EP4329893A1

EP4329893A1 - Cosmetic composition, cosmetic preparation containing said composition and their uses and the use of rna sodium salt

Info

Publication number: EP4329893A1
Application number: EP22729281.0A
Authority: EP
Inventors: Victoria NEYMANN; Vladimir ZEMSKOV; Robert NEYMANN
Original assignee: Vrfd Sa
Current assignee: Vrfd Sa
Priority date: 2021-04-29
Filing date: 2022-04-28
Publication date: 2024-03-06
Also published as: PL437731A1; WO2022229701A1

Abstract

The invention relates to a cosmetic composition containing: a) RNA sodium salt, b) hyaluronic acid, c) exopolysaccharides. The invention also relates to a cosmetic preparation containing said composition. The invention also relates to the uses of the cosmetic composition and the cosmetic preparation for skin care, in particular to improve skin appearance, skin elasticity, skin condition, hydration level, tone skin, smoothen skin, soften skin, reduce the appearance of imperfections and wrinkles. The invention also relates to the use of RNA sodium salt to prepare a cosmetic composition or cosmetic preparation, for skin care, in particular to improve skin appearance, skin elasticity, skin condition, hydration level, tone skin, smoothen skin, soften skin, reduce the appearance of imperfections and wrinkles.

Description

Cosmetic composition, cosmetic preparation containing said composition and their uses and the use of RNA sodium salt

The invention relates to a cosmetic composition and a cosmetic preparation containing said composition. The invention also relates to the use of the cosmetic composition and of the cosmetic preparation, in particular for skin care. The invention also relates to the use a RNA sodium salt.

For a long time, skin was seen only as a static shield that separated our body from the external environment. Today, however, it is known that skin is more than a physical barrier between the external and internal environment. The skin is an active immunocompetent organ that counteracts the effect of microorganisms activity and protects against trauma, UV radiation and various environmental toxins.

The concept of skin immunity and skin immune system was for the first time mentioned by Streilein (1983). Over the years, this concept was actively researched, what finally allowed to name skin as equal peripheral lymphoid organ (Ono and Kabashima, 2015). Actually, one of the key functions of the skin is to protect, and it does so by employing physical barriers, biomolecules, and an intricate network of resident immune and non-immune cells within the skin layers (Nguyen and Soulika, 2019).

The immune system is composed of specialised immunocompetent cells located in both major structural compartments: the epidermis and the dermis. Non-immune cell populations that constitute a general barrier against an invasion by foreign agents and organisms, which participate in shaping the immune response, also contribute to skin immunity.

The epidermis has a relatively simple histology, but the underlying dermis is anatomically more complex, with a greater cellular variety. The main immune cells found in epidermidis are Langerhans cells (LCs), whereas dermis comprises many specialised cells that compose the immune system residing in the skin in equilibrium, including various subpopulations of dendritic cells (DCs) subpopulations as well as subsets of T lymphocytes (including CD4+ T helper 1 (TH1) cells, TH2 and TH17 cells, T lymphocytes and NK T lymphocytes (NKT)). Moreover, it contains macrophages, mast cells, fibroblasts, which share the ability to produce cytokines with keratinocytes, as well as various nerve cells. The dermis is drained by lymphatic and vascular ducts through which migrating cells can be transferred. The effectiveness of the skin immune system strongly depends on the interaction between immune cells and keratinocytes and fibroblasts. This complex network ensures the proper surveillance, which can promptly induce immune response and efficient elimination of the external threat (Quaresma, 2019).

Langerhans' Cells (LCs) are found in the outer part of the skin, what makes them a first line fighters with ability to promote pro-inflammatory response (Shklovskaya et al., 2011). Upon stimulation, LCs can migrate through the dermal lymphatic vessels to the skin-draining lymph nodes and enter epidermal tight junctions to capture unwanted antigens. After completing maturation, they migrate to the T ceil area, where they are involved in crucial for capturing protein antigens and mediating antibody production via Th2 cell responses. Interestingly, permanent LCs depletion can result in decreased IgE serum levels, what significantly reduce skin defence mechanisms (Quaresma, 2019). The skin is a reservoir of approximately 20 billion T cells, which is nearly twice the number present in the entire blood volume (Clark et al., 2006). There is accumulating evidence supporting role of T cells residenting in the skin in establishing skin immunity and maintaining immune tolerance.

Depending on the role, there are several T cell subsets redistributed throughout the skin structure. For example, epidermal CD8+ αβ T cells are type of memory T cells, which live surrounded by keratinocytes and LCs, while CD8 and CD4 T cells are deposited in the tight epidermal-dermal junction in dermis and in capillaries and play a key role in the functioning of a healthy immune system. They assist B cells to make antibodies, activate the microbe killing capacity of macrophages and recruit other immune cells to infected or inflamed areas. (Moran, 2016). Th17 together with Th1 and Th2 cells are important effector cells in protecting protect skin against bacterial and fungal infections such as Candida albinos, Klebsiella pneumonia, and Staphylococcus aureus, as well as they can be involved in mediating certain inflammatory skin pathologies such as allergic inflammation or psoriasis (Matejuk, 2018). Keratinocytes are the major structural element of outer layer, which have important roles implicated in both innate and adaptive immunity (Nestle et al., 2009). Keratinocytes together with other immune cells, including neutrophils and epithelial cells create a major source of antimicrobial peptides (AMPs), acting as a first line of defence against pathogens colonization of the skin surface (Harder et al., 1997; Matejuk et al., 2010). Keratinocytes are involved in sensing pathogens, as they equipped with specialized receptors, known as Toll-like receptors (TLRs). Activation of TLRs on keratinocytes promotes Thl responses and production of interferons (IFNs) (Miller, 2008). Keratinocytes actively participate in the crosstalk with T cells, as depending on the stimuli and cellular environment, keratinocytes can induce T cells activation or antigen-specific tolerization. Not surprisingly, malfunction of keratinocytes may lead to pathological conditions, including autoimmunity.

Key function of fibroblasts is to produce structural proteins that constitute cutaneous tissue. They also coordinate with immune responses, allowing the migration of blood cells to sites of injury, and interact with other local cells, thereby potentiating immune responses to infectious agents. In addition, fibroblasts can also release a chemoattractant that is attract specialized immune cells such as dermal lymphocytes to the inflammation site. Moreover, similar to keratinocytes, fibroblasts can produce cytokines such as TGF-β and FGF-β, among others, and thus may influence the local immune response pattern (Quaresma, 2019).

The control of immune function over skin, a large and exposed organ, is challenging for the immune and effector system of the cell. If the immune response is insufficient, infections may occur, but if the immune response is too strong, it may develop into a chronic inflammation or autoimmunity. It is, therefore, a major challenge to control the extent of the immune response, in order to achieve immune homeostasis and maintain skin integrity. Proper cooperation between immune cells and adjacent keratinocytes and fibroblasts results in healthy skin. Various endogenous and exogenous factors, among which an important role is played by oxidative stress caused by the destructive effect of free radicals, may disrupt the proper functioning of the immune system of the skin, consequently leading to the disturbance of immune homeostasis and skin dysfunctions, expressed, inter alia, in slowing down the proliferation of keratinocytes, impaired regeneration functions, reduction skin's ability to heal wounds, premature skin aging due to decreased expression of Glycosaminoglycans (GAGs) in the skin, which, in addition to collagen fibres, are rich structural components of the extracellular matrix that modulate various biological processes, such as proliferation and migration, tissue repair, and immune responses (Rafael S. Aquino at al., 2010, Dong HunLee at al., 2016).

Various compositions and cosmetic preparations to be used on the skin are disclosed in prior art. Many of these compositions or preparations contain multiple synthetic ingredients designed to maintain a proper level of skin hydration, to increase the elasticity of the epidermis or to reduce irritation, redness and hyperkeratosis. Meanwhile, cosmetics containing natural or natural origin ingredients, as well as vegan ones, have recently become more and more popular, which is caused by the need of consumers focused on a healthy lifestyle, respect for nature and the elimination of unnecessary chemicals in food and cosmetics. Those gaining popularity includes for example various oils, butters and plant extracts that support basic functions of the skin and increase its hydration and smoothness. There is a growing interest in new natural compounds with antioxidant properties, enhancing the protective functions of the skin, improving its appearance and balancing skin dysfunctions. However, information based on scientific research on the use of natural ingredients to support proper immune function of the skin is insufficient.

When it comes to research into new cosmetic products, changing environmental conditions cannot be ignored. Processed food, high-stress life, air pollution or various forms of radiation resulting in increased levels of harmful free radicals and oxidative stress are multifactorial processes that damage the skin's immune system and disrupt its proper functioning. As a result of impaired self-defence, the skin loses its innate protective abilities, the balance of the skin microflora may be disturbed, leading to chronic dysbiosis. It is characterized primarily by an increase in the number of potentially pathogenic anaerobic bacteria and the development of inflammation, an increase in the number of acidophilic bacteria that acidify the pH of the skin, and a decrease in the number of protective bacteria. The above- mentioned factors ultimately result in skin hypersensitivity, chronic inflammation or allergy, a reduction in the natural ability to maintain proper hydration, loss of firmness, elasticity and uniformity of the skin, and consequently premature skin aging. Despite the large number of solutions available in the market that affect individual parameters of the skin, the use of active ingredients that synergistically support the proper functioning of the skin and its good condition remains crucial for the cosmetics industry. Therefore, there is a need for preparations of natural origin with proven effects to support the natural resistance of the skin to various harmful external factors, including pathogens and free radicals, preventing premature skin aging - a multifactorial process involving impairment of the functions of immunocompetent skin cells, as well as strengthening the repair and regenerative mechanisms of the skin.

Work on new cosmetic compositions focuses, among others, on the search for the new compositions of known ingredients for which enhanced action is observed, as well as adapting ingredients previously not used in the technical field of cosmetic technology for specific applications. The present invention is in the line with the trend observed in the technical field while providing a new synergistic combination of agents of natural origin in the cosmetic composition.

The present invention provides a novel composition of cosmetic agents and a formulation containing said composition which has a synergistic effect to be used for skin care. The composition according to the invention is essentially based on three ingredients of natural origin, two of which are agents used in cosmetics that are already known in the technical field, and one of them has not yet been studied or described for this use in depth. This agent is the sodium salt of ribonucleic acid (hereinafter referred to as RNA sodium salt, RNA-Na), in particular derived from baker's yeast. The standard cosmetic agents referred to, in turn, are hyaluronic acid and exopolysaccharides, in particular derived from marine organisms.

Hyaluronic acid is a popular ingredient in cosmetics to be used on the skin. Hyaluronic acid is a polysaccharide that is present in the human body and in all living organisms. It is present for example in the extracellular matrix of connective tissues, cartilage, synovial fluid, the vitreous body of the eye and the tear film, kidneys and vocal cords. It is also a component of body fluids and a building block of blood vessel walls. Most hyaluronic acid, over 50%, is found in the skin in the form of sodium hyaluronate. Ageing causes the amount of water in the skin to decrease, so it gradually loses its ability to bind water. As a result, the skin becomes dry and wrinkles appear. Hyaluronic acid has been used in aesthetic medicine since the 1980s as a filler and skin firming agent. Hyaluronic acid is an ingredient of various rejuvenating creams.

Raw materials of marine origin are used in skin care. Algae, sea salt, even pearls or caviar - cosmetics based on these products have a moisturising, firming and rejuvenating effect. Such raw materials also include exopolysaccharides produced by marine microorganisms. The marine environment forces the microorganisms that inhabit it to develop effective defence mechanisms. Under high salinity, high pressure and low temperatures, microorganisms need to produce certain substances to help them survive. These substances are sugar biopolymers, exopolysaccharides. Exopolysaccharides can stimulate the synthesis of hyaluronic acid, exhibiting a strong anti-wrinkle effect.

As regards nucleic acids, in turn, prior art comprises only scarce information on their use, either RNA or DNA, or salts thereof, in cosmetics for the skin. Their use in this regard is not commonly reported. Patent documents usually relate to RNA, including its sodium salt, as a beneficial auxiliary agent in a preparation/composition/formulation with other active ingredients being essential.

There is no document in prior art that would disclose all the ingredients of the present composition, even if compositions are disclosed in prior art that contain an ingredient of the present composition, either there are other active ingredients or other cosmetic agents in addition to the ingredient, or they are only referred to without the source of origin being referenced or their contribution to the cosmetic effect indicated.

The key ingredient of the inventive composition is the RNA sodium salt. Prior art disclose only few mentions regarding its use in the cosmetics industry. No studies are available on the effects of this ingredient on the skin.

The article entitled "Is sodium RNA the next big anti-aging ingredient?" The Derm Review, 28/08/20, available at https://thedermreview.com/sodium-rna/, discloses theoretical considerations of using RNA, specifically in the form of a sodium salt for use as an ingredient of cosmetics on the skin. According to said disclosure, the RNA sodium salt is supposed to have the following uses: anti-wrinkle, for improving skin tone (hyperpigmentation), anti-cellulite.

References in prior art patent documents usually relate to RNA, including its sodium salt, as an ingredient in a preparation/composition/formulation with other active ingredients being essential. For example, document EP0283893A1 discloses a cosmetic composition applied to the skin (a separate composition for day and night use) containing a mixture of active ingredients selected from: jojoba oil, hyaluronic acid, collagen, Retarderm, benzophenone 3 and ribonucleic acid with the following composition:

- cosmetic day preparation (cream B): Jojoba oil (2.50 - 4.50%); hyaluronic acid (0.04 - 0.06%), collagen (0.50 - 1.50%), Retarderm^® (1.50 - 3.50%); benzophenone 3 (0.10 - 0.30%),

- cosmetic night preparation (cream A): Jojoba oil (2.50 - 4.50%); hyaluronic acid (0.04 - 0.06%); collagen (0.50 - 1.50%); Retarderm^® (1.50 - 3.50%); benzophenone 3 (0.10 - 0.30%); ribonucleic acid (0.40 - 0.60%), wherein Retarderm^® has the following ingredients: vaseline, lanolin, PCA sodium salt, Polysorbate 85. Neither the origin, form or function of the RNA used in the formulation are disclosed. The disclosed preparations contain no exopolysaccharides. Studies of the efficacy of the use of ready-made cosmetic preparations in in-vivo tests according to this disclosure in improving skin quality were performed in two ways: as tests of the degree of hydration of the skin and its degree of strengthening, "fortifying" (elasticity).

Document EP2939657A1, in turn, discloses a composition to be applied on the skin having an anti- ageing effect that contains an aqueous medium as a base medium and one or more ingredients selected from the group consisting of L-ascorbic acid, a derivative of L-ascorbic acid and salts thereof as active ingredients. Nucleic acids such as deoxyribonucleic acid (DNA), potassium DNA, ribonucleic acid (RNA), sodium RNA are indicated therein as auxiliary ingredients. The anti-wrinkle agent added to the composition is preferably hyaluronic acid. The humectant used is reported to be acetylglucosamine.

There is no document in prior art that would disclose a composition consisting of a RNA sodium salt, in particular derived from baker's yeast, medium molecular weight hyaluronic acid and exopolysaccharides, in particular of marine microorganism origin. Surprisingly, it has been found that the combination of these ingredients has a synergistic effect and is beneficial for the skin in many regards.

Accordingly, the purpose of the invention is to provide a composition of three ingredients found to enhance the natural immunity of the skin, which so far have not been combined for cosmetic uses. It is also an object of the invention to provide preparations containing said composition. Another object of the invention is the use of a composition and/or preparation for skin care as well as the use of an RNA sodium salt for preparing cosmetic compositions or preparations for skin care. It has to be stressed out, that although there are reports in prior art on the presence of RNA sodium salt as an auxiliary ingredient of a multi-ingredient cosmetic composition, often without indicating its origin, no reliable and comprehensive research on the effect of this agent on the skin has been disclosed so far. Therefore, prior art references to the presence of RNA sodium salt as a merely complementary ingredient, one of many other ingredients of cosmetic preparations, cannot be perceived as a disclosure of the use of RNA sodium salt as an active ingredient for the preparation of a cosmetic composition or cosmetic preparations intended to be applied specifically on the skin. Only the present invention solves the technical problem associated with the use of RNA sodium salt, in particular derived from baker's yeast, for the claimed use.

The subject matter of the invention is a cosmetic composition containing: a) RNA sodium salt, b) hyaluronic acid, c) exopolysaccharides.

Preferably, the composition contains, on a dry mass: a) RNA sodium salt in an amount of 60-84.5% w/w of the total composition, b) hyaluronic acid in an amount of 15-39.5% w/w of the total composition, c) exopolysaccharides in an amount of 0.5-2.0% w/w of the total composition.

More preferably, the composition contains, on a dry mass: a) RNA sodium salt in an amount of 70.42% w/w of the total composition, b) hyaluronic acid in an amount of 28.12% w/w of the total composition, c) exopolysaccharides in an amount of 1.41% w/w of the total composition.

Preferably, the composition further contains at least one cosmetically acceptable additive and is then in the form of a cosmetic concentrate for dilution. Preferably, the at least one cosmetically acceptable additive is a substance selected from the group of solvents, carriers, moisturisers, fragrances, antioxidants, lubricants, glidants, emollients, preservatives, humectants. More preferably, the composition contains as additives sea water, apple water and a natural liposome system based on lecithin derived from organic soybean. Preferably, the natural liposome system is essentially a mixture of glycerine, lecithin, pentylene glycol, sodium hydroxide, tocopherol, and water.

Preferably, the composition according to the above description contains the following ingredients: a) RNA sodium salt in an amount of 4.00-6.50% w/w b) hyaluronic acid in an amount of 1.50-3.00% w/w c) exopolysaccharides in an amount of 0.07 to 0.20% w/w d) a natural liposome system based on lecithin derived from organic soybean in an amount of 25.00-37.00% w/w e) sea water in an amount of 10.00-20.00% w/w f) apple water in an amount of 25.00-50.00% w/w Preferably, the composition contains the following ingredients: a) RNA sodium salt in an amount of 5.85% w/w b) hyaluronic acid in an amount of 2.34% w/w c) exopolysaccharides in an amount of 0.12% w/w d) a natural liposome system based on lecithin derived from organic soybean in an amount of 33.22% w/w e) sea water in an amount of 15.20% w/w f) apple water in an amount of 43.27% w/w

Preferably, the exopolysaccharides are of marine origin. Preferably, the exopolysaccharides are essentially a mixture of a saccharide isomerate and benzyl alcohol.

Preferably, the RNA sodium salt is derived from baker's yeast. Preferably, the hyaluronic acid is in salt form. Preferably, the hyaluronic acid is also in the form of a hydrolysate. The hyaluronic acid in the form of a hydrolysate has preferably an average molecular weight between 100 and 300 kDa.

Preferably, the composition is characterised in that all its ingredients are of natural origin.

The invention also relates to a cosmetic preparation containing the composition as defined above. Preferably, the cosmetic preparation is in the form of a serum or cream and it is then characterised in that it contains the cosmetic composition in the form of a concentrate at a concentration of 4-6% w/w and excipients up to 100%. Preferably, the cosmetic preparation in the form of a serum or cream contains the cosmetic composition in the form of a concentrate at a concentration of 5.13% w/w and excipients up to 100%.

Preferably, the cosmetic preparation is in the form of a mask and it is then characterised in it contains the cosmetic composition in the form of a concentrate at a concentration of 0.6-1.0% w/w and excipients up to 100%. Preferably, the cosmetic preparation in the form of a mask contains the cosmetic composition in the form of a concentrate at a concentration of 0.855% w/w and excipients up to 100%.

Preferably, the cosmetic preparation is characterised in that the excipients are solvents, thickening agents, emollients, emulsifiers, humectants, antioxidants, free radical scavengers, pH adjusters, preservatives, fragrances, plant-derived extracts.

The invention also relates to the use of the cosmetic composition as defined above for skin care, in particular to improve skin appearance, skin elasticity, skin condition, hydration level, tone skin, smoothen skin, soften skin, reduce the appearance of imperfections - wrinkles and/or enlarged skin pores.

The invention also relates to the use of the cosmetic preparation as defined above for skin care, in particular to improve skin appearance, skin elasticity, skin condition, hydration level, tone skin, smoothen skin, soften skin, reduce the appearance of imperfections - wrinkles and/or enlarged skin pores.

The subject matter of the invention is also the use of RNA sodium salt to prepare a cosmetic composition or cosmetic preparations as defined above, for skin care, in particular to improve skin appearance, skin elasticity, skin condition, hydration level, tone skin, smoothen skin, soften skin, reduce the appearance of imperfections - wrinkles and/or enlarged skin pores,

As mentioned above, the hyaluronic acid is preferably in the form of a salt or hydrolysate. The effect of the hyaluronic acid depends on its molecular weight. Preferably, the hyaluronic acid according to the invention is a hydrolysed hyaluronic acid of medium molecular weight, preferably between 100 and 300 kDa. Such hyaluronic acid is commercially available e.g. as seven molecule hyaluronic acid in the form of a raw material under the market name PrimalHyal™ 300 from Givaudan France SAS. As per the characteristics of this product, hyaluronic acid enhances the natural immune system by stimulating specific antibacterial peptides, β-defensin 2 (DEFB2), by keratinocyte stimulation, proliferation and migration of fibroblasts in order to accelerate cell repair. It is obtained by biotechnological methods. For the purpose of the invention, the hyaluronic acid is preferably a product of natural origin. The hyaluronic acid according to the invention may be any other medium molecular hyaluronic acid available which, as per its characteristics, is regarded by a person skilled in the art to be suitable for preparing the inventive composition.

As mentioned, exopolysaccharides are essentially a saccharide isomerate, i.e. a mixture of polysaccharides, and benzyl alcohol as a preservative. The exopolysaccharides according to the invention are preferably produced by marine microorganisms. Examples of exopolysaccharides include the commercial product EPS SEAFILL from Codif produced by planktonic microorganisms from Aber Benoit (Brittany, France), a river where fresh and salt water from the sea meet. This agent is culturing in a bioreactor to obtain a pure, natural molecule consisting of a moiety of galactose and N-acetyl- glucosamine, which has no terrestrial equivalent. Its effects include improving skin renewal, elimination of dead cells on its surface, activation of the physical skin barrier, to provide a smoothing and softening effect, inhibition of the growth of bacteria on the skin's surface, enhancement of the recovery of optimal hydration.

Ribonucleic acid in the form of a sodium salt according to the invention is preferably obtained from yeast, more preferably from baker's yeast. Preferably, the RNA sodium salt is a compound registered under CAS number 9010-05-3, has a theoretical molar mass of 1,273.85, a theoretical molecular formula C₄₀H₅1N₁₅O₂₆P₃ Na, is available from Pharma Waldhof and obtained from pure culture yeast. It is a linear macromolecule (biopolymer) formed by way of polycondensation of various nucleotides. These single nucleotide residues are compounds of a heterocyclic base component with ribose containing one phosphate group as the linking bridge. The indicated formula is an arbitrarily designed example of a combination of four major nucleotides. The distribution of nucleic bases in the polynucleotide chain, however is irregular, and the molar composition of the nucleic bases has no constant proportion. In addition to these four main bases of adenine, guanine, cytosine and uracil, the RNA molecule contains many other bases, mainly methylated bases such as 5-methylcytosine and 5- methylguanine. As a result, the molecular weights of said RNA-Na range from about 3000 to 80,000. The inventive RNA sodium salt is an entirely natural component.

For the purposes of the present application, the composition denotes a dry or liquid, preferably dry, mixture of essential ingredients necessary to achieve the object of the invention. Preferably, the inventive composition is also in the form of a cosmetic concentrate, in which case it has at least one cosmetically acceptable additive. A cosmetic preparation, in turn, is a product containing a composition in a specific form adapted to be applied topically to the skin of various body parts. The cosmetic preparation is obtained by diluting the cosmetic composition in the form of a concentrate in a mixture of excipients.

The inventive composition contains the three ingredients described above. On a dry mass, the composition contains RNA sodium salt in an amount of 60-84.5% w/w, hyaluronic acid in an amount of 15-39.5% w/w, exopolysaccharides in an amount of 0.5-2.0% w/w, based on the total composition. A person skilled in the art will be able to adjust the amounts of the ingredients of the composition so as to obtain 100% w/w of the ingredients within the indicated ranges.

The inventive composition may be dissolved in at least one cosmetically acceptable additive to obtain the composition in the form of a cosmetic concentrate for obtaining cosmetic preparations. Preferably, the composition in the form of a cosmetic concentrate contains a cosmetically acceptable additive which is a substance selected from the group of solvents, surfactants, moisturizers, emollients, preservatives, humectants, or a mixture thereof. Cosmetically acceptable additives are known in the art and are presented for example in Erika Fink, „Kosmetyka. Przewodnik po substancjach czynnych i pomocniczych", MedPharmPolska, 2007 i/lub A. Marzec, „Chemia kosmetykow - surowce, półprodukty, preparatyka wyrobow", Dom Organizatora, Toruń, 2001.

Preferable additives include sea water (preferably from Odemer), apple water (preferably Fruitofood) and a natural liposome system. Liposomes are vesicles encapsulated in a lipid layer; they are formed from phospholipids in an aqueous environment, e.g. from lecithin, which may be derived from egg yolks, soybean, rape or sunflower seeds. Preferably, the liposomes for use in the invention are of natural origin, from lecithin from genetically unmodified soybean. Preferably, the natural liposome system is essentially a mixture of glycerine, lecithin, pentylene glycol, sodium hydroxide, tocopherol, and water. It is commercially available under the trade name NatipideEco from Lipoid Kosmetik.

The composition in the form of a cosmetic concentrate contains the following amounts of ingredients: RNA sodium salt in an amount of 4.00-6.50% w/w, hyaluronic acid in an amount of 1.50-3.00% w/w, exopolysaccharides in an amount of 0.07-0.20% w/w, a natural liposome system based on lecithin derived from organic soybean in an amount of 25.00-37.00% w/w, seawater in an amount of 10,00- 20.00% w/w, apple water in an amount of 25.00-50.00% w/w. A person skilled in the art will be able to adjust the amounts of the ingredients of the composition so as to obtain 100% w/w of the ingredients within the indicated ranges. The concentrate form of the cosmetic composition of three ingredients in the mixture of additives is obtained using the ratio of the composition to the mixture of additives such as, for example, approximately 1:5, 1:8, 1:10, 1:11, 1:12, 1:13. A cosmetic preparation is obtained by diluting a cosmetic composition in the form of a concentrate in a mixture of excipients. Cosmetic excipients are well known in the art and are presented for example in Erika Fink, „Kosmetyka. Przewodnik po substancjach czynnych i pomocniczych", MedPharm Polska, 2007 i/lub A. Marzec, „Chemia kosmetykow - surowce, półprodukty, preparatyka wyróbow", Dom Organizatora, Toruń, 2001. It will be within the general knowledge of a person skilled in the art to adapt the excipients to the specific form of the cosmetic preparation.

For example, if the cosmetic preparation is a serum, then the excipients will generally be solvents that increase the absorbability of the active ingredients, such as propanediol and/or pentylene glycol, thickening agents such as xanthan gum; humectants such as e.g. glycerine, glucose derivatives, xylitol derivatives; antioxidants such as tocopherol; radical scavengers such as e.g. phytic acid; pH-adjusting agents such as citric acid or sodium hydroxide solution; preservatives, such as e.g. sodium benzoate, gluconolactone, fragrances; diluents such as sterile water, sea water, apple water, witch hazel water, etc.

For example, if the cosmetic preparation is a cream, then the excipients will generally be solvents that increase the absorbability of the active ingredients, e.g. 1,3-propanediol, humectants such as glycerine, glucose derivatives, xylitol derivatives; thickening agents such as e.g. xanthan gum; emollients such as e.g. squalane or C8-C18 alkyl esters; viscosity regulators to produce emulsions such as e.g. glyceryl stearate; emulsifiers such as e.g. mixtures of C16-C22 alcohols and glucosides; antioxidants such as tocopherol; plant extracts such as e.g. olive or aloe vera, algae, green tea extracts; radical scavengers such as e.g. phytic acid; pH-adjusting agents such as citric acid; preservatives, such as e.g. sodium benzoate, gluconolactone; fragrances; diluents such as sterile water, sea water, apple water, etc.

If, in turn, the cosmetic preparation is a facial skin mask, then the excipients will generally be solvents which increase the absorbability of the active ingredients, e.g., 1,3-propanediol; humectants such as, e.g., glycerine, glucose derivatives, xylitol derivatives; thickening agents such as, e.g., xanthan gum; emollients such as, e.g., apricot kernel oil polyglyceryl-10 esters emulsifiers, e.g., mixtures of C16-C22 alcohols and glucosides; antioxidants, e.g., tocopherol or mixtures of tocopherols, plant extracts, e.g. refined palm fruit mesocarp oil from Amazonian Mauritia Flexuosa palm tree, Crithmum maritimum lipid extract; pH adjusters, e.g. citric acid or sodium hydroxide solution; preservatives, e.g. sodium benzoate, optionally preservative enhancers (co-preservatives) such as gluconolactone; antioxidants such as ascorbyl glucoside; fragrances; diluents such as sterile water, sea water, apple water, etc.

The inventive cosmetic preparation is obtained by admixing into a pre-prepared mixture of excipients the inventive composition in the form of a concentrate containing: a) RNA sodium salt in an amount of 4.00-6.50% w/w b) hyaluronic acid in an amount of 1.50-3.00% w/w c) exopolysaccharides in an amount of 0.07-0.20% w/w d) a natural liposome system based on lecithin derived from organic soybean in an amount of 25.00- 37.00% w/w e) sea water in an amount of 10.00-20.00% w/w f) apple water in an amount of 25.00-50.00% w/w

The cosmetic preparation in the form of a serum or cream contains the cosmetic composition in the form of a concentrate at a concentration of 4-6% w/w and excipients up to 100%. Thus, the mixture obtained from the excipients contains 4-6% w/w of the inventive composition in the form of a concentrate, preferably 4.5-5.5% w/w, also preferably about 5% w/w of the inventive composition in the form of a concentrate.

The cosmetic preparation in the form of a mask contains the cosmetic composition at a concentration of 0.6-1.0% w/w and excipients up to 100%. Thus, the mixture obtained from the excipients contains 0.6-1.0 % w/w of the inventive composition in the form of concentrate, preferably 0.7-0.9% w/w, also preferably about 0.8% w/w of the inventive composition in the form of a concentrate.

A person skilled in the art will be well aware that the values indicated above are to be regarded both precisely, but also as approximate values, i.e. +/- values differing from the values indicated herein by 10% will also fall within the scope of the invention.

The cosmetic composition, both in the form of a composition of the three essential ingredients and in the form of a concentrate, as well as the cosmetic preparation containing said composition, are used for skin care, in particular to improve the condition of the skin manifested in increased repair and regeneration capacity, to enhance natural microbiota functions, and, as a result, to reduce susceptibility to inflammation and acne formation, to maintain proper hydration, to improve elasticity, to smooth the skin and to reduce the perceptibility of wrinkles. The uses as above are due to the effect of the inventive composition consisting of an RNA sodium salt, hyaluronic acid, exopolysaccharides. Indeed, it has been observed that these three ingredients combined together in the inventive composition have synergistical properties. The other supplementary ingredients of the variants of the invention are merely auxiliary without substantially affecting the effect of the invention. Obviously, the use of liposome carriers in the composition/preparation, as a person skilled in the art will know, will allow to transport the ingredients of the composition into deeper layers of the skin, however, the composition exerts its effect on the skin in line with the object of the invention without this ingredient as well.

A preferable variant of the composition in the form of a concentrate containing a natural liposome system based on lecithin derived from organic soybean, preferably comprises sea water and apple water. The embodiments of the invention below relate to this variant of the composition referred to in the description as the composition in the form of a concentrate/cosmetic concentrate. Although synergy was observed for the three main ingredients of the composition, due to the type of tests conducted in order to demonstrate the effect of the said three ingredients, it was necessary to prepare a mixture which would be both dilutable in a culture medium for in-vitro tests and which would contain additives/excipients so as to reflect as much as possible the composition of the preparation already applied directly to the skin. Thus, the composition which was tested for its efficacy should not be regarded as limiting the scope of protection.

Both the composition and the preparation according to the invention contain no artificial preservatives and texturizers, petroleum-based ingredients such as mineral oils or paraffin, commonly used in traditional cosmetics and other synthetic ingredients such as parabens, silicones, chemical polymers, sulphates, phthalates and the like. The composition and the preparation according to the invention contain ingredients that are 100% natural and of natural origin (according to the Cosmos Natural standard).

The composition has the effect of enhancing proper defence functions of the skin by providing proven protection against oxidative damage. The inventive composition has a strong antioxidant effect, accelerates the synthesis and activity of Sirtuin-l under oxidative stress, thus contributing to the regulation of the cell cycle, while reducing the adverse effects of environmental and stress factors, and preventing the development of signs of accelerated ageing due to the consequences of a weakened self-defence system. The composition stimulates the regenerative capacity of the skin by significantly activating fibroblast proliferation. It significantly activates the production of glycosaminoglycans in human dermal fibroblasts that support the regulation of metabolite transport, so that it stimulates the bonding of the epidermis with the dermis and the maintenance of properskin structure, helps maintain proper hydration while preventing skin dryness and the formation of wrinkles. The composition significantly affects repair processes in the skin, increasing its wound healing capacity.

Thus, the technical problem solved by the present invention is providing of an entirely natural composition containing an RNA sodium salt, hyaluronic acid and exopolysaccharides, that have a synergistical effect, to be applied on the skin. The composition is not a simple mere aggregation of features. The combination of ingredients provides exceptional efficacy of the composition as regards skin care as a result of its cell proliferation enhancement effect, protection against physical and chemical factors, antioxidant effect, enhanced synthesis of glycosaminoglycans in human skin fibroblasts, increased efficacy of wound healing, ability to increase Sirtuin synthesis and activity under oxidative stress, which in turn slows down the ageing process. The technical problem has been solved by the invention, specifically by the unique combination of ingredients, in particular by including an RNA sodium salt in the composition. A series of in-vitro laboratory studies of the cytotoxicity and efficacy of the tested composition were conducted.

Example 1

A cosmetic composition was obtained in the form of a concentrate containing the following: Table 1.

The composition was obtained by mixing seawater and apple water at room temperature, followed by adding in sequence exopolysaccharides, RNA sodium salt, hyaluronic acid. Complete homogeneity of the mixture was achieved after adding each consecutive ingredient. A liposome system was added in the final step.

Example 2

Cosmetic preparations containing a cosmetic composition in the form of a concentrate at a concentration of 5.13% w/w and excipients are indicated in the tables below with the preparation steps of the formulation.

SERUM FOR OILY SKIN AND SKIN WITH IMPERFECTIONS Table 2

MOISTURISING CREAM Table 3

Example 3

A cosmetic preparation containing a cosmetic composition in the form of a concentrate at a concentration of 0.855% w/w and excipients are indicated in the table below with the preparation steps of the formulation.

REVITALISING MASK Table 4

Examples 4-10 below relate to the composition of the cosmetic composition according to the invention in the form of a concentrate, as in Example 1. The test samples were prepared by diluting the composition of example 1 in a culture medium to obtain the concentrations of the composition of Example 1 in the medium: a concentration of 5.13% w/w (approx. 5% w/w) as in the examples of preparations - Example 2 and a concentration of 0.855% w/w (approx. 8% w/w) as in the example of preparation - Example 3. In the test samples, the relation of the ingredients amounts of the inventive composition are as in Example 1. By diluting the composition of Example 1 in a culture medium to conduct the tests described in examples 4 to 10, concentrations of ingredients similar to proposed for cosmetic preparations (Examples 2 and 3) prepared for dermal administration has been achieved.

Example 4

Study of the cytotoxicity of the composition of Example 1 at concentrations of 5.13% and 0.855%

(an in-vitro study) A safety study of using the composition of Example 1 was conducted. For this purpose, the in-vitro cytotoxicity of the cosmetic composition was measured at a final concentration of the test composition of example 1 of 5.13% (as in example 2) and 0.855% (as in example 3).

A cytotoxicity study was conducted using human skin fibroblasts (ATCC-CRL-2703). The culture medium shall be sterile, with a pH of between 7,2 and 7,4, it has to meet the growth requirements of the selected cell line.

The cosmetic composition diluted in culture medium (to a final concentration of 5.13% and 0.855%) was tested against a negative control, which was culture medium supplemented with water.

Culture medium with the test composition of example 1, at concentrations of 5.13% and 0.855% was added to the 96 well plate containing cells in confluence. Cells were exposed to the product for 24 hours. At the end of incubation period, MTT colouring was performed in order to evaluate cell viability compared to untreated control (CTR, 100% viability for definition).

IC₅₀ as the product concentration determining cell mortality in 50% of cell population was calculated. For each tested product concentration, % cell viability is calculated according to the following:

% CELL VIABILITY = (OD540 treated *100)/OD540 negative control

Corresponding mortality is calculated by subtraction. Recorded mortality data are plotted against respective product concentrations to create a dose-response curve. By using curve equation IC₅₀ parameter (or IC₅₀, Inhibiting concentration 50), the product concentration that causes mortality in 50% of cell population, is calculated.

The results of the cytotoxicity measurements indicate that at the concentration of the test composition of example 1 of 5.13%, the cell viability is 87.37% (standard deviation of 0.02) compared with the negative control, for which viability was estimated at 103.84% (standard deviation of 3,4%).

For the final test concentration of the composition of example 1 of 0.855%, a cell viability of 96.91% (standard deviation of 0.04) compared with the negative control, for which cell viability was 99.60% (standard deviation of 2,97%) was found.

Example 5

Study of the effect of the composition of example 1 at concentrations of 5.13% and 0.855% on the cell proliferation activity (an in-vitro study)

In laboratory tests, the efficacy of using the composition of example 1 was evaluated by measuring the level of cell proliferation. For this purpose, proliferation was measured using human skin fibroblasts, in the GO phase of the cell cycle.

Test sample: the composition of example 1, at concentrations of 5.13% (as in example 2) and 0.855% (as in example 3) prepared in culture medium (DMEM). Control sample: Cell cultures without any treatment as negative control reference (CTR -) to calculate cell proliferation.

In-vitro cell proliferation was measured using the following testing technique:

The cells were plated in a plate of 96 with incomplete culture medium (missing bovine fetal serum). This induces the phase of cellular quiescence. After 24 hours, the concentrations of the product to be tested were prepared in complete medium, in order to restart the cells from the GO phase of the cell cycle. The negative control (CTR-) is set up with only complete medium.

The cells were exposed to the composition of example 1 for 24, 48 and 72 hours. In order to assess the level of cell proliferation, compared to the control sample (for which viability is by definition 100%), at the end of each incubation period the cells were washed with PBS buffer, followed by adding 200 μI of MTT solution prepared by dissolving 15 mg MTT in 30 ml of culture medium and incubating it for three hours at a temperature of 37°C and in a 5% CO₂ atmosphere. At the end of the incubation period, the MTT solution was removed and 200 μI of isopropanol was added.

Statistical analysis by T-test was conducted between the samples and the untreated negative control (CTR -). The variations are considered significant for p<0,05.

The results of cell proliferation measurement, based on the viability of the cells treated with the composition of example 1 at 24, 48 and 72 hours compared to the control sample demonstrate that the composition significantly increases the ability of cells to proliferate:

• after an incubation time of 24 h, the composition of example 1 demonstrates an efficacy in increasing the proliferation of the cells tested in the in-vitro assay for both concentrations tested: 5.13% and 0.855%. The increase in cell proliferation compared to the control sample is 34.8% and 90.5%, respectively.

• after an incubation time of 48 and 72 h, the composition of example 1 demonstrates an efficacy in increasing the proliferation of cells tested in the in-vitro assay of 45.4% at a concentration of 0.855% compared to the control sample for an incubation time of 48 h and by 34.5% compared to the control sample for an incubation time of 72 h.

Example 6

Study of the protective efficacy of the composition of example 1 against physical and chemical agents damaging cell cultures (an in-vitro test).

The test was carried out in order to evaluate the capability of the tested composition to contrast the cytotoxic effects of a stress agent after a repeated treatment for up to 72 hours. Protective efficacy is evaluated by means of the measure of cell viability after the induction of stress in presence and in absence of the tested composition. The analysis was conducted using human skin fibroblasts (ATCC-CRL-2703). The culture medium shall be sterile, with a pH of between 7,2 and 7,4, it has to meet the growth requirements of the selected cell line.

Preparation of the composition of example 1: the composition of example 1 was prepared by diluting it in the culture medium to obtain final concentrations of 5.13% (as in example 2) and 0.855% (as in example 3).

The culture medium without tested product is used as negative control (CTR-).

Positive control (CTR+) is represented by the culture medium with the stress agent H2O2 150 μM. Human skin fibroblast cells are plated in a 96-well plate with complete medium DMEM, supplemented with bovine fetal serum and antibiotics, and incubated at 37 and 5% CO2 for 24h.

After the incubation period, fresh culture medium containing the pro-oxidant agent (H2O2 150 μM) and the composition of example 1, at concentrations of 5.13% and 0.855% is added to each well. Cells treated with H2O2 150 μM are used as positive control, while untreated cells are used as negative control.

Cell exposure to test solution is prolonged and repeated for 72 hours. At end of the experimental period, cell viability is evaluated by means of MTT test.

The MTT (3,(4,5-dimethylthiazol-2)2,5 diphenyltetrazolium bromide) test is a standard, simple and accurate colorimetric method for cell viability assessment. The assay is based on the intracellular reduction of the yellow tetrazolium salts by the mitochondrial enzyme succinate dehydrogenase in blue/purple formazan crystals. The reaction may therefore take place only in metabolically active cells and the value of the optical density obtained by photometric reading can be correlated to the amount of viable cells.

At the end of each treatment the wells were rinsed with PBS (phosphate-buffered saline solution), stained with MTT solution 1 mg/mL and incubated for three hours at 36,5°C/5% CO2. Then the wells were treated with isopropanol and incubated for two hours at room temperature. After isopropanol incubation, absorbance readings were performed at 570 nm by microplate reader (isopropanol was used as blank for reading). For each test condition the ratio of the average optical density of the treated cultures on the average optical density of negative controls determines the viability rate. The protection %, intended as cell viability % increase compared to the positive control condition, treated only with the damaging agent, is also calculated.

Obtained results were subjected to statistical analysis by means of Student test. Variations (vs CTR- e CTR+) are considered statistically significant with p*<0.05.

Table 5

It was observed that cell cultures whose viability in the test with no added cell stress-inducing factor (negative control; CTR-) is 100%, while those treated with a cell stress-inducing factor (positive control CTR+) reduce their viability to 38%.

Cell cultures treated with the cell stress-inducing agent and with the composition of example 1 at concentrations of 5.13% and 0.855% have significantly higher cell viability compared to the positive control (CTR+) alone over a 72 hour exposure. The increase in cell protection for a concentration of the composition amounting to 5.13% was 40.7%, and at a concentration of the composition of 0.855% was even higher - 47.9%.

This indicates that, under the experimental conditions as above, the composition of example 1 shows enhanced protective efficacy against oxidative damage in a model using H₂O₂.

Example 7

Study of antioxidant properties of the composition of example 1 (in-vitro test).

The test allowed for determining the antioxidant properties of the composition of example 1 by evaluating its antiradical properties demonstrated by the DPPH assay (based on the reduction of 1,1- diphenyl-2-picrylhydrazyl; DPPH). Due to the presence of an odd electron it gives a strong absorption maximum at 515 nm. As this electron becomes paired off in the presence of a hydrogen donor, i.e. a free radical scavenging antioxidant, the absorption strength is decreased, and the resulting decolorization is stoichiometric with respect to the number of captured electrons.

The composition of example 1 was tested at final concentrations of 5.13% and 0.855%.

The antioxidant activity of the composition of example 1 was measured by quantifying the scavenging potency of a synthetic radical DPPH.

The composition of example 1 was added to a tray containing DPPH, and the decrease in the radiation absorption intensity at a wavelength of 515 nm over a 30 min experiment was measured. For each tested concentration, reduced DPPH concentration after monitored period spectrophotometrically measured was reported. The three replicate data and the mean reduction percentage for each concentration are shown.

Table 6

In the measurements conducted with the composition of example 1, a significant reduction (values expressed as percentage) of DPPH was observed at concentrations of 5.13% and 0.855%. The average percentage reduction in DPPH content for the concentration of the composition used amounting to 5.13% is 24.4%, while for the concentration of the composition of 0.855% it is 6.5%. The results obtained indicate the correlation between concentration of the composition and ability of the composition of example 1 to scavenge free radicals generated in the DPPH assay.

Example 8

Study of the efficacy of using the composition of example 1 on the level of glycosaminoglycan synthesis (an in-vitro test).

Glycosaminoglycans (GAGs) are unbranched, linear polysaccharides composed of repeating disaccharide units. These units consist of an amino sugar (galactosamine or glucosamine) and uronic acid (glucuronic or iduronic acid), which are mostly joined together by a 1,3-β or 1,4-β glycosidic bond. Both uronic acids and amino sugars may be sulphated and/or acetylated [3, 4], All glycosaminoglycans, except for hyaluronic acid, have sulphate moieties in their composition. They combine with proteins to form compounds known as proteoglycans.

Both free glycosaminoglycans and their complexes with proteins (proteoglycans) constitute a characteristic and commonly found component of the intercellular matrix. Glycosaminoglycans play a major part in both the physiology and pathology of individual cells and tissues. Glycosaminoglycans are found in the skin at the level of connective tissue only in the form of proteoglycans, i.e. in the form bound to structural proteins such as collagen and elastin. The role of these compounds in the skin consists in maintaining proper skin hydration, maintaining proper skin structure, bonding the epidermis to the dermis, regulating the transport of raw materials and metabolites, regulating osmotic pressure and maintaining an appropriate turgor. (Zastosowanie glikozaminoglikanow w preparatach kosmetycznych Anna KROMA, Agnieszka FELICZAK-GUZIK, Izabela NOWAK - Department of Chemistry, Adam Mickiewicz University in Poznan)

The following test is designed to evaluate the ability of the test preparation to enhance glycosaminoglycan synthesis in human skin fibroblasts. Measurements were made for concentrations of the composition of example 1 of 5.13% (as in example 2) and 0.855% (as in example 3) using different time intervals of exposure to the test composition of example 1: 24, 48 and 72 hours. Preparation of the test composition of example 1: The composition of example 1 was prepared by diluting it in culture medium so as to obtain final concentrations of 5.13% and 0.855%.

Cell culture without any treatment was used as negative control reference (CTR -).

At end of each experimental times, cell glycosaminoglycan neo-synthesis was determined.

Fibroblast cells were seeded onto a test plate (96-well) in complete culture medium (cells in confluence - this feature allows to maintain the cell number constant for the experiment duration time and avoids the influence of cell number in the observed trophism).). The plates were incubated for 24, 48 and 72 h in solutions of the test composition of example 1 at concentrations of 5.13% and 0.855%.

At the end of the incubation period, the medium was collected in order to determine the concentration of glycosaminoglycans (GAGs) produced and released by the cells. 50 μI of medium was used for each assay. Each measurement was conducted in triplicate.

The synthesis of glycosaminoglycans is determined by quantifying the dye bound therewith. Blyscan assay based on the capacity of binding a dye (1,9-dimethylmethylene blue) to sulphated proteoglycans and glycosaminoglycans (sGAGs) was used in the measurements above. The dye label used in the assay is 1,9-dimethylmethylene blue and the dye is employed under conditions that provide a specific label for the sulphated polysaccharide component of proteoglycans or the protein free sulphated glycosaminoglycan chains.

GAG concentration (determined as the amount in 50 μI of medium expressed in μg) is calculated by interpolating the data on the standard curve obtained with known and increasing glycosaminoglycan concentrations.

Obtained results were subjected to statistical analysis by means of Student test. The variation respect the untreated control are considered statistically significant for *p<0.05.

The following glycosaminoglycan synthesis data were obtained at various time points (72, 48 and 24 h) for concentrations of the test composition of example 1 of 5.13% and 0.855%. Data are presented as a mean of glycosaminoglycan synthesis in μg GAGs / per 50 μI of the medium.

Table 7

The results obtained indicate that cell cultures treated with the composition of example 1 at concentrations of 5.13% and 0.855% showed a significant increase in glycosaminogiycan ex novo synthesis compared to the control sample. The increase in the exposure time to the composition of example 1 resulted in an increased glycosaminogiycan synthesis for both concentrations tested. The greatest increases in glycosaminogiycan synthesis were obtained after a 48-hour exposure to the composition of example 1 and were as follows: a 63.6% increase for the concentration of the composition of 5.13% and a 64.6% increase for the concentration of the composition of 0.855%, compared to the negative control.

Example 9

Study of the repair effect of the composition of example 1 by testing the effect on the wound healing time (an in-vitro test).

The study described in this report concerns with the determination of the in vitro capability of the composition to promote wound healing induced in an in vitro experimental model represented by human skin Keratinocytes in monolayer. The repairing efficacy of the composition was assessed by its ability to promote the healing of damaged tissue in the in vitro system.

The test was performed using human skin fibroblasts (ATCC-CRL-2703) on which wounds were created and then exposing to the composition of example 1.

Preparation of the test composition of example 1: The test composition of example 1 was prepared by diluting it to final concentrations of 5.13% (as in example 2) and 0.855% (as in example 3).

Control (negative; CTR-): cell cultures without created wounds.

The culture medium containing the composition of example 1, at concentrations of 5.13% and 0.855%, was applied to a (96-well) test plate containing cells in confluence. The cells were exposed to these solutions for 24 hrs. After the incubation period, the (96-well) test plate was washed with PBS buffer, stained with 1 mg/ml MTT and incubated for three hours at a temperature of 36.7°C and in a 5% CO₂ atmosphere. The wells were then treated with isopropanol.

The plate was shaken on a rotatory plate for 30 minutes, in order to ensure that all the crystals have dissolved from the cells and have formed a homogeneous solution. The absorbance is measured at 540 nm on a microplate reader. The results are expressed as % cell viability compared to an untreated control cell culture.

For repair effect tests, human skin keratinocyte cells were used that were placed in a culture flask filled with Eagle's medium in the Dulbecco (DMEM) variant, with added 10% fetal bovine serum. The cells were incubated at a temperature of 37 °C under 5% CO₂ until complete confluence.

The artificial wound was created mechanically on a cell monolayer by moving the pipette tip over it. For each experimental condition 3 replica were performed.

The repair effect of the composition of example 1 was assessed by taking a series of photographic images of cell cultures at T0, T2 hrs, T8 hrs and T24 hrs following wound creation.

At each experimental time, cells were visually checked using (OPTIKA, XDS-2) at 10X and pictures with camera (TiEsseLab PrimoCam HD 5.0 with sensor Micron MT9P001) were collected. Pictures were analysed with Image J software. The distances between the wound margins for each picture were measured in μm and were recorded in 3 points of the wound length. The variation on wound margins were analysed by T-test. Some representative images of the wound healing progress are reported for the different experimental conditions at all the monitored times.

Measurements were made by measuring the distance between the edges of the wound created (measurement in μm).

Measurements of the distance between the wound edges were obtained as follows:

Table 8 The results obtained were used to determine the percentage change, i.e. reduction of the artificial wound edge distance for both concentration values of the test composition of example 1 and for the negative control (CTR-) in individual time intervals with respect to time T0 hrs.

Table 9

It was observed that treatment of cell cultures with the composition of example 1 significantly accelerates wound healing.

According to the obtained wound healing kinetics, the repair process in cells treated with the composition of Example 1 was more efficient for both concentrations of the composition used. Compared to the negative control, the distance between the edges of the wounds created was reduced by 3.1% and 3.9% for a time T2 hour and concentrations of 5.13% and 0.85% of the composition of example 1, respectively. The healing process was significantly increased for longer exposure times to the composition of example 1 applied and amounted to: 12.1% (using a composition concentration of 5.13%) and 23.5% (using a composition concentration of 0.855%) fortime T8 hours and to 36.8% (using a composition concentration of 5.13%) and 47.0% (using a composition concentration of 0.855%) for time T24 hours. The values obtained indicate a significant increase in wound healing efficiency compared to the control sample, in particular after 8 and 24 hours of exposure to the composition of Example 1.

Example 10

Study of the modulation on SIRT-1 expression by the composition in cell culture (an in-vitro test).

The ability of the test composition at concentrations of 5.13% and 0.855% to modulate SIRT-1 expression in human skin fibroblasts was measured.

Sirtuins, a sub-family of enzyme proteins, have histone deacetylase activity or monoribosyltransferase activity. They are located in the cytoplasm, cell nucleus, nucleolus, and mitochondria. The effect of Sirtuins involves suppression of ageing processes, cellular protection, sugar metabolism and regulation of the cell cycle. Unlike other known protein deacetylases, which simply hydrolyse acetyl-lysine residues, the Sirtuin-mediated deacetylation hydrolyses acetyl-lysine and NAD+. This hydrolysis reaction yields a deacetylated substrate, O-acetyl-ADP-ribose and nicotinamide, which inhibits Sirtuin activity itself. Research suggests that human Sirtuins may act as intracellular DNA-reducing regulatory proteins.

Sirtuin activity was assessed using a commercially available fluorometric kit based on a calibration curve plotted with known and increasing concentrations of the reaction product. Moreover, total HDAC (histone deacetylase inhibitors) activity was measured using the fluorometric kit.

Results were expressed as "HDAC activity" and "Sirtuin activity" (values expressed as pmol/well) in 50 μI of cell homogenate.

The obtained results were subjected to statistical analysis through the Student test. All the variations with regard to the negative and positive control were considered statistically significant for p<0.05. Preparation of the test composition of example 1: The composition of Example 1 was prepared by diluting it in culture medium so as to obtain final concentrations of 5.13% and 0.855%.

Negative control (CTR-): cell cultures.

Positive control (CTR+): cell cultures exposed to the stress-inducing agent H₂O₂ at a concentration of 150 μM.

The cell cultures were treated with a stress-inducing agent (for 72 hs) H₂O₂ at a concentration of 150 μM and then treated with the test composition of example 1, and the amount of SIRT 1 was measured. Cells were seeded onto a test plate (96-well), in complete culture medium (DMEM) with added bovine fetal serum. After 24 hours, the cells were treated with the stress-inducing agent H₂O₂ and with the composition of example 1, at concentrations of 5.13% and 0.855%. The samples were incubated for 72 h.

Sirtuin activity was assessed using a commercially available fluorescence spectroscopy kit based on a calibration curve. In addition, total HDAC activity was assessed using fluorescence spectroscopy.

Table 10

Cell cultures treated with the damaging agent showed a significant reduction in SIRT 1 expression, at an average level of 1.18 ng/ml. Cell cultures treated with the damaging agent and the composition of example 1, in particular at a concentration of 0.855% showed a significantly higher level of SIRT 1 expression, at 3.19 ng/ml compared to the positive control (CTR+) after 72 hrs of incubation as well as comparable to the negative sample (CTR-) (4.52 ng/ml), showing that the composition of example 1 can enhance the synthesis and activity of Sirtuin-1 under oxidative stress conditions.

Synergy of the inventive composition

In order to determine the synergistic effect of combining the three ingredients of the composition, appropriate experiments were carried out for the individual components of the composition, in particular the Study of the cell proliferation activity in cell culture and Study of glycosaminoglycan synthesis (an in-vitro tests).

To evaluate the results in terms of the occurrence of synergy the Kull's equation was used [Kull, F.C., et. al in Applied Microbiology 9:538-541 (1961)]: SI - synergy index

W_A-the weight fraction of the component A W_B-the weight fraction of the component B W_C-the weight fraction of the component C

R_A - result of the tested property for component A at a weight fraction W_A R_B - result of the tested property for component B at a weight fraction W_B R_C - result of the tested property for component C at a weight fraction W_C R_A+B+C - the result of the tested property for the composition of ingredient A, B i C.

According to the known approach to determining synergy from the Kull equation, if SI is greater than 1 it means that the combination of components meets the synergy. For the calculations the w value were taken from Example 1: for RNA sodium salt W_A = 0.7042% w/w, for hyaluronic acid W_B = 0.2812% w/w, for exopolysaccharides W_C = 0.0141% w/w.

Example 11

Study of the synergistic effect of the composition - cell proliferation activity

In order to demonstrate the synergy of the composition ingredients, study of the effect on the level of cell proliferation for three components of the composition according to the invention individually were performed. Test were carried out identical as Example 5 but taking individual ingredient in appropriate concentration as in Example 5.

Results are shown in Table 11 below.

Table 11

Si synergy index in any case is greater than 1. Therefore it has been shown that synergy exists in the composition according to the invention in relation to the significant increase of cell proliferation.

Example 12

Study of the synergistic effect of the composition - GAG synthesis

In order to demonstrate the synergy of the composition ingredients, study of the effect on the level of GAG synthesis for three components of the composition according to the invention individually were performed. Test were carried out identical as Example 8 but taking individual ingredient in appropriate concentration as in Example 8.

Results are shown in Table 12 below.

Table 12

Si synergy index is greater than 1. Therefore it has been shown that synergy exists in the composition according to the invention in relation to the increase of GAG synthesis.

Claims

Claims 1. A cosmetic composition containing: a) RNA sodium salt, b) hyaluronic acid, c) exopolysaccharides. 2. The composition according to claim 1 characterised in that it contains, based on dry mass: a) RNA sodium salt in an amount of 60-84.5% w/w of the total composition, b) hyaluronic acid in an amount of 15-39.5% w/w of the total composition, c) exopolysaccharides in an amount of 0.5-2.0% w/w of the total composition. 3. The composition according to claim 1 or 2, characterised in that it contains, based on dry mass a) RNA sodium salt in an amount of 70.42% w/w of the total composition, b) hyaluronic acid in an amount of 28.12% w/w of the total composition, c) exopolysaccharides in an amount of 1.41% w/w of the total composition. 4. The composition according to claim 1 or 2 or 3, characterised in that it further contains at least one cosmetically acceptable additive and is then in the form of a cosmetic concentrate for dilution. 5. The composition according to claim 4 characterised in that at least one cosmetically acceptable additive is a substance selected from the group of solvents, carriers, moisturisers, fragrances, antioxidants, lubricants, glidants, emollients, preservatives, humectants. 6. The composition according to claim 4 or 5 characterised in that it contains as additives sea water, apple water and a natural liposome system based on lecithin derived from organic soybean. 7. The composition according to claim 6 characterised in that the natural liposome system is essentially a mixture of glycerine, lecithin, pentylene glycol, sodium hydroxide, tocopherol, and water. 8. The composition according to any one of claims 4 to 7 characterised in that it contains the following ingredients: a) RNA sodium salt in an amount of 4.00-6.50% w/w b) hyaluronic acid in an amount of 1.50-3.00% w/w c) exopolysaccharides in an amount of 0.07 to 0.20% w/w d) a natural liposome system based on lecithin derived from organic soybean in an amount of 25.00-37.00% w/w e) sea water in an amount of 10.00-20.00% w/w f) apple water in an amount of 25.00-50.00% w/w 9. The composition according to claim 8 characterised in that it contains the following ingredients: a) RNA sodium salt in an amount of 5.85% w/w b) hyaluronic acid in an amount of 2.34% w/w c) exopolysaccharides in an amount of 0.12% w/w d) a natural liposome system based on lecithin derived from organic soybean in an amount of 33.22% w/w e) sea water in an amount of 15.20% w/w f) apple water in an amount of 43.27% w/w 10. The composition according to any one of claims 1 to 9 characterised in that the exopolysaccharides are of marine origin. 11. The composition according to any one of claims 1 to 10 characterised in that the exopolysaccharides are essentially a mixture of saccharide isomerate and benzyl alcohol. 12. The composition according to any one of claims 1 to 11 characterised in that the RIMA sodium salt is derived from baker's yeast. 13. The composition according to any one of claims 1 to 12 characterised in that the hyaluronic acid is in the form of a salt. 14. The composition according to any one of claims 1 to 13 characterised in that the hyaluronic acid is in the form of a hydrolysate. 15. The composition according to claim 14 characterised in that the hyaluronic acid is in the form of a hydrolysate with an average molecular weight of 100 to 300 kDa. 16. The composition according to any one of claims 1 to 15 characterised in that all its ingredients are of natural origin. 17. A cosmetic preparation containing the composition defined by any of the claims 1 to 16. 18. The cosmetic preparation according to claim 17 in the form of a serum or cream, characterised in that it contains a cosmetic composition as defined by any of claims 8 to 16 at a concentration of 4 to 6% w/w and excipients up to 100%.

19. The cosmetic preparation according to claim 18 in the form of a serum or cream, characterised in that it contains a cosmetic composition at a concentration of 5.13% w/w and excipients up to 100%. 20. The cosmetic preparation according to claim 17 in the form of a mask, characterised in that it contains a cosmetic composition as defined by any of claims 8 to 16 at a concentration of 0.6- 1.0% w/w and excipients up to 100%. 21. The cosmetic preparation according to claim 20 in the form of a mask characterised in that it contains a cosmetic composition at a concentration of 0.855% w/w and excipients up to 100%. 22. The cosmetic preparation according to any one of claims 17 to 21 characterised in that the excipients are solvents, thickeners, emollients, emulsifiers, humectants, antioxidants, free radical scavengers, pH adjusters, preservatives, fragrances, plant-derived extracts. 23. Use of the cosmetic composition as defined in any one of claims 1 to 16 for skin care, in particular to improve skin appearance, skin elasticity, skin condition, hydration level, tone skin, smoothen skin, soften skin, reduce the appearance of imperfections - wrinkles and/or enlarged skin pores. 24. Use of the cosmetic preparation as defined in any one of claims 17 to 22 for skin care, in particular to improve skin appearance, skin elasticity, skin condition, hydration level, tone skin, smoothen skin, soften skin, reduce the appearance of imperfections and wrinkles. 25. Use of an RNA sodium salt for the preparation of the cosmetic composition as defined in any one of claims 1 to 16 or cosmetic preparations as defined in any one of claims 17 to 22 for skin care, in particular to improve skin appearance, skin elasticity, skin condition, hydration level, tone skin, smoothen skin, soften skin, reduce the appearance of imperfections and wrinkles.