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Field of the invention

The present invention relates to a composition for the prevention and treatment of signs of skin aging.

The present invention relates to the nutraceutical and cosmetics sector and aesthetic dermatology.

Specifically the present invention relates to a composition for preventing or treating the signs of skin aging resulting from oxidative stress in cells.

Background

The aging of the human body is a natural gradual degenerative type process mainly caused by a combination of factors of genetic and environmental origin. Some factors or events such as the development of diseases, taking medicinal products and some unnecessary behaviour can accelerate the decay of the organism.

Some tissues, such as the epidermis, are more subject than others to the harmful action of environmental agents, mainly exposure to solar radiation and therefore they reveal the signs of aging of the organism more greatly.

Typically, over the years, the cells of the epidermis gradually lose their ability to proliferate, causing the skin to become thinner. This phenomenon is accompanied by a loss of elasticity and a thickening of the outer keratin layers whereas the slowing of cell proliferation causes an imbalance in the physiological water evaporation process and a reduction in collagen content.

These phenomena can be exacerbated by prolonged exposure to solar radiation in the absence of suitable protection with solar products and cause the early onset of wrinkles which in some cases can be accompanied by solar purpura and possibly precancerous lesions or tumours such as melanoma.

In relation to exposure to the sun’s rays, it has been found that exposure to UVB rays mainly causes damage to the more superficial layers of the skin. A typical example of this damage also associated with burning is presented in cases of sunburn. Vice versa, UVA rays have a greater capacity to penetrate into the skin’s layer and their exposure can cause early aging of the skin and the appearance of actinic or precancerous lesions.

Numerous studies have demonstrated how the exposure of the skin to UV radiation, in the absence of sun screens, determines the formation of highly toxic products such as free radicals and a reduction of the functionality of the epidermal stem cells themselves.

The presence of excess free radicals can also cause severe damage to the DNA, to the cell membranes, to the lipids and proteins of the skin’s stem cells and their progeny. In these conditions, the self-regeneration capacity of the epidermal structures is compromised with the consequent acceleration of skin aging.

It has also been demonstrated how photoaging and carcinogenesis mechanisms in the skin are the direct result of the effect of solar radiation. UV rays are in fact able to generate reactive oxygen species (ROS) and thus alter cellular homeostasis. These effects in turn alter the signal transduction pathways and the inflammatory cascade and induce the remodelling of the extracellular matrix (ECM) which contributes to making the skin lose elasticity. Therefore, exposure to UV radiation constitutes the main cause of oxidative stress and one of the main causes of skin aging.

Oxidative stress is determined by an imbalance between the production of ROS and their neutralization by the antioxidant systems of the cells. Non-neutralized ROSs promote different types of cell damage: lipoperoxidization which damages the cell membranes, the alteration of the structure and functionality of many enzymes, as well as promoting the oxidization of carbohydrates.

In response to the attacks of the ROSs, the skin has a protective system constituted by endogenous antioxidants of the enzymatic and non-enzymatic type. However, the cutaneous system of antioxidants becomes less efficient during aging and however is not able to stop continuous aggression from external agents, such as solar rays.

In an attempt to correct these phenomena, formulations have been prepared containing antioxidant substances.

For example, the use of preparations containing substances with antioxidant activity such as, for example, vitamin C and vitamin E or epigallocatechin gallate is known for the purpose of preventing or reducing skin damage induced by UV rays.

However, these substances have not provided particularly positive results from the cosmetic and aesthetic point of view.

There is currently a perceived need to provide preparations that prevent or reduce aesthetic damage to the skin caused by early chronological aging or by excessive or prolonged exposure to sunlight.

One of the aims of the invention therefore consists in providing a combination of active substances that are effective in the reduction of the signs of skin aging such as wrinkles or loss of elasticity of the skin.

Another aim of the invention consists in providing a composition for the prevention and treatment of the signs of skin aging that can be applied topically or administered orally whose use is almost free of side effects.

Summary of the invention

Within the scope of the technical field of the invention, the Applicant has found that by combining one or more active components available from a Withania somnifera extract with L-proline, an antiaging effect and a reduction in the typical signs of skin aging are obtained.

The inventors have also observed that the combination of biologically active components present or extracted from Withania somnifera with L-proline prevents or reduces oxidative stress in cells.

In view of the above aims, the present invention provides, according to a first aspect thereof, the non-therapeutic use of a composition comprising a Withania somnifera extract or withanolides in combination with L-proline or proline for reducing or delaying an aesthetic damage of the skin or signs of skin aging.

Within the scope of the applications in the cosmetic or aesthetic field, the composition of the invention is specifically indicated in the prevention or treatment of signs of skin aging, in particular the inherent signs caused by exposure to ultraviolet radiation, in particular solar radiations.

In accordance with this aspect, the composition of the invention can be specifically used in preventing or treating skin damage caused by prolonged exposure to solar radiations that determine early skin aging. Brief description of the figures

The characteristics and advantages of the present invention will more fully emerge from the appended figures wherein:

Figure 1 illustrates bar graphs representative of the effects on gene expression, named in ordinate as Fold Change, of some significant markers for aging and skin inflammation, in particular MMP2 and S100A7A, of L-proline (15mg/ml) of Withania somnifera (15 mg/ml) individually and of the combination thereof (15 mg/ml) on a 3D skin model, according to the test of Example 6.

Figure 2 illustrates a flow-chart of an embodiment of the production process of a powdered extract from a Withania somnifera plant as described in Example 7.

Detailed description of the invention

The present invention originates from the finding that by combining a plant extract of Withania somnifera with L-proline or proline, a synergistic action on the main skin markers of a skin model is obtained. This action makes it possible both to prevent/treat aesthetic skin damage and to prevent/treat damage to the epidermis caused by exposure to ultraviolet radiation.

Typically, the composition of the invention is therefore indicated in a non-therapeutic context in preventing and/or treating early skin aging, aging due to exposure to solar rays, in particular due to oxidative stress.

According to a first aspect of the invention, the use of a composition is provided as defined in the appended claim 1 .

Embodiments of the uses of the composition of the invention are defined in the appended dependent claims 2-10.

According to these aspects, the composition of the invention can be applied in delaying skin aging and/or signs of skin aging in particular following exposure to ultraviolet radiation.

According to another aspect, the invention relates to a composition comprising withanolides or an extract of Withania somnifera in combination with L-proline for use in reducing or delaying skin damage caused by exposure to ultraviolet radiation in particular the sun’s rays. According to this aspect of the invention, the composition is a pharmaceutical composition for use in therapy for preventing or treating skin diseases caused, originating or triggered by exposure to ultraviolet radiation, in particular dermatological affections selected from actinic lesions, precancerous skin lesions or a non-melanoma form of skin cancer.

The composition of the invention may be used in the prevention or non-therapeutic treatment of aging due to exposure to solar rays.

Furthermore, the composition of the invention may be used in preventing or treating signs of skin aging due to repeated exposure over time to ultraviolet radiations such as, for example, skin folds, wrinkles, skin thickening, skin sagging, dehydration and creasing of the skin on the body and in particular the face.

Typically, the composition of the invention may be used, both for uses in the medical field and non-therapeutic ones, in the treatment of skin damaged by exposure to UBA, UVB or UVC rays and in general in photoaging.

By way of example, UV-A radiation means radiation with a wavelength of 315 to 400 nm; UV-B radiation means radiation with a wavelength of 280 to 314 nm; UV-C radiation means radiation with a wavelength of 100 to 279 nm.

The biologically active components present in the composition both for medical and non-therapeutic use are the same. These components and other aspects of the composition of the invention are described in detail below.

In the composition of the invention a Withania somnifera extract or withanolides contained in this plant can be used.

Withania somnifera is a plant that belongs to the Nightshade family, Withania genus, with the common name of Ashwagandha. The plant and its extracts contain biologically active substances, mainly present in the roots among which the most representative or most active substances are non-steroidal lactones known as withanolides.

In the formulation of the composition of the invention it is preferable to use a Withania somnifera extract or alternatively withanolides which from a chemical point of view are typically steroidal lactones with the skeleton of ergostane present in the Withania somnifera plant.

By way of example, a Withania somnifera extract can contain from 0.5% to 15%, from 1 to 10% from 2 to 7% by weight of withanolides (drug) expressed as a drug/extract ratio.

Preferably, the withanolides described herein are of plant origin, however they may also be of synthetic origin, i.e. obtained by means of a chemical synthesis process. Suitable withanolides comprise withaferin A, Withanolide I, II, III, A, D, E, F, G, H, I, J, K, L in particular withanolide D, isopelletierine (alkaloid), Anefrin (alckaloid), Salpichrolide A, Nicandrenon-1 , Ixocarpalactone A and mixtures thereof.

Within the scope of the applications of the invention, withaferin A, withanolide D and the mixture thereof are suitable, in particular obtained from a Withania somnifera extract according to an embodiment described herein.

For the purposes of the present invention, a suitable vegetable/plant extract of Withania somnifera can be derived or extracted from the roots, leaves, fruits, berries or flowers of Withania somnifera or from two or more of these parts of the plant, preferably from the roots.

Preferably the vegetable plant extract described herein originates or is obtained from the roots of Withania somnifera and is advantageously in the form of powder, typically yellow/light brown coloured.

In some embodiments the Withania sominifera extract is in powdered or ground form, with powders/granules preferably with particle size passing through a 40 mesh sieve > 95% (p/p) or passing through an 80 mesh sieve > 85% (w/w). Examples of particles of Withamnia somnifera extract are reported in the following Examples 7 and 8.

Typically, for the conversion of the particle sizes into mesh, reference can be made to the following publication: https://www.sigmaaldrich.com/chemistry/stockroom- reagents/learning-center/technical-library/particle-size-con version.html.

The plant extract of the invention can be obtained by extraction from a part of the plant, in particular the roots using as the extraction means a physiologically acceptable or edible solvent. Within the scope of the invention, the term“edible” means a physiologically acceptable solvent that can be used or administered to a human being.

A suitable solvent for obtaining the plant extract is a physiologically acceptable and/or edible liquid in which the biologically active components are soluble and wherein they do not undergo any significant alteration such as to compromise the biological activity.

A suitable extract is obtained through aqueous extraction or organic extraction using ethanol or a hydro-alcoholic mixture.

In some embodiments the solvent is hydrophilic and is selected from water, ethanol, ethyl acetate or mixtures thereof and is preferably water.

It is possible to obtain a plant extract from Withania somnifera by resorting to conventional extraction techniques, e.g. by grinding in water or ethanol water mixture preferably while stirring or by resorting to suitable solid-liquid techniques suitable for separating/extracting one or more biologically active components from the plant tissues of the plants and of their fruits.

In certain embodiments the extraction of one or more biologically active components takes place by grinding a plant matrix or portion in a suitable solvent, e.g. a hydroalcoholic mixture.

For example, a suitable extraction envisages that the Withania somnifera roots are ground to provide a powder from which the extraction is performed using a suitable solvent such as water or a water-ethanol mixture, or ethanol, for an amount of time suitable to enrich the solvent with one or more biologically active components, e.g. 10 minutes. The extract can be sonified. And centrifuged for example at 3000 rpm for 4-6 minutes. The extract obtained is collected and can be diluted with water, alcohol, typically ethanol or a water-ethanol mixture, for example to obtain a final volume of 10 ml if starting with 1 gram of initial powder.

The extraction of the solvent of the biologically active components present in the plant tissues of the plant can thus take place by diffusion and osmosis.

The extract obtained from Withania somnifera may be fluid, soft or dry. For example, in the fluid extract 1 ml of extract contains biologically active components soluble in 1 g of herbal drug, in the soft extract the solvent is partially evaporated in particular until the extract wets a filter paper, in the dry extract the solvent is almost completely evaporated to obtain a powder.

In certain embodiments the extraction takes place using a weight ratio between solvent and plant matrix comprised between 1 : 10 and 10: 1 .

Further methods for obtaining Withania somnifera plant extract for the uses of the invention include extraction techniques through digestion, infusion, pressing, decoction, percolation, counter-current extraction, Soxhlet extraction, extraction with supercritical gases or ultrasound.

Within the scope of the invention, the term plant extract denotes an extract obtained by extraction from Withania somnifera or withanolides contained therein.

A further component of the composition of the invention is the proline of a non-polar amino acid also known as 2(S)-pyrrolidine-carboxylic acid. In particular, in the scope of the invention L-proline is used. As an alternative to proline or L- proline it is possible to use the hydroxyproline identified in the lUPAC as (2S,4R)-4- hydroxypyrrolidine-2-carboxylate.

The Applicants have unexpectedly observed that the combination of a Withania somnifera extract or withanolides contained therein with L-proline has anti-aging activity which at epidermal level manifests itself with an improvement to the appearance of the treated skin. This cosmetic or aesthetic effect manifests itself both in the case of topical skin treatment with the combination of Withania somnifera/withanolides and L-proline and in the case of oral administration of the combination or a composition that contains it.

The composition of the invention can therefore be intended for external use and for oral administration.

The composition of the invention can be authorized for sale as a cosmetic product for local application, or as a drug, a dietary or nutritional supplement intended for oral administration.

Typically, the composition of the invention comprises a carrier, diluent or physiologically and/or cosmetically acceptable excipient.

Typically, the physiologically or cosmetically acceptable carrier of the composition of the invention is an excipient, carrier or diluent suitable for topical application and/or oral administration.

Within the scope of the present invention the term“carrier” refers to an excipient, carrier, diluent or coadjuvant that may be present in the composition of the invention. Any carrier and/or excipient suitable for the desired form of preparation for administration is contemplated in the uses of the plant extract or active ingredients present therein described here. In certain embodiments, the composition of the invention contains one or more components of plant origin that are biologically active and substantially free from side effects, when administered orally or locally.

The carrier, diluent or physiologically or pharmaceutically acceptable excipient may be selected on the basis of the administration route for which the resulting pharmaceutical composition is intended.

According to some preferred embodiments the composition may be in the form for oral administration.

The compositions for oral administration may be in solid or liquid form. Typical compositions in solid form comprise tablets, capsules, powders, granules and pills. Typical compositions in liquid form comprise solutions, emulsions, suspensions and syrups. All the compositions also comprise controlled release forms thereof.

A preferred embodiment of the composition is the one in tablet form.

The tablets generally comprise a suitable carrier or excipient in which the plant extract is dispersed, typically in dry form.

In this case suitable excipients contained in the formulation are cellulose and derivatives thereof such as hydroxymethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxyethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl cellulose, cellulose acetate butyrate, cellulose acetate phthalate, and mixtures thereof.

Further examples of suitable excipients comprise polymers that are part of the family of lactam family such as pyrrolidone and derivatives thereof, for example polyvinylpyrrolidone, polyvinylpolypyrrolidone and mixtures thereof, inorganic salts such as calcium or dicalcium phosphate, lubricants such as for example magnesium stearate, triacylglycerides and mixtures thereof.

The tablet can further comprise one or more of the following excipients: bulking agents such as calcium phosphates, stabilizers such as crosslinked sodium carboxymethylcellulose, coating agents such as polyvinyl alcohol, polyethylene glycol, talc, anticaking agents such as magnesium salts of fatty acids; silicon dioxide.

The two biologically active synergistic components contained in the composition of the invention may be present in variable quantities, e.g. comprised from 0.0001 % by weight to 50% by weight, from 0.1 % by weight to 20% by weight, typically from 0.5 to 5% by weight.

According to certain embodiments, the composition of the invention further comprises one or more active ingredients such as vitamins, minerals, micronutrients and other active ingredients.

For example, biologically active suitable substances that can be included in the formulation of the composition are selected from the group comprising glycine, Moringa seeds or extract, N-acetylglucosamine, niacin, fermented Goji hyaluronic acid and mixtures thereof.

In particular, it was observed that N-acetylglucosamine increases the synergistic effect of antiaging and/or reduction of oxidative stress in the cells of the two basic components of the composition of the invention.

According to some embodiments the composition for oral administration is a functional food, a nutraceutical composition, a dietary product, a supplement or nutritional product or a medical device.

Functional food means any modified food or food ingredient that can provide a benefit or protection against a drawback or physiological affection, in addition to the traditional nutrients that it contains.

Nutraceutical product means an isolated or purified product from edible substances. A nutraceutical is such when it is demonstrated that it has a physiological benefit or that it provides protection against a drawback or physiological disorder.

Dietary or food supplement means a product that contains a vitamin, mineral, plant extract, amino acid, metabolyte, extract, concentrate or mixture of these ingredients. The amount administered and the frequency of administration of the composition will depend on the nature and gravity of the affection to be treated.

In some embodiments the administration route of the composition of the invention is the topical one. According to these embodiments the composition is for topical use and is applied to the skin.

The composition for topical application may be in solid, semi-solid or fluid form. Suitable formulations in solid form include creams, balms, pastes, ointments.

In some embodiments the formulation for local administration is in fluid form, e.g. in the form of lotions, jellies, shampoos, suspensions or semi-fluid e.g. in the form of gels and emulsions.

In some embodiments the compositions of the invention may comprise excipients commonly used in the formulation of cosmetic or pharmaceutical preparations, for local use, such as preservatives, anti-bacterial agents, stabilizers, emulsifiers, buffers, wetting agents, dyes and other excipients commonly used in cosmetic/pharmaceutical preparation techniques.

In one embodiment the formulation for local application is in emulsion form containing the extract carried in a suitable excipient. In some embodiments the composition for topical application comprises an excipient of the hydroxymethyl cellulose type and/or gelling agents suitable for the formulation and for the substances.

In applications in the cosmetic area, e.g. in preventing or treating skin aging consequent to exposure to UV rays, it is possible to apply an active cosmetic quantity of the composition of the invention to the affected area of skin once or more times a day, conveniently for a period of at least 2-3 months.

According to another aspect of the invention a cosmetic treatment method is provided which comprises the application onto the skin of an effective quantity of a composition for topical use as previously described.

The present invention will now be described making reference to the following examples that will be provided solely for illustrative purposes and must not be considered in the limiting sense of the present invention.

EXAMPLE No.1

Composition in tablet form containing the following active substances

Glycine 150 mg

L-Proline 150 mg

Withania somnifera L. Dunal root dry extract 150 mg

Of which Withanoids 3.75 mg

N-acetylglucosamine 100 mg

Moringa oleifera dry extract 100 mg

Niacin as niacinamide 54 mg

Goji (Lycium barbarum L.) dry extract 50 mg Hyaluronic acid 30 mg

Biotin 50 pg

Calcium phosphate 150-250 mg

Microcrystalline cellulose 100-150 mg

Crosslinked sodium carboxymethylcellulose 5-40 mg

Plant-based magnesium stearate 5-20 mg

Silicon dioxide 4-10 mg

EXAMPLE No.2

Tablet

L-Proline 200 mg

Withania somnifera L. Dunal root dry extract 200 mg

Of which Withanoids 5 mg

Biotin 300 pg

Hydroxy-propyl-cellulose . 10-70 mg

Crosslinked sodium carboxymethylcellulose . 10-60 mg

Calcium phosphate . 50-200 mg

Microcrystalline cellulose . 20-200 mg mono- and diglycerides of fatty acids . 5-14 mg

Silicon dioxide . 3-12 mg

EXAMPLE No.3

Soft gelatin capsule

L-Proline 50 mg

Withanolid A 5 mg

Soy oil ( Glycine max L.) . 100-400 mg

Soy lecithin ( Glycine max L.) . 5-10 mg mono- and diglycerides of fatty acids . 5-30 mg

Composition of the shell:

Gelatin . 20-145 mg

Glycerol . 5-70 mg EXAMPLE No. 4

Hard gelatin capsule

L-Proline 75 mg

Withania somnifera L. Dunal root dry extract

Titrated in Withanolid A - no less than 3% 100 mg

Maltodextrin . 5-200 mg

Silicon dioxide . 3-6 mg

Plant-based magnesium stearate . 1 -10 mg

Hard gelatin capsule . 1 capsule

EXAMPLE No. 5

Anti-aging face mask

INCI %

Water to reach 100

Sodium Benzoate 0.1 -1

Glycerin 5-10

Withania somnifera Root Extract 1 -5

Proline 1 -5

C10-18 Triglycerides 1 -5

Hydrogenated Vegetable Oil 1 -5

Octyldodecanol 1 -5

Octyldodecyl Myristate 1 -5

Caprylic/Capric T riglyceride 1 -5

Dimethicone 1 -5

Aluminum Starch Octenylsuccinate 1 -5

Cetyl Alcohol 1 -3

Glyceryl Stearate 1 -3

Phenoxyethanol 1 -3

Propylene Glycol 1 -3

Decylene Glycol 1 -3

Peg-75 Stearate 0.1 -1

Ceteth-20 0.1 -1 Steareth-20 0 1 -1

Parfum 0.1 -0.5

Dimethylmethoxy Chromanol 0 01 0.1 EXAMPLE No. 6

Comparative experimental tests

1. AIM OF THE COMPARATIVE EXPERIMENTAL TESTS

The following experimental procedure is a comparative study of the anti-aging activity through in vitro tests on full thickness skin (Henkel) of Withania somnifera and L-Proline.

Specifically, a comparison was performed on the efficacy of the individual active ingredients Withania somnifera and L-proline with respect to the combination thereof.

For further details, please refer to paragraph 2.1.

2. MATERIALS

2.1 Samples tested

2.2 Reactants and instruments used

2.3 Biological model used

The biological model used is represented by the Phenion ® Full-Thikness (FT) Skin Model supplied by Henkel. It is a full-thickness model obtained from the co-culture of keratinocytes and fibroblasts coming from a single donor, which simulates the histological and physiological properties of human skin. The model is presented in the form of a 1 .3 cm circular disc kept in culture for at least 10 days and that allows repeated treatments.

The Phenion® skin model comprising different types of cells, derived from human skin. The fibroblasts are cultivated within a collagen framework and form the connective tissue that will act as a base for the overlying layers of epithelial cells to mimic the in vivo conditions. During maturation the de novo synthesis of collagen and of the elastic matrix can be observed, consisting of proteins such as elastin and fibrillin-1 . The differentiation of the cells within the stratified multi-epithelium can be viewed through the detection of specific markers such as cytokeratin 10, filaggrin, involucrin and transglutaminase.

The expression of collagen IV and laminin in the borderline between compartments confirms the integrity of the basal membrane.

3. MATERIALS

The FT inserts (Phenion ® Full- Thickness (FT) must be treated in sterile conditions. Immediately after arrival the FT inserts were removed from the semi-solid transport medium according to the guidelines reported below.

By following these instructions, it is possible to cultivate the inserts in the air-liquid interface (ALI) up to 9 days after arrival.

3.1 Day 1 :

Seeding of inserts

a) Position in a Petri (100/20mm) 2 petri dishes (35/10mm);

b) Add a spacer filter inside each small Petri dish;

c) Distribute into each Petri dish 4 mL of ALI medium (brought to the temperature of 37°C, in a thermostated bath);

d) Position a Wartman paper filter on the spacer filter (the filters are supplied sterile and appropriately trimmed). The filters must be in contact with the culture medium but not be surmounted by it; e) Remove the cap placed to protect each insert for transport;

f) Take out the insert with the aid of two grippers and transfer it on top of the Wartman paper filter in the Petri-dish; the transfer causes slight mechanical stress therefore the insert must be handled with extreme care;

g) Close the 100/20 mm Petri-dish with the lid and transfer to an incubator at 37°C, 5% CO2;

The inserts normally reach Friday (6th day of differentiation). They are seeded as reported above and used for treatment on Monday (9th day of differentiation).

3.2 Day 2: Treatment with formulations of interest

The protocol envisages the stimulation of the FT inserts with Triton-X (45mg/ml_) in order to induce system inflammation.

a) Preparation of a stock solution of Triton-X (45mg/ml_) in ALI-medium;

b) Dilution of the stock in ALI-medium at the final concentration of 0.18mg/mL.

Validated protocols recommend the induction of inflammation with Triton-X in the amount of 0.12mg/cm ² .

c) Collect the inserts from the incubator and check their integrity;

d) Remove the waste medium from each Petri dish with the aid of an aspirator; e) Distribute 25uL of triton-X 0.18,g/mL solution on the insert (in duplicate). On the control inserts (negative control), distribute 25uL of ALI medium;

f) Distribute on each insert, with the aid of a pipette of each formulation 25pL.

Incubate for 24h at 37°C, 5% CO2.

3.3 Day 3: 1 st extraction step of the RNA and collection of the supernatants.

a) At the end of the incubation appropriately initial the Eppendorf tubes for the collection of the supernatants and the cryvials for the collection of a part (half) of the insert, which will be used for the subsequent analyses;

b) With the aid of an aspirator, gently aspirate the medium from each Petri-dish. c) Gently rinse each insert with 2m L of PBS from the top of the insert using a pipette. Repeat the operation twice;

d) Gently dry the insert on a piece of Wartman paper and split it into two portions with the aid of two sterile scalpels; e) Cut the second half further using the scalpel and incubate for about 3h in 1 5ml of Tri-reagent in ice for the extraction of the RNA;

f) At the end of the incubation in Tri-reagent homogenize the sample for about 2 minutes at 24,000rpm;

g) Transfer the homogenate into appropriately initialled eppendorf RNAse free tubes;

h) Add 300pL of Chloroform, vortex the Eppendorf 15” to promote phase inversion and leave the samples for 15 min at room temperature;

i) Centrifuge for 15 minutes, 12,000g at 4°C;

j) Using a p1000 transfer the aqueous layer into a clean Eppendorf tube and add 0.35 ml of isopropanol;

k) Store the samples at room temperature o.n.

Day 3: 2nd extraction step of the RNA and determination of the concentration of RNA extracted using spectrophotometric reading

a) Centrifuge to 12.000g x 10 min at 4°C;

b) Remove the supernatant (also by dabbing on absorbent paper) and wash the pellets (invisible) adding 1.5ml_ of 75% ethanol;

c) Vortex the sample and centrifuge to 7.500g x 5 min at 4°C;

d) Remove the washing ethanol using a p1000 and sequentially a p200; the white pellet will be visible on the bottom;

e) Give a spin and remove any other ethanol, resting on a sheet of absorbent paper;

f) Leave the pellet to dry briefly under the hood for about 15 minutes;

g) Re-suspend the pellet in 20pL of DEPC-treated water and, to promote dissolution, incubate for 15 minutes at 55 - 60 °C;

h) Store the samples at -20°C, if they are not used immediately.

The gene expression of the gene markers of skin-aging was evaluated through quantitative reverse transcription- polymerase chain reaction- qRT-PCR.

This analysis involved 3 sequential steps:

-Total RNA extraction; -Reverse transcription in cDNA;

-qRT-PCR.

At the end of the extraction, using a spectrophotometer (QiaExpert, Qiagen) the concentrations in pg/mL of total RNA were calculated extracted at the wavelength of 260 nm. Finally, the integrity of the RNA (2 pg/mL) was calculated using 1 % agarose gel electrophoresis.

The total RNA was converted into cDNA (complementary DNA), using the commercial“RT ² First Strand Kit” (Qiagen, Italy).

The samples were placed in a thermocycler (Stratagene Mx3000P Real Time PCR System, Agilent Technologies Italia S.p.A., Milano, Italy) and subjected to reverse transcription under the following conditions:

45°C for 15 minutes;

95°C for 5 minutes;

4°C hold.

At the end of the reverse transcription 91 pL of DEPC-treated water were added to the samples.

qRT-PCR represents an amplification and quantification method in real time for the amplified products monitoring the fluorescence emitted during the reaction.

For the RT-PCR amplification a custom kit containing anti-aging gene markers (Qiagen, Italia) was used and the amplification performed through RT2 SYBR® Green qPCR Mastermix (Qiagen, Italy).

The amplification was performed on a final volume of 25 uL under the following conditions:

95°C, 10min (Amplitaqactivation) (x1 ) for 40 cycles:

95°C, 15 sec (Denaturation)

60°C, 1 min (Annealing - extension);

Each analysis is performed in duplicate.

The data obtained were analysed according to the 2-AACt method and it was thus possible to calculate the relative gene expression values of interest, normalized with respect to the housekeeping gene and calibrated on the control sample (untreated cells):

AACt— ACt target-housekeeping (contl ol)-ACt target-housekeeping (treated cells) Assuming an amplification efficiency of 100% the 2-AACt was calculated.

4. STATISTICAL ANALYSIS

The statistical analysis was performed using GraphPad Prism software version 6.00 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com). Through the student test T the means and relative standard errors of all the formulations analysed were compared with respect to the positive control using a confidence interval of 95%. 5. RESULTS

The appended Figure 1 summarizes the following gene expression data of genetic markers related to aging phenomena, in particular skin aging.

The combination of Withania somnifera and L-proline can act synergistically on the stimulation of POT1 , SIRT1 , LMNA, CASP1 , COL1A1 , COL3A1 , KRT13, the increase in which is positively connected to anti-aging activity. At the same time it is able to synergistically inhibit the gene expression of MMP2 and S100A7A.

The aging process is complex and envisages a series of mechanisms that range from genetic programming to neuroendocrine and immune decline, inflammation and oxidative stress for excessive production of free radicals and incapacity of their detoxification by the other. Numerous experimental investigations highlight how genes such as SIRT1 or LMNA are also able to perform antioxidant activity with multiple mechanisms.

SIRT1 stimulates mitochondrial biogenesis, reduces the production of superoxide from the respiratory chain and improves the expression of manganese-dependent superoxide dismutase (SOD2) in the mitochondria, with the consequent increase in mitochondrial superoxide dismutase. Furthermore, SIRT1 accelerates the detoxification of ROS up-regulating the antioxidant cell enzymes, including SOD1 , catalase, glutathione peroxidase 1 and thioredoxin-1 , Li H. (2014) Sirtuin 1 (SIRT1 ) and Oxidative Stress. In: Laher I. (eds) Systems Biology of Free Radicals and Antioxidants. Springer, Berlin, Heidelberg.

Laminar proteins and LMNA play an important role in the regulation of SIRT1 , NF- KB and mTORCI pathways. These routes are known to be involved in the regulation of oxygen metabolism and oxidative stress, Takeshi Shimi and Robert D. Goldman, Nuclear Lam ins and Oxidative Stress in Cell Proliferation and Longevity . Adv Exp Med Biol. 2014 ; 773: 415-430. doi: 10.1007/978-1 -4899-8032-8 9.

Fig. 1 highlights the effects on gene expression (fold-change) of the main aging markers and skin inflammation of L-Proline (15mg/mL), of Withania somnifera (15mg/mL) and of the combination thereof (L-Proline+Withania somnifera, 30mg/mL) on a 3D skin model.

EXAMPLE 7

Analytical specifications of a dry powder extract of Withania somnifera to be used in the formulation of the composition of the invention

EXAMPLE 8

Production process of a powdered extract of Withania somnifera for the uses of the invention.

The extract of Withania somnifera (Ashwagandha) root was produced according to current Good Manufacturing Practices (GMP), as indicated in the appended Figure 2. For the production of the powdered Withania somnifera extract, the roots of the ashwagandha plant grown organically using GACP (Good Agricultural and Collection Practices) are collected. These roots are placed in quarantine and the quality roots (solid and bright, about 7 cm in length and with a diameter of 1 to 1.5 cm) are selected.

The selected roots are washed with water (reverse osmosis and demineralized) to remove sand, dust and undesired material in a washer designed specifically and then dried in hot air.

The dried roots are then ground to a fine powder in a pulverizer. Subsequently the fine pulverized powder is mixed with an equal amount of water and agitated for 2 hours. The liquid mixture thus obtained is transferred into a heating tank with more added water and continuous heating for 20 minutes under temperature and pressure control.

The slurry is cooled and then passed through a fine filter to remove undesired material such as waste particles and fibre.

The decoction obtained is placed in a vacuum dryer and dried at low temperatures until a humidity content of 2-3% is obtained. The final product obtained is powdered and is further subjected to micro-pulverization, with consequent free flow fine powder. This powder is then inserted into a vibrating sieve, to obtain the final powder of the desired particle size. The powder is packaged in double polyethylene food grade plastic and then inserted into watertight HDPE drums. The entire manufacturing process is performed in a clean room, following good manufacturing practices.

Inspections and tests are performed in various points during the production of KSM- 66. At the end of the process, the samples are collected and internal microbiological analyses, heavy metal analysis and finally phytochemical analysis are performed. With this manufacturing process a uniform and high quality product is obtained. EXAMPLE 9

Technical characteristics of a powdered Withania somnifera extract