IES84572Y1 - Cosmetic compositions containing lingonberry (vaccinium vitis idea) extracts - Google Patents

Description

COSMETIC COMPOSITIONS CONTAINING LINGONBERRY (VACCINIUM VITIS IDEA) EXTRACTS Field of the Invention The present invention relates to topical compositions for application to human skin and to their use in improving the condition and appearance of skin.

Background Art As the skin is the outermost aspect of our bodies it is subject to deterioration through environmental abuse (wind, air conditioning, central heating) or through the normal aging process on non-sun exposed areas (chronological aging) which may be accelerated by exposure of skin to sun (photoaging). As a result of this the consumer demands highly effective products to prevent the aging process.

In this respect consumers are increasingly seeking “anti-aging" cosmetic products which treat or delay the visible signs of aging skin such as wrinkles, lines. sagging, hyperpigmentation and age spots in addition to cosmetic products improving the appearance and condition of dry and flaky skin and to soothe irritated and itchy skin.

Skin care cosmetic and dermatological compositions for improving the condition and appearance of skin comprising botanical extracts are well known in the art.

Cranberries are well known for their health promoting activities. Arctic berry seed oils. including cranberry (Vaccinium oxycoccus) oil and lingonberry (Vaccinium vitis idea) seed oil. are commonly found in cosmetic products. Arctic berry seed oils contain predominantly fatty acids such as linoleic and alpha—|inolenic acids.

Cranberry seed oil is known in the art For use in cosmetic compositions. international Publication Number WO 00/72862 discloses isolated cranberry seed oil and components thereof in a form suitable for use as a foodstuff, dietary supplement, or a pharmaceutical composition. The isolated cranberry seed oil or compositions comprising one or a combination of components derived from the cranberry seed oil can be used as antioxidizing agents. for example.

Other cranberry extracts including cranberry juice and cranberry fruit extracts are known for use in cosmetic compositions. Japanese Patent no. JP2002275080 describes a skin care preparation comprising pressed juice of the cranberry fruit and/or the extract from the cranberry fruit together with one or more kinds of medicinally effective compounds. The skin care preparation has inhibitory effects on melanin formation.

JP2002003389 describes a cosmetic which acts as a radical scavenger having excellent radical scavenging properties. The composition comprises a pressed and filtered substance and/or extract of a cranberry fruit consisting essentially of an anthocyanine polymer.

Aqueous lingonberry extracts are different from the seed oil or cranberry extracts in their content of flavonoids and phenolic acids.

There is therefore a need for alternative effective cosmetic compositions for topical application to skin for treating and/or delaying the visible signs of aging and photodamaged skin such as wrinkles, lines, sagging, hyperpigmentation, skin dryness and age spots. lt is therefore an object ofthe invention to provide a cosmetic composition for treating and or preventing normal skin conditions due to chronological aging or photoaging, such as wrinkles, lines, sagging, hyperpigmentation and age spots. and/or of dry, flaky, itchyr irritated skin.

Summai_'y of the Invention Accordingly, the invention provides the use ofa therapeutically effective amount of an aqueous extract of a combination of lingonberry (Vaccinium vitis idea) fruit and leaf extract and/or derivatives thereof, in the manufacture of a cosmetic composition for treating skin conditions. The cosmetic composition is suitable for use in conditions selected from the group consisting of wrinkling, sagging, photodamaged skin, dry skin, flaky skin, irritated skin, itchy skin and age spots. f\_) l\_) (ft In a preferred embodiment, about 50% by weight of the lingonberry (Swedish cranberry) extract, and/or derivatives thereof, is present as the leaf extract and the other 50% as the fruit extract. Preferably at least 50% by weight of the extract is present as leaf extract. It has been surprisingly found that effective treatment and prevention of skin conditions clue to chronological aging or photoaging, such as wrinkles, lines, sagging, hyperpigmentation and age spots. and/or of dry, flaky, itchy. irritated skin may be obtained through the application of cosmetic compositions to the skin which comprise a specific synergistic combination of lingonberry (Swedish cranberry) fruit and leaf extract or derivatives thereof.

The cosmetic composition may comprise between 0.0001"/o to 50% by weight of lingonberry (Swedish cranberry) extract, preferably between 0.01% to l % by weight of lingonberry (Swedish cranberry) extract, most preferably between 0.1% to 5% by weight of lingonberry (Swedish cranberry) extract.

Such compositions are particularly useful for topical application to human skin for cosmetically treating and/or preventing skin conditions selected from the group consisting of wrinkling, sagging, photo—damaged skin, dry skin, flaky skin, irritated skin, itchy skin and age spots. The compositions are also useful for application to the skin as a cosmetic treatment which promotes epidermal regeneration and cellular renewal by chelating iron, protecting against free radicals but in particular protecting against UV irradiation, inducing epidermal differentiation and inhibiting collagenase.

Aqueous berry extracts are different from the seed oil fractions in their content of flavonoids and phenolic acids. They contain for example kaempferol, quercetin. myricetin, coumaric acid and ferulic acid etc. Marked differences in concentration of the biochemicals are found in different families and genera. Cranberry is one of the richest sources of flavonoids and in particular is rich in quercetin and myricetin. Type A proanthocyanidins are also particularly found in cranberries whereas the B types are found in other types of berries. Additionally compared with European cranberries (Vaccimum oxycoccus), Swedish cranberries or Lingonberries ( Vaccinium vitis idea) contain twice as much phenolics, higher quantities of secisolariciresinol, anthocyanins and cyanidins together with more resveratrol.

In one embodiment. the invention provides a topical composition comprising: (a) a therapeutically effective amount of an aqueous extract of a combination of lingonberry (Vaccinium v1’tis idea) fruit and leaf extract together with (b) a dermatologically acceptable carrier.

The composition according to the invention may comprise a dermatologically/cosmetically acceptable vehicle to act as a dilutant, dispersant or carrier for the lingonberry (Swedish cranberry) extracts. The carrier may be selected from the group consisting of water, liquid or solid emollients. silicone oils, emulsifiers, solvents, humectants, thickeners, powders, propellants and the like.

The carrier will usually form from 5% to 99.9%. preferably from 25% to 80% by weight of the composition, and can, in the absence of other cosmetic adjuncts. form the balance of the composition.

The composition may also comprise other specific skin—benefit actives such as sunscreens, moisturising agents, skin lightening agents. The vehicle may also further include adjuncts such as fragrances, opacifiers. preservatives, colourants, buffers and other anti—aging ingredients.

The topical composition according to the present invention may be prepared in a manner well known in the art for preparing skin care products. The active components are generally incorporated in a dermatologically acceptable carrier in conventional manner. The active components can suitably first be dissolved or dispersed in a portion of the water or another solvent or liquid to be incorporated in the composition.

The preferred compositions are oil-in-water or water-in-oil emulsions.

The composition may be in the form of conventional skin-care products such as a cream, gel or lotion or the like. The composition can also be in the form ofa cleanser eg. a bath or shower gel, possibly containing a delivery system for the actives to promote adherence to the skin during rinsing. The composition may also be a foundation or other colouring product e.g. mascara or lipstick. Most preferably the product is a “leave-on” topical product: a product to be applied to the skin without a deliberate rinsing step soon after its application to the skin.

The composition may be packaged in any suitable manner such as in a jar. a bottle. tube, roll—ball, or sachets.

The invention further provides a cosmetic method of treating/preventing skin conditions selected from the group consisting of wrinkling, sagging, photo-damaged skin, dry skin, flaky skin, irritated skin, itchy skin and age spots, the method comprising applying to the skin a topical composition comprising an extract of lingonberry (Swedish cranberry). The extract may comprise about 50 % by weight lingonberry fruit extract and about 50% by weight lingonberry leaf extract.

The method of the present invention may be carried out one or more times daily to the skin which requires treatment. The improvement in skin appearance will usually become visible after 3 to 6 months, depending on skin condition, the concentration of the active components used in the inventive method, the amount of composition used and the frequency with which it is applied. in general, a small quantity of the composition, for example from 0.1 to 5 ml is applied to the skin from a suitable container or applicator and spread over and/or rubbed into the skin using the hands or fingers or a suitable device. A rinsing step may optionally follow depending on whether the composition is formulated as a “leave-on" or a cleansing product.

The inventive compositions and methods thus provide anti-aging benefits which result in the promotion of smooth and supple skin with improved skin elasticity and a reduced or delayed appearance of wrinkles and aged skin with improved skin colour.

A general improvement in the appearance, texture and condition, in particular with respect to the radiance, clarity, and general youthful appearance of skin is achieved.

Thus the inventive methods and compositions provide a wide range of skin care benefits.

The term “treating” as used herein includes within its scope reducing, delaying and/or preventing the appearance of wrinkled, aged, photodamaged, dry and/or dry and irritated skin and generally enhancing the quality of skin by improving its appearance and texture. by preventing or reducing wrinkling and increasing flexibility. firmness. smoothness. suppleness and elasticity of the skin. The cosmetic compositions: methods and the uses ofthe lingonberry (Swedish cranberry) extracts according to the invention may be useful for treating skin which is already in a wrinkled, aged, photodamaged, dry and irritated condition or for treating youthful skin to prevent or reduce deteriorative changes due to the normal aging but particularly the photo aging process.

Detailed Description of the Invention Lingonberry (Swedish cranberry - Vaccinium vitis idea) extract Lingonberry (Vaccinium vitis idea) extract to be employed in accordance with the present invention is present in the topical composition in an effective amount derived from the fruit and /or from the leaf. Most preferably the combination of both extracts will be used and most preferably a 50:50 combination ofthe leaf and fruit extract will be used.

Lingonberry (Vuccinium vitis idea) extract comprises an aqueous extract rich in polyphenols, particularly flavonols and flavanols, and in oligosaccharides. Both leaf and fruit extract contain aromatic acid compounds such as hydroxybenzoic acid and hydroxycinnamic acid, typical flavanols are epicatechin whereas typical flavonols are quercitrin and quercetin. The leaf extract contains more flavonols compared with the fruit such that when combined nearly equal quantities of flavanols and flavonols are present in the lingonberry (Swedish cranberry) extracts. Nevertheless, the typical composition ofthe combined polyphenols that could be identified is: Compounds~ Hydroxybenzoic Phenol llydroxycinnamic Flavanol _______——_j — Fla_\/fonol Quercetin Due to the inherent biological variation of the polyphenols in plants these compositions should only be taken as a typical example and their concentrations will vary from batch to batch and year to year but approximately the same ratios between chemical classes are anticipated.

Lingonbcrry (Vaccirzizzm vms idea) extract to be employed in accordance with the present invention is present in the topical composition in an effective amount.

Normally the total amount ofthe active is present in an amount between 0,0001% and 50% by weight ofthe composition, More preferably the amount is from 0.01% to 10% and most preferably from 0.1% to 5% in order to maximise benefits at a minimum COSI.

In order that the present invention may be more readily understood. the following examples are given. by way of illustration only.

Skin aging is a complex process controlled by a number of factors broken down into intrinsic and extrinsic factors that include genetics, exposures to environmental stress (UV, mechanical stress), hormonal changes or metabolism. The combined results of these different factors are the deterioration ofthe structure ofthe skin, its function and its appearance (Rittie et al., 2002). The influence of the environment and UV irradiation in particular is very important in the process of skin aging (Rittie et al., 2002). lt is now known that these effects are directly related to the flattening of the dermal-epidermal junction, to the degradation of the fiber system of the skin, the extracellular matrix (ECM) and to the alteration ofepidermis.

The ECM is produced by fibroblasts and keratinocytes. It is composed of macromolecules including collagens, elastin, proteoglycans and fibronectin. Collagen and elastin are the major components of the ECM and are contained in tissues in the form of insoluble, rigid and stable fibers. They are tightly interwoven, organized in a network of microfibrils that imparts solidity and mechanical resistance to connective tissues. ECM thus acts like a physical support, but also carries out biological functions of intercellular exchange and communication that are essential for tight connection between the dermis and epidermis and the integrity of skin tissue.

Chronological aging or environmental stress (especially sunlight) destroys this balance. Cellular metabolic activity decreases and the renewal of collagen fibers are reduced. In parallel, enzymatic activities increase dramatically and result in an excessive and anarchic degradation of collagen fibers and a loss of structure in the ECM. The skin loses its suppleness and elasticity and becomes flaccid.

Since the work of McCord and Fridovich in 1969 on superoxide dismutasc. considerable interest has been focused on the relation between free radicals [superoxide (O2- and hydroxyl (OH—) anions and other reactive oxygenated species (RUSH and aging of the skin. The origin of free radicals may be endogenous (activation of arachidonic acid metabolism. activation of phagocytosis or accumulation of reduced metabolites) or exogenous (radiation, UV from sunlight, air pollution, tobacco smoke, pesticides and more).

All cells have varying degrees of a chemical and enzymatic anti-free radical arsenal.

This enables them to exert a natural protection against these highly reactive molecules and to optimally control their deleterious effects. Cellular responses may differ, however, depending on the extent of the oxidative stress. Low quantities of ROS stimulate cell proliferation and the over expression of antioxidant enzymes that can adapt and react to the stress. When produced in excessive quantities, on the other hand, ROS attack a series of biological substrates. These defense systems are often overwhelmed and cannot handle an overpowering radical flux. This saturation phenomenon is accentuated with age. In this case, ROS cause cell damage that is often irreversible and can even lead to cell death (McCord ct al.. I996). The major effects of this uncontrolled oxidative stress are intense, targeted and irreversible: oxidation of proteins and enzyme inactivation, DNA chain breaks and lipid peroxidation.

Example 1 Swedish cranberry fruit (Vaccinium vitis idea) extract is more effective than European cranberry fruit (Vaccinium oxycoccus) extract at protecting human keratinocytes from oxidative stress.

Active oxygen species and other free radicals are presumed to participate in actinic aging. In order to determine the protective activities of certain substances. cell viability tests were conducted. Cultures of human keratinocytes were artificially subjected to a stress by the hypoxanthine-xanthine oxidase system. The free radicals formed during this stress are responsible for increased cell death. The anti—oxidant activity of the product tested was determined by measuring cell viability.

Human keratinocytes were inoculated in 12-well plates at 2 ml of cell suspension per well. Cell cultures were incubated for 2 hours in the presence or absence of extracts at % and 2% and were subjected to a chemical aggression with a solution of hypoxanthine (160 pg/ml) — xanthine oxidase (l0 mU/ml). The protective effects of extracts towards chemically-induced free radicals were assessed by the spectrophotometric measurement of cell viability carried out with the M'l"l‘ method (3~(4.5-dimethyl thiazolyl)-2.5 diphenyl tetrazolium bromide) at 540 nm.

The results are expressed as percentage of efficacy.

A - B % efficacy = x 100 With: A untreated control — A treated control A = Control toxicity = A untreated control A untreated product —- A treated product B = Product toxicity = e—— A untreated product Tests were run in triplicate. The results are shown in table 1.

EFFICACY (%) TABLE I % Concentration 2% Concentration T Swedish Cranberry fruit 95% 100% European Cranberry fruit 27% 34% Lg _ Swedish cranberry fruit extracts (Vacc1'm‘zm1 vitis idea) are clearly more effective than European cranberry fruit extracts (Vaccmzzmiz oxycoccus) at protecting against oxidative stress.

Example 2 Demonstration of synergy of lingonberry (Swedish cranberry) leaf & fruit extracts on photoprotection of human kcratinocytes.

The aim of this study was to determine the ph0to—protective activity of lingonberry leaf & fruit extract compared to the lingonberry leaf extract and to the lingonberry fruit extract against UVB irradiation of human keratinocytes.

Keratinocytes were grown in KSFM medium (lnvitrogen) inoculated at a density of 70.000 cells/well in 24-well plates. Irradiation was with a « Biosun>> UV iamp (Vilbert-Lourmat, France). UVB intensities were 0.6J/cm2, respectively, in the presence of: — lingonberry leaf extract at 0.50% & l% - lingonberry fruit extract at 0.50% 8; 1% - lingonberry leaf& fruit extract at 0.50%. & l% After irradiation, the cells were kept at 37°C for 6 hours in a humid atmosphere containing 5% C02. The photo—protective effect of each product was estimated by measuring keratinocyte viability after staining with M.T.T. The results are expressed as percentage ofcell protection.

A - B Cell protecti0n/ control (%) = x 100 Abs non-irradiated control — Abs irradiated control with A: control toxicity = Abs non—irradiated control Abs non-irradiated product - Abs irradiated product B: product toxicity = Abs non—irradiated product Abs: absorbance. All experiments were run in triplicate.

The results are shown in table 2: Table 2: Photo-protective effect of LINGONBERRIES (Fruit, Leaves, Mix ”Leaves & Fruit”) Extracts after UV aggression Dosage Mean results Fruit + Leaves Mean results Fruit ( MP1) Mean (MFI + results MLI) / Leaves 2 (ML 1) ,5% l0 70 40 53 ,0053 "Synergy % 23 100 l .5 66 0.0041 Synergy In all further experiments only the effects ofthe combined extracts on cellular activity is shown although both individual extracts are effective.

Example 3 Effect of lingonberry (Swedish cranberry) leaf & fruit extract as a strong anti- radical agent.

The skin is the front line barrier of defense against environmental pollutants including chemical pollution (industrial pollution, exhaust gases) or solar radiation.

Environmental pollutant factors trigger a response from the defense system ofthe skin as a result of their oxidizing properties. They induce an oxidative stress and an imbalance in the antioxidant system of the skin (Afaq et al., 2001) due to the formation of free radicals. The body is naturally protected from these free radicals by chemical or enzymatic detoxification systems that may become overloaded. lt then mobilizes free radical traps that can stop these reactions. Free radicals produced by various types of diverse aggressions (stress, pollution, tobacco), are now considered as agents responsible for premature aging by causing, among other things. deterioration of tissues, damage to DNA and to the immune system.

The aim of this study was to determine the chelating capacity of Iingonberry (Swedish cranberry) leaf & fruit extract towards metal ions such as iron. The principle is based on quantifying free metal with a spectrophotometric assay. The assay ofthe chelation capacity of iron (iron solution at 10 mg/l) by Iingonberry (Swedish cranberry) leat‘& fruit extract was conducted with a colorimetric kit (SIGMA, method No. 565). The reagent ferrozine, a sulfonated derivative of diphenyltriazine, forms a water-soluble magenta complex with iron not complexed by Iingonberry (Swedish cranberry) leaf& fruit extract. Lingonberry (Swedish cranberry) leaf & Fruit extract was tested at 0.50%. l% and 2%.

The results are shown in table 3: ,_________ Table 3: Concentration Chelating activity (%) Lingonberry (Swedish cranberry) leaf& fruit extract at 1% presents a strong chelating activity towards iron.

Example 4 Effect of Iingonberry (Swedish cranberry) leaf & fruit extract on keratinocytes vitality after UV irradiation.

UVB radiation is responsible for phenomena of sunburn and immuno—suppression. lt also causes a number of skin disorders, the most important of which are skin cancers.

In addition, it damages DNA. UVA is the major component of the UV portion of the solar spectrum and is involved in skin aging, particularly slackening of the skin. It is The indirect also involved in the suppression of certain immunological functions. effects of UV/\ arise from photosensitization reactions following the absorption of .50% 27 % 48.

S’ T~i2"°/0 31 T”~~ _.‘ ____L.__.._}_,, , .-,--,- _ UVA by different cell ehromophores. UV radiation is also responsible for the formation of particular chemical species called reactive oxygen species (ROS) that have the capacity to create considerable damage directly at the level of the cell‘ such as the oxidation of proteins (Sander et al.. 2002) that lose their functionality. lipids or nucleic acids. Fortunately cells have defense systems that are effective against oxidative stress and combat a variety ofday to day aggressions.

The aim of this study was to determine the photo-protective activity of lingonberry (Swedish cranberry) leaf & fruit extract against UVB irradiation of human keratinocytes. Free radicals induced by UV radiation are responsible for an increase in cell death. The protective activity of the product studied can thus be determined by the spectrometric measurement of cell viability after staining with M.T.T. Keratinocytes were grown in KSFM medium (lnvitrogen) inoculated at a density of 50,000 cells/well in 12-well plates. Irradiation was with a « Biosun » UV lamp (Vilbert- Lourmat. France). UVB intensities were 0.l3J/cmz, respectively, in the presence of lingonberry (Swedish cranberry) leaf & fruit extract at 0.10%, 0.25%, 0.50%. and 1% .After irradiation, the cells were kept at 37°C for 24 hours in a humid atmosphere containing 5% C02. The photo-protective effect each product was estimated by measuring keratinocyte viability after staining with M.T.T.

The results are expressed as percentage of cell protection.

A-B ———-~x too Cell protection/ control (%) = A Abs non-irradiated control - Abs irradiated control with A: control toxicity = Abs non-irradiated control Abs non-irradiated product - Abs irradiated product B: product toxicity = Abs non-irradiated product All experiments were run in triplicate. The results are shown in table 4A |_..\ DJ Table 4: Concentration Cell protection / control (%) 0.10% + 31 .25% —_i— M + 47 0.50% + 63 1% + 75 ” Lingonberry (Swedish cranberry) leaf & fruit extract shows a clear protection olicells against UVB-induced free radicals.

Example 5 Effect of lingonberry (Swedish cranberry) leaf & fruit extract on keratinocytes DNA after UV irradiation.

The DNA molecule is a double helix composed ofa succession of deoxyribose rings (sugar) and phosphate groups. A purine or pyrimidine base is attached to each sugar residue. The basic unit of this structure is called a nucleotide, composed of a sugar, a phosphate group and one of the purine or pyrimidine bases. The purines are adenine (A) and guanine (G); the pyrimidines are cytosine (C) and thymine (T). Their precise succession determines what we call the DNA sequence. Deoxyribonucleic acid (DNA) is the support for genetic information, the "hard disk" of the cell. It is contained in the nucleus ofall cells of the organism and is responsible for storing and transmitting genetic information (Robert. i995). The integrity ofthe DNA molecule is under permanent threat. The molecule is in fact deteriorated by endogenous free radicals. diverse chemicals and above all by UV A, B and C radiation. Ionizing radiation results in a modification of nucleotide bases with the formation of pyrimidine dimers and strand lesions. These lesions prevent the correct operation of cells and cause genetic mutations which are often carcinogenic. There are DNA repair mechanisms to combat these aggressions. The most effective mechanism is excision repair. This process is carried out by a multi~enzyme system that recognizes pyrimidine dimers, cuts the damaged strand of DNA, eliminates it and stimulates the synthesis ofa new DNA strand. The new strand is identical to the fragment eliminated and is complementary to the intact opposite strand. In some cases. the lesion is not recognized or the repair mechanisms are incomplete. These defects result in a considerable risk of genetic mutation. This is why it is important to protect DNA strands by the use of antioxidants that can attenuate the risks of lesions.

In order to determine the protective effect of lingonberry (Swedish cranberry) leaf & fruit extract against UVB radiation, 21 DNA fragmentation test was used. UVB radiation causes chain breaks in DNA strands. This fragmentation can be visualized after extraction and electrophoresis of DNA. Our active ingredient must thus be capable of protecting DNA from breaks caused by solar radiation. Human keratinocytes were incubated at 37°C for 1 hour before UVB irradiation in an incubator containing 5% C02 in the presence or absence of lingonberry (Swedish cranberry) leaf & fruit extract at 1% (D0, T0h). lrradiations were with a << Biosun » UV lamp (Vilbert-Lourmat, France) with a UVB intensity of I80 m.l/cmz in the presence or absence of the product. The cells were then returned to the incubator (37°C. 5% CO2) (D0, Tl h). After 24 hours of incubation, DNA was extracted from the cells. DNA strand breaks were visualized after electrophoresis on agarose gels. and analyzed using the Bl0—PROFlL'i"7' system (Vilbcrt—Lourmat, France) (DI). The results are in table 5.

DNA protection TABLE 5 / Irradiated control (‘’/o) l ~-l Irradiated control ‘ 0 UVB + '1 % CONCENTR ATE Lingonberry (Swedish cranberry) leaf & fruit extract at 1% protects keratinocyte DNA from UVB—induced damage.

Example 6 Effect of lingonberry (Swedish cranberry) leaf & fruit extract on epidermal differen tiation.

The epidermis is a stratified epithelium composed of cells whose morphology is specific to each layer. During their migration towards the surface. kcratinocytes undergo biochemical and structural modifications that progressively transform them into keratinized cells: corneocytes that are eliminated by desquamation. In order to maintain a constant thickness, the epidermis is renewed by cell divisions of stem cells, followed by the differentiation of keratinocytes into corneocytes. The terminal differentiation of the corneocyte requires the formation of an intracorneocyte fibrous matrix, the production of intercellular lipids and the appearance ofa keratin envelope.

The fibrous matrix that replaces the cytoplasm and nucleus of the keratinocyte is formed from profilaggrin contained in keratohyalin grains of the stratum granulosum.

Profilaggrin is converted to filaggrin by a cascade of steps including dephosphorylation and proteolysis. Filaggrin enables the aggregation of cytokeratin filaments and is then degraded totally to form NMF, hygroscopic substances with a high capacity to bind water. The formation of the keratin envelope starts in the Malpighian body from precursor proteins (involucrin, keratolinin, loricrin) and with the participation of calcium and a membrane enzyme, transglutaminase that creates covalent bonds between these proteins. lnvolucrin is among the most important proteins since it is the protein skeleton of the plasma membrane of corneocytes. The keratin envelope makes corncocytes rigid and is responsible for the high mechanical resistance of the stratum corneum. Cadherins play an essential role not only in intercellular adherence. but also in the control of cell morphogenesis and differentiation. Cadherin-E participates in the regulation of proliferation and differentiation (Kee et al., 2001). It is expressed by Langerhans cells and keratinocytes, and enables the adhesion of these two cell types. It is localized in all cell layers ofthe epidermis, with predominance in differentiated layers (Serres, I997).

The aim of this study was to determine the effect of lingonberry (Swedish cranberry) leaf & fruit extract on the expression of messenger RNAs coding for transglutaminase—l, a marker of keratinocyte differentiation. The study was conducted on cultures of human keratinoeytes. Human keratinocytes were grown for 48 hours at 37°C in an incubator in an atmosphere of 5% CO2 in the presence of lingonberry (Swedish cranberry) |eaf& fruit extract at 0.50%. At the end of incubation. the cells were recovered and total RNA was extracted. The RNA was subjected to reverse transcriptase and the complementary DNA obtained was analyzed with PCR, using oligonucleotidcs complementary to a sequence coding for the genes of the proteins studied ttransglutaminase and Cadherin E). B-actin mRNA. the internal standard. was also analyzed in each experimental condition. The intensity of amplicon bands on agarose gels was quantified by image analysis (BlO—PROFlL® system. BIO-ID software. VILBERT-LOURMAT, France). The results are expressed as the ratio of intensity of the band of the gene analyzed over that of the internal standard (B—actin).

The results are shown in table 6 & 7.

The results are expressed as percentage of expression oftransglutaminase-1 mRNA.

TABLE 6 mRNA rate (°/o) Control 100 0.50% CONCENTRATE 1 18 Tested at 0.50%, the extract regulates the keratinocytes differentiation by increasing the expression oftransglutaminase-l and cadherin—E.

The results are expressed as percentage of expression of cadhcrin-E mRNA. mRNA rate (%) Control 100 TABLE 7 .50% CSNCENTRATE Lingonberry (Swedish cranberry) leaf & fruit extract at 0.50% supports the expression ofcadherin-E (+20%), marker ofthe keratinocyte differentiation.

Example 7 Effect of lingonberry (Swedish cranberry) leaf & fruit extract on collagenase (MMP-1) activity.

Collagen is the fibrous protein most abundant of conjunctive fabric, responsible for cutaneous lonicity. The activity anti-collagenase limits the degradation of collagen fibers. The aim of this study was to assess the anti—col|agenase activity of lingonberry (Swedish cranberry) leaf& fruit extract. One unit of collagenase is able to hydrolyze lumol of FALGPA (furylacryloyl-Leu-Gly—Pro—Ala; Sigma, F5l35) per minute at °C, pH 7.5 with calcium ion. This hydrolysis reaction involves a decrease of the absorbance at 324nm. If a product has an anti-collagenase activity, it will inhibit the reaction on the FALGPA substrate and the absorbance will not decrease.

The result is expressed as percentage of anti-collagenase activity.

AA(blank) — AA(sample) % activity = x I00 AA(blank) /\A(To) — AA(TlOmin) With 1 AA = ————z——— The results are shown in table 8.

TABLE 8 Anticollagenase activity (%) 1% C ONCENTRATE 3l 2% CONCENTRATE 47 % CONCFNTRATE 75 Tested at 2%, Lingonberry (Swedish cranberry) leaf & fruit extract limits the collagenase activity by 47%.

Example 8 Anti-wrinkle effect of lingonberry (Swedish cranberry) leaf & fruit extract.

The aim ofthis study was to quantify in vivo the anti-wrinkle efficacy ofthe products N°253 (placebo) and 694 (placebo + 3% of Lingonberry (Swedish cranberry) leaf & fruit extract) on the crow's feet. The study was conducted double blind on 24 volunteers presenting wrinkles on the crow's feet. The area of application for the products is randomized (left crow’s feet/right crow's feet). Replicas of the two crow's feet were made before and after 28 days oftwice daily application.

The anti-wrinkle effect of the products N°253 and 694 were analyzed by observing replicas with the help of a profilometer equipped with an image analy7er. Silicone polymer replicas were made before and after application ofthe product and examined by S.l.A. (silicone image analysis). Grazing light at an incidence of35° casts shadows on the surface of the impression that are then observed with a CCD camera connected to a computer. The field examined was 1 cm2. The gray level analysis of the resulting computer image provides the following parameters using QUANTIRIDES 99® (MONADERM, Monaco) software: - The number of wrinkles - The total wrinkled surface - The total length of wrinkles The results were calculated as a percentage using the mean of variation of wrinkle parameters observed under the effect of the product compared to D0: Pdt D28 — Pdt DO Variation / D0 (%) = With: Pdtooz Result before application of the product Pdtmg: Result after 28 days of product application The results are shown in table 9: Table 9: Anti-wrinkle effect of the products 253 and 694 ° Number of wrinkles Product 253 Product 694 D0 D28 D0 D28 M63“ 197 192 211 184 SEM 1 1 9 12 13 A / D0 _1 1% W’) 1% P M868 0.01 ° Total wrinkled surface Product 253 Product 094 D0 D28 D0 D28 M 63" 24.43 25.22 24.71 21.07 SEM 1.84 1.67 1 .57 _A_m17_._9%2 W_\_ A / D0 (0/°’ 9% 44% P 0.3024 0.0022 0 Total length Product 253 Product 694 D0 D28 D0 D28 M63“ 129.83 132.44 135.62 114.16 SEM 8.79 _ 7.78 8.22 8.36 A / D0 W") 7% .14% P 6 0.3492 0.0015 After 28 days of twice daily application. the product N°253 (placebo) shows no anti- wrinkle effect compared to D0.

After 28 days of twice daily application. the product N°694 (placebo + 3% of Lingonberry (Swedish cranberry) leaf & fruit extract) decreases significantly the different parameters characteristic of the skin aspect: - Number of wrinkles by l 1% (P=0.0l42) — Total wrinkled surface by 14% (P=0.0022) - Total length ofwrinkles by 14% (P400015) Example 9 Effect of lingonberry (Swedish cranberry) leaf & fruit extract on skin elasticity.

The aim of this study was to quantify in vivo the effect of the products N°253 (placebo) and 694 (placebo + 3% of Lingonberry (Swedish cranberry) leaf & fruit extract) on skin elasticity. The study was conducted double blind on the face of 24 volunteers. The area of application for the products is randomized (left face/right face). Measurements were made on the check before and after 28 days of twice—daily treatment using an SEM 575 Cutometer® (Courage & Khazaka, Germany).

The skin is sucked into the orifice of a probe by a constant vacuum for a constant period. Several successive suctions may be carried out. The depth of penetration of the skin in the probe is measured with two optical prisms placed at the opening ofthe probe (no friction or mechanical effect). Using the curves obtained, it is possible to calculate a number of parameters characteristic of the mechanical properties of the skin.

The following is a typical curve obtained during a measurement involving five I 1 I [ l I I I | I 35 40 successive 5 second suctions.

Deformation U L L. 40 ' (mm) [..,.a.__c_.aa::‘ _ . f Uv Ue Ua X k, Ue: Instantaneous deformation (elastic component) Uv: Delayed deformation (viscous component) Uf: Uv + Ue = extensibility or total elongation U’f: Final extensibility Ur: Immediate retraction x: Residual retraction temps 4(8) x : Residual deformation after 5 cycles Ua: Total retraction.

Among all the parameters calculated from the resulting curves, the following were selected to quantify changes in biomechanical properties resulting from the product - Ur/Ue = R5 If R5 increases, skin elasticity increases.

— Ua/Uf = R2 lf R2 increases, skin elasticity increases.

The results were calculated as percentage using the means of variations of skin elasticity parameters observed under the effect ofthe product compared to DO: Pdt D28 — Pdt D0 Variation / D0 (%) = —» x 100 Pdt D0 With: Pdtpoi Result before application of the product Pdtmg: Result after 28 days of product application The results are shown in table 10: Table 10: Effect ofthe products 253 and 694 on skin elasticity Parameter R2 Product 253 Product 694 DO 028’ D0 D28 M63“ 0.660 0.714 0.534 0.710 oooo Ht‘ MSEM 0.026 0.029 0.024 0023 J AIDO (%) 11% 15% 0 P 0.0550 _g0_12_1__ Parameter R5 Product 253 Product 694 [)0 D28 Do i D28 M93“ 0.572 0.597 0.575 0.639 '5” 0.031 0.029 9932 0.026 A I DO (°/°) 10% 17% P 0.2411 0.0323 After 28 days ofapplication, and in the conditions ofthis study: - The product N°253 (placebo) tends to increase the R2 and the R5 parameters, which represent the elasticity of the skin (+11%; +10%). This increase is non significant (P>0.05).

- The product N°694 (placebo + 3% of Lingonberry (Swedish cranberry) leaf& fruit extract) increases significantly the R2 and R5 parameters which represent the elasticity of the skin: - Increase ofthe R2 parameter by l5% (P=0.0l2l) - Increase of the R5 parameter by 17% (P=0.0323).

Example 10 In this example lingonberry (Swedish cranberry) extract were included in the inventive compositions.

The formulation below describes an oil in water cream incorporating the inventive composition which is suitable for the methods and uses according to the present invention. The percentages indicated are by weight of the composition unless stated otherwise.

W’[°/0 IIYDROGENATED POLYISOBUTFNE 4.5 CYCLOPENTASILOXANE 4.5 DIMETHICONE 7--5 Example 11 GLYCERYL STEARATE 2 C ETYI. ALCOHOL 2 PEG-40 STEARATE 1,25 SODIUM ACRYLATE/ACRYLOYLDIMETHYL l TAURATE CUPOLYMER TOCOPHERYL ACETATE 0.5 PRESERVATIVES QS PERFI IMF, QS LINGONBERRY VITAL CONCENTRATE V 3 WATER to 100 The formulation below describes a foundation incorporating the inventive composition which is suitable for the methods and cases according to the present invention. The percentages indicated are by weight of the composition unless stated otherwise.

Wt°/o CYCLOPENTASILOXANE PIGMENTS POLYGLYCERYL-4 ISOSTEARATE ISODODECANE -¥>~UI\O BUTYLENE GLYCOL BlS—PEG/PPG-l4/l4 DIMETHICONE HYDROGENATED POLYISOBUTENE BEESWAX MAGNESIUM SULPHATE PRESERVATIVES ”” LINGON BERRY VITAL CONCENTRATE K’.- WATER to 1 Example 12 The formulation below describes a body scent incorporating the inventive composition which is suitable for the methods and cases according to the present invention. The percentages indicated are by weight of the composition unless stated otherwise.

Wt‘)/0 DEN ATURED ALCOHOL PROPYLENE GLYCOL GLYCERINE BENZOPHENONE—2 PERFUME SOLUBILISER L») l\) O\ Q U3 U1 U1 LINGONBERRY VITAL CONCENTRATE WATER to 100 All the above topical compositions provide an effective cosmetic treatment to improve the appearance of wrinkled, aged, photo—clamaged, and/or irritated skin, when applied to skin that has deteriorated through the aging or photo-aging or when applied to youthful skin to help prevent or delay such deleriorative changes. All the compositions may be processed in conventional manner.

The words “comprises/comprising" and the words “having/including“ when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. l‘ .) ‘II lt is appreciated that certain features of the invention, which are, for clarity. described in the context of separate embodiments. may also be provided in combination in a single embodiment. Conversely, various features ofthe invention which are, for brevity, described in the context ofa single embodiment, may also be provided separately or in any suitable sub-combination.

Claims (5)

Claims

1. Use of a therapeutically effective amount of an aqueous extract of a combination of lingonberry (Vaccinium vitis idea) fruit and leaf extract and/or derivatives thereof, in the manufacture of a cosmetic composition for treating skin conditions.

2. Use according to claim 1 wherein about 50% by weight of the lingonberry (Vaccinium vitis idea) extract, and/or derivatives thereof, is present as the leaf extract and the other 50% as the fruit extract.

3. Use according to claim I or 2 wherein the composition comprises between 0,0001% to 50% by weight of lingonberry (Vaccinium vitis idea) extract, preferably between 0.01% to 10% by weight of lingonberry (Vaccinium vitis idea) extract, more preferably between 0.1% to 5% by weight of lingonberry (Vaccinium vitis idea) extract.

4. A topical composition comprising: (a) a therapeutically effective amount of an aqueous extract of a combination of lingonberry (Vaccinium vilis idea) fruit and leaf extract together with (b) a dermatologically acceptable carrier.

5. A topical composition substantially as hereinbefore described and/or with reference to the examples. TOMKINS & C().