Application of tea polyphenol serving as in-vivo micro-plastic agglomeration accelerant and in preparation of product for removing in-vivo micro-plastic accumulation

Abstract

The invention discloses application of tea polyphenol serving as an in-vivo micro-plastic agglomeration accelerant and in preparation of a product for removing in-vivo micro-plastic accumulation. It is found for the first time that tea polyphenol can promote microplastic agglomeration, accelerate discharge of microplastic in the body and effectively reduce accumulation of microplastic in the body, and harm of microplastic to human health can be well relieved. The tea polyphenol can be used as an in-vivo microplastic agglomeration accelerator to be added into various foods, accumulation of in-vivo microplastics is removed, discharge of the in-vivo microplastics is promoted, the similar effect can be achieved by drinking tea or other plant beverages containing the tea polyphenol, the application value of the tea polyphenol is greatly expanded, and the tea polyphenol has great popularization potential.

Description

Technical Field

[0001] The present invention belongs to the field of health foods, and particularly relates to the application of tea polyphenols as in vivo microplastic aggregation promoters in products for removing in vivo microplastic accumulation and alleviating toxicity. Background Art

[0002] With the rapid development of industrialization, the use of plastic products has shown an explosive growth. Plastic products decompose into tiny particles under physical, chemical, and biological actions, and plastic particles with a particle size less than 5 mm are defined as microplastics (MPs). MPs have become emerging environmental pollutants that are widespread and continuously generated globally. MPs are ubiquitous in air, soil, water bodies, food, and daily necessities, and humans inevitably ingest MPs. Excessive ingestion of MPs leads to oxidative stress and inflammatory responses, and significantly increases the risk of chronic diseases such as heart disease, stroke, and myocardial infarction (4.5-fold increase) and intestinal inflammation (1.5-fold increase). The MPs accumulated in the body pose a great threat to human health. However, there is still a lack of effective strategies to mitigate the toxicity caused by the accumulation of MPs in the body. Therefore, there is an urgent need for feasible solutions to reduce and prevent the damage of MPs to human health.

[0003] Existing technical reports on the adsorption and flocculation of MPs are generally limited to the in vitro environment of the environment and water bodies, but there are few reports on the research of removing the accumulation of MPs in the body. Removing MPs in the body requires the additive to be edible, not to cause additional harm to human tissues and organs, and to be resistant to gastrointestinal digestion, which greatly limits its sources. Some bacteria and fungi have been reported to be able to degrade or adsorb MPs, but they are not edible strains.

[0004] CN 117089486A reports an edible Lactobacillus plantarum that can adsorb MPs and is acid-resistant and bile-salt-resistant, but the cost of strain development is high, its colonization ability and utilization rate in the gastrointestinal tract are not high, its ability to remove MPs in the body (especially other organs except the intestine) is also limited, and it cannot repair the tissue damage caused by MPs, which limits its application scope and practical use value.

[0005] The ingestion and toxicity of MPs in the human body are closely related to their size. MPs larger than 150 μm can directly pass through the intestine without being absorbed, while MPs smaller than 10 μm can pass through the intestinal barrier and enter the blood and spread to other organs such as the liver, spleen, and kidney, leading to oxidative stress and inflammatory responses. An ideal method to mitigate the toxicity of MPs is to promote the aggregation of small-sized MPs particles to form non-toxic large-sized particles, thereby reducing their absorption in the body. At the same time, by removing the MPs accumulated in the body and reducing the oxidative stress and inflammatory responses induced by MPs, the toxicity can be further reduced, and the effective mitigation of the toxicity of MPs can be achieved. Summary of the Invention

[0006] Based on the above background, to solve the problem of the accumulation of microplastics in the body and their harm to human health, the present invention discovers that tea polyphenols can promote the aggregation of small-sized microplastics to form large-sized particle aggregates, accelerate the excretion of microplastics in the body, and alleviate the intestinal damage, reactive oxygen imbalance, and inflammatory factor disorder caused by microplastics. The research results of the present invention prove that natural tea polyphenols can be used to alleviate the toxicity of microplastics in the body. The technical solution adopted by the present invention is as follows:

[0007] Application of tea polyphenols as an accelerator for promoting the aggregation of microplastics in the body.

[0008] Furthermore, the tea polyphenols are a complex of polyhydroxyphenolic compounds in tea, including more than 30 kinds of phenolic substances. Among them, catechin compounds are the main components of tea polyphenols.

[0009] The tea polyphenols of the present invention can be tea polyphenol extracts (abbreviated as TP, tea polyphenol content > 96%), or catechin compounds. The tea polyphenol extracts can be obtained by purchasing commercially.

[0010] The catechin compounds are one or more of epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC), and gallic acid (GA), preferably EGCG.

[0011] The present invention discovers for the first time that tea polyphenols can promote the aggregation of microplastics in the body, aggregating small-sized microplastics (<10 μm) that are easily absorbed by the human body into large-sized particles (>150 μm) that are difficult to absorb, enabling them to be excreted from the human body through excretion, reducing the absorption and accumulation of microplastics in the body, and achieving the purpose of alleviating the toxicity of microplastics.

[0012] The tea polyphenols as an accelerator for promoting the aggregation of microplastics in the body can be added to various foods, health products, and drugs.

[0013] Furthermore, the present invention also provides:

[0014] Application of tea polyphenols in the preparation of products for clearing the accumulation of microplastics in the body.

[0015] Furthermore, the product is an edible food, such as food, drink, medicine, or health product, etc.

[0016] Furthermore, the present invention also provides the application of the tea polyphenols in the preparation of a drink for preventing the absorption of microplastics in the body, promoting the aggregation of microplastics in the body, and accelerating the excretion of microplastics from the body.

[0017] Specifically, the method of the application is to add 30-100 mg / L (preferably 50 mg / L) of tea polyphenols to a drink, and a drink for preventing the absorption of microplastics in the body, promoting the aggregation of microplastics in the body, and accelerating the excretion of microplastics from the body is prepared.

[0018] Furthermore, the present invention also provides:

[0019] The application of tea polyphenols in the preparation of health foods or drugs for promoting the aggregation of microplastics in the body, reducing the accumulation of microplastics in the body, and accelerating the excretion of microplastics in the body.

[0020] The present invention provides the application of the tea polyphenols in promoting the aggregation of microplastics in the body to form large particle aggregates. Furthermore, the tea polyphenols promote the aggregation of microplastics, accelerate the excretion of microplastics in the body, and reduce the content of microplastics in tissues and organs.

[0021] Furthermore, the present invention also provides:

[0022] The application of tea polyphenols in the preparation of health products or drugs for preventing or alleviating the toxicity of microplastics in the body. Furthermore, the toxicity of the microplastics includes intestinal damage, imbalance of reactive oxygen species, and disorder of inflammatory factors caused by microplastics in the body.

[0023] Microplastics can induce the intestinal tissue to generate excessive reactive oxygen species and pro-inflammatory factors, causing intestinal tissue damage, while tea polyphenols can be applied to scavenge the excessive reactive oxygen species and pro-inflammatory factors generated in the intestinal tissue induced by microplastics.

[0024] Therefore, the present invention provides the application of tea polyphenols in the preparation of scavenging the excessive reactive oxygen species and pro-inflammatory factors generated in the intestinal tissue induced by microplastics and improving cell survival rate.

[0025] Furthermore, the tea polyphenols are used to alleviate the cytotoxicity induced by microplastics.

[0026] Furthermore, foods containing tea polyphenols, such as tea leaves, tea-containing beverages, and other tea-containing foods, can also be applied as promoters of microplastic aggregation in the body or scavengers of microplastic accumulation in the body.

[0027] The innovation of the present invention lies in using natural tea polyphenols to promote the aggregation of microplastics, accelerating the excretion of microplastics in the body to reduce its accumulation in the body and alleviating the damage to organisms caused by microplastics. Furthermore, using tea polyphenols to scavenge the excessive reactive oxygen species and inflammatory reactions induced by microplastics can well alleviate the toxicity of microplastics to the body.

[0028] The tea polyphenols of the present invention can significantly promote the aggregation of small-sized microplastics (5 μm) into large-sized aggregates (over 150 μm) through hydrophobic interaction, electrostatic interaction, partitioning effect, etc., accelerate the excretion of microplastics in the body and reduce their accumulation, and alleviate the intestinal damage caused by microplastics. In addition, tea polyphenols reduce the toxic effects of microplastics in the body by scavenging excessive reactive oxygen species induced by microplastics and by inhibiting the content of pro-inflammatory factors. The present invention demonstrates that the application of plant polyphenols can reduce the chronic toxicity caused by microplastics, provides an innovative strategy for solving the harm of microplastic pollution to human health, and greatly expands the application scope of polyphenols.

[0029] The beneficial effects of the present invention are as follows:

[0030] (1) The cost of obtaining tea polyphenols is low. The tea polyphenols used in the present invention are all naturally occurring and widely distributed in nature, and are easily obtained and supplemented by the human body. The present invention can reduce the threat of microplastics to human health at a relatively low cost. Most importantly, the concentration of polyphenols used in the present invention is low, and the polyphenols ingested from daily tea drinking or other polyphenol-rich foods are sufficient to meet the requirements for promoting the excretion of microplastics in the body and reducing their toxicity.

[0031] (2) Tea polyphenols promote the aggregation of microplastics. The toxicity of microplastics to organisms is closely related to the particle size. In the present invention, tea polyphenols significantly promote the aggregation of microplastics in intestinal fluid, changing from 5 μm-sized particles to large particle aggregates over 150 μm. The promotion of microplastic aggregation by tea polyphenols reduces their absorption in the body and prevents the accumulation of microplastics in the body.

[0032] (3) Tea polyphenols accelerate the excretion of microplastics. The shorter the presence time of microplastics in the body, the less harmful they are to the body. In the present invention, polyphenols significantly accelerate the excretion of microplastics by the body, shorten their retention time in the body, and effectively reduce the toxicity of microplastics to the body.

[0033] (4) Tea polyphenols alleviate the toxicity of microplastics. Microplastics entering the body can cause oxidative stress and immune responses in the body, resulting in an imbalance of reactive oxygen species and pro-inflammatory factors, and harming the normal growth of the body. In the present invention, polyphenols effectively scavenge the excessive reactive oxygen species and pro-inflammatory factors induced by microplastics, significantly reduce the in vivo toxicity induced by microplastics, and alleviate the intestinal tissue damage, reactive oxygen species imbalance, and inflammatory factor disorder caused by microplastics.

[0034] The present invention discovers for the first time that tea polyphenols can promote the aggregation and excretion of microplastics in the body and effectively reduce the accumulation of microplastics in the body, which can well mitigate the harm of microplastics to human health. Tea polyphenols can be added as an accelerator for promoting the aggregation of microplastics in the body to various foods to promote the excretion of microplastics in the body and reduce their accumulation in the body. Drinking tea or other plant beverages containing polyphenols can achieve a similar effect, preventing or alleviating the chronic toxicity caused by microplastics, which greatly expands the application value of polyphenols and has great potential for popularization. Description of the Drawings

[0035] Figure 1 It is a comparative diagram of the deposition of EGCG promoting MPs-PS in aqueous solution in Example 1.

[0036] Among them, Figure A is the deposition diagram of MPs-PS alone in aqueous solution within 0 - 8 h, and Figure B is the deposition diagram of MPs-PS in aqueous solution within 0 - 8 h when EGCG and MPs-PS coexist.

[0037] Figure 2 It is a comparative diagram of EGCG promoting the aggregation of MPs-PS in solutions with different pH values in Example 2. Among them, Figure A is the distribution diagram of MPs-PS alone in solutions with different pH values, and Figure B is the aggregation diagram of MPs-PS in solutions with different pH values when EGCG and MPs-PS coexist. The white scale bar in Figures A and B represents 50 μm.

[0038] Figure 3 It is a diagram of the distribution and aggregation of polyphenols promoting MPs-PS in gastric and intestinal fluids in Example 2. Among them, Figure A is the structural formula of EGCG, ECG, EGC, EC, and GA, Figure B is the distribution diagram of PS alone, MPs-PS + ECG, MPs-PS + EGC, MPs-PS + EC, and MPs-PS + GA in gastric juice, and Figure C is the distribution diagram of PS alone, MPs-PS + ECG, MPs-PS + EGC, MPs-PS + EC, and MPs-PS + GA in intestinal juice. The white scale bar in Figures B and C represents 50 μm.

[0039] Figure 4 It is an electron micrograph of EGCG promoting the aggregation of MPs-PS in Example 2. Among them, Figure A is the electron micrograph of MPs-PS alone at different magnifications, and Figure B is the electron micrograph of the aggregation of MPs-PS at different magnifications after adding EGCG.

[0040] Figure 5 It is a graph of the potential changes of EGCG, MPs-PS, and MPs-PS + EGCG in Example 3. Among them, **, p < 0.01, compared with MPs-PS alone.

[0041] Figure 6It is the adsorption process diagram of MPs-PS to EGCG in Example 3. Among them, Figure A is the full-wavelength scanning diagram of EGCG alone, MPs-PS alone, and MPs-PS after adsorbing EGCG; Figure B is the standard curve diagram of different concentrations of EGCG and ultraviolet absorbance value; Figure C is the kinetic curve of MPs-PS adsorbing EGCG at different times, and the pseudo-first-order and pseudo-second-order kinetic curve fittings are carried out for the process of MPs-PS adsorbing EGCG respectively; Figure D is the isothermal curve of MPs-PS adsorbing different concentrations of EGCG, and the Langmuir and Freundlich isothermal curve fittings are carried out respectively.

[0042] Figure 7 It is the influence diagram of MPs-PS on cell viability and reactive oxygen species in Example 4. Among them, Figure A is the picture of the influence of different concentrations of MPs-PS on the adherent growth of HUVECs cells, stained with crystal violet; Figure B is the result diagram of the influence of different concentrations of MPs-PS on the viability of HUVECs cells, detecting cell viability with CCK-8; Figure C is the DCFH-DA staining diagram of the influence of different concentrations of MPs-PS on the reactive oxygen species of HUVECs cells; Figure D is the influence of different concentrations of MPs-PS on the intensity of reactive oxygen species of HUVECs cells, detecting the fluorescence value of reactive oxygen species of cells with DCFH-DA. The white scale bar in Figures A and C represents 100 μm. Different letters in Figures B and D indicate significant differences, P < 0.05, compared with the control group.

[0043] Figure 8 It is the diagram of EGCG alleviating the cytotoxicity of MPs-PS in Example 5. Among them, Figure A is the comparison diagram of the influence of different concentrations of EGCG on the viability of HUVECs cells, the inhibition of cell viability by MPs-PS, and the recovery of the cell viability inhibited by MPs-PS by different concentrations of EGCG; Figure B is the result diagram of the recovery of the cell viability inhibited by MPs-PS by EGCG after treating HUVECs cells with MPs-PS alone for 12 h and then adding 20 μmol / L EGCG for 12 h; Figure C is the diagram of the change of reactive oxygen species in HUVECs cells treated with MPs-PS plus different concentrations of EGCG; Figure D is the diagram of the change of the fluorescence intensity of reactive oxygen species in HUVECs cells treated with MPs-PS plus different concentrations of EGCG. Different letters in Figures A, B, and D indicate significant differences, P < 0.05, compared with the control group. The white scale bar in Figure C is 100 μm.

[0044] Figure 9It is the preparation of fluorescent MPs-PS and the result graph of its stability in Example 6. Among them, Figure A is the fluorescence photograph of the prepared fluorescent MPs-PS after being placed for 0, 1, 2, 3, 6, 8, 12, and 16 days. Figure B is the fluorescence intensity graph of MPs-PS at different days. Figure C is the infrared spectrogram of MPs-PS before and after being stained with Nile red. The white scale bar in Figure A is 50 μm.

[0045] Figure 10 It is the graph of EGCG promoting the excretion of MPs-PS in mice in Example 7. Among them, Figure A is the standard curve graph of the fluorescence intensity and concentration of different concentrations of fluorescent MPs-PS in the gastric and intestinal tissue extracts of untreated mice. Figure B is the result graph of EGCG reducing the content of MPs-PS in the stomach. Figure C is the result graph of EGCG reducing the content of MPs-PS in the colon. Figure D is the result graph of EGCG reducing the content of MPs-PS in the small intestine. Figure E is the line graph of the content of MPs-PS in the feces of mice at different days. The treatment time of mice is 7 days. Different letters in B-D indicate significant differences (p<0.05). **, p<0.01, compared with MPs-PS alone.

[0046] Figure 11 It is the result graph of EGCG scavenging excessive reactive oxygen species in the intestines of mice induced by MPs-PS in Example 8. Among them, Figure A is the result graph of EGCG scavenging excessive reactive oxygen species induced by MPs-PS in the colon. Figure B is the result graph of EGCG scavenging excessive reactive oxygen species induced by MPs-PS in the small intestine. The treatment time of mice is 7 days. Different letters in A and B indicate significant differences (p<0.05).

[0047] Figure 12 It is the result graph of EGCG reducing excessive pro-inflammatory factors in the intestines of mice induced by MPs-PS in Example 9. Among them, Figure A is the result graph of EGCG scavenging excessive IL-1β, IL-6, and TNF-α induced by MPs-PS in the colon. Figure B is the result graph of EGCG scavenging excessive IL-1β, IL-6, and TNF-α induced by MPs-PS in the small intestine. The treatment time of mice is 7 days. Different letters in A and B indicate significant differences (p<0.05). Detailed implementation manners

[0048] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts fall within the scope protected by the present invention.

[0049] Example 1 EGCG promotes the deposition of MPs-PS in aqueous solution

[0050] Tea polyphenols were dissolved in deionized water to prepare a stock solution of 10 mg / mL and stored in a refrigerator at 4 °C for later use. In this example, EGCG was selected as the representative of tea polyphenols.

[0051] The MPs were polystyrene (PS) with a particle size of 5 μm. The PS used was a typical representative of MPs and was obtained by purchasing commercially. It was prepared into a MPs-PS stock solution of 10 mg / mL with deionized water for subsequent experiments of the present invention.

[0052] To explore the effect of EGCG on the deposition of MPs-PS, two groups of experiments were set up:

[0053] 1) In a 4 mL system, MPs-PS was prepared into a solution of 2 mg / mL with deionized water and oscillated in a constant temperature shaker at 37 °C at 140 rpm / min. After 24 hours, the distribution map of MPs-PS in the aqueous solution was photographed, which was defined as the start of the control group. Then, it was left standing in a constant temperature incubator at 37 °C for 0.5, 1, 2, 4, 6, and 8 hours, and the distribution maps of MPs-PS in the aqueous solution at different times were photographed respectively as the control group, as shown in Figure 1 Figure A.

[0054] 2) In a 4 mL system, a mixed solution containing 2 mg / mL of MPs-PS and 50 mg / L of EGCG was prepared with deionized water and oscillated in a constant temperature shaker at 37 °C at 140 rpm / min. After 24 hours, the distribution map of MPs-PS in the aqueous solution was photographed, which was defined as the start of the experimental group. Then, it was left standing in a constant temperature incubator at 37 °C for 0.5, 1, 2, 4, 6, and 8 hours, and the distribution maps of MPs-PS in the aqueous solution at different times were photographed respectively as the experimental group, as shown in Figure 1 Figure B.

[0055] Figure 1 Figure A in shows that the MPs-PS was relatively evenly distributed in the aqueous solution, and only slight deposition occurred with the extension of the standing time, and the turbidity of the solution was relatively high. Figure 1 The results of Figure B in show that EGCG significantly accelerated the deposition rate of MPs-PS. Especially after 4 hours, the clarity of the solution was significantly better than that of the single MPs-PS solution. Figure 1 The results proved that at the same time, EGCG significantly promoted the deposition of MPs-PS in the aqueous solution. The promotion of MPs-PS deposition by EGCG was beneficial to reducing the fluidity of MPs-PS and decreasing its distribution in the body.

[0056] Example 2 Promotion of Aggregation of MPs-PS in Solutions with Different pH by Polyphenols

[0057] To study the effect of different pH values on the promotion of MPs-PS aggregation by EGCG, the pH values of deionized water were adjusted to 3, 5, 7, 9, and 11 using 0.1 M hydrochloric acid or sodium hydroxide, respectively. Then, in a 4 mL system, the following were prepared with solutions of different pH values: 1) a separate 2 mg / mL MPs-PS solution as the control group, as shown in Figure 2 Figure A of Figure 2 ; 2) a mixed solution containing 50 mg / L EGCG and 2 mg / mL MPs-PS as the experimental group, as shown in Figure 2 Figure B of Figure 2 . After oscillating in a constant temperature shaker at 37 °C at 140 rpm / min for 24 hours, 10 μL of the solution from each tube was dropped onto a glass slide. After natural drying, a fluorescence microscope (Olympus, IX73, Japan) was used to observe the effect of EGCG on promoting MPs-PS aggregation at different pH values.

[0058] Furthermore, artificial gastric juice and intestinal juice were used to simulate the in vivo gastrointestinal environment to explore the effect of different polyphenols on promoting MPs-PS aggregation.

[0059] Artificial gastric juice (pH 1.5) and intestinal juice (pH 7.4) were obtained by commercial purchase.

[0060] The experimental setup was similar to that of EGCG, except that deionized water was replaced with gastric juice and intestinal juice.

[0061] The distribution of MPs-PS in gastric juice is shown in Figure 3 Figure B, with the control group being separate MPs-PS and the experimental group being MPs-PS mixed with ECG, EGC, EC, and GA, respectively. The distribution of MPs-PS in intestinal juice is shown in Figure 3 Figure C, with the control group being separate MPs-PS and the experimental group being MPs-PS mixed with ECG, EGC, EC, and GA, respectively. After oscillating for 24 hours, a microscope was used to observe the promoting effect of ECG, EGC, EC, and GA on MPs-PS aggregation in gastric juice and intestinal juice. Figure 3 The results showed that separate MPs-PS were relatively dispersed in both gastric juice and intestinal juice, and the promoting effect of ECG, EGC, EC, and GA on MPs-PS aggregation in intestinal juice was significantly stronger than that in gastric juice. The results of polyphenols promoting MPs-PS aggregation in gastric juice and intestinal juice were consistent with the results of EGCG promoting MPs-PS aggregation at different pH values, indicating that polyphenols generally can promote MPs-PS aggregation, which significantly expands the application value of polyphenols.

[0062] In addition, to more clearly observe the promoting effect of EGCG on the aggregation of MPs-PS, scanning electron microscopy (SEM, Sigma 300, Oxford Xplore 50, Zeiss, Germany) was used to scan the samples of MPs-PS alone and MPs-PS with added EGCG. After shaking the MPs-PS alone (0.2 mg / mL) and MPs-PS (0.2 mg / mL) with EGCG (10 mg / L) for 24 hours, 10 μL of the mixed solution of MPs-PS alone and MPs-PS with EGCG was taken and dropped on a single-crystalline silicon wafer. After natural drying, it was further dried in a vacuum drying oven. Before the SEM test, the surface of MPs-PS was sputtered with gold to increase conductivity. Figure 4 Figure A shows that the MPs-PS alone is relatively dispersed, presenting spherical particles. Figure 4 In Figure B after adding EGCG, obvious aggregation of MPs-PS occurs, changing from relatively dispersed particles to large particle aggregates, and the particle size increases significantly.

[0063] Example 3 Adsorption process of MPs-PS to EGCG

[0064] Figure 5 The ζ-potential results of MPs-PS alone and MPs-PS + EGCG in aqueous solution show that before and after adding EGCG to MPs-PS, the surface potential changes from -46.9 mV to -39.1 mV. EGCG significantly changes the surface potential of MPs-PS. The change in the surface potential of MPs-PS by EGCG makes the original system of MPs-PS unstable, which is conducive to the aggregation of MPs-PS. The results of the surface potential indicate that there is an electrostatic interaction between EGCG and MPs-PS.

[0065] Furthermore, the process of MPs-PS adsorbing EGCG was studied. Ultraviolet absorption spectrum: Weigh 40 mg of MPs-PS into a 50 mL conical flask, add 20 mL of EGCG solution (50 mg / L), shake at 140 rpm / min at 37 °C for 24 hours, centrifuge at 3500 rpm / min for 10 minutes, and then use a UV-1800 spectrophotometer (PerkinElmer, Lambda 25, USA) to scan the ultraviolet absorption value in the supernatant in the range of 200 - 400 nm. Separate EGCG and MPs-PS were used as controls. Figure 6The results of Figure A show that EGCG has a maximum absorption peak at 275 nm, and the absorbance value of EGCG in the supernatant decreases after MPs-PS adsorbs EGCG. Further, the ultraviolet absorbance values of EGCG at different concentrations (5, 10, 20, 30, 40, 50 mg / L) were detected at 275 nm, and a standard curve of different concentrations of EGCG versus ultraviolet absorbance was plotted to calculate the adsorption capacity of MPs-PS for EGCG. The standard curve graph is as shown in Figure 6 Figure B. Combining Figure 6 Figures A and B, it was calculated that the concentration of EGCG decreased by 17.7% after MPs-PS adsorbed EGCG.

[0066] Kinetic curve: 40 mg of MPs-PS was placed in a 50 mL glass conical flask containing 20 mL of EGCG solution (50 mg / L) and shaken at 140 rpm / min at 37 °C. After adsorption for 0.5, 1, 2, 3, 6, 9, 12, 24, and 30 hours, 0.5 mL of the sample was taken and centrifuged, and the absorbance value of EGCG at different times was detected as the experimental group. The control group consisted of an EGCG solution without MPs-PS. The adsorption capacity of MPs-PS for EGCG was calculated by subtracting the absorbance value of the experimental group from that of the control group and combining the standard curve. The pseudo-first-order kinetic model (1) and the pseudo-second-order kinetic model (2) were used to fit the kinetic data after adsorption respectively. The two models are as follows:

[0067]

[0068] where q t (mg / g) represents the adsorption capacity of MPs-PS for EGCG at different times t (h). K 1 (h -1 ) and K 2 (g / mg·h) represent the pseudo-first-order and pseudo-second-order rate constants respectively.

[0069] Kinetics can estimate the efficiency and rate-limiting steps of the adsorption process. Figure 6 The results of Figure C show that after rapid adsorption in the early stage, the increasing trend of MPs-PS adsorption of EGCG tends to be stable over time until equilibrium is reached. The equilibrium adsorption capacity of MPs-PS for EGCG is 4.35 mg / g. In the pseudo-second-order kinetics, the determination coefficient (R 2 = 0.98) of MPs-PS exceeds that of the pseudo-first-order kinetics (R 2 = 0.82), indicating that the pseudo-second-order kinetic model is more suitable for explaining the adsorption process of MPs-PS for EGCG, suggesting that the surface active sites of MPs-PS are limited and chemical adsorption may be a rate-limiting step.

[0070] Furthermore, an isothermal curve model was used to predict the interaction between MPs-PS and EGCG after adsorption equilibrium occurred. Isothermal curve: 40 mg of MPs-PS was placed into 50 mL glass conical flasks containing 20 mL of EGCG solutions with different concentrations (5, 10, 20, 30, 40, 50 mg / L). After oscillating adsorption for 24 hours, 0.5 mL of the sample was taken for centrifugation, and the absorbance value of EGCG was measured as the experimental group. The control group consisted of EGCG solutions with the same concentrations without MPs-PS. The Langmuir isothermal model (3) and the Freundlich isothermal model (4) were used to fit the adsorption isotherm data respectively.

[0071]

[0072] where q m (mg / g) represents the maximum value of the adsorption capacity of MPs-PS for EGCG. K L (L / mg) and K F (L / mg) are the distribution coefficients of Langmuir and Freundlich respectively. 1 / n in the Freundlich model represents the adsorption intensity and heterogeneity of MPs-PS.

[0073] Figure 6 The results of Figure D show that the adsorption capacity of MPs-PS for EGCG increases with the increase of EGCG concentration. The Langmuir isotherm fails to simulate the real adsorption data, indicating that the adsorption is non-uniform. In contrast, the data fitted by the Freundlich model is more consistent with the experimental data, which means that the adsorption process of EGCG on MPs-PS includes multi-layer and monolayer adsorption behaviors on non-uniform surfaces. In addition, the adsorption isotherm of EGCG on MPs-PS is highly linear, and the R 2 value in the linear model is 1.00. The high linearity means that the partitioning effect is an important consideration in adsorption, indicating that the adsorption process is mainly physical adsorption. The adsorption of EGCG by MPs-PS includes physical adsorption and chemical adsorption, with physical adsorption being the main one. There are electrostatic interactions and partitioning effects during the process of EGCG promoting the aggregation of MPs-PS.

[0074] Example 4 Effects of MPs-PS on cell viability and reactive oxygen species

[0075] Human umbilical vein endothelial cells (HUVECs) in good growth state were cultured in DMEM medium containing 10% FBS, and the growth environment was 37 °C and 5% CO 2 . The digested HUVECs were inoculated onto 96-well plates at a density of 1×10 per well 4Cells (200 μL) were incubated for 24 hours. After removing the original medium, the cells were incubated for another 24 hours with media containing different concentrations (50, 100, 150, and 200 mg / L) of MPs-PS. The toxicity of MPs-PS to HUVECs cells was evaluated by crystal violet staining, CCK-8 assay, and reactive oxygen species detection.

[0076] After the treatment with MPs-PS, the cells were washed three times with PBS and stained with crystal violet. The staining results were observed and recorded using a fluorescence microscope. CCK-8 assay was used to evaluate the effect of MPs-PS on cell viability. After removing the medium and washing three times with PBS, 100 μL of serum-free solution containing 10% CCK-8 was added and incubated at 37 °C for 30 minutes. The absorbance at 450 nm was measured using a multifunctional microplate reader. The cell viability (5) was calculated as follows:

[0077]

[0078] where As is the absorbance value of the treatment with MPs-PS, Ac is the absorbance value without MPs-PS, and Ab is the absorbance value of the medium alone.

[0079] Reactive oxygen species detection: HUVECs cells were incubated in 24-well plates and treated with different concentrations of MPs-PS for 24 hours. The medium was removed and the cells were washed three times with PBS. Then, 0.5 mL of serum-free DMEM cell medium containing 10 μmol / L DCFH-DA staining solution was added and incubated in a cell culture incubator at 37 °C for 20 minutes. The cells were washed three times with serum-free DMEM medium to completely remove the DCFH-DA stain that did not enter the cells. Reactive oxygen species images of the cells were taken using a fluorescence microscope. In addition, after treating the cells with MPs-PS, the cells were digested with trypsin and collected. The cells were washed three times with PBS, the washing solutions were combined and centrifuged. The supernatant was discarded, 0.5 mL of diluted DCFH-DA staining solution was added, and incubated in a cell culture incubator at 37 °C for 30 minutes. Subsequently, the reactive oxygen species fluorescence intensity of HUVECs cells treated with different concentrations of MPs-PS was measured using a fluorescence spectrophotometer (Hitachi, F-4700, Japan) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The excitation and emission slits were 5 nm × 5 nm.

[0080] Figure 7 Figure A shows the crystal violet staining photographs, and the results show that MPs-PS significantly inhibited the adherent growth of HUVECs cells, and high concentrations of MPs-PS led to a sharp decrease in the number of adherent HUVECs cells. Figure 7Figure B shows the results of cell viability. The results indicate that, compared with the CK group, the viabilities of HUVECs cells treated with 50, 100, 150, and 200 mg / L MPs-PS were 85.07%, 77.91%, 60.65%, and 43.92%, respectively. Figure 7 Figure C shows the fluorescence detection photos of reactive oxygen species. The results show that MPs-PS led to a significant increase in the intensity of reactive oxygen species in HUVECs cells. Correspondingly, the reactive oxygen species in cells treated with 50, 100, 150, and 200 mg / L MPs-PS increased by 1.72, 2.53, 2.85, and 3.26 times, respectively ( Figure 7 Figure D). The above results indicate that the viability of HUVECs cells decreased significantly with the increase in the concentration of MPs-PS, while the content of reactive oxygen species increased with the increase in the concentration of MPs-PS. The excessive accumulation of reactive oxygen species in cells is closely related to cell apoptosis and necrosis. Removing the excessive reactive oxygen species induced by MPs-PS is a feasible method to reduce its toxicity.

[0081] Example 5 EGCG alleviates the cytotoxicity of MPs-PS

[0082] To determine the role of EGCG in reducing the cytotoxicity of MPs-PS, HUVECs cells were treated with 100 mg / L MPs-PS plus different concentrations (1, 5, 10, and 20 μmol / L) of EGCG, and the changes in cell viability and reactive oxygen species after co-treatment were measured respectively. Different concentrations of EGCG alone were also used to treat the cells. In addition, treating the cells with MPs-PS alone for 12 hours first and then adding EGCG for 12 hours was also used to determine the alleviation of the toxicity of MPs-PS by EGCG. The operation procedures for cell treatment refer to the methods for detecting viability and reactive oxygen species in Example 5.

[0083] Figure 8 Figure A shows the results of the viability of HUVECs cells with different concentrations of EGCG and MPs-PS added with different concentrations of EGCG. The results show that calculated with the CK viability as 100%, the viabilities of cells treated with 1, 5, 10, and 20 μmol / L EGCG were 97.71%, 97.36%, 96.09%, and 96.39%, respectively. Different concentrations of EGCG basically did not affect the viability of HUVECs cells. Figure 8The results of Figure A also showed that the cell viability under the treatment of 100 mg / L MPs-PS was 63.14%. After adding 1, 5, 10, and 20 μmol / L EGCG, the cell viability increased to 71.91%, 79.32%, 79.84%, and 82.55% respectively. The experimental results indicated that compared with MPs-PS alone, adding EGCG effectively improved the cell viability. In addition, when cells were first treated with MPs-PS for 12 hours and then incubated with EGCG for another 12 hours, the cell viability was also significantly increased, as shown in Figure 8 Figure B, EGCG could restore the cell viability inhibited by MPs-PS. Figure 8 Figure C shows the changes in reactive oxygen species (ROS) in HUVECs cells treated with MPs-PS plus different concentrations of EGCG. The results showed that untreated HUVECs cells served as the control group with almost no ROS signal. The cells treated with 100 mg / L MPs-PS were defined as the experimental group, which could lead to a significant increase in ROS in the cells. After adding EGCG to the experimental group, the ROS signal decreased significantly. Figure 8 Figure D shows the changes in the fluorescence intensity of ROS in HUVECs cells treated with MPs-PS plus different concentrations of EGCG. The results showed that compared with the control group, the fluorescence value of ROS in the experimental group increased by 2.53 times. Compared with the experimental group, adding 20 μmol / L EGCG reduced the ROS in the cells by 34.76%. The above results demonstrated that EGCG had a good effect in alleviating MPs-PS-mediated apoptosis and scavenging excessive ROS.

[0084] Example 6 Preparation and Stability of Fluorescent MPs-PS

[0085] To study the distribution of MPs-PS in mice, MPs-PS was first labeled with Nile red to prepare fluorescent MPs-PS. 0.5 g of MPs-PS was added to 20 mL of Nile red staining solution (0.5 mg / mL, preferably prepared with methanol), and the mixture was shaken at 140 rpm / min at 37 °C in the dark for 24 hours. Then, the mixed solution was centrifuged to remove the supernatant, and the precipitate was washed 3 times with ultrapure water to remove the staining agent on the surface of MPs-PS. The prepared MPs-PS was freeze-dried, and the changes in the peak shape and peak position of MPs-PS before and after staining were detected by microscopic Fourier transform infrared spectroscopy. To determine the stability of the prepared fluorescent MPs-PS, fluorescent images of fluorescent MPs-PS were taken at different times (0, 1, 2, 3, 6, 8, 12, and 16 days) under a red excitation wavelength using a fluorescence microscope, and the fluorescence intensity was analyzed using Image J software.

[0086] Figure 9Figure A of [Object Name] shows the fluorescence images of the prepared fluorescent MPs-PS on the 0th, 1st, 2nd, 3rd, 6th, 8th, 12th, and 16th days. The results show that the prepared fluorescent MPs-PS exhibit green fluorescence under red excitation light. Figure 9 Figure B of [Object Name] shows the fluorescence intensity graph of MPs-PS at different days, and Figure C shows the infrared spectra of MPs-PS before and after staining with Nile red. The results show that the structure of MPs-PS does not change before and after staining, and there is no significant difference in the fluorescence intensity of MPs-PS between the 0th day and the 16th day, indicating that the prepared fluorescent MPs-PS have good stability.

[0087] Example 7: EGCG Promotes the Excretion of MPs-PS in Mice

[0088] To study the excretion effect of EGCG on MPs-PS in vivo, 5-week-old male pathogen-free (SPF) C57BL / 6J mice were selected for the experiment. Throughout the study, the growth environment of the mice was a 12-hour / 12-hour light / dark cycle, a constant temperature of 25 ± 1°C, and a humidity of 50 ± 5%. The mice had free access to food and water.

[0089] Mouse treatment: After the mice were acclimated to the environment for one week, 6-week-old C57BL / 6J mice were weighed and randomly assigned to different groups, including the non-treatment group, the control group (treated with water only), the EGCG group, the fluorescent MPs-PS group, and the MPs-PS + EGCG group (6 mice in each group). The concentration of fluorescent MPs-PS used to treat the mice was 50 mg / kg (equivalent to 1 mg of MPs-PS per 20 g of mouse per day), which is close to half of the maximum exposure dose of mice and more in line with the actual situation of daily exposure to MPs in adults. Fluorescent MPs-PS at a concentration of 10 mg / mL was prepared with deionized water, and the gavage volume for each mouse was 200 μL. The concentration of EGCG used to treat the mice in the EGCG group was 50 mg / kg. For the MPs-PS + EGCG experimental group, MPs-PS and EGCG were premixed to the same final concentration as that of MPs-PS or EGCG alone and used to treat the mice together. The treatment was continued for 7 days, and feces were collected. After the treatment, the mice were humanely euthanized, and gastric and intestinal samples were immediately collected for measuring the changes in reactive oxygen species and inflammatory factors.

[0090] Preparation of tissue digestion solution: Weigh 1 g of proteinase K, 3.95 g of NH 4 HCO 3 and 5 g of SDS in 1 L of PBS solution with a pH of 7.4, dissolve them thoroughly, and filter through a 0.22 μm membrane.

[0091] Determination of MPs-PS content in the stomach and intestine: Take appropriate amounts of the stomach, colon, and small intestine of the untreated group of mice, blot the surface water, and accurately weigh 0.4 g of each tissue. Add 4 mL of tissue digestion solution, and use a grinder (RETSCH, MM 400, Germany) to grind the tissue into a homogenate. Digest the homogenate at 37 °C for 12 hours to obtain a clear tissue digestion solution. Prepare fluorescent MPs-PS solutions with different concentrations using the tissue digestion solution. Use a fluorescence spectrophotometer to detect the fluorescence value of MPs-PS at an excitation wavelength of 530 nm and an emission wavelength of 632 nm, and plot the standard curve between the fluorescence value and concentration of MPs-PS. Measure the fluorescence values of the homogenates of the stomach and intestinal tissues in other different treatment groups using the same method, and calculate the MPs-PS content in each tissue according to the standard curve.

[0092] Figure 10 Figure A shows the standard curve of the fluorescence intensity and concentration of fluorescent MPs-PS with different concentrations in the extracts of the stomach and intestinal tissues of untreated mice, which is used to quantify the amount of MPs-PS in the stomach and intestine under different treatment groups. Figure 10 Figures B - D respectively show the results of the content of MPs-PS reduced by EGCG in the stomach, colon, and small intestine. The results show that the fluorescent MPs-PS signals of the mice treated with water and EGCG are weak, and there is no significant difference between the two. Compared with the water group, the contents of MPs-PS in the stomach, colon, and small intestine of the mice treated with MPs-PS increased by 23.99, 23.09, and 50.24 times respectively. In contrast, EGCG significantly reduced the content of MPs-PS in the stomach and intestine, indicating that EGCG promoted the excretion of MPs-PS and reduced the accumulation of MPs-PS in the body. Figure 10 Figure E shows the line graph of the MPs-PS content in the feces of mice on different days. The results show that the MPs-PS content in the feces of mice increased significantly with the extension of the treatment time. From the 4th day, the MPs-PS content in the MPs-PS + EGCG group was significantly higher than that of the mice in the group treated with MPs-PS alone. The changing trend of MPs-PS in the feces of mice further proves that EGCG is beneficial to promoting the excretion of MPs-PS in the body. Generally speaking, EGCG promoting the excretion of MPs-PS in the body reduces the absorption time and total accumulation amount of MPs-PS in the body, thereby reducing the harm of MPs-PS.

[0093] Example 8: EGCG scavenges excessive reactive oxygen species in the intestine of mice induced by MPs-PS

[0094] Mice were treated according to the method of Example 7, and fresh intestinal tissues were collected. The fresh intestinal tissues were washed with PBS, 100 mg of intestinal tissue was weighed and placed in 1 mL of PBS solution, and the tissue was homogenized at 4°C. Centrifuge at 1000 rpm / min for 3 minutes at 4°C, and retain the supernatant for detection. The concentration of reactive oxygen species in the intestinal tissue was measured according to the instructions of the reactive oxygen species detection kit (Boster Biological Technology, Shanghai). The fluorescence spectrophotometer was used to quantify the intensity of reactive oxygen species in the intestinal tissue at an excitation wavelength of 535 nm and an emission wavelength of 610 nm.

[0095] Figure 11 Figure A shows the results of EGCG scavenging excessive reactive oxygen species induced by MPs-PS in the colon, and Figure B shows the results of EGCG scavenging excessive reactive oxygen species induced by MPs-PS in the small intestine. The results showed that MPs-PS treatment significantly increased the content of reactive oxygen species in the colon and small intestine of mice. Compared with the water treatment group, the content of reactive oxygen species in the colon and small intestine increased by 1.90 and 1.63 times, respectively. In contrast, EGCG significantly reduced the reactive oxygen species induced by MPs-PS. After adding EGCG, the content of reactive oxygen species in the colon and small intestine was close to that of the water treatment group, proving that EGCG effectively inhibited the imbalance of intestinal reactive oxygen species caused by MPs-PS.

[0096] Example 9 EGCG reduces excessive pro-inflammatory factors in the intestine of mice induced by MPs-PS

[0097] Mice were treated according to the method of Example 7, and fresh intestinal tissues were collected. The fresh intestinal tissues were washed with PBS, 100 mg of intestinal tissue was weighed and placed in 1 mL of PBS solution, and the tissue was homogenized at 4°C. Centrifuge at 5000 rpm / min for 15 minutes at 4°C, and retain the supernatant. According to the instructions of the ELISA kits for IL-1β, IL-6 and TNF-α (ELISA, Fankel, Shanghai), the absorbance values of inflammatory factors (IL-1β, IL-6 and TNF-α) in the colon and small intestine were measured at 450 nm using an enzyme-linked immunosorbent assay reader and quantified.

[0098] Oxidative stress and pro-inflammatory processes are considered to be interdependent, and oxidative stress can induce an inflammatory response. The pro-inflammatory factors IL-1β, IL-6 and TNF-α were selected to evaluate the effect of MPs-PS on the inflammatory response in the colon and small intestine of mice. Figure 12 Figure A shows the results of EGCG scavenging excessive IL-1β, IL-6 and TNF-α induced by MPs-PS in the colon, and Figure B shows the results of EGCG scavenging excessive IL-1β, IL-6 and TNF-α induced by MPs-PS in the small intestine. The results showed that PS significantly up-regulated the content of the three inflammatory factors in the colon and small intestine, while the addition of EGCG effectively inhibited the increase of IL-1β, IL-6 and TNF-α induced by MPs-PS in the colon and small intestine.Figure 12 Taking the small intestine in Group B as an example, compared with the water treatment group, the contents of IL-1β, IL-6 and TNF-α in the MPs-PS treatment group increased by 1.84 times, 1.77 times and 3.15 times respectively. Correspondingly, compared with MPs-PS alone, the contents of IL-1β, IL-6 and TNF-α in the small intestine treated with MPs-PS+EGCG decreased by 26.45%, 15.91% and 48.72% respectively. The above results prove that EGCG effectively inhibits MPs-PS-induced intestinal inflammation, which is of great significance for protecting the body from MPs toxicity.

[0099] The results of Example 1 show that EGCG can accelerate the deposition of MPs-PS in aqueous solution to reduce the mobility of MPs-PS and decrease the absorption of MPs-PS by the body.

[0100] The results of Example 2 show that polyphenols (EGCG, ECG, EGC, EC, GA) can promote the aggregation of small-sized MPs-PS to form large-sized aggregates, especially under neutral to alkaline conditions, which is beneficial to the aggregation and excretion of MPs-PS in intestinal fluid.

[0101] The results of Example 3 show that MPs-PS can adsorb EGCG, and the adsorption process includes physical adsorption and chemical adsorption, with physical adsorption being the main one, and there are electrostatic interactions and distribution effects between the two.

[0102] The results of Example 4 show that MPs-PS induction causes an outbreak and imbalance of reactive oxygen species in cells, reduces cell viability, and leads to apoptosis.

[0103] The results of Example 5 show that EGCG can effectively scavenge the excessive reactive oxygen species produced by MPs-PS-induced cells and improve cell viability.

[0104] The results of Example 6 show that the prepared fluorescent MPs-PS has good stability and can be used to study the distribution and accumulation rules of MPs in vivo.

[0105] The results of Example 7 show that MPs-PS can accumulate in large amounts in the intestine, and EGCG can effectively promote the excretion of MPs-PS in mice.

[0106] The results of Example 8 show that EGCG can significantly scavenge the excessive reactive oxygen species in the small intestine of mice induced by MPs-PS and restore the reactive oxygen species to normal levels.

[0107] The results of Example 9 show that EGCG can reduce the excessive pro-inflammatory factors in the small intestine of mice induced by MPs-PS and restore the inflammatory response to normal levels.

[0108] The present invention has been described in detail above using general descriptions and specific embodiments. However, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. Application of tea polyphenols as a microplastic aggregation promoter in vivo.

2. The use according to claim 1, characterized in that The tea polyphenols are a complex of polyhydroxyphenol compounds in tea leaves.

3. The use according to claim 2, characterized in that The tea polyphenols are tea polyphenol extracts or catechin compounds; the catechin compounds are one or more of epigallocatechin gallate, epigallocatechin, epicatechin gallate, epicatechin, and gallic acid.

4. Application of tea polyphenols in the preparation of products for removing microplastic accumulation in the body.

5. The use according to claim 4, characterized in that The tea polyphenols are used in the preparation of beverages that can prevent microplastic absorption in the body, promote microplastic aggregation in the body, and accelerate the discharge of microplastics from the body.

6. The use according to claim 4, characterized in that The tea polyphenols are used in the preparation of health foods or medicines that promote the aggregation of microplastics in the body, reduce the accumulation of microplastics in the body, and accelerate the excretion of microplastics in the body.

7. The application of tea polyphenols in the preparation of health products or medicines for preventing or alleviating the toxicity of microplastics in the body.

8. The use according to claim 7, characterized in that The microplastic toxicity includes intestinal damage, imbalance of reactive oxygen species and disorder of inflammatory factors caused by microplastics.

9. The use according to claim 7, characterized in that Application of tea polyphenols in the preparation of removing excess reactive oxygen and pro-inflammatory factors produced in intestinal tissue induced by microplastics in vivo and improving cell survival rate.

10. Foods containing tea polyphenols can be used as promoters of microplastic aggregation in the body or in the preparation of products for removing microplastic accumulation in the body.

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