Compound comprising extracellular vesicles derived from biological material not belonging to the animal kingdom and its applications

Abstract

Compound for relieving inflammation in an organism comprising nanometer-sized extracellular vesicles (EVs) derived from biological material not belonging to the animal kingdom, but preferably belonging to the plant and fungi kingdoms, and loaded with a bioactive or a mixture of bioactives, preferably of plant origin.

Description

[0001] "COMPOUND COMPRISING EXTRACELLULAR VESICLES DERIVED FROM BIOLOGICAL MATERIAL NOT BELONGING TO THE ANIMAL KINGDOM AND

[0002] ITS APPLICATIONS"

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This Patent Appl ication claims priority from Italian Patent Application No . 102023000025614 filed on November 30 , 2023 , the entire disclosure of which is incorporated herein by reference .

[0005] TECHNICAL FIELD

[0006] The present invention relates to a compound comprising extracellular vesicles (EVs ) derived from biological material not belonging to the animal kingdom . In particular, the present invention relates to a compound comprising nanometer-si zed extracellular vesicles (EVs ) derived from biological material belonging to the plant and fungi kingdoms . More in particular, the compound of the invention also comprises bioactives . The present invention also relates to the method for preparing said compound as well as its applications in the medical field and, speci fically, in anti-inflammatory and anti-oxidant therapies .

[0007] BACKGROUND ART

[0008] Extracellular vesicles (EVs ) comprise a wide variety of membrane-limited particles released from both eukaryotic and prokaryotic cells . Speci fically, EVs are small sphericalshaped particles consisting of a lipid bilayer . EVs are heterogeneous in terms of content , surface composition and si ze ; the definition of EVs includes , therefore , every vesicle having a diameter ranging between 30 nm and 5 pm actively released by cells .

[0009] Based on their s i ze , EVs may be classi fied into three sub-types : apoptotic bodies ( 1000 to 5000 nm) , macrovesicles ( 100 to 1000 nm) and finally exosomes ( 30 to 150 nm) . In the plant world, macro-vesicles may be derived from Exocyst-positive organelles (EXPOs ) , vacuoles and autophagosomes .

[0010] Regarding the EVs classi fied as exosomes , these are generated through a process of internal budding from multivesicular bodies . Furthermore , given their nanometric proportions , these vesicles are particularly stable in unfavorable environments in terms of pH and temperature .

[0011] The pivotal role of EVs in short- and long-distance intercellular communication, acting as messengers of biological information, has long been known . Indeed, by virtue of their ability to transport bioactives and to contain receptors for ligands exposed on the membrane of the target cells , EVs can af fect the behaviour of the target cells through multiple mechanisms of action .

[0012] Several studies have pointed out that the mechanism of action of EVs is determined by the interaction between its membrane proteins and the membrane proteins on animal or human target cells . The mechanism of action may occur by releasing EV bioactives into the cytosol of the target cell , as well .

[0013] EVs represent , therefore , natural transport systems of bioactives , the trans fer of which into the target cell may involve important variations in the activity, migration, proli feration, and survival of the cell . The natural ability of EVs to transport bioactive molecules may be used for transporting bioactives of interest . In this case , the bioactives of interest are loaded into the EVs lipid bilayer according to known techniques that are described below . EVs also represent a way of communicating between cells of the same organism, between cell s of di fferent organisms , but also an extra-species and extra-kingdom communication mechanism . Indeed, bacteria and fungi are able to produce EVs able to communicate with the host , and plant cells release EVs able to trans fer bioactives not only among cells of the same species , but also to cells of dif ferent kingdoms .

[0014] Thanks to their pivotal role as messengers of biological information and to all the unique features outlined above , EVs are of great interest both in the basic research and in future diagnostic, physiological and pharmacological applications . Indeed, referring to the pharmacological field, many studies are focused on evaluating their therapeutic use also in relation to the ef fects related to the bioactives contained therein .

[0015] Regarding the bioactives loaded into the EVs , these represent biomolecules exerting biological activities in the target cell , such as anti-inflammatory, immuno-stimulant , anti-oxidant , cytotoxic, anti-microbial or di f ferentiating activities . Indeed, bioactives are known to af fect the expression of genes and proteins by activating or inhibiting several metabolic pathways involved in various biological processes .

[0016] "Bioactives" means biomolecules of natural or synthetic origin having beneficial ef fects on the health of living organisms . These biomolecules may be extracted from plants ( e . g . , polyphenols , terpenes and phytosterols ) , but also derived from animals and microbial sources , or be chemically synthesi zed .

[0017] Speci fically, polyphenols are a highly diversi fied class of compounds including more than 8000 compounds divided into several sub-groups: phenolic acids, flavonoids, stilbenes and lignans. Polyphenols of natural origin accumulate in plant organs (roots, stems, fruits and flowers, depending on the characteristics of the species) , with greater abundance in the skin of fruits. Due to their chemical nature, polyphenols are known to be scavenger molecules of free radicals: reactive oxygen species (ROS) and reactive nitrogen species (RNS) . For this reason, they are already widely used in therapies against oxidative stress. At the cellular level, they have effects ranging from anti-inflammatory to anti-cancer and anti-diabetic. The mechanism of action of polyphenols on animal cells is strictly linked to signal pathways mediated by Ca2+as a secondary intracellular messenger, which leads to a temporary increase in Ca2+in the cell cytosol.

[0018] Whereas terpenes (e.g., tetraterpenes and triterpenes ) , which constitute the largest class of natural products, have a high anti-cancer potential. For example, ursolic acid, a pentacyclic triterpene hydroxy acid, has significant anabolic effects on skeletal muscles playing an essential role in the ageing process.

[0019] Phytosterols also have anti-inflammatory potential and are used in the treatment of periodontitis, oral ulcers and other related diseases.

[0020] Plant- and fungi- or yeast-derived EVs themselves exhibit anti-inflammatory activities in human and animal cells, regardless of the bioactive associated therewith.

[0021] However, to date, the innate anti-inflammatory properties of plant- and fungi- / yeast-derived EVs, which, when loaded with one or more bioactives, amplify the antiinflammatory response, are not being exploited. SUMMARY

[0022] It is therefore an obj ect of the present invention to provide a compound, its preparation method, and its use in the medical field capable of enhancing the anti-inflammatory ef fect in human and animal cells af fected by diseases involving the inflammatory process , including : immune- mediated inflammatory diseases ( IMIDs ) , tumors , heart issues , neurological and neurodegenerative disorders , rheumatological diseases , obesity, diabetes , liver diseases , pancreatitis , nephropathies , skin irritations and ophthalmias .

[0023] In accordance with these obj ects , the present invention thus relates to a compound for rel ieving inflammation in an organism, its preparation method, and its use in the medical field as defined in basic terms in the appended claims 1 , 10 and 14 , respectively .

[0024] Further preferred characteristics of the invention are set forth in the dependent claims .

[0025] The compound o f the invention is particularly advantageous in that the compound comprises nanometric extracellular ves icles derived from biological material not belonging to the animal kingdom and loaded with one or more bioactives . Indeed, loading one or more bioactives into the nanometric extracellular vesicles derived from biological material not belonging to the animal kingdom, in particular from biological material from plants and fungi or yeasts , allows to enhance the inhibition of inflammation in human and animal cells as the known anti-inflammatory ef fect of the bioactives combines with that of the plant and fungal nanometric extracellular vesicles .

[0026] Indeed, plant- and fungi- or yeast-derived EVs themselves exhibit anti-inflammatory ef fect in human and animal cells , regardless of the bioactive they are loaded with . Speci fically, plant- and fungi- or yeast-derived EVs act on cellular oxidative stress . "Oxidative stress" means an imbalance between the production of reactive oxygen species (ROS ) and the elimination thereof by protection mechanisms . The excess ROS produced may either oxidi ze biomolecules or structurally modi fy proteins and genes so as to trigger signal ing cascades that may lead to the onset and progression of inflammatory diseases .

[0027] Speci fically, the anti-inflammatory ef fect of the present compound is related to the release of Ca2+as a secondary intracellular messenger . Advantageously, the combination of plant and fungal nanometric extracellular vesicles with one or more bioactives triggers an enhanced anti-inflammatory ef fect , namely the release of intracellular Ca2+is greater than that of the individual components ( i . e . the bioactives and the plant and fungal nanometric extracellular vesicles ) when applied separately .

[0028] Furthermore , the present compound may comprise a mixture of bioactives loaded into the nanometric extracellular vesicle . This advantageously results in the possibility of stimulating a varied cellular response with speci fic ef fects ranging, for example , from antiinflammatory to anti-cancer and anti-diabetic .

[0029] Therefore , the compound of the invention has several advantages with respect to known compounds based on synthetic and animal-derived nanometric extracellular vesicles associated with a single bioactive , including : it is biocompatible , it does not create cellular cytotoxicity but rather aids the bio-absorption of the associated bioactives , and it promotes a varied cellular response and thus a high plasticity in pharmacological and physiological applications . With regard to the latter advantageous aspect , the compound of the invention may be incorporated into any formulation in liquid, solid, semisolid, gel , mousse and powder form . Said compound, included in any formulation, may be administered to a living human being or animal in the veterinary, medical , cosmetic or nutraceutical f ields by means of any appropriate route of administration, for example oral , nasal , enteral , topical , sublingual , intra-arterial , ocular, transdermal , vaginal , rectal routes , alone or combined with carriers .

[0030] BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The characteristics and advantages of the present invention will become apparent from the detailed description set forth below, the examples provided for illustrative and non-limiting purposes , and the accompanying Figures 1-5 , in which :

[0032] Figure 1 illustrates the proteins that are present ( and the cellular localization thereof ) in an example of a plant nanometric extracellular vesicle comprised in a compound of the present invention;

[0033] Figure 2 shows the cell viability of human cells treated with examples of compounds of the present invention as the concentration of the two constituents comprised in said compounds varies ;

[0034] Figure 3 shows the interaction between mitochondria belonging to human fibroblasts and an example of a compound of the present invention;

[0035] Figure 4 shows the cellular response via cytosolic intracellular Ca2+signal in human fibroblasts treated with examples of compounds of the present invention and with the two constituents of the compounds separately : in 4A and 4B the extracellular vesicles are derived from plants and the bioactive is a mix of polyphenols of plant origin in 4A and ursolic acid in 4B ; while in 4C and 4D the extracellular vesicles are derived from fungi and the bioactive is a mix of polyphenols of plant origin in 4C and ursolic acid in 4D; and

[0036] Figure 5 shows the mitochondrial oxidative stress in fibroblasts cultured in vitro in a state of inflammation and treated with examples of compounds according to the invention and with the two constituents of the compounds separately .

[0037] DESCRIPTION OF EMBODIMENTS

[0038] The present invention describes a compound comprising nanometer-si zed extracellular vesicles (EVs ) loaded with a bioactive or a mixture of bioactives , wherein the extracellular vesicles (EVs ) are from biological material not belonging to the animal kingdom .

[0039] For the purposes of the present invention, "not belonging to the animal kingdom" means any organism, both unicellular and multicellular, of the plant kingdom and the fungi / yeasts kingdom .

[0040] "Biological material" means any biological material derived from organisms not belonging to the animal kingdom .

[0041] The biological material may be fresh or may have been stored ( e . g . , frozen or refrigerated) .

[0042] In an embodiment of the invention, the biological material belongs to the plant kingdom, for example it is derived from Mal us Domesti ca .

[0043] Preferably, the biological material is extracted from the mesocarp of the fruit of Mal us Domesti ca . Advantageously, the nanometric extracellular vesicles derived from Mal us Domesti ca have , indeed, strong antiinflammatory and anti-oxidant power proven on several skin and immune system cell lines .

[0044] In another embodiment of the invention, the biological material belongs to the fungi or yeasts kingdom, for example it is derived from cultures of Saccharomyce cerevi siae .

[0045] Furthermore , the compound of the invention comprises a bioactive or a mixture of bioactives not belonging to the animal kingdom loaded into the nanometric extracellular vesicles . Speci fically, the bioactive or the mixture of bioactives is loaded into the lipid bilayer constituting the vesicle outer layer .

[0046] Preferably, the quantitative ratio expressed in grams between extracellular vesicles (EVs ) and the bioactive or the mixture of bioactives is in the range from 1 : 1 to 1000 : 1 , depending on the bioactive .

[0047] The bioactives may be of natural or synthetic origin .

[0048] Preferably, the bioactives comprised in the compound of the invention are of natural origin and, in particular, of plant origin .

[0049] The bioactives are selected from the group consisting of polyphenols , terpenes and phytosterols , and their combinations .

[0050] "Mixture of bioactives" means a combination of two or more bioactives either belonging to the same group of molecules , for example a mixture of polyphenols or a mixture of terpenes , or belonging to di f ferent groups of molecules , for example a mixture of two or more polyphenols and terpenes and / or phytosterols .

[0051] "Polyphenols" means , for example , molecules selected from the group consisting of phenolic acids, flavonoids, stilbenes and lignans. Preferably, the polyphenols are selected from the group consisting of kaempferol, myricetin, catechin, epicatechin, pro-anthocyanidins (Bl, B2, B4, B5, Cl) , phlorizin, quercetin, quercetin-glycoside, 3, 5, 7, 3' , 4'- pentaf lavonol-3-o-xyloside, 3, 5, 7, 3' , 4' -pentaf lavonol-3-o- rhamnoside, 3, 5, 7, 3' , 4' -pentaf lavonol-3-o-galactoside,

[0052] 3, 5, 7, 3' , 4' -pentaf lavonol-3-o-glucos ide, 3, 5, 7,3' , 4'- pentaf lavonol-3-o-arabinoside, 3, 5, 7, 3' , 4' -pentaf lavonol-3- o-rutinoside, anthocyanidins, glycosylated cyanidins, ideain, vanillin, vanillic acid, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, caffeic acid, gallic acid, ferulic acid, coumaric acid, hydroxybenzoic acid, cinnamic acid, salicylic acid, malonic acid, maleic acid, coniferyl alcohol, rutin, arbutin, isorhamnetin, resveratrol, t- and cis-piceid and their combinations.

[0053] In a preferred embodiment of the invention, the polyphenols are extracted from the fruit of Malus Domestica .

[0054] In a further embodiment of the invention, the compound comprises a mixture of polyphenols, namely two or more polyphenols, preferably plant-derived, more preferably extracted from Malus Domestica . For example, procyanidin Bl and ferulic acid.

[0055] "Terpenes" means, in particular, tetraterpenes and triterpenes . "Terpenes and phytosterols" means, for example, molecules selected from the group consisting of terpineol, alpha-f arnesene, farnesol, beta-f arnesene, limonene, nerolidol, linalool, beta-caryophyllene, sesquiterpene, eugenol (for example, isoeugenol, methyl eugenol) , citral, lupane, geraniol, ionone, guaiacol, myrcene, oleanolic acid, ursolic acid, corosolic acid, betulinic acid, pomolic acid, alpha- or beta-amyrins , uvaol, euscaphic acid, erythrodiol, oleanolic aldehyde, maslinic acid, betulin, betulinic aldehyde, beta-sitosterol, stigmasterol , isofucosterol, campesterol, cycloartanol and combinations thereof.

[0056] In a preferred embodiment of the invention, the terpenes and / or the phytosterols are extracted from the fruit of Malus Domestica .

[0057] In a further embodiment of the invention, the compound comprises a mixture of terpenes and / or phytosterols, namely two or more terpenes and / or phytosterols, preferably plant terpenes and / or phytosterols, more preferably terpenes and / or phytosterols extracted from the fruit of Malus Domestica. For example, a mixture of terpenes and phytosterols extracted from the fruit of Malus Domestica may comprise ursolic acid and beta-sitosterol.

[0058] In another embodiment of the invention, the compound of the invention comprises a mixture of polyphenols and terpenes and / or phytosterols, namely two or more polyphenols and terpenes and / or phytosterols, preferably plant-derived, more preferably extracted from the fruit of Malus Domestica.

[0059] In a second aspect, the present invention refers to a process for preparing the compound as described above.

[0060] In detail, the process of the invention comprises the following steps: a) isolating the nanometer-sized extracellular vesicles from biological material not belonging to the animal kingdom; b) loading the bioactive or the mixture of bioactives into the isolated extracellular vesicles (EVs) , obtaining the compound described above; and c) purifying the compound. Advantageously, the nanometric extracellular vesicles are isolated in step a ) of the present process from biological material not belonging to the animal kingdom by means of a method for isolating nanometer- and submicrometersi zed extracellular vesicles from biological material not belonging to the animal kingdom described in the Patent Application 102022000000719 .

[0061] In summary, the method for isolating nanometer- and submicrometer-si zed extracellular vesicles from biological material not belonging to the animal kingdom sequentially comprises a step (A) of pretreatment , obtaining a pretreated biological material , a step (B ) of enzymatic digestion, in which the pretreated biological material is subj ected to enzymatic digestion, obtaining an extracellular vesicles (EVs ) suspension, and a step ( C ) of isolation of extracellular vesicles (EVs ) from said extracellular vesicles (EVs ) suspension .

[0062] Therefore , at the end of step a ) , the isolated extracellular vesicles have a size less than 0 . 22 pm, preferably they have a si ze ranging between 40 nm and 220 nm .

[0063] The EVs isolated according to the method of the invention may be used fresh or stored at 4 ° C in freeze-dried form, or cryo- frozen using designated cryogenic or vitri fying reactants .

[0064] The subsequent step b ) of the present method relates to loading the bioactive or the mixture of bioactives into the isolated nanometric extracellular vesicles and may occur by means of di f ferent known techniques , including, for example , by means of passive incubation, hypotonic dialysis , sonication or extrusion . Specifically, the loading by means of passive incubation consists in incubating the extracellular vesicles with the bioactive or the mixture of bioactives at 37°C for periods ranging from 2 to 24 hours, stored in a suitable buffer. According to this technique, the loading efficiency depends on the polarity of the bioactive and is relatively low .

[0065] Regarding the sonication technique, this is performed by immersing a sonicating needle, suitable for the volume taken into consideration, into the solution containing the nanometric extracellular vesicles and the bioactive or the mixture of bioactives. The loading occurs through 6 sonication cycles of 4 minutes, 30 seconds on / off, with a cooling period of 2 minutes between cycles. The sonication cycles occur with the following parameters: amplitude 20%, room temperature, pulsed or continuous mode. This technique leads to an effective and quick loading of the bioactive or the mixture of bioactives. However, the aggregation of exosomes can occur, thus damaging the lipid structure.

[0066] Instead, the loading by means of hypotonic dialysis is performed by transferring the isolated nanometric extracellular vesicles and the bioactive (s) into the dialysis membranes (cellulose ester, molecular weight cutoff = 100-500 Da) . The membrane is placed in 200 mL of 10 mM phosphate buffer (pH 7.4) under stirring at RT for 2 to 24 hours. Although the loading efficiency is improved compared to the passive incubation technique, the vesicles undergo an undesired deformation.

[0067] As for the loading by means of the extrusion technique, this involves using a lipid extruder with a membrane having a porosity of 100-400 nm on which both the nanometric extracellular vesicles and the bioactive (s) are loaded. This technique allows to obtain both a high loading-efficiency and uniform vesicles. On the other hand, the vesicles loaded with the bioactives do not preserve the original properties (e.g., the zeta potential and the structure of the membrane proteins are altered) .

[0068] The selection of the loading technique depends on the nature of the bioactive and on the characteristics of the technique itself.

[0069] Regardless of the selected technique, at the end of step b) the compound of the present invention is obtained, which therefore comprises nanometer-sized extracellular vesicles loaded with the bioactive or the mixture of bioactives, the extracellular vesicles being from biological material not belonging to the animal kingdom.

[0070] Finally, step c) of the present process involves purifying the compound of the present invention, for example, through ultracentrifugation at 110000 g for 60 minutes.

[0071] A third aspect of the present invention relates to the use of the present compound as a medicament, for example, in the veterinary, medical, cosmetic or nutraceutical fields.

[0072] In this case, the compound may be administered, for example, in liquid, solid, semisolid, gel, mousse and powder form. Further, the administration may occur by ingestion and / or by direct intravenous and / or intramuscular administration and / or by topical and / or rectal and / or periocular application and / or by inhalation. Specifically, the compound of the present invention, when administered in any of the modes listed above, is bio-distributed towards the following systems: musculoskeletal, digestive, urinary, reproductive, lympho-hematopoietic, nervous and integumentary .

[0073] In an embodiment of the third aspect of the invention, the compound is used in anti-inflammatory and / or antioxidant therapies in the veterinary or human fields. Specifically, the compound may, therefore, be applied in every case of disease involving the inflammatory process, including, for example, immune-mediated inflammatory diseases (IMIDs) , tumors, heart issues, neurological and neurodegenerative disorders, rheumatological diseases, obesity, diabetes, liver diseases, pancreatitis, nephropathies, skin irritations and ophthalmias.

[0074] EXAMPLES

[0075] Some examples of compounds according to the invention and the interaction thereof with an exemplary target cell are reported below.

[0076] 1. Compounds according to the invention.

[0077] The biological material from which the extracellular vesicles comprised in the compound according to the invention are isolated is from the plant kingdom (Plant-Derived Nanovesicles, PDNVs) or from the fungi kingdom (Fungi- Derived Nanovesicles, FDNVs) .

[0078] For example, the plant extracellular vesicles (PDNVs) are from the Malus domestica variety (ADNVs) . In this regard, Figure 1 shows the distribution of proteins extracted from ADNVs in different cell compartments, where the first three terms are associated with the cell periphery, including the plasma membrane.

[0079] Alternatively, the fungal extracellular vesicles (FDNVs) are, for example, from cultures of Saccharomyce cerevisiae (ScDNVs) .

[0080] Regardless of the provenance of the biological material , the extracellular vesicles ADNVs and ScDNVs are isolated according to the method detailed in the Patent Application n° 102022000000719 and summari zed above .

[0081] After having been isolated, both ADNVs and ScDNVs are loaded with the bioactive , for example apple-derived ursolic acid, or with a mixture of polyphenols (hereinafter referred to as poly mix ) , for example phlori zin, quercetin-3-0- rutinoside and procyanidin Bl , derived from apples of the white renetta Canada variety .

[0082] Table 1 summari zes the four compounds according to the invention used in the experiments illustrated below .

[0083] TABLE 1

[0084] 2. Interacti on of the compounds according to the inventi on wi th human cell s

[0085] Figure 2 shows the cell viability of human cells treated with increasing concentrations of the four compounds according to the invention and listed in Table 1 .

[0086] As can be noted, the cell viability is not reduced by the treatment ; the compounds according to the invention are thus biocompatible and non-cytotoxic .

[0087] Figure 3 illustrates how the compounds according to the invention interact with human cell s . Specifically, Figure 3 shows human dermal cells ( fibroblasts ) after 12 hours of incubation with compound 1 of the invention (MitoTracker- labeled mitochondria ( long and narrow, shown by arrow 2 ) , Hoechst-labeled nucleus ( shown by arrow 3 ) and PKH26-labeled ADEVs (small and round, shown by arrow 1) ) .

[0088] As shown in Figure 3, the interaction between the compound of the invention and human cells occurs between the membrane proteins of the extracellular vesicles (i.e. the ADNVs) and the mitochondrial membrane proteins in human cells. Once they have entered the cell cytoplasm intact, ADNVs reach the mitochondria, establishing close connections .

[0089] Figure 4 compares instead the physiological response of fibroblasts treated with the compounds according to the invention and listed in Table 1 and treated separately with the individual constituents of the compound (i.e. ADNVs, ScDNVs, Ursolic Acid and poly mix) according to the invention .

[0090] Specifically, in Figure 4, the physiological response induced by treating the fibroblasts in vitro is quantified based on the release of free Ca2+into the cell cytosol.

[0091] In particular, Figure 4 shows the amplitude (AUG) of the Ca2+signal in fibroblasts treated according to different conditions, namely treated with the compounds according to the invention and, in comparison thereto, with the individual constituents of the compounds according to the invention.

[0092] The fibroblasts treatment conditions include: - the compounds according to the invention, i.e. ADNVs + poly mix in Figure 4A; ADNVs + Ursolic Acid in Figure 4B; ScNVs + poly mix in Figure 4C; ScNVs + Ursolic Acid in Figure 4D; and the respective individual constituents, i.e. ADNVs in Figures 4A and 4B; ScDNVs in Figures 4C and 4D; poly mix in Figures 4A and 4C; and ursolic acid in Figures 4B and 4D.

[0093] In Figure 4 it should be noted that the cellular response induced by treating the fibroblasts with the compounds according to the invention is greater than the response of the individual components when they are treated separately .

[0094] Regarding the anti-oxidant property, Figure 5 graphically illustrates the mitochondrial oxidative stress in human dermal cells ( fibroblasts ) cultured in vitro in a state of inflammation . Speci fically, the mitochondrial stress is referred to as the percentage of cells positive for the MitoSOX staining for detecting reactive oxygen species (ROS ) and reactive nitrogen species (RNS ) . As shown in Figure 5 , cells were subj ected to di f ferent culture conditions and compared with a control condition ( CTRL ) :

[0095] - with the compounds according to the invention, i . e . ScNVs + poly mix in Figure 5A; ScNVs + Ursolic Acid in Figure 5B ; ADNVs + poly mix in Figure 5C ; ADNVs + Ursolic Acid in Figure 5D;

[0096] - the respective individual constituents , i . e . ScDNVs in Figures 5A and 5B, ADNVs in Figures 5C and 5D; poly mix in Figures 5A and 5C ; and Ursolic acid in Figures 5B and 5D .

[0097] In Figure 5 it should be noted that the compounds according to the invention have anti-oxidant properties greater than the anti-oxidant properties of the individual components when applied separately, which leads to a decrease in the mitochondrial oxidative stress .

[0098] The advantages achieved by the compound according to the present invention become apparent from the foregoing description and from the examples indicated above .

Claims

CLAIMS1. Compound for relieving inflammation in an organism comprising nanometer-sized extracellular vesicles (EVs) derived from biological material not belonging to the animal kingdom and loaded with a bioactive or a mixture of bioactives, wherein the bioactive or the mixture of bioactives is selected from the group consisting of polyphenols, terpenes, phytosterols and their combinations .

2. Compound as claimed in claim 1, wherein the biological material is derived from the plant or fungi kingdoms.

3. Compound as claimed in claim 2, wherein the plant biological material is derived from Malus Domestica .

4. Compound as claimed in claim 2, wherein the fungal biological material is derived from cultures of Saccharomyce cerevisiae .

5. Compound as claimed in one of the previous claims, wherein the bioactive or the mixture of bioactives is of plant origin.

6. Compound as claimed in claim 5, wherein the bioactive or the mixture of bioactives is extracted from the fruit of Malus Domestica.

7. Compound as claimed in one of the previous claims, wherein the mixture of bioactives comprises two or more polyphenols or two or more terpenes and / or phytosterols or two or more polyphenols, terpenes and / or phytosterols .

8. Compound as claimed in one of the previous claims, wherein the quantitative ratio expressed in grams between extracellular vesicles (EVs) and the bioactive or the mixture of bioactives is in the range from 1:1 to1000 : 1.

9. Method for preparing the compound as claimed in any of the previous claims comprising the steps of: a) isolating nanometer-sized extracellular vesicles (EVs) from biological material not belonging to the animal kingdom; b) loading the bioactive or the mixture of bioactives into the isolated extracellular vesicles (EVs) , obtaining the compound as claimed in claims 1 to 8; and c) purifying the compound.

10. Method as claimed in claim 9, wherein the step a) comprises the steps of (A) pretreatment of the biological material, obtaining the pretreated biological material, a step (B) of enzymatic digestion, in which the pretreated biological material is subjected to enzymatic digestion, obtaining an extracellular vesicles (EVs) suspension, and a step (C) of isolation of extracellular vesicles (EVs) from said extracellular vesicles (EVs) suspension .

11. Method as claimed in claim 9 or 10, wherein the step b) occurs by means of passive incubation or hypotonic dialysis or sonication or extrusion.

12. Method for isolating extracellular vesicles (EVs) as claimed in one of the claims from 9 to 11, wherein the step c) occurs through ultracentrifugation at 110000 g for 60 minutes.

13. Compound according to claims from 1 to 8 for use as a medicament .

14. Compound as claimed in claim 13, wherein the compound is in liquid, solid, semisolid, gel, mousse and powder form.15 . Compound as claimed in claim 13 or 14 , wherein the compound is configured to be administered by ingestion and / or intravenously and / or intramuscularly and / or by topical and / or rectal and / or periocular application and / or inhalation .16 . Compound as claimed in claims from 13 to 15 for use in anti-inflammatory and / or anti-oxidant therapies in the veterinary or human fields .17 . Use of the compound as claimed in claims from 1 to 8 in the cosmetic and nutraceutical fields .

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