Extracellular vesicles from liquorice roots and their use

Abstract

Extracellular vesicles (EVs) extracted from liquorice roots from biological agriculture characterized in that said extracellular vesicles contain antioxidant compounds within the lipid membrane.

Description

[0001] EXTRACELLULAR VESICLES FROM LIQUORICE ROOTS AND THEIR USE

[0002] DESCRIPTION

[0003] Technical field of the invention

[0004] The present invention relates to extracellular vesicles derived from liquorice roots from biological agriculture and their use as natural carriers of bioactives, in particular for cosmetic and pharmaceutical use.

[0005] Technical background

[0006] As is well known, licorice (Glycyrrhiza glabra) belongs to the Fabaceae family (Leguminosae Papilionaceae) and is undoubtedly one of the most popular medicinal plants. In particular, licorice root is one of the oldest medicinal herbs globally, used as a therapeutic remedy for its numerous beneficial effects.

[0007] Licorice is native to the Middle East and the Mediterranean area and in Italy it is mainly found in Abruzzo, Basilicata, Calabria and Sicily. About 80% of the total Italian production is concentrated in Calabria. In this region, licorice is widespread in various areas with Mediterranean climate conditions characterized by long, hot and dry summers and mild winters, which reduce the transition seasons of spring and autumn to a few months.

[0008] The root, with its intense sweet taste, has been used in the food and medicinal fields to treat respiratory tract disorders, such as cough and bronchitis, and inflammation of the gastrointestinal tract, such as dyspepsia and gastritis.

[0009] The characteristic compounds of the licorice root are triterpene saponins, in particular glycyrrhizin, a molecule with a sweetening power fifty times greater than that of common sugar; in addition, it contains flavonoids (liquiretin, glabridin), polysaccharides and mineral salts. When taken orally, glycyrrhizin is metabolized by the intestinal bacterial flora to glycyrrhetinic acid (also known as glycyrrhizic acid). Glycyrrhizin reduces stomach acidity, preventing the formation of ulcers and improving the symptoms associated with gastric secretion disorders (e.g. reflux esophagitis). Some preclinical studies have also highlighted the ability of licorice flavonoids and polysaccharides to counteract Helicobacter pylori infection, which can facilitate the development of peptic ulcers.

[0010] For licorice, benefits have also been reported at the liver level, due to the antiinflammatory and antioxidant effects of glycyrrhetic acid and flavonoids. In particular, 18-glycyrrhetinic acid, commonly called glycyrrhetic acid, has many biological properties, such as anti-inflammatory, antioxidant and antimicrobial, which are essential for treating various human dermatological disorders related to inflammation, skin aging due to exposure to UV rays. Cortisol is a hormone that in normal conditions and following inflammatory stimuli is released at the tissue level, therefore also in the skin. Glycyrrhetic acid has an anti-inflammatory action as it activates a physiological mechanism capable of prolonging the activity of cortisol, thus increasing its effectiveness. Glycyrrhetic acid also inhibits the formation of inflammatory substances, such as cytokines and leukotrienes. Thanks to its properties, which are evident even at low doses, glycyrrhetic acid can be used as a natural alternative to cortisone. Glycyrrhetic acid is able to inactivate free radicals, such as those formed during exposure to UV rays, and at the same time inhibits lipid oxidation. Thanks to these properties and its soothing action, glycyrrhetic acid can be included in sunscreen formulations intended for sensitive and delicate skin. Glycyrrhetic acid also has anti-reddening and dermo-healing properties, which makes it useful following sun exposure, but also to treat atopic and seborrheic dermatitis.

[0011] Fungi such as Candida Albicans and viruses are inhibited by the application of formulations containing glycyrrhetic acid. Thanks to the antiseptic properties of glycyrrhetic acid, it is possible to treat skin conditions such as acne or mucous membranes such as cold sores with this active ingredient.

[0012] The biological effects of licorice extracts are mainly attributed to the presence of its metabolites but also to many vitamins including Vitamins C, E, B3 and B5.

[0013] A study on licorice roots identified a number of bioactives with various cosmeceutical activities, classified as anti-aging skin, photoprotective, hair care, and anti-acne.

[0014] It is also known that plant-derived extracellular vesicles are natural carriers for any bioactive compound. As used herein, the term "extracellular vesicles" or "EVs" refers to cell-derived nanoparticles, which are bounded or encapsulated by a phospholipid bilayer and which transport lipids, proteins, nucleic acids, and other molecules derived from the cell of origin. Conventionally, extracellular vesicles have a diameter in the range of 10-1000 nm.

[0015] Scientific research has shown that extracellular vesicles derived from plants have great potential in medicine, where it is necessary to act on the regulation of biological processes that are fundamental for correct metabolic functioning. In particular, significant potential has been found with respect to the immune response, in the development and regeneration of tissues and in the natural supplementation of all the bioactives that the body absolutely needs. It is also desirable that vesicles derived from edible vegetables and fruits can be used as natural carriers of therapeutic molecules with significant advantages such as: (i) absence of detectable toxicity; (ii) constitutive presence of a large variety of therapeutic bioactives and (iii) the possibility of producing them in large quantities and in a sustainable way for humans and the environment.

[0016] It is not known at the state of the art, the use of extracellular vesicles derived from liquorice roots from biological agriculture (not OGM). Extracellular vesicles play a key role in cell-cell communication in nature, both within an organ or system and at a distance; a phenomenon that operates both within the same species and between different species. Vesicles of both plant and animal origin are natural transporters of proteins, lipids and nuclear components, but with variations from species to species. It is known that plants contain intrinsic and unique compounds with physiologically relevant bioactivities, and therefore the molecular content of vesicles also varies depending on the plant from which they are released (Zhang et al. 2016). In their function of communication between cells and organs, extracellular vesicles have the ability to transfer their content into target cells via membranemembrane fusion, and it is therefore very likely that vesicles of plant origin can transfer their content into human cells, using the same mechanism.

[0017] In conclusion, extracellular vesicles derived from licorice roots constitutively contain bioactive molecules potentially effective in the treatment of skin ulcers, kidney and bladder diseases, gastritis, fever, lung disorders such as bronchial asthma and chronic bronchitis, and heart diseases, due to their biological properties, including antioxidant, antimicrobial, antitumor, antiaggregant activities.

[0018] However, the use of extracellular vesicles extracted from liquorice roots from biological agriculture and their use as natural carriers of bioactive compounds is not known.

[0019] Summary of the invention

[0020] The object of the present invention is therefore extracellular vesicles extracted from liquorice roots from biological agriculture containing at least one bioavailable substance inside the lipid membrane and their use as natural carriers of bioactive compounds, in particular for cosmetic and pharmaceutical use.

[0021] The present invention uses a selected subpopulation of extracellular vesicles (EV) extracted from liquorice roots from biological agriculture that have a diameter with average dimensions ranging between 143.8 ± 2.3 nm and 202.9 ± 3.9 nm.

[0022] According to the present invention, extracellular vesicles isolated from licorice roots from biological agriculture have been characterized based on their content of total antioxidants: glycyrrhizic acid and superoxide dismutase (SOD).

[0023] Therefore, according to a first aspect of the present invention, extracellular vesicles extracted from licorice roots from biological agriculture are defined, comprising a lipid membrane and containing within the lipid membrane at least one bioavailable antioxidant substance, as specified in the attached independent claim.

[0024] According to a further aspect, the use of such extracellular vesicles as cosmetic and pharmaceutical is defined, as specified in the independent claims of use.

[0025] The dependent claims outline particular and further advantageous aspects of the invention.

[0026] Brief description of the drawings These and other advantages of the invention will now be described in detail, with reference to the attached drawings, which represent an exemplary embodiment of the invention, in which:

[0027] - Figure 1 shows the graph of the dimensional characterization of extracellular vesicles isolated from licorice roots, according to the present invention;

[0028] - Figure 2 shows the graph of the comparison between the concentration of elastin in in vitro fibroblasts treated with extracellular vesicles extracted from licorice root in a CTR control, a first C1 and a second C2 concentration;

[0029] - Figure 3 shows the comparison of the expression of Matrix Metalloproteinase 9 in in vitro fibroblasts treated with extracellular vesicles extracted from licorice root.

[0030] Detailed Description

[0031] According to the present invention and according to the attached Figures, extracellular vesicles (EVs) were extracted from licorice roots from biological agriculture.

[0032] Biological agriculture is defined as agriculture that uses a cultivation technique and a way of producing food that respects natural life cycles. That is, without the use of chemical pesticides, synthetic fertilizers, antibiotics and other substances that are subject to strict restrictions. In addition, crops are rotated so that on-site resources are used efficiently; on-site resources are exploited, such as manure for fertilizer or feed produced on the farm. Furthermore, by definition, biological agriculture does not use genetically modified organisms (OGMs). On the contrary, plant and animal species that are resistant to disease and adapted to the environment are used. For this purpose, techniques such as the protection of useful insects, antagonists of parasites, are used; rustic, more resistant plants are chosen; mulching is practiced, which consists of covering the soil with hay or fresh grass to protect it from temperature changes and hinder the growth of weeds; green manure is used, that is, the sowing of some plants (clover, vetch, watercress, lamb's lettuce, spinach, rapeseed and so on) which, once in flower, are buried to fertilize the soil and protect it from erosion; crop rotation is practiced, which consists of alternating the cultivation of plants that improve the fertility of the soil, for example by enriching it with nitrogen, with plants that impoverish it, by removing nutrients; manure and biologic fertilizers such as compost are used, a mixture of soil, plant remains, wood ash and anything else that exists on the farm that is biodegradable and non-polluted.

[0033] The licorice roots used are obtained from specialized Italian companies certified for biological agriculture, carefully washed and subjected to mechanical extraction. The methodology through which extracellular vesicles are produced from licorice roots from biological agriculture is the subject of a different patent application No. 102024000006865 incorporated by reference, of the same applicant, unless specified below and includes the following phases: a. inspecting the vegetable raw material; b. transferring the vegetable raw material into plastic food containers and store it for short periods in the refrigerator at a temperature of +4°C, c. washing the vegetable raw material, d. preparing the vegetable raw material for extraction, e. extracting the first semi-finished product using automatic extractors, f. storing the first semi-finished product in plastic bottles, g. storing the first semi-finished product in the refrigerator, h. performing an initial filtration phase of the first semi-finished product in a filter with a mesh size between 0.2 mm and 1 mm, i. storing the filtered product not immediately used for a short period in the refrigerator at a temperature of +4°C, l. performing an initial centrifugation of the filtered product in centrifuge bottles at a speed of 2,000 x g for a period of 30 minutes, m. filtering the supernatant with a nylon filter with a mesh size of up to 100 pm to obtain a mixture of extracellular vesicles and discard the pellet, n. storing the mixture of extracellular vesicles for a short period in the refrigerator at a temperature of +4°C in plastic or borosilicate glass bottles, o. adding to the mixture of extracellular vesicles a quantity of maltodextrin between 4% and 35% by weight, p. drying the mixture of extracellular vesicles by atomization, q. collecting and storing the powdered product at room temperature in amber borosilicate glass containers.

[0034] Phase d) involves roots directly infused. The infusion process is performed with ultrafiltered water. From 1 to 1000 grams of dried raw material infused with water (from 2 to 10000 ml infusion). The infusion of dried / dehydrated plants, such as roots and leaves, is a phase that presents particular technical challenges, since the secretion of extracellular vesicles in these matrices requires specific conditions, such as temperature control, infusion time and type of solvent. In particular, the extraction of extracellular vesicles from plants with a low liquid content requires specific optimization, achieved by means of the method. The method according to the invention, aims to use a wide range of plant derivatives, including leaves, roots and fruits with a low liquid content, for the extraction of extracellular vesicles.

[0035] Advantageously, the infusion allows to better preserve the integrity of the exosomes and reduce the degradation of the bioactive components.

[0036] The drying phase p) is carried out according to the spray technique or "Spray Drying" that is one of the methods of choice for the production of powders starting from aqueous (and / or organic-aqueous) solutions or suspensions. This technique involves the atomization of a liquid in a chamber in which a hot gas recirculates.

[0037] Advantageously, the raw material used in the drying phase is a noble derivative extracted by infusion from licorice roots. The starting material is therefore richer in extracellular vesicles, which through the process can be isolated more easily, reducing waste and improving the quality of the final product.

[0038] Advantageously, the use of licorice roots from biological agriculture, specifically selected for their ability to produce high-quality extracellular vesicles, and the use of the “Spray Drying” step to ensure industrial scalability, imply the optimization of various technical parameters (e.g., drying temperature, air flow, and raw material concentration) by means of specific experiments and adjustments to obtain the desired product.

[0039] According to the method of the present invention, the drying step q) is performed according to optimized process parameters, namely:

[0040] -’’Drying Gas” Drying gas flow rate (m3 / h) = 30 - 35,

[0041] - ’’Inlet T” Inlet temperature (°C) = 115 - 140,

[0042] - ’’Spray Gas” Atomized gas flow rate (l / h) = 1000 - 1500,

[0043] - ’’Pump 1” Pump flow rate (ml / min): 6 -15, - “Outlet T” Outlet temperatures (°C) = 60 - 75.

[0044] To assess whether EVs isolated from licorice roots fall within the size range of extracellular vesicles, the isolated samples were analyzed by the Bradford assay (Pierce, Rockford, IL, USA) and Nanotracking particle analysis (NTA). As observed in Figure 1 , the isolated EVs show the typical distribution of extracellular vesicles, with mean sizes (diameter) ranging from 143.8 ± 2.3 to 202.9 ± 3.9 nm. The EVs were also characterized for their zeta potential. Zeta potential is the electrical potential on the slipping plane of nanoparticles that, when dispersed in a liquid medium, form a charge on the surface, the so-called double layer. This is compensated by the addition of counterions to the surface charge. If a particle moves in solution, the ions move with it, and a potential drop occurs between the different layers. This difference is called the zeta potential.

[0045] Zeta potential is the main force of interactions between particles, and it is very sensitive to the composition of the species present in the dispersion. The analysis of the Zeta Potential value allows to predict their stability: a high absolute value of zeta potential implies that the particles remain far from each other, avoiding agglomeration, aggregation and / or flocculation phenomena.

[0046] In the case of extracellular vesicles isolated from licorice roots, EVs have a zeta potential that varies between -27.27 mV and -23.99 mV, demonstrating their great stability in liquid solution.

[0047] The tests performed were performed in order to characterize the extracellular vesicles isolated from licorice roots fortheir content of total antioxidants, glycyrrhizic acid and superoxide dismutase (SOD). An antioxidant molecule is a molecule that slows down or prevents the formation of free radicals, protecting cells from the damage caused. Oxygen compounds with high oxidant activity (ROS) are produced during oxidation reactions in cells and can contribute to the development of various diseases such as Alzheimer's, Parkinson's and diabetes. The test used allows to evaluate the presence of both enzymatic antioxidants (e.g. catalase and superoxide dismutase) and non-enzymatic antioxidants (e.g. glutathione and vitamin C), analyzing the synergistic activity of the antioxidants present in the sample. In the case of EVs from liquorice roots, analyzing a range of vesicles between 106 and 1012, the total antioxidant content was found to be between 0.068 ± 0.005 and 58528 ± 0.5 pM.

[0048] In particular, the active ingredient that makes this plant so important is the presence of glycyrrhizic acid, also known as glycyrrhetic acid or glycyrrhizin. It has anti-inflammatory, anti-diabetic, antioxidant, anti-tumor, antimicrobial and antiviral properties. The experiments carried out measured the amount of glycyrrhizic acid in extracellular vesicles isolated from licorice roots, evaluating increasing concentrations of vesicles between 106e 1012and quantifying a glycyrrhetic acid content between 0.005 ± 0.011 and 15618 ± 6.0 mg / ml.

[0049] Superoxide dismutases are a class of enzymes involved in the mechanism of elimination of superoxide ions and free radicals, highly reactive elements responsible for oxidative stress and cellular aging. SOD, being a biological catalyst, can reduce ROS many times more and faster than other antioxidants without being consumed. Among the various beneficial effects on health, SOD has a powerful detoxifying and anti-inflammatory action, maintaining the oxidation-reduction balance that can be unbalanced by various factors. The anti-inflammatory action of SOD takes place above all on the respiratory tract and at the skin level, reducing the oxidative stress triggered by exposure to UV rays. The experiments carried out have quantified the concentration of SOD inside the extracellular vesicles isolated from licorice roots considering a range of vesicles between 106e 1012and we quantified a SOD content ranging from 0.605 ± 0.002 to 63445 ± 2.6 ll / rnl.

[0050] Among the in vitro effects, an increase in elastin production was measured. Elastin is produced by fibroblasts present in the dermis and is a protein that helps maintain the elasticity of the skin, stretching and contracting without suffering damage to its structure. As cells age, they undergo a progressive loss of elastin within the dermis, causing a loss of skin elasticity. Our body synthesizes elastin until about age 20, after which the loss of skin elasticity occurs and is mainly due to the fact that the damaged elastin is not replaced by functioning elastin. These changes in the dermis are then reflected in the formation of wrinkles and a decrease in skin tone over time. It has been shown that antioxidants present in plants help counteract the continuous loss of elastin in the skin. The experiments were carried out by treating fibroblasts in vitro for 24-48 hours with EVs isolated from licorice roots, to evaluate the production of elastin in the cellular supernatant. As shown in Figure 2, the analyses measured an increase in elastin in the fibroblast cell supernatant of 4% for a lower concentration (C1) and 7% for a higher concentration (C2), respectively. In addition, as shown in Figure 3, an increase in the expression of Matrix Metalloproteinase-9 was also measured. Matrix Metalloproteinase-9 is one of the most studied MMPs.

[0051] It is involved in the degradation of the extracellular matrix during the processes of skin wound healing and tissue remodeling. Various external stimuli, such as the environment, UV radiation, oxidative stress and nicotine, affect the tone of skin blood vessels, reducing the exchange of oxygen and nutrients between blood and tissues and accelerating the aging process. It has also been shown that metalloproteinase- 9 is involved in vascular remodeling, increasing the tone of blood vessels.

[0052] To evaluate the influence of EVs in increasing the expression of matrix metalloproteinase-9, fibroblasts in vitro were treated with two concentrations of licorice EVs and the expression of MM P-9 was assessed after 24 h of treatment. As reported in Figure 3, after 24 h of treatment, the expression of MMP-9 in the vesicle- treated cells was 156% higher than in the control cells.

[0053] These results indicate that licorice-extracted EVs predispose normal human fibroblasts to extracellular matrix (EMC) remodeling.

[0054] Conceivably, these data support the use of licorice-extracted EVs in certain cosmetic and pharmaceutical applications.

[0055] Conceivably, formulations comprising licorice root exosomes combined with selected ingredients enhance the efficacy of the final product.

[0056] An example of a formulation for cosmetic use is a soothing dermatological gel comprising EVs containing glycyrrhetic acid.

[0057] The composition for 100 ml of total gel includes in volumetric percentages:

[0058] - extracellular vesicles (extracted from the root licorice from biological agriculture “Glycyrrhiza glabra L.’y. 2-5%,

[0059] - aloe vera gel: 30-40%,

[0060] - distilled water: 30-50%

[0061] - vegetable glycerin: 5-10%

[0062] - other soothing plant extracts (e.g. chamomile, calendula): 2-5%

[0063] - ecological preservative: compliant with local regulations. The use of the compositions comprising glycyrrhetic acid in the formulations indicated above is for applications on sensitive skin and skin with couperose.

[0064] Rosacea (more commonly known as couperose) is a form of chronic benign dermatitis characterized by the presence of dilated capillaries, which mainly affects fair skin and especially affects the face of women. In the most severe stages of rosacea, the strong dilation of the blood vessels develops a greater influx of blood such as to cause the formation of acne or hypertrophy of the skin tissue. In these cases it is useful to resort to targeted pharmacological therapies, based on systemic drugs. The cosmetic composition according to an embodiment of the present invention contains components that are commonly used in the cosmetic composition. For example, the cosmetic composition may contain adjuvants and conventional vehicles, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances. In addition, other components in each formulation of the cosmetic composition can be appropriately selected without difficulty by experts in the field depending on the type or intended use of the cosmetic composition.

[0065] Another example of a cosmetic formulation is represented by a tensor effect cream in which the extracellular vesicles extracted from the licorice root from biological agriculture, contained in the formulation, act synergistically with other ingredients to stimulate skin renewal and counteract damage caused by free radicals.

[0066] The composition for 100 ml of total cream includes in volumetric percentages:

[0067] - extracellular vesicles (extracted from the root licorice from biological agriculture “Glycyrrhiza glabra L.’y. 10%,

[0068] - hyaluronic acid: 2% - coenzyme q10: 1%

[0069] - vitamin c (ascorbic acid): 1 %

[0070] - seaweed extract: 3%

[0071] - anti-aging peptides: 3%

[0072] - emulsifying and stabilizing components: 5%

[0073] - purified water: q.s. to 100%

[0074] Advantageously, the cosmetic composition is used for the purpose of improving the skin condition, elasticity, reducing wrinkles, regenerating the skin, reducing skin spots and discolorations.

[0075] Advantageously, the cosmetic composition combines active ingredients known for their anti-aging benefits with plant extracellular vesicles to improve their effectiveness and penetration into the skin.

[0076] Pharmaceutical compositions may include pharmaceutically acceptable vehicles, excipients, or diluents and the like. Vehicles, excipients, and diluents include lactose, dextrose, trehalose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, maltodextrin, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. Furthermore, an effective amount of the pharmaceutical composition of an embodiment of the present invention indicates an amount required for administration to be expected to have a pharmacological, wound healing or wound healing promoting effect in pharmaceutical forms such as plasters, bandages, sprays, creams, gel ointments, salves, mouthwashes and oral gels. The pharmaceutical composition according to the present invention can be used for gynecological and dermatological applications. In particular, the vegetal extracellular vesicles extracted from liquorice roots from biological agriculture can play a significant role in the prevention, protection and treatment of genital problems thanks to their beneficial properties for skin health and their anti-inflammatory and soothing action.

[0077] An example of a formulation as a pharmaceutical composition is a cream for gynecological use. The composition for 100 ml of cream in total includes in volumetric percentages:

[0078] - extracellular vesicles (extracted from the root of licorice from biological agriculture “Glycyrrhiza glabra L.” 2.5% - 5.5%,

[0079] - white petrolatum: 20% - 40%

[0080] - glycerin: 3% - 7%

[0081] - panthenol (provitamin B5): 1% - 3%

[0082] - emulsifying wax: 3% - 5%

[0083] - liquid paraffin: 10% - 20%

[0084] - purified water: Q.s. to 100%.

[0085] Further experiments were performed to identify and quantify bioactive compounds in licorice EVs. High-performance liquid chromatography (HPLC) analyses identified and quantified two key compounds in these EVs: glycyrrhizic acid and hesperidin (Table 1). Glycyrrhizic acid was further quantified with a significantly more sensitive method (HPLC) than the previous quantification obtained by spectrophotometric absorbance with a colorimetric kit. A more detailed explanation is provided below: • Glycyrrhizic acid: the main triterpene saponin of licorice root, with strong antiinflammatory, antiviral and antioxidant properties. This compound is known for its ability to modulate immune responses and protect the skin from oxidative stress,

[0086] • Hesperidin: flavonoid with high antioxidant activity, able to protect cells from premature aging and improve skin microcirculation, with beneficial effects in both cosmetic and therapeutic fields.

[0087] The use of the HPLC technique has allowed to obtain a precise and reproducible quantification, ensuring a high specificity and sensitivity in the analysis of the samples. This very detailed characterization of licorice EVs is the first to identify such bioactive compounds so specifically.

[0088] Tab e 1

[0089] Surprisingly, from the analyses performed the concentration of glycyrrhizic acid is between 0.5 mM and 1.5 mM and hesperidin is between 0.1 mM and 1.0 mM.

[0090] Although at least one exemplary embodiment has been presented in the summary and detailed description, it must be understood that there are a large number of variants falling within the scope of protection of the invention. Furthermore, it must be understood that the embodiment or embodiments presented are only examples that are not intended to limit in any way the scope of protection of the invention or its application or configurations. Rather, the summary and detailed descriptions provide the expert technician in the field with a convenient guide to implement at least one exemplary embodiment, it being clear that numerous variations can be made in the function and assembly of the elements described herein, without departing from the scope of protection of the invention as established by the attached claims and their technical-legal equivalents.

Claims

RIVENDICAZIONI1. Extracellular vesicles (EVs) extracted from liquorice roots from biological agriculture characterized in that said extracellular vesicles contain antioxidant compounds within the lipid membrane.

2. Extracellular vesicles (EVs) according to claim 1, wherein the total concentration of the antioxidant compounds is between 0.068 ± 0.005 pM and 58528 ± 0.5 pM for a number of vesicles between 106and 1012.

3. Extracellular vesicles (EVs) according to claim 1 or 2, wherein an antioxidant compound is glycyrrhizic acid and / or superoxide dismutase.

4. Extracellular vesicles (EVs) according to claim 3, wherein the concentration of glycyrrhizic acid is between 0.005 ± 0.011 mg / ml and 15618 ± 6.0 mg / ml for a number of vesicles between 106and 1012.

5. Extracellular vesicles (EVs) according to claims 3 and 4, wherein the concentration of superoxide dismutase is between 0.605 ± 0.002 ll / rnl and 63445 ± 2.6 ll / rnl for a number of vesicles between 106and 1012.

6. Extracellular vesicles (EVs) according to any of the previous claims, characterized in that a zeta potential is between -27.27 mV and -23.99 mV.

7. Composition comprising extracellular vesicles (EVs) according to any of the preceding claims, for use as cosmetic.

8. Composition comprising extracellular vesicles (EVs) according to any of the preceding claims, for use as drug.

Images