HK1262940A1 - Cosmetic preparation containing white truffle extract and cosmetic method thereof - Google Patents

Description

Cosmetic preparation containing white truffle extract and cosmetic method thereof

Technical Field

The present invention is in the field of cosmetics, more specifically in the field of skin care. The present invention relates to a method for preparing truffle (Tuber magnesium) extract by aqueous extraction, truffle extract obtainable by said method and cosmetic composition comprising said truffle extract.

The invention also relates to methods of reducing and/or correcting the signs of aging and photoaging of the skin, and of making the skin shiny.

The truffle extract can be used alone or in combination with other active substances.

Background

The skin is an important organ, consisting of several layers (dermis, proliferation layer and stratum corneum), covering the entire surface of the body and ensuring protective, sensory, immunological, metabolic or thermoregulatory functions. The skin undergoes aging as does other organs.

For example, the appearance of skin is altered by various types of internal (diseases and hormonal changes such as pregnancy) or external insults (environmental factors such as pollution, sunlight, ultraviolet radiation, pathogens, etc.). Wrinkles and fine lines, hyperpigmentation or hypopigmentation spots, dry or even dehydrated skin, thinned epidermis, elastosis, blemishes, age spots, etc. may then occur.

In the skin, pre-maturation aging (prematuring) is observed, appearing in the areas exposed to uv radiation.

With age, the appearance of skin changes. Dark spots, dull skin tone, uneven skin tone are significant effects associated with age. The loss of youthful color of the skin is often one of the first external signs of aging, and frequent exposure to the UV rays of the sun also causes the skin to appear dull and dry.

Fungi are known to be a large family of organisms. Some fruiting bodies of wild and culturable mushrooms contain pharmaceutical compounds for traditional medicine and cosmetics. During their evolution, fungi evolve various properties to resist environmental stress, and it is known that some fungal compounds may have the effect of regulating damage caused to organisms by the living environment and the natural environment. Thus, the search for new natural bioactive compounds from fungal sources has been intensified. For example, the inonotus obliquus mushroom contains enzymes such as superoxide dismutase (SOD), which is a key element in protecting cells against ROS.

Also, truffles are known to be effective in certain conditions, such as improvement of immunity and hyperlipidemia. Truffles belong to the phylum ascomycota and are underground fungi. Truffle fungi contain some truffle species. These truffle species are the fungal outgrowths that establish an ectomycorrhiza symbiosis with trees and shrubs and form fruit bodies.

Some truffle species are high value foods because of the characteristic aroma and deliciousness of the fruit body. These species are called "truffles" or "truffles", which are precious and expensive delicacies widely used in famous french and italian cuisine.

The general truffles belong to the ascomycetes, order truffles, and have been considered to be excellent edible fungi for many centuries and because of their particular aroma importance. Truffles are underground fungi that live in symbiosis with oak roots and form massive fruit bodies. In the truffle extraction process, several active substances are released. It has been found that non-specific stimulation of the immune system can be achieved.

In the description of the present invention, "truffle" refers to the following species: white Italy truffle (academic name: Tuber. italica (Tuber major pico)), black truffle (also known as Pelargo truffle from France, academic name: Tuber melanosporum (Tubermelanospora), Bouggium (Tuber uncinatum), Karaha truffle (Tuber feilii), lion truffle (Trefezia leonis), summer truffle (Tuberaestivum), winter truffle (Tuber brumal), Chinese truffle (Tuber china) or Tuber indicum), and Biankato or white truffle (Tuber borchi).

White truffle (truffle) is called foot or albaca truffle. The truffle grows several inches underground, symbiotic with the roots of hardwood trees such as oak, chestnut, hazel and hornbeam. Irregularly shaped, polyoma pellets range in size from about one inch to over one pound (although this size is rare). The firm flesh of white truffle is light yellow to light brown in color, and has white marbling throughout. Truffles are real earth's fruits, more rare and precious than any other edible root, tuber or mushroom. There are no other flavors on earth like them, which is perhaps why they are often described as coming from the world.

There have been many attempts to acclimatize these wild truffles, but they have grown for ten years. Some of these attempts have been successful, but the most reliable source is to find them in nature.

From the state of the art, many cosmetics are known to contain in some way plant-based raw materials, in the form of oils or extracts. In most cases, the known beneficial effects of individual plants are used to achieve the corresponding overall effect.

The use of white pine cone extracts in cosmetic compositions is known in the art. Most extracts are total or aqueous-alcoholic extracts. For example, chinese patent application CN-A-104856928 discloses an anti-wrinkle cosmetic wherein the active substance is one of or any combination of mixture A, echinaceA purpureA (echinaceA purpureA) extract and white pine dew extract, said mixture A being A mixture of trifluoroacetyl tripeptide-2 and glycerol and dextran. Us granted patent 6843995 discloses a cosmetic preparation comprising at least one aqueous extract of truffle in general (trufflaceae) and spray-dried champagne, wherein the active complex is provided in a cosmetically acceptable gel and stabilizer. Chinese patent CN-A-104666237 discloses A preparation method using A water-alcohol extract and the use of truffle active ingredients for the preparation of antioxidants and skin whitening products. However, no document discloses that an aqueous extraction process produces a completely safe and effective cosmetic ingredient from white truffle.

Although various anti-aging cosmetic products are available on the market for treating skin, there is still a need for effective topically applied cosmetic compositions that provide anti-aging or rejuvenating benefits to skin, hair and/or nails using natural ingredients as active substances. Non-natural, chemically synthesized products may be considered unsafe for the environment or individuals. In contrast, natural products are considered to be pure, mild, and superior to chemically synthesized products. Many natural-based products extracted from plants or herbs are known to contain antioxidants/free radical scavengers that can neutralize free radical damaging effects. In addition, it may contain substances that stimulate the synthesis and restoration of damaged connective tissue structures in the dermis and barrier functions in the epidermis.

There remains a need for cosmetic compositions that treat signs of aging, particularly the appearance of wrinkles, fine lines and sagging. It is therefore an object of the present invention to provide new compositions and methods for treating, alleviating and/or preventing the signs of aging or aging skin. It is a further object of the present invention to improve the overall appearance of aging or aged skin.

The foregoing description is presented only for the purpose of better understanding the nature of the problems faced in the art and should not be construed in any way as an admission of prior art, nor should any reference cited herein be construed as an admission that such references constitute "prior art" to the present application.

Disclosure of Invention

The main aspect of the present invention provides a method for obtaining an extract containing a fruit body of a white truffle mushroom (white truffle), comprising the steps of:

(i) adding water to white truffle to prepare a mixture;

(ii) agitating the mixture, maintaining the temperature from RT to below 80 ℃;

(iii) filtering the mixture to remove a solid portion to obtain the extract.

In another aspect, the present invention provides a white truffle extract obtained by the method of the present invention.

In another aspect, the present invention provides a cosmetic composition comprising a white truffle extract obtained by aqueous extraction, wherein said white truffle extract comprises a compound having a molecular weight of less than 60kDa in a physiologically acceptable medium.

In another aspect, the present invention provides a method of reducing and/or correcting the signs of aging and photoaging of the skin comprising topically applying to the skin a cosmetic composition comprising a white pine dew extract obtained by aqueous extraction, wherein the white pine dew extract comprises a compound having a molecular weight of less than 60kDa in a physiologically acceptable medium.

In another aspect, the present invention provides a method of reducing skin pigmentation and/or lightening skin comprising topically applying to the skin a cosmetic composition comprising a white truffle extract obtained by aqueous extraction, wherein said white truffle extract comprises a compound having a molecular weight of less than 60kDa in a physiologically acceptable medium.

Drawings

Further embodiments of the invention can be understood from the figures.

Figure 1 is a graphical representation of collagen I expression on fibroblasts assessed by immunofluorescence.

FIG. 2 is a graph showing melanin content on ex vivo skin biopsies (Fontana-Masson).

Figure 3 illustrates stimulation of LC3 (autophagy pathway), assessed on keratinocytes.

Fig. 4 illustrates stimulation of LC3 (autophagy pathway), assessed on ex vivo skin biopsies.

Detailed Description

Detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the invention, which can be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present invention.

Whenever a term is determined by reference to a range, that range is to be understood as specifically disclosing each element thereof. As non-limiting examples, a range of 1-10% should be understood to include 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%, as well as all values between 1 and 10%.

When two or more alternative elements are referred to as "selected from" a set of enumerated alternatives, it is meant that each alternative element may be any element of the set, independent of the characteristics of the other alternative elements.

As used herein, "%" refers to weight percent, which is the weight percent of one component relative to the total weight of the composition (i.e., including any carriers, solvents, fillers, or other components added prior to application to the skin), unless otherwise specified.

All terms used herein have their ordinary meaning unless otherwise specified. For the purposes of describing and claiming the present invention, the following terms are defined:

by "extract" is understood any substance or isolated preparation extracted from a natural source, regardless of the method or composition of extraction. The term is used in a broad sense and includes, for example, a solution in water or an organic solvent, an ingredient extracted from a natural substance with the solvent, or a specific ingredient of a natural substance.

By "aqueous extract" is understood a mixture of compounds obtained by extraction with water.

By "physiologically acceptable" it is understood that the active substances of the invention or compositions containing them are suitable for contact with the skin or mucous membranes without causing toxic or intolerable reactions.

By "signs of aging and photoaging of the skin" is meant all changes in the appearance of the skin and keratinous appendages caused by aging, such as thinning, sagging, dehydration and laxity of the skin, deep wrinkles and fine lines, loss of firmness, dullness, dermal atrophy or any other internal deterioration of the skin caused by exposure to ultraviolet radiation, such as age spots.

By "glossing the skin" is meant improving the complexion, radiance and/or clarity and/or restoring skin radiance or brightness.

The species "truffle" or "white truffle" or "trifolad' Alba Madonna" (italian "white maternal truffle") were found mainly in Langhe and Montferrat areas in the northern foot area of italy, most notably in rural areas around the city of Alba and Asti. White truffles are characterized by a very good and rare specific odor, a moisture of 81%, a protein content of about 10% and a sugar content of about 2%.

As used herein, "skin" refers to all the overlying tissues, mucous membranes and keratinous appendages that make up the skin, including hair, nails, eyelashes, and eyebrows.

By "topical application" is understood the application or application of a composition containing the white pine cone extract on the skin or mucosal surface.

Described herein are methods of obtaining an extract from ground truffle, cosmetic compositions comprising the extract, methods of reducing and/or correcting the signs of aging and photoaging of skin by topically applying the compositions comprising truffle extract to skin.

Antioxidants play an important role as health protection factors. Scientific evidence suggests that antioxidants reduce the risk of chronic diseases including cancer and heart disease. The main sources of naturally occurring antioxidants are whole cereals, fruits, flowers and vegetables. The plant antioxidant is vitamin C, vitamin E, carotene, phenolic acid, and flavone.

The truffle extract of the present invention is known to be rich in polyphenolic compounds, such as phenolic acids. All of these water soluble molecules known for their antioxidant activity contribute to providing the antioxidant efficacy of the white pine cone extract of the present invention. The total polyphenol content measured was 100mg/kg extract.

Polyphenolic compounds are considered to be potent antioxidant molecules. "polyphenolic compounds" are compounds found abundantly in natural plant food sources that have antioxidant properties. It refers to all polyphenols, meaning compounds comprising at least one aromatic ring of a diphenol, optionally the phenolic groups may be etherified or esterified. It is also referred to as "polyphenol" for short.

Polyphenols play an important role in maintaining health. Antioxidants generally help protect cells in the body from free radical damage, thereby controlling the rate of aging. Antioxidants can be mainly divided into three groups: carotenoids, allylic sulfides found in garlic and onions, polyphenols (also known as phenolics).

Polyphenols can be further divided into four categories: phenolic acids, flavones, lignans and stilbenes are further sub-groups based on the number of phenolic rings they contain and the structural elements that bind these rings to each other.

Phenolic acids are a class of phytochemicals called polyphenols, found in a variety of plant-based foods; the seeds and pericarp, as well as the vegetable leaves, are contained in the highest concentrations. Phenolic acids are readily absorbed through the intestinal wall and may be beneficial for health because they act as antioxidants to prevent cellular damage from free radical oxidation reactions. Many different phenolic acids are found in nature and can be divided into two categories: benzoic acid derivatives such as gallic acid, and cinnamic acid derivatives including caffeic acid and ferulic acid. Cinnamic acid is more common than benzoic acid.

The present invention provides a white pine dew extract obtained from ground white pine dew.

The truffle extract of the present invention may be obtained by aqueous extraction. A large number of compounds that may be biologically active are found in the white pine dew extract are water soluble.

In a preferred embodiment, the present invention provides a method of obtaining an extract from truffle (truffle), the method comprising:

(i) adding water to the ground white truffle to prepare a mixture;

(ii) agitating the mixture for 2 hours, maintaining the temperature from RT to below 80 ℃;

(iii) filtering the mixture to remove a solid portion to obtain the extract.

In a preferred embodiment, the milled albedo is immersed in water. The solution was gently immersed for a short period of time for 2 hours at a temperature from RT to below 80 ℃. The most preferred temperature is to maintain between RT and 50 ℃ to maintain the integrity of the molecule of interest, such as phenolic acid, and thus its ability to function as an antioxidant.

The extraction temperature is chosen according to the type of compound desired to be extracted, the structural characteristics of the botanical source (flowers, fruits, stems, seeds, leaves, roots), the quality and yield required for the extraction, and the economic feasibility of the process to scale up. Extraction of phenolic compounds from plant material is affected by the extraction temperature and time, which reflect the conflicting effects of dissolution and oxidative degradation of the analyte. However, many phenolic compounds are susceptible to hydrolysis and oxidation. The long extraction time and high temperature increase the chance of oxidation of phenols, which reduces the yield of phenols in the extract. Analysis at different temperatures showed that high temperatures degraded these types of molecules.

The water extraction can be carried out at room temperature or the water can be heated to not more than 80 ℃ while stirring. Preferably, the extraction is carried out by immersion in water heated to 50 ℃ for 2 hours. The crude solution was then subjected to mesh filtration to remove insoluble material. After mesh filtration, the aqueous or liquid fraction was collected. To remove the smaller residue of the aqueous extract, filtration may be performed by any method well known to those skilled in the art.

In a preferred embodiment, the purification process starts by performing sequential filtration using filters of decreasing pore size from 50-20 μm up to 0.5-0.2 μm to obtain the extract.

In a preferred embodiment, the purification process is followed by a double purification with carbon black.

In another preferred embodiment, the extract obtained comprises protein fragments and peptides, has a molecular weight of less than 60kDa, as evidenced by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The obtained extract was a clear and bright solution.

In another preferred embodiment, the extract is subsequently diluted with a solvent, such as water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxy or propoxy ethylene glycol, cyclic polyols or any mixture of these solvents, e.g. 30% butylene glycol and 2% 1, 2-hexanediol, at a concentration of 0.5g/kg to 1.5g/kg, preferably 1g/kg dry matter, keeping the pH between 4 and 5.

The diluted active substance is then sterilized by sterile filtration. The solution was then heated at 65 ℃ overnight for low temperature pasteurization.

Furthermore, the diluted active substances of the invention can be analyzed qualitatively and quantitatively. The characteristics are as follows:

-protein: 0.05-0.15 g/kg,

-a sugar: 0.35-1.05 g/kg,

-amino acids: 0.025-0.075 g/kg,

-phenolic compounds: 0.05-0.15 g/kg,

-said extract does not contain any ceramides or sphingolipids.

The protein content of the white truffle extract was analyzed by Lowry protein (Lowr)y OH, RosebroughNJ, Farr AL, Randall RJ (1951), "Protein measurement with the Folin phenol reagent", J.biol.chem.193 (1): 265-75) for quantifying the total protein content of said extract. The Lowry assay is a biochemical assay used to determine the total level of protein in solution. The Lowry method is based on Cu generated by oxidation of peptide bonds⁺Reaction with Folin-Ciocalteu reagent. The absorption of the sample was read at 550nm in a spectrophotometer. Protein content was determined using a BSA standard curve.

Amino acid content determination of white pine extract begins with the protocol disclosed by Moore et al (Moore et al, "Photometric chemical method for use in the chromatography of amino acids", Journal of Biological Chemistry 1948 Vol.176pp.367-388). The free amino acid content of the extract was evaluated by the reagent ninhydrin to disrupt the amino and carboxyl functions and then form a colored complex. The absorbance of the complex was read at 570nm on a spectrophotometer. The total amino acid content was determined using an amino acid pool (pool) standard curve.

The total sugar content of the truffle extract is determined colorimetrically by analysis as described by adaptation of Dubois et al (Dubois et al, "Colorimetric Method for Determination of Sugars and Related sustances", anal. chem.,1956,28(3), 350-. This analysis involves dissolving the starting material in concentrated sulfuric acid and then reacting with phenol to form a colored complex. The absorbance of the complex was read at 490nm in a spectrophotometer. The sugar content was determined using a glucose standard curve.

The polyphenol content of white pine dew extracts was determined using the Folin-Ciocalteu assay (Singleton et al (1999). "Analysis of total phenols and other oxidation substrates and antioxidants by microorganisms of Folin-Ciocalteu reagent", 299: 152). Polyphenolic compounds in the sample react with Folin-Ciocalteu reagent, which oxidizes to produce a blue color. The absorption of the sample was read at 760nm in a spectrophotometer. The content was expressed as gallic acid equivalent using a gallic acid standard curve.

SDS-PAGE electrophoresis was performed to evaluate the protein content of the extractsThe molecular weight of the substance. The white truffle extract is heated to 70 ℃ for 10 minutes in a denaturing sample buffer under reductive denaturing conditions. An antioxidant solution is added to the inner chamber (cathode) so that the reduced proteins are not re-oxidized during electrophoresis. Protein migration was performed using MES running buffer, as standardSharp as molecular weight marker. Protein staining was performed using silver staining.

The present application provides a cosmetic composition comprising a white truffle extract obtained by aqueous extraction, wherein said white truffle extract comprises a compound having a molecular weight of less than 60kDa and a physiologically acceptable medium.

The advantage of the extract according to the invention is that the small compounds are more stable and reproducible without sensitization.

In another preferred embodiment, the truffle extract is present at a concentration of about 0.01% to about 20% by weight, preferably about 0.1% to about 5% by weight, of the total weight of the composition.

In another embodiment, the white truffle extract is for cosmetic applications, more preferably, for topical application.

In another preferred embodiment, the present invention provides modified oral, parenteral or topical formulations by those skilled in the art, particularly for use in cosmetic or dermatological (dermatalogical) compositions. Advantageously, the compositions of the present invention are designed for topical application. Therefore, these compositions must contain a physiologically acceptable medium, i.e. compatible with the skin, covering all cosmetic and dermatological forms.

Further, the composition of the present invention is preferably in the form of an aqueous, hydroalcoholic or oily solution; oil-in-water emulsions, water-in-oil emulsions or multiple emulsions; creams, suspensions, powders suitable for application on the skin, mucous membranes, lips and/or keratinous appendages. These compositions may also be more or less liquid and be in the appearance of creams, lotions (positions), milks, serums, pomades, gels, pastes or mousses. It may also be present in solid form, such as a stick, or may be applied to the skin as an aerosol. It can also be used as a skin care and/or cosmetic product.

In another embodiment, the composition comprises additives conventionally used and necessary for its formulation, such as co-solvents (ethanol, glycerol, benzyl alcohol, damper (damper) … …), thickeners, diluents, emulsifiers, antioxidants, colorants, sunscreens, pigments, fillers, preservatives, fragrances, deodorants, essential oils, minor elements (oligo element), essential fatty acids, surfactants, film forming polymers, chemical filters or minerals, wetting agents or thermal waters, etc., which are conceivable within the scope of the present application. For example, mention may be made of (preferably natural) water-soluble polymers such as polysaccharides or polypeptides, methylcellulose or hydroxypropylcellulose-type cellulose derivatives, or even synthetic polymers, poloxamers, carbomers, silicones, PVA or PVP, in particular the polymers sold by the company Ashland.

It is to be fully understood that the active substances of the present invention may be used alone or in combination with other active ingredients.

Furthermore, the compositions which can be used according to the invention advantageously contain at least one further active substance. The following types of ingredients can be cited in a non-limiting manner: other peptide active substances, vegetable extracts, healing agents, anti-ageing agents, anti-wrinkle agents, soothing agents (soothing agents), anti-radical agents, anti-uv agents, substances stimulating dermal macromolecule synthesis or energy metabolism, moisturizers, antibacterial agents, antifungal agents, anti-inflammatory agents, anesthetics, substances regulating skin differentiation or skin pigmentation or depigmentation, and substances stimulating nail and hair growth.

In a more specific embodiment, the composition of the invention comprises:

-sunscreens, uv and ir screening agents;

-an anti-radical agent;

-DHEA (dehydroepiandrosterone);

-at least one cytochrome co-activating compound; and/or

-one (or more) aquaporin activating compounds; and/or

-one (or more) Sirtuin activating compounds; and/or

-one (or more) compounds that increase cell adhesion; and/or

-one (or more) compounds that increase the production of matrix proteins of the collagen or laminin type and the like;

-one (or more) HSP protein modulating compounds;

-one (or more) compounds that increase cellular energy;

-one (or more) pigmentation modulating compounds, such as yeast, amaranth, linseed, legume, cocoa, corn, soybean, sunflower, rapeseed or pea peptide extracts;

-one (or more) compounds improving the barrier function of the skin;

-one (or more) compound(s) protecting mitochondria;

-vitamin a, in particular retinoic acid, retinol propionate, retinol palmitate;

-vitamin B3, especially niacinamide, tocopherol nicotinate; ,

-vitamin B5, vitamin B6, vitamin B12, panthenol;

vitamin C, in particular ascorbic acid, ascorbyl glucoside, ascorbyl tetrapalmitate, magnesium ascorbyl phosphate and sodium;

-vitamin E, F, H, K, PP and coenzyme Q10, metalloproteinase inhibitors, Tissue Inhibitor Metalloproteinase (TIMP) activators;

amino acids, in particular arginine, ornithine, hydroxyproline dipalmitate, palmitoylglycine, hydroxylysine, methionine and derivatives thereof, N-acylated amino acids

Natural or synthetic peptides, including dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides and their lipophilic derivatives, isomers and complexes with other molecules such as metal ions (i.e. copper, zinc, manganese, magnesium and others), under commercial namesCHRONOGEN^TM、LAMINIXYL IS^TM、PEPTIDE Q10^TM、COLLAXYL^TM(patent FR2827170,)、PEPTIDE VINCI 01^TM(patent FR2837098,)、PEPTIDE VINCI 02^TM(patent FR2841781,)、ATPeptide^TM(patent FR2846883,) The sequence of the sold peptide is Arg-Gly-Ser-NH₂Synthetic peptide of (1), under the commercial name ATPeptide^TMByPeptides sold;

-extract of Artmia salina under the commercial name GP4G^TM(FR2817748,) Selling;

-vegetable peptide extracts, such as linseed extractsSubstance (Lipig two nine)^TMThe solution of the invention, as described in patent FR2956818,) Soybean extract, einkorn, grape, rapeseed, rice, corn, or pea;

yeast extracts, such as Dynagen^TM(patent FR2951946,) Or Actopontine^TM(the solution of the problem of the patent FR2944526,) Dehydroacetic acid (DHA);

natural or synthetic phytosterols (phystosterol);

- α -and β -hydroxy acids, silanols;

-sugar amine, glucosamine, D-glucosamine, N-acetyl-D-glucosamine, mannosamine, N-acetyl-mannosamine, galactosamine, N-acetyl-galactosamine;

-polyphenols, isoflavones, flavones, such as grape extract, pineapple extract, olive extract;

-lipids such as ceramides or phospholipids;

animal oils, such as squalene or squalene;

vegetable oils, such as almond oil, coconut oil, castor oil, jojoba oil, olive oil, rapeseed oil, peanut oil, sunflower oil, wheat germ oil, corn germ oil, soybean oil, cottonseed oil, alfalfa oil, poppy oil, pumpkin seed oil, evening primrose oil, millet oil, barley oil, rye oil, safflower oil, passion fruit oil (passion oil), hazelnut oil, palm oil, almond oil, avocado oil, calendula oil, ethoxylated vegetable oils or shea butter (shea butter), the above-mentioned compounds being either natural, such as peptide hydrolysates of plants, or synthetic, such as peptide compounds.

In another embodiment, the present application provides a cosmetic method to reduce and/or correct the signs of skin aging and photoaging comprising topically applying to the skin a composition comprising the white pine cone extract of the present application.

It is clear that the present invention is designed for use in mammals in general, and more specifically, in humans. The inventors have in fact identified biological activities that can be used to reduce and/or correct cutaneous signs of skin and of ageing and photoaging and to make the skin shiny.

In another aspect, the present application provides a cosmetic method to make skin shiny, wherein a cosmetic composition comprising the extract of truffle of the present invention is topically applied on the skin to be treated.

Embodiments for this cosmetic method are also from the above description.

Further advantages and features of the present invention are more fully seen in the illustrative, non-limiting examples provided.

Example 1 preparation of white truffle (Tuber melanosporum) extract

White truffle (fruiting body) was obtained from Alba, italy. 50g of ground white pine cone was placed in 1 liter of distilled water. The solution was heated for two hours at 50 ℃. Then, 20-50 μm filtration was performed to separate the solid truffle residue and the liquid fraction. The purification process starts by sequential filtration using filters with decreasing pore size from 50-20 μm up to 0.3-0.5 μm, then 0.25% carbon black (Cabot Norit SXplus) is added to the solution and mixed during 30 minutes at 50 ℃; then filtering and removing carbon by 0.3-0.5 mu m; an additional 0.25% carbon black (Cabot Norit SXplus) was added to the solution and mixed during 30 minutes at 50 ℃; then filtering with 0.3-0.5 μm filter to remove carbon. The filtrate was then diluted with 30% butanediol and 2% hexanediol to obtain an extract with 0.5-1.5g/kg dry matter.

The solution pH is adjusted to between 4 and 5 to increase the stability of the extract. After clarification and dilution, the filtrate was filter-sterilized with 0.2 μm filter pores under sterile conditions. The white truffle extract was analyzed using standard procedures. The obtained white truffle extract is characterized as follows: dry matter: 1 g/kg-protein: 0.1 g/kg-sugar: 0.70 g/kg-amino acid: 0.01g/kg and polyphenol compound 0.1 g/kg.

The protein content of the white truffle extract was determined by Lowry protein assay (Lowry et al,1951) to quantify the total protein content of the extract. The Lowry assay is a biochemical assay used to determine the total level of protein in solution. The Lowry method is based on Cu generated by oxidation of peptide bonds⁺Reaction with Folin-Ciocalteu reagent. The absorption of the sample was read at 550nm in a spectrophotometer. Protein content was determined using BSA (bovine serum albumin) standard curve.

The amino acid content determination of the extract starts with the protocol disclosed by Moore et al (1948). The free amino acid content of the extract was evaluated by the reagent ninhydrin to disrupt the amino and carboxyl functions and then form a colored complex. The absorbance of the complex was read at 570nm on a spectrophotometer. The total amino acid content was determined using an amino acid pool standard curve.

The total sugar content of the extract is determined colorimetrically by analysis as described by adaptation of Dubois et al (1956), "Colorimetric Method for Determination of Sugars and Related sustances", anal. chem.,1956,28(3), 350-. This analysis involves dissolving the starting material in concentrated sulfuric acid and then reacting with phenol to form a colored complex. The absorbance of the complex was read at 490nm in a spectrophotometer. The sugar content was determined using a glucose standard curve.

The polyphenol content of white pine dew extracts was determined using the Folin-Ciocalteu assay (Singleton et al (1999). "Analysis of total phenols and other oxidation substrates and antioxidants by microorganisms of Folin-Ciocalteu reagent", 299: 152). Polyphenolic compounds in the sample react with Folin-Ciocalteu reagent, which oxidizes to produce a blue color. The absorption of the sample was read at 760nm in a spectrophotometer. The content was expressed as gallic acid equivalent using a gallic acid standard curve.

SDS-PAGE was performed to assess the molecular weight of the protein of the extract. The white truffle extract is heated to 70 ℃ for 10 minutes in a denaturing sample buffer under reductive denaturing conditions. An antioxidant solution is added to the inner chamber (cathode) so that the reduced proteins are not re-oxidized during electrophoresis. Protein migration was performed using MES running buffer, as standardSharp as molecular weight marker. Protein staining was performed using silver staining.

The obtained extract contains peptides with a molecular weight of less than 60 kDa.

Example 2 preparation of white truffle (Tuber melanoidin) extract

Briefly, white truffle (fruit body) was obtained from Alba, italy. 100g of ground white pine dew was placed in 1 liter of distilled water. The solution pH was adjusted to 2 with hydrochloric acid. The solution was heated at 80 ℃ for two hours. Then, 20-50 μm filtration was performed to separate the solid truffle residue and the liquid fraction. The purification process starts by sequential filtration using filters with decreasing pore size from 50-20 μm up to 0.3-0.5 μm, then 1% carbon black (Cabot Norit SXplus) is added to the solution and mixed during 30 minutes at 50 ℃; then filtering with 0.3-0.5 μm filter to remove carbon. The filtrate obtained has a dry matter of 20g/kg, and the filtrate is then diluted to obtain an extract having a dry matter of 0.5-1.5 g/kg. The solution pH is adjusted to between 4 and 5 to increase the stability of the extract. After clarification and dilution, the filtrate was filter-sterilized with 0.2 μm filter pores under sterile conditions.

Example 3 preparation of white truffle (Tuber melanoidin) extract

Briefly, white truffle (fruit body) was obtained from Alba, italy. 100g of ground white pine dew was placed in 1 liter of distilled water. The solution pH was adjusted to 2 with hydrochloric acid. The solution was heated at 80 ℃ for two hours. Then, 20-50 μm filtration was performed to separate the solid truffle residue and the liquid fraction. The purification process starts by sequential filtration using filters with decreasing pore size from 50-20 μm up to 0.3-0.5 μm, then 0.25% carbon black (Cabot Norit SXplus) is added to the solution and mixed during 30 minutes at 50 ℃; then filtering and removing carbon by 0.3-0.5 mu m; an additional 0.25% carbon black (cabot norit SXplus) was added to the solution and mixed during 30 minutes at 50 ℃; then filtering with 0.3-0.5 μm filter to remove carbon. The filtrate obtained has a dry matter of 20g/kg, and the filtrate is then diluted to obtain an extract having a dry matter of 0.5-1.5 g/kg. The solution pH is adjusted to between 4 and 5 to increase the stability of the extract. After clarification and dilution, the filtrate was filter-sterilized with 0.2 μm filter pores under sterile conditions.

Example 4 preparation of white truffle (Tuber melanoidin) extract

Briefly, white truffle (fruit body) was obtained from Alba, italy. 100g of ground white pine cone was placed in 1 liter of a solution containing 10mM tetrasodium EDTA. The solution was heated at 50 ℃ for two hours. Then, 20-50 μm filtration was performed to separate the solid truffle residue and the liquid fraction. The purification process starts by sequential filtration using filters with decreasing pore size from 50-20 μm up to 0.3-0.5 μm, then 0.25% carbon black (Cabot Norit SXplus) is added to the solution and mixed during 30 minutes at 50 ℃; then filtering and removing carbon by 0.3-0.5 mu m; an additional 0.25% carbon black (Cabot NoritSXplus) was added to the solution and mixed during 30 minutes at 50 ℃; then filtering with 0.3-0.5 μm filter to remove carbon. The filtrate obtained has a dry matter of 20g/kg, and the filtrate is then diluted to obtain an extract having a dry matter of 0.5-1.5 g/kg. The solution pH is adjusted to between 4 and 5 to increase the stability of the extract. After clarification and dilution, the filtrate was filter-sterilized with 0.2 μm filter pores under sterile conditions.

Example 5 evaluation of the effects of white pine dew on skin aging by extracellular matrix evaluation on fibroblasts

The aim of this study was to show the effect of white pine dew extract on aging, as assessed by extracellular matrix (ECM), involving collagen I expression.

The scheme is as follows:

normal human fibroblasts were treated twice daily with the truffle extract solution of example 1,2, 3 or 4, diluted 1/200 in culture medium to a final concentration of 0.5% vol/vol for 48 hours.

For immunolabeling by anti-collagen I antibodies, the cells were washed and fixed with cold methanol. The cells were then incubated in the presence of specific anti-collagen I antibodies (Tebu, ref.600-401-103-0.5, rabbit polyclonal) and then with a suitable secondary antibody conjugated to a fluorescent dye. After mounting in the specified medium, the slide was observed with an epifluorescence microscope (Zeiss Axiovert200M microscope). By using6.3. The software (PerkinElmer, Inc.) analyzed the images to quantify fluorescence intensity.

As a result:

only 48 hours of treatment with the white pine syrup extract of example 1 diluted to 0.5% showed a highly significant increase in fibrinogen I expression on fibroblasts. The administration of the other extracts did not show any efficacy.

The results are graphically shown in FIG. 1.

To summarize:

0.5% white pine dew extract improved extracellular matrix on fibroblasts by stimulating collagen I. The extract of example 1 gave the best results.

Example 6 evaluation of the effect of white pine dew extract on skin lightening, evaluation of melanin content on in vitro skin biopsy

The purpose of this study was to show the effect of white pine cone extract on skin lightening, as assessed by melanin content using Fontan-Masson staining. Fontan-Masson staining is based on the ability of melanin to reduce ammoniacal silver nitrate solution to metallic silver (brown) without an external reducing agent.

The scheme is as follows:

normal human skin biopsies 6mm in diameter were maintained ex vivo in specific media (DMEM 1g/L, HAMF12, fetal bovine serum and antibiotics). The biopsies were treated twice daily for 48 hours with the white pine cone extract solutions of examples 1,2, 3 or 4, respectively, diluted 1/200 in PBS to a final concentration of 0.5% vol/vol, respectively. Control conditions were 1X PBS.

For Fontan-Masson staining, tissues were fixed and embedded in paraffin. The embedded skin biopsies are then cut and the sections are deparaffinized and rehydrated. Then, a stock solution containing ammonia and silver nitrate was applied to each slice. After distilled water washing, the biopsy was incubated with 5% sodium thiosulfate and washed again. After mounting in the specified medium, the slides were examined with Eclipse E600 microscope (Nikon). The number of brown/black pixels was quantified by analyzing the image using ImageJ software.

As a result:

treatment with the white pine dew extract of examples 1 and 4 diluted to 0.5% for 48 hours showed a significant reduction (Student's t-test) of melanin content on ex vivo skin biopsies. The administration of the other extracts did not show any efficacy.

The results are graphically shown in FIG. 2. The results for the extract of example 1 were observed to be better than the extract of example 4.

To summarize:

application of 0.5% white pine dew extract showed efficacy in skin lightening by reducing melanin content on ex vivo skin biopsies. The extract of example 1 gave the best results. The extract prepared in example 1 is the only extract that has an effect on both the extracellular matrix and the skin's shiny pathways. It is selected as the best candidate.

Example 7 evaluation of the Effect of the white pine cone extract of example 1 on the autophagy pathway, on keratinocytes and ex vivo skin biopsies

The aim of this study was to show the effect of white truffle extract on autophagy. Autophagy is a catabolic process in which autophago-lysosomes degrade most of the cytoplasmic contents (Cuervo AM et al, "Autophagy, nutrition and immunology", Mol accessories Med.33(1):2-13,2012). Here, LC3 (microtubule-associated protein 1A/1B-light chain 3), which is a structural protein involved in autophagosome formation, was evaluated (meledez a and Levine B. "Autophagy in c. elegans", wormbook.24:1-26,2009).

The scheme is as follows:

evaluation on keratinocytes

Normal human keratinocytes were treated twice daily for 48 hours with the white pine cone extract solution of example 1, which was diluted 1/200 in culture medium to a final concentration of 0.5% vol/vol.

For immunolabeling by LC3 antibody, the cells were washed and fixed with cold methanol. The cells were then incubated in the presence of specific LC3 antibodies (Cell Signaling, ref. pm036, rabbit polyclonal) and then with appropriate secondary antibodies conjugated with fluorescent dyes. After mounting in the specified medium, the slides were observed with an epifluorescence microscope (Zeiss Axiovert200M microscope). By using6.3. The software (PerkinElmer, Inc.) analyzed the images to quantify fluorescence intensity.

Evaluation on Ex vivo skin biopsy

Normal human skin biopsies 6mm in diameter were maintained ex vivo in specific media (DMEM 1g/L, HAMF12, fetal bovine serum and antibiotics). The biopsies were treated twice daily with the white pine cone extract solution of example 1 for 48 hours, said extract being 1/200 diluted in PBS to a final concentration of 0.5% vol/vol. Control conditions were 1X PBS.

For immunolabeling by the LC3 antibody, the tissues were fixed and embedded in paraffin. The embedded skin biopsies are then cut and the sections are deparaffinized and rehydrated. Then, a revealing (unmaking) protocol was performed, followed by the application of a specific anti-LC 3 antibody (Cell Signaling, PM036, rabbit polyclonal), followed by a suitable secondary antibody conjugated with a fluorescent dye. After mounting in the specified medium, the slide was observed with an epifluorescence microscope (Zeiss Axiovert200M microscope).

As a result:

treatment with 0.5% diluted white pine dew extract solution for 48 hours on both keratinocytes and ex vivo skin biopsies showed a highly significant increase in LC3 expression (Student's t-test).

To summarize:

0.5% white pine dew extract improved the autophagy pathway by stimulating LC 3.

Claims (16)

1. A method of obtaining an extract from white truffle (Tuber Magnatum), comprising:

(i) adding water to the ground white truffle to prepare a mixture;

(ii) agitating the mixture, maintaining the temperature from RT to 50 ℃;

(iii) filtering the mixture to remove a solid portion to obtain the extract.

2. The process of claim 1, wherein step (ii) is carried out at a temperature of 50 ℃.

3. The method of claim 1, wherein the extract of step (iii) is further clarified by sequential filtration through pores from about 50 μ ι η to about 20 μ ι η up to about 0.5 μ ι η to 0.2 μ ι η to obtain an extract.

4. The method of claim 1, wherein the white pine cone extract of step (iii) is subsequently diluted in a solvent selected from the group consisting of water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxy or propoxy ethylene glycol, cyclic polyols or any mixture of these solvents.

5. A white pine cone extract obtained by the method of claim 1.

6. The truffle extract of claim 5, comprising a compound having a molecular weight of less than 60 kDa.

7. The truffle extract of claim 5, wherein said truffle extract comprises dry matter of 0.5 to 1.5g/kg, protein of 0.05 to 0.15g/kg, sugar of 0.35 to 1.05g/kg, amino acids of 0.025 to 0.075g/kg, and phenolic compounds of 0.05 to 0.15 g/kg.

8. Cosmetic composition comprising the white truffle extract obtained according to the process of claim 1, wherein said white truffle extract comprises a compound having a molecular weight of less than 60kDa and a physiologically acceptable medium.

9. The cosmetic composition of claim 8, wherein the truffle extract is used at a concentration of about 0.01% to about 20% by weight of the total weight of the composition.

10. The cosmetic composition of claim 8, wherein the truffle extract is used at a concentration of about 0.1% to about 5% by weight of the total weight of the composition.

11. The cosmetic composition of claim 8, wherein the composition is in a form suitable for topical application.

12. The cosmetic composition of claim 8, wherein the composition is in the form of a gel, cream, ointment, lotion, or foaming product.

13. The cosmetic composition of claim 8, further comprising at least one other active.

14. The cosmetic composition of claim 8, further comprising at least one additional active selected from the group consisting of antioxidants, plant hydrolysates, synthetic peptide compounds, sunscreens, and anti-wrinkle agents.

15. A method of reducing and/or correcting the signs of skin aging and photoaging, comprising topically applying to the skin a cosmetic composition comprising the truffle extract obtained according to the method of claim 1, wherein said truffle extract comprises a compound having a molecular weight of less than 60kDa in a physiologically acceptable medium.

16. A method of lightening skin comprising topically applying to the skin a cosmetic composition comprising the truffle extract obtained according to the method of claim 1, wherein said truffle extract comprises a compound having a molecular weight of less than 60kDa in a physiologically acceptable medium.