Method for producing 4-aminobutyric acid from algae
09662515 · 2017-05-30
Assignee
Inventors
- Tim Köhler (Dorsten, DE)
- Jennifer Schild (Solingen, DE)
- Matthias Mentel (Dortmund, DE)
- Peter Lersch (Dinslaken, DE)
- Mike Farwick (Essen, DE)
- Christian Weitemeyer (Seeburg, DE)
Cpc classification
A61Q17/04
HUMAN NECESSITIES
C12P13/005
CHEMISTRY; METALLURGY
A61K8/44
HUMAN NECESSITIES
A61K36/04
HUMAN NECESSITIES
International classification
A61K36/04
HUMAN NECESSITIES
A61Q17/04
HUMAN NECESSITIES
A61K8/99
HUMAN NECESSITIES
A61K8/44
HUMAN NECESSITIES
C12P13/00
CHEMISTRY; METALLURGY
Abstract
The invention relates to a process for the production of 4-aminobutyric acid, also referred to as gamma-aminobutyric acid, in short GABA, to a process for the production of an extract comprising 4-aminobutyric acid, to a process for the preparation of a cosmetic formulation comprising 4-aminobutyric acid, and to the use of Cyanidium caldarium cells or cell components in cosmetic applications.
Claims
1. A process for the production of 4-aminobutyric acid, comprising: culturing Cyanidium caldarium cells containing 4-aminobutyric acid in an aqueous medium; disrupting the Cyanidium caldarium cells to obtain solid cell components containing said 4-aminobutyric acid; and incorporating the disrupted Cyanidium caldarium cells comprising solid cell components containing said 4-aminobutryic acid in a cosmetic formulation.
2. The process according to claim 1, wherein said Cyanidium caldarium cells are cultured at a temperature from 20 C. to 60 C.
3. The process according to claim 1, wherein said culturing provides an aqueous medium having a pH at 25 C. of from 1.5 to 6.
4. The process according claim 1, wherein said disrupting the Cyanidium caldarium cells is performed in an aqueous medium.
5. The process according to claim 1, wherein said disrupting the Cyanidium caldarium cells comprises a hot-water disruption in combination with an enzymatic cell disruption.
Description
(1) The following figures are part of the examples:
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EXAMPLES
Example 1
Culturing Cyanidium caldarium
(18) The preparative Cyanidium caldarium biomass production for obtaining the extract takes place in closed photobioreactors as described in detail in DE4411486, DE29607285, WO9105849, WO2010/149154 or DE102009028474A1 (WO2011/018082A2). The culturing method was adapted to the specific growth conditions of the red alga Cyanidium caldarium (SAG 16.91). Minimization of the shear stress by selecting suitable pump configurations, continuous light adaptation by a variable design of the light regime during culturing from 50 to 2000 E*m.sup.2s.sup.1 and adjusting a pH of <3 allows both high growth rates and reproducible biomass qualities to be achieved.
(19) The culture medium used consisted (per liter of distilled water) of 4.5 g (NH.sub.4).sub.2SO.sub.4, 0.6 g MgSO.sub.47H.sub.2O, 0.6 g KH.sub.2PO.sub.4, 0.03 g CaCl.sub.22H.sub.2O, 0.01 g FeSO.sub.4, 0.018 g ethylenediaminetetraacetic acid disodium dihydrate, 0.005 g H.sub.3BO.sub.3, 0.004 MnCl.sub.2, 0.00068 g ZnSO.sub.47H.sub.2O, 0.00052 g Na.sub.2MoO.sub.42H.sub.2O, 0.00008 g CuSO.sub.45H.sub.2O, 0.00008 g NaVO.sub.34H.sub.2O, 0.00064 g CoCl.sub.26H.sub.2O. The pH of the medium was brought to 2-3 by adding 1 N H.sub.2SO.sub.4 and/or by regulating the CO.sub.2 concentration. For a preculture, a 5 l Erlenmeyer flask was filled with 1 l of medium and inoculated with Cyanidium caldarium (SAG 16.91) stock culture. The culture was incubated at 30 C. underneath fluorescent tubes with 140 E*m.sup.2s.sup.1, with gentle shaking. The growth of the algae culture was monitored by absorption at 550 nm. As soon as the growth had reached the exponential phase (OD 1.0), the cells were harvested by centrifugation for 10 minutes at 5000 rpm, and the pellets were washed with distilled water. The wet biomass was stored at 20 C. until further use.
Example 2
Aqueous Extraction of Cyanidium caldarium
(20) Harvesting from photobioreactors is done as described for example in DE10136645 (EP 1277831) or WO9105849.
(21) The use of ultrasound for disrupting microalgae cells has been described and can be reproduced, inter alia, with reference to US2009069213, example 3 and US2008299147, example 3. Insoluble components were separated off by filtration with 200 m bag filter. 1.0% of Rokonsal BS is added to the aqueous extract for preservation purposes. The dry matter (105 C.) of the aqueous Cyanidium caldarium extract as determined by Sartorius MA30 Moisture Analyzer gave a value of 2.5%.
Example 3
Detection of GABA in Cyanidium caldarium
(22) GABA was detected by high-performance liquid chromatography (HPLC) following derivatization by ortho-phthalic dialdehyde (OPA). The HPLC apparatus was composed of Jasco PU 2080Plus pump, JascoAS 2055Plus autosampler, Jasco FP-2020Plus fluorescence detector and Jasco ChromPass 1.8.6.1 integrator (Jasco Germany GmbH, Gross-Umstadt, Germany). The stationary phase used was aZorbax Eclipse XDB-C18, 4.6150 mm, 3.5 m column (Agilent Technologies, Inc.) at a temperature of 40 C. The following gradient was used as the mobile phase (A: 2% acetonitrile, 2% tetrahydrofuran, 96%50 mM phosphoric acid, brought to pH 7.5 with NaOH); B: 65% acetonitrile, 35% water): 0-2 min 20% B constant, 2-8 min 20-40% B linear, 8-11 min 40-100% B linear, 11-38 min 100% B constant. The flow rate was 1.5 ml/min. 5 L of the analyte together with 15 L of OPA reagent (1 mL phthalic dialdehyde reagent, Solution Complete, Sigma-Aldrich P0532 and 0.5 L mercaptoethanol) were injected. The absorption wavelength was 340 nm and the emission wavelength 455 nm. The internal standard used was 1,6-diaminohexane. The analyte was prepared by diluting 1 ml of the aqueous Cyanidium caldarium extract to 100 ml with water.
(23) A typical value for GABA in the Cyanidium caldarium extract was 0.090%. This value corresponded to 3.3% of GABA based on Cyanidium caldarium dry matter.
Example 4
Gene Expression Analysis of Cyanidium caldarium Extract on Human Dermal Fibroblasts by Means of Gene Chip Study
(24) Method:
(25) In the present example, the effect of Cyanidium caldarium extract on the gene expression in fibroblasts (normal human dermal fibroblasts, NHDFs) was studied. To this end, primary human dermal fibroblasts (human dermal fibroblasts derived from neonatal skin (HDF), cryocenserved, Lifeline Cell Technology, obtained from CellSystems Biotechnologie Vertrieb GmbH, St. Katharinen, Germany) were first grown in Minimum Essential Medium (MEM) supplemented with Earle's salts (EMEM) (PAA Laboratories GmbH, Pasching, Austria) with the addition of 10% foetal calf serum (FBSfetal bovine serum, (Invitrogen Ltd, UK), 1% nonessential amino acids (NEAA (100)non essential amino acids, PAA, Pasching, Austria), 1% L-glutamine (100) (Invitrogen Ltd, UK) and 1% penicillin/streptomycin (5000 U/ml penicillin and 5000 g/ml streptomycin, Invitrogen Ltd, UK) at 37 C. and 5% CO.sub.2. For gene expression studies, the cells were sown in 6-well plates and grown to subconfluence (maximum 60%).
(26) Thereafter, the medium was drawn off from the cells and replaced by fresh medium with Cyanidium caldarium extract. The final concentration of Cyanidium caldarium extract in the medium was 100 ppm (=100 g/ml, based on Cyanidium caldarium extract dry matter). As a control, the cultures were grown without active substance, only with medium (vehicle). All cultures were performed in triplicate (3 biological replications).
(27) After the cells had been cultured for 24 hours, the medium was drawn off and the cells were lysed by the addition of RNeasy Lysis Buffer (Qiagen, Hilden, Germany). The total RNA was isolated following the manufacturer's instructions. In summary, the total RNA was isolated by means of RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNA quality was determined by the Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Kit (Agilent Technologies, Inc., Santa Clara, Calif., USA). The three biological replications were pooled, and the concentration of the pooled samples was determined by photometric measurement at 260/280 nm.
(28) Gene expression was analysed by means of Affymetrix GeneChip expression analyses including standard data evaluation following the manufacturer's instructions (Human Gene 1.0 ST Array, Expression Console Software, AFFYMETRIX, INC., Santa Clara, Calif., USA).
(29) The results are shown in Table 1
(30) TABLE-US-00001 TABLE 1 Synopsis of genes with at least 2-fold upregulation or downregulation in human dermal fibroblasts 24 h after the application of 100 ppm (=100 g/ml) Cyanidium caldarium extract (concentration based on extract dry matter). Regulation by 100 ppm of Gene Protein/function Cyanidium extract ELN Elastin; besides fibrillin main component of elastic fibres +2.4 FBN2 Fibrillin; besides elastin main component of elastic fibres +2.2 COL3A1 Collagen type III alpha-1chain; collagen type III is a fibrillary +1.8 collagen which is present in the skin in close association with the main collagen type I collagen. COL11A1 Collagen type XI alpha-1 chain +2.5 LUM Lumican; belongs to the family of the small leucine-rich +2.0 proteoglycans (SLRP; small leucine rich proteoglycan); interacts with collagen fibrils thereby regulating the interfibrillary distances ACAN Aggrecan; large aggregating proteoglycan; important +2.7 structural component of the extracellular matrix SDC2 Syndecan; a heparan sulphateproteoglycan; important for +2.1 cell proliferation, cell migration and cell matrix interactions CTGF Connective tissue growth factor; central factor for controlling +2.4 the formation of the extracellular matrix SULF1 Sulphatase; removal of sulphate residues from heparan +2.7 sulphate; signal transduction MMP1 Matrix metalloprotease 1; degradation of collagen types I, 5.7 II, III MMP3 Matrix metalloprotease 3; degradation of fibronectin, 2.1 laminin, collagentypes III, IV, IX, X
(31) The gene chip studies in human dermal fibroblasts demonstrated a uniform stimulation of elastin (ELN) and fibrillin (FBN2), the most important structural components of elastic fibres, by the addition of Cyanidium caldarium extract. Furthermore, the gene expression of important structural proteins of the connective tissue, collagen III and XI (COL3A1, COL11A1), was increased. Other proteins such as aggrecan (ACAN), syndecan (SDC2) and lumican (LUM) have a structure-imparting function or help with organizing interactions between various extracellular matrix (ECM) components and fibroblast cells. The genes of these proteins (LUM, ACAN, SDC2), too, were upregulated by a Cyanidium caldarium extract. The degradation of extracellular matrix components was prevented efficiently by the downregulation of matrix metalloprotease 1 (MMP-1) and matrix metalloprotease 3 (MMP-3). Moreover, Cyanidium caldarium extract is a potential mediator of the connective tissue growth factor (CTGF). To summarize, the described gene regulation demonstrates that the cosmetic according to the invention strengthens the overall structure of the ECM rather than merely the structure of the individual ECM components.
Example 5
Gene and Protein Expression Analysis of Cyanidium caldarium Extract on Human Dermal Fibroblasts by Means of qRT-PCR and ELISA
(32) Method:
(33) In the present example, the effect of Cyanidium caldarium extract on the gene and protein expression in fibroblasts (normal human dermal fibroblasts, NHDFs) was studied.
(34) To this end, primary human dermal fibroblasts (human dermal fibroblasts derived from neonatal skin (HDF), cryocenserved, Lifeline Cell Technology, obtained from CellSystems Biotechnologie Vertrieb GmbH, St. Katharinen, Germany) were first grown in Minimum Essential Medium (MEM) supplemented with Earle's salts (EMEM) (PAA Laboratories GmbH, Pasching, Austria) with the addition of 10% foetal calf serum (FBSfetal bovine serum, Invitrogen Ltd, UK), 1% nonessential amino acids (NEAA (100)non essential amino acids, PAA, Pasching, Austria), 1% L-glutamine (100) (Invitrogen Ltd, UK) and 1% penicillin/streptomycin (5000 U/ml penicillin and 5000 g/ml streptomycin/ml, Invitrogen Ltd, UK) at 37 C. and 5% CO.sub.2 until a suitable cell count had been achieved. For gene expression studies, the cells were sown in 6-well plates and grown to subconfluence (maximum 60%).
(35) Thereafter, the medium was drawn off from the cells and replaced by fresh medium with Cyanidium caldarium extract. The final concentration of Cyanidium caldarium extract in the medium was 100 ppm (=100 g/ml, based on extract dry matter). As a control, the cultures were grown without active substance, only with medium (vehicle) or with 10 ng/ml transforming growth factor beta 3 (TGF-3) as the positive control. All cultures were performed in triplicate (3 biological replications). To study the gene expression, the medium was drawn off after 24 hours' culturing, and the cells were lysed by addition of RNeasy Lysis Buffer (Qiagen, Hilden, Germany). The total RNA was isolated following the manufacturer's instructions. To summarize, the total RNA was isolated by means of the RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNA quality and quantity was determined by means of an Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Kit (Agilent Technologies, Inc., Santa Clara, Calif., USA).
(36) Each sample was analysed for the gene expression of collagen (COL1A1), elastin (ELN), matrix metalloproteinase-1 (MMP-1), fibrillin-2 (FBN2), connective tissue growth factor (CTGF), syndecan-2 (SDC2) and lumican (LUM) by means of quantitative real-time polymerase chain reaction (qRT-PCR). To this end, in each case 100 ng of total RNA were employed for the cDNA synthesis with the Super Script III First Strand Synthesis Super Mix (Invitrogen Ltd, UK) being employed in accordance with the manufacturer's instructions. The Quanti Tect SYBR Green PCR Kit (Qiagen, Hilden, Germany) was employed in accordance with the manufacturer's instructions for the PCR reaction. Besides the expression of the target genes, the expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, too, was determined quantitatively by way of reference. The following primer pairs were employed in each case for the target genes:
(37) TABLE-US-00002 COL1A1:60 C. Seq.IDNo.01 5-GCCTCGGAGGAAACTTTG-3 Seq.IDNo.02 5-GACCCATGGGACCTGAAG-3 ELN:56 C. Seq.IDNo.03 5-GCCCAGTTTGGCCTAGTG-3 Seq.IDNo.04 5-CAGCAGCACCGTATTTAG-3 MMP-1:60 C. Seq.IDNo.05 5-TGGGAGCAAACACATCTGA-3 Seq.IDNo.06 5-ATCACTTCTCCCCGAATCGT-3 FBN2:60 C. Seq.IDNo.07 5-TCGCCCGGCAGCAAACTCAG-3 Seq.IDNo.08 5-TCACACCGCTCACAGGGGCT-3 CTGF:60 C. Seq.IDNo.09 5-CAGGCTAGAGAAGCAGAGCC-3 Seq.IDNo.10 5-TGGAGATTTTGGGAGTACGG-3 SDC2:60 C. Seq.IDNo.11 5-CGTGGATCCTGCTCACCT-3 Seq.IDNo.12 5-CAATGGAGCTGTTGTCAAGG-3 LUM:56 C. Seq.IDNo.13 5-GGTTGAGCTGGATCTGTCCT-3 Seq.IDNo.14 5-AGTAGGATAATGGCCCCAGG-3 GAPDH:60 C. Seq.IDNo.15 5-ACCACAGTCCATGCCATCAC-3 Seq.IDNo.16 5-TCCACCACCCTGTTGCTGTA-3
(38) The PCR reactions were performed using an Opticon 1 (MJ Research, Waltham, Mass., USA). In all PCRs, duplicate determinations of the biological triplicates were carried out, and means of all measurements were calculated for the evaluation. The PCR protocol had the following profile: step 1), 15 minutes activation of the hot-start polymerase at 95 C.; step 2), 15 seconds denaturation at 94 C.; step 3), 30 seconds primer annealing at 56 C. or 60 C., depending on the primer pair; step 4), 30 seconds extension at 72 C. The number of cycles for steps 2)-4) was 44. The 2(-delta delta C(T)) method (Livak K J, and Schmittgen T D: Analysis of relative gene expression data using real time quantitative PCR and the 2 (-delta delta C(T)) method. Methods 2001, 25: 402-408) was employed for the relative comparison of the gene expression.
(39) To study the protein expression, the medium was drawn off after 24 hours of culturing, and the cells were lysed by ultrasonic treatment (10 seconds at 34-40 kHz). The total protein content of the sonicates was determined by the Bradford test (Bio-Rad Protein Assay, Bio-Rad Laboratories, Inc., Hercules, Calif., USA) following the manufacturer's instructions. Using ELISA technology, each sample was analysed for the protein content of elastin in the cell extract (human Elastin ELISA Kit, ABO Swiss Co., Ltd, Fujian, CN) and lumican (human Lumican ELISA Kit, ABO Swiss Co., Ltd, Fujian, CN) or procollagen (Procollagen Type I C-Peptide (PIP) EIA Kit, TAKARA BIO INC., JP) in the culture supernatant, following the instructions of the manufacturer in question. The absolute protein quantity was determined in relation to the total protein. The results are shown in
(40) To summarize, the gene regulation described corroborates the results of the gene chip analysis. The cosmetic according to the invention strengthens the overall structure of the ECM rather than merely its individual components.
Example 6
Effect of Cyanidium caldarium Extract on UVA-Stressed Human Epidermis Models
(41) Method:
(42) In the present example, the effect of Cyanidium caldarium extract on the gene and protein expression in epidermis skin models (reconstituted human epidermis) was studied.
(43) To this end, skin models (reconstructed epidermis of normal human keratinocytes, RHE/S/17, SkinEthic laboratories, Nice, F) were cultured for 24 hours at 37 C. and 5% CO.sub.2 in maintenance medium (SkinEthic laboratories, Nice, F). For gene and protein expression studies, the skin models were subsequently treated topically within each case 50 l of Cyanidium caldarium extract or the respective controls. After 1 hour, the sample excess was removed by blotting. The final concentration of Cyanidium caldarium extract in the medium was 750 ppm (=750 g/ml, based on extract dry matter). Culturing was performed without active substance or only with water (vehicle) by way of control. All cultures were performed in triplicate (3 biological replications). After 24 hours of culturing, the skin models were irradiated with a dose of 40 J/cm.sup.2 at 9.5 mW/cm.sup.2 UVA. Thereafter, the medium was exchanged, and the test substances or controls were reapplied. 24 hours after the second application, the skin models were transferred into RNAlater (RNA Stabilization Reagent, Qiagen, Hilden, Germany). The supernatant of the skin models (medium) was frozen (80 C.) with addition of Complete Protease Inhibitor Cocktail Tablets (F. Hoffmann-La Roche Ltd, Basle, CH) for further analysing the protein expression.
(44) Total RNA from the skin models was isolated by means of the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The RNA quality and quantity was determined using an Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Kit (Agilent Technologies, Inc., Santa Clara, Calif., USA).
(45) The gene expression of interleukin 1-alpha (IL1-), tumour necrosis factor-alpha (TNF-) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) of each sample was studied by means of quantitative real-time polymerase chain reaction (qRT-PCR). To this end, 100 ng of total RNA were employed in each case for the cDNA synthesis, with the Super Script III First Strand Synthesis Super Mix (Invitrogen Ltd, UK) being employed, following the manufacturer's instructions. The Quanti Tect SYBR Green PCR Kit (Qiagen, Hilden, Germany) was employed for the PCR reaction, following the manufacturer's instructions. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), too, was determined quantitatively by way of reference gene. The following primer pairs were employed in each case:
(46) TABLE-US-00003 NF-B: Seq.IDNo.17 5-CCTGGATGACTCTTGGGAAA-3 Seq.IDNo.18 5-TCAGCCAGCTGTTTCATGTC-3 TNF-: Seq.IDNo.19 5-CTCTGGCCCAGGCAGTCAGA-3 Seq.IDNo.20 5-GGCGTTTGGGAAGGTTGGAT-3 IL1-: Seq.IDNo.21 5-CACTCCATGAAGGCTGCATGG-3 Seq.IDNo.22 5-ACCCAGTAGTCTTGCTTTGTGG-3 GAPDH: Seq.IDNo.23 5-ACCACAGTCCATGCCATCAC-3 Seq.IDNo.24 5-TCCACCACCCTGTTGCTGTA-3
(47) The PCR reactions were performed using an Opticon 1 (MJ Research, Waltham, Mass., USA). In all PCRs, duplicate determinations of the biological triplicates were carried out, and means of all measurements were calculated for the evaluation. The PCR protocol had the following profile: step 1), 15 minutes activation of the hot-start polymerase at 95 C.; step 2), 15 seconds denaturation at 94 C.; step 3), 30 seconds primer annealing at 60 C.; step 4), 30 seconds extension at 72 C. The number of cycles for steps 2)-4) was 44. The 2(-delta delta C(T)) method (Livak K J, and Schmittgen T D: Analysis of relative gene expression data using real time quantitative PCR and the 2 (-delta delta C(T)) method. Methods 2001, 25: 402-408) was employed for the relative comparison of the gene expression.
(48) To study the protein expression, the supernatant of the skin models was drawn off and the total protein from the skin models was isolated in the form of a combined protocol in addition to the RNA by means of RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany). Specifically, the residual water phase was removed from the RNA isolation samples. 0.3 ml of 100% ethanol was added, and the samples were inverted and incubated at room temperature (IT) for 2-3 min. Thereafter, the samples were centrifuged at 2000g for 2 min at 4 C. The supernatant with the proteins was placed into a fresh tube (2 ml). The proteins were precipitated by addition of 1.5 ml of isopropanol and incubated at IT for 10 min. Thereafter, the samples were centrifuged at 12 000g for 10 min at 4 C. The pellet was taken up in 2 ml of guanidine/ethanol solution and incubated at IT for 20 min. Thereafter, the samples were centrifuged at 7500g for 5 min at IT. This wash step was repeated twice. After the addition of 2 ml of ethanol (100%) the samples were incubated at IT for 20 min. Thereafter, the samples were centrifuged at 7500g for 5 min at IT. The pellet was dried in the air for 5-10 min, taken up in 500 l of urea/DTT solution and incubated at IT for 1 h. To dissolve the proteins, the samples were heated at 95 C. for 3 minutes.
(49) The total protein content was determined by Bradford test (Bio-Rad Protein Assay, Bio-Rad Laboratories, Inc., Hercules, Calif., USA) following the manufacturer's instructions. By means of ELISA technique, each sample was analysed for the protein content of interleukin 1-alpha (IL1-) (HumanIL-1 ELISA Kit, (Invitrogen Ltd, UK) in the culture supernatant following the instructions of the respective manufacturer. The absolute protein quantity was determined in relation to the total protein.
(50) The results of this study are shown in
Example 7
Effect of Cyanidium caldarium Extract on Epidermal Stem Cells
(51) Method:
(52) In the present example, the activity of Cyanidium caldarium extract on epidermal stem cells (epidermal keratinocyte progenitors, HPEK) was studied.
(53) To this end, CnT-57 NHEK progenitor cells (normal human epidermal keratinocyte progenitor cells (HPEKp05)), derived from neonatal foreskin and cryoconserved at P2, CELLnTEC Advanced Cell Systems AG, Berne, Switzerland) were first cultured in supplemented CnT-57 medium (Progenitor Cell Targeted Epidermal Keratinocyte Medium based on low Bovine Pituitary Extract (BPE), CELLnTEC Advanced Cell Systems AG, Berne, Switzerland)+2% penicillin/streptomycin solution (5000 U/ml penicillin and 5000 g/ml streptomycin/ml, Invitrogen Ltd, UK) at 37 C. and 5% CO.sub.2 until a suitable cell density had been reached.
(54) To determine the colony forming efficiency (CFE), the cells were subsequently grown at low seeding density (800 cells/25 cm.sup.2) for 8 days in the presence of Cyanidium caldarium extract.
(55) The final concentration of Cyanidium caldarium extract in the medium was 100 ppm (=100 g/ml, based on algae dry matter). As a control, culturing was done without active substance, only with medium (vehicle). All the cultures were performed in triplicate (3 biological replications).
(56) After the cell culture medium had been drawn off, the cell colonies were stained with haematoxylin/eosin (CnT-ST-100 Stain Kit, CELLnTEC Advanced Cell Systems AG, Berne, Switzerland) following the manufacturer's instructions. The colony-forming units were counted under the microscope.
(57) For gene expression studies, the cells were furthermore grown at a seeding density of 3000 cells/25 cm.sup.2 for 7 days in the presence of Cyanidium caldarium extract until subconfluence (75%) had been reached.
(58) The final concentration of Cyanidium caldarium extract in the medium was 100 ppm (=100 g/ml, based on extract dry matter). As a control, the cultures were grown without active substance, only with medium (vehicle). All cultures were performed in triplicate (3 biological replications).
(59) To study the gene expression, the medium was drawn off and the cells were lysed by the addition of RNeasy Lysis Buffer (Qiagen, Hilden, Germany). Total RNA was isolated following the manufacturer's instructions. To summarize, the total RNA was isolated by means of the RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNA quality and quantity were determined by the Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Kit (Agilent Technologies, Inc., Santa Clara, Calif., USA).
(60) Each sample was analysed for the gene expression of CD34 (haematopoietic progenitor cell antigen), CTNNB1 (-catenine), ITGB1 (integrin beta-1), ITGA6 (integrin alpha-6), LRIG1 (leucine-rich repeats and immunoglobulin-like domains protein 1), DSG3 (desmoglein-3), MK167 (antigen KI-67) and KRT14 (keratin-14) by means of quantitative real-time polymerase chain reaction (qRT-PCR). To this end, 100 ng of total RNA were employed in each case for the cDNA synthesis, with the Superscript VILO cDNA Synthesis Kit (Invitrogen Ltd, UK) being employed following the manufacturer's instructions. The QuantiFast SYBR Green PCR Kit (Qiagen, Hilden, Germany) was employed for the PCR reaction, following the manufacturer's instructions. Besides the expression of the target genes, the expression of the B2M (2-microglobulin) gene, too, was determined quantitatively by way of reference. The following primer pairs were employed in each case for the genes:
(61) TABLE-US-00004 CD34: Seq.IDNo.25 5-CCACCAGAGCTATTCCCAAAAG-3 Seq.IDNo.26 5-GCGGCGATTCATCAGGAAAT-3 CTNNB1: Seq.IDNo.27 5-TGCCAAGTGGGTGGTATAGA-3 Seq.IDNo.28 5-TCAGATGACGAAGAGCACAGA-3 ITGB1: Seq.IDNo.29 5-ATGTGAATGCCAAAGCGAAG-3 Seq.IDNo.30 5-CTACCAACACGCCCTTCATT-3 ITGA6: Seq.IDNo.31 5-CTCTTCGGCTTCTCGCTG-3 Seq.IDNo.32 5-CCCGTTCTGTTGGCTCTCT-3 LRIG1: Seq.IDNo.33 5-GACCTGCCCTCCTGGAC-3 Seq.IDNo.34 5-GTAGGTTCGGCAAGTCCTCA-3 DSG3: Seq.IDNo.35 5-CGGCCTGCTGCTCCTTGGTC-3 Seq.IDNo.36 5-CACCGCTGTGCCTCTGGCAT-3 KI-67: Seq.IDNo.37 5-ATCGTCCCAGGTGGAAGAGTT-3 Seq.IDNo.38 5-ATAGTAACCAGGCGTCTCGTGG-3 KRT14: Seq.IDNo.39 5-GGCCTGCTGAGATCAAAGAC-3 Seq.IDNo.40 5-TCTGCAGAAGGACATTGGC-3 B2M: Seq.IDNo.41 5-GTGCTCGCGCTACTCTCTCT-3 Seq.IDNo.42 5-TTCAATGTCGGATGGATGAA-3
(62) The PCR reactions were performed using a StepOnePlus Real-Time PCR system (Life Technologies Corporation, CA, USA). For all PCRs, duplicate determinations of the biological triplicates were carried out, and means of all measurements were calculated for the evaluation. The PCR protocol had the following profile: step 1), 5 minutes activation of the hot-start polymerase at 95 C.; step 2), 10 seconds denaturation at 95 C.; step 3), 30 seconds primer annealing at 60 C. The cycle number of steps 2)+3) was 40. The 2(-delta delta C(T)) method (Livak K J, and Schmittgen T D: Analysis of relative gene expression data using real time quantitative PCR and the 2 (-delta delta C(T)) method. Methods 2001, 25: 402-408) was employed for the relative comparison of the gene expression.
(63) The results of this study are shown in
(64) To summarize, Examples 4 to 7 demonstrate that Cyanidium caldarium extract is a potent cosmetic for strengthening skin elasticity by the synthesis of elastin and the surrounding extracellular dermal structures such as lumican and collagen. Moreover, Cyanidium caldarium extract, when applied to UV-stressed skin, results, by virtue of its anti-inflammatory activity, in the restitution of the epidermal homeostasis of the skin. Furthermore, Cyanidium caldarium extract maintains the stem cell functions in the epidermis, and therefore assists in maintaining three of the most important functions by way of protection against skin ageing.
Example 8
Improving the Mechanical Properties of Hair by Treatment with Cyanidium Caldarium Extract
(65) A piece of human natural European hair tresses, remis, double-drawn, with a length of 23 cm (21 cm freehair) and a width of 2.0 cm, with a weight of 2.0 g and a dark brown colour, was wetted within 10 seconds under a running tap. Thereafter, 8 g of a 30% strength aqueous hydrogen peroxide solution were mixed with 4 g of Basler Blond Claire bleaching powder, 2 ml of a 25% strength aqueous ammonia solution were added, everything was remixed, and 8 g of the resulting paste were massaged into the piece of tresses with the aid of a comb and the hands, which had been protected. After an exposure time of 30 minutes at room temperature, the piece of tresses was washed for 2 minutes under running tap water with a temperature of approximately 35 C. and then dried with an electronic hair dryer for 3 minutes with simultaneous combing. The entire procedure was repeated once, starting with the wetting of the piece of tresses. Thereafter, 40 hairs were removed from the piece of tresses, and in each case the middle part of one hair with a length of 3 cm was crimped between two brass sleeves with internal plastic coating. The mean area of each individual hair was measured by means of Dia-stron FDAS760 fibre dimensional analysis system and UvWin PC application software. Thereupon, the hair samples were transferred into the sample cartridge of a Dia-stron Tensile Tester MTT 670 and in each case treated with fully demineralised water which had been brought to pH 7 with citric acid. Measurement of the individual hair strands with the single fibre method (extension 20%, rate 20 mm/min, gauge force 2, maximum force 200, break threshold 5, sample size 30 mm) was started. Thereafter, the hair samples were removed from the sample cartridge and, in a small plastic dish, covered with fully demineralised water. After 30 minutes, the water was removed and the hair samples were covered with leave-on hair conditioner (Table 2).
(66) TABLE-US-00005 TABLE 2 Test formulations. Data in percent by weight. Customary formulation known to the skilled worker were employed for preparing methods the formulations. 2.5% Cyanidium Raw material Vehicle caldarium extract A TEGINACID C (ceteareth-25) 0.5% 0.5% TEGO Alkanol 1618 2.0% 2.0% (cetearyl alcohol) Cyanidium caldarium extract 2.5% Water to 100% to 100% Methylchloroisothiazolinone, methyl 0.05% 0.05% isothiazolinone (Kathon CG) Lactic acid (10% in water) pH 4.0-4.5 pH 4.0-4.5
(67) After an exposure time of 30 minutes, each individual hair sample was washed for 6 seconds under the running water jet with a temperature of approximately 35 C. The hair samples were dried overnight at 22 C. and 50% relative atmospheric humidity. Thereafter, the measurement of the individual hair samples was repeated, starting with the transfer into the sample cartridge of the Dia-stron Tensile Tester MTT 670. The data are evaluated by means of a spreadsheet using the parameter modulus of elasticity (E module;
(68) As shown by the results of the tensile stress-elongation measurements, the addition of 2.5% Cyanidium caldarium extract to the leave-on hair conditioner caused an increase in the parameter E module by 25 N/mm.sup.2. This demonstrated that the tensile strength of damaged hair is increased by Cyanidium caldarium extract.
Example 9
Enhancement of the Skin Elasticity and Improvement of the Skin Structure by Cyanidium caldarium Extract
(69) To demonstrate the enhancement of the skin elasticity and improvement of the skin structure by the topical application of Cyanidium caldarium extract, a human randomized vehicle-controlled blind application monitoring was performed over a period of eight weeks.
(70) The panel comprised subjects aged 33-59 years (mean 478 years). Twenty subjects received vehicle formulation, 19 subjects the vehicle formulation comprising 1% Cyanidium caldarium extract and 21 subjects the vehicle formulation comprising 5% Cyanidium caldarium extract. The formulations were applied twice daily (in the morning and in the evening) to the inside of the forearm. 2 squeezes from a squeeze bottle (0.25-0.30 g) were applied by massaging in. The composition of the formulation is shown in Table 3:
(71) TABLE-US-00006 TABLE 3 Test formulations. Data in Percent by weight. Customary formulation methods known to the skilled worker were employed for preparing the formulations. Raw material Vehicle 1% Cyanidium 5% Cyanidium TEGO Care 450 (polyglyceryl-3 3.0 3.0 3.0 methylglucose distearate) TEGO Alkanol 18 (stearyl alcohol) 0.5 0.5 0.5 TEGIN M Pellets (glyceryl stearate) 1.0 1.0 1.0 TEGOSOFT CT (caprylic/capric 6.0 6.0 6.0 triglycerides) TEGOSOFT G 20 (octyldodecanol) 2.0 2.0 2.0 TEGOSOFT PC 41 (polyglyceryl-4 caprate) 2.0 2.0 2.0 Water 85.2 84.2 80.2 Xanthangum (Keltrol CG-SFT) 0.3 0.3 0.3 Cyanidium caldarium extract 1.0 5.0 Methylisothiazolinone, ethylhexylglycerol 0.1 0.1 0.1 (Euxyl K220) pH (NaOH) 5.5 5.5 5.5
(72) The application phase extended over eight weeks. The parameters measured were the skin elasticity, using a Cutometer MPA 580 (Courage+Khazaka electronic GmbH) and the consistency of the skin surface, using Visioscan VC 98 (Courage+Khazaka electronic GmbH), on the inside of the forearm at the time TO before the application phase and T8 after eight weeks. The data registered was the mean, which is calculated on the basis of the individual values of several repeated measurements. No formulation was applied on the evening before the respective measurement.
(73) As demonstrated by the human in-vivo study, a reduction in the skin elasticity parameter R1 was observed over the period of eight weeks. R1 is the remaining deformation of the skin after the first extension cycle and represents the ability of the skin to return to its original state. The parameter R1 increases greatly with age. The reduction of R1 was approximately 5 mm in the group of persons who applied 1% Cyanidium caldarium extract, while the reduction in the group of persons who applied 5% Cyanidium caldarium extract was three times as pronounced (
(74) A similar trend was found for the skin elasticity parameter R4. R4 is the minimum amplitude of the last of the three extension cycles of the skin and visualizes fatigue symptoms of the skin since the redeformation ability is reduced with every additional extension (
(75) An increase in the skin texture parameter tex was also found. The parameter tex describes the uniformity of the skin tone. An improved tex value is associated with improved skin appearance. A pronounced increase of the tex parameter value by 30 and 24 units was found, after eight weeks, both in the 1% Cyanidium and in the 5% Cyanidium group, respectively (
(76) Finally, a reduction of the skin surface parameter and of the volume parameter was found. The skin surface parameter describes the total surface of the measured skin, while the volume parameter represents the volume required for filling the wrinkles in the skin. In the case of a reduction of the depth and number of wrinkles, the skin surface parameter and the volume parameter should decrease by 7 units was found in the 1% Cyanidium group while the 5% Cyanidium group showed a greater reduction of this value by 15 units (
(77) The results of the human in-vivo study demonstrate that the use of the cosmetic formulation according to the invention results in an enhanced skin elasticity and skin firmness, an improvement of the skin structure and a reduction of the depth and number of wrinkles.
(78) Examples of Formulations
(79) Example of an after-Shave Lotion Formulation
(80) TABLE-US-00007 Percent by Raw material INCI weight ABIL Care 85 Bis-PEG/PPG-16/16 1.5 PEG/PPG16/16 dimethicone; caprylic/capric triglyceride TEGOSOFT CT Caprylic/capric triglyceride 3.0 Cyclomethicone Cyclomethicone 3.0 Tocopheryl acetate Tocopheryl acetate 0.5 Frescolate ML Menthyl lactate, H&R 0.5 TEGO SMO 80 V Polysorbate 80 0.5 Water Water to 100 Cyanidium caldarium extract 1.1 Glycerol 2.0 Ethanol 15.0 TEGO Carbomer 141 Carbomer 0.3 Xanthan gum Xanthan gum 0.1 Mineral oil (30 mPas) Mineral oil 1.6 Sodium hydroxide (10% in q.s. water) Preservative, perfume q.s.
Example of an O/W after-Shave Balm Formulation
(81) TABLE-US-00008 Percent by Raw material INCI weight AXOL C 62 pellets Glyceryl stearate citrate 1.50 TEGO Alkanol 1618 Cetearyl alcohol 1.00 TEGOSOFT CT Caprylic/capric triglyceride 5.00 TEGOSOFT P Isopropyl palmitate 2.00 Cyclomethicone Cyclomethicone 5.50 Glycerol Glycerol 5.00 Cyanidium caldarium extract 2.0 Water to 100 TEGO Carbomer 141 Carbomer 0.20 TEGOSOFT P Isopropyl palmitate 0.80 Sodium hydroxide (10% in water) q.s. Preservative, perfume q.s.
Example of a PEG- and Sulphate-Free Conditioning Shampoo Formulation
(82) TABLE-US-00009 Percent by Raw material INCI weight REWOTERIC AM C Sodium cocoamphoacetate 15.0 REWOPOL SB F 12 P Disodium lauryl sulfosuccinate 3.8 Water Water to 100 Cyanidium caldarium extract 0.5 TEGO Betaine F 50 Cocamidopropyl betaine 10.0 VARISOFT PATC Palmitamidopropyltrimonium 2.3 chloride REWOMID SPA Isostearamide MIPA 1.0 Citric acid Citric acid q.s. Preservative, perfume q.s.
Example of a Sprayable PEG-Free Hair Milk Formulation
(83) TABLE-US-00010 Percent by Raw material INCI weight Lactic acid, 80% Lactic acid 0.40 Water Water to 100 Cyanidium caldarium 1.50 extract TEGO Amid S 18 Stearamidopropyl dimethylamine 1.20 TEGIN G 1100 pellets Glycol distearate 0.60 TEGO Care PS Methyl glucose sesquistearate 1.20 TEGOSOFT DEC Diethylhexyl carbonate 0.30 Preservative, perfume q.s.
Example of a Leave-in Conditioning Foam Formulation with Heat Protection Activity
(84) TABLE-US-00011 Percent by Raw material INCI weight ABIL T Quat 60 Silicone quaternium-22 0.5 TAGAT CH 40 PEG-40 hydrogenated castor oil 0.5 TEGO Betaine 810 Capryl/capramidopropyl betaine 2.0 Water Water to 100 Cyanidium caldarium extract 1.0 TEGOCEL HPM 50 Hydroxypropyl methylcellulose 0.3 VARISOFT 300 Cetrimonium chloride 1.3 Citric acid, 30% q.s. Preservative, perfume q.s.
Example of a Sun Protective Caring Body Lotion Formulation
(85) TABLE-US-00012 Percent by Raw material INCI weight TEGO Care 165 Glyceryl stearate; PEG-100 stearate 3.5 TEGIN M pellets Glyceryl stearate 3.0 TEGO Alkanol 18 Stearyl alcohol 2.0 TEGOSOFT XC Phenoxyethyl caprylate 8.0 Sylvaclear WF1500V Polyamide-4, ArizonaChemical 4.0 TEGOSOFT APM PPG-3 myristyl ether 2.0 Ethylhexyl Ethylhexyl methoxycinnamate 3.0 methoxycinnamate Benzophenone-3 Benzophenone-3 5.0 Octocrylene Octocrylene 7.0 Menthyl anthranilate Menthyl anthranilate 4.0 TEGO Sun T 805 Titanium dioxide; 4.0 Trimethoxycaprylylsilane Xanthan gum Xanthan gum 0.2 Glycerol Glycerol 2.0 Water Water to 100 Cyanidium 1.0 caldarium extract Allantoin Allantoin 0.1 Tromethamin, 20% in Tris(hydroxymethyl)aminomethane q.s. water, Merck Preservative, perfume q.s.
Example of a Sun Care Soft Cream (Cold Processed) SPF 40+ Formulation
(86) TABLE-US-00013 Percent by Raw material INCI weight ABIL Care XL 80 Bis-PEG/PPG-20/5 PEG/PPG-20/5 2.5 dimethicone; methoxy PEG/PPG-25/4 dimethicone; caprylic/capric triglyceride TEGO Sun TDEC 45 Titanium dioxide; diethylhexyl 11.0 carbonate; polyglyceryl-6 polyhydroxystearate Ethylhexyl 8.0 methoxycinnamate Octocrylene 7.5 Butyl 3.0 methoxydibenzoylmethane Tocopheryl acetate 0.5 TEGO Carbomer 141 Carbomer 0.25 TEGO Carbomer Acrylates/C10-30 alkyl acrylate 0.25 341 ER crosspolymer Xanthan gum 0.2 TEGOSOFT P Isopropyl palmitate 2.0 Water Water to 100 Glycerol 3.0 Cyanidium caldarium 1.0 extract Sodium hydroxide (10% in q.s. water) Preservative, perfume q.s.
Example of an after-Sun Foam Formulation
(87) TABLE-US-00014 Percent by Raw material INCI weight TEGINACID H Glyceryl stearate; ceteth-20 3.60 TEGO Alkanol 18 Stearyl alcohol 1.20 TEGOSOFT CT Caprylic/capric triglyceride 3.60 TEGOSOFT liquid Cetearyl ethylhexanoate 1.80 TEGOSOFT P Isopropyl palmitate 1.80 TEGOSOFT DO Decyl oleate 3.00 Glycerol 1.80 Panthenol 0.60 Allantoin 0.10 Water Water to 100 Cyanidium caldarium 1.2 extract TEGO Betaine 810 Capryl/capramidopropyl betaine 7.00 Sodium hydroxide (10% in q.s. water) Preservative, perfume q.s.
Example of an O/W Cream Formulation
(88) TABLE-US-00015 Percent by Raw material INCI weight TEGO Care PSC 3 Polyglyceryl-3 dicitrate/stearate 3.00 TEGIN M pellets Glyceryl stearate 1.20 TEGO Alkanol 18 Stearyl alcohol 0.80 TEGOSOFT P Isopropyl palmitate 7.00 Prunus amygdalus dulcis oil 5.00 TEGOSOFT CT Caprylic/capric triglyceride 4.50 TEGOSOFT TIS Triisostearin 3.50 Water Water to 100 Cyanidium caldarium extract 2.0 Glycerol 3.00 Keltrol CG-SFT, CP Kelco Xanthan gum 0.30 Sodium hydroxide (10% in q.s. water) Preservative, perfume q.s.
Example of an Anti-Cellulite Body Lotion Formulation
(89) TABLE-US-00016 Percent by Raw material INCI weight TEGO Care LTP Sorbitan laurate; polyglyceryl-4 1.50 laurate; dilauryl citrate TEGOSOFT CI Cetearyl isononanoate 10.00 TEGOSOFT DEC Diethylhexyl carbonate 3.50 TEGOSOFT OP Ethylhexyl palmitate 1.10 TEGO Carbomer 140 Carbomer 0.15 TEGO Carbomer 141 Carbomer 0.15 Xanthan gum 0.10 Glycerol 3.00 Water Water to 100 Cyanidium caldarium extract 3.0 Sodium hydroxide (10% in q.s. water) Preservative, perfume q.s.
Example of a Moisturizing Anti-Cellulite Cream Gel Formulation
(90) TABLE-US-00017 Percent by Raw material INCI weight TEGOSOFT DC Decyl cocoate 8.00 TEGOSOFT OP Ethylhexyl palmitate 5.00 TEGOSOFT CR Cetyl ricinoleate 2.00 TEGO Alkanol 1618 Cetearyl alcohol 1.00 Tocopheryl acetate 0.50 TEGOSOFT PSE 141 G Sucrose stearate 2.00 TEGO Care CG 90 Cetearyl glucoside 0.50 Glycerol 4.00 Panthenol 0.50 Water Water to 100 Cyanidium caldarium extract 5.6 TEGO Carbomer 341 ER Acrylates/C10-30 alkyl acrylate 0.45 crosspolymer Sodium hydroxide (10% in q.s. water) Preservative, perfume q.s.
Example of a New-Generation Anti-Ageing Moisturizer Formulation
(91) TABLE-US-00018 Percent by Raw material INCI weight TEGO Care 450 Polyglyceryl-3 methylglucose 3.00 distearate TEGIN M pellets Glyceryl stearate 2.50 TEGO Alkanol 18 Stearyl alcohol 1.50 TEGOSOFT MM Myristyl myristate 1.00 TEGOSOFT DO Decyl oleate 8.00 TEGOSOFT OS Ethylhexyl stearate 9.00 Phytosphingosine SLC Salicyloyl phytosphingosine 0.10 Glycerol 3.00 Water Water to 100 Cyanidium caldarium extract 5.0 TEGO Carbomer 134 Carbomer 0.20 TEGOSOFT P Isopropyl palmitate 0.80 Sodium hydroxide (10% in q.s. water) Preservative, perfume q.s.
Example of a W/O Lotion Formulation
(92) TABLE-US-00019 Percent by Raw material INCI weight ISOLAN GPS Polyglyceryl-4 diisostearate/ 3.0 polyhydroxystearate/sebacate Paracera W 80, Paramelt Microcrystalline wax 0.1 B.V. Hydrogenated castor oil 0.1 TEGOSOFT DEC Diethylhexyl carbonate 7.0 TEGOSOFT TIS Triisostearin 3.0 Cyclomethicone 7.0 Glycerol 3.0 Magnesium sulphate 1.0 heptahydrate TEGO Cosmo C 100 Creatine 1.0 Skinmimics Ceteareth-25; glycerol; cetyl alcohol; 5.0 behenic acid; cholesterol; ceramide NP; ceramide NS; ceramide EOS; ceramide EOP; ceramide AP; caprooyl phytosphingosine; caprooyl sphingosine Water Water to 100 Cyanidium caldarium 4.5 extract Preservative, perfume q.s.
Example of a Winter Skin Moisturizer Formulation
(93) TABLE-US-00020 Percent by Raw material INCI weight ABIL EM 90 Cetyl PEG/PPG-10/1 2.0 dimethicone Hydrogenated castor oil 0.5 Paracera W 80, Paramelt Microcrystalline wax 0.5 B.V. TEGOSOFT CT Caprylic/Capric triglyceride 10.0 TEGOSOFT HP Isocetyl palmitate 10.0 TEGOSOFT DEC Diethylhexyl carbonate 5.0 Water Water to 100 Sodium chloride Sodium chloride 0.5 Cyanidium caldarium extract 15 Preservative, perfume q.s.