Extracellular vesicle isolated from cistanche tubulosa and use thereof

Abstract

Provided is use of an extracellular vesicle of Cistanche tubulosa in the preparation of a composition. The extracellular vesicle is prepared by means of the steps of wall breaking of Cistanche tubulosa, centrifugation to remove a precipitate, reaction with a polymer molecule, centrifugation to collect a precipitate, etc. The composition can protect nerves against amyloid damage, promote neurite extension, and prevent and treat amyloid-related neurological diseases or symptoms.

Description

Extracellular vesicles isolated from Cistanche tubulosa and their uses Technical Field

[0001] This invention relates to an extracellular vesicle and its uses, specifically to extracellular vesicles isolated from *Cistanche tubulosa*. The invention also relates to a method for preparing the extracellular vesicle and its use in protecting nerves, particularly in protecting nerves against amyloid damage and / or promoting synaptic extension. Background Technology

[0002] Extracellular vesicles (EVs) are nanoscale, bilayered lipid vesicles secreted by cells into the extracellular space. Their surface and internal space contain various biomolecules, such as nucleic acids, proteins, lipids, and secondary metabolites, which can serve as biomarkers and signaling factors, mediating intercellular signal transduction and communication to regulate physiological and pathological mechanisms. Based on their biogenesis, release pathways, and size, extracellular vesicles can be classified into several main subtypes, including microvesicles, exosomes, and apoptotic bodies. These subtypes differ in morphology, formation mechanisms, and biological functions. Researchers typically separate and purify them based on particle size. The purity and particle size distribution of the purified extracellular vesicles directly affect their subsequent physiological activity.

[0003] Among the major subtypes, exosomes are the smallest, typically ranging from 30 to 150 nanometers in diameter. Due to their endogenous and heterogeneous nature, exosomes can deliver bioactive substances to target cells through various pathways and sites, making them highly suitable as carriers. They have excellent development potential in basic research, industrial development, and clinical applications in the biomedical field.

[0004] Plant-derived exosomes are sometimes referred to as plant-derived exosome-like nanoparticles to distinguish them from animal-derived exosomes. The process of exosome production in plant cells is similar to that in animal cells. Multivesicular bodies (MVBs) are formed within the lumen of endosomes. These MVBs eventually fuse with the cell membrane, releasing their contained vesicles into the extracellular matrix. These vesicles belong to the exosome subtypes within extracellular vesicles. Extracellular vesicles produced by plant cells (especially exosome subtypes) participate in intercellular communication and physiological regulation within plants, enabling plants to cope with processes such as growth, development, stress responses, and pathogen defense. If extracted and isolated and used in animals, they may also produce specific physiological effects on animal cells.

[0005] It is noteworthy that plant-derived extracellular vesicles exhibit high heterogeneity, with their physiological functions varying depending on their origin. In particular, parasitic plants growing in harsh desert environments, such as Cistanche tubulosa, are expected to have extracellular vesicles that carry signaling molecules with strong stress regulation capabilities to cope with environmental stress, playing a key role in biological defense and environmental adaptation, and may also have excellent protective potential for animal cells.

[0006] Cistanche tubulosa is rich in phenylethanoid glycosides, iridoids, polysaccharides, and alkaloids. Traditional Chinese medicine believes it can tonify kidney yang and replenish essence and blood. Modern biomedicine widely uses Cistanche tubulosa extract for its antioxidant, anti-aging, neuroprotective, and anti-fatigue effects. However, past research has mostly focused on small molecules in the extract, and systematic development of the extracellular vesicles contained in Cistanche tubulosa and their specific contributions to the regulation of animal physiological functions is still lacking. Technical issues

[0007] Our team has been deeply involved in the field of Cistanche tubulosa for many years, having previously developed phenylethanoid glycoside preparations (CN1268341C) and successively explored various physiologically active applications. In recent years, we have further overcome the shortcomings of existing technologies regarding specific separation methods and application pathways, successfully obtaining extracellular vesicles from Cistanche tubulosa. Experiments have confirmed that extracellular vesicles isolated from Cistanche tubulosa can effectively protect nerves against amyloid protein damage and also effectively promote nerve synapse extension. We have discovered unique applications for these extracellular vesicles, including the prevention, treatment, or delay of amyloid protein-related neurological diseases or symptoms, as well as the regulation, maintenance, or improvement of nerve cell physiological functions. These discoveries fill gaps in published literature and patents, providing a highly valuable development direction for the biotechnology industry to find specific and high-efficiency materials for treating specific neurological diseases or symptoms affected by amyloid protein and protecting nerve physiological functions. Technical solutions

[0008] One objective of this invention is to provide an extracellular vesicle isolated from the parasitic plant Cistanche tubulosa, which has significant biological activity, particularly the physiological effects of protecting nerves against amyloid protein damage and / or promoting nerve synapse extension.

[0009] Another objective of this invention is to provide a method for preparing the extracellular vesicles, which obtains extracellular vesicles with high purity and uniform particle size through specific separation steps.

[0010] Another object of the present invention is to provide a use of the extracellular vesicles, in order to apply them to the preparation of pharmaceutical compositions to prevent, treat or delay amyloid-related neurological diseases or symptoms by means of their physiological effects of protecting nerve cells against amyloid damage and / or promoting nerve synapse extension.

[0011] Another object of the present invention is to provide a use of the extracellular vesicles to regulate, maintain or improve the physiological function of nerve cells by protecting nerve cells against amyloid damage and / or promoting the extension of nerve synapses.

[0012] Another object of the present invention is to provide a method for protecting nerve cells against amyloid damage and / or promoting synaptic extension, comprising administering an effective amount of the vesicles to an individual in need. This method may be used to prevent, treat or delay amyloid-related neurological diseases or symptoms, or to regulate, maintain or improve the physiological function of nerve cells.

[0013] Preferably, these neurological disorders or symptoms associated with amyloid include mild cognitive impairment, Alzheimer's disease, Parkinson's disease, Lewy body dementia, Guam Parkinson's-dementia complex, frontotemporal dementia, familial amyloid polyneuropathy, Huntington's disease, Dutch amyloidosis, cerebral amyloid angiopathy, Down syndrome, inclusion body myositis, age-related macular degeneration, or Pick's disease.

[0014] Preferably, the pharmaceutical composition prepared from the extracellular vesicles is administered via a method selected from one of the following groups: oral, intravenous, intramuscular, subcutaneous, mucosal, and transdermal administration.

[0015] Preferably, the extracellular vesicle is used in the form of a food composition, cosmetic composition, skin care composition, or feed composition to achieve the purpose of regulating, maintaining, or improving the physiological function of nerve cells.

[0016] Preferably, the physiological function of the nerve cells is affected by amyloid protein or by the growth of neural synapses.

[0017] Preferably, the physiological functions of the nerve cells maintain memory, clear thinking, improve reaction or enhance concentration.

[0018] These food compositions can be health foods, health care foods, functional foods, nutritional supplements, or special nutritional foods. Preferably, the food composition includes dairy products, processed meat products, breads, pasta products, biscuits, lozenges, capsules, fruit juices, teas, sports drinks, nutritional drinks, soups, powders, jellies, gummies, solid-liquid mixtures, or solid-liquid mixtures.

[0019] Preferably, the preparation method includes the following steps: (a) adding water to Cistanche tubulosa to break the cell wall and obtain a first Cistanche tubulosa juice; (b) centrifuging the first Cistanche tubulosa juice in a first stage and removing the precipitate to obtain a second Cistanche tubulosa juice, wherein the first stage centrifugation is performed at a speed of less than or equal to 6,000 × g; (c) centrifuging the second Cistanche tubulosa juice in a second stage and removing the precipitate, wherein the second stage centrifugation is performed at a speed of greater than or equal to 8,000 × g to obtain a supernatant of the Cistanche tubulosa juice; (d) filtering the supernatant; (e) adding polymer molecules to the filtered supernatant and mixing thoroughly to form a mixture; and (f) centrifuging the mixture and removing the precipitate to obtain extracellular vesicles of Cistanche tubulosa.

[0020] In step (b) above, the first stage of centrifugation is performed at a speed of 6,000 × g or less, for example, at a speed of 4,000 × g to 6,000 × g; preferably at a speed of 5,500 × g or less, for example, at a speed of 4,500 × g to 5,500 × g; more preferably at a speed of 5,200 × g or less, for example, at a speed of 5,000 × g. Any suitable centrifugation time can be used in the first stage of centrifugation, as long as it provides the desired effect of removing impurities. For example, the first stage of centrifugation can be performed at 5,000 × g for 5 to 10 minutes.

[0021] In step (c) above, the second-stage centrifugation is performed at a speed of 8,000 × g or higher, for example, from 8,000 × g to 12,000 × g; preferably, at a speed of 9,000 × g or higher, for example, from 9,000 × g to 11,000 × g; more preferably, at a speed of 9,500 × g or higher, for example, from 9,500 × g to 10,500 × g; and in some specific embodiments, at a speed of 10,000 × g. Similarly, any suitable centrifugation time can be used in the second stage, as long as it provides the desired impurity removal effect. For example, the second-stage centrifugation can be performed at 10,000 × g for 15 to 30 minutes. If necessary, the second-stage centrifugation can include two or more cycles of centrifugation and filtration until no precipitate remains.

[0022] In step (d) above, the supernatant is sequentially passed through a filter membrane of 0.4 μm or greater and a filter membrane of 0.25 μm or less. For example, in step (d), the supernatant may be initially filtered with a filter membrane of 0.4 μm to 0.5 μm (e.g., using a 0.45 μm filter membrane for initial filtration); then, the initially filtered supernatant may be further filtered with a filter membrane of 0.20 μm to 0.25 μm (e.g., using a 0.22 μm filter membrane for further filtration).

[0023] Preferably, in step (e) above, the amount of polymer molecule added is such that its volume percentage concentration in the mixture reaches 6-10%, and the polymer molecule is at least one of the following: polyethylene glycol, lectin, protamine sulfate, and sodium acetate. More preferably, in step (e) above, polyethylene glycol is used as the polymer molecule.

[0024] Preferably, in step (e) above, the mixing reaction is carried out at 4°C for 12 to 16 hours. Preferably, in step (f) above, the centrifugation is performed at a speed of less than or equal to 4000×g, for example, a speed of 2000×g to 4000×g; more preferably, at a speed of less than or equal to 3500×g, for example, a speed of 2500×g to 3500×g; even more preferably, at a speed of less than or equal to 3200×g, and in some specific embodiments, at a speed of 3000×g; in step (f) above, the centrifugation time is preferably 30 to 40 minutes. Attached Figure Description

[0025] Figure 1 shows the particle size data analyzed by a nanoparticle analyzer. The analyzed sample was extracellular vesicles of Cistanche tubulosa prepared in Example 2.

[0026] Figure 2 shows the cell viability analysis (MTT assay) data of the human neuroblastoma cell line SH-SY5Y, including the groups listed in Table 1, showing the cell viability of SH-SY5Y cells after 24 hours of interaction with different concentrations of Cistanche tubulosa extracellular vesicles.

[0027] Figure 3 shows the cell survival rate analysis data of the mouse neural crest-derived cell line Neuro-2a, including the groups listed in Table 1, showing the cell survival rate of Neuro-2a cells after 96 hours of interaction with extracellular vesicles of different concentrations of Cistanche tubulosa.

[0028] Figure 4 shows the cell viability analysis data of the human neuroblastoma cell line SH-SY5Y; Figure 4 includes the groups listed in Table 2.

[0029] Figure 5 shows the cell viability analysis data of the human neuroblastoma cell line SH-SY5Y (Part 2); Figure 5 includes the groups listed in Table 3; Figures 4 and 5 show the cell viability of SH-SY5Y cells after pre-reacting with different test samples for 1 hour, adding 20 μM β-amyloid 1-42 peptide (Aβ1-42), and culturing for 24 hours.

[0030] Figure 6 shows the results of the neurosynaptic growth experiment of the mouse neural crest-derived cell line Neuro-2a, specifically the cell photography image;

[0031] Figure 7 shows the second result of the neuro-2a neural synapse growth experiment, specifically the quantitative analysis of the synapse occurrence rate of Neuro-2a cells using image analysis. Figures 6 and 7 include the groups listed in Table 4, showing the changes in neural synapse growth after Neuro-2a cells were co-cultured with different test samples for 96 hours. Detailed Implementation

[0032] The detailed technical content and some specific embodiments of the present invention will be described below to enable those skilled in the art to understand the features of the present invention.

[0033] The detailed technology and preferred embodiments of the present invention will be described below to enable those skilled in the art to understand the features of the present invention; however, the present invention may be practiced in many different forms without departing from the spirit of the present invention, and the scope of protection of the present invention should not be construed as limited to those set forth in the specification. Furthermore, unless otherwise stated herein, the terms “a,” “the,” and similar terms used in this specification (especially in the claims described below) shall be understood to include both singular and plural forms; “effective amount” means a dose, when administered to an individual, that effectively reduces at least partially the effect of amyloid on the physiological function of the individual’s nerve cells, or at least partially improves the physiological function of nerve cells by promoting synaptic growth, or improves the individual’s amyloid-related neurological diseases or symptoms; “individual” means human or non-human mammal; “prevention” means the suppression or prevention of the onset of a specific condition, or the maintenance of good health in a susceptible individual or the establishment of tolerance to the disease in that individual; “treatment” should not be construed as treating an individual until complete recovery, but should include maintaining the progression of the disease or symptoms in an individual at a substantially static level, increasing the rate of recovery in an individual, reducing the severity of a specific condition, or improving the quality of life of a patient.

[0034] The numerical ranges used in this specification (e.g., 5 to 100) should be understood to include all rational numbers within that range and any range of rational numbers within that range. Therefore, the numerical ranges used in this specification include all possible combinations of values ​​between the listed minimum and maximum values. Furthermore, when the word "about" is used before a numerical value, it substantially means a difference of less than 10%, preferably less than 5%, from the stated value.

[0035] As mentioned above, abnormal accumulation of amyloid protein can affect the normal physiological functions of nerve cells, potentially causing amyloid-related neurological diseases or symptoms (such as mild cognitive impairment, Alzheimer's disease, Parkinson's disease, Lewy body dementia, Guam Parkinson's-Dementia complex, frontotemporal dementia, familial amyloid polyneuropathy, Huntington's disease, Dutch amyloidosis, cerebral amyloid angiopathy, Down syndrome, inclusion body myositis, age-related macular degeneration, or Pick's disease). The physiological functions of nerve cells (such as maintaining memory, clear thinking, improving reaction time, or enhancing concentration) may be affected by amyloid protein, or by the growth of neural synapses. The inventors of this study discovered that extracellular vesicles isolated from *Cistanche tubulosa*, after administration, can effectively protect nerve cells against amyloid protein damage. The extracellular vesicles isolated from Cistanche tubulosa of the present invention can be used for at least one of the following: prevention, treatment or delay of neurological diseases or symptoms related to amyloid protein, or for regulating, maintaining or improving the physiological function of nerve cells.

[0036] Therefore, this invention provides an extracellular vesicle derived from the parasitic plant *Cistanche tubulosa*, obtained through the following preparation steps: cell wall disruption, centrifugation to remove precipitate, reaction with polymer molecules, and centrifugation to collect the precipitate, thus separating it from *Cistanche tubulosa*. This extracellular vesicle can be used for at least one of the following: prevention, treatment, or delay of amyloid-related neurological diseases or symptoms, or for regulating, maintaining, or improving the physiological function of nerve cells.

[0037] The extracellular vesicles isolated from *Cistanche tubulosa* according to the present invention can be any part of *Cistanche tubulosa*, for example, the stem, flowers, or whole plant of *Cistanche tubulosa* can be used as raw material. In one embodiment of the present invention, the fleshy stem of *Cistanche tubulosa* is used as raw material.

[0038] The inventors discovered that by rationally configuring a specific combination of relevant parameters, the desired separation effect can be effectively achieved while maintaining the biological activity of extracellular vesicles. Preferably, the method for preparing the extracellular vesicles includes the following steps: (a) adding water to *Cistanche tubulosa* to break the cell wall and obtain a first *Cistanche tubulosa* juice; (b) centrifuging the first *Cistanche tubulosa* juice in a first stage and removing the precipitate to obtain a second *Cistanche tubulosa* juice, wherein the first stage centrifugation is performed at a speed of less than or equal to 6,000 × g; (c) centrifuging the second *Cistanche tubulosa* juice in a second stage and removing the precipitate, wherein the second stage centrifugation is performed at a speed of greater than or equal to 8,000 × g to obtain a supernatant of the *Cistanche tubulosa* juice; (d) filtering the supernatant; (e) adding polymer molecules to the filtered supernatant and mixing thoroughly to form a mixture; and (f) centrifuging the mixture and removing the precipitate to obtain extracellular vesicles of *Cistanche tubulosa*.

[0039] In the above preparation steps, to achieve the desired separation effect, in step (b) above, the first stage centrifugation is performed at a speed of less than or equal to 6,000 × g, for example, from 4,000 × g to 6,000 × g; preferably less than or equal to 5,500 × g, for example, from 4,500 × g to 5,500 × g; more preferably less than or equal to 5,200 × g, for example, from 5,000 × g. Any suitable centrifugation time can be used in the first stage centrifugation, as long as the desired impurity removal effect is achieved. For example, the first stage centrifugation can last for 5 to 10 minutes.

[0040] In step (c) above, the second-stage centrifugation is performed at a speed of 8,000 × g or higher, for example, from 8,000 × g to 12,000 × g; preferably, at a speed of 9,000 × g or higher, for example, from 9,000 × g to 11,000 × g; more preferably, at a speed of 9,500 × g or higher, for example, from 9,500 × g to 10,500 × g; and in some specific embodiments, at a speed of 10,000 × g. Any suitable centrifugation time can be used in the second stage, as long as it provides the desired impurity removal effect. For example, the second-stage centrifugation can be performed at 10,000 × g for 15 to 30 minutes. If necessary, the second-stage centrifugation can include two or more cycles of centrifugation and filtration until no precipitate remains.

[0041] In step (d) above, the supernatant is sequentially passed through a filter membrane of 0.4 μm or greater and a filter membrane of 0.25 μm or less. For example, in step (d), the supernatant may first be initially filtered with a filter membrane of 0.4 μm to 0.5 μm (e.g., using a 0.45 μm filter membrane for initial filtration); then, the initially filtered supernatant may be further filtered with a filter membrane of 0.20 μm to 0.25 μm (e.g., using a 0.22 μm filter membrane for further filtration).

[0042] Preferably, in step (e) above, the amount of polymer molecule added is such that its volume percentage concentration in the mixture reaches 6-10%, and the polymer molecule is at least one of the following: polyethylene glycol, lectin, protamine sulfate, and sodium acetate. More preferably, polyethylene glycol is used as the polymer molecule in step (e). The mixing reaction in step (e) is preferably carried out at 4°C for 12-16 hours.

[0043] In step (f) above, centrifugation is performed at a speed of 4000×g or less, for example, a speed of 2000×g to 4000×g; preferably, at a speed of 3500×g or less, for example, a speed of 2500×g to 3500×g; more preferably, at a speed of 3200×g or less, and in some specific embodiments, at a speed of 3000×g. In step (f), the centrifugation time is preferably 30 to 40 minutes.

[0044] The extracellular vesicles isolated from *Cistanche tubulosa* according to the present invention may be used in or for the preparation of at least one of the following: a pharmaceutical composition, a food composition, a cosmetic composition, a skincare composition, or a feed composition; or, the extracellular vesicles may be used in the preparation of at least one of the aforementioned compositions. The pharmaceutical composition may be in any suitable form without particular limitation, depending on the desired application and the appropriate dosage form. For example, but not limited to, the pharmaceutical composition may be administered to the individual in need via oral or non-oral administration (e.g., intravenous injection, intramuscular injection, subcutaneous injection, mucosal administration, or skin administration). Depending on the form of use and purpose, a suitable carrier may be selected to provide the pharmaceutical composition, including excipients, diluents, adjuvants, stabilizers, absorption delay agents, disintegrants, solubilizers, emulsifiers, antioxidants, binders, binders, thickeners, dispersants, suspending agents, lubricants, hygroscopic agents, etc.

[0045] Taking injectable or intravenous solutions suitable for intravenous, subcutaneous, or intramuscular injection as an example, the pharmaceutical composition provided according to the present invention may contain one or more components such as isotropic solutions, salt buffers (e.g., phosphate buffer or citrate buffer), solubilizers, emulsifiers, 5% sugar solutions, and other carriers, and may be provided in dosage forms such as intravenous infusions, emulsion intravenous infusions, dry powder injections, suspension injections, or dry powder suspension injections. Alternatively, the pharmaceutical composition may be prepared as a pre-injection solid, provided in a dosage form soluble in other solutions or suspensions, or in an emulsifiable dosage form, and the pre-injection solid may be dissolved in other solutions or suspensions or emulsified before being administered to the individual in need to provide the desired injectable.

[0046] Taking a dosage form suitable for oral administration as an example, the pharmaceutical composition provided according to the present invention may contain any pharmaceutically acceptable carrier that will not adversely affect the desired efficacy of the extracellular vesicles isolated from *Cistanche tubulosa* according to the present invention, such as: water, saline, glucose, glycerol, ethanol or analogues thereof, cellulose, starch, sugar bentonite, and combinations thereof. The pharmaceutical composition may be provided in a dosage form suitable for oral administration using any suitable method, such as: tablets (e.g., sugar-coated tablets), pills, capsules, granules, powders, fluid extracts, solutions, syrups, suspensions, tinctures, etc.

[0047] Taking a dosage form suitable for administration via mucosa as an example, the pharmaceutical composition provided by the present invention may be in the form of an eye, nasal cavity, oral cavity or other mucosa, specifically in the form of eye drops (e.g., solution, suspension, emulsion), eye ointment, spray, drops, spray, sublingual tablet, mucosal patch, gel, suppository, or film agent, foam agent, etc. for use on mucosal surface, but is not limited thereto.

[0048] Taking dosage forms suitable for transdermal administration as an example, the pharmaceutical compositions provided according to the present invention may be in the form of patches, lotions, creams, gels (e.g., hydrogels), pastes (e.g., dispersants, ointments), sprays, or solutions (e.g., suspensions) for direct external use, but are not limited thereto.

[0049] The pharmaceutical composition provided by the present invention can be administered at different frequencies, such as once a day, multiple times a day, or once every few days, depending on the individual's needs, age, weight, and health condition. The content ratio of extracellular vesicles isolated from *Cistanche tubulosa* in the pharmaceutical composition provided by the present invention can be adjusted according to actual application requirements.

[0050] As needed, the pharmaceutical composition, food composition, cosmetic composition, skincare composition, or feed composition provided according to the present invention may contain appropriate amounts of additives, such as flavoring agents, coloring agents, coloring agents, etc., which can improve the taste and visual appeal of the pharmaceutical composition, food composition, cosmetic composition, skincare composition, or feed composition when consumed, as well as buffering agents, preservatives, antiseptics, antibacterial agents, antifungal agents, etc., which can improve the stability and storability of the pharmaceutical composition, food composition, cosmetic composition, skincare composition, or feed composition.

[0051] The food composition provided by the present invention can be a health food, a health care food, a functional food, a nutritional supplement, or a special nutritional food, and can be made into products such as dairy products, processed meat products, bread, pasta products, biscuits, lozenges, capsules, fruit juices, teas, sports drinks, nutritional drinks, soups, powders, jellies, gummies, solid-liquid mixtures, or solid-liquid mixtures, but is not limited thereto.

[0052] The health foods, health supplements, functional foods, nutritional supplements, and special nutritional foods provided by this invention can be consumed at different frequencies, such as once a day, multiple times a day, or once every few days, depending on the individual's age, weight, and health condition. The content of extracellular vesicles in the health foods, health supplements, functional foods, nutritional supplements, and special nutritional foods provided by this invention can also be adjusted for specific groups, preferably to the recommended daily intake.

[0053] The recommended dosage, usage standards and conditions for specific groups (such as heart disease patients, diabetes patients, pregnant women, the elderly, adolescents or children, etc.), or suggestions for taking with other foods or medicines can be indicated on the outer packaging of health foods, health care foods, functional foods, nutritional supplements and / or special nutritional foods provided by this invention, so that users can take them at home without safety concerns without the guidance of a doctor, pharmacist or relevant personnel.

[0054] The cosmetic and skincare compositions provided by the present invention can be used to regulate, maintain, or improve the physiological function of nerve cells. The skincare compositions provided by the present invention can be in any suitable form without particular limitation. For example, the skincare compositions can be in the form of lotions, creams, gels (e.g., hydrogels), or solutions (e.g., serums, lotions) for direct external use, but are not limited thereto.

[0055] When the cosmetic composition or skincare composition provided according to the present invention is applied to regulate, maintain or improve the physiological function of nerve cells, the content of the extracellular vesicles may vary depending on the product type.

[0056] The feed composition provided according to the present invention may contain any other edible raw materials that will not adversely affect the desired benefits of the extracellular vesicles isolated from *Cistanche tubulosa* according to the present invention, such as emulsifiers, skim milk powder, soy protein, sugars, starch, processed starch, dextrin, and salts such as inorganic or organic salts. The feed composition may be provided in solid, liquid, emulsified, or other forms using any suitable method.

[0057] This invention also provides a method for protecting nerve cells against amyloid damage and / or promoting synaptic extension, comprising administering to an individual in need an effective amount of extracellular vesicles isolated from *Cistanche tubulosa*, wherein the extracellular vesicles may be comprised of or prepared as at least one of the following: a pharmaceutical composition, a food composition, a cosmetic composition, a skincare composition, or a feed composition. The nature, route of administration, form of administration, frequency of administration, and related applications of these compositions are as described above.

[0058] The present invention will now be further illustrated by the following embodiments. These embodiments are provided for illustrative purposes only and are not intended to limit the scope of protection of the present invention. The scope of protection of the present invention is as shown in the appended claims.

[0059] Example

[0060] Example 1: Preparation of phenylethanol glycoside preparation from Cistanche tubulosa

[0061] 1. Water extraction of Cistanche tubulosa

[0062] Take the fleshy stem of Cistanche tubulosa, slice it, and soak it in 8 times its volume of water for 1 hour. Then decoct for 2 hours and filter to collect the filtrate. Add 6 times its volume of water to the dregs and decoct the dregs twice, 1 hour each time, and filter again. Combine the three filtrates and concentrate under reduced pressure at 50°C to a specific gravity of 1.10. Then add ethanol to the concentrate, bringing the final ethanol volume percentage to 60%. Refrigerate for 12 hours, decant the supernatant, concentrate under reduced pressure at 50°C, and recover the ethanol to a specific gravity of 1.10 to obtain the crude extract.

[0063] 2. Resin column purification of phenylethanoid glycosides

[0064] Next, the crude extract was dissolved by heating with one volume of water and injected into a macroporous adsorption resin column. Elution was performed sequentially with four volumes of water and five volumes of 40% ethanol (i.e., an ethanol-water solution containing 40 ml of ethanol per 100 ml). The water eluent was then injected into the macroporous adsorption resin column and eluted with three volumes of water. The water eluent was discarded, and then eluted with four volumes of 40% ethanol. The two 40% ethanol eluents were collected, concentrated, and dried to obtain the Cistanche tubulosa phenylethanol glycoside preparation (code name CTE). Based on the dry weight of the fleshy stem of Cistanche tubulosa, the extraction rate was calculated to be approximately 8.65 wt%.

[0065] Example 2: Preparation of extracellular vesicle samples from Cistanche tubulosa

[0066] 1. Homogeneous juice from the fleshy stems of Cistanche tubulosa

[0067] Take the dried fleshy stem of Cistanche tubulosa and soak it in pure water to soften it (this step can be omitted if using fresh fleshy stem). Then add 3 times its weight of pure water (suggested ratio: weight of fleshy stem of Cistanche tubulosa to weight of pure water = 1:1~5). Use a machine to crush the fleshy stem of Cistanche tubulosa to obtain homogenized juice, and then filter it through a gauze filter.

[0068] 2. Polymer precipitation method for separating extracellular vesicles

[0069] At 4°C, the filtered juice from the fleshy stems of *Cistanche tubulosa* was centrifuged at 5,000 × g for 10 minutes. After centrifugation, the precipitate was discarded, and the supernatant was collected. The supernatant was then centrifuged at 10,000 × g for 30 minutes. After centrifugation, the precipitate was discarded, and the supernatant was collected. This centrifugation process was repeated until no precipitate was produced. The supernatant was filtered sequentially through a 0.45 μm filter membrane and a 0.22 μm filter membrane, and the filtered liquid was collected. The filtrate was mixed uniformly with a polyethylene glycol solution (the final polyethylene glycol concentration was 8% by volume) and reacted at 4°C for 16 hours. At 4°C, the reacted mixture was centrifuged at 3,000 × g for 30 minutes. After centrifugation, the supernatant was carefully removed, and the resulting precipitate was the extracellular vesicle (CTV).

[0070] Example 3: Counting extracellular vesicles of Cistanche tubulosa

[0071] Extracellular vesicles were captured and counted using a NanoSight NS300 nanoparticle analyzer. Hardware settings: built-in laser: 488 nm wavelength, 45 mW; camera: sCMOS.

[0072] The extracellular vesicle sample obtained in Example 2 was diluted with phosphate buffer solution and adjusted to the ideal measurement concentration of 20 to 100 vesicle particles per frame under the camera lens.

[0073] Adjust the settings according to the manufacturer's software user manual (MAN0541-01-EN-00, 2017): Increase image intensity until all grains are clearly visible, and maintain the grain signal below 20% of the saturation value. Ideally, the detection conditions should include as many vesicle grains as possible within the lens image, while meeting the software's interpretation of 10-100 red crosspoints and no more than 5 blue crosspoints, with no more than 10% of the red crosspoints not associated with obvious vesicle grains. Enable autofocus adjustment to avoid blurry grains.

[0074] Extracellular vesicles prepared in Example 2 were resuspended and diluted in phosphate-buffered saline (PBS). The diluted vesicle samples were injected into a nanoparticle analyzer under the following conditions: cell temperature: 25°C; syringe speed: 40 µl / s. Five video segments were recorded for each measurement, each lasting 1 minute. The recorded images were analyzed using the built-in NanoSight software NTA 3.1 Build 3.1.46, with a detection threshold of 5. During NTA measurements, the number of completed trajectories was consistently greater than the recommended minimum of 1000 to reduce data bias based on single large particles.

[0075] As shown in Figure 1, the extracellular vesicles (CTVs) prepared in Example 2 exhibit high purity and uniform particle size. Image analysis showed an average particle size of 130.1 nm and an extraction rate of 1.788 × 10⁻⁶. 11 granules / g (weight of the fleshy stem of Cistanche tubulosa).

[0076] Example 4: Test for extracellular vesicle cytotoxicity

[0077] 1. Cytotoxicity test of extracellular vesicles against human neuroblastoma cell line SH-SY5Y

[0078] The human neuroblastoma cell line SH-SY5Y was purchased from the American Type Culture Collection (ATCC), collection number CRL-2266.

[0079] The human neuroblastoma cell line SH-SY5Y was cultured in a 1:1 mixture of modified Eagle's Minimum Essential Medium Alpha (α-MEM) and Ham's F-12 medium containing 10% fetal bovine serum (FBS).

[0080] SH-SY5Y cells were suspended in culture medium, and the suspension was added to a 96-well cell culture dish so that each well contained 1.2 × 10⁶ cells. 4 After culturing SH-SY5Y cells overnight, the test samples shown in Table 1 were added and incubated for 24 hours. Finally, the changes in the cell viability of SH-SY5Y cells at different concentrations were detected by MTT assay, with the blank group as 100% standard. The results are shown in Figure 2.

[0081] As shown in Figure 2, when a concentration of 10 is added... 7 ~10 10 After culturing the test sample (i.e., the extracellular vesicles of Cistanche tubulosa prepared in Example 2, code name CTV) at a concentration of 102 / ml for 24 hours, the survival rate of SH-SY5Y cells in each group was higher than 90%, indicating that when CTV concentration is applied at 102... 10 Within the order of particles / ml (<10) 11 When the concentration is 10 ...

[0082] Table 1

[0083]

[0084] 2. Cytotoxicity test of extracellular vesicles against mouse neural crest-derived cell line Neuro-2a

[0085] The mouse neural crest-derived cell line Neuro-2a was purchased from the Bioresource Collection and Research Center (BCRC) in Taiwan, China, with the accession number 60026.

[0086] The mouse neural crest-derived cell line Neuro-2a was cultured in α-MEM medium containing 10% fetal bovine serum.

[0087] Neuro-2a cells were suspended in culture medium, and the suspension was added to a 96-well cell culture dish so that each well contained 1 × 10⁶ cells. 4 Neuro-2a cells were cultured overnight, and then the test samples of each group shown in Table 1 were added and incubated for 96 hours. Finally, the changes in the survival rate of Neuro-2a cells at different concentrations of the test samples were detected by MTT assay, with the blank group as 100% standard. The results are shown in Figure 3.

[0088] As shown in Figure 3, when a concentration of 10 is added... 7~10 10 After culturing the test sample (i.e., the extracellular vesicles of Cistanche tubulosa prepared in Example 2, code name CTV) at a concentration of 102 / ml for 96 hours, the survival rate of Neuro-2a cells in each group was higher than 80%, indicating that the application of CTV at a concentration of 102... 10 Within the order of particles / ml (<10) 11 At a concentration of 1000 granules / ml, it showed no significant toxicity to the neural crest-derived cell line Neuro-2a.

[0089] Example 5: Testing the efficacy of the sample against β-amyloid (Aβ) in nerve cells.

[0090] The human neuroblastoma cell line SH-SY5Y was cultured in a 1:1 mixture of α-MEM and Ham's F-12 medium containing 10% fetal bovine serum.

[0091] Quantitative analysis of SH-SY5Y cells (1.2 × 10⁻⁶) was performed. 4 Cells (each cell / well) were placed in 96-well cell culture dishes and cultured overnight in the aforementioned medium. The culture medium was then replaced with the medium containing the test samples shown in Table 2 for a pre-reaction time of 1 hour. Except for the blank group, 20 μM of β-amyloid 1-42 peptide (Aβ) was added to each well. 1-42 The cells were cultured for another 24 hours, and the MTT assay was used to detect the protective effect of each sample group against SH-SY5Y cells against amyloid. The blank group was used as the 100% standard. The results are shown in Figure 4.

[0092] Table 2

[0093]

[0094] As shown in Figure 4, compared to the control group, the addition of amyloid peptide Aβ... 1-42In the control group, the survival rate of SH-SY5Y cells decreased significantly (to approximately 40%), indicating that amyloid protein induces nerve cell death. Compared with the control group, the groups treated with the Cistanche tubulosa phenylethanoid glycoside preparations (CTE-10, CTE-30) prepared in Example 1 showed an increase in the survival rate of SH-SY5Y cells to approximately 50-60%. Compared with the control group, the groups treated with the main components of the Cistanche tubulosa phenylethanoid glycoside preparations, Act-50 and Iso-50, also showed an increase in the survival rate of SH-SY5Y cells to approximately 55-60%. Compared with the control group, the groups treated with the extracellular vesicles of Cistanche tubulosa (CTV-2, CTV-6, CTV-60) prepared in Example 2 also showed an increase in the survival rate of SH-SY5Y cells to about 52-53%, indicating that the tested Cistanche tubulosa extracellular vesicle samples have the effect of resisting amyloid protein damage to nerve cells.

[0095] The inventors observed that the survival rate improvement effect of SH-SY5Y cells was similar in the CTV-2, CTV-6, and CTV-60 groups, speculating that the extracellular vesicles of Cistanche tubulosa might be related to the 2×10⁻⁶ cells. 6 At dosages of granules / ml or higher, the effect of protecting nerve cells against amyloid damage has reached saturation. Therefore, we wanted to test the effect of Cistanche tubulosa extracellular vesicles on protecting nerve cells against amyloid damage at lower dosages.

[0096] SH-SY5Y cells were cultured in the same manner for a second experiment, with a quantitative sample of SH-SY5Y cells (1.2 × 10⁻⁶). 4 Cells (each cell / well) were placed in a 96-well cell culture dish and cultured overnight. The culture medium was then replaced with the medium containing the test samples shown in Table 3 for a pre-reaction time of 1 hour. Then, 20 μM of β-amyloid 1-42 peptide (Aβ) was added to each well. 1-42 The cells were cultured for another 24 hours, and the MTT assay was used to detect the protective effect of each sample group against SH-SY5Y cells against amyloid. The blank group in Table 2 was used as the 100% standard. The results are shown in Figure 5.

[0097] Table 3

[0098]

[0099] As shown in Figure 5, the addition of amyloid peptide Aβ... 1-42In the control group, the survival rate of SH-SY5Y cells decreased significantly (to approximately 49.2%), indicating that amyloid protein induces nerve cell death. Compared with the control group, in the groups treated with low doses (CTV-0.06, CTV-0.6) of Cistanche tubulosa extracellular vesicles prepared in Example 2, the survival rates of SH-SY5Y cells increased to approximately 59.4% and 67.5%, respectively, indicating that the tested Cistanche tubulosa extracellular vesicle samples, within 10... 4 At a level comparable to that, it is possible to protect nerve cells from damage caused by amyloid protein.

[0100] Example 6: Testing the efficacy of the sample in promoting synaptic extension of nerve cells

[0101] The mouse neural crest-derived cell line Neuro-2a was cultured in α-MEM medium containing 10% fetal bovine serum.

[0102] A quantitative sample of Neuro-2a cells (5,000 cells / well) was placed in 24-well cell culture dishes and cultured overnight. The culture medium was then replaced with α-MEM medium containing 2% fetal bovine serum and the test samples shown in Table 4, and cultured for 96 hours. Finally, the microtubules of the cells were labeled by immunofluorescence staining to help observe changes in neurites. At the same time, photographs were taken and recorded. The cell images are shown in Figure 6. The synaptic rate of the Neuro-2a cell line was quantified by image analysis, and the results are shown in Figure 7.

[0103] Table 4

[0104]

[0105] As shown in Figure 6, no obvious synaptic extension was observed in Neuro-2a cells in the control group. Compared with the control group, synaptic extension was observed in Neuro-2a cells treated with the Cistanche tubulosa phenylethanol glycoside preparation (CTE-30) prepared in Example 1. Compared with the control group, obvious synaptic extension was observed under a microscope in the groups treated with Cistanche tubulosa extracellular vesicles (CTV-0.06, CTV-0.6, CTV-6, CTV-60) prepared in Example 2. In particular, the neural synapses of the CTV-0.6, CTV-6, and CTV-60 groups gradually became more prominent, indicating that the length of neural synapses increased significantly with the increase of Cistanche tubulosa extracellular vesicle dosage.

[0106] As shown in Figure 7, the dosage of extracellular vesicles of Cistanche tubulosa is less than 0.6 × 10⁻⁶. 6 At a concentration of 1000 vesicles / ml, the effect of promoting the synaptic rate of Neuro-2a cells increased with increasing extracellular vesicle dosage, and the effect was further enhanced when the dosage of Cistanche tubulosa extracellular vesicles exceeded 0.6 × 10⁻⁶.6 When the concentration of *Cistanche tubulosa* was increased to a certain level (e.g., granules / ml), the effect of promoting the synaptic rate of Neuro-2a cells slowed down. The experimental results confirm that the extracellular vesicles of *Cistanche tubulosa* have the effect of promoting the growth of nerve cell synapses.

[0107] The experimental results above show that the extracellular vesicles isolated from Cistanche tubulosa of the present invention can indeed protect nerve cells from amyloid protein damage, and can also promote the growth of nerve cell synapses. Therefore, the extracellular vesicles of the present invention do have the effect of protecting nerves, and can be prepared into a pharmaceutical composition, a food composition, a cosmetic composition, a skin care product composition, or a feed composition, and given to individuals in need for at least one of the following purposes: prevention, treatment or delay of amyloid protein-related neurological diseases or symptoms, or for regulating, maintaining or improving the physiological function of nerve cells.

Claims

1. The use of extracellular vesicles isolated from Cistanche tubulosa in the preparation of a pharmaceutical composition, characterized in that, The pharmaceutical composition is used to prevent, treat or delay neurological diseases or symptoms associated with amyloid, and the pharmaceutical composition is administered orally, intravenously, intramuscularly, subcutaneously, via mucosal administration, via skin administration, or a combination thereof.

2. The use as described in claim 1, characterized in that, The neurological disorder or symptoms include mild cognitive impairment, Alzheimer's disease, Parkinson's disease, Lewy body dementia, Guam Parkinson's disease-dementia complex, frontotemporal dementia, familial amyloid polyneuropathy, Huntington's disease, Dutch amyloidosis, cerebral amyloid angiopathy, Down syndrome, inclusion body myositis, age-related macular degeneration, Pick's disease, or a combination of the foregoing.

3. A use of extracellular vesicles isolated from *Cistanche tubulosa* for regulating, maintaining, or improving the physiological function of nerve cells, characterized in that... The extracellular vesicles are used in the form of a food composition, a cosmetic composition, a skincare composition, or a feed composition.

4. The use as described in claim 3, characterized in that, The physiological function of this nerve cell is affected by amyloid protein or by the growth of neural synapses.

5. The use as described in claim 3, characterized in that, The physiological functions of this nerve cell include maintaining memory, clear thinking, improving reaction time, enhancing focus, or a combination thereof.

6. The use as described in any one of claims 1 to 5, characterized in that, The preparation method of this extracellular vesicle includes the following steps: (a) Add water to Cistanche tubulosa to break the cell wall and obtain the juice of Cistanche tubulosa. (b) The first tube of Cistanche deserticola juice was centrifuged in the first stage to remove the precipitate and obtain the second tube of Cistanche deserticola juice. The first stage of centrifugation was carried out at a speed of less than or equal to 6,000 × g. (c) The second stage of centrifugation of the second Cistanche tubulosa juice was carried out at a speed of ≥8,000×g to remove the precipitate, and the supernatant of the Cistanche tubulosa juice was obtained. (d) Filter the supernatant; (e) Add polymer molecules to the filtered supernatant and mix thoroughly to form a mixture; and (f) After centrifuging the mixture, the precipitate was collected to obtain the extracellular vesicles of Cistanche tubulosa.

7. The use as described in claim 6, characterized in that, In step (b), the first stage of centrifugation is performed at 5,000×g for 5 to 10 minutes.

8. The use as described in claim 6, characterized in that, In step (c), the second stage of centrifugation is performed at 10,000×g for 15 to 30 minutes.

9. The use as described in claim 6, characterized in that, In step (d), the supernatant is passed sequentially through a filter membrane with a diameter greater than or equal to 0.4 μm and a filter membrane with a diameter less than or equal to 0.25 μm.

10. The use as described in claim 6, characterized in that, In step (e), the polymer molecule is polyethylene glycol, lectin, protamine, sodium acetate, or a combination thereof.

11. The use as described in claim 10, wherein, In step (e), the polymer molecule is polyethylene glycol.

12. The use as described in claim 6, characterized in that, In step (e), the mixing reaction is carried out at 4°C for 12 to 16 hours.

13. The use as described in claim 6, characterized in that, In step (f), centrifugation is performed at 3,000 × g for 30 to 40 minutes.

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