LV15436B - A skin regenerating and skin protecting cosmetic active ingredient from undifferentiated cell biomass from genus dracocephalum ruyschiana and a method for its preparation - Google Patents

Abstract

A skin rejuvenating and protective cosmetic active substance which is a standardized cell biomass extract obtained from in vitro propagated plant cell biomass, wherein said in vitro propagated plant cells are undifferentiated D.ruyschiana callus cells. The active substance according to claim 1, wherein said calli are grown on a solid culture medium surface either in the dark or in a light photoperiod of 14-16 hours. The active substance according to claim 1, which is obtained from a cell culture of a D.ruyschiana suspension, in which the cells are grown in liquid culture medium either in the dark or in a light photoperiod of 14 to 16 hours. A method for the preparation of a skin rejuvenating and protective cosmetic active ingredient according to any one of the preceding claims, comprising the following sequential steps: i) growing in vitro undifferentiated D.ruyschiana callus cells on solid medium or liquid culture medium, ii) D. ruyschiana callus cell biomass is separated from the culture medium, (iii) the cell biomass is extracted with an ethanol concentration in the range of 30 to 70%, or with a mixture of ethanol, glycerol and water, where the ethanol, glycerol to water ratio is in the range of 20% / 20 % / 60% to 50% / 50% / 0% (v / v / v) and the ratio of biomass to solvent is in the range of 1: 3 to 1:10 (w / v). The method of claim 4, wherein the cells of step (i) are grown in the dark. The method of claim 4, wherein the cells according to step (i) are grown for a light photoperiod of 14-16 hours. The method of claims 5 and 6, wherein the method further comprises the step of (iv) combining an extract obtained from cells cultured in the dark and an extract obtained from cells cultured in a light photoperiod of 14-16 hours, wherein the ratio of extracts is in the range of 1: 1 to 1: 5 (v / v). A method according to any one of the preceding claims, wherein the D.ruyschiana biomass separated from the culture medium in step (ii) is lyophilized and subsequently extracted as described in step (iii) with an ethanol concentration in the range of 30 to 70%, or with a mixture of ethanol, glycerol and water, where the ratio of ethanol, glycerol to water is in the range of 20% / 20% / 60% to 50% / 50% / 0% (v / v / v) and the ratio of lyophilised biomass to solvent is in the range of 1:50 to 1: 100 (w / v). Use of an active substance according to claims 1 to 3 in the preparation of cosmetic compositions. Use of the active substance according to claim 9 in a concentration range from 0.25 to 2%. Use of the active substance according to Claim 9 or 10, in which the content of coffee acid derivatives in the active substance is in the range from 45 to 70% of the total flavonoid content in the extract.

Description

DESCRIPTION OF THE INVENTION Technical field

The invention relates to the production of an undifferentiated culture of plant cells and to the propagation of a large number of cells for the extraction of biologically active substances for use in cosmetics.

Prior art

The high demand for natural functional active substances of plant origin, the limited and more strictly regulated use of active substances of synthetic and animal origin have encouraged the use of biotechnological methods in the production of active substances in cosmetics. Plants grown in the wild or by traditional farming methods often produce low concentrations of the desired biologically active compounds. In addition, there are medicinal plants that cannot be cultivated, as well as medicinal plants that are rare or endangered, and the biologically active compounds produced by these plants are currently unavailable to the industry. Plant biotechnology, in particular, in vitro methods of culturing plant cells have become a promising way to produce biologically active substances of plant origin in a sustainable and consistent manner (Trehan S, Michniak-Kohn B, Berry K. Plant stem cells in cosmetics: current trends and future directions.Future Science OA. 2017; 3 (4): FS0226. doi: 10.4155 / fsoa-2017-0026).

The principles of forming plant cell cultures and propagating cells on a solid culture surface or by culturing in a liquid medium have been described above in scientific articles and patent applications (EP1736167B1). General processes for forming and culturing plant cell cultures are described in WO2004033673A2. However, the general principles of plant cell culture are not applicable to large-scale cell proliferation for production purposes and there is a need to develop specific technological processes. In order to maintain the cell culture in an undifferentiated state and to achieve efficient cell proliferation, it is necessary to adjust the auxin and cytokinin ratios for each cell line (each plant species). Modification of cell culture conditions and the use of different elicitors are essential for the production of specific biologically active compounds, the culture conditions and elicitors are specific to the plant species used as well as the target compounds to be obtained.

Plant cell biomass has so far been used efficiently to produce phenylpropanoids (in cell cultures derived from Ajuga repens) and verbascosides (cell cultures derived from Olea europea, Syringa vulgaris or Appia citrobara). The production of extracts standardized for verbascoside content from Syringa vulgaris cell cultures is used for the production of raw materials for medical and cosmetic products and is described in patent application EP1736167B1.

Obtaining cultures of meristem cells with a high content of coffee acid derivatives from the genera Achillea, Ajuga, Buddleja, Centella, Cynaris, Echinacea, Forsythia, Gardenia, Glycyrrhiza, Hippeastrum, Hoodia, Lamium, Leontopodium, Magnolia, Maronicera, Lonicera, Phytolacca, Plantago, Ruta, Stachys, Stapelia, Syringa, Teucriun or Zanthoxylurn are described in patent application EP2319914A1. Extracts derived from such cell cultures have been shown to have high antioxidant activity, collagen synthesis stimulating activity and skin pigmentation regulating properties (EP1736167B1).

Compositions of cosmetics, pharmaceuticals and food additives containing ingredients derived from cell cultures of the genera Dendrobium, Phalaenolopsis, Aniseilia, Polyrrhiza, Vanilla, Cattleya and Vanda are described in patent FR2978161B1.

The use of meristem cells of the genus Echinacea for skin rejuvenating applications in cosmetics is described in patent FR2978044B1.

Undifferentiated and dedifferentiated Lentopodium alpinum cells are used in cosmetic applications to restore skin homeostasis, stimulate skin metabolic activity (FR3031454A1).

Specific skin pore-tightening activity is attributed to an extract obtained from Marrubium vulgare dedifferentiated cells and standardized for forsitoside B content (FR3049194A1).

Patent application EP2817070A1 describes the use of a combined bilberry Rubus chamaemorus callus culture extract and cloudberry seed oil as a cosmetic active ingredient.

[Oil] Patent application JP2015505312A describes the use of dedifferentiated cells and plant cell extracts of plants of the genus Rosa in skin and hair care cosmetics to prevent the signs of aging.

The preparation of an extract as a biologically active substance from undifferentiated Dracocephalum ruyschiana cell biomass for use in cosmetic products has not yet been described.

[013] Also, the use of cell cultures derived from plants of the genus Dracocephalum for the production of coffee acid derivatives has not been described so far.

[014] Disclosure of the Invention

SUMMARY OF THE INVENTION

Skin rejuvenating and cosmetically active ingredient that protects the skin and is a standardized extract from in vitro propagated plant cell biomass consisting of in vitro propagated undifferentiated Dracocephalum ruyschiana cells. According to the discovery, D.ruyschiana callus is grown on a solid culture medium in the dark or in a 14-16 hour light photoperiod. According to another additional discovery, D.ruyschiana cells are grown in liquid culture medium in the dark or in a light photoperiod of 14-16 hours.

A method of preparing a skin rejuvenating and protective cosmetic active ingredient is described below, comprising the following sequential steps: (i) obtaining in vitro propagated undifferentiated D.ruyschiana cells grown on a solid culture medium or in a liquid culture medium, (ii) Separation of D.ruyschiana cell biomass from culture medium; (iii) extraction of cell biomass with ethanol at concentrations of 30 to 70% or with a mixture of ethanol, glycerol and water, where the ratio of ethanol, glycerol to water is in the range of 20% / 20% / 60% to 50% / 50% / 0% (volume / volume%). The cell biomass to solvent ratios range from 1: 3 to 1:10 (w / v). According to the invention, the D.ruyschiana cell biomass separated from the culture medium in step (ii) is lyophilized and further extracted with said solvent, as explained in step (iii), wherein the ratio of lyophilized biomass to solvent is in the range of 1:50 to 1: 100 (w / v). The in vitro propagated undifferentiated D.ruyschiana cells referred to in step (i) are cells grown in the dark or in a 14-16 hour light photoperiod. According to the invention, the method further comprises the step (iv) of combining extracts obtained from cells grown in the dark with extracts obtained from cells grown in a photoperiod of 14-16 hours in a ratio of 1: 1 to 1: 5 (v / v). ). The inventors propose the use of this skin rejuvenating and protective cosmetic active ingredient in cosmetics. The recommended concentrations of the ingredient to be used range from 0.25% to 2%. The active substance is standardized for the content of caffeic acid derivatives, as this group of compounds makes up 45-75% of the total amount of flavonoids in the cell biomass extract.

[016] Brief description of the drawings

Fig. 1 - a chromatogram illustrating the predominant compounds and their relative amounts in the D.ruyschiana cell biomass extract.

Fig. 2 - graph showing the effect of D.ruyschiana cell biomass extract on skin keratinocyte proliferation.

Fig. 3 - a graph showing the effect of D.ruyschiana cell biomass extract on dermal fibroblast proliferation.

Fig. 4. - Graph showing the effect of D.ruyschiana cell biomass extract on melanin synthesis in FM-55 and B16 melanocytes.

Fig. 5 - Graph showing the effect of D.ruyschiana cell biomass extract on collagen I and MMP-1 gene expression in skin fibroblasts.

[017] Detailed description of the invention

The first step of the invention involves the development of cell cultures from the surface vase portions of D.ruyschiana. This is done by cutting the surface vaso parts of the wild plant, washing them with water and treating them with bleach to obtain sterile tissue. The shredded tissue pieces are placed on a solid culture medium. According to the invention, the culture media containing trace elements (C0CI2X6H2O, CuSO4x5H2O, FeNaEDTA, H3BO3, KI, MnSO4xH2O, Na2MoO ₄ .2H ₂ O, ZnSO4.7H ₂ O), macro (cache, KH2PO4, KNO3, MgSO _4, NH4NO3) glycine, myoinositol, nicotinic acid, pyridoxine, thiamine, sucrose, plant agar and growth regulators (2,4-Dichlorophenoxyacetic acid, 6-Benzylaminopurine), the pH is adjusted to 5.6 - 5.8 with sodium hydroxide. However, other combinations of trace elements and macronutrients are possible. Callus formation from uncontaminated plant tissue explants is observed after a longer period of time. The calli are propagated by dividing and regularly moving (transplanting) to fresh growing plates. Calli with appropriate properties (fast and constant growth, suitable morphology and chemical composition] are selected after 10-15 growing cycles (inoculations).

[018] Suspension cell cultures are formed from selected calli by transferring them to a liquid culture medium. According to the invention, the culture medium consists of: trace elements (COCl2X6H2O, CuSO4x5H2O, FeNaEDTA, H3BO3, KI, МпЗСкхНгО, Na2MoŪ4.2H20, ZnSO4.7H2O], m , pyridoxine, thiamine, sucrose and growth regulators (2,4-Dichlorophenoxyacetic acid, 6-Benzylaminopurine], the pH is adjusted to 5.6 - 5.8 with sodium hydroxide, the callus to medium ratio ranges from 1: 5 to 1:10 (w / v). However, other combinations of trace elements and macronutrients are possible: fermentation in liquid culture medium is carried out at a temperature between 18 and 25 ° C using an orbital shaker with adequate aeration.The cells are regularly monitored for viability, division activity , as well as changes in sugar levels in the medium, and fresh culture medium is added regularly.

Extracts are prepared from callus or suspension cell cultures. After sufficient biomass growth has been achieved, the cells are mechanically separated from the culture medium (in the case of callus) or sedimented by centrifugation (in the case of cell suspension cultures). The cell biomass is extracted from the callus or suspension cultures with a mixture of ethanol and glycerol 20% / 20% / 60% to 50% / 50% / 0%] or with ethanol only (concentrations between 30% and 70%) (v / v) Extracts can be prepared from both fresh cell biomass, where the biomass to solvent ratio is from 1: 3 to 1:10 (w / v), or from lyophilized cell biomass, where the dry biomass to solvent ratio is from 1:50 to 1: 100 (w / v).

Chemical composition

The chemical composition of the extract obtained from D.ruyschiana cell biomass was analyzed using LC-TOF-HRMS method. Chromatographic analyzes were performed using a modular UHPLC system Agilent 1290 Infinity series (Agilent Technologies, Germany). Liquid chromatography was performed on a C18, 2.1x50 mm, 2.6 μm column with a mobile phase consisting of 0.1% formic acid (channel A] and acetonitrile (channel B] in a gradient mode at a flow rate of 0.3 mL / min. Injection volume was 20.0 pL High resolution mass spectra (HRMS] were recorded using an Agilent 6230 TOF LC / MS system with positive electron scattering ionization (ESI) Source parameters were: positive ionization mode, drying gas flow 12.0 L / min and temperature 320 ° C, spray pressure 40 psi, capillary voltage 3500 V and the fragmenter used was 130 V. One full mass spectrum was obtained in profile mode, mass range from m / z 70 to 2000. Data of all isolated and analyzed compounds were obtained using an individual chromatogram of the compound at their individual m / z values.The data were analyzed using MassHunter B07.00 software.

Surprisingly, chemical analyzes of plant cell biomass extracts showed high concentrations of caffeol derivatives. This group of compounds is predominant in extracts derived from cell cultures. The relative composition of the predominant caffeine derivatives is 45-70% of the total flavonoids in the whole extract. The high concentration of caffeine derivatives confirms the use of the extract as an active ingredient in skin rejuvenating and protective cosmetics.

Antiradical activity

Antiradical activity (ARA) was determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method using the 96-well broad screening method previously described by T. Heralds et al. {T.J. Herald, P. Gadgil and M. Tilley, High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging activity in sorghum bran and flour, Science of Food and Agriculture, ed. 92, Ip. 2326-2331, 2012) with minor changes.

Measurements of free radical scavenging activity were performed by mixing a 100 μΜ solution of DPPH in ethanol with extract or standard samples. The absorbance was read at 517 nm, together with control samples. The percentage of free radical scavenging activity of all extracts was calculated according to the following formula:

Binding [DPPH] = [(Ao-Ai / Ao)]> <100, where

Ao was the absorbance of the blank;

Ai was absorbed in the presence of the extract.

Antiradical activity was expressed in Trolox equivalents (Trolox μΜ / ml) based on the calibration curve (18.3 - 500 μΜ ml · ¹ ).

[024] Total phenols

Total phenols were analyzed using the Folin-Chicalto method and 96-well sample screening plates as described by the Herald et al. with minor, changes.

Readings were taken by mixing working Folin-Chicalto solution (1: 1 with water), sodium bicarbonate and ethanol extract or standard solutions. Absorbance readings were taken at 765 nm after 90 minutes of incubation. Total phenolic content (TPC) was expressed as gallic acid equivalents (g GAE mg / ml extract) based on the gallic acid (GA) calibration curve (concentration range 0.025 - 0.200 mg ml · ¹ ).

Absorption readings were performed on an Infinite M200 PRO (Tecan) at a bandwidth of 9 mm at 25 ° C.

Table 1. Antiradical activity and total phenols in D.ruyschiana cell biomass extract

Antiradical activity (Trolox equivalents, μΜ / ml) Total phenols (Rooster acid equivalents pg / ml) D.ruyschiana cell biomass extract 500.6 ± 10.08 193 ± 9.67

[027] Skin cell proliferation assay

Real-time living cell monitoring and visualization systems were used to evaluate the effect of the extract obtained from D.ruyschiana biomass on the division activity of HaCaT keratinocytes and dermal firbroblasts. HaCaT keratinocytes and primary fibroblasts were seeded in 96-well culture plates at a concentration of 2000 cells / plot. Cells were incubated in DMEM (Dulbeko modified germ medium, Millipore) culture medium supplemented with 10% fetal bovine serum and 100 u / ml penicillin, 100 pg / ml я

streptomycin (Biochrom) at 37 ° C, 5% CO2. The total volume of the culture medium is 100 μΐ. After 24 hours of pre-incubation in microplates, D.Ryschiana cell biomass extracts were added to HaCaT keratinocytes and dermal fibroblasts in microplates. Changes in cell division activity were analyzed over a 72-hour period in HaCaT cell culture and over a 48-hour period in dermal fibroblast culture. In the presence of 0.25% D.ruyschiana cell biomass extract, proliferation was increased by 24% compared to the solvent control during the first 24 hours of culture. Positive effects on cell division were also observed at 48 hours and 72 hours. In the case of dermal fibroblasts, the extract stimulated proliferation by 15% during 48 hours of culture.

[028] Analysis of melanin content in melanocytes

Changes in melanin content were analyzed in FM-55 and B16 melanocyte cell lines. Cells were incubated in DMEM (Dulbeko modified germ medium, Millipore) culture medium supplemented with 10% fetal bovine serum and 100 u / ml penicillin, 100 pg / ml streptomycin (Biochrom) at 37 ° C, 5% CO 2. Melanin production was stimulated by the addition of 0.2 mM L-tyrosine to melanocytes 24 hours before the addition of D.ruyschiana cell biomass extract. Melanocytes were incubated with D.ruyschiana extracts for 48 hours. After incubation, melanocytes were lysed with 1N NaOH and the melanin content was determined spectrophotometrically at 405 nm. The inventors unexpectedly observed that in the presence of D.ruyschiana biomass extract, the melanin content decreased by 41% in the FM-55 cell line and by 26% in the B16 cell line, a level of inhibitory activity comparable to that obtained by adding pure codic acid to the cells.

Expression of collagen I and matrix metalloproteinase 1 (MMP-1}

Changes in collagen I and MMP-1 gene expression were analyzed in dermal cell culture in the presence of D.ruyschiana cell biomass extract. Dermal fibroblasts were cultured in DMEM (Dulbeko modified germline medium, Millipore) culture medium supplemented with 10% fetal bovine serum and 100 u / ml penicillin, 100 pg / ml streptomycin (Biochrom) at 37 ° C, 5% CO 2 until 70% confluence. D.ruyschiana cell biomass extract was added to dermal fibroblast culture and incubated for 48 hours. After incubation, cells were lysed with Trizol reagent for RNA extraction. 1 pg of RNA was treated with DNase I, followed by complementary DNA (cDNA) synthesis. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed to relatively quantify changes in collagen I and MMP-1 gene expression. Relative quantification of changes in gene expression was performed using the method of Livak, Schmittgen 2001 2 ' ^длст . The amount of collagen I (collAl) and mmp-1 matrix RNA (mRNA) was normalized to the persistently expressed genes rpsl3 (ribosomal protein 13) and hprtl (hypoxanthine phosphoribosyltransferase) mRNA. The relative changes in collagen I and MMP-1 gene expression in the samples to which the extract was added were compared with the expression of each gene in the corresponding control samples (Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C (T) J Method. Methods. 2001 Dec; 25 (4): 402-8) Unexpected to the inventors, D.ruyschiana cell biomass extract stimulated collagen I expression in a dose-dependent manner, with a significant reduction (> 65%). collagen degrading matrix metalloproteinase I. The increase in collagen I expression and the suppression of MMP-1 expression indicate the applicability of D.ruyschiana cell biomass extract as an active substance in regenerating skin care products.

Examples of cosmetic product compositions containing D.ruyschiana cell biomass extract.

The following are examples of final compositions for facial skin care cosmetic products containing D.ruyschiana cell biomass extract.

[031] Example 1 - Rejuvenating face cream

Ingredient Volume% a) Water 59.80% b) Vitis vinifera (Grape) seed oil Caprylic / capric triglycerides Olivem-100 (ceateryl olivate, sorbitan olivate) Cetyl alcohol 20% 12% 4% 2% c) D.ruyschiana cell biomass extract Tocopherol acetate Potassium sorbate Gluconolactor, sodium benzoate 1% 0.5% 0.3% 0.4%

Component (a) is heated to 65 ° C; b) is heated to 65 ° C until the emulsifying waxes have completely melted and then, with vigorous stirring, a) is added to form an emulsion. Stirring is continued until the cream has cooled to a temperature of 35 ° C. Add c) to the mixture, stirring, until the cream is completely homogenized.

Example 2 - Regenerating serum for the skin around the eyes

Ingredients Volume,% a) Water 73.53% b) Squalene Cococaprylate Olivem-100 (ceateryl olivate, sorbitan olivate) Cetyl alcohol 10% 10% 3% 2% c) D.ruyschiana cell biomass extract Tocopherol acetate Potassium sorbate Gluconolactor, sodium benzoate Ubiquinone 0.25% 0.5% 0.3% 0.4% 0.02%

Component (a) is heated to 65 ° C; b) is heated to 65 ° C until the emulsifying waxes have completely melted and then, with vigorous stirring, a) is added to form an emulsion. Stirring is continued until the cream has cooled to 35 ° C. Add c) to the mixture, stirring, until the cream is completely homogenized.

Claims (11)

Claims A skin rejuvenating and protective cosmetic active substance which is a standardized cell biomass extract obtained from in vitro propagated plant cell biomass, wherein said in vitro propagated plant cells are undifferentiated D.ruyschiana callus cells. The active substance according to claim 1, wherein said calli are grown on a solid culture medium surface either in the dark or in a light photoperiod of 14-16 hours. The active substance according to claim 1, which is obtained from a cell culture of D.ruyschiana suspension, wherein the cells are grown in liquid culture medium either in the dark or in a light photoperiod of 14-16 hours. A method for preparing a skin rejuvenating and protective cosmetic active ingredient according to any one of the preceding claims, comprising the following sequential steps: (i) in vitro propagated undifferentiated D.ruyschiana callus cells are grown on solid medium or liquid culture medium, (ii) D.ruyschiana callus cell biomass is separated from the culture medium, (iii) The cell biomass is extracted with ethanol at concentrations ranging from 30 % to 70%, or with a mixture of ethanol, glycerol and water, where the ratio of ethanol, glycerol to water is in the range from 20% / 20% / 60% to 50% / 50% / 0% (v / v / v), and the ratio of biomass to solvent is in the range of 1: 3 to 1:10 (w / v). The method of claim 4, wherein the cells of step (i) are grown in the dark. The method of claim 4, wherein the cells according to step (i) are grown for a light photoperiod of 14-16 hours. The method of claims 5 and 6, wherein the method further comprises the step of (iv) combining an extract obtained from cells cultured in the dark and an extract obtained from cells cultured in a light photoperiod of 14-16 hours, wherein the extract the ratio is in the range of 1: 1 to 1: 5 (v / v). A method according to any one of the preceding claims, wherein the D.ruyschiana biomass separated from the culture medium in step (ii) is lyophilized and subsequently extracted as described in step (iii) with an ethanol concentration in the range of 30% to 70%, or with a mixture of ethanol, glycerol and water, where the ratio of ethanol, glycerol to water is in the range of 20% / 20% / 60% to 50% / 50% / 0% (v / v / v) and the ratio of lyophilized biomass to solvent is in the range from 1:50 to 1: 100 (w / v). Use of an active substance according to claims 1 to 3 in the preparation of cosmetic compositions. Use of the active substance according to claim 9 in a concentration range from 0.25% to 2%. Use of the active substance according to Claim 9 or 10, in which the content of coffee acid derivatives in the active substance is in the range from 45% to 70% of the total flavonoid content in the extract.