Diatom shell and fucoxanthin oral drug delivery system as well as preparation method and application thereof

Abstract

The invention discloses a diatom shell and fucoxanthin oral drug delivery system and a preparation method and application thereof, and the diatom shell and fucoxanthin oral drug delivery system (Frustule and FX) is obtained by compounding 400 [mu] g / ml of fucoxanthin solution and 50 [mu] g / ml of diatom shell suspension according to the volume ratio of 1: 10. Research results show that the FX for improving D-gal induced nerve aging relates to oxidation resistance and autophagy activation, and the FX has a remarkable effect in the aspects of aging improvement and nerve protection by activating an Nrf2-autophagy pathway; 2-5 [mu] g / ml of FX can significantly improve the survival rate of D-gal induced PC12 senescence cells and effectively inhibit the expression of a cell senescence marker SA-beta-gal, and shows good anti-aging potential; the FX with the concentration of 5 mu g / mL can obviously relieve oxidative stress induced by D-gal, so that oxidative damage of cells is relieved. The invention deeply discusses the improvement of the anti-aging and neuroprotective effects of FX on D-gal induced aging mice by the Frustule-coated FX, and the Nrf2-autophagy pathway still has an important effect on the improvement of aging and neuroprotection by the Frustule-coated FX.

Description

Technical Field

[0001] The present invention relates to a diatom frustule@fucoxanthin oral drug delivery system (Frustule@FX) and its preparation method and application. Fucoxanthin has a significant improvement and intervention effect on D-galactose (D-gal)-induced neurosenescence, belonging to the technical field of drug research and development. Background Art

[0002] Fucoxanthin (FX) is a xanthophyll carotenoid, mainly found in large brown algae and microalgae. FX contains carbon-carbon double bonds, epoxy groups and conjugated carbonyl groups. Due to the special allyl functional group contained in its molecular structure, it is believed to be one of the reasons for its anti-cancer, anti-diabetic, anti-obesity, anti-inflammatory, anti-angiogenic, anti-malarial properties and multiple protective activities; in addition, active compounds acting on the central nervous system must cross the blood-brain barrier to impart their therapeutic effects, making the permeability of this barrier a key factor in the development of drugs targeting this area. FX can pass through the blood-brain barrier and can be used to study various neurological diseases.

[0003] With the increasing global aging trend, aging and related neurological disorders will impose a significant economic and medical burden on human society. Studies have shown that aging can adversely affect the brain, leading to cognitive decline and an increased risk of neurodegenerative diseases. This is because brain tissue is particularly susceptible to the harmful effects of oxidative stress, which leads to the continuous production of large amounts of ROS. Nrf2 can regulate oxidative stress and exert anti-aging effects. Furthermore, autophagy plays a crucial role in eliminating proteins and organelles damaged by oxidative stress and aging. Existing research demonstrates that FX has safe and antioxidant properties, can cross the blood-brain barrier, and exerts neuroprotective effects, possessing high nutritional and pharmacological research value. Therefore, developing FX into a drug for the prevention or treatment of neurodegenerative diseases would have significant social value and application prospects.

[0004] However, FX contains α,β-conjugated carbonyl functional groups, making it structurally unstable under external conditions such as light, oxygen, high temperature, and low pH. FX has low solubility in water and degrades into fucoxanthinol when consumed directly. Consequently, its low bioavailability limits its clinical application. Improving FX's bioavailability and effectively integrating FX with drug delivery vehicles remain challenging research challenges in this field.

[0005] Diatom frustules are a rich, natural, and renewable biological resource. Their porous honeycomb structure can enhance drug adhesion in the intestine, prolong drug release, and improve the bioavailability of FX. Furthermore, delivery systems play a crucial role in oral administration by increasing drug bioavailability, achieving targeted and sustained release, and thereby enhancing drug efficacy and patient compliance. Compared to injection and gavage, oral administration (especially combined with a delivery system) is more suitable for patients on long-term medications and has greater clinical application value.

[0006] Based on this, the present invention provides new ideas and methods for the future development and clinical application of anti-aging drugs by in-depth research on the anti-aging mechanism of FX and the application of diatom shells as drug carriers. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to address the deficiencies in the existing technology and provide a diatom shell @ fucoxanthin oral drug delivery system (Frustule@FX) and its preparation method and application. Fucoxanthin (FX) can improve D-gal-induced neurosenescence involving antioxidant and autophagy activation. After diatom shells are loaded with fucoxanthin, they can enhance the anti-aging and neuroprotective effects of FX on D-gal-induced aging mice.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] One of the objectives of the present invention is to provide a diatom frustule@fucoxanthin oral delivery system (Frustule@FX), which is obtained by compounding a fucoxanthin (FX) solution and a diatom frustule (Frustule) suspension, wherein the concentration of the fucoxanthin solution is 25 to 1600 μg / ml, and the concentration of the diatom frustule suspension is 50 μg / ml.

[0010] In the above technical solution, the composite volume ratio of the fucoxanthin solution and the diatom frustule suspension is 1:10.

[0011] In the above technical solution, the fucoxanthin solution is obtained by dissolving fucoxanthin in olive oil; and the diatom frustule suspension is obtained by placing diatom frustules in deionized water and stirring.

[0012] In the above technical solution, the concentration of the fucoxanthin solution is preferably 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 400 μg / ml, 800 μg / ml and 1600 μg / ml; the concentration of the fucoxanthin solution is further preferably 400 μg / ml.

[0013] A second object of the present invention is to provide a method for preparing the diatom frustule@fucoxanthin oral drug delivery system, comprising the following steps: adding a fucoxanthin solution to a diatom frustule suspension for mixing, stirring the mixture for 12 hours, and then centrifuging at 3260g for 10 minutes. After the centrifugation, the solid precipitate is collected, and the solid precipitate is vacuum-freeze-dried at -20°C for 24 hours to obtain the diatom frustule@fucoxanthin oral drug delivery system (Frustule@FX).

[0014] The third object of the present invention is to provide a use of the diatom frustules@fucoxanthin oral drug delivery system in the preparation of drugs for treating and preventing neuroaging diseases.

[0015] In the above technical solution, the diatom frustules@fucoxanthin oral drug delivery system is orally administered, and the oral dosage is 200 mg / kg / d based on the mass of fucoxanthin.

[0016] A fourth object of the present invention is to provide a use of fucoxanthin in the preparation of drugs for intervening in neuroaging diseases, wherein the fucoxanthin solution obtained by dissolving the fucoxanthin in olive oil has a concentration of 25μg / ml, 50μg / ml, 100μg / ml, 200μg / ml, 400μg / ml, 800μg / ml and 1600μg / ml; the fucoxanthin solution is for oral administration in dosages of 50mg / kg / d, 100mg / kg / d and 200mg / kg / d.

[0017] In the above technical solution, the concentration of the fucoxanthin solution is 400 μg / ml; the fucoxanthin solution is for oral administration, and the oral dosage is 200 mg / kg / d based on the mass of fucoxanthin.

[0018] Compared with the existing technology, it has the following characteristics:

[0019] (1) This study investigated the anti-aging and neuroprotective effects of FX on D-gal-induced aging mice at both the cellular and mouse levels, and revealed its potential mechanism of action. The results showed that FX improved D-gal-induced neurosenescence, involving antioxidant and autophagy activation, and that FX had significant effects on improving aging and neuroprotection by activating the Nrf2-autophagy pathway. FX at a concentration of 2-5 μg / ml significantly increased the survival rate of D-gal-induced aging PC12 cells and effectively inhibited the expression of SA-β-gal, a cell senescence marker, demonstrating good anti-aging potential. FX at a concentration of 5 μg / mL significantly reduced D-gal-induced oxidative stress, thereby alleviating cellular oxidative damage.

[0020] (2) The present invention deeply explores the anti-aging and neuroprotective effects of Frustule@FX on D-gal-induced aging mice, and the Nrf2-autophagy pathway still plays an important role in Frustule@FX improving aging and neuroprotection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 To verify the effects of different concentrations of D-gal on PC12 cells in Example 1, Figure 1 a: The effect of D-gal at different concentrations and treatment times on the survival rate of PC12 cells was analyzed by CCK8; Figure 1 b shows the effect of different concentrations of D-gal on the SA-β-gal positive rate of PC12 cells after 24 h of treatment; Figure 1 c shows the effect of different concentrations of D-gal on SA-β-gal-labeled blue-stained senescent cells in PC12 cells after 24 h of treatment; **p<0.01, with the Control group as the control

[0022] Figure 2 To verify the effects of different concentrations of FX on PC12 cells in Example 1, Figure 2 a: The effects of different concentrations of FX on the survival rate of PC12 cells were analyzed by CCK8; Figure 2 b shows the effects of different concentrations of FX on the survival rate of senescent PC12 cells; Figure 2 c is the effect of FX on the SA-β-gal positive rate of PC12 senescent cells induced by D-gal; Figure 2 d shows the effects of different concentrations of FX on D-gal-induced senescence of PC12 cells. *p<0.05, **p<0.01, compared with the control group; #p<0.05, ##p<0.01, compared with the D-gal alone treatment group;

[0023] Figure 3 To verify the effects of different concentrations of FX on D-gal-induced oxidative stress in PC12 senescent cells in Example 1, Figure 3 a shows the effects of different concentrations of FX on ROS levels in aging PC12 cells; Figure 3 b shows the effects of different concentrations of FX on the MDA levels of senescent PC12 cells; Figure 3 c is the improvement of GCLM protein level in senescent PC12 cells by Western Blot analysis of different concentrations of FX, **p<0.01, compared with the control group; #p<0.05, ##p<0.01, compared with the D-gal alone treatment group;

[0024] Figure 4To verify the effect of FX on D-gal-induced autophagy of PC12 cells in Example 1, wherein Figure 4 a is the effect of FX on D-gal-induced autophagosome of PC12 cells observed by TEM; Figure 4 b is the effect of FX on the expression of autophagy-related proteins in aged PC12 cells analyzed by Western Blot, **p<0.01, compared with the Control group; #p<0.05, ##p<0.01, compared with the D-gal alone treatment group, and the cell autophagosome is indicated by a black arrow;

[0025] Figure 5 To verify the effect of FX on the expression of nuclear Nrf2 protein in D-gal-induced PC12 cells in Example 1 analyzed by Western Blot, **p<0.01, compared with the Control group; #p<0.05, ##p<0.01, compared with the D-gal alone treatment group;

[0026] Figure 6 To verify the effect of FX on the expression of Nrf2 gene in D-gal-induced PC12 cells in Example 1, wherein Figure 6 a is the expression of Nrf2 gene in PC12 cells inhibited by siRNA interference technology analyzed by Western Blot; Figure 6 b is the effect of FX on D-gal-induced PC12 siRNA-Nrf2 aging cells (Nrf2 low expression cells) survival rate analyzed by CCK8; Figure 6 c is the effect of FX on improving SA-β-gal positive rate of D-gal-induced PC12 siRNA-Nrf2 aging cells; Figure 6 d is the effect of FX on improving SA-β-galactosidase activity of D-gal-induced PC12 siRNA-Nrf2 aging cells; **p<0.01, compared with the Control group or NC, ##p<0.01, compared with the D-gal alone treatment group, &&p<0.01, compared with the Control (Nrf2- / -) alone treatment group;

[0027] Figure 7 To verify the effect of FX on D-gal-induced PC12 siRNA-Nrf2 autophagy in Example 1, wherein: Figure 7 a is the effect of FX on the expression of autophagy-related proteins LC3, p62 and Beclin1 in D-gal-induced PC12 siRNA-Nrf2 cells analyzed by Western Blot, Figure 7 b is the effect of FX on D-gal-induced PC12siRNA-Nrf2 Effect of cell autophagosomes; **p<0.01, compared with the Control group, the cell autophagosomes are pointed out by yellow arrows;

[0028] Figure 8 To verify the effects of FX on the pulling force, hair and suspension ability of aging mice in Example 2, Figure 8 a shows the effect of FX on the forelimb pulling force of aging mice; Figure 8 b shows the effect of FX on the suspension test in aged mice; Figure 8 c shows that FX improves the coat color of aging mice; **p<0.01, compared with the control group; #p<0.05, ##p<0.01, compared with the D-gal alone treatment group;

[0029] Figure 9 To verify the effect of FX on the learning and memory ability of aging mice in Example 2, Figure 9 a is the swimming trajectory of aging mice after FX intervention in the water maze test analyzed by the Viewpoint Zebrabox system; Figure 9 b is the percentage of swimming time spent by mice in each group in the target quadrant; Figure 9 c is the average frequency of mice in each group crossing the target quadrant; Figure 9 d is the escape latency of mice in each group; **p<0.01, compared with the control group; #p<0.05, ##p<0.01, compared with the D-gal alone-treated group;

[0030] Figure 10 To verify the effect of FX on the expression of SA-β-galactosidase in the brain of aging mice induced by D-gal in Example 2;

[0031] Figure 11 To verify the effect of FX on Nissl bodies in the hippocampus of aging mice in Example 2;

[0032] Figure 12 To verify the effect of FX on the expression of autophagy backbone proteins in the hippocampus of aged mice by immunohistochemistry in Example 2;

[0033] Figure 13 To verify the effect of FX on the tension and suspension of D-gal-induced Nfe212-eKO2 mice (Nrf2 gene knockout mice) in Example 2, wherein: Figure 13 a shows the effects of FX on the tensile force of D-gal-induced C57BL / 6J and Nfe212-eKO2 mice; Figure 13b Effects of FX on the D-gal-induced suspension test in C57BL / 6J and Nfe212-eKO2 mice; **p < 0.01, with the Control (Nfe212-eKO2) group as the control;

[0034] Figure 14 To verify the effect of FX on aging Nrf2 in Example 2 - / - Effects on the learning and memory abilities of mice: Figure 14 a) Analysis of Nrf2 after FX intervention in water maze test by Viewpoint Zebrabox system - / - Mouse swimming trajectory diagram; Figure 14 b is Nrf2 in each group - / - the percentage of swimming time mice spent in the target quadrant; Figure 14 c is Nrf2 in each group - / - the average frequency with which mice crossed the target quadrant; Figure 14 d is Nrf2 in each group - / - Escape latency of mice, **p<0.01, compared with the control group; #p<0.05, ##p<0.01, compared with the D-gal alone-treated group;

[0035] Figure 15 To verify the effect of FX on D-gal-induced Nrf2 in Example 2 - / - Effects of autophagy in the hippocampus of aging mice, including: Figure 15 a Western Blot analysis showed that FX could not activate senescent Nrf2 - / - Expression of autophagy-related proteins in mice; Figure 15 b TEM microscopy showed that FX could not improve aging Nrf2 - / - Autophagosomes in neurons of the hippocampus of mice, **p<0.01, the control group is the aging Nrf2 - / - Mouse control.

[0036] Figure 16 To verify the adsorption of fucoxanthin by diatom frustules in Example 3, Figure 16 a is the surface structure of diatom frustules observed from different angles using a scanning electron microscope; Figure 16 b is the before and after pictures of Frustule@FX; Figure 16 c is the change of adsorption rate of FX by diatom frustules;

[0037] Figure 17 To verify the effects of Frustule@FX on the pulling force, hair growth, and suspension ability of aging mice in Example 3, Figure 17 a shows the effects of FX and Frustule@FX on the pulling force of aging mice; Figure 17b shows the results of FX and Frustule@FX improving the suspension test in aging mice; Figure 17 c) Frustule@FX improves coat color changes in aging mice. **p<0.01, compared with the control group; #p<0.05, ##p<0.01, compared with the D-gal treatment group alone; &&p<0.01, compared with the D-gal treatment group alone H-FX group was the control;

[0038] Figure 18 To verify the effect of Frustule@FX on learning and memory in aging mice in Example 3, Figure 18 a is the swimming trajectory of aging mice after Frustule@FX intervention in the water maze test analyzed by the Viewpoint Zebrabox system; Figure 18 b is the escape latency of mice in each group; Figure 18 c) Percentage of swimming time spent in the target quadrant by each group of mice; (d) Frequency of crossing the target quadrant. **p < 0.01, compared with the Control (Frustule@FX) group; #p < 0.05, ##p < 0.01, compared with the D-gal (Frustule@FX) alone-treated group;

[0039] Figure 19 To verify the effect of Frustule@FX on the expression of autophagy-related proteins in the hippocampus of aging mice by immunohistochemistry in Example 3;

[0040] Figure 20 To verify the effect of Frustule@FX on SA-β-galactosidase activity in the brain of aging mice induced by D-gal;

[0041] Figure 21 To verify the effect of Frustule@FX on aging Nrf2 - / - The effects of pulling force and suspension ability on mice, Figure 21 a is the effect of Frustule@FX on aging Nrf2 - / - Effects of pulling force on mice; Figure 21 b Frustule@FX shows the effect of aging Nrf2 - / - Effect on the suspension ability of mice; **p<0.01, compared with the Control (Nrf2- / -) group;

[0042] Figure 22 To verify the effect of Frustule@FX on the learning and memory ability of aged Nrf2 knockout mice in Example 3, Figure 22a is the swimming trajectory of mice in the water maze test analyzed by the Viewpoint Zebrabox system; Figure 22 b is the percentage of swimming time spent by mice in each group in the target quadrant; Figure 22 c is the frequency of crossing the target quadrant; (d) escape latency of mice in each group; **p < 0.01, compared with the Control (Nrf2- / -) group; #p < 0.05, ##p < 0.01, compared with the D-gal (Nrf2- / -) alone treated group;

[0043] Figure 23 To verify the effect of Frustule@FX on D-gal-induced Nrf2 - / - Effects of autophagy in the hippocampus of aging mice, including: Figure 23 a Western Blot analysis shows that Frustule@FX cannot activate senescent Nrf2 - / - Autophagy in mice; Figure 23 b) TEM microscopy showed that Frustule@FX could not improve the aging Nrf2 - / - Neuronal autophagosomes in mice. The control group is aging Nrf2 - / - Mouse control; **p<0.01, the Control group was used as the comparison. DETAILED DESCRIPTION

[0044] The specific implementation of the technical solution of the present invention is described in detail below, but the present invention is not limited to the following description:

[0045] Verification Example 1: Study on the anti-aging effect of fucoxanthin on D-gal-induced PC12 cells

[0046] 1. Materials and instruments:

[0047] 1.1 Materials and Reagents

[0048] FX (purity >98%): Shanghai MacLean Biochemical Technology Co., Ltd. (China); D-gal: Solebo Biotechnology Co., Ltd. (China); PC12 cells: China Type Culture Collection (CTCC, China); horse serum (HBS), fetal bovine serum (FBS), RMPI-1640 medium, and trypsin (Trypsin-EDTA): Gibco Biotechnology Co., Ltd. (USA); dimethyl sulfoxide (DMSO): Sigma Biotechnology Co., Ltd. (USA); β-actin, Histone, Nrf2, GCLM, p62, LC3, and Beclin1, mouse and rabbit antibodies: Cell Signaling Technology Co., Ltd. (USA); cell β-galactosidase staining kit, reactive oxygen species test kit, nuclear and cytoplasmic protein extraction kit, MDA detection kit, protein concentration determination kit, cell lysate, and 5× protein loading buffer: Beyotime Biotechnology Co., Ltd. (China); 10% color gel fast kit, ultrasensitive EC chemiluminescence kit, and universal antibody diluent: Suzhou Xinsaimei Biotechnology Co., Ltd. (China).

[0049] 1.2 Instruments and Equipment

[0050] Microplate reader: Thermo Fisher Scientific, USA; ultrapure water system: Millipore, USA; high-speed refrigerated centrifuge (5430R): Shanghai Eppendorf; electronic constant temperature water bath (HH-501S): Changzhou Jincheng Chunlan Experimental Instrument Factory; chemiluminescence imaging system: Shanghai Qinxiang Scientific Instrument Co., Ltd.; constant temperature metal bath: Thermo Fisher Scientific, USA; ChemiScope Capture+ imaging system: Bio-Rad, USA; fluorescence microscope: NIKON (TI-S), Japan; Beckman Gallios flow cytometer: Beckman Incorporated, USA; transmission electron microscope (TEM): NIKON (TI-S), Japan.

[0051] 2. Experimental methods:

[0052] 2.1. Fucoxanthin preparation and cell culture:

[0053] Complete medium was prepared by thoroughly mixing 10% horse serum, 5% fetal bovine serum, and 85% RMPI-1640 medium. FX was dissolved in DMSO to prepare a 1 mg / mL FX stock solution, which was diluted to the desired concentration (0.5, 1, 2, or 5 μg / mL) using complete medium. FX concentrations were determined based on the CCK8 assay. PC12 cells were cultured aseptically in a cell culture incubator at 37°C in a 5% CO2 atmosphere and 95% air.

[0054] 2.2. Construction of cell aging model:

[0055] Experimental groups with different concentrations of D-gal (concentrations: 0, 10, 20, 30, 40 mg / mL) were set up, PC12 cells were evenly mixed, and 5×10 3 Each well was inoculated with 1640 cells / well in a 96-well cell culture plate. After culturing for 24 hours, the original culture medium in the well was absorbed and discarded. The blank control group was cultured with new serum-containing 1640 culture medium, and the other groups were cultured with a mixture of corresponding concentrations of D-gal and serum-containing 1640 culture medium, and then placed in a cell culture incubator for continued culture for 12 hours, 24 hours, and 48 hours. After that, 20 μL of CCK8 was added to each well and incubated for another 2 hours. The absorbance was measured at 450 nm using a microplate reader. Cell viability = (experimental well - blank well) / (control well - blank well) × 100%.

[0056] Culture cells in a 6-well plate, aspirate the cell culture medium, wash once with PBS, add 1 mL of β-galactosidase staining fixative, and fix at room temperature for 15 minutes. Aspirate the fixative again and wash the cells three times with PBS for 3 minutes each. Aspirate the PBS, add 1 mL of staining solution to each well, and incubate overnight at 37°C in the absence of CO2. Note: If crystals form, washing with 70% ethanol is recommended.

[0057] 2.3 ROS determination

[0058] Evenly plate PC12 cells in a six-well plate. When the cell density reaches 80%-95%, discard the old cell culture medium, rinse with PBS 1-2 times, and discard the PBS. Digest the cells with trypsin, transfer to a 1.5mL EP tube, and mark it. Centrifuge at 1200rpm for 5 minutes at room temperature and discard the supernatant. Prepare 2μL DCFH-DA stock solution: 3ml serum-free medium, mix by inverting (be careful to avoid light). Discard the medium and add 1mL serum-free medium to the blank tube.

[0059] Add 1 mL of the prepared probe solution to the remaining tubes and resuspend the cells (protect from light). Incubate at 37°C in the dark for 20 minutes. Centrifuge at 1200 rpm for 5 minutes at room temperature and discard the supernatant. Wash with 1 mL of cold PBS, centrifuge at 1200 rpm for 5 minutes at room temperature and discard the supernatant. Repeat twice (protect from light). Resuspend the cells in 500 μL of cold PBS, transfer to a flow cytometer, and analyze on the instrument. Set the flow cytometer parameters: select the FL1 or BL1 channel, excite at 488 nm, and measure emission at 530 nm.

[0060] 2.4, MDA detection:

[0061] PC12 cells can be lysed using lysis buffer. Use 0.1 mL of lysis buffer or homogenate per 1 million cells. After lysis, centrifuge at 10,000–12,000 g for 10 minutes, and remove the supernatant for subsequent assays. Lysis and other sample preparation steps should be performed in an ice bath. After cell sample preparation, protein concentration can be determined using a BCA protein concentration assay kit to facilitate calculation of MDA content per unit protein weight in tissue or cells. To a centrifuge tube or other suitable container, add 0.1 mL of lysis buffer as a blank control, 0.1 mL of the aforementioned standards of varying concentrations for standard curve generation, and 0.1 mL of sample for assay. Then, add 0.2 mL of MDA working solution. After mixing, heat at 100°C or in a boiling water bath for 15 minutes. Be careful to avoid splashing during heating. Cool to room temperature in a water bath, and centrifuge at 1,000 g for 10 minutes at room temperature. Transfer 200 μL of supernatant to a 96-well plate and measure absorbance at 532 nm using a microplate reader. For cell samples, after calculating the MDA content in the sample solution, the MDA content in the original sample can be expressed by the protein content per unit weight, such as μmol / mg protein.

[0062] 2.5、Western Blotting:

[0063] Immunoprecipitated and untreated proteins were loaded onto a 10% SDS-PAGE gel for electrophoresis. After electrophoresis, the proteins were electrophoretically transferred to a PVDF membrane. Immunoreactivity was then performed using antibodies against β-actin (mouse anti-mouse), GCLM (rabbit anti-mouse), p62 (rabbit anti-rabbit), LC3 (rabbit anti-rabbit), Beclin1 (rabbit anti-rabbit), Nrf2 (mouse anti-mouse), and histone (rabbit anti-rabbit) diluted 1:1000 in primary antibody diluent. The membrane was immersed in the diluted antibodies overnight. After three washes with TBST (7 minutes each), the membrane was incubated with the corresponding secondary antibody for 1 hour and then washed again with TBST (7 minutes each). Finally, the membrane was incubated in an ultrasensitive ECL chemiluminescent reagent for 2 minutes in the dark. Target protein bands were captured and quantified using the ChemiScopeCapture imaging system and Image J software, using β-actin and histone as references.

[0064] 2.6. siRNA interference:

[0065] The Nrf2 interference fragment synthesized by Shanghai Jima Company was used to interfere with the Nrf2 gene in the cell. The upstream sequence of the interference fragment is (5'to3')S:GCCUUGUACUUUGAAGACUTT;AS:AGUCUUCAAAGUACAAGGCTT. PC12 cells were cultured in 6-well plates. When the cell fusion rate reached 50-60%, OPTI-MEM medium was changed and the transfection reagent was prepared with X-tremeGENE siRNA transfection reagent according to the instructions. After 24 hours of transfection, FX was added and continued to act for 24 hours. Then, the changes in the levels of p62, Beclin1 and LC3 proteins were explored by Western Blot technology.

[0066] CCK8 was used to measure cell viability, and β-galactosidase was used to measure cell senescence.

[0067] 2.7 TEM observation of autophagy

[0068] First, fix PC12 cells with 2.5% glutaraldehyde at 4°C for 2 hours. Rinse with 0.1mol / L phosphate buffer three times, 15 minutes each time. Then, dehydrate with 50%, 70%, and 90% ethanol in sequence, 15 minutes each time. At 4°C, replace ethanol with a mixture of 90% ethanol and 90% acetone (1:1), and then replace with 90% acetone, 15 minutes each. Replace with pure acetone three times at room temperature, 20 hours each time. Subsequently, immerse the sample in a mixture of pure acetone and resin (such as Epon) and place it at room temperature for 3 hours. Then place it in pure resin overnight, and finally place it in a 37°C oven for 2 hours.

[0069] Place in a 45°C oven for 12 hours, and finally place in a 60°C oven for 24 hours. Use an ultrathin slicer to cut the embedded samples into ultrathin sections with a thickness of 50-100 nm. Stain the ultrathin sections with 3% uranyl acetate for 15 minutes, and then rinse with distilled water three times for 5 minutes each. Then stain with lead citrate for 10 minutes, and finally rinse with distilled water three times for 5 minutes each. Place the stained sections on a TEM for observation. Autophagosomes usually appear as double-membrane vesicles that enclose degraded cellular components. By recording the number, morphology, and distribution of autophagosomes, changes in autophagic activity can be evaluated.

[0070] 2.8 Statistical Analysis

[0071] Data were compiled using Microsoft Excel 2019, plotted using Origin 2018, and evaluated using SPSS 22.0. Results are expressed as mean ± standard deviation. All results were analyzed using one-way analysis of variance, Tukey-Kramer post-hoc test, and unpaired t-test. P < 0.05 indicated a significant difference, and P < 0.01 indicated a highly significant difference.

[0072] 3. Results and Analysis

[0073] 3.1. Optimization of conditions for constructing a senescence model for PC12 cells

[0074] In order to establish a PC12 cell aging model, the present invention screened suitable conditions by using D-gal at different concentrations and treatment times. The CCK8 method was used to detect the effects of 0, 10, 20, 30, and 40 mg / mL D-gal on the survival rate of PC12 cells at three time points: 12 h, 24 h, and 48 h. Figure 1 As shown in Figure a, cell viability decreased with increasing D-gal concentration. At the same concentration, cell viability decreased with increasing treatment duration, demonstrating a clear time-dependence. Ultimately, a 24-hour treatment with 30 mg / mL D-gal was determined as the condition for establishing a cell senescence model, achieving a cell viability of 54.9%. This condition was chosen to ensure sufficient viable cells for subsequent studies of the effects of FX on PC12 cells.

[0075] To verify the rationality of this condition, β-galactosidase staining experiments were performed on PC12 cells treated with different concentrations of D-gal for 24 hours. β-galactosidase staining (SA-β-gal) is a commonly used technique for detecting cell senescence. A positive reaction can show blue-stained senescent cells. The results showed that as the D-gal concentration increased, the number of blue-stained cells also increased ( Figure 1 c). It is worth noting that after 24 h of treatment with 30 mg / mL D-gal, the SA-β-gal positive rate of PC12 cells was 54.33% ( Figure 1 b), indicating that this condition is sufficient to induce senescence while ensuring sufficient normal cells. In summary, 30 mg / mL D-gal treatment for 24 h is a reasonable condition for establishing a PC12 cell senescence model.

[0076] 3.2 Fucoxanthin improves D-gal-induced PC12 cell senescence:

[0077] like Figure 3As shown in Figure a, to study the effect of FX on cell viability, CCK8 was used to detect cell viability at different concentrations of FX. The results showed that at 0.5, 1, 2, and 5 μg / ml, the cell viability was 99.47%, 99.02%, 95.85%, and 93.89%, respectively. However, when the FX concentration was greater than or equal to 10 μg / ml, the PC12 cell viability was less than 90%. To ensure the safety of FX to cells, the present invention used 0.5, 1, 2, and 5 μg / ml as the experimental concentrations of FX to study the effect of FX on D-gal-induced PC12 cell senescence.

[0078] like Figure 3 As shown in b, compared with the Control group, D-gal treatment significantly reduced the survival rate of PC12 cells, which decreased by 45.1% (p<0.01). As the concentration of FX increased, the survival rate of PC12 cells increased in a concentration-dependent manner. Among them, compared with the D-gal group, the cell survival rate increased by 1.65 times after treatment with 5μg / mL of FX (p<0.01). These results indicate that pretreatment with FX can improve the inhibitory effect of D-gal on PC12 cell proliferation. In order to further explore the improvement effect of FX on D-gal-induced PC12 cell senescence, the cell senescence state was explored by SA-β-gal staining. As shown in Figure 3 As shown in Figures c and 3d, the SA-β-gal positive rate in the D-gal-induced PC12 cell senescence model increased 5.17-fold compared to the control group (p < 0.01). However, after adding different concentrations of FX, the number of blue-stained (senescent) PC12 cells, as detected by SA-β-gal staining, decreased in a concentration-dependent manner. Specifically, treatment with 5 μg / ml of FX reduced the SA-β-gal positive rate by 60% compared to the D-gal group (p < 0.01). This suggests that FX can reverse D-gal-induced cellular senescence and reduce β-galactosidase expression.

[0079] From the above, we can see that 2-5 μg / ml of FX can significantly improve the survival rate of D-gal-induced PC12 senescent cells and effectively inhibit the expression of cell senescence marker SA-β-gal, showing good anti-aging potential.

[0080] 3.3 Fucoxanthin inhibits D-gal-induced oxidative stress in PC12 cells:

[0081] Oxidative stress is closely associated with aging and dysfunction in various biological systems. It is well known that the accumulation of ROS is a key factor in inducing oxidative stress and accelerating aging. In this study, the effects of FX on oxidative damage during D-gal-induced PC12 cell aging were evaluated by measuring ROS and MDA levels, as well as GCLM content, in PC12 cells.

[0082] like Figure 3 a and Figure 3 As shown in b, compared with the Control group, the levels of ROS and MDA in the D-gal-induced senescent PC12 cell model increased significantly by 2.60 times (p < 0.01) and 1.65 times (p < 0.01), respectively. Figure 3 As shown in Figure c, D-gal treatment resulted in a significant decrease in GCLM content by 29.93% in PC12 cells (p < 0.01). These results indicate that D-gal significantly increased the levels of oxidative stress-related indicators and successfully induced oxidative damage in PC12 cells.

[0083] Different concentrations of FX (0.5, 1, 2, and 5 μg / mL) were added to D-gal-induced senescent PC12 cells to further explore the intervention effect of FX on oxidative stress. Figure 3 FX treatment significantly reduced ROS levels and MDA content and promoted GCLM protein expression. Compared with the D-gal group, especially after 5 μg / mL FX treatment, ROS levels in cells decreased by 33.50% (p < 0.01) and MDA levels decreased by 40.71% (p < 0.01), while GCLM protein expression increased significantly by 1.37-fold (p < 0.01). These results indicate that FX can significantly alleviate D-gal-induced oxidative stress, thereby alleviating cellular oxidative damage.

[0084] 3.4 Fucoxanthin activates D-gal to induce autophagy in senescent PC12 cells:

[0085] Autophagy is an intracellular catabolic recycling system associated with lifespan and health extension. To evaluate the occurrence of autophagy in cells, this study used TEM to observe six groups of PC12 cells with different treatments. Figure 4As shown in Figure a, most autophagosomes in control PC12 cells exhibited regular morphology, typically presenting as double-membrane vesicles with relatively uniform size and a low number of autophagosomes. This is primarily due to the relatively stable metabolism and homeostasis of normal cells, resulting in fewer damaged proteins and organelles, which do not require extensive autophagy for clearance. In contrast, autophagosomes in D-gal-induced senescent PC12 cells exhibited more diverse morphologies. In addition to double-membrane autophagosomes, some exhibited irregular morphologies, such as multi-layered structures or those with partially damaged membranes. This may be due to a degree of disruption in the autophagic process within senescent cells, resulting in incomplete autophagosome formation and maturation. Furthermore, the number of autophagosomes increased significantly with cellular aging, indicating that autophagy is activated to clear these damaged components. However, with increasing FX concentration, the number of autophagosomes in senescent cells gradually decreased, and their structure became more regular. This suggests that FX can improve the autophagic structure of senescent PC12 cells.

[0086] like Figure 4 As shown in (b), Western blot analysis revealed that in D-gal-induced senescent PC12 cells, the level of LC3, a marker of autophagosome formation, decreased by 53.44% (p < 0.01), the level of the autophagy regulator Beclin1 decreased by 36.78% (p < 0.01), and the level of p62 protein increased by 1.12-fold (p < 0.01). However, after FX treatment, p62 protein levels decreased with increasing FX concentration, while LC3 and Beclin1 protein levels increased. These changes in autophagy-related markers suggest activation of the autophagy process. Notably, compared with the D-gal group, the high-concentration FX group decreased p62 by 41.60% (p < 0.01), while LC3 and Beclin1 levels increased by 2.11-fold (p < 0.01) and 1.46-fold (p < 0.01), respectively. This suggests that high-concentration FX can significantly activate the autophagy process, thereby alleviating D-gal-induced cellular senescence.

[0087] 3.5 Fucoxanthin regulates nuclear Nrf2 protein levels in aging PC12 cells:

[0088] In order to explore the mechanism of FX on D-gal-induced senescence of PC12 cells, Western Blot was used to detect the expression level of nuclear transcription factor Nrf2. Figure 5As shown, compared with the control group, the expression level of nuclear Nrf2 in D-gal-induced senescent cells decreased significantly by 44.00% (p < 0.01). However, in the D-gal-induced senescent cell model, the expression level of nuclear Nrf2 increased in a concentration-dependent manner with increasing FX concentration. Compared with the D-gal group, 5 μg / ml of FX increased the expression level of nuclear Nrf2 by 1.64 times (p < 0.01). These results indicate that FX can significantly reverse the decrease in the expression level of the nuclear transcription factor Nrf2 in D-gal-induced senescent PC12 cells, thereby improving the senescent state of cells by regulating nuclear Nrf2 expression.

[0089] 3.6 Effect of fucoxanthin on D-gal-induced PC12 siRNA-Nrf2 No effect on SA-β-galactosidase activity in senescent cells:

[0090] In order to explore whether Nrf2 deficiency affects the protective effect of FX on D-gal-induced PC12 cells, the present invention used siRNA interference technology to inhibit the expression of Nrf2 gene in PC12 cells and constructed a PC12 siRNA-Nrf2 Cell model. Western Blot analysis confirmed that the Nrf2 protein level in PC12 cells decreased by 78.11% compared with the NC group (p<0.01), indicating that Nrf2 expression in PC12 cells has been significantly inhibited ( Figure 6 a) After 24 h of incubation, 30 mg / mL of D-gal was added to the cells, followed by the addition of different concentrations of FX. siRNA-Nrf2 Changes in cell survival rate after FX intervention.

[0091] like Figure 6 As shown in b, there was no significant difference in cell survival rate in the Control (Nrf2- / -) group compared with the Control group. Compared with the Control (Nrf2- / -) group, the cell survival rate of the D-gal (Nrf2- / -) group decreased significantly by 52.55% (p<0.01). However, after intervention with a high concentration of 5 μg / mL FX, the cell survival rate still did not increase significantly (p>0.05), indicating that Nrf2 plays a role in FX improving D-gal-induced PC12 siRNA-Nrf2 It plays an important role in cell survival.

[0092] In addition, if Figure 6 As shown in c, the β-galactosidase staining experiment showed that the number of blue-stained cells in the D-gal (Nrf2- / -) group increased significantly, indicating that the degree of cell senescence was aggravated. Even after the addition of the highest dose of FX (5 μg / mL), the number of blue-stained PC12 siRNA-Nrf2The cell proportion continued to increase, and the number of senescent cells continued to rise. This further confirms that FX is unable to improve senescence in PC12 cells with low Nrf2 expression. Combined with these results, it is speculated that the improvement effect of FX on senescent PC12 cells is mediated by Nrf2.

[0093] 3.7 Effect of fucoxanthin on D-gal-induced PC12 siRNA-Nrf2 Cellular autophagy is mediated by Nrf2:

[0094] In order to further explore whether the improvement effect of FX on D-gal-induced PC12 cell senescence and autophagy is achieved through the Nrf2-autophagy pathway, the present invention used siRNA interference technology to significantly reduce the expression of Nrf2 gene in PC12 cells before the experiment, and detected the expression of Nrf2 gene in senescent PC12 cells by Western Blot. siRNA-Nrf2 Changes in the levels of autophagy-related proteins p62, Beclin1 and LC3 in cells.

[0095] like Figure 7 As shown in a, compared with the Control (Nrf2- / -) group, the p62 level in the D-gal Nrf2- / - group increased by 1.08 times (p<0.01), while the Beclin1 and LC3 levels decreased by 31.64% (p<0.01) and 50.92% (p<0.01), respectively. This indicates that in the case of Nrf2 deficiency, the autophagy process in D-gal-induced senescent cells is significantly inhibited. However, compared with the D-gal (Nrf2- / -) group, after intervention with a high concentration of FX (5 μg / mL), the autophagy process in senescent PC12 cells was significantly inhibited. siRNA-Nrf2 There was no significant change in the levels of p62, Beclin1 and LC3 proteins in the cells.

[0096] In order to observe the changes of autophagy at the cellular level, the present invention used TEM to observe ( Figure 7 b) PC12 in the Control (Nrf2- / -) group siRNA-Nrf2 The autophagosomes in the cells are relatively regular in shape, usually appearing as double-membrane vesicles with relatively uniform size and few in number. siRNA-Nrf2 The morphology of autophagosomes in cells is more diverse. In addition to autophagosomes with double-layer membrane structures, some irregular morphologies have also appeared, such as autophagosomes with multi-layer membrane structures or partially damaged membrane structures. In addition, the number of autophagosomes increases significantly with cell aging, indicating that the autophagic process is activated to remove damaged cellular components. However, the addition of high concentrations of FX has a negative effect on the aging of PC12 cells. siRNA-Nrf2The number and structure of autophagosomes in the cells did not change significantly, indicating that the loss of Nrf2 resulted in the inability of FX to improve the number and structure of autophagosomes in aging PC12 cells.

[0097] From the above, these results further confirmed that FX improved D-gal-induced PC12 cell senescence through the Nrf2-autophagy pathway.

[0098] It can be seen from Verification Example 1 that D-gal causes PC12 cell proliferation inhibition and aging, as well as oxidative imbalance, and inhibits autophagy. Specifically, D-gal significantly inhibits cell growth, increases the SA-β-gal positivity rate, increases the ROS content in PC12 cells, reduces the GCLM protein level, reduces the expression of Beclin1 and LC3, and increases the p62 protein level, thereby destroying the normal state of autophagosomes. However, 2-5 μg / mL of FX significantly enhanced cell proliferation and improved the state of senescent cells. At the same time, FX plays an anti-oxidative stress role in senescent PC12 cells, improves the state of autophagosomes and promotes autophagy activation. The ROS content gradually decreases with the increase of FX concentration, and the GCLM protein level increases, MDA expression is inhibited, the autophagy protein Beclin1 and LC3 levels increase, and the p62 level decreases. In addition, FX increases the nuclear Nrf2 protein level of senescent PC12 cells. However, FX cannot reverse the D-gal-induced senescent PC12 siRNA-Nrf2 Cell proliferation was inhibited and SA-β-gal positive rates were increased, but autophagy activation and improvement of autophagosome status were also not achieved. These findings suggest that FX exerts anti-aging and neuroprotective effects on senescent PC12 cells through the Nrf2-autophagy pathway.

[0099] Verification Example 2: Fucoxanthin activates Nrf2-autophagy to improve cognitive decline in aging mice induced by D-gal:

[0100] 1. Materials and instruments:

[0101] 1.1 Materials and Reagents

[0102] FX (purity >98%): Chengdu Purifa Technology Development Co., Ltd. (China); olive oil: Shanghai McLean Biochemical Technology Co., Ltd. (China); disposable sterile syringe: Chengdu Xinjin Shifeng Medical Instrument Co., Ltd. (China); 0.9% saline: Jiangsu Huaian Shuanghe Pharmaceutical Co., Ltd. (China); D-gal: Beijing Solebold Technology Co., Ltd. (China); paraformaldehyde fixative: Shanghai Aladdin Biochemical Technology Co., Ltd. (China); dimethyl sulfoxide (DMSO): Sigma-Aldrich (USA).

[0103] 1.2 Instruments and Equipment

[0104] Microplate Reader: Thermo Fisher Scientific (USA); Ultrapure water system (Simplicity): Millipore (USA); High-speed refrigerated centrifuge (5430R): Shanghai A4Lab (China); Electronic constant-temperature water bath (HH-501S): Changzhou Jincheng Chunlan Experimental Instrument Factory (China); qPCR instrument: Roche (Switzerland); Flow cytometer: Beckman (USA); Body fluorescence microscope, transmission electron microscope (TEM): NIKON (TI-S) (Japan).

[0105] 2. Experimental methods:

[0106] 2.1. Experimental animals and experimental procedures:

[0107] All C57BL / 6J mice (male, 8 weeks old) were purchased from Beijing Charles River Experimental Animal Technology Co., Ltd. (Beijing, China), and all Nrf2 knockout (Nrf2- / -) Nfe2l2-eKO2 mice (male, 8 weeks old) were purchased from Shanghai Southern Model Organism Technology Co., Ltd. (Shanghai, China) and were raised in the animal center of Ningbo University (Ningbo, China). The mice were fed with standard rodent feed and were raised under controlled temperature (21±2℃) and humidity (50±10%), 12h light and 12h dark cycle. All experimental procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the Animal-Related Experimental Animal Use and Protection Ethics Committee of the Health Science Center of Ningbo University (protocol code NBU20230255 and approval date 2023-01-04) approved.

[0108] The mice were free to obtain water and food, and after one week of adaptation, the C57BL / 6J mice were randomly divided into five groups (n=3): (1) Control, (2) D-gal, (3) D-gal L-FX, (4) D-gal M-FX and (5) D-gal H-FX. The D-gal (500mg / kg / d) injected subcutaneously was dissolved in sterile 0.9% saline solution. The Control group was injected subcutaneously with 0.9% saline, and orally with 0.9% saline (containing 9.6% DMSO) by gavage; the D-gal group of mice was injected subcutaneously with D-gal and orally with 0.9% saline (containing 9.6% DMSO) by gavage; the D-gal L-FX group, the D-gal M-FX group and the D-gal H-FX group of mice were injected subcutaneously with D-gal, and were respectively gavaged with 50, 100, 200mg / kg / d of FX (FX dissolved in olive oil) once a day, for nine weeks.

[0109] Separately, C57BL / 6J mice and Nfe2l2-eKO2 (Nrf2- / -) mice were randomly divided into four groups (n=4): (1) Control (C57BL / 6J), (2) Control (Nrf2- / -), (3) D-gal (Nrf2- / -), and (4) D-gal H-FX (Nrf2- / -). D-gal (500 mg / kg / d) was dissolved in sterile 0.9% saline solution for subcutaneous injection. Mice in the Control and Control (Nrf2- / -) groups received subcutaneous injections of 0.9% saline and oral administration of sterile 0.9% saline (containing 9.6% DMSO) by gavage. Mice in the D-gal (Nrf2- / -) group received subcutaneous injections of D-gal and oral administration of sterile 0.9% saline (containing 9.6% DMSO) by gavage. Mice in the D-gal H-FX group received subcutaneous injections of D-gal and oral administration of FX at 200 mg / kg / day by gavage once daily for nine weeks. Animal use and care were in accordance with the guidelines of the Ningbo University Animal Research Advisory Committee.

[0110] 2.2 Behavioral testing:

[0111] Mice were acclimated to the behavioral testing room 1 h before testing.

[0112] Suspension test: C57BL / 6 mice and Nfe2l2-eKO2 mice were placed on a 1.5 mm diameter horizontal wire, 25 cm above the ground. The mouse was placed on the wire, with only its front paws allowed to grasp the wire. Scoring criteria for the suspension test are shown in Table 1.

[0113] Table 1 Scoring system for the suspension ability test in behavioral experiments of aging model mice

[0114] Score Training situation Number of repetitions Mouse behavior 0 Fall immediately 1 Each mouse was treated 3 days before surgery The two hind legs cannot grasp the line 2 Conduct training Each mouse was tested 3 times Only one hind limb can grasp the line 3 Both hind legs can grasp the line

[0115] 2.3 Water Maze

[0116] The steps of the mouse water maze experiment mainly include preparing the water maze, training mice, testing memory, and analyzing data.

[0117] Prepare the water maze: Fill a closed pool with clear water to a depth of approximately 20-30 cm. Add a 10-15 cm buoyant platform to the water, keeping it approximately 1-2 cm above the surface. Ensure consistency in water quality and platform placement to avoid excessive fatigue or drowning of mice.

[0118] To train a mouse: Place the mouse at a starting point in the water maze and have it swim toward the exit (i.e., the platform). If the mouse successfully climbs onto the platform, allow it to remain on the platform for 10-15 seconds before returning it to its cage. If the mouse fails to reach the platform, remove it from the water, wait for it to remain on the platform for the same amount of time, and then return it to its cage. Repeat this process until the mouse can consistently find the platform.

[0119] In addition, mice should be subjected to stress management before the experiment, such as petting them for 1-2 minutes daily to prevent stress. For three to five consecutive days before the experiment, move the mice to the water maze experimental room and acclimate them for 3 hours daily. Connect and debug the behavioral video recording system to ensure video clarity and recording, and eliminate interference from reflections on the water surface. Fill the pool to a height of approximately 30 cm with clean water and maintain the water temperature at approximately 22°C using the water temperature maintenance device.

[0120] Testing Memory: After the mice have mastered the water maze task, they are tested. The platform is removed from the pool, and the mice are placed at the starting point of the water maze. Observe the mice's swimming trajectory. If they can find the exit of the water maze, they have spatial memory.

[0121] Data analysis: In each trial, the time (i.e., escape latency) was calculated from the time the mouse was placed in the water until it reached the hidden platform. After finding the hidden platform, the mouse spent 10 seconds there. The time to find the hidden platform was limited to 90 seconds. Mice that failed to find the hidden platform were gently placed on the platform for 10 seconds. In this case, the escape latency was recorded as 90 seconds. On the sixth day, a probe test without an escape platform was conducted. Mice were placed in the pool and allowed to swim for 120 seconds. The frequency with which each mouse crossed the target quadrant location and the time spent in the target quadrant were recorded.

[0122] 2.4. Collecting tissues:

[0123] Animals were anesthetized with CO2 and then perfused with 0.9% saline solution. Brains from three mice per group were rapidly dissected, and the hippocampi were harvested and stored at -80°C for future Western blot analysis. Additionally, brains from three mice per group were harvested for future immunofluorescence analysis and fixed in 4% paraformaldehyde for 48 h.

[0124] 2.5. Nissl body staining:

[0125] Mouse brain tissue sections are fixed with a fixative and then dehydrated with ethanol. Next, the samples are treated with Nissl stains (commonly methylene blue or modified Nissl stains). The dye binds to RNA within the cells, visualizing ribosomes. After staining, the samples are rinsed to remove excess dye and mounted on glass slides. The coloration of neuronal cell bodies and nuclei is observed under a microscope to study the morphology and structure of neural tissue.

[0126] 2.6. SA-β-galactosidase staining of mouse hippocampus:

[0127] Remove the frozen sections from -20°C, wash with PBS, and then fix with β-galactosidase staining fixative at room temperature for 15 minutes. Wash with PBS three times for 5 minutes each. Prepare the staining working solution by mixing β-galactosidase staining solution A and β-galactosidase staining solution B in a 1:1 ratio. Add the prepared staining working solution dropwise to the frozen sections, covering the tissue, and incubate at room temperature overnight. Afterwards, wash with PBS three times for 5 minutes each. Through the above steps, senescent cells in the mouse hippocampus can be effectively stained for β-galactosidase, and then aging-related changes can be observed and analyzed under a microscope.

[0128] 2.7 Immunohistochemistry

[0129] The fixed brain tissue was embedded in paraffin, and similar brain sections containing the hippocampus were selected based on the mouse brain atlas. The dewaxed brain sections were dewaxed in xylene and rehydrated in graded alcohol solutions. After rinsing with phosphate-buffered saline (PBS, pH 7.4), diluted primary antibodies (LC3, p62, and LC3) were added and placed at 4°C overnight and rinsed. Secondary antibodies (rabbit antibodies) were added and incubated at room temperature for 30 minutes. The sections were then developed with a colorimetric reagent, washed, counterstained with hematoxylin for 2 minutes, dehydrated, covered with neutral gum, and air-dried. The completed hippocampal sections were observed under a microscope and photographed.

[0130] 2.8. TEM observation of autophagy in the hippocampus:

[0131] First, fix the tissue with 2.5% glutaraldehyde at 4°C for 2 hours. Rinse with 0.1mol / L phosphate buffer three times, 15 minutes each time. Then, dehydrate with 50%, 70%, and 90% ethanol in sequence, 15 minutes each time. At 4°C, replace the ethanol with a mixture of 90% ethanol and 90% acetone (1:1), and then replace it with 90% acetone, each for 15 minutes. Replace it with pure acetone three times at room temperature, 20 hours each time. Subsequently, soak the sample in a mixture of pure acetone and resin (such as Epon) and place it at room temperature for 3 hours. Then place it in pure resin overnight, and finally place it in a 37°C oven for 2 hours.

[0132] Place in a 45°C oven for 12 hours, and finally in a 60°C oven for 24 hours. Use an ultramicrotome to cut the embedded sample into 50-100 nm thick ultrathin sections. Stain the ultrathin sections with 3% uranyl acetate for 15 minutes, then rinse with distilled water three times for 5 minutes each. Stain with lead citrate for 10 minutes, and finally rinse with distilled water three times for 5 minutes each. Observe the autophagosomes under a TEM.

[0133] 2.9 Statistical Analysis

[0134] Same as verification example 1.

[0135] 3. Results and Analysis

[0136] 3.1 Fucoxanthin improves tensile strength, hair and suspension ability in aging mice:

[0137] Aging is often accompanied by a decline in muscle strength, endurance, explosive power, coordination, and balance. In order to evaluate the effect of FX on the physical strength of D-gal-induced aging mice, the present invention used a tensile test. Figure 8 As shown in Figure a, the mean tensile force of C57BL / 6J mice in the control group was 2.13 N. However, the tensile force of mice in the D-gal group was reduced by 62.50% compared to the control group (p < 0.01). Notably, the tensile force of aged mice increased in a concentration-dependent manner under FX treatment. Compared to the D-gal group, the tensile force of aged mice increased 2.08-fold after oral administration of 200 mg / kg / day of FX (p < 0.01).

[0138] Suspension ability test is an important means to evaluate the neuromuscular coordination of mice. Figure 8 b. The mean suspension test score of mice in the Control group was 3 points. Compared with the Control group, the suspension test score of D-gal-induced aging mice decreased by 77.78% (p<0.01). However, under the intervention of FX, the scores of aging mice gradually increased with the increase of FX concentration. Among them, when 50 mg / kg / d (low concentration) FX was given, the suspension test score increased significantly by 2.0 times (p<0.01). Hair changes are one of the intuitive manifestations of improved physiological functions of aging mice. Figure 8 c, Control C57BL / 6J mice had thick, shiny, black hair, while D-gal-induced aging mice developed increased and sparse white hair. However, as FX concentrations increased, the thinning hair problem in aging mice gradually improved, and their coat color, brightness, and density approached those of normal mice.

[0139] 3.2 Fucoxanthin improves learning and memory abilities in aging mice:

[0140] The water maze is a behavioral test used to assess spatial learning and memory in mice. Memory declines with aging in humans and other species. This study used the water maze to test the effects of FX on D-gal-induced hippocampal-dependent learning and memory in aged mice during a spatial memory task.

[0141] like Figure 9 Trajectory analysis showed that mice in the control group gradually developed more direct swimming paths during training, tending towards linear or directional strategies, indicating that they were able to effectively use spatial information to locate the hidden platform. However, the swimming paths of mice in the D-gal group were more complex, often exhibiting marginal or random strategies, indicating that the spatial navigation ability of aging mice was severely impaired, making it difficult for them to effectively use spatial cues to determine the location of the platform. However, FX intervention gradually normalized the swimming paths of aging mice.

[0142] like Figure 9 b, The mean percentage of platform quadrant swimming time in normal mice in the control group was 33.23. Compared with the control group, the percentage of platform quadrant swimming time in mice in the D-gal group was reduced by 78.94% (p < 0.01). However, after FX administration, the percentage of platform quadrant swimming time in aged mice increased in a concentration-dependent manner.

[0143] like Figure 9 c. The mean number of platform strides in normal mice in the control group was 2.67. Compared with the control group, the number of platform strides in mice in the D-gal group decreased by 75.00% (p < 0.01). However, after FX administration, the number of platform strides in aged mice gradually increased, particularly after high-dose FX administration, which increased the number by 2.50-fold (p < 0.01).

[0144] like Figure 9 d. The mean escape latency of normal mice in the control group was 15.88. Compared with the control group, the escape latency of mice in the D-gal group was prolonged by 1.48 times (p < 0.01). However, after administration of FX, the escape latency of aged mice gradually decreased, especially after administration of high-concentration FX, which decreased the escape latency by 49.60% (p < 0.01). These results indicate that FX intervention can effectively improve spatial learning and memory abilities in D-gal-induced aged mice.

[0145] 3.3 Fucoxanthin reduces SA-β-galactosidase activity in the brain of D-gal-induced aging mice:

[0146] Increased SA-β-gal activity indicates that cells are undergoing senescence or are in an aging-related pathological state. In brain tissue, increased SA-β-gal activity is often closely associated with decreased neuronal function and neurodegenerative diseases. Therefore, the present invention utilizes SA-β-gal activity detection to evaluate the ameliorative effects of FX on D-gal-induced senescent cells in the hippocampus of aged mice.

[0147] like Figure 10 As shown, the blue-stained area of ​​the hippocampus of mice in the D-gal group was significantly more than that in the control group, indicating that long-term D-gal intake promotes the appearance of aging characteristics in mice. Compared with the D-gal group, FX inhibited the increase of blue-stained senescent cells in the hippocampus of aged mice in a dose-dependent manner. In particular, when 200μg / ml of FX (D-gal H-FX group) was used for intervention, the blue-stained area of ​​the hippocampus of mice induced by D-gal was significantly reduced. This shows that high concentrations of FX can effectively alleviate the aging effects induced by D-gal, thereby improving the aging of the mouse hippocampus.

[0148] 3.4. Fucoxanthin improves Nissl bodies in the hippocampus of D-gal-induced aging mice:

[0149] Nissl bodies are cellular structures in neurons that are rich in rough endoplasmic reticulum and ribosomes. Their status directly reflects the metabolic and anabolic function of neurons and can serve as an important marker for assessing neuronal health and anabolic function. The hippocampus is a key area of ​​the brain closely related to learning and memory. The morphology and distribution of its neurons are crucial for cognitive function, and Nissl staining can clearly highlight these features. In this study, Nissl body staining was used to investigate the effects of FX on D-gal-induced Nissl bodies in the mouse hippocampus.

[0150] like Figure 11 As shown, Nissl body staining results in the hippocampus of normal mice in the control group showed that the Nissl bodies in the cell bodies and dendrites of neurons were clearly stained dark blue or blue-purple, indicating normal protein synthesis function of neurons. Nissl body staining in the hippocampus of aged mice in the D-gal group was lighter. However, in D-gal-induced aged mice, after treatment with high concentrations of FX, Nissl body staining in the hippocampus gradually deepened. This suggests that FX can effectively improve the state of neurons in the hippocampus of aged mice, which may have a positive effect on the learning and memory abilities of aged mice.

[0151] 3.5 Fucoxanthin activates D-gal to induce autophagy in the hippocampus of aging mice:

[0152] When studying the therapeutic effect of FX on D-gal-induced aging mice, the results of immunohistochemistry in the hippocampus of mice revealed changes in autophagy-related proteins. Figure 12As shown, compared with the control group, the levels of Beclin1 and LC3 in the hippocampus of D-gal-induced aged mice decreased by 53.23% (p<0.01) and 55.27% (p<0.01), respectively, while the level of p62 increased significantly by 1.12 times (p<0.01). This indicates that the autophagy process is inhibited in the hippocampus of aged mice, resulting in a reduced efficiency in the clearance of damaged organelles and protein aggregates.

[0153] However, with increasing FX concentrations, autophagy was activated. In particular, when high-concentration FX was administered, the levels of Beclin1 and LC3 in the hippocampus increased by 1.65-fold (p<0.01) and 1.78-fold (p<0.01), respectively, while the level of p62 decreased significantly by 59.79% (p<0.01).

[0154] 3.6 Effect of Fucoxanthin on Nrf2 - / - Effects of Aging on Pulling Strength and Suspension Ability in Mice:

[0155] To further explore whether FX improves D-gal-induced aging in mice through the Nrf2-autophagy pathway, Nfe2l2-eKO2 gene knockout mice (Nrf2 - / - ).like Figure 13 As shown in a, C57BL / 6J (normal) mice in the Control group and Nrf2 - / - There was no significant difference in the tensile test between the mice. - / - The mean tensile strength of aging mice was 2.13N, and compared with the Control group, Nrf2 - / - The pulling force of aged mice decreased by 62.50% (p < 0.01). - / - ) group, Nrf2 - / - The pulling strength of aging mice continues to decline.

[0156] As shown in 13b, normal mice in the Control group and Nrf2 - / - There were no significant differences in the mice's suspension test; the mean suspension score for the control (Nrf2- / -) group was 3 points, while the suspension test score of D-gal-induced Nrf2- / - aged mice decreased by 77.78% compared to the control group (p < 0.01). Even administration of high concentrations of FX did not significantly improve the performance compared to the D-gal (Nrf2- / -) group.

[0157] From the above, we can see that FX has an effect on D-gal-induced Nrf2 - / -There was no improvement in the pulling strength and suspension ability of mice, indicating that Nrf2 may be an important target of FX in improving the pulling strength and suspension ability of aging mice.

[0158] 3.7 Effect of Fucoxanthin on Nrf2 - / - Effects of aging on learning and memory abilities in mice:

[0159] .like Figure 14 As shown, the mean percentage of platform quadrant swimming time in the control Nrf2- / - mice was 33.23. Compared with the control (Nrf2- / -) group, the percentage of platform quadrant swimming time in D-gal-induced Nrf2- / - aged mice decreased significantly by 78.40% (p<0.01). The mean number of platform crossings in the control (Nrf2- / -) group was 2.65, while this number decreased significantly by 74.84% (p<0.01) in D-gal-induced Nrf2- / - aged mice. The mean escape latency in the control (Nrf2- / -) group was 18.8, while the escape latency in D-gal-induced Nrf2- / - mice increased significantly by 1.49-fold (p<0.01). This suggests that D-gal leads to slower movement and reaction times and decreased memory in Nrf2- / - aged mice. However, compared with the D-gal(Nrf2- / -) group, Nrf2 - / - There were no significant differences in the percentage of swimming time in the platform quadrant, the number of platform crossings, and the escape latency between aged mice (p>0.05). This suggests that Nrf2 is involved in regulating FX-induced improvement of D-gal-induced learning and memory in mice.

[0160] 3.8 Effects of fucoxanthin on D-gal-induced autophagy in the hippocampus of Nrf2- / - aging mice:

[0161] The Nrf2-autophagy pathway may be an important pathway for treating neurological diseases caused by aging. Figure 15 As shown in a, compared with the Control (Nrf2- / -) group, D-gal significantly inhibited autophagy in Nrf2- / - mice, as evidenced by a 1.08-fold increase in p62 levels (p<0.01), while LC3 and Beclin1 decreased by 50.92% (p<0.01) and 31.64% (p<0.01), respectively. However, compared with the D-gal (Nrf2- / -) group, there was no significant difference in autophagy markers (such as p62, LC3, and Beclin1) in aged mice after high-concentration FX intervention, indicating that autophagy was not activated.

[0162] In TEM observation, changes in autophagosomes can provide intuitive evidence for evaluating autophagic activity. The following study evaluated the changes in the number and function of autophagic vacuoles in the hippocampus of D-gal mice under FX intervention. Figure 15 As shown in b, TEM showed that the hippocampus of normal mice in the control group had typical autophagosome structures. However, the autophagosomes in D-gal-induced aged mice showed irregular morphology and damaged membrane structure, indicating that the autophagy process was inhibited. However, FX failed to improve the Nrf2 - / - Autophagy status in the hippocampus of aging mice. This means that once Nrf2 is deficient, even with high concentrations of FX, the formation and function of autophagosomes in the hippocampus of mice are restricted, making it impossible to effectively remove damaged cellular components. This indicates that the absence of Nrf2 prevents FX from promoting D-gal-induced Nrf2 through the Nrf2-autophagy pathway. - / - Activation of autophagy in aging mice.

[0163] As shown in Example 2, FX can significantly improve the various physiological and behavioral characteristics of D-gal-induced aging mice, specifically by improving the pulling and suspension ability of mice, improving the hair condition, and enhancing the learning and memory ability of aging mice. In addition, FX can effectively reduce the activity of SA-β-galactosidase in the brain of D-gal-induced aging mice, improve the Nissl body structure in the hippocampus, and activate the autophagy process and improve the autophagy state. - / - The lack of significant effects in aging mouse models further confirms that the anti-aging effects of FX may be dependent on the Nrf2 signaling pathway, with significant effects observed at the cellular level and in C57BL / 6J mouse experiments. These findings further highlight the importance of the Nrf2-autophagy pathway in the anti-aging effects of FX on D-gal-induced aging mice.

[0164] Verification Example 3: Study on the neuroprotective effect on D-gal-induced mice based on the diatom frustule-fucoxanthin oral delivery system:

[0165] 1. Materials and instruments:

[0166] 1.1 Materials and Reagents

[0167] Diatom frustules: Shanghai MacLean Biochemical Technology Co., Ltd. (China); FX (purity >98%): Chengdu Purifa Technology Development Co., Ltd. (China); olive oil: Shanghai MacLean Biochemical Technology Co., Ltd. (China); disposable sterile syringe: Chengdu Xinjin Shifeng Medical Device Co., Ltd. (China); 0.9% saline: Jiangsu Huaian Shuanghe Pharmaceutical Co., Ltd. (China); D-gal: Beijing Solebold Technology Co., Ltd. (China); paraformaldehyde fixative: Shanghai Aladdin Biochemical Technology Co., Ltd. (China); dimethyl sulfoxide (DMSO): Sigma-Aldrich (USA).

[0168] 1.2 Instruments and Equipment

[0169] Microplate reader: Thermo Fisher Scientific (USA); ultrapure water system: Millipore (USA); high-speed refrigerated centrifuge (5430R): Shanghai Eppendorf (China); electronic constant temperature water bath (HH-501S): Changzhou Jincheng Chunlan Experimental Instrument Factory (China); qPCR instrument: Roche (Switzerland); Beckman Gallios flow cytometer: Beckman (USA); stereo fluorescence microscope, scanning electron microscope, and transmission electron microscope: NIKON (TI-S) (Japan).

[0170] 2. Experimental methods:

[0171] 2.1 Experimental Animals and Experimental Procedures

[0172] All C57BL / 6J mice (male, 8 weeks old) were purchased from Charles River Laboratory Animal Technology Co., Ltd. (Beijing, China), and all Nfe2l2-eKO2 mice (male, 8 weeks old) were purchased from Shanghai Model Organisms Science Co., Ltd. (Shanghai, China) and housed at the Ningbo University Animal Center (Ningbo, China). Mice were fed a standard rodent diet and housed under controlled temperature (21 ± 2°C) and humidity (50 ± 10%) with a 12-h light and 12-h dark cycle. All experimental procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

[0173] All animal experiments were approved by the Institutional Animal Care and Use Committee of Ningbo University Health Science Center (protocol code NBU20230255 and approval date 2023-01-04). FX and D-gal were dissolved in olive oil or sterile saline, respectively. Mice had free access to water and food. After one week of acclimation, C57BL / 6J mice were randomly divided into five groups (n = 3):

[0174] (1) Control, (2) D-gal Frustule, (3) D-gal Frustule@L-FX, (4) D-gal Frustule@M-FX, and (5) D-gal Frustule@H-FX. Subcutaneously injected D-gal (500 mg / kg / d) was dissolved in 0.9% saline solution. The control group received a subcutaneous injection of 0.9% saline and an oral administration of 0.9% saline (containing 9.6% DMSO) by gavage. The D-gal group received a subcutaneous injection of D-gal and an oral administration of 0.9% saline (containing 9.6% DMSO) by gavage. The D-galL-FX, D-galM-FX, and D-galH-FX groups received a subcutaneous injection of D-gal and an oral administration of FX at 50, 100, and 200 mg / kg / d, respectively, once a day for nine weeks.

[0175] In addition, C57BL / 6J mice and Nfe2l2-eKO2 mice were randomly divided into four groups (n=3): (1) Control, (2) Control (Nrf2- / -), (3) D-gal Frustule (Nrf2- / -), and (4) D-gal Frustule@H-FX (Nrf2- / -). D-gal (500 mg / kg / d) was dissolved in 0.9% saline solution for subcutaneous injection. Mice in the Control and Control (Nrf2- / -) groups received subcutaneous injections of 0.9% saline and oral administration of 0.9% saline (containing 9.6% DMSO). Mice in the D-gal (Nrf2- / -) group received subcutaneous injections of D-gal and oral administration of a 25 mg / kg / day diatom frustule solution (containing 9.6% DMSO). Mice in the D-gal H-FX group received subcutaneous injections of D-gal and oral administration of a mixture of a 25 mg / kg / day diatom frustule solution and 200 mg / kg / day FX once daily for nine weeks. Animal use and care were in accordance with the guidelines of the Ningbo University Animal Research Advisory Committee.

[0176] 2.2. Testing the adsorption capacity of diatom frustules on fucoxanthin:

[0177] Diatom frustules were pretreated by washing and drying to remove impurities. FX was dissolved in ethanol for storage and then diluted to a range of concentrations with deionized water before use. To synthesize Frustule@FX, 1 ml of FX solution (25, 50, 100, 200, 400, 800, and 1600 μg / ml) was added to 10 ml of a diatom frustule suspension (50 μg / ml). The mixture was stirred for 12 hours. Finally, the prepared Frustule@FX was collected by centrifugation at 3260 g for 10 minutes and stored in deionized water for further experiments. Following lyophilization, the obtained Frustule@FX sample was suspended in deionized water, frozen (-20°C for 24 hours), and then freeze-dried in a vacuum dryer for 24 hours. The amount of loaded FX was quantified using the absorbance of the supernatant collected at 450 nm using a microplate reader combined with a standard curve of FX solutions. The drug loading efficiency was defined as (amount of loaded FX / amount of FX in the initial FX loading solution) × 100%.

[0178] 2.3 Behavioral testing:

[0179] Same as verification example 2.

[0180] 2.4 Water Maze

[0181] Same as Verification Example 2.

[0182] 2.5. Collection of tissues:

[0183] Same as Verification Example 2.

[0184] 2.6. Nissl body staining:

[0185] Same as Verification Example 2.

[0186] 2.7 SA-β-galactosidase staining of mouse hippocampus:

[0187] Same as Verification Example 2.

[0188] 2.8 Immunohistochemistry

[0189] Same as verification example 2.

[0190] 2.9. TEM observation of autophagy in the hippocampus:

[0191] Same as Verification Example 2.

[0192] 2.10 Statistical Analysis

[0193] Same as verification example 2.

[0194] 3. Results and Analysis

[0195] 3.1. Diatom shells can absorb fucoxanthin:

[0196] like Figure 16 As shown in a, the hollow structure and porous honeycomb structure of diatom shells can be seen. These pores can adsorb FX, making it an ideal drug carrier. In order to evaluate the ability of diatom shells to adsorb FX, this study thoroughly mixed different concentrations of FX with 50μg / mL diatom shells. Figure 16 As shown in Figure b, the bottle marks 1, 2, 3, 4, 5, 6, and 7 represent FX concentrations of 25, 50, 100, 200, 400, 800, and 1600 μg / mL, respectively. When the initial concentration of FX was lower than 100 μg / mL, there was no obvious yellow color in the supernatant of the mixed culture, indicating that diatom frustules can effectively adsorb FX. When the initial concentration of FX was higher than 100 μg / mL, the adsorption rate of diatom frustules exceeded 80%. Figure 16 As shown in Figure c, when the FX concentration reaches 400 μg / mL, the adsorption rate of diatom frustules reaches the maximum. This ratio provides the optimal adsorption ratio for the preparation of Frustule@FX in subsequent experiments.

[0197] 3.2. Diatom frustules@fucoxanthin drug delivery system improves pulling strength, hair and suspension ability in aging mice:

[0198] By observing the pulling force, suspension ability and hair of mice, the effect of Frustule@FX on aging mice can be intuitively evaluated. Figure 17 As shown in the results, the scores of tension and suspension tests of the aged mice group without diatom shell addition and the aged mice group with diatom shell addition were significantly reduced, and the hair condition was also worse, and there was no significant difference between the two groups. Figure 17 a. The mean tensile strength of C57BL / 6J mice in the control (Frustule@FX) group was 2.13 N. Compared to the control (Frustule@FX) group, the tensile strength of mice in the D-gal (Frustule@FX) group was reduced by 62.03% (p < 0.01). As the concentration of Frustule@FX increased, the tensile strength of aged mice gradually increased. A significant improvement was observed at the intermediate Frustule@FX concentration (100 mg / kg / day). At this point, the tensile strength of the intermediate-concentration group increased by 1.10-fold compared to the D-gal (Frustule@FX) group (p < 0.01). Furthermore, when high-concentration Frustule@FX was administered, the tensile strength of aged mice significantly increased by 1.48-fold compared to the D-gal (Frustule@FX) group (p < 0.01).

[0199] like Figure 17b. The mean suspension test score of mice in the control (Frustule@FX) group was 3. Compared with the control (Frustule@FX) group, the suspension test score of aged mice induced by D-gal (Frustule@FX) decreased by 77.78% (p < 0.01). The suspension test scores of aged mice gradually improved with increasing Frustule@FX concentrations. A significant effect was seen at the moderate concentration of Frustule@FX, where the suspension scores of aged mice increased by 1.00-fold (p < 0.01). In particular, the high-concentration Frustule@FX group achieved the highest score, with a 2.00-fold increase compared to the D-gal Frustule group (p < 0.01).

[0200] like Figure 17 c. The fur of control C57BL / 6J mice was thick, shiny, and black, while the fur of D-gal-induced aged mice turned white, with noticeable bald patches. However, as the concentration of Frustule@FX increased, the fur of the aged mice increasingly resembled that of normal mice. In particular, at medium concentrations of Frustule@FX, the fur of the mice approached that of the control mice.

[0201] 3.3. Diatom frustules@fucoxanthin effectively improves spatial memory in aging model mice:

[0202] To investigate the effect of diatom frustules as FX carriers on spatial learning and memory ability in aging mice, this study conducted a water maze test on C57BL / 6J mice. Figure 18 a. During training, mice in the control group gradually developed a more direct swimming path, tending towards a linear or directional strategy, indicating that they were able to effectively use spatial information to find the hidden platform. However, the swimming paths of mice in the D-gal group were more complex, often exhibiting edge-based or random strategies. This suggests that the spatial navigation ability of aging mice was severely impaired, making it difficult for them to effectively use spatial cues to locate the platform. After administration of Frustule@FX, the swimming paths of aging mice returned to normal.

[0203] As shown in Figure 18b, the mean escape latency of mice in the control group was 15.88. Compared with the control group, the escape latency of mice in the D-gal group was prolonged by 1.42 times (p < 0.01). Administration of Frustule@FX gradually shortened the escape latency of aging mice. In particular, administration of high-concentration Frustule@FX reduced the escape latency by 55.46% (p < 0.01), and compared with administration of high-concentration FX alone, the escape latency was shortened by 14.03%.

[0204] like Figure 18 c. The mean percentage of platform quadrant swimming time for mice in the control group was 33.23. Compared with the control group, the percentage of platform quadrant swimming time in mice in the D-gal group decreased by 56.82% (p < 0.01). After administration of Frustule@FX, the percentage of platform quadrant swimming time in aging mice gradually increased. In particular, administration of high-concentration Frustule@FX increased the percentage of platform quadrant swimming time in aging mice by 3.26-fold (p < 0.01), and by 1.14-fold compared to administration of high-concentration FX alone.

[0205] like Figure 18 d. The mean number of platform crossings in the control group was 2.67. Compared with the control group, the number of platform crossings in the D-gal group was reduced by 75.00% (p < 0.01). After administration of Frustule@FX, the number of platform crossings in aging mice gradually increased. In particular, administration of high-concentration Frustule@FX increased the number of platform crossings by 1.88-fold (p < 0.01) and by 1.11-fold compared with administration of high-concentration FX alone.

[0206] These results indicate that Frustule@FX can more effectively improve spatial learning and memory abilities in D-gal-induced aging mice than FX alone.

[0207] 3.4 Effects of diatom frustules@fucoxanthin on D-gal-induced autophagy in the hippocampus of aging mice:

[0208] The hippocampus is a key area in the brain that is closely related to learning and memory. The maintenance of its neuronal health is inseparable from the participation of the autophagy process. In the study of the effect of Frustule@FX on the hippocampus of mice, immunohistochemistry was used to evaluate the expression changes of autophagy-related proteins. Figure 19 As shown, compared with the Control group, the levels of Beclin1 and LC3 in the hippocampus of D-gal-induced aged mice decreased by 53.23% (p<0.01) and 55.27% (p<0.01), respectively, while the p62 level increased significantly to 1.12 times (p<0.01). However, with the increase in the concentration of Frustule@FX, autophagy in the hippocampus of mice was activated. Especially when a medium concentration of Frustule@FX was administered, the levels of Beclin1 and LC3 in the hippocampus of mice increased by 1.45 times (p<0.01) and 1.53 times (p<0.01), respectively, and the p62 level decreased significantly by 77.53% (p<0.01). Combined Figure 12It can be seen that compared with the application of FX alone, Frustule@FX can better improve the autophagy-related indicators of aging mice and promote the positive effects of autophagy.

[0209] 3.5. Diatom frustules@fucoxanthin alleviates SA-β-galactosidase activity in the brains of D-gal-induced aging mice:

[0210] SA-β-gal is an important marker of aging, and the detection of its activity is of great significance for evaluating the degree of aging of tissues or cells. In this study, the effect of Frustule@FX on the activity of SA-β-gal in the brain of D-gal-induced aging mice was observed under a microscope. Figure 20 As shown, D-gal-induced aging in mice is manifested by increased SA-β-gal activity in the hippocampus and an increase in blue-stained cells, indicating that D-gal successfully induced aging in the mouse brain tissue. As Frustule@FX was administered to aging model mice, the blue-stained area decreased with increasing Frustule@FX concentrations. Furthermore, compared to the aging model group treated with FX alone ( Figure 10 ), Frustule@FX exhibited a better anti-aging effect. This suggests that Frustule@FX can more effectively inhibit SA-β-gal activity, thereby alleviating the degree of aging in the brain tissue of D-gal-induced aging mice.

[0211] 3.6 Effect of diatom frustules@fucoxanthin on D-gal-induced Nrf2 - / - Effects of pulling force and suspension ability on mice:

[0212] By observing the pulling force and suspension ability of mice, the effect of Frustule@FX on Nrf2 can be visually evaluated. - / - Effects on aging mice. Figure 21 As shown in a, there was no difference in pulling force and suspension ability between the Control (Nrf2- / -) group mice and the Control Frustule (Nrf2- / -) group mice. Figure 21 a, The mean tensile force of the Control Frustule(Nrf2- / -) group was 2.15N. Compared with the Control Frustule(Nrf2- / -) group, the tensile force of the D-gal Frustule(Nrf2- / -) group was reduced by 56.82% (p<0.01). However, after administration of high concentrations of Frustule@FX, Nrf2 - / - There were no significant differences in pulling strength between aged mice.

[0213] like Figure 21As shown in Figure b, the mean suspension test score for mice in the Control Frustule (Nrf2- / -) group was 2.83. Compared to the Control Frustule (Nrf2- / -) group, the D-gal Frustule (Nrf2- / -) group showed a 76.47% decrease in the suspension test score (p < 0.01). Administration of high-concentration Frustule@FX did not significantly affect the suspension test in Nrf2- / - aging mice.

[0214] From the above, it can be seen that Frustule@FX has no significant effect on the pulling force and suspension ability of Nrf2- / - aging mice.

[0215] 3.7. Diatom frustules@fucoxanthin cannot improve Nrf2 - / - Mouse memory:

[0216] like Figure 22As shown, there were no significant differences in water maze performance between control (Nrf2- / -) and control Frustule (Nrf2- / -) mice. Over the course of training, mice gradually developed more direct swimming paths, tending toward linear or directional strategies, indicating that they effectively utilized spatial information to locate the hidden platform. However, the swimming paths of mice treated with D-gal Frustule (Nrf2- / -) were more complex, often exhibiting edge-based or random strategies. This suggests that aging mice have severely impaired spatial navigation abilities and are unable to effectively use spatial cues to locate the platform. Administration of Frustule@FX restored the swimming paths of aging mice to normal. The mean percentage of time spent swimming in the platform quadrant for mice treated with Control Frustule (Nrf2- / -) was 33.00%. Compared with the Control Frustule (Nrf2- / -) group, the percentage of time spent swimming in the platform quadrant in the D-gal Frustule (Nrf2- / -) group was significantly decreased by 73.67% (p < 0.01). The mean number of platform crossings in the control Frustule (Nrf2- / -) group was 2.66, while the number in the D-gal Frustule (Nrf2- / -) group was significantly decreased by 58.50% (p < 0.01). The mean escape latency in the control Frustule (Nrf2- / -) group was 15.83, while the D-gal Frustule (Nrf2- / -) group significantly increased this by 1.88-fold (p < 0.01), indicating that D-gal leads to slower movement and reaction times in Nrf2- / - aged mice. However, administration of Frustule@FX showed no significant differences in the percentage of time spent swimming in the platform quadrant, the number of platform crossings, or the escape latency compared to the D-gal Frustule (Nrf2- / -) group in Nrf2- / - aged mice (p < 0.01). This suggests that Nrf2 deficiency limits the effects of Frustule@FX on improving spatial learning and memory in aged mice.

[0217] 3.8. Diatom frustules@fucoxanthin cannot activate Nrf2 - / - Autophagy in the mouse hippocampus:

[0218] In order to explore whether the Nrf2-autophagy pathway is an important pathway for treating neurological diseases caused by aging, the following experiments were conducted. Figure 23 As shown in a, compared with the Control (Nrf2- / -) group, D-gal-induced Nrf2 - / -The p62 protein level in the hippocampus of aged mice increased significantly by 1.12 times (p<0.01), while the Beclin1 and LC3 protein levels decreased by 36.78% and 53.42%, respectively (p<0.01). Compared with the D-gal Frustule group, Frustule@FX increased the p62 protein level in Nrf2- / - aged mice, while the Beclin1 and LC3 protein levels continued to decrease. This indicates that autophagy activation is inhibited and the autophagy process cannot be activated normally. Therefore, Frustule@FX cannot reverse the Nrf2 - / - Autophagy inhibition in aging mice.

[0219] like Figure 23 As shown in b, the autophagosomes in the hippocampal neurons of the Control (Nrf2- / -) group of mice were regular in shape. Compared with the Control (Nrf2- / -) group, the autophagosomes of D-gal-induced aged Nrf2- / - mice were irregular in shape, with a fuzzy double-layer membrane structure and complex contents. These abnormal morphologies may reflect the obstruction of the autophagosome formation and maturation process in aged mice. Even under high concentrations of Frustule@FX intervention, Nrf2 - / - These results indicate that Nrf2 is an important target for Frustule@FX to activate D-gal-induced autophagy in aged mice.

[0220] As can be seen from Verification Example 3, the hollow structure of the diatom shell and the porous structure on the surface were observed by scanning electron microscopy, which are conducive to the adsorption of FX; in behavioral experiments, Frustule@FX significantly improved the spatial learning and cognitive function of D-gal-induced aging mice. In addition, Frustule@FX can also activate the autophagy pathway in the hippocampus of aging mice and improve the state of autophagosomes. The SA-β-gal staining experiment further verified the reversal effect of Frustule@FX on D-gal-induced aging neurons in the hippocampus of mice. It is worth noting that this study found that Frustule@FX had no effect in Nrf2 gene knockout mice, which further highlights the important role of the Nrf2-autophagy pathway in neuroprotection in aging mice. The above results show that Frustule@FX can improve the movement, memory and learning ability of D-gal-induced aging mice, activate autophagy, and thus exert a neuroprotective effect on aging mice.

[0221] The present invention will be described below in conjunction with specific embodiments:

[0222] Example 1:

[0223] A diatom frustules@fucoxanthin oral drug delivery system is described. 1 ml of a 400 μg / ml fucoxanthin solution is added to 10 ml of a 50 μg / ml diatom frustules suspension. The mixture is stirred for 12 hours and then centrifuged at 3260 g for 10 minutes. The solid precipitate is then collected and freeze-dried at -20°C for 24 hours to obtain the Frustule@FX oral drug delivery system. The Frustule@FX oral drug delivery system is administered orally at a dosage of 200 mg / kg / day, based on the weight of the fucoxanthin.

[0224] Example 2:

[0225] A fucoxanthin solution for oral administration is obtained by dissolving the fucoxanthin in olive oil. The concentration of the fucoxanthin solution is 400 μg / ml. The fucoxanthin solution is for oral administration, and the oral dosage, calculated based on the weight of the fucoxanthin, is 200 mg / kg / d.

[0226] The above examples are only for illustrating the technical concept and technical features of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent transformation or modification made based on the essence of the present invention should be included in the scope of protection of the present invention.

Claims

1. A diatom frustule@fucoxanthin oral drug delivery system, characterized in that: The fucoxanthin solution is prepared by compounding a fucoxanthin solution and a diatom frustule suspension. The concentration of the fucoxanthin solution is 25-1600 μg / ml, and the concentration of the diatom frustule suspension is 50 μg / ml.

2. The diatom frustules@fucoxanthin oral drug delivery system according to claim 1, characterized in that: The composite volume ratio of the fucoxanthin solution and the diatom frustule suspension is 1:

10.

3. The diatom frustules@fucoxanthin oral drug delivery system according to claim 1, characterized in that: The fucoxanthin solution is obtained by dissolving fucoxanthin in olive oil; the diatom frustule suspension is obtained by placing diatom frustules in deionized water and stirring.

4. The diatom frustules@fucoxanthin oral drug delivery system according to claim 1, characterized in that: The concentrations of the fucoxanthin solution are 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 400 μg / ml, 800 μg / ml and 1600 μg / ml.

5. The diatom frustules@fucoxanthin oral drug delivery system according to claim 4, characterized in that: The concentration of the fucoxanthin solution is 400 μg / ml.

6. A method for preparing the diatom frustules@fucoxanthin oral drug delivery system according to claims 1 to 5, characterized in that: The following steps are involved: The fucoxanthin solution was added to the diatom frustule suspension and mixed. The mixture was stirred for 12 hours and then centrifuged at 3260 g for 10 minutes. After the centrifugation, the solid precipitate was collected and freeze-dried at -20°C for 24 hours to obtain the diatom frustule@fucoxanthin oral delivery system.

7. Provided is a use of the diatom frustules@fucoxanthin oral drug delivery system according to claims 1 to 5 in the preparation of drugs for treating and preventing neuroaging diseases.

8. Use of the diatom frustules@fucoxanthin oral drug delivery system according to claim 7 in the preparation of drugs for treating and preventing neuroaging diseases, characterized in that: The diatom frustules@fucoxanthin oral drug delivery system is administered orally, and the oral dosage is 200 mg / kg / d based on the mass of fucoxanthin.

9. A use of fucoxanthin in the preparation of a drug for intervention of neuroaging diseases, characterized in that: The fucoxanthin solution obtained by dissolving the fucoxanthin in olive oil has a concentration of 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 400 μg / ml, 800 μg / ml and 1600 μg / ml; the fucoxanthin solution is for oral administration at a dosage of 50 mg / kg / d, 100 mg / kg / d and 200 mg / kg / d.

10. The use of fucoxanthin in the preparation of drugs for intervention of neuroaging diseases according to claim 9, characterized in that: The concentration of the fucoxanthin solution is 400 μg / ml; the fucoxanthin solution is for oral administration, and the oral dosage is 200 mg / kg / d based on the mass of fucoxanthin.

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