RU2812891C2 - Compositions for topical application containing pichia anomala and retinol - Google Patents

Abstract

FIELD: cosmetic industry.

SUBSTANCE: group of inventions relates to a composition for topical application to human skin to increase the amount of hyaluronic acid produced by the skin and to a method of increasing the amount of hyaluronic acid produced by the skin. A composition for topical application to human skin to increase the amount of hyaluronic acid produced by the skin, containing a 5% aqueous solution of Pichia anomala extract, with the content of Pichia anomala extract in the composition from 0.065 to 0.26% by weight, and from 0.05 to 1% by weight of retinol, while the weight ratio of Pichia anomala extract and retinol in the composition for topical application is 1.3. A method of increasing the amount of hyaluronic acid produced by the skin upon contact with an extract of Pichia anomala, comprising bringing the skin into contact with a composition for topical application according to claim 1.

EFFECT: above composition effectively increases the amount of hyaluronic acid produced by the skin and stimulates the expression of HAS2, CD44.

6 cl, 7 tbl, 3 ex

Description

FIELD OF APPLICATION OF THE INVENTION

The present invention provides a method of treating skin by topically applying a combination of Pichia anomala extract and retinol to the skin. Additionally, a composition for external use is provided containing a combination of Pichia anomala extract and retinol.

PREREQUISITES FOR CREATION OF THE INVENTION

Hyaluronic acid is found in the skin in the peripheral zone of collagen and elastin fibers, as well as at the intersection of such fibers. Hyaluronic acid is localized not only in the dermis, but also in the intercellular spaces of the epidermis, especially in the intermediate spinous layer, but not in the stratum corneum (SC) or stratum granulosum. As we age, the concentration of hyaluronic acid in the skin decreases and it becomes separated from collagen and elastin. Skin containing reduced concentrations of hyaluronic acid also exhibits reduced water binding, which may be related to the changes seen in mature skin, including wrinkles, altered elasticity, decreased firmness, and decreased ability to maintain the skin's microvasculature. As a major GAG, hyaluronic acid can bind up to 1,000 times its own weight in water, helping skin retain and retain moisture. It is found throughout connective tissue and is synthesized mainly by fibroblasts and keratinocytes found in the skin.

Various treatments have been proposed to combat wrinkles and fine lines, including injecting hyaluronic acid. Exogenous hyaluronic acid injections are used as a temporary dermal filler in contouring procedures. However, the injected hyaluronic acid has a limited duration. On the other hand, penetration of exogenous hyaluronic acid into the skin has proven difficult to achieve through topical application.

Pichia is a genus of yeast in the family Saccharomycetaceae. More than 100 species of this genus are known. The best known species include Pichia anomala , Pichia guilliermondii , Pichia norvegensis and Pichia ohmeri .

Pichia anomala (formerly called Hansenula anomala ) can be found in unboiled milk and cheese. Yeast extracts of the genus Pichia have a high content of mannans, polysaccharides consisting of mannose monomers. Pichia anomala and mannans are known to be used to treat aging skin. See, for example, FR 2938768, FR 2906719, FR 2897266 and FR 2976490.

PRO-LIPISKIN® is a commercially available cosmetic ingredient containing Pichia anomala extract. It is produced using a strain of Pichia isolated from sugarcane. It can be purchased from Silab-France.

US Patent 2017/0172913A1 covers topical compositions containing combinations of Pichia anomala extract and chicory root extract that provide increased hyaluronic acid production, as well as methods for treating signs of skin aging and improving skin barrier protection and skin hydration.

Retinoids, including retinol, are well-known anti-aging active ingredients. They are used in various cosmetic products for topical application. Retinol is used, for example, in NEUTROGENA® Rapid Wrinkle Repair®, commercially available from Johnson & Johnson Consumer Inc., NEUTROGENA® Rapid Wrinkle Repair® is used to eliminate the appearance of skin wrinkles, smooth fine lines, improve skin texture and brighten skin tone . Retinol in particular has proven to be a highly effective and cost-effective cosmetic ingredient.

Pichia anomala extract and retinol act through different biological mechanisms and lead to improvements in various beneficial clinical effects. Retinol is known as the gold standard for anti-aging products and is known to act through a biological pathway involving the markers CRABP2 and HBEGF. Pichia anomala extract works through the hydration pathway by increasing the activity of the hyaluronic acid synthase 2 enzyme, resulting in beneficial clinical effects including hydration, skin barrier improvement, firmness, etc.

Although topical application of Pichia anomala and retinol extracts individually has been proposed in the art, applicants have now discovered that topical application of a combination of these two ingredients beneficially enhances the activity of retinol and the activity of Pichia anomala extract. This approach unexpectedly provides significant benefits to the skin, including improving, reducing, inhibiting or delaying the appearance of at least one sign of skin aging, as well as improving skin barrier protection and skin hydration. Accordingly, new treatments are now available for, for example, signs of skin aging.

SUMMARY OF THE INVENTION

The present invention relates to a composition for external use containing Pichia anomala extract and retinol.

The present invention also relates to a method for reducing the signs of skin aging, comprising topically applying to skin requiring treatment due to skin aging, a topical composition containing Pichia anomala extract and retinol.

The present invention further provides a method for improving skin barrier function and hydration, comprising topically applying to skin in need of improved skin barrier function and hydration a topical composition containing Pichia anomala extract and retinol.

DETAILED DESCRIPTION

The topical composition of the present invention improves the production of hyaluronic acid in the skin as a result of the synergistic effect of Pichia anomala extract and retinol.

It is believed that one skilled in the art, based on the description provided herein, will be able to use the present invention to its fullest extent. The specific embodiments presented below are to be considered as examples only and are not intended to limit the remainder of the description in any way.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention relates. In addition, all publications, patent applications, patents and other materials referenced herein are incorporated herein by reference. Unless otherwise indicated, percentages used to express amounts of ingredients are percentages by weight (i.e., % (w/w)). Likewise, weight ratios used to express the relative proportions of ingredients are also determined using weight percentages (i.e., weight ratios are calculated by dividing the weight percentage of one ingredient by the weight percentage of another ingredient). Unless otherwise stated, all ranges are subject to limits, for example the range "4 to 9" includes limits 4 and 9.

As used herein, “product” does not necessarily mean a finished product in packaged form. In one embodiment of the invention, the package is a container, such as a plastic, metal or glass tube or jar, containing the composition. The product may also have additional packaging, such as a plastic or cardboard box, in which the container is placed. In one embodiment, the product includes a composition of the present invention and contains instructions for the consumer to apply the composition to the skin or hair.

As used herein, the term "topical application" means direct application or distribution to the outer surface of the skin, scalp, or hair, for example, by hand or using an applicator such as a wipe, roller, or aerosol.

As used herein, the term “cosmetic” refers to an appearance-enhancing substance or preparation that maintains, restores, imparts, mimics or enhances physical attractiveness or produces an attractiveness or youth-enhancing effect, particularly with respect to the appearance of tissue or skin.

As used herein, the term “cosmetically acceptable” means that the ingredients that the term describes are acceptable for use in contact with tissue (e.g., skin or hair) without causing undesirable toxic effects, incompatibility, instability, irritation, allergic reactions, etc.

In some embodiments, the compositions of the present invention are useful for treating signs of skin aging. As used herein, the term “signs of skin aging” includes the presence of furrows and wrinkles, loss of elasticity, uneven skin tone and blotchiness. In a particularly preferred embodiment, a sign of aging is the presence of folds, wrinkles and/or loss of elasticity.

As used herein, the term “reversing signs of skin aging” refers to reducing, attenuating, preventing, correcting, or eliminating the presence of the signs of skin aging described above.

As used herein, the term “wrinkle” refers to fine lines, fine wrinkles or deep wrinkles. Examples of wrinkles include, but are not limited to, fine lines around the eyes (such as crow's feet), forehead and cheek wrinkles, glabellar furrows, and facial furrows around the mouth.

As used herein, the term “loss of elasticity” includes loss of elasticity or structural integrity of the skin or tissue, including, but not limited to, sagging, laxity, and laxity of the tissue. Loss of elasticity or integrity of tissue structure may result from a number of factors including, but not limited to, disease, aging, hormonal changes, mechanical trauma, environmental stress, or may result from the application of products such as cosmetics or pharmaceuticals to the tissue.

As used herein, the term “uneven skin tone” refers to a skin condition associated with diffuse or patchy pigmentation that can be classified as hyperpigmentation, such as post-inflammatory hyperpigmentation.

As used herein, the term "mottling" refers to a skin condition associated with redness or erythema.

As used herein, “increasing skin strength” means increasing skin strength or elasticity, preventing loss of skin strength or elasticity, or preventing or eliminating skin sagging, laxity, and laxity. The strength or elasticity of the skin can be measured using a cutometer. See Handbook of Non-Invasive Methods and the Skin, eds . J. Serup, G. Jemec & G. Grove, Chapter 66.1 (2006). Loss of skin elasticity or strength can be caused by a number of factors, including, but not limited to, aging, environmental damage, or the use of cosmetics.

As used herein, the term “improving skin texture” means smoothing the surface of the skin to eliminate ridges or depressions on the surface of the skin.

As used herein, the term “improving the appearance of skin wrinkles” means preventing, reducing the severity of, stopping the progression of, or reversing the formation of wrinkles and fine lines on the skin.

As used herein, “safe and effective amount” means an amount sufficient to produce the desired effect but low enough to prevent serious side effects. The safe and effective amount of a compound, extract or composition varies depending, for example, on the age, health status of the end user, as well as exposure to environmental conditions, duration and nature of treatment, the specific extract, component or composition used, the particular carrier used and similar factors.

As used herein, “skin requiring enhanced skin barrier function and hydration” refers to skin that is characterized by, without limitation, lack of hydration, lack of sebaceous gland secretions, cracks, dryness, itching, flaking, xeroderma, dehydration, lack of elasticity, lack of shine, dullness or lack of lipids.

As described herein, applicants have discovered that topical application of a combination of Pichia anomala extract and retinol provides unexpectedly good skin barrier function, skin hydration, and provides beneficial anti-aging effects to the skin.

Applicants have also discovered that, in particular, topical application of a composition containing a combination of Pichia anomala extract and retinol increases the concentrations of endogenous hyaluronic acid (HA) in the skin, which provides improvements in hydration and appearance in relation to at least one sign of skin aging. Topical application of such a composition can increase hyaluronic acid levels to values approaching those of youthful skin, thereby providing the structural framework of the skin necessary to reduce the signs of skin aging.

Pichia anomala

The topical composition contains one or more extracts of Pichia anomala . In particular, such extracts may be extracts obtained using one of the various strains of Pichia anomala isolated from the fruit or other aerial parts of the plant. Any cosmetically acceptable extract of Pichia anomala may be used.

One example of a suitable Pichia anomala extract is PRO-LIPISKIN, commercially available from Silab-France. It is produced from a strain of Pichia anomala , present on sugarcane.

Another example of a suitable Pichia anomala extract is obtained from a strain of Pichia anomala found in kiwi fruit or leaves.

The Pichia anomala extract may be presented as a solution containing a dry matter (extract) ranging from about 20% by weight, more particularly from 2 to 10% by weight, most particularly from 3 to 7% by weight.

Solvents for such solutions include water, alcohols, glycols, and the like. In one embodiment, the solvent constitutes at least about 90% by weight. water or at least about 95% of the mass. water.

Retinol

The topical composition also contains retinol. Retinol has the formula:

where R represents CH ₂ OH.

Quantities

Any suitable amount of Pichia anomala extract and retinol may be used in the compositions of the present invention. The compositions preferably contain safe and effective amounts of both ingredients. In particular, the amounts of Pichia anomala extract and retinol used are cosmetically acceptable and selected to achieve the desired treatment of a skin condition for a particular condition, such as signs of aging, decreased barrier function, or decreased hydration.

In certain preferred embodiments, the compositions contain from about 0.01 to about 1% by weight of Pichia anomala extract, more preferably from about 0.065 to about 0.26% by weight of Pichia anomala extract. In one embodiment, the composition contains about 0.13% by weight of Pichia anomala extract.

In some preferred embodiments, the compositions contain from about 0.01% to about 10% by weight, more preferably from about 0.05 to about 1% by weight of retinol. In one embodiment, the composition contains about 0.1% by weight of retinol.

In some embodiments, the weight ratio of Pichia anomala extract to retinol in the compositions is from about 0.5 to about 2. In one embodiment, the weight ratio of Pichia anomala extract to retinol in the compositions is about 1.3.

Compositions for topical application

The compositions of the present invention are applied topically to human skin or hair. Accordingly, the composition may further include a cosmetically acceptable topical carrier, which may comprise from about 50% to about 99.99% by weight. composition (eg, from about 80% to about 99% by weight of the composition). In a preferred embodiment of the invention, the cosmetically acceptable topical carrier comprises water.

The compositions may be formulated in a variety of product types, including, without limitation, lotions, creams, gels, sticks, aerosols, ointments, cleansing liquids and soap bars, shampoos and hair conditioners, hair fixatives, pastes, foams, powders , mousses, shaving creams, wipes, plasters, hydrogels, film-forming products, face masks and skin masks, films and decorative cosmetics such as makeup bases and mascaras. These types of products may contain several types of cosmetically acceptable carriers for topical use, including, but not limited to, solutions, suspensions, emulsions such as microemulsions and nanoemulsions, gels, solids, and liposomes. The following are non-limiting examples of such media. Other carriers may be included in the formulations by those skilled in the art.

The compositions used in the present invention may be formulated as solutions. Solutions typically include an aqueous or organic solvent (eg, about 50% to about 99.99%, or about 90% to about 99% cosmetically acceptable aqueous or organic solvent). Examples of suitable organic solvents include propylene glycol, polyethylene glycol, polypropylene glycol, glycerin, 1,2,4-butanetriol, sorbitol esters, 1,2,6-hexanetriol, ethanol, and mixtures thereof.

The compositions used as an object of the invention can be formulated as a solution containing a softener. Such compositions preferably contain from about 2% to about 50% softener(s). As used herein, the term “emollients” means substances used to prevent or eliminate dryness, for example, by preventing transepidermal water loss from the skin. Examples of such softeners include those known to those skilled in the art. Examples of particularly suitable softeners include vegetable oils, mineral oils, fatty acid esters, and the like.

A lotion can be made from such a solution. Lotions typically contain from about 1% to about 20% (for example, from about 5% to about 10%) emollient(s) and from about 50% to about 90% (for example, from about 60% to about 80 %) water.

Another type of product that can be prepared from a solution is a cream. Typically, the cream contains from about 5% to about 50% (for example, from about 10% to about 20%) emollient(s) and from about 45% to about 85% (for example, from about 50% to about 75% ) water.

The composition of the present invention may include water, or alternatively it may be anhydrous or may be an ointment that does not include water but contains organic and/or silicone solvents, oils, lipids and waxes. The ointment may contain a simple base of animal or vegetable oils or semi-solid hydrocarbons. The ointment may contain from about 2% to about 10% softener(s) and from about 0.1% to about 2% thickener(s).

The composition can be prepared in the form of an emulsion. If the topical carrier is an emulsion, then about 1% to about 10% (eg, about 2% to about 5%) of the topical carrier is emulsifier(s). Emulsifiers can be non-ionic, anionic or cationic. Examples of emulsions are known to those skilled in the art.

Lotions and creams can be prepared as emulsions. Typically, such lotions contain from 0.5% to about 5% emulsifier(s). Such creams typically contain from about 1% to about 20% (eg, from about 5% to about 10%) emollient(s); about 20% to about 80% (eg, 30% to about 70%) water; and about 1% to about 10% (eg, about 2% to about 5%) emulsifier(s).

Oil-in-water and water-in-oil skin care formulations based on a single-phase emulsion, such as lotions and creams, are well known in the cosmetic field and can be used in the claimed invention. Compositions of the "water-in-oil-in-water" or "oil-in-water-in-oil" type based on a multiphase emulsion are also used in the claimed invention. Essentially, such single- or multi-phase emulsions contain water, softeners and emulsifiers as the main ingredients.

The compositions of the present invention may also be formulated as a gel (eg, an aqueous, alcoholic, hydroalcoholic, or oily gel using a suitable gelling agent(s)). Suitable gelling agents for aqueous and/or alcoholic gels include, but are not limited to, natural gums, acrylic acid, and acrylate polymers and cellulose-derived copolymers (eg, hydroxymethylcellulose and hydroxypropylcellulose). Suitable gelling agents for oils (such as mineral oil) include, but are not limited to, hydrogenated butylene/ethylene/styrene copolymer and hydrogenated ethylene/propylene/styrene copolymer. In such gels, the content of these gelling agents is typically from about 0.1% to 5% by weight.

In one embodiment, the composition is a cream-gel. The aesthetic effect of the cream-gel is characterized by the creation of a watery, destructible, translucent texture and a light residual feeling on the skin after application. As used herein, the term “cream-gel” means a composition with a low content of oil droplets suspended in an aqueous gel matrix.

The compositions of the present invention may also be formulated as a solid composition (eg, a wax-based stick, a soap bar composition, a powder, or a wipe containing a powder).

The compositions may contain, in addition to the above components, a wide range of additional lipid-soluble materials and/or water-soluble materials conventionally used in skin and hair care compositions in concentrations known to those skilled in the art.

Additional cosmetically active agents

The compositions of the present invention may further contain any of various additional cosmetically active agents. Examples of suitable additional active agents include skin lightening agents, darkening agents, additional anti-aging agents, tropoelastin stimulants, collagen stimulators, anti-acne agents, skin radiance control agents, antimicrobial agents such as anti-yeast agents, antifungal and antibacterial agents, anti-inflammatory agents, antiparasitic agents, topical analgesics, sunscreens, photoprotectants, antioxidants, keratolytic agents, detergents/surfactants, humectants, nutrients, vitamins, energy-enhancing agents, antiperspirants, pore tighteners, deodorants, hair removers , hair growth promoting agents, hair growth retarding agents, thickeners, hydration enhancers, performance enhancers, anti-skin agents, skin conditioning agents, anti-cellulite agents, odor control agents such as odor masking agents or pH changing agents, etc. . P.

Examples of various suitable additional cosmetically acceptable active agents include hydroxy acids; benzoyl peroxide; D-panthenol; UV filters such as, but not limited to, avobenzone (Parsol 1789), disodium bisdisulisole (Neo Heliopan AP), diethylaminohydroxybenzoylhexyl benzoate (Uvinul A Plus), ecamsul (Mexoryl SX), methyl anthranilate, 4-aminobenzoic acid (PABA), cinoxate, ethylhexyl triazone (Uvinul T 150), homosalate, 4-methylbenzylidene camphor (Parsol 5000), octyl methoxycinnamate (Octinoxate), octyl salicylate (Octisalate), padimate O (Escalol 507), phenylbenzimidazole sulfonic acid (Ensulizole), polysilicone-15 (Parsol SLX), trolamine salicylate, bemotrizinol (Tinosorb S), benzophenones 1-12, dioxybenzone, drometrisoltrisiloxane (Mexoryl XL), iscotrizinol (Uvasorb HEB), octocrylene, oxybenzone (Eusolex 4360), sulisobenzone, bisoctrizol (Tinosorb M), titanium dioxide, zinc oxide; carotenoids; free radical scavengers; spin traps; Other retinoids and retinoid precursors such as retinoic acid and retinyl palmitate; ceramides; polyunsaturated fatty acids; essential fatty acids; enzymes; enzyme inhibitors; minerals; hormones such as estrogens; steroids such as hydrocortisone; 2-dimethylaminoethanol; copper salts such as copper chloride; peptides containing copper, coenzyme Q10; amino acids such as proline; vitamins; lactobionic acid; acetyl coenzyme A; niacin; riboflavin; thiamine; ribose; electron carriers such as NADH (nicotinamide adenine dinucleotide) and FADH2 (reduced form of flavin adenine dinucleotide); and other plant extracts such as oat extracts, aloe vera, feverfew, soybean, shiitake mushrooms, as well as their derivatives and mixtures.

In some preferred embodiments, the compositions contain a combination of Pichia anomala extract, retinol, and at least one additional skin moisturizing active agent.

In certain preferred embodiments, the compositions contain a combination of Pichia anomala extract, retinol and at least one additional agent to improve the appearance of at least one sign of skin aging. Examples of suitable adjuvant agents that improve the appearance of at least one sign of skin aging include, but are not limited to, tropoelastin stimulants, collagen stimulators, retinoids, hyaluronic acid, including cross-linked hyaluronic acid, chondroitin sulfate, including cross-linked chondroitin sulfate, dimethylaminoethanol, N ,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, alpha-hydroxy acids, polyhydroxy acids, sugar amines and combinations of two or more of these.

As used herein, the term “tropoelastin stimulants” refers to a class of compounds that have biological activity associated with enhancing the production of tropoelastin. In accordance with the present invention, tropoelastin stimulants include all natural or synthetic compounds capable of increasing the production of tropoelastin in the human body.

Examples of suitable tropoelastin stimulants include, but are not limited to, blackberry extracts, mackerel extracts, feverfew extracts, and bimetallic complexes containing copper and/or zinc moieties. The bimetallic complex containing copper and/or zinc components may be, for example, copper-zinc citrate, copper-zinc oxalate, copper-zinc tartrate, copper-zinc malate, copper-zinc succinate, copper-zinc malonate, copper-zinc maleate. zinc, copper-zinc aspartate, copper-zinc glutamate, copper-zinc glutarate, copper-zinc fumarate, copper-zinc glucarate, copper-zinc polyacrylate, copper-zinc adipate, copper-zinc pimelate, copper-zinc suberate, copper-zinc azealate zinc, copper-zinc sebacate, copper-zinc dodecanoate or combinations thereof. In a preferred embodiment, the tropoelastin stimulator is selected from blackberry extracts, mackerel extracts, feverfew extracts, and combinations thereof. In a particularly preferred embodiment, the tropoelastin stimulator is selected from blackberry extracts, feverfew extracts, and combinations thereof.

By "blackberry extract" is meant a mixture of compounds isolated from a plant of the genus Rubus , preferably Rubus fruticosus . In one embodiment, the compounds are isolated from the flowers of the plant. In a further embodiment, the compounds are isolated from the dried flowers of the plant. Such compounds can be isolated from one or more parts of a plant (eg, the whole plant, flower, seed, root, rhizome, stem, fruit and/or leaf of the plant). In a preferred embodiment, the blackberry extract is an extract of blackberry leaves. One particularly useful blackberry extract is prepared by extracting the leaves of Rubus fruticosus with a mixture of water and ethanol with the addition of about 5% to about 10% maltodextrin matrix and is commercially available from Symrise Inc., Teterboro, NJ, under the trade name SymMatrix .

The compositions of the present invention may include a cosmetically effective amount of one or more tropoelastin stimulants, such as the stimulants described above. The compositions preferably include (based on active ingredient content) from about 0.1% to about 10% tropoelastin stimulants, more preferably from about 0.5% to about 5% tropoelastin stimulants, and most preferably from about 0.5% to about 2% stimulants tropoelastin.

As used herein, the term “collagen stimulator” refers to compounds having biological activity associated with enhancing collagen production. As used herein, “non-retinoid collagen stimulators” include all natural or synthetic compounds that are not retinoids or retinoid derivatives that are capable of increasing collagen production in the human body.

Examples of suitable collagen stimulants include, but are not limited to, the following: retinoids, extractsfeverfew (Tanacetum parthenium)), extractsAsian centella (Centella asiatica) and extractsSiegesbeckia orientalis); soy extracts; peptides that stimulate collagen formation; ursolic acid and asiaticoside.

Centella asiatica , also known as Violette marronne on Reunion Island, gotu kola or Indian rosemary in India, Centella repanda in North America and talapetraca on the island of Madagascar, is a polymorphic herbaceous plant and belongs to the umbelliferae family ( Umbelliferae ( Apiaceae )), specifically to the subfamily Hydrocotyle . This wild plant is found throughout the tropics and prefers moist and shaded areas at an altitude of about 600 to 1200 meters above sea level. There are three species of Centella asiatica : common (Typica), Abyssinica (Abyssinica) and Florida (Floridana). The plant is known for its healing, soothing, analgesic, antidepressant, antiviral and antimicrobial properties. The biological activity of the plant is apparently associated with the presence of triterpene molecules in it. An acceptable extract of Centella asiatica under the trade name TECA is produced by Bayer Consumer HealthCare, Basel, Switzerland.

By " Siegesbeckia orientalis extracts" is meant any of various extracts of the Siegesbeckia orientalis plant, including Darutoside from Sederma (Croda International Group, Edison, NJ, USA).

Suitable peptides that stimulate collagen formation include the following: Matricine peptides (i.e., peptides derived from the destruction of extracellular matrix proteins such as collagen, elastin or proteoglycan), including palmitoyl pentapeptides such as MATRIXYL, manufactured by Sederma (Croda International Group , Edison, New Jersey); GHK (glycine-histidine-lysine) peptide copper complex, manufactured by Photomedex, Montgomeryville, Pennsylvania, under the trade name PROCYTE; palmitoyl derivative of GHK peptide, manufactured by Sederma (Croda International Group, Edison, NJ ) under the trade name Biopoeptide CL; biomimetic tetrapeptides available commercially from Unipex, Quebec, Canada, under the trade name Chronoline Tri Peptide; and palmitoyl tripeptide, available commercially from DSM, Basel, Switzerland, under the trade name Syn-Coll.

Ursolic acid is also known as pentacyclic triterpene acid, Prunol, Malol, Urson, beta-ursolic acid and 3-beta-hydroxy-urs-12-ene-28-oic acid. It is commercially available, for example, from Sigma-Aldrich, St. Louis, Missouri, USA.

Asiaticoside, also known as the chemical compound [6-[[3,4-dihydroxy-6-(hydroxymethyl)-5-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxymethyl ]-3,4,5-trihydroxyoxan-2-yl]-10,11-dihydroxy-9-(hydroxymethyl)-1,2,6a,6b,9,12a-hexamethyl-2,3,4,5,6 ,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylate), available commercially, inter alia, from Bayer Santé Familiale, Division Serdex, 69, Boulevard Victor Hugo, 93400, SAINT-OUEN, France.

The compositions of the present invention may include a cosmetically effective amount of one or more collagen stimulants. The compositions preferably include (based on active ingredient content) from about 0.1% to about 10% collagen stimulants, more preferably from about 0.5% to about 5% collagen stimulants, and most preferably from about 0.5% to about 2% stimulants collagen.

The compositions of the present invention may additionally contain at least one active skin lightening agent. Examples of suitable skin lightening active agents include, but are not limited to, tyrosinase inhibitors, melanin degradation promoting agents, melanosome transfer inhibitory agents including PAR-2 antagonists, exfoliants, sunscreens, retinoids, antioxidants, tranexamic acid, tranexamic cetyl ester hydrochloride acids, skin whitening agents, linoleic acid, disodium adenosine monophosphate, chamomile extract, allantoin, opacifiers, talcs and silicon dioxides, zinc salts, etc., and other agents described in Solano et al . Pigment Cell Res . 19 (550-571) and Ando et al. Int J Mol Sci 11 (2566-2575).

Examples of suitable tyrosinase inhibitors include, but are not limited to, vitamin C and its derivatives, vitamin E and its derivatives, kojic acid, arbutin, resorcinols, hydroquinone, flavones (e.g., licorice flavonoids, licorice root extract, mulberry root extract, Dioscorea coposita root extract, saxifrage extract, etc.), ellagic acid, salicylates and their derivatives, glucosamine and its derivatives, fullerene, hinokitiol, dicarboxylic acid, acetylglucosamine, 5,5'-dipropyldiphenyl-2,2'-diol (magnolignan), 4- (4-hydroxyphenyl)-2-butanol (4-HPB), combinations of two or more of them, etc. Examples of vitamin C derivatives include, but are not limited to, ascorbic acid and its salts, ascorbic acid 2-glucoside, sodium ascorbyl phosphate, magnesium ascorbyl phosphate and a natural extract enriched with vitamin C. Examples of vitamin E derivatives include, but are not limited to, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, delta-tocotrienol and mixtures thereof, tocopherol acetate, tocopherol phosphate and natural extracts enriched with vitamin E derivatives. Examples of resorcinol derivatives include, but are not limited to, resorcinol, 4-substituted resorcinols such as 4-alkylresorcinols such as 4-butylresorcinol (rucinol), 4-hexylresorcinol ( Synovea HR, Sytheon), phenylethylresorcinol (Symwhite, Symrise), 1-(2,4-dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylphenyl)-propane (nivitol, Unigen), etc., and also natural extracts enriched with resorcinols. Examples of salicylates include, but are not limited to, potassium 4-methoxysalicylate, salicylic acid, acetylsalicylic acid, 4-methoxysalicylic acid, and salts thereof. In some preferred embodiments, the tyrosinase inhibitors include 4-substituted resorcinol, a vitamin C derivative, or a vitamin E derivative. In more preferred embodiments, the tyrosinase inhibitor comprises phenylethylresorcinol, 4-hexylresorcinol, or ascorbyl-2-glucoside.

Examples of suitable melanin-degrading agents include, but are not limited to, peroxides and enzymes such as peroxidases and ligninases. In some preferred embodiments, the melanin inhibitory agents include peroxide or ligninase.

Examples of suitable melanosome transfer inhibitory agents include PAR-2 antagonists such as soy trypsin inhibitor or Bauman-Birk inhibitor, vitamin B3 and derivatives such as niacinamide, soy essence, whole soy, soy extract. In some preferred embodiments, the melanosome transfer inhibitory agents include soy extract or niacinamide.

Examples of exfoliants include, but are not limited to, alpha hydroxy acids such as lactic acid, glycolic acid, malic acid, tartaric acid, citric acid or any combination thereof, beta hydroxy acids such as salicylic acid, polyhydroxy acids such as lactobionic acid and gluconic acid, as well as mechanical exfoliants such as microdermabrasion. In certain preferred embodiments, the exfoliants include glycolic acid or salicylic acid.

Examples of retinoids other than retinol include, but are not limited to, retinal (the aldehyde form of vitamin A), retinyl acetate, retinyl propionate, retinyl linoleate, retinoic acid, retinyl palmitate, isotretinoin, tazarotene, bexarotene, adapalene, combinations of two or more of these, and the like. In some preferred embodiments, the retinoid is selected from the group consisting of retinal, retinyl acetate, retinyl propionate, retinyl linoleate, and combinations of two or more of these.

Examples of antioxidants include, but are not limited to, water-soluble antioxidants such as sulfhydryl compounds and their derivatives (eg, sodium metabisulfite and N-acetylcysteine, glutathione), lipoic acid and dihydrolipoic acid, stilbenoids such as resveratrol and its derivatives, lactoferrin, chelating agents iron and copper; and ascorbic acid and ascorbic acid derivatives (eg ascorbyl-2-glucoside, ascorbyl palmitate and ascorbyl polypeptide). Fat-soluble antioxidants suitable for use in the compositions of the present invention include, but are not limited to, butylated hydroxytoluene, retinoids, tocopherols ( eg , tocopherol acetate), tocotrienols, and ubiquinones. Natural extracts containing antioxidants suitable for use in the compositions of the present invention include, but are not limited to, extracts containing flavonoids and isoflavonoids, as well as their derivatives (for example, genistein and diadzein), extracts containing resveratrol, etc. Examples of such natural extracts include grape seed extract, green tea, black tea, white tea, pine bark, feverfew, parthenolide-free feverfew extract, oat extract, blackberry extract, mackerel extract, soybean extract, pomelo extract, wheat germ extract, hesperidin, grape extract, purslane extract, licochalcone, chalcone, 2,2'-dihydroxychalcone, primrose extract, propolis, etc.

The additional cosmetically active agent may be present in the composition in any suitable amount, for example in an amount from about 0.0001% to about 20% by weight. composition, for example from about 0.001% to about 10%, in particular from about 0.01% to about 5%. In certain preferred embodiments, in an amount of from 0.1% to 5%, and in other preferred embodiments, in an amount from 1% to 2%.

The compositions of the present invention may include a cosmetically effective amount of one or more anti-inflammatory compounds.

Examples of suitable anti-inflammatory agents include substituted resorcinols, (E)-3-(4-methylphenylsulfonyl)-2-propenenitrile (such as Bay 11-7082 from Sigma-Aldrich, St. Louis, MO), tetrahydrocurcuminoids ( such as Tetrahydrocurcuminoid CG from Sabinsa Corporation, Piscataway, NJ), extracts and substances derived from the following raw materials: Phellodendron amurense bark extract (PCE), undenatured soybean ( Glycine max ), feverfew ( Tanacetum parthenium ), ginger ( Zingiber officinale ), ginkgo ( Ginkgo biloba ), madecassoside (ingredient of Centella asiatica extract), mackerel ( Cotinus coggygria ), butterbur extract ( Petasites hybridus ), goji fruit ( Lycium barbarum ), milk thistle extract ( Silybum marianum ), honeysuckle ( Lonicera japonica ), Peru balsam ( Myroxylon pereirae ), sage ( Salvia officinalis ), cranberry extract ( Vaccinium oxycoccos ), amaranth oil ( Amaranthus cruentus ), pomegranate ( Punica granatum ), yerba mate ( Ilex paraguariensis leaf extract), white lily flower extract ( Lilium candidum ), olive leaf extract ( Olea europaea ), phloretin (apple extract), oat flour ( Aveena sativa ), Lifenol extract (hops: Humulus lupulus ), Bugrane P ( Ononis spinosa ), licochalcone (licorice: component of Glycyrrhiza inflate extract) , simrelifa (bisabolol and ginger extract), combinations of two or more of them, etc.

In one embodiment, the anti-inflammatory agent is resorcinol. Particularly suitable substituted resorcinols include 4-hexylresorcinol and 4-octylresorcinol, especially 4-hexylresorcinol. 4-Hexylresorcinol under the trade name SYNOVEA HR is commercially available from Sytheon, Lincoln Park, NJ, USA. 4-Octylresorcinol is commercially available from City Chemical LLC, West Haven, Connecticut, USA.

By "pyrethrum extracts" is meant extracts of the plant Tanacetum parthenium, which can be obtained, for example, in accordance with the detailed information set out in US patent application publication No. 2007/0196523 entitled PARTHENOLIDE FREE BIOACTIVE INGREDIENTS FROM FEVERFEW (TANACETUM PARTHENIUM) AND PROCESSES FOR THEIR PRODUCTION. One particularly useful extract of feverfew, in a form containing about 20% active feverfew, is commercially available from Integrated Botanical Technologies, Ossining, NY, USA.

In the skin care composition of the present invention, the ratio of the amounts of the combination of Pichia anomala extract and retinol to the amount of the anti-inflammatory compound may vary. For example, the extract and the anti-inflammatory compound may be present in a weight ratio (which is determined by dividing the amount by weight of the dry extract by the amount by weight of the anti-inflammatory compound) of about 0.001 to about 100, preferably from about 0.01 to about 10 or more preferably from about 0.25 to about 2.

A number of other materials may also be present in the compositions of the present invention. In certain preferred embodiments, the composition contains one or more topical components selected from the group consisting of surfactants, complexing agents, emollients, wetting agents, conditioners, preservatives, opacifiers, flavoring agents, and the like.

An emollient is defined as a compound that helps maintain the softness, smoothness, and pliability of the skin (for example, by being placed on the surface of the skin or in the stratum corneum and acting as a lubricant). Examples of acceptable emollients include those presented in Chapter 35, pp. 399-415 (Skin Feel Agents, G Zocchi) in the Handbook of Cosmetic Science and Technology (edited by A. Barel, M. Paye and H. Maibach, published in 2001 Marcel Dekker, Inc New York, NY), and include, but are not limited to, petrolatum, hexyldecyl stearate and vegetable, nut, and vegetable oils such as macadamia nut oil, rice bran oil, grape seed oil, palm oil, butter primrose, hydrogenated peanut oil and avocado oil.

By wetting agent is meant a compound intended to increase the water content in the upper layers of the skin (for example, hygroscopic compounds). Examples of acceptable moisturizers include those presented in Chapter 35, pp. 399-415 (G. Zocchi, Skin Feel Agents) in the Handbook of Cosmetic Science and Technology (edited by A. Barel, M. Paye and H. Maibach, published in 2001 Marcel Dekker, Inc New York, NY), and include, without limitation, glycerol, sorbitol or trehalose (e.g., α,α-trehalose, β,β-trehalose, α,β-trehalose) or a salt or ester thereof (e.g. trehalose-6-phosphate).

In one embodiment, the composition contains glycerin. For example, the composition contains at least 10% of the mass. glycerin. The composition may contain at least 12 wt.%. glycerin.

In another embodiment, the composition contains from 0.5 to 25 percent by weight glycerol. In one embodiment, the composition contains from 1 to 6 percent by weight glycerol.

In another embodiment, the composition has a pH of 6.5 or less. For example, the composition may have a pH of 5.5 or less.

In a specific embodiment, the composition contains at least 10% of the mass. glycerol and has a pH of 6.5 or less.

In another embodiment, the composition contains from 0.1 to 5 percent by weight cetearyl livate. In one embodiment, the composition contains from 0.1 to 2 percent by weight cetearyl livate.

In yet another embodiment, the composition contains from 0.1 to 5 percent by weight sorbitanolivate. In one embodiment, the composition contains from 0.1 to 2 percent by weight sorbitanolivate.

A suitable source of cetearyl olivate and sorbitan olivate is Olivem 1000, available from Hallstar Itali.

By surfactant we mean a surfactant intended for cleaning or emulsifying purposes. Examples of suitable surfactants include those presented in Chapter 37, pp. 431-450 (Classification of surfactants, by L. Oldenhove de Guertechin) in the Handbook of Cosmetic Science and Technology (ed. A. Barel, M. Paye and H. Maibach, published 2001 by Marcel Dekker, Inc., New York, NY), and include, but are not limited to, anionic surfactants such as sulfates, cationic surfactants such as betaines, amphoteric surfactants such as sodium cocoglycinate, nonionic surfactants such as alkyl polyglucosides.

Examples of suitable chelating agents include agents that are capable of protecting and preserving the compositions of this invention. The chelating agent is preferably ethylenediaminetetraacetic acid (EDTA), and more preferably tetrasodium EDTA, commercially available from Dow Chemical Company, Midland, Michigan, USA, under the trade name VERSENE 100XL.

Acceptable preservatives include, for example, parabens, quaternary ammonium compounds, phenoxyethanol, benzoates, DMDM hydantoin, organic acids and are present in the composition in an amount, based on the total weight of the composition, from about 0 to about 1 percent or from about 0.05 percent to about 0.5 percent.

Any of a number of conditioners that provide additional characteristics such as shine to the hair are suitable for use with the present invention. Examples include, but are not limited to, a volatile silicone conditioning agent having a boiling point at atmospheric pressure of less than about 220°C. Examples of suitable volatile silicones include, but are not limited to, polydimethylsiloxane, polydimethylcyclosiloxane, hexamethyldisiloxane, cyclomethicone fluids such as polydimethylcyclosiloxane manufactured by Dow Corning Corporation (Midland, Michigan, USA), commercially available under the trade name DC-345, and the mixtures, and preferably include cyclomethicone fluids. Other suitable conditioners include cationic polymers, including polyquaterniums, cationic guar gum, and the like.

Any of a number of commercially available pearlescent or opalescent agents are suitable for use in the composition. Examples of suitable pearlescent or opalescent agents include, but are not limited to, mono- or diesters of (a) fatty acids having from about 16 to about 22 carbon atoms and (b) ethylene or propylene glycol; mono- or diesters of (a) fatty acids having from about 16 to about 22 carbon atoms and (b) a polyalkylene glycol of the formula: HO-(JO) _a -H, where J is an alkylene group having from about 2 to about 3 carbon atoms; and a is 2 or 3; fatty alcohols containing from about 16 to about 22 carbon atoms; fatty acid esters of the formula: KCOOCH ₂ L, where K and L independently contain from about 15 to about 21 carbon atoms; inorganic solids insoluble in the shampoo composition, and mixtures of the above.

In accordance with the present invention, any fragrance composition suitable for application to the skin can be used.

In certain preferred embodiments, the present invention is in the form of a substrate containing a composition of the present invention. Any suitable substrate may be used. Examples of suitable substrates and substrate materials are described, for example, in US Patent Application Nos. 2005/0226834 and 2009/0241242, the contents of which are incorporated herein by reference in their entirety.

In certain preferred embodiments, the substrate is a wet wipe, glove, or face mask. Such embodiments preferably contain a water-insoluble substrate as defined in the above references. In certain embodiments, the water-insoluble substrate may be sized and shaped to cover the face of a human user to facilitate placement of the water-insoluble substrate on the user's face as a mask substrate. For example, the water-insoluble mask substrate may have openings for the user's mouth, nose, and/or eyes. In an alternative embodiment, the water-insoluble mask substrate may not have such holes. Such a holeless configuration may be suitable for embodiments of the invention in which the water-insoluble substrate is intended to be wrapped around areas of skin other than the face, or the water-insoluble substrate is intended to be used as a wet wipe. The water-insoluble substrate may have various shapes, such as a angled shape, such as a rectangle, or a curved shape, such as a round or oval. In certain embodiments, the substrate is a glove, such as that described in US Patent Application Published No. 2006/0141014, the contents of which are incorporated herein by reference in their entirety. In one embodiment of the present invention, the product includes a plurality of water-insoluble substrates of various shapes.

The present invention further includes a method of enhancing skin barrier function and/or hydration by applying a combination of Pichia anomala extract and retinol to skin requiring enhanced skin barrier function and/or hydration. This method includes, for example, topical application of a composition of the present invention containing a combination of Pichia anomala extract and retinol to the skin requiring improvement of skin barrier function and hydration. Such topical application can be applied to skin requiring care on any part of the body, such as the face, lips, neck, chest, back, arms, armpits, hands, feet and/or legs. The combination of Pichia anomala extract and retinol is preferably applied in an effective amount that results in the desired enhancement of skin barrier function.

The present invention further includes a method of improving the appearance of at least one sign of aging skin by applying a combination of Pichia anomala extract and retinol to skin requiring improvement of appearance of at least one sign of aging. This method includes, for example, topical application of a composition of the present invention containing Pichia anomala extract and retinol to skin requiring treatment of at least one sign of aging. Such topical application can be applied to skin requiring care on any part of the body, such as the face, lips, neck, chest, back, arms, armpits, hands, feet and/or legs. The combination of Pichia anomala extract and retinol is preferably applied in an effective amount that results in achieving the desired improvement in the appearance of at least one sign of skin aging.

Any suitable method of applying the composition to the skin requiring care can be used. For example, the composition may be applied directly from the packaging to the skin to be treated, applied by hand to the skin to be treated, or transferred to the skin from a substrate such as a wet wipe or mask, or a combination of two or more of the above. In other embodiments, the composition may be applied by pipette, tube, roll-on, aerosol, and patch, or may be added to a bath or otherwise in water for application to the skin, etc. The composition may be applied in a variety of ways and in a variety of forms, including, but not limited to , leave-in cream, mask and/or serum.

In certain embodiments, the methods of the present invention include applying to the skin at least two different compositions or products containing Pichia anomala extract and retinol. For example, the methods may include applying a first composition containing an extract of Pichia anomala and then applying a second composition containing a retinol that is different from the first composition to the skin to be treated.

In certain preferred embodiments, the first and second compositions may be independently selected from the group consisting of lotions, cleansers, masks, wipes, creams, serums, gels, and the like. In certain preferred embodiments, at least one of the first and the second composition is a cleanser, lotion, cream, essence or serum, and the other is a face mask or wet wipe. In other certain preferred embodiments, at least one of the first and second compositions is a cleanser and the other is a lotion or cream.

Compositions, formulations and products containing such compositions of the present invention can be prepared using techniques well known to those of ordinary skill in the art. These compositions can be used in the treatment of signs of skin aging such as wrinkles, loss of elasticity, uneven skin, including reduction of blemishes. The composition can be used on a regular basis and is substantially free of skin irritants.

The following non-limiting examples further illustrate the present invention.

Example 1

The production of hyaluronic acid (HA) by human skin explants was determined after they were treated with test compositions 1-4 containing varying amounts of Pichia anomala extract as follows.

First, a gel was prepared containing the following ingredients:

INCI name % wt. Phenoxyethanol; Methylparaben; Ethylparaben; Propylparaben 0.7 Carbomer 1 20% Sodium hydroxide solution Until pH=6 is reached Water 98.3

Compositions 1-4 were prepared with a compound of 2% wt. gel with varying amounts of a 5% aqueous solution of Pichia anomala extract along with additional ingredients. Pichia anomala was grown on a kiwi plant. The 5% aqueous solution was treated as a 100% stock solution.

Compositions 1-4 contained the ingredients shown in Table 1.

TABLE 1

INCI name Composition 1 (% mass) Composition 2 (% mass) Composition 3 (% mass) Composition 4 (% mass) gel 2 2 2 2 Cetearyl alcohol; Cetearyl glucoside 7 7 7 7 Isononyl isononanoate 8 8 8 8 Phenoxyethanol; Methylparaben; Ethylparaben; Propylparaben 0.7 0.7 0.7 0.7 5% Aq. Pichia anomala extract solution 2.5 (0.065% wt. Pichia anomala extract) 5 (0.13% wt. Pichia anomala extract) 7.5 (0.195% wt. Pichia anomala extract) 10 (0.26% wt. Pichia anomala extract) Water 79.8 77.3 74.8 72.3

HA production by compositions 1-4 was determined by immunohistopathological analysis on normal human skin explants from three donors (29, 30, 55 years old). Eight-mm-diameter flaps were cut from the explants, placed on pieces of sterile gauze, and placed in six wells with 3 ml of culture medium, one explant per well. The culture medium was sold under the trade name GIBCO DMEM/F-12 (ThermoFisher Scientific, Waltham, Massachusetts, USA, cat. no. 11514436) with 1% GIBCO penicillin-streptomycin (ThermoFisher Scientific, cat. no. 11528876) and 0.1 % amphotericin B was sold under the trade name FUNGIZONE (ThermoFisher Scientific, cat. no. 11510496).

For each test composition, 5 μl of the test composition was applied to the explant once a day for 5 days. An untreated explant sample was used as a control. On day 7, explants were removed, wiped with sterile gauze, then cut in half vertically and fixed in 4% paraformaldehyde (v/v). On day 8, explants were dehydrated and embedded in paraffin. Each test composition was tested three times.

Paraffinized slides were rinsed in xylene, and epitope retrieval was performed on a PT link instrument (Agilent, Santa Clara, CA, USA) using a target retrieval solution sold under the trade name ENVISION Flex, High pH (Dako, DM828, Agilent, . Santa Clara, California, USA). The slides were then washed with wash buffer sold under the trade name ENVISION (Dako, DM831, Agilent, Santa Clara, CA, USA) once for 10 min. Permeabilization and saturation were performed using phosphate buffered saline (PBS) 0.3% Triton/5% goat serum (Dako, Santa Clara, CA, USA, cat. no. CP3418/X090710-8) for 30 min followed by labeling with hyaluronic acid binding protein (HAPB from Calbiochem, cat. no. 385911, Millipore Sigma, St. Louis, NJ, USA) overnight at 4°C. The next day, the slides were washed three times with PBS for 5 minutes. Antibodies were detected using biotin binding protein covalently attached to a fluorescent tag sold under the trade name ALEXA FLUOR 488 streptavidin (Invitrogen™, cat. no. S11223), and nuclear staining was performed using DAPI solution (Dako, Santa Clara, State California, USA) in an amount of 5 µg/ml for 30 min at room temperature. The slides were then washed with PBS and mounted with Fluoprep mounting medium (BioMerieux UK, UK cat. no. 75521).

Images of skin sections were obtained using an Olympus IX70 fluorescence microscope (Olympus Corporation, Japan) coupled to an image analysis system (NIS-Elements AR software, Nikon instruments, Melville, NY, USA). Quantitative image analysis was performed using ImageJ software.

The results are shown in Table 2. The results are expressed as the average fluorescence intensity of the dermis (arbitrary units (arbitrary units)). The intensity of (green) fluorescence is proportional to HABP synthesis.

TABLE 2

Amount of 5% extract solution Pichia anomala Extract weight percentage Pichia anomala Delta HABP compared to untreated control Composition 1 2.5% 0.065% 10.36583333 Composition 2 5% 0.13% 17.73 Composition 3 7.5% 0.195% 15.23666667 Composition 4 10% 0.26% 15.4125

These results indicate that maximum skin HA production occurs at about 0.13 wt%. Pichia anomala extract. When this amount was exceeded, the production of HA leveled off.

Example 2

The production of hyaluronic acid (HA) by human skin explants was determined after treatment with four test compositions: composition 2 described in example 1 (containing Pichia anomala extract) , composition 5 (containing retinol) and compositions 6 and 7 (containing both) in accordance with the invention.

Composition 5 containing 0.1% wt. retinol and other ingredients are shown in Table 3.

TABLE 3 Composition 5

INCI name % wt. Water 62.18 Sodium acryloyldimethyltaurate/vinylpyrrolidone (VP) crosspolymer 0.5 Disodium salt EDTA 0.1 Glycerol 5 Butylene glycol 2 Sodium hyaluronate 0.1 Caprylyl glycol 0.5 stearyl alcohol; ceteareth-20. 3 cetearyl alcohol; ceteareth-20. 3 isohexadecane; 1.5 Stearyl ether PPG-15 4.5 pentaerythrityl tetraethylhexanoate; 7 OSH 0.1 Polyacrylamide; laureth-7; C13-14 isoparaffin 1 dimethicone; dimethicone crosspolymer; 5 dimethicone; trisiloxane; dimethicone; Dimethicone 3 Ascorbic acid 0.05 Phenoxyethanol 0.5 Chlorphenesin 0.2 Polyethylene; PTFE 0.5 Polysorbate 20 Retinol 0.23 Sodium hydroxide 0.04

Composition 5 was prepared as follows. Polysorbate 20 was removed; used retinol from the refrigerator; placed in an oven at 50°C until melted. Ascorbic acid premix. Ascorbic acid was mixed with 2% water and adjusted to pH ~ 5. Set aside for later addition at 40°C. Main phase. Add water (-2% for ascorbic acid premix) and begin mixing until smooth. Disodium EDTA was then added. While stirring at high speed, sodium acryloyldimethyltaurate/vinylpyrrolidone (VP) crosspolymer was added. Ensure homogeneity/complete hydration before proceeding. Heating was started to a target temperature of 73°C. Then glycerin and butylene glycol were added. At temperatures above 65°C, caprylyl glycol, chlorphenesin, phenoxyethanol and sodium hyaluronate were added. Oil phase. Isohexadecane, PPG 15 stearyl ester, pentaerythrityl tetraethylhexanoate, dimethicone were added; cross-linked dimethicone polymer, dimethicone; trisiloxane; dimethicone; dimethicone stearyl alcohol; ceteareth-20 and cetearyl alcohol; ceteareth-20. Heated to target temperature of 73°C with stirring. BHT was added 5 minutes before emulsification. Emulsification. After bringing the main phase and oil phase to ~73°C, the oil phase was slowly added to the water phase. Stirred for 10-15 minutes, after which polyacrylamide was added; laureth-7; C13-14 isoparaffin. Homogenize (Silverson, 4000 rpm) for 1 min. Cooling was started after mixing using a screw. Subsequent additions. At 40°C or lower, polyethylene was added; PTFE and premix 1 (ascorbic acid). The pH was measured and adjusted to the target pH value of 6.5-6.8. Under yellow light and argon atmosphere, retinol was added at 50°C and mixed thoroughly for 10-15 minutes. Leave to cool to 30-35°C.

Compositions 6 and 7 were prepared in the same way as composition 5, except that after the preliminary mixture of ascorbic acid, 2.5 wt.% was added. or 5% wt. accordingly, a 5% solution of the Pichia anomala extract described in example 1, and the amount of water was adjusted accordingly.

HA production was determined by immunohistopathological analysis as follows. Abdominal skin samples were taken from adults undergoing abdominoplasty. Informed consent was obtained from each patient, and all experimental steps were approved by the institutional review board (IRB). Subcutaneous fat was carefully removed and 0.93 ^cm2 skin biopsies were prepared under sterile conditions and acclimatized in DMEM/F12 (1:1) culture medium (Thermofisher Scientific, Bridgewater, NJ, USA, cat. no. 11320082) with heat inactivated 2% fetal bovine serum (Thermofisher Scientific, Bridgewater, NJ, cat. no. A3840202), 10 μg/ml insulin (Sigma Aldrich, Allentown, PA, USA, cat. no. I0516- 5ML), 10 ng/ml hydrocortisone (Sigma Aldrich, Allentown, PA, USA, cat. no. H 0135), 10 ng/ml EGF (Sigma Aldrich, Allentown, PA, USA, cat. no. 11376454001 ), 1x ABAM (Thermofisher Scientific, Bridgewater, NJ, USA, cat. no. 15240062) overnight in a humidified atmosphere containing 5% CO ₂ .

Explants were topically treated with 4 μl of the test composition daily for 5 days. An untreated explant sample was used as a control. On day 6, tissues were collected. The collected tissues were fixed in buffered formalin for 24 hours. After fixation, samples were dehydrated and paraffin-impregnated using a Leica TP 1020 automatic dehydrator (Leica Biosystems, Buffalo Grove, IL, USA). Samples were embedded using a Leica EG 1160 sample embedding station (Leica Biosystems). Sections of 5 μm thickness were prepared using a Leica RM 2125 type Minot microtome (Leica Biosystems) and the sections were mounted on histological slides sold under the trade name SUPERFROST. Microscopic studies were performed using a Leica DMLB (Leica Biosystems) or Olympus BX43 microscope (Olympus Corporation, Japan). Images were digitized by an Olympus DP72 digital camera with CellD storage software (Olympus Corporation, Japan). Hyaluronic acid staining was performed on paraffinized sections using biotinylated HABP (AMS Biotechnology Limited, Abingdon, UK, product code AMS.HKD.BC41) diluted 1:100 in PBS-BSA 0.3% for 1 hour at room temperature , were enhanced with the streptavidin/biotin system and detected using a peroxidase substrate sold under the brand name Vector VIP (Vector Laboratories, Burlingame, CA, USA, Cat. No. SK-4600). Staining was assessed by microscopic observation.

The results are shown in Table 4.

TABLE 4

Composition Increased HABP % increase compared to untreated Without processing 31,425 Composition 5 (0.1% wt. retinol) 44,775 42.48 Composition 2 (0.13% wt. Pichia anomala extract) 44.28 40.90 Composition 6 (0.1% retinol + 0.065% wt. Pichia anomala extract) 47,075 49.80 Composition 7 (0.1% retinol + 0.13% wt. Pichia anomala extract) 54.175 72.39

Treatment of tissue explants with compositions 6 and 7 containing combinations of Pichia anomala extract and retinol in accordance with the invention provided increased HA production compared to composition 5 containing only retinol or composition 2 containing only Pichia anomala extract.

Moreover, composition 7 containing 0.1% retinol and 0.13% wt. extract of Pichia anomala , provided an increase in HAPB by 72.39 percent compared to the untreated sample, while composition 2 containing 0.13% wt. extract of Pichia anomala , provided an increase of only 40.90 percent compared to the untreated sample, and composition 5 containing 0.1% wt. retinol, provided an increase of only 42.48 percent compared to the untreated sample.

Example 3

Gene expression for heparin-binding epidermal growth factor (HBEGF), hyaluronic acid synthase 2 (HAS 2) and CD44 was measured for composition 5 (0.1% wt. retinol), 5% solution of Pichia anomala extract described in example 1, and joint application of a composition of 5 and 5% solution of Pichia anomala extract.

First, abdominal skin samples were taken from adults undergoing abdominoplasty. Informed consent was obtained from each patient, and all experimental steps were approved by the institutional review board (IRB). Subcutaneous fat was carefully removed and 0.93 ^cm2 skin biopsies were prepared under sterile conditions and acclimatized in keratinocyte growth medium sold under the trade name KGM-GOLD BULLETKIT (Lonza, Walkersville, MD, USA), with 10% antibiotics overnight in a humid atmosphere containing 5% CO ₂ .

Skin explants were topically treated with 4 μl of the various formulations and left to incubate for 48 hours. An untreated explant sample was used as a control. In the case of combined application of the composition of 5 and 5% solution of Pichia anomala extract, 4 μl of each of them was used. At the end of the 48 hour period, the epidermis and dermis were separated by heating. Each was lysed in 400 μl of lysis buffer consisting of 100 parts RLT buffer (RNeasy mini kit, Qiagen, Valencia, CA, USA) and one part 2-mercaptoethanol inside a reinforced tube with screw cap and O-ring seal into the tube Ceramic fabric abrasion spheres (sold under the trade name PRECELLYS CKMix50-R, Bertin Corp, Rockville, MD, USA) were added. The tubes were shaken for 40 s at 6300 rpm. RNA was extracted from the solutions using the RNeasy mini kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions, and RNA was eluted in 25 μl of RNase-free water. Reverse transcription (RT) was performed using a Biosystems High Throughput Reverse Transcription Kit (ThermoFisher Scientific, Bridgewater, NJ, USA).

Gene expression analyzes were performed by quantitative real-time polymerase chain reaction (PCR) based on the SyBR green dye method. qPCR was performed on a real-time PCR system sold under the trade name CFX96 TOUCH (Bio Rad Laboratories, Inc., Hercules, California, USA). The expression of these genes was normalized to the expression of the constitutive human 18S RNA gene. Fold changes were calculated compared to untreated controls and ANOVA statistical test was performed.

The results obtained are shown in tables 5, 6 and 7.

TABLE 5

HBEGF expression Compositions Registered T-test Observed normalized to raw Expected additive effect % Synergy No processing 100 Composition 5 (0.1% retinol) 391 0.011330802 291 5% Pichia anomala extract solution 77 0.308217589 0 Composition 5+5% solution of Pichia anomala extract 594 0.001658771 494 291 69.76

Application of a combination of retinol and Pichia anomala extract resulted in an unexpected increase in HBEGF expression (494) compared to HBEGF expression obtained with retinol treatment alone (291). An increase of almost 70% was observed.

TABLE 6

HAS 2 expression Compositions Registered T-test Observed normalized to raw Expected additive effect % Synergy Without processing 100 Composition 5 (0.1% retinol) 122 0.406565333 22 5% solution of Pichia anomala extract 93 0.704495851 0 0.1% retinol + 5% Pichia anomala extract solution 137 0.244891449 37 22 68.18

The use of a combination of retinol and Pichia anomala extract resulted in an unexpected increase of almost 70% in the stimulation of HAS 2 expression (37) compared with the expected additive result of using retinol alone and Pichia anomala extract alone (22).

TABLE 7

CD44 expression Compositions Registered T-test Observed normalized to raw Expected additive effect % Synergy Without processing 100 Composition 5 120 0.407059542 20 5% solution of Pichia anomala extract 90 0.397819867 0 NTG RWR+Pichia anomala extract, 5% 165 0.11812913 65 20 225

Administration of a combination of retinol and Pichia anomala extract resulted in an unexpected stimulation of CD44 expression (65), compared with the expected additive effect of retinol alone and Pichia anomala extract alone (20). This increase was 225%.

Claims (6)

1. A composition for topical application to human skin to increase the amount of hyaluronic acid produced by the skin, containing a 5% aqueous solution of Pichia anomala extract, with the content of Pichia anomala extract in the composition from 0.065 to 0.26% by weight, and from 0.05 to 1 % by weight of retinol, with the weight ratio of Pichia anomala extract and retinol in the composition for topical application being 1.3. 2. The topical composition according to claim 1, wherein the Pichia anomala extract is obtained from a strain of Pichia anomala present in kiwi fruits or leaves. 3. The topical composition according to claim 1, wherein the Pichia anomala extract is obtained from a strain of Pichia anomala present in sugarcane. 4. Composition for topical application according to claim 1, additionally containing at least 10 wt.% glycerol. 5. The topical composition according to claim 1, having a pH level of 6.5 or less. 6. A method of increasing the amount of hyaluronic acid produced by the skin upon contact with an extract of Pichia anomala, including bringing the skin into contact with a composition for topical application according to claim 1.