Skin care active ingredient combination

Abstract

The invention relates to an active ingredient combination.

Description

[0001] Skin-care active ingredient combination

[0002] Cosmetic products generally serve not only to make one look beautiful and attractive, but also contribute significantly to increased self-esteem and well-being. Accordingly, a wide variety of cosmetic products are used for the daily cleansing and care of human skin.

[0003] Skin aging refers to the complex biological process of age-related changes in the skin. A distinction is made between intrinsic skin aging, caused by internal physiological and genetic factors, and extrinsic skin aging.

[0004] Extrinsic skin aging is caused by external factors such as environmental factors like UV light, chemical agents, mechanical stress, cigarette smoke, stress, or air pollution. Since UV radiation is the main cause of extrinsic skin aging, it is also referred to as "photoaging."

[0005] Extrinsic factors lead, for example, to wrinkles, sagging skin, loss of elasticity, and a dry appearance of the skin.

[0006] Intrinsic skin aging, also known as chronological skin aging, is caused by internal physiological and genetic factors and reflects degradation processes in the skin. These processes are mainly due to reduced proliferative activity of skin cells, reduced synthesis of matrix proteins, and an increase in the expression of matrix-degrading enzymes.

[0007] Aged cells exhibit resistance to apoptotic signals, leading to the accumulation in tissue of non-proliferating aged cells with altered gene expression patterns.

[0008] Skin aging often leads to the formation of wrinkles and lines, as well as a loss of elasticity and firmness.

[0009] P193695CH00 Skin aging and wrinkle formation can be significantly delayed through appropriate skin protection. Numerous options are available in the current state of the art, ranging from a healthy lifestyle to topically applied cosmetic and dermatological preparations.

[0010] US patent 584030 describes the combination of a) Glycoprotein 1, b) Glycoprotein 2, c) Ginseng extract, and d) Horsetail extract for stimulating the proliferation of fibroblasts and keratinocytes. It is desirable to provide preparations that are effective against extrinsic skin aging.

[0011] Gene expression refers to the formation of a gene product encoded by a gene, primarily proteins or RNA molecules. Gene expression consists of several individual processes. These include transcription, splicing, translation, and post-translational modification, as well as their regulatory mechanisms.

[0012] A disadvantage is that aged cells may exhibit insufficient gene expression. The following describes some genes whose gene expression can be impaired with age. The official gene symbols, as they appear in publicly accessible databases, are used below. See, for example, the database of the National Library of Medicine, accessible at www.ncbi.nlm.nih.gov / gene /

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[0020] Surprisingly, it has now been found that the expression of the above-mentioned genes could be brought back to comparable levels after cell aging by the present invention.

[0021] The invention is a combination of active ingredients comprising a) Paeonia Albiflora Root Extract, b) Calendula Officinalis Flower Extract, c) at least one glycoprotein 1, obtainable from the purified cytoplasmic fraction of yeasts (Saccharomyces), d) at least one glycoprotein 2, obtainable from the purified cytoplasmic fraction of Lactobacillus, e) ginseng extract, and f) horsetail extract (Equisetum Arvense extract).

[0022] The combination of components c), d), e) and f) is hereinafter referred to as GPVE extract.

[0023] The extract referred to therein as GP extract, comprising c), can be made from an aqueous cell extract of a purified cytoplasmic fraction of a natural, selected strain of the

[0024] P193695CH00 Yeast Saccharomyces cerevisiae are produced biotechnologically. The extract contains glycoproteins and a variety of cellular nutrients and factors.

[0025] The extract referred to as VE extract comprises d), e) and f) and can be obtained by a multi-stage biotechnological process using Lactobacillus casei.

[0026] Lactobacillus casei, which are particularly rich in lytic enzymes, are transferred to a fermentation culture medium where they can grow. This medium also contains protein-rich green microalgae. Under specific fermentation conditions, these lactobacilli are allowed to grow exponentially, producing enzymes that are released into the medium. These enzymes attack and rupture the cell walls of the algae, releasing their nutrient-rich cytoplasm into the medium.

[0027] As the fermentation process progresses, the nutrients in the medium are consumed, and when nutrients become scarce, their growth ceases and the lactobacilli eventually break down through autolysis. The cell walls open, and the cell contents, primarily proteins, enzymes, vitamins, etc., are released into the medium.

[0028] At this stage, the process is stopped, the entire medium is "harvested," algal residues are removed, and it is filtered. The resulting filtrate represents the complete cellular nutrient system (CNS) and is called Lactobacillus ferment.

[0029] This is supplemented with extracts of horsetail e) and ginseng root f) to form the final VE extract.

[0030] Glycoprotein 1, for example, is commercially available from DSM, Switzerland, and comprises a purified cytoplasmic fraction of Saccharomyces (SACCHAROMYCES CEREVISIAE EXTRACT). It consists of a mixture of amino acids, nucleic acids, nucleotides, carbohydrates, lipids, trace elements, vitamins, and phosphatase enzymes.

[0031] Glycoprotein 2, for example, is commercially available from Sederma, France, and comprises a purified cytoplasmic fraction from Lactobacillus, including a mixture of amino acids, nucleic acids, nucleotides, carbohydrates, lipids, trace elements, vitamins and phosphatase enzymes (e.g. LACTOBACILLUS FERMENT).

[0032] P193695CH00 Ginseng extract is obtained, for example, by extraction with a hydrophilic solvent (especially water, ethanol, glycol, or any mixture thereof) of the root of Panax ginseng. It contains saponins, sterols, carbohydrates, pectin, vitamins, minerals, and lipids (e.g., PANAX GINSENG ROOT EXTRACT).

[0033] Horsetail extract is obtained, for example, by extracting the whole Equisetum arvense plant with a hydrophilic solvent (e.g., water, ethanol, glycol, or any mixture thereof). It contains silicates, flavonoids, saponins, caffeic acid, and ferulic acid (e.g., EQUISETUM ARVENSE EXTRACT).

[0034] Paeonia albiflora root extract is an extract of the root of the milk-flowered peony. Such extracts can be obtained, among other places, under the trade name VOLUNAGE from the company Silab.

[0035] Calendula Officinalis Flower Extract is an extract of the marigold (Calendula officinalis) and can be obtained under the trade name EPIGENOMYL from the company Silab.

[0036] With regard to the overall combination of active ingredients, it is advantageous if the proportion of Paeonia Albiflora Root Extract is 20 wt.% to 40 wt.%, preferably 22 wt.% to 36 wt.%.

[0037] With regard to the overall combination of active ingredients, it is advantageous if the proportion of Calendula Officinalis Flower Extract is from 15 wt.% to 50 wt.%, preferably from 20 wt.% to 47 wt.%.

[0038] With regard to the overall combination of active ingredients, it is advantageous if the proportion of c) Glycoprotein 1 is from 1 wt.% to 10 wt.%, preferably from 2 wt.% to 7 wt.%.

[0039] With regard to the overall combination of active ingredients, it is advantageous if the proportion of d) Glycoprotein 2 is from 2 wt.% to 20 wt.%, preferably from 6 wt.% to 15 wt.%.

[0040] With regard to the overall combination of active ingredients, it is advantageous if the proportion of ginseng extract is from 1 wt.% to 10 wt.%, preferably from 2 wt.% to 7 wt.%.

[0041] P193695CH00 With regard to the overall combination of active ingredients, it is advantageous if the proportion of horsetail extract is from 1 wt.% to 10 wt.%, preferably from 2 wt.% to 7 wt.%.

[0042] Another object of the invention is the use of the combination of active ingredients in cosmetic preparations.

[0043] Another aspect of the invention is the use of the active ingredient combination to restore the expression rates of genes in aged cells compared to non-aged cells.

[0044] Another aspect of the invention is the use of the active ingredient combination for the rejuvenation of aged cells.

[0045] The proportion of extracts according to the invention is defined as the ratio of the mass of the pure extract, without solvents or extraction agents, to the total mass of the preparation.

[0046] Another object of the invention is a cosmetic or dermatological preparation comprising the active ingredient combination according to the invention.

[0047] Advantageously, the total proportion of the active ingredient combination in the described preparation is from 0.001 wt.% to 5 wt.%, preferably from 0.01 wt.% to 2.5 wt.% and particularly preferably from 0.03 wt.% to 1.0 wt.%, based on the total weight of the preparation.

[0048] Where weight percentages (wt%) are given below without reference to a specific composition or mixture, these percentages always refer to the total weight of the preparation. Where ratios of components / substances / groups of substances are disclosed below, these ratios refer to the weight ratios of the components / substances / groups of substances mentioned.

[0049] Where weight percentage ranges for the components of the cosmetic emulsion are specified below, the disclosure of the present application also includes all individual values ​​in steps of 0.1 wt.% within these weight percentage ranges.

[0050] P193695CH00 The terms “according to the invention”, “advantageous according to the invention”, “advantageous in the sense of the present invention”, etc., always refer, within the scope of the present disclosure, to both the preparation according to the invention and the use according to the invention.

[0051] Unless otherwise stated, all tests were conducted under standard conditions. "Standard conditions" means 20°C, 1013 hPa, and a relative humidity of 50%.

[0052] When the term skin is used, it preferably refers to human skin.

[0053] Any viscosity values ​​given in this disclosure refer to measurements taken at 25°C in a 150 ml wide-mouth bottle (VWR No.: 807-001) using the Rheomat R 123 from proRheo. The Rheomat R 123 from proRheo GmbH is a rotational viscometer, meaning that a measuring element rotates within the substance being measured. The force required to rotate the measuring element within the sample at a predetermined speed is measured. The viscosity is then calculated from this torque, the rotational speed of the measuring element, and the geometric dimensions of the measuring system. The measuring element used is No. 2 (article no. 200 0192), with a rotational speed range of 62.5 min⁻¹.

[0054] The preparations according to the invention can be in known forms and types. One known form of the preparations is leave-on preparations, such as creams, lotions, or body milks. These are frequently formulated as emulsions, in particular W / O, O / W, O / W / O, or W / O / W emulsions. The preparations can also be dispersions, gels, aqueous or alcoholic solutions, serums, oils, wipe impregnation media, tinctures, or ointments. The extracts can also advantageously be applied to the skin in the form of, or integrated into, wipes, plasters, bandages, patches, or pads.

[0055] It is advantageous if the combination of active ingredients is used in combination with other components.

[0056] According to the invention, it is advantageous if phenoxyethanol is additionally included. It is also advantageous according to the invention if ethylhexylglycerin is additionally included. It is also advantageous according to the invention if glycerin is additionally included. It is also advantageous according to the invention if sodium dehydroacetate is additionally included.

[0057] P193695CH00. It is also advantageous according to the invention if potassium sorbate is additionally included. It is also advantageous according to the invention if glycoproteins are additionally included. Last but not least, it is advantageous if water is included as a cosmetic carrier.

[0058] Comparative experiments and examples

[0059] The following examples are intended to illustrate the present invention without limiting it. Unless otherwise stated, all quantities, proportions, and percentages are based on the weight and total quantity or total weight of the preparations.

[0060] To illustrate the advantageous effect of the invention, the following preparations were provided:

[0061] The preparations were investigated in a study regarding their effects on gene expression following artificial cell aging. The experimental procedures are described below:

[0062] 1. Preparation of a keratinocyte cell bank

[0063] Human epidermal progenitor keratinocytes (HPEKs) from 3 healthy donors (aged between 18 and 30 years and tested negative for HIV, HCV, HBV and COVID) were cultured in CB-TAK-GM (keratinocyte growth medium, serum,

[0064] P193695CH00 (animal components and non-physiological factor-free medium). The cells were stored, their growth was subjected to quality control, and they were used for further in vitro experiments.

[0065] 2. Growth screening / cytotoxicity test

[0066] HPEKs were used to perform growth inhibition / cytotoxicity assays and to determine subtoxic concentrations. Cells were expanded, counted, and seeded into 96-well microplates and incubated overnight in an actively humidified biomedical CO2 incubator at 37 °C and 5% CO2.

[0067] The following day, all 7 test samples were diluted to a broad concentration range, and the cells were cultured for 2 days, either in the presence or absence of test samples. The cells were incubated with WST-8 solution, and the optical density (ODs) was recorded using a multimode plate reader.

[0068] All replicates were performed in triplicate and the data were expressed as a percentage of the untreated control with mean + / - SEM.

[0069] Cell types: HPEKs

[0070] 3. In-vitro aging of HPEKs

[0071] Aging in the metabolomics medium

[0072] Protocol:

[0073] • Stored HPEKs from early passages were expanded, counted and seeded for one day in 75 cm2 flasks in normal keratinocyte growth medium (CB-TAK-GM) to allow the cells to attach.

[0074] • The growth medium was replaced with or without a comparison sample / combination of active ingredients against metabolomic medium (CB-TAK-MM).

[0075] • The cells were cultured for 5 days. Every other day, the cells were exchanged for fresh CB-TAK-MM, with or without a control sample / drug combination.

[0076] • The cells were detached, counted in an automated cell counter (LUNA-II™, Logos Biosystems, Korea) and re-seeded for a second passage in 75 cm2 flasks in CB-TAK-MM without a control / drug combination.

[0077] P193695CH00 • The following day, the cells were exchanged for fresh metabolomic medium (CB-TAK-MM) and cultured for a further 5 days with or without a control sample / drug combination, with the cells being exchanged for fresh medium every other day.

[0078] • The cells were (de)passaged for passage 3 according to the steps described above.

[0079] • The cells were separated, centrifuged in a 15 ml Falcon tube, the supernatant was discarded, and the cells were lysed in an RNA extraction lysis buffer (Bio-Rad) and stored in a freezer at -80 °C (Thermo Scientific TSC series) until the RNA was extracted.

[0080] 4. Cultivating HPEKs in normal growth medium

[0081] Protocol:

[0082] • Stored HPEKs from early passages were expanded, counted and seeded for one day in 75 cm2 flasks in normal keratinocyte growth medium (CB-TAK-GM) to allow the cells to attach.

[0083] • The following day, the cells were replaced with fresh normal medium, either with or without a control sample / drug combination.

[0084] • The cells were cultured for 5 days. Every other day, the cells were replaced with fresh CB-TAK-GM, either with or without extracts.

[0085] • The cells were detached, counted in an automatic cell counter (LUNA-II™, Logos Biosystems, Korea) and re-seeded for a second passage in 75 cm2 flasks in CB-TAK-GM without a control / drug combination for 1 day to allow the cells to adhere to the culture dishes.

[0086] • The following day, the cells were replaced with fresh medium (CB-TAK-GM) and cultured for another 5 days with or without a control sample / drug combination.

[0087] • The cells were passaged for passage 3 according to steps de as described above.

[0088] • The cells were separated, centrifuged in a 15 ml Falcon tube, the supernatant was discarded, and the cells were lysed in an RNA extraction lysis buffer (Bio-Rad) and stored in a freezer at -80 °C (Thermo Scientific TSC series) until the RNA was extracted.

[0089] P193695CH00 5. Differential gene expression using bulk RNA barcoding and sequencing technology (BRB-seq)

[0090] Differential gene expression was performed using a new method described in Genome Biology volume 20, Article number: 71 (2019).

[0091] Stored lysates were subjected to RNA extraction (Bio-Rad), quantified in nanodrops (Thermo Nanodrop 2000) where an initial quality control was performed, and further processed for RNA quality control on the BioAnalyzer (Agilent Technologies) with a random check of 24 samples for a 96-well format.

[0092] For RNA sequencing, library preparation was performed using the MERCURIUS BRB Sequencing Library Preparation Kit (Alitheagenomics, Switzerland) and subjected to Illumina sequencing. The measurement volume was 5 million values ​​per sample, which should yield a gene expression profile of 15,000 to 20,000.

[0093] Bioinformatic analysis

[0094] After Illumina sequencing, the sequencing reads (FASTA files) were demultiplexed using the BRB-seq tools. Counting matrices were generated by matching them to the human genome (hg38) using STAR and HTSeq (version 0.9.1). Gene expression analysis was performed using the iDEP platform. Hierarchical clustering and K-mean clustering were carried out using the 2000 most variable genes, centered by subtracting the mean and dividing by the standard deviation. For K-mean clustering analysis, genes were normalized by the mean center. For differential enrichment analysis, a false-detection rate of 0.1 and a minimum folding change of 2 were specified as parameters for DESeq2 analysis in the iDEP platform. Gene ontology analysis was also performed on the iDEP platform. Plot generation and further analyses were performed using R, version R 4.0.2.

[0095] The results were presented as differences in gene expression. mRNA sequencing was performed using total RNA from HPEKs treated with extracts in both metabolomic and normal growth medium.

[0096] The following table lists the expressions in the form of log2Foldchange values. This represents the log2 transformation of the change in the

[0097] P193695CH00 represents gene expression between two conditions. The change is the ratio of the expression levels of a gene in one condition compared to another. The Iog2 transformation is applied to stabilize the variance and make the values ​​more interpretable.

[0098] The conditions are specified in the table. The first log2FoldChange value refers to the change in gene expression between a metabolically treated control sample and a non-metabolically treated control medium (normal medium). Negative values ​​indicate a decrease in gene expression during cell growth under metabolic conditions. However, when the active ingredient combination is used (see table above), only a minimal change is observed compared to the non-metabolically treated control medium. Thus, the invention ensures that gene expression is maintained even in metabolically treated cells, or that the cell activity for expressing these genes is restored. Consequently, the cells could be reset to the activity of unaged cells for the genes under investigation.

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[0101] P193695CH00 Further examples:

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Claims

Patent claims 1. Containing a combination of active ingredients: a) Paeonia Albiflora Root Extract, b) Calendula Officinalis Flower Extract, c) At least one glycoprotein 1, obtainable from the purified cytoplasmic fraction of yeast (Saccharomyces), d) At least one glycoprotein 2, obtainable from the purified cytoplasmic fraction of Lactobacillus, e) Ginseng extract, and f) Horsetail extract (Equisetum Arvense extract).

2. Active ingredient combination according to claim 1, characterized in that the proportion of Paeonia albiflora root extract is 20% to 40% by weight, preferably 22% to 36% by weight, based on the total weight of the Active ingredient combination.

3. Active ingredient combination according to one of the preceding claims characterized in that the proportion of Calendula Officinalis Flower Extract is from 15 wt.% to 50 wt.%, preferably from 20 wt.% to 47 wt.%, based on the total weight of the active ingredient combination.

4. Active ingredient combination according to one of the preceding claims characterized in that the proportion of c) glycoprotein 1 is from 1 wt.% to 10 wt.%, preferably from 2 wt.% to 7 wt.%, based on the total weight of the active ingredient combination ati ion .

5. Active ingredient combination according to one of the preceding claims characterized in that the proportion of d) glycoprotein 2 is from 2 wt.% to 20 wt.%, preferably from 6 wt.% to 15 wt.%, based on the total weight of the active ingredient combination.

6. Active ingredient combination according to one of the preceding claims characterized in that the proportion of ginseng extract is from 1 wt.% to 10 wt.%, preferably from 2 wt.% to 7 wt.%, based on the total weight of the active ingredient combination.

7. Active ingredient combination according to one of the preceding claims characterized in that the proportion of horsetail extract is from 1 wt.% to 10 wt.% P193695CH00 wt.% is, preferably from 2 wt.% to 7 wt.%, based on the total weight of the active ingredient combination.

8. Cosmetic preparation comprising the combination of active ingredients according to any one of claims 1 to 7.

9. Cosmetic preparation according to claim 8 characterized in that the total proportion of the active ingredient combination in the preparation is from 0.001 wt.% to 5 wt.%, preferably from 0.01 wt.% to 2.5 wt.% and particularly preferably from 0.03 wt.% to 1.0 wt.%, based on the total weight of the preparation.

10. Use of the combination of active ingredients according to any one of claims 1 to 7 in cosmetic preparations.

11. Use of an active ingredient combination according to any one of claims 1 to 7 to restore the expression rates of genes of aged cells compared to non-aged cells.

12. Use of an active ingredient combination according to any one of claims 1 to 7 for the rejuvenation of aged cells. P193695CH00

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