RU2825635C2 - Topical compositions containing pichia anomala and n-acetylglucosamine - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to a composition for increasing the amount of hyaluronic acid produced by the skin and a method for increasing the amount of hyaluronic acid produced by the skin. Composition for local application applied on human skin to increase the amount of skin-produced hyaluronic acid containing (i) 2 wt.% of 5% aqueous solution of Pichia anomala extract and (ii) 2 wt.% of N-acetylglucosamine, in which the weight ratio of the Pichia anomala extract and N-acetylglucosamine in the topical composition is 0.026. Method for increasing the amount of skin-produced hyaluronic acid upon contact with the extract of Pichia anomala, involving bringing the skin into contact with a composition containing (i) 2 wt.% of 5% aqueous solution of Pichia anomala extract and (ii) 2 wt.% of N-acetylglucosamine, in which the weight ratio of Pichia anomala extract and N-acetylglucosamine in said composition is 0.026.

EFFECT: composition described above effectively increases the amount of hyaluronic acid produced by the skin.

8 cl, 5 tbl, 2 ex

Description

FIELD OF APPLICATION OF THE INVENTION

The present invention provides a method for skin care by topical application to the skin of a combination of Pichia anomala extract and N-acetylglucosamine. Additionally, a composition for topical application is provided, containing a combination of Pichia anomala extract and N-acetylglucosamine.

PREREQUISITES FOR THE 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, particularly in the intermediate spinous layer, but not in the stratum corneum (SC) or granular layer. With age, the concentration of hyaluronic acid in the skin decreases and it dissociates from collagen and elastin. Skin containing lower concentrations of hyaluronic acid also exhibits decreased water binding, which may be related to the changes observed in mature skin, including wrinkles, altered elasticity, decreased firmness, and a decreased ability to support the skin's microvasculature. As one of the major primary glycosaminoglycans (GAGs), hyaluronic acid can bind up to 1,000 times its own weight in water, helping the skin retain and store moisture. It is found throughout connective tissue and is synthesized primarily by fibroblasts and keratinocytes found in the skin.

Various methods have been proposed to combat wrinkles and fine lines, including hyaluronic acid injection. Injection of exogenous hyaluronic acid is used as a temporary dermal filler in contour plastic procedures. However, injected hyaluronic acid has a limited lifespan. On the other hand, penetration of exogenous hyaluronic acid into the skin has proven difficult to achieve with topical application.

Pichia is a genus of yeast in the family Saccharomycetaceae. There are over 100 known species in this genus. The most well-known species include Pichia anomala, Pichia guilliermondii, Pichia norvegensis, and Pichia ohmeri.

Pichia anomala (formerly Hansenula anomala) can be found in unboiled milk and cheese. Extracts of the Pichia yeast have high levels of mannans, polysaccharides composed of mannose monomers. Pichia anomala and mannans are known to be used in the care of 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 obtained using a strain of Pichia isolated from sugar cane. It is available from Silab-France.

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

N-acetylglucosamine is a sugar amine and a building block for GAGs. It is used in a number of cosmetic skin care products, such as NEOSTRATA Skin Active Retinol+NAG Complex, marketed by NeoStrata Company Inc.

U.S. Patent No. RE41,339 discloses a variety of N-acetyl aldosamines, N-acetylamino acids, and other N-acetyl compounds, including N-acetylglucosamine, for topical application to the skin, nails, and hair. Such compounds provide a variety of anti-aging benefits, such as alleviation of thinning skin, fragile skin, fine lines, wrinkles, etc.

Pichia anomala extract and N-acetyl glucosamine act on the basis of different biological mechanisms and lead to improvement in various beneficial clinical effects. N-acetyl glucosamine is known as one of the substrates required for HA production. Pichia anomala extract works through the hydration pathway by increasing the activity of hyaluronic acid synthase 2 enzyme, which leads to beneficial clinical effects including hydration, improved skin barrier, increased strength, etc.

Although the art has suggested topical application of Pichia anomala extract and N-acetylglucosamine separately, the applicants have now discovered that topical application of a combination of these two ingredients in relatively small amounts still unexpectedly has a superior effect on hyaluronic acid production. This approach provides significant skin benefits, including improvement, reduction, inhibition or delay in the appearance of at least one sign of skin aging, as well as improvement in skin barrier protection and skin hydration, using cost-effective amounts of actives, especially Pichia anomala extract. Accordingly, new ways of, for example, targeting signs of skin aging are now available.

SUMMARY OF THE INVENTION

The present invention relates to a composition for topical application comprising an extract of Pichia anomala and N-acetylglucosamine.

The invention also relates to a composition for topical application containing about 0.052% by weight of Pichia anomala extract and about 2% by weight of N-acetylglucosamine.

The present invention also relates to a method for treating a sign of skin aging, comprising topical application to skin requiring care due to skin aging, of a composition for topical application containing an extract of Pichia anomala and N-acetylglucosamine.

The present invention further provides a method for improving skin barrier function and hydration, comprising topical application to skin in need of improvement of skin barrier function and hydration, of a composition for topical application containing Pichia anomala extract and N-acetylglucosamine.

The invention also provides a method for 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 containing an extract of Pichia anomala and N-acetylglucosamine.

DETAILED DESCRIPTION

The topical composition of the present invention improves the production of hyaluronic acid in the skin as a result of the synergistic action of Pichia anomala extract and N-acetylglucosamine.

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 should be considered only as examples, which in no way limit the rest of the description.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In addition, all publications, patent applications, patents and other materials mentioned herein are incorporated herein by reference. Unless otherwise specified, percentages used to express amounts of ingredients are weight percentages (i.e., % (w/w)). Likewise, weight ratios used to express 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 the other ingredient). Unless otherwise specified, all ranges are inclusive of their extremes, such as the range "4 to 9" inclusive of the extremes of 4 and 9.

In this document, "product" does not necessarily mean a finished product in packaged form. In one embodiment of the invention, the packaging 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 said container is placed. In one embodiment, the product includes the composition of the present invention and contains instructions for the consumer on how to apply the composition to the skin or hair.

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

As used herein, the term "cosmetic" refers to a substance or preparation that enhances appearance and maintains, restores, imparts, imitates or enhances physical attractiveness or produces an attractiveness or youthfulness effect, especially with respect to the appearance of tissue or skin.

As used herein, the term "cosmetically acceptable" means that the ingredients to which the term refers are acceptable for use in contact with tissues (e.g., skin or hair) without causing undesirable toxic effects, incompatibility, instability, irritation, allergic reaction, or the like.

In some embodiments, the compositions of the present invention are suitable for affecting the 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, the sign of aging is the presence of lines, wrinkles, and/or loss of elasticity.

As used herein, the term “reversing signs of skin aging” refers to reducing, alleviating, 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 (e.g., crow's feet), forehead and cheek wrinkles, frown lines, and fine lines around the mouth.

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

In this document, the term "uneven skin tone" refers to a skin condition associated with diffuse or patchy pigmentation that can be classified as hyperpigmentation, such as postinflammatory hyperpigmentation.

In this document, the term "maculation" refers to a skin condition associated with redness or erythema.

As used herein, "increasing the strength of the skin" means increasing the strength or elasticity of the skin, preventing loss of strength or elasticity of the skin, or preventing or eliminating sagging, laxity, or looseness of the skin. 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 elasticity or strength of the skin can be caused by a number of factors including, but not limited to, aging, environmental damage, or the application of cosmetics to the skin.

As used herein, the term "skin texture improvement" 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 wrinkles in the skin” means preventing, reducing the appearance, stopping the progression of, or reversing the formation of wrinkles and fine lines in the skin.

As used herein, the term "safe and effective amount" means an amount sufficient to initiate the desired effect, but low enough to prevent serious side effects. A safe and effective amount of a compound, extract or composition will vary depending on, for example, the age, health condition of the end user, as well as exposure to environmental conditions, the duration and nature of exposure, the specific extract, component or composition used, the specific carrier used and similar factors.

In this document, the phrase "skin requiring enhanced skin barrier function and hydration" refers to skin that is characterized by, but not limited to, lack of hydration, lack of sebum, cracking, dryness, itching, flaking, xeroderma, dehydration, lack of elasticity, lack of luster, dullness, or lack of lipid content.

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

The applicants also found that topical application of a composition containing a combination of Pichia anomala extract and N-acetylglucosamine increases the concentration of endogenous hyaluronic acid (HA) in the skin above the concentration achieved by using Pichia anomala extract alone, even at higher amounts. Topical application of such a composition can increase hyaluronic acid concentrations to values approaching those of young skin, thereby providing the structural framework of the skin necessary to reduce the signs of skin aging.

Pichia anomala

The composition for topical application comprises 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 an acceptable Pichia anomala extract is PRO-LIPISKIN, commercially available from Silab-France. It is produced from a strain of Pichia anomala found in sugar cane.

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

The Pichia anomala extract may be presented in the form of a solution containing dry matter (extract) in the range of 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, etc. In one embodiment, the solvent comprises at least about 90% by weight water or at least about 95% by weight water.

N-acetylglucosamine

The topical composition also contains N-acetylglucosamine. N-acetylglucosamine has the structure:

Quantities

Any suitable amounts of Pichia anomala extract and N-acetyl glucosamine 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 N-acetyl glucosamine used are cosmetically acceptable and selected to achieve the desired effect on the skin in a particular condition, such as signs of aging, reduced barrier function or reduced hydration.

In certain preferred embodiments, the compositions comprise from about 0.01 to about 1% by weight of Pichia anomala extract, more preferably from about 0.025 to about 0.25% by weight of Pichia anomala extract. In one embodiment, the composition comprises about 0.052% by weight of Pichia anomala extract.

In certain preferred embodiments, the compositions comprise from about 0.01% by weight to about 10% by weight, more preferably from about 0.5% to about 5% by weight of N-acetylglucosamine. In one embodiment, the composition comprises about 2% by weight of N-acetylglucosamine.

In some embodiments, the weight ratio of Pichia anomala extract to N-acetylglucosamine in the compositions is from about 0.01 to about 0.05. In one embodiment, the weight ratio of Pichia anomala extract to N-acetylglucosamine in the compositions is about 0.026.

Compositions for topical application

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

The compositions can be prepared in the form of a variety of product types, including, but not limited to, lotions, creams, gels, sticks, aerosols, ointments, cleansing liquids and bars of soap, shampoos and hair conditioners, hair fixatives, pastes, foams, powders, mousses, shaving creams, wipes, patches, hydrogels, film-forming products, face masks and skin masks, films, and decorative cosmetics such as makeup bases and mascaras. These types of products can contain several types of cosmetically acceptable carriers for topical application, including, but not limited to, solutions, suspensions, emulsions such as microemulsions and nanoemulsions, gels, solids, and liposomes. Non-limiting examples of such carriers are provided below. Other carriers can be incorporated into the compositions by those skilled in the art.

The compositions useful in the present invention can be formulated as solutions. Solutions typically include an aqueous or organic solvent (e.g., from about 50% to about 99.99% or from about 90% to about 99% of a cosmetically acceptable aqueous or organic solvent). Examples of acceptable organic solvents include propylene glycol, polyethylene glycol, polypropylene glycol, glycerol, 1,2,4-butanetriol, sorbitan esters, 1,2,6-hexanetriol, ethanol, and mixtures thereof.

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

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

Another type of product that can be prepared from a solution is a cream. Typically, a cream contains about 5% to about 50% (e.g., about 10% to about 20%) of the softener(s) and about 45% to about 85% (e.g., about 50% to about 75%) of 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% of emollient(s) and from about 0.1% to about 2% of thickener(s).

The composition may be prepared as an emulsion. If the topical carrier is an emulsion, about 1% to about 10% (e.g., about 2% to about 5%) of the topical carrier is(are) emulsifier(s). Emulsifiers may be nonionic, anionic, or cationic. Examples of emulsions are known to those skilled in the art.

Lotions and creams may 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% (e.g., about 5% to about 10%) emollient(s); from about 20% to about 80% (e.g., 30% to about 70%) water; and from about 1% to about 10% (e.g., about 2% to about 5%) emulsifier(s).

Oil-in-water and water-in-oil compositions for skin care 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. Water-in-oil-in-water or oil-in-water-in-oil compositions based on a multi-phase emulsion are also used in the claimed invention. In essence, such single- or multi-phase emulsions contain water, softeners and emulsifiers as the main ingredients.

The compositions of the present invention can also be formulated as a gel (e.g., an aqueous, alcoholic, hydroalcoholic, or oil gel using 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 copolymers, cellulose derivatives (e.g., 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% by weight 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, degradable, translucent texture and a light residual sensation on the skin after application. In this document, 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 (e.g., as a wax 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 fat-soluble materials and/or water-soluble materials traditionally 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 comprise any of various additional cosmetically active agents. Examples of suitable additional active agents include skin lightening agents, darkening agents, additional anti-aging agents, tropoelastin stimulators, 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, photoprotectors, antioxidants, keratolytic agents, detergents/surfactants, humectants, nutrients, vitamins, energy-enhancing agents, antiperspirants, pore tightening agents, deodorants, hair removers, hair growth enhancers, hair growth retarding agents, thickeners, hydration enhancers, efficacy enhancers, anti-coarsening agents, skin conditioning agents, anti-cellulite agents, for odor control, such as odor-masking agents or pH-changing agents, etc.

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), bisdisulizole disodium (Neo Heliopan AP), diethylaminohydroxybenzoylhexyl benzoate (Uvinul A Plus), ecamsule (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, drometrizole trisiloxane (Mexoryl XL), iscotrizinol (Uvasorb HEB), octocrylene, oxybenzone (Eusolex 4360), sulisobenzone, bisoctrizole (Tinosorb M), titanium dioxide, zinc oxide; Carotenoids; Free radical scavengers; Spin traps; Retinoids and retinoid precursors such as retinol, 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; Copper-containing peptides, 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, aloe vera, feverfew, soybean, shiitake mushroom extracts, and their derivatives and mixtures.

In some preferred embodiments, the compositions comprise a combination of Pichia anomala extract and N-acetylglucosamine and at least one additional skin moisturizing active agent.

In certain preferred embodiments, the skin care compositions comprise a combination of Pichia anomala extract, N-acetyl glucosamine, and at least one additional agent for improving the appearance of at least one sign of skin aging. Examples of suitable additional agents that reduce at least one sign of skin aging include, but are not limited to, tropoelastin stimulators, collagen stimulators, retinoids, hyaluronic acid, including cross-linked hyaluronic acid, dimethylaminoethanol, N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, alpha hydroxy acids, polyhydroxy acids, and combinations of two or more thereof.

In this document, the term "tropoelastin stimulators" refers to a class of compounds that have a biological activity associated with enhancing the production of tropoelastin. According to the present invention, tropoelastin stimulators include all natural or synthetic compounds that are capable of increasing the production of tropoelastin in the human body.

Examples of suitable tropoelastin stimulators include, but are not limited to, blackberry extracts, smoke tree extracts, feverfew extracts, and bimetallic complexes containing copper and/or zinc components. The bimetallic complex containing copper and/or zinc components can be, for example, copper-zinc citrate, copper-zinc oxalate, copper-zinc tartrate, copper-zinc malate, copper-zinc succinate, copper-zinc malonate, copper-zinc maleate, 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, copper-zinc sebacate, copper-zinc dodecanoate or combinations thereof. In a preferred embodiment, the tropoelastin stimulator is selected from blackberry extracts, smoke tree 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 dried flowers of the plant. Such compounds can be isolated from one or more parts of the plant (e.g., the whole plant, a flower, a seed, a root, a rhizome, a stem, a fruit, and/or a leaf of the plant). In a preferred embodiment, the blackberry extract is an extract of blackberry leaves. One particularly suitable blackberry extract is obtained by extracting Rubus fruticosus leaves 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, N.J., under the trade name SymMatrix.

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

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

Examples of suitable collagen stimulators include, but are not limited to, the following: retinoids, including retinol, retinaldehyde, and retinoic acid; feverfew (Tanacetum parthenium) extracts; Centella asiatica extracts; and Siegesbeckia orientalis extracts; soy extracts; collagen-stimulating peptides; ursolic acid; and asiaticoside.

Centella asiatica, also known as Violette marronne in Reunion Island, gotu kola or Indian pennywort in India, Centella repanda in North America and talapetraca in Madagascar, is a polymorphic herbaceous plant and belongs to the 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 1,200 meters above sea level. There are three species of Centella asiatica: common (Typica), Abyssinian (Abyssinica) and Florida (Floridana). The plant is known for its healing, sedative, analgesic, antidepressant, antiviral and antimicrobial properties. The biological activity of the plant is apparently related to the presence of triterpene molecules. An acceptable extract of Centella asiatica under the trade name TECA is produced by Bayer Consumer HealthCare, Basel, Switzerland.

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

Suitable collagen stimulating peptides include the following: matrikine peptides (i.e., peptides derived from the degradation of extracellular matrix proteins such as collagen, elastin, or proteoglycan), including palmitoyl pentapeptides such as MATRIXYL, available from Sederma (Croda International Group, Edison, NJ); a copper complex of GHK (glycine-histidine-lysine) peptide available from Photomedex, Montgomeryville, PA, under the trade name PROCYTE; the palmitoyl derivative of GHK peptide available from Sederma (Croda International Group, Edison, NJ) under the trade name Biopoeptide CL; biomimetic tetrapeptides available from Unipex, Quebec, Canada, under the trade name Chronoline Tri Peptide; and palmitoyl tripeptide, commercially available 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 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), is commercially available from, among others, 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 stimulators. The compositions preferably include (by active substance content) from about 0.1% to about 10% collagen stimulators, more preferably from about 0.5% to about 5% collagen stimulators, and most preferably from about 0.5% to about 2% collagen stimulators.

The compositions of the present invention may further comprise at least one active skin lightening agent. Examples of suitable active skin lightening agents include, but are not limited to, tyrosinase inhibitors, melanin degradation stimulating agents, melanosome transfer inhibiting agents including PAR-2 antagonists, exfoliants, sunscreens, retinoids, antioxidants, tranexamic acid, tranexamic acid cetyl ester hydrochloride, skin whitening agents, linoleic acid, disodium adenosine monophosphate, chamomile extract, allantoin, opacifiers, talcs and silicas, zinc salts, and the like, as well as 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 Composita root extract, saxifrage extract, etc.), ellagic acid, salicylates and their derivatives, glucosamine and its derivatives, fullerene, hinokitiol, dioic acid, 5,5'-dipropyldiphenyl-2,2'-diol (magnolignan), 4-(4-hydroxyphenyl)-2-butanol (4-HPB), combinations of two or more thereof, and the like. 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 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), and the like, as well as 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, 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-inhibiting agent comprises a peroxide or ligninase.

Examples of suitable melanosome transfer inhibiting agents include PAR-2 antagonists such as soybean trypsin inhibitor or Bowman-Birk inhibitor, vitamin B3 and derivatives such as niacinamide, soybean essence, whole soybean, soybean extract. In some preferred embodiments, the melanosome transfer inhibiting agents include soybean 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 of any of these, beta hydroxy acids such as salicylic acid, polyhydroxy acids such as lactobionic acid and gluconic acid, and mechanical exfoliants such as microdermabrasion. In certain preferred embodiments, exfoliants include glycolic acid or salicylic acid.

Examples of retinoids include, but are not limited to, retinol (the alcohol form of vitamin A), 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 thereof, and the like. In some preferred embodiments, the retinoid is selected from the group consisting of retinol, retinal, retinyl acetate, retinyl propionate, retinyl linoleate, and combinations of two or more thereof. In some more preferred embodiments, the retinoid is retinol.

Examples of antioxidants include, but are not limited to, water-soluble antioxidants such as sulfhydryl compounds and derivatives thereof (e.g., sodium metabisulfite and N-acetylcysteine, glutathione), lipoic acid and dihydrolipoic acid, stilbenoids such as resveratrol and derivatives thereof, lactoferrin, iron and copper chelating agents, and ascorbic acid and ascorbic acid derivatives (e.g., ascorbyl-2-glucoside, ascorbyl palmitate, and ascorbyl polypeptide). Oil-soluble antioxidants suitable for use in the compositions of this invention include, but are not limited to, butylated hydroxytoluene, retinoids (e.g., retinol and retinyl palmitate), tocopherols (e.g., 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 derivatives thereof (e.g., genistein and diadzein), extracts containing resveratrol, and the like. Examples of such natural extracts include grape seed extracts, green tea, black tea, white tea, pine bark extracts, feverfew, feverfew extract that does not contain parthenolide, oat extract, blackberry extract, smoke tree extract, soybean extract, pomelo extract, wheat germ extract, hesperidin, grape extract, purslane extract, licochalcone, chalcone, 2,2'-dihydroxychalcone, primrose extract, propolis, and the like.

The additional cosmetically active agent may be present in the composition in any suitable amount, for example in an amount of from about 0.0001% to about 20% by weight of the 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 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 available from Sigma-Aldrich, St. Louis, Mo.), tetrahydrocurcuminoids (such as Tetrahydrocurcuminoid CG available from Sabinsa Corporation, Piscataway, N.J.), extracts of and substances derived from the following: Phellodendron amurense bark extract (PCE), undenatured soybean (Glycine max), feverfew (Tanacetum parthenium), ginger (Zingiber officinale), ginkgo (Ginkgo biloba), madecassoside (an ingredient in Centella asiatica extract), smoke tree extract (Cotinus coggygria), butterbur extract (Petasites hybridus), goji berries (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), 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), simrelief (bisabolol and extract ginger), 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 is commercially available under the trade name SYNOVEA HR from Sytheon, Lincoln Park, NJ, USA. 4-Octylresorcinol is commercially available from City Chemical LLC, West Haven, CT, USA.

By "feverfew extracts" is meant extracts of the plant Tanacetum parthenium, which may be prepared, for example, in accordance with the detailed information set forth in U.S. Patent Application Publication No. 2007/0196523, entitled PARTHENOLIDE FREE BIOACTIVE INGREDIENTS FROM FEVERFEW (TANACETUM PARTHENIUM) AND PROCESSES FOR THEIR PRODUCTION. One particularly suitable feverfew extract in a form containing about 20% active feverfew is commercially available from Integrated Botanical Technologies, Ossining, NY, U.S.A.

In the skin care composition of the present invention, the ratio of the amounts of the combination of the extract of Pichia anomala and N-acetylglucosamine 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 based on the weight of the dry extract by the amount based on the weight of the anti-inflammatory compound) from about 0.001 to about 100, preferably from about 0.01 to about 10, 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 comprises one or more topical components selected from the group consisting of surfactants, complexing agents, emollients, humectants, conditioners, preservatives, opacifiers, flavoring agents, and the like.

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

By humectant is meant a compound intended to increase the water content of the upper layers of the skin (e.g., hygroscopic compounds). Examples of suitable humectants include those presented in Chapter 35, pp. 399-415 (G. Zocchi, Skin Feel Agents) in Handbook of Cosmetic Science and Technology (eds. A. Barel, M. Paye, and H. Maibach, published 2001 Marcel Dekker, Inc New York, NY), and include, but are not limited to, glycerin, 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 comprises glycerin. For example, the composition comprises at least about 4% by weight glycerin. The composition may comprise at least about 10% by weight glycerin.

In another embodiment, the composition has a pH of about 6.5 or less. For example, the composition may have a pH of about 5.5 to about 6.

In a specific embodiment, the composition comprises about 0.052% by weight of Pichia anomala extract and about 2% by weight of N-acetylglucosamine.

The composition may further comprise from about 1 to about 2% by weight of a mixture of cetearyl olivate and sorbitan olivate, from about 4 to about 5% by weight of glycerin and at least about 65% by weight of water, and have a pH of less than about 6.

By surface-active agent is meant a surface-active agent intended for cleansing or emulsifying purposes. Examples of suitable surface-active agents include those presented in Chapter 37, pp. 431-450 (Oldenhove de Guertechin, Classification of surfactants) in Handbook of Cosmetic Science and Technology (ed. A. Barel, M. Paye and H. Maibach, published 2001 Marcel Dekker, Inc New York, NY) and include, without limitation, 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 those which 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, Mich., U.S.A., 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 hair are suitable for use in 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 from Dow Corning Corporation (Midland, Mich., U.S.A.), commercially available under the trade name DC-345, and mixtures thereof, 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 opacifying agents are suitable for use in the composition. Examples of suitable pearlescent or opacifying 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) polyalkylene glycol of the formula: HO-(JO) _a -H, wherein 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: _KCOOCH2L , wherein K and L independently contain from about 15 to about 21 carbon atoms; inorganic solids insoluble in the shampoo composition, and mixtures of the listed substances.

Any fragrance compositions suitable for application to the skin can be used in accordance with the present invention.

In certain preferred embodiments, the present invention is in the form of a substrate comprising a composition of the present invention. Any suitable substrate may be used. Examples of suitable substrates and substrate materials are described, for example, in U.S. Patent Application Publication 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 wipe, a glove, or a face mask. Such embodiments preferably comprise a water-insoluble substrate defined as set forth in the references cited above. 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 mouth, nose, and/or eyes of the user. In an alternative embodiment, the water-insoluble mask substrate may not have such openings. Such a configuration without openings may be suitable for embodiments of the invention in which the water-insoluble substrate is intended to be wrapped around areas of skin that are not on the face, or the water-insoluble substrate is intended to be used as a wipe. The water-insoluble substrate may have various shapes, such as a shape with corners, 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 U.S. Patent Application Publication 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 varying shapes.

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

The present invention further comprises a method for reducing the improvement in the appearance of at least one sign of skin aging by applying to the skin requiring an improvement in the appearance of at least one sign of skin aging a combination of Pichia anomala extract and N-acetyl glucosamine. This method comprises, for example, topical application of a composition of the present invention comprising a combination of Pichia anomala extract and N-acetyl glucosamine to the skin requiring an effect on at least one sign of skin aging. Such topical application can be performed on the skin in need of care in any part of the body, such as the skin of the face, lips, neck, chest, back, arms, armpits, hands, feet and/or legs. The combination of Pichia anomala extract and N-acetyl glucosamine 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 for applying the composition to the skin in need of care may be used. For example, the composition may be applied directly from the package to the skin in need of care, applied by hand to the skin in need of care, or transferred to the skin from a substrate such as a wet wipe or mask, or a combination of two or more of these. In other embodiments, the composition may be applied by means of a pipette, tube, roller, aerosol, and patch, or may be added to a bath or other water for application to the skin, etc. The composition may be applied in various ways and in various forms, including, but not limited to, a 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 comprising Pichia anomala extract and N-acetylglucosamine. For example, the methods may include applying a first composition comprising Pichia anomala extract, followed by applying a second composition comprising N-acetylglucosamine, which is different from the first composition, to the skin in need of treatment.

In certain preferred embodiments, the first and second compositions can be independently selected from the group consisting of lotions, cleansers, masks, wipes, creams, serums, gels, etc. In certain preferred embodiments, at least one of the first and second compositions is a cleanser, lotion, cream, essence, or serum, and the other is a facial mask or 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 to treat signs of skin aging such as wrinkles, loss of elasticity, "uneven skin" including reducing blemishes. The composition can be applied on a regular basis and is substantially free of skin irritants.

In one embodiment, the invention also provides a method for increasing the amount of hyaluronic acid produced by the skin upon contact with an extract of Pichia anomala, comprising contacting the skin with a composition comprising an extract of Pichia anomala and N-acetyl glucosamine. Thus, a low concentration of the extract of Pichia anomala can be used, such as less than about 0.1 or less than about 0.08, such as about 0.052% by weight, and still provide good production of HA. N-acetyl glucosamine can function as an activity enhancer for the extract of Pichia anomala in inducing HA production by the skin.

The following non-limiting examples provide further illustration of the present invention.

Example 1

The production of hyaluronic acid (HA) by human skin explants was determined after their treatment with test compositions 1-4 containing different amounts of Pichia anomala extract, as follows.

First, a gel was prepared containing the ingredients listed in Table 1.

TABLE 1

INCI name % mass. 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 by combining 2 wt.% gel with various amounts of 5% aqueous solution of Pichia anomala extract together with additional ingredients. Pichia anomala was grown on kiwi plant. The 5% aqueous solution was treated as 100% stock solution.

Compositions 1-4 contained the ingredients shown in Table 2.

TABLE 2

INCI name Composition 1 (% wt.) Composition 2 (% wt.) Composition 3 (% wt.) Composition 4 (% wt.) 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% aqueous solution of Pichia anomala extract 2.5 (0.065% wt. of Pichia anomala extract) 5 (0.13% wt. of Pichia anomala extract) 7.5 (0.195% wt. of Pichia anomala extract) 10 (0.26% wt. of Pichia anomala extract) Water 79.8 77.3 74.8 72.3

The production of GC by compositions 1-4 was determined by immunohistopathological analysis on normal human skin explants from three donors (29, 30, 55 years). Eight-mm diameter flaps were cut from the explants, placed on pieces of sterile gauze and placed in six-well plates 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, MA, USA, Cat. #11514436) with 1% GIBCO penicillin-streptomycin (ThermoFisher Scientific, Cat. #11528876) and 0.1% amphotericin B, which was sold under the trade name FUNGIZONE (ThermoFisher Scientific, Cat. #11510496).

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

Paraffin-embedded slides were washed with 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, CA, USA). Slides were then washed with ENVISION wash buffer (Dako, DM831, Agilent, Santa Clara, CA, USA) once for 10 min. Permeabilization and loading were performed using phosphate-buffered saline (PBS) with 0.3% Triton/5% goat serum (Dako, Santa Clara, CA, USA, Cat# CP3418/X090710-8) for 30 min followed by labeling with hyaluronic acid binding protein (HAPB from Calbiochem, Cat# 385911, Millipore Sigma, St. Louis, NJ, USA) overnight at 4 °C. The following day, slides were washed three times with PBS for 5 min each. Antibodies were detected using a biotin binding protein covalently attached to a fluorescent label sold under the trade name ALEXA FLUOR 488 streptavidin (Invitrogen ^TM , Cat# S11223) and nuclear staining was performed using DAPI (Dako, Santa Clara, CA, USA) at 5 μg/ml for 30 min at room temperature. Slides were then washed with PBS and mounted with Fluoprep mounting medium (BioMerieux UK, UK Cat# 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 3. The results are expressed as the average dermal fluorescence intensity (in arbitrary units (AU)). The intensity of (green) fluorescence is proportional to the amount of hyaluronic acid synthesized.

TABLE 3

% by weight of 5% solution of Pichia anomala extract % wt. of Pichia anomala extract Increase in HAPB compared to untreated control Composition 1 2.5 0.065 10.37 Composition 2 5 0.13 17.73 Composition 3 7.5 0.195 15.24 Composition 4 10 0.26 15.41

These results show that the maximum production of HA by the skin occurs when using about 0.13% by weight of Pichia anomala extract. When this amount is exceeded, the production of HA levels off.

Example 2

The production of hyaluronic acid (HA) by human skin explants was determined after their treatment with compositions 5-7 according to the invention, containing Pichia anomala extract and N-acetylglucosamine.

Compositions 5-7 contained the ingredients shown in Table 4.

TABLE 4

Phase INCI name Composition 5 (% wt.) Composition 6 (% wt.) Composition 7 (% wt.) A Water 69.4 69.4 69.4 A Hydroxyethyl acrylate copolymer/
sodium acryloyldimethyl taurate 1,2 1,2 1,2 B Sclerotium resin 0.4 0.4 0.4 B Glycerol 4.0 4.0 4.0 C Cetearylolivate, sorbitanolivate 1.5 1.5 1.5 C Chlorphenesin 0.2 0.2 0.2 D Water 15 9 9 D N-acetylglucosamine 3.0 1.0 2.0 D Glycerol 1 1 1 D Glyceryl dilaurate 0.5 0.5 0.5 D Steareth-10 0.5 0.5 0.5 E Dimethicone/Dimethicone Crosspolymer 2.5 2.5 2.5 E Dimethicone 3.0 3.0 3.0 E Dimethicone; dimethiconol 2 2 2 F 5% solution of Pichia anomala extract 1 (0.026% wt. Pichia anomala extract) 3 (0.078% wt. Pichia anomala extract) 2 (0.052% wt. Pichia anomala extract) F Ethylhexylglycerin; Phenoxyethanol 0.8 0.8 0.8 G 20% citric acid solution Until pH=5.5-6 is reached Until pH=5.5-6 is reached Until pH=5.5-6 is reached G 20% sodium hydroxide solution Until pH=5.5-6 is reached Until pH=5.5-6 is reached Until pH=5.5-6 is reached

The compositions were prepared as phases. Phase A was prepared by combining water and hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer in a mixing vessel with agitation by a propeller. Mixing was continued until complete dispersion and a smooth, homogeneous consistency was obtained. Phase B was prepared by pre-mixing the sclerotium resin and glycerin until homogeneous and then adding this mixture to the main vessel and re-mixing until a smooth, homogeneous consistency was obtained. The main vessel was subsequently heated to 70-75 °C. Next, for Phase C, chlorphenesin, cetearyl olivate and sorbitan olivate were added to the main vessel using a Greerco high-speed homogenizer and mixed until a smooth, homogeneous mass was formed. The main vessel was subsequently cooled to 60 °C while mixing with agitation blades. Phase D was prepared by combining water and N-acetyl glucosamine in a separate kettle and heating to 65°C, then adding glycerin, glycerol dilaurate, and steareth-10, mixing until smooth, and holding at 65°C. Once Phase D was smooth, it was added to the main kettle at 65°C and mixed until smooth using vortex mixing. The mixture was subsequently cooled to 40°C. In a second additional kettle, Phase E was prepared by combining dimethicone/dimethicone crosspolymer and dimethicone, mixing until smooth, and then adding to the main kettle at 40°C, followed by the addition of dimethicone/dimethiconol. Phase F was added to the main kettle with a solution of Pichia anomala extract, ethylhexylglycerin, and phenoxyethanol at 40°C or less and mixed until smooth. Finally, the main kettle was cooled to 30 °C. If necessary, the pH was adjusted to 5.5-6 using 20% citric acid and/or 20% sodium hydroxide, if necessary (phase G).

The compositions were evaluated in the skin explant model described in Example 1 for changes in HAPB production relative to the untreated control. The results are shown in Table 5.

TABLE 5

% wt. of Pichia anomala extract % wt. N-acetylglucosamine Increase in HAPB compared to untreated control Composition 5 ---0.026--- 3 14.35 Composition 6 --0.078---- 1 12.34 Composition 7 ----0.052-- 2 16.15

Although each of Formulations 5-7 contained 0.078 wt% or less of Pichia anomala extract, all of them stimulated HA production in the explants at high concentrations in the presence of N-acetyl glucosamine. Formulation 7 provided particularly good results. Formulation 7 contained only 0.052 wt% of Pichia anomala extract with 2 wt% of N-acetyl glucosamine, but provided an increment of HABP over the untreated sample of 16.15, which was unexpectedly higher than the increment of HABP over the untreated sample observed in Formulation 1 in Example 1 (10.37), which contained approximately the same amount of Pichia anomala extract (0.065 wt%).

Claims (8)

1. A composition for topical application applied to human skin to increase the amount of hyaluronic acid produced by the skin, comprising (i) 2% by weight of a 5% aqueous solution of Pichia anomala extract and (ii) 2% by weight of N-acetylglucosamine, in which the weight ratio of Pichia anomala extract and N-acetylglucosamine in the composition for topical application is 0.026. 2. A composition for topical application according to claim 1, wherein the Pichia anomala extract is obtained from a strain of Pichia anomala present on kiwi fruits or leaves. 3. A composition for topical application according to claim 1, wherein the Pichia anomala extract is obtained from a strain of Pichia anomala present on sugar cane. 4. A composition for topical application according to claim 1, additionally containing at least 4% by weight of glycerin. 5. A composition for topical application according to claim 1, having a pH value of 6.5 or less. 6. A composition for topical application according to claim 1, further comprising from 1 to 2% by weight of a mixture of cetearyl lylate and sorbitan lylate, from 4 to 5% by weight of glycerin and at least 65% by weight of water and having a pH of less than 6. 7. A method for increasing the amount of hyaluronic acid produced by the skin upon contact with an extract of Pichia anomala, comprising contacting the skin with a composition containing (i) 2% by weight of a 5% aqueous solution of the extract of Pichia anomala and (ii) 2% by weight of N-acetylglucosamine, in which the weight ratio of the extract of Pichia anomala and N-acetylglucosamine in said composition is 0.026. 8. The method according to claim 7, wherein the composition further comprises from 1 to 2% by weight of a mixture of cetearyl lylate and sorbitan lylate, from 4 to 5% by weight of glycerin and at least 65% by weight of water and has a pH of less than 6.