WO2014041528A2 - A composition - Google Patents

Abstract

A composition which can be oral or topical for treating the cutaneous signs of ageing comprising collagen and a combination of Reactive Oxygen Species (ROS) scavengers/antioxidants such Vitamin C, disulfides, tocopherols/tocotrienols, carotenoids and coenzyme Q₁₀ and collagen pre-cursors and use of such compositions for treating cosmetic ageing.

Description

A Composition

Introduction This invention relates to a composition for the preventative and/or curative treatment of cutaneous signs of ageing and in particular to an oral composition for such treatment. The invention also relates to a cosmetic method for the treatment of cutaneous signs of ageing. Background of the Invention

Collagen is one of the most plentiful and naturally occurring proteins in the human body and accounts for more than 30% of human protein mass. A fibrous protein, collagen is the main component of connective tissue and connects and supports body tissues such as skin, ligaments and cartilage and the organic component of bone.

From the mid 20's, the body's own natural collagen production starts to decline at a rate of 1.0% - 1.7% per annum. The collagen is noWeplenished while there is an even more pronounced decline in menopausal women due to reduced oestrogen levels.

Human skin is made up of three major components - collagen, hyaluronan

(hyaluronic acid) and elastin fibres which provide the skin with the scaffolding for structure and provide it with strength and elasticity. Accordingly, due to age related decreased collagen production, skin loses suppleness, wrinkles and sags.

Skin cells also exhibit signs of ageing due to oxidative damage by free radicals. Free radicals cause damage to all the components of the skin cell including the collagen, the lipid element and the DNA element by starting chemical chain reactions such as lipid peroxidation or by oxidising DNA or proteins. Damage to DNA can cause mutations and possibly cancer if not reversed by DNA repair mechanisms while damage to collagen results in the breakdown of the collagen fibres and consequent increased signs of ageing.

In light of the above, significant effort has been expended in developing methods of improving the appearance and feel of human skin using anti-ageing products focused on collagen and free radicals. More radical methods of counteracting the signs of ageing such as plastic surgery and other invasive techniques are also being used more and more with the associated surgical risks.

However, most anti-ageing products for the preventative or curative treatment of the cutaneous signs of ageing are topical products which claim to address the problem by acting on the exterior surface of the skini 'Such known topical applications have many limitations and in many cases fail to achieve the claimed results. It is also known that, when ingested, certain ingredients can provide improvements in skin appearance and texture. For example, Vitamin E and Vitamin C when taken orally are known to provide protection against UV skin damage while similar benefits have been observed with ingestion of carotenoids such as lycopene and β-carotene. Nevertheless, there remains a need for compositions that provide improved preventative and/or curative treatment of cutaneous signs of ageing.

Summary of the Invention

According to the invention, there is provided a composition for treating the cutaneous signs of ageing comprising:

collagen, and

a combination of Reactive Oxygen Species (ROS) scavengers/antioxidants selected from the group comprising Vitamin C, disulfides, Vitamin E, carotenoids and coenzyme Q-io-

Preferably, the combination of ROS scavengers/antioxidants comprises Vitamin E and coenzyme Qi₀ More preferably, the combination of ROS

scavengers/antioxidants further comprises Vitamin C. Most preferably, the combination of ROS scavengers/antioxidants further comprises a disulfide.

Suitably, the disulfide comprises a-lipoic acid.

Preferably, the combination of ROS scavengers/antioxidants further comprises a carotenoid. More preferably, the carotenoid comprises lycopene. Alternatively, the carotenoid comprises astaxanthin. Advantageously, the composition further comprises a giycosamine source.

Preferably, the giycosamine source comprises hyaluronic acid. Alternatively, the giycosamine source comprises aTamarindus Indicia extract. In one embodiment of the invention, the composition is augmented with a collagen pre-cursor. Preferably, the collagen pre-cursor comprises L-proline and/or glycine.

Suitably, the composition comprises β-carotene. Advantageously, the composition comprises Resveratrol.

Suitably, the composition comprises L-carnitine.

Preferably, the collagen comprises marine collagen.

In a preferred embodiment of the invention, the composition comprises

from about 20 weight % to about 40 weight % collagen;

from about 0.5 weight % to about 3.0 weight % Vitamin E;

from about 0.2 weight % to about 2.0 weight % coenzyme Q ₀;

from about 10 weight % to about 25 weight % Vitamin C;

from about 5.0 weight % to about 20 weight % a-lipoic acid;

from about 0.5 weight % to about 2.0 weight % lycopene, and

from about 0.0003 weight % to about 2.0 weight % astaxanthin. Preferably, the composition comprises from about 25 weight % to about 35 weight % collagen and from about 1 .0 weight % to about 2.5 weight % Vitamin E.

Suitably, the composition comprises from about 0.003 weight % to about 2 weight % coenzyme Qio and from about 0.2 weight % to about 2 weight % coenzyme Q ₀.

Preferably, the composition comprises from about 0.75 weight % to about 1 .5 weight % coenzyme Qi₀ and from about 15 weight % to about 20 weight % Vitamin C. Suitably, the composition comprises from about 10 weight % to about 15 weight % a- lipoic acid. Advantageously, the composition comprises from about 0.75 weight % to about 1.5 weight % lycopene.

Preferably, the composition comprises from about 0.1 weight % to about 2.0 weight % astaxanthin. More preferably, the composition comprises from about 0.2 weight % to about 1 .0 weight % astaxanthin.

Advantageously, the composition comprises from about 0.1 weight % to about 2.0 weight % β-carotene.

Suitably, the composition comprises from about 0.1 weight % to about 2.0 weight % β-carotene. Preferably, the composition comprises from about 0.1 weight % to about 2.0 weight % L-proline. More preferably, the composition comprises from about 0.2 weight % to about 1.0 weight % L-proline. Preferably, the composition comprises from about 0.05 weight % to about 1.0 weight % glycine. More preferably, the composition comprises from about 0.1 weight % to about 0.5 weight % L-proline.

In a preferred embodiment of the invention, the composition is an oral composition.

Preferably, the collagen and ROS scavengers/antioxidants are present in the composition at sufficient concentrations to deliver daily dosages sufficient to achieve an anti-ageing effect. More preferably, the composition further comprises a collagen pre-cursor present at sufficient concentration to promote collagen production.

Suitably, the daily dosage comprises about 500mg collagen, about 250mg Vitamin C, about 200mg a-lipoic acid, about 100mg hyaluronic acid, about 40mg Vitamin E, about 1.5mg lycopene, about 10mg coenzyme Qio and about 2mg astaxanthin.

Preferably, the daily dosage comprises about 70mg collagen pre-cursors. More preferably, the collagen pre-cursor comprises L-proline and/or glycine. Most preferably, the daily dosage of collagen pre-cursor comprises 50mg L-proline and 20mg glycine. The invention also extends to a cosmetic method for providing a cutaneous anti- ageing benefit comprising administering a composition as hereinbefore defined.

In a further embodiment, the invention also extends to use of a composition comprising collagen and a combination of Reactive Oxygen Species (ROS) scavengers/antioxidants selected from the group comprising Vitamin C, disulfides, Vitamin E, carotenoids and coenzyme Qio in the treatment of cutaneous ageing.

Preferably, the combination of ROS scavengers/antioxidants comprises Vitamin E and coenzyme Q_10. More preferably, the combination of ROS

scavengers/antioxidants further comprises Vitamin C. Most preferably, the combination of ROS scavengers/antioxidants further comprises a disulfide.

Advantageously, the disulfide comprises a-lipoic acid.

Suitably, the combination of ROS scavengers/antioxidants further comprises a carotenoid. Preferably, the carotenoid comprises lycopene, astaxanthin or β- carotene. Preferably, the composition further comprises a glycosamine source. More preferably, the glycosamine source comprises hyaluronic acid.

Alternatively, the glycosamine source comprises aTamarindus Indicia extract. In a preferred embodiment, the composition comprises a collagen pre-cursor. Preferably, the collagen pre-cursor comprises L-proline and/or glycine.

In a further embodiment, the composition comprises Resveratrol and/or L-carnitine.

Preferably, the collagen comprises marine collagen.

The invention therefore provides a composition for treating the cutaneous signs of ageing comprising collagen, and a combination of Reactive Oxygen Species (ROS) scavengers/antioxidants selected from the group comprising Vitamin C, disulfides, tocopherols/tocotrienols, carotenoids and coenzyme Q₁₀. The collagen and ROS scavengers/antioxidants are present in the compositions at sufficient concentrations to deliver a daily dosage sufficient to achieve the anti-ageing effect. The invention also extends to a cosmetic method for providing cutaneous anti-ageing benefits selected from the group comprising increased collagen generation, improved skin elasticity, reduced appearance of wrinkles and reduced sagging comprising administering an oral composition as hereinbefore defined. The invention also extends to the use of a composition as hereinbefore defined to provide cutaneous anti-ageing benefits selected from the group comprising increased collagen generation, improved skin elasticity, reduced appearance of wrinkles and reduced sagging. The oral compositions of the invention function as nutraceuticals - nutritional supplements that have cosmetic or health benefits including the prevention and treatment of ageing and diseases. More particularly, the compositions of the invention are highly efficacious at reducing the signs of ageing when administered orally. The anti-ageing compositions are typically made up of a unique combination of bio-marine collagen, hyaluronic acid and an ROS scavenger/antioxidant blend formed from a-lipoic acid, Vitamin C, Vitamin E, astaxanthin, lycopene and coenzyme Qi₀ for treating either preventative^ or curatively individuals suffering from cutaneous signs of ageing.

The mechanical properties of the skin, such as elasticity, suppleness and strength are supported by an underlying network of cells containing collagen fibres and elastin tissue permeated with hyaluronic acid. This scaffolding of cells can become damaged and flattened by degradation giving rise to the loss of the skin's contractile properties so that the skin becomes thinner in thickness and looser, resulting in wrinkling, fine and deep wrinkling, static wrinkling and furrowing of the skin and skin roughness. The skin also sags, droops and suffers volume loss. Ultimately, the loss of skin structure and volume prevents the skin from being able to contract back to its initial position following expression.

Surprisingly, the Applicant has identified a unique combination of compounds made up of collagen, an optimal blend of co-operable ROS scavengers/antioxidants and hyaluronic acid at doses which interact to provide the body with the precursors required for the formation of new collagen and the replenishment of defective collagen at extracellular level giving the desired anti-ageing outcome.

It should be noted that although antioxidants used in the present invention may already be known as effective quenchers of free radicals to prevent skin damage, the compositions of the invention have a greater efficacy than previously known products whilst also exhibiting surprising improved collagen production, reduced wrinkles and improved ATP production . In short, the compositions of the invention boost the formation and repair of collagen both inside and outside the cells to tackle the signs of ageing resulting from weakened skin structure due to oxidative stress of the cells, excessive unprotected sun exposure and ultimately an age-related diminishing supply of collagen in the subcutaneous layer of the dermis of the skin. Other demonstrable benefits include, inter alia, reduced oxidative damage by free radicals by destroying free radical cells, stimulation of new skin cell generation at the basal level, increased hydration levels with use and defence of collagen and elastin fibres against harmful UV rays.

The anti-ageing benefits of the compositions of the invention, particularly when taken as a nutritional supplement, can be summarised as follows:

The appearance of fine lines, expression lines and wrinkles is corrected; Improved skin firmness;

Immediate skin hydration;

Improved skin texture for a brighter, more radiant complexion; - Skin cell renewal, cell turnover and collagen formation optimised;

- Anti-oxidant protection against damage from sun exposure, pollutants and oxidative stress by free radicals;

- Visible signs of ageing minimised.

Detailed Description of the Invention

Collagen

As indicated above, degradation of collagen leads to wrinkles that accompany ageing. The marine collagen containing compositions of the present invention have the following beneficial effects on collagen in the skin:

- stimulates the synthesis of new collagen and elastin fibres to improve skin density and structure;

- increases cell proliferation;

- increases fibroblast activity in skin cells (fibroblasts are responsible for making new collagen);

- improves epidermal turnover and increases keratinocyte proliferation;

- daily supplementation significantly increases the amount of collagen in the basement membrane; - prolongs the life of the skin cells by slowing down the natural ageing process; - improves the formation of basement membrane components which are essential for skin health;

- reduces the appearance of fine lines and wrinkles; - improves skin quality and moisture balance;

- makes skin on the face and body feel softer and more supple.

Marine collagen is the preferred form of collagen employed in the compositions of the present invention in order to achieve the above benefits. Marine collagen exhibits reduced allergenicity and exhibits superior bioactivity and the high in-vivo absorption rate believed to be required to achieve the aforementioned benefits.

A form of collagen suitable for use in the formulations of the invention is Naticol 4000 (Trade Mark) available from Weishardt which is made up of 93-95% protein principally in the form of collagen peptides, approximately 6% water and 2% mineral salts. The formulations of the invention preferably contain from about 20 weight % collagen to about 40 weight % collagen and more preferably from about 25 weight % collagen to about 35 weight % collagen. ROS Scavengers/Antioxidants

The compositions of the present invention also include highly efficacious ROS scavengers and antioxidants. It has been found that a combination of ROS scavengers/antioxidants in accordance with the invention is more effective than a single antioxidant and, although the Applicant does not wish to be bound by any theorem, it is believed that the combination of ROS scavengers/antioxidants employed in the anti-ageing compositions exhibit a synergistic effect at least at the levels employed. The following types of ROS scavengers/antioxidants are suitable for use in achieving the preventative and/or curative benefits with the anti-ageing compositions: Vitamin C (which as outlined below also assists in collagen synthesis); disulfides;

tocopherols/tocotrienols; carotenoids; Coenzyme Q₁₀. An especially preferred combination of antioxidants found to be particularly efficacious in the anti-ageing compositions is a combination of Vitamin C, a-lipoic acid (a disulfide), Vitamin E (a tocopherol), lycopene (a carotenoid), coenzyme Q ₀ and astaxanthin (a carotenoid). Vitamin C

Vitamin C is a monosaccharide oxidation-reduction (redox) catalyst found in both animals and plants.

The destruction of collagen is largely due to exposure to sunlight and Vitamin C deficiency. However, Vitamin C is one of the vitamins humans are most deficient in due to depleted soils and the fact the body utilises vitamin C when fighting the free radicals associated with pollution.

The vitamin C in the composition of the invention serves as a potent and powerful antioxidant which scavenges free radicals. In addition to its direct antioxidant effects, the Vitamin C in the composition of the invention functions in the conversion of the collagen precursor, procollagen, into collagen. Moreover, studies have confirmed that Vitamin C has a critical role in collagen synthesis. More particularly, prolonged exposure of cultures of human connective-tissue cells to ascorbate induced an eight-fold increase in the synthesis of collagen with no increase in the rate of synthesis of other proteins (See Regulation of Collagen Synthesis by

Ascorbic Acid (prolyl hydroxylation/lysyl hydroxylation/human skin fibroblasts) S. MURAD, D. GROVE, K. A. LINDBERG, G. REYNOLDS, A. SIVARAJAH, AND S. R. PINNELL, Division of Dermatology, Department of Medicine, Duke University Medical Center, Durham, North Carolina February 9, 1981). In addition, the hydroxylation of amino acids to form procollagen is dependent on Vitamin C as a cofactor. The Vitamin C is therefore vital in the formation of new collagen and is a co-factor for collagen synthesis.

In short, the Vitamin C in the compositions of the invention is crucially important for the preservation of skin cells by preserving collagen integrity and elasticity and destroying free radicals. Other benefits include protection against cancer,

acceleration of wound healing, promotion of immunity against disease and potent anti-toxin action. The preferred form of Vitamin C in the compositions of the invention is ascorbic acid when the compositions are used in tablet or capsule form as this form of Vitamin C is known to produce an eightfold increase in collagen synthesis. Moreover, the ascorbic acid also acts as a natural preservative of the other constituents within the tablets or capsules protecting the formulations from oxidation and preventing rancidity.

The preferred dosage of Vitamin C in the compositions of the invention is about 250mg per day. The formulations of the invention typically comprise from about 10 weight % to about 25 weight % Vitamin C, preferably from about 15 weight % to about 20 weight % Vitamin C.

Vitamin C suitable for use in the formulations of the invention is available from

Vitanutrition (Trade Mark). g-lipoic acid a-lipoic acid is the preferred disulfide antioxidant employed in the compositions of the invention, a-lipoic acid is highly effective at neutralizing free radicals. Moreover, a- lipoic acid functions in water and fat and can recycle other spent antioxidants such as vitamin C and glutathione - an antioxidant synthesised in the human body that also helps the body eliminate potentially harmful substances.

Accordingly, the α-lipoic acid in the present formulations increases the formation of glutathione, improves improve blood flow and enhances immune function by restoring the levels of glutathione.

Although the Applicant does not wish to be bound by any theorem, it is believed that the α-lipoic acid assists in restoring cellular signalling processes that would otherwise decay in older blood vessels and also reduces mitochondrial decay in cells which is also closely linked to the symptoms of ageing.

As α-lipoic acid can cross the blood-brain barrier, it is also thought to protect brain and nerve tissue by preventing free radical damage.

Although α-lipoic acid is made by the body and can be found in very small amounts in foods in the diet, the α-lipoic acid containing compositions of the invention provide the body with the micronutrient in sufficient quantity to achieve anti-ageing benefits of the invention. The Applicant has found that a daily dose of about 200mg in the compositions of the invention achieves the beneficial anti-ageing effects.

The formulations of the invention typically comprise from about 5 weight % to about 20 weight % a-lipoic acid, preferably from about 10 weight % to about 15 weight % a-lipoic acid. a-lipoic acid suitable for use in the formulations of the invention is available from Vitanutrition (Trade Mark).

Vitamin E (d-a-tocopherol)

Vitamin E is the collective name for a set of eight related tocopherols and

tocotrienols which are fat-soluble vitamins with antioxidant properties. In the present invention, d-a-tocopherol, the natural form of Vitamin E is the preferred form as it has been found to exhibit the highest bioavailability while the human body preferentially absorbs and metabolises this natural form of Vitamin E over the synthetic form.

In the compositions of the invention, Vitamin E serves as a powerful antioxidant which prevents free radicals from turning into LDL (bad cholesterol) and assists in lowering blood cholesterol by helping to metabolise polyunsaturated fats.

The vitamin also protects fats in the body and the diet from destruction and rancidity by preventing free radicals taken into the body from reacting with the fats which can also result in cell damage. Vitamin E can also protect against lung damage caused by air pollution, tissue damage from radiation exposure, tumour growth and the destruction of Vitamin A which all contribute to the anti-ageing properties of the compositions of the invention. In short, deficiency of Vitamin E has been associated with premature ageing and the compositions of the invention therefore prevent premature ageing associated with Vitamin E deficiency. Moreover, the preferred natural Vitamin E employed in the invention improves blood circulation to improve skin health, complexion and cell turnover.

The compositions of the invention provide a preferred daily dosage of 40mg Vitamin E.

The formulations of the invention typically comprise from about 0.5 weight % to about 3 weight % Vitamin E, preferably from about 1.0 weight % to about 2.5 weight % Vitamin E.

A form of Vitamin E suitable for use in the formulations of the invention is d-a- tocopheryl succinate available as Covitol (Trade Mark) from BASF.

Lycopene

Lycopene, a phytochemical, is a bright red carotenoid pigment found in tomatoes and some other red fruits. Lycopene is a potent carotenoid antioxidant broken down by the body often during the process of absorption into the bloodstream from the small intestine from which it makes its way to specific tissues and organs to protect against the type of oxygen damage that can harm DNA.

In the compositions of the invention, lycopene improves skin health by protecting the skin cells from damage from free radicals. Accordingly, the formulations of the invention are particularly beneficial to smokers and to those exposed to

environmental pollutants. Accordingly, the lycopene carotenoid provides the body with anti-ageing skin benefits including stronger cell junctions (improved cell metabolism and cell communication so that skin cells operate more efficiently, facilitating better repair and regeneration), ultraviolet light blocking and reduced facial redness.

Accordingly, due to its impact at the cellular level, use of lycopene in the formulations of the invention greatly improves the structure and health of collagen in skin cells and indeed throughout the body.

As with the other components of the invention, the Applicant has identified an ideal daily dosage of lycopene, in combination with the other components, to achieve desirable anti-ageing benefits. The desired daily dosage of lycopene is 1.5mg.

The formulations of the invention typically comprise from about 0.5 weight % to about 2 weight % lycopene, preferably from about 0.75 weight % to about 1.5 weight % lycopene. A form of lycopene suitable for use in the formulations of the invention is Lycored (Trade Mark) which contains 10% lycopene.

Another carotenoid anti-oxidant suitable for use in the formulations of the invention β-carotene. β-carotene is a pro-vitamin A carotenoid while an advantage of dietary beta-carotene is that the body only converts as much as is required to Vitamin A (retinol). β-carotene can be used alone or in combination with lycopene.

The formulations of the invention typically comprise from about 0.1 weight % to about 2 weight % β-carotene, preferably about 2 weight % β-carotene.

A suitable daily dosage of β-carotene is about 30mg. Astaxanthin

Astaxanthin is also a carotenoid antioxidant. In the present invention, astaxanthin is naturally sourced from the Haematococcus Pluvialis microalgae.

Due to astaxanthin's potent antioxidant activity, the Applicant believes it to be beneficial in cardiovascular, immune, inflammatory and neurodegenerative diseases. It has also been demonstrated that it may protect body tissues from oxidative and ultraviolet damage through its suppression of NF-κΒ activation. Astaxanthin is also an efficient absorber of specific ultraviolet sunlight rays that contribute to skin ageing and cancer. Accordingly, in the present invention, by penetrating the skin cells and protecting each dermal layer from free radical related damage, astaxanthin reduces moisture loss, promotes smoothness and elicits cellular renewal.

Astaxanthin has additional health benefits in the compositions of the invention. For example, it alleviates medical conditions by enhancing the immune response and decreasing DNA damage. Astaxanthin's free radical-scavenging activity also protects lipids from peroxidation and reduces oxidative damage of LDL-cholesterol to reduce arterial plaque formation. Like a-lipoic acid, astaxanthin can cross the blood- brain barrier in mammals and extend its powerful antioxidant protection to the central nervous system which is highly susceptible to oxidative damage from free radicals because of its richness in unsaturated fatty acids.

A clinical research study by Dr. Debasis Bagchi at Creighton University has demonstrated the potency of astaxanthin which was shown to eliminate free radicals 6,000 times more potently than Vitamin C, 800 times more than coenzyme Qi₀, 550 times more than Vitamin E and green tea, 75 times more than a-lipoic acid and 20 times more than β-carotene.

Accordingly, in the present invention, a dosage of 2mg per day is provided in order to achieve the aforementioned anti-ageing benefits. The formulations of the invention typically comprise from about 0.0003 weight % to about 2 weight % astaxanthin, preferably from about 0.01 weight % to about 2 weight % astaxanthin, and more preferably from about 0.2 weight % to about 1.0 weight % astaxanthin. Most preferably, the formulation comprises about 0.3% astaxanthin.

A suitable form of astaxanthin is available from Cambridge Commodities Ltd. Coenzyme Q-m Coenzyme Qi ₀ is an oil-soluble, naturally-occurring vitamin-like substance found in every cell in the body. Coenzyme Qio plays a key role in producing energy in the form of ATP in the mitochondria. Accordingly, coenzyme Qio performs a critical anti- ageing role in the formulations of the invention as the functional loss of mitochondria represents an inherent part in the cutaneous ageing process.

Coenzyme Q₁₀ positively influences the age affected cellular metabolism and combats the signs of ageing at the cellular level. As a consequence, the oral supplementation or topical application of coenzyme Qio in the formulations of the invention is beneficial for human skin due to the improved mitochondrial function.

The antioxidant nature of coenzyme Qi o derives from the aforementioned energy carrier function whereby free radicals are quenched to prevent propagation of lipid peroxidation. Indeed, the antioxidant role of the molecule as an antioxidant has been widely studied (see Pro- and Antioxidant Functions of Quinones and Quinone Reductases in Mammalian Cells, F. F. Nord, Enrique Cadenas, Paul Hochstein, Lars Emster, 22 NOV 2006, Advances in Enzymology and Related Areas of Molecular Biology, Volume 65). Moreover, the antioxidant activity not only protects lipids, but also collagen and elastin from oxidative breakdown to prevent skin ageing. The coenzyme Qio of the formulations also protects against oxidative stress-induced cell death and enhances the synthesis of basement membrane components in dermal and epidermal cells whilst it has also been proven to help maintain a healthy cardiovascular system and assist in the treatment of migraine. The Applicant has identified that an optimal dosage in the formulations of the invention in which coenzyme Qio is combined with other antioxidants is 10mg per day.

The formulations of the invention typically comprise from about 0.003 weight % coenzyme Q₁₀ to about 2 weight % coenzyme Q₁₀, preferably from about 0.2 weight % to about 2 weight % coenzyme Q₁₀, and more preferably from about 0.75 weight % to about 1.5 weight % coenzyme Qio.

Suitable forms of coenzyme Q-io are available from Cambridge Commodities Limited.

Hyaluronic acid

The anti-ageing formulations of the invention also contain at least one source of giycosamine. In a preferred embodiment of the invention, hyaluronic acid has been found to be a highly efficacious giycosamine. Hyaluronic acid or hyaluronan is a polysaccharide distributed widely throughout connective, epithelial, and neural tissues. A gel like substance, it is one of the chief components of the extracellular matrix along with collagen and elastin - the average 70 kg (154 lbs.) person has roughly 15 grams of hyaluronic acid in their body, one- third of which is turned over (degraded and synthesized) daily.

Hyaluronic acid contributes significantly to cell proliferation and migration and is a major component of the synovial fluid. Hyaluronic acid increases the viscosity of the synovial fluid and together with lubricin is one of the fluid's main lubricating components.

Like collagen, hyaluronic acid is also a major component of skin where it is involved in tissue repair. However, when skin is exposed to excessive UVB rays, it becomes inflamed (sunburn) and the production of hyaluronic acid by the cells of the dermis is halted so that the rate of degradation of hyaluronic acid is increased and the degradation products accumulate in the skin.

While it is abundant in extracellular matrices, hyaluronic acid also contributes to tissue hydrodynamics, movement and proliferation of cells, and participates in a number of cell surface receptor interactions, notably those including its primary receptors, CD44 and RHAMM. High concentrations of hyaluronic acid in the brains of young rats, and reduced concentrations in the brains of adult rats suggest hyaluronic acid plays an important role in brain development. Skin wound healing is a complex process and includes many interacting processes initiated by haemostasis and the release of platelet-derived factors. The following stages are inflammation, granulation tissue formation, re-epithelisation and remodelling. Hyaluronic acid plays a multifaceted role in mediation of these cellular and matrix events. Accordingly, in the compositions of the present invention, hyaluronic acid is used as a highly effective antioxidant and stimulating agent for collagen synthesis and cell proliferation and cytotaxis and to fight the ageing process. The Applicant has identified that a daily dosage of about 100mg in combination the aforementioned components provides the desirable anti-ageing benefits.

The formulations of the invention typically comprise from about 3.0 weight % to about 10 weight % hyaluronic acid, preferably from about 5 weight % to about 8 weight % hyaluronic acid

A form of hyaluronic acid suitable for use in the formulations of the invention is available from Cambridge Commodities Ltd. Powder forms of hyaluronic acid are preferred.

A naturally occurring polysaccharide which may be suitable for use in the formulations of the invention in place of hyaluronic acid is an extract of Tamarindus Indicia commercially available as Xilogel (Trade Mark) from Indena. Collagen pre-cursors

In one embodiment of the invention, the compositions are augmented with collagen pre-cursors which encourage fibroblast production. More particularly, additional collagen pre-cursors assist in further boosting endogenous collagen production in vivo whilst employing relatively low levels of collagen in the compositions.

Accordingly, unlike known collagen containing products which often contain extremely high doses of collagen, up to 10,000mg the bulk of which is simply enzymatically degraded in the gut, due to the combination of collagen, ROS scavengers/anti-oxidants and collagen pre-cursors, the compositions of the present embodiment are particularly efficacious at achieving anti-ageing benefits.

Suitable collagen pre-cursors are those amino acids required for the endogenous production of collagen.

Preferred amino acid pre-cursors which are formed into collagen proteins include L- proline and glycine which can be used alone but preferably in combination as glycine and proline are the major amino acids require for collagen production. As a result of the presence of proline and glycine, a three dimensional stranded structure is assembled in vivo, with glycine and proline as its principal components. This is not yet collagen but its precursor, procollagen.

As indicated above, the vitamin C present in the compositions plays crucial role in the synthesis of collagen and is considered a "Co-Factor". The primary sequence of collagen (its amino acid sequence) is largely composed of triplet repeat sequences in which the first residue is usually the small hydrophobic residue glycine, the second is usually proline, and the third is usually hydroxyproline which is synthesised from the proline. More particularly, proline is hydroxylated to hydroxyproline in an enzymatic reaction that occurs after the collagen polypeptide is synthesized.

The pre-cursors are incorporated into the compositions at therapeutically effective levels.

The formulations of the invention typically comprise from about 0.1 weight % to about 2 weight % L-proline, preferably from about 0.2 weight % to about 1 weight % L-proline and most preferably about 0.6 weight percent L-proline and from about 0.05% weight % to about 1 weight % glycine, preferably from about 0.1 weight % to about 0.5 weight % glycine and most preferably about 0.2 weight percent glycine

The Applicant has identified that a daily dosage of about 70mg of collagen precursors is particularly beneficial. For example, a combination of a daily dosage of 50mg L-proline and 20mg glycine in combination with the aforementioned

components is particularly efficacious at producing the desirable anti-ageing benefits. Resveratrol

Resveratrol can also be included if desired in the compositions of the invention. Resveratrol is a type of natural plant phenol and is classed as a polyphenolic compound. Resveratrol is found in the skin of red grapes and from the roots of the Japanese Knotweed. As a compound it can assist in further helping slow down the ageing process of the cell. Resveratrol also acts as an antioxidant, and also as an anti-inflammatory while it can also lower fat levels within the liver and is responsible for raising HDL "good" cholesterol.

L-carnitine

The compositions can also optionally include L-carnitine. L-Carnitine is a nonprotein amino acid that is found is the heart and skeletal muscle and helps carry fatty acids across the mitochondria of the cell, thus providing heart and skeletal cells with energy.

A decline in mitochondrial function is thought to contribute to the ageing process. A daily dosage of about 10Omg can be incorporated into the compositions of the invention.

Although the composition of the invention could be applied topically, it is preferred that the composition be employed in the form of an edible product for maximum benefit and the composition has been formulated to provide optimal anti-ageing benefits when consumed and metabolised.

The edible composition can take any suitable form including food products and nutritional supplements or nutraceuticals. Suitable nutritional supplement forms include capsules, pills, tablets and powders while other edible forms include bars, beverages, confectionery and cereals.

Where the compositions of the invention are in the form of nutritional supplements such as pills, capsules and tablets, the active ingredients described above can be combined with excipients such as, inter alia, bulking agents, fillers, disintegrants, preservatives, sweeteners etc known in the art as required.

Examples

The invention will now be described further having regard to the following non- limiting Examples and drawings in which:

Figure 1 is a graph illustrating the results of the human dermal fibroblast cell proliferation assay of Example 3 demonstrating that formulations of the invention result in increased skin cell production;

Figure 2 is two micrographs showing the increased level of HDFa of Example 3 cells following treatment with the anti-ageing formulation of the invention compared with the Control; Figure 3 is a graph showing the reduced levels of intracellular H₂0₂ achieved as outlined in Example 4 employing the formulation of the invention compared with the Control; Figure 4 is a graph showing increased levels of ATP production achieved as outlined in Example 5 with the compositions of the invention compared with a Control;

Figure 5 is a graph showing the increased levels of Collagen I production achieved as outlined in Example 6 with the compositions of the invention compared with the Control;

Figure 6 is two micrographs showing the improved protective effect against UVB exposure as outlined in Example 7 achieved with formulations of the invention compared with the Control;

Figure 7 is a graph showing the improved cell viability achieved with cells treated with compositions of the invention as outlined in Example 8 compared with the Control;

Figure 8 is a graph showing the improved benefits achieved by pre-treatment with the anti-ageing compositions of the invention when compared with the Control as outlined in Example 9 prior to UVB exposure on intracellular H₂0_2l and Figure 9 is a graph showing improved ATP production on pre-treatment of cells with the anti-aging composition as outlined in Example 10 prior to UVB exposure compared with a Control. The materials listed in Table 1 below were employed in the following Examples:

Table 1

Materials

Reagent Supplier

Agarose Invitrogen

Antibiotic-Antimycotic (100X) Gibco

Beta Actin Real Time PCR Primer mix Invitrogen

Co-Enzyme Q10 Sigma-Aldrich

Collagen Biomarine boosting complex Martin BioTech

Chloroform Sigma

Col1A1 Real Time PCR Primer Mix Invitrogen

ColA2 Real Time PCR Primer Mix Invitrogen

ColA4 Real Time PCR Primer Mix Invitrogen

DMEM (low glucose) GIBCO

DMSO Sigma

DNase Reaction Buffer Invitrogen dNTP mix Invitrogen

DTT (0.1M) Invitrogen

Elastin Real Time PCR Primer Mix Invitrogen

Eppendorf tubes Greiner Bio-One Ethanol BDH

F12 Medium Gibco

First Strand Buffer (5X) Invitrogen

Foetal Calf Serum (FCS) Gibco

Formaldehyde BDH

Glycine Sigma-Aldrich

Hyaluronic Acid (HA)

Hydrochloric acid BDH

Hydrocortisone Sigma-Aldrich

Hydrogen Peroxide BDH

ITS Sigma-Aldrich

Isopropanol Sigma

Low Serum Growth Supplement (LSGS) Gibco

Medium 106 Gibco

Methanol BDH

Molecular weight marker Invitrogen

MTT

NaOH Sigma-Aldrich

Oligo-dT Primers Invitrogen

Reverse Transcription PCR Reagents Invitrogen

RNAse OUT Invitrogen

Superscript II Invitrogen

Triton X- 100 Boehringer

Trizol Reagent Gibco Trypan Blue Solution Invitrogen

Trypsin/EDTA (1X) Gibco

Vitamin E Sigma

Example 1

An oral composition in the form of a capsule was prepared as follows Table 2

Anti-ageing Capsule Composition Ingredients

Per Daily Description

Capsule Dose 2 x

Capsules

Quantity

250mg 500mg Marine Collagen Complex

125mg 250mg Ascorbic Acid (Vitamin C), white

crystalline powder

100mg 200mg Alpha Lipoic Acid, yellow

crystalline powder

50mg 100mg Hyaluronic Acid Powder

20mg 40mg dl-alpha-Tocopherol Acetate

(50%) Powder [Vitamin E]

0.75mg 1.5mg Lycopene [from Solarium

Lycopersicum] Powder (10%

Lycopene)

5.00mg 10mg Coenzyme Qi₀ [Ubiquinone,

Ubidecarenone], crystalline

powder

lOOmg 2mg Astaxanthin [Haematococcus

Pluvialis] P.E. (5% Astaxanthin)

Per Unit: Description

Quantity

5.50mg Dicalcium Phosphate Dihydrate,

fine powder

mg Magnesium Stearate Powder

(vegetable origin)

mg Amorphous Fumed Silica Powder

Per Unit- Description Quantity

1 Capsules Size 00 - gelatine (clear)

Note: (all weights +/- 10%); Capsule weight (+/- 5%) 730mg total weight including capsule 850mg

The ingredients of the formulation were mixed, blended and encapsulated using conventional techniques, equipment and methods known to those skilled in the art to produce compositions of the invention having the benefits outlined above.

Accordingly, the 850mg total weight capsule of the present Example has the following weight % make-up:

Collagen 29.4%

Hyaluronic acid 5.8%

a-lipoic acid .7%

Lycopene (10% material) 0.8%

Astaxanthin (1.5% material) 0.3%

Coenzyme Qi₀ 0.5%

Vitamin C 14.8%

Vitamin E 1.5%

Example 2

A second Example of the formulation of the invention was made up of the ingredients as outlined in Table 3 below. Table 3

Anti-ageing Capsule Composition Ingredients

*The marine collagen, L-proline and glycine can be blended in a single complex *^*Can be substituted with succinate form. Vitamin E is employed in its natural form **^*lncluded as 50mg Resveratrol containing 20% trans-Resveratrol Example 3: Fibroblast/Production

The positive impact of the formulation of Example 1 on dermal fibroplasts (the main cells responsible for manufacturing collagen and other proteins in the skin) in vitro was demonstrated as follows:

Primary human dermal fibroblasts were grown under standard conditions in vitro.

HDFa are human dermal fibroblasts isolated from normal human adult dermis.

HDFa cells are a well-established system for in vitro analysis of fibroblast growth, migration and collagen metabolism in wound healing. Accordingly, the cells were ideal for assessing the efficacy of the formulations of the invention.

The cells were cultured as follows:

Initiating cultures from cryopreserved HDFa cells

The HDFa vial was removed from liquid nitrogen storage and thawed in a 37°C water bath. A cell count of viable cells/ml was determined with 10μΙ of trypan blue and pipetting 10μΙ of a cell suspension/ trypan blue mix (1 :1 ) onto a Countess Chamber slide. The cell suspension was diluted to 2.5^*10⁴ viable cells/ml in supplemented medium 106 supplemented with a LSGS vial and 5ml antibiotic-antimycotic. 5ml of cell suspension was added to each 25 cm² culture flask. HDFa cells were incubated at 37°C in an aseptic, humidified 95% air/ 5% C0₂ atmosphere.

Cell Maintenance

Following establishment of the initial culture, spent media was aspirated off and replaced with 10ml fresh supplemented Medium 106 72hours (hrs) later. Flasks were returned to the incubators. For subsequent subcultures, medium was changed every 48hrs.

Subculturing HDFa Cells

At 80-90% confluency, the medium was aspirated off cells and replaced with 3mls of 1X trypsin, gently washed over the cells to ensure full coverage before immediately aspirating off the trypsin. Cells were kept at room temperature (RT) for 1-2 minutes (mins) with gently agitation to detach cells. Once cells have begun to detach 3ml of supplemented Medium 106 was added to the flask to neutralise the trypsin. After gently pipetting up and down, the suspension was removed to a 50ml tube. Another 3mls of medium was added to the flask, swirled to collect any remaining cells and removed to the tube. 7ml of fresh Medium 106 was added to a T75 flask and 3ml of cell suspension was added to the flask before returning flasks to the incubators. MTT Assay

The MTT assay was performed as follows. HDFa cells were grown on 6 well plates and treated with the anti-ageing supplement composition for 48hrs. MTT stock solution (5mg/ml in PBS) was diluted in medium to a 10% solution. After washing twice with PBS, 1ml MTT was added to each well and the plate was incubated for 3hrs at 37°C. The dye was then aspirated from the wells, replaced with 500μΙ of DMSO and pipetted to dissolve the formazan crystals. The absorbance readings were taken at 590nm.

Cell Treatments

Stock solutions of the formulation of Example 1 were prepared and, where

necessary, dilutions were performed using cell culture media to achieve the required concentrations. Cells were seeded in plates at 1^*10⁶cells/well. Phase contrast microscopy was used to determine the confluency of cells. At 80% confluency cells cell were serum-starved for 24hrs and then treated with 3mls of either fresh serum- free medium or 3mls of treatment solutions. TGF-β was included as a positive control as TGF-β induces the formation of collagen.

Results: After seeding, HDFa cells were cultured for 4 days prior to treatment with the formulation of the invention or with 5ng/ml TGF-β for 48hrs. Control cells displayed characteristics typical of fibroblast cells; cells were elongated spindle- shaped cells, oriented in a parallel array. As shown in Figure 1 , the formulation of the invention complex significantly increased the number of skin cells produced when treated. In particular, the rate at which dermal fibroblasts divided leading to increased numbers of fibroblasts was significantly increased. Increased fibroblast levels leads to increased collagen production in the human skin. In addition, as shown in Figure 2, visually in a phase contrast micrograph taken with a CCD camera and a Nikon microscope at a magnification of x100, the formulation of the invention resulted in an increase in fibroblast numbers compared with the control.

Example 4: Determination of Intracellular H?02

Cells were first prepared as outlined in Example 3 above. Preparation of cytosolic extracts The cells were washed twice with ice-cold PBS. 200μΙ of ice-cold lysis buffer (0.1% Triton X-100 in 0.05M sodium phosphate buffer, pH7.4) was added to the cells.

Cells were gently detached using a cell scraper and cell lysates were transferred to sterile pre-chilled Eppendorf tubes. Tubes were placed on ice with vortexing every 3mins for 15mins to facilitate complete cell lysis. Homogenates were centrifuged at 14,000g at 4°C for 15mins. Supernatants were carefully aspirated and transferred to sterile pre-chilled Eppendorf tubes. Samples were analyzed immediately for hydrogen peroxide content as outlined below. BCA Assay

The BCA working solution was prepared by mixing 10ml of reagent A with 200μΙ of reagent B. Using a 96 well plate 200μΙ of the working solution was added to each well containing 20μΙ of protein sample or standard, mixed and incubated in darkness at 37°C for 30mins. Absorbencies were read at 540nm using Wallac Victor plate reader and protein concentrations were calculated from the BSA standard curve.

Determination of intracellular H2O2 by Amplex Red Assay

50μΙ supernatants or standards (0-1.25μΜ H₂O₂) were placed in 96-well opaque black microplate. The reaction was initiated by adding 50μΙ of Amplex Red reaction mixture (0.1 mM Amplex red+0.2U/ml horseradish perodixase (HRP) in 0.05M sodium phosphate buffer, pH 7.4). After 10 seconds initial mixing, fluorescence was measured kinetically every 30 seconds for 10mins, in a scanning microplate reader equipped with a kinetic trace using excitation and emission wavelengths of 530 and 590nm, respectively. The H2O2 content was calculated against a standard curve prepared from H₂0₂ solutions of known concentrations and normalized with the protein content of supernatants.

Results: As shown in Figure 3, the formulation of the invention demonstrated a significant decrease in H2O2 production immediately following the addition of the Amplex Red reagent and this trend continued throughout the assay. The formulation of the invention therefore significantly reduced the intracellular H₂0₂ levels. Example 5: ATP Synthesis

Preparation of cells for ATP Assay Cells as prepared in Example 3 were washed twice with ice-cold PBS before 400μΙ 1X trypsin-EDTA solution was added to cells, swirled to ensure complete coverage and aspirated off before incubation at RT to facilitate cell detachment. Once detached, 600μΙ supplemented Medium 106 was added to neutralize the trypsin. The cell suspension was transferred into sterile Eppendorf tubes and centrifuged at 800g for 3mins at 4°C. The supernatant was carefully discarded. Cell pellets were re-suspended with 1ml pre-warmed PBS to remove residual culture medium. The cell suspension was centrifuged at 800g for 3mins at 4°C. The supernatant was discarded and cell pellet was shock-fcozen in liquid nitrogen and stored at -80°C until analysis.

Determination of ATP content by luciferase-luciferin assay

The pellet was re-suspended in 100μΙ ice cold PBS. 50μΙ of a 25-fold dilution of cell suspensions was transferred in triplicates into a black microtiter plate. A 50-fold dilution of cell suspension was used for protein determination by BCA assay.

Intracellular ATP content was determined using the ATP Bioluminescence Assay Kit HSII (Roche) according manufacturers' instructions. ATP content was normalized for protein concentration of the cell suspension. Results: ATP production was measured in cell lysates obtained from the HDFa cells treated with the formulation of the invention. As shown i Figure 4, a statistically significant increase in ATP synthesis was detected indicating an increased mitochondrial activity, resulting in increased ATP levels.

Example 6: Collagen I Production RNA Preparation And Quantification Cells were prepared as outlined in Example 3. After treatment for 48hrs, the medium was aspirated off and cells were washed with non-sterile PBS. 1 ml Trizol was added to each well and cells were scraped from the plate surface, transferred to Eppendorf tubes and incubated at RT for 5mins. 20ΌμΙ chloroform was added to the tubes, vigorously vortexed for 20 seconds and incubated at RT for 2-3mins. Eppendorfs were centrifuged at 12000g for 15mins at 4°C. The aqueous phase was transferred to fresh Eppendorfs. 500μΙ isopropanol was added to the tubes, inverted and incubated at RT for 10mins and centrifuged at 12000g for 10mins at 4°C. The supernatants were removed and RNA pellets were washed with 1 ml 75% ethanol (EtOH). Tubes were vortexed briefly and centrifuged at 7500g for 5mins at 4°C. . EtOH was removed and RNA pellets were air-dried for 5-10mins. RNA pellet was re- suspended in 30μΙ RNase-free water and incubated at 60°C for 10-15mins. Samples were briefly centrifuged and stored at -80°C.

The nanodrop was employed to quantify the RNA yield. 1 μΙ RNA sample was added to 4μΙ RNase-free water, vortexed and centrifuged briefly. 2μΙ of sample was placed on the arm of the nanodrop and measured at 260nm and 280nm. RNA stocks were prepared at SOOng/μΙ in a 20μΙ volume.

Real Time Polymerase Chain Reaction (RT-PCR)

- cDNA Synthesis

Master Mix I was prepared (Table 4) and 2μΙ of the mix was added to tubes containing RNA before adjusting to a final volume of 12μΙ with RNase-free water.

Table 4

Master Mix I Preparation

Tubes were incubated at 65°C for 5mins in a thermal cycler, quickly chilled on ice and briefly centrifuged. Master Mix II (Table 5) was prepared and 7μΙ was added to each tube.

Table 5

Master Mix II Preparation

Tubes were placed in a thermal cycler at 42°C for 2mins before 1 μΙ Superscript II RT was added. The thermal cycler program was started (Table 6). Samples were stored at -20°C.

Table 6

Thermal Cycle Program for cDNA synthesis

- RT-PCR

1 μΙ cDNA sample was placed in labelled Eppendorfs on ice. The master mix was prepared (Table 7). The number of reactions was calculated as below:

[#samples + #standards + #non-template controls(ntc) + QC]n repeats = X reactions Table 7

Real-Time PCR Master Mix

If only one target gene was analysed a multiplex reaction was set up with the control. Otherwise all reactions were run separately to the control. Once the master mix was prepared and briefly vortexed, 19μΙ was added to Eppendorf tubes containing the cDNA. Eppendorfs were vortexed and pulse centrifuged before 9.5μΙ solution was added to wells on a 384 well plate in duplicate. The RT-PCR program was initiated selecting the absolute quantification setting, detectors were selected depending on the target probe and wells were designated unknown or ntc, indicating the correct probes. Once the program was successfully completed threshold levels and baseline were set and the data was exported for analysis.

Results: RT-PCR analysis of Collagen I levels was performed following treatment of HDFa cells with anti-aging supplement components. As shown in Figure 5, the formulation of the invention induced a 1.5 fold increase in expression.

Example 7: Analysis of the protective effect of pre-treatment with formulations on UVB exposure

HDFa cells were cultured for 4 days as previously described prior to treatment with the formulation of the invention and a control for 48hrs. For UV exposure, cells were placed under UVB light (254nm) for 1 hr. As shown in Figure 6 (a phase contrast micrograph taken with a CCD camera and a Nikon microscope at a magnification of x100), control cells displayed typical characteristics of fibroblast cells - cells were typically spindle shaped but there appeared to be less cells present and no cell-to- cell contact was visible as a result of UV damage.

However, increased cell numbers were visible and cells retained their typical fibroblast morphology when treated with the formulations of the invention. Example 8: Effect of pre-treatment with anti-ageing supplement components prior to exposure of HDFa cells to UVB radiation on cellular viability.

HDFa cells were cultured for 4 days as previously described prior to treatment with the formulation of the invention and a control for 48 hrs. Cellular viability was assessed by means of MTT assays. As shown in Figure 7, a significant increase in cellular viability was evident with the formulation of the invention.

Example 9: Effect of pre-treatment with anti-aging supplement components prior to UVB exposure on intracellular H?Q?

The effect of the anti-ageing supplement of the invention on intracellular H₂0₂ was investigated. As shown in Figure 8, the formulation resulted in significant decreases in intracellular H₂0₂ compared to control.

Example 10: Effect of pre-treatment with anti-aging supplement components prior to UVB exposure of HDFa cells on ATP synthesis.

Analysis of ATP synthesis following pre-treatment with the anti-ageing composition of the invention and subseguent exposure to UVB radiation as previously described indicated significant increases in the ATP levels for cells treated with the composition compared to the control as shown in Figure 9. Example 11 : Clinical Trial

A clinical trial was performed on a sample of 60 users provided with the formulation as described in Example 1. The users ingested a daily dose of two capsules over an extended period. The following results were observed by users under the headings below:

Skin Hydration

- 89% of end-users rated their skin to be greatly hydrated within one continuous month of use

Lines/Wrinkles (rhytides)

- 81 % of users reported a significant improvement in the appearance of Class I fine lines surrounding the peri-orbital area (eyes)

- 70% of users reported an improvement in Class II wrinkles in the area of the naso-labial folds

- 76% of end- users rated an improvement in the appearance of facial static lines Volume (facial pads)

- 90% of end users stated that there was a noticeable and favourable increase in the volume in the facial pads Other Observations

- 55% of end-users stated that they had not needed to take their regular pain- relieving medication for on-going joint pain - even though the user had taken the supplement for the beauty indication, the users also benefited for joint care.

Overall, the compositions of the invention therefore demonstrated increased cellular viability and an increase in fibroblast proliferation which, in vivo, slows the ageing process since fibroblasts are responsible for the production of collagen, one of the key components of healthy, youthful skin, forming the basis of the protein scaffold which endows structure and support to the skin.

RT-PCR analysis of collagen levels within the supplement treated cells also showed an increase in collagen levels. Significant antioxidant activity was demonstrated by the ability of the formulation of the invention to significantly decrease intracellular H₂O₂ levels. H₂0₂ is a ROS and a large body of evidence exists which indicates that damage to the cellular

constituents by ROS is a major driving force for the ageing process. The formulations of the invention also protected the mitochondria from UVB radiation, with treated fibroblasts demonstrating significantly decreased H₂O₂ levels compared to the control. Accordingly, the compositions of the invention are adapted to remove UVB induced ROS. Mitochondria are central components of our cells, generating the majority of the cells energy in the form of ATP from nutrients through a process of aerobic respiration which utilises the oxygen molecule. Unfortunately, through their normal activity, mitochondria generate unstable molecules known as ROS that harm both the mitochondrion itself and other cellular components. The resulting damage accumulates over time and is thought to play a significant role in aging. As the mitochondria are damaged by ROS, ATP synthesis decreases. Treatment of fibroblasts with the formulations of the invention resulted in increased ATP

production. Accordingly, the formulations of the invention are adapted to clearly protect the mitochondria from ROS and promote mitochondrial activity.

The in vivo clinical data reinforced the results achieved in vitro.

The invention is not limited to the embodiments herein described which may be varied in construction and detail without departing from the scope of the invention.

Claims

Claims

1. A composition for treating the cutaneous signs of ageing comprising:

collagen, and

a combination of Reactive Oxygen Species (ROS) scavengers/antioxidants selected from the group comprising Vitamin C, disulfides, Vitamin E, carotenoids and coenzyme Q₁₀.

2. A composition as claimed in Claim 1 wherein the combination of ROS scavengers/antioxidants comprises Vitamin E and coenzyme Q_10.

3. A composition as claimed in Claim 2 wherein the combination of ROS scavengers/antioxidants further comprises Vitamin C. 4. A composition as claimed in Claim 3 wherein the combination of ROS scavengers/antioxidants further comprises a disulfide.

5. A composition as claimed in Claim 4 wherein the disulfide comprises a-lipoic acid.

6. A composition as claimed in Claim 4 or Claim 5 wherein the combination of ROS scavengers/antioxidants further comprises a carotenoid.

7. A composition as claimed in Claim 6 wherein the carotenoid comprises lycopene.

8. A composition as claimed in Claim 6 wherein the carotenoid comprises astaxanthin.

9. A composition as claimed in any of Claims 1 to 8 wherein the composition further comprises a glycosamine source.

10. A composition as claimed in Claim 9 wherein the glycosamine source comprises hyaluronic acid. 1 1. A composition as claimed in Claim 9 wherein the glycosamine source comprises aTamarindus Indicia extract.

12. A composition as claimed in any of Claims 1 to 1 1 wherein the composition comprises a collagen pre-cursor. 3. A composition as claimed in Claim 12 wherein the collagen pre-cursor comprises L-proline and/or glycine.

14. A composition as claimed in any of Claims 1 to 13 wherein the composition comprises β-carotene.

15. A composition as claimed in any of Claims 1 to 14 wherein the composition comprises Resveratrol.

16. A composition as claimed in any of Claims 1 to 15 wherein the composition comprises L-carnitine.

17. A composition as claimed in any of Claims 1 to 11 wherein the collagen comprises marine collagen.

18. A composition as claimed in Claim 1 wherein the composition comprises: from about 20 weight % to about 40 weight % collagen;

from about 0.5 weight % to about 3.0 weight % Vitamin E;

from about 0.2 weight % to about 2.0 weight % coenzyme Qi₀;

from about 10 weight % to about 25 weight % Vitamin C;

from about 5.0 weight % to about 20 weight % a-lipoic acid;

from about 0.5 weight % to about 2.0 weight % lycopene, and

from about 0.0003 weight % to about 2.0 weight % astaxanthin. 9. A composition as claimed in Claim 8 wherein the composition comprises from about 25 weight % to about 35 weight % collagen.

20. A composition as claimed in Claim 18 or Claim 19 wherein the composition comprises from about 1.0 weight % to about 2.5 weight % Vitamin E.

21. A composition as claimed in any of Claims 18 to 20 wherein the composition comprises from about 0.003 weight % to about 2 weight % coenzyme Q₁₀.

22. A composition as claimed in Claim 21 wherein the composition comprises from about 0.2 weight % to about 2 weight % coenzyme Q-io-

23. A composition as claimed in Claim 22 wherein the composition comprises from about 0.75 weight % to about 1 .5 weight % coenzyme Qi o-

24. A composition as claimed in any of Claims 18 to 23 wherein the composition comprises from about 15 weight % to about 20 weight % Vitamin C. 25. A composition as claimed in any of Claims 18 to 24 wherein the composition comprises from about 10 weight % to about 15 weight % a-lipoic acid.

26. A composition as claimed in any of Claims 18 to 25 wherein the composition comprises from about 0.75 weight % to about 1 .5 weight % lycopene.

27. A composition as claimed in any of Claims 18 to 26 wherein the composition comprises from about 0.1 weight % to about 2.0 weight % astaxanthin.

28. A composition as claimed in Claim 27 wherein the composition comprises from about 0.2 weight % to about 1.0 weight % astaxanthin.

29. A composition as claimed in any of Claims 18 to 28 wherein the composition comprises from about 0.1 weight % to about 2.0 weight % β-carotene.

30. A composition as claimed in any of Claims 18 to 29 wherein the composition comprises from about 0.1 weight % to about 2.0 weight % β-carotene.

31. A composition as claimed in any of Claims 18 to 30 wherein the composition comprises from about 0.1 weight % to about 2.0 weight % L-proline.

32. A composition as claimed Claim 31 wherein the composition comprises from about 0.2 weight % to about 1.0 weight % L-proline. 33. A composition as claimed in any of Claims 18 to 32 wherein the composition comprises from about 0.05 weight % to about 1.0 weight % glycine.

34. A composition as claimed in Claim 33 wherein the composition comprises from about 0.1 weight % to about 0.5 weight % L-proline.

35. A composition as claimed in any of Claims 1 to 25 wherein the composition is an oral composition.

36. A composition as claimed in Claim 35 wherein the collagen and ROS scavengers/antioxidants are present in the composition at sufficient concentrations to deliver daily dosages sufficient to achieve an anti-ageing effect.

37. A composition as claimed in Claim 36 wherein the composition further comprises a collagen pre-cursor present at sufficient concentration to promote collagen production.

38. A composition as claimed in Claim 36 or Claim 37 wherein the daily dosage comprises about 500mg collagen, about 250mg Vitamin C, about 200mg a-lipoic acid, about 100mg hyaluronic acid, about 40mg Vitamin E, about 1.5mg lycopene, about 10mg coenzyme Qi₀ and about 2mg astaxanthin.

39. A composition as claimed in Claim 38 wherein the daily dosage comprises about 70mg collagen pre-cursors. 40. A composition as claimed in Claim 39 wherein the collagen pre-cursor comprises L-proline and/or glycine.

41. A composition as claimed in Claim 40 wherein the daily dosage of collagen pre-cursor comprises 50mg L-proline and 20mg glycine.

42. A cosmetic method for providing a cutaneous anti-ageing benefit comprising administering a composition as defined in any of Claims 1 to 32.

43. Use of a composition comprising collagen and a combination of Reactive Oxygen Species (ROS) scavengers/antioxidants selected from the group comprising Vitamin C, disulfides, Vitamin E, carotenoids and coenzyme Q₁₀ in the treatment of cutaneous ageing.

44. Use as claimed in Claim 43 wherein the combination of ROS

scavengers/antioxidants comprises Vitamin E and coenzyme Qi₀

45. Use as claimed in Claim 44 wherein the combination of ROS

scavengers/antioxidants further comprises Vitamin C. 46. Use as claimed in Claim 45 wherein the combination of ROS

scavengers/antioxidants further comprises a disulfide.

47. Use as claimed in Claim 46 wherein the disulfide comprises a-lipoic acid. 48. Use as claimed in Claim 46 or Claim 47 wherein the combination of ROS scavengers/antioxidants further comprises a carotenoid.

49. Use as claimed in Claim 48 wherein the carotenoid comprises lycopene. 50. Use as claimed in Claim 48 wherein the carotenoid comprises astaxanthin.

51. Use as claimed in Claim 48 wherein the carotenoid comprises β-carotene.

52. Use as claimed in any of Claims 43 to 51 wherein the composition further comprises a glycosamine source.

53. Use as claimed in Claim 52 wherein the glycosamine source comprises hyaluronic acid.

54. Use as claimed in Claim 53 wherein the glycosamine source comprises aTamarindus Indicia extract.

55. Use as claimed in any of Claims 43 to 54 wherein the composition comprises a collagen pre-cursor.

56. Use as claimed in Claim 55 wherein the collagen pre-cursor comprises L- proline and/or glycine. 57. Use as claimed in any of Claims 43 to 56 wherein the composition comprises Resveratrol.

58. Use as claimed in any of Claims 43 to 57 wherein the composition comprises L-carnitine.

59. Use as claimed in any of Claims 43 to 58 wherein the collagen comprises marine collagen.