Cosmetic composition comprising a vesicle concentrate comprising an aqueous solution of lipid vesicles for use in cosmetic products, cosmetic products comprising said cosmetic composition and methods for producing same

Abstract

The cosmetic composition comprises a vesicle concentrate consisting of an aqueous solution of vegetable lipid vesicles or lipid vesicles of plant origin having walls containing phospholipids, phytosphingosine, phytosterols, β-phytosteryl sulfate and optionally other derivatives of phytosterols, and ceramides and / or pseudoceramides, for use in the formulation of cosmetic products for the care of the skin and hair, and for decorative purposes for the face, eyes, eyelashes, lips and body. The vesicle concentrate may also contain lysophospholipids, esters of phytosterols, fatty acids, moisturizers, emollients, active lipophilic molecules, active hydrophilic molecules, thickeners and film-forming molecules. A cosmetic product comprises the vesicle concentrate cosmetic composition and other cosmetically acceptable ingredients.

Description

[Technical field]

[0001] The present invention relates to cosmetic compositions comprising concentrates of biomimetic lipid vesicles that are used to prepare various types of cosmetic products.

[0002] Said cosmetic products are skin and hair care products as well as decorative products for the face, eyes, eyelashes, lips and body.

[0003] More specifically, the present invention relates to cosmetic products comprising a concentrate of complex colloidal lipid vesicles (liposomes) containing active ingredients, the wall structure of said vesicles being composed of phospholipids, phytosphingosines, phytosterols, β-phytosteryl sulfate and other phytosterol derivatives, ceramides and / or pseudoceramides (synthetic molecules that mimic the structure of natural ceramides), with the optional addition of fatty acids, moisturizers, emollients, etc., and with the optional presence of active lipophilic molecules.

[0004] Furthermore, the present invention provides an original solution to the problem of preparing vesicles (liposomes) and their incorporation in a stable form into cosmetic formulations. [Background technology]

[0005] As is known, vesicles (or liposomes) are supramolecular structures composed of phospholipids, found everywhere in biological systems, which, due to their amphiphilic structure, self-assemble in an aqueous environment according to a bilayer wall structure in which the hydrophilic parts are in contact with the aqueous environment, while the hydrophobic parts are hidden inside the bilayer.

[0006] In vivo, these vesicles are made of a supramolecular shell or membrane consisting of a simple bilayer of phospholipids and have the functions of secretion (exocytosis), transport, and internalization (endocytosis) of hydrophilic compounds, and play an important role in intercellular communication.

[0007] Moreover, it is known that in vivo, vesicles are able to embed within their membrane a variety of compounds that find a suitable environment within the bilayer, as well as membrane proteins and other lipophilic substances.

[0008] Biomimetic lipid vesicles can be produced using synthetic phospholipids or phospholipids obtained by purifying phospholipids extracted from plant or animal sources.

[0009] It is known that the moisture permeability and mechanical stability of vesicles depend on the composition of the lipid mixture forming the membrane, and said properties can be tuned by using lipids with different degrees of unsaturation or by inserting sterols into the membrane.

[0010] Biomimetic lipid vesicles are microstructures that contain one lipid bilayer or multiple lipid bilayers arranged in concentric or tubular configurations. In the literature, various amphiphilic molecules have been used to prepare liposomes, and methods for their tailoring can be employed to control the size and morphology of the liposomes. Active molecules are either encapsulated in the aqueous space or inserted into the lipid bilayer, but the exact location of these active molecules within the vesicle depends on the physicochemical structure and lipid composition. In the literature, biomimetic lipid vesicles have demonstrated great potential as a system to release active ingredients for personal care. A wide range of molecules, including peptides and proteins, have been incorporated into biomimetic colloidal lipid vesicles through various preparation systems. Due to their high biocompatibility, these molecules are one of the most efficient systems to facilitate the delivery of active ingredients to the skin and skin treatments.

[0011] In synthetic vesicles, the bilayer organization can be single (so-called SUV / LUV: Small / Large Unilamellar Vesicles) or multiple (SMV / LMV: Small / Large Multilamellar Vesicles), forming large and small unilamellar or multilamellar lamellae, with more or less thick shells and large or small sizes, respectively.

[0012] The size of synthetic lipid vesicles depends on the method of their production. The state of the art offers different methods to produce synthetic lipid vesicles, mainly using ultrasound, extrusion through porous membranes, hydration of thin phospholipid films or microfluidic techniques. Although these methods do not allow for high density vesicles, they allow precise sizing of the vesicles, making them the method of choice in academic and pharmaceutical fields.

[0013] Particularly in the pharmaceutical and biological fields, liposomes are of great importance for their ability to release their contents into cells by tissue penetration. Inside the cells, the active principles contained in the vesicles can exert their pharmacological action. Tissue penetration is facilitated through the manufacture of liposomes with nanometric sizes (diameter <100 nm), so that such formulations are extremely precise and tailored for such purposes. They are administered in controlled scenarios and undergo rigorous testing. In fact, they are either injectable formulations (as in the case of so-called mRNA vaccines) or are used for in vitro internalization of genetic material, through interaction with the cell membrane.

[0014] Today, consumers tend to prefer personal care products that make daily life healthier and more comfortable, improve social relations and contribute to personal well-being. For these reasons, today the quality of new products for personal care is becoming increasingly higher with the introduction of new sophisticated and functional ingredients. There is a particularly strong trend towards the formulation of products that take into account environmental sustainability and the renewable nature of raw materials. In this respect, plant-based ingredients and compounds taken from the plant kingdom are particularly popular.

[0015] This consumer need and new scientific knowledge led the Applicant to research and develop an innovative project for formulating cosmetic products containing biomimetic vesicles.

[0016] The cosmetics industry is interested in vesicle systems due to the fact that, similar to what occurs in the body, they can act as efficient carriers for hydrophilic and lipophilic active molecules. The literature is known for their ability to encapsulate hydrophilic compounds in the aqueous core and to insert lipophilic compounds into the lipid bilayer shell. Furthermore, it is known that the compounds forming the vesicles / liposomes can be chosen from among biologically derived substances that mimic the lipid components present in the epidermis. They are able to integrate and improve the barrier properties of the epidermis against external factors and dehydration.

[0017] The properties of encapsulating active ingredients can be exploited in the creation of personal care products, especially skin care products. Indeed, products are offered on the market and described in the literature that feature the inclusion of liposomal vesicles in face creams and other topical products. Such products contain, among others, phospholipids, ceramides, fatty acids and sterols, which are essential ingredients for the preparation of liposomes and have different functions, but the presence and integrity of the vesicle structure in cosmetics has not been demonstrated. The preparation of vesicles is a process that requires a perfect control of their composition, the temperature to which the phospholipid layer is exposed and the stirring intensity, so that the in situ formation of vesicles in the final composition is far from guaranteed. In a market environment where the desired properties of cosmetics and personal care products, especially luxury products, must be demonstrated and supported by scientific evidence, companies need to be provided with methods and technologies that allow them to realize such demands.

[0018] In fact, the vesicular structure is maintained by weak supramolecular interactions and can be easily disrupted by processing energy, temperature, and the use of emulsifiers and solvents, resulting in various multiphase systems, such as oil-in-water emulsions.

[0019] The cosmetic scenario differs from that of the pharmaceutical sector, since it requires the formulation of many ingredients, often combined with a perfect understanding of heterogeneous production processes, resulting in a pleasant and at the same time functional texture. Moreover, methods described in the scientific and patent literature are suitable for the production of nanometer-sized (<100 nm) liposomes and vesicles, characterized by improved in vivo tissue penetration. Such a feature is counterproductive in the dermocosmetics sector, where structures deliberately produced with a size below 100 nm are considered nanomaterials, an undesirable feature that often causes anxiety for users.

[0020] Cosmetic formulators must deal with a wide variety of formulations, while producing products containing intact liposomes / vesicles poses challenges.

[0021] For example, the use of traditional secondary emulsifiers mixed into the vesicles is incompatible with the phospholipid system, destroying its order and compromising its efficacy. To provide the final product with optimal characteristics, such as ease of use and sensory comfort, one possible formulation strategy is to insert the vesicles into a polymer gel. Such a composite structure has the unique feature of improving the stability of the vesicles in the polymer system and making them more resistant than free vesicles in solution. Furthermore, when the active molecules are placed in the core of the vesicles and the vesicles are contained in the gel web, both the web and the bilayer optimally maintain the different active ingredients during transport. Finally, the controlled release of the active ingredient over time minimizes the problem of "burst release", i.e. overdosing of the active ingredient due to too rapid release.

[0022] Korean Patent Publication No. 101440383 describes a cosmetic composition containing osmosels in the form of vesicles, which contain a lipid complex of squalene and a human adipose stem cell culture extract. The osmosels are obtained by emulsifying squalene, phospholipids, phytospingosine, ceramide, cholesterol, phytosterol, and β-sitosterol at high pressure to form a gel-type lipid complex. By adding this complex to an aqueous solution containing the extract at room temperature, vesicles containing the extract complexed with squalene in a closed bilayer structure are obtained. The osmosels essentially contain a lipid composition that forms a cell membrane prepared to stabilize the heat-labile human adipose stem cell culture extract and facilitate its percutaneous absorption (paragraph 0026). According to this document, the osmosels are characterized by a size in the range of 20-500 nm, preferably 50-250 nm, most preferably 100-200 nm, with an average of 120 nm. According to the literature, when the average droplet size of osmocell exceeds 500 nm, the skin permeability and formulation stability become very low (paragraph 0029). [Prior art documents] [Patent documents]

[0023] [Patent Document 1] Korean Patent Publication No. 101440383 Summary of the Invention [Problem to be solved by the invention]

[0024] The objective of the present invention is to produce cosmetic products containing biomimetic vesicles that function in skin care, with maintained vesicle integrity and demonstrated beneficial activity on epidermal health. [Means for solving the problem]

[0025] The above object is achieved by making a cosmetic composition comprising a vesicle concentrate as defined in claim 1 and by using said cosmetic composition comprising said vesicle concentrate in the formulation of a cosmetic product as defined in claim 13.

[0026] The vesicles are specifically formulated in a processing step that combines and processes only the ingredients necessary for vesicle preparation according to a manner that ensures vesicle formation in high yield and concentration, and is subsequently mixed with other cosmetically acceptable and compatible ingredients through a process suitable for preserving the vesicles' properties.

[0027] The walls of the vesicles are preferably composed of phospholipids (including but not limited to phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylinositol phosphate, phosphatidylinositol diphosphate, phosphatidylinositol triphosphate, etc., each with varying degrees of saturation / unsaturation), monoacyl derivatives of phospholipids (including but not limited to monoacylphosphatidylcholine), steroid derivatives (including but not limited to phytosterol, β-sitosterol, phytosteryl isostearate, β-phytosteryl sulfate, etc.), ceramides and analogs (sphingosine acylates and / or "pseudoceramides"), sphingosine and analogs, aliphatic oils (including but not limited to squalane), fatty acids (including but not limited to oleic acid), fatty alcohols (including but not limited to octyldodecanol), antioxidants, preservatives, and, optionally, active lipophilic molecules. The fatty alcohol and steroid components may optionally be modified with silane functional groups, including but not limited to triethoxysilane functional groups.

[0028] The process used to obtain the cosmetic product according to the invention is defined in claim 15.

[0029] In particular, the vesicle concentrate of the cosmetic composition according to the invention can be obtained by molten (liquid crystalline) vesicle components (phase A) in a plant-derived moisturizer (including, but not limited to, glycerol, diglycerol, isosorbide, butylene glycol) to obtain a homogeneous single-phase system, and introducing an aqueous phase to said system at a temperature higher than the solidification temperature of the lipid phase, thereby organizing the liquid crystalline lipid phase into a multilamellar vesicle structure.

[0030] The active ingredients that can be incorporated into the vesicle concentrate (phase B) are preferably of plant origin and include stearyl glycyrrhetinic acid, Magnolia acuminata bark extract, Betula alba bark extract, Centella asiatica leaf extract, Jania rubens extract, tocopherol acetate, hydroxypinacolone retinoate, linolenic and linoleic acid, ascorbyl tetraisopalmitate, 3-o-ethyl ascorbic acid, niacinamide, fructooligosaccharides, Beta vulgaris (beet) root extract, tranexamic acid, Sophora japonica leaf extract, hexylresorcinol, undecylenoyl phenylalanine, salicylic acid, Salix alba bark extract, zinc pyrithione, Brassica napus flower extract, Candida bombyx mori extract, glyceryl stearate ... These include, but are not limited to, kola / canola oil ferment extract, undecylenic acid and derivatives, climbazole, quillaja saponaria bark extract, cypress fruit extract, burdock root extract, 10-hydroxydecanoic acid, sebacic acid, zinc PCA, caprylic acid, fig fruit / leaf extract, nettle extract, hydrolyzed yeast protein, sausage tree fruit extract, ginkgo biloba leaf extract, basil hairy root culture extract, caffeine, neolascogenin, lascogenin, betaine, oat, zanthoxylum piperitum fruit extract, rapeseed sterols, willow herb flower / leaf / stem extract, hydrogenated starch hydrolysate, ascophyllum nodosum extract, asparagopsis armata extract.

[0031] The concentration of the lipid component of the vesicles may range from 15 to 40% by weight of the vesicle concentrate.

[0032] The aqueous phase comprises water and may optionally contain humectants (including, but not limited to, glycerol, diglycerol, isosorbide, butylene glycol), salts, preservatives, acidity regulators (including, but not limited to, lactic acid, citric acid, sodium hydroxide, arginine), rheology modifiers, hydrophilic actives, and other hydrophilic cosmetically acceptable compounds.

[0033] The system is then brought below the solidification temperature of the lipid phase, resulting in a pasty textured material with rheological properties of a pseudoplastic fluid (shear thinning, i.e. becoming more fluid under the action of increasing shear rate) or a viscoelastic solid (at low strain the elastic modulus is higher than the viscous modulus, at high strain these moduli are reversed). The size distribution of the vesicles thus prepared ranges from 0.6 to 1.0 microns, with an average size of 0.8 to 1 micron (measured by laser diffraction), depending on the intensity of the shear forces applied during the manufacturing process of the concentrate.

[0034] With particular reference to Example 1 and the following examples, the vesicle concentrate can be obtained by feeding the ingredients of phase A one at a time into a jacketed mixer equipped with a turbine (rotor-stator), blades for room temperature mechanical stirring, and wipers. The mixture is then mechanically stirred at room temperature until a homogeneous and smooth dispersion of the ingredients is obtained. Phase A is gradually heated to 80-100°C in 10°C increments over a period of 1 hour to melt the solid ingredients, resulting in a paste-like texture and a slightly yellowish translucent dispersion. Phase B is then gradually added to the mixer while keeping the system at a constant temperature, while Phase A is brought to 60°C with continued mechanical stirring. After the addition of Phase B is complete, the composition is kept stirred for 30 minutes. Finally, turbulent stirring is applied using a turbine and the temperature is gradually reduced to room temperature to obtain a paste-like concentrate that is white / yellowish in appearance. The vesicle concentrate thus obtained can be diluted with water and other ingredients to obtain the formulations of Example 4 and the following examples.

[0035] Cosmetic products are obtained by combining the vesicle concentrate with water and hydrophilic ingredients. The hydrophilic ingredients include, for example, humectants (including but not limited to glycerol, diglycerol, isosorbide, butylene glycol), salts, preservatives, acidity regulators (including but not limited to lactic acid, citric acid, sodium hydroxide, arginine), rheology modifiers (including but not limited to polyquaternium-37, polyacrylates, polysaccharides, xanthan gum, bentonite), hydrophilic actives, and other cosmetically acceptable hydrophilic compounds. In addition, the formulation may optionally contain cosmetically acceptable lipophilic ingredients such as oils, waxes, silicones, and derivatives. The concentrate is included in the cosmetic product in a proportion of 0.1 to 99.99% by weight of the final product and is mixed with other cosmetic ingredients under conditions that do not damage the integrity of the vesicles.

[0036] In summary, the method for obtaining a cosmetic product according to claim 13 or 14 comprises the steps of: preparing plant vesicles or plant-derived biomimetic vesicles of size between 0.6 and 1.0 microns, the walls of which consist of a lipid phase containing the above components, by carrying out the following sub-steps: o preparing a composition containing a vesicle concentrate by melting the walls of said vesicles to a liquid crystalline state so as to obtain a homogeneous single-phase system; o introducing an aqueous phase into said vesicle concentrate composition at a temperature above the freezing temperature of the lipid phase of the vesicles; o cooling the vesicle concentrate composition at a temperature below the solidification temperature of the lipid phase of the vesicles to obtain a paste-like material having the rheological properties of a pseudoplastic fluid or a viscoelastic solid and a vesicle size distribution of 0.6 to 1.0 microns; and mixing the paste-like textured vesicle concentrate composition with water and other cosmetic ingredients to obtain a final cosmetic product containing the vesicle concentrate in a desired weight percent.

[0037] It is important to point out that the compositions prepared according to the present invention preferably contain only raw materials of plant origin.

[0038] Phytosterol is a general term for cholesterol-like molecules derived from plants. This category includes beta-sitosterol sulfate, which is chemically identical to cholesterol sulfate of animal origin, and the ester phytosterol isostearate.

[0039] The use of phytosterols in sulfate form gives the compositions of the invention different characteristics than simple cholesterol of animal or plant origin. For more information, see the review by Strott and Higashi, "Cholesterol Sulfate and Human Physiology: The Full Story - Journal of Lipid Research, 2003(44):1268-1278." In this review, it is explained that cholesterol sulfate is a component of cell membranes and is found in human plasma. Surprisingly, cholesterol sulfate exhibits a regulatory function, which suggests an analogy with compounds classified as hormones. In particular, cholesterol sulfate plays a role in the differentiation of keratinocytes and the formation of the epidermal barrier. Cholesterol sulfate is thought to play an important role in the proper functioning of the stratum corneum and the normal delamination of dead cells. A decrease in the cholesterol sulfate content in the stratum corneum reduces the stability of the bilayer and the proportion of lipids arranged in a liquid lateral packing. This results in a decrease in the elasticity of the lipid phase, which prevents the proper orientation of the lipid lamellae of the corneocytes. Cholesterol sulfate has been demonstrated to have a regulatory effect on the transcriptional activity of specific keratinocyte genes.

[0040] As explained in the introduction, a technique of using liposomes to transport active molecules and facilitate their penetration into cells is also known, as disclosed in Korean Patent Publication No. 101440383, which discloses an osmocell containing a human adipose stem cell medium extract.

[0041] Surprisingly, it was found that an improvement in the skin condition was observed as early as 6 hours after application of the cream according to the invention (see comments in FIG. 9), which is in contradiction to the action of the active ingredient contained in the vesicles. It is also interesting to note that the moisturizing properties return to the initial values ​​one week after the application of the cream according to the invention has ceased (see points 5.1.2 and 5.1.3).

[0042] These observations can only be explained by the consideration that the vesicle concentrates (and formulations containing them in different proportions) allow the deposition of a continuous film of non-volatile components (lipid shell of the vesicles) whose lateral size is in the order of microns. Such evidence is substantiated by sensory observations on the texture of the product (feel of a film that softens and nourishes the skin) and by instrumental observations (transmission electron microscope (TEM) images of vesicles after deposition and evaporation of the aqueous components, see figures 16 and 17). The vesicle structure is maintained in the presence of active ingredients, and the film-forming and nourishing abilities are maintained even in the presence of active ingredients, allowing an immediate effect observable within a few hours. In addition to the short-term film effect, long-term effects can be obtained by repeated exposure to specific active ingredients that improve the functionality of the concentrates, on a time scale that coincides with changes in cellular metabolism (2-4 weeks in clinical trials).

[0043] The present invention further relates to the formulation of an aqueous cosmetic product for decorative application to the face, eyes, eyelashes, lips and body, comprising said vesicle concentrate and cosmetically acceptable pigments.

[0044] Finally, the invention relates to the manufacture of a cosmetic powder obtained by combining the vesicle concentrate with a powder and removing the aqueous phase in an additional evaporation process.

[0045] One of the main functions of the skin is to protect the organism from environmental factors (e.g. UV radiation, microorganisms, physical and chemical agents). The entire surface of our skin is covered by a basal lipid layer that forms a protective layer. This lipid layer is called the cutaneous sebum film, is composed mainly of lipids (95%) and has an acidic pH (4.9).

[0046] The integrity of the sebum film is of paramount importance for maintaining soft and moisturized skin, protecting the body from external microorganisms, reducing the risk of infection, and regulating the absorption and excretion of substances through the skin.

[0047] In fact, said lipids ensure water dispersion reduction, electrolyte dispersion reduction, natural defense against microorganisms and binding action for keratinocytes.

[0048] The efficacy of formulations containing vesicle concentrates has been demonstrated in the areas of skin barrier strengthening, skin rejuvenation, anti-aging (or pro-aging) action, increased skin moisture retention, immediate results, improved skin elasticity and firmness, reduced appearance of wrinkles, promotion and increase of collagen I synthesis, promotion and increase of aquaporin 3 channel activity and retinol-like activity.

[0049] The efficacy is demonstrated in the formulation examples of vesicle concentrates and derived cosmetic products disclosed at the end of this specification. [Brief description of the drawings]

[0050] [Figure 1] Figure 1 shows images obtained by transmission electron microscopy (TEM) of the vesicle concentrate of Example 1. These images show the multilamellar structure and the characteristic size of the vesicle structures produced according to the invention (left side: scale bar 500 nm). [Diagram 2] Figure 2 shows images obtained by transmission electron microscopy (TEM) of the vesicle concentrate of Example 1. These images show the multilamellar structure and the characteristic size of the vesicle structures produced according to the invention (left side: scale bar 200 nm). [Diagram 3] FIG. 3 shows an image of intact vesicles for the vesicle concentrate of Example 2, obtained by transmission electron microscopy of frozen samples (cryo-TEM). [Figure 4] Figure 4 shows an image obtained by transmission electron microscopy of frozen samples (cryo-TEM) for the cream formulation of Example 4 containing 10% by weight of the vesicle concentrate of Example 2. The vesicle structure is maintained. [Diagram 5] Figure 5 is a graph showing the relationship between type I collagen production and in vitro evaluation in cell culture. [Figure 6] The left panel of Figure 6 is a chart showing the percentage increase in collagen type I production relative to the control blank sample (set at 100%) and the positive control TGFβ, and the right panel of Figure 6 is an immunofluorescence image of a skin tissue section, showing that the majority of newly formed collagen type I is localized in the upper layers of the dermis close to the epidermis. [Figure 7] FIG. 7 is a graphical representation showing the percentage increase in AQP3 compared to blank control (100%) and bactiol as a positive control (***=p-value −0.001%). [Figure 8] The left panel of Figure 8 is a graph showing the percentage increase in AQP3 compared to a control blank (set at 100%) and two positive controls (bactiol and retinoic acid), and the right panel of Figure 8 is an immunohistofluorescence image of a section of a skin tissue slice where the AQP3 water channel is localized. [Figure 9] FIG. 9 is a graphical representation of skin moisturization values ​​measured in treated and untreated areas at different time points, expressed as percentage change compared to the respective basal values ​​at time 0. [Figure 10] FIG. 10 is a graph showing the skin elasticity parameter R0 and the skin resilience R2. [Figure 11] FIG. 11 is a graphical representation showing the mean percentage change in parameter R0 (compactness) after 2 and 4 consecutive weeks (t2weeks, t4weeks) of twice daily application of the face cream being evaluated in the treated area (face) relative to the initial value before treatment (baseline, tb) compared with the similar value at the same measurement time point in the untreated area (forearm). [Figure 12] FIG. 12 is a graph showing the mean percent change in parameter R2 (skin elasticity) from the baseline value (tb) before treatment at treated areas on the face after 2 and 4 consecutive weeks (t2weeks, t4weeks) of daily application (twice a day) of the test product, compared with the similar value at the untreated control area (forearm). [Figure 13]FIG. 13 shows photographs of the same facial areas of volunteer n.1 and volunteer n.2 taken tb (before treatment), 2 weeks and 4 weeks after product use. [Figure 14] FIG. 14 is a graphical representation of the mean percentage change in wrinkle visibility index in treated facial areas after 2 consecutive weeks and 4 consecutive weeks of product application (twice daily) versus baseline (tb). [Figure 15] FIG. 15 is a graphical representation showing the percentage recovery of TEWL values ​​after injury (skin stripping) with different vesicle concentrate-containing formulations and placebo versus injured skin not treated with a product. [Figure 16] Figure 16 shows what happens to the skin after applying a cream containing vesicle concentrate, the first image on the right side of Figure 16 is an image showing that during application, the vesicles are separated by the moisture in the cream. The second image on the right side of Figure 16 is an image showing that after natural evaporation of the moisture, the vesicles flatten out and form a protective layer. The two photographs on the left side of Figure 16, cryo-TEM and TEM, show its effect on the skin. [Figure 17] FIG. 17 is a diagram illustrating the advantages of micrometer-sized vesicles (left side) over nanometer-sized vesicles (right side). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Figures 1 and 2 show images obtained by transmission electron microscopy (TEM) of the vesicle concentrate of Example 1. These images show the multilamellar structure and the characteristic size of the vesicle structures produced according to the invention (left: scale bar 500 nm, right: scale bar 200 nm).

[0052] FIG. 3 shows an image of intact vesicles for the vesicle concentrate of Example 2, obtained by transmission electron microscopy of frozen samples (cryo-TEM).

[0053] Figure 4 shows an image obtained by transmission electron microscopy of frozen samples (cryo-TEM) for the cream formulation of Example 4 containing 10% by weight of the vesicle concentrate of Example 2. The vesicle structure is maintained.

[0054] In particular, experimental tests were performed on Example 1, as summarized below.

[0055] 1) Evaluation of the efficacy of the vesicle concentrate according to Example 1 The efficacy of the vesicle concentrate according to Example 1 was demonstrated by an extensive testing strategy using a fairly comprehensive set of in vitro, ex vivo and in vivo (clinical trial) tests.

[0056] Activity of vesicle concentrates According to observations, the vesicle concentrate according to Example 1, when used in cosmetic formulations, appears to be effective in the following respects: -Strengthen skin barrier -Protection and repair of the human skin barrier -Skin rejuvenation -Anti-age / pro-age activity -Immediate increase in skin moisturization -Improve skin elasticity and firmness in a short time - Reduces the visibility of wrinkles -Improvement of overall skin condition -General noticeable immediate effect -Highly effective retinol-like activity

[0057] Benefits of Vesicle Concentrate To demonstrate the efficacy of the vesicle concentrate according to Example 1, specific indicators related to skin barrier and skin rejuvenation activity were used.

[0058] Efficacy related to skin barrier / regeneration / rejuvenation activity For in vitro studies in cell cultures and ex vivo studies in human skin explants, different indicators were chosen: aquaporin 3 (AQP3) production and procollagen I synthesis.

[0059] Evaluation of non-cytotoxic concentrations for use in cell culture: MTT for keratinocytes (HaCaT) Preliminary cytotoxicity tests (MTT assay on HaCaT cells treated with increasing concentrations of vesicle concentrates for 48 h) were performed to select the maximum non-cytotoxic doses of vesicle concentrates (0.011% and 0.017%) to be used in the following in vitro studies.

[0060] Type I collagen Collagen is the major structural protein in the extracellular matrix and is present in various connective tissues of animals. Type I collagen is one of the most representative (most abundant) collagens in the dermis and is an essential "scaffolding" protein that gives strength and structure to the skin.

[0061] Collagen I production decreases with age, resulting in a loss of skin smoothness and elasticity and increased wrinkle formation.

[0062] Procollagen is a precursor of collagen. Type I procollagen is a new collagen synthesized in the dermis. Increasing collagen synthesis prevents skin aging and improves skin health.

[0063] Production of collagen type I – in vitro evaluation in cell cultures using two different concentrations of the vesicle concentrate according to Example 1

[0064] Example 1 In this test, the synthesis of new collagen I was monitored through the quantification of type I procollagen using a specific antibody by enzyme-linked immunosorbent assay (ELISA) test in cell cultures made from a monolayer of normal human dermal fibroblasts (NHDFs).

[0065] Two aqueous dispersions containing 0.01% and 0.017% of the vesicle concentrate according to Example 1 were applied to the culture medium for 24 hours. The peptide TGFβ (transforming growth factor beta 1), a natural growth factor involved in tissue repair, was used as a positive control. Three independent experiments were carried out in triplicate each.

[0066] result As shown in Figure 5, the vesicle concentrate significantly increased the synthesis of procollagen I by 20% in both tested dilutions (*=p-value 0.05, **=p-value 0.01, ***=p-value 0.001), indicating the generation of type I collagen, especially its in vitro evaluation in cell culture.

[0067] Type I collagen production – ex vivo evaluation in cell cultures using three different concentrations of the vesicle concentrate according to Example 1 To determine the effect of the vesicle concentrate in promoting collagen type I synthesis, biopsies taken from human skin tissue explants were treated with three different concentrations of the vesicle concentrate in glycerol (1%, 5%, and 20%). TGFβ (transforming growth factor beta 1), a natural factor involved in tissue regeneration, was used as a positive control, while control samples contained glycerol only.

[0068] The abundance of collagen type I was detected by immunohistofluorescence staining, and the intensity of fluorescence was calculated by ImageJ® software. Three independent experiments were performed in triplicate.

[0069] result As shown in Figure 6 (* = p-value 0.05, ** = p-value 0.01), the vesicle concentrate, even at the lowest concentration tested (1%), increased collagen I production by 40%, an effect that was not dose-dependent and comparable to the results with the positive control.

[0070] In FIG. Left: Chart showing the percentage increase in collagen type I production relative to the control blank sample (set at 100%) and the positive control TGFβ. Right: Immunofluorescence image of a skin tissue section showing the majority of newly formed type I collagen localized in the upper layers of the dermis close to the epidermis.

[0071] Thus, by monitoring the production of type I procollagen both in vitro and ex vivo, we demonstrated that vesicle concentrates, even at low concentrations, significantly stimulate new collagen synthesis.

[0072] Aquaporin 3 (AQP3) Aquaporin 3 (AQP3) is the protein product of the human gene AQP3. It is expressed in many tissues, including skin, and functions as a membrane transporter of water and glycerol. Aquaporin 3 (AQP3) is one of the most important pathways for water and glycerol in the epidermis, regulating skin moisturization to maintain a good healthy balance in the skin.

[0073] Aquaporins (AQPs) are essential for maintaining water homeostasis. Aquaporin 3 channels (AQP3) are highly expressed in the basal layer of normal skin keratinocytes and are the most important passive transporters of water and glycerol in the epidermis, regulating skin moisture balance and maintaining good skin health and functionality (e.g., elasticity). A significant decrease in dermal water content and skin elasticity was observed in mice in which AQP3 expression was suppressed.

[0074] AQP3 therefore plays an important role in maintaining skin moisture and proper function.

[0075] Production of AQP3 - in vitro evaluation in cell culture using vesicle concentrates according to example 1 at two different concentrations. In this study, the efficacy of the vesicle concentrate in increasing the expression of aquaporin 3 was evaluated against the known active ingredient bakuchiol, which was used as a positive control.

[0076] Bactiol is known as a natural active ingredient with many cosmetic properties (anti-wrinkle, anti-acne, promotes elastin production, reduces dark spots), making it a safer natural alternative to retinol with similar anti-aging effects. In this study, bactiol was chosen as the positive control due to its ability to promote the synthesis of AQPS, as shown in the literature (DOI:10.1111 / ICS.12117).

[0077] Two aqueous dispersions of vesicle concentrate (0.011% and 0.017%) were applied for 24 hours to cell cultures made from a monolayer of human keratinocytes (Human Keratinocytes-HaCaT).

[0078] The amount of expressed AQPS was evaluated via specific antibodies using ELISA plates. Three independent experiments were performed in triplicate each. The results were calculated as the average.

[0079] result The vesicle concentrate (Figure 7) showed excellent activity in increasing AQP3 at both dilutions tested (0.010% and 0.017%) in the dose-response model, with effects very similar to those of bactiol at the commonly used concentration.

[0080] The histogram shows the percentage increase of AQP3 compared to the blank control (100%) and bactiol as a positive control (***=p-value -0.001%).

[0081] Production of AQP3 - Ex vivo evaluation in cell culture using three different concentrations of the vesicle concentrate according to Example 1. To confirm the promotion of AQP3 by the vesicle concentrate resulting from the in vitro studies described above, biopsies from human tissue explants were treated by topical application of three different concentrations of the vesicle concentrate (Example 1) in glycerol (1%, 5%, 20%). The expression of AQPS was detected by immunohistofluorescence staining. Three independent experiments were performed in triplicate each and the results were calculated as the average. Retinol and bactiol, known activators of AQP3, were selected as positive controls.

[0082] result The graphs in Figure 8 (* = p value p 0.05, ** = p value 0.01, *** = p value 0.001) show the intensity of fluorescence detected by ImageJ® software. All tested concentrations of vesicle concentrates significantly increased the production of AQP3 channels. More interestingly, the increase in AQP3 by 5% vesicle concentrate was more than three times higher than that of retinol and almost two times higher than that of bactiol.

[0083] Figure 8: Left: The graph shows the percentage increase of AQP3 compared to the control blank (set at 100%) and two positive controls (bactiol and retinoic acid). Right: Immunohistofluorescence image of a section of skin tissue section where AQP3 water channels are localized.

[0084] From in vitro and ex vivo studies on aquaporin 3 channels, we can infer the following: Vesicle concentrates significantly increased aquaporin 3 channels in in vitro and ex vivo studies. In this parameter, the vesicle concentrate was much more effective than retinol and bactiol, both of which are well-known AQP3 activators.

[0085] Clinical studies of vesicle concentrate systems Based on the excellent results of in vitro and ex vivo experiments, a testing strategy for the clinical evaluation of vesicle concentrates was established.

[0086] Specific research endpoints were selected to evaluate the anti-aging effects of the vesicle concentrate, with a particular focus on efficacy in skin barrier and skin rejuvenation.

[0087] Two different comprehensive studies confirmed the results summarized below. -The first study investigated the functionality of increasing the proportion of vesicle concentrate (Example 1) in the base formula of a face cream. In a second study, multiple vesicle concentrate systems containing different amounts of components were investigated to test the flexibility of the vesicle concentrates to be considered as platforms rather than as cosmetic active compounds created in specific formulations.

[0088] 4.1 Clinical trials with increasing percentage concentrations of the vesicle concentrate of Example 1 In this first study, five basic face cream formulations were prepared by increasing the percentage of vesicle concentrate: 0% (placebo), 1%, 5%, 10%, and 20%. Clinical evaluation studies were conducted on 50 volunteers (ages 40-65, female) divided into five groups of 10, each of which was assigned one of the five formulations.

[0089] In the first session, the product was applied to the volunteers' faces under controlled conditions at a clinical center, after which the volunteers continued to use the product by applying it twice a day (morning and evening) for four consecutive weeks (28 days).

[0090] To evaluate the anti-aging efficacy of the vesicle concentrates, specifically their effect on the skin barrier and their skin regenerating / rejuvenating activity, three different evaluations were selected. 4.1.1 Moisture retention (based on keratinometric evaluation) 4.1.2 Skin elasticity (by skin viscoelasticity measurement) 4.1.3 Wrinkle visibility (based on digital image analysis)

[0091] Instrumental measurements were performed on the face (test sites) and on an untreated site on the forearm (control site) of volunteers treated with the products. Measurements were performed at different time points (short-term: 6 hours after the first application, mid-term: 14 days, long-term: 28 days) according to the scheme in the table below and compared with the baseline values ​​obtained at time 0.

[0092] [Table 1]

[0093] Measurements after 2 and 4 weeks were taken approximately 4 hours after the last application of product.

[0094] Considering the effects of temperature and relative humidity on the skin, volunteers were placed under controlled conditions (range: 22±2°C and 50±5%, respectively) for at least 20 min before measurements.

[0095] 4.1.1 Skin moisturizing effect (assessed by keratinometry) Skin moisturization is a direct general parameter that allows the overall control of the well-being and functionality of the skin. Alterations in the skin barrier due to physical, chemical or ageing factors result in a decrease in the skin moisturization level.

[0096] In this study, the moisturizing effect of the vesicle concentrate according to Example 1 was evaluated by measuring the moisturizing state of the skin after a single application and after repeated daily applications for 14 days using an instrument (Corneometer CM825, Courage+Khazaka). The collected data were statistically evaluated through mean comparison using a two-tailed t-test.

[0097] result FIG. 9 shows the results in graphical form, showing the skin moisturization values ​​measured in treated and untreated areas at different time points, expressed as percentage change compared to the respective basal values ​​at time 0.

[0098] The graph shows the percentage change in skin moisturization after a single application (t6h) and after two consecutive weeks of twice daily application of the test products (t2weeks) compared to the baseline values ​​(tb) for the treated area (face) and the control untreated area (forearm) at the same measurement time points.

[0099] The quadratic correlation functions calculated for the treated area values ​​after 6 hours and 14 days are also shown along with their respective formulas.

[0100] The vesicle concentrate was shown to have a significant moisturizing effect on the outer layer of the skin: at all concentrations, the mean moisturizing levels of the facial areas treated with the vesicle concentrate were significantly increased (p<0.05) compared to the basal values ​​6 hours after a single application of the test product and after 2 weeks of daily application, whereas no significant changes were detected in the control untreated areas.

[0101] Just one application significantly increased the skin's moisturizing value, and this increase remained constant after two weeks of application. This indicates the immediate benefit of the vesicle concentrate, which has the ability to "fuse" with the skin and create a kind of unique "shield" that physically protects it. In fact, at two different measurement time points (after 6 hours and 14 days), almost the same moisturizing value was detected in all groups (without a cumulative effect). This indicates that the vesicle concentrate does not interact with the skin's metabolic system, but rather physically interacts with the skin.

[0102] This effect is also confirmed by the virtually linear correlation of the dose-effect response found for the different tested percentages of vesicle concentrate (1%, 5%, 10%, 20%). The moisturization level achieved is directly proportional to the amount of vesicle concentrate in both series (measurements after 6 hours and after 14 days). [Data are quadratic distributed (R 2 = 0.9813 and R 2 =0.9632), but in both correlations the coefficient of the quadratic term is negligible, so they are very close to a linear distribution (see Figure 9).

[0103] No minimum dose was detected to trigger this effect, nor did it plateau within the range of concentrations tested.

[0104] All these results regarding moisturization were confirmed in a second study described below, which assessed them as such in the indices examined.

[0105] Thus, the vesicle concentrate was shown to be effective in significantly moisturizing the outer skin layers at all tested rates. The effect is immediate: after a single application of the cream containing said vesicle concentrate on the face (measured after 2 and 6 hours), a desired increase in the moisturizing level is achieved, which lasts for the entire period of application of the product on the skin. This result explains the immediate advantage of the vesicle concentrate, since it does not interact with the skin metabolism, but interacts and fuses with the skin, thus creating a kind of unique "microfilm on the skin" that physically protects the skin. In fact, 7 days after the cessation of use of the various cosmetic products containing the vesicle concentrate under investigation, the moisturizing value of the skin returns to its initial baseline value. This effect is also confirmed by an excellent linear dose-response relationship: the increase in moisturizing values ​​is directly proportional to the tested doses of vesicle concentrate (1%, 5%, 10%, 20%). No minimum amount of vesicle concentrate was found to be necessary to exert a moisturizing effect, and no plateau of efficacy was observed above a given dose.

[0106] 4.1.2 Skin elasticity: Density and elastic properties (by skin viscoelasticity measurements) The effect of the vesicle concentrate according to Example 1 in improving skin firmness and elasticity was tested under the same study conditions.

[0107] The objective of this study was to evaluate the effectiveness of multiple face creams containing increasing amounts of vesicle concentrate (0% placebo, 1%, 5%, 10%, 20%) in improving skin elasticity after repeated daily application for a total of 4 weeks compared to an untreated site (forearm).

[0108] The method is based on the instrumental evaluation of the ability of the skin to return to its original position after suctioning it under standard conditions. The principle of the measurement is the suctioning of the skin of the test area obtained through a recessed section applied through a circular opening (diameter 2 mm) of the instrumental probe (Cutometer MPA580, Courage+Khazaka). An operating mode was applied at a constant low pressure level, where the skin is sucked by the probe by applying a uniform vacuum for a specific time, and the skin returns to its original position when the pressure is released. The experimental conditions are as follows: -Suction time = 1 second - Release time = 1 second -Pressure = 450mbar The measurements were repeated for three consecutive cycles.

[0109] The cream was applied to the volunteers' faces, and the volar side of the forearm served as an untreated control. To ensure that measurements were always taken at the same location at different time points, a reference mask was used for both treated and untreated areas. Facial measurements were taken in the cheek area. To randomize the conditions, 25 volunteers were measured in the right cheek and volar area, and the remaining 25 volunteers were measured in the left area.

[0110] Measurements were performed on both treated and untreated areas before product application (basal, tb), 2 weeks after application (t2w) and another 2 weeks after application (after a total of 4 weeks, t4w).

[0111] In this skin viscoelasticity study, we considered the percentage increase / decrease in R0 and R2 (Figure 10), the two most important parameters in this type of study. · The parameter of skin elasticity R0 (figure 10) determines the maximum depth (in mm) that the skin penetrates into the probe when a vacuum is applied (R0=Uf). It indicates the suppleness of the skin and reflects the stiffness / flexibility of the skin. It describes the passive behavior of the skin in response to an applied force. An improvement in skin compactness usually implies a decrease in R0 (less skin changes due to suction). The skin elasticity R2 parameter determines the ability of the skin to return to its original state after the vacuum is turned off. It reflects the overall elasticity (recovery / deformation) of the skin and is calculated according to the following formula: R2=Ua / Uf. Where (Figure 10): Uf is the maximum skin elongation at the end of the vacuum application period (i.e., Uf = R0); Ua is the total recovery of the skin towards its original position one second after the vacuum is removed (also defined as "total elastic recovery / deformation"), expressed as a percentage.

[0112] Therefore, the parameter R2 reflects the elastic properties of the skin and represents the ratio between the magnitude of deformation after suction and the ability to return to the initial position (after release / after suction). The higher the recovery (time-delayed recovery), the better the elastic properties of the skin. As skin elasticity improves, the value of R2 typically increases. The maximum penetration depth (mm) of the skin to the probe when a vacuum is applied is defined as R0 (=Uf). The ability of the skin to return to its initial state after the vacuum is stopped is defined as R2:Ua / Uf, where Uf is the maximum stretch of the skin within the probe at the end of the suction period (Uf=R0) and Ua is the total recovery of the skin towards its initial state after 1 second under normal pressure.

[0113] result At all concentrations tested (20%, 10%, 5%, 1%), the mean R0 of facial skin treated with vesicle concentrates (FIG. 11) was significantly reduced (p<0.05) compared to baseline after 2 and 4 weeks of twice daily application.

[0114] For both series of data (2-week and 4-week applications), an excellent, virtually linear dose-response relationship was found when comparing the change in R0 with the increasing percentage of vesicle concentrate in the test face cream. [Again, the data were quadratically distributed (R 2 = 0.9975 and R 2=0.987), but in both correlations the coefficient of the quadratic term is so small that it can be neglected, making the relationship very close to a linear one in effect.

[0115] The mean R2 values ​​for skin in the facial area treated with 20%, 10%, 5%, and 1% vesicle concentrates (FIG. 12) were significantly increased (p<0.05) compared to the initial baseline values ​​after 2 and 4 weeks of twice daily application.

[0116] Again, an excellent linear dose-response relationship was observed when comparing the percentage change in R2 with increasing amounts of vesicle concentrate of the test formulation in the two data series (2 weeks: R 2 =0.9943, 4 weeks:R 2 =0.9464).

[0117] On the other hand, in the control area (untreated forearm), no statistically significant changes were observed in both R0 and R2.

[0118] For both parameters R0 and R2, no significant changes (decreases and increases, respectively) were observed between the values ​​after 4 weeks of product use and those observed after the first 2 weeks for all tested amounts of vesicle concentrate.

[0119] Similar to the moisturizing studies, these results reflect the immediate effect of the vesicle concentrate on the skin: high efficacy in improving skin elasticity is achieved over a relatively short application period (2 weeks).

[0120] The graph in Figure 11 shows the mean percentage change in parameter R0 (compactness) after 2 and 4 consecutive weeks (t2weeks, t4weeks) of twice-daily application of the face cream under evaluation, in treated areas (face), relative to the initial value before treatment (baseline, tb), compared with the analogous value at the same measurement time point in an untreated area (forearm). The quadratic correlation curve and the correlation equation for both series (2w and 4w) are also shown.

[0121] The histogram in Figure 12 shows the mean percent change in parameter R2 (skin elasticity) from baseline pre-treatment values ​​(tb) at treated areas on the face after 2 and 4 consecutive weeks (t2weeks, t4weeks) of daily application (twice a day) of the test products, compared to the corresponding values ​​at the untreated control area (forearm). The linear correlation curves and correlation equations for both series (2w and 4w) are also shown. Therefore, the vesicle concentrate was shown to be effective in linearly improving the skin firmness / suppleness (parameter R0) and elastic properties (parameter R2) of the treated facial skin. Excellent linear dose-response relationships were observed for both parameters R0 and R2. -After just 2 weeks of use, the product was highly effective in improving the elastic properties of the skin, confirming its short-term effectiveness with visible results in a short period of time.

[0122] 4.1.3 Wrinkles: Wrinkle visibility coefficient (based on image analysis) Healthy skin has a fine network of roughly linear grooves on its surface, known as "skin microstructure" or "skin texture". This texture consists of parallel grooves of various depths that intersect with each other to form rectangles, squares, diamonds, trapezoids and triangles.

[0123] This complex network of fine lines firstly increases the effective surface area of ​​the human body by 10% to 50% of the apparent surface area, which in the long run improves the exchange between the epidermis and the environment, improving respiration, heat dissipation, and excretion of sweat and sebum so that optimal moisture levels are maintained.

[0124] Furthermore, this texture contributes to the extensibility of the skin.

[0125] The appearance of skin microstructures changes with age: the highly regular groove structure in healthy, young skin is gradually replaced by a more disordered structure characterized by a decrease in the density of fine lines and an increase in their depth.

[0126] The efficacy of the vesicle concentrate in restoring the normal characteristics of microroughness, reducing the appearance of fine lines, improving skin smoothness and the overall appearance of the skin was tested.

[0127] The method chosen was based on the prominence of wrinkles in facial photographs (high-quality standard photographs taken in polarized parallel light conditions in the frontal and / or ±45° profile - Headscan Dynamics, Orion TechnoLab, France) (Figure 13). Dedicated software ensured the acquisition of images under reproducible lighting and photography conditions. The photography system employed was made with a Nikon camera equipped with a 24 Mpx sensor. The continuous spectrum illumination had a color temperature of 4500k° and a CRI of 96. The system was also equipped with an internal 24 color chart that allowed real-time control of colorimetric measurements and the proper presentation of the images.

[0128] The potential efficacy of face cream base formulations containing increasing concentrations of vesicle concentrate (0% placebo, 1%, 5%, 10%, 20%) in improving (reducing) the visibility of wrinkles was evaluated in a cohort of 50 volunteers (white females, 44-66 years old) by testing each formulation on 10 volunteers. Each group applied their assigned formulation twice daily for 4 consecutive weeks, during which three measurements (images taken) were taken before product application (tb), at a midpoint after 2 weeks of application (t2w), and at the end of 4 weeks of use (t4w). The lateral outer periocular area was selected as the facial area to be measured (Figure 13).

[0129] Using software dedicated to the analysis of digital images (FrameScanV3, Orion Technolab, France), which allows the assessment of the intensity of facial wrinkles by measuring the contrast of the images (number of fine lines, wrinkle dimensions, contrast with the surrounding wrinkles), various parameters were calculated from these images, obtained in the same facial area of ​​each volunteer at different treatment times.

[0130] They then calculated an index of wrinkle visibility, which correlates strongly with clinical perception. Visibility coefficient = (wrinkle occupancy rate) x (wrinkle / skin contrast) x 100 This coefficient is a numerical assessment of wrinkle appearance and visual perception, rather than a measurement of wrinkle depth (as is available with 3D analysis).

[0131] In this experiment, the apparent reduction in wrinkles corresponds to a reduction in the value of the coefficient of prominence, as shown in FIG. 13, which shows photographs of the same facial areas of volunteer n.1 and volunteer n.2 taken tb (before treatment), 2 weeks and 4 weeks after product use. Software for image analysis allows these differences to be calculated by measuring the contrast. A qualitative assessment of the wrinkle reduction can already be assessed by visual (clinical) observation of the images at different time points.

[0132] result Figure 14 graphically summarizes the results of this study, showing the mean percentage change in wrinkle visibility index in treated facial areas after 2 consecutive weeks and 4 consecutive weeks of product application (twice daily) relative to baseline (tb). We also present the linear correlation curves for both the 2-week and 4-week series values ​​and their equations.

[0133] In the treated facial areas of all formulations containing the vesicle concentrate, the wrinkle visibility index after 2 weeks of daily application of the test product was significantly reduced relative to the initial baseline value. Thus, the vesicle concentrate was effective in significantly reducing the visibility of wrinkles at all tested concentrations, an effect that was achieved already after a relatively short period of use (2 weeks).

[0134] This reduction in the appearance of wrinkles correlates very well with the amount of vesicle concentrate applied to the skin in both data series (after 2 weeks: R 2 =0.9973;After 4 weeks:R 2 =0.9927).

[0135] In the case of this clinical study, no further reduction in the wrinkle visibility index was observed after an additional 2 weeks of product application (the difference between the values ​​after 2 and 4 weeks for each concentration of vesicle concentrate was not statistically significant).Once again, this result proves the "fast" effect associated with the "physical" benefits that the vesicle concentrate provides to the skin. The vesicle concentrate system was effective in significantly reducing the appearance of wrinkles at all concentrations tested. The reduction in the appearance of wrinkles correlates linearly with the amount of vesicle concentrate applied to the skin. This beneficial effect was achieved in a relatively short period of two weeks, again illustrating the "rapid effectiveness" that correlates with the beneficial physical interaction of the vesicle concentrate with the skin.

[0136] These results regarding wrinkle visibility were confirmed in a second study described below in this paper, which also assessed the indicators examined as such.

[0137] Overall, in all three indices examined in the study (moisturizing effect, skin firmness, and wrinkle visibility), Vesicle concentrates were effective at all doses tested with an excellent linear dose-response relationship. No plateau was detected. No dose threshold for efficacy is required.

[0138] The specific indicators chosen for these studies (all direct or indirect indicators of healthy skin) relate to the ability of the vesicle concentrate to fully integrate into the skin, i.e., its barrier capacity, in that it is a biomimetic to the skin, enhancing the skin's protective capacity, expanding its inherent functions, and inducing rapid renewal / rejuvenation activity with easily perceptible skin tone improvement and wrinkle reduction results.

[0139] Overall, the vesicle concentrate is a flexible medium with a wide range of uses, which when added to cosmetic formulations will have a beneficial effect on the skin barrier in proportion to the amount used, thus allowing cosmetic formulators the freedom to use it in a wide range of cosmetic applications. These benefits of a thick cream can also be obtained in a "light" serum containing the vesicle concentrate.

[0140] Effective in strengthening the skin barrier In this second clinical study, several vesicle concentrate systems with different amounts of components were formulated and studied to explore the flexibility of vesicle concentrates in order to understand them as a platform, rather than as a compound cosmetic active ingredient with a specific formulation.To this end, the two indicators already examined in the above study (moisturization and wrinkle visibility) were evaluated again, and in addition, more specific indicators were evaluated to evaluate the protection of the skin barrier and the improvement of damage.

[0141] The test formulations in this study were identical but had different vesicle concentrate systems within them, each containing different amounts of the system's components. Thus, four base face cream formulas were prepared, three of which contained 10% of each of the different vesicle concentrates. -Anti-Age PCS OI8293: Example 2 -Empty PCS OI8304: Example 1 -Alternative Anti-Aging PCS OI8292: Example 2 Part 2 - A fourth formulation without vesicle concentrate as placebo.

[0142] The clinical trial was conducted on 40 volunteers (40-65 years old, female) divided into four groups of 10, with each group assigned one of the four formulations.

[0143] Regarding TEWL index, this study was conducted as follows:

[0144] For moisturization and visibility parameters, the first facial application was performed under controlled conditions in a clinical center.

[0145] Volunteers continued to use the product as instructed by applying it twice daily (morning and evening) for two consecutive weeks (14 days), after which they discontinued use of the test product and any other cosmetic products on their face for seven days.

[0146] Instrumental measurements of moisturization and visibility coefficients were performed on the face (test area) and on an untreated area of ​​the forearm (control area) of volunteers treated with the assigned product at different time points: immediately before product application (tb), 2 hours after the first application of the product (t2h), after daily application (twice a day) of the product for 2 weeks (14 days) (t2w) and 7 days after cessation of application of the product and other cosmetic products (t3w).

[0147] Considering the effects of temperature and relative humidity on the skin, volunteers were placed in controlled conditions (range: 22±2°C and 50±5%, respectively) for at least 20 min before measurements.

[0148] 5.1.1 Trans Epidermal Water Loss (TEWL) 5.1.2 Moisturizing properties 5.1.3 Wrinkle visibility

[0149] 5.1.1 Improving the skin barrier Protection against environmental stress - immediate effect measured via TEWL (Transepidermal Water Loss) after stripping.

[0150] TEWL is a measure of the integrity of the skin's "barrier function." Increases in TEWL are measured under controlled conditions by inducing an initial mild injury to the skin (15 consecutive skin strips).

[0151] The effectiveness of the vesicle concentrate in protecting / repairing such altered skin barrier was evaluated by comparing the short-term (30, 60, 120 min) TEWL recovery rate of skin treated with the vesicle concentrate after injury with that of untreated skin after injury, and also with the behavior of a placebo formulation not containing the vesicle concentrate.

[0152] result The results of this study are summarized in a graph in Figure 15. The graph shows the percentage recovery of TEWL values ​​after injury (skin stripping) with different vesicle concentrate-containing formulations and placebo, relative to injured skin not treated with any product.

[0153] All test formulations enabled rapid restoration of the skin barrier (TEWL reduction) at significantly higher levels than the placebo formulation (not containing vesicle concentrate) tested for comparison.

[0154] 5.1.2 Increased moisturization As described for the first study, the moisturizing efficacy of the test formulation was again tested by instrumental measurement of skin moisture (Corneometer CM825, Courage+Khazaka) after a single application (2 h), after repeated daily applications for 14 days, and after 7 days of cessation of treatment.

[0155] result The data on skin hydration obtained in the experiment were evaluated as statistically valid and are presented in the following table showing the percentage difference of each tested product relative to the basal value at the different measurement time points.

[0156] As can be readily seen, all three vesicle concentrate-containing cream formulations were highly effective, increasing moisturization values ​​immediately and then remaining constant throughout the entire use of the product, an increase that was significantly higher than that achieved by the placebo of the same formulations but without the vesicle concentrate.

[0157] The slight differences detected relative to the untreated area (forearm) were not statistically significant.

[0158] One week after cessation of application, moisture retention essentially returns to initial values.

[0159] [Table 2]

[0160] When comparing the hydration increase values ​​at the different measurement time points achieved by formulation OI8304 in the present study (+30.51% at 2 hours, +28.57 at 14 days) with the same formulation tested in the first study (10% vesicle concentrate: +24.59% at 6 hours, +23.91% at 14 days), it can be seen that these results overlap well, taking into account that they were obtained at different times from two different small groups of volunteers (10 each).

[0161] 5.1.3 Improving the appearance of facial skin Reduce wrinkle visibility coefficient As described in the first clinical study above, the objective here is to evaluate the effect on restoring the normal characteristics of skin microstructure, reducing the appearance of fine lines, improving skin smoothness and the overall appearance of the skin.

[0162] To summarise what has been explained in more detail above, the method is based on measuring the prominence of wrinkles in photographic images of the face acquired with a system (HeadScanDynamics, Orion TechnoLab, France) capable of acquiring standard photographs of the front face and ±45° profile under polarised parallel light. Dedicated software ensured the acquisition under reproducible lighting and photographing conditions.

[0163] From these images, a wrinkle visibility index is calculated that correlates highly with clinical perception of wrinkles. Visibility coefficient = (wrinkle occupancy rate) x (wrinkle / skin contrast) x 100 result

[0164] The table below summarizes the results of this part of the study.

[0165] [Table 3]

[0166] Again, it should be noted that the test vesicle concentrates performed approximately the same, with all concentrates significantly reducing the appearance of wrinkles, while the placebo was less effective.

[0167] Upon cessation of product use, the conspicuity coefficient returns to approximately baseline values, similar to what was observed for this parameter in the first study.

[0168] Overall, no significant differences were observed between the different series of vesicle concentrates tested for the three indicators examined in this study, confirming that the composition of vesicle concentrates can be modified to suit various cosmetic applications while maintaining high efficacy.

[0169] Figure 16 shows what happens to the skin after application of a cream containing a vesicle concentrate according to the invention. During application, the water content of the cream causes the vesicles to break apart (first image on the right). After natural evaporation of the water, the vesicles flatten out and form a protective film (second image on the right). The two photographs on the left, Cryo-TEM and TEM, show its effect on the skin.

[0170] Figure 17 shows a comparison between microvesicles on the left and nanovesicles on the right. Assuming a constant rate of water evaporation, micrometer-sized (0.6-1.0 μm) vesicles similar to those according to the present invention form a much more effective barrier than those formed by nanometer-sized (diameter <100 nm) vesicles.

[0171] It appears that the barrier effect provided by the vesicles is in addition to that provided by active ingredients that can be transported by the vesicles which require longer periods (2-4 weeks) to be effective, i.e. active ingredients such as panthenol, oat kernel protein, ectoine, 3-o-ethyl ascorbic acid, and niacinamide.

[0172] [Table 4]

[0173] [Table 5]

[0174] [Table 6]

[0175] [Table 7]

[0176] [Table 8]

[0177] [Table 9]

[0178] The contents of the components (a) to (e) in the different Examples 1 to 3 are always in the range of 15 to 40% as shown in claim 1.

[0179] [Table 10]

[0180] [Table 11]

[0181] [Table 12]

[0182] [Table 13]

[0183] [Table 14]

[0184] Example 8 provides a mixture obtained by mixing phase A and phase B through a powder mixer, drying the treated powder in a ventilated oven at 50° C. until completely dry, followed by passing it through a sieve with mesh number 120 and compressing it with a godet.

Claims

1. 1. A cosmetic composition comprising a vesicle concentrate comprising an aqueous solution of lipid vesicles, The lipid vesicles are multilamellar vesicles ranging in size from 0.6 to 1.0 microns and have walls formed from the following components: (a) Phospholipids (b) Phytosphingosine (c) Phytosterols (d) β-phytosteryl sulfate and, optionally, other phytosterol derivatives. (e) Ceramide and / or pseudoceramide A cosmetic composition, characterized in that the weight content of components (a) to (e) is in the range of 15 to 40% of the vesicle concentrate.

2. 2. The cosmetic composition of claim 1, wherein all ingredients are botanical ingredients or derived from plants.

3. The cosmetic composition according to claim 1 or 2, further comprising a lysophospholipid as component (f).

4. The cosmetic composition according to any one of claims 1 to 3, further comprising an ester of phytosterol as component (g).

5. A cosmetic composition according to any one of claims 1 to 4, further comprising as component (h) a fatty acid, in particular one or a mixture of oleic acid, palmitic acid, coconut oil fatty acids.

6. The cosmetic composition according to any one of claims 1 to 5, further comprising as component (i) a humectant, in particular one or a mixture of glycerol, xylitol, sorbitol, isosorbide, butylene glycol.

7. 7. The cosmetic composition according to any one of claims 1 to 6, further comprising as component (l) an emollient, in particular one or a mixture of phytosqualane, phytosqualene, emisqualane, caprylyl / capric triglyceride, cetyl palmitate, cetearyl ethylhexanoate octyldodecanol, dimethicone.

8. A cosmetic composition according to any one of claims 1 to 7, further comprising as component (m) a preservative, in particular any one or a mixture of sorbitan caprylate, 1,2-hexylene glycol, hydroxyacetophenone.

9. 9. The cosmetic composition according to any one of the preceding claims, further comprising as component (n) a lipophilic active molecule, in particular one or a mixture of tocopheryl acetate, tocopheryl nicotinate, retinol, tocotrienol, ethyl ferulate, ethyl linoleate.

10. 10. The cosmetic composition according to any one of the preceding claims, further comprising as component (o) a hydrophilic active molecule, in particular ectoine, d-panthenol, NMF factors, glucosyl ascorbic acid, sodium ascorbyl phosphate, myo-inositol, acetyl glucosamine, alanyl glutamine, acetyl cysteine, or a mixture thereof.

11. 11. The cosmetic composition according to any one of the preceding claims, further comprising as component (p) a thickening agent, in particular one or a mixture of xanthan gum, cellulose gum, hyaluronan, glucan, network polymer of sodium acrylate, polyquaternium-37, carrageenan, alginic acid derivatives, guar gum derivatives.

12. The cosmetic composition according to any one of the preceding claims, further comprising as component (q) a film-forming molecule, in particular one or a mixture of corn protein, oat protein, rice protein, hydrolyzed rice protein.

13. A cosmetic composition according to any one of claims 1 to 12, Cosmetic products containing other cosmetically acceptable ingredients.

14. The cosmetic product according to claim 13, comprising 0.1 to 60% by weight of said cosmetic composition.

15. A method for obtaining a cosmetic product according to claim 13 or 14, comprising the steps of: Preparing plant vesicles or plant-derived biomimetic vesicles of size between 0.6 and 1.0 microns, the walls of which consist of a lipid phase comprising the component of any one of claims 1 to 12, comprising the following sub-steps: o preparing a composition comprising a vesicle concentrate by melting the walls of said vesicles to a liquid crystalline molten state so as to obtain a homogeneous single-phase system; o introducing an aqueous phase into the vesicle concentrate composition at a temperature above the freezing temperature of the lipid phase of the vesicles; o cooling the vesicle concentrate composition below the solidification temperature of the lipid phase of the vesicles to obtain a paste-like textured material having the rheological properties of a pseudoplastic fluid or a viscoelastic solid, with a vesicle size distribution of 0.6-1.0 microns. - mixing the resulting paste-textured vesicle concentrate composition with water and other cosmetic ingredients to obtain a final cosmetic product containing the desired weight percent of the vesicle concentrate.

16. The vesicle concentrate comprises: - adding the vesicle wall components (Phase A) one at a time to a jacketed mixer equipped with a turbine (rotor-stator), mechanical agitator and wipers; - mechanically stirring the mixture at room temperature to obtain a smooth and homogenous dispersion of the components; heating phase A gradually in increments of 10°C over a period of 1 hour to a temperature of 80-100°C in order to dissolve the solid components and obtain a slightly yellowish translucent dispersion with a paste-like texture; Bringing phase A to 60° C. with continued mechanical stirring; - gradually adding the ingredients to be added to the vesicles (phase B) to the mixer while keeping the temperature constant; - stirring the composition for 30 minutes after the addition of Phase B is complete; - activating the turbine to turbulently agitate the mixture and gradually lowering the temperature to room temperature to obtain a concentrate having a white or yellowish pasty appearance.

17. The other cosmetic ingredient comprises a cosmetic powder, 16. The method of claim 15, comprising a final drying step to remove water and obtain a powder cosmetic product comprising said vesicle concentrate composition and a cosmetic powder.

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