CN113670875B - Method for screening antioxidant by using delicious euglena - Google Patents
Method for screening antioxidant by using delicious euglena Download PDFInfo
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Abstract
The invention discloses a method for screening an antioxidant by utilizing a delicious euglena, which comprises three steps of breeding the delicious euglena, establishing an ROS model of the delicious euglena and verifying the model, wherein the establishment of the ROS model of the delicious euglena comprises the steps of testing the selection of the delicious euglena, determining lymphocytes producing ROS, determining a transparent cell separation method and determining a positive control compound H 2 O 2 Determining concentration and acting time, and performing in-vitro stability and reproducibility experiments on ROS. The method has good application prospect in screening the antioxidant by utilizing the euglena gracilis model, and the euglena gracilis belongs to a body cavity animal with closed-tube circulation, so that the metabolism of an organism can be reflected more truly; the cell membrane of the transparent cell of the kouchima is thin, the fluorescent marker and the antioxidant can easily permeate into the transparent cell, and the oxidation resistance result can be better displayed; the method has the advantages of easy culture of the pungent sipunculus nudus, low cost and simple experimental operation.
Description
Technical Field
The invention belongs to the technical field of screening antioxidants, and particularly relates to a method for screening antioxidants by using a delicious euglena.
Background
With the improvement of living standard, people pay more attention to physical health. Active oxygen is an important substance affecting biological signaling, and its accumulation is associated with the occurrence of various chronic diseases such as aging and cardiovascular diseases. Many pure compounds, foods and dietary supplements are currently being extensively studied for their antioxidant activity, and new antioxidants (i.e., radical scavengers) are being screened for continued depth.
At present, antioxidant screening evaluation models mainly comprise in-vitro evaluation and in-vivo evaluation methods, wherein the in-vitro evaluation mainly comprises a chemical evaluation method and a cell model evaluation method, and the in-vivo evaluation method mainly comprises an animal evaluation method. The chemical evaluation method cannot truly simulate the physiological environment, the effects of drug transmembrane entering cells and the like are not considered, and the cell model evaluation method does not consider factors such as in-vivo metabolism and the like, so that the biggest defect of the in-vitro evaluation method is that the in-vivo environment cannot be truly simulated, and the actual antioxidant effect of antioxidants cannot be truly reflected. In vivo antioxidant animal model size mouse model and zebra fish model mainly comprise mice, and the method is expensive and time-consuming and is not suitable for early screening of foods and dietary supplements. The zebra fish model has the defects of small individual, high injection difficulty, little blood, incapability of extracting and the like. Therefore, it is necessary to screen an antioxidant screening evaluation model which has the advantages of not only an in vitro cell model but also an in vivo animal model.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a method for screening antioxidants by using the delicious euglena, which solves the problems that a chemical evaluation method cannot truly simulate physiological environment, medicines enter cells through a membrane and the like are not considered, in vivo metabolism and the like are not considered in a cell model evaluation method, and a rat animal model method is expensive, time-consuming, difficult to operate and the like.
In order to achieve the above purpose, the present invention provides the following technical solutions: a method for screening antioxidants using a palatable euglena comprising the steps of:
s1: and (3) culturing the delicious euglena: healthy and delicious euglena with the weight of 4.0+/-0.5 g is picked and put into a plastic square tray for feeding, and sandy soil with the thickness of 5 cm is paved in the tray.
S2: building a delicious euglena ROS model:
a1: test selection of the delicious euglena: the method comprises the steps of selecting a delicious euglena with a weight of 4.0+/-0.5 g, extracting euglena lymph with a 1mL syringe, observing the types of blood cells of the lymph under a microscope, distinguishing female and male delicious euglena, and selecting male delicious euglena as a test animal.
A2: determination of ROS-producing lymphocytes: selecting a developed and mature sipunculus nudus individual, extracting sipunculus nudus lymph liquid by a 1mL syringe, centrifuging, diluting into 106cell/mL blood cell suspension by PBS, taking 200uL, adding 50 mu LDCFH-DA working solution, gently mixing, incubating in a dark place, washing twice by PBS after incubation in a dark place, then gently resuspending blood cells by 200uL of PBS, and observing the blood cells presenting green light under a fluorescence microscope so as to determine the blood cells for measuring the ROS content.
A3: clear cell isolation method determination: a gradient was made with 15% and 45% Pereol (v/v) in each of lmL in a centrifuge tube, and 5mL of blood cell suspension was added to the gradient, after centrifugation for a period of time, the cell status of each layer was observed, and the layer where the clear cells were located was determined.
A4: positive control compound H 2 O 2 Determination of concentration and duration of action: configuring H with different gradient concentrations 2 O 2 Liquid, 50uL of H with different concentration is injected into the body cavity of the sipunculus 2 O 2 Extracting lymph from each Sipunculus nudus after multiple interval, centrifuging to collect Sipunculus nudus transparent blood cells, and diluting to 10 7 Determining the ROS content of the transparent blood cells according to the A2 step method, and determining H by taking the standard that the survival rate of the sipunculus nudus is more than 95% and the change range of the ROS content of the transparent cells is less than 5% at different time points 2 O 2 Concentration and duration of action.
A5: ROS in vitro stability: with 200. Mu. Mol/L H 2 O 2 After the stress of the asteroid, respectively taking the coelomic fluid of the asteroid after the gradient interval time, centrifugally collecting transparent blood cells, diluting to 10 7 And (3) placing the sample in an environment with a certain temperature, respectively measuring the ROS content of the transparent cells at different intervals, and determining the placing time by taking the ROS content variation range as a standard, namely measuring the operation time of the ROS content test.
A6: reproducibility experiments: to verify reproducibility of the ROS-producing Starworm model, 50ul of 200. Mu. Mol/L H was injected into the body cavity of the Starworm using the method determined in step A4 2 O 2 Extracting lymph after a certain interval, centrifuging to collect Sipunculus nudus transparent blood cells, diluting to 10 7 And measuring the ROS content of the transparent blood cells, repeating the steps for a plurality of times, calculating the RSD value, and examining the repeatability of the method.
S3: model verification:
a1: antioxidant verification effect: the ROS model is divided into 2 groups of test groups and control groups, 10 test groups of the Sipunculus nudus are internally injected with 200uL of 2mg/mL of vitamin C, the control groups of the Sipunculus nudus are internally injected with the same amount of physiological saline, and after a certain interval of time, the ROS content of transparent blood cells of the Sipunculus nudus body cavity is observed.
A2: ROS clearance calculation: ROS clearance (%) = (control ROS content-test ROS content)/control ROS content x 100%.
Preferably, the ratio of soil to sand in sandy soil paved in a dish in the cultivation of the sipunculus nudus in the step S1 is 3:1, the cultivation condition is dry dew for 12 hours per day, water flooding is carried out for 12 hours, the salinity of the cultivation seawater is 25+/-1 ppt, the temperature is 25+/-1 ℃, and diatoms are fed during cultivation.
Preferably, the transparent cells in the determination of the lymphocytes producing ROS in the step A2 in the step S2 in the establishment of the delicious euglena ROS model are ROS-producing cells.
Preferably, in the determination of the transparent cell separation method of the step A3 in the establishment of the S2 step of the delicious euglena ROS model, a gradient is made by using 15% and 45% of Pereol liquid, and the mixture is centrifuged at 4000rpm/min for 10min.
Preferably, the positive control compound H of the step A4 in the step S2 of the establishment of the delicious Phascotaomica ROS model 2 O 2 In the determination of concentration and duration of action, H 2 O 2 The concentration of the liquid is increased to the upper layer gradient of 50 mu mol/L, and the concentration is as follows: 0. Mu. Mol/L, 50. Mu. Mol/L, 100. Mu. Mol/L, 150. Mu. Mol/L, 200. Mu. Mol/L, 250. Mu. Mol/L, 300. Mu. Mol/L, 350. Mu. Mol/L, 400. Mu. Mol/L and 450. Mu. Mol/L, and the time for measuring the ROS content by extracting lymph fluid from each of the Sipunculus nudusRespectively injection H 2 O 2 And (3) after 3, 6, 12, 24 and 48 hours, and determining H by taking the standard that the survival rate of the sipunculus nudus is more than 95% and the variation range of the ROS content of the transparent cells is less than 5% 2 O 2 Concentration and duration of action.
Preferably, in the in vitro stability of the ROS in the step A5 in the step S2 of the establishment of the delicious Phascrocarum ROS model, 200 mu mol/L of H is injected 2 O 2 And taking the coelomic fluid of the sipunculus nudus after 3h, 6h, 9h, 12h, 15h and 18h respectively, centrifuging to collect transparent blood cells, and after standing for 0h, 0.5h, 1h, 2h, 3h, 4h, 5h and 6h at the temperature of 25 ℃, measuring the ROS content of the transparent cells respectively, and determining the time by taking the ROS content change range less than 5% as a standard.
Preferably, in the step A6 repeatability experiment in the establishment of the S2-step delicious euglena ROS model, the time for extracting the lymph fluid is 3h, 6h, 9h, 12h, 15h and 18h respectively, and the number of repetitions of the repeatability experiment is at least 5.
Preferably, in the antioxidant verification effect of step A1 in the step S3 model verification, the ROS content of the transparent blood cells of the body cavity of the sipunculus nudus is measured after 1h, 3h, 6h and 12h respectively by using the ROS clearance as an index, and the clearance is calculated.
Compared with the prior art, the invention has the beneficial effects that:
1. the method has good application prospect in screening antioxidants by utilizing the sipunculus nudus, and the sipunculus nudus belongs to the sipunculus nudus animal phylum, is a marine body cavity animal with closed tube circulation, belongs to living animal cells, and can truly reflect organism metabolism.
2. The method has good application prospect in screening the antioxidant by utilizing the sipunculus nudus, has simpler morphological result, belongs to lower animals, reduces the interference of complex life phenomena, and is favorable for researching the basic metabolism mechanism of life bodies.
3. The method for screening the antioxidant by using the sipunculus nudus has good application prospect, the coelomia of the sipunculus nudus is filled with a large amount of lymphocytic cells, the blood cells are single in type and only contain transparent cells and granular cells, and the transparent cells and the granular cells are easily separated by a centrifugal method, so that the transparent cells of a research object are easily obtained.
4. The method has good application prospect in screening the antioxidant by utilizing the sipunculus nudus, the cell membrane of the transparent cell of the sipunculus nudus is thin, the fluorescent marker and the antioxidant are easy to permeate into the transparent cell, and the antioxidant result can be displayed better.
5. The method for screening the antioxidant by utilizing the sipunculus nudus has good application prospect, and the sipunculus nudus is easy to breed, low in cost and simple in experimental operation.
Drawings
FIG. 1 is a schematic flow diagram of the present invention;
FIG. 2 is a schematic representation of the ROS fluorescent pattern of the clear cell of the present invention;
FIG. 3 is a schematic representation of the invention without H 2 O 2 Color schematic of stress transparent cells under fluorescence microscope;
FIG. 4 is a diagram of the H of the present invention 2 O 2 Color schematic of stress transparent cells under fluorescence microscope;
FIG. 5 shows the different concentrations of H according to the present invention 2 O 2 Schematic diagram of the change of the ROS content of the transparent cells under the action time;
FIG. 6 shows the different concentrations of H according to the invention 2 O 2 A diagram of the survival rate of the sipunculus nudus under the action time;
FIG. 7 is a schematic of the in vitro stability of the transparent cell ROS of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1-7, the present invention provides a technical solution: a method for screening antioxidants using a palatable euglena comprising the steps of:
s1: and (3) culturing the delicious euglena: healthy and delicious euglena with the weight of 4.0+/-0.5 g is picked and put into a plastic square tray for breeding, sandy soil with the thickness of 5 cm is paved in the tray, the proportion of soil to sand in the sandy soil is 3:1, the breeding condition is that dry dew is carried out for 12 hours per day, water flooding is carried out for 12 hours, the salinity of breeding seawater is 25+/-1 ppt, the temperature is 25+/-1 ℃, and diatoms are fed during breeding.
S2: building a delicious euglena ROS model:
a1: test selection of the delicious euglena: the method comprises the steps of selecting a delicious euglena with a weight of 4.0+/-0.5 g, extracting euglena lymph with a 1mL syringe, observing the types of blood cells of the lymph under a microscope, distinguishing female and male delicious euglena, and selecting male delicious euglena as a test animal.
A2: determination of ROS-producing lymphocytes: selecting mature Sipunculus nudus individuals, extracting Sipunculus nudus lymph liquid with 1ml syringe, centrifuging, diluting with PBS to 10 6 After 200uL of 50 mu LDCFH-DA working solution is added into the cell/mL blood cell suspension, the mixture is incubated in a dark place for 30min at 37 ℃ after being gently mixed, the incubation in a dark place is performed, the mixture is washed twice with PBS after being incubated in a dark place, and then the blood cells are lightly resuspended with 200uL of PBS, and the blood cells presenting green light are observed under a fluorescence microscope to determine the blood cells for measuring the ROS content.
A3: clear cell isolation method determination: gradient was made with 15% and 20% Pereol solution (v/v) lmL in centrifuge tubes, and 5ml of blood cell suspension was added to the gradient, centrifuged at 4000rpm for 10min, and the cell status of each layer was observed to determine the layer where the clear cells were located.
A4: positive control compound H 2 O 2 Determination of concentration and duration of action: configuring H with different gradient concentrations 2 O 2 Liquid H 2 O 2 The concentration of the liquid is increased to the upper layer gradient of 50 mu mol/L, and the concentration is as follows: 0. Mu. Mol/L, 50. Mu. Mol/L, 100. Mu. Mol/L, 150. Mu. Mol/L, 200. Mu. Mol/L, 250. Mu. Mol/L, 300. Mu. Mol/L, 350. Mu. Mol/L, 400. Mu. Mol/L and 450. Mu. Mol/L, and lymph fluid was extracted from each of the Sipunculus nudus for measurementThe time of ROS content was injection H 2 O 2 Extracting lymph from each of the Sipunculus nudus at 3, 6, 12, 24 and 48 hr, centrifuging to collect Sipunculus nudus transparent blood cells, and diluting to 10 7 Determining the ROS content of the transparent blood cells according to the A2 step method, and determining H by taking the standard that the survival rate of the sipunculus nudus is more than 95% and the change range of the ROS content of the transparent cells is less than 5% at different time points 2 O 2 Concentration and duration of action.
A5: ROS in vitro stability: with 200. Mu. Mol/L H 2 O 2 After the stress of the sipunculus nudus, respectively taking coelomic fluid of the sipunculus nudus after 3h, 6h, 9h, 12h, 15h and 18h respectively after gradient interval time, centrifugally collecting transparent blood cells, diluting to 10 7 And (3) placing the sample in an environment with a certain temperature, respectively measuring the ROS content of the transparent cells at different intervals, wherein the specific time is 0h, 0.5h, 1h, 2h, 3h, 4h, 5h and 6h, the temperature is 25 ℃, and the placing time is determined by taking the change range of the ROS content as a standard and the ROS content is less than 5%, namely the operation time for measuring the ROS content.
A6: reproducibility experiments: to verify reproducibility of the ROS-producing Starworm model, 50uL of 200. Mu. Mol/L H was injected into the body cavity of the Starworm using the method determined in step A4 2 O 2 After a certain interval of time, extracting lymph fluid from 3h, 6h, 9h, 12h, 15h and 18h respectively, centrifuging to collect Sipunculus nudus transparent blood cells, diluting to 10 7 The ROS content of the transparent blood cells is measured, the ROS content is repeated at least 5 times, the RSD value is calculated, and the repeatability of the method is examined.
S3: model verification:
a1: antioxidant verification effect: the ROS model is divided into 2 groups of test groups and control groups, 10 test groups of Sipunculus nudus are respectively injected with 200uL of 2mg/mL vitamin C, the control groups of Sipunculus nudus are respectively injected with the same amount of physiological saline, and after a certain interval of time, the specific time is 1h, 3h, 6h and 12h, the transparent blood cell ROS content of the Sipunculus nudus body cavity is observed.
A2: ROS clearance calculation: ROS clearance (%) = (control ROS content-test ROS content)/control ROS content x 100%.
The working principle and the using flow of the invention are as follows: after centrifugation in centrifuge tubes with 15% and 45% Pereol (v/v) solution lmL each, the clear cells were layered in the upper layer, as shown in FIG. 2, where a is ROS clear cells and b is ROS-free clear cells; sipunculus coelomis injection 50ul of H with different concentration 2 O 2 After 3H, 6H, 9H, 12H, 15H, 18H, 21H and 24H of solutions (0, 50, 100, 200, 400 and 800. Mu. Mol/L), the transparent cells showed color changes under a fluorescence microscope as shown in FIGS. 3 and 4, the transparent blood cells showed ROS content as shown in FIG. 5, and the color changes were observed when H 2 O 2 When the concentration is 200 mu mol/L, the ROS content is stable within 3-18 h; the mortality rate of the sipunculus nudus is shown in FIG. 6, and the survival rate is more than 95% within the concentration of 200 mu mol/L; with 200. Mu. Mol/L H 2 O 2 After the star worms are stressed for 3 hours, 6 hours, 9 hours, 12 hours, 15 hours and 18 hours, the star worm coelomic fluid is taken and placed for 0, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours, and then the ROS content of the transparent cells is measured as shown in figure 7, and in 3 hours, the ROS content of the transparent cells is changed by less than 5 percent, so that the experiment is completed in 3 hours; in addition, the data obtained through the reproducibility experiments were prepared as follows in table 1:
TABLE 1
As can be seen from Table 1 above, the stability of the Coccida ROS model was all acceptable within 18 hours, but better within 12 hours.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A method for screening antioxidants by using a delicious euglena, which is characterized in that: the method comprises the following steps:
s1: and (3) culturing the delicious euglena: picking healthy and delicious euglena with the weight of 4.0+/-0.5 g, putting the euglena into a plastic square tray for feeding, and paving sandy soil with the thickness of 5 cm in the tray;
s2: building a delicious euglena ROS model:
a1: test selection of the delicious euglena: selecting a delicious euglena with the weight of 4.0+/-0.5 g, extracting euglena lymph by using a 1mL syringe, observing the types of blood cells of the lymph under a microscope, distinguishing female and male delicious euglena, and selecting the male delicious euglena as a test animal;
a2: determination of ROS-producing lymphocytes: selecting mature Sipunculus nudus individuals, extracting Sipunculus nudus lymph liquid with 1mL syringe, centrifuging, diluting with PBS to 10 6 Taking 200uL of cell/mL blood cell suspension, adding 50uL of DCFH-DA working solution, gently mixing, incubating in a dark place, washing twice with PBS after incubation in a dark place, then gently resuspending the blood cells with 200uL of PBS, and observing the blood cells presenting green light under a fluorescence microscope so as to determine the blood cells for measuring the ROS content;
a3: clear cell isolation method determination: preparing a gradient by using 15% and 45% Pereol liquid (v/v) lmL in a centrifuge tube, adding 5ml of blood cell suspension on the gradient, centrifuging at 4000rpm for 10min, observing the cell condition of each layer, and determining the layer where transparent cells are located;
a4: positive control compound H 2 O 2 Determination of concentration and duration of action: configuring H with different gradient concentrations 2 O 2 Liquid, 50ul of different concentrations of H are injected into the body cavity of the sipunculus 2 O 2 Extracting lymph from each Sipunculus nudus after multiple interval, centrifuging to collect Sipunculus nudus transparent blood cells, and diluting to 10 7 Determining the ROS content of the transparent blood cells according to the A2 step method, and determining H by taking the standard that the survival rate of the sipunculus nudus is more than 95% and the change range of the ROS content of the transparent cells is less than 5% at different time points 2 O 2 Concentration and duration of action;
a5: ROS in vitro stability: with 200. Mu. Mol/L H 2 O 2 After the stress of the asteroid, respectively taking the coelomic fluid of the asteroid after the gradient interval time, centrifugally collecting transparent blood cells, diluting to 10 7 Placing in a certain temperature environment, and measuring the ROS content of transparent cells at different intervalsDetermining the standing time, namely the operation time for measuring the ROS content by taking the ROS content variation range less than 5% as a standard;
a6: reproducibility experiments: to verify reproducibility of the ROS-producing Starworm model, 50uL of 200. Mu. Mol/L H was injected into the body cavity of the Starworm using the method determined in step A4 2 O 2 Extracting lymph after a certain interval, centrifuging to collect Sipunculus nudus transparent blood cells, diluting to 10 7 Measuring the ROS content of the transparent blood cells, repeating for a plurality of times, calculating an RSD value, and examining the repeatability of the method;
s3: model verification:
a1: antioxidant verification effect: the ROS model tasty euglena falls into 2 groups: the method comprises the steps of (1) injecting 200uL 2mg/mL vitamin C into a test group and a control group in which 10 astrosoma in the test group, injecting the same amount of physiological saline into the control group, and observing the ROS content of transparent blood cells in the coeloma of the astrosoma after a certain period of time;
a2: ROS clearance calculation: ROS clearance (%) = (control ROS content-test ROS content)/control ROS content x 100%.
2. A method of screening for antioxidants using a palatable euglena as claimed in claim 1 wherein: the ratio of soil to sand in sandy soil paved in a dish in the cultivation of the sipunculus nudus in the step S1 is 3:1, the cultivation condition is dry dew for 12 hours per day, water flooding is carried out for 12 hours, the salinity of cultivation seawater is 25+/-1 ppt, the temperature is 25+/-1 ℃, and diatoms are fed during cultivation.
3. A method of screening for antioxidants using a palatable euglena as claimed in claim 1 wherein: in the step S2, in the determination of the lymphocyte which generates the ROS in the step A2 in the establishment of the ROS model of the Phascophyta, the transparent cell is the ROS-generating cell.
4. A method of screening for antioxidants using a palatable euglena as claimed in claim 1 wherein: the positive control compound H of the step A4 in the S2 step of the establishment of the ROS model of the Phascota stipuloides 2 O 2 In the determination of concentration and duration of action, H 2 O 2 The concentration of the liquid is increased to the upper layer gradient of 50 mu mol/L, and the concentration is as follows: 0. Mu. Mol/L, 50. Mu. Mol/L, 100. Mu. Mol/L, 150. Mu. Mol/L, 200. Mu. Mol/L, 250. Mu. Mol/L, 300. Mu. Mol/L, 350. Mu. Mol/L, 400. Mu. Mol/L and 450. Mu. Mol/L, and the time for measuring the ROS content by extracting lymph fluid from each of the Sipunculus is H injection 2 O 2 Last 3, 6, 12, 24 and 48h.
5. A method of screening for antioxidants using a palatable euglena as claimed in claim 1 wherein: in the S2 step, 200 mu mol/L H is injected in the in-vitro stability of the ROS in the A5 step in the establishment of the S2 step of the Coccipitalis ROS model 2 O 2 And taking the coelomic fluid of the sipunculus nudus after 3h, 6h, 9h, 12h, 15h and 18h respectively, centrifuging to collect transparent blood cells, and respectively measuring the ROS content of the transparent cells after standing for 0h, 0.5h, 1h, 2h, 3h, 4h, 5h and 6h at the temperature of 25 ℃.
6. A method of screening for antioxidants using a palatable euglena as claimed in claim 1 wherein: in the reproducibility experiment of the step A6 in the establishment of the S2-step delicious euglena ROS model, the time for extracting the lymph fluid is respectively 3h, 6h, 9h, 12h, 15h and 18h, and the repetition time of the reproducibility experiment is at least 5 times.
7. A method of screening for antioxidants using a palatable euglena as claimed in claim 1 wherein: in the antioxidant verification effect of the step A1 in the step S3 model verification, the ROS clearance is taken as an index, and after 1h, 3h, 6h and 12h respectively, the ROS content of the transparent blood cells of the body cavity of the Sipunculus nudus is measured, and the clearance is calculated.
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