Extracellular vesicles derived from alpine rose and their use

Abstract

Extracellular vesicles (EVs) extracted from alpine rose petals from biological agriculture characterized in that said alpine rose petals contain antioxidant compounds within the lipid membrane.

Description

[0001] EXTRACELLULAR VESICLES DERIVED FROM ALPINE ROSE AND THEIR USE

[0002] DESCRIPTION

[0003] Technical field of the invention

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

[0005] Background art

[0006] As is known, the Alpine rose or mountain rose (Rosa pendulina) is a species of wild rose found in the mountains of central and southern Europe.

[0007] It has a woody, weak stem, with a prostrate shape and a reddish color. It is rare to find thorns along the stem, which, if present, are generally recognized by their straight shape. They are generally very fragile and disappear almost completely on the highest branches. The root is fasciculated, very deep and branched. It is possible to find this rose in sparse woods, pastures and rocky places, at an altitude ranging from 500 to 2600 meters. The flowers have five free petals, bright pink or red-purple in color. The fruit, which is more specifically called rosehip, appears as a large red berry. The fruits of the Rosa pendulina are used throughout Europe and the United States to prepare tea, jellies, jams and syrups. It is interesting to note how the properties of rose hips, which contain vitamins C, A, B1 , B2 and P, are enhanced by intense cold. Due to the abundant presence of these vitamins, rose hips can be used in cases of avitaminosis. In addition, they can be considered excellent tonics. In fact, some of these vitamins promote immune defenses. The simplest way to benefit from these properties is to eat these fruits directly, being careful not to ingest the seeds and fluff that covers them, otherwise it is possible to obtain excellent jams or infusions.

[0008] In particular, on the slopes of the Schmiedthof farm of the Frener family, at an altitude of 1400 meters above sea level, the fresh mountain air nourishes strong and robust plants, while the naturally fertile soil enriches them with healthy elements. In this area the optimal cultivation of the Alpine rose takes place, with particular attention to the lunar and planetary phases, which can have a strengthening effect on the herbs. The Alpine rose is widespread in Europe, Asia, North Africa and naturalized in America and Australia. It appears as a shrub or a small tree with woody stems full of large curved red thorns. The rose essence and aromas used in perfumery, cosmetics and pastry making are extracted from the petals. The petals have astringent properties and the leaves are antidiarrheal, the fruits are rich in vitamin C and are diuretic, sedative, astringent and vermifuge.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] It is also known that extracellular vesicles can act as cosmetics.

[0013] The use of extracellular vesicles derived from Alpine rose petals 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 operating both within the same species and between different species. Vesicles of both plant and animal origin are natural carriers 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.

[0014] In conclusion, extracellular vesicles derived from Alpine rose petals constitutively contain bioactive molecules potentially effective in the treatment of intestinal tract disorders, rheumatism, colds and feverish states. In particular, rose petals on the skin have a soothing and anti-aging action.

[0015] However, the use of extracellular vesicles extracted from Alpine rose petals from biological agriculture and their use as natural carriers of bioactive compounds is not known.

[0016] Summary of the invention

[0017] The object of the present invention is therefore extracellular vesicles extracted from dried Alpine rose petals 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 use and topical gynecological use.

[0018] The present invention uses a selected subpopulation of extracellular vesicles (EV) extracted from Alpine rose petals from biological agriculture that have a diameter with average dimensions ranging between 150 ± 0.5 nm and 190.7 ± 4.6 nm.

[0019] Alpine rose petals are rich in Vitamin C, a powerful antioxidant that counteracts the action of free radicals, strengthens the immune system and increases the production of skin collagen; essential fatty acids, such as linoleic acid, important for skin health, brain function and regulation of inflammation; phytosterols that help reduce LDL cholesterol levels in the blood; pectin that aids digestion and promotes intestinal health; tannins, powerful astringents with anti-inflammatory properties that reduce inflammation and promote wound healing.

[0020] According to the present invention, extracellular vesicles isolated from Alpine rose petals from biological agriculture have been characterized based on their total antioxidant content.

[0021] Therefore, according to a first aspect of the present invention, extracellular vesicles isolated from Alpine rose petals 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 appended independent claim.

[0022] According to a further aspect, the use of such extracellular vesicles as cosmetic and topical gynecological use is defined as specified in the independent use claims.

[0023] The dependent claims outline particular and further advantageous aspects of the invention.

[0024] Brief description of the drawings

[0025] 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:

[0026] - Figure 1 illustrates the graph of the dimensional characterization of extracellular vesicles isolated from Alpine rose petals, according to the present invention;

[0027] - Figure 2 shows the comparison of the in vitro fibroblast cell proliferation between the control, the oxidized cells and after treatment, with extracellular vesicles of Alpine rose and H2O2,

[0028] - Figure 3 shows the graph of the analysis of mitochondrial superoxide anion (graph A) and intracellular ROS (graph B) and the comparison between the fluorescence intensities (in a CTR control, a first C1 and a second C2 vesicle concentration,

[0029] - Figure 4 shows the graph of the analysis of mitochondrial superoxide anion (graph A) and intracellular ROS (graph B) and the comparison between the fluorescence intensities (in a CTR control, a first C1 and a second C2 vesicle concentration.

[0030] Detailed description

[0031] According to the present invention and according to the attached Figures, extracellular vesicles (EVs) were extracted from Alpine rose petals 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 organic 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 methodology by which extracellular vesicles were produced from Alpine rose petals obtained from biological agriculture is the subject of a different patent application No. 102024000006865 by the same applicant and includes the following phases: a. inspecting the plant raw material; b. transferring the plant raw material into plastic food containers and storing it for short periods in the refrigerator at a temperature of +4°C, c. washing the plant raw material, d. preparing the plant 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. keeping the first semi-finished product in the refrigerator, h. carrying out 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. carrying out 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 in plastic or borosilicate glass bottles for a short period of time in the refrigerator at a temperature of +4°C, o. adding a quantity of maltodextrin between 4% and 35% by weight to the mixture of extracellular vesicles, 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] The drying phase p) is carried out according to the spray technique or “Spray Drying” i.e. 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.

[0035] According to the method of the present invention, the drying phase q) is carried out according to optimized process parameters, i.e.:

[0036] -’’Drying Gas” Drying gas flow rate (m3 / h) = 30 - 35,

[0037] - ’’Inlet T” Inlet temperature (°C) = 115 - 140,

[0038] - ’’Spray Gas” Atomized gas flow rate (l / h) = 1000 - 1500,

[0039] - ’’Pump 1” Pump flow rate (ml / min): 6 -15,

[0040] - “Outlet T” Outlet temperatures (°C) = 60 - 75.

[0041] To evaluate whether the isolated EVs from Alpine rose petals, extracted from biological agriculture, fell within the size range of extracellular vesicles, the isolated samples were analyzed by the “Bradford assay” (Pierce, Rockford, IL, USA) and the “Nanotracking particle analysis” (NTA). As observed in Figure 1 , isolated EVs show the typical distribution of extracellular vesicles, with average dimensions (diameter) ranging from 150 ± 0.5 nm to 190.7 ± 4.6 nm. EVs were also characterized for their zeta potential. Zeta potential is the electric potential on the slipping plane of nanoparticles that, dispersed in a liquid medium, form a charge on the surface, the so-called double layer.

[0042] This is compensated by adding 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.

[0043] The zeta potential turns out to be the main force of interactions between particles, and 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.

[0044] In the case of extracellular vesicles isolated from Alpine rose petals, EVs have a zeta potential that varies between -31.73 mV and -29.53 mV, demonstrating their great stability in liquid solution.

[0045] The tests carried out were performed in order to characterize the extracellular vesicles isolated from Alpine rose petals for their content of total antioxidants, vitamins and soothing substances.

[0046] In particular, rose petals on the skin have a soothing and anti-aging effect on the skin. Alpine rose petals are rich in Vitamin C, a powerful antioxidant that counteracts the action of free radicals, strengthens the immune system and increases the production of collagen in the skin; essential fatty acids, such as linoleic acid, which is important for skin health, brain function and regulation of inflammation; phytosterols, which help reduce LDL cholesterol levels in the blood; pectin, which aids digestion and promotes intestinal health; tannins, powerful astringents with antiinflammatory properties that reduce inflammation and promote wound healing. 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 Alpine rose petals, a range of vesicles between 106and 1012were analyzed, characterizing a total antioxidant content between 0.248 ± 0.005 pM and 78663 ± 2.5 pM.

[0047] Vitamin C is a very powerful antioxidant that can neutralize the action of free radicals and combat the signs of aging by stimulating collagen production. This vitamin makes the skin bright, evens out skin tone by acting on skin blemishes and compacting the texture. Vitamin C, due to its intrinsic characteristic of powerful antioxidant, is an excellent ally in fighting oxidative damage caused by UV rays.

[0048] The experiments carried out measured the amount of Vitamin C in vesicles isolated from Alpine rose petals, evaluating increasing concentrations of vesicles between 106and 1012and quantified a Vitamin C content between 0.020 ± 0.001 nM and 1.046 ± 0.005 nM.

[0049] For centuries, natural products have been used in skin care as sources of antioxidants capable of eliminating free radicals that damage the skin and accelerate its aging. Reactive oxygen species (ROS) are formed during numerous biochemical processes, but are also generated by other physical factors (sun and pollution). In this redox imbalance, the action of low molecular weight antioxidants, such as vitamin C, is fundamental and plays a significant role in the prevention and repair of skin damage induced by ROS. EVs isolated from rose petals can be used as effective antioxidants, acting against photo / chrono-aging and protecting the skin from damage caused by free radicals produced by the sun and pollution. The product can be used to counteract the onset of expression wrinkles, providing toning, firming and illuminating effects.

[0050] To evaluate the capacity of EVs isolated from Alpine rose petals, fibroblasts in vitro were pretreated with different concentrations of vesicles and then subjected to oxidative stress with H2O2 for two hours (H2O2 concentrations ranging from 250 to 600 pM). After 24-48 h, cell proliferation was measured. As can be seen in Figure 2, pretreatment with Alpine rose EVs effectively limits the damage induced by oxidative stress, restoring cell proliferation to 58% (C1) and 70% (C2) compared to oxidized cells (5%). Furthermore, following treatment with EVs isolated from Alpine rose petals, mitochondrial superoxide anion levels were measured. Mitochondrial superoxide anion is the oxygen free radical mainly produced at mitochondrial level as a by-product of mitochondrial respiration. The anion is very reactive and is quickly transformed into H2O2, which in turn contributes to increasing cellular oxidative stress.

[0051] The experiments evaluated the protective effect of vesicles isolated from Alpine rose petals, treating fibroblasts in vitro first with concentrations of EVs between 106and 1012and subsequently with induction of oxidative stress with H2O2. For the analysis of mitochondrial superoxide anion and intracellular ROS, two fluorimetric kits were used and the fluorescence intensity of the treated samples was analyzed, in comparison with cells subjected only to oxidative stress.

[0052] As shown in the graphs in Figure 3, the analysis of fluorescence intensity allowed to establish that the treatment with EVs isolated from Alpine rose petals can mitigate the harmful effects induced by H2O2 by reducing the levels of mitochondrial superoxide anion by 12% for the C1 dose and by 28% for the C2 dose (Figure 3 graph A). The same trend was also observed for the levels of intracellular ROS, with a reduction of 21% for C1 EVs and 31% after pretreatment with C2 EVs (Figure 3 graph B).

[0053] In addition, the extracellular hyaluronic acid expression was also evaluated, i.e. how EVs isolated from rose petals can regulate the expression of hyaluronic acid, even in the presence of oxidative stress.

[0054] Hyaluronic acid is a fundamental component of human connective tissues and skin. In connective tissues, such as joints and cartilage, it helps keep structures lubricated, while at the skin level it helps maintain the elasticity, softness, hydration of the skin, protecting it from damage induced by external stimuli. Hyaluronic acid is especially known for its hydrating properties; in fact, a molecule of hyaluronic acid is able to bind and retain up to a liter of water. Furthermore, hyaluronic acid is able to soothe irritation and redness, promoting wound healing and slowing down skin aging. It has been shown that oxidative stress induced by exposure to UV rays acts on the stability of extracellular hyaluronic acid, influencing skin texture and aging. In this regard, we evaluated how EVs from rose petals can regulate the expression of hyaluronic acid, even in the presence of oxidative stress. The expression of hyaluronic acid in the cell supernatant was quantified using a commercially available ELISA kit. As can be seen in Figure 4 graph A, in in vitro fibroblasts treated with EVs there is a higher concentration of hyaluronic acid, compared to the untreated control, for both concentrations used (C1 , C2).

[0055] Specifically, an increase in concentration of 191% is observed for the C1 concentration and of 381% for the C2 concentration. In the case of cells subjected to oxidative stress after pretreatment (Figure 4 graph B) the C1 concentration increases the expression of hyaluronic acid by 75% and the C2 concentration by 102%.

[0056] Advantageously, these data support the use of EVs extracted from Alpine rose petals from biological agriculture, in certain cosmetic applications.

[0057] An example of a formulation for cosmetic use is a lotion comprising EVs extracted from Alpine rose petals from biological agriculture.

[0058] The composition per 100 ml of hyaluronic acid lotion for skin hydration includes in volumetric percentages: a. Active phase

[0059] - extracellular vesicles (extracted from Alpine rose petals from biological agriculture “Rosa pendulina L.’’) 0.1% - 5%,

[0060] - preservative (e.g. ecological preservative, preservative compliant with local regulations): according to the supplier's instructions, b. Aqueous phase:

[0061] - demineralized water: 60- 85%

[0062] - vegetable glycerin: 2-7%

[0063] - hyaluronic acid (1% solution): 1-3% c. Oily phase:

[0064] - vegetable oil (e.g. jojoba oil, sweet almond oil): 5-15%

[0065] - emulsifier (e.g. emulsifying wax): 2-10%Additivi:

[0066] - Vitamin E (tocopherol): 0.1-2%

[0067] - Perfume or essential oils (optional): according to personal preference.

[0068] Extracellular vesicles extracted from Alpine rose petals are known for their moisturizing and nourishing properties for the skin; in addition, they can effectively reach the deeper layers of the skin, where they can moisturize and nourish more effectively.

[0069] These vesicles can reduce transepidermal water loss as they form a protective layer on the surface of the skin, helping to reduce transepidermal water loss. This helps to keep the skin hydrated by locking moisture within the skin layers.

[0070] Another example of a cosmetic formulation is a lubricating gel for topical gynecological use. In this composition, the extracellular vesicles extracted from Alpine rose petals from biological agriculture, contained in the formulation, act synergistically to provide deep hydration and protection of the delicate skin of the intimate areas.

[0071] The composition per 100 ml of total gel includes in volumetric percentages:

[0072] - extracellular vesicles (extracted from Alpine rose petals “Rosa pendulina L.” from biological agriculture): 1 % - 2%

[0073] - hyaluronic acid: 1 % - 3%

[0074] - glycerin: 3% - 7%

[0075] - carbopol: 0.5% - 1.5%

[0076] - triethanolamine: 0.3% - 0.7% - preservatives: 0.3% - 0.7%

[0077] - purified water: q.s. to 100%.

[0078] Hyaluronic acid is an effective moisturizer that can retain moisture in the skin, keeping it hydrated and reducing the appearance of fine lines and wrinkles, helps protect the skin from damage caused by free radicals and UV rays, and helps keep the skin looking young and healthy, reducing the risk of premature aging damage.

[0079] Advantageously, the cosmetic compositions are used for the purpose of regenerating and protecting the skin, reducing inflammation and improving its barrier function.

[0080] Advantageously, the cosmetic composition combines active ingredients known for their benefits with plant extracellular vesicles to improve their effectiveness and penetration into the skin.

[0081] Although at least one exemplary embodiment has been presented in the summary and detailed description, 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 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

CLAIMS1. Extracellular vesicles (EVs) extracted from alpine rose petals from biological agriculture characterized in that said alpine rose petals 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 0.248 ± 0.005 pM and 78663 ± 2.5 pM for a number of vesicles between 106and 1012.

3. Extracellular vesicles (EVs) according to claim 1 or 2, wherein an antioxidant compound is Vitamin C.

4. Extracellular vesicles (EVs) according to claim 3, wherein the concentration of Vitamin C is between 0.020 ± 0.001 nM and 1.046 ± 0.005 nM for a number of vesicles between 106and 1012.

5. Extracellular vesicles (EVs) according to any of the previous claims characterized in that a zeta potential is between -31.73 mV and -29.53 mV.

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

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

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