COMBINATION OF AT LEAST TWO ACTIVE INGREDIENTS FOR COSMETIC USE

TECHNICAL FIELD

The present invention relates to a combination of at least two, preferably all, of three active ingredients, a cosmetic composition comprising the combination, and uses of the combination.

BACKGROUND ART

The mid 20' s is the first stage when people begin to notice the signs of aging. Typical signs of aging include fine lines on the skin, in particular on the face. Also, many women are concerned about uneven texture and tone of the skin.

The skin is continuously in contact with the environment, and therefore is more subject to environmental aggressions than other body tissues. Constant exposure of the skin to physical stimuli (UV, heat radiation and the like) and toxic chemical stimuli (atmospheric pollutants, tobacco and the like) creates oxidative stress which damages important cellular components.

ROS, or Reactive Oxygen Species, is created in reactions including the stimulation of a biological molecule by UV radiation. Too much exposure to UV light on the skin initiates a substantial amount of oxidative reactions which overtake the antioxidative capacity of the skin and increases the cellular ROS level accompanied by activation of many ROS-sensitive signaling pathways (Sander CS, Chang H, Hamm F, Eisner P, and Thiele JJ, Role of oxidative stress and the antioxidant network in cutaneous carcinogenesis, Int. J. Dermatol. 43 (2004) 326-335). Since this reaction proceeds according to a set of repeating and propagating steps, this can consequently damage the cutaneous tissues.

Chronic exposure to UV radiation results in damage including altered gene structures, altered gene activities, and altered protein structures and functions (Rittie L and Fisher DJ, UV- Light-Induced Signal Cascades and Skin Aging, Aging Research Reviews. 1 (2002) 705-720). This speculatively leads to irregularities in enzymatic reactions due to impairment of active proteins, degradation of dermal proteins such as collagen, failure in energy production due to alteration of mitochondria, and cell death due to membrane disruption.

In terms of skin, marked histological changes in the extracellular matrix (ECM) can be seen, leading to clinically photo-aged skin with loss of rigidity and elasticity. The skin appears rough and leathery with deep coarse winkles and uneven pigmentation on the surface. It is known that altered balance in the synthesis of ECM proteins (collagen, elastin, glucoproteins and the like) as well as their degradation by matrix metalloproteinases (MMPs) contribute to the dermal remodeling of the photo-aged skin (Kossodo S, Wong WR, Simon G, and Kochevar IE, Effects of UVR and UVR-induced cytokines on production of extracellular matrix proteins and proteases by dermal fibroblasts cultured in collagen gels. Photochem. Photobiol . 79 (2004) 86-93). MMPs are induced by various extracellular stimuli, e.g., UV or infrared radiation, growth factors, cytokines, and are assumed to be involved in the degradation of the epidermal basement membrane and wrinkle formation (Amano S, Ogura Y, Akutsu N, Matsunaga Y, Kadoya K, Adachi E, and Nishiyama T, Protective effect of matrix metalloproteinase inhibitors against epidermal basement membrane damage: Skin equivalents partially mimic photo-ageing process, Br. J. Dermatol. 153, 37-46., 2005).

DISCLOSURE OF INVENTION

The above changes due to oxidative stress contribute to the development of photo-aging and appearance on the skin surface as fine lines and wrinkles. Therefore, protection against oxidative stress will be no doubt beneficial for cosmetic products.

In particular, the above oxidative stress is one of the main causes of premature aging, which may be accompanied with rough microrelief on the skin. Therefore, a cosmetic product which is capable of protecting against oxidative stress will be useful to prevent rough mirorelief on the skin, which will give the skin a youthful appearance.

On the other hand, ATP, or Adenosine Tri-Phosphate, is a fundamental energy form for humans. This metabolic fuel is used for many body functions like muscle contraction. ATP is essential to cell metabolism and gives the cells sufficient energy to work efficiently. ATP is a molecule that contains chemical energy- produced in mitochondria primarily by consuming glucose and oxygen. Mitochondria use ADP (Adenosine Di-Phosphate) and phosphate to make ATP through multiple processes starting from glycolysis. The last stage known as the electron transport chain produces a large quantity of ATPs. In this stage, protons are accumulated in the intermembrane space of mitochondria as potential energy so that the mitochondria can generate the power to produce ATP.

The activation of mitochondria to promote ATP production will result in cell energizing which will be useful for cosmetic . products because the boost in cellular energy will stimulate all cellular functions of the human body, and in particular the skin.

Thus, an objective of the present invention is to provide a new anti-aging active ingredient (s) which can protect the body, in particular the skin, from oxidative stress; energize cells; and protect microrelief of the skin; such that it will be useful for cosmetic products such as anti-aging cosmetic products, in particular for the skin.

The objective of the present invention can be achieved by a combination of at least two, preferably three, of specific active ingredients, i.e., (a) an extract of amalaki, (b) an extract of pomegranate, and (c) at least one carotenoid. The above combination can function as an active ingredient for an anti- oxidizing agent, a cell energizing agent and a skin microrelief protecting agent.

It is preferable that the extract of amalaki is a fruit extract of amalaki, and that the extract of pomegranate is a fruit extract of pomegranate . The carotenoid (c) can be chosen from carotenoids of natural or synthetic origin. Further, the carotenoid can be chosen from carotenoids with provitamin A activity and carotenoids without provitamin A activity. The carotenoid with provitamin A activity can be chosen from α-carotene and β-carotene. On the other hand, the carotenoid without provitamin A activity can be chosen from zeaxanthin, lutein and lycopene. The preferable carotenoid is lycopene .

The above combination can be included in a cosmetic composition. The amount of the extract of amalaki, the amount of the extract of pomegranate, and the amount of the carotenoid can be independently from 0.0001 to 5wt% (dry basis) of the total weight of the cosmetic composition.

The cosmetic composition is preferably intended for application onto the skin. The cosmetic composition is preferably intended for an anti-aging cosmetic product.

The cosmetic composition can further comprise at least one selected from the group consisting of anti-oxidants, anti-aging agents and hydrating agents. The anti-oxidant may be selected from the group consisting of ascorbic acid, ascorbic acid derivatives, gallic acid, gallic acid derivatives, tocopherols, phenols and polyphenols. The anti-aging agent may be selected from the group consisting of α-hydroxy acids, β-hydroxy acids, carboxylic acids, carboxylic acid derivatives, retinoids, vitamin and plant extracts.

Another aspect of the present invention is the use of a combination of at least two, preferably three, of (a) an extract of amalaki, (b) an extract of pomegranate, and (c) at least one carotenoid, for the manufacture of an anti-oxidizing agent, a cell energizing agent, a skin microrelief protecting agent, and a cosmetic composition.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows graphs showing the bityrosine formation in Examples 1 to 4 and Comparative Examples 1 to 4. Fig. 2 shows photographs of Control and Example 6 with regard to fibroblasts.

Fig. 3 shows photographs of Control and Example 6 with regard to keratinocytes .

Fig. 4 shows a graph showing the score of the skin surfaces treated and not treated by Example 7 as well as the variation of the skin surfaces.

Fig. 5 shows photographs of the imprints of the skin surfaces treated and not treated by Example 7.

BEST MODE FOR CARRYING OUT OF THE INVENTION

The applicant surprisingly discovered that a combination of at least two, preferably all, of (a) an extract of amalaki, (b) an extract of pomegranate, and (c) at least one carotenoid can function as a strong anti-oxidizing agent, a cell energizing agent, and a skin microrelief protecting agent.

The above combination will be useful as a new anti-aging ingredient, which will be preferably contained in a cosmetic composition.

(Amalaki Extract)

Amalaki is a plant which is native to India and belongs to the genus Emblica of Euphorbiaceae . Amalaki has a nomenclature of Phyllanthus emblica. It is also called Emblica officinalis. Amalaki may be called Emblic myrobalan, and Indican gooseberry, in English, and may be called "Amura", "komikasou", "yukan" or "anmaroku" in Japanese.

The extract of Amalaki used in the present invention is not limited, but is preferably an extract from the fruit of Amalaki. The pulp or flesh of the fruit of Amalaki and/or the skin of the fruit of Amalaki can be extracted. The pulp or flesh of the fruit and/or the skin of the fruit of Amalaki may be dried. In order to enhance extraction efficacy, the pulp or flesh of the fruit and/or the skin of the fruit of ^~ Amalaki may preferably crushed or slashed.

The process for obtaining an extract of Amalaki is not limited. For example, the fruit of Amalaki may be extracted by using various solvents. The extraction may be performed at room temperature or an elevated temperature. The solvent may be stirred, if necessary.

As the solvents for the extraction, mention may be made of water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1- butanol, 2-butanol and the like), liquid polyhydric alcohols (ethyleneglycol, 1,3-butylene glycol, propyleneglycol, glycerin and the like) , ketons (acetone, methylethylketone and the like) , esters (ethyl acetate, butyl acetate and the like), acetonitriles, hydrocarbons (hexane, heptane, liquid paraffin, and the like) , and ethers (ethylether, tetrahydrofuran, propylether and the like) . Polar solvents such as water and lower alcohols are preferable. Water and ethanol are most preferable. These solvents may be used alone or in combination.

The extract thus obtained may be used as is or may be used after purification, concentration, dilution, and the like. The dried product of the extract may be used.

As the extract of Amalaki, Emblica® marketed by Rona, New York, USA, may be used.

In the cosmetic composition according to the present invention, the extract of Amalaki (dry content) can be used in a quantity from 10 ^~4 % to 5% of the total weight of the composition, preferably from 10 ^"3 % to 3%, and more preferably from 10 ^~2 % to 2%, and even more preferably from 10 ^"1 % to 1%, of the total weight of the composition.

The extract of Amalaki has a free radical (hydroxyl radicals and superoxide anion) scavenging effect. Further, the extract of Amalaki is capable of chelating iron and copper ions which are some of the main causes of oxidation in human body. In human body, exposure to UV light can induce the release of iron and copper ions from ion-storage proteins such as ferritin in the body. These ions can further react with hydrogen peroxide to produce free radicals which are highly active and damage body tissues including the skin. Since the extract of Amalaki can chelate iron and copper ions, it exhibits anti-oxidative effects.

The anti-oxidative effects of the extract of Amalaki would be complementary (or additionally) enhanced by a combination of an extract of pomegranate and/or a carotenoid.

(Pomegranate Extract)

Pomegranate has a nomenclature of Punica granatum. Pomegranate is native to the region of South Asia such as Iran, Nothern India and the Himalayas.

The extract of pomegranate used in the present invention is not limited, but is preferably an extract from the fruit of pomegranate. The pulp or flesh of the fruit of pomegranate and/or the skin of the fruit of pomegranate can be extracted. The pulp or flesh of the fruit and/or the skin of the fruit of pomegranate may be dried. In order to enhance extraction efficacy, the pulp or flesh of the fruit and/or the skin of the fruit of pomegranate may preferably crushed or slashed.

The process for obtaining an extract of pomegranate is not limited. For example, the fruit of pomegranate may be extracted by using various solvents. The extraction may be performed at room temperature or an elevated temperature. The solvent may be stirred, if necessary.

As the solvents for the extraction, mention may be made of water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1- butanol, 2-butanol and the like) , liquid polyhydric alcohols (ethyleneglycol, 1,3-butylene glycol, propyleneglycol, glycerin and the like) , ketons (acetone, methylethylketone and the like) , esters (ethyl acetate, butyl acetate and the like), acetonitriles, hydrocarbons (hexane, heptane, liquid paraffin, and the like), and ethers (ethylether, tetrahydrofuran, propylether and the like) . Polar solvents such as water and lower alcohols are preferable. Water and ethanol are most preferable. These solvents may be used alone or in combination.

The extract thus obtained may be used as is or may be used after purification, concentration, dilution, and the like. The dried product of the extract may be used.

As the extract of pomegranate, Amiporine® marketed by Alban Muller Intl. may be used.

In the cosmetic composition according to the present invention, the extract of pomegranate (dry content) can be used in a quantity from 10 ^~4 % to 5% of the total weight of the composition, preferably from 10 ^'3 % to 3%, and more preferably from 10 ^"2 % to 2%, and even more preferably from 10 ^"1 % to 1%, of the total weight of the composition.

The extract of pomegranate has antioxidant properties, such as the reduction of lipid peroxydation and the scavenging free radicals. Further, the extract of pomegranate can increase the activity of detoxifying enzymes such as catalase and SOD, and the concentration of glutathione. The antioxidant effects of the extract of pomegranate may be attributed to flavonoids and polyphenols in the extract. Furthermore, the extract of pomegranate may play protective roles in UVA-mediated cellular modifications and UVB-mediated oxidative damages on keratinocytes . The extract of pomegranate can inhibit UVB-mediated decrease in cell viability, UVB-mediated decrease in intracellular glutathione content, and UVB-mediated increase in lipid peroxidation. The above suggest that the extract of pomegranate can protect the skin from UV-induced oxidative stress and consequently photo-aging.

The anti-oxidative effects of the extract of pomegranate would be complementary (or additionally) enhanced by combination with an extract of Amalaki and/or a carotenoid.

Furthermore, the extract of pomegranate also has effects for energizing cells, in particular keratinocytes, which are based on the activation of mitochondria in the cells. The activation of mitochondria in cells causes the promotion of ATP production in the mitochondria. The produced ATP will energize or boost the cells. Thus, the cell energizing effect is in particular preferable to skin cells because the skin cells are given power to function as young cells, and therefore, they can combat with the signs of aging.

The cell energizing effects of the extract of pomegranate are complementary (or additionally) enhanced by a combination of an extract of Amalaki and/or a carotenoid.

In order to maintain its overall healthy condition, the skin, a multicellular organ, has to be well-conditioned within itself. Through cell communication, skin cells can review what is happening to other skin cells and detect differences in environmental conditions to respond accordingly. This communication involves either sending signals to neighboring cells through channel proteins to generate a coordinated response or isolating groups of cells from the rest of the community to maintain the integrity of the skin.

There are different types of channel proteins for cell communication. They are located on the cell membrane and transfer particular substances between cells or to the outer environment from the inside of the cells.

Aquaporins (AQPs) are a family of transmembrane proteins forming channels that primarily transport water across the cell plasma membrane. They transport water by osmosis and small solutes by concentration gradients passively. This water communication between cells and the extra cellular matrix provides a short circuit for water between the base of the epidermis and the stratum corneum in order to maintain water content.

So far, 13 APQs are known. Among the known APQs, AQP3 is expressed abundantly in human keratinocyte plasma membranes. So regulating AQP3 may hold a key to maintaining overall skin hydration and homeostasis of the skin cells, thus providing healthy skin conditions. The extract of pomegranate can increase the level of AQP3. Therefore, the extract of pomegranate would contribute to the hydration of the skin which will help the skin to combat with the signs of aging such as fine lines caused by dehydration.

(carotenoid)

According to the present invention, the expression carotenoid is understood to mean both a carotenoid with provitamin A activity and a carotenoid without provitamin A activity. Among the carotenoids with provitamin A activity, mention may be made of, by way of example, β-carotene or α-carotene. Among the carotenoids without provitamin A activity, mention may be made of, by way of example, zeaxanthin, lutein or lycopene.

The carotenoid used according to the present invention may be of natural or synthetic origin. The expression natural origin is understood to mean the carotenoid, in the pure state or in solution regardless of its concentration in said solution, obtained from a natural component. The expression synthetic origin is understood to mean the carotenoid, in the pure state or in solution regardless of its concentration in said solution, obtained by chemical synthesis.

When the carotenoid is of natural origin, it may be obtained from a plant material derived from a whole plant cultured in vivo or derived from in vitro culture. The expression in vivo culture is understood to mean any culture of the conventional type, that is to say in the soil in the open air or in a greenhouse, or alternatively with no soil. The expression in vitro culture is understood to mean all the techniques known to persons skilled in the art which artificially allow the production of a plant or of a portion of a plant. The selection pressure imposed by the physicochemical conditions during the growth of plant cells in vitro makes it possible to obtain a standardized plant material which is available throughout the year unlike plants cultured in vivo.

Preferably according to the present invention, a plant derived from in vivo culture is used. Any method of extraction known to persons skilled in the art may be used to prepare the carotenoid used according to the present invention. The carotenoid may be in an alcoholic, in particular ethanolic, solution. The carotenoid may also be in a lipid (oil) or lipid-alcohol solution.

The preferred carotenoids according to the present invention are β-carotene and lycopene. Most preferably, lycopene is used.

Lycopene is a natural pigment which is found in ripe fruits, particularly in tomatoes. Its structure is close to that of β- carotene. It may be in cis or trans form.

Lycopene in an extract of tomato (Solanum lycopersicum) may be used. By way of example, a lycopene-rich tomato extract prepared by the company Lycored and marketed under the name Lyc-O-Mato® may ^¬ be used. Alternatively, lycopene of tomato, which is encapsulated with a polycaprolactone polymer, marketed under the name of Mexoryl® SAQ by CHIMEX may be used.

It is also possible to use, according to the present invention, any preparation containing lycopene with the objective of improving the bioavailability of the latter and any novel raw material containing lycopene obtained from a novel method of manufacture .

The quantity of carotenoid which can be used according to the present invention of course depends on the desired effect and may therefore vary to a great extent.

In the cosmetic composition according to the present invention, the carotenoid in the pure state is in a quantity representing from 10 ^~4 % to 5%, preferably from 10 ^"3 % to 3%, more preferably from 10 ^'2 % to 2%, and even more preferably from 10 ^" H to 1%, of the total weight of the composition.

Of course, persons skilled in the art, if they use the carotenoid in the form of a solution, a plant extract for example, know how to adjust the quantity of solution which they use in their composition so that the final quantity of carotenoid in the composition is consistent with the above-defined quantities which can be used.

A carotenoid such as lycopene has anti-oxidant properties such that it has a high ability for quenching singlet oxygen. The anti-oxidative effects of a carotenoid would be complementary (or additionally) enhanced by extract (s) of Amalaki and/or pomegranate

Furthermore, a carotenoid also has effects for energizing cells, in particular keratinocytes, which are based on the activation of mitochondria in the cells. The activation of mitochondria in cells causes the promotion of ATP production in the mitochondria. The produced ATP will energize or boost the cells. Thus, the cell energizing effect is in particular beneficial to skin cells because the skin cells are given power to function as young cells, and therefore, they can combat the signs of aging.

The cell energizing effects of a carotenoid are complementary (or additionally) enhanced by a combination of extract (s) of Amalaki and/or pomegranate.

As mentioned above, there are different types of channel proteins for cell communication.

A Gap Junction is a channel that is located between cells and regulates the passage of ions such as K ⁺ and Ca ²⁺ and small metabolites such as glucose, and is important for overall homeostasis of the skin cell functions such as differentiation of epidermis .

A protein called connexin is the smallest unit in a Gap Junction. A Gap Junction of human keratinocyte comprises mostly connexin 43. So regulating connexin 43 may be the key to maintaining overall homeostasis of skin cells, thus maintaining a healthy skin condition. Connexin 43 is expressed in differentiating human keratinocytes. Substantial levels of connexin 43 are detected in proliferating cultures of normal human keratinocytes. Lycopene can increase connexin 43 gene expression. Thus, lycopene may have an important protective role and enhancive role in keratinocyte proliferation through gap junction communication. Thus, a carotenoid such as lycopene would help the skin to combat the sing of aging by promoting keratinocyte proliferation.

(Cosmetic Composition)

The cosmetic composition according to the present invention comprises the above combination of the components (a) , (b) and (c) , as an anti-aging active ingredient. Accordingly, the cosmetic composition according to the present invention can exert anti- aging effects.

The cosmetic composition according to the present invention is preferably suitable for topical application to the skin. It may ^¬ be provided in all the galenic forms normally used for this type of application, in particular in the form of an aqueous or oily solution, an oil-in-water or water-in-oil or multiple emulsion, a silicone emulsion, a microemulsion or nanoemulsion, an aqueous or oily gel or a liquid, pasty or solid anhydrous product.

The cosmetic composition according to the present invention may be fluid to a greater or lesser degree and may have the appearance of a white or colored cream, an ointment, a milk, a lotion, a serum, a paste, a mousse or a gel. It may be optionally applied to the skin in aerosol form. It may also be provided in solid form, for example in the form of a stick. It may be used as a care product and/or as a make-up product for the skin. Preferably, the cosmetic composition according to the present invention is intended for an anti-aging cosmetic product.

In a known manner, the cosmetic composition of the present invention may also contain the customary adjuvants in the cosmetic field, such as hydrophilic or lipophilic gelling agents, preservatives, antioxidants, anti-aging agents, solvents, perfumes, fillers, UV screening agents, pigments, odor absorbers and coloring matter. The cosmetic composition of the present invention preferably further comprises at least one selected from the group consisting of antioxidants, anti-aging agents, and hydrating agents, in addition to the above components (a) , (b) and (c) .

As the antioxidants, mention may be made of ascorbic acid, ascorbic acid derivatives, gallic acid, gallic acid derivatives, tocopherols, phenols and polyphenols (e.g., flavonoids (e.g., catechin, rutin, and quercetin) , carnosic acid and carnosol, optionally substituted (2, 5-dihydroxyphenyl) carboxylic acid and (2, 5-dihydroxyphenyl) alkylenecarboxylic acid as well as their derivatives such as salts, esters and amides, caffeic acid and its esters or amides, and tannic acid and its esters and amides).

As the anti-aging agents, mention may be made of α-hydroxy acids (e.g., lactic acid, glycolic acid, and citric acid), β-hydroxy acids (e.g., salicylic acid, and 5-n-octanoyl salicylic acid), carboxylic acids (e.g., acetic acid, and palmitic acid), carboxylic acid derivatives (e.g., retinyl propionate), retinoids (e.g., retinol), vitamins (e.g., vitamin C) and plant extracts.

As the hydrating agents, mention may be made of trehalose and derivatives thereof, hyaluronic acid and derivatives thereof, glycerol, pentanediol, sodium pidolate, serine, xylitol, sodium lactate, glycerol polyacrylate, ectoin and derivatives thereof, chitosan, oligo- and polysaccharides, cyclic carbonates, N-lauroyl pyrrolidone carboxylic acid, and N-α-benzoyl-L-arginine .

The quantities of these various adjuvants are those conventionally used in the cosmetic field, and are for example from 0.001 to 20% of the total weight of the composition. These adjuvants, depending on their nature, can be introduced into the fatty phase or into the aqueous phase of the cosmetic composition. These adjuvants, and their concentrations, should be such that they do not adversely affect the advantageous properties of the plant extracts, or of the carotenoid used in the present invention.

When the cosmetic composition according to the present invention is an emulsion, the proportion of the fatty phase may range from 5 to 80% by weight, and preferably from 5 to 50% by weight relative to the total weight of the composition. The fatty substances, the emulsifiers and the co-emulsifiers used in the composition in the form of an emulsion are chosen from those conventionally used in the cosmetic field. The emulsifier and the co-emulsifier are preferably present in the composition in a proportion ranging from 0.3 to 30% by weight, and preferably from 0.5 to 20% by weight relative to the total weight of the composition.

As fatty substances which can be used in the present invention, it is possible to use oils and in particular mineral oils (e.g., liquid paraffin), oils of plant origin (e.g., avocado oil, and soybean oil), oils of animal origin (e.g., lanolin), synthetic oils (e.g., perhydrosqualene) , silicone oils (e.g., cyclomethicone) and fluorinated oils (e.g., perfluoro-polyethers) . It is also possible to use, as fatty substances, fatty alcohols such as cetyl alcohol, fatty acids, waxes and gums, and in particular silicone gums.

As emulsifiers and co-emulsifiers which can be used in the present invention, mention may be made of, for example, esters of fatty acids and of polyethylene glycol such as PEG-100 stearate, PEG-50 stearate and PEG-40 stearate; esters of fatty acids and of polyols such as glyceryl stearate, sorbitan tristearate, oxyethylenated sorbitan stearates which are available under the trade names Tween® 20 or Tween® 60, for example; and mixtures thereof.

As hydrophilic gelling agents, there may be mentioned in particular carboxyvinyl polymers (carbomer) , acrylic copolymers such as acrylate/alkyl acrylate copolymers, polyacrylamides, polysaccharides, natural gums and clays, and, as lipophilic gelling agents, mention may be made of modified clays such as bentones, metal salts of fatty acids and hydrophobic silica.

The cosmetic composition according to the present invention finds application in particular in the prevention and/or treatment of the signs of skin aging, in particular of photo-aging, because the above combination of at least two, preferably all, of the components (a) , (b) and (c) can function as an anti-oxidizing agent, a cell energizing agent, a skin microrelief protecting agent . The present invention also relates to the use of the above combination of at least two, preferably all, of the components (a) , (b) and (c) for the manufacture of a cosmetic composition, in particular for preventing or treating the signs of skin aging, in particular of photo-aging.

It also relates to the use of the above combination of at least two, preferably all, of the components (a) , (b) and (c) for the manufacture of an anti-oxidizing agent, a cell energizing agent and a skin microrelief protecting agent.

EXAMPLES

The present invention will be described in more detail by way of examples, which however should not be construed as limiting the scope of the present invention.

1. Anti-Oxidation Effect

[Formulations]

Four types (eye cream, cream, emulsion and concentrate) of formulations containing the combination of lycopene (Mexoryl® SAQ) , the fruit extract of amalaki (Emblica®) , and the fruit extract of pomegranate (Amiporine®) were prepared as Examples 1 to 4. The concentration of the active ingredient (the above combination) in the eye cream, cream and emulsion was 0.1%, while that in the concentrate was 0.5%.

The formulations corresponding to Examples 1 to 4 excluding the active ingredient were prepared as Comparative Examples 1 to 4.

Tables 1 to 4 show the compositions of Examples 1 to 4 and Comparative Examples 1 to 4.

Lycopene: Mexoryl® SAQ, CHIMEX

Amalaki fruit extract: Emblica®, Rona

Pomegranate fruit extract: Amiporine®, Alban Muller Intl. [Table 1]

Eye Cream

[Table 2]

Cream

[Table 3]

Emulsion

[Table 4]

Concentrate

[Test]

2g of each of Examples 1 to 4 and Comparative Examples 1 to 4 was mixed in 20ml of a methanol solution containing tyrosine in a concentration of 200μg/ml and diesel particles in a concentration of 50μg/ml. The solution was exposed to UVA radiation at a power of 20 Joules/cm ² . After the exposure, the amount of bityrosine formed in the solution was measured, and the inhibition rate was evaluated. The results were shown in Table 5.

[Table 5]

The above results are also shown in Figure 1.

The formation of bityrosine is caused by oxidation due to UV radiation in the presence of diesel particles. The above results show that the combination of lycopene, the fruit extract of amalaki, and the fruit extract of pomegranate significantly inhibits the oxidation of tyrosine. Therefore, the above combination has excellent antioxidant power.

2. Cell Energizing Effect

[Test]

Fibroblasts and keratinocytes were cultivated in normal cell culture conditions (DMEM, 10% Fetal calf serum, L-glutamine 2mM, and Penicillin 50U/ml and Streptomycin 50μg/ml) until they formed a confluence. At confluence, the cells of the fibroblasts and keratinocytes were treated for 24 hours without (Control) or with

(a) 10μg/l of the fruit extract of amalaki (Comp. Ex. 5),

(b) 100μg/l of the fruit extract of amalaki (Comp. Ex. 6),

(c) 0.001% of the fruit extract of pomegranate (Comp. Ex. I) ₁ (d) 0.01% of the fruit extract of pomegranate (Comp. Ex. 8),

(e) 0.0001% of lycopene (Comp. Ex. 9),

(f) 0.001% of lycopene (Comp. Ex. 10),

(g) 10μg/l of the fruit extract of amalaki, 0.001% of the fruit extract of pomegranate, and 0.0001% of lycopene (Example 5), or (h) 100μg/l of the fruit extract of amalaki, 0.01% of the fruit extract of pomegranate, and 0.001% of lycopene (Example 6).

Lycopene: Mexoryl® SAQ, CHIMEX

The fruit extract of amalaki: Emblica®, Rona

The fruit extract of pomegranate: Amiporine®, Alban Muller Intl.

Mitochondria in the fibroblasts and the keratinocytes were labeled with MitoTracker® for their presence (green in a color photograph) and with MitoFluor™ for membrane potential (red in a color photograph) , and photos of the fibroblasts and the keratinocytes were obtained. The activity of mitochondria was calculated equal to 1000/red fluorescence. The photo-images of the fibroblasts and the keratinocytes were superimposed to give yellow color for visual analysis. Color intensity was analyzed and compared to Control, Comparative Examples 5 to 10, and Examples 5 and 6, by using INCeIl Analyzer™ 1000. The results were shown in Tables 6 and 7.

[Table 6]

The photos of Control and Example 6 are shown in Figure 2.

[Table 7]

The photos of Control and Example 6 are shown in Figure 3.

The above results show that, with regard to fibroblasts, a significant increase (11%) of mitochondria activity was observed, and that, with regard to keratinocytes, a significant increase (20%) of mitochondria activity was observed. Since mitochondria are "energy factories" of cells, boosting their activity indicates that the energy flow inside the cells is increased.

3. Microrelief Protection Effect

[Formulation]

A concentrate containing O..5wt% of lycopene, 0.5wt% of the fruit extract of amalaki, and 0.5wt% of the fruit extract of pomegranate was prepared as Example 7. The composition of the concentrate is shown in Table 8. [ Table 8 ]

Lycopene: Mexoryl® SAQ, CHIMEX

Amalaki fruit extract: Emblica®, Rona

Pomegranate fruit extract: Amiporine®, Alban Muller Intl.

[Test]

9 subjects (Caucasian women and men of age 18 to 55) were recruited, and two areas were chosen on their back. A skin imprint was obtained by using a synthetic polymer.

On the first day, the above concentrate was applied on one of the two areas in an amount of 2mg/cm ² . The other area was not treated. Then, both areas were exposed to UVA and UVB at 1.5 MED (Minimum Erythemal Dose) . On the second day, the same treatment as the first day was performed for all subjects. The subjects were then left untreated until the eighth day. A skin imprint was obtained by using a synthetic polymer, in order to evaluate the evolution of microrelief on the skin. A variation percentage was calculated after scoring visually (14 point scale, 7 being the initial) the imprint by experts, in accordance with the formula: Variation (%) = { (Score-7 ) /7 } *100. The average score and the variation percentage for the area treated by Example 7 and the non-treated area were compared. The results are shown in Table 9.

[Table 9]

The above results are also shown in Figure 4. The imprints of the area before the first day (Day 0) and that after the eighth day (Day 8) are shown in Figure 5.

After exposure to UV for two days, the microrelief of the area treated with Example 7 and that treated with nothing were damaged compared to that before the exposure. However, the microrelief of the area treated with Example 7 was not as evolved significantly- compared to the non-treated area. Therefore, it was found that Example 7 protected the microrelief on the skin.