RO135101A0 - Emollient cream for dry skin with grape skin/seed extract (ecological culture of vitis vinifera l., feteasca neagra variety) - Google Patents

Abstract

The invention relates to an emollient cream for dry skin and to a process for preparing it, with applicability in the cosmetic or dermatological field, mainly for anti-wrinkle purposes. According to the invention, the cream consists of 7% (volume/ mass) hydroalcoholic extract from grape skin/seeds of ecologically cultivated Vitis vinifera L., Feteasca Neagra variety, in a cream base as a mixture of lanolin, cetyl alcohol and sodium lauryl sulphate. According to the invention, the process consists of the steps of: 1. extraction of the active compounds from the grape skin/seed from fresh fruit or biomass, in ethanol-water extraction solvent, by maceration at room temperature for 24 h, of which the first 3 h under mechanical stirring, followed by filtration, the resulting extract having a content of 2.5 to 3.5 mg EAG/ml (skin) and 4.5 to 7.5 mg EAG/ml (seeds), respectively, polyphenolic compounds, 3...4.5 mg EQt/ml (skin), and 4.2...7.7 mg EQt/ml (seed) flavonoids, antioxidant activity in the range of 0.8...1.5 mg EAA/ml (skin), and 10...12.5 mg EAA (seed), respectively, antibacterial and antifungal activity, 2. preparation of the cream base by mixing, at 50°C, 15% cetyl alcohol, 20% lanolin, 1% sodium lauryl sulphate and for the rest, distilled water, with continuous homogenization up to cooling, 3. incorporation of the liquid extract into the cream base, resulting in a moisturizing revitalizing anti-wrinkle cream product for a wide range of dry skin types.

Description

SOFTENING SKIN SOFTEN CREAM WITH SKIN / ITEM EXTRACT (ECOLOGICAL CULTURE VITIS VINIFERA L, BLACK GIRL VARIETY)

The invention relates to an EMOLYING CREAM FOR DRY SKIN WITH SKIN / KIND EXTRACT (ECOLOGICAL CULTURE VITIS VINIFERA L „NATURE FETEASCA BLACK) with applicability in the cosmetic or dermatological field, mainly for anti-wrinkle purposes. According to the invention, the cream contains lanolin, cetyl alcohol, sodium lauryl sulfate, hydroalcoholic extract from the skin and / or grape seeds from organically grown Vitis vinifera L., Romanian variety Fetească Neagră. The combination of cream components results in a product that deeply nourishes and prevents dry skin (moisturizing, revitalizing, antimicrobial effect), repairs and smoothes fine lines of expression, prevents aging (anti-wrinkle effect).

The local variety of Fetească Neagră is the grape variety from Romania with the highest quality potential, with a special aroma and a content of fine tannins. This valuable variety is very old, known since the time of the Dacians, well adapted to both growing conditions (plain / hill) and climatic, being resistant to both frost and drought. Representative for the Fetească Neagră variety is the fact that it is self-fertile (early variety), being thus cultivated in pure, ecological plantations (organic culture), without modifying the phytochemical profile of the grape. According to patent RO129328A2 [Ranca, A.M. and Negraru, A., 2015] (Ecological process for cultivating the black fetal vine variety), which refers to a process for the ecological cultivation of the Black fetal vine variety applied in a wine farm, phytosanitary treatments and applied to several vine varieties in order to make them more efficient and adapt to the conditions of water scarcity, however, have the disadvantage that they are made with a series of synthetic chemicals which, even if they are systemic relevant legislation, alters the properties of grapes. By applying the above-mentioned invention, several advantages are specified, among which the obtaining of natural grapes, without chemical residues. The patent states that the use of synthetic products for phytosanitary treatments is prohibited and that plant health is ensured in a preventive manner, allowing only products based on simple mineral salts (copper, sulfur, sodium silicate) or plant extracts, within the limits of the rules established by the relevant legislation (EC Regulations no. 834/2007 and no. 889/2008).

Grapes, pomace and wines from the red varieties of Vitis vinifera L. are important sources of natural antioxidants, due to the high concentration of various phenolic compounds [Balea, Ș.S., et al., Oxide. Med. Cell. Longev., 2018; Manach, C., et al., Am. J. Clin. Nutr., 2004, 79, 727]. Approximately 70% of the phenolic compounds have been reported to remain in the pomace [Mattos, G.N., et al., J. Sci. Food Agric., 2017, 97, 1055]. Most grape polyphenols come from the skin and seeds and include two main classes: flavonoids (anthocyanins, flavonols, flavan-3-oils, flavones and chalcones) and nonflavonoids (phenolic acids, stilbenes, tannins, coumarins and neolignans) [Xu, I ., et al., Food Sci. Nutr., 2016, 4, 125]. Moreover, in the case of polyphenolic compounds, it has been found that they have antioxidant properties, but can also act as prooxidants, as they can induce the generation of free radicals [Cotoras, M., et al., Molecules, 2014, 19, 21154], Evaluation comparative antioxidant activity for unfermented and fermented pomace, indicated a significantly positive correlation of antioxidant activity with tannin concentration, total anthocyanin content, flavan-3-ol monomers and stilbenes [Balea, Ș.S., et al., Oxide. Med. Cell. Longev., 2018], in 2014 a report on the evaluation of the safety of use in cosmetics of 24 ingredients derived from grapes (Vitis vinifera L.) [Fiume, M.M., et al., Int. J. Toxicol., 2014, 33, 48S] specified the frequent use of ingredients such as skin extract - "Vitis Vinifera (Grape) Skin Extract" - and pips - Vitis Vinifera (Grape) Seed Extract ", as conditioning agents of the skin, and some of these ingredients may function as antioxidants, flavoring agents and / or colorants. Phenols occupy the third place in terms of abundance in grapes (after carbohydrates and acids), fully extractable phenols being present in varying proportions depending on the anatomical part of the fruit: less than 10% in the pulp, 60-70% in the seeds and 28 -35% in the skin [Fiume, MM, et al., Int. J. Toxicol., 2014, 33, 48S]. It is also mentioned that the amount of constituents present in the extract depends on the solvent used for extraction and the variety / variety of Vitis vinifera L. used. The main constituents of grape seeds are phenolic compounds represented, based on standardized grape seed extracts, in a proportion of 92-95% of oligomeric proanthocyanidins [National Toxicology Program, Summary of Data for Chemical Selection, 2000]. Seed extract works as an anti-aging, anti-dandruff, antifungal, antimicrobial, antioxidant, flavoring, light stabilizing, oral care / health agent and sunscreen agent. The skin extract works as an antioxidant, colorant and flavoring agent. Both extracts fall into the chemical class of botanical products and botanical derivatives [Personal Care Products Council, On-Line INFOBASE Ingredient Database, 2012].

The extraction of polyphenols is mainly dependent on two factors, the dissolution of each polyphenolic compound at the cellular level in the matrix of plant material and the diffusion in the external environment (solvent) [Kallithraka, S., et al., Phytochem. Anal., 1995, 6, 265; Nawaz, H., et al., Sep. Purif. Technol., 2006, 48, 176], Initially, extractions were performed with organic solvents but although these extraction procedures were effective, the extracts were not safe for human use due to the potential toxic effects of the residual solvent; solvents such as hexane and methanol combinations were used [Santos-Buelga, C., et al., Food Chem., 1995, 53, 197], ethanol-benzene combinations [Kofujita, H., et al., Wood Sci . Technol., 1999, 33, 223], ethyl acetate [Bonilla, F., et al., Food Chem., 1999, 66, 209] and sulfur dioxide [Cacace, J.E., et al., J. Agric. Food Chem., 2002, 50, 5939]. These solvents are toxic to the human body depending on the dose used, these potential health effects being one of the reasons for researching methods that would reduce the use of organic solvents in the extraction procedure [Nawaz, H., et al., Sep. Purif. Technol., 2006, 48, 176]. The selection of ethanol as an organic solvent was based on the fact that ethanol, when mixed with water, improves the solubility of bioactive components compared to pure water [Nawaz, H., et al., Sep. Purif. Technol., 2006, 48, 176]. The optimal solution consists of a mixture of 50% ethanol - 50% water (v / v); In addition, ethanol is a safe biological solvent,

unlike the organic solvents mentioned above. The presence of ethanol in the mixture contributes to the penetration of the solvent into the hydrophobic areas of the kernel matrix and to the precipitation of soluble kernel proteins that would interfere with the filtration procedure [Shi, J., et al., J. Food Agric. Environ., 2003, 1,42], Although numerous studies have indicated that the use of acetone and methanol leads to high extraction yields of individual polyphenols, hydroethanolic mixtures have shown a higher degree of recovery compared to total polyphenols [Bogdan, C., et al., Antioxidants (Basel), 2020, 9, 502; Nieto, J.A., et al., Foods, 2020, 9, 604; Li, J., et al., J. Food Sci. Technol., 2019, 56, 4879; Moldovan, M., et al., Pharmacy, 2020, 68, 15],

Anti-wrinkle / anti-aging creams are predominantly skin care products based on moisturizers, the segment of topical products formulated with the addition of natural components showing a growing interest from consumers regarding health and appearance [Soto, ML, et al., Cosmetics, 2015, 2, 259; Noian, K.A., et al., J. Drugs Dermatol., 2012, 11,220; Sharma, B., et al., Int. J. Pharm. Pharm. Sci., 2012, 4, 57], The growth of this market segment is also based on the demand of the population who opt for less invasive, non-surgical alternatives to slow down the effects of aging on the skin. In terms of active compounds, current trends in cosmetics have two directions: a return to the past and a new beginning, with the use of traditional ingredients and the benefits of modern research and clinically tested products [Cohen-Letessier, A., Ann. Dermatol. Venereol., 2009, 136, S367]. Natural polyphenols, through their confirmed antioxidant activity relative to radical oxygen species, are of constant interest in the cosmetic field for anti-aging purposes or for nutraceutical applications. Grapes provide active ingredients capable of counteracting the symptoms of epidermal aging, such as phenolic components, which can improve protection against solar radiation and epidermal antioxidant activity [Soto, M.L., et al., Cosmetics, 2015, 2, 259; Lorencini, M., et al., Aging Res. Rev., 2014, 15, 100]. Grape-based creams / derivatives are effective in improving premature aging [Sharif, A., et al., Int. J. Cosmet. Sci., 2015, 37, 253] and could reduce the occurrence of skin cancer and delay the photoaging process [Saraf, S., et al., Pharmacogn. Rev., 2010, 4,

1], Therapy using bioactive compounds associated with grape-derived products has been proposed for the treatment of extrinsic skin aging processes [Sanchez, V., et al., Ars Pharm., 2008, 49, 309; Ratz-Lyko, A., et al., Phytother. Res., 2015, 29, 509], The new phytoproducts for skin rejuvenation are of increased interest, in a study that evaluated the marketed anti-aging creams, the grape seed extract being one of the most used ingredients [Cronin, H., and colab., J. Cosmet. DermatoL, 2010, 9, 218],

For the treatment of dry skin, creams based on synthetic chemical compounds, some toxic, carcinogenic (paragen category, isothiazolinones, bronopol, dimethylol hydantoin or diazolidinyl urea, etc.) are generally used. Given the general preference of consumers for natural products and the care given by international forums regarding the prohibition of synthetic compounds in the composition of cosmetics used daily / weekly, the invention also provides consumers with a solution related to chemophobia. Another aspect would be the use of the extract from the local variety, highly appreciated, Fetească Neagră, a variety without genetic modifications, secular, cultivated in ecological vineyards, on soils without historical pollution, drip irrigation and periodic natural treatments, without artificial fertilizers. The special chemical composition of this variety, Fetească Neagră, with a remarkable content of resveratrol (the elixir of youth ”) and piceid (stilbenoid glucoside - major derivative of resveratrol), as shown by the quantitative HPLC analysis, with anti-aging effect, could be an invigorating safe alternative and much better received by consumers in Romania and other countries. [citation needed]

They are known from the prior art products / compositions such as:

WO 2012/056052 Al [Moro Gonzalez, L.C., et al. 2012] (Polyphenolic grape extract and cosmetic product comprising said extract ') / PCT / ES2010 / 070689, which refers to a polyphenolic grape extract characterized by having the following technical specifications: a total polyphenolic index (TPI) between 100 and 1000; a polyphenolic content expressed as gallic acid equivalents between 2 and 25 mg gallic acid / L; and a pH between 2 and 4. The invention also relates to a cosmetic composition comprising said extract and also to its use for the production of cosmetic articles. As a raw material

EN 135101 AO It is possible to use different grape varieties to obtain the extract, although red grapes are especially preferred. These extracts usually come from the skin after pressing the grapes. From the grape polyphenolic extract obtained and characterized as described above, cosmetic compositions were made for topical administration, using conventional methods, including a moisturizer. It contains the polyphenolic extract described in a concentration of 2-10% by weight, but also at least one vehicle consisting of cosmetically acceptable excipients, such as rose oil, caviar extract, jojoba oil and germ wheat.

Document ES2228284B1 [Quilis, L.J. and Casani, AS, 2003] (Composiciones que compren un extracto de hollejo, semillas y raspas de uva negra) refers to the natural extract from the skin, seeds and remains of black grapes, rich in antioxidant compounds (polyphenols and anthocyanins), which can be used in the preparation of inactive foods enriched with natural antioxidants, as well as in the preparation of dietary, pharmaceutical or cosmetic compositions. The concentrated natural extract can be obtained by percolation which involves in a first stage a solid-liquid extraction process (using as raw material a mixture of skin, seeds and remains of black grapes, and as solvent water) to obtain a crude extract; separating the fraction comprising the antioxidant components (polyphenols and anthocyanins) from the crude extract to obtain a concentrated extract, and, optionally, a process of drying or removing the solvent to obtain the concentrated extract in the form of a dry powder. In a particular embodiment, a cosmetic composition provided by this invention includes a vehicle comprising one or more acceptable cosmetic excipients; The authors of the present invention have observed that said extract is absorbed percutaneously, allowing its use in topical administration. An example of such a cream-like cosmetic composition contains: dry extract of Vitis vinifera, glycolic extract of Fucus vesiculosus, glycolic extract of Centella asiatica, allantoin, glycolic extract of Ginko biloba, vitamin E, O / A emulsion.

WO 2010/056675 A2 [Jacobs, J., June Jacobs Labs, 2012] (Antioxidant compositions for the cleansing and conditioning of skin ”) / PCT / US2009 / 063891, refers to antioxidant cosmetic compositions for skin care

skin formulated to combat diseases associated with free radical damage and oxidative stress. The compositions contain one of several active agents, including an agent derived from one or more of the plant species: Lycium barbarum, Punica granatum, Vitis vinifera, Aspalathus linearis and Camellia Sinensis, as well as cosmetically acceptable vehicles; these cosmetic compositions find their applicability in improving the appearance of aged or damaged skin. In one embodiment, the active agent is liquid, finely ground powder or an extract derived from a component of one or more of the plant varieties listed above. Accordingly, a composition may include as active agents, components of any or all of the above plant varieties in any quantity or combination, to achieve the effect of rejuvenating the skin by topical application. For example, in certain variations, a composition may contain from about 0.001% to about 20% of an extract derived from one or more of the species Lycium barbarum, Punica granatum, Vitis vinifera, Aspalathus linearis and / or Camellia Sinensis, as an active ingredient. The compositions may include a cosmetically acceptable vehicle to act as a diluent, dispersant or carrier for the active ingredients, so as to facilitate their distribution and absorption when the composition is applied to the skin. Such vehicles may be water, liquid or solid emollients, other solvents, humectants, thickeners, powders and perfumes; for example, a suitable carrier may include glycerin, butylene glycol, propylene glycol, water, various oils (jojoba, almond, soybean, sunflower, apricot, etc.) and the like.

WO 2011/055222 A2 [Bergamini, E., et al. 2011] (Composition comprising an extract from polygonum cuspidatum and a polyphenolic extract from red vitis vinifera) / PCT / IB2010 / 002812, refers to a composition that includes a water-soluble extract of Polygonum cuspidatum and an extract (red grape variety) of Vitis vinifera and its uses. The composition according to the invention comprises an extract of Polygonum cuspidatum containing frans-resveratrol and a polyphenolic extract of Vitis vinifera in relative proportions between 1% and 25% (mass percentages) being claimed for dermatological use (anti-aging). In the said patent, the term extract is understood as meaning a phyto-composite comprising all the components present in Vitis vinifera red ”corresponding to their natural balance. Preferably, the extraction is carried out using a “hydroalcoholic” method that does not introduce chemical compounds that are harmful to human health; therefore, this definition includes polyphenols that are produced naturally (for example, by extraction from red wine or red grapes). The topical composition may preferably be formulated as a cream, gel or ointment. An example of a cream composition includes: glycerin monostearate, cetyl alcohol, mineral oil, sesame oil, glycerin, red polyphenolic extract of Vitis vinifera, Polygonum cuspidatum extract, resveratrol added, water.

RO121004B1 [Maftei, E.A., et al. 2006] (Cosmetic composition with anti-cellulite action and reduction of fat deposits) refers to a cosmetic composition with anti-cellulite action and reduction of fat deposits, with plant extracts, which consists in that it consists of 3 ... 8 parts extract hydroalcoholic acid of Vitis vinifera, containing at least 2.5% dry matter, the parts being expressed by weight. The cosmetic composition, according to the invention, due to the plant bioactive complex, ensures the hydration of the epidermis and stops the aging processes of the skin, due to the association of the plant bioactive complex with cosmetic adjuvants and water. An example of anti-cellulite, moisturizing cream consists of: anhydrous lanolin, cetyl alcohol, yellow wax, ammonium diethanol phosphate mist, ivy hydroalcoholic extract, vine hydroalcoholic extract, sorghum hydroalcoholic extract, vegetable oil, olive oil distilled water, preservatives / antioxidants.

Document RO127720B1 [Postescu, I.D., et al. 2016] (Photochemoprotective gel and its preparation process) refers to a composition for a gel with photochemoprotective properties, intended for topical application for cosmetic use, in order to protect the skin from the harmful effects of ultraviolet radiation, and a preparation process his. The invention combines a natural extract, obtained from grape seeds, the Burgundy Mare variety, and simple and cheap ingredients, which ensure a good penetration of the extract into the skin (high molecular weight carboxyvinyl polymer, for example, carbopol, triethanolamine, glycerin, p- methyl hydroxybenzoate). The pharmaceutical composition of the product presented in the invention is maintained

RO 135101 AO long-term physical and chemical stability. The effects of the photochemoprotective gel on the skin are multiple: it reduces the effect of free radicals generated by exposure to ultraviolet radiation on skin components (nucleic acids, lipids, proteins), inhibits the appearance of mutations in normal cells and improves DNA repair. In another aspect, the invention relates to a process for the preparation of a photochemoprotective gel defined above, in which over a high molecular weight carboxyvinyl polymer, dispersed in a mixture of water and glycerin, and kept at rest for hydration , gradually add, under stirring, triethanolamine dissolved in water, and over this mixture is added a mixture of methyl and propyl esters of p-hydroxybenzoic acid, dissolved in alcohol, and a fluid extract of grape seeds obtained from 1 part in weight of dried and ground seeds in the form of a fine powder, taken up in 10 parts by volume of ethanol-water solution, 60% (v / v), which is then refluxed on a water bath, cooled to room temperature and filtered, then make up to 100 parts with distilled water and mix. The process for obtaining the extract from red grape seed {Vitis vinifera L.), Burgundy Mare variety, is as follows: 1 part by weight of seeds, dried and ground in the form of a fine powder, were taken up by 10 parts by volume of ethanol solution - water, 60% (v / v), and the mixture was refluxed on the water bath for a while. After cooling (at room temperature) and filtering off the solid fraction, the filtrate was analyzed for content in total polyphenols, proanthocyanidins, anthocyanins, and antioxidant activity (AO). The extraction yield, in total polyphenols, was between 9.2 and 9.8 g EAG / L (EAG = gallic acid equivalent). Experimental research was performed on the tolerance of the photochemoprotective gel with grape seed extract. It was applied in patch tests to healthy volunteer subjects, the results obtained being very good, without local reactions and at a distance of intolerance. 1> 1 1

Document RO122894B1 [Burghelea, B., et al. 2010] (Process for obtaining a powdered grape seed extract with a high proanthocyanidin content ') refers to a process for obtaining a powdered grape seed extract with a high content of proanthocyanidins, an extract that can be used in the pharmaceutical, food and cosmetic fields. The process

according to the invention consists in treating the grapes with water and / or ethyl alcohol, at a temperature of up to 120 ° C and a pressure of 1 atm, for 45 ... 60 min, after which the polyphenolic extract in alcohol solution ethyl, rich in proanthocyanidins, is concentrated, centrifuged and filtered to give an extract which is then purified and enriched by passing through a reverse phase chromatographic column and subsequently obtaining a powder by lyophilizing the total liquid extract. or enriched liquid extract. The results of toxicological experiments on animals as well as its pharmaceutical presentation (fine non-hygroscopic powder, with high stability under normal atmospheric conditions), recommend the polyphenolic extract as safe for use as an ingredient in cosmetics.

The technical solutions in the processes / products presented above have the following disadvantages:

A total polyphenolic content expressed as milligrams equivalent of gallic acid per mL extract (mg-EAG / mL) significantly reduced (WO patent 2012/056052) compared to that determined in the case of the extract claimed in the present invention; also the pH values of the polyphenolic grape extract used as an ingredient in cosmetic compositions (moisturizer type) are not relatively close (patent WO 2012/056052) to the average value (4.7) of the “natural pH” of the human epidermis [Lambers, H., et al., Int. J. Cosmet. Sci., 2006; Rogiers, V., Skin Pharmacol. Appl. Skin Physiol., 2001].

The use of one or more active ingredients (natural extracts), although based on natural products (eg rose oil, caviar extract, goose germs, glycolic extracts of Fucus vesiculosus, Centella asiatica, etc.) may induce specific interactions with the compounds. phytochemicals from Vitis vinifera extract ', does not specify the compatibility of other natural active ingredients added with grape skin extract (patent WO 2012/056052, patent ES2228284B1, patent WO 2010/056675 A2, patent WO 2011/055222 A2, patent R0121004B1).

Solid-liquid extraction using only water as solvent (even in the case of using a mixture of skin, seeds and grape residues as raw material) leads to low extraction yields of polyphenolic compounds compared to the use of hydroalcoholic mixtures; the values of the total polyphenolic index or of the total polyphenolic content in the case of liquid or powdered Vitis vinifera extract are not specified (patent ES2228284B1).

The wide concentration range (eg 0.001-20%, 1-25%) specified for the use of Vitis vinifera extract as the active ingredient as such or in combination with other natural extracts may induce problems in selecting a vehicle that is acceptable cosmetic to act as a diluent, dispersant or carrier for the active ingredients, so as to facilitate their distribution and absorption when the composition is applied to the skin (patent WO 2010/056675 A2, patent WO 2011/055222 A2).

It does not mention the performance of studies / clinical tests in the preliminary phase of cosmetic compositions that include extracts of Vitis vinifera as such (from the skin / seeds or combinations thereof) or in combination with other natural extracts (patent ES2228284B1, patent WO 2010/056675 A2 , patent WO 2011/055222 A2, patent RO121004B1).

The use of synthetic chemical compounds (eg triethanolamine, p-hydroxybenzoic acid methyl / propyl esters) in the preparation of cosmetic compositions (although there are insufficient scientific data on the behavior of each such ingredient) may induce potential risks related to the formation of chemical contaminants. (e.g. Nitrozo compounds (NOC)) [Fiume, MM, et al., Int. J. Toxicol., 2015; Elder, R.E. (ed), J. Am. Coli. Toxicol., 1983; Fiume, M.M., et al., Int. J. Toxicol., 2017] (patent RO127720B1).

The technical problem solved by the invention consists in obtaining an emollient cream for cosmetic and / or dermatological use that can be used as a product containing natural bioactive compounds, with moisturizing, anti-wrinkle, antifungal and antibacterial properties.

The composition according to the invention eliminates the mentioned disadvantages by the fact that an emollient cream is used for application on dry skin, with a wide spectrum, with the basic formula; lanolin, cetyl alcohol, sodium lauryl sulfate and hydroalcoholic extract from grape skin / kernels (ecological culture of Vitis vinifera L., Fetească Neagră variety), which is characterized by an extract content of 7% (volume / mass).

The hydroalcoholic extracts from the skin / kernel are characterized in that they are obtained by maceration at room temperature, in a percentage of 4% dry mass / volume. The hydroalcoholic extract of the skin is characterized in that it has a total polyphenol content in the range of 2.50-3.50 mg-EAG / mL, a total flavonoid content in the range of 3.00-4.50 mg-EQt / mL ( EQt = quercetin equivalent), an antioxidant activity in the range of 0.80-1.50 mg-EAA / mL (EEA = ascorbic acid equivalent), a moderate in vitro antimicrobial inhibitory action on selected Gram-positive bacteria with corresponding values of minimum inhibitory concentration in the range of 820-4300 pg / mL, a low antifungal efficacy compared to the selected strains. The hydroalcoholic extract from grape seeds is characterized by the fact that it has a total content of polyphenols in the range of 4.50-7.50 mg-EAG / mL, a total content of flavonoids in the range of 4.20-7.70 mg-EQt / mL, an antioxidant activity in the range of 10.00-12.50 mgEAA / mL, a broad spectrum of action, in vitro, on selected Gram-positive bacteria, with corresponding values of the minimum inhibitory concentration in the range of 850-4000 pg / mL, a selective and moderate antifungal efficacy against selected strains.

By applying the invention the following advantages are obtained:

Plant material from organic farming is used as a source of bioactive ingredients; thus eliminating potentially toxic sources adsorbed by the plant, both by air (conventional spraying with pesticides or other chemical phytosanitary agents) and by rooting from soils with potential historical toxicity or fertilized with various synthetic chemicals. The lack of pesticides remaining in the anatomical parts of plants used as a source of raw materials is a desideratum of the pharmaceutical and cosmetic field, especially due to the therapeutic uses of phytocompounds. Ecological culture is the solution for solving the issues related to meeting the demand for natural products, which do not involve the use of synthetic chemicals.

The process of obtaining the product is easy and has minimal costs (maceration at room temperature), well defined technically (fixed percentage of active ingredient in the base cream) and does not involve the generation of potentially toxic by-products / intermediates, and component extraction technologies / active cosmetic principles involve

RO 135101 A0.

minimum energy consumption according to the principles of environmentally friendly technologies ”. This creates the premises for an easy-to-apply product technology.

Bioactive ingredients are used whose phytochemical, antibacterial and antifungal properties have been demonstrated through complex analytical investigations; thus, the following are considered to be advantages: (a) the use of the ethanol-water mixture, as a solvent for highly efficient extraction of bioactive compounds with beneficial properties for dry skin types; (b) a clear definition of the domains of CTP (total polyphenol content), CTF (total flavonoid content) and global AA (antioxidant activity) respectively; compared to other existing technical solutions mentioned in this patent application, the CTP obtained for skin / stone extracts is significantly higher (for CTF and AA there are no comparative data for similar extracts from the autochthonous variety Fetească Neagră); (c) data on the antibacterial and antifungal activity of the skin / stone extracts of the Fetească Neagră variety, no data are specified / available regarding these aspects.

The basic cream used to incorporate bioactive ingredients (skin extracts / pips) is prepared according to the Romanian Pharmacopoeia, 10th edition, and contains as main vehicle lanolin (fat extracted and purified from sheep's wool), recognized for its structure and composition that mimics " the lipid matrix of the stratum corneum. This creates the premises for use for a wide range of dry skin types.

The compatibility of the products proposed for patenting with dry skin has been demonstrated by clinical trials in human subjects according to Regulation (EC no. 1223/2009 on cosmetics; the results obtained were good, no irritant or inflammatory side effects were detected in the subjects test.

In a concrete embodiment of the invention the steps are followed:

Stage 1 - the extract is obtained from the skin, respectively from the grape seeds of the Fetească Neagră variety, organically cultivated. The skin / kernels (from fresh fruit or fresh biomass resulting from various processing processes) were separated by hand, then dried in an oven with ventilation at 40 ° C for 48 h. The samples thus prepared were stored at room temperature. , in closed containers, protected from

RO 135101 A0 „humidity and light. The method chosen for extraction was maceration at room temperature (22-23 ° C, for 24 hours, by contact with a solvent consisting of a 50% (v / v) hydroalcoholic solution. Solid-liquid ratio. - hydroalcoholic solvent) used in the extraction was 4% mass / volume.The operation took place in a closed container, magnetic stirring was applied for the first 3 hours, and in the next 21 hours the contact with the solvent used for maceration was maintained. was separated by filtration, and the filtrate (skin / stone extract) was kept cold (4-5 ° C) for bio-physico-chemical analysis, respectively until the moment of incorporation into the base cream (step 3).

Stage 2 - prepare the base cream for dry skin, according to the Romanian Pharmacopoeia 10th edition [Romanian Pharmacopoeia, ed. a X-a, 2015] with the following composition: 15.00 g cetyl alcohol, 20.00 g lanolin, 1.00 g sodium lauryl sulfate, q.as.ad 100.00 distilled water. The cetyl alcohol, lanolin and sodium lauryl sulphate are melted in a water bath at 50 ° C. After melting, distilled water is gradually added under continuous stirring at the same temperature of 50 ° C. Mix / mix continuously until cool.

Step 3 - the incorporation of the extract was done by slow mixing of the liquid skin / core extract of the Fetească Neagră variety in the base cream prepared according to step 2. A proportion of 7 mL extract / 100 g of base cream was used.

The extracts obtained in stage 1 and the creams that are the object of the invention were characterized by bio-physico-chemical analysis processes. Thus, for hydroalcoholic extracts from grape skin / seed, Fetească Neagră variety, were determined experimentally: pH and electrical conductivity, total content of polyphenolic compounds, total content of flavonoids, antioxidant activity, antimicrobial activity and a profile was made chromatographic analysis of compounds with bioactive potential. Comparative tests were performed between the extracts used, the base cream and the creams which are the object of the invention by vibrational spectroscopy in IR and Raman; through these experimental investigations, the aim was to identify some structural changes, but also to highlight the incorporation of some compounds with bioactive potential from the extracts, in the skin / core cream extract proposed for patenting (Figure 1 and Figure 2). Determination of Resveratrol (cis, trans) and Piceid (cis, trans) by High Performance Liquid Chromatography (HPLC) in the hydroalcoholic extracts of skin / pips of Vitis vinifera L., Romanian variety Fetească Neagră, was performed using the method described by Roșa M. Lamuela-Raventos et al., In the article Direct HPLC Analysis of cis- and trans-Resveratrol and Piceid Isomers in Spanish Red Vitis vinifera Wines [Roșa M. Lamuela-Raventos et al., Journal of Agricultural and Food Chemistry, 1995, 43 (2), 281-283]. The standard against which the values were reported was the cis-, transresveratrol standard.

The pH and electrical conductivity determinations of the extracts used for the preparation of the creams object of the invention showed values in the range 4.4 - 4.7 for pH (skin extract) and 5.4 - 5.7 for pH (kernel), respectively in range 290 310 pS / cm (skin extract) and 155 - 175 pS / cm (core) for electrical conductivity.

The determination of the total polyphenol content was performed by ultraviolet-visible absorption spectrometry, Folin-Ciocâlteu method, as described in the Phytochemical Profiles, Antioxidant and Antibacterial Activities of Grapes (Vitis vinifera L.) Seeds and Skin from Organic and Conventional Vineyards [Radulescu , C. et al., PLANTS-Basel, 2223-7747, 2020, 9, 11, 1470], which is based on a chemical process of oxidation of polyphenols in the presence of Folin-Ciocâlteau reagent. The readings were taken at a wavelength of 765 nm, in tanks with an optical path length of 10 mm, and the total polyphenol content was determined using a calibration curve in gallic acid as a reference antioxidant, the values being expressed in milligrams equivalent to gallic acid per mL extract (mg-EAG / mL).

The determination of the total flavonoid content was performed according to the procedure described in the Influence of Extraction Method on Chemical Composition from Red Grapes Skin Extract [Nicolescu, CM, et al., Journal of Science and Arts, 1844-9581, 2019, 1, 201, 208] , the applied method being spectrophotometric based on the formation of complex combinations in the reaction of the Al ³⁺ ion with the carbonyl and hydroxyl groups in the flavonoid structure. The readings were taken at a wavelength of 510 nm, in tanks with an optical path length of 10 mm, and the values of the total flavonoid content were determined using a calibration curve using quercetin as the reference flavonoid, the values are expressed in milligrams quercetin equivalent per milliliter extract (mg-EQt / mL).

Antioxidant activity was determined by the Prieto method, as described in the Phytochemical Profiles, Antioxidant and Antibacterial Activities of Grapes (Vitis vinifera L.) Seeds and Skin from Organic and Conventional Vineyards, [Radulescu, C. et al., PLANTS-Basel, 2223-7747, 2020, 9, 11, 1470], which is based on the formation of a colored phosphomolybdenum complex, the readings being made at a wavelength of 700 nm, in tanks with an optical path length of 10 mm. The calibration curve was plotted using ascorbic acid as a reference, and the values of antioxidant activity were expressed in ascorbic acid equivalent per mL extract (mg-EAA / mL).

Study of antimicrobial activity. Biological material: The extracts from the skin and kernels of the grapes were obtained with the provision of sterility conditions for the materials used and for the working environment as a whole. A number of six bacterial strains, respectively three fungal strains from the Culture Collection of the Multidisciplinary Research Institute for Science and Technology - Valahia University ”from Târgoviște were tested on growth inhibition in the presence of antimicrobial compounds in hydroalcoholic extracts. grapes. The stems were chosen so as to come from different habitats (food, soil, air vineyard of grape origin) and belong to a diverse number of genera. It is known that many strains of Bacillus are widely used in biotechnology, being considered GRAS (Generally Recognized As Safe) by the Food and Drug Administration (FDA). Also, many Bacillus tiilpini have beneficial traits for promoting and protecting plant growth and have a great potential to be used as agro-inoculants, because they are spore formers and easy to formulate and preserve [Sicuia, OA, et al. , Acta Hortic., 2017, 1164, 257], Numerous species of lactic acid bacteria (LAB), bacteria with multiple applications in the food industry, are part of the intestinal microbiota of humans and animals [Vaughan, EE, et al., FEMS Microbiology Review , 2005, 29, 477], which was also the basis for the selection of the above-mentioned bacterial strains, in the context of the subject matter of the patent proposal. The mold strains proposed in the study belong to genes potentially producing skin mycotoxins [Dantigny, P., et al., CRC Press, Taylor & Francis Group, 2021] and potentially present in the skin microbiota [Hammoudi, N., et al., Sci Rep, 2021, 11, 3777], Bacterial strains have been isolated and characterized by classical microbiological techniques. Isolation of pure bacterial cultures was performed from different habitats (food of animal origin and food of plant origin, air), after successive transplanting, so as to avoid contamination of microorganisms used as a test. Agarized meat broth (pH = 6.8 ± 0.2) was used to grow the bacteria, and the incubation was performed at 37 ± 0.2 ° C. Macroscopic and microscopic characterization of the colonies was done according to Bergey's Manual of Systematic Bacteriology. Isolated pure cultures were maintained on agarized nutrient broth at 4 ° C. Isolation of fungal strains from natural environments was achieved by inoculation (1 mL) of serial dilutions (10 ' ³ -10' ⁶ ) in Petri dishes with Potato - Dextrose - Agar (PDA) medium. The plates were incubated at 28 ° C for one week, then the fungal colonies were isolated as pure cultures. The identification of the fungus genus was performed by macroscopic and microscopic examinations, depending on the color of the colony, its shape, the presence of hyphae, conidia, conidiophores and the arrangement of spores.

Determination of antimicrobial activity of grape extracts. The determination of the antimicrobial activity of hydroalcoholic extracts from grapes (skin, seeds) was performed in solid nutrients, by applying the diffusimetric method (Kirby-Bauer), which allowed the simultaneous determination of the sensitivity spectrum of microorganisms (bacterial strains and molds, respectively). , as well as the value of the minimum inhibition concentration (MIC). Two methods were used to highlight the antimicrobial activity: the technique of discs impregnated with extracts (disc diffusimetric technique), standardized, method recommended by NCCLS (National Committee for Clinical Laboratory Standardization) and diffusimetric technique with wells. For isolated bacterial strains, a volume of fresh culture (20-50 pL - depending on the diameter of the Petri dish), with an optical density at 600 nm (D0 ₆ oo) between 0.2 and 0.4, was dispersed in plates with medium Meat broth with agar. After laying the plates in the vicinity of a gas bulb (for 10-15 min), the impregnated discs were placed on their surface for 1 hour in the extracts from the skin / seeds, previously prepared

according to the described protocol. The discs were placed approximately 15 mm from the periphery of the plate and 30 mm from each other, respectively. The plates were incubated at 37 ° C for 48 h. The extracts with antibacterial action showed a clear area (halo) around the colony, due to the inhibition of the growth of the strain of bacteria tested. Measurements of the growth inhibition area were performed. To determine the antifungal activity of grape extracts, on the surface of petri dishes with nutrient medium (malt must) agarized seeded "in cloth" with a standardized inoculum from each strain of mold tested, wells were stamped (5 mm diameter) at approx. 15 mm from the periphery of the plate and 30 mm from each other, respectively. In each well, without exceeding the edges, the grape extracts (10 pL) were distributed as such (2 extracts of 2 samples each on a plate). The plates were incubated at 25 ° C for 3 days. The reading consisted, as in the case of determining the antibacterial activity, in assessing the size of the areas of inhibition induced by extracts, areas where microbial colonies were absent. All determinations were performed in triplicate. The results were expressed in the form of average values obtained by performing the arithmetic mean of the diameters corresponding to the 3 tests. The value of the minimum inhibition concentration of the extracts obtained from the grapes was determined by the dilution method in liquid medium. A standardized inoculum of the tested strain was seeded in a discontinuous gradient of extract concentrations (skin / pips) in nutrient broth tubes. After proper incubation, at 37 ± 0.5 ° C for 48h, MIC values were read by macroscopic observation of the tubes. The extract concentration corresponding to the tube with the lowest concentration, which inhibited the visible growth of the microbial culture, represented the MIC value (pg / mL) for that extract. For the preparation of the standardized inoculum microbial strains aged 18-24 hours were used, taking identical colonies that were suspended in sterile saline - the representative inoculum; the inoculum size was standardized on a spectrophotometric basis, by means of the optical density at the wavelength of 600 nm, DOeoo = 0.2-0.4).

For the creams obtained, in vivo clinical studies were performed, the results of the study being inserted in the Report on the safety of the cosmetic product, according to Annex I of Regulation (EC) no. 1223/2009 on cosmetic products. in this respect, Bulletins of analysis (No 209928 / 27.11.2020, 209929 / 27.11.2020) and Reports on the safety of the cosmetic product were issued for: point 1 (Quantitative and qualitative composition of the cosmetic product), point 5 (Use normal and reasonably foreseeable), point 6 (Exposure to the cosmetic product), point 7 (Exposure to the substances), point 8 (Toxicological profile of the substances) and point 10 (Information on the cosmetic product), to the manufacturer Hyperici Farm SRL, of to the accredited laboratory / person. Thus, tests were performed to verify the compatibility of cosmetic products EMOLYING CREAM FOR DRY SKIN WITH SKIN / KIND EXTRACT (ECOLOGICAL CULTURE VITIS VINIFERA THE BLACK GIRL VARIETY) by single dermal application under occlusive conditions (p human subjects aged between 15 and 65 years, based on consent and voluntary acceptance (in compliance with the legislation governing the conduct of clinical trials, the Rules of Good Practice in Clinical Trials and the agreement of the National Ethics Commission). The study aimed to verify the acceptability of the skin and to evaluate the cosmetic qualities of the emollient cream for dry skin proposed for patenting, after application for 10 days. The results of the study were good and showed that there were no irritant or inflammatory side effects in the tested subjects. The report dated 19.11.2020, was accompanied by a protocol, study and results assumed by the accredited laboratory. The reports were issued to serve the competent control bodies to supervise the placing on the market of these emollient creams with seed extract / skin from the organic culture of Fetească Neagră. It is also specified in accordance with Article 13 of Regulation (EC) No. 1223/2009 of the European Parliament on cosmetic products, that the product must be notified through the Cosmetics Notification Portal (CPNP).

Evaluation of structural changes by vibrational spectroscopy

The analysis of the investigated samples (extract, cream base and cream containing the extract) was performed with the least possible manipulation to avoid contamination, chemical reagents used for fixation, binding or elution can lead to relocation of trace elements and change of native chemical composition and structural evidence [Turker-Kaya, S., et al., Molecules, 2017, 22, 168], The main barrier to the administration of dermal drug principles is the stratum corneum that forms the outer layer of the epidermis. The stratum corneum (SC) consists of several layers of partially overlapping corneocytes that are surrounded by the cell shell and are embedded in a lipid matrix [Carrer, V., et al., Pharmaceutics, 2018, 10, 73], SC permeability depends on the interaction between the skin, the drug product / principle and the vehicle components. Due to the high degree of impermeability of the cornified sheath of corneocytes, the lipid matrix SC is the real barrier in the case of diffusion of the substance through the skin [Carrer, V., et al., Pharmaceutics, 2018, 10, 73], The structure of lanolin mimics the lipid matrix of SC having similar composition and properties (cholesterol and its derivatives, ceramides, free fatty acids).

In addition to the influence associated with environmental factors, daily cleansing with soap or surfactants can affect the skin barrier. Surfactants not only remove / wash away impurities and excess sebum, but can also remove some of the intercellular mixture of lipids [Prasch, T., et al., J. Cosmet. Sci., 2000, 22, 371]. Even when the amount of lipids removed is reduced, the selective removal of free fatty acids and cholesterol esters can lead to a significant change in the composition of intercellular lipids. In addition, surfactants enter the stratum corneum, adsorb on the keratin of the corneocytes and mix with the intercellular lipids resulting in an impairment of the skin barrier [Prasch, T., et al., J. Cosmet. Sci., 2000, 22, 371], in the cosmetic field, efforts are made to avoid these undesirable effects. in the case of skin that has damaged barrier properties, mixtures of physiological lipids slowly enter the epidermis and reconstruct the barrier in a natural way by accelerated metabolism in lamellar bodies and play a role in the recovery of scaly skin following the action of surfactants [Prasch, T. , et al., J. Cosmet Sci., 2000, 22, 371],

Polyphenolic extracts are characterized by the presence of numerous hydroxyl aromatic structures that give them the ability to form complexes with proteins. The hydrolyzable tannins together with the aforementioned structures also contain carbonyl groups, especially in the form of esters, which can be hydrolyzed to acids in aqueous systems [dos Santos, G.F., et al., Spectrochim. Acta A Mol. BiomoL Spectrosc., 2016, 153, 94],

RO 135101 AO

Spectral bands in the range 3500-3100 cm ' ¹ (intense band in the spectrum of skin extract centered at -3294 cm' ¹ and ~ 3279 cm ' ¹ in the spectrum of cream with skin extract, respectively) can be attributed to the cumulative stretching vibrations of the groups -OH, characteristic aspect of polyphenolic extracts [Fernandez, K., et al., J. Agric. Food Chem., 2007, 55, 7294; Unsalan, O., et al., J. Raman Spectrosc., 2009, 40, 562; dos Santos, GF, et al., Spectrochim. Acta A Mol. Biomol. Spectrosc., 2016, 153, 94; Smith, BC, Infrared Spectral Interpretation; A Systematic Approach, 1999]. Usually in this spectral range polyphenolic extracts have vibration bands similar to acids; however, the amount of vibrational contributions of the -OH groups is actually recorded. The spectral band located at ~ 2978 cm ' ¹ (maximum average intensity present in the spectrum of the skin extract) can be associated with the solvent (ethanol) being due to the stretching vibrations of the OH groups [dos Santos, GF, et al., Spectrochim. Acta A Mol. Biomol. Spectrosc., 2016, 153, 94], Distinct spectral maxima from 2916 and 2849 cm ' ¹ can be attributed to the conformation of the lipid alkyl chain (lanolin) being associated with asymmetric and symmetric CH2 tensile vibrations [Carrer et al., Pharmaceutics, 2018 , 73]; both maxima can be clearly observed in the spectrum of the cream base and that of the cream with skin extract. Shifting the maximum position from 2849 cm ' ¹ to higher wave numbers generally indicates an increase in the degree of chain disorder (from orthorhombic, hexagonal to crystalline liquid conformation); a maximum behavior can have a maximum of 2916 cm ^-1 although symmetrical stretching vibrations are more sensitive to conformational changes [Carrer et al., Pharmaceutics, 2018, 73], the spectral maximum located at -1736 cm ^-1 can be associated carbonyl stretching vibrations (C = O) [Westfall, A., et al., Antioxidants, 2020, 9, 486; Pasieczna-Patkowska, S. and Olejnik, T., Ann. Univ. Mariae CurieSklodowska, AA-Chemia, 2013, 68, 95] being present both in the spectrum of the cream-base (low intensity) and of the cream with skin extract (medium intensity).

The average spectral maximum localized at 1641 cm ' ¹ can be associated with the aromatic C = C stretching vibrations present in the condensed tannins [dos Santos, GF, et al., Spectrochim. Acta A Mol. Biomol. Spectrosc., 2016, 153, 94] as well as C = O tensile vibrations and the presence of unsaturated bonds in flavonoid structures

[Turker-Kaya, S., et al., Molecules, 2017, 22, 168, Smith, BC, Infrared Spectral Interpretation: A Systematic Approach, 1999; Noh, C., et al., Adv. Sci. Technol. Eng. Syst. J., 2017, 2, 435]; the presence of this maxim suggests the presence of both flavones and flavanones [Noh, C., et al., Adv. Sci. Technol. Eng. Syst. J., 2017, 2, 435]. Low intensity bands from 1448, 1390 and 1375 cm ' ¹ can be associated with CH bending vibrations of CH ₂ and CH _{3 groups} (polysaccharides associated with cell walls, lipids and proteins) [Turker-Kaya, S., et al., Molecules, 2017, 22, 168], C = CC stretching vibrations associated with the aromatic nucleus [Agatonovic-Kustrin, S., et al., Mod. Chem. Appl., 2013, 1, 110], bending vibrations associated with aromatic cycles (flavonoids) [Oliveira, RN, et al., Materia (Rio J.), 2016, 21, 769] and OH deformation vibrations in the plane of compounds polyphenols [Agatonovic-Kustrin, S., et al., Mod. Chem. Appl., 2013, 1, 110], in the spectral range 900-1160 cm ' ¹ (maxima of increased intensity, distinct especially in the spectrum of skin extract - 1043 and 1084 cm' ¹ - but also of the spectrum of the base-cream and cream with extract at -1060 cm ' ¹ ) spectral maxima may be associated with C-0 stretching vibrations of glycosidic moieties and to a lesser extent C-0 aromatic stretching vibrations [Westfall, A., et al., Antioxidants, 2020, 9, 486], The spectral range 1150-1400 cm ' ¹ shows multiple bands of variable intensity attributed to CO tensile vibrations and COH bending vibrations associated with phenols, esters, carboxylic acids and alcohols [Westfall, A., et al., Antioxidants, 2020, 9, 486; Coates, J., Interpretation of Infrared Spectra, A Practicai Approach, 2000, 10815]; Variables of variable intensity present mainly in the spectrum of skin extract and skin extract cream. The spectral maximum located at 1085 cm ' ¹ can be attributed to the aromatic CH deformation vibrations in the plane [Fernandez, K., et al., J. Agric. Food Chem., 2007, 55, 7294; Ping, L., et al., Ind Crops Prod., 2012, 40, 13; Jensen, JS, et al., J. Agric. Food Chem., 2008, 56, 3493] and C-0 deformation vibrations (secondary alcohols, aliphatic esters) [Turker-Kaya, S., et al., Molecules, 2017, 22, 168; Agatonovic-Kustrin, S., et al., Mod. Chem. Appl., 2013, 1, 110], OH and C-OH stretching vibrations (component polysaccharides of cell walls) can be associated with the maximum present at 1044 cm ' ¹ [Agatonovic-Kustrin, S., et al., Mod. Chem. Appl., 2013, 1, 110], The spectral maximum of average intensity from 877 cm ' ¹ can be associated

RO 135101 A0 A out-of-plane aromatic CH bending vibrations [dos Santos, GF, et al., Spectrochim. Acta A Mol. Biomol. Spectrosc., 2016, 153, 94] and CO (monosaccharide) stretching vibrations [Turker-Kaya, S., et al., Molecules, 2017, 22, 168] and CC [Oliveira, RN, et al., Matter ( Rio J.), 2016, 21, 769]. The spectra of the base cream and the cream with skin extract also show spectral maxima associated with cetyl alcohol located at ~ 1640, 1464 (CH ₂ bending vibrations associated with the aliphatic chain), 1060 (CO stretching vibrations) and 719 cm ' ¹ (vibrations OH stretching of alcohol groups) [Argimon, M., et al., J. Braz. Chem. Soc., 2016; Elmowafy, M., et al., J. Drug Deliv. Sci. TechnoL, 2018, 45, 230; Pasieczna-Patkowska and S., Oleinik, T., Ann. Univ. Mariae Curie-Sklodowska, AA-Chemia, 2013, 68, 95].

The FTIR spectrum of the seed extract provides a range of spectral information especially in the spectral ranges 3350-2900 cm ' ¹ and 1650-850 cm' ¹ . The broad, intense spectral band, centered at ~ 3330 cm ' ¹ can be attributed to the components of the extraction solvent and to the hydroxyl groups in the structure of the phenolic compounds (stretching vibrations of the hydroxyl groups). The maxima present at 2978 and 2901 cm ¹ can be associated with asymmetric CH stretching vibrations associated with the methyl and methine groups, respectively [daRocha, JC, et al., Int. J. Electrochem. Sci., 2012, 7, 11941; Brezoiu et al., 2019, Coates, J., Interpretation of Infrared Spectra, A Practice Approach, 2000, 10815], The spectral band from 1643 cm ' ¹ is associated with the aromatic character, CH stretching vibrations [Heredia-Guererro, JA, et al., FPLS, Plant Biophysics and Modeling, 2014, 5, 305; Lucarini, M., et al., Foods, 2020, 9, 10] and C = O conjugate tensile vibrations [daRocha, JC, et al., Int. J. Electrochem. Sci., 2012, 7, 11941], The footprint region 1500-800 cm ' ¹ shows numerous maxima of variable intensity associated with different modes of vibration. Although it is a spectral field rich in information, it is difficult to analyze due to its complexity; in this spectral range the maxima present may be associated with alcohols, sugars, organic acids, phenolic compounds [Lucarini, M., et al., Foods, 2020, 9, 10], in the region 1390-1310 cm ' ¹ (defined maxima present at 1373 and 1315 cm ' ¹ ) spectral bands can be associated with CO-H angular deformation vibrations in phenols [daRocha, JC, et al., Int. J. Electrochem. Sci., 2012, 7, 11941], out-of-plane methylene bending vibrations (polysaccharides, pectins) [Nogales-Bueno,

J., et al., Food Chem., 2017, 232, 602], methylene and C-0 shear vibrations [Lucarini, M., et al., Foods, 2020, 9, 10; Nogales-Bueno, J., et al., Food Chem., 2017, 232, 602] as well as the tensile vibrations of the pyran ring (carbohydrates) [Tociu, M., et al., UPB Scientific Bulletin B, 2019, 81 ]. The maximum of ~ 1161 cm ' ¹ is associated with ester C-0 stretching vibrations [Mohansrinivasan, V., et al., Braz. Arch. Biol. Technol., 2015, 58, 540; Canbay, HS, et al., SDU J. Science (E-Journal), 2011, 6, 140; Tociu, M., et al., UPB Scientific Bulletin B, 2019, 81] and aromatic CH [Nogales-Bueno, J., et al., Food Chem., 2017, 232, 602] attributed to phenolic compounds. High and medium intensity spectral bands from 1043 and 877 cm ' ^{1, respectively,} can be associated with CH deformation vibrations associated with the aromatic nucleus [daRocha, JC, et al., Int. J. Electrochem. Sci., 2012, 7, 11941] and aromatic CH bending vibrations, respectively [dos Santos, GF, et al., Spectrochim. Acta A Mol. Biomol. Spectrosc., 2016, 153, 94].

The FTIR spectra of the base cream (CB) and the seed extract cream (CES) show multiple similar spectral characteristics suggesting an advanced incorporation of the extract into the CB; the low proportion (7%) of the extract in the formulation also determines the overlap / combination of the intense maxima associated with the CB components (lanolin, cetyl alcohol, sodium lauryl sulfate) with the spectral maxima associated with the extract [Westfall, A., et al., Antioxidants, 2020, 9, 486; Coates, J., Interpretation of Infrared Spectra, A Practicai Approach, 2000, 10815]. Intense spectral band, centered at ~ 3340 cm ' ¹ present in both the CB and CES spectra can be attributed to OH stretching vibrations associated with the extraction solvent, cetyl alcohol and OH groups associated with the extract compounds, and intense maxima from 2915 and 2828 cm ' ¹ can be associated with asymmetric and symmetrical methylene tensile vibrations, respectively [Coates, J., Interpretation of Infrared Spectra, A Practice Approach, 2000, 10815], attributed mainly to lanolin, but also to sodium lauryl sulfate [SpectraBase, John Wiley & Sons Inc., Lanolin, 2021; SpectraBase, John Wiley & Sons Inc., Sodium Lauryl Sulfate, 2021]. in the spectral range 1800-1300 cm ^{1 the} spectra CB and CES present a series of spectral maxima of variable intensity, attributed especially to the vehicle (CB), but also to the seed extract, present at: 1736 cm ' ¹ (stretching vibrations associated with carbonyl groups - aldehyde, ester

- attributed to the compounds in the extract and lanolin) [SpectraBase, John Wiley & Sons Inc., Lanolin, 2021], -1638 cm ' ¹ (C = C tensile vibrations associated with phenolic compounds and the influence of their aromatic character) [Heredia-Guererro, JA, et al., FPLS. Plant Biophysics and Modeling, 2014, 5, 305; Coates, J., Interpretation of Infrared Spectra, A Practice Approach, 2000, 10815], 1464 cm ' ¹ (asymmetric methyl bending vibrations associated with the alkyl skeleton - cetyl alcohol, lanolin - conjugated with aromatic ring stretching vibrations - phenolic compounds from extract) [SpectraBase, John Wiley & Sons Inc., Lanolin, 2021; SpectraBase, John Wiley & Sons Inc., Cetyl alcohol, 2021; Coates, J., Interpretation of Infrared Spectra, A Practice Approach, 2000, 10815], 1375 cm ' ¹ (COH angular deformation vibrations from phenols conjugated with dimethyl / isomethyl stretching and bending vibrations) [SpectraBase, John Wiley & Sons Inc ., Lanolin, 2021; daRocha, JC, et al., Int. J. Electrochem. Sci., 2012, 7, 11941, Coates, J., Interpretation of Infrared Spectra, A Practicai Approach, 2000, 10815]. Low intensity spectral maxima from 1172 and 1060 cm ' ¹ may be associated with the overlap of maxima due to the extract (ester C-0 tensile vibrations and aromatic CH) and CB components (CC tensile vibrations of aliphatic chains) [SpectraBase, John Wiley & Sons Inc., Lanolin, 2021; SpectraBase, John Wiley & Sons Inc., Sodium Lauryl Sulfate, 2021; SpectraBase, John Wiley & Sons Inc., Cetyl alcohol, 2021],

The presence of resveratrol (cis, trans) and piceid (cis, trans) was investigated by HPLC-UV / DAD in the hydroalcoholic extracts of the skin / seeds of Vitis vinifera L. The chromatograms showed, for the analyzed extracts, at 285 nm, c / s -resveratrol, and at 306 nm, trans-resveratrol. The quantitative values, expressed as mean ± standard deviation (n = 3), were 3.65 ± 0.42 and 10.14 ± 1.02 pg / g s.u. c / s-resveratrol for the skin extracts, respectively Feteasca Neagră kernels, and for transresveratrol, the values were, respectively, of 2.92 + 0.01 and 1.72 ± 0.49 pg / g s.u. The values obtained for the glucoside stilbenoids, trans-piced, respectively c / s-piceid in the hydroalcoholic extracts from the skin / core are: 13.48 + 1.01 and 29.01 ± 2.71 pg / g s.u. (transpiced), respectively 3.01 ± 0.55 and 9.40 ± 0.51 pg / g s.u. (c / s-piced).

Antibacterial activity of extracts from the skin and seeds. The hydroalcoholic extracts, from grape skin / seeds (Vitis vinifera L.), the Fetească Neagră variety from ecological culture, showed antibacterial action against the tested Gram-positive strains. The differentiated antibacterial effect manifested by the skin / stone extract relative to the bacterial strains tested was evaluated by the diameter of inhibition of bacterial growth and determination of MIC (Figure 3). The diameter of the growth inhibition zone was within the limits of 8-16 mm, the values presented (Figure 3) representing the arithmetic mean of three determinations, including the diameter of the disc impregnated with extract (6 mm) related to the method of highlighting antibacterial activity. discs). The diameters of the areas of growth inhibition (mm) by the hydroalcoholic extract from the skin, in the case of the Fetească Neagră variety cultivated in an ecological system, varied in the order: (Lactococcus sp./CCB1, 0 ± 0,5)> (Lactobacillus sp./ CCB5, 15 ± 0.41)> (Bacillus sp./CCB3, 10 ± 1.41) = (Bacillus sp./CCB4, 10 ± 0.71) = (Leuconostoc sp./CCB7, 10 ± 0.82) > (Streptococcus sp./CCB6, 9 ± 0.82). The diameters of the areas of growth inhibition (mm) by the hydroalcoholic extract from the seeds, in the case of the Fetească Neagră variety cultivated in an ecological system, varied in the order: (Lactococcus sp.lCCBA, 16 ± 0.41)> (Streptococcus sp./CCB6 , 14 + 0.71)> (Bacillus sp./CCB3, 10 + 0.82) = (Leuconostoc sp./CCB7, 10 ± 0.71)> (Bacillus sp./CCB4, 8 ± 0.82) = (Lactobacillus sp./CCB5, 8 ± 0.71). The comparative analysis of the data shows that the hydroalcoholic extract obtained from the seeds showed a higher intensity of antibacterial activity (compared to the bacterial strains CCB1 and CCB6, for which the minimum inhibition concentration was significantly lower compared to the corresponding values of the skin extract, for the same strains tested) and a relatively wider spectrum (antibacterial activity manifested for all six bacterial strains tested), compared to the extract obtained from the skin (Figure 3).

The values of the minimum inhibition concentration of the extracts obtained from the grape skin varied between 820 and 4300 pg / mL, thus highlighting the antagonistic activity of the extracts compared to the growth of most of the tested bacterial strains. The MIC values of the extracts obtained from the grape seeds varied between 850 and 3600 pg / mL. Substances with antibacterial activity (relative to the strains tested) were present mainly in grape seeds, higher values of antibacterial activity (quantified as values of the diameter of inhibition of bacterial growth, MIC values and

RO 135101 AQ (spectrum of inhibition of bacterial growth), compared to the corresponding values recorded for hydroalcoholic extracts in the skin being correlated with the total content of polyphenolic compounds, the total content of flavonoids and antioxidant activity, clearly higher in the case of hydroalcoholic extracts from seeds. The standardization of hydroalcoholic extracts obtained from the skin and kernels of grapes and the knowledge of their MIC values, is of particular practical importance in order to propose applications for different fields, applications in which bacteriostatic and bactericidal activity to be scientifically substantiated in relation to a particular bacterial strain.

The antibacterial and anti-inflammatory potential of natural, plant-derived compounds has been reported in many studies. Thus, in vitro and in vivo research has shown that mixtures of essential oils, oleoresin, flavonoids, alkaloids, phenolic compounds, tannins, glycosides are effective in the treatment of acne due to their antimicrobial and anti-inflammatory activities [Azimi, H., et al., Phytotherapy, 2012, 83, 1306; Fisk, W.A. et al., Phytother. Res. 2014, 28, 1137], Emerging evidence indicates that essential oils derived from plants and / or their major compounds may be a plausible alternative treatment for acne, a prevalent skin disorder in both adolescents and adults. In this context, an antimicrobial combination eugenol / β-pinene / 2-phenoxyethanol / potassium sorbate was included in the formulation of an anti-acne cream base [Saviuc, C., et al., Int. J. Mol. Sci., 2017, 18, 175]. The bacterial strains proposed in this patent have the potential for use in pharmaceutical applications, given that B. subtilis is a ubiquitous organism, which is often found in the skin microbiome.

Antifungal activity of extracts from the skin and seeds. The hydroalcoholic extracts from the skin and seeds of grapes (Vitis vinifera L., Fetească Neagră variety) showed zero to moderate antifungal activity against Rhizopus spp., Penicillium spp. And Aspergillus spp. Tested. The diameters of the growth inhibition zones of the tested fungal strains were smaller compared to those that showed the same extracts, compared to the strains of bacteria isolated from natural environments, varying between (6 ± 0.42) and (8 ± 0, 51) mm. As there is no standardized antimicrobial screening protocol, the technique for determining antimicrobial activity has significantly influenced

RO 135101 ΑΟ ^ ή categorically the results. Thus, the disc diffusimetric method provided much better results compared to the well diffusimetric method. Highlighting the antimicrobial activity of grape extracts, in conjunction with their antioxidant activity, provides the necessary scientific basis for the isolation and purification of bioactive chemical compounds from grape extracts, compounds that could be used, without being limited to these applications, in therapy. , in order to solve the phenomenon of antibiotic resistance, as well as in the food industry, in order to replace chemical preservatives and prevent food spoilage, respectively in order to design nutraceuticals and health benefits.

Claims (4)

demand The product EMOLYING CREAM FOR DRY SKIN WITH SKIN / ITEM EXTRACT (ECOLOGICAL CULTURE VITIS VINIFERA L "BLACK GIRL VARIETY) is characterized in that: 1. The emollient cream for dry skin consists of a mixture of lanolin, cetyl alcohol, sodium lauryl sulfate (base cream) and hydroalcoholic extract obtained from the skin / kernel of organically grown grapes of Vitis vinifera L., Fetească Neagră variety , the percentage of extract used under the conditions of the claim being 7% (volume / mass). The hydroalcoholic extract of the skin / kernel of organically grown grapes of Vitis vinifera L., the Fetească Neagră variety, used in the claim, is characterized in that it is obtained by maceration for 24 hours (of which 3 hours with shaking magnetic), at room temperature (22-23 ° C), in a percentage of 4% dry mass / volume of hydroalcoholic solvent (50% v / v). Hydro-alcoholic grape skin extract of organically grown grapes of Vitis vinifera L., the Fetească Neagră variety used in the claim, is characterized in that it has a total content of polyphenolic compounds in the range of 2.50-3.50 mg-EAG / mL (EAG = gallic acid equivalent), a total flavonoid content in the range of 3.00-4.50 mg-EQt / mL (EQt = quercetin equivalent), an antioxidant activity in the range of 0.80-1.50 mg-EAA / mL (EEA = ascorbic acid equivalent), a moderate antimicrobial inhibitory action, in vitro, on selected Gram-positive bacteria, with corresponding values of the minimum inhibitory concentration in the range 820-4300 pg / mL, a low antifungal efficacy compared to the selected strains. Hydro-alcoholic grape seed extract of organically grown Vitis vinifera L., the Fetească Neagră variety used in the claim, is characterized in that it has a total content of polyphenolic compounds in the range of 4.50-7.50 mg / mL mg- EAG / mL (EAG = gallic acid equivalent), a total flavonoid content in the range of 4.20-7.70 mg-EQt / mL (EQt = quercetin equivalent), an antioxidant activity in the range of 10.00-12.50 mg -EAA / mL (EEA = ascorbic acid equivalent), a broad spectrum of action, in vitro, on selected Gram-positive bacteria, with corresponding values of the minimum inhibitory concentration in the range 850-4000 pg / mL, a selective and moderate antifungal efficacy against of the selected strains.