Selenium-enriched spirulina carbon dots as well as preparation method and application thereof

Abstract

The invention discloses selenium-enriched spirulina carbon dots as well as a preparation method and application thereof, and belongs to the technical field of medicines. The selenium-rich spirulina powder is obtained by taking selenium-rich spirulina growing to a logarithmic phase as a raw material, centrifuging, collecting and freeze-drying. Uniformly mixing the spirulina platensis with a catalyst and deionized water according to a mass ratio of 1: (0.1-0.8): 20, then putting the mixture into a high-temperature reaction kettle to react at 140-260 DEG C for 1.5-5.5 hours, dialyzing the reaction liquid for 24 hours after the reaction is finished, centrifuging, and freeze-drying the dialysate to obtain the selenium-enriched spirulina platensis carbon dots with the yield of 70-95%. The selenium-enriched spirulina carbon dots provided by the invention not only have the inhibition capability of alpha-amylase, alpha-glucosidase, cholesterol esterase and pancreatic lipase and the cholate binding capability, but also can improve the endogenous antioxidant defense capability of model organisms while removing exogenous oxidizing substances, so that the glycolipid metabolism balance is efficiently regulated and controlled.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and biotechnology, and relates to a selenium-enriched spirulina carbon dot, its preparation method, and its application. Background Technology

[0002] Selenium-enriched spirulina is a high-quality nutritional supplement that efficiently converts inorganic selenium into organic selenium through biotransformation technology. Its efficacy in antioxidation, immune regulation, and thyroid health maintenance has been proven, demonstrating a clear biological activity basis. However, its application value has not yet been fully explored, primarily limited to low-value-added areas such as food ingredients and animal feed additives. In the high-value fields of biomedicine and biotechnology, the application of selenium-enriched spirulina remains unexplored, failing to fully realize its unique physiological functional advantages.

[0003] Carbon dots, as a novel zero-dimensional carbon-based nanomaterial with a size of less than 10 nm, have become a research frontier in nanoscience and technology due to their excellent optical properties, good biocompatibility, low toxicity, and tunable luminescence characteristics. Carbon dots with near-infrared emission properties exhibit great application potential in the biomedical field due to their strong tissue penetration and low background interference, and are widely used in areas such as biosensing, in vivo imaging, drug delivery, and synergistic therapy.

[0004] Current research on spirulina-derived carbon dots primarily focuses on its versatility as a high-nitrogen biomass, concentrating on utilizing its proteins and nitrogen elements as carbon and nitrogen sources to prepare carbon dots and exploring their applications in light-emitting devices or general sensing. Existing research has failed to combine the unique organic selenium bioactive library of selenium-enriched spirulina with the advantages of the carbon dot nanoplatform, creating a significant technological gap. Summary of the Invention

[0005] To address the aforementioned technical problems and deficiencies, this invention provides a method for preparing selenium-enriched spirulina carbon dots and their applications.

[0006] In a first aspect, the present invention provides a method for preparing selenium-enriched spirulina carbon dots, comprising: mixing selenium-enriched spirulina, water and a catalyst, performing a hydrothermal reaction, and naturally cooling to room temperature to obtain a reaction solution; dialyzing the reaction solution to obtain a dialysis medium; concentrating the dialysis medium and freeze-drying it to obtain selenium-enriched spirulina carbon dots; The catalyst is selected from any one of NaOH, CH3COOH, and H2SO4.

[0007] Furthermore, in the method for preparing selenium-enriched spirulina carbon dots provided by the present invention, the preparation of selenium-enriched spirulina is as follows: an inorganic selenium compound is added to the spirulina culture system; the spirulina with added inorganic selenium compound is subjected to intermittent light culture to grow to the logarithmic growth phase, thereby obtaining selenium-enriched spirulina.

[0008] Furthermore, in the method for preparing selenium-enriched spirulina carbon dots provided by the present invention, the inorganic selenium compound is Na2SeO3; The temperature for the intermittent light culture was 25 ℃.

[0009] Furthermore, in the method for preparing selenium-enriched spirulina carbon dots provided by the present invention, the mass ratio of selenium-enriched spirulina, water and catalyst is 1:0.1~0.8:20.

[0010] Furthermore, in the method for preparing selenium-enriched spirulina carbon dots provided by the present invention, the hydrothermal reaction temperature is 140~260 ℃ and the time is 1.5~5.5 h; The dialysis time was 24 hours.

[0011] Secondly, the present invention provides a selenium-enriched spirulina carbon dot, which is prepared by the above-mentioned method for preparing selenium-enriched spirulina carbon dots.

[0012] Thirdly, this invention provides the application of selenium-enriched spirulina carbon dots in the preparation of drugs that regulate the balance of glucose and lipid metabolism.

[0013] Fourthly, this invention provides the application of selenium-enriched spirulina carbon dots in the preparation of anti-aging and longevity-promoting drugs.

[0014] Fifthly, this invention provides the application of selenium-enriched spirulina carbon dots in the preparation of drugs against oxidative stress.

[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: This invention utilizes NaOH, CH3COOH, and H2SO4 as catalysts to prepare selenium-enriched Spirulina carbon dots with yields as high as 70%–95% using a hydrothermal method at 140–260 °C. This yield is significantly higher than that of other biomass carbon dots prepared using hydrothermal methods. The carbon dots prepared by this invention not only possess the ability to efficiently scavenge O2, but also… - H2O2, ·ABTS +The spirulina carbon dots exhibit broad-spectrum antioxidant capacity and, by inhibiting α-amylase, α-glucosidase, pancreatic lipase, and cholesterol esterase, also possess bile salt binding capacity, thus exerting hypoglycemic and lipid-lowering effects. These selenium-enriched spirulina carbon dots are of biomass origin and possess good biocompatibility. They extended the median lifespan of N2-type and high-sugar diet N2-type Caenorhabditis elegans nematodes by 10.81% and 59.46%, respectively. They also increased the activities of nematode antioxidant enzymes (including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT)) by 109.74%, 66.47%, and 177.03%, respectively, and reduced the content of malondialdehyde, a lipid peroxidation product, by 33.01%. Furthermore, the carbon dots significantly reduced glucose and triglyceride levels in nematodes. A high-sugar diet induces oxidative stress, and excessive reactive oxygen species (ROS) attack the mitochondria of pancreatic β-cells, leading to impaired β-cell function and a reduction in initial insulin secretion. Simultaneously, oxidative stress activates inflammatory signaling pathways and inhibits the IRS-1 / PI3K / Akt insulin signaling pathway, thereby triggering insulin resistance. In response, the body initiates compensatory mechanisms, prompting the pancreas to secrete more insulin, resulting in hyperinsulinemia. This, on the one hand, fails to effectively promote glucose uptake by peripheral tissues, leading to glucose accumulation in the blood, while simultaneously promoting hepatic gluconeogenesis and lipogenesis, resulting in elevated triglyceride and total cholesterol levels. On the other hand, hyperinsulinemia inhibits the breakdown of fat into glycerol and fatty acids in adipose tissue, reducing the release of free fatty acids. These free fatty acids are absorbed in large quantities by the liver, further promoting hepatic triglyceride synthesis, leading to a decrease in high-density lipoprotein cholesterol (HDL-C) and an increase in low-density lipoprotein cholesterol (LDL-C), ultimately causing dyslipidemia. Based on the close causal relationship between oxidative stress and glucose and lipid metabolism disorders, selenium-enriched spirulina carbon dots can not only remove exogenous oxidative substances, but also enhance the endogenous antioxidant defense capacity of nematodes, thereby effectively regulating glucose and lipid metabolism. Attached Figure Description

[0016] Figure 1 The images show the infrared spectrum and zeta potential of carbon dots from selenium-enriched Spirulina. (A) is the infrared spectrum, and (B) is the zeta potential.

[0017] Figure 2 The image shows the fluorescence emission spectrum of carbon dots from selenium-enriched Spirulina. (A) represents SeSP1, (B) represents SeSP2, and (C) represents SeSP3.

[0018] Figure 3 The images show transmission electron microscopy (TEM) images and relative frequency distribution diagrams of carbon dots from selenium-enriched Spirulina. (A) is the TEM image, and (B) is the relative frequency distribution diagram.

[0019] Figure 4Photographs of selenium-enriched spirulina carbon dot solutions under different lighting conditions.

[0020] Figure 5 This image shows the in vitro antioxidant activity of selenium-enriched spirulina carbon dots, spirulina carbon dots, and selenium-enriched spirulina powder. (A) represents ·ABTS. + Sweep rate, (B) is O2 - Scavenging rate, (C) is the H2O2 scavenging rate, (D) is the ·OH scavenging rate.

[0021] Figure 6 The diagram shows the effects of selenium-enriched spirulina carbon dots on lowering blood sugar and lipids. In the diagram, (A) represents the inhibition rate of α-glucosidase, (B) the inhibition rate of α-amylase, (C) the inhibition rate of cholesterol esterase, (D) the inhibition rate of pancreatic lipase, (E) the binding rate of sodium taurocholate, and (F) the binding rate of sodium glycocholate.

[0022] Figure 7 Survival curves of *C. elegans* N2 treated with selenium-enriched *Spirulina* carbon dots and *C. elegans* N2 cultured in high sugar. (A) represents *C. elegans* N2, and (B) represents *C. elegans* N2 cultured in high sugar.

[0023] Figure 8 The figure shows the effect of selenium-enriched Spirulina carbon dots on the oxidative stress level of *C. elegans* N2 cultured under high sugar conditions. In the figure, (A) represents the SOD, GSH-Px, CAT, and MDA activities of *C. elegans* N2 cultured under high sugar conditions, and (B) represents the SOD, GSH-Px, CAT, and MDA activities of *C. elegans* N2 cultured under high sugar conditions.

[0024] Figure 9 The glucose and triglyceride contents of *C. elegans* N2 culturated with selenium-enriched *Spirulina pulvinata* carbon dots, *Spirulina pulvinata* carbon dots, and selenium-enriched *Spirulina pulvinata* powder were measured. (A) represents glucose content, and (B) represents triglyceride content.

[0025] In the figure above, SeSP1 represents the selenium-enriched spirulina carbon dots prepared in Example 2, SeSP2 represents the selenium-enriched spirulina carbon dots prepared in Example 3, SeSP3 represents the selenium-enriched spirulina carbon dots prepared in Example 4, SeSP represents the selenium-enriched spirulina carbon powder prepared in Example 1, SP1 represents the spirulina carbon dots prepared in Example 5, VC represents ascorbic acid, and Control represents the blank control. Detailed Implementation

[0026] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0027] Example 1 This embodiment provides a method for preparing selenium-enriched spirulina powder.

[0028] Spirulina platensis was inoculated into Zarrouk medium for selenium enrichment treatment, specifically, 1 mL of a 1.6 mg / mL solution was added on days 1, 3, and 5 of the Spirulina platensis culture. -1 The *Spirulina platensis* was cultured in a Na₂SeO₃ solution at 25 °C in an artificial climate chamber according to a 12-hour dark / 12-hour day cycle until the logarithmic phase, yielding selenium-enriched *Spirulina platensis*. The selenium-enriched *Spirulina platensis* was collected by centrifugation, freeze-dried, and then yielded selenium-enriched *Spirulina platensis* powder (SeSP).

[0029] Example 2 This embodiment provides a method for preparing selenium-enriched spirulina carbon dots.

[0030] 1 g of selenium-enriched spirulina powder and 0.4 g of NaOH were uniformly dispersed in 20 mL of deionized water, then transferred to a high-temperature reactor and reacted at 230 °C for 180 min. After cooling to room temperature, the dialysis medium was concentrated by rotary evaporation and freeze-dried to obtain selenium-enriched spirulina carbon dots (SeSP1) with a yield of 75.4%.

[0031] Example 3 This embodiment provides a method for preparing selenium-enriched spirulina carbon dots.

[0032] 1 g of selenium-enriched spirulina powder and 0.8 g of CH3COOH were uniformly dispersed in 20 mL of deionized water, then transferred to a high-temperature reactor and reacted at 190 °C for 120 min. After cooling to room temperature, the dialysis medium was concentrated by rotary evaporation and freeze-dried to obtain selenium-enriched spirulina carbon dots (SeSP2) with a yield of 91.2%.

[0033] Example 4 This embodiment provides a method for preparing selenium-enriched spirulina carbon dots.

[0034] 1 g of selenium-enriched spirulina powder and 0.2 g of H2SO4 were uniformly dispersed in 20 mL of deionized water, then transferred to a high-temperature reactor and reacted at 160 °C for 100 min. After cooling to room temperature, the dialysis medium was concentrated by rotary evaporation and freeze-dried to obtain selenium-enriched spirulina carbon dots (SeSP3) with a yield of 85.7%.

[0035] Example 5 This embodiment provides a method for preparing spirulina carbon dots.

[0036] 1 g of spirulina powder and 0.4 g of NaOH were uniformly dispersed in 20 mL of deionized water, then transferred to a high-temperature reactor and reacted at 230 °C for 180 min. After cooling to room temperature, the dialysis medium was concentrated by rotary evaporation and freeze-dried to obtain spirulina carbon dots (SP1).

[0037] SeSP1, SeSP2, SeSP3, SeSP and Spirulina powder were compressed into tablets using the KBr method and then measured at 4000~450 cm⁻¹. -1 The infrared spectrum. (From) Figure 1 As can be seen from A in the figure, selenium-enriched spirulina grows at 540 cm. -1 The characteristic C-Se peak appeared. Chlorophyll a, as a major component of spirulina, exhibits infrared absorption peaks at 1458, 1547, 1650, 1733, 2854-2929, and 2067 cm⁻¹ for the -CN stretching vibration, -C=N, -C=C-, ester-C=O, saturated -CH stretching vibration, and benzene ring-CH, respectively. -1 In contrast, in the infrared spectrum of selenium-enriched spirulina carbon dots, 540 cm⁻¹ -1 The characteristic peaks of -C-Se are still retained. Furthermore, since most of the selenium in the hydrothermally synthesized carbon dots exists in a high-valence oxidation state, the -Se=O peak at 882 cm⁻¹ is also present. -1 The characteristic peaks at 1031, 1403-1451, and 2861-2971 cm⁻¹ are clearly visible. -1 The -CO, -CN, and -CH stretching vibrations appeared successively. More importantly, the characteristic peaks of -C=C- and the anhydride -C=O formed by the high-temperature dehydration of the carboxyl gene in selenium-enriched spirulina appeared at 1650 and 1733 cm⁻¹. -1 This demonstrates that the present invention successfully prepared selenium-enriched spirulina carbon dots. Figure 1 As shown in (B), the SeSP1, SeSP2 and SeSP3 prepared by this invention are all negatively charged, with Zeta potentials of -13.40±1.47 mV, -13.20±1.34 mV and -9.88±1.02 mV, respectively.

[0038] SeSP1, SeSP2, and SeSP3 were dissolved in deionized water to achieve a final concentration of 1 mg·mL. -1 The fluorescence emission spectra of carbon dots at excitation wavelengths of 300–400 nm were collected using a fluorescence spectrophotometer. Figure 2It is known that SeSP1, SeSP2 and SeSP3 all have wavelength-dependent excitation characteristics, further confirming the successful synthesis of selenium-enriched Spirulina carbon dots.

[0039] Dissolve SeSP1 in deionized water to a final concentration of 1 mg·mL⁻¹. -1 The microstructure was observed using transmission electron microscopy, and the particle size of the carbon dots was quantitatively analyzed using ImageJ. SeSP1 has a spherical structure ( Figure 3 (A) has a particle size of 5.71 ± 0.32 nm. Figure 3 (B) in the middle.

[0040] Deionized water and 1 mg·mL⁻¹ were tested under room temperature sunlight and 365 nm ultraviolet light, respectively. -1 Photograph of SeSP1 aqueous solution. (From...) Figure 4 As can be seen, SeSP1 is a transparent solution at room temperature, but exhibits blue fluorescence after being excited by 365 nm ultraviolet light.

[0041] Example 6 This embodiment demonstrates the in vitro antioxidant capacity of selenium-enriched spirulina carbon dots.

[0042] This invention uses 1.0 mg·mL -1 ABTS of selenium-enriched spirulina carbon dots, selenium-enriched spirulina, and spirulina carbon dots + O2 - The scavenging experiments of H2O2 and OH were conducted, with ascorbic acid as a positive control.

[0043] Depend on Figure 5 From (A), we can see that SeSP1, SeSP2, and SeSP3 affect ABTS. + The scavenging rates exceeded 67.33%, 84.79%, and 91.15%, respectively, demonstrating extremely high free radical scavenging capabilities. Whether in vitro or in vivo, the function of antioxidant enzymes or the scavenging of free radicals begins with O2. - The decomposition of SeSP1, SeSP2, and SeSP3 for O2. - The clearance rate showed a concentration-dependent increase, ranging from 35% to 52%. SeSP1, SeSP2, and SeSP3 all showed the highest clearance rates of 37.72% ± 3.46%, 49.89% ± 2.54%, and 52.77% ± 0.80%, respectively. Figure 5 (B) in the middle). O2 - The decomposition will form H2O2 and O2, by Figure 5As shown in (C), the scavenging ability of SeSP1, SeSP2, and SeSP3 for H2O2 is comparable to that of the positive control ascorbic acid, with a scavenging rate exceeding 90%, confirming that they can decompose H2O2 into H2O and O2. This indicates that the carbon dots of selenium-enriched spirulina can effectively interrupt the intermediate steps of free radical chain reactions and prevent their conversion into OH. SeSP1, SeSP2, and SeSP3 also showed a scavenging rate of approximately 40% for OH. Figure 5 (D) indicates that selenium-enriched spirulina carbon dots possess a strong ability to combat ultimate oxidative damage. Compared with SeSP1, SeSP2, and SeSP3, spirulina carbon dots (SP1) and selenium-enriched spirulina powder (SeSP) not only have a higher scavenging rate for H2O2, but also exhibit stronger resistance to ABTS. + O2 - The OH scavenging rate of the composite material is much lower than that of selenium-enriched spirulina carbon dots. This is because the high-temperature environment of the hydrothermal method can decompose and reduce organic selenium (such as selenoprotein) in selenium-enriched spirulina and reconstruct it into nano-element selenium. The antioxidant capacity of nano-element selenium far exceeds that of organic selenium. In addition, the carbon skeleton of selenium-enriched spirulina carbon dots is itself an antioxidant material. When nano-selenium is incorporated into it, the two can produce a synergistic effect in electron transfer and other aspects, making the antioxidant performance of the composite material stronger than that of a single component. The carbon dots transform the raw material into nanoparticles of about 5.71 nm, greatly increasing the density of active sites, which can more fully and quickly contact and scavenge free radicals.

[0044] Example 7 This embodiment provides the detection results of the hypoglycemic and lipid-lowering activity of selenium-enriched spirulina carbon dots.

[0045] (1) Assay for α-glucosidase inhibition effect: Add 0.4 mL of α-glucosidase solution (0.5 U / mL) and 0.05 mL of 1 mg·mL to a 5 mL centrifuge tube. -1 Selenium-enriched spirulina carbon dots were incubated at 37 ℃ for 15 min; 0.4 mL of 5 mM PNPG solution was added, and the mixture was incubated at 37 ℃ for 30 min, followed by termination of the reaction with 1 mL of 1 M Na2CO3 solution. Acarbose was used as a control, and each group was replicated three times. 200 μL of the reaction solution was transferred to a 96-well plate, and the absorbance was measured at 405 nm using a microplate reader to calculate the inhibition rate.

[0046] (2) Determination of α-amylase inhibition rate: 0.1 mL of α-amylase solution (2 U / mL) and 0.2 mL of 1 mg·mL⁻¹ were added sequentially to a 5 mL centrifuge tube. -1Selenium-enriched spirulina carbon dots were incubated at 37 ℃ for 15 min; then 0.2 mL of 1% starch solution was added, and the mixture was incubated at 37 ℃ for 20 min. Finally, 0.5 mL of DNS solution was added and the mixture was boiled for 15 min to terminate the reaction. Acarbose was used as a control, and each group was replicated three times. After cooling, 200 μL of the solution was transferred to a 96-well plate, and the absorbance was measured at 540 nm using a microplate reader to calculate the inhibition rate.

[0047] (3) Determination of cholesterol esterase inhibition effect: Prepare acetonitrile solution of PNPB (4 mM), store at -20 ℃ and use immediately. Prepare PB buffer (containing sodium taurocholate to a final concentration of 5.16 mM and NaOH to a final concentration of 0.1 M) and adjust the pH to 7 for later use. Add 0.05 mL of cholesterol esterase enzyme solution (20 μg·mL⁻¹) to a 5 mL centrifuge tube. -1 ) and 0.05 mL 1 mg·mL -1 Selenium-enriched spirulina carbon dots and 1 mL of PB solution were incubated at 25 °C for 10 min, followed by the addition of 0.02 mL of 4 mM PNPB solution and incubation at 25 °C for 30 min. Orlistat was used as a control, and each group was repeated three times. After the experiment was completed, the absorbance of each well was recorded at 405 nm to calculate the inhibition rate.

[0048] (4) Determination of pancreatic lipase inhibition rate: Prepare a 2% polyvinyl alcohol solution, heat to dissolve, and bring the volume to 100 mL. Mix polyvinyl alcohol and olive oil in a 3:1 ratio and vortex for 10 min to prepare an emulsion. Add 2 mL of PBS, the same volume of emulsion, and 1 mL of 1 mg·mL⁻¹ solution to a 10 mL test tube. -1 Selenium-enriched spirulina carbon dots were incubated at 37℃ for 10 min, and 0.5 mL of a 2 mg·mL⁻¹ concentration was added to the test tube. -1 The enzyme solution was incubated for another 15 minutes. To terminate the reaction, 5 mL of 95% ethanol was added to each tube. For the control group, the reaction was stopped after adding ethanol, and then pancreatic lipase was added. The terminated liquid was poured into an Erlenmeyer flask, a few drops of phenolphthalein were added, and titration was performed using sodium hydroxide (0.025 mol / L). Titration was stopped when the liquid just turned slightly pink. Orlistat was used as a control, and each group was repeated three times. The volumes used were recorded and calculated.

[0049] (5) Investigation of bile salt binding effect in simulated in vivo environment: First, a standard curve of bile salt was plotted. Bile salt was dissolved in PBS solution. 1 mL of sodium glycocholate solution and sodium taurocholate solution of different concentrations (concentrations of 0.04, 0.08, 0.16, 0.24, 0.32, 0.40 mM) prepared with standard products and 2 mL of sulfuric acid (60%) were added to the test tubes. The mixture was heated at 60 °C for 30 min, and the test tubes were placed in ice water to cool for 10 min. 200 μL of each test tube was aspirated into a 96-well plate, and the absorbance at 387 nm was measured and recorded. The standard curve was obtained by plotting the absorbance and concentration. The binding process of bile salt in the gastrointestinal environment was simulated in vitro. Pepsin solution was prepared with PBS at pH 6.3. Selenium-enriched spirulina carbon dots (1 mg·mL⁻¹) were added to the test tubes. -1 ) and 3 mL of pepsin (10 mg·mL) -1 Add 1 mL of hydrochloric acid (0.01 M) and mix well in a shaker at 37°C for 1 h to simulate the digestive process in a gastric environment. Then add 4 mL of trypsin (10 mg / mL) to the test tube. -1 The pH was adjusted to 6.3 to simulate the digestive process in an intestinal environment, and the shaking process described above was repeated. 4 mL of sodium taurocholate solution (0.5 mM) was added to one test tube, and the shaking process was repeated. 4 mL of sodium glycocholate solution (0.4 mM) was added to another set of test tubes, and the shaking process was repeated. Each set was repeated three times. The reacted liquid was centrifuged at 4000 rpm for 15 min. 1 mL of the supernatant from each centrifuge tube was added to 2 mL of 60% sulfuric acid and heated at 60 °C for 30 min. The test tubes were then cooled in ice water for 10 min. 200 μL was aspirated from each test tube and the absorbance at 387 nm was measured and recorded in a 96-well plate. The binding rates of sodium taurocholate and sodium glycocholate were calculated using cholestyramine as a control.

[0050] Depend on Figure 6 As shown in (A), the inhibition rate of α-glucosidase by selenium-enriched spirulina carbon dots exceeded that of the positive control acarbose, reaching 66.67%–87.06%. This significantly slowed down the rate of glucose production and absorption, indicating that selenium-enriched spirulina carbon dots can effectively lower blood sugar. The inhibition rates of α-amylase by SeSP1, SeSP2, and SeSP3 were 46.67%–72.38%, indicating that selenium-enriched spirulina carbon dots can effectively prevent the initial breakdown of starch and reduce the total amount of substrate required for α-glucosidase action. Figure 6 In addition, the inhibition rates of SeSP1, SeSP2, and SeSP3 on cholesterol esterase and pancreatic lipase were also close to those of the control group orlistat, reaching as high as 42.74%~65.41% and 44.33%~51.00%, respectively. Figure 6(C) and (D) in the text indicate that selenium-enriched spirulina carbon dots not only prevent most dietary fats from being effectively broken down and ultimately excreted in feces, but also reduce the absorption of cholesterol in the intestines. The binding rates of selenium-enriched spirulina carbon dots to sodium taurocholate and sodium glycocholate are close to or even exceed those of the positive control cholestyramine, reaching 42.68%~59.84% and 58.54%~77.82% respectively. Figure 6 (E) and (F) in the figure indicate that selenium-enriched spirulina carbon dots can exert a cholesterol-lowering effect through the classic pathway of "binding and excreting bile acids, activating liver cholesterol metabolism, and ultimately promoting blood cholesterol clearance".

[0051] Example 8 This embodiment provides the biosafety evaluation results of selenium-enriched spirulina carbon dots.

[0052] L4 stage N2 type Caenorhabditis elegans was transferred to a solution containing 1 mg·mL⁻¹ -1 Add 1 mL of PBS buffer and 10 μL of 5-fluorouracil (1.25 mg / mL) to each well of a 24-well plate containing SeSP1 and SeSP2. -1 The plates were then rotated every two days, and the number of dead nematodes was recorded. Survival curves were plotted, and the mean, median, and longest lifespan were calculated. N2 type Caenorhabditis elegans without any sample was used as a blank control group.

[0053] Depend on Figure 7 As shown in (A), SeSP1 and SeSP2 significantly prolonged the lifespan of *C. elegans* N2. The mean, median, and longest lifespan of *C. elegans* N2 in the SeSP1 group were 12.56±0.28, 10.25±0.35, and 26.00±1.41 days, respectively, which were extended by 12.14%, 10.81%, and 8.33% compared with the blank control group (Table 1). SeSP2 also had a similar effect ( Figure 7 In (A) of the group, the mean, median and longest lifespan of the N2 type Caenorhabditis elegans were extended by 7.68%, 10.81% and 6.25% respectively compared with the control group (Table 1), which preliminarily indicates that SeSP1 and SeSP2 have good biocompatibility.

[0054] Table 1. Lifespan statistics of selenium-enriched Spirulina carbon dots after feeding N2 type Caenorhabditis elegans (high-sugar diet) with Caenorhabditis elegans carbon dots.

[0055] Example 9 This embodiment provides the effects of selenium-enriched Spirulina carbon dots on the lifespan, oxidative stress, and energy metabolism of Caenorhabditis elegans on a high-sugar diet.

[0056] (1) Lifespan: N2 type Caenorhabditis elegans was cultured on NGM plates containing OP50 and supplemented with 40 mM glucose for 3 days to simulate a high-glucose diet. Then it was transferred to carbon dots without selenium-enriched Spirulina and those containing 1 mg·mL⁻¹. -1 SeSP1 and SeSP2 were cultured in 24-well plates for 48 h. The survival rate was determined and lifetime analysis was performed.

[0057] like Figure 7 As shown in (B), the life-extending effects of SeSP1 and SeSP2 were also effective in high-glucose cultured *C. elegans* N2. SeSP1 extended the mean, median, and longest lifespan of *C. elegans* N2 cultured in high-glucose culture by 46.56%, 59.46%, and 51.61%, respectively, compared to the control group. The effect of SeSP2 was similar to that of SeSP1, extending the mean, median, and longest lifespan of *C. elegans* N2 cultured in high-glucose culture by 40.49%, 54.05%, and 51.61%, respectively, compared to the control group. These results indicate that selenium-enriched *Spirulina* carbon dots not only have good biocompatibility but also extend the lifespan of *C. elegans* N2 cultured in high-glucose culture.

[0058] (2) Oxidative stress: Synchronized L1-stage N2 type Caenorhabditis elegans were subjected to carbon doping (1 mg·mL⁻¹) for 10 days under normal culture and high glucose culture. -1 After exposure, N2 type Caenorhabditis elegans were collected, and the activities of SOD, CAT, GSH-Px and MDA content were measured.

[0059] Depend on Figure 8 It was found that SeSP1 and SeSP2 significantly enhanced the antioxidant enzyme activity and reduced the level of malondialdehyde (MDA), a lipid peroxidation product, in both normal and high-glucose cultures of *C. elegans*. These results combined indicate that selenium-enriched *Spirulina* carbon dots can not only scavenge exogenous oxidizing substances, such as... ABTS + , O2 - H2O2 OH can also enhance the endogenous antioxidant defense of N2-type Caenorhabditis elegans, and these two pathways lay the foundation for the efficient regulation of glucose and lipid metabolism.

[0060] (3) Energy metabolism: N2 type Caenorhabditis elegans was cultured on NGM plates containing OP50 and exogenously supplemented with 40 mM glucose and 1 mg·mL⁻¹. -1 SeSP1, SeSP2, SP1, and SeSP were subjected to a simulated high-sugar diet for 3 days. The glucose and triglyceride contents of the nematodes were measured for evaluation.

[0061] like Figure 9As shown, SeSP1, SeSP2, SP1, and SeSP all significantly reduced glucose and triglyceride levels in *C. elegans* N2 cultured in high glucose. Specifically, SeSP1 and SeSP2 reduced glucose levels in *C. elegans* N2 cultured in high glucose by 46.95% and 38.84%, respectively, while SP1 and SeSP only reduced them by 21.98% and 22.52%, respectively. Figure 9 (A)). Compared with the 75.39% and 70.54% reduction in triglycerides achieved by SeSP1 and SeSP2, the triglyceride reductions after SP1 and SeSP treatment were 46.23% and 38.04%, respectively. Figure 9 (B)). The above results indicate that selenium-enriched spirulina carbon dots have a significantly better effect on regulating glycolipid metabolism than spirulina carbon dots and selenium-enriched spirulina powder.

[0062] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A method for preparing selenium-enriched spirulina carbon dots, characterized in that, include: After mixing selenium-enriched spirulina, water, and a catalyst, a hydrothermal reaction was carried out, and the mixture was naturally cooled to room temperature to obtain a reaction solution. The reaction solution was dialyzed to obtain a dialysis medium. The dialysis medium was concentrated and freeze-dried to obtain selenium-enriched spirulina carbon dots. The catalyst is selected from any one of NaOH, CH3COOH, and H2SO4.

2. The method for preparing selenium-enriched spirulina carbon dots according to claim 1, characterized in that, The preparation of the selenium-enriched spirulina is as follows: an inorganic selenium compound is added to the spirulina culture system; the spirulina with the added inorganic selenium compound is cultured under intermittent light until it grows to the logarithmic growth phase, thus obtaining the selenium-enriched spirulina.

3. The method for preparing selenium-enriched spirulina carbon dots according to claim 2, characterized in that, The inorganic selenium compound is Na2SeO3; The temperature for the intermittent light culture was 25 °C.

4. The method for preparing selenium-enriched spirulina carbon dots according to claim 1, characterized in that, The mass ratio of the selenium-enriched spirulina, water, and catalyst is 1:0.1~0.8:

20.

5. The method for preparing selenium-enriched spirulina carbon dots according to claim 1, characterized in that, The hydrothermal reaction temperature is 140~260 ℃, and the time is 1.5~5.5 h; The dialysis time was 24 hours.

6. A type of selenium-enriched spirulina carbon dots, characterized in that, It is prepared by the method for preparing selenium-enriched spirulina carbon dots according to any one of claims 1 to 5.

7. The application of the selenium-enriched spirulina carbon dots as described in claim 6 in the preparation of drugs that regulate glucose and lipid metabolism balance.

8. The application of the selenium-enriched spirulina carbon dots as described in claim 6 in the preparation of anti-aging and longevity-promoting drugs.

9. The use of the selenium-enriched spirulina carbon dots as described in claim 6 in the preparation of drugs for anti-oxidative stress.

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