CN111610322A - Method for determining repair degree of purple potato extract anthocyanin to oxidative damage in zebra fish body - Google Patents
Method for determining repair degree of purple potato extract anthocyanin to oxidative damage in zebra fish body Download PDFInfo
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Abstract
The invention discloses a method for determining the in vivo oxidative damage repair degree of purple potato extract anthocyanin to zebra fish, which comprises the steps of firstly establishing a zebra fish oxidative damage model; then measuring SOD activity and malondialdehyde content of the zebra fish embryo, the young fish and the adult fish after oxidative damage, and judging whether the zebra fish is oxidized or damaged; measuring main indexes of oxidative damage in the zebra fish body after oxidative damage, including ROS generation level, lipid peroxidation degree and apoptosis degree; and finally adding the purple potato extract anthocyanin solution into an artificial seawater culture medium for establishing the zebra fish oxidative damage model, carrying out oxidative damage repair on the zebra fish, and determining the strength of the purple potato extract anthocyanin solution on the zebra fish oxidative damage repair function. The determination method provided by the invention can accurately determine the degree of the anthocyanin extracted from the purple potato on the repair of the oxidative damage in the zebra fish body, and can be applied to the actual repair of the oxidative damage in the zebra fish body.
Description
Technical Field
The invention relates to the technical field of in vivo oxidative damage repair of zebra fish, in particular to a method for determining the in vivo oxidative damage repair degree of anthocyanin extracted from purple potatoes to zebra fish.
Background
Normally, the animal body has a dynamic balance of oxidation and antioxidation, i.e. the generation and elimination of free radicals in the body maintain the dynamic balance. However, when the body is in a specific environment, such as food composition changes, radioactive irradiation, heavy metal exposure, bacterial and viral infections, emotional and emotional instability, the body can be in an oxidative stress state.
Anthocyanins (anthocyanines) are water-soluble pigments widely present in plants, belong to flavonoid compounds, are powerful antioxidants, can protect human bodies from damage by harmful substances such as free radicals, and have certain functions in the aspects of preventing DNA cracking, estrogenic activity, enzyme inhibition, promoting production factors and the like to regulate immune response, anti-inflammatory activity, lipid peroxidation, cell membrane reinforcement and the like. However, the anthocyanin extract of different plants has different repair capability on the oxidative damage in the animal body, whether the anthocyanin extract of purple potato can repair the oxidative damage of the animal body, and the degree of the anthocyanin extract of purple potato to repair the oxidative damage of different animal bodies need to be determined in advance before practical application. At present, no specific index for evaluating the degree of oxidative damage exists at home and abroad.
Disclosure of Invention
In view of the existing problems, the invention aims to provide a method for measuring the repair degree of purple potato anthocyanin to the oxidative damage in zebra fish bodies, and a set of indexes for evaluating the oxidative damage degree are provided in the method.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the method for determining the repair degree of the anthocyanin of the purple potato extract to the oxidative damage in the zebra fish body is characterized by comprising the following steps,
s1: respectively establishing oxidation damage models of zebra fish embryos, juvenile fishes and adult fishes, and correspondingly setting a blank control group for comparison;
s2: determining SOD enzyme activity and malondialdehyde content of zebra fish embryos, juvenile fish and adult fish by using a superoxide dismutase activity determination kit and a malondialdehyde content determination kit, and determining the in vivo oxidative damage degree of the zebra fish after oxidative damage;
s3: measuring main indexes of oxidative damage in the zebra fish body, including ROS generation level, lipid peroxidation degree and apoptosis degree;
s4: adding 15-50 mg/L purple potato extract anthocyanin solution into an artificial seawater culture medium for establishing a zebra fish oxidative damage model, and performing oxidative damage repair on the zebra fish;
s5: detecting SOD activity, malondialdehyde content, ROS generation level, lipid peroxidation degree and apoptosis degree in the zebra fish embryo, juvenile fish and adult fish after the oxidative damage is repaired, and judging the degree of the oxidative damage repair of the zebra fish by the purple potato extract anthocyanin solution.
Further, the specific operation steps of establishing the oxidation damage model of the zebra fish embryo, the juvenile fish and the adult fish in the step S1 include,
s11: randomly dividing 7-8hpf zebra fish embryos and 4dpf zebra fish juvenile fish into a blank control group and a lipopolysaccharide treatment group, putting the blank control group and the lipopolysaccharide treatment group into a 12-hole culture plate, respectively carrying out oxidative stress on the 7-8hpf embryos and the 4dpf juvenile fish of the lipopolysaccharide treatment group for 14h and 24h by using 2.5ppm LPS, and then cleaning and re-suspending by using a fresh artificial seawater culture medium;
s12: adult zebrafish 4mpf were randomly divided into a blank control group and a lipopolysaccharide-treated group, and after exposing adult zebrafish 4mpf of the lipopolysaccharide-treated group to 0.1ppm LPS for 7 days, they were washed with fresh artificial seawater medium and resuspended.
Further, the specific operation steps of the SOD enzyme activity determination in step S2 include: adding physiological saline into zebra fish embryo and young fish to make homogenate, adding enzyme action substrate and buffer solution, incubating at 37 deg.C for 20min, and measuring SOD enzyme activity at 450nm wavelength of enzyme labeling instrument;
cutting tail of adult zebra fish, collecting blood, standing the blood at room temperature for 4h, sucking upper yellow supernatant, adding enzyme substrate and buffer solution, incubating at 37 deg.C for 20min, and measuring SOD enzyme activity at 450nm wavelength of enzyme labeling instrument.
Further, the specific operation steps of the determination of the content of malondialdehyde in step S2 include: adding malonaldehyde extract into zebra fish embryo and young fish to obtain homogenate, incubating at 95 deg.C for 40min, and measuring malonaldehyde content at 530nm wavelength of microplate reader;
cutting liver of adult zebra fish, adding malondialdehyde extractive solution, homogenizing, incubating at 95 deg.C for 40min, and measuring malondialdehyde content at 530nm wavelength of microplate reader.
Further, in step S3, the level of ROS production in zebrafish is determined using 2',7' -dichlorofluoroxanthate probe.
Further, the specific operation of measuring the ROS generation level in the zebra fish by using the 2',7' -dichlorofluorescence yellow diacetate probe comprises the following steps: and (3) treating the blank control group and the zebra fish subjected to oxidative damage with 20 mu g/mL of 2',7' -dichloro fluorescent yellow diacetate respectively, culturing for 1h at 28.5 ℃ in the dark, rinsing with artificial seawater for several times after 1h, anesthetizing with 0.02% tricaine, taking a picture under a fluorescent microscope, and calculating the fluorescence intensity of the pictures.
Further, in step S3, the degree of lipid peroxidation in zebrafish was measured using a 1, 3-bis (diphenylphosphino) propane probe.
Further, the specific operation steps of utilizing the 1, 3-bis (diphenylphosphino) propane probe to measure the lipid peroxidation degree in the zebra fish body comprise: and (3) treating the blank control group and the zebra fish subjected to oxidative damage with 25 mu g/mL 1, 3-bis (diphenylphosphino) propane respectively, culturing for 1h at 28.5 ℃ in the dark, rinsing with artificial seawater for several times after 1h, anesthetizing with 0.02% tricaine, placing under a fluorescence microscope for photographing, and calculating the fluorescence intensity.
Further, in step S3, the degree of apoptosis in zebrafish was measured using an acridine orange probe.
Further, the specific operation steps of determining the apoptosis degree of the zebra fish in vivo after oxidative damage by using the acridine orange probe comprise: and (3) taking a blank control group and the zebra fish after oxidative damage, respectively treating the blank control group and the zebra fish with 2.5 mu g/mL acridine orange, culturing the zebra fish in the dark at 28.5 ℃ for 30min, then leaching the zebra fish with artificial seawater for a plurality of times, anesthetizing the zebra fish with 0.02% tricaine, placing the zebra fish under a fluorescence microscope for photographing, then photographing the zebra fish under the fluorescence microscope, and calculating the fluorescence intensity.
The invention has the beneficial effects that:
1. according to the method, the repairing degree of the purple potato extract anthocyanin to the oxidative damage in the zebra fish body can be completely and rapidly detected by establishing the zebra fish oxidative damage model, and the method is expected to be popularized to the determination of other sources of anthocyanin.
2. According to the method for determining the in-vivo oxidative damage repair degree of the purple potato extract anthocyanin to the zebra fish, a set of indexes which can be used for evaluating the oxidative damage degree are provided, wherein the indexes comprise SOD enzyme activity, malondialdehyde, ROS generation level, lipid peroxidation degree and apoptosis degree, and can be used as specific evaluation indexes for evaluating other oxidative damage degrees.
Drawings
FIG. 1 is a bar graph showing the effect of LPS treatment on SOD enzyme activity of zebra fish embryos and young fish according to the present invention;
FIG. 2 is a bar graph showing the effect of LPS treatment on SOD enzyme activity in adult zebra fish according to the present invention;
FIG. 3 is a bar graph showing the effect of LPS treatment on the malondialdehyde content in zebra fish embryos and young fish;
FIG. 4 is a bar graph showing the effect of LPS treatment on the malondialdehyde content in adult zebra fish according to the invention;
FIG. 5 is a fluorescent photomicrograph of a 2',7' -dichlorofluorescein diacetate probe of the present invention at a time of ROS production levels in zebrafish embryos and larvae after oxidative damage;
FIG. 6 is a bar graph of the effect of LPS treatment of the present invention on ROS production levels in zebrafish embryos and juvenile fish;
FIG. 7 is a fluorescence micrographic image of the invention using 1, 3-bis (diphenylphosphino) propane probe to determine the degree of lipid peroxidation in the body of zebra fish embryos and larvae after oxidative damage;
FIG. 8 is a bar graph showing the effect of LPS treatment on the degree of lipid peroxidation in zebrafish embryos and larvae;
FIG. 9 is a fluorescence micrographic image of the invention using acridine orange probe to determine the degree of apoptosis in zebra fish embryos and larvae after oxidative damage;
FIG. 10 is a bar graph showing the effect of LPS treatment of the present invention on the degree of apoptosis in zebrafish embryos and larvae.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following further describes the technical solution of the present invention with reference to the drawings and the embodiments.
The method for determining the repair degree of the anthocyanin of the purple potato extract to the oxidative damage in the zebra fish body comprises the following steps,
s1: establishing a zebra fish oxidative damage model;
specifically, 7-8hpf (hour after fertilization) zebra fish embryos and 4dpf (day after fertilization) zebra fish larvae are randomly divided into a blank control group (NC) and a lipopolysaccharide treatment group (LPS), the blank control group (NC) and the lipopolysaccharide treatment group (LPS) are placed in a 12-hole culture plate, the zebra fish 7-8hpf embryos and the 4dpf larvae in the lipopolysaccharide treatment group are subjected to oxidative stress for 14 hours and 24 hours respectively by using 2.5ppm LPS, and then the zebra fish embryos and the 4dpf larvae are washed and resuspended by using a fresh artificial seawater culture medium.
Adult zebrafish 4mpf (months after fertilization, months fertilization, mpf) were randomly divided into a blank control group (NC) and a lipopolysaccharide-treated group (LPS), and the adult zebrafish 4mpf of the lipopolysaccharide-treated group was exposed with 0.1ppm LPS for 7 days, washed with fresh artificial seawater medium and resuspended.
S2: determining SOD enzyme activity and malondialdehyde content of zebra fish embryos, juvenile fish and adult fish by using a superoxide dismutase activity determination kit and a malondialdehyde content determination kit, and determining the in vivo oxidative damage degree of the zebra fish after oxidative damage treatment;
specifically, collecting blank control group and LPS oxidative damage treated embryo and young fish, adding physiological saline to make homogenate, adding substrate and buffer solution, incubating at 37 deg.C for 20min, and measuring SOD enzyme activity (U/mg) at 450nm wavelength of enzyme labeling instrument;
collecting blank control group and LPS oxidized injury treated adult fish, cutting tail, collecting blood, standing blood at room temperature for 4 hr, sucking upper yellow supernatant, adding substrate and buffer solution, incubating at 37 deg.C for 20min, and measuring SOD enzyme activity (U/mg) at 450nm wavelength of enzyme labeling instrument;
the measurement results are as follows:
embryo SOD enzyme Activity
Blank control group: SOD activity 3.32(U/mg)
Lipopolysaccharide makes module: SOD activity 3.98(U/mg)
Juvenile fish SOD enzyme activity
Blank control group: SOD activity 7.9554(U/mg)
Lipopolysaccharide makes module: SOD activity 13.1366(U/mg)
Adult fish SOD enzyme activity
Blank control group: SOD activity 10.75(U/mg)
Lipopolysaccharide makes module: SOD activity 14.78(U/mg)
Comparing the SOD enzyme activity results of the blank control group and LPS treated zebra fish embryo and young fish to obtain histogram, and the result is shown in figure 1; the SOD enzyme activity results of the blank control group and LPS treated adult zebra fish are prepared into bar chart, and the results are shown in figure 2.
Further, collecting blank control group and LPS oxidative damage treated embryo and young fish, adding malondialdehyde extract, homogenizing, incubating at 95 deg.C for 40min, and measuring malondialdehyde content (nmol/mg) at 530nm wavelength of microplate reader;
collecting blank control group and LPS oxidative damage treated adult fish, cutting liver, adding malondialdehyde extract, homogenizing, incubating at 95 deg.C for 40min, and measuring MDA (malondialdehyde) content (nmol/mg) at 530nm wavelength of microplate reader;
the measurement results are as follows:
embryo MDA content
Blank control group: MDA content 2.49(nmol/mg)
Lipopolysaccharide makes module: MDA content 3.32(nmol/mg)
Young fish MDA content
Blank control group: MDA content 4.47(nmol/mg)
Lipopolysaccharide makes module: MDA content 6.93(nmol/mg)
MDA content of adult fish
Blank control group: MDA content 6.89(nmol/mg)
Lipopolysaccharide makes module: MDA content 9.92(nmol/mg)
Comparing and analyzing MDA contents of the blank control group and LPS-treated zebra fish embryo and juvenile fish into histogram, and the result is shown in figure 3; the blank control group and the LPS-treated adult zebra fish have MDA content which is prepared into a histogram for comparative analysis, and the result is shown in figure 4.
Superoxide dismutase (SOD) is an important antioxidant enzyme in organisms, and can prevent damage of cell structures by free radicals. The higher the SOD activity, the stronger the oxidative stress in the organism.
Exogenous contaminants induce a large number of oxygen radicals produced by zebrafish, which if not removed in time, will bind to unsaturated fatty acids in the biofilm, causing lipid peroxidation and thus formation of lipid peroxides such as malondialdehyde. Therefore, MDA is considered to be one of the most representative indexes reflecting body oxidative damage. The higher the Malondialdehyde (MDA) content, the more severe the attack of the body cells by free radicals.
As can be seen by combining the attached figures 1-4, for zebra fish embryos, the SOD enzyme activity and the MDA content of the zebra fish embryos are not significantly different from those of a control group after being treated by LPS; the SOD enzyme activity and the MDA content of the zebra fish juvenile fish treated by LPS are obviously improved compared with those of the NC group and respectively reach 1.63 times and 1.55 times of those of the NC group; for adult zebra fish, after LPS treatment, the SOD enzyme activity and the MDA content of the adult zebra fish are obviously improved compared with those of a control group and respectively reach 1.38 times and 1.46 times of those of an NC group.
S3: measuring main indexes of oxidative damage in zebra fish embryos and juvenile fish bodies, including ROS generation level, lipid peroxidation degree and apoptosis degree;
one of the most important markers of oxidative stress in the body is the accumulation of Reactive Oxygen Species (ROS) in the body. Oxygen radicals can attack unsaturated fatty acids in biological membranes, initiating lipid peroxidation, and forming lipid peroxidation products. Three probes, namely 2',7' -dichloro-fluorescent yellow diacetate (DCFH-DA), 1, 3-bis (diphenylphosphino) propane (DPPP) and Acridine Orange (AO), can be used for detecting the ROS generation level, the lipid peroxidation degree and the apoptosis condition in zebra fish bodies respectively so as to judge whether the molding is successful or not.
Specifically, the ROS generation level in zebrafish embryos and juvenile fish is measured by using a 2',7' -dichlorofluorescein diacetate probe:
taking a blank control group and the zebra fish embryo and the young fish after oxidative damage, respectively treating the blank control group and the zebra fish embryo and the young fish with 20 mu g/mL of 2',7' -dichloro-fluorescent yellow diacetate, culturing the zebra fish embryo and the young fish for 1h at 28.5 ℃ in the dark, rinsing the zebra fish embryo and the young fish with artificial seawater for a plurality of times after 1h, anesthetizing the zebra fish embryo and the young fish with 0.02% tricaine, and then placing the zebra fish embryo and the young fish under a fluorescence microscope for photographing, wherein the results are shown:
embryonic ROS production level (DCFH-DA Probe detection)
Blank control group: DCF fluorescence intensity 665.225(au)
Lipopolysaccharide makes module: DCF fluorescence intensity 1695.998(au)
Juvenile fish ROS production level (DCFH-DA Probe detection)
Blank control group: DCF fluorescence intensity 774.856(au)
Lipopolysaccharide makes module: DCF fluorescence intensity 1329.457(au)
The generation level of ROS is an important index for judging oxidative stress, and the DCFH-DA probe does not fluoresce, but after the DCFH-DA probe is combined with ROS in a living body to react, the DCFH-DA probe is cracked into DCF and can emit green fluorescence. The greater the fluorescence intensity, the higher its ROS level and the more severe the oxidative damage. The ROS production levels of zebrafish embryos and juvenile fish were histogram analyzed and the results are shown in fig. 6. As can be seen from the attached figure 5, the fluorescence intensity of the LPS-treated zebra fish embryos and the juvenile fish is very different from that of the control group, and the fluorescence intensity of the LPS group is 2.55 times and 2.49 times that of the NC group respectively, which shows that the ROS level can be remarkably improved by the LPS-treated zebra fish embryos and the juvenile fish.
Further, the lipid peroxidation degree in the zebra fish embryo and the juvenile fish is measured by using a 1, 3-bis (diphenylphosphino) propane probe:
taking a blank control group, and the zebra fish embryo and the young fish after oxidative damage, respectively treating the blank control group and the zebra fish embryo and the young fish with 25 mu g/mL of 1, 3-bis (diphenylphosphino) propane, culturing for 1h at 28.5 ℃ in the dark, rinsing with artificial seawater for several times after 1h, anesthetizing with 0.02% tricaine, placing under a fluorescence microscope for photographing, wherein the result is shown in the attached figure 7, photographing under the fluorescence microscope, and calculating the fluorescence intensity, wherein the calculation results are respectively:
degree of embryo lipid peroxidation (DPPP Probe test)
Blank control group: DPPP fluorescence intensity 524.67(au)
Lipopolysaccharide makes module: DPPP fluorescence intensity 1613.573(au)
Degree of lipid peroxidation in juvenile fish (DPPP Probe test)
Blank control group: DPPP fluorescence intensity 1031.873(au)
Lipopolysaccharide makes module: DPPP fluorescence intensity 1802.793(au)
The degree of lipid peroxidation is also an important index for judging oxidative stress, and DPPP emits blue fluorescence after reacting with lipid peroxide in vivo, and the higher the fluorescence intensity, the higher the degree of lipid peroxidation in vivo. The lipid peroxidation degree of the zebra fish embryo and the juvenile fish is prepared into a histogram for comparative analysis, and the result is shown in figure 8. As can be seen from FIG. 8, the fluorescence intensities of the LPS-treated zebra fish embryos and the juvenile fish are very different from those of the control group, and the fluorescence intensities of the LPS group are 3.08 times and 2.67 times of those of the NC group, respectively, which indicates that the LPS-treated zebra fish embryos and the juvenile fish can cause significant lipid peroxide accumulation.
Furthermore, the degree of apoptosis in zebra fish embryos and juvenile fish is measured by using an acridine orange probe.
Specifically, the method comprises the following steps: respectively treating a blank control group and the zebra fish embryo and the young fish after oxidative damage with 2.5 mu g/mL acridine orange, culturing at 28.5 ℃ in the dark for 30min, then leaching with artificial seawater for several times, anesthetizing with 0.02% tricaine, and then taking a picture under a fluorescence microscope, wherein the results are shown in the attached figure 9, and the fluorescence intensity is calculated as follows:
embryo DNA Damage level (AO Probe test)
Blank control group: AO fluorescence intensity 646.538(au)
Lipopolysaccharide makes module: AO fluorescence intensity 1765.676(au)
DNA damage level of juvenile fish (AO Probe test)
Blank control group: AO fluorescence intensity 729.483(au)
Lipopolysaccharide makes module: AO fluorescence intensity 2088.045(au)
The apoptosis degree is also an important index for judging oxidative stress, and the organism is oxidized and damaged to cause damage to DNA in cells. The AO probe emits green fluorescence when combined with DNA and red fluorescence when combined with RNA. The apoptosis degree of zebra fish embryo and juvenile fish is prepared into histogram for comparative analysis, and the result is shown in figure 10. As can be seen from FIG. 10, the fluorescence intensities of the LPS-treated zebra fish embryos and the juvenile fish are very different from those of the control group, and the fluorescence intensities of the LPS group are 2073 times and 1.92 times of those of the NC group respectively, which indicates that the LPS-treated zebra fish embryos and the juvenile fish can cause significant apoptosis.
In conclusion, the LPS treatment of the zebra fish embryo and the young fish is an effective oxidative damage modeling method, and the oxidative damage model of the zebra fish embryo is detected by a fluorescence intensity method, so that the systemic oxidative damage, namely ROS (reactive oxygen species) generation level, lipid peroxidation degree and apoptosis are obviously different from those of a control group.
For the oxidation damage model of the zebra fish juvenile fish and adult fish after organ differentiation, the fluorescence detected by adopting the fluorescence intensity method is not systemic and is only concentrated on a certain organ part, and the quantitative data is unreliable. Therefore, only the physiological index method in step S2 is used to determine the activity and content of SOD enzyme, and the determination result shows that both the activity of SOD enzyme and the content of MDA are significantly different from those of the control group.
S4: adding 15-50 mg/L purple potato extract anthocyanin solution into an artificial seawater culture medium for establishing a zebra fish oxidative damage model, and performing oxidative damage repair on the zebra fish;
specifically, 7-8hpf zebrafish embryos are divided into 6 groups: blank control group (NC), oxidation modeling group (LPS), positive control group (Vc) and three dosage groups of low, medium and high anthocyanin;
the culture medium of the blank control group is an artificial seawater culture medium, the oxidation making module group is only treated by LPS, the positive control group (Vc) is added with LPS and subjected to synergistic treatment by Vc with the final concentration of 30mg/L, the anthocyanin low, medium and high dose groups are respectively added with LPS and the purple potato extract anthocyanin with the concentrations of 15mg/L, 30mg/L and 50mg/L for synergistic treatment, and the concentration of the LPS is 2.5 ppm. After 14h of treatment the drug was removed and cultured to 24 hpf.
4dpf of zebra fish larvae were divided into 6 groups: blank control group (NC), oxidation modeling group (LPS), positive control group (Vc) and three dosage groups of low, medium and high anthocyanin;
the culture medium of the blank control group is an artificial seawater culture medium, the oxidation making module group is only treated by LPS, the positive control group (Vc) is added with LPS and subjected to synergistic treatment with Vc with the final concentration of 30ppm, the anthocyanin low, medium and high dose groups are respectively added with LPS and purple potato extract anthocyanin with the concentration of 15mg/L, 30mg/L and 50mg/L for synergistic treatment, the concentration of the LPS is 2.5ppm, and the medicine is removed after 24 hours of treatment.
Adult zebrafish of 4mpf were selected for the experiment, and the male and female were randomly divided into a blank control group (NC), an oxidation-induced disease module (LPS), and an anthocyanin group. The blank control group was cultured in artificial seawater, the oxidation module group was treated with LPS only, and the anthocyanin group was treated with LPS and 15mg/L purple potato extract anthocyanin in a synergistic manner, with the concentration of LPS being 0.1 ppm. Feeding 10 zebra fish in each group for three times every day, and replacing fresh culture medium after feeding. The drug was removed after treatment 7 d.
S5: detecting SOD activity, malondialdehyde content, ROS generation level, lipid peroxidation degree and apoptosis degree in the zebra fish embryo, juvenile fish and adult fish after the oxidative damage is repaired, and judging the degree of the oxidative damage repair of the zebra fish by the purple potato extract anthocyanin solution.
The fluorescence intensity method is adopted to detect the ROS generation level, the lipid peroxidation degree and the apoptosis condition in the zebra fish embryo body so as to determine the oxidative damage repair degree of the anthocyanin extracted from the purple potato to the zebra fish embryo body, and the result is as follows:
ROS level of production (DCFH-DA Probe test)
Blank control group: DCF fluorescence intensity 674.865(au)
Oxidizing to form a module: DCF fluorescence intensity 1681.65(au)
Positive control group (Vc): DCF fluorescence intensity 1100.28(au)
Anthocyanin repair group: low: DCF fluorescence intensity 1329.457(au)
The method comprises the following steps: DCF fluorescence intensity 1276.387(au)
High: DCF fluorescence intensity 1236.357(au)
Degree of lipid peroxidation (DPPP Probe test)
Blank control group: DPPP fluorescence intensity 597.49(au)
Oxidizing to form a module: DPPP fluorescence intensity 1442.919(au)
Positive control group (Vc): DPPP fluorescence intensity 1345.831(au)
Anthocyanin repair group: low: DPPP fluorescence intensity 1469.457(au)
The method comprises the following steps: DPPP fluorescence intensity 1265.899(au)
High: DPPP fluorescence intensity 1214.798(au)
DNA Damage level (AO Probe test)
Blank control group: AO fluorescence intensity 546.538(au)
Oxidizing to form a module: AO fluorescence intensity 1830.776(au)
Positive control group (Vc): AO fluorescence intensity 1190.009(au)
Anthocyanin repair group: low: AO fluorescence intensity 945.718(au)
The method comprises the following steps: AO fluorescence intensity 1074.842(au)
High: AO fluorescence intensity 1130.025(au)
From the results, the purple potato extract anthocyanin repair group can reduce the oxidation damage degree of the zebra fish embryo model and obviously reduce the fluorescence intensity of the zebra fish embryo repair group as the Vc of the positive control group, which indicates that the purple potato extract anthocyanin can really and obviously repair the oxidation damage in the zebra fish embryo body. DCF fluorescence intensity and DPPP fluorescence intensity show that with the increase of anthocyanin concentration, the repair capacity to oxidative damage caused by oxygen free radicals and lipid peroxidation products is improved, but the result of AO fluorescence intensity is just opposite, and the repair capacity of low-concentration anthocyanin to apoptosis is stronger.
Further, the kit is used for detecting the SOD enzyme activity and the MDA content of the zebra fish juvenile fish so as to determine the oxidative damage repair degree of the anthocyanin in the purple potato extract, and the specific result is as follows:
SOD enzyme activity
Blank control group: 8.9554(U/mg)
Oxidizing to form a module: 13.1366(U/mg)
Positive control group: 10.4381(U/mg)
Anthocyanin repair group: low: 10.3788(U/mg)
The method comprises the following steps: 11.0972(U/mg)
High: 11.0565(U/mg)
MDA content
Blank control group: 4.84(nmol/mg)
Oxidizing to form a module: 6.39(nmol/mg)
Positive control group: 5.18(nmol/mg)
Anthocyanin repair group: low: 4.93(nmol/mg)
The method comprises the following steps: 5.26(nmol/mg)
High: 5.09(nmol/mg)
Compared with the modeling group, the anthocyanin repair group extracted from the purple potato can obviously reduce the SOD enzyme activity and the MDA content of the zebra fish juvenile fish as the positive control Vc, which indicates that the anthocyanin can obviously repair the oxidative damage in the zebra fish juvenile fish. Wherein, the repairing capability is strongest by adding 15mg/L anthocyanin, and the SOD activity and the MDA content are lower than those of the positive control Vc.
Further, cutting the tail of the adult zebra fish to obtain blood, and cutting the liver. Placing the blood at room temperature for 4h, sucking the upper yellow supernatant, weighing the liver, homogenizing, and detecting the SOD activity of the serum and the MDA content of the liver by using the kit to determine the oxidative damage repair degree of the anthocyanin in the purple potato extract, wherein the specific result is as follows:
SOD enzyme activity
Blank control group: SOD enzyme activity 10.65(U/mg)
Oxidizing to form a module: SOD enzyme activity 14.96(U/mg)
Anthocyanin repair group: SOD enzyme activity 11.29(U/mg)
MDA content
Blank control group: MDA content 6.84(nmol/mg)
Oxidizing to form a module: MDA content 9.89(nmol/mg)
Anthocyanin repair group: MDA content 7.76(nmol/mg)
Compared with a modeling module, the SOD enzyme activity and MDA content of the adult zebra fish can be obviously lower than those of the modeling module by adding 15mg/L of the purple potato extract anthocyanin, and can be respectively reduced by 23.29% and 24.30%, which indicates that the purple potato extract anthocyanin can really and obviously repair the oxidative damage in the adult zebra fish.
In conclusion, the purple potato extract anthocyanin has the highest repairing degree on the oxidative damage of the zebra fish embryos and the young fishes, wherein the repairing capability on the oxidative damage in the zebra fish bodies is the strongest when 15mg/L of the purple potato extract anthocyanin is added.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. The method for determining the repair degree of the anthocyanin of the purple potato extract to the oxidative damage in the zebra fish body is characterized by comprising the following steps,
s1: respectively establishing oxidation damage models of zebra fish embryos, juvenile fishes and adult fishes, and correspondingly setting a blank control group for comparison;
s2: determining SOD enzyme activity and malondialdehyde content of zebra fish embryos, juvenile fish and adult fish by using a superoxide dismutase activity determination kit and a malondialdehyde content determination kit, and determining the in vivo oxidative damage degree of the zebra fish after oxidative damage;
s3: measuring main indexes of oxidative damage in the zebra fish body, including ROS generation level, lipid peroxidation degree and apoptosis degree;
s4: adding 15-50 mg/L purple potato extract anthocyanin solution into an artificial seawater culture medium for establishing a zebra fish oxidative damage model, and performing oxidative damage repair on the zebra fish;
s5: detecting SOD activity, malondialdehyde content, ROS generation level, lipid peroxidation degree and apoptosis degree in the zebra fish embryo, juvenile fish and adult fish after the oxidative damage is repaired, and judging the degree of the oxidative damage repair of the zebra fish by the purple potato extract anthocyanin solution.
2. The method for determining the degree of restoration of oxidative damage in zebra fish by anthocyanins extracted from purple potato as claimed in claim 1, wherein: step S1, the concrete operation steps of establishing the oxidation damage model of zebra fish embryo, juvenile fish and adult fish include,
s11: randomly dividing 7-8hpf zebra fish embryos and 4dpf zebra fish juvenile fish into a blank control group and a lipopolysaccharide treatment group, putting the blank control group and the lipopolysaccharide treatment group into a 12-hole culture plate, respectively carrying out oxidative stress on the 7-8hpf embryos and the 4dpf juvenile fish of the lipopolysaccharide treatment group for 14h and 24h by using 2.5ppm LPS, and then cleaning and re-suspending by using a fresh artificial seawater culture medium;
s12: adult zebrafish 4mpf were randomly divided into a blank control group and a lipopolysaccharide-treated group, and after exposing adult zebrafish 4mpf of the lipopolysaccharide-treated group to 0.1ppm LPS for 7 days, they were washed with fresh artificial seawater medium and resuspended.
3. The method for determining the degree of repairing the oxidative damage of the purple potato extract anthocyanin to the zebra fish as claimed in claim 2, wherein the specific operation steps of the SOD enzyme activity determination in the step S2 comprise: adding physiological saline into zebra fish embryo and young fish to make homogenate, adding enzyme action substrate and buffer solution, incubating at 37 deg.C for 20min, and measuring SOD enzyme activity at 450nm wavelength of enzyme labeling instrument;
cutting tail of adult zebra fish, collecting blood, standing the blood at room temperature for 4h, sucking upper yellow supernatant, adding enzyme substrate and buffer solution, incubating at 37 deg.C for 20min, and measuring SOD enzyme activity at 450nm wavelength of enzyme labeling instrument.
4. The method for determining the repair degree of the anthocyanins extracted from the purple potato to the oxidative damage in the zebra fish as claimed in claim 2, wherein the specific operation steps of the determination of the content of the malondialdehyde in the step S2 comprise: adding malonaldehyde extract into zebra fish embryo and young fish to obtain homogenate, incubating at 95 deg.C for 40min, and measuring malonaldehyde content at 530nm wavelength of microplate reader;
cutting liver of adult zebra fish, adding malondialdehyde extractive solution, homogenizing, incubating at 95 deg.C for 40min, and measuring malondialdehyde content at 530nm wavelength of microplate reader.
5. The method for determining the degree of restoration of oxidative damage in zebra fish by anthocyanins extracted from purple potato as claimed in claim 1, wherein: in step S3, the ROS production level in zebrafish is determined using 2',7' -dichlorofluoroxanthate probe.
6. The method for determining the degree of restoration of oxidative damage in zebra fish by anthocyanins from a purple potato extract as claimed in claim 5, wherein: the specific operation of utilizing the 2',7' -dichloro-fluorescent yellow diacetate probe to measure the ROS generation level in the zebra fish body comprises the following steps: and (3) treating the blank control group and the zebra fish subjected to oxidative damage with 20 mu g/mL of 2',7' -dichloro fluorescent yellow diacetate respectively, culturing for 1h at 28.5 ℃ in the dark, rinsing with artificial seawater for several times after 1h, anesthetizing with 0.02% tricaine, taking a picture under a fluorescent microscope, and calculating the fluorescence intensity of the pictures.
7. The method for determining the degree of restoration of oxidative damage in zebra fish by anthocyanins extracted from purple potato as claimed in claim 1, wherein: in step S3, the lipid peroxidation degree in zebra fish is measured using a 1, 3-bis (diphenylphosphino) propane probe.
8. The method for determining the degree of restoration of oxidative damage in zebra fish by anthocyanins extracted from purple potato as claimed in claim 7, wherein: the specific operation steps for determining the lipid peroxidation degree in the zebra fish body by using the 1, 3-bis (diphenylphosphino) propane probe comprise: and (3) treating the blank control group and the zebra fish subjected to oxidative damage with 25 mu g/mL 1, 3-bis (diphenylphosphino) propane respectively, culturing for 1h at 28.5 ℃ in the dark, rinsing with artificial seawater for several times after 1h, anesthetizing with 0.02% tricaine, placing under a fluorescence microscope for photographing, and calculating the fluorescence intensity.
9. The method for determining the degree of restoration of oxidative damage in zebra fish by anthocyanins extracted from purple potato as claimed in claim 1, wherein: in step S3, the degree of apoptosis in zebrafish is measured using acridine orange probe.
10. The method for determining the degree of restoration of oxidative damage in zebra fish by anthocyanins from a purple potato extract as claimed in claim 9, wherein: the specific operation steps for determining the apoptosis degree of the zebra fish in vivo after oxidative damage by using the acridine orange probe comprise: and (3) treating the blank control group and the zebra fish subjected to oxidative damage with 2.5 mu g/mL acridine orange respectively, culturing for 30min at 28.5 ℃ in the dark, then leaching with artificial seawater for several times, anesthetizing with 0.02% tricaine, then taking a picture under a fluorescence microscope, then taking a picture under the fluorescence microscope, and calculating the fluorescence intensity.
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