Application of gastrodin-derived nanovesicles in preparation of drugs for improving depression

Abstract

The application discloses application of nano vesicles from Gastrodia elata Blume in preparation of a medicine for improving depression. It is found through a large amount of experimental researches that the nano vesicles from Gastrodia elata Blume can improve the movement times and / or time of a subject, reduce or relieve depression-like behaviors, repair blood-brain barrier damage, reduce or relieve memory damage, improve spatial memory capacity and the like. In addition, the nano vesicles from Gastrodia elata Blume have the advantages of low toxicity, high biocompatibility, no induction of immunogenicity, scalable preparation and the like.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and in particular relates to the application of Gastrodia elata-derived nanovesicles in the preparation of drugs to improve depression. Background Technology

[0002] Depression is a common and serious mental disorder, mainly characterized by persistent low mood, loss of interest, anhedonia, sleep and appetite disturbances.

[0003] Currently, first-line antidepressants in clinical practice include selective serotonin reuptake inhibitors (SSRIs, such as fluoxetine and paroxetine) and serotonin-norepinephrine reuptake inhibitors (SNRIs, such as venlafaxine). However, these drugs generally suffer from slow onset of action (usually 2-4 weeks or more), limited remission rates (about one-third of patients still have significant residual symptoms after treatment), numerous adverse reactions (such as sexual dysfunction, weight gain, withdrawal reactions), and drug resistance in some patients. Furthermore, poor long-term medication adherence and high relapse rates after discontinuation are also pressing clinical challenges that need to be addressed.

[0004] Therefore, developing novel antidepressant drugs or treatment strategies that are highly biosafe, have a faster onset of action, better efficacy, and fewer side effects is of great clinical significance. Summary of the Invention

[0005] To address at least some of the technical problems in the prior art, this invention provides the application of Gastrodia elata-derived nanovesicles in the preparation of drugs for improving depression. Specifically, this invention includes the following:

[0006] In a first aspect, the present invention provides the use of Gastrodia elata-derived nanovesicles in the preparation of a drug for improving depression, wherein the particle size of the Gastrodia elata-derived nanovesicles is 200-350 nm and the potential is -40-(-15) mV.

[0007] In some embodiments, the application of the Gastrodia elata-derived nanovesicles according to the present invention in the preparation of a medicament for improving depression, wherein the improvement includes at least one of the following: (1) Increase the number of exercises and / or the duration; (2) Reduce or alleviate depressive-like behaviors; (3) Repairing damage to the blood-brain barrier; (4) Reduce or alleviate memory impairment; (5) Improve spatial memory ability.

[0008] In some embodiments, the application of the Gastrodia elata-derived nanovesicles according to the present invention in the preparation of a medicament for improving depression is described, wherein the Gastrodia elata-derived nanovesicles are prepared by the following method: (1) Take Gastrodia elata, homogenize it, and then centrifuge it at a differential speed to obtain crude extract; (2) The crude extract was purified by density gradient centrifugation to obtain the Gastrodia elata-derived nanovesicles.

[0009] In some embodiments, the application of the Gastrodia elata-derived nanovesicles according to the present invention in the preparation of a medicament for improving depression is described, wherein the Gastrodia elata-derived nanovesicles are prepared by the following method: (1) Take Gastrodia elata, homogenize it at 0-10℃, and obtain crude extract by differential centrifugation at 400-100000 g; (2) The crude extract was centrifuged together with 0.5-1.4 M sucrose and 1.6-2.5 M sucrose at 0-10°C at a speed of 80000-120000 g for 0.1-3 h, and the intermediate liquid was taken to obtain the Gastrodia elata-derived nanovesicles.

[0010] In some embodiments, the use of Gastrodia elata-derived nanovesicles according to the present invention in the preparation of a medicament for improving depression, wherein the medicament further comprises a pharmaceutically acceptable carrier.

[0011] In some embodiments, the application of the Gastrodia elata-derived nanovesicles according to the present invention in the preparation of a medicament for improving depression, wherein the pharmaceutically acceptable carrier comprises at least one of a diluent, filler, absorbent, wetting agent, binder, disintegrant, lubricant, sweetener, preservative, and antioxidant.

[0012] In some embodiments, the application of the Gastrodia elata-derived nanovesicles according to the present invention in the preparation of a medicament for improving depression, wherein the dosage form of the medicament includes injection, oral liquid, capsule, tablet, granule or gel.

[0013] In a second aspect, the present invention provides a pharmaceutical composition for improving depression, comprising gastrodia-derived nanovesicles having a particle size of 200-350 nm and a potential of -40-(-15) mV.

[0014] A third aspect of the present invention provides the use of Gastrodia elata-derived nanovesicles in the preparation of a medicament for the combined treatment of depression with other drugs, wherein the particle size of the Gastrodia elata-derived nanovesicles is 200-350 nm and the potential is -40-(-15) mV.

[0015] A fourth aspect of the present invention provides a method for promoting the proliferation of neuronal cells in vitro, comprising: (I) Microglia were cultured using nanovesicles derived from Gastrodia elata, and the supernatant was collected. The nanovesicles derived from Gastrodia elata had a particle size of 200-350 nm and a potential of -40-(-15) mV. (II) Use the supernatant to culture neuronal cells.

[0016] Extensive experimental research has revealed that Gastrodia elata-derived nanovesicles can increase the frequency and / or duration of physical activity in subjects, reduce or alleviate depressive-like behaviors, repair blood-brain barrier damage, reduce or alleviate memory impairment, and improve spatial memory. Furthermore, the Gastrodia elata-derived nanovesicles of this invention possess advantages such as low toxicity, high biocompatibility, non-induction of immunogenicity, and scalability for large-scale preparation. Attached Figure Description

[0017] Figure 1 The results show the particle size determination of nanovesicles derived from Gastrodia elata. In this figure, A represents the particle size and light intensity distribution, and B represents the correlation curve.

[0018] Figure 2 The results show the potential measurement of nanovesicles derived from Gastrodia elata, where A represents the zeta potential distribution and B represents the phase diagram.

[0019] Figure 3 This is an electron micrograph of nanovesicles derived from Gastrodia elata.

[0020] Figure 4 The results of the microglia BV2 uptake experiment on Gastrodia elata-derived nanovesicles are shown, where GENPs represent Gastrodia elata-derived nanovesicles.

[0021] Figure 5 A and B are the results of intestinal and brain uptake experiments of nanovesicles derived from Gastrodia elata, respectively.

[0022] Figure 6 The nanovesicles derived from Gastrodia elata were shown to inhibit ROS levels in microglia. GENPs-L, GENPs-M, and GENPs-H represent low, medium, and high doses of Gastrodia elata-derived nanovesicles, respectively.

[0023] Figure 7 The results show that nanovesicles derived from Gastrodia elata can promote the proliferation of HT22 cells.

[0024] Figure 8 These are the results of a cell permeability experiment.

[0025] Figure 9 The graph shows the mouse open field trajectory and bar chart, where A is the mouse open field trajectory, B is the open field heatmap, and C and D are the total distance the mouse traveled in the open field, the distance it traveled in the central area, the number of times it entered the central area, and the time it spent in the central area, respectively.

[0026] Figure 10 A and B represent the results of the mouse sucrose water preference and tail suspension experiment, respectively.

[0027] Figure 11The results of the mouse water maze experiment are shown below. In the figure, A is the movement trajectory of the mouse in the water maze, B is the motion heat map of the water maze, and C is the time spent in the target quadrant (i.e., the quadrant where the original platform is located) on the day of the test. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0031] In this invention, the term "improvement" includes an improvement in the condition of a disease or dysfunction before or after its onset. This improvement or prevention, measured by any standard technique, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to an untreated control group under equivalent conditions. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and reduction (whether partial or complete). Such improvements include, but are not limited to, the following depression-related conditions: increased frequency and / or duration of physical activity; reduced or alleviated depressive-like behaviors; repair of blood-brain barrier damage; reduced or alleviated memory impairment; improved spatial memory, etc.

[0032] application One aspect of the present invention provides the application of Gastrodia elata-derived nanovesicles in the preparation of a medicament for improving depression, wherein the Gastrodia elata-derived nanovesicles are the sole active ingredient, and their particle size is 200-350 nm, preferably 210-340 nm, even more preferably 220-330 nm, further preferably 230-320 nm, more preferably 240-310 nm, and even more preferably 250-300 nm, for example 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300 nm, and the potential of the Gastrodia elata-derived nanovesicles is -40 to -15 mV, preferably -38 to -16 mV. The mV is preferably -36-(-17) mV, even more preferably -34-(-18) mV, more preferably -32-(-19) mV, and even more preferably -30-(-20) mV, for example -30, -29.5, -29, -28.5, -28, -27.5, -27, -26.5, -26, -25.5, -25, -24.5, -24, -23.5, -23, -22.5, -22, -21.5, -21, -20.5, -20 mV.

[0033] In this invention, the gastrodia-derived nanovesicles are nanoscale membrane-structured vesicles actively secreted by gastrodia. They possess a lipid bilayer structure and contain active ingredients such as proteins, nucleic acids, and gastrodia-specific metabolites, which are secreted through exosomes, microvesicles, and apoptotic bodies. The inventors analyzed the composition of the gastrodia-derived nanovesicles and found that they contain little to no gastrodin.

[0034] In a preferred embodiment, the Gastrodia elata-derived nanovesicles of the present invention are prepared by the following method: (1) Take Gastrodia elata, homogenize it, and then centrifuge it at a differential speed to obtain crude extract; (2) The crude extract was purified by density gradient centrifugation to obtain the Gastrodia elata-derived nanovesicles.

[0035] In a further preferred embodiment, the Gastrodia elata-derived nanovesicles of the present invention are prepared by the following method: (1) Take Gastrodia elata tubers (preferably sliced ​​or diced) with a moisture content of not less than 70%, homogenize them at 0-10℃ (preferably 0-9℃, more preferably 0-8℃, further preferably 0-7℃, more preferably 1-6℃, for example 1, 2, 3, 4, 5, 6℃), filter to obtain a homogenate, add protease inhibitors (such as, but not limited to, leucinogen, PMSF, sodium azide, etc.), and adjust the pH to 6-8 (preferably 6.1-7.9, more preferably 6.2-7.8, further preferably 6.3-7.7, more preferably 6.4-7.6, even more preferably 6.5-7.5, for example 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5) using a pH adjuster (such as, but not limited to, Tris-HCl solution) and sterilize for 400-100000 hours. Crude extract was obtained by differential centrifugation at speeds of 300-500 g (preferably 310-490 g, more preferably 320-480 g, further preferably 330-470 g, more preferably 340-460 g, and even more preferably 350-450 g, such as but not limited to 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450 g), 700-900 g (preferably 710-890 g, more preferably 720-880 g, further preferably 730-870 g, more preferably 740-860 g, and even more preferably 750-850 g, such as 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850 g), 9000-11000 g. Centrifuge at 9100-10900 g (preferably 9200-10800 g, even more preferably 9300-10700 g, more preferably 9400-10600 g, even more preferably 9500-10500 g, e.g., 9500, 9600, 9700, 9800, 9900, 10000, 10100, 10200, 10300, 10400, 10500 g) for 5-40 min (preferably 7-38 min, even more preferably 10-36 min, even more preferably 12-34 min, more preferably 14-32 min, even more preferably 16-30 min, e.g., 16, 18, 20, 22, 24, 26, 28, 30 min), and then ultracentrifuge at 90000-110000 g for 0.5-2 h (preferably 0.5-1.9 h). h, preferably 0.5-1.8 h, even more preferably 0.5-1.7 h, more preferably 0.5-1.6 h, and even more preferably 0.5-1.5 h, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5 h); (2) The crude extract is mixed with 0.5-1.4 M (preferably 0.6-1.4 M, more preferably 0.7-1.3 M, even more preferably 0.8-1.2 M, such as 0.8, 0.9, 1, 1.1, 1.2 M) sucrose and 1.6-2.5 M (preferably 1.7-2.4 M, more preferably 1.8-2.3 M, even more preferably 1.8-2.2 M, such as 1.8, 1.9, 2, 2.1, 2.2 M) sucrose at 0-10°C (preferably 0-9°C, even more preferably 0-8°C, further preferably 0-7°C, even more preferably 1-6°C, such as 1, 2, 3, 4, 5, 6°C) at a rate of 80,000-120,000 g (preferably 82,000-118,000 g, even more preferably 84,000-116,000 g, even more preferably 86,000-114,000 g) Centrifuge at a speed of 0.1-3 h (preferably 0.2-2.8 h, even more preferably 0.3-2.6 h, further preferably 0.4-2.4 h, more preferably 0.5-2.2 h, even more preferably 0.5-2 h, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 h) for 0.1-3 h (preferably 0.2-2.8 h, even more preferably 0.3-2.6 h, further preferably 0.4-2.4 h, more preferably 0.5-2.2 h, even more preferably 0.5-2 h, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 h) and take the intermediate liquid to obtain the Gastrodia elata-derived nanovesicles.

[0036] In a preferred embodiment, the raw material is *Gastrodia elata* with a moisture content of not less than 70%, rather than dried *Gastrodia elata*. The specific criteria for determining whether *Gastrodia elata* has a moisture content of not less than 70% and whether it is dried are known in the art. For example, the moisture content can be determined using the drying method according to the moisture determination method (General Rule 0832, Method II) in the Chinese Pharmacopoeia (2020 edition). Specifically, *Gastrodia elata* with a moisture content of not less than 70% refers to *Gastrodia elata* that, after being sampled, pulverized, and dried to constant weight at 105°C, has a moisture content of not less than 70%, preferably 70%-85%, and even more preferably 75%-80% (w / w).

[0037] In a preferred embodiment, the medicament further comprises a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is involved in transporting or delivering the medicament from one organ or part of the body to another organ or part of the body. Each carrier is "acceptable," meaning it is compatible with other components of the formulation (e.g., Gastrodia elata-derived nanovesicles) and does not harm the patient. The pharmaceutically acceptable carrier includes at least one of diluents, fillers, absorbents, wetting agents, binders, disintegrants, lubricants, sweeteners, preservatives, and antioxidants. Examples of diluents include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, and dispersion media; fillers include, but are not limited to, starch, lactose, mannitol, and microcrystalline cellulose; absorbents include, but are not limited to, calcium sulfate, dicalcium phosphate, and calcium carbonate; wetting agents include, but are not limited to, water and ethanol; binders include, but are not limited to, hydroxypropyl methylcellulose, povidone, and microcrystalline cellulose; disintegrants include, but are not limited to, croscarmellose sodium, croscarmellose, surfactants, and low-substituted hydroxypropyl cellulose; lubricants include, but are not limited to, magnesium stearate, talc, polyethylene glycol, sodium dodecyl sulfate, micronized silica gel, and talc; sweeteners include, but are not limited to, sucralose, acetylsupan, saccharin, sucrose, xylitol, mannitol, sorbitol, glucose, fructose, and aspartame; preservatives include, but are not limited to, parabens, chlorobutanol, phenol, and sorbic acid; and antioxidants include, but are not limited to, ascorbic acid and methionine.

[0038] In this invention, depression is prevented or improved by administering a therapeutically effective amount of the Gastrodia elata-derived nanovesicles to subjects. Subjects include, but are not limited to, mammals, including but not limited to, humans, mice, rabbits, cats, dogs, cattle, sheep, and pigs.

[0039] In this invention, there are no particular limitations on the method of administration of the drug. Representative methods of administration include, but are not limited to, oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous) and local administration. Accordingly, the drug of this invention can be formulated into various clinically acceptable dosage forms, including but not limited to injections, oral solutions, capsules, tablets, granules, and gels.

[0040] The present invention also provides a pharmaceutical composition for improving depression, comprising the Gastrodia elata-derived nanovesicles described in this invention.

[0041] The present invention further provides the application of Gastrodia elata-derived nanovesicles in the preparation of drugs for the combined treatment of depression with other drugs, including but not limited to fluoxetine, paroxetine, venlafaxine, etc.

[0042] method One aspect of the present invention provides a method for promoting the proliferation of neuronal cells in vitro, comprising: (I) Microglia were cultured using the Gastrodia elata-derived nanovesicles described in this invention, and the supernatant was collected; (II) The neuronal cells are cultured using the supernatant.

[0043] In some embodiments, the method of the present invention for promoting neuronal cell proliferation in vitro is a non-therapeutic method, such as screening neuronal proliferation-promoting drugs, evaluating the efficacy of neuronal proliferation-promoting drugs, neuronal cell production, and studying the microglia-neuron interaction mechanism. Those skilled in the art will also understand that microglia cultured from Gastrodia elata nanovesicles can also serve as a cell model for further evaluation of its efficacy in combination with other drugs. In some embodiments, the method of the present invention for promoting neuronal cell proliferation in vitro is a therapeutic method, i.e., for therapeutic use. Therefore, the present invention also provides the application of the above method in the preparation of medicaments for treating depression.

[0044] This invention also provides a method for in vitro repair of cell barrier damage, comprising: obtaining barrier-damaged cells (e.g., but not limited to bEnd.3 cells), contacting the cells with nanovesicles derived from Gastrodia elata, and detecting the repair status of the cell barrier damage. The detection can be performed using methods and apparatus known in the art, and is not particularly limited thereto.

[0045] In some embodiments, the method of the present invention for repairing cell barrier damage in vitro is a non-therapeutic method, such as for screening drugs for repairing cell barrier damage or for studying the mechanism of cell barrier damage.

[0046] Example The following shows the preparation, characterization, and application of nanovesicles derived from Gastrodia elata.

[0047] 1. Preparation and Characterization 1.1 Preparation Take an appropriate amount of Gastrodia elata with a water content of not less than 70%, wash it clean with distilled water, and air dry it. Cut the Gastrodia elata into 3-5 cm thick slices, and homogenize it in a homogenizer with 1*PBS (calcium and magnesium-free). The temperature is controlled at 4℃ throughout the homogenization process. Pass the homogenate through a silk cloth to obtain Gastrodia elata homogenate. Immediately add protease inhibitors (leucopeptide, PMSF, sodium azide), and then adjust the pH of the Gastrodia elata homogenate to 7.0 with 1 M Tris-HCl. Centrifuge at 4℃, 400 g, 800 g, and 10000 g for 20 min in sequence, and collect the supernatant. Centrifuge at 4℃ for 100000 g and collect the precipitate after 1 h. Dissolve the precipitate with 20 mM Tris-HCl to obtain crude Gastrodia elata extract.

[0048] The crude extract of Gastrodia elata was centrifuged together with 1 M sucrose and 2 M sucrose (4℃, 100000 g, 1 h), and the intermediate liquid was collected to obtain a nanovesicle solution derived from Gastrodia elata.

[0049] 1.2 Characterization The sample was diluted 100-fold with deionized water and ultrasonically dispersed to prevent agglomeration. 1 mL was injected into the sample cell and placed in the instrument. Dynamic light scattering was employed, with the temperature (25℃) and material refractive index parameters set. By analyzing the fluctuations in scattered light intensity, the average particle size and polydispersity index were automatically calculated. Results are as follows: Figure 1 As shown in A and B, the average diameter of the nanovesicles derived from Gastrodia elata is 272.8 nm.

[0050] After diluting the sample, it was injected into a dedicated sample cell equipped with electrodes using a syringe. An electric field was applied, and the velocity of the charged particles was measured based on electrophoretic light scattering or phase analysis techniques to calculate the zeta potential. Each sample was repeated three times, and the average value was taken. Results are as follows... Figure 2 As shown in A and B, the potential of the nanovesicles derived from Gastrodia elata is -27.54 mV.

[0051] Electron microscopy observation: The sample was diluted 100-fold with deionized water and ultrasonically dispersed to avoid agglomeration. A small amount was dropped onto a silicon wafer (or copper mesh), allowed to dry naturally, and then sputter-coated with gold to increase conductivity. The sample was placed in the electron microscope chamber, and the accelerating voltage (e.g., 5-15 kV) and working distance were set. High-magnification morphology images were obtained using secondary electron or transmission electron imaging. Results are as follows: Figure 3 As shown, they are roughly spherical, with most particles being independent and a few agglomerated, which is consistent with the microscopic identification characteristics of nanovesicles.

[0052] 2. Application 2.1 Intake Experiment PKH26-labeled Gastrodia elata-derived nanovesicles were then administered to BV2 cells and mice via gavage to determine the absorption of Gastrodia elata-derived nanovesicles in vivo and in vitro. After co-culturing Gastrodia elata-derived nanovesicles with BV2 cells for 24 h, the in vitro uptake effect was observed using confocal microscopy. Fluorescence imaging results of mouse brain tissue at 0 h, 12 h, and 24 h were observed using a small animal imaging system.

[0053] In vitro results as follows Figure 4 As shown, nanovesicles derived from Gastrodia elata can be taken up by BV2 cells. In vivo results are as follows... Figure 5 As shown in A and B, Gastrodia elata-derived nanovesicles were taken up by the brain after 24 hours.

[0054] 2.2 Reactive Oxygen Species Experiment BV2 cells 1×10 5Cells were seeded per well in 6-well plates and cultured for 24 h. Five groups were formed: a blank control group, an LPS model group, and a Gastrodia elata-derived nanovesicle administration group (low, medium, and high doses were 5, 10, and 20 μg / ml, respectively). All groups were incubated together for 24 h. ROS levels in BV2 cells were detected using a reactive oxygen species (ROS) detection kit. The fluorescent probe DCFH-DA can penetrate the cell membrane and be oxidized by intracellular ROS into fluorescent DCF, which was detected using fluorescence microscopy.

[0055] In situ probe loading: Dilute DCFH-DA 1:1000 with blank culture medium, discard the original culture medium in each well, add 1 ml of diluted DCFH-DA to each well, place in a 37℃ constant temperature incubator for 30 min, discard DCFH-DA, wash three times with PBS, add 2 ml of serum-free culture medium to each well, and take pictures under a fluorescence microscope in the dark for detection.

[0056] The results are as follows Figure 6 As shown, Gastrodia elata-derived nanovesicles can enhance the antioxidant capacity of BV2 cells and reduce the damage to cells caused by oxidative stress. Gastrodia elata-derived nanovesicles can inhibit the activation of inflammatory cells and reduce the ROS of inflammatory cells, thereby alleviating the inflammatory response in a concentration-dependent manner.

[0057] 2.3 Cell co-culture BV2 cells were fed at a rate of 1×10 5 HT22 cells were seeded in six-well plates at a rate of 5 × 10⁶ cells / well. 4 BV2 cells were seeded into 96-well plates. Different concentrations of Gastrodia elata-derived nanovesicles were added to the plates containing BV2 cells, and an inflammation model was established using LPS. After 24 h, the supernatant of each well was collected and centrifuged at 1000 rpm for 5 min to remove excess impurities. The supernatant was then added to the plates containing HT22 cells and incubated for 24 h. After incubation, the supernatant was removed, and the prepared CCK8 solution was added. After 1 h, the absorbance was measured and recorded at 450 nm using a microplate reader.

[0058] The results are as follows Figure 7 As shown, nanovesicles derived from Gastrodia elata can indirectly promote the proliferation of HT22 neurons by influencing BV2 microglia.

[0059] 2.4 bEnd.3 Cell permeability bEnd.3 cells were fed at a rate of 1×10 5Five groups were inoculated into the upper chamber of a 24-well Transwell microplate, with blank culture medium in the lower chamber. The groups were: blank, LPS model, and Gastrodia elata-derived nanovesicles (5, 10, and 20 μg / ml). The nanovesicles were incubated for 6 days, with the medium changed every two days. Once the nanovesicles had fully grown and densely covered the chamber, LPS and Gastrodia elata-derived nanovesicles were used to induce drug delivery. After 24 hours, the culture medium was discarded. A 1 μg / ml fluorescein isothiocyanate (FITC) solution prepared with PBS was added to the upper chamber, and PBS was added to the lower chamber. After 2 hours, the PBS from the lower chamber was aspirated and the absorbance was measured using a microplate reader at an excitation wavelength of 458 nm and an emission wavelength of 528 nm. The leakage was calculated using a FITC concentration standard curve.

[0060] The results are as follows Figure 8 As shown, Gastrodia elata-derived nanovesicles repaired LPS-induced bEnd.3 cell barrier damage and reduced FITC leakage.

[0061] 2.5 In vivo experiments Wild-type male C57BL / 6 mice (6-8 weeks old) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and first acclimatized for one week in the SPF-grade mouse house of the Animal Center of Changchun University of Traditional Chinese Medicine. During this period, the ambient temperature was controlled at a constant 22℃, and a 12-hour light-12-hour dark circadian rhythm was adopted, with free access to food and water. The experiment lasted for 3 weeks. In the first week, the mice were randomly divided into six groups: blank control group, LPS model group, low-dose Gastrodia elata-derived nanovesicle group, high-dose Gastrodia elata-derived nanovesicle group, negative control group, and fluoxetine positive control group. Pretreatment was performed by gavage from 14:00 to 15:00 every day, with the blank control group receiving PBS by gavage as well as the LPS model group. In the second week, intraperitoneal injection was performed from 10:00 to 11:00 every morning. Except for the blank control group which received PBS, the other groups received LPS at a concentration of 1 mg / kg. Treatment was performed by gavage of the corresponding drugs from 14:00 to 15:00 every afternoon. In the third week, modeling and drug administration were discontinued, and behavioral tests such as open field, sucrose water, and tail suspension were initiated.

[0062] The results were obtained by analyzing the mouse's movement trajectory, distance traveled, number of times it entered the center, and duration of stay within a 40×40 cm white frame. Figure 9 As shown in the AF, the amount of movement of mice in the LPS model group and the negative control group was significantly reduced, and they mostly moved around the walls. However, the amount of movement in the low-dose group, the high-dose group and the fluoxetine positive drug group was significantly improved, and the number of times they entered the central area and the time spent there were relatively higher than those in the model group and the negative control group. The high-dose group and the positive drug group showed significant improvement.

[0063] Mice underwent an adaptation experiment with 2% whole sucrose and half-sugar half-water. After 12 hours of deprivation of water and food, the intake of sucrose and drinking water in each group of mice was recorded. The results are as follows: Figure 10 As shown in Figure A, the LPS model group and the negative control group mice showed significantly reduced sucrose water intake, exhibiting anhedonia. In contrast, the intake in the low-dose, high-dose, and fluoxetine positive control groups tended to be similar to that of the blank control group, indicating a therapeutic effect on LPS-induced depressive-like behavior. The TST experiment results are as follows... Figure 10 As shown in Figure B, the tail of the mouse was fixed to the suspension device at 1 / 3, ensuring that the mouse's head was perpendicular to the ground and could not touch the surrounding objects. The struggle and immobility time of the mouse within 5 minutes were recorded. It was found that the immobility time of the LPS model group and the negative control group was longer and the degree of struggle was less than that of other groups. This indicates that the nanovesicles derived from Gastrodia elata and the positive drug fluoxetine have an ameliorative effect on the despair behavior caused by LPS.

[0064] After three days of training, the time it took for the mice to find the platform on the last day was recorded. The results are as follows: Figure 11 As shown in the AC, the model group mice took a relatively long time to find the platform, the negative control group could not find the platform within the specified time, while the drug-treated group could find and climb the platform more quickly. This indicates that the nanovesicles derived from Gastrodia elata and the positive drug fluoxetine have a good therapeutic effect on memory impairment caused by LPS and improve the spatial memory ability of mice.

[0065] All data in this embodiment represent at least three independent experiments and are expressed as mean ± SEM. Statistical calculations were performed using software. Statistical comparisons were conducted using one-way ANOVA and Dunnett's post-hoc test. P <0.05 indicates a statistically significant difference. Here, # indicates a difference compared to the control group, * indicates a difference compared to the model group, and * indicates a difference in... P <0.05, ** indicates P <0.01, *** indicates P <0.001, **** indicates P <0.0001, # indicates P <0.05, ## indicates P <0.01, ### indicates P <0.001, #### indicates P <0.0001.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of Gastrodia elata-derived nanovesicles in the preparation of drugs for improving depression, characterized in that, The nanovesicles derived from Gastrodia elata have a particle size of 200-350 nm and a potential of -40-(-15) mV.

2. The application of the Gastrodia elata-derived nanovesicles according to claim 1 in the preparation of drugs for improving depression, characterized in that, The improvement includes at least one of the following: (1) Increase the number of exercises and / or the duration; (2) Reduce or alleviate depressive-like behaviors; (3) Repairing damage to the blood-brain barrier; (4) Reduce or alleviate memory impairment; (5) Improve spatial memory ability.

3. The application of the Gastrodia elata-derived nanovesicles according to claim 1 in the preparation of drugs for improving depression, characterized in that, The Gastrodia elata-derived nanovesicles were prepared by the following method: (1) Take Gastrodia elata, homogenize it, and then centrifuge it at a differential speed to obtain crude extract; (2) The crude extract was purified by density gradient centrifugation to obtain the Gastrodia elata-derived nanovesicles.

4. The application of the Gastrodia elata-derived nanovesicles according to claim 1 in the preparation of drugs for improving depression, characterized in that, The Gastrodia elata-derived nanovesicles were prepared by the following method: (1) Take Gastrodia elata, homogenize it at 0-10℃, and obtain crude extract by differential centrifugation at 400-100000 g; (2) The crude extract was centrifuged together with 0.5-1.4 M sucrose and 1.6-2.5 M sucrose at 0-10°C at a speed of 80000-120000g for 0.1-3 h, and the intermediate liquid was taken to obtain the Gastrodia elata-derived nanovesicles.

5. The application of the Gastrodia elata-derived nanovesicles according to claim 1 in the preparation of a drug for improving depression, characterized in that, The drug further includes a pharmaceutically acceptable carrier.

6. The application of the Gastrodia elata-derived nanovesicles according to claim 5 in the preparation of a drug for improving depression, characterized in that, The pharmaceutically acceptable carriers include at least one of the following: diluents, fillers, absorbents, wetting agents, binders, disintegrants, lubricants, sweeteners, preservatives, and antioxidants.

7. The application of the Gastrodia elata-derived nanovesicles according to claim 1 in the preparation of a drug for improving depression, characterized in that, The dosage forms of the drug include injections, oral solutions, capsules, tablets, granules, or gels.

8. The application of Gastrodia elata-derived nanovesicles in the preparation of drugs for the combined treatment of depression with other drugs, characterized in that... The nanovesicles derived from Gastrodia elata have a particle size of 200-350 nm and a potential of -40-(-15) mV.

Images