Extracellular vesicles from green tea and their use

Abstract

Extracellular vesicles (EVs) extracted from green tea leaves from biological agriculture characterized in that said green tea leaves contain antioxidant compounds within the lipid membrane.

Description

[0001] EXTRACELLULAR VESICLES FROM GREEN TEA AND THEIR USE

[0002] DESCRIPTION

[0003] Technical field of the invention

[0004] The present invention relates to extracellular vesicles derived from green tea from biological agriculture and their use as natural carriers of bioactives, for cosmetic use, in particular neurocosmetics, pharmaceutical use and as a medical device for topical use.

[0005] Background art

[0006] As is well known, tea is a drink obtained by infusing the leaves of the Camellia sinensis plant of the Theaceae family. It has been cultivated since ancient times in India, China and Japan; more recently in Indonesia, the Caucasus, East Africa and South America. Currently, there are different types of tea (including white tea, black tea, green tea, oolong tea), which differ from each other in the processing method, which is the most important factor for differentiating the finished product.

[0007] Green tea, well known for its beneficial effects on the human body and especially for its antioxidant action, is obtained by subjecting the tea leaves to a thermal process called "stabilization" - which can be performed with dry heat (roasting) or moist heat (vaporization) - which inhibits the enzymes responsible for oxidation and therefore allows the leaves to maintain their characteristic green color (oxidation is a chemical reaction that occurs spontaneously in tea leaves, darkening their color).

[0008] Green tea contains very high quantities of polyphenols, substances with a beneficial and antioxidant role for the human body; in particular, the most characteristic and main polyphenol responsible for the properties of green tea is Epigallocatechin gallate (also known by the acronym EGCG, it is present in quantities equal to 71 mg per 100 ml of beverage, higher than that contained in 100 ml of black tea). The benefits for human health that can derive from the consumption of green tea are truly many. This beverage has already been and still is the subject of several studies, whose aim is to increasingly understand its possible healing role. Most of the benefits of green tea would seem to depend on the presence of large quantities of catechins, capable of strengthening antioxidant defenses and therefore reducing damage to cells, with positive effects on all tissues. In particular, green tea is said to have anti-tumor properties (it protects good cells from mutations and inhibits the growth of cancerous cells) and to be beneficial to the cardiovascular system (it inhibits the formation of clots, reduces the presence of fats and cholesterol in the blood and slows the development of atherosclerosis and coronary heart disease) and the respiratory system (it is particularly useful against the negative effects of smoking). Green tea also accelerates the metabolism of fats and sugars, facilitating weight loss, and facilitates diuresis, proving useful in cases of water retention, cellulite and urinary tract infections. Thanks to the combined action of the polyphenolic derivatives (flavonoids, chlorogenic acid, caffeic acid, tannins) it contains, this type of tea is said to have neuroprotective effects and could play a role of some importance in the prevention and treatment of neurodegenerative diseases. Green tea also appears to be able to combat hypertension and prevent osteoporosis (its powerful remineralizing action increases bone density in menopausal women).

[0009] There are also several studies that attribute anti-stress and anti-inflammatory effects to green tea. Although the benefits for human health that can derive from the consumption of green tea are truly many, it is good to keep in mind that green tea contains caffeine and, therefore, if consumed in large quantities it can cause anxiety, nervousness and insomnia. Green tea can also affect the functioning of the thyroid. Some studies have also highlighted that at certain concentrations the polyphenols of green tea can induce oxidative stress and liver toxicity (particular caution must therefore be exercised in the consumption of concentrated tea extracts).

[0010] Specifically, the Italian green tea "Assolo" belongs to an exclusive crop of "Via del Te, from biological agriculture. In the uncontaminated territory of Vai d'Ossola, near Lake Maggiore, a vast production of fine Italian tea has flourished in the Premosello plantation, which is home to 20,000 Camellia sinensis plants. It is the second largest in Europe and the first in Italy to guarantee a significant and constant production of tea.

[0011] Cultivation excludes the use of synthetic products, insecticides or pesticides. Weeding and harvesting are done manually with the straw mulching technique. The method of processing the leaves determines the difference between one tea and another. The decisive step is the oxidation phase, which distinguishes between nonoxidized, oxidized and partially oxidized teas. As already mentioned, in the processing of green tea, unlike black tea, there is no oxidation phase. After a rapid withering, the leaves are stabilized through a heating phase that inhibits the enzymes responsible for oxidation.

[0012] Green teas, therefore, are the only ones to have a zero oxidation level, with a consequent much higher catechin content than other teas (for example black tea). Having a higher catechin content means having a greater antioxidant activity.

[0013] 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 that are bounded or encapsulated by a phospholipid bilayer and that 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.

[0014] Scientific research has shown that plant-derived extracellular vesicles have great potential in medicine, where it is necessary to act on the regulation of biological processes that are fundamental for proper 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.

[0015] 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 large quantities and in a sustainable way for humans and the environment. It is also known that extracellular vesicles can act as neurocosmetics. A neurocosmetic is a substance that, when applied to the skin, can influence the cutaneous nervous system or, in general, act as cutaneous messengers. In fact, the skin, in addition to being the first physical barrier against external agents, is directly connected to the central nervous system and can therefore be considered the sensory organ par excellence. For this reason, in the case of cosmetic applications, it is also important to evaluate the ability of extracellular vesicles to influence the production of serotonin and melatonin by fibroblasts. Serotonin and melatonin are important regulators of the sleep-wake rhythm. Serotonin is a neurotransmitter that helps regulate mood, memory and cardiovascular functions. Serotonin is found in the central nervous system, in platelets and in the mucosa of the gastrointestinal tract, just think that 95% of the serotonin in our body is found in the intestine. In fact, the cells that make up the intestinal lining release serotonin that regulates the peristaltic waves that move food in the digestive tract and the secretion of enzymes. It also has a strong anti-aging effect on the skin and regulates proper skin hydration.

[0016] Melatonin is particularly known as a hormone that regulates sleep-wake rhythms, but it also has a powerful antioxidant and anti-inflammatory action, and also regulates blood pressure. Our brain, in response to darkness, produces melatonin; when the retinas detect darkness, a signal is sent to the brain and from there to the pineal gland which produces melatonin. Melatonin is produced throughout the body for local use, from the gastrointestinal tract, where it regulates the health and function of the intestinal epithelium and strengthens the immune system of the intestine and relaxes the gastrointestinal muscles. Furthermore, on the skin it protects against oxidative stress and damage induced by ultraviolet radiation. Melatonin has antioxidant and anti-inflammatory properties, acts as a broadspectrum photoprotector, with a significant impact on reducing premature skin aging and improving the distinctive features of photo-aged skin.

[0017] However, the use of extracellular vesicles derived from green tea from biological agriculture (non-OGM) is not known at the state of the art. Extracellular vesicles have a key role in cell-cell communication in nature, both within an organ or an apparatus, 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 membrane-membrane fusion, and it is therefore very likely that vesicles of plant origin can transfer their content into human cells, using the same mechanism.

[0018] In conclusion, extracellular vesicles derived from green tea, in particular from tea leaves, contain multiple antioxidant molecules that exert beneficial effects on health, strengthening antioxidant defenses and preserving tissue health. In fact, green tea is very rich in antioxidants and phenolic compounds that counteract the formation of free radicals, responsible for cellular aging, reduces cholesterol levels and promotes the thermo-combustion of fatty acids, carrying out a powerful detoxifying and invigorating action.

[0019] However, the use of extracellular vesicles extracted from tea leaves from biological agriculture and their use as natural transporters of bioactive compounds is not known.

[0020] Summary of the invention

[0021] The object of the present invention is therefore extracellular vesicles extracted from green tea leaves 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. The present invention uses a selected subpopulation of extracellular vesicles (EV) extracted from green tea from biological agriculture that have a diameter with average dimensions ranging between 169.4 ± 1.8 and 217.6 ± 5.6 nm.

[0022] According to the present invention, the extracellular vesicles isolated from green tea leaves biological origin have been characterized based on their total antioxidant content, in particular the content of total polyphenols and that of Epigallocatechin gallate.

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

[0024] According to a further aspect, the use of such extracellular vesicles as a cosmetic, in particular neurocosmetic, pharmaceutical and as a medical device for topical use is defined, as specified in the independent use claims. The dependent claims outline particular and further advantageous aspects of the invention.

[0025] Brief description of the drawings

[0026] 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 illustrates the graph of the dimensional characterization of extracellular vesicles isolated from green tea leaves, according to the present invention;

[0028] - Figure 2 illustrates the comparison of the expression of matrix type I collagen in in vitro fibroblasts treated with extracellular vesicles extracted from green tea leaves from biological agriculture and the control, - Figure 3 shows the comparison graph between the concentration of serotonin (A) and melatonin (B) in in vitro fibroblasts treated with extracellular vesicles extracted from biological green tea leaves in a CTR control, a first C1 and a second C2 concentration.

[0029] Detailed description

[0030] According to the present invention and according to the attached Figures, extracellular vesicles (EVs) were extracted from green tea leaves from biological agriculture.

[0031] Biological agriculture means an 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 they have flowered, 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 organic fertilizers such as compost are used, a mixture of soil, plant remains, wood ash and anything else that exists in the company that is biodegradable and nonpolluted.

[0032] In particular, the tea leaves that are the subject of this invention are those of Italian green tea "Assolo" that belong to an exclusive crop in the Vai d'Ossola area, near Lake Maggiore, where the production of fine Italian tea is present, in the Premosello plantation, which hosts 20,000 Camellia Sinensis plants. The Italian company is specialized and certified for biological agriculture, the tea leaves are carefully washed and dried. The methodology by which the extracellular vesicles were extracted from biological tea leaves 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 plant raw material; b. transferring the plant raw material into food containers and storing it at room temperature; c. washing the plant raw material, d. carrying out an initial processing of the plant raw material for extraction, i.e. an infusion phase of the green tea leaves in ultrafiltered water for 24 hours; e. extracting the first semi-finished product, i.e. the first infusion or extraction juice, using professional automatic extractors; the model used has a screw that rotates at 40 RPM per minute so as not to tear the plant cells but to squeeze them by mimicking chewing, f. store the first semi-finished product in plastic bottles such as High Density Polyethylene (HDPE), Polyethylene Terephthalate (PET), Polypropylene (PP), Polycarbonate (PC), to avoid the release of toxic substances, g. store at -40 for approximately 6 months or at -80 for one year, h. perform an initial filtration phase of the first semi-finished product in a filter with a mesh size between 0.2 mm and 1 micron l. perform an initial centrifugation of the filtered product in centrifuge bottles of the polypropylene or PPCO type at a speed of 2,000 x g for 30 minutes, m. filter 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. add to the mixture of extracellular vesicles a quantity of maltodextrin between 4% and 35% by weight, p. dry the mixture of extracellular vesicles by atomization (spray drying or “SPRAY-DRYING”), q. collect and store the powdered product at room temperature in tightly closed amber borosilicate glass containers, away from heat sources, not exposed to light and humidity.

[0033] Phase d) involves leaves 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 low liquid content, for the extraction of extracellular vesicles.

[0034] Advantageously, the infusion allows to better preserve the integrity of the exosomes and reduce the degradation of the bioactive components.

[0035] The drying phase p) is carried out according to the spray technique or “Spray Drying” which 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.

[0036] Advantageously, the raw material used in the drying phase is a noble derivative extracted by infusion from green tea leaves. 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.

[0037] Advantageously, the use of green tea leaves 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) through specific experiments and adjustments to obtain the desired product.

[0038] According to the method of the present invention, the drying step q) is performed according to optimized process parameters, namely:

[0039] - “Drying Gas” Drying gas flow rate (m3 / h) = 30 - 35,

[0040] - ’’Inlet T” Inlet temperature (°C) = 115 - 140,

[0041] - ’’Spray Gas” Atomized gas flow rate (l / h) = 1000 - 1500, - ’’Pump 1” Pump flow rate (ml / min): 6 -15,

[0042] - “Outlet T” outlet temperatures (°C) = 60 - 75.

[0043] To evaluate whether the EVs isolated from green tea leaves fell within the size range of extracellular vesicles, the isolated samples were analyzed by means of the “Bradford assay” (Pierce, Rockford, IL, USA) and the “Nanotracking particle analysis” (NTA). As seen in Figure 1 , isolated EVs show the typical distribution of extracellular vesicles, with average sizes (diameter) ranging from 169.4 ± 1.8 to 217.6 ± 5.6 nm. 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 zeta potential.

[0044] 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.

[0045] In the case of extracellular vesicles isolated from green tea leaves, EVs have a zeta potential that varies between -30.49 mV and -28.42 mV, demonstrating their great stability in liquid solution.

[0046] The tests performed were performed in order to characterize the extracellular vesicles isolated from green tea leaves for their total antioxidant content. Green tea has multiple antioxidant molecules that exert beneficial effects on health, strengthening antioxidant defenses and preserving tissue health. In fact, green tea is very rich in antioxidants and phenolic compounds that counteract the formation of free radicals, responsible for cellular aging, reduces cholesterol levels and promotes the thermo-combustion of fatty acids, carrying out a powerful detoxifying and invigorating action. To characterize the cargo of the vesicles, the EVs from tea leaves were analyzed for total antioxidant capacity and Epigallocatechin gallate content.

[0047] 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 pathologies.

[0048] The test used allows to evaluate the presence of both enzymatic (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 green tea leaves, we analyzed a range of vesicles between 106 and 1012, characterizing a content of total phenolic compounds between 0.102 ± 0.005 pM and 84703 ± 2.5 pM.

[0049] In particular, Epigallocatechin gallate (EGCG) is the best studied catechin derivative so far. Its amount in tea depends on the variety, cultivation method and processing of the leaves. Green tea catechins have a ROS scavenging activity, which makes them potential candidates for anti-photoaging therapy.

[0050] The experiments carried out measured the amount of EGCG in vesicles isolated from Italian green tea, evaluating increasing concentrations of vesicles between 106 and 1012 and quantifying an EGCG content between 8.5 ± 1.3 nM and 11.2 ± 0.04 nM.

[0051] In addition, numerous experiments were performed to evaluate whether EVs isolated from green tea leaves could act as neurocosmetics, i.e. as substances that applied to the skin can influence the cutaneous nervous system or in general act as cutaneous messengers.

[0052] The experiments carried out tested the ability of extracellular vesicles isolated from green tea leaves to influence the production of serotonin and melatonin by fibroblasts.

[0053] The experiments performed quantified the concentration of serotonin and melatonin in samples of EVs isolated from green tea leaves from biological agriculture. The analysis was conducted in a concentration range between 106 and 1012, quantifying serotonin values between 4.60 ± 0.01 pg / ml and 801.716 ± 0.001 ng / ml and melatonin between 0.4 ± 0.02 pg / ml and 412.132 ± 0.003 ng / ml.

[0054] Further experiments were performed by treating fibroblasts in vitro with EVs isolated from green tea, to evaluate the production of type I collagen. As shown in Figure 2, the analyses measured an increase of 23% in cellular collagen expression after 24 hours of treatment with EVs isolated from green tea, compared to the untreated control (CTR).

[0055] Furthermore, as shown in Figure 3, experiments were carried out by treating fibroblasts in vitro with increasing concentrations of EVs isolated from biological green tea, to evaluate the release of serotonin and melatonin in the cell supernatant. As reported in Figure 3 graph A, in the cell culture medium there is an increase in serotonin released equal to 146% and 237% respectively for the lowest dose (C1) and highest (C2) of EVs used. Instead, melatonin measured in cells treated with C1 increases by 4%, while for C2 by 7% compared to the untreated control (Figure 3 graph B).

[0056] Overall, these results indicate that EVs extracted from green tea from biological agriculture may have potential applications in promoting skin and connective tissue health, thanks to their rich composition of bioactive compounds and the ability to stimulate collagen production. Specifically, the presence of EGCG contributes to induce beneficial effects on health, including antioxidant and anti-inflammatory effects, while the presence of serotonin and melatonin suggests potential beneficial effects on mood and sleep regulation. Finally, the production of type I collagen by fibroblasts and induced by EVs from green tea leaves suggests a potential anti-aging and regenerative effect on skin and connective tissue.

[0057] Advantageously, these data support the use of EVs extracted from biological green tea, in certain cosmetic applications, in particular neurocosmetics, and pharmaceuticals and as a medical device for topical use.

[0058] Advantageously, formulations comprising exosomes extracted from green tea combined with selected ingredients enhance the efficacy of the final product.

[0059] An example of a formulation for neurocosmetic use is represented by a neurocosmetic cream comprising EVs extracted from biological green tea.

[0060] The composition for 100 ml of total neurocosmetic cream includes in volumetric percentages:

[0061] - extracellular vesicles (extracted from green tea leaves from biological agriculture “Camellia Sinensis”, aloe vera “Aloe barbadensis" or chamomile “Matricaria chamomilla’’) 1% - 3% and - Auxiliary ingredients (in indicative percentages):

[0062] Jojoba oil: 15% - 25%

[0063] Shea butter: 5% - 10%

[0064] Hyaluronic acid: 0.5% - 1.5%

[0065] Centella asiatica extract: 2% - 4%

[0066] Vitamin E (tocopherol): 0.2% - 0.5%

[0067] Glycerin: 3% - 7%

[0068] Beeswax: 2% - 4%

[0069] Purified water: Q.s. at 100%

[0070] Extracellular vesicles (extracted from green tea leaves) rich in bioactive compounds, can help protect and regenerate the skin, as well as provide benefits for the skin's nervous system. Plant extracellular vesicles derived from green tea leaves, aloe vera or chamomile are particularly suitable for their antioxidant, antiinflammatory and soothing properties.

[0071] Additional auxiliary ingredients: jojoba oil and shea butter are selected for their moisturizing, emollient and nourishing properties. They help keep the skin soft, elastic and protected from harmful external agents; hyaluronic acid is known for its moisturizing and anti-aging properties, helps keep the skin hydrated, reducing the appearance of fine lines and wrinkles; Centella Asiatica extract is known for its regenerative and soothing properties on the skin, can help reduce inflammation, promote healing of damaged tissue and improve the overall appearance of the skin; Vitamin E (Tocopherol) is a powerful antioxidant that helps protect the skin from damage caused by free radicals and UV rays and helps keep skin looking young and healthy. This formulation combines the beneficial effects of plant extracellular vesicles with the moisturizing, nourishing and antioxidant effects of other ingredients, providing a complete neurocosmetic cream to improve the health and appearance of the skin. The percentages of the ingredients are provided with some flexibility to adapt to the manufacturer's preferences and regulatory requirements.

[0072] Another example of a cosmetic formulation is a pediatric zinc paste to protect and soothe the sensitive skin of children. In this composition, the extracellular vesicles extracted from biological green tea leaves, contained in the formulation, act synergistically with other ingredients to provide a protective and soothing barrier against irritation and redness, help reduce inflammation, relieve itching and promote healing of damaged skin.

[0073] The composition for 100 ml of total paste includes in volumetric percentages:

[0074] - extracellular vesicles (extracted from green tea leaves from biological agriculture “Camellia Sinensis”): 0.1 % -5%

[0075] - zinc oxide: 10- 30%

[0076] - vegetable glycerin: 5- 15%

[0077] - distilled water: 30- 55%- emulsifying and stabilizing components: 5%

[0078] - sweet almond oil: 5- 25%

[0079] - cellulose gel (to adjust the consistency): q.s.

[0080] - ecological preservative: compliant with local regulations.

[0081] A further example of a cosmetic composition is a dermatological cream for the protection and treatment of damaged or sensitive skin.

[0082] The composition for 100 ml of total dermatological cream includes in volumetric percentages: - plant extracellular vesicles (extracted from green tea leaves from biological agriculture “Camellia Sinensis”, from aloe vera “Aloe barbadensis" and from chamomile “Matricaria chamomilla"'. 1% - 3%

[0083] Auxiliary Ingredients (approximate percentages):

[0084] - Coconut oil: 20% - 30%

[0085] - Shea butter: 5% - 10%

[0086] - Hyaluronic acid: 0.5% - 1.5%

[0087] - Calendula extract: 2% - 4%

[0088] - Vitamin E (tocopherol): 0.2% - 0.5%

[0089] - Glycerin: 3% - 7%

[0090] - Beeswax: 2% - 4%

[0091] - Purified water: Q.s. to 100%

[0092] The extracellular vesicles green tea leaf extracts are rich in lipids, proteins and other bioactive compounds that can help protect and regenerate damaged or sensitive skin. These vesicles can act as carriers of active ingredients, providing them with greater efficacy and better penetration into the skin.

[0093] Additional Auxiliary Ingredients: Jojoba Oil and Shea Butter are rich in essential fatty acids and vitamins that moisturize and nourish the skin, protect the skin from drying out and external aggressors, keeping it soft and supple; Hyaluronic Acid is an effective moisturizer that can lock moisture into the skin, keeping it hydrated and reducing the appearance of fine lines and wrinkles; Calendula Extract has soothing and anti-inflammatory properties that can help reduce inflammation and calm sensitive or damaged skin; Vitamin E (Tocopherol) is a powerful antioxidant that helps protect the skin from damage caused by free radicals and UV rays and helps keep the skin young and healthy, reducing the risk of premature aging damage.

[0094] In addition, extracellular vesicles extracted from bilogical green tea leaves, containing bioactive and anti-inflammatory compounds, can help reduce inflammation and promote healing of skin affected by dermatitis.

[0095] An example of a medical device is a composition for topical use comprising biological green tea vesicles, to be used against dermatitis, in particular for atopic dermatitis and seborrheic dermatitis.

[0096] The composition per 100 ml of total topical use composition includes in volumetric percentages:

[0097] -vegetable extracellular vesicles (extracted from green tea leaves from biological agriculture “Camellia Sinensis”, 1% - 2.5%

[0098] - Auxiliary Ingredients (indicative percentages):

[0099] - Sweet almond oil: 20% - 30%

[0100] - Shea butter: 5% - 10%

[0101] - Beeswax: 2% - 5%

[0102] - Hyaluronic acid: 0.5% - 1%

[0103] - Panthenol (provitamin B5): 1% - 3%

[0104] - Chamomile extract: 1% - 2%

[0105] - Purified water: Q.s. to 100%

[0106] The extracellular vesicles extracted from green tea leaves contain bioactive compounds such as catechins and polyphenols, known for their anti-inflammatory, antioxidant and soothing properties. These compounds can help reduce inflammation and promote healing of skin affected by dermatitis. Additional auxiliary ingredients: Sweet almond oil and Shea butter ingredients are selected for their emollient and moisturizing properties, they help keep the skin soft, hydrated and protected from external irritants; Beeswax is used for its emollient and protective properties. It forms a protective layer on the skin that helps prevent moisture loss and keep the skin hydrated; Hyaluronic acid and panthenol are known for their moisturizing and soothing properties on the skin, they help reduce irritation and promote the regeneration of damaged tissue; Chamomile extract is known for its anti-inflammatory and soothing properties on the skin, it can help reduce redness and irritation associated with dermatitis.

[0107] In particular, the formulation provides a combination of active and auxiliary (ancillary) ingredients to effectively treat dermatitis, providing relief from symptoms and promoting healing of the affected skin. The percentages of the ingredients are provided with some flexibility to adapt to the manufacturer's preferences and regulatory requirements.

[0108] Advantageously, this composition is suitable for use as a medical device for several reasons:

[0109] 1. Active components and targeted therapeutic actions: Plant extracellular vesicles extracted from biological green tea leaves are rich in bioactive compounds such as catechins and polyphenols. These compounds are known for their anti-inflammatory, antioxidant and soothing properties. In particular, the anti-inflammatory action can help reduce inflammation and promote healing of skin affected by dermatitis, both atopic and seborrheic, thus helping to improve symptoms. The effectiveness of these compounds in the treatment of dermatitis is well documented, which justifies their use as part of a topical composition against this skin condition.

[0110] 2. Skin compatibility and balanced formulation: The auxiliary ingredients included in the composition, sweet almond oil, shea butter, beeswax, hyaluronic acid, panthenol and chamomile extract, are known for their moisturizing, emollient, soothing and regenerative properties on the skin. These ingredients not only support the action of the active compounds extracted from green tea leaves, but also help to keep the skin hydrated and reduce dryness and irritation associated with dermatitis, improve its barrier function, which can be compromised in people with dermatitis.

[0111] 3. Design for topical use and safety of use: The composition is designed for topical use, which means that it is applied directly to the affected skin. This allows for a targeted delivery of the active ingredients to the affected area, thus maximizing the therapeutic benefits and minimizing systemic side effects.

[0112] Furthermore, the percentages of the various ingredients are controlled to ensure an optimal balance between efficacy and skin tolerability, thus contributing to the safety of use of the product.

[0113] 4. Skin healing support: The combination of active compounds and auxiliary ingredients aims not only to reduce the symptoms of dermatitis, but also to promote the healing of damaged skin. This is particularly important in the treatment of chronic skin conditions such as atopic and seborrheic dermatitis, where the restoration of the skin barrier function is essential for the control of the disease. 5. Controlled percentages of ingredients: The percentages of the various ingredients are carefully calibrated to ensure an optimal balance between efficacy and skin tolerability. For example, green tea vesicles are present in a concentration of 1 % to 2.5%, which is sufficient to provide the desired benefits without risk of skin irritation.

[0114] In summary, this topical composition comprising extracellular vesicles extracted from green tea leaves obtained from biological agriculture is suitable for use as a medical device against dermatitis thanks to the presence of active ingredients known for their anti-inflammatory properties, its balanced formulation, targeted therapeutic actions and safety of use and other components that promote skin health and healing.

[0115] Advantageously, cosmetic compositions are used for the purpose of regenerating and protecting the skin, reducing inflammation and improving its barrier function.

[0116] Advantageously, the cosmetic composition combines active ingredients known for their benefits with plant extracellular vesicles to improve their effectiveness and penetration into the skin.

[0117] 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 the 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, ointments, mouthwashes, oral gels. The pharmaceutical composition according to the present invention can be used for gynecological and dermatological applications. In particular, the plant extracellular vesicles extracted from green leaves from biological agriculture can play a significant role in the prevention, protection and treatment of genital problems due to their beneficial properties for skin health and their anti-inflammatory and soothing action.

[0118] An example of a formulation as a pharmaceutical composition is represented by an ovule for gynecological use. The composition for a single ovule (1000 mg) total includes in volumetric percentages:

[0119] - extracellular vesicles (extracted from green tea leaves from biological agriculture “Camellia Sinensis”): 3.5% - 5.5%,

[0120] Auxiliary Ingredients (indicative percentages):

[0121] - solid semi-synthetic glycerides: 70% - 80%

[0122] - lactic acid: 1% - 3%

[0123] - gelatin: 5% - 15%

[0124] - liquid paraffin: 0.5% - 2%.

[0125] Further experiments were conducted to quantify the absence of theophylline (CAS 58-55-9) in extracellular vesicles (plant-derived extracellular vesicles, PDEVs) obtained from tea leaves, using the I.M. method (Ref. HPLC n.0015). The analysis showed the absence of theophylline in the final product. The HPLC (High-Performance Liquid Chromatography) is a high-precision analytical technique used to identify and quantify chemical compounds in a complex matrix. In our case, this method confirmed that our manufacturing process is able to effectively remove theophylline, a natural alkaloid present in tea with stimulant effects on the nervous system.

[0126] The absence of theophylline in the PDEVs obtained indicates that our method guarantees a purer and safer product, eliminating a molecule that could have contraindications in some applications, while maintaining the functional benefits of extracellular vesicles.

[0127] Further experiments were conducted to quantify the presence of epigallocatechin gallate (EPCG) and hesperidin (Table 1).

[0128] Table 1

[0129] EGCG and hesperidin were quantified in extracellular vesicles (PDEVs) obtained from tea leaves using high-performance liquid chromatography (HPLC), a technique that allows the separation and quantification of compounds based on their chemical and physical properties. This method ensures high specificity and sensitivity, allowing accurate quantification of these flavonoids in complex samples such as plant extracts.

[0130] The presence of bioactive compounds in plant-derived extracellular vesicles represents a growing field of interest for their potential applications in nutraceutical and pharmaceutical fields. In this study, we quantified the presence of epigallocatechin gallate (EGCG) and hesperidin in tea leaf-derived extracellular vesicles (PDEVs), using high-performance liquid chromatography (HPLC). This technique, based on the separation of compounds based on their chemical-physical properties, guarantees high specificity and sensitivity, allowing an accurate quantification of flavonoids in complex matrices such as plant extracts.

[0131] The identification of EGCG and hesperidin in PDEVs is a particularly important result, since it demonstrates that the isolation and purification process of these vesicles allows to preserve and concentrate bioactive metabolites known for their beneficial properties. The ability of vesicles to deliver these flavonoids could improve their stability, bioavailability and biological activity, making them highly promising natural delivery systems.

[0132] EGCG is the main polyphenol in green tea and is widely studied for its antioxidant, anti-inflammatory and neuroprotective properties. Numerous evidences suggest its role in the modulation of oxidative stress, cardiovascular protection and regulation of cellular pathways involved in proliferation and apoptosis, with possible implications in the prevention of neurodegenerative and oncological diseases.

[0133] Hesperidin, a flavonoid widely present in citrus fruits, is known for its vasoprotective, anti-inflammatory and antioxidant properties. Clinical studies highlight its potential in the modulation of endothelial function and in the protection of the microcirculation, with applications in the cardiovascular and dermatological fields.

[0134] Therefore, the detection of EGCG and hesperidin in PDEVs obtained from tea leaves confirms the potential of these extracellular vesicles as natural vehicles for the delivery of bioactive compounds. Their ability to stabilize and transport flavonoids opens new perspectives for the development of innovative formulations in the nutraceutical, cosmetic and pharmacological fields.

[0135] Although at least one exemplary embodiment has been presented in the summary and detailed descriptions, it should be understood that there are a large number of variations that fall within the scope of the invention. Furthermore, it should be understood that the embodiment or embodiments presented are merely examples that are not intended to limit in any way the scope of the invention or its application or configurations. Rather, the summary and detailed descriptions provide the person skilled in the art with a convenient guide to implement at least one exemplary embodiment, it being understood that numerous variations may be made in the function and assembly of the elements described therein, without departing from the scope of the invention as set forth in the appended claims and their technical-legal equivalents.

Claims

CLAIMS1. Extracellular vesicles (EVs) extracted from green tea leaves from biological agriculture characterized in that said green tea leaves contain antioxidant compounds within the lipid membrane.

2. Extracellular vesicles (EVs) according to claim 1, configured to be obtained by infusing green tea leaves in ultrafiltered water for 24 hours and drying carried out according to the spray drying technique.

3. Extracellular vesicles according to claim 1 or 2, wherein the total concentration of the phenolic compounds is between 0.102 ± 0.005 pM e 84703 ± 2.5 pM for a number of vesicles between 106and 1012.

4. Extracellular vesicles (EVs) according to claim 3, wherein the at least one phenolic compound is Epigallocatechin gallate.

5. Extracellular vesicles (EVs) according to claim 4, wherein the concentration of Epigallocatechin gallate is between 0.85 ± 1.3 nM and 11.2 ± 0.04 nM for a number of vesicles between 106and 1012.

6. Extracellular vesicles (EVs) according to any of the previous claims, further comprising serotonin in a concentration between 4.60 ± 0.01 pg / ml and 801.716 ± 0.001 ng / ml and melatonin in a concentration between 0.4 ± 0.02 pg / ml and 412.132 ± 0.003 ng / ml.

7. Extracellular vesicles (EVs) according to any of the previous claims characterized in that a zeta potential is between -30.49 mV and -28.42 mV.

8. Composition comprising extracellular vesicles (EVs) according to any of the preceding claims, for use as cosmetic9. Composition comprising extracellular vesicles (EVs) according to claim 7, for use as neurocosmetic.

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

11. Composition comprising extracellular vesicles (EVs) according to any of the preceding claims, for use as a medical device.

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