WO2019175829A1

WO2019175829A1 - A skin regenerating and skin protecting cosmetic active ingredient from undifferentiated cell biomass from genus dracocephalum ruyschiana and a method for its preparation

Info

Publication number: WO2019175829A1
Application number: PCT/IB2019/052083
Authority: WO
Inventors: Anna Ramata-Stunda; Martins Boroduskis; Elza Kaktina; Baiba Silamikele
Original assignee: Alternative Plants, Sia
Priority date: 2018-03-16
Filing date: 2019-03-14
Publication date: 2019-09-19
Also published as: LV15436A; LV15436B

Abstract

The present invention relates to production of cosmetic active ingredient as a standardized extract rich in caffeoyl derivatives obtained from undifferentiated cell biomass of Dracocephalum ruyschiana. Cosmetic active ingredient is aimed at skin protection and skin regeneration. A method for the preparation of a skin regenerating and skin protecting cosmetic active ingredient obtained from in vitro propagated undifferentiated D.ruyschiana cells is also proposed.

Description

A skin regenerating and skin protecting cosmetic active ingredient from undifferentiated cell biomass from genus Dracocephalum ruyschiana and a method for its preparation

Technical field

This invention relates to establishment and large scale propagation of undifferentiated plant cell cultures for extraction of biologically active substances for cosmetic use.

Prior art

High demand for natural plant based functional ingredients, limitations and more strict regulations associated with synthetic and animal derived ingredients has promoted the application of biotechnological methods to produce cosmetic active ingredients. Plants in the wild or cultivated using traditional agricultural techniques often produce low levels of desired bioactive compounds. Moreover, there are medicinal plants that are impossible to farm and such that are rare or threatened, making valuable ingredients that they produce inaccessible to the industry. Plant biotechnology, specifically, plant cell cultivation in vitro has become a promising tool to sustainably and consistently produce plant derived biologically active ingredients (Trehan S, Michniak-Kohn B, Beri K. Plant stem cells in cosmetics: current trends and future directions. Future Science OA. 20l7;3(4):FSO226. doi: l0.4l55/fsoa-20l7-0026).

Principles of establishment of plant cell cultures and propagation of plant cells on solid and in liquid cultivation medium have been described before in scientific publications and patent applications (EP1736167B1). Overall processes related to establishment and cultivation of plant cells have been described in patent application W02004033673A2. However, general plant cell cultivation principles are not applicable to large scale cell propagation for manufacturing purposes and specific technological processes need to be developed. For maintenance in undifferentiated cell state and effective propagation ratios of auxins and cytokinins need to be adjusted for each cell line (each plant species). Modifications in cell cultivation conditions and use of elicitors are crucial to produce specific biologically active compounds and are specific to used plant species as well as desired target compounds.

Plant cell biomass has been effectively used to produce phenylpropanoids (cells derived from Ajuga repens) and verbascoside (cells derived from Olea europea , Syringa vulgaris or Appia citrobara). Production of extracts, standardized regarding verbascoside content, from Syringa vulgaris cell cultures for use in medicine and cosmetics has been described in patent application EP1736167B1. Preparation of meristem cells with high content of caffeic acid derivatives from cell cultures belonging to the genera Achillea, Ajuga, Buddleja, Centella, Cynaris, Echinacea, Forsythia, Gardenia, Gfycyrrhiza, Hippeastrum, Hoodia, Laminm, Leontopodium, Lippia, Lonicera,Magnolia, Marrubium, Passi flora, Phytolacca, Plantago, Ruta, Stachys, Stapelia, Syringa, Teucriun, or Zanthoxylum has been described in patent application EP2319914A1. Extracts of such cell cultures have been reported to have high antioxidative activity, collagen production stimulating activity and pigmentation regulating characteristics (EP1736167B1).

Cosmetic, pharmaceutical and dietary supplement compositions containing ingredients produced in cell cultures of the genera Dendrobium , Phalaenolopsis , Anisellia , Polyrrhiza , Vanilla , Cattleya and Vanda have been described in FR2978161B1.

Use of Echinacea merismatic cells for skin regenerating cosmetic applications has been claimed in patent FR2978044B 1.

Undifferentiated and dedifferentiated Lentopodium alpinum cells are used for cosmetic applications to restore skin homeostasis, increase metabolic activity of the skin (FR3031454A1).

Specific skin pore tightening activity has been claimed for Marrubium vulgare dedifferentiated plant cell extract that is standardized based on forsythoside B content (FR3049194A1).

Patent application EP2817070A1 describes combined use of cloudberry, Rubus chamaemorus callus culture extract and cloudberry seed oil as cosmetic active ingredients.

Patent application JP2015505312A claims use of Rosa sp. dedifferetiated plant cell and plant cell culture extracts for skin and hair cosmetic use to prevent signs of ageing.

In cell cultures various compounds can be produced in significantly higher concentrations than in wild plants. Such compounds as rosmarinic acid and chlorogenic acid can be more efficiently produced in plant cell cultures than in plants (Razzaque, A. & Ellis, B E. Plants (1977) 137: 287. https://doi.org/l 0.1007/BF00388164). However often just the increased production of a single compound can be achieved, not the production of several synergistically acting biologically active compounds. In the recent publications highest rosmarinic procuction achieved in cell cultures varies, but rarely exceeds 1,5-2, 5% RA/dry biomass (Ching-Fen Wu, Anastasia Karioti, Doris Rohr, Anna Rita Bilia, Thomas Efferth, Production of rosmarinic acid and salvianolic acid B from callus culture of Salvia miltiorrhiza with cytotoxicity towards acute lymphoblastic leukemia cells, Food Chemistry, Volume 201, 2016, Pages 292-297, https://doi.org/10_· 1016/j .foodchem.2016.01 054; Bektas Tepe & Atalay Sokrnen (2007) Production and optimisation of rosmarinic acid by Satureja hortemis L. callus cultures, Natural Product Research, 21 : 13, 1133-1 144, DPI: 10 1080/14786410601 130737).

Similarly, production of caffeic and chlorogenic acids in cell cultures have been reported with variable success, but there are no case where production of these compounds is induced simultaneously.

So far, it has not yet been reported to formulate an extract obtained from undifferentiated cell biomass from Dracocephalum ruyschiana as biologically active ingredients for cosmetic use. Neither it has been reported before that cells derived from genus Dracocephalum have been used for production of mixtures of rosmarinic acid, caffeic acid and chlorogenic acid.

Disclosure of the invention

Summary of the invention

A skin regenerating and skin protecting cosmetic active ingredient is proposed, the ingredient being a standardized extract of in vitro propagated plant cell biomass, where said in vitro propagated plant cells are undifferentiated Dracocephalum ruyschiana cells, where said active ingredient comprising (weight/volume): rosmarinic acid in the range of 250 - 900 itg/ml; chlorogenic acid in the range of 40 - 100 pg/ml; caffeic acid 4 - 10 ug/ml. According to one embodiment the D. ruyschiana calli are grown on solidified cultivation medium either in dark or l4-l6h light photoperiod. According to another embodiment the D. ruyschiana cells are grown in liquid cultivation medium either in dark or l4-l6h light photoperiod.

Further a method for the preparation of a skin regenerating and skin protecting cosmetic active ingredient is proposed, the method comprising the following consecutive steps: (i) providing an in vitro propagated undifferentiated D.ruyschiana cells grown in solidified or liquid cultivation medium, (ii) separating of D.ruyschiana cell biomass from the cultivation medium; (iii) extracting of cell biomass with ethanol having concentration ranging from 30% to 70%, or with mixture of ethanol, glycerol and water, where ratios of ethanol, glycerol and water are within the range of 20%/20%/60% to 50%/ 50%/ 0% (volume/volume %). The biomass to solvent ratios are from 1 :3 to 1 : 10 (w/v). According to yet another embodiment D.ruyschiana cell biomass separated from the cultivation medium at step (ii) is freeze-dried and further extracted with said solvent explained at step (iii) above, wherein freeze dried biomass to solvent ratios are from 1 :50 to 1 : 100 (w/v). The in vitro propagated undifferentiated D.ruyschiana cells provided at step (i) are cells grown in dark or in l4-l6h light photoperiod. According to yet another embodiment the method further comprises the step (iv) of combination of extracts derived from cells grown in dark with extracts derived from cells grown in l4-l6h photoperiod at ratios within the range of 1 : 1 to 1 :5 (volume/volume). The inventors propose to use of said skin regenerating and skin protecting cosmetic active ingredient for the preparation of cosmetic compositions. The use of the ingredient is proposed at concentration ranging from 0,25% to 2%. The active ingredient is standardized regarding content of caffeic acid, rosmarinic acid and chlorogenic acids, so that this mixture of compounds constitutes 60-85% of the total phenolic compounds in the cell biomass extract.

The active ingredient obtained from undifferentiated D.ruyschiana cell culture according to the invention is characterized by the capability to produce rosmarinic acid in the range 30-40 mg/g dry biomass, chlorogenic acid in the range 4-6 mg/g dry biomass, caffeic acid in the range 0.4- 0.6 mg/g dry biomass.

Brief description of drawings

Fig. 1 is a graph showing the effect of D.ruyschiana cell biomass extract on proliferation of skin keraiinocyi.es

Fig. 2 is a graph showing the effect D.ruyschiana cell biomass extract on proliferation of dermal fibroblasts

Fig. 3 is a graph showing the effect of D.ruyschiana cell biomass extract on expression of collagen I and MMP-1 genes in dermal fibroblasts.

Detailed description of invention

First stage of invention includes establishment of cell cultures from aerial parts of D.ruyschiana. This is done by chopping aerial parts of wild plant, washing with water and treating them with bleach to obtain sterile tissue. Chopped tissues are placed on solid cultivation medium. According to one embodiment of the invention, the cultivation medium contains microelements (CoCl₂x6H₂0, CuS0₄x5H₂0, FeNaEDTA, H3BO3, KI, MnS0₄xH₂0, Na₂Mo0₄.2H₂0, ZnS0₄.7H₂0), macroelements (CaCl₂, KH₂R0₄, KNO3, MgS0₄, NH₄N0₃), glycine, myo-inositol, nicotinic acid, pyridoxine, thiamine, sucrose, plant agar and growth regulators (2,4~Diehlorophenoxyacetic acid, 6~Benzylaminopurine), pH adjusted with sodiu hydroxide to 5.6 - 5.8. However, it is obvious that other combination of microelements and macroelements are also possible. Formation of calli from uncontaminated plant tissue explants is observed after a suitable period of time. Calli are propagated by transferring them regularly to fresh cultivation plates. Calli with suitable characteristics (fast and constant proliferation, appropriate morphology and chemical composition) are selected after 10-15 cultivation cycles (transfers).

Suspension cell cultures are established form selected calli by inoculating growth medium. The medium composition used according to one embodiement: microelements (C0CI2X6H2O, CUS0_4X5H₂0, FeNaEDTA, H3BO3, KI, MnS0₄xH₂0, Na₂Mo0₄.2H₂0, ZnS0₄.7H₂0), macroelements (CaCh, KFhPCri, KNO3, MgS0₄, NFENO3), glycine, myo-inositol, nicotinic acid, pyridoxine, thiamine, sucrose and growth regulators (2,4-Dichlorophenoxyacetic acid, 6- Benzylaminopurine), pH adjusted with sodium hydroxide to 5.6 - 5.8, at calli to medium ratios of 1 :5 to 1 : 10 (w/v). However, it is obvious that other combination of microelements and macroelements are also possible. Fermentation in liquid cultivation medium is performed at temperature ranging from 18 to 25 °C, with orbital agitation under adequate oxygenation. Fresh cultivation medium is added regularly and cell cultures monitored by assessment of cell viability, proliferative activity and changes in sugar content in cultivation medium.

Extracts are prepared from either callus cultures or suspension cell cultures. After sufficient amount of biomass is grown cells are separated from cultivation medium by detaching (in case of calli) or sedimentation or centrifugation (in case of suspension cultures). Cell biomass from callus cultures or suspension cell cultures is extracted with mixture of ethanol and glycerol (ethanol/glycerol/water ratios ranging from 20%/20%/60% to 50%/ 50%/ 0%) or only ethanol (concentrations ranging from 30% to 70%), v/v. Extracts are prepared either from fresh cell biomass where fresh biomass and solvent ratios are from 1 :3 to 1 : 10 (w/v), or from freeze dried biomass where dry biomass and solvent ratios are from 1 :50 to 1 : 100 (w/v).

Chemical composition

Chemical composition of D.ruyschiana cell biomass extracts obtained was analysed using LC- TOF-HRMS method in multireaction monitoring regime. Chromatographic analyses were performed on a tandem mass spectrometer Micromass Quatro microTM API (Waters) and UHPLC system Acquity UPLC (Waters). Separations were achieved by using a C18 (Acquity BEH) column with a mobile phase composed of 0.1 % formic acid (channel A) and acetonitrile (channel B) in a gradient mode at a flow rate of 0.25 mL/min. The injection volume was 5.0 pL. For mass spectrometry negative electrospray ionization (ESI) was used; the source parameters were: drying gas flow 800 L/h and temperature 400 °C, capillary voltage 3.0 kV and applied fragmentor was 130 V.

Chemical analyses showed high concentrations of rosmarinic acid, chlorogenic acid and caffeic acids in obtained plant cell biomass. The concentrations of the compounds are given in the table 1. These compounds are dominating in the extracts obtained from cell cultures. The relative composition of combination of dominating compounds is 60-85% of the total phenolic compounds in the whole extract. High concentrations and the combination of these specific compounds affirms the applicability of the extracts as active ingredients in skin regeneration and protection products.

Table 1. Rosmarinic acid, chlorogenic acid and caffeic acid concentration in D.ruyschiana cell biomass

Table 2. Determined concentrations of rosmarinic acid, chlorogenic acid and caffeic acid in extracts derived from D.ruyschiana cell biomass (sample - extracts derived from plant cell biomass using various solvents and biomas to solvent ratios).

Antiradical activity

Antiradical activity (ARA) was determined by 2,2-diphenyl-l-picrylhydrazyl (DPPH) radical scavenging assay using high-throughput 96-well plate method as described by Herald 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, vol. 92, pp. 2326-2331, 2012) with slight modifications. The measurements of radical scavenging activity were done by mixing of 100 mM DPPH solution in ethanol with extract or standard samples. The absorbance was measured at 517 nm, along with the blank. The percentage of radical scavenging activity of all extracts was calculated from the following formula:

To scavenging [DPPH] =[ (A o-A i/Ao) ] ^ 100 , where

Ao was the absorbance of the blank;

Ai was the absorbance in the presence of the extract.

Antiradical capacity was expressed as Trolox equivalents (Trolox mM/ml), based on calibration curve (18,3 - 500 mM ml ¹).

Total phenolic content

Total phenolic content was analyzed using Folin-Ciocalteu assay using high-throughput 96- well plate method as described by Herald et al. with slight modifications.

The measurement was conducted by mixing working Folin-Ciocalteu solution (1 : 1 with water), sodium bicarbonate and ethanolic extract or standard solutions. The absorbance was measured after 90 minutes of incubation at 765 nm, along with the blank. TPC was expressed as gallic acid equivalents (g GAE mg/ml extract), based on gallic acid (GA) calibration curve (range 0.025 - 0.200 mg ml ¹).

Absorbance readings were done on Infinite M200 PRO (Tecan) instrument. Bandwidth 9 mm, temperature 25 °C.

Table 3. Antiradical activity and total phenolic content of D.ruyschiana cell biomass extract

Analysis of skin cell proliferation

To assess effect of D.ruyschiana biomass extract on proliferative activity of HaCaT keratinocytes and dermal fibroblasts live cell monitoring and time-lapse imaging was used . HaCaT keratinocytes and primary dermal fibroblasts were seeded in 96-well cultivation plates at concentration 2000 cells per well. Cells were incubated in DMEM (Dulbecco’s modified Eagles medium, Millipore) medium supplemented with 10% fetal bovine serum and 100 u/ml penicillin, 100 pg/ml streptomycin (Biochrom), in 37 °C, 5 % C0₂ atmosphere. Total volume of media used for cultivation - 100 mΐ. After 24h preincubation D.ruyschiana cell biomass extracts were added to the microplates of HaCaT keratinocytes and dermal fibroblasts. Changes in cell proliferation were analyzed for 72h in HaCaT cell culture, and for 48h in dermal fibroblast cell culture. In presence of 0.25% D.ruyschiana cell biomass extract proliferation was increased by 24% compared to solvent control, during first 24h of cultivation. Positive impact on cell proliferation was observed at 48h and 72h as well. In case of dermal fibroblasts, extract stimulated proliferation by 15% during 48h of cultivation.

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

Changes in gene expression of collagen I and MMP-l in dermal cell culture in presence of D.ruyschiana cell biomass extract was analyzed. Dermal fibroblasts were cultivated in DMEM ( Dulbecco’s modified Eagles medium, Millipore) medium supplemented with 10% fetal bovine serum and 100 u/ml penicillin , 100 pg/ml streptomycin (Biochrom), in 37 °C, 5 % C0₂ atmosphere until confluence of 70%. Extract of D.ruyschiana cell biomass culture was added to dermal fibroblast culture and incubated for 48h. After incubation cells were lysed with Trizol for RNA extraction. 1 pg of RNA was used for DNAse I treatment and subsequent cDNA synthesis. RT-qPCR analysis was performed for relative quantification of collagen I and MMP- 1 gene expression changes. Relative quantification of gene expression is done using 2 ^AACT method by Livak and Schmittgen, 2001. mRNA of collagen I ( collAl ) and mmp-1 genes is normalized to the mRNA of constitutively expressed housekeeping genes rpsl3 (ribosomal protein 13) and hprtl (hypoxanthine phosphoribozyltranspherase). Relative changes in expression of collagen and MMP-l in samples treated with plant extracts is compared to the expression of each gene in appropriate control samples (Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402-8). ETnexpectedly to inventors D.ruyschiana cell biomass extract stimulated collagen I expression in a dose dependent manner, moreover there was remarkable decrease (>65%) in expression of collagen degrading matrix metalloproteinase I. Increase in collagen I expression and suppression of MMP-l gene expression affirms the applicability of D.ruyschiana cell biomass extract as an active ingredient in regenerating skin care products. Examples of cosmetic product compositions containing D.ruyschiana cell biomass extract. Below are given examples of facial cosmetic products containing D.ruyschiana cell biomass extract.

Example 1 regenerating facial cream

Component Volume % a) Aqua 59,80%

Vitis vinifera (Grape) Seed Oil 20%

Caprylic/capric triglycerides 12% b) Olivem-100 (Cetearyl Olivate, Sorbitan Olivate) 4%

Cetyl alcohol 2%

D.ruyschiana cell biomass extract 1% Tocopherol acetate 0,5% c) Potassium sorbate 0,3%

Gluconolactone, Sodium Benzoate 0,4%

Component a) is heated to 65°C; b) is likewise heated to 65 °C till emulsifying waxes are completely melted, and then added while vigorously stirring to mixture a) to form emulsion.

Stirring is continued until the cream has cooled down to 35 °C. Then mixture c) is added while stirring and the cream is homogenized.

Example 2 reseneratins serum for periocular skin

Component Volume, % a) Aqua 73,53%

Squalane 10%

, . Coco-caprylate 10%

Olivem-100 (Cetearyl Olivate, Sorbitan Olivate) 3%

Cetyl alcohol 2%

D.ruyschiana cell biomass extract 0,25%

Tocopherol acetate 0,5%

Potassium sorbate

C) 0,3%

Gluconolactone, Sodium Benzoate 0,4%

Ubiquinone 0,02%

Component a) is heated to 65°C and mixture b) is likewise heated to 65 °C till emulsifying waxes are completely melted, and then added while vigorously stirring to mixture a) to form emulsion. Stirring is continued until the formulation has cooled down to 35 °C. Then mixture c) is added while stirring and formulation is homogenized.

Thus, it has been experimentally proven that composition obtained according to the invention comprising (weight/volume): rosmarinic acid in the range of 250 - 900 pg/ml; chlorogenic acid in the range of 40 - 100 pg/ml and caffeic acid 4 - 10 gg/ml has a skin regenerating and skin protecting effect.

Claims

1. A skin regenerating and skin protecting cosmetic active ingredient, being a standardized extract of in vitro propagated plant cell biomass, wherein said in vitro propagated plant cells are undifferentiated D.ruyschiana cells, said active ingredient comprising (weight/volume):

- rosmarinic acid in the range of 250 - 900 pg/rnl;

- chlorogenic acid in the range of 40 - 100 pg/ml and

- caffeic acid 4 - 10 pig/ml.

2. The active ingredient according to claim 1, wherein it is derived from D.ruyschiana calli culture, where said calli are grown on solidified cultivation medium either in dark or l4-l6h light photoperiod.

3. The active ingredient according to claim 1, wherein it is derived from D.ruyschiana suspension cell culture, where cells are grown in liquid cultivation medium either in dark or 14- l6h light photoperiod.

4. A method for the preparation of a skin regenerating and skin protecting cosmetic active ingredient as per any preceding claims, comprising the following consecutive steps:

(i) providing an in vitro propagated undifferentiated D.ruyschiana cells grown in solidified or liquid cultivation medium,

(ii) separating of D.ruyschiana cell biomass from the cultivation medium;

(iii) extracting of cell biomass with ethanol having concentration ranging from 30% to 70%, or with mixture of ethanol, glycerol and water, where ratios of ethanol, glycerol and water are within the range of 20%/20%/60% to 50%/50%/0% (v/v/v), wherein biomass to solvent ratios are from 1 :3 to 1 : 10 (w/v).

5. The method according to claim 4, wherein the in vitro propagated undifferentiated D.ruyschiana cells provided at step (i) are cells grown in dark.

6. The method according to claim 4, wherein the in vitro propagated undifferentiated D.ruyschiana cells provided at step (i) are cells grown in l4-l6h light photoperiod.

7. The method according to claims 5 and 6, wherein the method further comprises the step (iv) of combination of extracts derived from cells grown in dark with extracts derived from cells grown in l4-l6h photoperiod at ratios within the range of 1 : 1 to 1 :5 (v/v).

8. The method according to any preceding claims, wherein D.ruyschiana cell biomass separated from the cultivation medium at step (ii) is freeze-dried and further extracted at step (iii) with ethanol having concentration ranging from 30% to 70%, or with mixture of ethanol, glycerol and water, where ratios of ethanol, glycerol and water are within the range of 20%/20%/60% to 50%/ 50%/ 0% (v/v/v), wherein freeze dried biomass to solvent ratios are from 1 :50 to 1 : 100 (w/v).

9. Use of the active ingredient as per any claims 1 to 3 for the preparation of cosmetic compositions.

10. Use of the active ingredient according to claim 9, at concentration ranging from 0,25% to 2%.

11. Use of the active ingredient according to claim 9 or 10, wherein the content of caffeoyl derivatives in the active ingredient are ranging from 45-70% of the total flavonoids in the extract.