GB2440117A

GB2440117A - Skin care composition comprising a trienzyme activity

Info

Publication number: GB2440117A
Application number: GB0614349A
Authority: GB
Inventors: Jan Kusmirek
Original assignee: ELEMIS Ltd
Current assignee: ELEMIS Ltd
Priority date: 2006-07-19
Filing date: 2006-07-19
Publication date: 2008-01-23
Anticipated expiration: 2026-07-19
Also published as: GB2440117B; GB0614349D0

Abstract

A cosmetic composition is provided comprising a suitable carrier and at least one thiol protease; at least a first serine protease derived from Aspergillus melleus; and at least a second serine protease. The thiol protease may be selected from papain, ficin, bromelain and actinidin. The first serine protease is derived from Aspergillus melleus, and is preferably Aspergillus melleus proteinase. The second serine protease is selected from the group consisting of subtilisin, trypsin and chymotrypsin. The synergistic combination of these three proteolytic enzyme activities - the trienzyme activity - in the compositions of the invention facilitates improved cosmetic epidermal conditioning and resurfacing. Methods of using the compositions and methods of reconstituting the desired trienzyme activity in situ are also provided.

Description

1 2440117

SKINCARE COMPOSITIONS COMPRISING A TRIENZYME ACTIVITY

FIELD

The invention relates to cosmetic skin care compositions that are intended to rejuvenate the skin and thereby reverse the aging process. More specifically, the invention relates to skincare compositions comprising proteolytic enzymes.

BACKGROUND

In modern society great emphasis has been placed on improving health and particularly in rejuvenation of the appearance. Over the last half century, the expansion of the so-called middle classes has meant that many people are working longer hours and becoming increasingly exposed to stressful working surroundings. In combination with these social stresses, well-documented environmental stresses are increasing with greater amounts of UV light penetrating the atmosphere and increasing levels of atmospheric pollution, especially in urban areas. The combination of work and environmental stress, together with diets that are often high in processed foods, leads to a detrimental effect upon the skin and in some cases the appearance of premature aging.

Whilst it is allegedly possible to delay the progression of skin aging by radical changes in diet, lifestyle and exposure to sunlight and environmental pollutants, there remains a significant demand for the cosmetic reversal of existing damage.

Cosmetic procedures that exist for addressing this demand can be expensive, radical and invasive. For example, laser skin resurfacing, chemical peels or even surgical intervention. However, more often than not, consumers wish to avoid harsh treatments and seek a more gentle approach to rejuvenation of the skin, and more particularly acquisition of a more youthful facial appearance.

The epidermis represents the outer, non-vascular layer of the skin. It comprises a horny keratinised layer of dead material over live squamous layer and basal layer cells. Exposure to environmental pollutants, UV radiation and changes in the biology of the live skin layers can affect the level of keratinisation of the horny outer layer. Circumstances of metabolic imbalance or cellular damage can cause localised or widespread hyperkeratinisation, which can cause the skin to appear patchy, rough and aged. Hence, in order to promote rejuvenation of the appearance of the skin, it is desirable to reduce the thickness of the hyperkeratinised areas, a process termed resurfacing', and promote cellular turnover in the live squamous and basal cell layers.

In recent times been a trend towards more naturally derived cosmetic products based on herbal or traditional remedies. In the field of dematological rejuvenation, FR-A-2815852 (Peyronel et al.) exemplifies this trend as it describes a composition comprising papaya extracts, together with several other ingredients such as lemon and olive extracts. Papaya fruit is known to contain the proteolytic enzyme, papain, which has been suggested for use in exfoliation and skin conditioning. However, such naturally derived compositions are difficult to standardise and can be prone to large batch to batch variations or to natural variations in the starting materials. There is a need for a combinatorial approach whereby standardised natural extracts are used in combination with biotechnologically derived ingredients. This ensures safety and efficacy can be guaranteed in the product and consumer confidence in the product is maintained for repeat purchases.

Stabilised formulations of proteolytic enzymes (proteases) have been utilised in the field of detergents for many years. US-A-3557002 (McCarty) describes stabilised compositions of proteolytic enzymes including papain and subtilisin and their use in detergents and mouthwashes. However, the cosmetic uptake of such proteolytic compositions has been slow as proteases are known to elicit immunogenic responses and to cause irritation when included in compositions that come into contact with the skin -for example, in biological washing powders. Hence, any protease activity included in a cosmetic composition must be relatively mild and optionally supplemented by other factors, which serve to reduce or moderate skin irritation.

Cosmetic compositions that have included proteases have generally been intended for topical application to the skin solely for the purpose of controlling hair growth (see US-A-2004/O1 61483, Tsuji et al.). These compositions have a depilatory action, but it is widely appreciated that such compositions are unsuitable for extended use or overnight. Also, such compositions are often unsuitable for use on areas of sensitive skin such as the face.

Proteases have also been utilised clinically in what are known as debridement compositions. Enzymatic debridement is step of the degrading damaged and necrotic tissues in wounds, typically following burn injury. Topically applied compositions comprising proteases (see WO-A-9523614, Constantine et al.) provide an alternative to surgical debridement. The composition described in Constantine et al. comprises proteases such as papain and/or subtilisin A together with a combination of antibiotics. It is apparent that the intention of debridement is to rapidly digest dead and dying skin tissue and, as such, is a far more intensive and rigorous procedure compared to desired cosmetic methods of gentle resurfacing and reconditioning of normal healthy skin. Indeed, it would be inconceivable to apply a debridement composition to normal healthy skin for cosmetic purposes.

It is clear, therefore, that there is a need for improved cosmetic compositions and methods that are directed toward improving epidermal resurfacing and skin conditioning without eliciting irritation and an allergic response. Such compositions need to be off-the-shelf products, which are easy to use and avoid the need for complex handling by the end user. Ideally, such compositions should be able to be used confidently by a consumer in a manner similar to their existing skincare regimen. In addition, such products should also be suitable for use by professionals in the field of beauty treatments and cosmetic skin care.

SUMMARY

The present invention provides a cosmetic composition comprising a synergistic combination of proteolytic enzymes that promote skin cell turnover and renewal of the epidermis. The compositions of the invention advantageously allow for resurfacing and renewal of the epidermis without harsh peeling or chemical burning or other highly allergenic procedures. Further, the compositions of the invention can remain in contact with the skin for a prolonged period of time without the need for active removal or neutralisation. Hence, the cosmetic compositions of the invention provide the significant advantages of ease of use and improved tolerance for the user.

Accordingly, in a first aspect the present invention provides a cosmetic composition comprising: at least one thiol protease; at least a first serine protease derived from Aspergillus melleus; and at least a second serine protease.

It can be seen that the composition of the invention comprises two distinct types, or classes, of proteolytic enzymic activity from at least three distinct sources.

Hence, the property of this synergistic combination is referred to herein as the trienzyme' activity. Nevertheless, it will be appreciated that the trienzyme activity is not limited to only three proteolytic enzymes being present in the compositions of the invention, rather, it refers to the combination of three contributory proteolytic activities being present in these cosmetic compositions.

In specific embodiments of the invention the thiol protease is a fruit derived protease selected from papain, ficin, bromelain and actinidin. Suitably the first serine protease derived from Aspergifius melleus is Aspergillus melleus proteinase (seaprose). Suitably, the second serine protease can be selected from subtilisin, trypsin and chymotrypsin.

In one embodiment of the invention, the cosmetic composition comprises: between about 0.001 and about 0.100 wt% of subtilisin; between about 0.005 and about 0.200 wt% of papain; and between about 0.001 and about 0.100 wt% of Aspergillus melleus protease, wherein wt% is the percentage by weight of the composition as a whole.

In a specific embodiment of the invention in use, the composition comprises: about 0.005 wt% of subtilisin; about 0.010 wt% of papain; and about 0.015 wt% of Aspergillus melleus protease, wherein wt% is the percentage by weight of the composition as a whole.

In a further specific embodiment of the invention in use, the composition comprises: about 0.001 wt% of subtilisin; about 0.100 wt% of papain; and about 0.003 wt% of Aspergillus melleus protease, wherein wt% is the percentage by weight of the composition as a whole.

The cosmetic compositions of the invention typically comprise a cosmetically suitable carrier. Such carriers may include one or more of a solvent (such as water and/or glycerine), an emulsifier, an emollient, a preservative, a humectant and any other component suitably included in a cosmetic base or chassis.

The cosmetic composition of the invention may further comprise one or more alpha hydroxyl acids (AHAs), suitably the AHAs are of botanical origin. Preferred AHAs are comprised within a botanical extract of a flower selected from at least one of Freesia alba; Gardenia tahitensis; Hibiscus sabdariffa; and Lilium candidum. A combination of two or more of the aforementioned flower extracts can be advantageously used to enhance the skin smoothing and resurfacing properties of the present composition.

The cosmetic composition of the invention may further comprise an antioxidant.

S Suitably, the antioxidant can be selected from one or more of vitamin C (ascorbic acid); Moringa seed extract; Poria cocos root extract; and vitamin E (tocopherol).

In a specific embodiment of the invention the cosmetic composition further comprises a sun blocking agent.

Typically the cosmetic composition of the invention is in the form of a cream; a lotion; a serum; a face wash; a shampoo; a foam; a skin tonic; an ointment; or a gel. However, the skilled person will appreciate that any composition suitable for administering the active trienzyme combination of the invention to the epidermis can be used.

A second aspect of the invention provides for a cosmetic method for promoting smoothing and renewal of the epidermis of an individual. The method of the invention comprises applying a composition or a combination of compositions to the epidermis of the individual, wherein the composition or combination of compositions comprises: at least one thiol protease; at least a first serine protease derived from Aspergillus melleus; and at least a second serine protease.

In a preferred embodiment of the invention the at least one thiol protease, at least a first serine protease derived from Aspergillus melleus, and at least a second serine protease are comprised within a single cosmetic composition. In an alternative embodiment of the invention, the at least one thiol protease, at least a first serine protease derived from Aspergillus melleus, and at least a second serine protease are comprised within separate cosmetic compositions and are applied to the epidermis of the individual sequentially -i.e. such that the trienzyme activity is reconstituted at the point of action on the skin's surface.

All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic diagram with the locations of the sites of measurement (X) used in the standardised visioscan measurement area of facial skin.

DETAILED DESCRIPTION

To achieve the benefits associated with the compositions of the invention a synergistic combination of protease activities have been combined. Together, these proteolytic activites serve to promote self renewal of the skin, via a gentle targeted degradation of the keratin in the upper layers of the epidermis. The combination of these combined enzymic activities allows for a smaller than expected amount of total protease content in the composition as a whole, thereby improving tolerance and minimising the risk of irritation and allergic reaction.

Proteases are carbonyl hydrolases which generally act to cleave peptide bonds of proteins or peptides. As used herein, "protease" means a proteolytic enzyme of either naturally occurring or recombinant origin.

Serine proteases are endopeptidases that are characterised by the presence of a serine residue in the active site of the enzyme and a pH optima around neutrality. Serine proteases can participate in a wide range of functions in the body, including blood clotting, inflammation as well as digestive enzymes. Serine proteases include subtilisin, trypsin and chymotrypsin. In accordance with the invention the compositions comprise at least one serine protease derived from Aspergillus melleus, preferably such a protease is Aspergillus melleus proteinase (also sometimes referred to as seaprose). Suitably Aspergilius melleus proteinase is obtained from compositions such as Prozyme 6 (Amano Enzyme Inc., Nagoya, Japan) or combined with subtilisin in Prozymex HBt LS 9142 (Laboratoire Serobiologiques, Pulnoy, France).

Thiol proteases are endopeptidases that are characterised by the presence of a thiol group at the active site of the enzyme, usually supplied by a cysteine residue. According to the invention preferred thiol proteases are those of plant derived origin and may include papain (from papaya), ficin (from figs), bromelain (from pineapple) and actinidin (from kiwi fruit). Thiol proteases are readily available in recombinant or extracted form, for example from Sigma-Aldrich (Poole, Dorset, United Kingdom).

The cosmetic compositions of the invention comprise a triezyme combination with the combined activity of thiol and serine proteases. In use, the inventive compositions are applied topically to the skin. Usually, the compositions of the invention are applied to the skin of the face, hands and neck, however other skin surfaces may be subjected to the cosmetic treatment. The only contraindication is that particularly sensitive areas of skin, such as inflamed skin or the skin close to the eyes be avoided. The compositions of the invention are usually applied directly to the skin surface manually by the user as is conventional.

It is envisaged that the compositions of the invention should contain sufficient proteolytic activity to enable between about 0.1 units and about 100 units of total serine protease activity (as measured by the benzoyl-L-arginine ethyl ester (BAEE) assay) to be applied to the skin per dose, wherein a dose is an amount of between about 2 ml and about 10 ml of product. More preferably, the serine protease activity is in the range of between 5 units and 60 units per application dose, even more preferably below 12 units per application dose. The compositions of the invention should contain a total thiol protease activity of less than around 100, 000 units per dose (as measured by the standard papain casein digestion assay), preferably between 500 units and 75,000 units per dose, more preferably between 2000 units and 60,000 units per dose, most preferably below 40,000 units perdose.

The cosmetic compositions according to the present invention can suitably include all active materials and appropriate components conventionally known for such formulations. Such components include, for instance, gelling agents, anionic polymers, thickeners, surfactants, hydrating agents, emollients, chelating agents, antioxidants, preservatives, buffering compounds, perfumes, fillers, colourings, volatile or non-volatile, modified or non-modified silicones and reducing agents. The relative proportions of the different components are suitably those that are used in conventional cosmetic formulations. It is envisaged that the person skilled in the art will naturally select the optionally present materials and incorporate these into the composition according to the invention in such a manner that the advantageous properties associated with the desirous trienzyme activity are not significantly modified or eliminated. It is further envisaged that certain components may fulfil more than one criteria, for instance, glycerine is known to act as both a solvent and a humectant.

As mentioned above, a variety of components may be present in the compositions of the present invention; foremost of these components is likely to be water, as a solvent. Amounts of water may range from about 1% to about 95%, preferably from about 25% to about 90%, optimally from about 60% to about 85% by weight of the composition. Other suitable solvents include glycerine and benzyl alcohol.

The cosmetic compositions of the present invention may include one or more emulsifiers, for example, if the composition is in the form of a lotion, cream, gel or serum. Suitable emulsifiers that can be used in the present invention include, for example, one or more alkoxylated fatty alcohols, C14 alcohols, alkylpolyglycosides, C,4.20 alkylgiucoside, saponifiers, alkyl sulfates, monoalkyl and dialkyl phosphates, alkyl sulphonates, acyl isothionates, cetyl alcohol, stearyl alcohol, sorbitans, stearic acid, glyceryl stearate or any combinations thereof. When used in the present compositions, the emulsifier is present in about 1% to about 10 % by weight of the composition. For embodiments of the invention where the composition is in the form of a cream or lotion, the emulsifier is typically present in an amount about 3% to about 8% by total weight of the composition.

Certain oil in water emulsion based compositions of the invention may also include one or more stabilisers to stabilise the emulsion. Suitable stabilisers include, for example, alcohols, alkoxylated alcohols, fatty alcohols, glyceryl esters, such as, glyceryl stearate, gums, soaps, synthetic polymers, waxes, or any combinations thereof. Particularly suitable emulsion stabilisers include a stearyl alcohol or glyceryl stearate.

The compositions of the invention may further include one or more emollients to soften and soothe the skin. Suitable emollients comprise, for example, one or more fats and oils (hydrogenated or non-hydrogenated), such as, vegetable oil, castor oil, coconut oil and cocoa butter, shea butter (Butyrospermum parkii), as well as esters, such as, tricapryl citrate and isononyl isonanoate, or any combinations thereof. The one or more emollients are present in the lotion composition in an amount about 0.01 wt. % to about 15 wt. %. Preferably, the one or emollients are present in an amount about 1 wt. % to about 10 wt. % and more preferably about 4 wt. % to about 6 wt. %.

The compositions of the invention may include one or more humectants. A humectant is a component that absorbs or retains moisture. Suitable humectants that can be used in the present composition include, for example, urea, pyroglutamic acid, amino acids (e.g. glutamic acid), polyols or other compounds with hygroscopic properties, or any combinations thereof. typically, the humectant is a polyol, such as a sorbitol. In a composition of the invention in the form of a serum, described in detail below, the primary humectants present include methyl-gluceth-20 and propylene glycol. When present, the humectant is typically in an amount about 0.1 wt. % to about 10 wt. %. Typically, the humectant is present in an amount about 0.5 wt. % to about 8 wt. %, of the total weight of the composition.

Suitable preservatives for use in the compositions of the present invention include, for example, one or more alkanols such as phenoxy ethanol, ethylenediaminetetraacetic acid (EDTA) salts, EDTA fatty acid conjugates, isothiazolinone, parabens such as methylparaben and propylparaben, propylene glycols, sorbates, urea derivatives such as diazolidinyl urea and imidazolidinyl urea, quaternary ammonium salts, or any combinations thereof. When present, the preservatives are typically in an amount of between about 0.1 wt. % to about 2 wt. % of the total weight of the composition. Preferably, the preservatives are present in an amount about 0.3 wt. % to 1 wt. % of the total weight of the composition.

Suitable chelating agents for inclusion in the compositions of the invention may include, for example EDTA derivatives, or any combinations thereof. Preferably, the one or more chelating agents is disodium EDTA, present in the composition in an amount about 0.01 wt. % to about 0.2 wt. %. Preferably, chelating agent is present in an amount about 0.05 wt. % to about 0.15 wt. %, of the total weight of the composition.

The composition may include one or more buffering compounds that serve to adjust and maintain the pH of the composition. Suitable buffering agents include, for example, one or more adipic acids, glycines, citric acids, calcium hydroxides, magnesium aluminometasilicates, triethanolamine, or any combinations thereof.

When present, the buffering agent should be present at a level suitable to ensure stable pH in the composition throughout its normal shelf life and period of use.

The combination of selected buffering agents is of importance in the compositions of the present invention as the proteolytic activity of the trienzyme component is dependent, at least in part, upon the pH of the composition. The preferred pH range for the compositions of the present invention between about 6.5 and about 8.0, more preferably between about 7.0 and 7.5.

The present cosmetic compositions may also include one or more surfactants.

Surfactants are of particular importance in compositions of the invention that are for the purposes of skin washes, such as soaps or face washes. Suitable surfactants include, for example, anionic, noriionic, cationic, amphoteric, or any combinations thereof. Preferably the one or more surfactants are non-ionic surfactants. Suitable nonionic surfactants include, for example, one or more alkoxylated alcohols, ethoxylated alcohols, propoxylated alcohols, inter-dispersed ethoxylated-propoxylated alcohols, copolymers, fatty acids, alkyl phenols, polyglycosides, polyglucosides, n-alkylpyrrolidones, block copolymers, or any combinations thereof.

A lipid, or oily, phase can be suitably incorporated into the compositions of the invention, for instance in lotion or ointment embodiments of the invention. The lipid phase can consist of up to about 50% of the total weight of the agent depending upon the application and can contain an oil or a wax or mixtures thereof, or fatty acids, fatty alcohols and fatty acid esters. The oils can be selected from animal oils, vegetable oils, mineral oils or synthetic oils or liquid silicones and especially paraffin oils, isoparaffins or polyolefines. similarly suitable waxes can be of animal, fossil, vegetable, mineral or synthetic origin.

The compositions of the invention need not be restricted to homogenous mixtures but can also include compositions which include compartmentalised phases. Typically such two or more phase compositions are oil-in-water emulsions, although inclusion of microparticles such as microbeads and/or liposomes is possible. In optional compositions of the invention the trienzyme activity can be localised within such microparticles and only activated' during use -i.e. during application to the skin.

The compositions of the invention may further include one or more hydrophilic or lipophilic sun screen filters that absorb in the UV-A and UV-B range. Suitable filtering compounds are well known in the art and could include, for example cinnamates. Following the resurfacing and renewal activity stimulated in the skin of the user by the trienzyme activity of the present invention, it will be appreciated that in the short term the skin can be more sensitive to UV radiation exposure. Hence, it is optional to include sun filters in those compositions of the invention that are intended for daytime use. Alternatively, the compositions of the invention can be used in combination with a separate sun filter containing preparation.

The cosmetic compositions of the invention can also include naturally derived extracts and components that serve to enhance the trienzyme activity further or simply to provide alternative or complimentary properties to the composition as a whole. Such extracts can include essential oils for aromatherapy and other benefits. Botanical extracts can include extracts in the form of aqueous preparations, alcoholic extracts, oil based extracts or dried powders. Typical plant based extracts include Aloe vera extract, yeast extracts, Poria cocos extract, Oryza sativa powder, Jojoba seed oil, Acerola fruit extract, Moringa seed extract, and Babassu oil.

EXAMPLES

There follow four compositions of the invention that are suitable for topical application to the skin. Each composition comprises the trienzyme activity that typifies the primary aspects of the present invention. These compositions are suitable for retail sale to the consumer for private use. In addition, the compositions are suitable for professional use as so-called spa treatments, usually offered within health-spa clinics. The disclosed compositions are suitable for stand-alone use or as part of a combined trienzyme programme.

Exam le 1 -Cosmetic serum formulation comprising trienzyme activity

Table 1

Component Function % by weight of total composition Glycerine Denaturant/humectant/solvent 3.000 Methyl gluceth-20 Humectantimoisturiser 2.000 C14-22 alcohols Emulsion stabiliser 1.400 Propylene glycol Humectant/solvent 1.248 Hydroxypropyl starch Bulking agent 1.000 phosphate Cl 2-20 atkylglucoside Surf actarit/em ulsifier 0.600 phenoxyethanol Preservative 0.504 Benzyl alcohol Preservative 0.400 Sorbitol Humectant/plasticiser 0.399 Tromethamine Buffer 0.300 Disodium EDTA Chelating agent 0.150 Citric acid Buffer/chelating agent 0.120 Methylparaben Preservative 0.113 Xanthan gum Binder 0.100 Malic acid Buffer 0.060 Magnesium ascorbyl Antioxidant 0.050 phosphate Tocopherol Antioxidant 0.050 Hydrogenated castor oil Emollient/emulsifier 0.048 Stearyl alcohol Emollient/emulsifier 0.039 Butylparaben Preservative 0. 028 Ethylparaben Preservative 0.028 Tartaric acid Buffer 0.020 Kaolin Absorbent/abrasive 0.020 Aspergillus melleus proteinase Skin conditioner 0.015 Propylparaben Preservative 0.015 lsobutylparaben Preservative 0.014 Papain Skin conditioner 0.010 Subtilisin Skin conditioner 0.005 Water Solvent balance The serum formulation described above is suitable for smoothing the skin's surface to improve the complexion. Since the skin's natural cell renewal is stimulated, imperfections appear diminished.

S

Example 2 -Cosmetic serum formulation comprising trienzyme activity and botanically derived alpha hydroxyl acids (Al-lAs)

Table 2

Component Function % by weight of total composition GlycerineDenaturantJhumectant/solvent 3.000 Methyl gluceth-20 Humectant/moisturiser 2.000 C14-22 alcohols Emulsion stabiliser 1.400 Propylene glycol Humectant/solvent 1.248 Hydroxypropyl starch Bulking agent 1.000 phosphate Cl 2-20 alkylglucoside Surfactant/em ulsifier 0.600 Phenoxyethanol Preservative 0.504 Benzyl alcohol Preservative 0.400 Sorbitol H umectant/plasticiser 0.399 Tromethamine Buffer 0.300 Disodium EDTA Chelating agent 0.150 Citric acid Buffer/chelating agent 0.120 Methylparaben Preservative 0.113 Xanthan gum Binder 0.100 Malic acid Buffer 0.060 Magnesium ascorbyl Antioxidant 0.050 phosphate Tocopherol Antioxidant 0.050 Hydrogenated castor oil Emollient/emulsifier 0.048 Stearyl alcohol Emollient/emulsifier 0.039 Butylparaben Preservative 0. 028 Ethylparaben Preservative 0.028 Tartaric acid Buffer 0.020 Kaolin Absorbent/abrasive 0.020 Aspergillus melleus proteinase Skin conditioner 0.015 Propylparaben Preservative 0.015 Isobutylparaben Preservative 0.014 Papain Skin conditioner 0.010 Extract of Freesia alba flower AHA source 0.009 Extract of Gardenia tahitensis AHA source 0.009 flower Extract of Hibiscus sabdariffa AHA source 0.009 flower Extract of Lilium candidum AHA source 0.009 flower Subtilisin Skin conditioner 0.005 Water Solvent balance Standardised aqueous extracts of the flowers of Freesia alba, Gardenia tahitensis, Hibiscus sabdariffa and Lilium candidum can be obtained from Alban Muller International (Vincennes, France). The synergistic combination of AHAs present in the flower extracts can assist in providing a non-abrasive exfoliating action to the skin. Together with the trienzyme activity in the serum, the AHAs further contribute to the advantageous skin conditioning properties of the composition.

Example 3-Cosmetic skin cream com osition comprising trienzyme activity

Table 3

Component Function % by weight of total composition Glycerine Den aturant/humectant/solvent 8.032 Glyceryl stearate SE Emulsifier 4.000 Isononyl isononanoate Emollient 2.000 Ethyl macadamiate Emollient 1.997 Caprylic/capric triglyceride Emollient/solvent 0.750 Shea butter Viscosity agent 0.654 Propylene glycol Humectantlsolvent 0.538 Cetyl alcohol Emollient/emulsifier 0.500 Sodium polyacrylate Binder 0.500 Stearic acid Emulsifier 0.500 Benzyl alcohol Preservative 0.400 Phenoxyethanol Preservative 0.360 Behenyl alcohol Emollient 0.240 Hydrogenated vegetable oil Emollient 0.200 Disodium EDTA Chelating agent 0.150 Methylparaben Preservative 0.088 Hydrogenated castor oil Emollient 0.048 Stearyl alcohol Emollient 0.039 Butylparaben Preservative 0.020 Ethylparaben Preservative 0.020 Tocopheryl acetate Antioxidant 0.020 Aspergillus melleus proteinase Skin conditioner 0.015 Propylparaben Preservative 0.012 Isobutylparaben Preservative 0.010 Papain Skin conditioner 0.010 Subtilisin Skin conditioner 0.005 Tocopherol Antioxidant 0.002 Malic acid Buffer 0.001 Water Solvent balance The above formulation is particularly suitable as a night cream and, thus, is intended for long-term application (i.e. 6-12 hours). This treatment stimulates the skin's natural cell renewal cycle, actively smoothing and enhancing the skin whilst cellular respiration as at its optimum.

Example 4 -Cosmetic facial wash composition comprising trienzyme activity

Table 4

Component Function % by weight of total composition Surf actant / viscosity Sodium lauroyl sarcosinate 4.500 controlling Glycerin humectant / solvent 3.175 Cocamidopropyl betaine Surfactant 3.000 Antistatic / binding / film Acrylates copolymer 2.400 forming Polysorbate 20 Emulsifier I surfactant 2.000 Glycol distearate Emollient / emulsifier 1.250 Sodium lactate Buffer / humectant 1.200 Coco-glucoside Surfactant 1.160 Tromethamine Buffering 0.900 Glyceryl oleate Emollient I emulsifier 0.860 Dicaprylyl ether Solvent 0.825 Phenoxyethanol Preservative 0.720 Benzyl alcohol Preservative 0.400 Glyceryl stearate Emollient 0.200 Methylparaben Preservative 0.160 Disodium EDTA Chelating agent 0.100 Papain Skin conditioner 0.100 Propylene glycol Humectant 0.100 Butylparaben Preservative 0.040 Ethylparaben Preservative 0.040 Isobutylparaben Preservative 0.020 Stearyl alcohol Emollient 0.001 Aspergillus melleus proteinase Skin conditioner 0.030 Subtilisin Skin conditioner 0.001 Water Solvent balance This composition is suitable as a daily face wash that stimulates the skin's cell renewal cycle, and also actively smoothes by sloughing away dead skin cells which can dull the complexion.

ExamDle 5 -In vivo skin resurfacing activity of a tnenzvme containing comDosition The effect of a trienzyme serum described above (see Example 2) on facial skin smoothness was assessed in a user study. In the study, subjects used the trienzyme serum composition at home according to the manufacturers instructions for 28 consecutive days. The skin surface micro topography was measured by image analysis of skin surface images taken using a Visioscan VC 98 (Courage & Khazaka, GmbH). Measurements were taken at day 0 before the study started and after 14 and 28 days of use.

The study: A total of 40 normal female volunteer subjects aged between 25 and 55 years were recruited for the study. Details of the age of the subjects are given in Table 5. The mean subjects taking part in the user study was 40 years, age range 25 to 55 years.

Table 5

__________ __________ __________ __________

Subject Age Subject Age 1 55 21 46 2 45 22 33 3 38 23 46 4 35 24 37 46 25 31 6 37 26 45 7 33 27 35 8 51 28 44 9 48 29 35 44 30 39 11 49 31 44 12 43 32 39 13 40 33 33 14 43 34 34 44 35 38 16 41 36 29 17 50 37 46 18 32 38 50 19 35 39 40 25 40 32 Mean = 40.3 Upper Age 55 Lower Age =25 All subjects fulfilled the inclusion and exclusion criteria as detailed in the protocol, these were as follows: Inclusion criteria - * Female subjects within the age range 25 -55 years.

* Caucasian or light skinned individuals.

* Subjects in good general health with no significant concurrent illnesses.

* Subject has signed the consent form after the nature of the study has been fully explained.

Exclusion criteria - * Pregnant or lactating females.

* Subject is using any topical or systemic medication likely to interfere with the study. (e.g. drugs with a known effect on the skin).

* Subject has taken part in a study involving the test site during the previous 4 weeks (28 days).

* Subject has history of skin disease likely to interfere with the study (e.g. facial dermatoses).

* Subject has history or evidence of alcohol or drug abuse in the previous 2 years.

* Subject is unlikely to comply with the conditions of the study as detailed in the protocol.

Test oroduct use Forty (40) subjects took part in this part of the study. Subjects used the enzyme serum at home according to the manufacturer instructions for 28 consecutive days.

Subiect restrictions Subjects were instructed to continue with their normal washing and make up procedures. Subjects however were not permitted to use any other facial moisturisers, exfoliating products or facial treatment products. In addition subjects could not use sun beds or sunbathe during the study. Subjects did not apply the test product or other moisturisers or makeup to the test area of the cheek on the assessment days. Subjects were permitted to use eye and lip products on the assessment days.

Visioscan measurements The topography of the skin at the side of the face in the cheek area was assessed using the Visioscan VC 98 (Courage & Khazaka, GmbH). The device uses the SELS (Surface Evaluation of Living Skin) evaluation method developed by Tronnier et al. (Adv. Exp. Med. Biol. (1999) 455:507-16). The parameters measured, included skin smoothness. The site of measurement was standardised by reference to anatomical features.

Standardisation of Visioscan measurement site The measurement site was defined by a rectangle 2cm x 5cm in area. The 2cm edge was on a line from the corner of the eye to the point where the ear is attached to the head. The corner of the rectangle was at a point 1.5cm from the corner of the eye.

The sites of measurement were standardised by reference to the 2cm by 5cm rectangle shown in Figure 1.

Three measurements were taken within the test area at points defined by reference to marks on the border of the rectangle, 1cm on the 2cm edge and 1.25cm, 2.5cm and 3.75cm on the 5cm edge.

Visioscan measurements The SELS parameters measured were roughness (SER), scaling (SESC), smoothness (SESM) and wrinkling (SEW). The site of measurement was standardised by reference to anatomical features. Three images were taken at separate sites on the face before, during and after treatment. The images were taken as per protocol in a vertical line from the side of the eye. The analysis has been carried out on the third (lower) image taken from the middle of the cheek area. It was noted during the study that in some subjects the first (upper) image and to a lesser extent the middle image showed fine velous hair. As this may have interfered with the subsequent computer image analysis it was decided to use only the lower mid cheek image. The SELS values before, during and after treatment were compared using a non-parametric multiple comparison procedure based on the Tukey test using Unistat version 5.5 for Windows.

All of the 40 subjects recruited for the study attended every assessment and were deemed to have completed the study.

Visioscan measurements The results of the Visioscan measurements are summarised as follows:

Table 6

SER Roughness Before During After Mean 1.61 1.90 1.91 Std Dev 0.70 0.70 0.60 Although a small increase in the roughness value was seen, there was a large variation in the measurement as indicated by the high standard deviation. The statistical analysis indicated that the difference between before and after measurements was not statistically significant (p = 0.0569).

Table 7

SESC Scaling Before During After Mean 1.14 1.09 1.12 StdDev 0.59 0.47 0.43 There was little difference in the mean values for scaling. The statistical analysis indicated that the difference between before and after measurements was not statistically significant (p = 0.6438).

Table 8

SESM Smoothness Before During After Mean 38.34 50.82 53.12 StdDev 5.11 7.18 8.72 There was an increase in smoothness during the course of the study, the mean value increasing by approximately 38%. The statistical analysis indicated that the difference between before and after measurements was statistically significant (p 0.0001).

Table 9

SEW Wrinkling Before During After Mean 27.73 24.87 25.34 Std Dev 1.83 1.83 2.15 There was small decrease in wrinkling during the course of the study, the mean value decreasing by approximately 8%. The statistical analysis indicated that despite the relatively small change the difference between before and after measurements was statistically significant (p = 0.0002).

Results The results of the Visioscan analysis of the skin surface micro topography indicated that use of the Enzyme Serum did lead to changes in the skin surface.

An increase in the parameter SELS Smoothness (SESM) and a decrease in the SELS parameter Wrinkling (SEW) was noted. Both of these changes were statistically significant (SESM, p = 0.0001; SEW, p = 0.0002) indicating that use of the trienzyme serum composition for as little as four consecutive weeks led to smoother skin.

Although particular embodiments of the invention have been disclosed herein in detail, this has been done by way of example and for the purposes of illustration only. The aforementioned embodiments are not intended to be limiting with respect to the scope of the appended claims, which follow. It is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.

Claims

CLAIMS

1. A cosmetic composition comprising a suitable carrier and: at least one thiol protease; at least a first serine protease derived from Aspergillus melleus; and at least a second serine protease.

2. The cosmetic composition of claim 1, wherein the thiol protease is selected from the group consisting of papain, ficin, bromelain and actinidin.

3. The cosmetic composition of claim 1, wherein the at least a first serine protease derived from Aspergillus melleus is Aspergillus melleus proteinase.

4. The cosmetic composition of claims 1 and 2, wherein the at least a second serine protease is selected from the group consisting of subtilisin, trypsin and chymotrypsin.

5. The cosmetic composition of any previous claim comprising: between about 0.001 and about 0.100 wt% of subtilisin; between about 0. 005 and about 0.200 wt% of papain; and between about 0.001 and about 0.100 wt% of Aspergillus melleus protease, wherein wt% is the percentage by weight of the composition as a whole.

6. The cosmetic composition of claim 5 comprising: about 0.005 wt% of subtilisin; about 0.010 wt% of papain; and about 0.015 wt% of Aspergillus melleus protease, wherein wt% is the percentage by weight of the composition as a whole.

7. The cosmetic composition of claim 5 comprising: about 0.00 1 wt% of subtilisin; about 0.100 wt% of papain; and about 0.003 wt% of Aspergillus melleus protease, wherein wt% is the percentage by weight of the composition as a whole.

8. The cosmetic composition of any previous claim, further comprising one or more alpha hydroxyl acids (AHAs).

9. The cosmetic composition of claim 8, wherein the AHAs are of botanical origin.

10. The cosmetic composition of claim 9, wherein the AHAs are comprised within a botanical extract of a flower selected from at least one of the group consisting of Freesia alba; Gardenia tahitensis; Hibiscus sabdariffa; and Lilium candidum.

11. The cosmetic composition of any previous claim, further comprising an antioxidant.

12. The cosmetic composition of claim 11, wherein the antioxidant is selected from one or more of the group consisting of vitamin C (ascorbic acid); Moringa seed extract; Poria cocos root extract; and vitamin E (tocopherol).

13. The cosmetic composition of any previous claim further comprising a sun blocking agent.

14. The cosmetic composition of any previous claim wherein the composition is in a form selected from the group consisting of a cream; a lotion; a serum; a face wash; a shampoo; a skin tonic; an ointment; and a gel.

15. A cosmetic method for promoting smoothing and renewal of the epidermis of an individual, the method comprising applying a composition or a combination of compositions to the epidermis of the individual, wherein the composition or combination of compositions comprises: at least one thiol protease; at least a first serine protease derived from Aspergillus melleus; and at least a second serine protease.

16. The cosmetic method of claim 15, wherein the at least one thiol protease, at least a first serEne protease derived from Aspergillus melleus, and at least a second serEne protease are comprised within a single cosmetic composition.

17. The cosmetic method of claim 15, wherein the at least one thiol protease, at least a first serine protease derived from Aspergillus melleus, and at least a second serine protease comprised within separate cosmetic compositions and are applied to the epidermis of the individual sequentially.

18. A cosmetic composition substantially as herein described in the examples.