Fructus alpiniae oxyphyllae-derived extracellular vesicle-like nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses fructus alpiniae oxyphyllae-derived extracellular vesicle-like nanoparticles as well as a preparation method and application thereof. The preparation method comprises the following steps: soaking a fructus alpiniae oxyphyllae raw material, breaking walls, crushing, centrifugally filtering, concentrating by tangential flow, centrifuging in multiple stages, filtering and the like. According to the preparation method provided by the invention, differential centrifugation is combined with a tangential flow concentration technology, so that the extracellular vesicle-like nanoparticles can be efficiently and completely enriched and purified from the fructus alpiniae oxyphyllae. The whole process is carried out at low temperature, so that the biological activity of the vesicles is protected to the greatest extent, the use of an organic solvent is avoided, and the naturalness and safety of the product are ensured. According to the fructus alpiniae oxyphyllae-derived nanoparticles, by activating IRS-1, promoting PI3K activation, increasing PIP3 generation and activating Akt, GSK-3beta is subjected to phosphorylation to inactivate GSK-3beta, excessive phosphorylation and accumulation of Tau protein are reduced, and plaque formation is delayed. In a diabetic cognitive impairment model, the nanoparticles have a remarkable neuroprotective effect and can improve the cognitive function and reduce inflammatory response.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an extracellular vesicle-like nanoparticle extracted from Alpinia oxyphylla, its preparation method, and its application in preventing or treating diabetic cognitive impairment, promoting neuronal cell survival, inhibiting insulin resistance, and improving cognitive function. Background Technology

[0002] Diabetic cognitive impairment (DCI) is a common chronic neurological complication of diabetes, characterized by acquired cognitive decline, behavioral abnormalities, and accompanying brain morphological changes and neurophysiological dysfunction. A 2025 study indicated that the prevalence of cognitive impairment in patients with type 2 diabetes was as high as 41.70%, rising to 70.70% in patients with diabetes for more than 7 years. This cognitive impairment not only significantly impacts patients' daily living abilities but may also lead to increased needs for personal care, higher hospitalization rates, and a higher incidence of depression. Furthermore, cognitive decline weakens patients' ability to manage their disease, further affecting glycemic control, thus creating a vicious cycle that complicates treatment and care. Therefore, early diagnosis and treatment of diabetes-related cognitive impairment are crucial for delaying cognitive decline and improving patient outcomes.

[0003] Alpiniae Oxyphyliae Fructus, the dried, mature fruit of the ginger family Alpiniae Oxyphyliae, was first recorded in Chen Zangqi's *Compendium of Materia Medica*. It is pungent and warm in nature; although its astringent taste is not mentioned, its medicinal properties are extensively documented in various herbal texts throughout history. Alpiniae Oxyphylla is warm in nature and pungent in taste, entering the spleen and kidney meridians, and has the effects of warming the spleen and stopping diarrhea, warming the kidneys and consolidating essence, and reducing urination. Extracellular vesicles (EVs) are tiny vesicles secreted by cells, containing various proteins, lipids, and nucleic acids. They mediate intercellular communication and play important physiological functions. Almost all types of eukaryotic and prokaryotic cells can secrete EVs. Plant-derived EVs are morphologically similar to mammalian EVs, but research on their composition and function is relatively limited. In recent years, studies have found that plant EVs are an important component of the plant's innate immune system and have antifungal effects. Furthermore, plant EVs exhibit cross-species regulatory functions, not only regulating the physiological functions of mammalian cells but also intervening in and preventing disease processes, and possessing potential therapeutic effects for certain diseases. These studies indicate that plant-derived EVs, as a novel type of natural product, may be a promising candidate source for new drug development. Traditional Chinese medicine (TCM) is largely derived from plants, offering advantages such as low cost and minimal side effects. However, research on TCM-derived EVs is still in its early stages, and future in-depth research is expected to further explore their application potential in the medical field.

[0004] Alpinia oxyphylla, a traditional Chinese medicine, has the effects of warming the spleen and stopping diarrhea, warming the kidneys and consolidating essence and reducing urination. However, there is little research on its application in the treatment of cognitive impairment in diabetes and its related active ingredients. Whether it is feasible to extract extracellular vesicles from Alpinia oxyphylla, and whether the extracted Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles have biological activity, remains unknown.

[0005] Current treatments for cognitive impairment in diabetes have limitations, including limited efficacy of medications, numerous side effects, difficulty in reversing cognitive impairment progression, and inability to repair damaged nerve function. Summary of the Invention

[0006] In view of this, in order to solve the technical problems existing in the prior art, the present invention provides an Alpinia oxyphylla-derived extracellular vesicle-like nanoparticle, which uses differential centrifugation combined with tangential flow concentration technology to efficiently and completely enrich and purify extracellular vesicle-like nanoparticles from Alpinia oxyphylla, providing a new strategy for the research and development of plant-derived extracellular vesicles and the research and treatment of cognitive impairment in diabetes, and has important scientific significance and clinical application value. This invention also provides a method for its preparation and application.

[0007] On one hand, the present invention provides a method for preparing Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles, the preparation method comprising the following steps:

[0008] Step 1) Raw material pretreatment: Soak 300g-1000g of dried Alpinia oxyphylla raw material in a pre-cooled phosphate buffer solution and then crush it to obtain juice;

[0009] Step 2) Centrifugation and filtration: Centrifuge the juice obtained in step 1) at a centrifugal force of 1000-4000g for 20-40min, take the supernatant and filter it through a 1-5μm filter membrane, and collect the supernatant;

[0010] Step 3) Tangential flow concentration: The supernatant obtained in step 2) is concentrated by tangential flow to 10-40% of its original volume to obtain a concentrated solution;

[0011] Step 4) Centrifugation of concentrate: Centrifuge the concentrate obtained in step 3), collect the supernatant and filter it;

[0012] Step 5) Ultracentrifugation: Place the filtrate obtained in step 4) into an ultracentrifuge for ultracentrifugation and collect the precipitate;

[0013] Step 6) Precipitation and filtration: After resuspending the precipitate obtained in step 5) with phosphate buffer solution, filter it through a 0.22-0.30 μm filter membrane to obtain Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles.

[0014] Further, in step 4), the centrifugation of the concentrate is carried out at 0-8°C, with a centrifugal force of 8000-15000g and a centrifugation time of 40-100min; the filtration is carried out through a 0.45μm filter membrane.

[0015] Furthermore, the ultracentrifugation of the filtrate in step 5) is carried out at 0-8°C, with a centrifugal force of 80,000-150,000g and a centrifugation time of 40-100min.

[0016] Furthermore, the Alpinia oxyphylla raw material mentioned in step 1) is Alpinia oxyphylla slices.

[0017] On the other hand, the present invention also provides an Alpinia oxyphylla-derived extracellular vesicle-like nanoparticle, which is prepared by the preparation method described in claim 1. The particle size of the Alpinia oxyphylla-derived extracellular vesicle-like nanoparticle is distributed between 50-500 nm, and carries Alpinia oxyphylla-derived bioactive molecules.

[0018] This invention further provides applications of the above-mentioned Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles, specifically including:

[0019] Application of the Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles in the preparation of drugs for the prevention or treatment of cognitive impairment in diabetes.

[0020] Application of the Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles in the preparation of a promoter for the survival of hippocampal neurons.

[0021] Application of the Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles in the preparation of inhibitors of insulin resistance response in nerve cells.

[0022] The application of the Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles in the preparation of cognitive function improvers.

[0023] Application of Alpinia oxyphylla extracellular vesicle-like nanoparticles in the preparation of IRS-1 / PI3K / Akt signaling pathway activators.

[0024] Compared with the prior art, the technical solution of the present invention has at least the following beneficial effects:

[0025] 1) The preparation method of this invention includes soaking, cell wall disruption and pulverization, centrifugation and filtration, tangential flow concentration, multi-stage centrifugation and filtration of Alpinia oxyphylla raw material. The preparation method provided by this invention uses differential centrifugation combined with tangential flow concentration technology, which can efficiently and completely enrich and purify extracellular vesicle-like nanoparticles from Alpinia oxyphylla. The entire process is carried out at low temperature, which maximizes the protection of the bioactivity of the vesicles, avoids the use of organic solvents, and ensures the naturalness and safety of the product.

[0026] 2) The Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles prepared in this invention can promote the proliferation of HT22 cells or inhibit the insulin resistance and cognitive impairment response of HT22 cells at the cellular level (in vitro level). By activating the IRS-1 / PI3K / Akt signaling pathway, regulating GSK-3β activity, reducing abnormal phosphorylation and accumulation of Tau protein, and inhibiting the formation of neurofibrillary tangles, the pathological characteristics of diabetic cognitive impairment are improved. The Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles exert a therapeutic effect on diabetic cognitive impairment through multi-target action (IRS-1 / PI3K / Akt signaling pathway, GSK-3β, Tau protein, etc.), with significant effects. Attached Figure Description

[0027] Figure 1 This is a particle size distribution diagram of the Alpinia oxyphylla-derived extracellular vesicles (AOF-EVLPs) of this invention;

[0028] Figure 2 This is a graph showing the purity test results of the Alpinia oxyphylla-derived extracellular vesicles (AOF-EVLPs) of this invention;

[0029] Figure 3 This is a transmission electron microscope image of the Alpinia oxyphylla-derived extracellular vesicles (AOF-EVLPs) of this invention;

[0030] Figure 4 This is a diagram showing the metabolite categories of the Alpinia oxyphylla-derived extracellular vesicles (AOF-EVLPs) of this invention;

[0031] Figure 5 This is a graph showing the results of HT22 cell internalization in this invention (where A is the raw data from flow cytometry; B is the statistical result of cell uptake rate at different time points; and C is the statistical result of average fluorescence intensity of cells at different time points).

[0032] Figure 6 This is a confocal scanning microscope image of the Alpinia oxyphylla-derived extracellular vesicles (AOF-EVLPs) of the present invention.

[0033] Figure 7 This is a graph showing the results of the CCK-8 assay used in this invention to detect the proliferation of RAW264.7 cells by AOF-EVLPs;

[0034] Figure 8 This is a diagram showing the establishment of the HT22 cell IR model induced by ddxm and the screening of the optimal concentration of AOF-EVLPs to improve IR.

[0035] Figure 9 This is a graph showing the RNA levels of various molecules in HT22 cells according to the present invention (A is IRS-1, B is PI3K, C is Akt, D is GSK-3β, E is Tau).

[0036] Figure 10 This is a graph showing the protein levels of various molecules in HT22 cells according to the present invention (A is the protein expression levels of IRS-1, PI3K, Akt, GSK-3β, and Tau in DXM-stimulated cells treated with AOF-EVLPs analyzed by Western blotting; B is the relative protein expression level of IRS-1; C is the relative protein expression level of PI3K; D is the relative protein expression level of Akt; E is the relative protein expression level of GSK-3β; F is the relative protein expression level of Tau).

[0037] Figure 11 This is a graph showing the effect of the AOF-EVLPs of this invention on the expression level of Tau protein.

[0038] Figure 12 This is a diagram showing the effect of the AOF-EVLPs of this invention on glycogen deposition in IR. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings, which form part of this specification. The principles of the invention are illustrated by means of embodiments, and other aspects, features, and advantages of the invention will become apparent from this detailed description. In the accompanying drawings, the same or similar parts in different figures are indicated by the same reference numerals.

[0040] On one hand, the present invention provides a method for preparing Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles, the method comprising the following steps:

[0041] Step 1) Raw material pretreatment: Soak 300g-1000g of dried Alpinia oxyphylla raw material in a pre-cooled phosphate buffer solution and then crush it to obtain juice;

[0042] Step 2) Centrifugation and filtration: Centrifuge the juice obtained in step 1) at a centrifugal force of 1000-4000g for 20-40min, take the supernatant and filter it through a 1-5μm filter membrane, and collect the supernatant;

[0043] Step 3) Tangential flow concentration: The supernatant obtained in step 2) is concentrated by tangential flow to 10-40% of its original volume to obtain a concentrated solution;

[0044] Step 4) Centrifugation of concentrate: Centrifuge the concentrate obtained in step 3), collect the supernatant and filter it;

[0045] Step 5) Ultracentrifugation: Place the filtrate obtained in step 4) into an ultracentrifuge for ultracentrifugation and collect the precipitate;

[0046] Step 6) Precipitation and Filtration: The precipitate obtained in Step 5) is resuspended in phosphate buffer solution and then filtered through a 0.22-0.30 μm filter membrane to obtain Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles. The preparation method of this invention includes soaking, cell wall disruption and pulverization, centrifugation and filtration, tangential flow concentration, multi-stage centrifugation, and filtration of Alpinia oxyphylla raw materials. The preparation method provided by this invention uses differential centrifugation combined with tangential flow concentration technology, which can efficiently and completely enrich and purify extracellular vesicle-like nanoparticles from Alpinia oxyphylla. The entire process is carried out at low temperature, maximizing the protection of the vesicle's biological activity, avoiding the use of organic solvents, and ensuring the naturalness and safety of the product. The Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles obtained by the preparation method of this invention activate IRS-1, promote PI3K activation, increase PIP3 generation, activate Akt, and then phosphorylate GSK-3β to inactivate it, reducing Tau protein hyperphosphorylation and accumulation, and delaying plaque formation. Furthermore, the preparation method of this invention extracts from Alpinia oxyphylla raw materials, resulting in Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles with higher purity. In a diabetic cognitive impairment model, these Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles have significant neuroprotective effects, improving cognitive function and reducing inflammatory responses, providing new strategies and methods for the prevention or treatment of diabetic cognitive impairment.

[0047] In practice, the pH value of the phosphate buffer solution described in step 6) is 7.2-7.6.

[0048] The centrifugation of the concentrate in step 4) is carried out at 0-8℃, with a centrifugal force of 8000-15000g and a centrifugation time of 40-100min; the filtration is carried out through a 0.45μm filter membrane.

[0049] The ultracentrifugation of the filtrate in step 5) is carried out at 0-8℃, with a centrifugal force of 80000-150000g and a centrifugation time of 40-100min.

[0050] The raw material for Alpinia oxyphylla mentioned in step 1) is Alpinia oxyphylla slices.

[0051] On the other hand, the present invention also provides an Alpinia oxyphylla-derived extracellular vesicle-like nanoparticle, which is prepared by the preparation method described in claim 1. The particle size of the Alpinia oxyphylla-derived extracellular vesicle-like nanoparticle is distributed between 50-500 nm, and carries Alpinia oxyphylla-derived bioactive molecules.

[0052] This invention further provides applications of the above-mentioned Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles, specifically including:

[0053] Application of Alpinia oxyphylla extracellular vesicle-like nanoparticles in the preparation of drugs for the prevention or treatment of cognitive impairment in diabetes;

[0054] Application of Alpinia oxyphylla extracellular vesicle-like nanoparticles in the preparation of a promoter for the survival of hippocampal neurons;

[0055] Application of Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles in the preparation of inhibitors of insulin resistance response in nerve cells;

[0056] Application of Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles in the preparation of cognitive function improvers;

[0057] Application of Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles in the preparation of IRS-1 / PI3K / Akt signaling pathway activators. This invention demonstrates that Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles possess excellent neuroprotective activity and show potential in various applications, such as: significantly promoting the survival of hippocampal neurons; inhibiting insulin resistance in neurons; and ultimately improving cognitive function, particularly showing outstanding effects in the prevention and treatment of diabetic cognitive impairment. This provides a novel material basis and candidate for developing new drugs for related neurological diseases.

[0058] The *Alpinia oxyphylla*-derived extracellular vesicle-like nanoparticles prepared in this invention can promote the proliferation of HT22 cells or inhibit the insulin resistance and cognitive impairment responses of HT22 cells at the cellular level (in vitro). By activating the IRS-1 / PI3K / Akt signaling pathway, regulating GSK-3β activity, reducing abnormal phosphorylation and accumulation of Tau protein, and inhibiting neurofibrillary tangles formation, they improve the pathological characteristics of diabetic cognitive impairment. These *Alpinia oxyphylla*-derived extracellular vesicle-like nanoparticles exert a therapeutic effect on diabetic cognitive impairment through multi-target action (IRS-1 / PI3K / Akt signaling pathway, GSK-3β, Tau protein, etc.), with significant efficacy. In other words, the *Alpinia oxyphylla*-derived extracellular vesicle-like nanoparticles can be formulated as a simple experimental preparation for exploring the physiological metabolic processes of HT22 cells. The cell proliferation promoters or cellular cognitive impairment inhibitors claimed in this invention are not intended to eliminate the cause or lesion; that is, they are applications in the preparation of HT22 cell proliferation promoters or HT22 cell cognitive impairment inhibitors for non-therapeutic purposes.

[0059] Example 1

[0060] Example 1 of this invention provides a method for preparing Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles, which includes the following steps:

[0061] Step 1) Raw material pretreatment: Soak 300g of dried Alpinia oxyphylla slices in a pre-cooled phosphate buffer solution and then crush them to obtain juice;

[0062] Step 2) Centrifugation and filtration: Centrifuge the juice obtained in Step 1) at 4℃ and 3000g for 30 minutes, take the supernatant and filter it through a 4μm filter membrane, collect the supernatant and take the supernatant.

[0063] Step 3) Tangential flow concentration: The supernatant obtained in step 2) is concentrated by tangential flow to 20% of its original volume to obtain a concentrated solution;

[0064] Step 4) Centrifugation of concentrate: Centrifuge the concentrate obtained in step 3) at 10000g for 60min, take the supernatant and filter it through a 0.22μm filter membrane;

[0065] Step 5) Ultracentrifugation: Place the filtrate obtained in step 4) into an ultracentrifuge and ultracentrifuge at 4°C and 100,000g for 70 minutes, and collect the precipitate.

[0066] Step 6) Precipitation filtration: Place the precipitate obtained in step 5) into each centrifuge tube with 1 mL of pre-cooled phosphate buffer solution, resuspend it in the phosphate buffer solution, filter it through a 0.22 filter membrane to obtain the resuspended solution, and store it in a freezer at -80℃ for later use.

[0067] Example 2: Validation of characterization of Alpinia oxyphylla-derived extracellular vesicles (AOF-EVLPs)

[0068] The Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles (AOF-EVLPs) prepared in Example 1 above were characterized in various ways to confirm their particle size, purity, morphology and chemical composition.

[0069] (1) Characterization of particle size distribution of Alpinia oxyphylla-derived extracellular vesicles (AOF-EVLPs)

[0070] A suitable amount of the AOF-EVLPs sample prepared in Example 1 was taken, and its particle size distribution was detected using a nanoflow cytometer (NanoFCM). The detection results are as follows: Figure 1 As shown, the AOF-EVLPs exhibit a concentrated particle size distribution with an average particle size of 86.25 nm. This result demonstrates that the AOF-EVLPs extracted using the method of this invention have a particle size range consistent with the standard extracellular vesicle particle size range reported in the literature (typically 30-150 nm), indicating successful extraction of the target vesicle population.

[0071] (2) Purity characterization of AOF-EVLPs

[0072] The purity of AOF-EVLPs was verified using a Triton X-100 membrane disruption assay. Specifically, the AOF-EVLPs sample was divided into several portions and treated with different concentrations (0%, 0.01%, 0.05%, 0.1%, and 0.5%) of Triton X-100 to dissolve the vesicle membrane structure. The number of particles before and after treatment in each group was counted using nanoflow cytometry. The results are as follows: Figure 2 As shown, the number of particles decreased significantly after treatment with Triton X-100. This phenomenon indicates that the vast majority of particles extracted by the present invention are vesicles with membrane structures, rather than protein aggregates or other non-membrane structure impurities, proving the high purity of the product obtained by the extraction method of the present invention.

[0073] (3) Morphological characterization of AOF-EVLPs

[0074] The microstructure of AOF-EVLPs was observed using transmission electron microscopy. A small sample of AOF-EVLPs was negatively stained and then observed under an electron microscope. The results are as follows: Figure 3As shown, under transmission electron microscopy, the AOF-EVLPs exhibit a typical spherical or teacup-shaped morphology with surface depressions. The vesicle membrane structure is clear and intact, with sharp and distinct boundaries. The entire field of view has a clean stained background and significant contrast, further confirming the high purity of the sample and the absence of significant impurities. This observation confirms that the AOF-EVLPs extracted in this invention possess complete and standard extracellular vesicle morphological characteristics.

[0075] (4) Chemical composition characterization of AOF-EVLPs

[0076] A comprehensive analysis of the chemical composition of AOF-EVLPs was performed using non-targeted metabolomics technology. The results are as follows: Figure 4 As shown in Figure A, the AOF-EVLPs are rich in various bioactive components, mainly including lipids, organic acids, amino acids and their derivatives, alkaloids, and benzene compounds. This composition indicates that AOF-EVLPs effectively enrich and retain various active substances in Alpinia oxyphylla, providing a material basis and explanation of its mechanism of action for its subsequent physiological functions in pharmaceuticals, health products, and other fields.

[0077] Furthermore, a detailed classification and proportion analysis of lipid components was performed, and the results are as follows: Figure 4 As shown in Figure B, the lipid composition of the AOF-EVLPs is predominantly composed of free fatty acids, approximately 90%. Other components include glycerides (0.91%), lysophosphatidylcholine (LPC, 4.55%), lysophosphatidylethanolamine (LPE, 1.82%), and sphingolipids (2.73%). This unique lipid profile, particularly the high proportion of free fatty acids, reveals the distinctive chemical properties of AOF-EVLPs compared to extracellular vesicles from other sources, which are closely related to their unique stability and functional activity.

[0078] Conclusion: The above characterization results collectively demonstrate that the AOF-EVLPs extracted from Alpinia oxyphylla using the preparation method of this invention (Example 1) not only possess standard extracellular vesicle characteristics (including suitable nanoscale particle size, intact membrane structure, and typical teacup-shaped morphology), but also exhibit extremely high purity and a unique chemical composition. In particular, its rich content of various active ingredients demonstrates its potential as a carrier of active substances from Alpinia oxyphylla, providing a solid basis for the development of innovative drugs or health products based on plant-derived extracellular vesicles.

[0079] Example 3: Validation experiment on the internalization and uptake of Alpinia oxyphylla-derived extracellular vesicles (AOF-EVLPs) by mouse hippocampal neurons (HT22).

[0080] 1. Experimental Objective

[0081] Example 3 aims to verify that Alpinia oxyphylla-derived extracellular vesicles (AOF-EVLPs) can be efficiently internalized and absorbed by mouse hippocampal neurons (HT22 cells), which is a functional prerequisite for the subsequent application of AOF-EVLPs in the treatment or improvement of nervous system diseases.

[0082] 2. Preparation of fluorescent dye-labeled AOF-EVLPs (DiI-AOF-EVLPs)

[0083] Take 500 μL of a concentration of 6 × 10 11 A suspension of AOF-EVLPs particles / mL was placed in a sterile EP tube, and 500 μL of phosphate buffer (PBS buffer, pH 7.4) was added and gently mixed. Then, 3 μL of DiI cell membrane fluorescent dye (1,1'-octadecyl-3,3,3',3'-tetramethylindole carboxycyanine perchlorate, DiI; Waltham, MA, USA) was added to the mixture, and vortexed to mix thoroughly.

[0084] The above mixture was incubated at 37°C in the dark for 30 minutes to ensure sufficient binding of the dye to the vesicle membrane. After incubation, the EP tube was placed in an ultracentrifuge and centrifuged at 100,000g for 70 minutes at 4°C. After centrifugation, the supernatant was carefully discarded to remove unbound free DiI dye.

[0085] The precipitate was gently resuspended in 1 mL of pre-chilled phosphate-buffered saline (PBS buffer, pH 7.4) and centrifuged again at 4°C and 100,000 g for 70 minutes. This washing process was repeated a total of 3 times to ensure complete removal of unbound dye. Finally, the labeled vesicle precipitate was resuspended in 1 mL of phosphate-buffered saline (PBS buffer, pH 7.4) to obtain a concentration of 3 × 10⁻⁶. 11 DiI-AOF-EVLPs solution with particles / mL was aliquoted and stored at -80°C in the dark for later use.

[0086] 4. Flow cytometry analysis of the internalization and uptake of AOF-EVLPs in HT22 cells:

[0087] HT22 mouse hippocampal neurons in good growth condition were distributed at a concentration of 1 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of 1000 cells per well in 6-well plates and incubated overnight at 37°C in a 5% CO2 incubator to allow for full cell adhesion.

[0088] Discard the old culture medium, and add DiI-AOF-EVLPs (concentration of 3×10⁻⁶) to each well of the experimental group. 11 Fresh, complete culture medium (particles / mL) was used to ensure full contact between vesicles and cells. A cell group without DiI-AOF-EVLPs was set up as a negative control. Cells were co-cultured in an incubator for 2 hours, 4 hours, and 8 hours, as shown in Table 1 below.

[0089]

[0090]

[0091] Table 1

[0092] After incubation, discard the culture medium and gently wash the cells three times with pre-chilled phosphate-buffered saline (PBS) to thoroughly remove uninternalized vesicles. Digest and collect the cells with trypsin, resuspend in PBS, centrifuge and wash, and finally resuspend the cells in 400 μL of PBS. Pass the cells through a 300-mesh sieve to prepare a single-cell suspension, and immediately perform flow cytometry analysis (BDFACSymphony). TM The excitation wavelength was 549 nm and the emission wavelength was 565 nm. At least 10,000 cell events were collected for each sample, and the data were analyzed using FlowJo V10.8.1 software. The fluorescence background threshold was set with negative control cells, and the percentage of DiI positive cells (i.e., uptake rate) and mean fluorescence intensity (MFI) of the experimental group cells were calculated to quantitatively evaluate the internalization efficiency and extent of AOF-EVLPs.

[0093] Experimental results: The experimental results are as follows Figure 5 As shown (where, Figure 5 A is a scatter plot representing flow cytometry findings; Figure 5 B is a graph showing the statistical results of the uptake rate of DiI-AOF-EVLPs by HT22 cells at different time points; Figure 5 (C represents the statistical results of the average fluorescence intensity of the cell population at different time points). After co-culturing with HT22 cells, DiI-AOF-EVLPs were effectively internalized by the cells. With the extension of co-culture time (from 2h to 8h), the uptake rate and average fluorescence intensity of DiI-AOF-EVLPs by HT22 cells showed a significant increasing trend. Figure 5 B, Figure 5 As shown in C, after 8 hours of co-culture, most HT22 cells internalized DiI-AOF-EVLPs, and the vesicle load in each cell was extremely high, indicating that AOF-EVLPs can be efficiently and rapidly internalized and absorbed by neuronal cells.

[0094] 5. Confocal scanning microscopy was used to detect the internalization and uptake of AOF-EVLPs by HT22 cells:

[0095] HT22 mouse hippocampal neurons in good growth condition were distributed at 5 × 10⁵ cells per well. 4 Cells were seeded at a density of 1000 cells per well in 6-well plates and incubated overnight at 37°C in a 5% CO2 incubator to allow for full cell adhesion.

[0096] Discard the old culture medium, and add DiI-AOF-EVLPs (concentration of 3×10⁻⁶) to each well of the experimental group. 11 Fresh, complete culture medium (particles / mL) was used to ensure full contact between vesicles and cells. A cell group without DiI-AOF-EVLPs was set up as a negative control. Cells were co-cultured in an incubator for 2 hours, 4 hours, 8 hours, and 24 hours, as shown in Table 1 below.

[0097]

[0098]

[0099] Table 1

[0100] After incubation, discard the culture medium and gently wash the cells three times with pre-cooled phosphate-buffered saline (PBS buffer, pH 7.4) to thoroughly remove any uninternalized vesicles. Add 1 ml of paraformaldehyde to each well to fix cells for 20 minutes. Discard the paraformaldehyde and wash three times with phosphate-buffered saline (PBS, pH 7.4). After shaking several times on a shaker, treat cells with permeabilizing agent (0.1% Triton X-100) at room temperature for 1 hour. Wash three times with PBS (PBS, pH 7.4). Add 1 μL of FITC-labeled phalloidin stock solution to 1 mL of PBS (containing 1% BSA) buffer to obtain 1× working solution. Add 100 μL of the prepared FITC-labeled phalloidin working solution to each well to cover the cells. Incubate at room temperature in the dark for 30 minutes. Wash three times with PBS (PBS, pH 7.4). Add 20-50 μL of DAPI to each well and incubate in the dark for 10 minutes. Finally, wash three times with PBS (PBS) and immediately pass the cells through a confocal scanning microscope (Oxford). Instruments Andor (BC43, UK) captured fluorescence images of cells to analyze the uptake, distribution, and interaction of AOF-EVLPs with the cytoskeleton in HT22 cells.

[0101] Experimental results: The experimental results are as follows Figure 6As shown, DiI-AOF-EVLPs can be effectively internalized by HT22 cells after co-culturing. With the extension of co-culture time (from 2h to 24h), the uptake rate of DiI-AOF-EVLPs by HT22 cells showed a significant increasing trend; and after 24 hours of co-culture, most HT22 cells had internalized DiI-AOF-EVLPs, and the vesicle load in each cell was extremely high, indicating that AOF-EVLPs can be efficiently and rapidly internalized and absorbed by neuronal cells.

[0102] 6. Conclusion: This Example 3 demonstrates that the Alpinia oxyphylla-derived extracellular vesicles (AOF-EVLPs) provided by this invention can be efficiently internalized and absorbed by mouse hippocampal neurons (HT22 cells), with a significant degree of internalization, and the internalization efficiency is time-dependent. This result provides important in vitro experimental evidence for AOF-EVLPs as a carrier of active ingredients for crossing the blood-brain barrier and treating nervous system diseases.

[0103] Example 4: Bioactivity Validation of Alpinia oxyphylla-derived Extracellular Vesicles (AOF-EVLPs)

[0104] Example 4 verified the proliferation effect of AOF-EVLPs on the mouse hippocampal neuronal cell line HT22 and its effect on improving the dexamethasone (ddxm)-induced insulin resistance (IR) model through a series of in vitro cell experiments. It also explored the molecular mechanism by which AOF-EVLPs exert their effects through the IRS-1 / PI3K / Akt / GSK-3β signaling pathway, thereby inhibiting the overexpression of Tau protein and promoting glycogen synthesis.

[0105] 1. Effects of AOF-EVLPs on the proliferation activity of HT22 cells

[0106] To verify the effect of AOF-EVLPs on the proliferation activity of HT22 cells, the CCK-8 assay was used. The specific steps were as follows: Mouse hippocampal HT22 cells in logarithmic growth phase were seeded at an appropriate density in 96-well plates. After cell adhesion, different numbers of AOF-EVLPs (final concentrations of 1×10⁻⁶) were added. 6 1×10 7 1×10 8 1×10 9 and 1×101 0 Cells were co-cultured with microplatelets (particles / mL) and a control group without AOF-EVLPs was set up, as shown in Table 2 below. After 12h, 24h and 48h of culture, CCK-8 reagent was added to each well and the cells were incubated in a cell culture incubator for a certain period of time. The absorbance (OD value) of each well was measured at a wavelength of 450nm using a microplate reader.

[0107]

[0108] Table 2

[0109] Experimental results Figure 7 As shown, compared with the control group, after 24 hours of treatment with AOF-EVLPs, 1×10 8 1×10 9 and 1×101 0 The HT22 cell proliferation activity was significantly promoted in all particle / mL concentration groups, indicating that AOF-EVLPs have good biological activity in promoting neuronal cell proliferation.

[0110] 2. Establishment of a dexamethasone-induced IR model in HT22 cells and screening of the optimal concentration of AOF-EVLPs

[0111] First, a dexamethasone (ddxm)-induced insulin resistance (IR) model in HT22 cells was established. The cytotoxicity of dexamethasone was assessed using a CCK-8 assay kit; concentrations ≤4 μM of dexamethasone did not produce significant toxicity to HT22 cells (see attached diagram in the instruction manual). Figure 8 (As shown in Figure A). Subsequently, the glucose consumption rate in the cell culture medium was measured using the hexokinase method to evaluate insulin sensitivity. After treating HT22 cells with 4 μM dexamethasone for 24 h and 48 h, treatment with 4 μM dexamethasone for 24 h was determined as the optimal condition for inducing the IR model in subsequent experiments, at which point the glucose consumption rate decreased the most and the model stability was optimal (see attached figures in the instruction manual for results). Figure 8 B, Figure 8 C).

[0112] Based on the successful establishment of the IR model, in order to screen the optimal concentration of AOF-EVLPs to improve IR, different concentrations of 1×10⁻⁶ were added to the model. 6 Up to 1×101 0 Intervention was performed using AOF-EVLPs with particles / mL, and glucose consumption rate was measured after 24 hours. The group design is shown in Table 3 below.

[0113]

[0114] Table 3

[0115] The results show (see Figure 8 D, Figure 8 E), compared with the normal control group, the glucose consumption rate in the model group was significantly reduced (P<0.001). 24 hours after AOF-EVLPs intervention, 1×10⁻⁶ glucose was added... 81×10 9 and 1×101 0 The particles / mL concentration group significantly reversed the decrease in glucose consumption rate induced by dexamethasone (***P<0.001), indicating that AOF-EVLPs in this concentration range can effectively and significantly improve glucose consumption rate (***P<0.001).

[0116] 3. AOF-EVLPs improve insulin resistance (IR) in HT22 cells through the IRS-1 / PI3K / Akt / GSK-3β signaling pathway, thereby inhibiting Tau protein expression.

[0117] To explore the molecular mechanism by which AOF-EVLPs improve intravascular coagulation (IR), quantitative PCR (qPCR) and Western blotting were used to detect the mRNA and protein expression levels of key genes in the IRS-1 / PI3K / Akt signaling pathway, as well as GSK-3β and Tau proteins. Furthermore, Tau protein expression levels were detected using immunofluorescence staining. An IR model was established in HT22 cells induced with 4 μM dexamethasone, and simultaneously treated with the optimal concentration of AOF-EVLPs (1 × 10⁻⁶). 8 ~1×101 0 Intervention was performed using particles / mL. After 24 hours, the mRNA and protein expression levels of IRS-1, PI3K, Akt, GSK-3β, and Tau proteins in cells were detected by qPCR (real-time quantitative PCR) and WB (Western blotting). The expression level of Tau protein in cells was detected by cell immunofluorescence labeling technology. The experimental groups are shown in Table 4.

[0118] Group Preprocessing AOF-EVLPs treatment concentration control group none 0 Dexamethasone model group (ddxm) 4μMddxm, 24h 0 AOF-EVLPs treatment group 4μMddxm, 24h <![CDATA[1×10 10 particles / mL]]>

[0119] Table 4

[0120] The results are as follows Figure 9 As shown (A represents IRS-1, B represents PI3K, C represents Akt, D represents GSK-3β, and E represents Tau protein). Compared with the control group, the mRNA expression levels of IRS-1, PI3K, and Akt were significantly decreased in the dexamethasone model group, while the mRNA expression levels of GSK-3β and Tau protein were significantly increased. Compared with the dexamethasone model group, after AOF-EVLPs treatment, the mRNA expression levels of IRS-1, PI3K, and Akt were increased in the AOF-EVLPs treatment group, while the mRNA expression levels of GSK-3β and Tau protein were significantly inhibited.

[0121] These results indicate that AOF-EVLPs can effectively improve dexamethasone-induced insulin resistance in HT22 cells by activating the IRS-1 / PI3K / Akt signaling pathway and inhibiting GSK-3β activity. This, in turn, effectively inhibits the overexpression of Tau, a key downstream protein in dexamethasone-induced insulin resistance. In other words, AOF-EVLPs can effectively inhibit dexamethasone-induced Tau protein overexpression by improving insulin resistance.

[0122] The results are as follows Figure 10 As shown, A represents the protein expression levels of IRS-1, PI3K, Akt, GSK-3β, and Tau in DXM-stimulated cells treated with AOF-EVLPs, analyzed by Western blotting; B represents the relative protein expression level of IRS-1; C represents the relative protein expression level of PI3K; D represents the relative protein expression level of Akt; E represents the relative protein expression level of GSK-3β; and FTAu represents the relative protein expression level of Tau. Compared with the control group, the protein expression levels of IRS-1, PI3K, and Akt were significantly decreased in the dexamethasone model group, while the protein expression levels of GSK-3β and Tau were significantly increased. In quantitative PCR (qPCR), we found that at 1×10 10 In the AOF-EVLPs treatment group at a particle / mL concentration, the expression of these genes (IRS-1, PI3K, Akt, GSK-3β, Tau) all showed stable and statistically significant differences (****, p<0.0001), indicating that AOF-EVLPs have a significant effect on these molecules at this concentration. Therefore, in the WB (Western blotting) experiment, we paid particular attention to high concentrations of AOF-EVLPs (1×10⁻⁶ particles / mL). 10 The AOF-EVLPs treatment group (particles / mL) was used to assess its effect on protein expression. Compared with the dexamethasone model group, after treatment with AOF-EVLPs (1×10⁻⁶ particles / mL), the AOF-EVLPs (1×10⁻⁶ particles / mL) group showed a significant reduction in protein expression. 10 In the treatment group (particles / mL), the protein expression levels of IRS-1, PI3K, and Akt were increased, while the protein expression levels of GSK-3β and Tau were significantly inhibited.

[0123] These results demonstrate that AOF-EVLPs possess the ability to regulate the IRS-1 / PI3K / Akt signaling pathway, thereby inhibiting GSK-3β activity. AOF-EVLPs treatment reversed dexamethasone-induced insulin resistance in HT22 cells and significantly reduced the dexamethasone-induced increase in Tau protein expression. In other words, AOF-EVLPs significantly inhibit dexamethasone-induced Tau protein overexpression by enhancing insulin sensitivity.

[0124] The results are as follows Figure 11 As shown, compared with the control group (NC group), the protein expression level of Tau protein was significantly increased in the dexamethasone model group. Compared with the dexamethasone model group (DXM group), the protein expression level of Tau protein was significantly inhibited in the AOF-EVLPs treatment group after treatment.

[0125] These results indicate that AOF-EVLPs can effectively inhibit the overexpression of the key protein Tau in dexamethasone-induced diabetic cognitive impairment, meaning that AOF-EVLPs can play a role in treating diabetic cognitive impairment by effectively inhibiting the overexpression of Tau protein induced by dexamethasone.

[0126] 4. Effects of AOF-EVLPs on glycogen synthesis in HT22 cells

[0127] Glycogen synthesis is a crucial indicator of insulin function. To verify the ameliorative effect of AOF-EVLPs on glucose metabolism in an insulin resistance model, periodate-Schiff (PAS) staining was used to detect intracellular glycogen deposition levels. In a 4 μM dexamethasone-induced HT22 cell IR model, PAS staining was performed 24 hours after intervention with the optimal concentration of AOF-EVLPs.

[0128] The results are as follows Figure 12 As shown, the normal control group (NC group) showed obvious purplish-red staining (indicating abundant glycogen deposition), while the purplish-red staining in the dexamethasone model group (DXM group) was significantly lighter (indicating reduced glycogen deposition). That is, when dexamethasone was used alone, the purplish-red staining of glycogen deposition in the DXM group was lighter than that in the control group (NC group). After treatment with AOF-EVLPs, the purplish-red staining depth in the cells was significantly restored, indicating an increase in glycogen synthesis. This result directly demonstrates that AOF-EVLPs can effectively improve dexamethasone-induced glycogen synthesis impairment in HT22 cells and reverse insulin resistance.

[0129] In summary, the comprehensive results of Example 4 demonstrate that the Alpinia oxyphylla-derived extracellular vesicles (AOF-EVLPs) of this invention can significantly promote the proliferation of HT22 neurons and effectively improve dexamethasone-induced insulin resistance by activating the IRS-1 / PI3K / Akt / GSK-3β signaling pathway, thereby inhibiting the abnormal expression of Tau protein and promoting glycogen synthesis, showing its potential for application in the preparation of drugs for the prevention or treatment of insulin resistance and related neurodegenerative diseases.

[0130] The present invention has been described in detail above. The above description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of this application should still fall within the scope of the present invention.

Claims

1. A method for preparing a shikuangzi-derived extracellular vesicle-like nanoparticle, characterized in that: The preparation method comprises the following steps: Step 1) raw material pretreatment: 300-1000 g of dried Fructus Alpiniae Oxophyllae is soaked in a pre-cooled phosphate buffered saline solution and then subjected to wall breaking and crushing to obtain juice; Step 2) centrifugal filtration: the juice obtained in step 1) is centrifuged at a centrifugal force of 1000-4000 g for 20-40 min, the supernatant is collected and filtered through a filter membrane with a pore size of 1-5 μm; Step 3) tangential flow concentration: the supernatant obtained in step 2) is subjected to tangential flow concentration, and the concentration is 10-40% of the original volume to obtain a concentrated solution; Step 4) centrifugation of the concentrated solution: the concentrated solution obtained in step 3) is centrifuged, and the supernatant is collected and filtered; Step 5) ultracentrifugation: the filtrate obtained in step 4) is subjected to ultracentrifugation in an ultracentrifuge, and the precipitate is collected; Step 6) filtration of the precipitate: the precipitate obtained in step 5) is resuspended in a phosphate buffered saline solution and filtered through a filter membrane with a pore size of 0.22-0.30 μm to obtain Fructus Alpiniae Oxophyllae-derived extracellular vesicle-like nanoparticles.

2. The method for preparing Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles according to claim 1, characterized in that: The centrifugation of the concentrated solution in step 4) is performed at 0-8 ℃, the centrifugal force is 8000-15000 g, and the centrifugation time is 40-100 min; the filtration is performed through a filter membrane with a pore size of 0.45 μm.

3. The method for preparing Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles according to claim 1, characterized in that: The ultracentrifugation of the filtrate in step 5) is performed at 0-8 ℃, the centrifugal force is 80000-150000 g, and the centrifugation time is 40-100 min.

4. The method for preparing Alpinia oxyphylla-derived extracellular vesicle-like nanoparticles according to claim 1, characterized in that: The Fructus Alpiniae Oxophyllae raw material in step 1) is a Fructus Alpiniae Oxophyllae decoction piece.

5. A Schisandra chinensis -derived extracellular vesicle-like nanoparticle, characterized in that: The Fructus Alpiniae Oxophyllae-derived extracellular vesicle-like nanoparticles are prepared by the preparation method of claim 1, the particle size distribution of the Fructus Alpiniae Oxophyllae-derived extracellular vesicle-like nanoparticles is between 50-500 nm, and the Fructus Alpiniae Oxophyllae-derived extracellular vesicle-like nanoparticles carry Fructus Alpiniae Oxophyllae-derived bioactive molecules.

6. Use according to claim 5, characterized in that: The Fructus Alpiniae Oxophyllae-derived extracellular vesicle-like nanoparticles are used for preparing a medicament for preventing or treating diabetic cognitive impairment.

7. Use according to claim 5, characterized in that: The Fructus Alpiniae Oxophyllae-derived extracellular vesicle-like nanoparticles are used for preparing a brain hippocampal neuron cell survival promoter.

8. Use according to claim 5, characterized in that: The Fructus Alpiniae Oxophyllae-derived extracellular vesicle-like nanoparticles are used for preparing a nerve cell insulin resistance response inhibitor.

9. Use according to claim 5, characterized in that: The Fructus Alpiniae Oxophyllae-derived extracellular vesicle-like nanoparticles are used for preparing a cognitive function improver.

10. Use according to claim 5, characterized in that: The Fructus Alpiniae Oxophyllae-derived extracellular vesicle-like nanoparticles are used for preparing an IRS-1 / PI3K / Akt signal pathway activator.

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