 The UV-visible absorption spectra of DPBF at different irradiation times in Experimental Example 1 of the present application;

[0042] Figure 3 The effect of different concentrations of astaxanthin on the UV-visible absorption spectrum of DPBF in Experimental Example 2 of this application;

[0043] Figure 4 This is a curve diagram of the DPBF reduction rate of methylene blue at different concentrations and different irradiation times in Experimental Example 3 of this application;

[0044] Figure 5 This is a curve diagram of DPBF reduction rate variation at different concentrations of DPBF at different irradiation times in Experimental Example 4 of this application;

[0045] Figure 6 This is the quenching rate of singlet oxygen by astaxanthin at different concentrations in different reaction media in Experimental Example 5 of this application.

[0046] Explanation of the accompanying symbols: 1. Xenon lamp; 2. 660 nm filter; 3. Sample: MB+DPBF+astaxanthin; 4. UV-visible absorption spectrometer; 5. UV-visible absorption spectrum. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Example 1

[0049] This embodiment provides a method for evaluating the anti-photooxidation ability of astaxanthin, comprising the following steps:

[0050] (1) Astaxanthin is dissolved in dimethyl sulfoxide to prepare a mother solution with a concentration of 400 μmol / L, and then diluted with anhydrous ethanol to prepare an astaxanthin reserve solution, wherein the concentration of the astaxanthin reserve solution is 0-6.0 μmol / L, wherein the astaxanthin, DPBF and methylene blue used are all commercially available.

[0051] The DPBF mother solution with a concentration of 0.2 mg / mL was dissolved in anhydrous ethanol to prepare a DPBF-ethanol solution.

[0052] A methylene blue (MB) stock solution with a concentration of 3 mg / mL was dissolved in deionized water to prepare a methylene blue-water solution.

[0053] (2) Add 1 mL of astaxanthin stock solution, 1 mL of DPBF-ethanol solution, and 50 μL of methylene blue-water solution into a 3 mL cuvette in sequence, and then add ethanol-water mixed solution (V 乙醇 :V 水 =3:0) to 3 mL, blow and mix the cuvette evenly, add a lid on the cuvette, and seal it with a sealing film. The concentration of astaxanthin is 2.0 μmol / L, the concentration of DPBF is 6 μmol / L, and the concentration of methylene blue is 6 μmol / L.

[0054] Following the same operation, anhydrous ethanol was used instead of the astaxanthin stock solution as a control.

[0055] (3) Figure 1 As shown, in a dark environment, a xenon lamp 1 is used as a light source, and after the stray light is removed by a 660nm filter 2, the sample 3 containing astaxanthin and the control sample 3 are irradiated for 0s, 30s, 60s, 90s, and 120s, and the sample 3 is scanned by a UV-visible absorption spectrometer 4 to obtain a UV-visible absorption spectrum. Figure 5 , observe the changes in the absorption peak of DPBF near 380-410nm under different irradiation times.

[0056] (4) According to the change of DPBF absorption peak, the initial reduction rate of DPBF probe in astaxanthin-containing samples and control samples was calculated, and the quenching constant k of astaxanthin for singlet oxygen was calculated by substituting it into the formula: r .

[0057] The quenching constant k of astaxanthin for singlet oxygen r The calculation formula is as follows:

[0058] S0 / S=1+k r [A] / (k d +k Q [P])

[0059] Among them, S0 represents the initial reduction rate of the DPBF probe of the sample without astaxanthin;

[0060] S represents the initial reduction rate of the DPBF probe in the sample containing astaxanthin;

[0061] [A] represents the initial concentration of astaxanthin, and [P] represents the initial concentration of DPBF;

[0062] k d represents the decay rate constant of singlet oxygen itself. In different solvents, k d Different, in water it is 3×10 5 s -1 , in ethanol it is 6.7×10 4 s -1 When the reaction solution is a mixed solution, the k of each solvent is adjusted according to the ratio of the solvent. d The values ​​are weighted summed to calculate the total decay rate constant; the reaction solution in this embodiment is ethanol, k d Take 6.7×10 4 s -1 ;

[0063] k Q The rate constant of the reaction between singlet oxygen and DPBF is 8x10 8 mol -1 s -1 ;

[0064] By k r The value of k is used to judge the scavenging ability of astaxanthin on singlet oxygen. r The larger the value, the stronger the scavenging ability of astaxanthin for singlet oxygen.

[0065] Example 2

[0066] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 1 in that the concentration of methylene blue in step (2) is 12 μmol / L.

[0067] Example 3

[0068] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 1 in that the concentration of methylene blue in step (2) is 9 μmol / L.

[0069] Example 4

[0070] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 1 in that the concentration of methylene blue in step (2) is 4 μmol / L.

[0071] Example 5

[0072] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 1 in that the concentration of methylene blue in step (2) is 2 μmol / L.

[0073] Example 6

[0074] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 1 in that the concentration of DPBF in step (2) is 12 μmol / L.

[0075] Example 7

[0076] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 1 in that the concentration of DPBF in step (2) is 10 μmol / L.

[0077] Example 8

[0078] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 1 in that the concentration of DPBF in step (2) is 8 μmol / L.

[0079] Example 9

[0080] This embodiment provides a method for evaluating the anti-photooxidation ability of astaxanthin, which differs from Embodiment 1 in that the concentration of astaxanthin in step (2) is 0 μmol / L, and anhydrous ethanol is used as a control sample.

[0081] Example 10

[0082] This embodiment provides a method for evaluating the anti-photooxidation ability of astaxanthin, which is different from that of Embodiment 1 in that the concentration of astaxanthin in step (2) is 0.5 μmol / L.

[0083] Embodiment 11

[0084] This embodiment provides a method for evaluating the anti-photooxidation ability of astaxanthin, which is different from that of Example 1 in that the concentration of astaxanthin in step (2) is 1 μmol / L.

[0085] Example 12

[0086] This embodiment provides a method for evaluating the anti-photooxidation ability of astaxanthin, which is different from that of Example 1 in that the concentration of astaxanthin in step (2) is 1.5 μmol / L.

[0087] Embodiment 13

[0088] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 9 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 2:1.

[0089] Embodiment 14

[0090] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 10 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 2:1.

[0091] Embodiment 15

[0092] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 11 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 2:1.

[0093] Example 16

[0094] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 12 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 2:1.

[0095] Embodiment 17

[0096] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 1 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 2:1.

[0097] Embodiment 18

[0098] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 9 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 1:2.

[0099] Embodiment 19

[0100] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 10 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 1:2.

[0101] Embodiment 20

[0102] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 11 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 1:2.

[0103] Embodiment 21

[0104] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 12 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 1:2.

[0105] Embodiment 22

[0106] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which is different from that of Embodiment 1 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 1:2.

[0107] Embodiment 23

[0108] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 9 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 0:3.

[0109] Embodiment 24

[0110] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 10 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 0:3.

[0111] Embodiment 25

[0112] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 11 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 0:3.

[0113] Embodiment 26

[0114] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which differs from Embodiment 12 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 0:3.

[0115] Embodiment 27

[0116] This embodiment provides a method for evaluating the photo-oxidation resistance of astaxanthin, which is different from that of Embodiment 1 in that the volume ratio of ethanol to water in the ethanol-water mixed solution in step (2) is 0:3.

[0117] Test Example 1

[0118] In Example 9, anhydrous ethanol was used as a control sample, and the UV-visible absorption spectra of DPBF at different irradiation times were obtained without the addition of astaxanthin. Figure 2 shown.

[0119] from Figure 2It can be seen that the absorbance of DPBF at 410 nm decreases with the increase of illumination time. This is because the illumination causes the methylene blue in the reaction solution to produce singlet oxygen, and DPBF captures the singlet oxygen and reacts with the singlet oxygen, which reduces the absorption of DPBF at 410 nm.

[0120] The present application utilizes ultraviolet-visible absorption spectroscopy to detect changes in the absorbance value of DPBF at 410 nm, which can reflect changes in the singlet oxygen content in the solution, thereby establishing a detection method for the singlet oxygen scavenging ability of astaxanthin, which has low requirements for experimental equipment, is simple and convenient, and has strong operability; and uses DPBF and methylene blue as the main detection reagents, which are cheap and easy to obtain, effectively reducing the detection cost.

[0121] Test Example 2

[0122] In Example 1 and Examples 9-12, the absorption peak of DPBF between 380-440nm when the light was irradiated for 120s was statistically analyzed to obtain the UV-visible absorption spectra of DPBF with different concentrations of astaxanthin as shown in the figure below: Figure 3 shown.

[0123] from Figure 3 It can be seen that the absorbance of DPBF at 380-440nm increases with the increase of astaxanthin concentration. This is because astaxanthin removes singlet oxygen, thereby reducing the decrease of DPBF. Therefore, compared with the treatment without astaxanthin, the absorption peak of DPBF increases significantly after adding astaxanthin.

[0124] Test Example 3

[0125] In Examples 1-5, anhydrous ethanol was used as a control sample to obtain the changes in the DPBF reduction rate at different concentrations of methylene blue and different irradiation times. Figure 4 shown.

[0126] from Figure 4 It can be seen that the absorption reduction rate of DPBF at 380-440nm increases with the increase of methylene blue concentration, indicating that methylene blue with a higher concentration (6-9μmol / L) is easily excited under light with a wavelength of 660nm, thereby producing more singlet oxygen, thereby providing sufficient singlet oxygen for the test system; when the concentration of methylene blue is less than 6μmol / L, the reduction rate of DPBF changes little, indicating that less singlet oxygen is produced and it is not suitable for subsequent detection; and when the concentration of methylene blue is 12μmol / L, too much singlet oxygen is produced, which is not conducive to the subsequent astaxanthin clearance experiment. The amount of singlet oxygen produced can be controlled by changing the concentration of methylene blue, which is convenient for studying the scavenging ability of astaxanthin for singlet oxygen of different concentrations.

[0127] Test Example 4

[0128] In Example 1 and Examples 6-8, anhydrous ethanol was used as a control sample to obtain the changes in the DPBF reduction rate at different concentrations of DPBF and different irradiation times. Figure 5 shown.

[0129] from Figure 5 It can be seen that the absorption reduction rate of DPBF at 380-440nm decreases with the increase of DPBF concentration, indicating that under the same other conditions, the increase of DPBF concentration reduces the detection sensitivity of singlet oxygen. In the detection of astaxanthin's ability to scavenge singlet oxygen, the concentration of DPBF is selected to be 6-10μmol / L, which is conducive to ensuring the sensitivity of detection.

[0130] Test Example 5

[0131] In Examples 1, 9-12, 13-17, 18-22, and 23-27, mixed solutions of different ethanol and water volume ratios were used as reaction media to obtain the relationship curves of the quenching rate of singlet oxygen with different astaxanthin concentrations. Figure 6 shown.

[0132] As can be seen from the figure, the quenching rate of singlet oxygen increases with the increase of astaxanthin concentration, and when the ethanol-water mixed solution with a volume ratio of ethanol to water of 3:0 is used as the reaction medium, the quenching rate of singlet oxygen by astaxanthin is higher. When the ethanol-water mixed solution with a volume ratio of ethanol to water of 2:1 is used as the reaction medium, the quenching rate of singlet oxygen by astaxanthin is higher, so our preferred reaction conditions are a volume ratio of ethanol to water of 3:0 or 2:1.

[0133] Principle and advantages of use: This application uses methylene blue as a photosensitizer and DPBF as a probe. Under light conditions, methylene blue produces singlet oxygen. DPBF captures singlet oxygen and reacts with singlet oxygen to reduce the absorption of DPBF at 380-440nm. The change in the absorbance value of DPBF at 380-440nm is detected by ultraviolet-visible absorption spectroscopy to obtain the concentration change value of DPBF, and then the quenching constant k of astaxanthin for singlet oxygen is calculated according to the formula r , thereby achieving a quantitative evaluation of astaxanthin's ability to scavenge singlet oxygen.

[0134] The method for detecting and evaluating the anti-photooxidation ability of astaxanthin established by this patent has low requirements for experimental equipment and has the advantages of being simple, convenient and highly operational. It uses DPBF and methylene blue as the main detection reagents, which are cheap, easy to obtain and highly recognized, effectively reducing the detection cost.

[0135] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for evaluating the photo-oxidation resistance of astaxanthin, characterized in that: An experimental scheme and evaluation method for evaluating astaxanthin's resistance to light oxidation were established: using methylene blue as a photosensitizer, using xenon lamp irradiation, adding a 660nm filter, and generating singlet oxygen under light conditions; using DPBF as a probe to detect singlet oxygen, adding different concentrations of astaxanthin, and using UV-visible absorption spectroscopy to detect changes in the DPBF absorption peak at 380-440nm to determine the scavenging effect of astaxanthin on singlet oxygen; The sample without astaxanthin was used as the control, and the quenching constant k of astaxanthin for singlet oxygen was calculated according to the following formula: r : S0 / S=1+k r [A] / (k d +k Q [P]) Among them, S0 represents the initial reduction rate of the DPBF probe of the sample without astaxanthin; S represents the initial reduction rate of the DPBF probe in the sample containing astaxanthin; [A] represents the initial concentration of astaxanthin, and [P] represents the initial concentration of DPBF; k d represents the decay rate constant of singlet oxygen itself; k Q represents the rate constant of the reaction between singlet oxygen and DPBF; By k r Determine the singlet oxygen scavenging ability of astaxanthin.

2. A method for evaluating the anti-light oxidation ability of astaxanthin according to claim 1, characterized in that: The following steps are involved: (1) dissolving astaxanthin in an organic solvent to prepare a mother solution, and then diluting it with anhydrous ethanol to prepare an astaxanthin reserve solution; dissolving DPBF in anhydrous ethanol to prepare a DPBF-ethanol solution, and dissolving methylene blue in deionized water to prepare a methylene blue-water solution; (2) Add astaxanthin reserve solution, DPBF-ethanol solution, and methylene blue-water solution into a cuvette, fill it up with ethanol-water mixed solution, and seal the cuvette; use anhydrous ethanol as a control; (3) After the samples containing astaxanthin and the control samples were exposed to light, the samples were scanned using a UV-visible spectrophotometer to observe changes in the absorption peak of DPBF at 380-440 nm; (4) According to the change of DPBF absorption peak, the initial reduction rate of DPBF probe in astaxanthin-containing samples and control samples was calculated, and the quenching constant k of astaxanthin for singlet oxygen was calculated by substituting it into the formula: r .

3. A method for evaluating the anti-light oxidation ability of astaxanthin according to claim 2, characterized in that: The final concentrations of DPBF and methylene blue in step (2) are 6 μmol / L and 6 μmol / L, respectively.

4. A method for evaluating the light-oxidation resistance of astaxanthin according to claim 2, characterized in that: In the step (2), the volume ratio of ethanol to water in the ethanol-water mixed solution is 67%-100%.

5. A method for evaluating the anti-light oxidation ability of astaxanthin according to claim 2, characterized in that: The concentration of astaxanthin in the cuvette of step (2) is 0.5-2.0 μmol / L.

6. A method for evaluating the anti-photooxidation ability of astaxanthin according to claim 2, characterized in that: The concentration of DPBF in the cuvette of step (2) is 6-10 μmol / L, and the concentration of methylene blue is 6-9 μmol / L.

7. A method for evaluating the light-oxidation resistance of astaxanthin according to claim 2, characterized in that: In the step (3), a xenon lamp is used as the light source for inducing methylene blue to produce singlet oxygen, and the light intensity is adjustable.

8. A method for evaluating the light-oxidation resistance of astaxanthin according to claim 7, characterized in that: In the step (3), a filter is used to remove stray light from the light emitted by the xenon lamp, and the wavelength of the transmitted light is 660nm.

9. A method for evaluating the light-oxidation resistance of astaxanthin according to claim 2, characterized in that: The illumination time of the sample in the step (3) is 0 to 120 seconds.

10. The method for evaluating the anti-photooxidation ability of astaxanthin according to claim 2, characterized in that: The step (4) is to calculate the quenching constant k of astaxanthin for singlet oxygen. r k in the formula d 3×10 in water 5 s -1 , in ethanol it is 6.7×10 4 s -1 ;k Q 8x10 8 mol -1 s -1 .

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