Extracellular vesicle isolated from cistanche tubulosa and use thereof

Abstract

An extracellular vesicle derived from a parasitic plant Cistanche tubulosa, isolated from Cistanche tubulosa by means of preparation steps comprising wall breaking, centrifugation to remove a precipitate, reaction with polymer molecules, and centrifugation to collect a precipitate. The extracellular vesicle can be used for at least one of the following: protecting retinal pigment epithelial cells against oxidative pressure injury and protecting corneal epithelial cells against high osmotic pressure injury, thereby achieving the use of eye protection.

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 the eye, particularly in protecting eye cells against oxidative stress and / or hyperosmotic stress. 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, where 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 them 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 utilizes Cistanche tubulosa extracts for their antioxidant, anti-aging, neuroprotective, and anti-fatigue effects. However, past research has largely 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 remains 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 lack of specific separation methods and application pathways in existing technologies, successfully obtaining extracellular vesicles from Cistanche tubulosa. Experiments have confirmed that extracellular vesicles isolated from Cistanche tubulosa can effectively protect retinal pigment epithelial cells against oxidative stress damage, leading to the discovery of unique applications for these extracellular vesicles, including the prevention or treatment of eye diseases or symptoms related to oxidative stress, and the regulation, maintenance, or improvement of the physiological functions of eye cells affected by oxidative stress. Experiments have also confirmed that extracellular vesicles isolated from Cistanche tubulosa can effectively protect corneal epithelial cells against high osmotic pressure damage, leading to the discovery of unique applications for the prevention, treatment, or delay of dry eye syndrome, as well as the regulation, maintenance, or improvement of dry eye. These findings fill gaps in literature disclosure and patent applications, providing a valuable direction for the biotechnology industry to find specific and high-performance materials for specific eye diseases, symptoms, and physiological effects caused by oxidative stress damage and / or high osmotic pressure damage (such as dry eye syndrome and dry eye). 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 in protecting ocular cells against oxidative stress damage and / or protecting ocular cells against high osmotic pressure damage.

[0009] Another objective of this invention is to provide a method for preparing the extracellular vesicles, thereby obtaining 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 for the prevention or treatment of eye diseases or symptoms related to oxidative stress, by virtue of their physiological function of protecting eye cells against oxidative stress damage.

[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 ocular cells affected by oxidative stress by protecting ocular cells against oxidative stress damage.

[0012] Another object of the present invention is to provide a method for protecting ocular cells against oxidative stress damage, comprising administering an effective amount of the vesicles to an individual in need, the method being used to prevent or treat ocular diseases or symptoms associated with oxidative stress, or to regulate, maintain or improve the physiological function of ocular cells affected by oxidative stress.

[0013] Another object of the present invention is to provide a use of the extracellular vesicles, by virtue of their physiological function of protecting ocular cells against high osmotic pressure damage, in the preparation of pharmaceutical compositions for the prevention, treatment or delay of dry eye syndrome.

[0014] Another object of the present invention is to provide a use of the extracellular vesicles to regulate, maintain or improve dry eyes by protecting ocular cells against the physiological effects of high osmotic pressure damage.

[0015] Another object of the present invention is to provide a method for protecting ocular cells against high osmotic pressure damage, comprising administering an effective amount of the vesicles to an individual in need, the method being used to prevent, treat or delay dry eye syndrome, or to regulate, maintain or improve dry eye.

[0016] Preferably, these oxidative stress-related eye diseases or symptoms include macular degeneration, macular holes, retinopathy, or glaucoma. Preferably, the macular degeneration is age-related macular degeneration, which includes dry macular degeneration or wet macular degeneration. Preferably, the retinopathy includes diabetic retinopathy, retinitis pigmentosa, retinal disease, retinal artery and vein occlusion, proliferative vitreoretinopathy, or central serous chorioretinopathy.

[0017] Preferably, the dry eye syndrome or dry eye is associated with at least one of the following groups of factors: smoke, particulate matter, dry air, air conditioning, prolonged use of electronic devices, contact lens wear, aging, hormonal changes, inflammation, autoimmune diseases, refractive surgery, and medications.

[0018] 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.

[0019] Preferably, the extracellular vesicle is used in the form of a food composition, cosmetic composition, skincare composition, or feed composition to regulate, maintain, or improve the physiological function of eye cells affected by oxidative stress. Preferably, the aforementioned oxidative stress is caused by chemical molecules or blue light. More preferably, the eye cells are retinal pigment epithelial cells.

[0020] Preferably, the extracellular vesicles are 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 dry eyes.

[0021] These food compositions can be health foods, health care foods, functional foods, nutritional supplements, or special nutritional foods. Preferably, the food composition is a dairy product, processed meat product, bread, pasta product, biscuit, lozenge, capsule, fruit juice, tea, sports drink, nutritional drink, soup, powder, jelly, gummy, solid-liquid mixture, or solid-liquid mixture beverage.

[0022] Preferably, the preparation method comprises 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 not greater than 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 not less than 8,000×g to obtain a supernatant of the Cistanche tubulosa juice; (d) adding polymer molecules to the filtered supernatant and mixing thoroughly to form a mixture; (e) adding polymer molecules and mixing thoroughly; and (f) centrifuging the mixture and removing the precipitate to obtain extracellular vesicles of Cistanche tubulosa.

[0023] The first stage of centrifugation in step (b) above shall be performed at a speed not exceeding 6,000 × g, for example, at a speed of 4,000 × g to 6,000 × g; preferably at a speed not exceeding 5,500 × g, for example, at a speed of 4,500 × g to 5,500 × g; more preferably at a speed not exceeding 5,200 × g, for example, at a speed of 5,000 × g. Any suitable centrifugation time may be used in the first stage of centrifugation, as long as the desired impurity removal effect is achieved. For example, the first stage of centrifugation may be performed at 5,000 × g for 5 to 10 minutes.

[0024] The second stage of centrifugation in step (c) above is performed at a speed of not less than 8,000 × g, for example, at a speed of 8,000 × g to 12,000 × g; preferably at a speed of not less than 9,000 × g, for example, at a speed of 9,000 × g to 11,000 × g; more preferably at a speed of not less than 9,500 × g, for example, at a speed of 9,500 × g to 10,500 × g; and in some specific embodiments, a speed of 10,000 × g is used. Similarly, any suitable centrifugation time can be used in the second stage, as long as it can provide the desired effect of removing impurities. For example, the second stage of centrifugation can be performed at 10,000 × g for 15 to 30 minutes. If necessary, the second stage of centrifugation can include two or more cycles of centrifugation and filtration until no precipitate remains.

[0025] In step (d) above, the supernatant is sequentially passed through a filter membrane with a diameter of not less than 0.4 μm and a filter membrane with a diameter of not more than 0.25 μm. 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).

[0026] Preferably, the amount of polymer molecule added in step (e) 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).

[0027] Preferably, the mixing reaction in step (e) is carried out at 4°C for 12 to 16 hours. Preferably, step (f) is centrifuged at a speed not exceeding 4000×g, for example, 2000×g to 4000×g; more preferably, at a speed not exceeding 3500×g, for example, 2500×g to 3500×g; even more preferably, at a speed not exceeding 3200×g, and in some specific embodiments, at a speed of 3000×g; the centrifugation time in step (f) is preferably 30 to 40 minutes.

[0028] The detailed technical content and some specific embodiments of the present invention will be described below so that those skilled in the art can understand the features of the present invention. Attached Figure Description

[0029] 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.

[0030] Figure 2 shows the cell viability analysis (MTT assay) data of the human retinal pigment epithelial cell line APRE-19, including the groups listed in Table 1, showing the cell viability of APRE-19 cells after 48 hours of interaction with different concentrations of Cistanche tubulosa extracellular vesicles.

[0031] Figure 3 shows the cell viability analysis data of the human retinal pigment epithelial cell line APRE-19, including the groups listed in Table 2. It shows the cell viability of APRE-19 cells after 24 hours of pre-reaction with different test samples, and then after 24 hours of co-treatment with the test samples and 0.1 mM tert-butyl hydroxyperoxide (t-BHP).

[0032] Figure 4 shows the APRE-19 cell survival rate analysis data, including the groups listed in Table 3, showing the cell survival rate of APRE-19 cells after 24 hours of combined treatment with different test samples and blue light irradiation.

[0033] Figure 5 shows the APRE-19 cell survival rate analysis data, including the groups listed in Table 4. It shows the cell survival rate of APRE-19 cells after 24 hours of pre-reaction with extracellular vesicles of different concentrations of Cistanche tubulosa and then 24 hours of blue light irradiation.

[0034] Figure 6 shows the cell viability analysis (MTT assay) data of the human corneal epithelial cell line 2.040 pRSV-T, including the groups listed in Table 5, showing the cell viability of 2.040 pRSV-T cells after 24 hours of interaction with different concentrations of Cistanche tubulosa extracellular vesicles.

[0035] Figure 7 shows the cell viability analysis data of the human corneal epithelial cell line 2.040 pRSV-T, including the groups listed in Table 6, showing the cell viability of 2.040 pRSV-T cells after 24 hours of interaction with high-osmotic medium containing different test samples. Detailed Implementation

[0036] 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 can 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 stated in the specification. Furthermore, unless otherwise stated herein, the terms “a,” “the,” and similar terms used in this specification (especially in the claims) shall be understood to include both singular and plural forms; “effective amount” means, when administered to an individual, a dose that, when applied to that individual, can effectively at least partially reduce the effects of oxidative stress on the physiological function of that individual’s ocular cells, or improve that individual’s oxidative stress-related eye diseases or symptoms, or can effectively at least partially reduce that individual’s dry eye sensation, or improve that individual’s dry eye 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 sensitive 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 an individual’s disease or symptoms at a substantially static level, increasing an individual’s rate of recovery, reducing the severity of a specific condition, or improving a patient’s quality of life.

[0037] 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.

[0038] As mentioned above, oxidative stress can affect the physiological function of eye cells, potentially causing oxidative stress-related eye diseases or symptoms (such as macular degeneration, macular holes, retinal diseases, or glaucoma). The inventors of this invention have discovered that extracellular vesicles isolated from *Cistanche tubulosa*, upon administration, can effectively protect eye cells against oxidative stress damage. It is believed that the extracellular vesicles isolated from *Cistanche tubulosa* of this invention can be used for at least one of the following: prevention or treatment of oxidative stress-related eye diseases or symptoms, or for regulating, maintaining, or improving the physiological function of eye cells affected by oxidative stress.

[0039] As mentioned above, dryness of the eyes (which may be caused by one or more factors such as smoke, particulate matter, dry air, air conditioning, prolonged use of electronic devices, contact lens wear, aging, hormonal changes, inflammation, autoimmune diseases, refractive surgery, or medications) can lead to an increase in the osmotic pressure of the local environment around the eyes. High osmotic pressure can affect the physiological health of eye cells (especially corneal epithelial cells) and may even develop into dry eye syndrome. The inventors of this invention have discovered that extracellular vesicles isolated from *Cistanche tubulosa* can effectively protect eye cells against high osmotic pressure damage after administration. It is believed that the extracellular vesicles isolated from *Cistanche tubulosa* of this invention can be used for at least one of the following: prevention, treatment, or delay of dry eye syndrome, or for regulating, maintaining, or improving dry eyes.

[0040] 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 precipitates, reaction with polymer molecules, and centrifugation to collect the precipitates, thus separating it from *Cistanche tubulosa*. This extracellular vesicle can be used for at least one of the following purposes: protecting retinal pigment epithelial cells against oxidative stress damage and protecting corneal epithelial cells against high osmotic pressure damage, thereby achieving the purpose of eye protection.

[0041] 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.

[0042] The inventors discovered that by rationally configuring specific technical combinations of relevant parameters, the desired separation effect can be achieved while maintaining the biological activity of extracellular vesicles. Preferably, the method for preparing the extracellular vesicles comprises 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 not greater than 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 not less than 8,000×g to obtain a supernatant of the Cistanche tubulosa juice; (d) adding polymer molecules to the filtered supernatant and mixing thoroughly to form a mixture; (e) adding polymer molecules and mixing thoroughly; and (f) centrifuging the mixture and removing the precipitate to obtain extracellular vesicles of Cistanche tubulosa.

[0043] In the above preparation steps, to achieve the desired separation effect, the first stage centrifugation in step (b) is performed at a speed not exceeding 6,000×g, for example, a speed between 4,000×g and 6,000×g; preferably, a speed not exceeding 5,500×g, for example, a speed between 4,500×g and 5,500×g; more preferably, a speed not exceeding 5,200×g, for example, a speed of 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.

[0044] The second stage of centrifugation in step (c) above is performed at a speed of not less than 8,000 × g, for example, at a speed of 8,000 × g to 12,000 × g; preferably, at a speed of not less than 9,000 × g, for example, at a speed of 9,000 × g to 11,000 × g; more preferably, at a speed of not less than 9,500 × g, for example, at a speed of 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 effect of removing impurities. For example, the second stage of centrifugation can be performed at 10,000 × g for 15 to 30 minutes. If necessary, the second stage of centrifugation can include two or more cycles of centrifugation and filtration until no precipitate remains.

[0045] In step (d) above, the supernatant is sequentially passed through a filter membrane with a diameter of not less than 0.4 μm and a filter membrane with a diameter of not more than 0.25 μm. 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).

[0046] Preferably, the amount of polymer molecule added in step (e) 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.

[0047] The aforementioned step (f) involves centrifugation at a speed not exceeding 4000×g, for example, a speed of 2000×g to 4000×g; preferably, a speed not exceeding 3500×g, for example, a speed of 2500×g to 3500×g; more preferably, a speed not exceeding 3200×g, and in some specific embodiments, a speed of 3000×g is used. The centrifugation time in step (f) is preferably 30 to 40 minutes.

[0048] The extracellular vesicles isolated from *Cistanche tubulosa* according to the present invention can be included in or used to prepare 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 can be used in the preparation of at least one of the aforementioned compositions. The pharmaceutical composition can 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 can be administered to the individual in need via oral or non-oral routes (e.g., intravenous injection, intramuscular injection, subcutaneous injection, mucosal administration, skin administration). Depending on the form of use and purpose, a suitable carrier can 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] The outer packaging of health foods, health care foods, functional foods, nutritional supplements and / or special nutritional foods provided by this invention can indicate 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 them with other foods or medicines, so that users can take them at home without safety concerns without the guidance of a doctor, pharmacist or relevant personnel.

[0058] The cosmetic and skincare compositions provided by the present invention can be used for at least one of the following: regulating, maintaining, or improving the physiological function of eye cells affected by oxidative stress, and regulating, maintaining, or improving dry eyes. 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.

[0059] When the cosmetic composition or skincare composition provided according to the present invention is applied to regulate, maintain or improve the physiological function of eye cells affected by oxidative stress, or to regulate, maintain or improve dry eyes, the content of the extracellular vesicles may vary depending on the product type.

[0060] 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.

[0061] This invention also provides a method for protecting ocular cells against oxidative stress damage, comprising administering to an individual in need an effective amount of extracellular vesicles isolated from *Cistanche tubulosa*, wherein the extracellular vesicles may be contained in 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 formulations, routes of administration, forms of administration, frequency of application, and related applications of these compositions are as described above.

[0062] 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 invention. The scope of protection of the invention is as defined in the claims.

[0063] Example

[0064] Example 1: Preparation of phenylethanol glycoside preparation from Cistanche tubulosa

[0065] 1. Water extraction of Cistanche tubulosa

[0066] 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.

[0067] 2. Resin column purification of phenylethanoid glycosides

[0068] 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%.

[0069] Example 2: Preparation of extracellular vesicle samples from Cistanche tubulosa

[0070] 1. Homogeneous juice from the fleshy stems of Cistanche tubulosa

[0071] 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.

[0072] 2. Polymer precipitation method for separating extracellular vesicles

[0073] 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).

[0074] Example 3: Counting extracellular vesicles of Cistanche tubulosa

[0075] Extracellular vesicles were captured and counted using a NanoSight NS300 nanoparticle analyzer. Hardware settings: built-in laser: 488 nm wavelength, 45 mW; camera: sCMOS.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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).

[0080] Example 4: Test for extracellular vesicle cytotoxicity

[0081] 1. Cytotoxicity test of extracellular vesicles on human retinal pigment epithelial (hRPE) cells.

[0082] Human retinal pigment epithelial cells APRE-19 were purchased from the Bioresource Collection and Research Center (BCRC) in Taiwan, Taiwan, with accession number 60383.

[0083] Human retinal pigment epithelial cells APRE-19 were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium containing 15 mM 2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES) and 10% fetal bovine serum (FBS).

[0084] APRE-19 cells were suspended in culture medium and added to a 96-well cell culture dish, so that each well contained 6,000 APRE-19 cells. After culturing for 24 hours, the test samples of each group shown in Table 1 were added and treated for 48 hours. Finally, the changes in APRE-19 cell viability under different concentrations of test samples were detected by MTT assay. The results are shown in Figure 2.

[0085] As shown in Figure 2, when a concentration of 10 is added... 5 ~10 8 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 48 hours, the survival rate of APRE-19 cells in each group was higher than 90%, indicating that when the CTV concentration is 102, the cell viability is better than that of the test sample (i.e., the extracellular vesicles of Cistanche tubulosa prepared in Example 2, code name CTV). 8 Within the order of particles / ml (<10) 9 When the concentration is 10 ...

[0086] Table 1

[0087]

[0088] Example 5: Testing the efficacy of the sample in resisting oxidative stress in retinal pigment epithelial cells (Part 1)

[0089] Human retinal pigment epithelial cells APRE-19 were cultured in a 1:1 mixture of DMEM and Ham's F-12 medium containing 15 mM HEPES and 10% fetal bovine serum.

[0090] A quantitative amount of APRE-19 cells (6,000 cells / well) were placed in 96-well cell culture dishes and cultured in the aforementioned medium for 24 hours. Then, the medium was replaced with the medium containing the test samples shown in Table 2 for a pre-reaction of 24 hours. Next, the medium was replaced with the medium containing the test samples shown in Table 2 and 0.1 mM tert-butyl hydroxyperoxide (t-BHP) as an oxidative stress inducer, and the mixture was treated together for 24 hours. Finally, the protective effect of each sample group against oxidative stress on APRE-19 cells was detected by the MTT assay. The results are shown in Figure 3.

[0091] Table 2

[0092]

[0093] As shown in Figure 3, compared to the control group, the survival rate of APRE-19 cells in the control group with added oxidative stress inducers decreased significantly (down to 70%), indicating that oxidative stress induces retinal pigment epithelial cell death. Compared to the control group, the groups treated with the Cistanche tubulosa phenylethanol glycoside preparations (CTE-10, CTE-50) prepared in Example 1 showed an increase in the survival rate of retinal pigment epithelial cells, but this increase was not statistically significant. Compared to the control group, the groups treated with the Cistanche tubulosa extracellular vesicles (CTV-10, CTV-20, CTV-200) prepared in Example 2 showed a significant increase in the survival rate of retinal pigment epithelial cells, reaching a statistically significant level, indicating that the tested Cistanche tubulosa extracellular vesicle samples have an effect on resisting oxidative stress damage to retinal pigment epithelial cells.

[0094] Example 5: Testing the efficacy of the sample in resisting oxidative stress in retinal pigment epithelial cells (Part 2)

[0095] Human retinal pigment epithelial cells APRE-19 were cultured in a 1:1 mixture of DMEM and Ham's F-12 medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin.

[0096] In the first experiment, a quantitative amount of APRE-19 cells (5,000 cells / well) were placed in a 96-well cell culture dish and cultured until the cells adhered. Then, the test samples shown in Table 3 were added and cultured for another 24 hours under blue light (approximately 300 Lux, 460 nm). Finally, the protective effect of each sample group against oxidative stress on APRE-19 cells was detected by the MTS assay. The results are shown in Figure 4.

[0097] Table 3

[0098]

[0099] As shown in Figure 4, compared to the unexposed group, the survival rate of APRE-19 cells in the blue light-exposed control group decreased to approximately 90%, indicating that blue light induces retinal pigment epithelial cell death. Compared to the control group, the groups that added the Cistanche tubulosa phenylethanoid glycoside preparations (CTE-50, CTE-100, CTE-200) prepared in Example 1 during blue light irradiation showed an increase in retinal pigment epithelial cell survival, but only CTE-200 reached a statistically significant level. Compared to the control group, the groups that added the Cistanche tubulosa extracellular vesicles (CTV-100, CTV-200, CTV-400) prepared in Example 2 during blue light irradiation did not show a significant change in retinal pigment epithelial cell survival.

[0100] The inventors considered that the extracellular vesicles of Cistanche tubulosa and the phenylethanol glycoside preparations of Cistanche tubulosa have different components. The molecules carried by the extracellular vesicles may require time to regulate intracellular signal transmission. Therefore, they adjusted the experimental design and treated the test samples for 24 hours before irradiating the cells with blue light, and then carried out the subsequent experimental steps.

[0101] APRE-19 cells were cultured in the same manner for a second experiment. A quantitative amount of APRE-19 cells (5,000 cells / well) were placed in a 96-well cell culture dish and cultured until the cells adhered. The test samples shown in Table 4 were added for a pre-reaction of 24 hours, and then cultured for another 24 hours under blue light (approximately 300 Lux, 460 nm). Finally, the protective effect of each sample group against oxidative stress on APRE-19 cells was detected by the MTS assay. The results are shown in Figure 5.

[0102] Table 4

[0103]

[0104] As shown in Figure 5, compared to the untreated group, the control group, which only had culture medium without samples added during the sample treatment, saw its APRE-19 cell survival rate decrease to approximately 75% after 24 hours of blue light irradiation, indicating that blue light induces retinal pigment epithelial cell death. Compared to the control group, the groups treated with *Cistanche tubulosa* extracellular vesicles (CTV-100, CTV-200, and CTV-400) prepared in Example 2 for 24 hours, followed by 24 hours of blue light irradiation, showed a cell survival rate exceeding 80%. Furthermore, the CTV-200 and CTV-400 groups achieved statistical significance, demonstrating that the tested *Cistanche tubulosa* extracellular vesicle samples, after pretreatment, have an effect on resisting oxidative stress damage to retinal pigment epithelial cells.

[0105] The experimental results above show that the extracellular vesicles isolated from Cistanche tubulosa of the present invention can indeed protect retinal pigment epithelial cells from oxidative stress damage. Therefore, the composition of the present invention does have the effect of protecting the eyes. It can be prepared as 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 or treatment of eye diseases or symptoms related to oxidative stress, or for regulating, maintaining or improving the physiological function of eye cells affected by oxidative stress.

[0106] Example 6: Test for Extracellular Vesicle Cytotoxicity

[0107] Cytotoxicity test of extracellular vesicles on human corneal epithelial cells (hCEC)

[0108] The human corneal epithelial cell line 2.040 pRSV-T was purchased from the American Type Culture Collection (ATCC), collection number CRL-11515.

[0109] 2,040 pRSV-T cells were routinely cultured in keratinocyte-serum-free medium containing 5 ng / ml recombinant human epidermal growth factor (EGF), 0.05 mg / ml bovine pituitary extract, 0.005 mg / ml insulin, and 500 ng / ml hydrocortisone. The medium was then spread on a coating solution containing 0.01 mg / ml fibronectin, 0.03 mg / ml bovine type I collagen, and 0.01 mg / ml bovine serum albumin (4.5 ml / 75 cm²). 2 ) The culture dish after 24 hours of treatment.

[0110] When performing cytotoxicity testing, first apply the above coating solution (4.5 ml / 75 cm). 2The cells were cultured in 96-well plates for 24 hours, and then 2.040 pRSV-T cells were suspended in trypsin-EDTA to ensure that each well contained 6,000 2.040 pRSV-T cells. After culturing for 24 hours, culture medium containing the test samples shown in Table 5 was added and incubated for another 24 hours. Finally, the changes in cell viability of 2.040 pRSV-T cells under different concentrations of test samples were detected by MTT assay. The results are shown in Figure 6.

[0111] As shown in Figure 6, when a concentration of 10 is added... 5 ~10 10 After culturing the test samples (i.e., the extracellular vesicles of Cistanche tubulosa prepared in Example 2, code-named CTV) for 24 hours, the survival rate of 2.040 pRSV-T cells in each group was higher than 90%, indicating that the CTV concentration ≤ 1×10⁻⁶ was optimal. 10 At a concentration of 2,040 particles / ml, it does not cause toxicity to human corneal epithelial cells (2.040 pRSV-T).

[0112] Table 5

[0113]

[0114] Example 7: Testing the efficacy of the sample against corneal epithelial cell damage caused by high osmotic pressure.

[0115] Human corneal epithelial cells (2.040 pRSV-T) were routinely cultured in keratinocyte-serum-free medium containing 5 ng / ml recombinant human epidermal growth factor, 0.05 mg / ml bovine pituitary extract, 0.005 mg / ml insulin, and 500 ng / ml hydrocortisone. The medium was then spread on a coating solution containing 0.01 mg / ml fibronectin, 0.03 mg / ml bovine type I collagen, and 0.01 mg / ml bovine serum albumin (4.5 ml / 75 cm²). 2 ) The culture dish after 24 hours of treatment.

[0116] The above coating solution was added to a 96-well cell culture dish at a volume of 19.2 μL / well and treated for 24 hours. 2.040 pRSV-T cells were suspended with trypsin-EDTA. A quantitative amount of 2.040 pRSV-T cells (6,000 cells / well) was placed in the above culture dish and cultured for 24 hours. Then, high-osmotic pressure DMEM / F12 medium (500 mOsM) containing the test samples shown in Table 6 was added and incubated for 24 hours. Finally, the changes in the survival rate of 2.040 pRSV-T cells under different concentrations of test samples were detected by cell viability analysis. The results are shown in Figure 7.

[0117] Table 6

[0118]

[0119] As shown in Figure 7, compared to the control group, the survival rate of 2,040 pRSV-T cells in the control group with high osmotic pressure (500 mOsM) DMEM / F12 medium decreased significantly (to 68%), indicating that high osmotic pressure induces corneal epithelial cell death. Compared to the control group, the groups treated with Cistanche tubulosa phenylethanoid glycoside preparations (CTE-10, CTE-50, CTE-100, CTE-200) showed a statistically significant increase in corneal epithelial cell survival. The experimental data trend shows that the Cistanche tubulosa phenylethanoid glycoside preparations exhibit a dose-dependent protective effect within the concentration range of 10–100 μg / ml, gradually saturating. At concentrations exceeding 100 μg / ml and reaching 200 μg / ml, the protective effect slightly decreased.

[0120] Compared with the control group, the survival rate of corneal epithelial cells in the groups treated with extracellular vesicles of Cistanche tubulosa (CTV-10, CTV-20, CTV-100, CTV-200, CTV-1000, CTV-10000) was significantly improved, reaching a statistically significant level. This indicates that the test samples have an effect on resisting high osmotic pressure damage to corneal epithelial cells. The experimental data trend shows that the extracellular vesicles of Cistanche tubulosa, upon reaching 10... 7 At concentrations above 1000 particles / ml, the effect of protecting corneal epithelial cells can be achieved, and saturation occurs quickly, even when the dose is increased to 10 ... 9 ~10 10 With a concentration of 10 ...

[0121] The experimental results above show that the extracellular vesicles isolated from Cistanche tubulosa of the present invention can indeed protect corneal epithelial cells from high osmotic pressure damage. Therefore, the composition of the present invention does have the effect of protecting the eyes. It can be prepared as 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 dry eye syndrome, or for regulating, maintaining or improving dry eyes.

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 or treat eye diseases or symptoms related to oxidative stress, and the pharmaceutical composition is administered orally, intravenously, intramuscularly, subcutaneously, via mucosal administration, via skin administration, or a combination of the foregoing.

2. The use as described in claim 1, characterized in that, The eye disease or symptom is macular degeneration, macular hole, retinal disease, glaucoma, or a combination of the above.

3. The use as described in claim 2, characterized in that, The macular degeneration is age-related macular degeneration, which is dry macular degeneration and / or wet macular degeneration.

4. The use as described in claim 2, characterized in that, The retinopathy is diabetic retinopathy, retinitis pigmentosa, retinal artery and / or vein occlusion, proliferative vitreoretinopathy, central serous chorioretinopathy, or a combination of the foregoing.

5. The use of extracellular vesicles isolated from Cistanche tubulosa for regulating, maintaining, or improving the physiological function of ocular cells affected by oxidative stress, 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.

6. The use as described in claim 5, characterized in that, This oxidative stress is caused by chemical molecules or blue light.

7. The use as described in claim 6, characterized in that, These ocular cells are retinal pigment epithelial cells.

8. A 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 dry eye syndrome, and the pharmaceutical composition is administered orally, intravenously, intramuscularly, subcutaneously, via mucosal administration, via skin administration, or a combination of the foregoing.

9. A method for using extracellular vesicles isolated from Cistanche tubulosa for regulating, maintaining, or improving dry eyes, 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.

10. The use as described in any one of claims 1 to 9, characterized in that, The method for preparing 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 no more than 6,000×g. (c) The second stage of centrifugium was centrifuged to remove the precipitate. The second stage of centrifugation was carried out at a speed of not less than 8,000×g to obtain the supernatant of the Cistanche tubulosa juice. (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.

11. The use as described in claim 10, characterized in that, Step (b) Centrifuge at 5,000×g for 5 to 10 minutes.

12. The use as described in claim 10, characterized in that, Step (c) Centrifuge at 10,000×g for 15–30 minutes.

13. The use as described in claim 10, characterized in that, Step (d) involves passing the supernatant sequentially through a filter membrane with a diameter of not less than 0.4 μm and a filter membrane with a diameter of not more than 0.25 μm.

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

15. The use as described in claim 14, characterized in that, The polymer molecule in step (e) is polyethylene glycol.

16. The use as described in claim 10, characterized in that, The mixing reaction in step (e) was carried out at 4°C for 12–16 hours.

17. The use as described in claim 10, characterized in that, Step (f) Centrifuge at 3,000×g for 30–40 minutes.

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