Astaxanthin-based method for reducing microplastic bioaccumulation

Abstract

This invention discloses a method for reducing microplastic bioaccumulation based on astaxanthin, relating to the field of microplastic technology. The method includes the following steps: co-exposing astaxanthin and microplastics to organisms. The beneficial effects of this invention are: it can effectively inhibit the bioaccumulation of microplastics in organisms, solving the problem of microplastics easily accumulating in the body and causing health hazards. It has advantages such as high biocompatibility and simple operation, and has broad application prospects in preventing excessive accumulation of microplastics in the body.

Description

Technical Field

[0001] This invention relates to the field of microplastics technology, and more specifically to a method for reducing microplastic bioaccumulation based on astaxanthin. Background Technology

[0002] Since the mid-20th century, plastics have been widely used in food packaging and containers, synthetic textiles, rubber tires, pipes, and electronic equipment due to their waterproof and pressure-resistant properties, as well as their low cost and ease of production. However, plastic products that enter the environment and are not recycled are further degraded into smaller plastic particles by physicochemical factors such as light, heat, oxygen, mechanical stress, and ultraviolet radiation. These particles, typically less than 5 mm in diameter, are considered microplastics. In addition, there are primary microplastics, including small particles added directly to toothpaste, facial cleanser, and laundry detergent, which can directly enter the environment. During the COVID-19 crisis, the widespread use of masks also led to a large influx of microplastics into the environment. Studies have shown that for every mask exposed to the environment, more than 1.5 million microplastics are released into the aquatic environment. Due to their recalcitrant nature, small particle size, and ability to adsorb environmental pollutants, microplastics were listed as the second major scientific problem in environmental and ecological science research at the Second United Nations Environment Assembly in 2016, alongside global climate change, ozone depletion, and ocean acidification.

[0003] Microplastics are currently widespread in the environment, detected in water, soil, and airborne dust, and also found in organisms such as springtails, shellfish, and fish. This means that microplastics can enter the human body through inhalation or bioaccumulation through the food chain. Studies have shown that microplastics have been detected in the human placenta, blood, breast milk, and brain. Microplastic bioaccumulation can cause oxidative stress, immune responses, gut microbiota imbalance, and damage to tissue cell barriers. Furthermore, large-scale accumulation of microplastics in organisms has been reported to be associated with malnutrition, affecting the quantity and quality of offspring, and thrombosis. Recently, scientists reported a possible link between microplastics and inflammatory bowel disease (IBD), with a positive correlation between the concentration of microplastics in feces and the severity of IBD. This phenomenon provides some theoretical basis for understanding the impact of microplastics on human health. Currently, the health risks of microplastics to organisms mainly stem from their long-term, large-scale accumulation in the body, but effective methods to reduce microplastic bioaccumulation are still lacking.

[0004] Astaxanthin (AST) is a carotenoid derived from green algae, yeast, crustaceans, and bird feathers. Its chemical name is 3,3′-dihydroxy-4,4′-diketo-β,β′-carotene, and its molecular formula is C2. 40 H 52O4, due to its conjugated double bonds and the unsaturated ketone and hydroxyl groups at its ends, can attract unpaired electrons from free radicals or donate electrons to them, thus scavenging free radicals and exerting an antioxidant effect. Studies have shown that astaxanthin's antioxidant capacity is more than 10 times that of other carotenoids and more than 100 times that of vitamin E, exhibiting extremely strong antioxidant capabilities. Currently, astaxanthin is widely used as an antioxidant in health products and cosmetics, demonstrating good biocompatibility. For example, patent application number 201710260583.5 discloses an astaxanthin health product with antioxidant functions. However, there is currently no existing technology to use astaxanthin to reduce microplastic bioaccumulation. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a new method for applying astaxanthin to reduce microplastic bioaccumulation.

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

[0007] A method for reducing microplastic bioaccumulation based on astaxanthin includes the following steps: co-exposing astaxanthin and microplastics to organisms.

[0008] Beneficial effects: This invention can effectively inhibit the endocytosis of microplastics, that is, inhibit the bioaccumulation of microplastics in organisms, and solve the problem of microplastics accumulating in the body and causing health hazards. It has the advantages of high biocompatibility and simple operation, and has broad application prospects in preventing excessive accumulation of microplastics in the body.

[0009] Preferably, the concentration of astaxanthin is 2.5-20 μM, and the organism is a mammalian cell.

[0010] Beneficial effects: Astaxanthin within the scope of this invention has no toxic side effects on mammalian cells and is biosafe.

[0011] Preferably, the mammalian cell is a mouse mononuclear macrophage J774A.1.

[0012] Preferably, the method for culturing mouse mononuclear macrophages J774A.1 includes the following steps: placing J774A.1 cells in DMEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin antibiotics, and culturing them in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

[0013] Preferably, the microplastic is polystyrene.

[0014] Beneficial effects: Astaxanthin can reduce the bioaccumulation of polystyrene by about 50%, showing good inhibition efficiency and has broad application prospects in preventing excessive accumulation of microplastics in the body.

[0015] Preferably, the concentration of the polystyrene is 25-200 μg / mL.

[0016] Preferably, the culture time is 24 hours.

[0017] Preferably, the safety detection method for astaxanthin includes the following steps: diluting the concentration of AST to 2.5-20 μM using DMEM medium, treating model cells for 24 hours, discarding the culture medium for culturing model cells, then adding 50-100 μL of CCK-8 fluorescent dye diluted 5-10 times with DMEM medium to each well of a 96-120 well plate, incubating in a cell culture incubator at 37°C and 5% CO2 in the dark for 1-4 hours, and measuring its absorbance at 450 nm using an ELISA reader.

[0018] Preferably, the effect of microplastic bioaccumulation is detected after co-exposure.

[0019] Preferably, flow cytometry and fluorescence microscopy are used to detect the bioaccumulation effect of microplastics.

[0020] The advantages of this invention are: it can effectively inhibit the endocytosis of microplastics, that is, inhibit the bioaccumulation of microplastics in organisms, solve the problem of microplastics accumulating in the body and causing health hazards, and has the advantages of high biocompatibility and simple operation. It has broad application prospects in preventing excessive accumulation of microplastics in the body.

[0021] Astaxanthin within the scope of this invention has no toxic side effects on mammalian cells.

[0022] Astaxanthin can reduce the bioaccumulation of polystyrene by about 50%, demonstrating good inhibitory efficiency and showing broad application prospects in preventing excessive accumulation of microplastics in the body. Attached Figure Description

[0023] Figure 1 This is a diagram showing the effect of astaxanthin on the viability of mouse mononuclear macrophage J774A.1 cells in an embodiment of the present invention;

[0024] Figure 2 This is a flowchart illustrating the exposure method in an embodiment of the present invention;

[0025] Figure 3 This is a flow cytometry graph showing the efficiency of astaxanthin in inhibiting the bioaccumulation of microplastics (green fluorescent label) in an embodiment of the present invention.

[0026] Figure 4 This is a diagram showing the inhibitory effect of astaxanthin on the bioaccumulation of microplastics (green fluorescent label) detected by fluorescence microscopy in an embodiment of the present invention.

[0027] Figure 5This is an embodiment of the present invention. Figure 4 A semi-quantitative plot of the efficiency of astaxanthin in reducing the bioaccumulation of microplastics (green fluorescent marker) (ratio of fluorescent cells in 500-800 cells). Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0030] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0031] (1) Cell culture: J774A.1 cells were placed in DMEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin antibiotics and cultured for 24 hours in a cell culture incubator at 37°C and 5% CO2.

[0032] AST biosafety assay: After treating mouse mononuclear macrophages J774A.1 cells with 2.5-20 μM AST for 24 hours, the biosafety of AST was evaluated using the Cell Counting Kit-8 (CCK-8) kit, purchased from Biosharp. The specific steps were as follows: After cell treatment, the old culture medium was discarded, and then 100 μL of CCK-8 fluorescent dye diluted 10-fold with DMEM medium was added to each well of a 96-well plate. The plates were incubated at 37°C and 5% CO2 in the dark for 1-4 hours, and the absorbance at 450 nm was measured using a microplate reader.

[0033] Results analysis, such as Figure 1 As shown, within the concentration range of 2.5-20 μM, AST did not have significant toxic side effects on mammalian cells—mouse monocytes J774A.1.

[0034] (3) Astaxanthin and microplastics were used to expose cells together: Astaxanthin was purchased from Sigma-Aldrich, and 100nm polystyrene (PS) with a 488nm fluorescent label was purchased from Tianjin Bestlé Chromatography Technology Development Center. Figure 2The exposure method shown was as follows: mouse mononuclear macrophages J774A.1 were treated with AST and PS simultaneously for 24 hours. The concentration of AST used was 2.5-20 μM and the concentration of PS was 25-200 μg / mL.

[0035] (4) Detection of Bioaccumulation Inhibition Effect: The bioaccumulation inhibition effect was detected using two techniques: flow cytometry and inverted fluorescence microscopy. The specific steps for flow cytometry are as follows: After cell treatment, discard the old culture medium, wash once with PBS, and collect cells from the six-well plate into flow cytometry loading tubes using 500 μL PBS. Detect the fluorescence intensity using the 488 nm laser channel of the flow cytometer; the fluorescence intensity represents the amount of PS entering the cells. The specific steps for inverted fluorescence microscopy are as follows: After cell treatment, discard the old culture medium, wash three times with PBS, dilute 10 mg / mL Hochest 33342 stock solution 1:10000, add 1 mL to each well of the six-well plate, incubate in the dark for 20 min, wash three more times with PBS, and capture images of Hochest 33342 staining and PS fluorescence labeled with 488 nm excitation light under a fluorescence microscope. Process the images using ZEN 3.6 and calculate the ratio of cells with obvious fluorescence within 500-800 cells.

[0036] Results analysis showed that 2.5-20 μM AST was co-exposed with 25-200 μg / mL PS, with 15 μM AST and 50 μg / mL and 100 μg / mL PS as examples. Figure 3 As shown, flow cytometry results indicated that 15 μM AST reduced the bioaccumulation of 50 μg / mL PS by 45.74% and the bioaccumulation of 100 μg / mL PS by 47.10%, significantly inhibiting the bioaccumulation of PS.

[0037] Results analysis, such as Figure 4 As shown, after adding 15 μM AST, regardless of whether the treatment was with 50 μg / mL or 100 μg / mL PS, the number of fluorescent cells was significantly reduced, meaning that the amount of green fluorescently labeled polystyrene entering the cells was significantly reduced. Figure 4 (Fluorescence comparisons were made between the groups treated with 50 μg / mL PS alone and those treated with both 50 μg / mL PS and 15 μM AST, as well as between the groups treated with 100 μg / mL PS alone and those treated with both 100 μg / mL PS and 15 μM AST). Figure 5 As shown, the ratio of cells with obvious fluorescence in 500-800 cells was calculated, and 15 μM AST significantly reduced the bioaccumulation of 50 μg / mL PS and 100 μg / mL PS.

[0038] In this embodiment, microplastic polystyrene and the selected test model—J774A.1 cells—were used. However, the implementation of the present invention may also include other microplastics and other model organisms, and this should not be used to limit the scope of protection of the present invention.

[0039] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for reducing microplastic bioaccumulation in vitro, characterized in that: Includes the following steps: Astaxanthin and microplastics were co-exposed to mouse mononuclear macrophages J774A.1; the concentration of astaxanthin was 15 µM. The microplastic is 100 nm polystyrene.

2. The method for reducing microplastic bioaccumulation in vitro according to claim 1, characterized in that: The method for culturing mouse mononuclear macrophages J774A.1 includes the following steps: J774A.1 cells are placed in DMEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin antibiotics and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h.

3. The method for reducing microplastic bioaccumulation in vitro according to claim 1, characterized in that: The concentration of the polystyrene is 25-200 μg / mL.

4. The method for reducing microplastic bioaccumulation in vitro according to claim 3, characterized in that: The concentration of the polystyrene is 25 μg / mL.

5. The method for reducing microplastic bioaccumulation in vitro according to claim 3, characterized in that: The concentration of the polystyrene is 50 μg / mL.

6. The method for reducing microplastic bioaccumulation in vitro according to claim 3, characterized in that: The concentration of the polystyrene is 100 μg / mL.

7. The method for reducing microplastic bioaccumulation in vitro according to claim 3, characterized in that: The concentration of the polystyrene is 200 μg / mL.

8. The method for reducing microplastic bioaccumulation in vitro according to claim 1, characterized in that: The safety testing method for astaxanthin includes the following steps: Dilute the concentration of AST to 2.5-20 µM using DMEM medium, treat model cells for 24 hours, discard the culture medium for culturing model cells, then add 50-100 µL of CCK-8 fluorescent dye diluted 5-10 times with DMEM medium to each well of a 96-120 well plate, incubate in a cell culture incubator at 37 ℃ and 5% CO2 in the dark for 1-4 hours, and measure its absorbance at 450 nm using an ELISA reader.

9. The method for reducing microplastic bioaccumulation in vitro according to claim 1, characterized in that: The bioaccumulation effect of microplastics was assessed after co-exposure.

10. The method for reducing microplastic bioaccumulation in vitro according to claim 9, characterized in that: The bioaccumulation effect of microplastics was detected using flow cytometry and fluorescence microscopy.

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