Topical compositions for skin application for the treatment and prevention of the formation of skin spots

Abstract

Topical compositions for skin application comprising as active ingredient a) a fluid extract from branches and / or twigs of Vitis Vinifera L. b) a fluid extract from the bark of Abies alba Mill. c) a fluid extract from the bark of Pinus sylvestris L. in combination with suitable excipients and / or diluents. in which each of said fluid extracts includes or consists of the extraction product dissolved in the same solvent mixture consisting of water and 1,3 propanediol from which said product was extracted. Such topical compositions can be used in the cosmetic field for the treatment and prevention of the formation of skin spots.

Description

[0001] 1

[0002] "Topical compositions for skin application for the treatment and prevention of the formation of skin spots".

[0003] DESCRIPTION

[0004] FIELD OF THE INVENTION

[0005] 5 The present invention relates to mixtures of fluid extracts with depigmenting activity obtained from waste plant material from viticulture and / or wood processing, the related preparation process, in particular cosmetic compositions containing them, as well as the related use in the treatment and prevention of the formation of skin spots.

[0006] 10 STATE OF THE ART

[0007] Skin hyperpigmentation is a common condition characterized by excessive production of melanin, the pigment responsible for skin color. This condition can manifest itself in several forms, including freckles, melasma, nevi, senile and sun-spots (solar lentigo) and can be influenced by a variety of factors, including UV exposure, aging, hormonal

[0008] 15 imbalances and genetic predisposition.

[0009] In particular, sun exposure is a common cause of skin pigmentation. The body produces more melanin to defend itself from the sun's UV rays. UV radiation also produces free radicals that activate biological agents that impact melanocytes, the cells responsible for pigment creation [Thawabteh AM, JibreenA, Karaman D, Thawabteh

[0010] 20 A, Karaman R. Skin Pigmentation Types, Causes and Treatment-A Review. Molecules. 2023 Jun 18;28(12):4839. dot: 10.3390 / molecules28124839. PMID: 37375394; PMCID: PMC 10304097], The addition of real antioxidants in lightening formulations is, therefore, related to the evidence that melanogenesis is induced by the pro-oxidant action of UV radiation. Ingredients capable of exerting antioxidant action also have

[0011] 25 the possibility of reducing the photo-oxidation of the melanin already formed.

[0012] Daily use of broad-spectrum sunscreens with high SPF is critical to prevent further UV damage and to protect hyperpigmented skin from future damage.

[0013] Apart from the preventive action, which consists of the application of UV filters and the exfoliating action that removes surface pigmentation with hydroxy acids (for

[0014] 30 example alpha-hydroxy acids (AHA) and polyhydroxy acids (PHA)), it is possible to act according to a cosmetic strategy by exploiting the following activities: 2

[0015] Inhibition of melanogenesis, in particular of the tyrosinase enzyme that catalyzes the first phase of transformation of the amino acid tyrosine into melanin;

[0016] Inhibition of melanin transfer;

[0017] 5 Antioxidant, since oxidation is one of the most relevant processes in hyperpigmentation

[0018] Melanogenesis is the process by which melanin pigments are produced, within melanocytes, specialized skin cells present in the basal layer of the epidermis. This

[0019] 10 process is crucial for protecting the skin from damage caused by the sun's ultraviolet (UV) rays and for determining the colour of the skin itself.

[0020] It is possible to summarize the complex process of melanogenesis in three main phases:

[0021] 1. Stimulation: Melanogenesis can be stimulated by various factors, mainly by

[0022] 15 UV rays. When the skin is exposed to sunlight, melanocyte stimulating factor (MSF) is released, which activates melanocytes to initiate melanin production.

[0023] 2. Melanin Production: Within melanocytes, melanogenesis occurs in specialized compartments called melanosomes. These are vesicles within which the precursors of melanin are transformed into melanin, thanks to the action of the

[0024] 20 enzyme tyrosinase. The tyrosinase enzyme catalyzes the hydroxylation of L- tyrosine to L-DOPA, which is further oxidized to DOPAquinone. Dopaquinone is then converted to different forms of melanin, such as eumelanin (which gives the skin a dark color) and pheomelanin (which gives the skin a lighter color).

[0025] 3. Melanin transfer: Once produced within the melanosomes, melanin is

[0026] 25 transferred from melanocytes to keratinocytes, the skin cells that make up the upper layers of the epidermis. In the epidermis each melanocyte interacts through the dendrites with the keratinocytes, allowing the transfer of mature melanosomes into the cytoplasm of the keratinocytes.

[0027] The transfer of melanosome to keratinocytes is not only important to protect DNA

[0028] 30 from UV damage but also for the main pathological process of skin pigment disorders such as melasma, spots due to skin aging and vitiligo.

[0029] Once they reach the keratinocytes melanosomes are degraded and melanin is distributed around the nucleus of the cells, offering protection against damage caused 3 by UV rays. [D Mello, S.A.N.; Finlay, G.J.; Baguley, B.C.; Askarian-Amiri, M.E. Signaling Pathways in Melanogenesis. Int. J. Mol. Sci. 2016, 17, 1144. https : / / doi. org / 10.3390 / ijmsl 7071144} .

[0030] Topical treatments for skin hyperpigmentation disorders are aimed at lightening a

[0031] 5 defined skin area, using active substances conveyed in preparations suitable for use. In the plant world there are numerous examples of compounds that, through the various mechanisms mentioned above, may be useful in cosmetics for the treatment of skin hyperpigmentation.

[0032] For example, Vitis vinifera L. (Vitaceae), the vine, is one of the most abundant fruit

[0033] 10 crops, the most common species of the family, accounting for 90%. The vine is widespread mainly in southern Europe, western Asia and America.

[0034] The vine is a climbing shrub plant, with a typically Mediterranean twisted stem, now cultivated in all areas of the globe with a Mediterranean climate. There are sparse branches, developed in length, called pampini when they are herbaceous, branches

[0035] 15 when they are lignified. Flowering occurs between May and June; the flowers develop into an inflorescence called a cluster or racemate.

[0036] The extract from grapevine seeds is known to contain viniferines which are a family of natural compounds found in different plant sources and such compounds chemically belong to the family of stilbenoids. There are different types of viniferines and the

[0037] 20 most common are resveratrol dimers. These compounds have potential use in cosmetic products due to their antioxidant and anti-inflammatory properties [Par£, S., et al. (2022). Highly efficient bioconversion of trans-resveratrol to 8-viniferin using conditioned medium of grapevine callus suspension cultures. International Journal of Molecular Sciences, 23(8), 4403. https: / / doi.org / 10.3390 / ijms23084403} that make

[0038] 25 them suitable for use especially in anti-aging cosmetics [Ferreira, M., Magalhaes, M., Oliveira, R., Lobo, J., & Almeida, I. (2021). Trends in the use of botanicals in antiaging cosmetics. Molecules, 26(12), 3584. https: / / dot. org / 10.3390 / molecules26123584} .

[0039] Viniferine has also been studied for its potential use in the treatment of

[0040] 30 hyperpigmentation, a-viniferin, present in C. sinica, has been approved as an antimelanogenic agent for topical application in skin hyperpigmentation [Yun, C., et al. (2018). A-viniferin improves facial hyperpigmentation via accelerating feedback termination of camp / pka-signaled phosphorylation circuit in facultative 4 melanogenesis. Theranostics, 8(7), 2031-2043. https: / / doi.org / 10.7150 / thno.24385\. Viniferin has been shown to accelerate the termination of the phosphorylation circuit feedback signaled by cAMP / PKA in optional melanogenesis, leading to an improvement in facial hyperpigmentation.

[0041] 5 Abies alba (Pinaceae), the white spruce, is a species of gymnosperm extremely present in Europe, particularly in Central and Eastern Europe, with marginal groups in the southern regions of Europe, the Balkans and the Pyrenees. Spontaneously present in all regions of continental Italy, with optimum in the mountain range, it is usually associated with beech in the coolest and wettest seasons. In Belluno area it is spread

[0042] 10 unevenly throughout almost the entire territory; the species can arrive, with single specimens stunted up to 2100 m in altitude. It is a tree that can reach up to 60 metres in height, one of the tallest in Europe.

[0043] The bark of the trunk is silvery-grayish, smoother in young trees and darker and cracked, with pink fractures in older trees [Portal della Flora d 'Italia / Portal to the

[0044] 15 Flora of Italy. Available at http: / dryades.units.it / floritaly\.

[0045] The chemical composition of the bark of white spruce is mainly characterized by the presence of triterpenoids and flavonoids, including lignans, of which the most abundant are lariciresinol and taxiresinol, flavonoids and phenolic acids, including procyanidins and, to a lesser extent, phenolic compounds [Vek V, Smidovnik T, Humar

[0046] 20 M, Poljansek I, Oven P. Comparison of the Content of Extractives in the Bark of the Trunk and the Bark of the Branches of Silver Fir (Abies alba Mill.). Molecules. 2023; 28(1):225. htins; / / doi.Qrg / 103390 / motocules2801022S\. [Ancuceanu R, Hovanet MV, Miron A, Anghel Al, Dinu M. Phytochemistry, Biological, and Pharmacological Properties of Abies alba Mill. Plants (Basel). 2023 Aug 3; 12(15):2860. dot:

[0047] 25 10.3390 / plantsl2152860. PMID: 37571016; PMCID: PMC10421038],

[0048] Pinus sylvestris L. (Pinaceae), the wild pine, is the most common and most widespread species of the genus Pine, with a wide Eurasian-boreal distribution that reached Italy during the glacial period coming from Siberia, and which today is widespread throughout the Alpine arc, with optimum in the internal valleys with a more continental

[0049] 30 climate where it forms secondary forests on rather primitive soils, such as on consolidated gravels; elsewhere it has often been introduced with reforestation [Portal to the Flora of Italy. Available at http: / dryades. units. it / floritaly\ . Commonly, it reaches on average 23-27 m in height, 50-80 cm in diameter and is a fairly long-lived tree, 5 which can live about 500 years. The bush pine bark is scaly at the top of the trunk and thicker at the base. \Krakau, UK., Liesebach, M., Aronen, T, Lelu-Walter, MA., Schneck, V. (2013). Scots Pine (Pinus sylvestris L). In: Pdques, L. (eds) Forest Tree Breeding in Europe. Managing Forest Ecosystems, vol 25. Springer, Dordrecht.

[0050] 5 mips: / 7do^^

[0051] Multiple authors have described hydroalcoholic extracts of pine bark as particularly rich in polyphenols, the most abundant of which is taxifolin. [Amalinei R. et at, Polyphenol-rich extract from Pinus sylvestris L. bark - chemical and antitumor studies, Rev. Med. Chir. Capital Med. Nat., Ia§i - 2014 - vol. 118, no. 2],

[0052] 10 Pinus sylvestris bark extract showed good antioxidant activity compared to the reference in all tested models.

[0053] There are some studies in the literature on the cosmetic efficacy of wild pine extracts and their associations. A phase 3 randomized double-blind study described the efficacy of topical use of 1% Centella Asiatica and 0.5% Finns sylvestris for the treatment of

[0054] 15 scars. Efficacy was determined by measuring the decrease in scar height at TO, 8 and 12 weeks of treatment compared to placebo. The advantage of the use of the treatment compared to placebo was mainly revealed in the first 8 weeks, while at the last endpoint the difference of the treatment compared to placebo was not statistically significant.

[0055] 20 A single-arm phase 2 study described the efficacy of topical use of 1% Centella Asiatica and 0.5% Finns sylvestris for the treatment of post-operative scars in paediatric patients. Patients applied 0.1 ml / cm2of cream 2 times daily. Efficacy was tested across multiple variables (size of scar; change in staining; overall response). Regarding the color variation, 95.2% of patients with burns and 72% with scars

[0056] 25 experienced evident skin whitening (p=0.05) \De Armas L, ZelenkovaH., The efficacy of a cream with Centella Asiatica and Finns Sylvestris to treat hypertrophic scars and keloids - resume of a clinical observation, (2012) Kosmetische Medizin 33(4): 122- 129], \E. Romo et at, The effectiveness of cream with Centella Asiatica and Pinus Sylvestris to treat scars and burns. Clinical trail. Dermatology Kliniczna 2012, 14 (2):

[0057] 30 105-110 ISSN 1730-7201],

[0058] Das et al. described in a review the effects of taxifolin, a polyphenol more abundant than Pinus sylvestris bark extract: in vitro studies have shown antioxidant activity and capillary protective action. The antioxidant and protective efficacy of taxifolin has 6 been shown to be 3.4 times greater than quercetin at the 100 mg / kg dose and 4.9 times at the 300 mg / kg dose. [ / )=>' / •> A, Baidya I< Chakraborly T, Samanla AK, Roy S. Pharmacological basis and new insights oftaxifolin: A. comprehensi ve revie w. Biomed. Pharmacother. 2021 Oct; 142: 112004. doi: 10. 1016f.biopha.2021.112004. Epub 2021

[0059] 5 Aug 10. PMID: 34388527],

[0060] SUMMARY OF THE INVENTION

[0061] The Applicant has now developed a mixture of three fluid extracts that was effective in inhibiting the action of tyrosinase, demonstrating a synergy of efficacy between

[0062] 10 classes of different molecules that could act with different mechanisms of action.

[0063] Based on a careful analysis of different plant matrices, the Applicant has produced three extracts and their characteristic compounds are as follows:

[0064] • a fluid extract from branches and / or twigs of Vitis Vinifera in which the main compounds are: catechins, proanthocyanidins, stilbenes (Viniferins and derivatives,

[0065] 15 resveratrol and derivatives)

[0066] • a fluid extract from the bark of Abies alba in which the main compounds are: catechins, lignans (Lariciresinol and Taxiresinol)

[0067] • a fluid extract from the bark of Pinus sylvestris, in which the main compounds are: proanthocyanidins, catechins, taxifolin and derivatives.

[0068] 20 Object of the present invention are topical compositions for skin application containing as active ingredient a) a fluid extract from the branches and / or twigs of Vitis Vinifera L. b) a fluid extract from the bark of Abies alba Mill. c) a fluid extract from the bark of Pinus sylvestris L.

[0069] 25 in combination with suitable excipients and / or diluents, wherein each of said fluid extracts includes or consists of the relevant extraction product dissolved in the extraction solvent mixture consisting of water and 1,3 propanediol,

[0070] A further object of the present invention is therefore the process of extraction of said fluid extracts a), b) and c) contained in the topical compositions object of the invention

[0071] 30 which comprises the following steps:

[0072] I. Drying the starting plant material at a temperature less than or equal to 40°C and up to a humidity less than or equal to 10%. 7

[0073] II. Grinding of the dried product from step I. until a homogeneous powder with a size less than or equal to 6 mm is obtained;

[0074] III. Preparing a solvent mixture including the addition under agitation of 1,3- propanediol to water until a homogeneous solution is obtained in which 1,3-

[0075] 5 propanediol is 70 percent m / m and water is 30 percent m / m on the total weight of that solvent mixture

[0076] IV. Mixing the dried powder obtained in step II. with the solvent mixture prepared in step III. in weight ratios of dried powder / solvent mixture equal to 1 : 10

[0077] V. Treating the mixture of step IV in an ultrasonic bath for 1 hour at room

[0078] 10 temperature:

[0079] VI. Eliminating solid residues resulting from step V. by pressing with a screw press or pneumatic press or by filtering with at least two vacuum filtrations using filters with porosity up to 0.22 microns.

[0080] 15 A further object of the present invention is the cosmetic and non-cosmetic use of the mixture of liquid extracts object of the present invention for the prevention and treatment of the formation of skin spots.

[0081] Finally, a further object of the present invention are topical compositions for skin application, comprising a mixture of the following extracts:

[0082] 20 a) an extract from branches and / or twigs of Vitis Vinifera L. b) an extract from the bark of Abies alba Mill. c) an extract from the bark of Finns sylvestris L: as well as their cosmetic use and not in the treatment or prevention of the formation of skin spots.

[0083] 25 Such skin spots or dyschromias are due to aging, sun exposure, acne, hormonal changes or medications.

[0084] DESCRIPTION OF FIGURE 1

[0085] Figure 1 describes the release of IL- la (pg / ml) by the reconstructed human epidermis following to different treatments in which NC is a negative control, SLS sodium lauryl

[0086] 30 sulfate and * indicates p< 0.05.

[0087] DETAILED DESCRIPTION OF THE INVENTION 8

[0088] For the purposes of the present invention, the definitions "comprising, containing" do not exclude the presence of additional components / steps in addition to those listed after such definitions.

[0089] The definition “consisting of, constituted by,” excludes the presence of additional

[0090] 5 components / steps beyond those expressly listed after these definitions.

[0091] By extraction product is meant "the phytocomplex" i.e. the set of molecules directly extracted from biomass of plant origin.

[0092] By topical composition for skin application, is meant a formulation that is suitable to

[0093] 10 be applied to the skin in situ or where dyschromias are present and where they are more likely to form.

[0094] With the term "active ingredient" or "active" or "functional active", any ingredient, molecule, substance or mixture of compounds can be defined, capable of measurably stimulating a change in one of the skin parameters being evaluated, where such

[0095] 15 changes are significantly different from the application of the vehicle alone. By the term carrier or diluent is meant the set of excipients that contribute to the topical composition for skin application object of the invention and can affect the skin permeability of the active ingredients. However, it should be considered that even a simple "vehicle" without active ingredients is not a completely inert cosmetic product.

[0096] 20 The synergy between vehicle and active ingredients is not predictable in advance and the choice of the right vehicle guarantees / supports the effectiveness and stability of the cosmetic product as a whole, especially in the case of active ingredients of plant origin. For the purposes of the present invention, the term "eudermic extraction mixture" and / or "solvent mixture" and / or "extraction mixture" means an extraction solvent

[0097] 25 mixture in a process for preparing a liquid extract, which comprises at least one cosmetic grade ingredient that performs the function of solubilizing and extracting molecules / active ingredients (which make up the phytocomplex) from a plant drug. At the same time, this "eudermic extraction mixture" acts as a vehicle for the phytocomplex contained therein.

[0098] 30

[0099] The topical compositions for skin application object of the invention comprise as active ingredient the components a), b) and c) wherein 9 a) is a fluid extract from branches and / or twigs of Vitis Vinifera L. b) is a fluid extract from the bark of Abies alba Mill. c) is a fluid extract from the bark of Finns sylvestris L. in combination with suitable excipients and / or diluents, wherein each of said fluid

[0100] 5 extracts comprises or consists of the extraction product dissolved in the same solvent mixture consisting of water and 1,3 propanediol, from which said product has been extracted.

[0101] Preferably, the topical compositions for skin application object of the invention are

[0102] 10 ointments, oils, lotions, gels, creams, oil-in-water or water-in-oil emulsions.

[0103] In the topical compositions for skin application object of the present invention, the sum of (a) + (b) + (c) preferably is between 0.03 and 3% by weight of the total weight of said topical compositions, more preferably between 0.3 and 1.5% by weight of the total weight of said topical compositions.

[0104] 15 In the topical compositions for skin application object of the invention the weight ratio between components a) b) and c) is preferably 1 : 1 : 1.

[0105] The solvent mixture in the skin compositions object of the present invention preferably consists of 30% by weight of water, while 70% of 1,3 propanediol.

[0106] The topical compositions for skin application may contemplate at least one further

[0107] 20 active d) selected in the class consisting of Niacinamide, Tranexamic acid, Hyaluronic acid or a salt thereof, one or more sunscreens.

[0108] By sunscreens are meant those of a conventional type such as, for example, Bis-ethyl- ethyloxy-phenol, methoxyphenyl-triazines or t-butylmethoxy-dibenzoyl-methane.

[0109] The preparation examples of the extracts object of the present invention and the

[0110] 25 efficacy tests in inhibiting tyrosinase and the composition of such extracts are given for illustrative purposes only.

[0111] EXAMPLE 1: SELECTION OF PLANT MATRICES AND PRODUCTION OF

[0112] FLUID EXTRACTS

[0113] 30

[0114] In the first phase of the study, the Applicant selected some plant species that met the following requirements:

[0115] 1. that these species were present on the Italian territory 10

[0116] 2. that the plant material was waste material came from wood processing and / or the forestry and / or agri-food sector.

[0117] 3. that by means of a simple and green extraction process, a phytocomplex comprising molecules characterized by belonging to one of the following

[0118] 5 families of molecules could be obtained: Proanthocyanidins, ellagic acid and derivatives, flavonoids, stilbenes, polyphenols in general.

[0119] Table 1 Selected plant maternal

[0120] Species Part of the plant

[0121] Extract Code Species (botanical name) (common name) present in the waste

[0122] T279 English Oak Quercus robur L. bark

[0123] T272 Saffron Crocus sativus L. flowers without pistils

[0124] T269 Blueberry Vaccinium myrtillus L. seeds and peel

[0125] T271 Scutellaria Scutellaria alpina L. aerial parts

[0126] T282 Beech Fagus sylvatica L. bark

[0127] T278 Red spruce Picea abies L. bark

[0128] T280 Oak Quercus petraea Liebl. bark

[0129] Rhododendron

[0130] T270 Rhododendron leaves ferrugineum L.

[0131] T277 White spruce Abies alba Mill. bark

[0132] T267 Grape marc Vitis vinifera L. fruit of the vine

[0133] T266 Chestnut Castanea sativa Mill. leaves

[0134] T281 Wild pine tree Finns sylvestris L. bark

[0135] T265 Pomegranate Punica granatum L. pericarp

[0136] 10

[0137] The process that the Applicant used to prepare the fluid extracts is the same for each plant matrix indicated in Table 1 and comprises the following steps:

[0138] 1) DRUG GRINDING 11

[0139] After drying the starting plant material at a temperature of less than or equal to 40°C and up to a humidity of less than or equal to 10%, the plant drug is ground to a particle size of between 1 and 6 mm

[0140] 2) ADDITION OF THE SOLVENT MIXTURE

[0141] 5 The solvent mixture composed of 70% (m / m) of 1,3-propanediol and 30% (m / m) of water is added to the plant drug. The w / w drug: solvent ratio is 1 : 10.

[0142] 3) EXTRACTION MACHINE

[0143] The extraction takes place in an ultrasonic bath at room temperature for 1 h.

[0144] 4) SEPARATION OF THE DRUG FROM THE SOLVENT MIXTURE

[0145] 10 The drug is separated from the solvent mixture by pressing (screw or pneumatic press) or by filtration with a buchner funnel.

[0146] 5) FILTRATION

[0147] Proceed with progressive filtration with Buchner funnel or filter press until final filtration with 0.22 or 0.4 micron porosity filters.

[0148] 15 EXAMPLE 2: EVALUATION OF ANTI-TYROSINE ACTIVITY

[0149] For the development of the anti -tyrosinase activity evaluation protocol to be tested on extracts and raw materials reported in table 1, what was reported in the work of Zuo et al. was adapted. Chin Med (2018) 13:51.

[0150] INTRODUCTION

[0151] 20 The enzyme tyrosinase is an oxidase that catalyses two reactions involved in melanin synthesis: the hydroxylation of tyrosine to DOPA (monophenolase) and the oxidation of DOPA (diphenolase) to the corresponding quinone. DOPA quinone is endowed with high chemical reactivity and, once formed, gives rise to a series of spontaneous transformations, including oxidoreductive exchange with oxidative cyclization and

[0152] 25 polymerization.

[0153] Preliminary tests were conducted by incubating L-DOPA, used as a substrate, with the tyrosinase enzyme.

[0154] The L-Dopa solution in the presence of tyrosinase turns its colour from transparent to red, an indication of oxidation to reactive quinone. It was subsequently evaluated how

[0155] 30 the inclusion of active ingredients with hypothetical antityrosinase activity inhibits the transformation of the reactive quinone, decreasing the absorbance values at 475 nm. As a first approach, kojic acid was evaluated, a commercially used cosmetic active ingredient for lightening activity. 12

[0156] Reagents:

[0157] - Standard L-Dopa (Sigma-Aldrich) in Phosphate Buffer (pH 6.4)

[0158] - Fungal Tyrosinase (Sigma-Aldrich) in Phosphate Buffer (pH 6.4)

[0159] - Kojic acid (ACEF) in DMSO (1000-100 pg / ml)

[0160] 5 Fluid extracts tested:

[0161] T265: Pomegranate - pericarp

[0162] T281 : Wild Pine - Bark

[0163] T266: Chestnut - leaves

[0164] T267: Grapevine - Grape marc

[0165] 10 T277: White spruce - bark

[0166] T270: Rhododendron - Leaves

[0167] T278: Red spruce - bark

[0168] T280: Oak - bark

[0169] T282: Beech - bark

[0170] 15 T271 : Skutellaria - aerial parts

[0171] T269: Blueberry - seeds and peel

[0172] T272: Saffron - flowers without pistils

[0173] T279: Farnia - bark

[0174] The fluid extracts were tested as such.

[0175] 20 Details of the sample preparation performed during the assay are reported.

[0176] Control: 2.8 ml of L-Dopa solution was incubated with 0.1 ml of tyrosinase solution. Absorbance at 475 nm is associated with 100% of tyrosinase activity.

[0177] Samples: 2.8 ml of L-Dopa solution was incubated with 0.1 ml of kojic acid solution, and extracted as such. Subsequently 0.1 ml of tyrosinase was added.

[0178] 25 To subtract interference from the natural staining of the extracts, the absorbance was read for each sample tested prior to the addition of tyrosinase.

[0179] Samples were incubated at 30°C for 6 minutes and then the absorbance at 475 nm was read for the calculation of IC50 (concentration of sample tested that allows to obtain the inhibition of 50% of tyrosinase activity.)

[0180] 30 In table 2 are shown the results of the antityrosinase activity of the fluid extracts analyzed. 13

[0181] Th&fe 2 Results y'the annte‘Os;i:ass achvi?)- qftrze fl?«d extmcto axafy'sed

[0182] IC50

[0183] Extract (mg / ml)

[0184] Kojic acid 0.019

[0185] T265 : Pomegranate - pericarp 5.170

[0186] T281: Wild Pine - bark 12.830

[0187] T266: Chestnut - leaves 14.130

[0188] T267 : Grapevine - Grape marc 14.340

[0189] T277: White spruce - bark 17.450

[0190] T270: Rhododendron - leaves 22.120

[0191] T278: Red spruce - bark 35.700

[0192] T280: Oak - bark 38.380

[0193] T282: Beech - bark 64.110

[0194] T271 : Skutellaria - aerial parts 69.700

[0195] T269: Blueberry - seeds and peel 71.790

[0196] T272: Saffron - flowers without pistils 106.910

[0197] T279: Famia - bark 122.460

[0198] The most interesting species are: white spruce, red spruce, oak, wild pine, chestnut, pomegranate and vine, while blueberry, rhododendron, skullcap, saffron, fern and

[0199] 5 beech are among the least active species.

[0200] EXAMPLE 3

[0201] Compared to the plant matrices tested in Example 2, the Applicant replaced the grape marcs, which are difficult to process and microbiologically stabilize, with another waste from the cultivation of the same species, Vitis vintfera, the branches derived

[0202] 10 from pruning the plant.

[0203] On the most effective extracts from the first screening (Wild Pine, White spruce, Pomegranate, Chestnut, Vine) a second efficacy test was performed as performed in example 2 to confirm the preliminary results obtained and with the replacement of the marc extract with one from vine branches. 14

[0204] Activity ratio compared to

[0205] Extract Activity mg KAE / g* kojic acid

[0206] EF bush pine bark batch T281 51.56 1 / 19

[0207] EF vine branches batch T334 47.23 1 / 21

[0208] EF white spruce bark batch T277 46.18 1 / 22

[0209] EF pomegranate pericarp batch T265 45.74 1 / 22

[0210] EF chestnut leaves batch T266 43.06 1 / 23 equivalents g

[0211] For the tyrosinase enzyme inhibition activity test, the reference compound kojic acid (known depigmenting agent) was selected and the results were expressed in mg equivalents of compound per gram of extract.

[0212] 5 Extracts T281, T334 and T277 show better tyrosinase enzyme inhibitory activity than extracts T265 and T266, as well as demonstrating very similar action to each other. The extract with the greatest action was T281 in each case.

[0213] From this test, the Applicant has selected the three extracts that are the object of the present invention:

[0214] 10 a) a fluid extract from the branches and / or twigs of Vitis Vinifera L. b) a fluid extract from the bark of Abies alba Mill. c) a fluid extract from the bark of Pinus sylvestris L.

[0215] EXAMPLE 4: ANTIOXIDANT AND TYROSINASE INHIBITING ACTIVITY

[0216] 15 OF INDIVIDUAL EXTRACTS

[0217] The antioxidant and tyrosinase enzyme inhibitory activity of the selected extracts was assessed

[0218] The samples were dissolved in a vehicle compatible with the enzymatic reaction and then tested. 15

[0219] Below are the results of the three extracts, selected in example 3, used for the development of a skin depigmenting ingredient (mixture) object of the present invention in association with suitable excipients and / or diluents, for cosmetic purposes.

[0220] 5 Samples: Fluid extracts

[0221] -EF vine branches batch T334 (5),

[0222] -EF wild pine bark batch T281 (19),

[0223] -EF white spruce bark batch T277 (17)

[0224] Results antioxidant activity:

[0225] 10 • DPPH Assay

[0226] The test allows to determine the antioxidant power by reacting the sample to be analysed with a solution of DPPH [2,2-diphenyl-l-picrylhydrazyl] and analysing the decrease in the radical peak by UV. The solution with DPPH appears purple when the compound is in radical form. Antioxidant compounds that are capable of transferring

[0227] 15 a hydrogen atom to the radical, cause a discoloration of the solution which testifies to the fact of eliminating the radical from the DPPH molecule. The UV-Vis is then analyzed for the decrease in absorbance at 517nm corresponding to the absorption of the DPPH radical after a predetermined incubation time. This decrease (discoloration) is proportional to the antioxidant capacity present in the sample. The assay is

[0228] 20 conducted in an organic (alcohol) environment and is widely used.

[0229] • ABTS Assay

[0230] The test allows the antioxidant power of different biological matrices to be determined by the reaction between the test sample with a cation radical. The colored, stable cation radical is generated by oxidation of the diammonium salt of 2,2'-azino-bis(3-

[0231] 25 ethylbenzothiazoline-6-sulfonic acid (ABTS) by a solution of potassium persulfate (K2S2O8).

[0232] This cation radical has an absorption peak at 734 nm. Antioxidant compounds that are capable of transferring a hydrogen atom or an electron to the cation radical, cause a discoloration of the solution since they remove the radical from the original compound. 16

[0233] The UV-Vis is then analyzed for the decrease in the peak at 734 nm corresponding to the absorption of the ABTS cation radical after a predetermined incubation time.

[0234] This decrease (discoloration) is proportional to the antioxidant capacity present in the sample.

[0235] 5 Similar to the previous one, the difference is that it is carried out in an aqueous environment at physiological pH, thus aiming to mimic what may occur in the cytosol or in body compartments.

[0236] • CUPRAC Assay

[0237] The Cupric Reducing Antioxidant Capacity (CUPRAC) assay is a method that allows

[0238] 10 the measurement of the total antioxidant capacity of a sample. The main reagent, copper, is reduced by the antioxidants present in the sample to then undergo chelation by the neocuproin, which allows a faster reaction by raising the redox potential of the reagent.

[0239] Antioxidant capacity was measured by a spectrophotometric assay.

[0240] 15 This test uses a system consisting of a reagent that contains copper and oxidises the antioxidant substances present in the sample.

[0241] This test compared to the previous ones is able to measure both hydrophilic and lipophilic antioxidants quite effectively.

[0242] • FRAP Assay

[0243] 20 The FRAP assay is a colorimetric method based on the ability of various antioxidants to reduce the ferric tri pyridyl triazine complex (Fe +3-TPTZ) to the colored ferrous form (Fe +2-TPTZ), at low pH values developing an intense blue coloration.

[0244] The reaction is monitored by spectrophotometrically measuring the change in absorbance at 593nm, directly related to the activity of the antioxidants present.

[0245] 25 It is an assay that is based on the change in color resulting from the reduction of Fe .3+ to Fe2+.

[0246] Table 4 shows the results obtained from the average of the 3 experiments conducted for each assay. 17

[0247] Ta&fe 4 ffesu / ts of tAe anifaxfatant actvV / ey assays of extracts 7384, T277 and 7287. 7?2'-tro / :?x e< / « / va / er» fsy

[0248] Sample

[0249] (mgTE / g) (mgTE / g) (mgTE / g) (mgTE / g)

[0250] EF vine branches 21.82+0.34 30.72+1 .04 21.36+0.33 18.63+0.47 batch T334

[0251] (5)

[0252] EF white spruce bark 31.45+0.96 42.24+1.76 26.55+1.13 24.26+2.59 batch T277

[0253] (17)

[0254] EF bush pine bark batch 82.68+1.04 114.20+3.42 77.99+0.28 61.11+1.38

[0255] T281 (19)

[0256] For each antioxidant activity test, trolox was selected as the reference compound and

[0257] 5 the results were expressed as mg of compound per gram of extract. This means that, for example, 1g of vine extract corresponds to 21.82 mg of trolox, a soluble analogue of vitamin E, known for its antioxidant activity.

[0258] The extracts show good antioxidant activity in all the models used. The extract that shows the most activity is that of Pino Silvestre T281.

[0259] 10 EXAMPLE 5: PHYTOCHEMICAL CHARACTERIZATION OF SELECTED

[0260] EXTRACTS

[0261] Example 5.1 Characterization of the T334_vine branch extract

[0262] SAMPLE PREPARATION AND METHOD OF ANALYSIS

[0263] Phytochemical characterization of T334 extract was obtained by LC-DAD-MSn. The

[0264] 15 fluid extract was diluted 1 to 10 in water. Solutions were sonicated and centrifuged prior to analysis. An agilent SB-C18 column (50x4.6mm, 1.8pm) was used for the chromatographic separation, the flow was set at 0.75 ml / min, the mobile phases were 18

[0265] 1% formic acid water (A), acetonitrile (B) and methanol (C). The gradient is as follows: min 0 95:5:0 (% A:B:C), min 5 85: 15:0 (% A:B:C), min 15 80: 18:2 (%

[0266] A:B:C), min 18 50:40: 10 (% A:B:C), min 25 20:70: 10 (% A:B:C), min 30 0:85: 15 (%

[0267] A:B:C), min 35 0:85: 15 (% A:B:C), min 40 0: 100:0 (% A:B:C), min 45 0: 100:0 (%

[0268] 5 A:B:C).

[0269] At the exit of the column, the flow is divided by a T-connector into two and one half is sent to a DAD detector and the other to a mass spectrometer with an ion-trap analyzer. The chromatograms were acquired at different wavelengths, 280, 250, 330,

[0270] 350 nm recording the UV spectra by DAD and at the same time analyzed in mass in

[0271] 10 positive and negative mode.

[0272] Trans epsilon viniferine was used as the reference substance.

[0273] RESULTS

[0274] Table 5 Quond / fcotfon of rhe compounds detected t / ie 7334 / tod extract

[0275] 451 459 905.5 905.5 905.5 453.5

[0276] TR 9.13 9.13 16.3 16.7 17.7 16.9

[0277] 15 trans- Stilbenoide

[0278] Campione Viriiferifurar! Scirpusin tetramero Stilbenoide Stilbenoide t Viniferina tot A R2-viniferina etramero tetr amero mg / 100 ml. mg / lOOmt mg / lOQmt mg / lOOmL mg / lOOmL mg / 100 ml Tot mg / lOOml.

[0279] 1.11 t 27.974 10.943 72.9

[0280] All the compounds identified for their structural properties, such as the presence of multiple cycles and phenols, are particularly interesting for their antioxidant and

[0281] 20 enzymatic action and are excellent candidates for the study of their biological activity.

[0282] Example 5.2 Characterization of White Spruce Bark and Scots Pine Bark

[0283] Extracts

[0284] Samples:

[0285] -EF wild pine bark batch T281,

[0286] 25 -EF fir bark batch T277

[0287] The fluid extracts were diluted 1 :5 in water. For qualitative purposes, extraction approaches were also used using SPE (Cl 8). 19

[0288] An Agilent SB-C18 column (50x4.6mm, 1.8pm) was used for the chromatographic separation, the flow was set at 0.75 ml / min, the mobile phases were 1% formic acid water (A), acetonitrile (B) and methanol (C).

[0289] The gradient is as follows: min 0 95:5:0 (% A:B:C), min 2.5 85: 15:0 (% A:B:C), min

[0290] 5 12 80: 18:2 (% A:B:C), min 15.5 50:40: 10 (% A:B:C), min 19 20:70: 10 (% A:B:C), min 21 0:85: 15 (% A:B:C), min 23 0:85: 15 (% A:B:C), min 24 0: 100:0 (% A:B:C), min 26 0: 100:0 (% A:B:C).

[0291] At the exit of the column, the flow is divided by a T-connector into two and one half is sent to a DAD detector and the other to a mass spectrometer with an ion-trap

[0292] 10 analyzer. The chromatograms were acquired at different wavelengths, 280, 250, 330, 350 nm recording the UV spectra by DAD and at the same time analyzed in mass in positive and negative mode.

[0293] As reference substances for quantification, PAC-B2, resveratrol, catechin, epicatechin, chlorogenic acid, rutin were used, quantifying by calibration line.

[0294] 15 The results obtained for each extract are shown in Tables 6 and 7 below.

[0295] Tufefe 6 of the compounds detected ;n the fu / rf extract 7277- White spruce - feurft

[0296] _ Compound _ ug / mL _ Catechin _ 9.78 _ Lariciresinol-hexoside _ 53.10 Taxiresinol pyrcatecolglyceryl ether

[0297] 35.26 _ derivative 1 _ Taxiresinol pyrcatecolglyceryl ether

[0298] 43.30 _ derivative 2 _ Taxiresinol -rhamnoside 51.67

[0299] Tdbfe 7 Quonftffcutfon of the compounds detected / n the fesrf extract 7282- Scots p / ne- tot

[0300] Compound ug / niL

[0301] PAC B2 10.60

[0302] Catechin 6.90

[0303] Taxifolin hexoside 9.78

[0304] Taxifolin 148.95

[0305] EXAMPLE 6 STUDIES ON THE INHIBITORY ACTIVITY OF THE

[0306] MIXTURE OF EXTRACTS a), b), c) ON THE FACTORS THAT PROMOTE

[0307] 20 MELANOGENESIS. 20

[0308] 6.1. Introduction

[0309] The study concerns the in vitro evaluation of the soothing potential of samples against the inflammatory reaction induced by moderate irritants such as sodium lauryl sulfate (SLS), using a three-dimensional model of the human epidermis reconstructed in vitro.

[0310] 5 The inhibition of the release of the cytokine IL- la caused by SLS, in the presence of the tested substance compared to untreated epidermis samples, was investigated by means of specific ELISA tests.

[0311] Interleukin or IL-1 is a highly active proinflammatory cytokine and mutations or interactions involving its maturation or the antagonist IL-1 receptor can produce

[0312] 10 inflammatory conditions especially on the skin. Among skin cells keratinocytes produce them in considerable amounts and especially IL- la, which induces an inflammatory response on the skin.

[0313] For example, IL-1 a released following UVB exposure, stimulates the production of the keratinocyte growth factor KGF which in turn promotes the expression of

[0314] 15 tyrosinase. Therefore, the combination of KGF and IL-1 a are among the factors capable of promoting melanogenesis through melanin deposition. For this reason IL- la was selected as the inflammatory parameter for the present study, in addition to tyrosinase expression.

[0315] In fact, in addition to the preliminary in vitro test reported in example 4 that

[0316] 20 demonstrates the antioxidant activity of the three individual extracts a), b) and c), we conducted the following studies reported in:

[0317] • Example 6.2, where the tyrosinase inhibitory activity of the individual extracts was studied with the process of the invention and of the mixture of extracts a), b) and c) in a 1 : 1 : 1 ratio compared to that of kojic acid.

[0318] 25 • Example 6.3, where the overall anti-inflammatory activity on reconstructed human epidermis was evaluated as a function of IL- la produced following insult caused by an inflammatory agent alone or in combination with the mixture of extracts a), b), c) in a 1 : 1 : 1 weight ratio with a view to their application in the cosmetic field.

[0319] 30 EXAMPLE 6.2: Tyrosinase inhibitory activity of mixture a)+b)+c) and comparison with that of kojic acid and of the individual extracts a), b) and c) 21

[0320] In the choice of species and supply chains, aspects related to environmental sustainability were also taken into account. The three different plant biomasses selected derive from up-cycling chains created in the territory of the Veneto region, giving value to three different waste, the pruning branches of the vine and the barks of

[0321] 5 pine and fir, supporting small local companies in the wine and forestry sectors.

[0322] The procedure for analysis of tyrosinase activity is the same as that described in example 2

[0323] Reagents used:

[0324] - Standard L-Dopa (Sigma-Aldrich) in 50 mM pH 6.8 Phosphate Buffer (100 pg / mL)

[0325] 10 -Mushroom tyrosinase (Sigma-Aldrich) in 50 mM pH 6.8 phosphate buffer (170 pg / ml indicated activity >1000 units per mg of solid)

[0326] -Kojic acid in DMSO at different concentrations (1000-100 pg / ml)

[0327] Samples tested

[0328] Fluid extracts

[0329] 15 - Fluid extract white spruce bark fluid extract batch T354

[0330] - Fluid extract bark pine fluid extract batch T355

[0331] - Fluid extract vine branches Fluid extract batch T356

[0332] - Mixture of the three fluid extracts mentioned above in 1 : 1 : 1 ratio

[0333] The four extracts were further diluted 1 :2, 1 :3, 1 :5 in water.

[0334] 20 The protocol used for the preparation of the samples is shown

[0335] Control solution: 2.8 ml of L-Dopa solution was incubated with 0.1 ml of tyrosinase solution. Absorbance at 475 nm is associated with 100% of tyrosinase activity.

[0336] Kojic acid: 2.8 ml of L-Dopa solution was incubated with 0.1 ml of kojic acid solution at the different concentrations reported above. Subsequently 0.1 ml of tyrosinase was

[0337] 25 added.

[0338] Fluid extracts: 2.8 ml of L-Dopa solution were incubated with 0.1 ml of extract as such and diluted with water in the ratio 1 :2, 1 :3, 1 :5. Subsequently 0.1 ml of tyrosinase was added.

[0339] The various samples were incubated at 30°C for 6 minutes and then the absorbance at

[0340] 30 475 nm was read. Absorbance control is an index of 100% tyrosinase activity.

[0341] The inhibition of tyrosinase activity is calculated as: [(Control Abs - Detected Abs) xlOO] / Control Abs 22

[0342] RESULTS

[0343] The results obtained are shown in the following table 8

[0344] Tanfe S

[0345] AttiyitS tirosinasira %

[0346] Adda csgics Img / ml, 75.91.

[0347] Acids eogieo SSOug / nil 58.64

[0348] Adds csgtcs 200-ug / niL 33.18

[0349] Adda csgto lOOmg / ml 21,86

[0350] Extralti AttlvitA (irosmastea M sstratm Vite r am i tai quale 63,78 t-xrjue s a- s ’ :c , ' 86,39 estratto site rami 1:3 48,48 esvi'atm vHg ram: 1 :5 37,58 extratto Piiw tai quale 48,64

[0351] 1:2 44.85 gsixatm plao 1:3 pmo 1:3 28.64 mam AsmW tai qu. 43.39

[0352] Watto abate 1:2 29,85

[0353] 24 79 20,61

[0354] Mix sward ul quale' 68,64 mix estmtti 1:2 51'36 mmWmtti 1:5 44-24 nux estritti 1 :3 3^06

[0355] 5 The data allow the following considerations to be made. A proportional response is observed between the amount of the extracts under study and the inhibitory activity on the enzyme. However, if we want to compare and study the MIX composition that consists of a mixture in equal part of the three starting extracts, we can consider the inhibitions obtained at 1 :3 dilution for the individual ingredients and the mix extracts

[0356] 10 as such as a reference.

[0357] With the different dilutions the extract maintains a certain activity unlike the standard and the individual extracts which, with the 1 :5 dilution lose a lot of activity. 23

[0358] If the 1 :5 dilution of the extract mix is analysed and compared with the 1 :5 dilutions of all tested compounds, including the reference standard (200 pg / mL) it can be assumed that there is an effect / factor linked to the coexistence of the three actives, whereby the enzyme inhibition activity on tyrosinase is preserved even at very low

[0359] 5 concentrations.

[0360] This effect of preserving activity even at very low concentrations is very important, especially considering that in the formulation of a cosmetic product, the active ingredients, as they are, undergo dilution.

[0361] Applicant has surprisingly found that a mixture of three extracts as described in claim

[0362] 10 1 demonstrates greater tyrosinase enzyme inhibitory activity than individual extracts and greater than the reference standard compound, at the same dilution.

[0363] EXAMPLE 6.3: In vitro evaluation of the soothing efficacy of the mixture of extracts a) b) and c)

[0364] 6.3.1 Methodology

[0365] 15

[0366] The objective of this study was to evaluate the effects of extracts a), b) and c) in a 1 : 1 : 1 ratio in protecting skin cells and inhibiting the inflammatory reaction caused by moderate irritants, such as sodium lauryl sulphate (SLS) on a multilayer reconstructed human epidermis model. Inhibition of SLS-induced IL-la cytokine release was

[0367] 20 assessed by specific Enzyme-linked immunosorbent assay (ELISA) following exposure to the mixture of extracts and compared to untreated skin.

[0368] For this a reconstructed artificial human skin model (SkinEthic™ RHE, Episkin, Lyon, France) was employed, comprising normal human keratinocytes growing according to a three-dimensional cell culture model.

[0369] 25 A preliminary study was also carried out on the cytotoxicity to MTT(3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)), on the mixture of extracts using PBS (Phosphate Buffered Saline) as a negative control to assess the compatibility of the sample with the experimental conditions by assessing the cell viability of the sample after 24 hours of exposure. In this preliminary test, propanediol

[0370] 30 was also tested in order to rule out any adverse effects related to the use of this solvent used in the production of the extracts.

[0371] Epidermal units were pretreated for 60 minutes with 0.4% SLS to induce IL-la synthesis and release. The tissues were then washed with PBS and then 40 pl of the 24 mixture of the three undiluted extracts was applied. As a negative control, the epidermal units were treated with PBS, while the units treated only with SLS were used as a positive control. Units treated only with the mixture of extracts were used as a further control. The test was performed in triplicate. Exposure was carried out for 24

[0372] 5 h at 37 °C, 5% CO2. At the end of the exposure period, the sample was removed by washing with PBS, and the culture medium was harvested for IL-la assay.

[0373] IL-la was determined in the culture medium of the treated and untreated epidermis, using a direct ELISA test. The colorimetric signal is directly proportional to the amount of cytokine present in the culture medium. Samples were read at 450 nm. The

[0374] 10 sensitivity soil is less than 10 pg / ml.

[0375] 6.3.2 Results

[0376] The preliminary MTT cytotoxicity test confirms the compatibility of all samples tested with the selected experimental model. Cell viability after 24 hours of exposure was 100±0.7%, 86.2±0.4%, and 74.1±0.5% for PBS (negative control), grape, pine, and

[0377] 15 red spruce extract mixture (1 : 1 : 1), and propanediol (the solvent used for plant extraction), respectively.

[0378] The results of the in vitro test are shown in Figure 4. Results are expressed in pg / ml of IL-la released after the different treatments (PBS as negative control, SLS 0.4% as positive control, SLS 0.4%+40 pL of extract mixtures and 40 pL of extract mixtures

[0379] 20 without SLS). Incubation of PBS on reconstructed human epidermis produces a low amount of IL-la (17.1 pg / ml), as expected. Furthermore, incubation of mixtures of vine branch extracts, pine bark, and red spruce (1 : 1 : 1) without SLS-induced damage has no significant impact on the inflammatory parameter, and the value of released IL- la remains rather low (45.4 pg / ml). SLS-induced inflammation is almost 20 times

[0380] 25 higher than the negative control (442 pg / ml) and is significantly inhibited by addition of the extract mixture (337 pg / ml). In conclusion, after a 24 hour incubation, the mixture of extracts produced a significant (-24%) (p<0.05) reduction of IL-la release induced by SLS damage.

[0381] 30 EXAMPLE 7: COSMETIC FORMULATIONS

[0382] Some cosmetic compositions object of the present invention are given for illustrative but non-limiting purposes. 25

[0383] Cosmetic Formulation 1 :

[0384] Component range [% w / w] as needed up to

[0385] Aqua 100

[0386] Oils (vegetable or mineral) Waxes, fats (e.g., long chain alcohols) 0.1-3

[0387] Silicones 0.5-1.5

[0388] Humectants 2-5

[0389] Rheological modifiers 0.1-2

[0390] Preservatives 1.5-3

[0391] Chelating Agent 0.1-0.3

[0392] Antioxidants 0.03-0.5

[0393] Hyaluronic acid 0.01-1

[0394] Fluid Extract a) 0.01-1

[0395] Fluid extract b) 0.01-1

[0396] Fluid extract c) 0.01-1

[0397] Niacinamide 2-6

[0398] Tranexamic Acid 1-3

[0399] Cosmetic Formulation 2:

[0400] 5

[0401] Component range [% w / w]

[0402] Aqua as needed up to

[0403] 100 _

[0404] Oils (vegetable or mineral) Waxes, fats 20-25 (e.g., long chain alcohols)

[0405] UV filters 10-25

[0406] Silicones 0.5-10

[0407] Humectants 1-10

[0408] Rheological modifiers 1-5

[0409] Emulsifiers 0.5-3

[0410] Preservatives 0.1-3

[0411] Scent 0.1-1

[0412] Chelating Agent 0.1-0.3

[0413] Antioxidants 0.03- 0.5

[0414] Fluid Extract a) 0.01-1

[0415] Fluid extract b) 0.01-1

[0416] Fluid extract c) 0.01-1

[0417] Niacinamide 0.5-5

Claims

26CLAIMS1. Topical compositions for skin application comprising as active ingredient a) a fluid extract from branches and / or twigs of Vitis Vinifera L. b) a fluid extract from the bark of Abies alba Mill.5 c) a fluid extract from the bark of Pinus sylvestris L. in combination with suitable excipients and / or diluents, wherein each of said fluid extracts comprises or consists of the extraction product dissolved in the same solvent mixture consisting of water and 1,3 propanediol, from which said product has been extracted.10 2. Topical compositions for skin application according to claim 1, wherein the sum of a) + b) + c) is comprised between 0.03 and 3% by weight on the total weight of said topical compositions, preferably between 0.3 and 1.5% by weight on the total weight of said topical compositions.

3. Topical compositions according to claim 1 or 2, wherein the weight ratio between15 the components a) b) and c) is 1 : 1 : 1.

4. Topical compositions for skin application according to any one of claims 1-3, wherein the solvent mixture consists of 30% by weight of water, while 70% consists of 1,3 propanediol.

5. Cosmetic compositions according to any one of claims from 1 to 3, further20 comprising at least one active ingredient d) selected from the class consisting of Niacinamide, Tranexamic acid, Hyaluronic acid, one or more sunscreens.

6. Preparation process of the liquid extracts a) b) and c) contained in the topical compositions for skin application according to any one of claims 1-5 comprising the following steps:25 I. Drying the starting plant material at a temperature less than or equal to 40°C and up to a humidity less than or equal to 10%II. Grinding and sifting the dried product from step I. until a homogeneous powder with sizes less than or equal to 6 mm is obtained;27III. Preparing a solvent mixture comprising: the addition under stirring of 1,3- propanediol to water until obtaining a clear solution in which the 1,3- propanediol is 70% m / m and water is 30% m / m on the total weight of said solvent mixture5 IV. Mixing the dried powder obtained in step II. with the solvent mixture prepared in step III. in weight ratios of dried powder / solvent mixture equal to 1 : 10V. Treating the mixture of step IV in an ultrasonic bath for 1 hour at room temperature:10 VI. Eliminating solid residues resulting from step V. by pressing with a screw press or pneumatic press or by filtering with at least two vacuum filiations using filters with porosity up to 0.22 microns.

7. Topical compositions for skin application, comprising a mixture of the following extracts:15 a) an extract from branches and / or twigs of Vitis Vinifera L. b) an extract from the bark of Abies alba Mill. c) an extract from the bark of Pinus sylvestris L.

8. Cosmetic use of the topical compositions for skin application according to any one of claims from 1 to 5 or according to claim 7, for the treatment and prevention of the20 formation of skin spots.

9. Topical compositions according to any of claims 1-5 or 7 for use in treating or preventing the formation of skin spots.

10. Cosmetic use according to claim 8 or the topical compositions for use according to any one of claims from 1 to 5 and 7, wherein said skin spots are due to ageing, sun25 exposure, acne, hormonal variations, drugs.

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