FR3160892A1 - Cosmetic treatment based on pycnidione - Google Patents

Abstract

Cosmetic treatment based on pycnidione The treatment is intended for the skin and its appendages to improve their general condition and/or sensory comfort. It can be chosen from a treatment for the signs of chronological or premature aging, a moisturizing treatment, a treatment for dull and/or tired skin, a treatment for sensory discomfort of the skin, a treatment for oily and/or acne-prone skin, and/or a hair treatment. Pycnidione can be provided in the form of an extract of the biomass of an in vitro culture of a microorganism capable of producing pycnidione as a secondary metabolism.

Description

Cosmetic treatment based on pycnidione

The present invention relates to a non-therapeutic cosmetic treatment of the skin and its appendages, implemented in particular topically.

It targets the cosmetic, dermatological and hygiene and personal care product industries, intended for people and animals, which manufacture and implement active ingredients and final formulas.
These industries are increasingly looking for new treatments to offer to improve an unsightly condition of the skin and/or its appendages or to improve their sensory comfort.
In general, it will involve preventing or treating unsightly imperfections or discomfort due to chronological or premature aging of the skin and/or its appendages, such as surface homogeneity defects (wrinkles, fine lines, roughness, etc.), color homogeneity defects (spots, redness, etc.) and radiance of the complexion (loss of radiance, luminosity), flaccidity (appearance of jowls, drooping eyelids, etc.), or sensory discomfort (tingling, tightness, etc.).
It can also involve treating skin or appendages that are not necessarily mature, for example in the case of a preventive treatment, or even treatments aimed at all skin types such as moisturizing treatments, improving the radiance of the complexion, or sensory discomfort that can be due to factors other than aging, such as pollution or an unhealthy lifestyle.
In the context of a cosmetic treatment, it will in no case be a question of offering a curative treatment for diseased skin.

The aim of the present invention is to propose a new cosmetic treatment for the skin (including the scalp and lips) and/or its appendages (including in particular the hair, body hair, eyelashes and eyebrows, nails), this treatment also advantageously having the possibility of implementing one or more active compounds obtained by a biotechnological route in a logic of “natural” products and sustainable development.

To this end, it proposes the use of pycnidione for a non-therapeutic cosmetic treatment of the skin (including the scalp and lips) and its appendages to improve their general condition and/or sensory comfort.

Pycnidione has been described as having anti-plasmodial activity, anti-cancer activity and more recently anti-bacterial activity, particularly anti-dandruff via an action on Malassezia-type bacteria (WO2010/061185).

Test results are given further in the description showing the advantageous use of pycnidione, for a non-therapeutic cosmetic treatment of the skin and/or its appendages, in particular a treatment of the signs of chronological or premature aging, a moisturizing treatment, a treatment of dull and/or tired skin (including dark circles and bags under the eyes), a treatment of sensory discomfort of the skin, a treatment of oily and/or acne-prone skin (treating a pro-acne condition of the skin) and/or a treatment of brittle and/or split hair.

More particularly and advantageously, the treatment according to the invention is suitable for:
- maintain or improve the mechanical and viscoelastic properties of the skin including the scalp;
- strengthen the dermis-epidermis junction (DEJ);
- combat the harmful effects of glycation (anti-glycation treatment);
- combat the harmful effects of free radicals (antioxidant or anti-free radical treatment, photoprotection);
- prevent or reduce roughness, fine lines and wrinkles of the skin;
- prevent or reduce sagging of the skin (jowls, drooping eyelids);
- prevent or reduce pigmentation defects; and/or
- stimulate the production of hyaluronic acid.
Other cosmetic activities or benefits may also be envisaged by those skilled in the art.

Pycnidione has a bis-tropolone-like chemical structure, corresponding to the following formula (I) in its natural form (stereochemistry described in the scientific publication “Total synthesis and computational investigations of sesquiterpene tropolones alleviate stereochemical inconsistencies and resolve an ambiguous biosynthetic relationship”, C. Bemis et al. J. Am. Chem. Soc. 2021, 143, 6006−6017):

(I)

According to the invention, the term "pycnidione" also includes dehydroxypycnidione (in which the hydroxyl group of the 10-carbon ring is replaced by a hydrogen) and eupenifeldin (stereoisomer of pycnidione). Pycnidione is preferred.

According to the invention, the term "pycnidione" includes pycnidione in a suitable, physiologically acceptable salt form.

Pycnidione can be produced by chemical synthesis, or it can be derived from a natural source and obtained by in-vitro biofermentation.

The following examples of microorganisms (fungi) can be used to produce pycnidione as a secondary metabolite by in-vitro biofermentation culture: Neosetophoma cerealis (synonym of Coniothyrium cereale , Coniothyrium cerealis , Neosetophoma samarorum , strain RKDO834), Neosetophoma sp. (Strain MSX50044), Theissenia rogersii (strains 9203120, 92031201, OS-F69284 (ATCC 74390)), Phoma sp. ( Strain ​ ​ ​ ​ Phomopsis tersa (strain FS441).

Such strains are available from collections or banks, for example for Neosetophoma cerealis from the Central Bureau for Fungal Biodiversity (Centraalbureau voor Schimmelcultures, CBS) under number CBS 963.68.

Preferably according to the invention, the pycnidione is provided in the form of an extract of the biomass of an in vitro culture of a microorganism capable of producing pycnidione as a secondary metabolite. This extract can be purified to increase the pycnidione content. Preferably the extract is produced by a strain of Neosetophoma cerealis . According to a particular example, the extract produced in vitro contains as secondary metabolites a set of tropolones, of which at least 80%, preferably at least 90% by weight, correspond to pycnidione, the remainder consisting of epolone A and/or epolone B which are tropolones such as pycnidione.

When the in-vitro production route by biofermentation is chosen, pycnidione is produced in the biomass under suitable and optimal culture conditions for the microorganism.

Cultivation may be carried out on any suitable synthetic medium or natural medium provided it contains appropriate carbon sources, nitrogen sources, and inorganic salts. If necessary, the medium may be appropriately supplemented with vitamins and other nutrients. Examples of general carbon sources include (but are not limited to) sugars such as glucose, maltose, fructose, sucrose, and starch, alcohols such as glycerol and mannitol, amino acids such as glycine, alanine, and asparagine, and oils and fats such as soybean oil and olive oil. Examples of nitrogen sources include organic nitrogen-containing compounds such as soybean powder, corn liquor, beef extract, peptone, yeast extract, amino acid mixtures, and fish powder, as well as inorganic nitrogen compounds such as ammonium salts and nitrates. Micronutrients in the form of inorganic salts may also be used, for example, calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, copper sulfate, manganese chloride, zinc sulfate, cobalt chloride, and various phosphates.
The microorganism can be cultured at a suitable culture temperature within a range that allows its growth and efficient production of pycnidione. The preferred culture temperature is 10°C to 32°C, and more preferably 20°C to 25°C. The pH at the start of culture is preferably about 6 to 8, and the culture time is from one day to a few weeks.
The culture can be stopped when a sufficient amount of pycnidione production is reached, preferably the maximum possible amount.
The culture can be carried out in solid or liquid phase under appropriate agitation in a bioreactor.

Appropriate treatment of the biomass (once separated from the supernatant) then allows the recovery of an extract containing pycnidione. Any separation method usually used in in vitro culture to extract a metabolite from biomass can be used to recover this extract, such as extraction with a suitable solvent in which the metabolite can be solubilized.

More preferably according to the invention, to extract the pycnidione from the biomass, extraction using a solvent is used and, more preferably, a physiologically acceptable solvent is also used, in particular a glycolated solvent, for example pentylene glycol or butylene glycol, or a mixture thereof. A clarification step to remove any remaining cellular debris is also carried out if necessary at the end of the treatment.

This extract comprising pycnidione in an appropriate quantity can be advantageously used as is for the implementation of the invention. The extract obtained can also be purified to increase the pycnidione content, until, if desired, purified pycnidione is obtained at a high purity level, for example having a pycnidione content greater than 80%. The purification of pycnidione can be carried out by chromatography or any other suitable means known to those skilled in the art.

If the biomass extraction solvent is not a physiologically acceptable solvent, and consequently if the extract cannot be used as such, the latter is freed from all traces of non-physiologically acceptable solvent by an appropriate treatment, and the residue of the extract obtained is then taken up in a physiologically acceptable medium so that it can be used at the appropriate dosage of pycnidione in the cosmetic treatment according to the invention.

“Physiologically acceptable” means according to the invention that the compositions are suitable for topical or transdermal use, in contact with the mucous membranes, nails, scalp, hair, fur and skin of mammals and more particularly humans, compositions which can be ingested or injected into the skin, without risk of toxicity, incompatibility, instability, allergic response, and others. This “physiologically acceptable medium” forms what is conventionally called the excipient of the composition.

Preferably according to the invention, the physiologically acceptable medium may be an alcoholic, glycolic, aqueous, hydroglycolic or hydroalcoholic medium, or formed by a water-in-oil emulsion, an oil-in-water emulsion, or a microemulsion. Preferably, the physiologically acceptable medium is glycolic according to the invention.

The "effective" amount of pycnidione for use in the treatment according to the invention depends on the intended use of the composition containing it, in particular face, hands, body, hair/scalp and it also depends on various factors, such as age, the condition of the person or the extent of the disorder. An effective amount means a non-toxic amount sufficient to obtain the desired effect.

As an indication, in a composition intended for a consumer for a treatment according to the invention, the pycnidione, to be present in an effective quantity, can range in the composition used relative to the total weight thereof from 0.000001% (0.001 ppm) to 10% (100,000 ppm), preferably from 0.000001% (0.01 ppm) to 1% (10,000 ppm), more preferably from 0.00001% (0.1 ppm) to 0.1% (1000 ppm), preferably from 0.0001% (1 ppm) to 0.1% (100 ppm), even more preferably from 0.001% (10 ppm) to 0.05 (50 ppm), depending on the destination of the composition and the desired effect, more or less pronounced, the number of applications recommended.

All percentages and ratios used in this application are by weight of the total composition and all measurements are made at 25°C, unless otherwise specified.

Also for information purposes, for cosmetic facial treatment, the European Cosmetics Directive has set a standard application quantity of 2.72 mg/cm²/day/person for a cream-type composition and 0.5 mg/cm²/day/person for a body lotion-type composition.

According to other particularities, the cosmetic treatment according to the invention can be combined with one or more other treatments targeting the skin or hair, such as light therapy, heat or aromatherapy treatments.

According to the invention, it is possible to propose multi-compartment devices or kits intended for implementing the method described above, and which could comprise, by way of example, and without this being limiting, in a first compartment a composition containing pycnidione, and in a second compartment an additional active agent acting jointly, the compositions contained in said first and second compartments being considered here as a combination composition for simultaneous, separate or spread out use over time, in particular in one of the treatments defined above.

According to other advantageous characteristics, pycnidione can be used according to the invention in combination with one or more additional active ingredients at effective concentrations advantageously making it possible to offer a synergistic effect or to reinforce activity.

The additional active ingredients may be chosen, for example, from lightening, anti-redness, anti-blemish, slimming, calming active ingredients for the treatment of sensitive, reactive skin, filtering radiation, in particular UVA, UVB, IR, visible light including blue light, moisturizing, humectant, exfoliating, smoothing, toning, anti-aging, anti-wrinkle and fine lines active ingredients, improving mechanical and elastic properties, radiance of the complexion, detoxifying active ingredients, anti-hair regrowth or on the contrary promoting growth, anti-dandruff, acting on the skin barrier, anti-acne, acting on sebum secretion, mattifying, unifying, anti-inflammatory, anti-oxidant, anti-free radical, anti-glycant, eye contour (anti-dark circles and anti-puffiness), promoting blood circulation, peptides, vitamins, ceramides, hyaluronic acid, etc. These active ingredients may be synthetic or obtained from plant materials, such as plant extracts or products of plant culture, fermentation, bacterial culture or enzyme culture.
The "International cosmetic ingredient dictionary &handbook" published by "the Cosmetic, Toiletry, and Fragrance Association, Inc.", Washington, DC describes a wide variety, without limitation, of cosmetic and pharmaceutical ingredients commonly used in the skin care industry, which are suitable for use as additional ingredients in the compositions according to the present invention, as long as they are physically and chemically compatible with the other ingredients of the composition and especially with the actives of the present invention. Furthermore, the nature of these additional ingredients must not unacceptably alter the benefits of the invention. These additional ingredients may be synthetic or natural such as plant extracts or be derived from a biotechnological process.
Other active ingredients for skin, scalp or hair care that are particularly useful in combination with the composition according to the invention can be found in the commercial documentation of Crodarom, Alban Muller International and Sederma, and on the website www.croda.fr.

Other examples include the following commercial active ingredients: betaine, glycerol, Actimoist Bio 2™ (Active organics), AquaCacteen™ (Mibelle AG Cosmetics), Aquaphyline™ (Silab), AquaregulK™ (Solabia), Carciline™ (Greentech), Codiavelane™ (Biotech Marine), Dermaflux™ (Arch Chemicals, Inc), Hydra'Flow™ (Sochibo), Hydromoist L™ (Symrise), RenovHyal™ (Soliance), Seamoss™ (Biotech Marine), Argireline™ (trade name for Lipotec's acetyl hexapeptide-3), spilanthol or an extract of Acmella oleracea known as Gatuline Expression™, an extract of Boswellia serrata known as Boswellin™, Deepaline PVB™ (Seppic), Syn-AKE™ (Pentapharm), Ameliox™, Bioxilift™ (Silab), PhytoCellTec™Argan (Mibelle), Papilactyl D™ (Silab), Preventhelia™ (Lipotec), or one or more of the following active ingredients sold by Sederma: Subliskin™, Venuceane™, Moist 24™, Vegesome Moist 24™, Essenskin™, Juvinity™, Revidrat™, Resistem™, Chronodyn™, Kombuchka™, Chromocare™, Calmosensine™, Glycokin factor S™, Biobustyl™, Idealift™, Ceramide 2™, Ceramide A2™, Ceramide HO3™, Legance™, Intenslim™, Prodizia™, Beautifeye™, PacifeelTM, Zingerslim™, Meiritage™, Sebuless™, Apiscalp™, Rubistem™, Citystem ^TM , Neonyca ^TM , NG Shea Butter Unsaponifiables ^TM , Majestem ^TM , Hydronesis ^TM , Poretect ^TM , Amberstem ^TM , Synchrolife™, Sylverfree™, Feminage™, Ameyezing™, BB-Biont™, Revitalide™, Mel[o]stem™, Luceane ^TM , or mixtures thereof.

Among the plant extracts (in the form of conventional extracts or prepared by an in vitro method) which can be used as additional active agents, mention may be made, in addition and in particular, of ivy extracts, for example of climbing ivy ( Hedera helix ), Bupleurum chinensis , Bupleurum falcatum , arnica ( Arnica montana L.), rosemary ( Rosmarinus officinalis N.), marigold ( Calendula officinalis ), sage ( Salvia officinalis L.), ginseng ( Panax ginseng ), ginko biloba, St. John's wort ( Hyperycum perforatum ), butcher's broom ( Ruscus aculeatus L.), ulm ( Filipendula ulmaria L.), orthosiphon ( Orthosiphon stamincus Benth .), artichoke ( Cynara scolymus ), algae ( Fucus vesiculosus ), birch ( Betula alba ), green tea, kola nut ( Cola nipida ), horse chestnut, bamboo, Centella asiatica , heather, fucus, willow, pilosella, escin extracts, cangzhu extracts, crysanthellum indicum extracts, plants of the Armeniacea genus, Atractylodis platicodon , Sinnomenum, pharbitidis , Flemingia , from Coleus such as C. forskohlii , C. blumei , C. esquirolii , C. scutellaroides , C. xanthantus and C. barbatus , as an extract from the roots of Coleus barbatus , extracts from Ballote, Guioa, Davallia, Terminalia, Barringtonia, Trema, Antirobia, Cecropia, Argania, Dioscoreae such as Dioscorea opposita or Mexican, extracts of Ammi visnaga , Siegesbeckia, in particular Siegesbeckia orientalis , plant extracts of the Ericaceae family, in particular extracts of blueberries ( Vaccinium angustifollium ) of Arctostaphylos uva ursi , Aloe vera, plants containing sterols (in particular phytosterols), Manjistha (extract of plants of the genus Rubia, in particular Rubia cordifolia ), Guggal (extract of plants of the genus Commiphora, in particular Commiphora mukul), an extract of kola, chamomile, red clover, Piper methysticum (Kava Kava from Sederma), Bacopa monieri (Bacocalmine™, Sederma) and sea whip, Glycyrrhiza glabra , mulberry, melaleuca (tea tree), Larrea divaricata , Rabdosia rubescens , Euglena gracilis , Fibraurea recisa hirudinea , Chaparral sorghum , sunflower, Enantia chlorantha , Mitracarpus of the genus Spermacocea, Buchu barosma , Lawsonia inermis L., Adiantium capillus-veneris L., Chelidonium majus , Luffa cylindrica , “Japanese Mandarin” ( Citrus reticulata blanco var. unshiu), Camelia sinensis , Imperata cylindrical , Glaucium flavum , Cupressus sempervirens , Polygonatum multiflorum , Loveyly hemsleya , Sambucus nigra , Phaseolus lunatus , Centaurium , Macrocystis pyrifera , Turnera diffusa , Anemarrhena asphodeloides , Portulaca pilosa , Humulus lupulus , Arabica coffee, Ilex paraguariensis , Globularia cordifolia , Oxydendron arboreum , Albizzia julibrissin , Zingiber zerumbet smith, Astragalus membranaceus , Atractylodes macrocephalae , Plantago lanceolata , Leontopodium alpinum (or eldelweiss), Mirabilis jalapa , Apium graveolens , Marrubium vulgare , Buddleja davidii Franch. , Monarda didyma , Lavandula angustifolia or orchids.

The compositions according to the present invention may comprise peptides, including, but not limited to, di-, tri-, tetra-, penta- and hexapeptides and their derivatives. According to a particular embodiment, the concentration of the additional peptide, in the composition, varies between 1x10-7% and 20%, preferably between 1x10-6% and 10%, preferentially between 1x10-5% and 5%, by weight.
For the purposes of the present invention, the term "peptide" herein refers to peptides containing ten or fewer amino acids, their derivatives, isomers, and complexes with other species such as a metal ion (e.g., copper, zinc, manganese, magnesium, and others). The term "peptides" refers to both natural peptides and (bio)synthetic peptides. It also refers to compositions containing peptides that occur in nature and/or are commercially available.

Non-limiting examples of dipeptides suitable for use in the present invention include Carnosine (beta-AH), YR, VW, NF, DF, KT, KC, CK, KP, KK, TT, PA, PM or PP.
Non-limiting examples of tripeptides include RKR, HGG, GHK, GGH, GHG, GKH, KPK, KFK, KavaK, KβAK, KabuK, KacaK, KPK, KMOK, KMO ₂ K, PPL, PPR, SPR, QPA, LPA or SPA.
Non-limiting examples of tetrapeptides are KTFK (SEQ ID NO: 1), GQPR (SEQ ID NO: 2), RSRK (SEQ ID NO: 3), KTAK (SEQ ID NO: 4), KAYK (SEQ ID NO: 5), KFYK (SEQ ID NO: 6), TKPR (SEQ ID NO: 7), AVPG (SEQ ID NO: 8), VPGA (SEQ ID NO: 8). 9), LKLE (SEQ ID NO: 10), ELED (SEQ ID NO: 11) or LLAN (SEQ ID NO: 12).
Non-limiting examples of pentapeptide are KTTKS (SEQ ID NO: 13) and KTSKS (SEQ ID NO: 14).
Non-limiting examples of hexapeptides are GKTTKS (SEQ ID NO: 15) or VGVAPG (SEQ ID NO: 16).
Other peptides that can be used in the context of the present invention may be chosen from, without this list being limiting: lipophilic derivatives of peptides, preferably palmitoyl and myristoyl derivatives, and complexes with the metal ions mentioned above (eg copper complex of the tripeptide HGG).
Preferred dipeptides include, for example, N-Palmitoyl-β-Ala-His, Pal-KT, Pal-RT (Sederma). Preferred tripeptides include, in particular, the copper derivative of HGG (Lamin™, Sigma), Pal-GHK, lipospondin (N-Elaidoyl-KFK) and its conservatively substituted analogs, N-Acetyl-RKR-NH2 (Peptide CK+), Pal-KavaK, Pal-KβAlaK, Pal-KabuK, Pal-KacaK, Pal-KMO2K, N-Biot-GHK (Sederma) and their derivatives.

Mention may also be made here of the anti-aging tripeptides of general formula X–Pro*–Pro*–Xaa–Y described in application WO2015/181688 with Xaa chosen from Leu, Arg, Lys, Ala, Ser, and Asp, at the N-terminal end, X chosen from H, -CO-R1 and -SO2-R1 and at the C-terminal end Y chosen from OH, OR1, NH2, NHR1 or NR1R2, R1 and R2 being, independently of one another, chosen from an alkyl, aryl, aralkyl, alkylaryl, alkoxy and aryloxy group, which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated and/or sulfur-containing, said group being able to have in its skeleton a heteroatom in particular O, S and/or N, and Pro* corresponding to Proline, an analog or a derivative thereof; including for example Myr-PPL-OH and Myr-PPR-OH.

Mention may also be made here of the pro-pigmenting and/or pro-MEC dipeptides and tripeptides of general formula X–(Xaa1)n–Pro*–Xaa2–Y described in application WO2014/080376, with n=0, 1 or 2, Xaa1 a hydrophobic amino acid chosen from Ala, Val, Met, Leu, Iso, Phe, Pro, and the analogues or derivatives thereof; or a polar amino acid chosen from Ser, Thr, Tyr, Asp, Glu and the derivatives and analogues thereof; and when n=2 the two Xaa1 amino acids may be identical or different; Xaa2 a hydrophobic amino acid chosen from Ala, Val, Met, Leu, Iso, Phe, and the analogues or derivatives thereof; a basic amino acid chosen from Arg, Lys, His, and the derivatives and analogues thereof; at the N-terminal end of the peptide, X is chosen from H, -CO-R1 and -SO2-R1; at the C-terminal end of the peptide, Y is chosen from OH, OR1, NH2, NHR1 or NR1R2, R1 and R2 being, independently of one another, chosen from an alkyl, aryl, aralkyl, alkylaryl, alkoxy and aryloxy group, which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated and/or sulfur-containing, said group being able to have in its skeleton a heteroatom in particular O, S and/or N; Pro* corresponding to Proline, an analog or a derivative thereof; including for example the peptides Pal-SPR, Pal-PPR, Pal-QPA, Pal-LPA, Myr-SPA, Pal-PM, Pal-PA and Pal-PP.

Tetrapeptide derivatives that may be used in the present invention include, but are not limited to, Ela-KTAK (SEQ ID NO: 17), Ela-KAYK (SEQ ID NO: 18), Ela-KFYK (SEQ ID NO: 19), Pal-GQPR (SEQ ID NO: 20) or Pal-KTFK (SEQ ID NO: 21).
Useful pentapeptide derivatives include, but are not limited to, Pal-KTTKS (SEQ ID NO: 22), Pal-KTSKS (SEQ ID NO: 23), Pal-YGGFXaa (SEQ ID NO: 24) with Xaa being Leu or Pro.
Useful hexapeptide derivatives include, but are not limited to, Pal-HLDIIXaa (SEQ ID NO: 25) with Xaa being Trp, Phe, Tyr, Tic, 7-hydroxy-Tic or Tpi, or a mixture thereof, Pal-GKTTKS (SEQ ID NO: 26), Pal VGVAPG (SEQ ID NO: 27).

The preferred compositions available commercially and offered by Sederma:
- tripeptides or derivatives include in particular Biopeptide-CL™, Maxilip™, or Procapil™ comprising GHK;
- tetrapeptides or derivatives include: RIGIN™, Eyeliss™, which contain Pal-GQPR (SEQ ID NO: 20) and an excipient;
- pentapeptides or derivative such as Matrixyl ™ source of Pal-KTTKS (SEQ ID NO: 22).
We can also cite:
- the mixture Pal-GHK and Pal-GQPR (SEQ ID NO: 20) (Matrixyl™ 3000), and
- the mixture Pal-GHK and Pal-VGVAPG (SEQ ID NO: 27) (Biobustyl™).

The following commercial peptides may also be mentioned as additional active ingredients:
- Vialox™, Syn-ake™ (β-Ala-Pro-Dab-NH-Bzl) or Syn-Coll™ (Pal-Lys-Val-Lys-OH) sold by the company Pentapharm,
- Argireline™ (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 (SEQ ID NO: 28) (INCI Name = Acetyl hexapeptide-3), Leuphasyl™ (Tyr-D-Ala-Gly-Phe-Leu (SEQ ID NO: 29)), Aldenine™ (Gly-His-Lys), Trylagen™ (INCI Name = Pseudoalteromonas Ferment Extract, Hydrolyzed Wheat Protein, Hydrolyzed Soy Protein, Tripeptide-10 Citrulline (product of the reaction of citrulline and Tripeptide-10 (synthetic peptide consisting of aspartic acid, isoleucine and lysine)), Tripeptide-1), Eyeseryl™ (Ac-β-Ala-His-Ser-His (SEQ ID NO: 30)), Serilesine™ (Ser-Ile-Lys-Val-Ala-Val (SEQ ID NO: 31)) or Decorinyl™ (INCI Name: Tripeptide-10 Citrulline = product of the reaction of Citrulline and Tripeptide-10 (synthetic peptide consisting of aspartic acid, isoleucine and lysine) sold by the company Lipotec,
- Collaxyl™ (Gly-Pro-Gln-Gly-Pro-Gln (SEQ ID NO: 32)) or Quintescine™ (Cys-Gly) sold by the company Vincience,
- Cytokinol™LS (casein hydrolyzate) sold by Laboratoires Serobiologiques/Cognis,
- Kollaren™ (Gly-His-Lys), IP2000™ (Pal-Val-Tyr-Val) or Meliprene™ sold by the European Institute of Cell Biology,
- Neutrazen™ (Pal-His-D-Phe-Arg-NH2) sold by the company Innovations, or
- BONT-L-Peptide™, Timp-Peptide™ or ECM Moduline™ (INCI Name = Palmitoyl Tripeptide-28: product of the reaction of palmitic acid and Tripeptide-28 (synthetic peptide consisting of arginine, lysine and phenylalanine) sold by the company Infinitec Activos.

It is also possible to combine the invention with one or more cyclic peptides, in particular those extracted from flax seed oil described in the Applicant's patent application FR1850845.

More specifically, pycnidione can be combined with at least one of the compounds chosen from vitamin compounds, group B C, E, F, D and A in particular, compounds such as niacinamide or tocopherol, retinoid compounds such as retinol, hyaluronic acid, hexamidine, α-lipoic acid, resveratrol, or DHEA, peptides, ceramides, UV or IR filters, chemical or physical, which are classic active ingredients used in cosmetic topical compositions.

The present invention also provides the use of pycnidione for the manufacture of a composition for a cosmetic treatment, as described above.

A composition for use according to the invention may be applied to the face, body, décolleté, hair and scalp in any form or vehicle known to those skilled in the art, defined according to the function and/or the site of application, in particular in the form of a solution, dispersion, emulsion, paste or powder, individually or in a premix or be carried individually or in a premix by vectors such as macrocapsules, microcapsules or nanocapsules, macrospheres, microspheres, or nanospheres, liposomes, oleosomes or chylomicrons, macroparticles, microparticles or nanoparticles, macrosponges, microsponges or nanosponges, microemulsions or nanoemulsions, or adsorbed on powdery organic polymers, talcs, bentonites, spores or exines and other mineral or organic supports.

In cosmetics in particular, applications may be proposed, in particular in the ranges of skin care for the face and/or body and/or hair and/or scalp and makeup-care ranges, in particular eyelashes and eyebrows.

For example, the form of the composition may be a lotion, cream, butter, milk, solid form, mousse, gel, deodorant, antiperspirant, shampoo, conditioner, hair mask, face mask, shower gel, etc.

The formulas may be included in personal care and/or beauty product lines, including skin care, cleansing, makeup, makeup removal, sunscreen, artificial tanning, pre-shave, shaving, or after-shave, slimming, moisturizing, humectant, emollient, conditioning, exfoliating, astringent, depilatory, antiperspirant or antiperspirant, deodorant, air freshener, perfumes, etc.

The composition may also be incorporated into a non-woven or woven material, made of natural or synthetic fibers, wool, or any material intended to come into contact with the skin and which may be used in clothing, in particular tights and socks, shorts, day or night underwear, handkerchiefs, masks, patches or fabrics, in order to exert its cosmetic effect through this skin/textile contact and allow continuous topical delivery (cosmetotextiles).

In the particular case of a cosmetic hair treatment according to the invention, the composition may comprise at least one surfactant which may be chosen from anionic, non-ionic, amphoteric and/or cationic surfactants.

Suitable anionic surfactants include alkyl sulfates, alkyl ether sulfates, alpha-olefin sulfonates, sulfosuccinates, isethionates, acylamides, acylglutamates, alkyl ether carboxylates and alkyl phosphates. The alkyl group preferably comprises between 6 and 30, more preferably between 8 and 20, in particular between 10 and 14, and especially 12 carbon atoms. Alkyl ether sulfates and/or alkyl sulfates are preferred, in particular alkali metals, e.g. their sodium and/or ammonium salts. Lauryl ether sulfate and/or lauryl sulfate are particularly preferred anionic surfactants.

Suitable nonionic surfactants include fatty alcohol acid or amide ethoxylates, alkanolamides and alkoxylated alkanolamides, monoglyceride ethoxylates, sorbitan ester ethoxylates, alkyl polyglycosides, ethylene glycol monoesters, ethylene glycol diesters, and mixtures thereof.

Suitable amphoteric surfactants include alkylimino-dipropionates, alkylamphoglycinates, alkylamphoproprionates, alkylamphoacetates (mono- and di-), N-alkyl beta-aminoproprionic acids, alkylpolyaminocarboxylates, phosphorylated imidazolines and mixtures thereof.

Suitable cationic surfactants include alkyl quaternaries, benzyl quaternaries, quaternary esters, ethoxylated quaternaries, alkylamines and mixtures thereof. The alkyl group preferably comprises between 6 and 30, more preferably between 8 and 22, and in particular between 10 and 20 carbon atoms.

The cationic surfactant may also be a polyquaternium (or polyquat) material. Polyquats include acrylamide- and/or dimethylallylammonium chloride-based polymers such as Polyquaternium 6, Polyquaternium 7, etc. Polymeric quaternium ammonium salts of guar gum, such as guar hydroxypropyltrimonium chloride, may be used. Polymeric quaternium ammonium salts of cellulose such as Polyquaternium 10 and the like, and polymeric quaternium ammonium salts of starch, may also be used.

The hair composition may also contain at least one secondary surfactant. If present, the secondary surfactant may be selected from a non-ionic, amphoteric, betaine and/or cationic surfactant.

Suitable betaines include alkyl betaines, alkylamido betaines, alkyl sultaines, alkylamidosultaines and mixtures thereof. Alkylamido betaines are preferred. The alkyl group preferably comprises between 6 and 30, more preferably between 8 and 20, and in particular between 10 and 14 carbon atoms.

The hair composition may further comprise lubricating agents such as talc, magnesium stearate and mineral oil, wetting agents, emulsifying and suspending agents, preservatives such as methyl- and propylhydroxy-benzoates, sweetening agents and flavoring agents.

It may include one or more other standard ingredients or carriers commonly used in hair care products, including shine enhancers, moisturizers, herbal additives, hair strengtheners, vitamin additives, colorants, hair thickeners, fixing and styling agents, ultraviolet absorbers, silicone oils, essential oils and perfumes, thickening or viscosity improving agents, detergents, stabilizers, emollients, chelating or sequestering agents, preservatives, disinfectants, antioxidants/free radical scavengers, antistatic agents, conditioning agents, detangling ingredients, emulsifying or dispersing agents, stimulants, softeners, solvents, carrier, and others.

It may include additional hair active ingredients, as support or complement to activity, in particular the following active ingredients marketed by Sederma:
Active ingredient acting on the scalp:
- Apiscalp ^TM : Extract of Apium graveolens (celery) seeds, titrated in senkyunolide and obtained by supercritical _CO2 extraction. Its function is to alleviate dandruff and relieve itchy scalp. It moisturizes and reduces sebum production.
- Hairspa ^TM : : Combination of Lactitol and xylitol in glycerin with the function of soothing and moisturizing the scalp. It helps combat scalp discomfort: dryness, itching, dandruff,
Irritations, by rebalancing the skin microflora.
Active ingredient acting on the quality or color of the hair:
- FruitBio ^TM : Complex of α-hydroxy acids, obtained by lactic fermentation with the addition of malic and citric acids, combined with green tea extract. This active ingredient smooths the hair cuticles and green tea softens, for smoother and softer hair.
- Heliogenol ^TM : Hydroglycolic extract of sunflower meal titrated in polyphenols. It has a strong activity against free radicals. It protects and repairs natural and colored hair against the aggression of shampoo and UV rays.
- Ceramide A2 ^TM : Chemical analogue of ceramide 2, a natural molecule in hair. Its function is to restructure, smooth and strengthen the hair structure, to restore volume, shine and vigor to damaged hair.
- Sylverfree ^TM : Hydroglycerin solution of palmitoyl prolyl proline (Pal-PP). It helps fight hair graying.
Active ingredient acting on hair growth:
- Capigen ^TM : is composed of homotaurine, which is a synthetic analogue of Taurine, a natural amino acid, a bacterial filtrate rich in peptides and a sulfomucopolysaccharide extract of marine origin. It is a hair tonic stimulant and helps prevent hair loss.
- Procapil ^TM : Combination of a vitamin matrikine (biotinyl-GHK) with apigenin (a natural flavonoid) and oleanolic acid of natural origin. It helps strengthen and rejuvenate the hair follicle to slow hair loss. Hair anchoring is promoted by strengthening the metabolism and structure of the follicle. Oleanolic acid inhibits 5α-reductase, apigenin improves microcirculation and biotinyl-GHK stimulates cellular metabolism.
- Kelisoft ^TM : Chelidonine, a highly purified plant molecule in an excipient. Its function is to reduce hair growth.

The hair composition may also comprise an additional anti-dandruff active agent selected from one of the following: nystatin, cuprimyxin, tolnaftate, candicidin, haloprogin, iodochlorohydroxyquine, clotrimazole, undecylenic acid, proprionic acid, caprylic acid, benzoic acid, salicylic acid, griseofulvin, amphotericin B, ketoconazole, miconazole, filipin, hamycin, natamycin, rimocidin, bifonazole, butoconazole, econazole, fenticonazole, isoconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, fluconazole, isavuconazole, itraconazole, posaconazole, ravuconazole, terconazole, voriconazole, abafungin, amorolfine, butenafine, naftifine, terbinafine, anidulafungin, caspofungin, micafungin, iclopirox olamine, flucytosine, crystal violet, piroctone olamine, zinc pyrithione, selenium sulfide, tar and tea tree oil, preferably selected from ketoconazole, zinc pyrithione (ZPT), piroctone olamine, octopirox, salicylic acid, selenium sulfide, coal tar, azelaic acid, grimpazole, salicylic acid, undecylenic acid, and mixtures thereof.

The present invention thus covers a method of cosmetic, non-therapeutic topical treatment for improving the appearance and general condition of the skin and its appendages, as well as its sensory comfort, comprising the topical application to the skin of a subject in need of an effective amount of pycnidione, the treatment being as defined above and in the examples given below.

DETAILED DESCRIPTION

The present invention will be better understood in light of the detailed description which follows of exemplary embodiments.

1- Example of preparation of an extract containing pycnidione

Microorganism used: strain of Neosetophoma cerealis which can be obtained from the Central Bureau for Fungal Biodiversity (Centraalbureau voor Schimmelcultures, CBS).
A pre-inoculum step is performed using cryopreserved spores or mycelium, inoculated into a pre-inoculum-specific culture medium (medium that contains a gelling agent, sugar, and nitrogen in the form of peptone or yeast extract). Several pre-inoculum steps are performed to amplify the biomass.
When the pre-inoculum has grown sufficiently, the biomass produced is used to inoculate the bioreactor in a culture medium based on rice and microelements. In the bioreactor, also called a producer, the biomass is amplified with the concomitant production of pycnidione.
After 7 days of cultivation in the producer, pycnidione production is at its maximum.
The biomass is then harvested using a solid/liquid separation technique such as centrifugation, decantation by centrifugation, or filtration through a filter press. The supernatant is removed.
The biomass thus harvested is mixed with a solvent intended to form a physiologically acceptable excipient, consisting of a mixture (20/80 w/w) of pentylene glycol (PTG) and butylene glycol (BG), with stirring for 1 hour at 60°C to extract the pycnidione in this excipient.
After extraction, cellular debris is removed by clarification using a known solid/liquid separation technique, as mentioned above.
Analysis of the dry extract makes it possible to determine a final concentration in the extract of total tropolones of 1200 ppm, including approximately 1100 ppm of pycnidione, the target active molecule according to the invention, and the remainder of the tropolones comprising in particular Epolone A and Epolone B.

2- Example of preparation of an active ingredient for the treatment according to the invention

The PTG/BG extract prepared in point 1 below, comprising a physiologically acceptable excipient, constitutes an active ingredient which can be used in a cosmetic formula at a % by weight defined according to the desired effect, more or less pronounced, as explained above in the description. Different cosmetic formulas using this cosmetic ingredient are described further in the description in point 4.

3- In vitro efficacy tests

The tests were carried out with either the dry extract of the biomass prepared in point 1 above diluted in DMSO, or pure pycnidione (approximately 82%) also diluted in DMSO. The ppm indicated in the tables below correspond to the ppm of the pycnidione molecule.

All statistical analyses were performed with a Student's t-test to determine the significance of the results of pairwise comparisons (control versus treated).

3.1- Stimulation of hyaluronic acid synthesis

Hyaluronic acid is an essential component of the skin. It is present in the dermis and epidermis and, thanks to its unusual three-dimensional structure, is able to absorb a thousand times its weight in water and thus occupy a very large volume relative to its molecular weight. The intracellular spaces of the extracellular matrix are filled with water-swollen hyaluronic acid, which gives the skin good viscoelastic properties.
By stimulating the synthesis of hyaluronic acid, hydration is maintained or increased for more resilient and elastic skin, with a smoother and more comfortable texture to the touch, without tingling or tightness.

Protocol :

Human keratinocytes (KH) are cultured to sub-confluence and then brought into contact with the active ingredient according to the invention. Following this contact, the synthesis of hyaluronic acid is evaluated using an ELISA-type assay. An estimation of the quantity of cells by the Hoechst method makes it possible to homogenize the results.

Results

Variation in hyaluronic acid production by keratinocytes compared to control.

Pycnidione (ppm) Variation (%); significance Control / Reference Extract 0.3 +347%; p<0.01 1 +314%; p<0.01 3 +422%; p<0.01 Purified Pycnidione 1 +87%; p<0.01 5 +110%; p<0.01 10 +129%; p<0.01

The results show that pycnidione significantly increases the synthesis of hyaluronic acid in keratinocytes.

3.2- Strengthening of the dermo-epidermal junction (DEJ)

The DEJ ensures cohesion between the epidermis and the dermis. During skin aging, whether normal or accelerated by external factors, there is a decrease in the synthesis of DEJ components, particularly laminins and collagen VII, leading to disorganization with repercussions on the elasticity and resilience of the skin and the loss of its dynamism.

Protocol

KH cells are cultured to sub-confluence and then placed or not (for control cases) in contact with the active ingredient according to the invention. Following this contact, the synthesis of collagen VII and laminins is evaluated using ELISA kits. An estimation of the quantity of cells by the Hoechst method allows the results to be standardized.

Results

Variation in the production of laminins and collagen VII by keratinocytes compared to control:

Pycnidione (ppm) Laminins
Variation (%); significance Collagen VII
Variation (%); significance Control / Reference Reference Extract 0.3 +70%; p<0.01 +98%; p<0.01 1 +79%; p<0.01 +52%; p<0.01 3 +132%; p<0.01 +59%; p<0.01

The results show that pycnidione significantly increases the production of laminins and collagen VII in keratinocytes.

3.3- Anti-glycation:

The human body needs sugar to produce its energy. However, the sugar that is consumed is never fully used by the body for this beneficial purpose. A residual portion of this absorbed sugar will react non-enzymatically with the amino groups of proteins, nucleic acids or lipids to create advanced glycation end products, called glycotoxins or AGEs for "Advanced Glycosylated End-products" (AGEs), which accumulate over time in the tissues. The functional, enzymatic, and structural properties of glycated molecules are altered, and this has consequences on the proper functioning of cells or the body.

In the skin, AGEs are found not only in the dermis but also in the epidermis, up to the stratum corneum . Tissues are affected more when their proteins have a long lifespan, such as collagen, elastin, fibronectin and laminins. This has the consequence of altering the mechanical and elastic properties of the extracellular matrix (ECM) of the dermis, which becomes less flexible, more rigid, but also more flaccid and less reactive.

Additionally, within skin cells, mitochondrial proteins are also glycated, causing decreased efficiency in the production of adenosine triphosphate (ATP) and impaired energy production.

AGEs therefore represent a major source of cellular dysfunction and their accumulation tends to alter the properties and performance of tissues and cells: reduced flexibility, reduced mobility, reduced reactivity, reduced energy production. On the face, glycation leaves its mark, for example, dark circles under the eyes, puffy bags, dull complexion and drawn features, less reactive, tired, flabby skin. A study also showed that the quantity of AGEs and hair quality were linked. A reduction in protein content and an increase in AGEs content lead to poorer hair quality with a decrease in resistance to breakage.

Glycation therefore visually translates on the skin by the appearance of wrinkles and fine lines, dull, flaccid, lackluster, tired-looking skin, skin lacking tone and suppleness, and on the hair by poorer quality hair, split ends and brittleness.

Protocol

The study of non-enzymatic glycation is carried out between a model protein, serum albumin, which serves as a target, and an edible reducing sugar derived from fruit. The protein is progressively glycated (linked to the sugar) irreversibly, in the presence or absence of the product according to the invention. This modification is monitored by fluorescence.

Results

Variation in AGE production compared to control:

Pycnidione (ppm) Variation (%); significance Control / Reference Extract 1 -32.8%; p<0.01 3 -65.5%; p<0.01 6 -87.6%; p<0.01

The results show the strong anti-glycating potential of pycnidione.

3.4- Decrease in the synthesis of pro-inflammatory molecules:

The skin is subjected to constant stress (exposure to UV rays, smoke, pollutants, etc.), some of which cause a direct or indirect inflammatory response. The uncontrolled or constant inflammatory response, although of low intensity, leads to the production of cytokines intended to attract or stimulate other cells, which causes cascading reactions. The pro-inflammatory microenvironment thus formed leads to a modification of the skin's homeostasis and gradually leads to the modification or even destruction of the biomolecules of cells and tissues. It also leads to the disruption of the integrity of the skin barrier. Thus, the mediators of inflammation, IL-6 and IL-8, are known to induce premature aging phenomena via micro-inflammations. In addition, sensitive and irritated skin is characterized by an abnormally high secretion of these cytokines.
By reducing the production of these cytokines following stress, the invention demonstrates a preventive anti-inflammatory effect. The cells are better protected. From a cosmetic point of view, the treatment according to the invention helps prevent discomfort, such as tightness, and unsightliness, such as micro-redness of the skin, for example due to external aggressions such as pollution, radiation or cold.

Protocol

Normal human keratinocytes (NHK) are seeded and placed in contact or not with the active ingredient to be tested at different concentrations for 48 hours and are irradiated with UVB. Non-irradiated plates are treated in parallel. The quantities of IL-6 and IL-8 synthesized are measured in the culture supernatants by ELISA assays. An estimation of cell viability is carried out at the end of culture using Hoechst labeling on fixed cell lawns (estimation of cell number).

Results

Variation in IL-6 and IL-8 synthesis after keratinocyte stress compared to control:

Pycnidione (ppm) IL-6
Variation (%); significance IL-8
Variation (%); significance Control / Reference Reference Extract 0.3 -47%; p<0.01 / 1 -49%; p<0.05 -47%; p<0.01

The results show that pycnidione induces a decrease in IL-6 and IL-8 synthesis after stress (UVB condition).

3.5- Decrease in intracellular production of oxygenated free radicals (OR):

It is now recognized that it is essentially the accumulation of oxygenated free radicals that is at the origin of the biochemical disorders themselves responsible for aging. Whether they are generated by UV rays (via a complex cascade of reactions), by the various pollutants that interact with the skin chronically or by natural aging, these free radicals will cause occasional and short-term skin inflammation, and throughout the body and over a longer period skin aging. Under so-called normal conditions, on young, healthy skin that is relatively protected from external aggressions, there is a balance between the production of oxygenated free radicals in the body and the antioxidant defenses responsible for neutralizing them. These defense systems are made up of either enzymes (SOD, catalane, glutathione peroxidase), or classic antioxidants (vitamin C, vitamin E, glutathione, carotene, etc.). When an imbalance occurs between production and neutralization, there is an overabundance of oxygenated free radicals causing oxidative stress. This will result in damage to DNA, proteins and cellular lipids, which will undergo oxidation, causing alterations in their structure and function. This results in the appearance of signs of premature aging, damage to the dermis and epidermis with loss of their mechanical and viscoelastic properties, thinning of the skin, sagging of the skin, fine lines, wrinkles, spots, etc.

Protocol

Normal human fibroblasts (NHF) are seeded and placed in contact with the product for 24 hours. After removal of the active ingredient, the cells are rinsed and loaded with the DCFH-DA probe, which becomes fluorescent, after metabolization by cellular enzymes, in the presence of RLO. This fluorescence is further increased when a stressor (H ₂ O ₂ ) is applied to the cells. An active product will reduce basal and induced fluorescence (RLO production).
An estimation of cell viability is carried out in parallel using Hoechst labeling (DNA intercalating reagent).

Results

Variation in RLO synthesis by fibroblasts compared to control, with and without oxidative stress:

Pycnidione (ppm) Non-stressed case
Variation (%); significance Stressed case ( _{H2O2 )}
Variation (%); significance Control / Reference Reference Extract 0.1 -44%; p<0.01 -42%; p<0.01 0.3 -33%; p<0.01 -54%; p<0.01 1 -57%; p<0.01 -69%; p<0.01

Pycnidione _{significantly} decreases the generation of reactive oxygen species (ROS) following _H2O2 stress but also basally (unstressed condition) without dose effect.
Pycnidione helps combat the appearance of intracellular RLOs known for their involvement in skin aging.

3.6- Anti-Lipoperoxidation:

Lipoperoxidation or lipid peroxidation corresponds to the peroxidation of lipid membranes surrounding cells and mitochondria, leading to their weakening and the generation of oxygenated free radicals by autocatalysis, amplifying the damage.

Protocol

Lipid peroxidation is assessed using a test to monitor the occurrence of peroxidation (measurement of the amount of conjugated dienes formed) of lipid membranes in the form of liposomes after oxidative UVA radiation. Reduction by an antioxidant is sought. The product is put into this test after irradiation.

Results

Variation of lipoperoxidation compared to control:

Pycnidione (ppm) Variation (%); significance Control / Reference Extract 0.3 -51%; p<0.01 1 -61%; p<0.01 3.33 -82%; p<0.01 Purified Pycnidione 5 -65%; p<0.01 10 -84%; p<0.01

The results show the strong potential of pycnidione to counter lipoperoxidation and therefore its harmful effects on cell membranes and their dynamism.

3.7- Slowing down the proliferation of C. acnes :

The skin microbiota, a very complex ecosystem composed of a set of living microorganisms (bacteria, yeasts, viruses and parasites), has several functions: defense role, skin barrier role and immune system regulator. It is important to protect its balance by preventing, for example, that certain species by developing excessively cause damage to the skin. This is the case for example C. acnes ( Cutibacterium acnes ), the acne bacterium. The latter, although part of the normal microflora of the skin, by multiplying too quickly, will promote the proliferation and migration of keratinocytes, participate in the formation of radical species such as superoxide anion and cause a cascade of reactions which results in the production of pro-inflammatory molecules and thus contribute to the development of acne.

By slowing down or inhibiting the growth of c.acnes , the epidermis is protected against acne. This effect is particularly interesting for cosmetic treatment of so-called oily and acne-prone skin. This type of skin often corresponds to oily skin in adolescence due to excess sebum for hormonal reasons. By preventing or limiting the proliferation of the C. acnes bacteria, we prevent the appearance of waste in the hair sheath in the hair follicle where the bacteria develops anaerobically by feeding on this sebum and producing waste, including dead cells, then leading to obstruction of the hair follicle sheath (blackheads), inflammation and finally acne pimples, the treatment of which is a matter of dermatology. Reducing the quantity of C.acnes therefore helps prevent the development of a pro-acne state.

Protocol
Suspensions of C. acnes of equivalent density are cultured in a suitable medium in the presence (test case) or absence (control case) of the product to be tested, under anaerobic conditions, at 37°C. After 48 to 72 hours of incubation, the growth of the bacterial population is measured by reading the optical density (OD) at 600nm.

Results

Variation in C. acnes proliferation compared to control:

Pycnidione (ppm) Variation (%); significance Control / Reference Extract 1.88 -101%; p<0.01 3.75 -100%; p<0.01 7.5 -100%; p<0.01 15 -99%; p<0.01 30 -99%; p<0.01 Purified Pycnidione 1 -57%; p<0.01 4 -99%; p<0.01 8 -101%; p<0.01

The results show that pycnidione induces a significant slowdown in the proliferation of C. acnes bacteria responsible for a pro-acne state.

4- Examples of cosmetic formulas

Various cosmetic formulas are described below. Additional active ingredients, where appropriate supporting and/or complementing the activity of the active ingredient according to the invention, may be added in the appropriate phase depending on their hydrophobic or hydrophilic nature. These ingredients may be of any category depending on their function, the place of application (body, face, neck, bust, hands, hair, eyelashes, eyebrows, body hair, etc.), the desired final effect and the target consumer, for example an antioxidant, moisturizing, nourishing, protective, smoothing, remodeling, volumizing, acting on the radiance of the complexion, anti-spot, anti-dark circle, anti-glycation, slimming, soothing, muscle relaxant, anti-redness, anti-stretch mark, etc. They are mentioned above in the description.

Ingredient according to the invention: ingredient prepared in paragraph 2) above (extract prepared in vitro containing approximately 1100 ppm of pycnidione in a mixture of butylene glycol and pentylene glycol).

Recommended effective amount of ingredient: for example between 1% and 15%, preferably between 1% and 10%, more preferably between approximately 1% and 5%, which corresponds to between approximately 10 ppm and 60 ppm of pycnidione, indicated at 3% (approximately 35 ppm) in the example formulas below.

4.1- Light cream formula, suitable for the face

Preparation :

Weigh Part A. Weigh and mix Part B. Slowly add Part B to Part A while stirring and mix well for 30 min. Heat Part A+B to 75°C in a water bath. Weigh and heat Part C to 75°C in a water bath. Add Part C to Part A+B while stirring vigorously. Add Part D to Part A+B+C until approximately 45°C. Add Part E to the previous part while stirring. Add Part F, if applicable, to the previous part and mix well.
The result is a light, non-sticky cream that can be used, for example, as a facial cream for a relaxing massage application.

4.2- Cream formula suitable for a day cream

Preparation :

Weigh Part A, mix well under normal stirring. Weigh and mix Part B. Pour Part B into Part A under normal stirring for one hour. Heat Part A+B to 75°C in a water bath. Weigh and heat Part C to 75°C in a water bath. Add Part C to Part A+B, stirring quickly. Weigh and add Part D to the previous part at approximately 45°C, mix well. Weigh and add Part E to the previous part, mix well. If applicable, weigh and add Part F, to the previous part, mix well.

The result is a white, opaque cream with a light, soft feel that can be used as a daytime treatment. The active ingredient Crystalide® is used as an additional ingredient to help repair the skin and give it a radiant complexion.

4.3- Cream formula, suitable for oily, acne-prone skin

Preparation :

Weigh Part A and mix. Weigh and mix Part B. Add Part B to Part A, stirring for 1 hour. Heat Part A+B to 75°C in a water bath. Weigh and heat Part C to 75°C in a water bath. Add Part C to Part A+B, stirring vigorously. Weigh and mix Part D. Add Part D to the previous part, mixing well. Add Part E, mixing well. If applicable, add Part F, mixing well.

The result is an opaque, off-white viscous cream suitable for oily skin thanks to its dry feel and rapid absorption. The combination with the Fruitbio ^TM ingredient allows for the addition of a gentle chemical exfoliant to further smooth the skin's texture.

4.4- Cream formula, suitable for hands

Preparation :

Weigh Part A and mix under normal stirring. Weigh and mix Part B. Pour Part B into Part A under normal stirring for one hour. Activate Part A+B by stirring vigorously for 1 min. Add Part C into Part A+B, stir well. Heat Part A+B+C to 75°C in a water bath. Weigh and heat Part D to 75°C in a water bath. Weigh and add Part E to Part D well before creating the emulsion. Add Part D+E to the previous part, mix well under rapid stirring. Weigh and mix Part F. Add Part F to the previous part at approximately 45°C, mix well. Weigh and add Part G to the previous part, mix well. Weigh and add Part H to the previous part, mix well.

The result is a cream that is perfectly suited to the treatment of dry hands thanks to the ingredient according to the invention and the additional combination with a ceramide, a natural lipid constituent of the skin.

4.5- Cream formula, suitable for face and neck

Preparation :

Weigh Part A. Weigh and mix Part B. Slowly add Part B to Part A while stirring and mix well for 30 min. Heat Part A+B to 75°C in a water bath. Weigh and heat Part C to 75°C in a water bath. Add Part C to Part A+B while stirring vigorously. Add Part D to Part A+B+C while stirring. If necessary, add Part E to the previous part while stirring. Mix well during the cooling phase.

The result is a pale yellow cream, ideal for the face and neck, which is quickly absorbed by the skin.

4.6- Cream formula, suitable for a day cream for the décolleté

Preparation :

Weigh Part A and mix under normal stirring. Weigh and mix Part B. Pour Part B into Part A under normal stirring for one hour. Activate Part A+B by stirring vigorously for 1 minute. Weigh Part C. Add Part C to Part A+B, stir well. Heat Part A+B+C to 75°C in a water bath. Weigh and heat Part D to 75°C in a water bath. Weigh and add Part E to Part D before creating the emulsion. Add Part D+E to the previous part, mix well under rapid stirring. Weigh and add Part F to the previous part, mix well. Weigh and add Part G to the previous part, mix well.

The result is an opaque cream emulsion with a soft and light texture, quickly absorbed by the skin, suitable for a large area such as the décolleté.

4.7- Cream lotion formula

Preparation :

Weigh Part A, mix well under normal agitation. Weigh and mix Part B. Add Part C to Part B, stir well. Weigh and add Part D to B+C, mix well. Weigh Part E, mix well. Adjust the pH of B+C+D to pH +/-5.40 with Part E, mix well. Slowly add Part B+C+D+E to Part A, stirring rapidly.

A white opaque fluid emulsion is obtained, forming a super concentrated lotion comparable to an E/O emulsion with a rich creamy feel.

4.8- Hydrogel formula

Preparation :

Heat part A to 80°C in a water bath with stirring for 1 hour. Weigh part B and mix well. Slowly add part B to part A while stirring at 80°C in a water bath. Allow part A+B to heat to 80°C for 1 hour in a water bath with stirring. Add part C to part A+B in a water bath at 80°C. Add part D to part A+B+C and mix well. Add part E if necessary and mix well. Pour immediately into the molds, at a warm temperature.

The result is a hydrogel capable of providing an instant cooling and soothing sensation while the active ingredient(s) work deep down to soothe and protect the skin over the long term. The addition of Crodateric CAB 30™ as a mild surfactant allows the skin to be gently cleansed with water at the end of use.

4.9- Concentrated serum formula

Preparation :

Weigh Part A, mix well under normal stirring. Weigh and mix Part B. Pour Part B into Part A under normal stirring for one hour. Weigh and add Part C to Part A+B, under rapid stirring. Weigh Part D, mix well. Add Part D to the previous part, stir well. Weigh and add Part E to the previous part, mix well. If applicable, weigh and add Part F to the previous part, mix well.

The result is an emulsion that forms a super-concentrated serum that resembles a gel and has the feel of a rich cream. In combination, the ingredient according to the invention and the Synchrolife ^TM ingredient provide a complete regenerating night treatment.

4.10- Dough formula

Preparation :

Weigh Part A and heat to approximately 65°C. Weigh Part B. Add it to Part A and mix well while stirring. Weigh Part C and add it to Part A+B while stirring. Weigh Part C and add it to the previous part at 50°C. Weigh and add Part D. Mix well. If necessary, weigh and add Part E, mix well. Pour immediately into the pot.

The result is an opaque beige paste that can be used as a concealer and as a screen against urban pollution.

4.11- Cream formula, suitable for daytime care of the eye contour

Preparation :

Weigh and mix Part A while stirring. Weigh Part B. Add Part B to Part A while stirring normally. If applicable, weigh Part C. Add Part C to Part A+B and mix well. Weigh Part D. Weigh Part D and mix well while stirring normally. Add Part A+B+(C) to Part D while stirring quickly and mix well.

The result is an opaque, off-white viscous cream, perfectly suited to eye contour care, firming and illuminating this area. The FeminageTM active ingredient reinforces the treatment by acting on sagging skin.

4.12- Spray formula

Preparation :

Weigh Part A. Weigh and mix Part B. Pour Part B into Part A under normal stirring. Weigh and add Part C into Part A+B, stir well. Weigh and add Part D to the previous part, mix well. Weigh and mix Part E. Add Part E to the previous part, mix well. If applicable, weigh and add Part F to the previous part, mix well.

The result is a clear, pale yellow liquid that can be used as a spray with a light texture. The addition of the BB-Biont ^TM ingredient also helps regulate the microbiome and smooth out acne marks. The formula is particularly suitable for use on mask-wearing skin.

4.13- Lotion formula, suitable for the body

Preparation :

Weigh Part A, mix well. Heat Part A to 75°C in a water bath. Weigh Part B, mix well. Add Part B to Part A while stirring rapidly for one hour at 75°C in a water bath. Mix Part A+B while stirring very vigorously for 1 minute. Weigh and heat Part C to 75°C in a water bath. Add Part C to Part A+B while stirring vigorously. Weigh Part D, mix well. Adjust the pH =5.80+/-0.10 with Part D, at approximately 45°C, mix well. Add Part E to the previous part, mix well. If necessary, add Part F to the previous part, mix well.

The result is an opaque fluid cream with a fresh, silky texture. It can be used as an after-hair removal lotion, for example, in combination with the active ingredient Kelisoft ^TM for hair regrowth.

4.14- Solid stick formula

Preparation :

Weigh and heat Part A to 75°C in a water bath. Weigh Part B and add to Part A. Weigh and mix Part C. Heat Part C to 75°C in a water bath. Add Part C to Part A+B while stirring rapidly. Weigh Part D and mix. Add Part D to the previous part at approximately 50°C while stirring rapidly. Pour to form the stick into a mold immediately, at approximately 45°C which becomes solid at room temperature.

The result is a solid, opaque, light yellow stick. It can be used for a quick and convenient touch of care. The combination of the ingredient according to the invention with the PoreTect™ ingredient allows for a specific moisturizing, anti-aging, and care action for enlarged pores. As a daytime treatment, the Solaveil™ CT-300 ingredient also protects against damage that can be caused by UV rays.

4.15- Rich cream formula with a whipped texture

Preparation :

Weigh Part A, mix well. Weigh Part B, mix well. Add Part B to Part A, stirring rapidly for one hour. Heat Part A+B to 75°C in a water bath. Weigh and heat Part C to 75°C in a water bath. Add Part C to Part A+B, stirring vigorously. Weigh Part D, mix well. Add Part D to the previous part, at about 45°C, stir well. Add Part E to the previous part, stir well. Add Part F, if applicable, to the previous part, stir well.

After 24 hours of rest, stir the emulsion with a U-shaped paddle for one hour.

The result is a pale yellow, opaque, viscous cream with a whipped, mousse-like texture. It is rich yet leaves the skin with a comfortable, non-greasy feel.

4.16- Formula for roll-on applicator for lips

Preparation :

Weigh Part A and heat to about 75°C. Add Part B and mix well for 30 minutes while stirring. Weigh Part C and heat to about 75°C. Add Part C to Part A+B while stirring at high speed. Weigh Part D and mix well. Adjust the pH to 6.00-6.20 with Part D when the temperature is below 45°C, mix well. Weigh and add Part E to the previous part, mix well. Weigh Part F and add it to the previous part, mix well.

The result is a dark red fluid emulsion compatible with a “roll-on” type application which offers a restorative, moisturizing and protective cosmetic treatment thanks to its active ingredients, and an aesthetic visual effect of volume and shine to the lips.

4.17- Shampoo formula

Preparation :

Weigh Part A. Mix well. Add the ingredients of Part B, one after the other, to Part A+B while stirring. Mix well. Weigh Part C, mix well. Add Part C to Part A+B with normal stirring. Add Part D to the previous part with normal stirring. Add Part E to the previous part while stirring normally.

The result is a clear, pale yellow fluid gel that forms a shampoo to gently cleanse and treat hair and scalp.

4.18- Leave-on spray formula for hair application

Preparation :

Weigh Part A and mix. Weigh Part B and mix well. Weigh Part C and mix well. Mix Part B into Part C. Pour Part B+C into Part A with normal helical stirring. Add Part D to the previous part with normal helical stirring.

We obtain an opaque off-white fluid emulsion with a texture suitable for application by spraying to be applied lightly to the hair and scalp.

4.19- Mask formula for hair application

Preparation :

Weigh and heat Part A to 85°C in a water bath. Weigh and heat Part B to 90°C in a water bath. Weigh Part C and mix well. Pour Part C into Part A with normal stirring. Disperse Part B into Part A + C with vigorous stirring, mix well. If necessary, pour Part D, at approximately 40°C, into the previous part while stirring. If necessary, add Part E, mix well. If necessary, add Part F to the previous part, mix well. Add Part G, mix well. Add Part H, mix well.

A yellow opaque viscous emulsion is obtained, forming a thick cream suitable for a silicone-free hair and scalp mask which can include a range of active ingredients such as the active ingredient according to the invention for a complete cosmetic treatment.

4.20- Serum formula for hair application

Sprinkle the carbomer into the water and let it swell for 30 minutes. Weigh Part B. Add Part B to Part A while stirring. Weigh and mix Part C. Neutralize with Part C to Part A + B. Homogenize well while stirring. Weigh and mix Part D. Pour Part D into Part A + B + C while stirring. Add Part E and mix well. Add Part F and mix well.

We obtain a colorless fluid gel forming a serum with a light, non-greasy texture.

Claims (15)

Use of pycnidione for non-therapeutic cosmetic treatment of the skin and its appendages to improve their general condition and/or sensory comfort. Use according to claim 1, characterized in that the pycnidione is provided in the form of an extract of the biomass of an in vitro culture of a microorganism capable of producing pycnidione as a secondary metabolite, said extract being able to be optionally purified to increase the pycnidione content. Use according to claim 2, characterized in that the extract comes from the biomass of an in vitro culture of a strain of Coniothyrium cerealis . Use according to any one of the preceding claims, characterized in that the pycnidione is added to a physiologically acceptable medium. Use according to claim 4, characterized in that the physiologically acceptable medium corresponds to the biomass extraction solvent. Use according to any one of the preceding claims, characterized in that the treatment is topical. Use according to any one of the preceding claims, characterized in that the treatment is chosen from:

• treatment of the signs of chronological or premature aging,
• a moisturizing treatment,
• a treatment for dull and/or tired skin,
• treatment of sensory discomfort of the skin,
• treatment for oily and/or acne-prone skin, and/or

• a hair treatment.

Use according to any one of the preceding claims, characterized in that the treatment is suitable for maintaining or improving the mechanical and viscoelastic properties of the skin including the scalp. Use according to any one of the preceding claims, characterized in that the treatment is a treatment suitable for preventing or reducing roughness, fine lines and wrinkles of the skin. Use according to any one of the preceding claims, characterized in that the treatment is suitable for preventing or reducing sagging of the skin. Use according to any one of the preceding claims, characterized in that the treatment is suitable for stimulating the production of hyaluronic acid. Use according to any one of the preceding claims, characterized in that the treatment is suitable for strengthening the dermis-epidermis junction (DEJ). Use according to any one of the preceding claims, characterized in that the treatment is suitable for combating the harmful effects of glycation. Use of any one of the preceding claims, characterized in that the treatment is suitable for combating the harmful effects of free radicals. Use of any one of the preceding claims, characterized in that the hair treatment is suitable for treating brittle or split hair, and/or preventing and/or treating pigmentation defects.