RO135039A2 - Associations of a grape waste extract with plant extracts for preparing dermatocosmetic products - Google Patents

Abstract

The invention relates to a dermatocosmetic product with a superior biological effect for antioxidant protection against environmental aggression factors and skin recovery following chemical aggression and dehydration. According to the invention, the product consists, in mass percentage, of 1...8% pomace extract, 0.1...4% marigold extract, up to 5% chestnut extract, as well as cosmetic excipients for conditioning it as cream, gel/hydrogel or lotion.

Description

ASSOCIATIONS OF AN EXTRACT FROM GRAPE WASTE WITH EXTRACTS

FROM PLANTS FOR THE PREPARATION OF DERMATOCOSMETIC PRODUCTS

Technical field:

The present invention relates to associations of the active principles of the waste extract of Vitis vinifera with those of the plant extracts of the species Calendula officinalis and Aesculus hippocastanum, in variable proportions, and their use in the preparation of topical products applicable in skin regeneration and antioxidant protection. compared to external pollutants.

Known state of the art:

In the field of spectacular development of the dermatocosmetic field, a major objective aims to achieve therapeutic schemes and products with complex efficacy, based on mechanisms for regulating the functionality of skin tissue at the cellular and molecular level. Among the solutions with positive impact due to the efficiency / side effects ratio, the single use or combination of active principles such as saponins, flavones and isoflavones, anthocyanins, polyphenolicarboxylic acids from different plant species, with the maximization of biological action, is highlighted. The literature data provide information on dermatocosmetic applications of grape, marigold or chestnut extracts, as follows:

U.S. Patent No. 6,074,481 Bl "Process for the extraction, purification and concentration of polyphenolic compounds from whole grapes, seeds and pomace", describes a new process for obtaining a concentrated polyphenolic product used as an antioxidant in food, beverages and nutraceuticals in cosmetics.

Patent WO2005 / 036988 of the "Process for obtaining a grape seed extract with a low content of monomeric polyphenols" describes a process consisting in the separation of seeds and husks immediately after squeezing to avoid / stop the fermentation process in order to obtain a concentrated extract in monomeric polyphenols.

Patent EP 0096481 Al "Extraction and intensification of anthocyanins from grape pomace and other materials" describes a method of obtaining a colored product, which can be used as a dye in the food industry. The anthocyanin material is extracted with sulfur dioxide and treated enzymatically, then treated with acetaldehyde to enhance color.

U.S. Pat. No. 6,238,673 Bl. "Method for obtaining polyphenolic compositions with a high content in flavones" describes a process for obtaining a dry polyphenolic compound which involves separating the liquid grape extract with a non-oxidizing medium which ^ ^ tÎâÎî ^ from the separation medium cWith eluedf and / U recovery of the fraction containing polyphenols in proportion of 25% w / w and flavones in proportion of 2% w / w from the content of polyphenols.

Patent US8524293 Bl “Method for producing a composition similar to red grape tea”, describes a method of obtaining a nutraceutical beverage for human use (can be consumed as a tea), from a dry mixture of red grapes (husks, seeds , bunches) by natural fermentation. The invention consists in following the natural methods of processing grapes: crushing, fermentation, drying, boiling and steaming, completely excluding the use of any catalysts, chemical agents or artificial additives, with an antioxidant drink with many benefits on the human body.

Patent EP 2875822 of the "Extraction of polyphenols from white grape residue" refers to a method for obtaining extracts with antioxidant and antibacterial properties from white grape residues, with applicability in the cosmetic, pharmaceutical or food industry. Patent FR2902334A1 "Use of marigold extract for the preparation of a cosmetic and / or dermatological composition for topical application to combat the signs of skin aging", describes the use of marigold extract in formulations with applications in ameliorating the effects of photoaging by acting as a metalloproteinase inhibitor.

EP0848612B1 'Use of Calendula glycosides for the treatment of psoriasis' relates to compounds and plant extracts containing compounds indicated as having an inhibitory effect on cell proliferation. More specifically, the invention relates to glycosidic compounds derived from Calendula species, plant glycosides having a cytostatic effect on cells and their use as cytostatic agents, especially in the treatment of psoriasis.

US5997876A "Ointment for burns and wounds" refers to the use in the treatment of wounds and burns of an ointment containing extracts of Chelidonium majus; Plantago major; Chamomile matricaria; Achillea millefolium; Calendula officinalis; Hypericum perforatum; Eucalyptus globulus; Oleum olivarum and Cera flava.

Patent CN113O35OA / 1995, “Dermatological preparations”, describes an external topical preparation based on escinol containing one, two or more of its salts.

U.S. Patent 2010/0310689 The “Method for Producing a Chestnut Seed Extract” describes a method of obtaining an extract from the seeds of a plant belonging to the Hippocastanaceae family, such as Aesculus hippocastanum or Aesculus turbinate. As it is known, an extract of Hippocastanaceae or a triterpene saponin isolated from this extract (called escin), has excellent anti-inflammatory and astringent effects. Chestnut extract has also been used as a raw material for cosmetic compositions.

recent studies have shown that a chestnut extract has an effect of preventing or improving wrinkles or loss of skin elasticity.

WO 2008 / 093958A1 "Method for obtaining a wild chestnut leaf extract" describes the process for obtaining a chestnut extract with inhibitory activity on matrix metalloproteinases (MMP) and a composition containing the extract as an active ingredient in the treatment or prevention of diseases. dependent on MMP, including periodontal disease.

As presented, the waste resulting from the vinification process can be a convenient source of bioactive substances in terms of spread, simplicity of technological processes for obtaining active principles and capitalization of antioxidant and anti-inflammatory properties. Also, the extracts of marigold (Calendula off.) And chestnut (Aesculus hippocastanum) have therapeutic potential in skin diseases and are used singly or in combination with other plant extracts in the development of original products.

The present invention relates to the association of the active principles in grape waste (Vitis vinifera) - rich in anthocyanins, flavones and resveratrol with those of Calendula officinalis and Aesculus hippocastanum, in varying proportions, to enhance the antioxidant effect and correlative anti-inflammatory and skin regeneration action. The derived products according to the invention are conditioned by double or multiple combinations of the above-mentioned extracts in semi-solid cosmeceutical preparations: gel, ointment, creams, emulsions.

Most patents relating to skin care products refer to total plant extracts or therapeutic combinations with skin action in general, without the demonstration of specific molecular targets, or with unique targets, without ensuring a complex efficiency at this level. We highlight the need for configuration in the up-to-date approach to skin regeneration of associations with multiple targets of biological action, based on compounds with antioxidant, anti-inflammatory, cell restorative properties, for cell regeneration and structural recovery of extracellular proteins but also for homeostatic rebalancing of dermo-epidermal processes subjected to pro-oxidative stress. Simultaneous modulation of different related metabolic pathways creates an optimized efficacy spectrum and reduces the impact of adverse reactions.

The technical problem solved by the present invention consists in obtaining bioactive phytocomplexes with a well-characterized composition in terms of the ratio of active structures - therapeutic efficiency, in order to streamline applications in antioxidant, anti-inflammatory and skin regenerating therapies, impacting harmful factors from medium.

The extraction processes follow a well-defined content of flavonoids and polyphenols from the three associated plant sources (marigold - tescovina, respectively chestnut - tescovina or marigold - chestnut - tescovina), in proportions rigorously determined by specific tests, in vitro: multiple antioxidant profile highlighted on standardized keratinocyte and fibroblast lines and potential for dermo-epidermal regeneration through protein matrix homeostasis and cell tum-over stimulation.

Disclosure of the invention:

According to the invention, the pomace extract is obtained by pressing the grapes under conditions of controlled temperature (20-25 ° C) and constant pressure, maceration - fermentation with control of sugars and temperature (22-28 ° C) for 7-10 days, pressing with the help of the pneumatic press at a maximum pressure of 1.2 bar and separation of the pomace as a by-product of vinification. The subsequent stages of the technological flow refer to vacuum drying, crushing and water solubilization. Grape waste extract is obtained (Vi tis viniferay rich in flavonoids (0.133 g / 100 g extract), polyphenols, including resveratrol (0.45 g / 100 g extract). It is combined in skin tissue regimens with propylene glycol extracts of marigold and chestnut, with a content of flavonoids (0.119g / 100g marigold extract; 0.235g / 100g chestnut extract), polyphenols (0.29g / 100g marigold extract; 0.53g / 100g chestnut extract).

The derived products, according to the invention, are obtained by conditioning the combinations of plant extracts, by double or multiple combinations, in various cosmeceutical forms: gel, emulsion, creams, lotions.

Relevant tests for specific biological activity and targeting dermato-cosmetic applicability:

Tescovina extract (abbreviated TES) is associated with Calendula officinalis extract (marigold - abbreviated Gb) and Aesculus hippocastanum (chestnut - abbreviated Cs). The combinations of the three tested plant extracts were made as follows:

the BL BUT A2 B2 C2 Thess 1 1 9 1 1 9 Gb 9 1 1 Cs 9 1 1

A. Multiple antioxidant profile highlighted on standardized keratinocyte and fibroblast lines

A.l. The antioxidant / antiradical action in the acellular system was determined by ·

give spectrophotometry that monitors the reduction of the DPPH radical

picrylhydrazyl). Antiradical activity (AAR) has been defined as the amount of antioxidant required to decrease the initial concentration of DPPH * by 50% and represents the effective concentration, EC50.

Evaluation of the antiradical effect of the tested active principles

P roba ES b S A1_T ES: Gb (1: 9) B1_T ES: Gb (1: 1) C1_T ES: Gb (9: 1) A2_ TES: Cs 1: 9) B2_T ES: Cs (1: 1) C2_ TES: Cs (9: 1) E C50 (nD .98 .67 .24 1.57 2.34 3.87 3.29 4.08 5.0 3

The most pronounced antiradical effect is noted for the combinations Al_TES: Gb (1: 9) and Bl_TES: Gb (1: 1), demonstrating a significant redox potential.

A.2. Evaluation of antioxidant activity in the dermo-epidermal system

Stimulation of the antioxidant system in order to induce cellular protection against pro-oxidant aggression was studied on both types of dermo-epidermal cells, under conditions of pro-inflammatory stimulation associated with bacterial invasion (LPS) and oxidative stimulation associated with inflammation (PMA). Evaluation of intracellular oxidative stress was performed by flow cytometry analysis methods aimed at monitoring reactive oxygen species (hydrogen peroxide and superoxide anion-labeling with DCFH-DA, respectively HE) and determination of intracellular glutathione. The determination of the antioxidant impact of the extracts was also performed by determining the enzymatic activity of two oxidative enzymes of phase I, respectively catalase (reaction of decomposition of hydrogen peroxide in water and 02) and superoxide dismutase (monitoring the inhibition of cytochrome c reduction process by the superoxide radical ).

The active principles studied individually or in combination intervene on the activity of oxidative enzymes by maintaining the cellular redox balance, thus counteracting the attack of free radicals, both by eliminating superoxide anions under the action of SOD and CAT-catalyzed hydrogen peroxide. An activating effect on SOD, have the combinations of extracts Bl (71248%), C1 (7I55%), B2 (7i57%) and C2 (7i209%). Mixtures of Al (7U01%), B1 (7I2O6%), A2 (7i30%) and B2 (7i214%) catalyze the decomposition of H2O2 (by CAT activation), having a multiple oxidative-reducing role at the enzymatic level.

Individually studied plant extracts, namely TES, Gb and Cs, reduce intracellular hydrogen peroxide (| 21%, 114%, 135%) in keratinocytes in the presence of LPS and PMA stimuli. Mixtures B1, Cl, A2 and B2 have a reducing effect on hydrogen peroxide with superoxide anion accumulation.

The antioxidant compounds from the three studied plant extracts intervene in _x regulation of intracellular redox at the dermal level (normally stimulated human fibroblast $ ^ - ^^

pro-inflammatory with bacterial polysaccharide) as follows: chestnut extract, marigold and grape waste extract reduce especially hydrogen peroxide, without major impact at the level of superoxide anion; combinations A2, more than Al and B2 more than B1 have a concerted effect on both hydrogen peroxide and the intracellular superoxide anion, while combination C2 acts mainly on hydrogen peroxide.

There is a stimulation of intracellular glutathione in keratinocytes treated with TES and Gb extracts. Among the mixtures of studied active principles, a significant effect in terms of stimulating intracellular glutathione in stimulated keratinocytes, are A 1 (^ 32%), Bl (725%), A2 (780%), B2 (773%) and C2 (/ and 17%), being effective in combating the intrinsic oxidative stress produced by the inflammation associated with bacterial infections. There is a potentiating effect of intracellular glutathione in the case of fibroblasts stimulated and treated with chestnut extract (767%), TES extract (746%), A2 (738%), B2 (755%) and CI (758%), thus manifesting a strong intrinsic antioxidant effect.

B. Potential for dermo-epidermal regeneration through homeostasis of the protein matrix and stimulation of cellular tum-over.

B.l. Evaluation of the activity of MMP 2 and 9 secreted by keratinocytes in the extracellular culture medium:

Evaluation of metalloproteinase activity was performed from growth medium collected after treatment of normal human keratinocytes and normal human fibroblasts, cultured as follows: 24h adhesion, 48h treatment under normal conditions of development in the presence and absence of investigated compounds, as well as in a nonspecific inflammation model induced by treatment with LPS and PMA.

Method for estimating the concentration of gelatinases (MMP2 and MMP9) in the conditioned environment based on the ability of these enzymes to regenerate after electrophoretic migration in gelatin copolymerized polyacrylamide-SDS gels and removal of SDS by repeated washings with Triton X- 100, the enzymes thus exerting their proteolytic activity on the copolymerized substrate during 18 h of incubation at 37 ° C in a development buffer.

As in the case of epidermal cells treated with single bioactive extracts, the enzymatic activity of MMP 9 and MMP 2 is inhibited in the case of keratinocytes treated with the mixture of TES and Gb in a ratio of 1: 1 - (Bl) and 9: 1 - (CI) , in all three stimulation conditions. Also, the same inhibitory effect is noticed in the case of keratinocytes stimulated proifol ^^ tter.

and bacterial and treated mixture TES: Cs in a ratio of 1: 9 (C2). The results obtained the protective role of the active principles tested individually or in combination, against the degradation of extracellular matrix proteins, contributing to maintaining the integrity of skin tissue, in conditions of an inflammatory response associated with bacterial attack.

following the evaluation of the enzymatic activity of matrix metalloproteinases secreted in the extracellular culture medium by dermal fibroblasts in the presence of bioactive extracts, TES and Cs extracts are noted that reduce MMP9 activity and their association in a 1: 1 ratio (B2), induces a pronounced inhibitory effect on the two matrix enzymes involved in the degradation of structural components in the dermis. The association of the active principles from the TES and Gb extracts in a ratio of 1: 9 (Al) generates in the pro-inflammatory and bacterially stimulated fibroblasts a decrease of the enzymatic activity of the matrix metalloproteinases.

From the analysis of the experimental data, significant changes of collagen biosynthesis are observed after 48 hours of treatment, and among the studied compounds are TES and Gb extracts, which generate an increase of 30% and 28% of intracellular collagen in LPS-stimulated fibroblasts and PMA. In the case of fibroblasts treated with the mixture between the two extracts, TES and Gb in a ratio of 1: 9 - Al, an increase in the amount of collagen of up to 50% is observed. At the level of fibroblasts treated with TES and Cs extracts associated in a ratio of 1: 9 A2, respectively 1: 1, there is a considerable increase in intracellular collagen (105% - A2 and 57% - B2), thus demonstrating their role in maintaining tissue homeostasis. cutaneous and in the prevention of degenerative processes.

Considering the presented results, as well as the potentiation of the effectiveness of the complementary active principles from the three extracts in skin regeneration, the following variants of combinations of active principles were selected:

• Cl_TES: Gb (9: 1) - stimulates intrinsic and extrinsic antioxidant protection by activating intracellular glutathione and reducing oxygen free radicals; inhibits epidermal degradative processes • B2_TES: Cs (1: 1) - activates intracellular antioxidant defense enzymes, catalase and superoxide dismutase, reducing oxygen free radicals.

• TES mixture: Gb: Cs (9: 1: 9) - antioxidant and dermo-restitutive

The advantages of the invention in relation to the prior art are the following:

- extraction processes of simple active components / extracts, with superior technological yields, in mild but strictly controlled conditions, with solvents from the category of “green chemistry”;

- Innovative combinations with proven potentiating effect for maximized therapeutic efficacy in skin regeneration through multicomponent action in complementary antioxidant, anti-inflammatory and cell recovery mechanisms.

The products obtained according to the invention consist in the association of the pomace extract obtained according to the technology described with other types of marigold and chestnut preparations in well defined proportions, for a superior biological effect, cumulative, synergy of the active principles contained.

Embodiment no. 1: Dermatocosmetic product, containing as biologically active ingredients tescovina extract 1.00- ^ 8.00% and marigold extract 0.10- ^ 4.00% processions of excipients for cosmetic use, conditioned in the form of cream, gel / hydrogel, lotion, including in the variant that contains natural / organic ingredients, usable in antioxidant protection against skin aggression factors in the environment - anti-pollution product.

Embodiment no. 2: Dermatocosmetic product, containing as biologically active ingredients tescovina extract 1.00 ^ -8.00%, associated with marigold extract 0.10 ^ -4.00% and chestnut extract 0.3- ^ 5.00% together with excipients for cosmetic use, conditioned in the form of cream, gel / hydrogel, lotion, including the version containing natural / organic ingredients, usable in skin recovery after chemical aggression (detergents, etc.) and dehydration, etc.

BIBLIOGRAPHY

US 6544581 Bl, Process for extraction, purification and enrichment of polyphenolic substances from whole grapes, grape seeds and grape pomace (2003).

WO 2005/036988 Al, Process for producing a grape seed extract having a low content of monomeric polyphenols, (2005).

EP 0096481 Al, Extraction and intensification of anthocyanins from grape pomace and other material (1983)

US 6,238,673 Bl, Method of producing high flavonol content polyphenol compositions (2001)

US8524293 Bl, Method for producing ared grape tea-like composition (2013)

EP 2875822 Al, Polyphenolextrakt aus weisser traubenmaische (2012)

FR2902334A1, Use of a souei extract in a cosmetic composition (2007)

EP0848612B1, Use of calendula glycosides for the treatment of psoriasis (2003)

US5997876A, Bum and wound ointment (1997)

CN1130350A / 1995, Dermatologic preparation

US 2010/0310689 Al, Method for Producing Hippocastanaceae Plant Seed Extract (2 (102 ^^^

WO 2008 / 093958A1, Method of producing horse chestnut leaf extract (2007)

Claims (3)

demand 1. Combination of plant extracts characterized in that it combines 1 ... 9 parts of pomegranate extract with 1 ... 9 parts of marigold extract and / or 1 ... 9 parts of chestnut extract, to induce an antiradical effect and significant redox potential, namely the stimulation of intrinsic and extrinsic antioxidant protection by activating intracellular glutathione and reducing oxygen free radicals, especially in conditions of bacterial attack and associated "in situ" inflammation. Dermatocosmetic products characterized in that the combination of extracts defined according to claim 1, consists in the combination of 1 ... 9 parts of pomegranate extract with 1 ... 9 parts of marigold extract alone or in combination with other bioactive ingredients, excipient groups for cosmetic use, conditioned in the form of cream, gel or lotions, intended for all skin types, applicable in antioxidant protection, as a prevention of the aggression of harmful environmental factors: temperature fluctuations, UV radiation, etc. Dermatocosmetic products characterized in that the combination of extracts defined according to claim 1, consists in combining 1 ... 9 parts of pomegranate extract with 1 ... 9 parts of marigold extract and 1 ... 9 parts of chestnut extract, alone or in combination with other bioactive ingredients, packs of cosmetic excipients, conditioned in the form of cream, gel or lotions, intended for skin regeneration, being applicable in skin recovery following chemical aggression (detergents, solvents, cleaning chemicals) and dehydration.