阅读说明：本技术 一种利用可口革囊星虫筛选抗氧化剂的方法 (Method for screening antioxidant by using phascolosoma esculenta ) 是由 周逢芳 蔡彬新 阮少江 黄伟卿 李筝筝 于 2021-08-19 设计创作，主要内容包括：本发明公开了一种利用可口革囊星虫筛选抗氧化剂的方法,包括可口革囊星虫养殖、可口革囊星虫ROS模型建立和模型验证三个步骤,其中可口革囊星虫ROS模型建立包括试验可口革囊星虫挑选、产ROS的淋巴血细胞确定、透明细胞分离方法确定、阳性对照化合物H-(2)O-(2)浓度及作用时间的确定、ROS体外稳定性和重现性实验六个步骤。通过利用可口革囊星虫模型进行筛选抗氧化剂具有很好的应用前景,可口革囊星虫属于闭管式循环的体腔动物,能较真实的反应机体代谢；可口革囊星虫透明细胞细胞膜薄,荧光标记物及抗氧化剂容易渗透入透明细胞内,可以更好的对抗氧化性结果进行显示；可口革囊星虫容易养殖,成本低,且实验操作简单。(The invention discloses a method for screening an antioxidant by using phascolosoma esculenta, which comprises the three steps of phascolosoma esculenta culture, phascolosoma esculenta ROS model establishment and model verification, wherein the phascolosoma esculenta ROS model establishment comprises test phascolosoma esculenta selection, determination of ROS-producing lymphocyte, determination of a transparent cell separation method, and positive control compound H 2 O 2 Determination of concentration and action time, and ROS in-vitro stability and reproducibility experiment. The method has good application prospect by utilizing the Phascolosoma esculenta model to screen the antioxidant, and the Phascolosoma esculenta belongs to a closed-tube type circulating coelomic animal and can truly reflect the metabolism of an organism; the membrane of the phascolosoma esculenta transparent cell is thin, and a fluorescent marker and an antioxidant are easy to permeate into the transparent cell, so that an anti-oxidation result can be better displayed; the Phascolosoma esculenta is easy to breed, has low cost and simple experimental operationAnd (3) singly.)

1. A method for screening an antioxidant by using phascolosoma esculenta is characterized by comprising the following steps: the method comprises the following steps:

s1: culturing Phascolosoma esculenta: selecting healthy Phascolosoma esculenta with the weight of 4.0 +/-0.5 g, putting the Phascolosoma esculenta into a plastic square plate for breeding, and paving sandy soil with the thickness of 5 cm in the plate;

s2: establishing an ROS model of phascolosoma esculenta:

a1: test Phascolosoma esculenta selection: selecting Phascolosoma esculenta with the weight of 4.0 +/-0.5 g, extracting the lymph of the Phascolosoma esculenta by using a 1mL needle cylinder, observing the types of lymph blood cells under a microscope, distinguishing female Phascolosoma esculenta from male Phascolosoma esculenta, and selecting male Phascolosoma esculenta as a test animal;

a2: determination of ROS-producing lymphocytes: selecting mature sipunculus nudus individual, extracting sipunculus nudus lymph fluid with 1mL syringe, centrifuging, and diluting with PBS to 10%⁶Taking 200uL of blood cell suspension, adding 50uL of DCFH-DA working solution into the blood cell suspension, gently mixing the solution uniformly, incubating the solution in a dark place, washing the solution twice by PBS after incubating in a dark place, gently suspending the 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: respectively lmL putting 15% and 45% Pereol solution (v/v) into a centrifugal tube to prepare a gradient, adding 5ml of blood cell suspension on the gradient, centrifuging for 10min at 4000rpm, observing the cell condition of each layer, and determining the layer where the transparent cells are located;

a4: positive control Compound H₂O₂Determination of concentration and action time: h with different gradient concentrations₂O₂Injecting 50ul of H with different concentrations into the body cavity of the sipunculus₂O₂Collecting lymph from each sipunculus nudus after multiple time intervals, centrifuging to collect sipunculus nudus clear blood cells, and diluting to 10%⁷Determining the ROS content of the hyaline blood cells according to the method of the step A2, and determining H by taking the survival rate of the sipunculus nudus at different time points as the standard that the survival rate is more than 95 percent and the ROS content of the hyaline blood cells is less than 5 percent₂O₂Concentration and duration of action;

a5: ROS stability in vitro: with 200. mu. mol/L H₂O₂Stressing the sipunculus nudus, centrifuging to collect transparent blood cells, and diluting to10⁷Placing the cell in an environment with a certain temperature, respectively measuring the ROS content of the transparent cell at different intervals, and determining the placing time, namely the operation time for measuring the ROS content, by taking the ROS content variation range smaller than 5% as a standard;

a6: reproducibility test: to verify the reproducibility of the model for producing ROS in a Sipunculus sp, 50uL of 200. mu. mol/L H was injected into the body cavity of the Sipunculus sp using the method determined in step A4₂O₂Collecting lymph at certain intervals, centrifuging to collect blood cells of sipunculus nudus, and diluting to 10 deg.C⁷Measuring the ROS content of the transparent blood cells, repeating the measurement for multiple times, calculating the RSD value, and inspecting the reproducibility of the method;

s3: and (3) model verification:

a1: the antioxidant verification effect is as follows: ROS model phaeocystis punctatus were divided into 2 groups: the test group and the control group are 10 animals each, 200uL of vitamin C2 mg/mL is injected into the body of the sipunculus nudus of the test group, the same amount of normal saline is injected into the body of the sipunculus nudus of the control group, and the ROS content of transparent blood cells in the body cavity of the sipunculus nudus is observed after a certain time;

a2: ROS clearance calculation: ROS clearance (%) (control ROS content-test ROS content)/control ROS content × 100%.

2. The method for screening antioxidants using phascolosoma esculenta according to claim 1, wherein: the ratio of soil to sand in the sandy soil laid in the Phascolosoma esculenta culture middle tray in the step S1 is 3:1, the culture conditions are that the water is exposed for 12 hours and flooded for 12 hours every day, the salinity of the culture seawater is 25 +/-1 ppt, the temperature is 25 +/-1 ℃, and diatoms are fed during culture.

3. The method for screening antioxidants using phascolosoma esculenta according to claim 1, wherein: in the determination of ROS-producing lymphocytes in the step A2 in the establishment of the ROS model of phascolosoma esculenta in the step S2, the hyaline cells are ROS-producing cells.

4. The method for screening antioxidants using phascolosoma esculenta according to claim 1, wherein: in the determination of the A3 transparent cell separation method in the S2 step Phascolosoma esculenta ROS model establishment, 15% and 45% Pereol solution is used for preparing a gradient, and the gradient is centrifuged at 4000rpm/min for 10 min.

5. The method for screening antioxidants using phascolosoma esculenta according to claim 1, wherein: a4 step positive control compound H in the S2 step Phascolosoma esculenta ROS model establishment₂O₂In the determination of the concentration and duration of action, H₂O₂The concentration of the solution is increased towards the upper layer by 50 mu mol/L, and the concentration is as follows in sequence: 0. 50, 100, 150, 200, 250, 300, 350, 400 and 450 μmol/L, and the time for extracting lymph from each sipunculus to determine the ROS content is injection H₂O₂Last 3, 6, 12, 24 and 48 h.

6. The method for screening antioxidants using phascolosoma esculenta according to claim 1, wherein: in the ROS in-vitro stability of the step A5 in the ROS model establishment of the Phascolosoma esculenta ROS at the step S2, 200 mu mol/L of H is injected₂O₂Then, collecting the coelomic fluid of the sipunculus before centrifuging for 3, 6, 9, 12, 15 and 18 hours, collecting the transparent blood cells, and respectively measuring the ROS content of the transparent cells after placing for 0, 0.5, 1, 2, 3, 4, 5 and 6 hours at the temperature of 25 ℃.

7. The method for screening antioxidants using phascolosoma esculenta according to claim 1, wherein: in the repeated experiments of the A6 step in the ROS model establishment of the Phascolosoma esculenta at the step S2, the time for extracting the lymph fluid is respectively 3, 6, 9, 12, 15 and 18 hours, and the repeated times of the repeated experiments are at least 5 times.

8. The method for screening antioxidants using phascolosoma esculenta according to claim 1, wherein: in the antioxidant verification effect of the step A1 in the model test of the step S3, the ROS clearance is used as an index, the ROS content of the transparent blood cells in the body cavities of the sipunculus nudus is measured after 1h, 3h, 6h and 12h respectively, and the clearance is calculated.

Technical Field

The invention belongs to the technical field of antioxidant screening, and particularly relates to a method for screening an antioxidant by utilizing phascolosoma esculenta.

Background

With the improvement of living standard, people pay more attention to physical health. Active oxygen is an important substance affecting biological signal transduction, and accumulation thereof is associated with the occurrence of various chronic diseases such as aging and cardiovascular diseases. The antioxidant activity of many pure compounds, foods and dietary supplements is now widely studied and new antioxidants (i.e., free radical scavengers) are being screened.

At present, antioxidant screening and evaluation models mainly comprise in-vitro evaluation methods and in-vivo evaluation methods, wherein the in-vitro evaluation methods mainly comprise a chemical evaluation method and a cell model evaluation method, and the in-vivo evaluation methods mainly comprise an animal evaluation method. The chemical evaluation method cannot truly simulate physiological environment, does not consider the action of a drug entering cells through a membrane and the like, and the cell model evaluation method does not consider the factors of in vivo metabolism and the like, so 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 the antioxidant cannot be truly reflected. In vivo antioxidant method animal models, rat models, zebrafish models, mainly mice, are expensive and time-consuming and are not suitable for initial screening of food and dietary supplements. The zebra fish model has the defects of small size, high injection difficulty, little blood, incapability of extraction and the like. Therefore, it is necessary to select an antioxidant screening and evaluating model having advantages of not only an in vitro cell model but also an in vivo animal model.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a method for screening an antioxidant by using phascolosoma esculenta, which solves the problems that a chemical evaluation method cannot truly simulate a physiological environment, the transmembrane entering of a drug into a cell and the like is not considered, a cell model evaluation method does not consider the metabolism in vivo and the like, and a rat and mouse model method is expensive, time-consuming, difficult to operate and the like.

In order to achieve the purpose, the invention provides the following technical scheme: a method for screening antioxidants by using Phascolosoma esculenta, comprising the steps of:

s1: culturing Phascolosoma esculenta: selecting healthy Phascolosoma esculenta with the weight of 4.0 +/-0.5 g, putting the Phascolosoma esculenta into a plastic square plate for breeding, and paving sandy soil with the thickness of 5 cm in the plate.

S2: establishing an ROS model of phascolosoma esculenta:

a1: test Phascolosoma esculenta selection: selecting Phascolosoma esculenta with weight of 4.0 + -0.5 g, extracting stellate lymph with 1mL syringe, observing lymph blood cell type under microscope, distinguishing female and male Phascolosoma esculenta, and selecting male Phascolosoma esculenta as test animal.

A2: determination of ROS-producing lymphocytes: selecting a mature stellera individual, extracting stellera lymph fluid by using a 1mL needle cylinder, centrifuging, diluting the stellera individual into 106cell/mL blood cell suspension by using PBS, adding 200uL of the blood cell suspension into 50 mu L of the working solution of LDCFH-DA, gently mixing the solution uniformly, incubating the solution in a dark place, washing the solution twice by using PBS, then gently suspending the blood cells by using 200uL of PBS, and observing the blood cells presenting green light under a fluorescence microscope so as to determine the blood cells for determining the ROS content.

A3: clear cell isolation method determination: a gradient was prepared by placing lmL each in 15% and 45% Pereol (v/v) solutions in a centrifuge tube, adding 5mL of blood cell suspension to the gradient, and after centrifuging for a while, observing the cell condition of each layer to determine the layer in which the clear cells are located.

A4: positive control Compound H₂O₂Determination of concentration and action time: h with different gradient concentrations₂O₂Injecting 50uL of H with different concentrations into the body cavity of the sipunculus₂O₂Collecting lymph from each sipunculus nudus after multiple time intervals, centrifuging to collect sipunculus nudus clear blood cells, and diluting to 10%⁷Determining the ROS content of the hyaline blood cells according to the method of the step A2, and determining H by taking the survival rate of the sipunculus nudus at different time points as the standard that the survival rate is more than 95 percent and the ROS content of the hyaline blood cells is less than 5 percent₂O₂Concentration and duration of action.

A5: ROS stability in vitro: with 200. mu. mol/L H₂O₂Stressing the sipunculus nudus, respectively taking the body cavity of the sipunculus nudus after gradient interval timeCollecting transparent blood cells by liquid centrifugation, and diluting to 10%⁷And placing the cell in an environment with a certain temperature, respectively measuring the ROS content of the transparent cells at different intervals, and determining the placing time, namely the operation time of the ROS content measurement test, by taking the ROS content variation range smaller than 5% as a standard.

A6: reproducibility test: to verify the reproducibility of the ROS-producing Trypanosoma model, 50ul of 200. mu. mol/L H was injected into the body cavity of the Trypanosoma using the method determined in step A4₂O₂Collecting lymph at certain intervals, centrifuging to collect blood cells of sipunculus nudus, and diluting to 10 deg.C⁷And measuring the ROS content of the transparent blood cells, repeating the measurement for multiple times, calculating the RSD value, and inspecting the reproducibility of the method.

S3: and (3) model verification:

a1: the antioxidant verification effect is as follows: the ROS model phascolosoma esculenta is divided into 2 groups of test groups and 10 control groups, vitamin C with the concentration of 200uL 2mg/mL is injected into the body of the test group phascolosoma esculenta, the same amount of normal saline is injected into the body of the control group phascolosoma esculenta, and the ROS content of the coelomic hyaline blood cells of the phascolosoma esculenta is observed after a certain interval of time.

A2: ROS clearance calculation: ROS clearance (%) (control ROS content-test ROS content)/control ROS content × 100%.

Preferably, the ratio of soil to sand in the sandy soil laid in the Phascolosoma esculenta culture medium tray in the step S1 is 3:1, the culture conditions are that the water is exposed for 12 hours every day, the water is flooded for 12 hours, the salinity of culture seawater is 25 +/-1 ppt, the temperature is 25 +/-1 ℃, and diatoms are fed during culture.

Preferably, the transparent cells in the determination of ROS-producing lymphocytes in the step A2 in the establishment of the ROS model of phascolosoma esculenta at the step S2 are ROS-producing cells.

Preferably, in the determination of the A3 clear cell separation method in the S2 step Phascolosoma esculenta ROS model establishment, 15% and 45% Pereol solution is used for making a gradient, and the gradient is centrifuged at 4000rpm/min for 10 min.

Preferably, the positive control compound H at the A4 step in the ROS model establishment of the Phascolosoma esculenta at the S2 step₂O₂Determination of concentration and duration of actionH₂O₂The concentration of the solution is increased towards the upper layer by 50 mu mol/L, and the concentration is as follows in sequence: 0. 50, 100, 150, 200, 250, 300, 350, 400 and 450 mu mol/L, and the lymph fluid is extracted from each of the sipunculus and injected with H₂O₂The later stages are 3, 6, 12, 24 and 48H, and H is determined by taking the standard that the survival rate of the sipunculus nudus is more than 95 percent and the ROS content of the transparent cells is less than 5 percent₂O₂Concentration and duration of action.

Preferably, in the ROS in-vitro stability of A5 step in the ROS model establishment of the Phascolosoma esculenta at the S2 step, after 200 mu mol/L of H2O2 is injected, the coelomic fluid of the phascolosoma esculenta is taken for centrifugation and collected to obtain the clear blood cells after 3, 6, 9, 12, 15 and 18 hours, and the ROS content of the clear cells is respectively determined after 0, 0.5, 1, 2, 3, 4, 5 and 6 hours at the temperature of 25 ℃. And determining the time by taking the ROS content variation range less than 5 percent as a standard.

Preferably, the time for extracting lymph fluid to determine the ROS content in the A6 repeated experiment in the ROS model building of the Phascolosoma esculenta at the S2 step is respectively 3, 6, 9, 12, 15 and 18 hours, and the repeated times of the repeated experiment are at least 5 times.

Preferably, in the antioxidant verification effect of the step A1 in the model test of the step S3, the ROS clearance is used as an index, the ROS content of the transparent blood cells in the body cavities of the sipunculus nudus is measured after 1h, 3h, 6h and 12h respectively, and the clearance is calculated.

Compared with the prior art, the invention has the beneficial effects that:

1. the method has good application prospect by utilizing the Phascolosoma esculenta to screen the antioxidant, and the Phascolosoma esculenta belongs to the phylum Astroichopus, is a marine and closed-tube type circulating coelomic animal, belongs to living animal cells, and can truly reflect the metabolism of organisms.

2. The method has good application prospect by utilizing the phascolosoma esculenta to screen the antioxidant, has simple morphological result, belongs to lower animals, reduces the interference of complex life phenomena, and is beneficial to researching the basic metabolic mechanism of a life body.

3. The method has good application prospect by utilizing the phascolosoma esculenta to screen the antioxidant, the phascolosoma esculenta body cavity is filled with a large number of lymphocytes, the types of the blood cells are single, only transparent cells and granular cells are contained, the transparent cells and the granular cells are easy to separate through a centrifugation method, and the transparent cells of a research object are easy to obtain.

4. The method has good application prospect by utilizing the phascolosoma esculenta to screen the antioxidant, the phascolosoma esculenta transparent cell membrane is thin, the fluorescent marker and the antioxidant are easy to permeate into the transparent cell, and the antioxidant resistance result can be better displayed.

5. The method has the advantages that the method has good application prospect by utilizing the phascolosoma esculenta to screen the antioxidant, the phascolosoma esculenta is easy to breed, the cost is low, and the experimental operation is simple.

Drawings

FIG. 1 is a schematic flow diagram of the present invention;

FIG. 2 is a schematic representation of the fluorescence of the clear cell ROS of the present invention;

FIG. 3 shows the present invention without H₂O₂Color schematic of stressed clear cells under fluorescence microscope;

FIG. 4 is H of the present invention₂O₂Color schematic of stressed clear cells under fluorescence microscope;

FIG. 5 shows different concentrations H according to the present invention₂O₂And schematic diagram of ROS content change of the hyaline cells under action time;

FIG. 6 shows different concentrations H according to the present invention₂O₂And the survival rate of the sipunculus nudus under the action time;

FIG. 7 is a schematic representation of the ROS stability of the hyaline cells of the present invention in vitro.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1-7, the present invention provides a technical solution: a method for screening antioxidants by using Phascolosoma esculenta, comprising the steps of:

s1: culturing Phascolosoma esculenta: selecting healthy Phascolosoma esculenta with the weight of 4.0 +/-0.5 g, putting the Phascolosoma esculenta into a plastic square tray for breeding, paving sandy soil with the thickness of 5 cm in the tray, wherein the ratio of the soil in the sandy soil to the sand is 3:1, the breeding conditions are exposure for 12 hours each day, flooding for 12 hours, the salinity of the breeding seawater is 25 +/-1 ppt, the temperature is 25 +/-1 ℃, and feeding diatoms during breeding.

S2: establishing an ROS model of phascolosoma esculenta:

a1: test Phascolosoma esculenta selection: selecting Phascolosoma esculenta with weight of 4.0 + -0.5 g, extracting stellate lymph with 1mL syringe, observing lymph blood cell type under microscope, distinguishing female and male Phascolosoma esculenta, and selecting male Phascolosoma esculenta as test animal.

A2: determination of ROS-producing lymphocytes: selecting mature Sipunculus nudus individual, extracting Sipunculus nudus lymph with 1ml syringe, centrifuging, and diluting with PBS to 10⁶And (2) taking 200uL of the blood cell suspension, adding 50 mu L of LDCFH-DA working solution into the blood cell suspension, gently mixing the solution uniformly, incubating the solution in a dark place for 30min, incubating the solution in a dark place at the temperature of 37 ℃, washing the solution twice by PBS, gently suspending the 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 determining the ROS content.

A3: clear cell isolation method determination: lmL of Pereol liquid (v/v) with 15 percent and 20 percent are respectively put in a centrifugal tube to form a gradient, 5ml of blood cell suspension is added on the gradient, the centrifugal tube is centrifuged for 10min at 4000rpm, the cell condition of each layer is observed, and the layer where the transparent cells are positioned is determined.

A4: positive control Compound H₂O₂Determination of concentration and action time: h with different gradient concentrations₂O₂Liquid, H₂O₂The concentration of the solution is increased towards the upper layer by 50 mu mol/L, and the concentration is as follows in sequence:0. 50, 100, 150, 200, 250, 300, 350, 400 and 450 mu mol/L, and 50ul of H with different concentrations is injected into the body cavity of the stellera₂O₂Collecting lymph from each Sipunculus nudus at multiple intervals of 3, 6, 12, 24 and 48 hr, centrifuging to collect Sipunculus nudus clear blood cells, and diluting to 10%⁷Determining the ROS content of the hyaline blood cells according to the method of the step A2, and determining H by taking the survival rate of the sipunculus nudus at different time points as the standard that the survival rate is more than 95 percent and the ROS content of the hyaline blood cells is less than 5 percent₂O₂Concentration and duration of action.

A5: ROS stability in vitro: with 200. mu. mol/L H₂O₂Stressing the sipunculus nudus, centrifuging to collect transparent blood cells after gradient interval time of 3, 6, 9, 12, 15 and 18h, diluting to 10⁷And placing the cell in an environment with a certain temperature, and respectively measuring the ROS content of the hyaline cells at different intervals, wherein the specific time is respectively 0, 0.5, 1, 2, 3, 4, 5 and 6 hours, the temperature is 25 ℃, and the placing time, namely the operation time for measuring the ROS content, is determined by taking the ROS content variation range less than 5% as a standard.

A6: reproducibility test: to verify the reproducibility of the model for producing ROS in a Sipunculus sp, 50uL of 200. mu. mol/L H was injected into the body cavity of the Sipunculus sp using the method determined in step A4₂O₂After certain time intervals, namely 3, 6, 9, 12, 15 and 18 hours respectively, extracting lymph fluid, centrifugally collecting blood cells of the sipunculus nudus, and diluting to 10⁷And measuring the ROS content of the transparent blood cells, repeating for at least 5 times, calculating the RSD value, and inspecting the reproducibility of the method.

S3: and (3) model verification:

a1: the antioxidant verification effect is as follows: dividing the ROS model phascolosoma esculenta into 2 groups of test groups and 10 control groups, injecting 200uL of 2mg/mL vitamin C into the body of the test group phascolosoma esculenta, injecting the same amount of normal saline into the body of the control group phascolosoma esculenta, and observing the content of ROS in coelomic clear blood cells of the phascolosoma esculenta after certain time intervals, namely 1h, 3h, 6h and 12h respectively.

A2: ROS clearance calculation: ROS clearance (%) (control ROS content-test ROS content)/control ROS content × 100%.

The working principle and the using process of the invention are as follows: using 15% and 45% Pereol solution (v/v) lmL in each centrifuge tube, after centrifugation, dividing the tube into three layers, wherein the transparent cells are at the upper layer, as shown in FIG. 2, wherein a is ROS transparent cells, and b is ROS-free transparent cells; the sipunculid is injected into the body cavity of the sipunculid with 50ul of different concentrations H₂O₂After 3, 6, 9, 12, 15, 18, 21 and 24H in the solutions (0, 50, 100, 200, 400 and 800. mu. mol/L), the color of the clear cells was changed under a fluorescence microscope as shown in FIG. 3 and FIG. 4, the ROS content of the clear blood cells is shown in FIG. 5, and when H is₂O₂When the concentration is 200 mu mol/L, the ROS content is stable within 3h-18 h; the death rate of the sipunculus nudus is shown in figure 6, and the survival rate is more than 95% within the concentration of 200 mu mol/L; with 200. mu. mol/L H₂O₂After the sipunculus nudus is stressed for 3, 6, 9, 12, 15 and 18 hours, the coelomic fluid of the sipunculus nudus is placed for 0, 0.5, 1, 2, 3, 4, 5 and 6 hours, the ROS content of the transparent cell is determined as shown in figure 7, and the ROS content of the transparent cell changes by less than 5% within 3 hours, so that the experiment needs to be completed within 3 hours; the data obtained from further reproducibility experiments were generated as follows:

TABLE 1

As can be seen from Table 1 above, the Phascolosoma esculenta ROS model is stable within 18h, but is more stable within 12 h.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.