PREPARATION FOR PROTECTING AGAINST EXTRINSIC AND INTRINSIC SKIN AGING

Abstract

Disclosed is a cosmetic or dermatological preparation which comprises one or more extracts of Saxifraga oppositifolia (purple saxifrage) and/or one or more extracts of Soldanella alpina (alpine snowbell). The use of these extracts to combat extrinsic and intrinsic skin aging is likewise described.

Claims

1. A cosmetic or dermatological preparation, wherein the preparation is suitable for topical application to human skin and for cosmetic and/or dermatological use, is present as at least one of a dispersion, gel, aqueous and/or alcoholic solution, serum, oil, tincture, ointment, salve or wipe impregnation media and comprises, based on a total weight of the preparation, from 0.0001% to 15% by weight of (i) one or more extracts of Soldanella alpina, as well as one or more components selected from fats, oils, waxes, moisturizing and/or humectant substances, alcohols, polyols, polymers, electrolytes, organic solvents, silicones, foam stabilizers, substances for preventing foaming, dyes, coloring pigments, thickeners.

2. The preparation of claim 1, wherein the preparation further comprises from 0.0001% to 15% by weight of (ii) one or more extracts of Saxifraga oppositifolia.

3. The preparation of claim 2, wherein the preparation comprises from 0.0001% to 1% by weight of (i) and/or from 0.0001% to 1% by weight (ii).

4. The preparation of claim 1, wherein (i) comprises at least one water-soluble extract obtained by hydro-alcoholic extraction of plant material.

5. The preparation of claim 4, wherein the plant material comprises plant leaves and/or plant stems.

6. The preparation of claim 1, wherein (i) comprises at least one oil-soluble extract obtained by supercritical fluid extraction of plant material.

7. The preparation of claim 6, wherein the plant material comprises plant leaves and/or plant stems.

8. The preparation of claim 1, in which all of the one or more extracts (i) are water-soluble or all of the one or more extracts (i) are oil-soluble.

9. The preparation of claim 1, wherein the preparation further comprises (iii) one or more extracts of Chlamydocapsa sp.

10. The preparation of claim 2, wherein the preparation further comprises (iii) one or more extracts of Chlamydocapsa sp.

11. The preparation of claim 1, wherein the preparation further comprises one or more extracts selected from Artemisia umbelliformis extract, Rubus idaeus (raspberry) extract, Plantago lanceolata leaf extract, Saccharomyces cerevisiae extract, Equisetum arvense extract, Evernia furfuracea (tree moss) extract.

12. The preparation of claim 10, wherein the preparation further comprises one or more extracts selected from Artemisia umbelliformis extract, Rubus idaeus (raspberry) extract, Plantago lanceolata leaf extract, Saccharomyces cerevisiae extract, Equisetum arvense extract, Evernia furfuracea (tree moss) extract.

13. The preparation of claim 1, wherein the preparation is present as an oil.

14. The preparation of claim 1, wherein at least one extract of the one or more extracts (i) is oil-soluble, and the preparation comprises jojoba oil.

15. The preparation of claim 1, wherein at least one extract of the one or more extracts (i) is water-soluble, and the preparation comprises maltodextrin.

16. A cosmetic or dermatological preparation, wherein the preparation is suitable for topical application to human skin and for cosmetic and/or dermatological use, is present as at least one of a dispersion, gel, aqueous and/or alcoholic solution, serum, oil, tincture, ointment, salve or wipe impregnation media and comprises, based on a total weight of the preparation, from 0.0001% to 0.5% by weight of (i) one or more extracts of Soldanella alpina, as well as one or more components selected from fats, oils, waxes, moisturizing and/or humectant substances, alcohols, polyols, polymers, electrolytes, organic solvents, silicones, foam stabilizers, substances for preventing foaming, dyes, coloring pigments, thickeners, the one or more extracts of Soldanella alpina comprising at least one water-soluble extract obtained by hydro-alcoholic extraction of plant leaves and/or plant stems and/or comprising at least one oil-soluble extract obtained by supercritical fluid extraction of plant leaves and/or plant stems.

17. The preparation of claim 16, wherein the preparation further comprises from 0.0001% to 0.5% by weight of (ii) one or more extracts of Saxifraga oppositifolia, which one or more extracts comprise at least one water-soluble extract obtained by hydro-alcoholic extraction of plant leaves and/or plant stems and/or comprise at least one oil-soluble extract obtained by supercritical fluid extraction of plant leaves and/or plant stems.

18. The preparation of claim 17, wherein the preparation further comprises (iii) one or more extracts of Chlamydocapsa sp.

19. The preparation of claim 16, wherein the preparation is present as an oil.

20. The preparation of claim 16, wherein at least one of the one or more extracts (i) is oil-soluble and the preparation comprises jojoba oil and/or at least one of the one or more extracts (i) is water-soluble and the preparation comprises maltodextrin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] In the accompanying drawings:

[0080] FIGS. 1 and 2 are bar diagrams which graphically represent the results set forth below under the heading Mitochondrial function under oxidative stress;

[0081] FIGS. 3A-3C are bar diagrams which graphically represent the results set forth below under the heading Protection of mitochondrial DNA upon UV irradiation;

[0082] FIG. 4 is a bar diagram which graphically represents the results set forth below under the heading Delaying the aging process of human keratinocytes (intrinsic skin aging);

[0083] FIGS. 5 and 6 are bar diagrams which graphically represent the results set forth below under the heading Formation of epidermal skin following UV irradiation;

[0084] FIG. 7 is a bar diagram which graphically represents the results set forth below under the heading Activation of the proteasome in HaCaT cells following UV stress;

[0085] FIGS. 8A, 8B are bar diagrams which graphically represent the results set forth below under the heading Inhibiting the release of the matrix metalloproteinases MMP-1 and MMP-3 following UVA stress in human dermal fibroblasts;

[0086] FIGS. 9A, 9B are bar diagrams which graphically represent the results set forth below under the heading Colony formation by primary epidermal keratinocyte precursor cells following UV irradiation or treatment with hydrogen peroxide; and

[0087] FIG. 10 is a bar diagram which graphically represents the results set forth below under the heading Formation of spherical colonies by dermal precursor cells following UV irradiation.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0088] The particulars shown herein are by way of example and for purposes of illustrative discussion of the embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the present invention. In this regard, no attempt is made to show details of the present invention in more detail than is necessary for the fundamental understanding of the present invention, the description in combination with the drawings making apparent to those of skill in the art how the several forms of the present invention may be embodied in practice.

[0089] Hereinbelow, the advantageous properties of the preparation according to the invention are illustrated by reference to the results of various tests. In all of the tests, the aforementioned water-soluble extract products AlpinEffect Saxifraga WS and AlpinEffect Soldanella WS were used. The percentages refer to extract actually used (i.e. without the additives present in these products such as, for example, maltodextrin).

[0090] 1. Mitochondrial Function Under Oxidative Stress

[0091] It is known that mitochondria are responsible for generating energy in human cells. They are located in the cytoplasm and serve the cells as “batteries”, in order to produce, store and distribute energy. The human cell contains on average 1500 mitochondria. Cells with a high metabolic output (e.g. muscles or the liver) contain more mitochondria. The mitochondria move in the cytoplasm according to the demand of the cell. They are equipped with their own DNA and can therefore replicate independently irrespective of cell division. Without the mitochondria, the cell is incapable of functioning and no life is possible. If these powerhouses of the cells do not work correctly, this can accelerate aging processes in the skin. Defects in the mitochondria, in particular also in the mitochondrial DNA, can therefore accelerate aging.

[0092] Protection of the mitochondrial functionality against extrinsic disturbance factors, e.g. against UV radiation and oxidative stress, and/or the guarantee of the integrity of the mitochondria is therefore an effective protection against skin aging.

[0093] The following test shows that 10 mM hydrogen peroxide solution reduces the membrane potential of the mitochondria as a measure of healthy, metabolically active skin cells by about 50%. Hydrogen peroxide is therefore an example of extrinsic disturbance factors.

[0094] In the presence of both the Saxifraga and the Soldanella extract, the reduction in the membrane potential as a result of hydrogen peroxide is only about 25%, i.e. the extracts according to the invention protect the skin cells effectively against oxidative stress and therefore against extrinsic skin aging.

[0095] For carrying out the tests, primary human epidermal keratinocytes, more precisely “transit amplifying cells”, were used.

[0096] In order to analyze the influence of the extract on mitochondrial functionality following oxidative stress, a JC-1 mitochondria membrane potential assay was carried out. In healthy cells, the metabolically active mitochondrial potential is high and the dye JC-1 spontaneously forms complexes (J aggregates) and as a result displays an intensive red fluorescence. On the other hand, the dye JC-1 remains in the uncomplexed, monomeric form in damaged cells with a low membrane potential. The ratio of the fluorescence intensity of J aggregates to JC-1 monomers serves as a measure for the functionality of the cell.

[0097] The test results are shown graphically in FIGS. 1 and 2. These Figures show the effectiveness and protective function of the extracts according to the invention against extrinsic disturbance factors.

[0098] The concentration of Saxifraga oppositifolia used in the test was 0.00024% by weight and that of Soldanella alpina was 0.00024% by weight (based on the weight of the cell medium).

[0099] 2. Protection of Mitochondrial DNA Upon UV Irradiation

[0100] The following assay shows the protective effect of the extracts according to the invention as regards mitochondrial DNA upon UV irradiation.

[0101] Located on the mitochondrial DNA (mtDNA) are some, if not all, of the genes for the enzymes of the respiratory chain, as well as genes which are responsible for the structure and reproduction of the mitochondria. Damage to the mtDNA can arise very easily, it is present in unprotected form in the mitochondria and is exposed there to the free radicals which can be formed during the production of energy. Damage to the mtDNA can therefore lead to a severe impairment of cellular energy production.

[0102] One of the most frequent types of damage to the mtDNA, which is detected in the assay below, is referred to as “common deletion”.

[0103] HaCaT cells are cells of a specific human keratinocyte cell line. Cultivated keratinocytes (HaCaT) are incubated for 48 h with various compounds and then stressed for 1 h with UVB radiation (1.5 mJ/cm.sup.2). The cells are then collected and lyzed for the purposes of DNA extraction.

[0104] With the help of the intensity of the common deletion band, expressed as a ratio of the common deletion compared to standard, the protection against damage by UV light can be determined.

[0105] The following compounds were investigated:

TABLE-US-00001 % reduction in the DNA Fraction % damage induced by UVB Plant extracts by wt. (common deletion test) Figure Saxifraga oppositifolia 0.002%  −46% 3A Soldanella alpina 0.002%  −52% 3A Saxifraga + Saxifraga = −100% 3B Soldanella 0.0003%  Soldanella = 0.00036%  Saxifraga + Saxifraga =  −92% 3C Soldanella + snow 0.00033%  algae Soldanella = 0.0004%  snow algae = 0.0022% 

[0106] FIG. 3A shows that Soldanella alpina extract reduces the UV-induced damage of the mitochondrial DNA in keratinocytes (HaCaT cells) by 52% and Saxifraga oppositifolia extract by 46% relative to the control.

[0107] The combination of the extracts of Saxifraga oppositifolia and Soldanella alpina reduces the UV-induced damage by 100% (FIG. 3B). Even at a fraction of 0.0003%-0.00036% of extracts used according to the invention, a significant protection is observed which, as FIG. 3B shows, can be increased to 100% upon increasing the concentration and combining the extracts.

[0108] The combination with a further preferably used algae or plant extract, Chlamydomonas (Chlamydocapsa sp. (snow algae)), also leads to an almost 100% protection against damage induced by UV radiation (FIG. 3C).

[0109] 3. Protection of Cellular DNA Upon UV Irradiation

[0110] DNA damage leads to an impairment of cellular functions and ultimately to skin aging. UV radiation is the essential factor in the case of premature (extrinsic) skin aging.

[0111] 3D epidermal cultures were investigated using primary human keratinocytes and 3D prime culture medium. After 18 days, the cultures were treated for 1 hour with 100 μl either of a solution of Soldanella alpina extract in medium or medium on its own (untreated controls). In the case of an irradiation of 1200 mJ/cm.sup.2 (total UV), the samples were then exposed to 4 hours of UVB/UVA light (20%/80%). The samples were then fixed, embedded into paraffin, cut and stained with thymine dimers using a monoclonal antibody. Images were then recorded using a fluorescence microscope.

[0112] Result:

[0113] In the untreated control, no thymine dimers were observed. In UV-exposed controls, nuclear thymine dimers were clearly visible in the lower layers of the samples. In contrast to this, no dimers were visible in the cells treated with Soldanella alpina (0.1%) and UV-treated cells. This shows that the extract from Soldanella alpina prevents the formation of UV-induced DNA damage in the cells of an epidermal three-dimensional skin model and thus prevents extrinsic skin aging or photoaging.

[0114] 4. Delaying the Aging Process of Human Keratinocytes (Intrinsic Skin Aging)

[0115] Besides the protective function against extrinsic factors, the extracts used according to the invention also exhibit an effectiveness as regards delaying the aging process of human keratinocytes (intrinsic skin aging).

[0116] In order to demonstrate this, human keratinocytes in an early growth phase (transit amplifying cells) were incubated in a defined medium, in which these cells age within four weeks, with and without Soldanella extract and the proliferation rate of the cells was determined as a measure of their aging.

[0117] In order to assess the intrinsic anti-aging function of the extracts, the cells were cultivated in CnT-07 medium (progenitor cell targeted medium) or in CnT-AG1 medium, a completely defined aging medium in which the keratinocytes age over a period of four weeks. The signs of aging include a reduction in the proliferation rate. The proliferation was measured every week using an automated cell counter. During the first two weeks of aging, the proliferation rates of the keratinocytes which have been treated with plant extracts were similar to those of the cells growing in the CnT-AG1 medium alone. After an aging process of 3 to 4 weeks, however, the differences increased. In the 4.sup.th week, the proliferation rate of the cells with Soldanella alpina extract (0.00024%) compared to the CnT-AG1 control was more than 40% higher.

[0118] FIG. 4 shows that the proliferation of the cells in the aging medium in the presence of the Soldanella alpina extract after 2 and 4 weeks is higher than in the aging medium without extract. Since aged cells no longer replicate as quickly and thus have a lower proliferation rate than young cells, this shows that the intrinsic aging of the cells is slowed by the Soldanella alpina extract.

[0119] 5. Formation of Epidermal Skin Following UV Irradiation

[0120] The new formation of epidermal skin is also destroyed by UV irradiation.

[0121] The renewing of the epidermis is ensured by the epidermal stem cells and their direct descendants, the “transit amplifying cells”. If these cells lose their ability for skin renewal, be it through aging or as a result of external stress, e.g. UV radiation, then skin renewal in general slows and the quality, that is to say the thickness of the epidermis, reduces and signs of skin aging become visible.

[0122] Test Design:

[0123] An important indicator of the keratinocyte progenitor cells function is the possibility to form a coated, 3D epidermal structure. In order to assess the ability of the plant extracts used according to the invention to protect against the loss of the progenitor cells function, keratinocytes were left to grow to 80% confluence in culture medium and then treated for 24 hours with 0.002% Soldanella alpina extract or with 0.002% Saxifraga oppositifolia extract. The cells were then subjected to UVB/UVA light. Unilluminated samples served as a negative control. One hour after the UV treatment, the cells were sown out for the construction of 3D models using the standard protocol. On the 18.sup.th day of the airlift culture, 3D model samples were taken, embedded into formalin, fixed into paraffin, cut and stained. Histological cuts were then evaluated under the light microscope. Duplicate images were taken and for each position, the epidermal thickness (from the basal layer down to the horny layer) was measured. The result found was that both extracts used according to the invention conserve the skin renewal potential of human skin cells under UV stress. Without pretreatment of the skin cells with one of the two extracts, the cells lose their ability to form in vitro an intact three-dimensional epidermis if they are subjected to UV radiation. The extracts therefore protect the ability of the “transit amplifying cells” for skin renewal under UV stress. The epidermal thickness of the skin is retained, as shown in FIGS. 5 and 6.

[0124] 6. Activation of the Proteasome in HaCaT Cells Following UV Stress

[0125] In a further assay, the activation of the proteasome in HaCaT cells following UV stress was investigated.

[0126] The proteasome is a protein complex which degrades in the cytoplasm and in the cell core (in the case of eukaryotes) proteins that are defective and no longer required to give fragments. The proteasome is therefore a constituent of protein quality control.

[0127] UV stress inhibits the proteolytic activity of the proteasome, which leads to the accumulation of proteins that are defective and/or no longer required and ultimately impairs cellular functions. If defective or oxidized proteins aggregate, this can lead to the formation of the aging pigment lipofuscin.

[0128] FIG. 7 shows the results following the treatment of the proteasomes with the combination according to the invention of three plant extracts (Saxifraga oppositifolia, Soldanella alpina and Chlamydocapsa sp.): the activity of the proteasomes in HaCaT cells following UV irradiation (70 mJ/cm.sup.2 UVB+10 J/cm.sup.2 UVA) is again increased or reactivated, and specifically by 48%.

[0129] The concentrations (% by weight) of the extracts were: Saxifraga 0.00033%, Soldanella 0.00044% and Chlamydocapsa 0.0022%.

[0130] 7. Inhibition of the Release of the Matrix Metalloproteinases MMP-1 and MMP-3 Following UVA Stress in Human Dermal Fibroblasts

[0131] Further tests were carried out as regards the inhibition of the release of the matrix metalloproteinases MMP-1 and MMP-3 following UVA stress in human dermal fibroblasts.

[0132] Matrix metalloproteinases are proteases which degrade components of the extracellular matrix. UV light can stimulate the expression of matrix metalloproteinases.

[0133] MMP-1 is a collagenase, i.e. a collagen-degrading proteinase. Besides various collagens, MMP-3 also degrades fibronectin, elastin and laminin and plays a central role in the modulation of connective tissue and in wound healing.

[0134] A preparation according to the invention which comprises a combination of extracts of Saxifraga oppositifolia (0.00135%) and Soldanella alpina (0.0018%) inhibits the UV-induced expression of MMP-1 and MMP-3 significantly and as a result prevents light-induced skin aging, as is evident from FIGS. 8A and 8B.

[0135] 8. Colony Formation by Primary Epidermal Keratinocyte Precursor Cells Following UV Irradiation or Treatment with Hydrogen Peroxide

[0136] The combination according to the invention of the extracts of Saxifraga oppositifolia, Soldanella alpina and Chlamydomonas sp. protects the proliferation potential of epidermal stem cells under UV and oxidative stress, as measured by the ability of the cells to form colonies in vitro (Colony Forming Efficiency=CFE). For this, the following experiments were carried out:

[0137] UV Irradiation:

[0138] Primary epidermal keratinocyte precursor cells were sown out in the presence of the extract combination in CnT-07 culture medium and left to grow for 48 hours. Each sample is then subjected to an irradiation with a UVA and UVB light source (1200 mJ or 1800 mJ). A test with nonirradiated sample was likewise carried out. After the illumination, the cells for the CFE assays were sown out with a low density and cultivated. The cultures are then fixed and stained and the colonies were counted. The CFE evaluations were carried out three times. Untreated cells were used as control.

[0139] Hydrogen Peroxide:

[0140] Using a similar experimental design to that described for the UV irradiation, the CFE upon peroxide exposure was evaluated.

[0141] As the results depicted graphically in FIG. 9A show, the extract combination used according to the invention protects the proliferation potential of the epidermal stem cells significantly against UV radiation. For example, the number of colonies formed doubles in the presence of the extracts compared to the untreated control at 1200 mJ/cm.sup.2. Likewise, the extracts protect against the negative influences of peroxides (10 μM) on the proliferation ability of the epidermal stem cells (FIG. 9B).

[0142] 9. Formation of Spherical Colonies by Dermal Precursor Cells Following UV Irradiation

[0143] The plant extracts used according to the invention protect not only epidermal, but also dermal stem cells against the negative effects of UV radiation on their proliferation ability.

[0144] Dermal precursor cells (isolated from dermal papilla) were cultivated in monolayers in the presence or absence of the extracts for a period of 2 days and then irradiated with a UVA and UVB light source with a dose of 1200 mJ or 1800 mJ. The formation of spherical colonies was evaluated after about 5 days of the cultivation.

[0145] The proliferation ability of the dermal stem cells was ascertained by reference to their ability to form spherical colonies (spheres). Whereas in the control (without extracts) the number of spheres formed under UV irradiation (1200 mJ or 1800 mJ) dropped by 50% and 62%, respectively, the number of spheres in the presence of the extracts increased by up to 100% (FIG. 10).

[0146] Epidermal and dermal stem cells and their descendants are essentially involved in the routine maintenance and renewal of the corresponding skin layer. The protection of these cells against external stressors such as UV radiation or free radicals therefore contributes significantly to the retention of youthful, healthy and beautiful skin.

[0147] The following non-limiting examples are intended to further illustrate the present invention.

Example 1

[0148] Care Oil-Containing Preparation

TABLE-US-00002 INCI % by wt. PRUNUS AMYGDALUS DULCIS (SWEET ALMOND) OIL 91.0370 HELIANTHUS ANNUUS (SUNFLOWER) SEED OIL 4.0000 SIMMONDSIA CHINENSIS (JOJOBA) SEED OIL 1.1000 SILYBUM MARIANUM ETHYL ESTER 1.0000 FRAGRANCE (PERFUME) 1.5000 PHENOXYETHANOL 0.6000 CAPRYLIC/CAPRIC TRIGLYCERIDE 0.5000 TOCOPHERYL ACETATE 0.1000 C10-18 TRIGLYCERIDES 0.0995 BRASSICA CAMPESTRIS (RAPESEED) SEED OIL 0.0495 ALGAE EXTRACT 0.0100 SAXIFRAGA OPPOSITIFOLIA EXTRACT 0.0040 SOLDANELLA ALPINA EXTRACT 0.0016 100.0000

[0149] The preparation can advantageously be supplemented with further plant oils, oil-soluble vitamins and/or antioxidants.

Example 2

[0150] W/O Emulsion

TABLE-US-00003 INCI % by wt. WATER (AQUA) 42.400 GLYCERIN 15.170 CAPRYLIC/CAPRIC TRIGLYCERIDE 10.500 BUTYLENE GLYCOL 6.150 COCO-CAPRYLATE/CAPRATE 4.000 CYCLOPENTASILOXANE 3.500 DIMETHICONE 3.000 GLYCERYL STEARATE 2.000 CETYL ALCOHOL 2.000 MALTODEXTRIN 1.900 PEG-40 STEARATE 1.800 POTASSIUM CETYL PHOSPHATE 1.600 BUTYROSPERMUM PARKII (SHEA BUTTER) 1.500 PHENOXYETHANOL 0.900 SORBITAN TRISTEARATE 0.700 CARBOMER 0.500 FRAGRANCE (PERFUME) 0.350 CALCIUM ALUMINUM BOROSILICATE 0.338 ETHYLHEXYLGLYCERIN 0.300 SODIUM ACRYLATE/SODIUM 0.247 ACRYLOYLDIMETHYL TAURATE COPOLYMER PROPANEDIOL 0.200 SODIUM HYDROXIDE 0.176 ISOHEXADECANE 0.163 PENTYLENE GLYCOL 0.150 TITANIUM DIOXIDE 0.135 ISOMALT 0.092 SODIUM HYALURONATE 0.070 TRITICUM VULGARE (WHEAT) FLOUR LIPIDS 0.050 POLYSORBATE 80 0.046 PEG-8 0.025 SILICA 0.023 CHLAMYDOMONAS EXTRACT 0.006 SOLDANELLA ALPINA EXTRACT 0.004 SAXIFRAGA OPPOSITIFOLIA EXTRACT 0.003 CITRIC ACID 0.002 POTASSIUM SORBATE 0.001 100.000

[0151] This preparation can advantageously additionally comprise one or more of the following exemplary components:

[0152] Cellular Complex (La Prairie, Switzerland), peptides, plant extracts, extracts of plant stem cells, biopolymers, vitamins, antioxidants.

[0153] While the present invention has been described with reference to exemplary embodiments, it is understood that the words which have been used herein are words of description and illustration, rather than words of limitation. Changes may be made, within the purview of the appended claims, as presently stated and as amended, without departing from the scope and spirit of the present invention in its aspects. Although the present invention has been described herein with reference to particular means, materials and embodiments, the present invention is not intended to be limited to the particulars disclosed herein; rather, the present invention extends to all functionally equivalent structures, methods and uses, such as are within the scope of the appended claims.