WO2012120290A2

WO2012120290A2 - Cosmetic formulations

Info

Publication number: WO2012120290A2
Application number: PCT/GB2012/050486
Authority: WO
Inventors: Katja Svennevig; Espen KVALE
Original assignee: Sana Pharma As; Gardner, Rebecca
Priority date: 2011-03-04
Filing date: 2012-03-05
Publication date: 2012-09-13
Also published as: WO2012120290A3; EP2694030A2

Abstract

The present invention relates to a topical skin care formulation comprising a) β-(1,3/1,6)- glucan and b) a protein-containing plant extract, wherein said plant is of the family Fagaceae; or c) a protein-containing plant extract, wherein said plant is of the genus Vigna. The present invention also relates to a topical skin care formulation comprising β-(1,3/1,6)-glucan and an oxidoreductase. The present invention also relates to an oral composition comprising (i) borage seed oil and astaxanthin; or (ii) vitamin C, collagen hydrolysate, chondroitin sulphate and glucosamine, and also to kits comprising such oral compositions and optionally such topical skin care formulations. The present invention also relates to cosmetic skin treatment method in which these oral compositions and/or topical formulations are used.

Description

Cosmetic Formulations

The present invention relates to cosmetic compositions used in cosmetics, cosmeceuticals and nutraceuticals, in particular to topical skin care formulations and oral compositions which reduce the visible signs of aging.

Aged skin exhibits wrinkles, lacks elasticity, moisture, firmness and density and possesses blemishes and areas of atypical pigmentation. A variety of biological mechanisms are implicated in skin aging, including the degradation of

macromolecules such as elastin and collagen. Skin thickness and strength is determined to a large part by the collagen content in the skin's connective tissue. The content of collagen in the skin decreases by about 1 % each year in adults and to a greater extent after menopause in women. The cosmetic industry is a large market and there are countless products on offer which are marketed as capable of reducing the visible signs of aging. Cosmetic formulations and compositions include general skin creams, lotions, sera, suncreams and makeup as well as similar products designed for specific use on certain areas of the skin, e.g. the hands, face, neck, decolletage and around the eyes.

The effectiveness of anti-aging cosmetics depends in part on the active ingredient or ingredients contained therein. Commonly used ingredients in anti-aging cosmetics include antioxidants, exfoliants, moisturizing agents, proteins and peptides, enzymes and cofactors. Extracts from plants or parts thereof are used in cosmetic compositions since they contain a multitude of active ingredients. Algal, fungal and bacterial extracts are also sometimes used.

The effects of the cosmetic compositions can also depend on the concentration of the ingredients and their mode and timings of application. Many skin care companies recommend using a specific treatment program with their products.

Despite great demand, many such products and treatments have not been proven to give lasting or major positive effects. For many, skin aging has a psychological impact and causes concern. Despite the current availability of cosmetic formulations and compositions which are marketed as reducing the visible signs of skin aging, there remains a need for new compositions that are effective.

The present inventors have identified a novel combination of components for a topical skin care formulation with efficacy in the reduction of the visible signs of skin aging. As well as or as an alternative to reducing the visible signs of skin aging, the formulations may moisturise the skin, lessen the feelings of skin 'tightness' after bathing, provide a protective barrier to UV or other environmental factors, reduce the visibility of pores, reduce the build up of sebum and/or the development of blemishes.

In a first aspect, the present invention provides a topical skin care formulation comprising:

a) 3-(1 ,3/1 ,6)-glucan;

b) soy protein; and/or

c) hydrolysed rice brain protein; and/or

d) an oxidoreductase.

The term "topical" means that the skin care formulation is suitable and intended for application to the skin of a human subject.

Optionally, the topical skin care formulation comprises:

a) 3-(1 ,3/1 ,6)-glucan;

b) soy protein; and

c) hydrolysed rice brain protein.

Preferably the topical skin care formulation comprises:

3-(1 ,3/1 ,6)-glucan; and

an oxidoreductase,

and optionally also soy protein and/or hydrolysed rice brain protein.

More preferably, the topical skin care formulation comprises

a) 3-(1 ,3/1 ,6)-glucan;

b) soy protein;

c) hydrolysed rice brain protein; and d) an oxidoreductase.

Glucans are a heterogeneous group of glucose polymers found in the cell walls of plants, bacteria and fungi. The basic structural unit in beta-glucans as described herein is a backbone chain and side chains comprising or consisting of β(1→3)- linked glucosyl units. Depending upon the source and method of isolation, beta- glucans have various degrees of branching and of linkages in the side chains. The frequency and type of linkage in the side chains determine the molecule's biological activity. Beta-glucans of fungal and yeast origin are normally insoluble in water, but can be made soluble either by acid hydrolysis or by derivatisation introducing foreign groups like -phosphate, -sulphate, -amine, -carboxymethyl and so forth to the molecule. In Europe, Asia and USA, beta-glucans especially from Bakers' yeast have long been employed as feed additives for animals and as dietary supplements for humans.

The glucans used in the formulations of the present invention are β-(1 ,3)-glucans, i.e. glucans containing β-(1 ,3)-linkages.

The glucan may be derived from any known source including but not limited to yeast, fungi, algae, grasses, moss, bacteria, seaweed and poaceae (gramineae) such as but not limited to oat, wheat, corn, millet and barley.

The glucans of the present invention may consist essentially only of β-(1 ,3)-linked glucose residues. Such glucans include curdlan, which is derived from

Agrobacterium biobar, a non pathogenic bacteria and zymosan, which is derived from yeast.

Preferably however the glucans are mixed-linkage glucans, i.e. comprising β-(1 ,3)- linked glucose residues in addition to glucose residues linked via other β-linkages, such as β-(1 ,6)-linkages, β-(1 ,4)-linkages and β-(1 ,2)-linkages.

Glucans derived from poaceae (gramineae) are typically in the form of a soluble fiber. The bonds between the D-glucose or D-glucopyranosyl units in these glucans are either β-1 ,3 linkages or β-1 ,4 linkages. This type of beta-glucan is also referred to as a mixed-linkage β-(1/3, 1 /4)-glucan. Another β-(1 ,3/1 ,4)-glucan which may be used in the formulations of the present invention is lichenan (moss starch), which is obtained from mosses such as Cetraria islandica.

Preferably, the glucans are 3-(1 ,3/1 ,6)-glucans, i.e. glucans containing substantially no β-(1 ,4)-linkages. These glucans have a beta-(1 ,3)-backbone, i.e. the backbone comprises beta-(1 ,3)-linked glucopyranose units. Such glucans include essentially linear glucans such as but not limited to laminarin, which is derived from brown algae, Chrysolaminarin, which is derived from photosynthetic heterokonts and Pleuran, which is an insoluble polysaccharide derived from Pleurotus ostreatus.

Alternatively, the β-(1 ,3)-glucans of the present inventions may have branched structures. The β-(1 ,3)-glucans may have a beta-(1 ,3)-backbone with multiple single glucose residues each attached thereto by a β-(1 ,6)-linkage. Such single glucose residues do not constitute a "side chain" since they are single residues only and are not made up of beta-(1 ,3)-linked glucopyranose units. Examples of such glucans include schizophyllan, which is derived from the fungus Schizophyllum commune, scleroglucan, which is derived from the fungus Sclerotium rolfsii and lentinan, which is derived from the shiitake (Lentinula edodes) mushroom. Particularly preferred scleroglucans are those with a molecular weight of 10⁵ to 10 x 10⁶, more preferably 2 x 10⁶ to 10 x 10⁶ determined from the readily measured Staudinger Index η using the following Mark-Houwink equation MW= [η/4.45 x 10^"7] ^{/ 49}.

Preferably, however, the glucans have one or more beta-1 ,3 side chains comprising or consisting of β-linked D-glucosyl units, i.e. side chains attached to the backbone via a beta-1 ,6 linkage and where the side chains are made up of beta-1 ,3 linked glucopyranose units. The side chain comprises 2 or more, typically 3, 4, 5, 10 or more beta-1 ,3 linked glucopyranose units.

Preferably the β-(1 ,3/1 ,6)-glucan is a yeast glucan, more preferably from

Saccharomyces cerevisiae.

The yeast glucans used in the present invention may be in their natural state, e.g. in whole yeast, or they might be processed in the sense that either the glucans are isolated from other cell components, the glucans are derivatised and/or that the chemical structure is altered as compared to the naturally occurring structure. A derivatised glucan would preferably contain the following groups: sulphate, amine, acetate, phosphate, phosphonate or carboxymethyl. Further alterations of the chemical structure of the glucans will typically comprise reductions in length of the backbone and/or in length or complexity of branches and/or side chains.

Preferred derivatised glucans for use in the present invention are

carboxymethylated glucans. Particularly preferred are the soluble

carboxymethylated glucan products described in US 6,342,486 and the commercial carboxymethyl glucan products from the company Mibelle.

Preferably the glucan is not in its natural state, i.e. not present as whole cells or even a whole cell wall fraction, but processed to be partially isolated from other cell wall components which it is found with in nature, for example proteins and chitin. For instance, acid or alkali treatment or enzymatic treatments may be used to prepare glucans for use in the formulations of the present invention.

The glucans of the invention include soluble and particulate glucans, both of which are effective. Preferably the glucans are particulate. Methods of solubilizing glucans are known in the art, such but not limited to treatment of the glucan with formic acid.

Examples of useful beta-glucans include, but are not limited to, particulate and soluble yeast cell wall glucans as described in PCT/IB95/00265 and EP 0759089. Depending upon yeast strain and type, glucan constitutes up to 25 % of the yeast cell wall dry weight. During the process of isolating beta-glucan from yeast the outer layer of mannoprotein is removed as well as most of the inner content of the cell, leaving a "ghost" particle, or whole glucan particle, constituting the beta-glucan layer. An example of such beta-glucans include, but is not limited to, the beta- (1 ,3/1 ,6)-glucan product marketed as APG 3-6 by the company Biothera. If the beta-glucan is isolated from autolysed yeast, the cell wall is more collapsed giving a crumpled ghost particle.

Other yeasts which provide a source for the glucan include Brewers yeast, Candida sp. like Candida albicans, Candida cloacae, Candida tropicalis, Candida utilis,, Hansenula sp. like Hansenula wingei, Hansenula ami, Hansenula henricii and Hansenula americana, Histoplasma sp., Kloeckera sp., Kluyveromyces sp. like Kluyveromyces lactis, Kluyveromyces fragilis, Kluyveromyces polysporus, Pichia sp., Rhodotorula sp., Saccharomyces sp. like Saccharomyces delbruekii,

Saccharomyces rosei, Saccharomyces microellipsodes, Saccharomyces carlsbergensis or different Saccharomyces strains like Saccharomyces cerevisiae R4 (NRRL Y-15903) and R4 Ad (ATCC No. 74181 ), Schizophyllum sp.,

Schizosaccharomyces sp. like Schizosaccharomyces pombe, Torula sp. and Torulopsis sp.. It is convenient to use processed products or cell extracts in the formulations of the present invention. These products may be hydrolysed or autolysed cells, partially or completely purified cell walls. All these products are available in various forms suited to different types of use: liquid, semi-paste, paste, fine powder, oil-coated powder, micro-granulated powder, to mention only some.

Products containing isolated carbohydrate components may be combination products of two or more components (e.g. from the yeast cell wall), for example a combination of glucan and mannan. Mannan is a polysaccharide containing a high proportion of mannose sub-units. Preferably it is made up of D-mannose, D-glucose and D-galactose at a ratio of approximately 3:1 :1 .

The glucan may be mixed with other components e.g. other parts of the cell wall such as mannans or components not being part of the cell walls, like vitamins or minerals and other agents frequently used in the cosmetic industry.

As mentioned above, β-glucans comprise β-linked D-glucose residues. The β- glucans of the present invention may contain one or more non-glucose residues, particularly mannose residues. Thus, the glucans of the present invention may be of a mixed chain structure. However, the number of glucose residues is far higher than the number of non-glucose residues. Typically, if non-glucose residues are present within the glucan molecule, the ratio of non-glucose residues (e.g.

mannose) : glucose residues does not exceed 1 : 10 and preferably does not exceed 1 : 20. In addition to the (1 ,3)-linked side chains, the glucans preferably also have one or more (1 ,6)-linked side chains. Optionally the glucans present in the formulations may have been treated by acid or enzyme or any other suitable method to significantly reduce or eliminate the number of repetitive (1 ,6)-linked glucose molecules within the glucan, or occur naturally with low levels of (1 ,6)-linkages. These (1 ,6)-linked glucose molecules are mainly in a beta-conformation, and would normally be found in the side chains of the beta-glucan molecule. The number of beta-(1 ,6)-linked glucose moieties can vary from one to a significant proportion of the glucose moieties depending on the source of glucan. The resulting glucans have beta-(1 ,3)-main chains and beta- (1 ,3)-side chains which are linked thereto through a single beta-(1 ,6)-linkage which is not cleaved off by the elimination treatment. These products can be particulate, semi-soluble or soluble. These modified glucan molecules are preferably derived from yeast, more preferably S. cerevisiae.

Thus, in one embodiment, the glucans are essentially free of repetitive beta-(1 ,6)- linked glucosyl units. Thus, a single (1 ,6)-linkage at the branch points does not provide 'repetitive' beta (1 ,6)-linked glucosyl units. A glucose residue in a (1 ,3)- backbone to which a sidechain or single glucose residue is attached via a β-(1 ,6)- linkage is thus a β-(1 ,3,6)-linked glucose residue. By 'essentially free' is meant less than 2%, preferably less than 1 % of the total glucosyl units. In one embodiment, preferably less than 10% more preferably less than 5%, most preferably less than 3% or 2% of the glycosidic bonds in the glucan molecule will be (1 ,6)-linkages.

Some treatments, such as enzyme treatments, may leave up to 4 beta-(1 ,6)-linked glucosyl units uncleaved in the side chains. Such molecules are also 'essentially free' of repetitive beta-(1 ,6)-linked glucosyl units.

In one embodiment, preferably the glucans contain 80% to 98% β-(1 ,3)-linked glucosyl resides, 1 % to 10% β-(1 ,6)-linked glucosyl residues and 1 % to 10% β- (1 ,3,6)-linked glucosyl residues. More preferably the glucans contain 88% to 98% β-(1 ,3)-linked glucosyl resides, 1 % to 6% (more preferably 1 -3%) β-(1 ,6)-linked glucosyl residues and 1 % to 6% (more preferably 3-6%).

The glucan which can be used in relation to the present invention could be in the form of a single, extracted fraction or two or more different fractions with different molecular weights.

Preferred sources for the glucan for the present application are cell walls from Saccharomyces cerevisiae. Of these, the particulate yeast products of Biotec Pharmacon ASA and the particulate yeast product NBG of Sana Pharma AS are particularly preferred. The particulate yeast product NBG is most preferred.

The molecular structure of NBG is as follows:

R= H or (CeHe-ioOsJi-so; n= 35-2000;

NBG (Norwegian Beta Glucan) is a product derived from Bakers Yeast

(Saccharomyces cerevisiae). The product is a natural underivatized (in terms of chemical modifying groups) particulate beta-(1 ,3/1 ,6)-glucan, characterised by NMR and chemical analysis to consist of polymers of beta-1 ,3-linked D-glucose containing side-chains of beta-(1 ,3)- and beta-(1 ,6)-linked D-glucose.

Typical values for the chemical composition of NBG are as follows:

Component % by weight Typical range %

CARBOHYDRATES Min 75 75-80

LIPIDS Max 5 3-5 NITROGEN Max 1 .4 0.8-1 .2

ASH Max 12 8-10

TOTAL SOLID Min 95 95-98

A further preferred glucan for use in the present invention is one which is substantially purified compared to NBG. Such insoluble glucans include 75 to 98% carbohydrate and a lower % by weight of lipids and/or ash than those shown in the above table. Further preferred glucan products are insoluble and have both (1 ,3)- and (1 ,6)-linked side chains, i.e. have not been treated to effect selective removal of (1 ,6)-linked residues.

The preferred particulate beta-glucan may be prepared in the following way:

By repeated extractions in alkali and acid of dry Saccharomyces cerevisiae, for example according to the procedure described in US Patent No. 5,401 ,727

(incorporated herein by reference). The extraction process described removes cytoplasmic components inside the yeast cells as well as the mannose containing polysaccharides and proteoglycans which are on the cell surface. The product prepared according to this procedure consists of a beta-(1 ,3/1 ,6)-glucan with a particle size of 2-5 micrometers. By way of example, the chemical structure of this micro-particulate beta-(1 ,3/1 ,6)-glucan may be characterized by 83% beta-(1 ,3)- linked glucose, 6% beta-(1 ,6)-linked and 5% beta-(1 ,3,6)-linked glucose, and it is a beta-(1 ,3)-glucan chain with beta-(1 ,3,6)-linked glucose as the branch points.

The particulate glucans for use in the present invention have a molecular weight in the range of 5000 Da to 1 ,000,000 Da, preferably in the range of 25 kDa to 500 kDa, more preferably in the range of 150 kDa to 300 kDa and most preferably about 250 kDa.

The β-(1 ,3)-glucan used in the formulations of the present invention may also be isolated from yeast cell walls by conventional methods known by those of ordinary skill in the art, including the method disclosed in US 5223491 . The glucan may have a particle size of about 1 .0 microns or less, preferably about 0.2 microns or less. An example of a method which can be used to obtain glucans with such small particle size is described in US 5576015. The amount of water-insoluble glucan to be used and the specific components of the composition will depend upon the nature of the product and its intended use. Preferably, the effective amount of glucan will range from about 0.001 w/w % to about 10 w/w % of the composition, more preferably from about 0.1 w/w % to about 4 w/w %. Some preferred concentration ranges are as follows: for body lotions and creams: 0.1 -2%; for skin care creams and gels: 0.1 -7%; for eye care products: 0.1 -5%; for concentrated products such as creams, serums, unidoses, etc: up to 10%; as a co-active agent: 0.1 -7%; and for decorative cosmetics (powder form): 0.001 -0. 015%. Another example of a glucan which can be used in the formulations of the present invention is a glucan prepared in accordance with the process as described and claimed in US Patent No. 6,242,594. This document describes the isolation of a microparticulate poly-(l,3)-beta-D-glucopyranosyl-(l,6)- beta-D-glucopyranose from Saccharomyces cerevisiae. The glucan has the a general 1 ,3 linkage structure and has a molecular weight of between about 1 .2 million and 2.2 million Daltons. It is an amorphous powder slightly, soluble in most aqueous and organic solvents but sparingly soluble in DMSO.

The particulate glucans described above may be solubilised as described in WO/2001 /062283 (incorporated herein by reference).

The reduction of the beta-(1 ,6)-linked glucosyl residues may be achieved in one of the following ways: i) Enzymatic treatment, for example as described in Norwegian Patent No. 300692:

The side chains of beta-(1 ,6)-linked glucose in the micro-particulate product prepared as in US Patent No. 5,401 ,727 are selectively removed by enzyme treatment with an enzyme which specifically acts on beta-(1 ,6)-linkages in a poly- glucose chain. The micro-particulate product (0.2 grams) is suspended in 40 ml 50 mM ammonium acetate buffer at pH 5.0 and mixed with 20 units of the beta-(1 ,6)- glucanase enzyme. The mixture is continuously stirred for 6 hours at 37 degrees Celsius and the action of the enzyme stopped by boiling for 5 minutes. The residual enzyme treated particles are washed repeatedly in sterile distilled water by centrifugation and re-suspension. The resulting product is a branched beta-(1 ,3)- glucan with beta-(l,3)-glucan side chains connected by beta-(1 ,6)-linked at the branching points, and being essentially free of beta-(1 ,6)-linked glucose in the side chains which extend from the branching points.

The key step being incubation of a particulate glucan with a beta-(1 ,6)-glucanase enzyme at 32 to 40 ^<€ for 3 to 9 hours. ii) Formic Acid treatment: For example, a micro-particulate product prepared as in US Patent No. 5,401 ,727 may be suspended in formic acid and heated. The suspension is cooled and free formic acid removed. Thus, formic acid can be used to both reduce the number of beta-(1 ,6)-linked glucosyl residues in the glucan and to solubilise the glucan.

An optional glucan containing formulation for use in the invention is a mixture of soluble beta-glucan molecules with molecular weights (MW) >6000 daltons that interact to give a higher order conformation. For example, a mixture of linear beta- (1 ,3)-glucan chains with a numerical MW >6 kDa, preferably with a MW ranging from 6-30 kDa, together with branched high molecular weight beta-(1 ,3)-glucans (e.g. MW >15 kDa) with beta-(1 ,3)-linked side chain(s) extending from within the main chain as shown above.

Preferably, the glucans have an average molecular weight of single chains of about 20 kDa, with a range from about 6 to about 30 kDa, preferably from about 15 to about 25 kDa. In the single strain format the glucans may exist as a mixture of conformations including random coils, gel matrices or aggregates, triple helices and single helices. When in aqueous solution the molecules may take part in interchain interactions giving a high molecular weight appearance of up to 5 000 kDa when analysed by gel performance chromatography. Preferred compositions are those that form a gel like appearance in aqueous solution, demonstrating complex intermolecular interactions.

Other structures and/or structural conformations within the family of beta-(1 ,3)- glucans can be readily identified or isolated by a person of ordinary skill in the art following the teaching of this invention. The above is thus a guideline to achieve a highly potent product, but is not a limitation towards even more potent products. Isolated structural elements of the complex mixture may have improved effects over the presently exemplified formulations when administered.

Glucans have been described as components in skin care formulations, e.g. in EP1 165020 B1 .

Preferably, the total amount of glucan present in the formulation is 0.15% to 10%, more preferably 0.25% to 5%, most preferably about 0.3% by weight of the formulation.

Preferably, the glucan is present in the form of NBG of Sana Pharma AS, which comprises approximately 75% by weight of β-glucan. In this case, preferably the total amount of glucan-containing product in the formulation is 0.2% to 10%, more preferably 0.3 to 5%, most preferably about 0.4% by weight of the formulation.

Soy protein is well-known in the field as protein extracted from the soybean

(Glycine soja) plant. The term "soy protein" includes within its scope unmodified soy protein, hydrolyzed soy protein, i.e. soy protein hydrolysate, soy protein extract, hydrolyzed soy protein extract, soy peptides and mixtures thereof. Mixtures include mixtures of different peptide or protein fractions or extracts. Hydrolyzed proteins are proteins that have been broken down into smaller units (e.g. peptides).

The soy peptides in the present formulations are preferably shorter than 300 amino acids in length, preferably shorter than 100 amino acids, more preferably shorter than 50 amino acids, most preferably shorter than 30 amino acids. Preferably the soy protein is hydrolyzed soy protein. Preferably the soy protein is a purified soy protein.

The above soy protein products are known in the field as useful in the formulation of body, hand, face, and neck products and other skin and hair care products, as well as mascara. Soy protein products are typically used as skin conditioning agents or emulsifying agents. Soy protein products suitable for cosmetic applications are known to one of ordinary skill in the art and any such product can be used in the present formulations. The soybean protein used in the present formulations may be prepared from any soybean species. Soy protein is made from de-hulled, de-fatted soybean meal. Methods of preparing soybean protein suitable for cosmetic applications would be well-known to one of ordinary skill in the art and any such method could be used. For instance, soy protein concentrate can be prepared by various processes including but not limited to an aqueous alcohol wash process, an acid wash process and a water wash process with heat denaturation. Soy protein hydrolysates can be prepared by any method known in the art, for instance via enzymatic or chemical degradation of soy protein. For instance, a process for preparing hydrolyzed products of soy protein is disclosed in US 4,757,007.

The use of rice products in cosmetics is known. Bran is the hard outer layer of grain and consists of combined aleurone and pericarp. Rice bran is a by-product of the rice milling process and it contains various antioxidants. Hydrolyzed rice bran protein has been used in cosmetic products as a skin and hair conditioning agent.

The term "hydrolyzed rice bran protein" includes within its scope hydrolyzed rice bran protein extract, rice bran peptides and mixtures thereof. Mixtures include mixtures of different peptide fractions or extracts. Hydrolyzed proteins are proteins that have been broken down into smaller units (e.g. peptides). Rice bran peptides are preferably shorter than 300 amino acids in length, preferably shorter than 100 amino acids, more preferably shorter than 50 amino acids, most preferably shorter than 30 amino acids. Preferably the hydrolyzed rice bran protein is purified. Hydrolyzed rice bran protein products suitable for cosmetic applications are known to one of ordinary skill in the art and any such product can be used in the present formulations.

The hydrolyzed rice bran product used in the present formulations may be prepared from any rice species. Methods of preparing hydrolyzed rice bran protein suitable for cosmetic applications would be well-known to one of ordinary skill in the art and any such method could be used. Rice bran protein hydrolysates can be prepared by enzymatic or chemical degradation of soy protein. For instance, processes for preparing hydrolyzed products of rice bran protein using a microbial protease is disclosed in Silpradit et a!., (2010) As. J. Food Ag-lnd. 3(02): 221 -231 . An oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule to another. Within skin, oxidoreductases serve to increase the efficiency of antioxidants which reduce collagen and elastin degradation. Oxidoreductases have been used in skin care to provide skin protection by providing for prolonged antioxidant activity and by reducing the presence of free radicals. An example of an oxidoreductase suitable for use in a cosmetic product is disclosed in US

10/655,780. The oxidoreductase used in the present formulations may be derived from any natural or recombinant source, but is preferably a yeast-derived oxidoreductase. Mixtures of a plurality of oxidoreductases may be used in the present formulations.

As mentioned above, preferably the topical skin care formulation comprises a) 3-(1 ,3/1 ,6)-glucan;

b) soy protein;

c) hydrolysed rice brain protein; and

d) an oxidoreductase. The combination of hydrolyzed rice bran protein, soy protein and oxidoreductases can reduce dark circles and puffiness around the eyes, improve micro-circulation, strengthen native collagen and elastin matrix, and reduce the presence of free radicals to delay the visible signs of premature aging. The composition inhibits elastase and collagenases, thereby reducing the effects of chronic UV radiation on the connective tissue, which helps the skin retain its natural firmness.

Oxidoreductases reduce the amount of free radicals. This results in a visible reduction of puffiness around the eyes.

In the case where hydrolyzed rice bran protein, soy protein and oxidoreductases are all present in the formulations of the invention, preferably the soy protein, hydrolysed rice bran protein and oxidoreductase components are not provided separately in the formulation but are instead provided in the form of a single product, which is preferably REGU^®-AGE, available from Pentapharm. REGU^®- AGE is a clear to slightly opalescent, yellowish solution with a pH of 5.3 to 6.3. The optimal pH range for REGU^®-AGE in cosmetic formulations is 5.5 to 8.0 and REGU -AGE should be processed below 40 °C. REGU -AGE is preferably incorporated into the aqueous phase of a cosmetic formulation. REGU^®-AGE is compatible with ethanol at concentrations of up to 20%. REGU^®-AGE possesses >10 Collagenase inhibitory units of activity per ml, >5 elastase inhibitory units of activity per ml and >1000 oxidoreductase units of activity per ml (all assessed as per photometric assay).

REGU^®- AGE comprises 0.1 % to 1 % by weight soybean protein, 5% to 10% by weight hydrolysed rice bran protein and 0.1 % to 1 % oxidoreductases.

As well as soy protein, hydrolysed rice bran protein and oxidoreductases, REGU^®- AGE contains the solvents water (>50% by weight of the REGU^®-AGE composition) and glycerin (10% to 25% by weight of the REGU^®- AGE composition), together with sodium benzoate (0.1 % to 1 % by weight of the REGU^®- AGE composition) and preservatives (totalling 0.1 % to 1 % by weight of the REGU^®-AGE composition. The preservatives include phenoxyethanol, methylparaben, ethylparaben,

propylparaben, butylparaben, isobutylparaben and sodium dextran sulphate, each of which is present at <0.1 % by weight of the REGU^®-AGE composition.

Preferably, the total amount of soy protein present in the formulation is 0.00125% to 5% by weight of the formulation, more preferably 0.00125% to 3%, still more preferably 0.00125% to 1 %, yet more preferably 0.00125% to 0.2%, yet more preferably 0.002% to 0.05%, most preferably about 0.015%.

Preferably, the total amount of hydrolysed rice bran protein present in the formulation is 0.0625% to 5% by weight of the formulation, more preferably

0.0625% to 3%, still more preferably 0.0625% to 2%, yet more preferably 0.0625% to 1 %, yet more preferably 0.1 % to 0.5%, most preferably about 0.225%.

Preferably, the total amount of oxidoreductase present in the formulation is

0.00125% to 5% by weight of the formulation, more preferably 0.00125% to 3%, still more preferably 0.00125% to 1 %, yet more preferably 0.00125% to 0.2%, yet more preferably 0.002% to 0.05%, most preferably about 0.015%. If REGU -AGE is present in the formulation then preferably it is present at a level of 1 .25 to 20% by weight of the formulation, more preferably 2% to 15%, still more preferably 2% to 5%, most preferably about 3%. In a second aspect, the present invention provides a topical skin care formulation comprising:

a) 3-(1 ,3/1 ,6)-glucan; and

b) a protein-containing plant extract, wherein said plant is a rosid. Preferably, the present invention provides a topical skin care formulation comprising:

a) 3-(1 ,3/1 ,6)-glucan; and

b) a protein-containing plant extract, wherein said plant is of the genus Vigna. Alternatively preferred, the present invention provides a topical skin care formulation comprising:

a) 3-(1 ,3/1 ,6)-glucan; and

b) a protein-containing plant extract, wherein said plant is of the family Fagaceae. The term "topical" means that the skin care formulation is suitable and intended for application to the skin of a human subject.

The β-(1 ,3/1 ,6)-glucan is as described anywhere above. The definitions, description and preferred embodiments discussed above in relation to the first aspect provided by the present invention apply mutatis mutandis to this second aspect provided by the present invention. Thus, preferably, the total amount of glucan present in the formulation is 0.15% to 10%, more preferably 0.25% to 5%, most preferably about 0.3% by weight of the formulation. Preferably, the glucan is present in the form of NBG of Sana Pharma AS, which comprises approximately 75% by weight of β-glucan. In this case, preferably the total amount of glucan- containing product in the formulation is 0.2% to 10%, more preferably 0.3 to 5%, most preferably about 0.4% by weight of the formulation. The rosids are a clade of flowering plants. The topical skin care formulations of the present invention comprise a protein-containing extract from such plants.

The term "protein-containing plant extract" includes within its scope plant extracts containing protein, hydrolyzed protein i.e. protein hydrolysate, protein extract, hydrolyzed protein extract, peptides and mixtures thereof. Mixtures include mixtures of different peptide or protein fractions or extracts. Hydrolyzed proteins are proteins that have been broken down into smaller units (e.g. peptides). Preferably, the plant is of the family Fabaceae or Fagaceae. More preferably, the plant is of the genus Vigna or Fagus.

Members of the Vigna genus include but are not limited to Vigna aconitifolia, Vigna angularis, Vigna caracalla, Vigna debilis, Vigna dinteri, Vigna lanceolata, Vigna lanceolata, Vigna luteola, Vigna marina, Vigna maritima, Vigna mungo, Vigna o- wahuensis, Vigna parkeri, Vigna radiata, Vigna speciosa, Vigna subterranea, Vigna trilobata, Vigna umbellata, Vigna unguiculata and Vigna vexillata. Most preferably, the Vigna is Vigna aconitifolia. Members of the Fagus genus include but are not limited to Fagus crenata, Fagus engleriana, Fagus grandifolia, Fagus hayatae, Fagus japonica, Fagus

longipetiolata, Fagus lucida, Fagus mexicana, Fagus orientalis, Fagus sylvatica, and Fagus taurica. Most preferably the Fagus is Fagus sylvatica. Methods of obtaining plant extracts containing protein are well-known in the art, as are protein-containing plant extract products. The plant extract used in the formulations of the present invention may be obtained by any known method and any plant extract product may be used, provided that the plant extract contains protein. Preferably the protein-containing plant extract contains at least one soluble protein fraction.

The plant extract used in the formulations of the present invention may be a whole plant extract or an extract from a particular part of plant such as but not limited to the roots, stem, leaves, flower, bud and seed. Preferably, the plant extract is a seed extract or a bud extract. Most preferably the protein-conatining plant extract is a Vigna aconitifolia seed extract or a Fagus sylvatica bud extract. The seeds of Vigna aconitifolia contain mainly carbohydrates (56 to 62%), proteins (22 to 24%), water (1 1 %), lipids (1 .9%) and polyphenols (1 .3%).

Vigna aconitifolia seed extract is a botanical alternative to retinol in terms of its effect on the skin but with a higher tolerance and stability. A Vigna aconitifolia seed extract suitable for use in the present application is disclosed in US 2005/0019301 . Preferably, the protein-containing Vigna aconitifolia seed extract is present in the topical formulations of the present invention as Vit-a-Like LS 9737 (Vit-a-Like) from Laboratoires Serobiologiques, Division de Cognis France.

Vit-a-Like is a yellow liquid with characteristic odour. It is soluble in water and insoluble in fats and oils. Vit-a-Like is preferably incorporated into cosmetic formulations at a temperature below 60 °C in the final phase or at room temperature for cold processing. The optimal pH for Vit-a-Like is 3 to 8. Vit-a-Like contains 5 to 10% by weight of seed extract, 1 to 5% by weight of sodium cocoyl glutamate and water (up to 100%).

The Fagus sylvatica bud extracts used in the topical skin care formulations may be obtained by any method known in the art, for instance by using a solvent such as water, methanol, ethanol, propanol, butanol, 1 ,3-butylene glycol or mixtures thereof. Fagus sylvatica bud extracts have been demonstrated to improve cutaneous moistruization and reduce the appearance of wrinkles. A Fagus sylvatica bud extract suitable for use in the present application is disclosed in US 2003/0072827.

Preferably, the protein-containing Fagus sylvatica bud extract is present in the topical formulations of the present invention as Gatuline^® RC or Gatuline^® RC BIO from Gattefosse. Gatuline is prepared from Fagus sylvatica buds which are frozen soon after being gathered. The buds are later thawed and rehydrated. Ultra high frequencies are used to increase the buds' internal temperature quickly, which deactivates the enzymes in the buds' tissues and allows for the extraction of the active substances orignally contained in the fresh plant. The extraction process does not involve any chemical or solvent. Gatuline RC is a light brown-amber liquid, the optimum pH for which is 4.5 to 5.5. Gatuline RC Bio is a brown liquid, the optimum pH for which is 3.5 to 5.5. Gatuline is preferably incorporated into cosmetic formulations at room temperature towards the end of the manufacturing process.

The compositions of Gatuline RC and Gatuline RC Bio are identical apart from the preservatives present therein. The preservative present in Gatuline RC is phenonip (0.45%) and the preservatives in Gatuline RC Bio are benzoic acid (0.2%) and benzyl alcohol (0.3%). Gatuline RC is the preferred Gatuline product for use in the formulations of the present invention.

Both Gatuline RC and Gatuline RC Bio comprise at least 50% by weight water and 10% to 25% by weight Fagus sylvatica extract.

Preferably, the topical skin care formulations of the present invention further comprise hyaluronic acid. Hyaluronic acid is a glycosaminoglycan which attaches to collagen and elastin to form cartilage. This product is increasingly popular in cosmetic formulations which claim to hydrate the skin. Hyaluronic acid is widely available and is typically obtained by fermentation of plant substrates. Biosynthesis is usually performed by a Streptococcus species. Restylane is a common trade name for the product. Juvederm is a bacterial hyaluronic acid injectable filler, similar to Restylane. Preferably, the hyaluronic acid is present in the topical skin care formulaitons of the present invention in the form of 1% BT hyaluronic acid solution from the company Solabia.

Preferably, the total amount of protein-containing rosid plant extract present in the formulation is 0.15% to 5%, more preferably 0.2% to 3.75%, still more preferably 0.2% to 1 .25%, most preferably about 0.3% or about 0.5% by weight of the formulation.

If the rosid plant extract is from a plant of the family Fabaceae, preferably from the genus Vigna and more preferably from the species Vigna acontifolia, then preferably the total amount of rosid plant extract present in the formulation is from 0.15% to 0.5%, more preferably 0.2% to 0.4%, most preferably about 0.3% by weight of the formulation.

When the protein-containing plant extract is Vit-a-Like described above then preferably the total amount of Vit-a-Like present in the formulation is 3% to 5%, most preferably about 4% of the formulation. Vit-a-Like comprises 5 to 10% of Vigna aconitifolia seed extract, therefore, when said extract is present in the formulation in the form of Vit-a-Like, the total amount of rosid plant extract present in the formulation is from 0.15% to 0.5%, more preferably 0.2% to 0.4%, most preferably about 0.3% by weight of the formulation.

If the rosid plant extract is from a plant of the family Fagaceae, preferably from the genus Fagus and more preferably from the species Fagus sylvatica, then preferably the total amount of rosid plant extract present in the formulation is from 0.2% to 5%, more preferably 0.2% to 3.75%, still more preferably 0.2% to 1 .25%, most preferably about 0.5% by weight of the formulation.

When the protein-containing plant extract is a Fagus sylvatica bud extract such as

Gatuline RC described above then preferably the total amount of Gatuline RC present in the formulation is 2% to 20%, more preferably 2% to 15%, still more preferably 2% to 5%, most preferably about 3% by weight of the formulation.

Gatuline RC comprises 10% to 25% by weight of Fagus sylvatica bud extract.

Therefore, when said extract is present in the formulation in the form of Gatuline

RC, the total amount of rosid plant extract present in the formulation is from 0.2% to 5%, more preferably 0.2% to 3.75%, still more preferably 0.2% to 1 .25%, most preferably about 0.5% by weight of the formulation. The same preferred ranges apply when Gatuline RC is used.

Optionally, the total amount of hyaluronic acid present in the formulation is from 0.01 % to 0.03%, most preferably about 0.01 % by weight of the formulation. When the hyaluronic acid is present in the formulation in the form of 1 % BT hyaluronic acid solution, the total amount of said 1 % solution present in the formulation is from 1 % to 3%, most preferably about 1 % by weight of the formulation. Preferably, the total amount of hyaluronic acid present in the formulation is from 0.001 % to 0.003%, most preferably about 0.001 % by weight of the formulation. When the hyaluronic acid is present in the formulation in the form of 1 % BT hyaluronic acid solution, the total amount of said 1 % solution present in the formulation is from 0.1 % to 0.3%, most preferably about 0.1 1 % by weight of the formulation.

Ingredients generally used in cosmetics can also be included in the topical formulations of the present invention, as desired. Such optional additional ingredients include but are not limited to aqueous ingredients, water, powdered ingredients, surfactants, moisturizing agents, viscosity donors, viscosity controllers, binding agents, texture modifiers, lubricants, disintegrants, anti-cracking agents, UV absorbers, colourants, humectants, emulsifiers, skin softeners/conditioning agents, emollients, anti-oxidants, dispersing agents, thickeners, emulsion stabilizers, fixatives, perfumes, adjuvants, polyols, preservatives and cosmetic vectors.

Cosmetic vectors include film formers, liposomes, cyclodextrins, micelles, macroparticles, microparticles, nanoparticles, macrocapsules, microcapsules and nanocapsules. The formulations may also be adsorbed or grafted onto organic polymers or mineral supports.

Preservatives are commonly used in cosmetics and personal care products to prevent product damage caused by microorganisms and to protect the product from inadvertent contamination by the consumer during use. The optional additional ingredients must be added in quantities and quality standards that do not affect the efficacy of the formulation and must result in a dermatologically compatible formulation. One of ordinary skill in the art will be aware of a multitude of optional additional ingredients for topical skin care formulations and will be able to select the ingredients required for his desired purpose. Different additional ingredients may be required for different types of skin care formulation, for instance, a greater number of water-soluble ingredients may be required for a cosmetic serum or gel than for a cosmetic cream.

Optionally, the topical skin care formulation is a formulation selected from the group consisting of a face cream, a face serum, a face lotion, an eye cream, an eye gel, a suncream, an aftersun lotion, a neck cream, a body lotion, a hand cream, a hand serum, a hand lotion, a hand gel and a makeup product.

Preferably, the topical skin care formulation comprising

a) 3-(1 ,3/1 ,6)-glucan;

b) soy protein; and/or

c) hydrolysed rice brain protein; and/or

d) an oxidoreductase,

is a face cream.

Preferably, the topical skin care formulation comprising

a) 3-(1 ,3/1 ,6)-glucan; and

b) a protein-containing plant extract, wherein said plant is a rosid,

is a face serum or an eye gel. More preferably, when the rosid plant extract is an extract from a plant of the Fabaceae family then said formulation is a face serum and when the rosid plant extract is an extract from a plant of the Fagaceae family then said formulation is an eye gel.

Preferred additional ingredients include water and the following:

Glycerin, which is a humectant, emulsifier and skin softener derived from vegetable oil.

Capric/caprylic triglyceride, which is an emollient commonly used in skin products and soaps. Capric/caprylic triglyceride is a combined triester, a blend of capric and caprylic acids. It is also known as fractionated coconut oil. Its most popular use is as an emollient for calming and softening the skin due to the fact that it is capable of rapid skin penetration. It is also an effective dispersing agent. Caprylic/capric triglyceride is not greasy and has a very light texture. It is also rapidly absorbing, and is highly skin-compatible, due to the fact that it will not aggravate sensitive skin types. It also does not leave behind visible oil remnants on the skin. Since it has an extended shelf life, it does not necessitate special storage or handling practices, beyond being stored in a dry, cool location. Optionally, capric/caprylic triglyceride is present in the form of Crodamol GTCC, a commercial product from Croda. Hydrolysed waxes derived from candelilla and/or jojoba and/or rice bran, together with glyceryl stearate, ceteraryl alcohol & sodium stearoyi lactylate. The preferred hydrolysed waxes contain polyglyceryl-3 esters. Such compounds are natural emulsifiers which are free from polyethylene glycol. These natural emulsifiers significantly increase the softness of topical skin care formulations and boosts the moisturizing capacity of the formulation. The sodium stearoyi lactylate is broken down by the skin releasing lactic acid which, as well as being a natural humectant, improves the barrier function and stimulates ceramide production. In addition, the hydrophilized waxes form a soft, smooth film on the skin's surface and prevent the skin from water loss. Yet, the skin can perfectly breathe as the wax does not prevent gaseous exchange.

Optionally, the hydrolysed waxes are present in the form of the commercial product Emulium^® Kappa from the Gattefosse. Emulium^® Kappa is capable of absorbing six times its weight in water.

As an alternative to using hydrolysed waxes derived from candelilla and/or jojoba and/or rice bran, together with glyceryl stearate, ceteraryl alcohol & sodium stearoyi lactylate, high amounts of silicone gums and elastomers can be used in the formulation.

Butyrospermum Parkii (Shea Butter), which is a commonly used emollient in cosmetic products. It is a slightly yellowish or ivory coloured natural fat extracted from the seed of the African shea tree by crushing and boiling. It is widely used in cosmetics as a moisturizer and salve. Optionally, the shea butter is present in the form of Lipex® Sheasoft, a commercial product from AarhusKarlshamn.

Simmondsia Chinensis (Jojoba) seed oil, which is an emollient. It is the liquid wax produced in the seed of the jojoba (Simmondsia chinensis) plant, a shrub native to southern Arizona, southern California and north-western Mexico. The oil makes up approximately 50% of the jojoba seed by weight. Unlike common vegetable oils, jojoba oil is chemically very similar to human sebum. Jojoba oil is commonly used an ingredient in cosmetics and personal care products, especially skin care and hair care. Canola Oil, which is a general-purpose emollient commonly used in skin care creams and lotions. It contains natural phytosterols and tocopherols. When used in cosmetic formulations, canola oil may be present as fractionated canola oil. Canola oil has documented skin calming properties. It has a high oxidative stability, which gives it an extended shelf life in formulations. Optionally, the canola oil is present in the form of Akorex L, a commercial product from AarhusKarlshamn.

A straight-chain alcohol selected from cetearyl, cetyl, myristyl and behenyl alcohols, or a branched-chain alcohol such as but not limited to isostearyl alcohol. Such alcohols are thickeners and emulsion stabilizers. Cetearyl alcohol, which is a mixture of mostly of cetyl and stearyl alcohols is preferred. Stearyl alcohols are fatty alcohols that occur naturally in small quantities in plants and animals. Cetearyl alcohol and the other fatty alcohols keep an emulsion from separating into its oil and liquid components. These ingredients are also used to alter the thickness of liquid products. Optionally, these alcohols are present in the form of Kalcol 6850P, a commercial product from Kao Chemicals.

Microcrystalline cellulose or cellulose gum, which are viscosity controllers and binding agents. Microcrystalline cellulose is derived from high quality wood pulp. While cellulose is the most abundant organic material, microcrystalline cellulose can only be derived from a special grade of alpha cellulose. In addition to its use as a binder/diluent, microcrystalline cellulose also has some lubricant and disintegrant properties. It is a natural emulsion stabilizer, a viscosity controller and anti-cracking agent. Optionally, the microcrystalline cellulose or cellulose gum is present in the form of Avicel^®PC61 1 , a commercial product from FMC BioPolymer.

Vitamin E, which is a general term for tocopherols and tocotrienols. Vitamin E compounds are fat-soluble antioxidants which stop the production of reactive oxygen species formed when fat undergoes oxidation. Optionally, vitamin E is present in the form of Vitamin E acetate (dl-a-Tocopheryl Acetate).

Xanthan gum, which is a binding agent and a texture modifier. Xanthan gum is a cream coloured, odourless powder derived from the fermentation of corn sugar. It is used in a wide variety of cosmetics and personal care products including makeup, skincare products and toothpaste. Xanthan gum is added to cosmetics and personal care products as a natural binding agent, an emulsion stabilizer, a skin conditioning agent, a surfactant and to increase the viscosity of liquid. Optionally, the xanthan gum is present in the form of Satiaxane CX91 1 , a commercial product from Cargill.

Phenoxyethanol and/or benzoic acid and/or dehydroacetic acid which are preservatives. Phenoxyethanol is an organic chemical compound, a glycol ether often used in dermatological products such as skin creams and sunscreen. It is a colourless oily liquid. It is also used as a fixative for perfumes. It is moderately soluble in water. Benzoic acid is a colourless crystalline solid and the simplest aromatic carboxylic acid. Dehydroacetic acid is a pyrone derivative. Optionally all of phenoxyethanol, benzoic acid and dehydroacetic acid are present, in which case they may be present in the form of Euxyl^® K703, a commercial product from

Schulke & Mayr GmbH.

Preferred perfumes include natural perfumes such as orange and pomegranate perfumes. Perfumes would not typically be used in the eye gel formulations of the present invention. Octyldodecyl Myristate, which is a fluid emollient for oils and emulsions. It provides a rich feel, improves the spread ability on the skin and is non comedogenic. It can also reduce dry feel of emulsions containing vegetable oils and durably improves comfort. Preferably the octyldodecyl myristate is present in the present

formulations in the form of MOD, a commercial product from Gattefosse.

Actinidia Chinensis (Kiwi) Fruit Water, which is a preservative-free plant water. Kiwi water can enhance skin radiance and contribute to a better skin hydration.

Preferably, the kiwi fruit water is extracted by flash distillation or hyper frequencies. Preferbaly the kiwi water is 100% kiwi water, i.e. pure kiwi water. Such pure kiwi waters provide the skin with essential nutrients. Preferably, the kiwi water is present in the form of Original Extract Kiwi, a commercial product from Gattefosse.

Sodium Ascorbyl Phosphate, which is a vitamin C derivative. It is known for use in cosmetic products, and has been discloses as acting as an in-vivo antioxidant, promoting collagen formation and lightening the skin. Sodium Ascorbyl Phosphate is cleaved enzymatically in the skin to release active vitamin C. It is water-soluble and is stable in cosmetic formulations. Preferably the sodium ascorbyl phosphate is present in the form of STAY-C^® 50, a commercial product from DSM Nutritional Products.

Helianthus Annuus (Sunflower) Seed Oil, which is an emollient and a carrier of another cosmetic ingredient; rosemary extract (below). Sunflower Seed Oil is derived from the seed of sunflower plants and contains large amounts of vitamin E, softens and protects the skin.

Rosemarinus Officinalis (Rosemary) Leaf Extract, which stimulates scalp circulation, encourages hair growth, purifies and rejuvenates skin.

Preferably the sunflower seed oil and the rosemary lead extract are present in the formulation in the form of a combined product, Rosemary Antioxidant 14%

Diterpene Phenols, a commercial product from Flavex Naturextrakte GmbH.

Acrylates/Cio-3o alkyl acrylate crosspolymer, which is an emulsifier and a non-toxic thickener suitable for use in formulations which demand superior appearance, exceptional clarity, smooth flow (shear-thinning) and suspending or stabilizing properties. It delivers effective performance across a broad pH range, with greatest viscosity efficiency at pH 5.0. The polymer imparts a light, cushiony feel in applications, helping to promote an enhanced tactile experience in use. Preferably the acrylates/Cio-3o alkyl acrylate crosspolymer is present in the form of Pemulen TR-1 , a commercial product from Lubrizol.

Sodium Hydroxide, which functions as a pH adjuster. Preferably the sodium hydroxide is present in the present formulations in the form of solid caustic soda micropearls, which are readily available.

Argan oil, which is is the nut oil of the Argania Spinosa (Argan) tree, which grows in south west Morocco. Argan oil has twice as much vitamin E as olive oil and is rich in antioxidants, which help neutralise free radicals and prevent skin damage. It is 80% unsaturated and contains eight essential fatty acids including Omega 6, which cannot be produced by the body. It helps prevent loss of moisture from the skin. It contains rare plant sterols, which have anti-inflammatory properties. It also contains saponins, which soften the skin and ease acne and eczema and it restores the skin's water lipid layer, helping to minimise wrinkles. Preferably, the Argan oil is present in the form of organic Argan oil.

Hamamelis virginiana (Witch Hazel) leaf extract, which is derived from the leaves of the Witch Hazel plant. It is a known astringent and anti-inflammatory agent. It soothes and heals skin. Preferably the Witch Hazel leaf extract is present in the form of Witch Hazel Didtillate SB, a commercial product from Crodarom.

Malva sylvestris (Mallow) flower extract, which has some anti-inflammatory and soothing properties for the skin, as well as some potential antioxidant benefits. Preferably the Malva sylvestris (Mallow) flower extract is present in the form of Mallow GL, a commercial product from Crodarom.

All combinations of the additional optional ingredients discussed above in the formulations of the present invention are explicitly provided. The skilled man will readily be able to determine the amounts of the additional optional ingredients required for his desired purpose.

Table 1 displays the preferred amounts of the above optional additional ingredients when they are present in the topical skin care formulations, which are preferably face creams, comprising:

a) 3-(1 ,3/1 ,6)-glucan;

b) soy protein; and/or

c) hydrolysed rice brain protein; and/or

d) an oxidoreductase. Table 2 displays the preferred amounts of the above optional additional ingredients when they are present in the topical skin care formulations, which are preferably face serums or eye gels, comprising:

a) 3-(1 ,3/1 ,6)-glucan; and

b) a protein-containing plant extract, wherein said plant is a rosid. Table 1

Table 2

Table 2 (continued)

If any components are present in the formulation in the form of a product in which they are not the sole component then it is within the competence of the skilled man to calculate the amount of the product to include in order to provide the preferred amount of said component in the formulation.

Preferably, the topical skin care formulation of the present invention comprising

a) 3-(1 ,3/1 ,6)-glucan;

b) soy protein; and/or

c) hydrolysed rice brain protein; and/or

d) an oxidoreductase

also comprises water, glycerin, caprylic /capric triglyceride, Candelilla/Jojoba/Rice Bran Polyglyceryl-3 Esters & Glyceryl Stearate & Ceteraryl Alcohol & Sodium StearoyI lactylate, Butyrospermum Parkii Butter, Simmondsia Chinensis (Jojoba) Seed Oil, Canola Oil, Cetearyl Alcohol, Microcrystalline Cellulose & Cellulose Gum, Tocopherol (Vitamin E, Xanthan Gum, Phenoxyethanol, Benzoic Acid, Dehydroacetic Acid and Parfum as descrived above. Preferably such a formulation is a face cream.

Preferably, if the rosid plant extract is an extract from a plant of the Fabaceae family then the topical skin care formulation of the present invention comprises, in addition to said extract and β-(1 ,3/1 ,6)-glucan as described above, hyaluronic acid, water, glycerin, caprylic /CapricTriglyceride, Butyrospermum Parkii Butter, Octyldodecyl Myristate, Simmondsia Chinensis (Jojoba) Seed Oil, Actinidia Chinensis (Kiwi) Fruit Water, Cetearyl Alcohol, Microcrystalline Cellulose & Cellulose Gum, Sodium Ascorbyl Phosphate, Sunfower seed oil, Rosemarinus Officinalis Leaf Extract, Acrylates/C 10-30 Alkyl Acrylate Crosspolymer, Tocopherol (Vitamin E), Xanthan Gum, Phenoxyethanol, Benzoic Acid, Dehydroacetic Acid, Sodium Hydroxide and Parfum as described above, preferably at the levels described above. Preferably such a formulation is a facial serum.

Preferably, if the rosid plant extract is an extract from a plant of the Fagaceae family then the topical skin care formulation of the present invention comprises, in addition to said extract and β-(1 ,3/1 ,6)-glucan as described above, hyaluronic acid, water, glycerin, caprylic /CapricTriglyceride, Octyldodecyl Myristate, Simmondsia Chinensis (Jojoba) Seed Oil, Actinidia Chinensis (Kiwi) Fruit Water, Argan oil, Cetearyl Alcohol, Microcrystalline Cellulose & Cellulose Gum, Witch Hazel leaf water, Mallow Flower extract, Acrylates/C 10- 30 Alkyl Acrylate Crosspolymer, Tocopherol (Vitamin E), Xanthan Gum, Phenoxyethanol, Benzoic Acid, Dehydroacetic Acid and Sodium Hydroxide as described above, preferably at the levels described above. Preferably such a formulation is an eye gel.

The topical skin care formulations of the invention may be manufactured using any technique known in the art. In a further aspect, the present invention provides a method of preparing a topical skin care formulation comprising admixing:

a) 3-(1 ,3/1 ,6)-glucan;

b) soy protein; and/or

c) hydrolysed rice brain protein; and/or

d) an oxidoreductase.

In a further aspect, the present invention provides a method of preparing a topical skin care formulation comprising admixing:

a) 3-(1 ,3/1 ,6)-glucan; and

b) a protein-containing plant extract, wherein said plant is a rosid.

The preferences, definitions and embodiments described above in relation to the formulations of the invention apply equally to these methods of preparing said

formulations.

In a further aspect, the present invention provides a cosmetic skin treatment method in which a topical skin care formulation as defined anywhere herein is applied topically to the skin of a human subject.

Preferably the skin care formulation reduces wrinkle number or depth or prevents an increase in wrinkle number or depth, reduces skin roughness or prevents an increase in skin roughness, increases skin elasticity or prevents a loss of skin elasticity, increases skin firmness or prevents a loss of skin firmness, increases skin density or prevents a loss of skin density, increases skin moisture content or prevents a loss of skin moisture content, reduces blemishes or areas of atypical pigmentation. Particularly preferably the topical skin care formulation reduces wrinkle depth, reduces skin roughness, increases skin firmness, increases skin elasticity or prevents the opposite effect from occurring. The skin care formulation may be applied on an as-needed or as-desired basis, for example, once-monthly, once-weekly, daily, or more than once daily. Similarly, application can be every other day, week, or month, every third day, week, or month, every fourth day, week, or month, and the like. Application can be multiple times per day. The frequency of application may depend on the type of formulation in question, i.e. a face cream, suncream, eye gel, face serum etc.

Preferably, however, the skin care formulation is applied once or twice daily. Most preferably the skin care formulation is applied twice daily.

The human subject may apply any amount of the formulation upon each application. The amount of formulation applied may depend on the type of formulation in question, i.e. a face cream, suncream, eye gel, face serum etc as well as the area of skin to which it is to be applied. Clearly, a greater quantity of formulation will be applied to the entire body than to a part thereof and a greater quantity of formulation will be applied to the face of a subject than specifically to the area surrounding the eyes. The degree of wrinkling, blemishing or other visible sign of skin aging in a given area may also influence the amount of formulation to be applied per application. Those using these products are familiar with the amount it is appropriate to apply and this will also depend on skin dryness and the time taken to rub in the product and how much surface residue is desired or acceptable. Typically, the amount applied will be sufficient to cover the desired area.

In a further aspect, the present invention provides an oral composition comprising either:

(i) borage seed oil and astaxanthin; or

(ii) vitamin C, collagen hydrolysate, chondroitin sulphate and glucosamine.

An oral composition is a composition suitable for and intended to be consumed orally. The term "oral composition" includes within its scope a tablet, chewable tablet, caplet, capsule, gelcap, powder and softgel liquid. Preferably the oral composition is a capsule or a tablet.

Preferably, if the oral composition is formulated into a capsule then it comprises borage seed oil and astaxanthin. Still more preferably said borage seed oil and astaxanthin are present in the filling of the capsule rather than in a capsule coating. If the oral composition is a tablet then preferably it comprises vitamin C, collagen hydrolysate, chondroitin sulphate and glucosamine.

These oral compositions are intended to provide a per os cosmetic treatment of the skin. Preferably the compositions provide or contribute to a reduction in the visible signs of skin aging and/or the other benefits discussed above in the context of topical formulations.

Borage seed oil is derived from the seeds of the Borago officinalis (borage) plant. Borage oil is a clear yellow to greenish oil with characteristic odour. It is a pure, virgin oil produced only by cold press and filtration without using any solvents, additives, antioxidants or other synthetic agents.

Borage seed oil is the highest known plant-based source of gamma-linolenic acid (GLA, 18:3, cis 6,9,12-octadecatrienoic acid), for which borage is the highest known plant-based source. The seed oil content is typically between 20.0% and 38% by weight, however, a specific extraction process may offer purified products with 50% or greater GLA content. Borage seed oil also contains the fatty acids palmitic acid (9.0% to 12.0%), stearic acid (3.0% to 5.0%), oleic acid (15.0% to 20.0%), linoleic acid (35.0% to 42.0%), eicosenoic acid (3.0% to 5.5%), docosenoic acid (1 .0% to 4.0%) erucic acid (1 .5% to 3.5%), and nervonic acid (about 1 .5%) and others. The oil is often marketed as "starflower oil" or "borage oil".

Unsaturated fatty acids are important for the skin barrier function and skin moisture. GLA has been used in oral and topical cosmetic products and has been demonstrated to improve skin moisture, firmness and elasticity.

Methods of isolating borage seed oil are well-known to those of ordinary skill in the art. Similarly, borage seed oil products suitable for use in oral compositions would be well- known to one of ordinary skill in the art.

Any borage seed oil may be used in the oral compositions of the present invention, e.g. as sold by the company Sanmark. Preferably the borage oil is present in the form of Borage Oil 20% GLA cold pressed, a commercial product from Sanmark, which contains about 20% by weight GLA or in the form of Refined Borage Oil min 22% GLA, which is a commercial product from Northstar Lipids containing a minimum of 22% GLA by weight. Exposure to sunlight leads to increased formation of reactive oxygen species (ROS) and can damage skin. Fat-soluble antioxidants can be stored in the skin where they act as natural pigments and protect the skin against the harmful effects of the sun's rays, however, this natural protection is depleted with age and UV exposure.

Antioxidants help to prevent the accumulation of ROS. For this reason they are useful in topical and oral cosmetic products and food supplements. Astaxanthin has potent antioxidant activity.

The astaxanthin used in the present oral compositions can be obtained from any known source. For instance, astaxanthin may be prepared from the ink coloured yeast

Xanthophyllomyces dendrorhous, or prepared synthetically. Preferably, however, astaxanthin is produced by algae such as but not limited to Haematococcus pluvialis. Haematococcus algae can contain up to 30 g of astaxanthin per kg of dried algae.

Preferably, the astaxanthin is present in the form of an algal extract, more preferably an algal extract containing a minimum of 10% astaxanthin.

Methods of isolating astaxanthin and algal extracts are well-known to those in the field. Similarly, a variety of astaxanthin-containing commercial products are available and would be known to the skilled man. Any such product can be used as the source of astaxanthin in the present oral compositions. Preferably, the oral compositions of the present invention contain astaxanthin in the form of BioAstin® SCE, a commercial product from Cyanotech.

Vitamin C is essential for the formation of collagen in the skin. Vitamin C's role in collagen production is that it is a co-enzyme for three enzymes that are important for stabilizing the structure of collagen. Vitamin C deficiency is not considered common nowadays because of the ample supply of vitamin C-rich foods, but in a large population survey conducted in the United States there has been revealed that as much as 10 to 14% of adults have vitamin C deficiency. In particular, smokers have little vitamin C in the body due to the oxidative stress smoking creates. People with a poor diet also have increased risk of having low levels of vitamin C and may therefore have additional needs for vitamin C supplementation. Vitamin C is widely available and methods of isolating vitamin C and commercial products containing vitamin C would be well-known to one of ordinary skill in the field. Vitamin C isolated by any means and any vitamin C-containing product can be used in the oral compositions of the present invention. Preferably, the vitamin C is prepared from fruit solids. More preferably, vitamin C is provided in the form of Acerola C, an acerola (cherry) juice powder which is produced by low temperature spray-drying. Acerola C is a free- flowing, fine powdered product with high hygroscopic and thermoplastic characteristics. Acerola C contains 45% to 60% by weight of fruit solids and contains 16.0 to 18.0% vitamin C, typically 17% vitamin C.

Alternatively, the Vitamin C is preferably present in the form of Amla Extract 50%, a commercial product from Green Chem containing the fruit extract of Phyllanthus emblica , which contains at least 50% by weight Vitamin C as determined by HPLC.

Collagen hydrolysate is the peptidic result of hydrolysis of collagen protein. Hydrolyzed collagen is produced from collagen found in the bones, skin, and connective tissue of animals such as cattle, pigs, horses, and fish. The process of hydrolysis involves breaking down the molecular bonds between individual collagen strands using heat and either acid or alkali solutions.

Hydrolyzed collagen can be formulated into topical creams and is also commonly used in nutraceutical products. Hydrolyzed collagen may has excellent organoleptic properties, and may be tasteless and odourless.

The term "hydrolyzed collagen" includes within its scope hydrolyzed collagen protein, hydrolyzed collagen extract, collagen peptides and mixtures thereof. Mixtures include mixtures of different peptide fractions or extracts. Hydrolyzed proteins are proteins that have been broken down into smaller oligomeric units (peptides). The hydrolysis process results in reducing the collagen proteins of about 300,000 Daltons (Da) into small peptides. Preferably these peptides have an average molecular weight of between 500 and 10,000 Da, more preferably 2000 and 5000 Da.

Methods of preparing hydrolyzed collagen suitable for cosmetic applications would be well- known to one of ordinary skill in the art and any such method could be used. Collagen hydrolysates can be prepared by enzymatic or chemical degradation of collagen. The collagen hydrolysate used in the oral compositions of the present are preferably prepared by an enzymatic process. Optionally, the hydrolysed collagen is purified, preferably however it is present in the form of an extract, more preferably a cartilage extract, most preferably a marine cartilage extract.

The hydrolysed collagen may be made from any collagen type, for instance type I collagen or type II collagen, preferably type II collagen. The collagen may be obtained in the form of an extract from the bones, cartilage, skin, and connective tissue of animals such as cattle, pigs, horses, and marine animals, including fish. Preferably, the collagen is obtained in the form of a marine cartilage extract.

Hydrolyzed collagen products suitable for cosmetic applications would be well-known to one of ordinary skill in the art and any such product can be used in the present

compositions.

Chondroitin sulfate is a sulfated glycosaminoglycan (GAG) composed of a chain of alternating sugars (N-acetylgalactosamine and glucuronic acid). It is usually found attached to proteins as part of a proteoglycan. A chondroitin chain can have over 100 individual sugars, each of which can be sulfated in variable positions and quantities.

Chondroitin sulfate is an important structural component of cartilage. Chondroitin sulfate is a common component of dietary supplements for the treatment of osteoarthritis.

The oral compositions of the present invention contain chondroitin sulphate from any source. Preferably, the chondroitin sulphate is obtained from cartilage. The chondroitin sulfate may be present in the oral compositions of the present invention in a purified form or, preferably, in the form of an extract, more preferably a cartilage extract, most preferably a marine cartilage extract.

Various methods of isolating chondroitin sulfate are very well-known in the art and the skilled man would be aware of them. Any such method can be used to prepare the chondroitin sulfate for inclusion in the oral compositions of the present invention.

Chondroitin sulphate products suitable for cosmetic applications would be well-known to one of ordinary skill in the art and any such product can be used in the present

compositions. Glucosamine (C₆H₁₃N0₅) is an amino sugar and a prominent precursor in the biochemical synthesis of glycosylated proteins and lipids. Glucosamine is part of the structure of the polysaccharides chitosan and chitin, which compose the exoskeletons of crustaceans and other arthropods, cell walls in fungi and many higher organisms. Glucosamine is a precursor in the biosynthesis of cartilage ground substance components.

Methods of preparing glucosamine suitable for cosmetic applications would be well-known to one of ordinary skill in the art and any such method could be used to prepare glucosamine for inclusion in the present compositions. Typically, glucosamine is produced by the hydrolysis of crustacean exoskeletons or, less commonly by fermentation of a grain such as corn or wheat.

The oral compositions of the present invention contain glucosamine from any source. Preferably, the glucosamine is obtained from cartilage. The glucosamine may be present in the oral compositions of the present invention in a purified form or, preferably, in the form of an extract, more preferably a cartilage extract, most preferably a marine cartilage extract. The glucosamine may be present together with other hexosamines.

Glucosamine products suitable for cosmetic applications would be well-known to one of ordinary skill in the art and any such product can be used in the present compositions.

Preferably the hydrolysate, chondroitin sulphate and glucosamine components are not provided separately in the oral composition but are instead provided in the form of a single product, which is preferably obtained from cartilage, more preferably in the form of a cartilage extract, still more preferably in the form of a marine cartilage extract. Most preferably the hydrolysate, chondroitin sulphate and glucosamine components are present in the form of Protein M+, available from Copalis.

Protein M+ is a marine cartilage extract containing hydrolysed collagen II (45 to 60% by weight of the Protein M+ composition), chondroitin sulphate (45 to 60% by weight of the Protein M+ composition) and hexosamines which include glucosamine (10 to 12% by weight of the Protein M+ composition). Thus, protein M+ combines the most important components of connective tissues. Given its hydrolysed form, Protein M+ is soluble in the aqueous phase and is fully digestible.

Preferably, the total amount of borage oil present in the oral compositions is 0.5mg to 7500mg, more preferably ^"l OOmg to 1500mg, most preferably about 500mg e.g. 487 to 488 mg. Given the use of a borage oil with 20% GLA content, this would provide an oral composition with 0.1 mg to 1500mg, more preferably 20mg to 300mg, most preferably about 100mg e.g. 97 to 98 mg GLA. Given the use of a borage oil with 22% GLA content, this would provide an oral composition with 0.1 1 mg to 1650mg, more preferably 22mg to 330mg, most preferably about 1 10mg e.g. 107 to 108 mg GLA.

Preferably the GLA is present in the oral composition at a minimum concentration of 13% to 25% more preferably 16.5% to 21 %, most preferably about 20% by weight of the composition. If the borage oil is included in the form of a commercial product with a minimum GLA content of 22% then preferably said commercial product is present in the oral composition at a concentration of 60 to 98%, more preferably 75% to 90%, most preferably about 91 % by weight of the composition.

Preferably, the total amount of astaxanthin present in the oral composition is 0.05mg to 12.5 mg, more preferably 1 mg to 5mg, most preferably 2mg. If the astaxanthin is present in the form of a Haematococcus pluvialis or other extract containing about 10%

astaxanthin then the oral composition preferably comprises 0.5mg to 125mg, more preferably 10mg to 50mg, most preferably about 20mg extract.

Preferably the astaxanthin is present in the oral composition at a concentration of 0.1 % to 1 % more preferably 0.2% to 0.5%, most preferably about 0.37% by weight of the composition. If the astaxanthin is included in the form of a commercial product containing about 10% astaxanthin then preferably said commercial product is present in the oral composition at a concentration of 1 to 10%, more preferably 2% to 5%, most preferably about 3.7% by weight of the composition.

Preferably, the total amount of vitamin C present in the oral composition is 12mg to 2000mg, more preferably 20mg to 500mg, still more preferably about 60mg or about 80mg, most preferably about 68mg. If the vitamin C is present in the oral composition in the form of an Acerola or other extract comprising about 17% vitamin C then the oral composition preferably comprises 70mg to 1 1750mg, more preferably 1 15mg to 3000mg, still more preferably about 350mg or about 470mg, most preferably about 400mg Acerola extract. Another particularly preferred embodiment is wherein the oral composition comprises 380mg to 420mg of such an extract. If the vitamin C is present in the oral composition in the form of an Phyllanthus emblica fruit or other extract comprising about 50% vitamin C then the oral composition preferably comprises 24mg to 4000mg, more preferably 40mg to ^"l OOOmg, still more preferably about 120mg or 160mg, most preferably about 136mg extract.

Preferably the vitamin C is present in the oral composition at a concentration of 1 .7% to 12.75% more preferably6% to 10.2%, most preferably about 9% by weight of the composition. If the vitamin C is included in the form of a commercial product containing about 17% vitamin C then preferably said commercial product is present in the oral composition at a concentration of 10 to 75%, more preferably 35% to 60%, most preferably about 53% by weight of the composition.

Preferably the amount of collagen hydrolysate present in the oral composition is 0.5mg to 600mg, more preferably 50mg to 300mg, most preferably about 140mg. Preferably the collagen hydrolysate is present in the oral composition at a concentration of 4% to 42% more preferably 1 1 % to 30%, still more preferably 14% to 21 % most preferably about 17% by weight of the composition.

Preferably the amount of chondroitin sulphate present in the oral composition is 0.5mg to 600mg, more preferably 50mg to 300mg, most preferably about 140mg. Preferably the chondroitin sulphate is present in the oral composition at a concentration of 4% to 42% more preferably 1 1 % to 30%, still more preferably 14% to 21 % most preferably about 17% by weight of the composition.

Preferably the total amount of hexosamines, which include glucosamine, present in the oral composition is 0.1 mg to 120mg, more preferably 10mg to 60mg, most preferably about 27.5mg. Preferably the hexosamines, which include glucosamine, are present in the oral composition at a concentration of 1 % to 10% more preferably 2.5% to 6%, still more preferably 3% to 4% most preferably about 3.6% by weight of the composition. If the collagen hydrolysate, chondroitin sulphate and hexosamines including glucosamine are present in the form of the combined product Protein M+, then preferably the total amount of Protein M+ present in the oral composition is 1 mg to ^"l OOOmg, more preferably ^"l OOmg to 500mg, still more preferably 238mg to 263mg, most preferably about 250mg. Preferably the Protein M+ is present in the oral composition at a concentration of 10% to 70% more preferably 25% to 50%, still more preferably 31 % to 35% most preferably about 33% by weight of the composition.

If the oral composition is a capsule then it preferably comprises borage seed oil and astaxanthin present at the above levels, still more preferably at the above levels within the filling of the capsule. A preferred total capsule weight is 700mg to 750mg, e.g. about 728mg. Of this the majority of the weight is the capsule filling i.e. excluding the capsule shell/coating. Typically the capsule filling weighs 450mg to 550mg, e.g. about 535mg. Typically, the shell/coating weighs 150mg to 250mg, e.g. about 193mg. The above- mentioned concentrations in terms of % by weight of the composition are the weight of the ingredient as a percentage of the filling weight, not of the total composition weight which includes the weight of the shell.

If the oral composition is a tablet then preferably it comprises vitamin C, collagen hydrolysate, chondroitin sulphate and hexosamines including glucosamine present at the above levels. A preferred table weight is 500 to 1000mg, e.g. about 750mg.

The oral compositions may be manufactured using a number of techniques known in the art. The ingredients described herein are preferably present in the oral composition of the invention in an amount sufficient to provide the daily dosage (amount consumed per day, if known) when the recommended number of oral compositions are consumed per day.

The oral compositions of the invention can optionally comprise supplementary substances such as but not limited to vitamins, minerals, salts, condiments, colorants, and

preservatives. Non-limiting examples of supplementary minerals include calcium, phosphorous, potassium, sodium, iron, chloride, boron, copper, zinc, manganese, iodine, selenium and the like. Non-limiting examples of supplementary vitamins include, various B vitamins, vitamin D, and vitamin K. Additional supplements may also be included, e.g., niacin, pantothenic acid, insulin, folic acid, biotin, amino acids, and the like. The oral compositions may also comprise one or more solidifying, bulking and

agglomerating agents (collectively referred to herein as "solidifying agent(s)"). The solidifying agent(s) are used both in tableting and in generating solid-like carriers, such as beadlets, which are capable of transforming oils into stable agglomerates suitable for granulation, blending, and compression required for tableting. Examples of solidifying agents useful in the preparation of the oral compositions include, but are not limited to, sucrose, glucose, fructose, starches (e.g., corn starch), syrups (e.g., corn syrup), and ionic and nonionic polymers including, but not limited to, PEGs and other poly ether-like alkoxy cellulosics (HPMC), gellan, carrageenans, guar, hyaluronates, alginates, chondroitin sulfate, pectins, and proteins e.g. gelatin. Other solidifying agents known to those skilled in the field may also be used in the preparation of the compositions of the present invention. The amount of solidifying agent(s) will vary, depending on the other components contained in the composition, but will generally comprise the majority weight and volume of the composition.

If the oral composition is a capsule then preferably it also contains the following additional ingredients:

Vitamin A, or equivalents or precursors thereof. Vitamin A may be provided in an active form but is preferably provided in the form of a Vitamin A precursor, more preferably a carotenoid, most preferably beta-carotene. Precursors are converted to active forms by the body. β-Carotene is an organic compound classified as a terpenoid. It is a strongly- coloured red-orange pigment abundant in plants and fruits. Given that Vitamin A may be provided in many different forms, amounts of vitamin A are typically measured in Retinal Equivalents (RE).

Any known method of obtaining Vitamin A or equivalents or precursors thereof from any known source can be used. Isolation of beta-carotene from fruits abundant in carotenoids is commonly performed using column chromatography. The separation of beta-carotene from the mixture of carotenoids is based on the polarity of a compound. Beta-carotene is a non-polar compound, so it is separated with a non-polar solvent such as hexane. Being highly conjugated, it is deeply colored, and as a hydrocarbon lacking functional groups, it is very lipophilic. A large variety of Vitamin A containing and Vitamin A precursor containing products, including β-Carotene containing products, are available and would be well-known to one of ordinary skill in the field. Preferably, β-Carotene is present in the oral compositions of the invention in the form of CaroCare® Nat^-Carotene 30% S from DSM Nutritional Products. This product is a viscous oil containing micronized crystals of β-Carotene obtained by fermentation from Blakeslea trispora, dispersed in sunflower oil. It has a minimum β- carotene content of 30% by weight. The product is slightly soluble in oils and fats.

Alternatively, a preferred β-carotene containing product for use in the oral compositions of the present invention is Betatene (20%) OLV from Cognis. 40% β-Carotene-containing products are also available.

Preferably, the oral composition comprises 60 to 1500 Retinol Equivalents (RE) of Vitamin A, more preferably 100 to 350 RE, most preferably about 200 RE. Preferably, the Vitamin A is present in the form of β-Carotene and the oral composition comprises 0.05 to 2.0 mg, more preferably 0.3 to 0.6 mg, most preferably about 0.4 mg, e.g. 0.46 to 0.47 mg of β- Carotene. If the β-Carotene is present in the form of a 20% β-Carotene-containing commercial composition, then preferably the oral composition comprises 0.25 mg to 10 mg, more preferably 1 .5 mg to 3.0 mg, most preferably about 2.0 mg, e.g. about 2.3 mg of that commercial composition.

Alternatively preferred, the oral composition comprises 60 to 1500 Retinol Equivalents (RE) of Vitamin A, more preferably 250 to 500 RE, most preferably about 400 RE.

Preferably, the Vitamin A is present in the form of β-Carotene and the oral composition comprises 0.1 to 4.0 mg, more preferably 0.6 to 1 .2 mg, most preferably about 0.8 mg e.g. 0.93 mg of β-Carotene. If the β-Carotene is present in the form of a 40% β-Carotene- containing commercial composition, then preferably the oral composition comprises 0.25 mg to 10 mg, more preferably 1 .5 mg to 3.0 mg, most preferably about 2.0 mg, e.g. about 2.3 mg of that composition. If the β-Carotene is present in the form of a 20% β-Carotene- containing commercial composition, then preferably the oral composition comprises 0.5 mg to 20 mg, more preferably 3.0 mg to 6.0 mg, most preferably about 4.0 mg e.g. 4.6 to 4.7 mg of that commercial composition.

Thickening agents, preferably a silica compound, more preferably a fumed silica may also be present in the oral compositions. Aersosil 300 from Degussa Corporation is a preferred example of such a fumed silica. This composition is a fluffy white powder made up of nanometer-sized particles with silanol groups on the surface.

Vitamin E, which is a general term for tocopherols and tocotrienols. Vitamin E compounds are fat-soluble antioxidants which stop the production of reactive oxygen species formed when fat undergoes oxidation. Preferably, vitamin E is present in the form of d alpha tocopherol 1000 iu/g IP.

Zinc is optionally present in the oral compositions. Zinc maintains normal cell division in the skin since it is required for the normal function of many enzymes involved in cell division. Zinc is also used in some antioxidant reactions. Zinc is preferably present in the form of tri zinc citrate which can be prepared by a variety of known methods and can be purchased from a large number of suppliers, all of which would be readily determined by the skilled man.

Copper is optionally present in the oral composition. Copper is required for the formation of connective tissue in the skin, particularly for the formation of a network of collagen and elastin. Lack of copper leads to fragile skin. Preferably, copper is present in the form of copper (ii) sulphate, which is a readily available ingredient.

Other preferred additional ingredients include glazing agents, such as but not limited to tricalcium phosphate, magnesium stearate and isomalt, which is a well-known sugar substitute.

The skilled artisan will understand how to determine the appropriate amount of ingredients to be added to a given oral composition for the purpose according to the present invention. Such factors that may be taken into account include the type of composition, the average consumption of specific types of compositions by different individuals, and the

manufacturing conditions under which the composition is prepared. According to certain aspects of the invention, the ingredients can be added at any time during the manufacture and/or processing of the composition. This includes, without limitation, incorporation within the composition, or as a coating applied to the oral composition. Table 3 displays the preferred amounts of optional additional ingredients when they are present in the compositions of the invention (the preferred amounts of Vitamin A are discussed above):

Table 3

^* RE = retinol equivalent

A preferred tablet weight is 750mg. A preferred capsule filling weight is 535mg. Thus, preferred concentrations of ingredient in terms of % by weight of the tablet or capsule filling are readily determinable from the preferred mg amounts in Table 3 and these preferred tablet and capsule filling weights.

If any components are present in the composition in the form of a product in which they are not the sole component then it is within the competence of the skilled man to calculate the amount of the product to include in order toprovide the preferred amount of said component in the composition.

If the oral composition is a capsule then preferably the capsule comprises borage oil, Vitamin A (preferably in the form of β-carotene), astaxanthin, fumed silica and vitamin E as discussed above. Still more preferably, these components are present in the filling of the capsule. If the oral composition is a tablet then preferably the tablet comprises vitamin C, collagen hydrolysate, chondroitin sulphate, hexosamine including glucosamine, zinc citrate, copper sulphate, isomalt, tricalcium phosphate and tricalcium stearate as discussed above.

The oral compositions of the present invention may also comprise additional excipients useful in preparing and finishing the compositions. Such excipients may include timed- release polymer coating agents useful in prolonging dissolution of the active agents in the digestive tract. Examples of such polymers include, but are not limited to ionic and nonionic polymers, such as PEGs and other poly ether-like alkoxy cellulosics (HPMC), gellan, carrageenans, Eucheuma gelatenae, starch, hyaluronates, chondroitin sulfate, pectins, and proteins. Since the xanthophyll/carotenes are highly pigmented, coating technology may be applied to the oral composition in order to provide a product of uniform color. Examples of color coating agents may include, but are not limited to, polymers, colorants, sealants and surface active agents including, not limited to, fatty acids and esters, di- and triglycerides, phospholipids including mono- and di-alkyl glyceryl phosphates, nonionic agents (like sugars, polysaccharides, e.g., HPMC and polysorbate 80) and ionic agents.

The skilled man will understand how to determine the appropriate amount of ingredients to be added to a given oral composition coating for the purpose according to the present invention.

In particular, the capsules of the present invention may be coated. Preferred coating ingredients are gelatin and glycerol (a humectant). Iron oxide paste (a colouring agent) is optionally present. Table 4 displays the preferred amounts of these ingredients when they are present in the coatings of a capsule of the invention: Table 4

A preferred capsule shell/coating weight is 193mg. Thus, preferred concentrations of ingredient in terms of % by weight of the capsule shell/coating are readily determinable from the preferred mg amounts in Table 4 and this preferred capsule shell/coating weight.

All combinations of the additional optional ingredients discussed above in the oral compositions of the present invention are explicitly provided.

The oral compositions of the present invention may be manufactured using any technique known in the art. In a further aspect the present invention provides a method of preparing an oral composition as defined above comprising admixing either:

(i) borage seed oil and astaxanthin; or

(ii) vitamin C, collagen hydrolysate, chondroitin sulphate and glucosamine.

The preferences, definitions and embodiments described above in relation to the oral compositions apply equally to this method of preparing said compositions.

In a further aspect, the present invention provides a kit comprising:

(i) a first oral composition comprising

borage seed oil and astaxanthin; and

(ii) a second oral composition comprising

vitamin C, collagen hydrolysate, chondroitin sulphate and glucosamine. In a further aspect, the present invention provides a kit comprising:

(i) a first oral composition comprising

borage seed oil and astaxanthin; and/or

(ii) a second oral composition comprising

vitamin C, collagen hydrolysate, chondroitin sulphate and glucosamine; and

(iii) a topical skin care formulation as defined anywhere herein.

The preferences, definitions and embodiments described above in relation to the oral compositions and topical skin care formulations apply equally to all features of the above mentioned kits.

Preferably the kit comprises the first and second oral compositions as well as a topical skin care formulation defined anywhere herein. Preferably, the first oral composition is a capsule and the second oral composition is a tablet. Preferably, the kit comprises as twice as many capsules as tablets.

The topical skin care formulations will be provided in a suitable container.

In a further aspect the present invention provides a cosmetic skin treatment method in which an oral composition as defined herein is orally consumed by a human subject.

Preferably the oral composition reduces wrinkle number or depth or prevents an increase in wrinkle number or depth, reduces skin roughness or prevents an increase in skin roughness, increases skin elasticity or prevents a loss of skin elasticity, increases skin firmness or prevents a loss of skin firmness, increases skin density or prevents a loss of skin density, increases skin moisture content or prevents a loss of skin moisture content, reduces blemishes or areas of atypical pigmentation. Particularly preferably the oral composition reduces wrinkle depth, reduces skin roughness, increases skin firmness, increases skin elasticity or prevents the opposite effect from occurring.

The oral composition may be consumed as regularly as the human subject wishes, provided that the recommended daily allowances of the components therein are not exceeded. The frequency of consumption may depend on the type of composition in question, i.e. a tablet or capsule and the component ingredients therein.

Consumption can be on an as-needed or as-desired basis, for example, once-monthly, once-weekly, daily, or more than once daily. Similarly, consumption can be every other day, week, or month, every third day, week, or month, every fourth day, week, or month, and the like. Consumption can be multiple times per day. Preferably two of the first oral compositions defined at (i) above, which are preferably capsules, and one of the second oral compositions defined at (ii) above, which is preferably a tablet, are consumed per day.

Preferably, the daily amount of borage oil consumed is 1 mg to 15000mg, preferably 5mg to 15000mg, more preferably 200mg to 3000mg, most preferably about ^"l OOOmg or about 975mg. Optionaly, a borage oil product with about 20% GLA content is used in the oral compositions of the invention. Thus, a preferred daily consumed amount of GLA is 1 mg to 3000mg, more preferably 40mg to 600mg, most preferably about 200mg or about 195mg. An alternative preference is that a borage oil product with about 22% GLA content is used in the oral compositions of the invention, in which case a preferred daily consumed amount of GLA is 1 .1 mg to 3300mg, more preferably 44mg to 660mg, most preferably about 220mg or about 214.5mg.

Preferably, the daily amount of astaxanthin consumed is 0.1 mg to 25mg, more preferably 2mg to 10mg, most preferably 4mg. Preferably, astaxanthin is present in the form of a Haematococcus pluvialis extract containing about 10% astaxanthin. Thus, a preferred daily consumed amount of Haematococcus pluvialis extract is 1 .Omg to 250mg, more preferably 20mg to 100mg, most preferably about 40mg.

Preferably, the daily amount of vitamin C consumed is 12mg to 2000mg, more preferably 20mg to 500mg, still more preferably about 60mg or 80mg, most preferably about 68mg. Preferably, vitamin C is present in the oral composition in the form of an Acerola or other extract comprising about 17% vitamin C. Thus, a preferred daily consumed amount of such extract is 70mg to 1 1750mg, more preferably 1 15mg to 3000mg, most preferably about 350mg or 400mg. Alternatively, vitamin C is preferably present in the oral composition in the form of an Phyllanthus emblica fruit or other extract comprising about 50% vitamin C. Thus, a preferred daily consumed amount of such extract is 24mg to 4000mg, more preferably 40mg to 1000mg, still more preferably about 120mg or 160mg, most preferably about 136mg.

Preferably the daily amount of collagen hydrolysate consumed is 0.5mg to 600mg, more preferably 50mg to 300mg, most preferably about 140mg. Preferably the daily amount of chondroitin sulphate consumed is 0.5mg to 600mg, more preferably 50mg to 300mg, most preferably about 140mg. Preferably the amount of hexosamines, which include glucosamine, consumed is 0.1 mg to 120mg, more preferably 10mg to 60mg, most preferably about 27.5mg.

If the collagen hydrolysate, chondroitin sulphate and hexosamines including glucosamine are present in the form of the combined product Protein M+, then preferably the total daily amount of Protein M+ consumed is 1 mg to 1000mg, more preferably 100mg to 500mg, most preferably about 250mg.

Preferably, 120 to 3000 Retinol Equivalents (RE) of Vitamin A, more preferably 200 to 700 RE, most preferably about 400 RE are consumed per day. Preferably, the Vitamin A is present in the form of β-Carotene and 0.1 to 4.0 mg, more preferably 0.6 to 1 .2 mg, most preferably about 0.8 mg, e.g. 0.93 mg of β-Carotene is consumed per day. If the β- Carotene is present in the form of a 20% β-Carotene-containing commercial composition, then preferably 0.5 mg to 20 mg, more preferably 3 mg to 6.0 mg, most preferably about 4.0 mg, e.g. 4.6 to 4.7 mg of that commercial composition is consumed per day.

Alternatively preferred, 120 to 3000 Retinol Equivalents (RE) of Vitamin A, more preferably 500 to 1000 RE, most preferably about 800 RE are consumed per day. Preferably, the Vitamin A is present in the form of β-Carotene and 0.2 to 8.0 mg, more preferably 1 .2 to 2.4 mg, most preferably about 1 .6 mg, e.g. 1 .86 mg of β-Carotene is consumed per day. If the β-Carotene is present in the form of a 20% β-Carotene-containing commercial composition, then preferably 1 .0 mg to 40 mg, more preferably 6.0 mg to 12.0 mg, most preferably about 8.0 mg, e.g. 9.2 to 9.3 mg of that commercial composition is consumed per day. If the β-Carotene is present in the form of a 40% β-Carotene-containing commercial composition, then preferably 0.5 mg to 20 mg, more preferably 3 mg to 6.0 mg, most preferably about 4.0 mg, e.g. 4.6 to 4.7 mg of that commercial composition is consumed per day. Table 5 displays the preferred daily consumed amounts of the preferred optional additional components of the oral compositions (the preferred daily consumed amounts of Vitamin A are discussed above):

Table 5

^* RE = retinol equivalent

In a further aspect, the present invention provides a cosmetic skin treatment method in which a first and a second oral composition as defined above are orally consumed by a human subject simultaneously, sequentially or separately.

Preferably, two capsules (as discussed above at (i)) and one tablet (as discussed above at (ii)) are consumed per day in order for the human subject to consume the above mentioned preferred daily amounts of the above mentioned oral composition components. However, in principle any number of capsules and number of tablets can be consumed per day, provided that the recommended daily allowances of the components therein are not exceeded. The skilled man will be able to establish, given the quantities of the

components in the different oral compositions, how many of each type of composition should be consumed in order for the desired amount of each component to be consumed over a specified period.

In cosmetic skin treatment methods in which a first and second oral compositions are consumed, the first and second oral compositions can be consumed simultaneously on an as-needed or as-desired basis as defined above. Alternatively, consumption of the first and second oral compositions can be sequential, such that they are consumed at a predetermined rate or at pre-determined times relative to each other, for instance the second oral composition being consumed 1 , 2, 3, 4, 6, 8, 12 or 24 hours, or any number of days, weeks or months after the consumption of the first oral composition. Similarly, the second consumption of the first oral composition may be at any specified time following the first consumption of the second oral composition and so on. Alternatively still, the first and second oral compositions may be consumed separately, i.e. over periods and at rates or at specified time points which are independent from the periods, rates or time points of consumption of the other oral composition.

In a further aspect, the present invention provides a combined cosmetic skin treatment method in which a topical skin care formulation as defined anywhere above is applied topically to the skin of a human subject and in which an oral composition as defined above is orally consumed by said subject simultaneously, sequentially or separately but as part of a combined treatment regimen.

In a further aspect, the present invention provides a combined cosmetic skin treatment method in which a topical skin care formulation as defined anywhere above is applied topically to the skin of a human subject and in which both a first and second oral composition as defined above (at i and ii) is orally consumed by said subject

simultaneously, sequentially or separately but as part of a combined treatment regimen.

All preferences, definitions and embodiments described above in relation to the other aspects of this invention apply equally to these combined cosmetic skin treatment methods. Where a method comprises the application or consumption of three or more topical formulations or oral compositions then the terms "simultaneously", "sequentially" and "separately" mean that each topical formulation or oral composition may be applied or consumed simultaneously with, sequentially with or separately to any one or more of the other topical formulations and oral compositions applied or consumed as part of the method.

Preferably, one tablet and two capsules are consumed per day at any point in time in relation to each other and the topical skin care formulation is applied once or twice daily at any point in time in relation to the consumption of the oral compositions.

The components of each formulation, whether topical or oral, combine to provide surprisingly effective skin care products. Although numerous ingredients are known as components of skin care products, the efficacy of the particular combinations of the present invention could not be predicted. Preferably the active components in each formulation combine synergistically and are thus present not only in active amounts but in synergistic amounts.

The art contains very little teaching about the benefits of a combined topical and oral approach to skin care and so the combined methods and products disclosed herein represent a particularly surprising and effective solution to the problems of skin care, protection and aging. Particularly preferably, there is a synergistic effect on skin care between the topical and oral formulations described herein.

The formulations and compositions described herein may comprise, consist essentially of, or consist of any of the elements as described herein.

Various documents including, for example, publications and patents, are recited throughout this disclosure. All such documents are, in relevant part, hereby incorporated by reference. The citation of any given document is not to be construed as an admission that it is prior art with respect to the present invention. To the extent that any meaning or definition of a term in this written document conflicts with any meaning or definition of the term in a document incorporated by reference, the meaning or definition assigned to the term in this written document shall govern.

Referenced herein are trade names for components including various ingredients utilized in the present invention. The inventors herein do not intend to be limited by materials under a certain trade name. Equivalent materials (e.g., those obtained from a different source under a different name or reference number) to those referenced by trade name may be substituted and utilized in the descriptions herein.

The present invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification within the scope of the appended claims.

The following examples are provided to describe the invention in greater detail. They are intended to illustrate, not to limit, the invention. These Examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Examples

Materials and Methods

Direct measurement of wrinkles

Direct measurement of wrinkles is performed using the Phaseshift Rapid in vivo

Measurement of Skin (3D Primos®Compact) device from GFMesstechnik. This device permits measurement of cutaneous relief parameters, i.e. 2D and 3D measurements of wrinkles can be made on a zone of interest. 32 profiles are obtained per zone, each of which reveals three parameters. Firstly, the average roughness (Ra) of the profile, a decrease in which characterizes a smoothing effect. Secondly, the average relief (Rz) of the profile, a decrease in which characterizes an anti-wrinkle effect. Thirdly the amplitude (Rt) of the profile, a decrease in which characterizes an anti-wrinkle effect.

Measurement of skin biomechanical properties

Measurement of skin biomechanical properties is performed using a SEM 575 Cutometer® device from COURAGE & KHAZAKA. The technique consists of skin aspiration by a measurement probe. In the method, the skin is sucked into the orifice of a probe by constant vacuum pressure (300 mbar) for a set length of time. The depth to which the skin penetrates the probe is measured by two optical prisms located at the opening of the probe's orifice to eliminate the effects of friction and mechanical strain. A cycle of inhalation (3 seconds) and relaxation (2 seconds) is performed three times. A profile is thereby obtained, which is visible on computer screen. The profile presents a firmness parameter (R0), which represents the passive behaviour of the skin with regard to the applied strength (an decrease in this parameter translates as firmer skin). The device also permits evaluation of variations in the biological elasticity of superficial cutaneous layers via three elasticity parameters (R2, R5 and R7). The more the value of any of the elasticity parameters tends to a value of 1 , the more elastic the skin.

Subjects

Subjects are all healthy and have given written consent. Unless otherwise specified, female patients are over 40 years old and display wrinkles on crow's feet area and slackened facial skin. Pregnant or nursing women or those expecting to get pregnant during the study are excluded from the study, as are those with cutaneous pathology on the studies zone, those who have started, stopped or changed any hormonal treatment including oral contraception for less than 1 .5 months, those undergoing any potentially interfering treatment, those with facial cosmetic surgery, those with excessive sunlight/UV ray exposure in the previous month, those with known allergies to the product and those having undergone general aesthetic in the month before the study. Throughout the studies only usual cleansing products are authorised on the face. No excessive exposure to sunlight or UV is authorised.

Example 1 : Preparation of topical skin care formulations

The following topical formulations were prepared:

A) Face Cream:

B) Facial Serum:

C) Eve Gel:

Example 2: Evaluation of the anti-aqeinq effects of topical skin care formulations on skin

Each of the studies below was a double blinded study. Results obtained were for the treated zone versus the non-treated zone versus placebo. The zone treated in each case is a hemi-facial zone. In all cases a daily log was kept to evaluate the products' tolerance. No undesirable events occurred in any of the subjects.

A) Face Cream

The efficacy of the face cream on reducing the visible signs of aging was evaluated on 18 female subjects presenting wrinkles on day 28 and 19 such subjects on day 56. A face cream ("product") was prepared in accordance with Example 1 . The placebo was the formulation prepared in accordance with Example 1 minus the active ingredients, i.e. minus the glucan, soy protein, hydrolysed rice bran protein and oxidoreductases. 100ml samples of the face cream were used. i) Measurement of anti-wrinkle effects of Face Cream by direct measurement of wrinkles on the crow's foot region (statistically significant effects are shown in underlined bold text):

The data demonstrates that the product according to the present invention performed more effectively than the placebo in all three measures and at all time points, the sole exception being the Rt measurement on day 56 for which the placebo worked demonstrated the same effect. The majority of the effects for the product were statistically significant, no effects for the placebo were statistically significant. ii) Measurement of effects of Face Cream on skin biomechanical properties:

While none of the effects in the above table were shown to be statistically significant, the data clearly demonstrates that the product outperforms the placebo in all measures and at all time points. The improvement on skin firmness (R0) of the product compared to the placebo was statistically significant, i.e. the face cream product performed significantly better than placebo.

B) Face Serum

The efficacy of the face serum on reducing the visible signs of aging was evaluated on 21 female subjects presenting wrinkles on day 28 and 19 such subjects on day 56.

A face serum ("product") was prepared in accordance with Example 1 . The placebo was the formulation prepared in accordance with Example 1 minus the active ingredients, i.e. minus the glucan, protein-containing rosid plant extract and hyaluronic acid. 50ml samples of the face serum were used. i) Measurement of anti-wrinkle effects of Face Serum by direct measurement of wrinkles on the crow's foot region region (statistically significant effects are shown in underlined bold text):

The data demonstrates that the product according to the present invention performed more effectively than the placebo in all three measures and at all time points. The product's effects were statistically significant at day 56 for all measures. No effects for the placebo were statistically significant.

C) Eve Gel

The efficacy of the eye gel on reducing the visible signs of aging was evaluated on 21 female subjects presenting wrinkles on day 28 and 22 such subjects on day 56.

An eye gel was prepared in accordance with Example 1 . The placebo was the formulation prepared in accordance with Example 1 minus the active ingredients, i.e. minus the glucan, protein-containing rosid plant extract and hyaluronic acid. 50ml samples of the eye gel were used. i) Measurement of anti-wrinkle effects of Eye Gel by direct measurement of wrinkles on the crow's foot region region (statistically significant effects are shown in underlined bold text):

The data demonstrates that the product according to the present invention performed more effectively than the placebo in all three measures at day 56. At day 56, the product's effects were statistically significant in all three measures.

Example 3: Preparation of oral compositions

The following oral compositions were prepared: i) Tablet:

QS = a sufficient quantity

Thus, each tablet comprises:

10 mg zinc citrate (33mg of a 32% zinc citrate product), 1 .3% by weight of the tablet; 1 mg copper sulphate (4mg of 25% copper sulphate product), 0.13% by weight of the tablet;

68mg vitamin C (400mg of a 17% vitamin C product), 9.06% by weight of the tablet. ii) Capsule: a) Fill Materials

^* RE = retinol equivalent

Thus, each capsule comprises:

A minimum GLA content of 107.25mg, 20% by weight of the capsule filling;

2mg astaxanthin, 0.37% by weight of the capsule filling;

0.4656mg beta-carotene, 0.087% by weight of the capsule filling. b) Coating Materials

Amount of commercial

product present

Ingredient Amount per capsule (mg) in formulation

(% by weight)

Gelatin (present as BP

Bovine 150 Bloom Bone/Hide 68.4252 132.0071

Blend)

Glycerol 31 .5748

60.9147

(present as glycerol BP)

Total weight: 193 Example 4: Evaluation of the anti-ageing effects of topical skin care formulations and oral compositions on skin

Each of the studies below was a double blinded study. Results obtained were for the treated zone versus the non-treated zone versus placebo. The zone treated in each case is a hemi-facial zone. Oral products are consumed orally. The placebo treatment comprises the consumption of the oral products and application of the facial cream minus the active ingredients, i.e. minus the glucan, soy protein, hydrolysed rice bran protein and oxidoreductases. 100ml samples of the face cream are used. Boxes of 27 capsules and boxes of 27 tablets were used. Subjects consumed two capsules and one tablet per day. In all cases a daily log was kept to evaluate the products' tolerance. No undesirable events occurred in any of the subjects. i) Measurement of anti-wrinkle effects of direct measurement of wrinkles on the crow's foot region:

The efficacy of the combination of a face cream prepared in accordance with Example 1 ("product") and tablets and capsules (together "supplement") prepared in accordance with Example 3 was evaluated on 21 female subjects presenting wrinkles on day 28 and on day 56 (statistically significant effects are shown in underlined bold text):

Kinetic Δ (mean ± SEM) Δ% on mean

Product + Δ (D28-D0) -3.2 ± 1 .0 -12%

Supplement Δ (D56-D0) -2.5 ± 1 .1 -9%

Ra (in μπι)

Placebo + Δ (D28-D0) -1 .7 ± 0.8 -7%

Supplement Δ (D56-D0) -1 .3 ± 0.8 -6%

Product + Δ (D28-D0) -15.5 ± 4.7 -11%

Supplement Δ (D56-D0) -12.4 ± 5.2 -9%

Rz (in μπι)

Placebo + Δ (D28-D0) -8.3 ± 3.8 -7%

Supplement Δ (D56-D0) -5.4 ± 3.6 -5%

Product + Δ (D28-D0) -20.6 ± 9.7 -9%

Supplement Δ (D56-D0) -15.7 ± 9.9 -7%

Rt (in pm)

Placebo + Δ (D28-D0) -10.5 ± 1 1 .8 -6%

Supplement Δ (D56-D0) -4.3 ± 1 1 .8 -2% The data demonstrates that the combination of product and supplement according to the present invention performed more effectively than the combination of placebo and supplement in all three measures and at all time points. The effects of the combination of product and supplement were statistically significant at day 28 for all measures and at day 56 for all measures except Rt. ii) Measurement of skin biomechanical properties.

The efficacy of the combination of a face cream prepared in accordance with Example 1 ("product") and tablets and capsules (together "supplement") prepared in accordance with Example 3 was evaluated on 22 female subjects presenting wrinkles on day 28 and on day 56 (statistically significant effects are shown in underlined bold text).

The data demonstrates that the combination of product and supplement according to the present invention performed more effectively than the combination of placebo and supplement in measures of firmness and elasticity at all time points. The effects of the combination of product and supplement were statistically significant in all measures at all time points.

The above data in relation to the face cream and placebo topical products with/without oral supplements can be presented as follows for direct comparison (statistically significant effects are shown in underlined bold text): Placebo + Product +

Kinetic Placebo Product

Supplement Supplement

Ra (in μηι) Δ (D28-D0) -1 % -7% -6% -12%

Δ% on mean Δ (D56-D0) -5% -6% -9% -9%

Rz (in μηι) Δ (D28-D0) -3% -7% -6% -11%

Δ% on mean Δ (D56-D0) -5% -5% -8% -9%

Rt (in μηι) Δ (D28-D0) -3% -6% -7% -9%

Δ% on mean Δ (D56-D0) -5% -2% -5% -7%

Firmness (R0) Δ (D28-D0) +12% -32% -2% -38%

Δ% on mean Δ (D56-D0) +15% -4% -4% -16%

Elasticity (R7) Δ (D28-D0) -1 1 % 26% +1 % 46%

Δ% on mean Δ (D56-D0) -9% 15% +2% 24%

This comparison demonstrates that the face cream performs better than placebo in all but measures of skin ageing (anti-wrinkle effects (Ra, Rz and Rt), firmness and elasticity) and at all time points other than in the sole case of Rt at day 56, wherein the placebo performs as effectively as the face cream. The face cream's anti-wrinkle effects are mainly statistically significant. The combination of face cream and oral supplement further improved the anti-wrinkle, firmness and elasticity effects compared to placebo at all time points. The effects of the combination of face cream and oral supplement were statistically significant in all measures at all time points with the sole exception of Rt at day 56.

Furthermore, the combination of face cream and oral supplement outperformed the combination of topical placebo and oral supplement in all measures at all time points. The additional consumption of oral supplement with the placebo increased the effects in the vast majority measures and time points when compared to the placebo alone. Statistically significant effects were observed for the combination of placebo and oral supplement in measures of anti-wrinkle effect, skin firmness and skin elasticity.

In conclusion, the data presented herein demonstrates the efficacy of the topical and oral products of the present invention and the further surprisingly superior efficacy when these topical and oral products are used in combination.

Claims

1 . A topical skin care formulation comprising:

a) 3-(1 ,3/1 ,6)-glucan; and

b) a protein-containing plant extract, wherein said plant is of the family Fagaceae; or c) a protein-containing plant extract, wherein said plant is of the genus Vigna.

2. The topical skin care formulation of claim 1 , wherein the plant of the family Fagaceae is a plant is of the genus Fagus,

3. The topical skin care formulation of claim 2, wherein the plant of the genus Fagus is Fagus sylvatica.

4. The topical skin care formulation of claim 2 or claim 3, wherein the formulation is an eye gel.

5. The topical skin care formulation of claim 1 , wherein the plant of the genus Vigna is Vigna aconitifolia.

6. The topical skin care formulation of claim 5, wherein the formulation is a face serum.

7. The topical skin care formulation of any one of claims 1 to 6, further comprising

hyaluronic acid.

8. A topical skin care formulation comprising:

3-(1 ,3/1 ,6)-glucan; and

an oxidoreductase,

optionally also comprising soy protein and/or hydrolysed rice brain protein.

9. The topical skin care formulation of claim 8 comprising;

a) 3-(1 ,3/1 ,6)-glucan;

b) soy protein;

c) hydrolysed rice brain protein; and

d) an oxidoreductase.

10. The topical skin care formulation of claim 8 or claim 9, wherein the formulation is a face cream.

1 1 . The topical skin care formulation of any one of claims 1 to 10, wherein the glucan is

derived from yeast.

12. The topical skin care formulation of any one of claims 1 to 1 1 , wherein the glucan

comprises about 0.3% by weight of the formulation.

13. An oral composition comprising:

(i) borage seed oil and astaxanthin; or

(ii) vitamin C, collagen hydrolysate, chondroitin sulphate and glucosamine.

14. A kit comprising:

(i) a first oral composition comprising borage seed oil and astaxanthin; and

(ii) a second oral composition comprising vitamin C, collagen hydrolysate, chondroitin sulphate and glucosamine.

15. The kit of claim 14 further comprising:

(iii) the topical skin care formulation of any one of claims 1 to 12.

16. The oral composition or kit of any one of claims 13 to 15, wherein the collagen

hydrolysate, chondroitin sulphate and glucosamine are obtained from a marine cartilage extract.

17. A cosmetic skin treatment method in which the topical skin care formulation of any one of claims 1 to 12 is applied to the skin of a human subject.

18. A cosmetic skin treatment method in which an oral composition as defined in claim 13 or 16 is orally consumed by a human subject.

19. The cosmetic skin treatment method of claim 18, in which a first and a second oral

composition as defined in claim 13 or claim 16 are orally consumed by a human subject simultaneously, sequentially or separately.

20. A combined cosmetic skin treatment method, in which the topical skin care formulation of any one of claims 1 to 12 is applied topically to the skin of a human subject and in which an oral composition as defined in claim 13 or claim 16 is orally consumed by said subject simultaneously, sequentially or separately as part of a combined treatment regimen.

21 . The combined cosmetic skin treatment method of claim 20, in which a topical skin care formulation of any one of claims 1 to 12 is applied topically to the skin of a human subject and in which both a first and second oral composition as defined in claim 13 or claim 16 are orally consumed by said subject simultaneously, sequentially or

separately as part of a combined treatment regimen.