WO2011113785A2

WO2011113785A2 - A cosmetic composition for rejuvenating skin appearance

Info

Publication number: WO2011113785A2
Application number: PCT/EP2011/053779
Authority: WO
Inventors: Daniela Montanari; Manuela Guglielmo
Original assignee: Labo Cosprophar Ag
Priority date: 2010-03-15
Filing date: 2011-03-14
Publication date: 2011-09-22
Also published as: WO2011113785A3; CH700735B1

Abstract

The present invention relates to a biologicallyactive complex, comprising a combination of at least one biologically active component at the level of mitochondrial DNA and at least one biologically active component at the level of the nuclear DNA, its use for the cosmetic anti- aging treatment of the skin and cosmetic compositions containing it.

Description

TITLE

A cosmetic composition for rejuvenating skin appearance

TECHNICAL FIELD

The present invention falls within the field of cosmetic composition for topical use for skin care and it relates to a cosmetic composition to rejuvenate skin appearance.

Particularly, the present invention relates to a biologically active complex, comprising a combination of at least one biologically active component at the level of mitochondrial DNA and at least one biologically active component at the level of the nuclear DNA, its use for the cosmetic anti-aging treatment of the skin and cosmetic compositions thereof.

Background of the invention

Skin aging is the process by which our body shows signs of its transformation over time: the skin is, in fact, the organ that most visibly changes with all the years passing by.

When cells grow old, DNA accumulates an amount of damage that exceeds the repair speed and results in a reduction of protein synthesis. Since proteins perform many vital functions in the cell, the cell is slowly undermined and eventually dies. When in a tissue, such as skin, a sufficient number of cells reach such a state, the same tissue will be compromised and signs of aging begin to show.

Skin aging is a physiological involution process, influenced by both individual and genetic factors and environmental ones, as well as by the personal lifestyle.

The genetic pool, together with endocrine factors, controls and regulates the intrinsic skin aging, also known as the chrono-ageing. Genetic factors, as opposed to the environmental ones, remain constant throughout life. They represent a kind of 'biological clock' that marks the time of aging of all cells of the human body.

The skin aging originating from genetic factors starts very slowly, after the age of 25, with great variability among people, and is visibly manifested by the age of 40.

The intrinsic aging leads to a slowdown in the production of cells, collagen and elastin, the loss of tissue, fat and muscles, a decreased sweating and sebum production, and finally to a damage at the level of DNA. The main changes derive from a detectable loss of turgor and elasticity of the skin. Wrinkles caused by intrinsic aging are rather fine; the skin gets thinner, becomes more transparent and has a tendency to sag.

Environmental factors, on the other hand, such as incorrect exposure to the sun, pollution, incorrect lifestyle (excessive alcohol consumption and an unbalanced diet) and smoking, cause in increased production of free radicals and activation of metalloproteinases, and are responsible for the morphological and structural changes typical of extrinsic aging, or photo-aging. Typical signs are very deep wrinkles, spots and thickened skin.

A large number of anti-aging cosmetic formulations currently on the market aim to improve or preserve over time the efficiency of the physiological mechanisms of skin cells.

In particular, many anti-aging cosmetic products available on the market incorporate active ingredients that exert activity against free radicals such as vitamin E, beta-carotene, vitamin C derivatives, coenzyme Q, and others.

Free radicals (ROS) originate from oxidation reactions and are unstable molecular species, because they have one or more free electrons. Therefore they are able to react with organic substances with particular ease, leading to phenomena of cellular degeneration, with changes in the biological structure, particularly evident in the dermal layer. ROS are generated in many processes within the cell. Membrane proteins, cytosolic enzymes (e.g. cyclooxygenase), lipid metabolism in peroxisomes all involve a small production of free radicals, but most of the ROS (estimated at 90%) are produced by mitochondria during oxidative phosphorylation.

During this process, NADH or FADH2 are used to generate a proton gradient between the matrix and intermembrane space. Normally, the electrons generated from NADH pass through the respiratory chain and are used to produce water and to transfer protons in the intermembrane space, generating a membrane potential. Sometimes, however, these electrons can react with a molecule of oxygen to give superoxide anion O2^" Oxygen can be converted to hydrogen peroxide (H2O2) by the manganese-dependent superoxide dismutase enzyme (located in the matrix) or by the copper/sulfur-dependent superoxide dismutase enzyme (located in the cytoplasm and the intermembrane space). Hydrogen peroxide is more stable than O2^" and can diffuse through the membrane and outside the mitochondria, to the nucleus or can be inactivated by the glutathione peroxidase enzyme and catalase enzyme. However, H2O2 in the presence of reduced transition metals, can also be converted into highly reactive hydroxyl radical (.OH) species. In general, as the mitochondria are the main source of ROS in the cell, they are also more affected by the cellular oxidative stress.

The so-called "mitochondrial theory of aging" states that responsible for the degenerative aging is a random accumulation of oxidative damage to biological macromolecules, caused by free radicals and, in particular, by reactive oxygen species (ROS). ROS are normally neutralized by enzymes such as various forms of superoxide dismutase and glutathione peroxidase and catalase as well as by non-enzymatic "scavengers" like glutathione, coenzyme Q and other compounds. Under oxidative stress conditions, i.e. under conditions of excess of the capacity for self- protection of the cell, ROS can strike at random nucleic acids, proteins and lipids. Mitochondrial macromolecules would be more vulnerable molecules because the mitochondria, which metabolize about 90% of the cellular oxygen, are a favored source of ROS. MtDNA is particularly vulnerable because it is naked, that is devoid of histone-type proteins, and physically close to the main source of ROS. Mitochondrial DNA is present in multiple copies in the mitochondria and is closely associated with various proteins.

Due to the presence of ROS in the vicinity of the inner mitochondrial membrane, the loss of the protective role of histones and the limited reparative capacity of mtDNA with respect to that of nuclear DNA, mitochondrial DNA (mtDNA) is much more vulnerable to oxidative damage.

Consequently, during the life mtDNA is damaged repeatedly and over time the produced mitochondria become increasingly unable to produce energy.

As the cell ages, changes occur continuously, partly because of harmful by-products (reactive species, or radicals) of the oxygen we breathe. The body, however, has a DNA repair intrinsic function to cope with the damage caused by these products. However, in aged cells, the repair activity becomes inefficient. This is due to the fact that the enzymes involved in the repair are blocked along their path to reach the site of the damage inside the mitochondria. Almost half of the enzyme activity is stopped outside the mitochondria and can not reach the DNA to be repaired.

For these reasons, presumably, the defects in mitochondrial DNA accumulate faster than in nuclear DNA.

Somatic mitochondrial mutations are the primary cause of energy decline that characterizes senescence. The reduced functionality of the respiratory chain enzymes not only decreases ATP synthesis but also increases the production of oxygen radicals in a vicious cycle of respiratory impairment, accumulation of free radicals and mitochondrial DNA mutations. Mitochondria are the first energy source of cells and are therefore essential for the maintenance of their health and longevity.

At present a need is felt for biological activity components-based cosmetic preparations with skin anti-aging action capable of acting at the level of cellular mechanisms governing the regulation of cellular aging processes. SUMMARY OF THE INVENTION

The applicant has found that combining at least biological active ingredient at the level of mitochondrial DNA with at least one biological active ingredient at the level of nuclear DNA, it is possible to obtain a biologically active complex capable of rejuvenating the skin's appearance.

In particular, it is possible to limit the appearance and visibility of the signs of skin aging and preserve cellular longevity, cooperating in the rejuvenation of the skin, bringing together in a biologically active complex, one or two selected biological components which act primarily at the level of mitochondrial DNA with one or two selected biological components which act primarily at the level of nuclear DNA.

Particularly it has been found that combining at least one specific biological active ingredient at the level of mitochondrial DNA with at least one biological active ingredient in nuclear DNA a significally greater capability of rejuvenating the skin's appearance than of the individual components taken alone is achieved.

In accordance with a first aspect, the present invention provides a biologically active complex preventing and/or delaying skin aging, characterized in that it comprises

A) at least one biologically active ingredient at the level of mitochondrial DNA selected from a) a Pentapeptide including serine, cysteine, isoleucine, asparagine, threonine amino acids, or b) a hydrolized soy protein or a mixture thereof

and

B) at least one biologically active ingredient at the level of nuclear DNA selected from c) teprenone or d) an extract from Myrtus communis or a mixture thereof.

The biological complex according to the invention provides the activity and functionality of the cells of the epidermis and dermis to be maintained and extended, to provide a proper cell renewal and to prevent reactions that damage the same cells, causing their premature aging and death.

Thus, the biological complex according to the present invention determines a delay of the cellular aging and promotes cellular longevity. In one preferred embodiment, the present invention provides a biologically active complex with preventing and/or delaying skin aging, characterized in that it comprises:

a) a Pentapeptide including serine, cysteine, isoleucine, asparagine, threonine amino acids;

b) a hydrolyzed soy protein;

c) teprenone and

d) an extract from Myrtus communis.

Specifically, application on the skin of a biologically effective amount of the four biologically active ingredients (i.e. a - d ) produces a skin rejuvenation effect particularly strong.

In another embodiment there is provided a biologically active complex for the rejuvenation of the skin comprising a) a Pentapeptide including serine, cysteine, isoleucine, asparagine, threonine amino acids, b) a hydrolyzed soy protein containing amino acids and proteins with molecular weight lower than 5 kDa, c) teprenone and d) an extract from Myrtus communis

The present invention also provides an anti-aging cosmetic composition for skin application comprising a biologically active complex in a cosmetically effective quantity and in a cosmetically acceptable vehicle.

The present invention also provide the use of the biologically active complex or a cosmetic composition containing it for the cosmetic anti- aging treatment of the skin.

The present invention also provide a cosmetic treatment for skin care comprising the application to the skin of at least one of cosmetic composition according to the present invention comprising a biologically active complex as defined above. DETAILED DESCRIPTION OF THE INVENTION

To facilitate the understanding of this invention the meaning of some terms, expressions as used in the contest of the invention are included. Pentapepide in the contest of this invention is Pentapeptide-28 [INCI name].

Extract from Myrtus communis in the contest of the present invention is

Hydrolyzed Myrtus Communis Leaf Extract [INCI name].

The following abbreviations are used in the text:

INCI international Nomenclature Cosmetic Ingredient,

CAS number =Chemical abstract Number,

MtDNA=mitochondrial DNA,

NAD or NADH=nicotinammide adenine dinucleotide,

ATP=Adenosine triphosphate,

FAD or FADH =Flavina adenine dinucleotide,

UVB= Ultraviolet B. Each of the active ingredients (i.e. a - d) of the biologically active complex of the invention performs its own specific action at the level of the skin cell components.

In particular, the biologically active ingredients of the group A) perform a protective action on the structure and functionality of mitochondrial DNA, while preserving their activity over time.

Thus, one of the biolologically active ingredients of the group A is the hydrolyzed soy protein [INCI name] and having CAS number 68607- 88-5 number, which is typically rich in bio-mimetic peptides against the respiratory chain proteins.

The hydrolyzed soy protein is obtained by acidic, alkaline, or enzymatic hydrolysis of soy composed primarily of amino acids, peptides, and proteins. It may contain impurities consisting chiefly of carbohydrates and lipids along with smaller quantities of miscellaneous organic substances of biological origin.

According to one embodiment, the hydrolyzed soy protein contains amino acids and proteins with MW (molecular weight) lower than 5kDa, as well as polyphenols and carbohydrates.

According to one embodiment, the soy hydrolyzed protein contains all eight essential amino acids found in animal proteins, i.e. Phenylalanine, Isoleucine, Leucine, Lysine, Methionine, Threonine, Tryptophan, Valine.

Typically, the soy hydrolyzed protein is obtained by enzymatic hydrolysis and purification achieved, for example, by ultrafiltration. The protein MW was analyzed by electrophoresis.

The hydrolyzed soy protein according to the invention is commercial available.

The soy hydrolyzed protein performs a specific action at the level of mitochondrial DNA by acting mainly through the mechanisms of mitochondrial sirtuins SIRT-3 gene expression. This gene, as well as the SIRT-4 gene, are able to protect the mitochondria and is important for the maintenance of their health and longevity. When the efficiency of the mitochondria begins to decrease, the energy of the cells is reduced and the processes of aging and degeneration of mitochondria are triggered.

Thanks to the action of SIRT-3 genes but also possibly of SIRT-4, the mitochondria remain active longer, providing more energy to the cells and extending their longevity.

In particular, it was also noted that the SIRT-3 gene is localized at the level of the mitochondria inner membrane, and plays an important role in the energy metabolism. Moreover it takes part in the adaptive thermogenesis, and decreases the membrane potential and ROS production and stimulates the production and regeneration of mitochondrial antioxidants

The activity of soy hydrolyzed component within the complex of the invention, was demonstrated by in vitro test tests. Thus for example, human fibroblasts were treated and untreated with 1 % soy hydrolyzed protein for 24 and 48h. The mitochondrial sirtuins SIRT-3 expression was evaluated by immunofluorescence as compared with the control.

The results were as follows:

after 24h: + 10.23% higher expression of SIRT-3

after 48h: + 30,16% higher expression of SIRT-3

Using skin obtained from biopsies, a further test was performed by treating it or not with 1 % of soy hydrolyzed component.

Immunofluorescence reveals the production of SIRT-3 and then the gene expression of the gene for the Sit-in 3.

Photographically, it is noted that the treated skin shows a greater expression of the SIRT-3 gene than the control after 48h: + 20,7% higher expression of SIRT-3.

The hydrolyzed soy protein, in addition to being provided with a specific activity at the mitochondrial level, is particularly active in promoting activity of Cytochrome C oxidase. Cytochrome C is a small hemoprotein found in the inner membrane of mitochondria and is part of the cytochromes C class. It is a soluble protein, unlike other cytochromes, and is an essential component of the electron transport chain.

Cytochrome C is involved in the 3rd stage of cellular respiration, the oxidative phosphorylation, in which the Cytochrome C passes 4 electrons to the oxygen molecule to form 2 water molecules.

The reaction is catalyzed by Cytochrome C oxidase (or complex IV) which is the last enzyme complex involved in the electron transport chain: 4 ferrocytochrome C + O2 + 4 H⁺→ 4 ferricytochrome C + 2 H₂O.

Also known as cytochrome a3 or Warburg's respiratory ferment, the enzyme is an integral protein of inner mitochondrial membrane and contains a porphyrinic group containing Fe²⁺ ions (if the cytochrome is reduced) or Fe³⁺ ions (if the cytochrome is oxidized). The reduction of oxygen is accompanied by the extrusion of four protons from the intramitochondrial compartment. The consumption of four hydrogen ions per oxygen molecule and the translocation of four additional hydrogen ions across the inner mitochondrial membrane are responsible for maintaining a gradient of p_H on both sides of the membrane, a gradient on which the phosphorylation of ADP to ATP and the production of energy in the cell depend. Also for this reason, the presence of components of hydrolized soy protein in the complex of the invention results in an increased production of energy in the form of ATP and protection against damage induced by ROS on mitochondria, thereby preserving cellular and skin vitality and longevity.

To assess the activity of the hydrolyzed soy protein in promoting the activity of Cytochrome C oxidase an in vitro test was performed on fibroblasts which were treated or not, with 1 % of hydrolyzed soy protein.

After 3 and 24 incubation hours the activity of Cytochrome C was assessed and compared with the control. Cells treated with hydrolyzed soy protein indicated by the Applicant significantly increase the activity of Cytochrome C oxidase in the following amounts as compared to control:

after 3 h: + 132% in respect to control

after 24 h: + 623% in respect to control.

The soy hydrolyzed component therefore reveals a stimulating activity on the mitochondrial respiratory chain at the level of Cytochrome C oxidase, in order to increase energy skin lastingly.

The increase in enzyme activity actually means an increased cellular respiration and a resulting increased energy production.

To demonstrate the increased synthesis of ATP a second test in vitro was also carried out.

Fibroblasts were treated with 1 % hydrolyzed soy protein. Then ATP was quantified by bioluminescence after 1 and 3 hours incubation.

Soy hydrolyzed component significantly increases the synthesis of

ATP by 40% after 1 hour. The assessment also shows that the increase of ATP persists up to 3 hours, with +28% of ATP produced.

One of the biologically active ingredients of group A), which is active at the level of mitochondrial DNA, includes a Pentapeptide protecting cells from oxidative damage on the mitochondrial DNA.

The Pentapeptide, according to the invention, which includes serine, cysteine, isoleucine, asparagine and threonine amino acids, is known as Pentapeptide-28 [INCI name] and it is a commercially available compound.

Typically, the Pentapeptide of the invention which includes the following five amino acids: serine, cysteine, isoleucine, asparagine and threonine is produced by synthesis.

The Pentapeptide performs a particularly evident activity at the level of aconitase, a protein involved in cellular respiration during the first step of the Krebs' cycle. This activity is particularly appreciated because the damage to mitochondrial DNA is directly responsible for the deficiency of energy production at the cellular level, and therefore for the cellular aging.

Some scientific studies have shown that proteins related to mitochondrial DNA are able to protect it against oxidative damage.

The protective effect of the Pentapeptide present in the formulation of the complex of the invention, on the mtDNA was evaluated by an in vitro test.

For this purpose, human keratinocytes were cultured with or without 1 % of Pentapeptide for 48 hours, and subjected to repeated stress (with UVB radiation) so that it resulted in the formation of ROS and cellular aging was induced: they have been exposed to UVB radiation 3 times at a power output of 75mJ/cm². The mtDNA of aged keratinocytes undergoes specific alterations with the formation of deletions, fragments of mtDNA. These deletions serve as a marker of mutations in mtDNA.

The PCR (Polymerase Chain Reaction) technique allows the frequency of the deletion to be analyzed. PCR is followed by electrophoresis run allowing the separation and visual identification (colored band) of fragments of mtDNA. The higher intensity of the band, the higher the frequency of the deletion.

The deletions are analyzed in the cell extract of the non-irradiated keratinocytes (control), in irradiated keratinocytes and in irradiated keratinocytes treated with the active at 1 %.

The Pentapeptide has been shown to determine the total protection of mtDNA: 100% with absence of the formation of deletions.

A second test evaluated the activity of the enzyme Aconitase. The

Aconitase enzyme plays a key role in cellular respiration, the main way of energy production. It is one of the enzymes of the Krebs' cycle and converts citric acid into isocitric acid.

Fibroblasts were incubated for 72 h in the presence of 1 % of the Pentapeptide. Then, the activity of mitochondrial Aconitase enzyme was assessed and compared to the control.

The result was as follows:

1 % Pentapeptide: + 26% Aconitase activity compared to control.

Biologically active ingredients of the group B perform an action at the level of nuclear DNA slowing down premature aging of the skin through the action of preventing damage to nuclear DNA.

One of the biologically active ingredients of the group B active is a vegetal extract of Myrtus communis, a Mediterranean shrub rich of oligo- galaturonans.

Vegetal extract of Myrtus communis, according to this invention, is Hydrolyzed Myrtus Communis Leaf Extract [INCI name] is the hydrolysate of Myrtus Communis Leaf Extract derived by acid, enzyme or other method of hydrolysis.

Typically, the vegetal extract is obtained from leaves of myrtle and includes an active fraction rich in carbohydrates, mainly galacturonic acid, but also glucose, arabinose, galactose, rhamnose, xylose, fructose. The vegetal extract has been shown to be particularly active in carrying out a protective action on cellular DNA.

In particular, it was noted that the vegetal extract Myrtle, when added to cultured cells, induces the production of sirtuins by activating SIRT-1 gene and a decrease in the expression of cellular senescence factors.

The sirtuins (silent information regulators) are a family of enzymes involved in the regulation of gene expression, in the cellular apoptosis, in the metabolism of fatty acids, in regulating the extension of cellular life. The sirtuins are associated with genes that coordinate and optimize the functions of cells because these latter struggle to survive in a stressful environment, such as in the case of skin cells, continuously exposed to attacks by external agents.

The SIRT1 gene belongs to this family, and encodes for a protein of the same name, which has the task of keeping the DNA young and making sure that only the "genes of youth" are active maintaining the stability of the genome. The SIRT-1 controls the correct gene attitude in the cells of each tissue, by coordinating and optimizing cellular functions.

The SIRT-1 protein is a histone deacetylase and requires the presence of NAD+ to perform its gene regulation activity in response to changes in the redox state of the cell.

In response to a high dose of stress (UVB radiation), SIRT1 interacts with the p53 protein, causing a repression of transcription and cell apoptosis in order to prevent the proliferation of abnormal cells, whereas in response to a low dose of stress, SIRT-1 interacts with the Ku70 protein in order to inhibit cell proliferation and promote the process of DNA repair.

SIRT-1 is therefore a key regulator of defense and cell survival in response to stress and photo-aging.

SIRT-1 exerts its effect at nuclear level in all major cells of the dermis and epidermis, particularly in the basal compartment responsible for the maintenance of cell turnover (regeneration and renewal of the skin).

SIRT-1 is involved in cell division and metabolism, in DNA repair, in apoptosis, in the cell cycle, in the heterochromatin formation, in the detoxification of superoxide anion.

The sirtuins, and then SIRT-1 itself, in human skin are found both in the fibroblasts and keratinocytes, in the area between the lower epidermis, the upper dermis and the dermo-epidermal junction. About 20 youth markers are located in this area, which change with aging and are implicated in the formation of wrinkles, in loss of compactness, in uneven pigmentation, in skin dryness, and other typical signs of aging.

The basal and suprabasal epidermal cells, as well as the dermal cells, express the SIRT-1 protein in the nucleus. Furthermore, immunofluorescence studies have shown that the SIRT1 is expressed in normal epidermal keratinocytes and dermal fibroblasts cultured in vitro.

SIRT1 has an additional protective role against environmental stresses, for example by UV, and other factors of skin aging.

The induction of the production of SIRT1 in aged human skin cells by Sirtuin-activating compounds according to an aspect of the invention, causes a reduction in the expression of senescence markers and extension of life span of the same cells.

In addition, human fibroblasts treated with a Sirtuin-activator compound and irradiated with UVB radiations are characterized by a reduction of the DNA degradation as compared to control, indicating an increase in the integrity of genetic information contained in all the nuclear DNA by the action performed by sirtuins.

The increase in cell viability in cells having induced expression of SIRT-1 varies from 9 to 28% (this depends on the age of the cells) as compared to a control where the SIRT-1 is not induced.

The SIRT-1 gene plays an important role in the metabolic changes associated with aging. The SIRT-1 gene is involved in the regulation of p53 protein, a factor involved in cellular apoptosis. Under conditions of cellular stress and when DNA is damaged, the gene for p53 is acetylated and activated. SIRT-1 is capable of determining deacetylation of p53 and blocking the transcription of this gene thus extending the cellular longevity.

It was observed that human fibroblasts aged by consecutive cellular replication steps have a reduction of the sirt-1 gene and its SIRT-1 protein expression (-23% with respect to the control). Another important role performed by the extract of Myrtus communis leaves lies in its capability to limit protein glycation, a phenomenon typical of the aging process.

With protein glycation a reaction is indicated by which sugars bind to certain groups of proteins. The products of glycation, once attached to the body protein, are responsible for any damage to the tissues. In particular, the glycation reaction leads to the formation of products which form abnormal and irreversible intermolecular bridges between the macromolecules constituting the extracellular matrix (collagen). The so glycated proteins are involved in aging of the skin, after the alteration of the mechanical properties of the dermis, such as its elasticity.

To demonstrate the properties of the extract of Myrtle and its effect on cellular longevity its effect on the synthesis of SIRT-1 was quantified through determination on human fibroblasts both normal and aged.

In particular, an efficacy study was conducted by Western blot analysis on human fibroblasts normal (control) and senescent after treatment with H2O2.

Fibroblasts incubated with appropriate medium are both inoculated and not inoculated, with different concentrations of the active in question. After 4 days the cell extracts are homogenized and stored at -80°C before analyzing the SIRT-1 .

Then the Western Blot assay is executed, which allows half- quantification of the amount of SIRT-1 produced, by means of electrophoresis, immunotyping and visualization via image analysis.

The increase in the synthesis of SIRT-1 induced by the active compared to the untreated control for normal fibroblasts was as follows: 0,25% of active: + 2%

0,5% of active: + 11 %

1 % of active: + 36% The increase in the synthesis of SIRT-1 compared to the untreated control for aged fibroblasts was as follows:

0,25% of active: + 11 %

0,5% of active: + 20%

1 % of active: + 31 %

To assess the ability of galacturonic acid to intervene in the glycation process, its ability to limit the glycation reaction of collagen was determined.

Glycated and non-glycated collagen is incubated, and not incubated, with the active in question, and the reduction of the glycation process is evaluated by measuring the fluorescence emitted by glycated products, by means of a spectrofluorometer, after incorporation in the samples of a solution of glycol aldehyde.

The percentage reduction in the glycation process of collagen by the active compared to glycated collagen without active is as follows:

% active: -16%

2,5% active: -33%

5% active: -40%

According to one embodiment, the Myrtle extract contains galacturonic acid, typically at a high concentration such as between 20 and 70% by weight in the total weight of the extract, and more preferably between 30 and 50% by weight.

The experiments carried out confirmed that the extract from the Myrtus communis and particularly its main component the galacturonic acid, act on cellular mechanisms involved in the determination of cell and tissue longevity. This stimulates the expression of the longevity gene Sirt-1 and control intercellular signals, which are essential in maintaining the proliferative capacity of cells.

The biologically active ingredients of group B include Teprenone (Geranylgeranone). The Teprenone [INCI name], also known as 5,9, 13,17-Nonadecatetraen-2- one 6,10, 14, 18-Tetramethyl[IUPAC name] with CAS number 6809-52-5, is an active ingredient of synthetic origin which is commercially available or may be prepared following the procedures described, in international patent application WO/2002/003981 , the contents of which are incorporated herein by reference.

The teprenone is a biologically active compound that retards cellular aging, substantially slowing the reduction of the cell telomere length, thereby contributing to increase cellular longevity.

It was found that the action of preventing signs of skin aging, regeneration of skin cells and protection against the stress of Teprenone, is due only in part to his activity for the stabilization of telomeres, a terminal region of the chromosome, consisting of highly repeated DNA, that does not codify for any protein product, and that plays a crucial role in preventing the loss of information during the duplication of chromosomes.

Typically, the administration of the Teprenone at the skin level improves intercellular communication and delays cellular senescence while extending the life span.

This action performed by the Teprenone can be partially ascribed to a stimulating action on the synthesis of thioredoxin, a small protein with a strong antioxidant action that controls intracellular redox status, increasing cellular and tissue resistance to stress and increasing the functionality of prenylated proteins allowing increased protection of telomeres.

The active ingredients of biologically active complex may be present in varying proportions within the biologically active complex. Thus for example they may range between 0.0001 and 50% by weight of total biologically active complex.

Particularly, each of the 4 biologically active ingredients described above may be present for example between 0.0001 and 30% by weight of total biologically active complex, depending on which type of activity is to prevail on the other.

According to one aspect of the invention the biologically active complex can be dispersed in a cosmetic base or in a cosmetically acceptable vehicle to produce a cosmetic composition capable of reactivating the longevity, vitality and regeneration of the epidermis and dermis cells and/or to prolong cell longevity and assist the rejuvenation of the skin.

According to another aspect of the invention, a cosmetic composition for rejuvenation of the skin appearance is provided, which includes a biologically active complex, including the synergistic combination of

a) at least one biologically active ingredient at the level of mitochondrial DNA selected from among a) a Pentapeptide including serine, cysteine, isoleucine, asparagine, threonine amino acids, b) a hydrolized soy protein conveniently containing peptides with molecular weight lower than 5 kDa or mixture thereof

and

b) at least one biologically active ingredient at the level of nuclear DNA selected from among c) teprenone and d) an extract from

Myrtus communis or a mixture thereof.

The cosmetically effective amount of the complex of the invention to be administered depends on many factors including age, state of skin and severity of the skin damages along with the route and frequency of the administration.

Cosmetically effective amount as used herein means a sufficient amount of biologically active complex of invention to provide the desired effect. The complex of the invention is used in the cosmetic composition of this invention in cosmetically effective concentration to achieve the desired effect.

Thus, according to one embodiment, the biologically active complex of the invention may be present in a concentration ranging between 1 and 50% by weight, preferably between 3 and 35% by weight, more preferably between 5 and 15% by weight of the total weight of the cosmetic composition.

According to another embodiment, the biologically active complex of the invention is present in a concentration ranging between 0.0001 and 1 % the total weight of the cosmetic composition, preferably between 0.01 and 0.05% of the total weight of the cosmetic composition.

The dispersion of the biologically active complex with biological activity in the cosmetic base may be made by the consumer at the moment of using it.

Alternatively a cosmetic composition ready to use can be provided containing a cosmetically effective quantity of said complex, which is already dispersed in a suitable cosmetically acceptable vehicle.

According to one embodiment, the cosmetic composition of the invention comprises one or more suitable cosmetically acceptable vehicles and/or adjuvant substances, and/or cosmetic agents. Typical cosmetic agents used for the formulation of cosmetic composition of the invention include one or more substances chosen from among oils, thickeners, preservatives, water, alcohols, glycerin, stabilizers, antioxidants, antibacterial agents, humectants, emulsifiers, waxes.

According to one embodiment, the composition of the invention can be in the solid form, for example in the form of cream, for example, for direct application on the skin of the face or body, in the form of a stick to apply, for example, to lips, in the form of trans-dermal patches or make-up (foundation cream, powder, blush, eye shadow, mascara, eye pencil, lip pencil, etc.). Alternatively, the cosmetic composition can be in liquid form, for example in the form of lotion such as hydrophilic lotions, hydroalcoholic lotions, or as a cosmetic milks, oleolites, shampoo, bath foam, emulsions, suspensions, solutions applied directly or by spray. The cosmetic composition may also be in semi-solid form such as both oil in water and water in oil emulsion, serum, gel hydrophilic and lipophilic, hydrophilic or oil-based make-up removers.

Such a liquid forms can be applied directly to the skin or can be optionally dispersed into a suitable cosmetically acceptable vehicle at the moment of using it.

According to another embodiment, the cosmetic composition further comprises one or more among thickeners, preservatives, water, alcohols, glycerin, stabilizers, antioxidants and antibacterial cosmetically acceptable in quantities matching those applicable to common cosmetic formulations.

According to one embodiment, the biologically active ingredient of the biologically active complex may be present in varying amounts in the cosmetic compositions, for example, the extract from Myrtus communis may be present in the amount of from 0.0001 to 30% by total weight of the cosmetic composition, more preferably from 0.5 to 10% even more preferably from 1 to 5% by total weight of cosmetic composition.

In another preferred embodiment the extract of myrtus communis may be present in the amount ranging between 0.001 to 0.005% and preferably from 0.010 to 0.030% by total weight of the cosmetic composition.

The hydrolyzed soy protein may be present in an amount of from

0.0001 to 30% by total weight of the cosmetic composition, more preferably from 0.5 to 10% even more preferably from 1 to 5% by total weight of the cosmetic composition. In another preferred embodiment, the hydrolyzed soy protein may be present in the amount of from 0.001 to 0.010% by total weight of the cosmetic composition.

Teprenone may be present in an amount of from 0.0001 to 30% by weight, more preferably from 0.5 to 10% even more preferably from 1 to 5% by total weight of the cosmetic composition. In another preferred embodiment, the teprenone may be present in the amount of from 0.001 to 0.010% by total weight of the cosmetic composition.

The Pentapeptide according to the invention may be present in an amount of from 0.0001 to 30% by total weight of the cosmetic composition, more preferably from 0.5 to 10% even more preferably from 1 to 5% by total weight of the cosmetic composition.

In another preferred embodiment, the Pentapeptide may be present in the amount of from 0.00001 to 0.00020% by total weight of the cosmetic composition.

In the composition of the invention excipients may be incorporated typically used in the formulation of cosmetic products such as oils, glycerin, emollients, emulsifiers, dispersants in amounts typically used for cosmetic compositions.

In the case of formulations in the form of solution, suspension, lotion, or semifluid ones, water is present as a diluent or solvent, optionally mixed with other liquids used for the formulation of cosmetic compositions such as alcohols such as ethyl alcohol, glycols such as ethylene glycol.

In the formulation of cosmetic composition of the invention other active substances may be present such as vitamins, especially vitamin A, vitamin E and their derivatives, vegetal active extracts such as extracts from green tea leaves, or other cosmetically active ones such as lecithin, coenzyme Q, hyaluronic acid, fruit acids, exfoliating substances.

According to another aspect of the invention, there is provided the use of a cosmetic composition previously described in the treatment of aged skin that typically presents with wrinkles, tonus and smoothness loss, dull, lower density of the dermis and sagging skin.

According to another aspect, the present invention provides a method of cosmetic treatment comprising the application on the skin of a cosmetically active and acceptable quantity of a cosmetics composition for the rejuvenation of the skin appearance comprising the synergistic combination of A) at least one biologically active ingredient at the level of mitochondrial DNA consisting of Pentapeptide including serine, cysteine, isoleucine, asparagine, threonine amino acids, a hydrolized soy protein or a mixture thereof, with B) at least one biologically active ingredient at the level of the nuclear DNA consisting of Teprenone, a vegetal extract of Myrtus communis or a mixture thereof.

Biological data

The in vitro studies were performed using the following biologically active complex compositions according to the invention.

Composition 1

Myrtus communis leaf extracts 0.5%

Hydrolyzed Soy Protein 0.5%

Pentapeptide 0.5%

Teprenone 0.0025%

Composition 2

Myrtus communis leaf extracts 1 .0%

Hydrolyzed Soy Protein 1 .0%

Pentapeptide 1 .0%

Teprenone 0.0025%

Composition 3

Myrtus communis leaf extracts 2.0%

Hydrolyzed Soy Protein 2.0% Pentapeptide 2.0%

Teprenone 0.0025%

Composition 4

Teprenone 0.0025%

The in vitro studies were conducted in cultured human fibroblasts derived from skin, not senescent (negative control), and fibroblasts for which cellular aging has been induced experimentally, through repeated exposure to two damaging agents, such as: H₂O₂ and UV radiation.

Following a treatment of these cultures with the above compositions of active complex being tested, the protective effectiveness against the damaging agents was assessed by an analysis of cell viability and the functionally of the cells anabolism function (collagen synthesis) and in decreasing the oxidative damage to cellar membranes (lipoperoxidation) which all are marker of cellular aging. The results were compared with the results obtained from the positive control cells (senescent cells, treated only with the damaging agent) and that with untreated control cells (normal cells, negative control).

The results of cells viability study, showing a mean increase of cell viability of senescent cell cultures treated with composition 1 -4 and of non-senescent fibroblast compared to untreated senescent cell cultures (CTR H₂O₂ and CTR UV), are illustrated in Table 1 .

These results show that the treatment with the tested compositions protect the cell by the cell mortality induced by the damage agents (i.e. H₂O₂ and UV).

TABLE 1 Cell Viability Study

Tested product VS. CTR H₂O₂ vs. CTR UV

Non-senescent fibroblasts + 26.58% + 25.71 % Composition 1 + 27.61 % + 27.54%

Composition 2 + 35.07% + 28.26%

Composition 3 + 44.78% + 34.78%

Composition 4 + 20.15% + 20.15%

The results of collagene study, showing a mean increase of collagene synthesis of senescent cell cultures treated with composition 1 -4 and of non-senescent fibroblast compared to untreated senescent cell cultures (CTR H₂O₂ and CTR UV), are illustrated in Table 2.

These results show that the treatment with the tested compositions protect the cells by the collagen synthesis decrease induced by the damage agents (i.e. H2O2 and UV). TABLE 2 Collagene synthesis study

The results of study of cell aging (LPO), showing a mean increase of lipoperoxidation level of senescent cell cultures treated with composition 1 -4 and of non-senescent fibroblast compared to untreated senescent cell cultures (CTR H2O2 and CTR UV), are illustrated in Table 3

These results show that the treatment with the tested compositions protect the cells by lipid peroxidation induced by the damage agents (i.e. H2O2 and UV). TABLE 3 Cell Viability Study

Furthermore the above results also have shown that the response of senescent cell cultures to the treatment with compositions 1 to 3 was higher than senescent cell response to treatment with composition 4, made up of only Teprenone.

Thus, the above results provide evidence for a synergistic effect between the four components (vegetal extract of Myrtus Communis, Hydrolyzed soy protein, Pentapeptide, Teprenone) present in the compositions 1 to 3, in comparison with the sole Teprenone present in composition 4.

Clinical Instrumental Study

The efficacy of the cosmectic treatment of the present invention in attenuating the signs of skin ageing was dimostrated by a clinical- instrumental study carried out on 30 healthy female (age between 40 and 55 years old) showing clinical signs of skin ageing due to chronological or photoageing. After 8 and 30 days of treatment the product efficacy was evaluated using non invasive bioengineering technique to quantify: wrinkledness, elasticity and skin brightness . The instrumental analysis is integrated with the clinical analysis carried out by the dermatologist.

Brightness parameter was evaluated by examining the histogram of colour dispersion using AdobeR PhotoshopR (ver. 7.0). The image analysis was performed on the images taken before and after treatment. The photographic images were taken using a professional digital reflex camera NIKON D300 digital camera (Nikon Corporation Tokyo, Japan) equipped with a macro-objective (AF-S) Micro NIKKOR 60mm f/2.8G ED, Nikon Corporation Tokyo, Japan) and a flash system (Kit R1 C1 , Nikon Corporation.

Skin elasticity measurement was based on the suction/elongation method and the subsequent release of the skin inside the opening of the instrument device. During the suction/elongation phase the instrument generates, in fact, constant negative pressure (450 mbar) able to aspirate the skin inside the measurement probe. The suction phase was followed by the release phase, in which the pressure inside the probe is switched to 0 mbar allowing the skin recovery after the elongation phase. An optical measurement system, evaluates the depth of the skin inside the probe in the two phases of the measurement, the obtained data are then elaborated and showed graphically and numerically in order to calculate the viscoelastic properties of the skin. The instrument used for measuring is the CUTOMETERR MPA 580 (Courage+Khazaka, electronic GmbH).

Skin thickness (epidermidis+dermis) was evaluated by means of high frequency ecography. The evaluation is based on the physical principle of high frequency ultrasound emission by a transducer. Ultrasound emission was discontinuous: firstly the transducer emits the ultrasounds and after then it detect the reflected echoes before to proceed to further emission. When the ultrasound beam reaches the skin, it crosses regions structurally different, the beam was then partially transmitted and partially reflected at the interface between adjacent structures. Are so generated echoes of different amplitude that the transducers convert in an electrical signal that intensity was evaluated by a microprocessor and visualized on a display. The use for 30 days of the cosmetic treatment containing the active complex of the invention as described in the Example 5 determined an eutrophic effect for the skin of the healthy voluntary. The results of the clinical instrumental studies, showing the improvement of the skin elasticity in 93.3% of the subjects with a mean variation of +12.5% and lightness in 100% of subject with a mean variation of +10.6%, are illustrated in Figure 1 .

To this eutrophic effect followed an amelioration of the aesthetical aspect of the superficial skin relief. Infact, the profilometric analysis shows an improvement of the skin smoothness (in 100% of subjects, meanvariation: +20.2%) and a decrease of both the skin wrinkledness (in 100% of subjects, mean variation: -10.4%) and the wrinkle volume (in 100% of subjects, mean variation: -16.0%). Consumer test

The perceived efficacy of a cosmetic anti-ageing treatment according to the invention to make face skin younger, to reduce wrinkle visibility and to make skin brighter and more compact was evaluated by a Consumer test. Volunteers received a cosmetic composition containing the complex of the invention as described in the Example 5 and questionnaire with specific questions on a range of skin parameters. Volunteers were instructed to compile the questionnaire after the observation of their own skin to understand if their skin was benefited from the treatment after both 24 hours of product application and a period of 8, 20 and 30 days of product use.

The subjects expressed their opinion which are summarised in the graph of Figure 2. Particularly skin brighter in 99% of the subjects, skin is more elastic in 94% of the subject and more smoother in 94% of the subjects. 53% of the subjects also recorded that the wrinkles are reduced and the 67% of the subjects declared the treatment have anti-aging efficacy.

The following examples are provided merely for illustrative purposes of the present invention and should not be construed by limiting the scope of protection, as apparent from the attached claims.

In the following examples, unless otherwise stated the ingredients

1 ) "Extract of leaves of myrtrus communis" means hydrolyzed myrtus communis leaf extract

2)"Pentapeptide" means Pentapeptide-28

Examplel

A biologically active complex to be dispersed in a suitable cosmetically acceptable vehicle.

Example 2

Cosmetic composition containing the biologically active complex having the following formulation:

WATER just enough

to 100

CYCLOPENTASILOXANE 28.0%

GLYCERINE 3.0%

SILICONES 3-4%

EMULSIFIERS 2.5-4%

XYLITOL 1.0%

STEARIC ACID 0.8%

LIMNANTE OIL 0.8%

VITAMIN E 0.4%

EXTRACT OF LEAVES OF MYRTUS 0.0001-30%

COMMUNIS

TEPRENONE 0.0001-30%

A HYDROLIZED SOY PROTEIN 0.0001-30% PENTAPEPTIDE 0,0001-30%

SODIUM CHLORIDE 0.2%

ALLANTOIN 0.1 %

LECITHIN 0.1 %

HYALURONIC ACID 0.07%

XANTHAN GUM 0.07%

SHEA BUTTER 0.10%

SILICA 0.05%

GREEN TEA 0.01 %

HYDROXYETHYLCELLULOSE 0.01 %

PERFUME just enough

SOLUBILIZERS just enough

CHELATORS just enough

PRESERVATIVES just enough

ANTIOXIDANTS just enough

Example 3

A cosmetic composition containing the biologically active complex for direct application to the skin or dispersible in a suitable cosmetically acceptable wehicle having the following formulation:

GLYCERYL STEARATE 5.00%

Cetearyl (12) OE 1-5 %

POLYSORBATE 80 0.5-1 %

CETYL ALCOHOL 1-3 %

OCTYLDODECANOL 1-3 %

Ce-10 TRIGLYCERIDES 10-20 %

DIMETHYLPOLYSILOXANE 0.01-0.1 %

WHEAT GERM OIL 0.8 %

TOCOPHERYL ACETATE 0.1 %

EXTRACT OF LEAVES OF 0.0001-30%

MYRTUS COMMUNIS

TEPRENONE 0.0001-30%

PENTAPEPTIDE 0.0001-30%

HYDROLIZED SOY PROTEIN 0.0001-30% ANTIOXIDANTS just enough

PRESERVATIVES

WATER just enough to 100

Example 4

A cosmetic composition containing the biologically active complex application to the skin of the face having the following formulation

Example 5

A cosmetic composition containing the biologically active complex for direct application to the skin of the face or dispersible in a suitable cosmetically acceptable vehicle having the following formulation:

WATER just enough to 100

PPG-1-PEG-9 LAURYL GLYCOL

ETHER 0,36000-1 ,80000

IMIDAZOLIDINYL UREA 0, 17600-0,88000 GLYCERIN 0.11400-0,57000

XANTHAN GUM 0,08000-0,40000

CAPRYLIC/CAPRIC TRIGLYCERIDE 0,07056-0,35280

TOCOPHERYL ACETATE 0,02000-0, 10000

PENTAPEPTIDE-28 0,00002-0,00010

HYDROLYZED MYRTUS COMMUNIS LEAF EXTRACT 0,00672-0.03360

HYDROLYZED SOY PROTEIN 0,00190-0,00950

TEPRENONE 0,00144-0,00720

METHYLCHLOROISOTHIAZOLINON

E 0,00034-0,00168

METHYLISOTHIAZOLINONE 0,00011-0,00058

Claims

1 . A biologically active complex characterized in that it comprises:

A) at least one biologically active ingredient at the level of mitochondrial DNA selected from a) a pentapeptide including serine, cysteine, isoleucine, asparagine, threonine amino acids, or b) a hydrolyzed soy protein or a mixture thereof

and

B) at least one biologically active ingredient at the level of nuclear

DNA selected from c) teprenone or d) an extract from Myrtus communis or a mixture thereof.

2. A biologically active complex according to claim 1 , comprising a) a pentapeptide including serine, cysteine, isoleucine, asparagine, threonine amino acids, b) a hydrolyzed soy protein, c)

Teprenone, and d) an extract from Myrtus communis.

3. A biologically active complex according to claim 1 or 2 wherein said hydrolyzed soy protein includes Phenylalanine, Isoleucine, Leucine, Lysine, Methionine, Threonine, Tryptophan, Valine amino acids.

4. A biologically active complex according to any of claims 1 -3 wherein said vegetal extract from Myrtus communis contains the

galacturonic acid.

5. An anti-aging cosmetic composition for skin application comprising a biologically active complex according to any of claims 1 -4 in a cosmetically effective quantity and in a cosmetically acceptable vehicle.

6 A topical cosmetic composition according to claim 5 in a solid, semi-fluid or fluid form.

7. Use of a complex or a cosmetic composition according to any of claims 1 -6, for the cosmetic anti-aging treatment of the skin.

8. The use according to claim 7 to regenerate, revitalize and increase longevity, repair or protect the skin cell layers.

9. The use in accordance with claim 8 to prevent or delay aging and photoaging of the skin.

10. A cosmetic treatment for skin care comprising the application to the skin of at least one of a cosmetic composition comprising a biologically active complex according to any of claims 1 to 4.