Tetraselmis Extract

Abstract

The present invention relates to a novel extract and a method for obtaining such an effective Tetraselmis extract which was found to have advantages in dermatological treatments and can be employed as a topical cosmetic.

Claims

1. A Tetraselmis suecica extract comprising the following based on the extract dry weight: a) total minerals ≥10 wt. % of the total composition, b) mannitol ≥5 wt. % of the total composition, c) total galactose, which is the sum of free and bound galactose, ≥3 wt. % of the total composition, d) total glucose, which is the sum of free and bound glucose ≥4 wt. % of the total composition, e) total amino acids ≥3 wt. % of the total composition, f) total nitrogen ≥2 wt. % of the total composition.

2. A Tetraselmis suecica extract according to claim 1 comprising the following based on the extract dry weight: a) total minerals 11 to 25 wt. % of the total composition, b) mannitol 6 to 15 wt. % of the total composition, c) total galactose, which is the sum of free and bound galactose, 4 to 15 wt. % of the total composition, d) total glucose, which is the sum of free and bound glucose, 4 to 10 wt. % of the total composition, e) total amino acids 4 to 10 wt. % of the total composition, f) total nitrogen 3 to 5 wt. % of the total composition.

3. Method of obtaining a Tetraselmis extract comprising the step of extracting a cell material comprising viable, freeze-dried or dried cells of Tetraselmis, with a liquid extractant selected from the group consisting of 2-propanone, ethanol, water, methanol, isopropanol and mixtures of two or more of these extractants, and wherein the extraction comprises: a) exposition of the cell material to the extractant for up to 8 h at a temperature higher than 60° C., and b) removal of the cell material to obtain the extract.

4. A method according to claim 3, wherein the Tetraselmis extract comprises the following based on the extract dry weight: a) total minerals ≥10 wt. % of the total composition, b) mannitol ≥5 wt. % of the total composition, c) total galactose, which is the sum of free and bound galactose, ≥3 wt. % of the total composition, d) total glucose, which is the sum of free and bound glucose ≥4 wt. % of the total composition, e) total amino acids ≥3 wt. % of the total composition, f) total nitrogen ≥2 wt. % of the total composition.

5. Method according to claim 3, wherein the Tetraselmis classification is Tetraselmis sp.

6. A combination composition comprising Tetraselmis extract according to claim 1 further comprising niacinamide.

7. Combination composition according to claim 6, wherein the weight ratio range of Tetraselmis extract to niacinamide is 1:10000 to 1:1, wherein all weights are calculated based on dry weights.

8. A Tetraselmis extract concentrate comprising: a) 0.5 to 80 wt. % Tetraselmis extract according to claim 1, calculated based on dry weights, b) 0.5 to 90 wt. % water, c) 0.5 to 90 wt. % liquid carrier, d) optionally 0.1 to 5 wt. % of one or more preservative or preservative system.

9. A liquid Tetraselmis extract concentrate comprising: a) 0.5 to 10 wt. % Tetraselmis extract according to claim 1, calculated based on dry weights, b) 30 to 70 wt. % water, c) 20 to 50 wt. % glycerin, d) 5 to 20 wt. % 1,2-pentanediol, e) optionally 0.1 to 5 wt. % of one or more preservative or preservative system.

10. Solid Tetraselmis extract concentrate comprising: a) 0.5 to 10 wt. % Tetraselmis extract according to claim 1, calculated based on dry weights, b) 0.5 to 8 wt. % water, c) 50 to 98 wt. % solid carrier.

11. A method of treating or preventing dysfunctions of human hair and/or skin, seborrhoeic dermatitis, acne vulgaris, wound healing, tissue regeneration, postinflammatory hyperpigmentation, inflammatory related diseases, dandruff or Pityriasis versicolor comprising administering to a subject in need thereof a Tetraselmis extract according to claim 1.

12. A method of treating a skin disease comprising administering a dermatological or therapeutic product comprising a Tetraselmis extract according to claim 1 and optionally auxiliary substances.

13. Non-therapeutic or cosmetic product comprising a Tetraselmis extract according to claim 1 and optionally auxiliary substances and/or perfumes, wherein the cosmetic product is a skin and/or hair care product.

14. Cosmetic product according to claim 13, wherein the amount of Tetraselmis extract or Tetraselmis extract concentrate calculated based on dry weights, in the product is 0.0001 to 10 wt. %.

15. A method of using a Tetraselmis extract according to claim 1 for application on, caring, cleansing, sun-protecting or protecting the skin or for reduction of sebum.

16. A method of using a Tetraselmis extract according to claim 1 for; a) stimulation of cutaneous junctions, b) stimulation of cutaneous antimicrobial peptides, c) reduction of COX-2 gene expression and prostaglandin mediated effects, d) reduction of post-inflammatory hyperpigmentation, e) stimulation of filaggrin.

17. A method of using a Tetraselmis extract according to claim 1: a) for improvement of epidermal integrity of the skin, b) for prevention of external stimuli such as air pollution or particulate matter induced effects, c) for prevention of skin barrier dysfunction.

18. Cosmetic product according to claim 13 further comprising one or more of the following: other sebum reducing agents, anti-acne agents, anti-dandruff agents, other anti-inflammatory agents, TRPV1 antagonists, anti-itch agents, anti-microbial agents, especially anti-Propionibacterium acnes agents, anti-Malassezia agents.

19. (canceled)

20. A method of using the cosmetic product according to claim 18 for: a) application on, caring, cleansing, sun-protecting or protecting the skin or for reduction of sebum, b) stimulation of cutaneous junctions, c) stimulation of cutaneous antimicrobial peptides, d) reduction of COX-2 gene expression and prostaglandin mediated effects, e) reduction of post-inflammatory hyperpigmentation, f) stimulation of filaggrin, g) improvement of epidermal integrity of the skin, h) prevention of external stimuli such as air pollution or particulate matter induced effects; or i) prevention of skin barrier dysfunction.

Description

EXPERIMENTAL SECTION

Example 1: Preparation of a Tetraselmis suecica Extract

[0200] 3 g freeze-dried Tetraselmis suecica biomass and 30 g of water were mixed and stirred for 2 hours at 80° C. The liquid extract was separated from the biomass, 30 g of water was added to the extracted biomass and the mixture was stirred for another 2 hours at 80° C. The liquid was separated from the biomass by centrifugation, both extract solutions were combined, and the water was removed by freeze-drying. The extractions were performed with 3 different biomass batches.

[0201] For comparison, an aqueous extract according to the description in US2010143267 A1 was prepared from the same 3 biomass batches and water was removed by freeze-drying.

TABLE-US-00001 TABLE 1 Tetraselmis suecica extract obtained by extraction at room temperature and at 80° C. Condition of extraction Mean yield Appearance 80° C. 38.4 ± 0.2% Beige greenish solid Room temperature 40.0 ± 0.9% Intensive dark green solid (18 to 23° C.)

[0202] Extraction upon heating gives a well comparable, very slightly lower, high extraction yield when compared to extraction at room temperature, but it surprisingly gives a much lighter colored extract which is especially advantageous for the use as cosmetic ingredient as consumers prefer low colored products. Heat treatment furthermore has the additional advantage that enzymes in the biomass are inactivated which is especially advantageous when using viable or non-inactivated biomass. Additionally, microbiological contamination by bacteria, fungi or yeasts, which is especially challenging for extractions with water or extractant systems with high water content at low temperatures is prevented by extracting at higher temperatures (>50° C.)

TABLE-US-00002 TABLE 2 Composition of Tetraselmis suecica extract obtained by extraction at 80° C. Mean content Mean content [wt.-%] [wt.-%] (Extract (Extract obtained at obtained Room temperature Substance class at 80° C.) (18 to 23° C.)) Sum of minerals 20.3 ± 0.6  21.4 ± 0.8  Containing but not limited to: Sodium Na.sup.+ 5.7 ± 0.2 5.7 ± 0.3 Potassium K.sup.+ 3.6 ± 0.1 3.6 ± 0.2 Magnesium Mg.sup.2+ 0.7 ± 0.1 0.7 ± 0.0 Calcium Ca.sup.2+ 0.8 ± 0.1 0.9 ± 0.0 Chloride Cl.sup.− 6.9 ± 0.2 7.1 ± 0.2 Sulfate SO.sub.4.sup.2− 2.1 ± 0.1 2.6 ± 0.1 Phosphate PO.sub.4.sup.3− 0.4 ± 0.2 0.9 ± 0.4 Mannitol 11.8 ± 0.9  11.6 ± 0.7  Total galactose (free and bound)* 9.7 ± 0.7 7.8 ± 0.4 Total glucose (free and bound)* 7.0 ± 0.8 3.5 ± 0.0 Sum of amino acids 8.4 ± 0.8 9.2 ± 0.7 Containing but not limited to: Glutamic acid 2.88 ± 0.30 2.77 ± 0.27 Alanine 1.11 ± 0.08 1.10 ± 0.10 Arginine 0.80 ± 0.30 0.17 ± 0.02 Ornithine 0.54 ± 0.28 1.26 ± 0.17 Citruline 0.53 ± 0.23 0.74 ± 0.03 Asparagine 0.39 ± 0.04 0.19 ± 0.02 Taurine 0.39 ± 0.04 0.35 ± 0.04 Lysine 0.37 ± 0.04 0.49 ± 0.02 Aspartic acid 0.27 ± 0.10 0.76 ± 0.03 Proline 0.14 ± 0.02 0.18 ± 0.02 Glutamine 0.12 ± 0.06 0.07 ± 0.02 Total Nitrogen** 4.22 ± 0.17 4.91 ± 0.16 *determined after hydrolysis and derivatization by GC **determined by nitrogen analyzer

[0203] In comparing the values for the extract at RT and at 80° C., it is apparent that certain components are conserved whereas others are shifted respective the lower temperature extraction. In particular, the content of galactose and glucose differs significantly (is increased from 7.8 to 9.7 wt.-% and 3.5 to 7.0 wt-%, respectively). Also, the content of certain amino acids is significantly enhanced, such as Arginine and Asparagine (0.8 from 0.17 wt.-% and 0.39 from 0.19 wt.-%, respectively). Some other amino acids are selectively decreased such as Aspartic acid which drops from 0.76 wt.-% to 0.27 wt.-% and Ornithine which drops from 1.26 wt.-% to 0.54 wt.-%. Overall most mineral compounds are conserved except for phosphate.

Example 2: Preparation of Liquid Versions of Tetraselmis suecica Extract

[0204] To 4.6 g Tetraselmis suecica extract dry matter obtained by extraction at 80° C. according to Example 1, 97 g water, 79 g glycerin (99.5%), 0.5% sodium benzoate and 0.2% potassium sorbate (both based on the total weight of the liquid mixture) were added, and the pH of the mixture was adjusted with help of lactic acid to 4.5 giving a yellow beige to light brownish solution, refractive index (n.sub.20/D): 1.396, mannitol content: 0.29%.

[0205] For another liquid version, 25 g Tetraselmis suecica extract dry matter obtained by extraction at 80-90° C. according to Example 1, was dissolved in 483 g water and 392 g glycerin (99.5%) and 100 g of 1,2-pentanediol (Hydrolite-5) were added. A light yellow-greenish, clear to slightly turbid solution was obtained; color according to L*a*b* color system: L* 88.4, a* −13.6, b* 47.5, pH 7.6, mannitol content: 0.28%.

Example 3: Effect of Tetraselmis suecica Extract (Dried) on the Total Lipid Content of Ex Vivo Human Sebaceous Glands

[0206] Organ culture of human sebaceous glands micro-dissected from human skin explants was performed to evaluate the modulatory activity of Tetraselmis suecica extract prepared according to the description given in Example 1 on the sebum level. The extract is employed in the dried form.

[0207] After removal of the epidermis of the full thickness skin sample, the sebaceous glands were carefully removed using micro-scissors and scalpel. The micro-dissected sebaceous glands were then pooled in groups of 8 and cultured up to day 6 in a 24 well plate immersed in 500 μl of modified Williams' E medium. After 24 hours of acclimation the culture medium was changed and substituted with the medium containing the extract to be tested. The medium was renewed at day 3 and 5 of culture. At day 6 the glands were collected and used for the quantification of lipids and proteins. In order to make the estimated productivity of the glands comparable, which are variable in biomass, their total sebum content was estimated and divided by the proteins extracted from the gland tissue, obtaining the ratio between the produced sebum and the tissue proteins (i.e. mg of lipids/mg of proteins).

[0208] To do so, each sebaceous glands group was homogenized in 100 μl of isopropyl alcohol to extract lipids and let the proteins undissolved. After centrifugation the supernatant containing the extracted sebum was collected and analyzed. The remaining pellet was dried using a vacuum dry evaporator and then minced in presence of 50 μl of protein lysis buffer. After an appropriate incubation time, this extractive mixture was centrifuged, and the supernatant was collected and analyzed. The lipids dissolved in isopropyl alcohol and the proteins dissolved in the lysis buffer were quantified by infrared spectroscopy using a Direct Detect IR Spectrometer (Millipore). The total lipid amount was obtained by normalizing the quantified lipids upon the quantified proteins (i.e. mg of lipids/mg of proteins). The amounts of normalized lipids, i.e. the sebum produced by each group of sebaceous glands, obtained from the treated groups was compared to that of the untreated control group and the modulatory activity was calculated in percentage. As positive control, a 5 μM Capsaicin treatment was included in the experimental design. Capsaicin is an active component of chili peppers suitable to inhibit sebogenesis [Tóth et al., J. Invest. Derm. (2009), 129: 329-339]. For statistical analysis, differences among groups were evaluated by one-way anova with permutation test followed by Dunnett's permutation test.

[0209] To better understand the response to the extract, a viability test was performed in parallel at day 1 and day 6 of organ culture. Resazurin was added to the wells (1:11) and let incubate for 2 hours. At the end of the incubation an aliquot of the medium was read with a fluorometer (excitation: 560 nm, emission: 590 nm). The medium was then replaced with normal medium for 2 hours in order to eliminate residual resazurin. After this the medium was replaced again with medium containing the test samples. The viability in each well was measured as the difference in percentage between day 6 and day 1.

[0210] To evaluate donor responsiveness and interindividual variability the extract was tested on sebaceous glands obtained from skin samples of three different donors.

TABLE-US-00003 TABLE 3 Effect of Tetraselmis suecica water-extract (dried)on lipids and viability of micro-dissected human sebaceous glands Parameter Test sample Donor 1 Donor 2 Donor 3 Reduction of lipids 5 μM (=1.5 ppm) 11 28 14 at day 6 versus Capsaicin untreated [%]* 0.3 ppm extract 19 33 18 (extraction at 80° C.) Viability [%] Untreated 93 100 81 5 μM (1.5 ppm) 92 99 85 Capsaicin 0.3 ppm extract 108 101 83 (extraction at 80° C.) *All results were statistically significant versus untreated with p < 0.01
In the present cell tests, ex vivo and in vitro, and generally for biological tests, the dried form of the Tetraselmis extract is employed to avoid side effects resulting from solvents, glycerin or the preservative system.

[0211] The results show that Tetraselmis suecica water extract (dried) obtained by extraction at 80° C. is surprisingly a highly effective reducer of the normalized total lipids, i.e. sebum content of human sebaceous glands without affecting their viability. It is more effective than the positive control capsaicin and this even at a 5-fold lower concentration. Furthermore, the sebaceous glands obtained from all three donors responded to the extract (donor responsiveness: 100%).

Example 4: Synergistic Effect of Tetraselmis suecica Extract (Dried) and Niacinamide on the Total Lipid Content of Ex Vivo Human Sebaceous Glands

[0212] The same experimental set-up as described in Example 3 was used to evaluate the combination of Tetraselmis suecica extract and niacinamide for synergistic activity.

[0213] Kull's equation for calculation of the synergism index SI was used:

SI=C×D/A+C×E/B

With

[0214] A=lipid reduction by Tetraselmis suecica extract at concentration x
B=lipid reduction by niacinamide at concentration y
C=lipid reduction by the combination of Tetraselmis suecica extract at concentration x/2 and niacinamide at concentration y/2
D=Factor for Tetraselmis suecica extract=>0.5 (due to half concentration tested in the combination)
E=Factor for niacinamide=>0.5 (due to half concentration tested in the combination)
A SI=1 is obtained for additive activity of the two combined components, whereas a SI<1 proves antagonistic activity (observed efficacy is lower than additive) and SI>1 proves synergistic activity (observed efficacy is higher than additive). Results of this experiment are summarized in Table 4.

TABLE-US-00004 TABLE 4 Tetraselmis suecica extract and niacinamide on the total lipid content of ex vivo human sebaceous glands Parameter Test sample Donor 1 Reduction of lipids at day 6 0.3 ppm extract 11 versus untreated [%]* 100 ppm niacinamide 6 0.15 ppm extract + 13  50 ppm niacinamide Untreated 81 Viability [%] 0.3 ppm extract 79 100 ppm niacinamide 78 0.15 ppm extract + 81  50 ppm niacinamide *All results were statistically significant versus untreated with p < 0.01 SI = 13 × 0.5/11 + 13 × 0.5/6 = 1.674

[0215] The obtained SI of 1.674 clearly proves that a combination of Tetraselmis suecica water extract and niacinamide surprisingly synergistically reduce the total lipids content, i.e. sebum level of human sebaceous glands.

Example 5: Effect of Tetraselmis suecica Extract (Dried) on the Gene Expression of Human Sebocytes

[0216] Dermal primary human sebocytes (from face (T-zone) localization, Caucasian donor, purchased from Zen-bio) were cultivated in sebum basal medium at 5% CO.sub.2 at 37° C. according to the supplier instructions. Sebocytes were treated for 24 hours with Tetraselmis suecica water extract obtained according to Example 1 by extraction at 80° C. at 0.01% and 0.1% or DMSO as vehicle control. Each experiment was performed in triplicate. Genomic target expression levels in extract treated cells were measured by RT-qPCR comparing to DMSO treatment.

[0217] Total RNA with miRNAs from sebocytes stimulated with the extract over 24 h, was extracted and purified using Qiaquick RNA Isolation Kit (from Quiagen), following the manufacturer's instructions. For mRNA target quantitation, total RNA was reverse-transcribed with the Superscript VILO cDNA Synthesis Kit (ThermoFisher) according to the manufacturer's instructions. The purity of the isolated RNA was determined by spectrophotometry: ratio 260/280≥1.5 to 2 (RNA extract is free of protein contamination). RQ values were calculated and the results were normalized to endogenous control GAPDH expression. Statistical analysis was performed using two-tailed unpaired T-test (*p-value<0.05). Results of this experiment are summarized in Table 5.

TABLE-US-00005 TABLE 5 Modulation of gene expression of primary human sebocytes after treatment with Tetraselmis suecica water dry extract Not treated 0.01% Genes/RQ values per (DMSO extract 0.1% extract treatment control) (p value) (p value) SREBF1 (SREBP-1) [sterol 1.00 ± 0.04 0.81 ± 0.03 0.59 ± 0.03 regulatory element binding (p < 0.05) (p < 0.001) transcription factor 1] DGAT1 [diacylglycerol O- 1.01 ± 0.08 0.81 ± 0.04 0.69 ± 0.03 acyl-transferase 1] (n.s.) (p < 0.05)  MGAT1 [mannosyl (alpha- 1.00 ± 0.05 0.76 ± 0.01 0.52 ± 0.04 1,3-)-glycoprotein beta-1,2-N- (p < 0.05) (p < 0.01)  acetylglucosaminyltransferase] SCD [stearoyl-CoA 1.01 ± 0.09 0.92 ± 0.06 0.59 ± 0.02 desaturase] (n.s.) (p < 0.05) PTGS2 (COX-2) 1.01 ± 0.09 0.44 ± 0.02 0.20 ± 0.01 [prostaglandin-endoperoxide (p < 0.01) (p < 0.001) synthase 2] NR1H3 (LXRa) [nuclear 1.01 ± 0.10 0.81 ± 0.07 0.64 ± 0.03 receptor subfamily 1 group H] (n.s.) (p < 0.05)  APOC1 1.01 ± 0.07 0.73 ± 0.05 0.76 ± 0.07 [apolipoprotein C1] (p < 0.05) (p = 0.067) ACAT1 1.01 ± 0.09 0.66 ± 0.05 0.73 ± 0.06 [acetyl-CoA acetyltransferase (p < 0.05) (p = 0.064) 1] APPL1 [adaptor protein, 1.00 ± 0.04 0.63 ± 0.05 0.73 ± 0.07 phosphotyrosine interacting (p < 0.01) (p < 0.05)  with PH domain and leucine zipper 1] ADIPOR1 1.00 ± 0.07 0.72 ± 0.02 0.72 ± 0.06 [adiponectin receptor 1] (p < 0.05) (p < 0.05) 

[0218] The results clearly show that Tetraselmis suecica water extract repress a large majority of genes involved in fatty acid, triglycerides and cholesterol production and thereby reduce the lipid, i.e. sebum production of sebocytes. The major pathways analyzed and known to regulate lipid synthesis and storage and sebaceous gland size, are involved.

[0219] Tetraselmis suecica water dry extract is able to modulate genes involved in lipid production and storage such as: fatty acid (SREBF1, SCD, APOC1), triglycerides (DGAT1) and cholesterol (ACAT1) and to regulate dedicated pathways including: adiponectin (ADIPOR1, APPL1), LXR/RXR/PPARA (SREBPF1, NH1H3 (LXRa), ACAT1, and prostaglandin (PTSG2 (COX2)).

[0220] Treatment with Tetraselmis suecica water extract at 0.01% statistically downregulated 7 genes (APOC1, SREBPF1, APPL1, ADIPOR1, MGAT1, ACAT1, PTSG2 (COX-2)).

[0221] Treatment with Tetraselmis suecica water dry extract at 0.1% statistically downregulated 8 genes (SREBF1, NR1H3 (LXRa), APPL1, ADIPOR1, DGAT1, MGAT1, SCD, PTSG2 (COX-2)).

[0222] The extract is able to reduce lipid production by repressing genes involved in fatty acid production (SREBF1, SCD, APOC1), diglycerides (MGAT1), triglycerides (DGAT1) and cholesterol (ACAT1).

[0223] IGF-I plays a key role in the induction of lipid synthesis in human sebocytes. In SEB-1 sebocytes, IGF-I increases lipogenesis by the induction of SREBF1 which preferentially regulates genes of fatty acid synthesis.

[0224] SCD is highly expressed in the sebaceous gland; SCD is a Δ9 fatty acid desaturase that primarily catalyzes the conversion of the saturated fatty acids palmitic acid (16:0) and stearic acid (18:0) into the cis-monounsaturated fatty acids (MUFA) palmitoleic acid (16:1n7) and oleic acid (18:1n9), respectively. The MUFA serve as important esterification substrates in the formation of triglycerides, cholesterol esters and wax esters, which are components of sebum.

[0225] Transgenic mice that overexpressed the APOC1 had hypoplastic sebaceous glands and hypertriglyceridemia.

[0226] DGAT1 catalyzes the final and rate-limiting step in triglyceride synthesis.

[0227] MGAT1 is involved in the synthesis of protein-bound and lipid-bound oligosaccharides. Acyl-CoA:monoacylglycerol acyltransferase (MGAT) genes are best known for their role in fat absorption in the intestine. MGAT1 has been shown to exhibit MGAT activity in mammalian cell lines, specific for catalyzing diacylglycerol synthesis by incorporating fatty acyl-CoA into diacylglycerol.

[0228] ACAT1 is an enzyme that catalyzes the formation of cholesteryl ester from free cholesterol and is highly expressed in the sebaceous gland, where it allows for the incorporation of cholesteryl esters into cytoplasmic lipid droplets.

[0229] The major pathways that induce lipid production and storage are LXR/RXR/PPARA and adiponectin. Indeed, it was shown that treatment of SZ95 sebocytes with LXR ligands enhanced accumulation of lipid droplets in the cells and lipid synthesis was markedly enhanced in sebocytes treated with adiponectin. Thus, NR1H3 (LXRa) that codes for a nuclear receptor responsible for activation of LXR/RXR/PPARA pathway is repressed by the extract.

[0230] The adiponectin receptor ADIPOR1 and its ligand APPL1 are responsible for activation of adiponectin pathway, are also reduced. In 3D culture of sebocytes, lipid synthesis was markedly enhanced in sebocytes treated with adiponectin.

[0231] The pronounced effect was surprisingly observed on the gene expression level of PTGS2 (COX-2). PTGS2 (COX-2) plays a major role in sebocyte function. Transgenic mice that overexpressed cyclooxygenase-2 (COX-2) exhibited an increased sebum level due to sebaceous gland hyperplasia. Thus, reduction of PTGS2 (COX-2) can be expected to lead to reduction of sebaceous gland size and sebum production.

Example 6: In Viva Sebum Reduction by Tetraselmis suecica Extract (Dried)

[0232] A randomized, split-face study with 2 groups of 15 Caucasian volunteers each was performed. Volunteers applied the test products at home on both semi-foreheads twice daily (mornings and evenings) for 4 weeks. Test product was a hydrodispersion gel with and without 0.05% Tetraselmis suecica extract prepared by extraction at 80° C. according to Example 1. As positive control/reference a combination of 2% niacinamide and 1% D-panthenol (Z. D. Draelos et al. J. Cosmet. Laser Ther. 2006, 8:2, 96-101) formulated in hydrodispersion gel was used. Read-outs were Casual sebum level (sebumeter), sebum surface percentage and number of active pores (both Visioscan® equipped with a Sebufix® foil) and readings were performed at baseline (t.sub.0) and after 4 weeks (t.sub.1).

TABLE-US-00006 TABLE 6 Mean modulation of casual sebum level, sebum surface percentage and number of active pores Test product/ Hydrodispersion gel Modulation with T0 T1 t.sub.1 versus t.sub.0 Signif- Casual sebum Level (baseline) (4 weeks) in % icance No active (Placebo) 176.1 ± 40.4 166.5 ± 42.5 −5.5 n.s. 0.05% Tetraselmis 186.4 ± 36.5 166.2 ± 40.4 −10.8 p < suecica extract 0.05 2% Niacinamide and 180.4 ± 40.0 152.4 ± 48.1 −15.6 p < 1% D-panthenol 0.05 Sebum surface percentage No active (Placebo) 10.3 ± 6.3 10.3 ± 7.3 −0.4 n.s. 0.05% Tetraselmis 12.2 ± 8.6  8.4 ± 6.6 −31.3 p < suecica extract 0.05 2% Niacinamide and 10.1 ± 8.2  9.0 ± 5.8 −11.7 n.s. 1% D-panthenol Number of active pores No active (Placebo) 175.3 ± 47.0 149.5 ± 52.4 −14.7 n.s. 0.05% Tetraselmis 174.8 ± 61.9 129.8 ± 64.8 −25.7 p < suecica extract 0.05 2% Niacinamide and 154.4 ± 50.9 136.6 ± 65.7 −11.6 n.s. 1% D-panthenol n.s. = non-significant

[0233] The results clearly proof the potent sebum reducing activity of Tetraselmis suecica water extract also in vivo. The extract is the only active which significantly reduced all three read-outs (casual sebum level by −10.8%, sebum surface percentage by −31.3% and number of active pores by −25.7%). The positive control/reference, the combination of 2% Niacinamide and 1% D-panthenol, significantly reduced the casual sebum level by −16.5%, but failed to give a significant reduction of the sebum surface percentage and the number of active pores. The placebo had no significant effect on any of the 3 read-outs.

Example 7: Effect of Tetraselmis suecica Extract (Dried) on the Gene Expression of Human Keratinocytes

[0234] Neonatale humane epidermal keratinocytes (nHEK) were cultivated in EpiLife medium (Gibco) including HKGS-Kit (Gibco) at 5% CO.sub.2 at 37° C. according to the supplier instructions.

[0235] The cells were treated for 24 hours with Tetraselmis suecica water extract obtained according to Example 1 by extracting at 80° C. at 0.025% or medium as vehicle control. Genomic target expression levels in extract treated cells were measured by RT-qPCR comparing to medium treatment.

[0236] RNA isolation took place using RNeasy® Mini Kit, Qiagen. Total RNA concentrations were measured using μCuvetteG 1.0 and BioPhotometer, Eppendorf by measuring the absorption at 260 nm. Purity control values, like E260/280 and E 260/230 were calculated simultaneously. Reverse transcription was done using high capacity RNA-to-cDNA Kit, Applied Biosystems, according to the supplier instructions. Samples were treated in the PCR Thermocycler, Biometra.

[0237] For the fast real-time PCR, cDNA was diluted with RNase-free water and TaqMan™ Fast Universal PCR Master Mix, Applied biosystems. Quantitative Real-Time PCR was done using StepOne Plus Fast Real Time PCR Instrument, Applied biosystems. Analysis was done with StepOne-Software and 2-ΔCT Method (normalized to endogenous control HTRP1 expression).

[0238] For upregulations RQ values≥2.0 and for downregulations RQ values<0.5 are considered to be relevant.

TABLE-US-00007 TABLE 7a Modulation of gene expression of human epidermal keratinocytes after treatment with 0.025 wt. % Tetraselmis suecica water extract (prepared by re-dissolving the dried extract) Indicated relevance but not RQ Genes limited to value AQP3 [Aquaporin 3] water/glycerol-transporting 4.3 protein KRT1 [Keratin 1] Differentiation 12.8 KRT10 [Keratin 10] Differentiation 14.2 FLG [(Pro)Filaggrin] Differentiation 2.3 FLG2 [Filaggrin Family Member 2] Differentiation 4.3 SBSN [Suprabasin] Differentiation 7.3 CSP14 [Caspase 14] Differentiation 10.9 DMKN [Dermokine] Differentiation 10.6 LIPN [Lipase Family Member N] Differentiation 14.3 TGM5 [Transglutaminase 5] Differentiation 5.0 S100A10 [S100 Calcium Binding Differentiation 3.2 Protein A10] S100A11 [S100 Calcium Binding Differentiation 3.2 Protein A11] S100A7 [S100 Calcium Binding Differentiation/antimicrobial 6.5 Protein A7] peptide S100A8 [S100 Calcium Binding Differentiation/antimicrobial 5.7 Protein A8] peptide S100A9 [S100 Calcium Binding Differentiation/antimicrobial 8.2 Protein A9] peptide SPRR1A [Small Proline Rich Late differentiation 6.5 Protein 1A] SPRR1B [Small Proline Rich Late differentiation 8.5 Protein 1B] SPRR2C [Small Proline Rich Late differentiation 2.6 Protein 2C] SPRR4 [Small Proline Rich Protein Late differentiation 2.9 4] DSG1 [Desmoglein-1] Desmosomes 13.4 DSG3 [Desmoglein-3] Desmosomes 5.1 DSC1 [Desmocollin 1] Desmosomes 10.8 DSP [Desmoplakin] Desmosomes 3.8 EVPL [Enyoplakin] Desmosomes 4.5 PKP1 [Plakophilin 1] Desmosomes 4.2 CDH1 [Cadherin-1] Adherens junctions 3.0 CTNNB1 [Catenin Beta 1] Adherens junctions 2.5 CSTA [Cytostain A] Desmosome stability 2.9 CLDN1 [Claudin 1] Tight junctions 7.6 CLDN7 [Claudin 7] Tight junctions 6.2 OCLN [Occludin] Tight junctions 7.5 CGN [Cingulin] Tight junctions 8.5 TJP1 [Tight junction protein ZO-1] Tight junctions 3.0 F11R [F11 Receptor] Tight junctions 3.1 F11R [F11 Receptor] Tight junctions 3.1 GJA1 [Gap Junction Protein Alpha Gap junctions 2.9 1] CXCR1 [C-X-C Motif Chemokine Re-epithelialization 6.9 Receptor 1] Serpine1 Re-epithelialization 3.2

[0239] The results clearly show that Tetraselmis extract surprisingly upregulates many genes involved in epidermal junctions, such as desmosomal (“mechanical”), tight, adherens and gap junctions relevant for cell-to-cell adhesion and allowance of the exchange of ions, second messengers, and small metabolites between adjacent cells in skin cells. These adhesion structures are essential not only for the maintenance of cell structure and integrity, but also for tissue development and morphogenesis. Mutations within the desmosome are e.g. the underlying cause of many skin fragility disorders.

[0240] Furthermore, genes relevant for differentiation, re-epithelialization and water/glycerol-transport are modulated by treatment with Tetraselmis extract.

[0241] In another experiment, the cells were treated for 24 hours with Tetraselmis suecica water dry extract obtained according to Example 1 from a different microalgae biomass batch either by extracting at 80° C. or at room temperature (18-23° C.) at 0.025% or medium as vehicle control. Genomic target expression levels of selected genes in extract treated cells were measured by RT-qPCR comparing to medium treatment as described above.

TABLE-US-00008 TABLE 7b Modulation of gene expression of human epidermal keratinocytes after treatment with 0.025% Tetraselmis suecica water extract (prepared by re-dissolving the dried extract) RQ value Genes Extract 80° C. Extract Room temp. KRT1 [Keratin 1] 8.0 1.0 KRT10 [Keratin 10] 7.9 1.0 CSP14 [Caspase 14] 4.0 1.0 SPRR1A [Small Proline 4.0 2.0 Rich Protein 1A] SPRR1B [Small Proline 4.0 4.0 Rich Protein 1B] DSG1 [Desmoglein-1] 8.0 2.0 DSC1 [Desmocollin 1] 4.0 1.0 DSP [Desmoplakin] 2.0 1.0 CTNNB1 [Catenin Beta 1] 2.0 1.0 CLDN1 [Claudin 1] 4.0 2.0 OCLN [Occludin] 4.0 4.1 CGN [Cingulin] 4.0 4.0

[0242] Results show that 6 genes (KRT1, KRT10, CSP14, DCS1, DSP, CTNNB1) were upregulated by the extract prepared at 80° C. whereas the extract prepared at room temperature had no effect. 6 of the selected genes (SPRRA1, SPRR1B, DSG1, CLDN1, OCLN, CGN) were upregulated by both extract.

Example 8: Effect of Tetraselmis suecica Extract (Dried) on the Gene Expression of AMPs

[0243] HaCaT keratinocytes were cultivated in EpiLife medium (Gibco) at 5% CO.sub.2 at 37° C.

[0244] The cells were treated for 24 hours with Tetraselmis suecica water dry extract obtained according to Example 1 by extracting at 80° C. at 0.05% or medium as vehicle control. Genomic target expression levels in extract treated cells were measured by RT-qPCR comparing to medium treatment.

[0245] RNA isolation took place using RNeasy® Mini Kit, Qiagen. Total RNA concentrations were measured using μCuvetteG 1.0 and BioPhotometer, Eppendorf by measuring the absorption at 260 nm. Purity control values, like E260/280 and E 260/230 were calculated simultaneously. Reverse transcription was done using RNA-to-cDNA Kit, Applied Biosystems, according to the supplier instructions. Samples were treated in the PCR Thermocycler, Biometra.

[0246] For the fast real-time PCR, cDNA was diluted with RNase-free water and TaqMan™ Fast Universal PCR Master Mix, Applied biosystems. Quantitative Real-Time PCR was done using StepOne Plus Fast Real Time PCR Instrument, Applied biosystems. Analysis was done with StepOne-Software and 2-ΔCT Method (normalized to endogenous control HTRP1 expression).

[0247] For upregulations RQ values≥2.5 and for downregulations RQ values<0.5 are considered to be relevant.

TABLE-US-00009 TABLE 8 Modulation of gene expression of AMPs in HaCaT keratinocytes after treatment with 0.05% Tetraselmis suecica water dry extract Genes RQ value DEFB1 [beta-Defensin 1] 20.8 DEFB103A; DEFB103B [beta-Defensin 103 A/ 3.1 103B] ADM [Adrenomedulin] 3.1 S100A7 [Psoriasin, S100 calcium binding 7.4 protein A7]

[0248] The results clearly show that Tetraselmis extract surprisingly also upregulates the gene expression of antimicrobial peptides such as beta-defensins, adrenomedullin and psoriasin in skin cells.

Example 9: Cyclooxygenase (COX)-2 Inhibition Assay

[0249] COX-2 is the inducible rate limiting enzyme for prostaglandin, e.g. PGE2, synthesis. COX-2/PGE2 are expressed by keratinocytes and sebocytes. The test substance, Tetraselmis suecica water dry extract, is dissolved in assay buffer (Tris-HCl pH 8.0, 100 mM) and is given into a 96-well half area microplate. The co-enzyme HEME, the fluorometric substrate 10-acetyl-3,7-dihydroxy-phenoxanin (ADHP) and COX-2 are added. The half area microplate is incubated for 2 minutes at 600 rpm on a microplate shaker. Afterwards the substrate arachidonic acid is added. COX-2 converts arachidonic acid into the prostaglandin endoperoxide G2 (PGG2). PGG2 is reduced to the corresponding alcohol PGH2. During this reaction ADHP results in fluorescent resorufin. Resorufin is quantified at an extinction wavelength of 535 nm and an emission wavelength of 590 nm.

[0250] The inhibition of COX-2 activity in the presence of test substances was calculated according to the following equation:

[00001]$\mathrm{Inhibition}\mathrm{of}\mathrm{COX}\text{-}2\left[\%\right]=100-\left(\frac{\begin{array}{c}\mathrm{Resorufin}\mathrm{test}\mathrm{substance}-\\ \mathrm{Resorufin}\mathrm{control}\mathrm{without}\mathrm{COX}\text{-}2\end{array}}{\begin{array}{c}\mathrm{Resorufin}\mathrm{control}-\\ \mathrm{Resorufin}\mathrm{control}\mathrm{without}\mathrm{COX}\text{-}2\end{array}}\times 100\right)$

The abbreviations have the following meanings:

[0251] Resorufin test substance:

Resorufin concentration of the wells with test substance and with COX-2

[0252] Resorufin control:

Resorufin concentration of the wells without test substance, but with COX-2

[0253] Resorufin control without COX-2:

Resorufin concentration of the wells without test substance and without COX-2
Results are mean values from at least 2 independent experiments.

TABLE-US-00010 TABLE 9 COX-2 Inhibition by Tetraselmis suecica water extract (dried) Test concentration COX-2 inhibition versus control 0.025% 34 ± 5%

[0254] The results show that Tetraselmis extract also inhibits COX-2 enzyme activity.

Example 10: Ex Vivo Human Skin—Filaggrin

[0255] Skin samples of approx. 8×3 mm (Ø×thickness) were cultured in an air-liquid interface in a perforated ring of stainless steel in contact with culture medium (modified Williams' E medium) up to day 6. After 24 acclimation, Tetraselmis suecica water dry extract obtained according to Example 1 by extracting at 80° C. at 0.3 ppm or medium as vehicle control were topically applied on the human skin explants (6 explants per each treatment). At day 6 of culture, the skin samples were embedded in an appropriate medium and frozen in liquid nitrogen. Quantitative analysis of filaggrin was performed on cryostat sections submitted to specific immunofluorescence staining. Twelve skin sections per treatment were immunostained with the selected antibody (filaggrin: rabbit polyclonal, SantaCruz). Each section was photographed by using a fluorescence microscope and the resulting image was analyzed. The amount of antigen present in each slide was evaluated by evaluating the intensity and the distribution of the stain within the epidermis (without stratum corneum). The obtained data was then normalized upon the length of the basal lamina.

TABLE-US-00011 TABLE 10 ex vivo human skin-filaggrin modulation Filaggrin Control/ Tetraselmis Stimulation vs score vehicle extract control Mean value 40 107 163% SEM 7 15

[0256] The results clearly show, that Tetraselmis suecica extract increases the epidermal filaggrin level of ex vivo human skin after topically application.

Example 11: Ex Vivo Human Skin—Particle Matter (PM) Induced Barrier Damage

[0257] To evaluate the protective effect of Tetraselmis suecica water extract (dried) on cutaneous barrier damages induced by diesel particulate matter, ex vivo human skin explants used. Standard Reference Material® 1650b was obtained from the US National Institute of Standards and Technology (NIST) and is intended for use in evaluating analytical methods for the determination of selected polycyclic aromatic hydrocarbons (PAHs) and nitro-substituted PAHs (nitro-PAHs) in diesel particulate matter and similar matrices. It was collected from the heat exchangers of a dilution tube facility following 200 engine hours of particulate accumulation. Several direct injection four-cycle diesel engines, operating under a variety of conditions were used to generate this particulate material and it should be representative of heavy-duty diesel engine particulate emissions. Skin samples of approx. 8×3 mm (Ø×thickness) were cultured in a perforated ring of stainless steel in contact with culture medium (modified Williams' E medium) at environmental humidity up to day 3. 1650b was suspended in PBS. Hydrodispersion gels without (placebo) and with 0.01 and 0.05% Tetraselmis suecica water dry extract were formulated (Table 12). After 24 h acclimation, the formulations were topically applied on the skin explants before the application of the particulate matter 1650b outside the incubator for 90 min in order to let them dry. Afterwards the diesel particulate was topically applied at 10 μg/cm2 on the treated and untreated skin. Application of formulations and treatment with 1650b was renewed every day.

[0258] On day 3, the skin explants were harvested to evaluate skin barrier properties. Rhodamine B cannot penetrate the intact skin, thus the more rhodamine B is detectable inside the epidermis the more damaged the skin barrier is. Therefore, skin explants were stained with rhodamine B, cryo-fixed and cut at the cryostat for consequent image acquisition and analysis. The analysis of rhodamine B fluorescence was performed within the epidermis area. For each skin explant two sections were taken and fluorescent images acquired. For each image the upper dermis was analyzed by evaluating the fluorescence through Image-J application (NIH, USA). The obtained value was then normalized upon the dimension of the selected area.

TABLE-US-00012 TABLE 11 Formulation 0.01% 0.05% Tetra- Tetra- selmis selmis Raw material INCI Placebo extract extract Water Water (Aqua) Ad 100 Ad 100 Ad 100 Hydrolite-5 Pentyleneglycol 2 2 2 PCL liquid 100 Cetearyl Octanoate 3 3 3 Lanette O Cetearyl Alcohol 2 2 2 Mineral Oil 5°E Mineral Oil 3 3 3 Eutanol G Octyldodecanol 4 4 4 Abil 350 Dimethicone 0.5 0.5 0.5 Pemulen TR1 Acrylates/C10-30 Alkyl 0.2 0.2 0.2 Acrylate Crosspolymer Ultrez-21 Acrylates/C10-30 Alkyl 0.05 0.05 0.05 Acrylate Crosspolymer Sodium Sodium Hydroxide 0.50 0.5 0.5 hydroxide Sol. 10% Tetraselmis Tetraselmis suecica extract — 0.01 0.05 suecica extract pH value 5.5

TABLE-US-00013 TABLE 12 Ex vivo human skin results-rhodamine B penetration 0.01% 0.05% Tetra- Tetra- Rhodamine B selmis selmis penetration Placebo + extract + extract + [L*/pixel] Untreated 1650b 1650b 1650b 1650b Mean 0.244 0.934 0.798 0.650 0.516 SEM 0.034 0.151 0.149 0.069 0.074 Stimulation versus — 283 untreated [%] Reduction versus — — 15 30 45 1650b [%] Reduction versus — — — 19 35 placebo [%]

[0259] As expected, topical treatment of human skin explants with particulate matter 1650b significantly increased the penetration of rhodamine B into the skin and thus cutaneous barrier damage.

[0260] The placebo led to a reduction of rhodamine B penetration by 15% versus 1650b treatment alone. Tetraselmis suecica extract led to a dose dependent reduction of rhodamine B penetration of 30 and 45% versus 1650b treatment alone and of 19 and 35% versus placebo+1650b treatment.

Example 12: TEER Assay

[0261] Transepithelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell culture models of epithelial monolayers. TEER values are strong indicators of the integrity or strength of the cellular barriers. Increased resistance of a tissue is a result of higher density. Therefore, increased resistance relates to an improved skin barrier.

[0262] Neonatal humane epidermal keratinocytes (nHEKs) were seeded in a concentration of 1.5×10.sup.5 cells per inserts in 0.47 cm.sup.2 cell culture inserts. After incubation with cell culture medium for four days, Tetraselmis suecica extract prepared according to the description given in Example 1 was systemically applied for eight days within the cell culture media in a final volume as listed below. Following the substance treatment, the TEER was determined. Cell culture medium was used as control.

TABLE-US-00014 TABLE 13 TEER essay results Medium Medium 0.025% control control Tetraselmis Readout Blank* start end suecica extract Readings 31 42 36 66 45 37 69 42 37 75 Mean value 31 43 36.67 70 Standard 1.73 0.58 4.58 deviation TEER [Ω cm.sup.2] 14.57 20.21 17.23 32.90 *Insert without cells

[0263] The results clearly show, that Tetraselmis suecica extract increased the TEER value when compared to untreated (medium control).

Example 13: Formulation Examples

[0264] In formulations 1-22 the following two perfume oils PFO1 and PFO2 were each used as fragrance (DPG=dipropylene glycol).

TABLE-US-00015 TABLE 14 Perfume oil PFO1 with rose smell (amounts in parts b.w.) Component Amount Acetophenone, 10% in DPG 10.00 n-Undecanal 5.00 Aldehyde C14, so-called (peach aldehyde) 15.00 Allylamyl glycolate, 10% in DPG 20.00 Amyl salicylate 25.00 Benzyl acetate 60.00 Citronellol 80.00 d-Limonene 50.00 Decenol trans-9 15.00 Dihydromyrcenol 50.00 Dimethylbenzylcarbinyl acetate 30.00 Diphenyloxide 5.00 Eucalyptol 10.00 Geraniol 40.00 Nerol 20.00 Geranium oil 15.00 Hexenol cis-3, 10% in DPG 5.00 Hexenyl salicylate cis-3 20.00 Indole, 10% in DPG 10.00 Alpha-ionone 15.00 Beta-ionone 5.00 Lilial ® (2-methyl-3-(4-tert-butyl-phenyl)propanal) 60.00 Linalool 40.00 Methylphenyl acetate 10.00 Phenylethyl alcohol 275.00 Styrolyl acetate 20.00 Terpineol 30.00 Tetrahydrolinalool 50.00 Cinnamyl alcohol 10.00 Total: 1,000.00

TABLE-US-00016 TABLE 15 Perfume oil PFO2 with white blossom and musk smell (amounts in parts b.w.) Component Amount Benzyl acetate 60.00 Citronellyl acetate 60.00 Cyclamenaldehyde (2-methyl-3-(4-isopropylphenyl) 20.00 propanal Dipropylene glycol (DPG) 60.00 Ethyllinalool 40.00 Florol (2-isobutyl-4-methyltetrahydro-2H-pyran-4-ol) 30.00 Globanone ® [(E/Z)-8-cyclohexadecen-1-one] 180.00 Hedione ® (methyldihydrojasmonate) 140.00 Hexenyl salicylate, cis-3 10.00 Vertocitral (2,4-dimethyl-3-cyclohexenecarboxaldehyde) 5.00 Hydratropaaldehyde, 10% in DPG 5.00 Isodamascone (1-(2,4,4-trimethyl-2-cyclohexen- 5.00 1-yl)-2-buten-1-one, 10% in DPG Isomuscone (cyclohexadecanone) 40.00 Jacinthaflor (2-methyl-4-phenyl-1,3-dioxolane) 10.00 Cis-jasmone, 10% in DPG 20.00 Linalool 50.00 Linalyl acetate 30.00 Methyl benzoate, 10% in DPG 25.00 para-Methyl cresol, 10% in DPG 10.00 Nerol 20.00 Phenylpropylaldehyde 5.00 2-Phenylethyl alcohol 82.00 Tetrahydrogeraniol 13.00 2,2-Dimethyl-3-cyclohexyl-1-propanol 80.00 Total: 1,000.00

TABLE-US-00017 TABLE 16 Cosmetic formulations (amounts in parts b.w.) Ingredients 1 2 3 4 5 6 7 8 9 10 11 Tetraselmis suecica extract 2.5% in glycerin/water 2 1 5 1 0.4 1.5 3 Water, Glycerin, Tetraselmis suecica extract Spray-dried Tetraselmis suecica extract containing 1 0.5 1 0.2 95% maltodextrin, 5% extract matter Maltodextrin, Tetraselmis suecica extract Actipone Alpha-Pulp 0.1 1 Aqua, Butylene Glycol, Malic Acid, Actinidia Chinensis Fruit Extract, Citrus Aurantium Dulcis Juice, Citrus Paradisi Juice, Pyrus Malus Juice, Trideceth-9, Prunus Amydalus Dulcis Seed Extract Allantoin 0.1 Allantoin Aloe Vera Gel Conc. 10:1 0.2 Aloe Barbadensis(Aloe) Leaf Juice Aluminium Stearate 1.2 Aluminium Stearate Asebiol 3 Water, Pyridoxine HCL, Niacinamide, Glycerin, Panthenol, Hydrolyzed Yeast Protein, Threonine, Allantoin, Biotin Beta-Arbutin 1 Arbutin Arlypon ® F 2 Laureth-2 Asensa ® SC 220 2 Polyethylene Azelaic acid 0.5 Azelaic acid Biotive L-Arginine 0.6 Arginine Biotive Troxerutin 0.5 Troxerutin (—)-alpha-Bisabolol 0.1 Bisabolol Carbopol Aqua SF-1 Polymer 5 Acrylates Copolymer Carbopol ® Ultrez-10 0.2 0.2 0.2 0.3 Carbomer Citric acid 10% in water 0.2 0.5 Citric acid, water Colour 0.04 Crinipan ® AD 0.3 Climbazole Cutina ® AGS 1.5 Glycol Distearate Cutina ® PES 2 Pentaerythrityl Distearate D-Panthenol 0.5 Panthenol Dehyton K 8 8 Cocamidopropyl Betaine Dow Corning 200(100cs) 2 2 0.5 Silicone Fluid Dimethicone Dracorin ® CE 5 2.5 Glyceryl Stearate Citrate Dracorin GOC 2.5 Glyceryl Oleate Citrate, Caprylic/Capric Triglyceride Dragocalm ® 1 Water (Aqua), Glycerin, Avena Sativa (Oat) Kernel Extract Dragoderm ® 0.5 Glycerin, Triticum Vulgare (Wheat) Gluten, Water (Aqua) Dragosan ® W/O P 8 Sorbitan Isostearate, Hydrogenated Castor Oil, Ceresin, Beeswax (Cera Alba) Dragosantol ® 100 0.2 Bisabolol Dragosine ® 0.2 0.2 Carnosine Dragoxat ® 89 5 7 1 5 Ethylhexyl Isononanoate Disodium EDTA 0.1 0.1 0.1 0.1 0.1 0.05 0.05 Disodium EDTA Emulsiphos ® 2 1.5 Potassium Cetyl Phosphate, Hydrogenated Palm Glycerides Estearina L2SM GS 2 Stearic Acid, Palmitic Acid Ethanol 2 Ethanol Extrapone ® Aloe vera 1 Water (Aqua), Aloe Barbadensis, Propylene Glycol, Alcohol Extrapone Eucalyptus 1 Aqua, Propylene Glycol, Eucalyptus Globulus Leaf Extract Extrapone Iris B 0.5 Aqua, Propylene Glycol, PEG-40 Hydrogeanted Castor Oil, Trideceth-9, Bisabolol, Iris Germanica Root Extract Extrapone ® Witch Hazel 1 Propylene Glycol, Hamamelis Virginiana (Witch Hazel) Water, Water (Aqua), Hamamelis Virginiana (Witch Hazel) Extract Food Color Brown E172 + E171 Powder 2 1.5 Titanium Dioxides (CI77891), Iron Oxides (CI77492), Iron Oxides (CI77491), Iron Oxides (CI77499) Food Color Titanium Dioxide Powder E171 3 Titanium Dioxides (CI77891) Frescolat ® MGA 0.5 Menthone Glycerin Acetal Frescolat ® ML 0.3 0.2 0.3 Menthyl Lactate Frescolat Plus 0.2 Menthol, Menthyl Lactate Frescolat ® X-Cool 0.2 Menthyl Ethylamido Oxalate Genapol ® LRO Liquid 37 Sodium Laureth Sulfate Glycerin 3 3 4.5 3 1.5 3 Glycerin Hydrolite ® 5 3 2 Pentylene Glycol Hydroviton-24 ® 1 Water (Aqua), Pentylene Glycol, Glycerin, Lactic Acid, Sodium Lactate, Serine, Urea, Sorbitol, Sodium Chloride, Allantoin Hydroviton ® Plus 2290 2 Water (Aqua), Pentylene Glycol, Glycerin, Fructose, Urea, Citric acid, Sodium Hydroxide, Maltose, Sodium PCA, Sodium Chloride, Sodium Lactate, Trehalose, Allantoin, Sodium Hyaluronate, Glucose Isoadipate 2 2 Diisopropyl Adipate Isodragol ® 1 Triisononanoin Jojoba Oil 0.3 Simmondsia Chinensis (Jojoba) Seed Oil Kaolin 10 Kaolin Keltrol ® CG-RD 0.2 0.1 0.3 0.2 0.3 1.2 Xanthan Gum Kojic acid 0.5 Kojic Acid KP-545 1 Cyclopentasiloxane Acrylates/Dimethicone Copolymer Lanette ® 16 1.5 2 Cetyl Alcohol Lanette ® 22 3 Behenyl Alcohol Lanette ® O 5 2 Cetearyl Alcohol Magnesium Sulfate 0.7 Magnesium Sulfate Mineral Oil 5 Paraffinum Liquidum Neo Heliopan ® 303 4 10 Octocrylene Neo Heliopan ® 357 2 4 2 Butylmethoxydibenzoyl-methane Neo Heliopan ® AP 15% Lösung, neutralisiert mit L-Arginin 6.7 Aqua, Disodium Phenyl Dibenzimidazole Tetrasulfonate, Arginin Neo Heliopan ® AV 7.5 Ethylhexyl Methoxycinnamate Neo Heliopan ® BB 3 Benzophenone-3 Neo Heliopan ® E 1000 1 Isoamyl p.Methoxycinnamate Neo Heliopan ® HMS 7 10 Homosalate Neo Heliopan ® OS 3 5 5 Ethylhexyl Salicylate Neo Heliopan ® Hydro 20% Lösung, neutralisiert mit 10 3.5 Biotive Arginine Aqua, Phenylbenzimidazole, Sulphonic Acid, Arginin Neo-PCL Water Soluble N 1.5 2 Trideceth-9, PEG-5 Ethylhexanoate, Water (Aqua) Neutral oil 2 Caprylic/Capric Triglyceride Niacinamide 0.5 3 0.3 0.75 Niacinamide Ozokerite Wax 2389 2 Ozokerite Parfume oil PFO1 or PFO2 0.05 0.3 1 0.3 0.3 0.5 0.3 0.1 0.5 Parfum Passion Fruit Oil 1 Refined Passiflora Edulis seed oil PCL-Liquid 100 3 2 5 Cetearyl Ethylhexanoate PCL-Solid 1 2 Stearyl Heptanoate, Stearyl Caprylate Pemulen ® TR-2 0.6 0.15 Acrylates/C10-30 Alkyl Acrylate Crosspolymer Phenethyl Alcohol 0.2 Phenethyl Alcohol Phytoconcentrole ® 1 Shea Butter, Glycine Soja (Soybean) Oil, Butyrospermum Parkii (Shea Butter) Plantacare PS 10 5 Sodium Laureth Sulfate, Lauryl Glucoside Polymer JR 400 0.4 Sodium Laureth Sulfate, Lauryl Glucoside Retinol 0.1 Retinol Salicylic acid 0.5 0.3 Salicylic Acid Sodium Ascorbyl Phosphate 1 Sodium Ascorbyl Phosphate Sodium Chloride 0.1 Sodium Chloride Sodium Hydroxide 10% Solution 1 0.5 2 0.2 1.9 1.1 Sodium Hydroxide 10% Solution Softisan 100 6 Hydrogenated Coco-Glycerides Solubilizer 3 PEG-40 Hydrogenated Castor Oil, Trideceth-9, Propylene Glycol, Water (Aqua) Sulfetal LA 12 Ammonium Lauryl Sulfate SymCalmin ® 1 0.1 0.5 Butylene Glycol, Pentylene Glycol, Hydroxyphenyl Propamidobenzoic Acid SymClariol ® 0.1 1 0.2 0.38 Decylene Glycol SymDecanox HA 1 2 Caprylic/Capric Triglyceride, Hydroxymethoxyphenyl Decanone Symdiol ® 68 1 0.5 0.5 0.5 0.8 1,2-Hexanediol, Caprylyl Glycol SymFinity ® 1298 0.05 Echinacea Purpurea Extract SymGlucan ® 1 2 Water (Aqua), Glycerin, Beta-Glucan SymHair ® Force 1631 2 Pentylene Glycol, Isochrysis galbana Extract SymHelios ® 1031 0.3 Benzylidene Dimethoxydimethylindanone SymLift 2 Water, Trehalose, Glycerin, Pentylene glycol, beta-Glucan, Hordeum Vulgare Seed Extract, Sodium Hyaluronate, 1,2- Hexanediol, Caprylyl glycol, Sodium Benzoate, Maltodextrin SymMatrix 0.2 Maltodextrin, Rubus Fruticosus (Blackberry) Leaf Extract SymMollient S 1 Cetearyl Nonanoate SymMollient ® W/S 1 2 1.5 2 Trideceth-9, PEG-5 Isononanoate, Water (Aqua) SymOcide ® C 0.1 o-Cymen-5-ol SymOcide ® PC 1 Phenoxyethanol, Caprylyl Glycol, SymOcide ® PH 1 Phenoxyethanol, Hydroxyacetophenone, Caprylyl Glycol, Water (Aqua) SymOcide ® PS 0.8 0.8 Phenoxyethanol, Decylene Glycol, 1,2-Hexanediol SymOcide ® PT 0.8 Phenoxyethanol, Tropolone SymPeptide ® 225 1 Glycerin, Water (Aqua), Myristoyl Pentapeptide-11 SymRelief ® 100 0.1 Bisabolol, Zingiber Officinale (Ginger) Root Extract SymRelief ® S 0.1 Bisabolol, Hydroxymethoxyphenyl Decanone SymRepair ® 100 1 Hexyldecanol, Bisabolol, Cetylhydroxyproline Palmitamide, Stearic Acid, Brassica Campestris (Rapeseed) Sterols SymSave ® H 0.5 0.5 0.5 0.5 Hydoxyacetophenone SymSitive ® 1609 1 0.5 Pentylene Glycol, 4-t-Butylcyclohexanol SymVital ® AgeRepair 3040 0.1 Zingiber Officinale (Ginger) Root Extract SymWhite ® 377 0.5 Phenylethyl Resorcinol Vitacel CS 20 FC 3 Cellulose Vitamin A Palmitate 0.1 Retinyl Palmitate Vitamin E Acetate 0.5 0.2 0.5 0.25 Tocopheryl Acetate Willow bark extract 0.1 Salix Alba Extract Xiameter PMX-345 6 Cyclopentasiloxane, Cyclohexasiloxane Zetesol LA-2 26 Ammonium Laureth Sulfate Water Ad to 100 1 = Skin calming balm for sensitive oily skin 2 = Tinted Face Balm, SPF 15 3 = Rinse-off purifying mask for greasy skin 4 = Night cream W/O 5 = Facial Cleansing gel 6 = Face tonic for oily skin 7 = Anti-dandruff hair shampoo for greasy hair 8 = Sunscreen fluid for acne prone skin, SPF 30 9 = Skin lightening day care fluid O/W for impure oily skin 10 = Anti-acne skin cream 11 = 3 in 1 Skin purifying Wash + Scrub + Mask

TABLE-US-00018 TABLE 17 Cosmetic formulations 12 to 22 (amounts in parts b.w.) Ingredients 12 13 14 15 16 17 18 19 20 21 22 Tetraselmis suecica extract 2.5% in glycerin/water 1.2 3 0.2 2 0.4 1 3 Water, Glycerin, Tetraselmis suecica extract Spray-dried Tetraselmis suecica extract containing 2 0.8 0.3 1 95% maltodextrin, 5% extract matter Maltodextrin, Tetraselmis suecica extract Actipone ® White Tea GW 1 Aqua, Glycerin, Camellia Sinensis Leaf Extract Actipone ® Witch Hazel 3 1 Hamamelis Virginiana Bark/Leaf/Twig Extract, Alcohol, Hamamelis Virginiana Water Actipone ® Black Currant GW 1 Aqua, Glycerin, Ribes Nigrum Juice Amisoft ® CS-11/CS-11(F) 0.5 Sodium Cocoyl Glutamate Andiroba Oil, refined 0.3 Carapa Guaianensis Seed Oil Aristoflex ® AVC 0.5 Ammonium Acryloyoldimethyltaurate/VP Copolymer 5-Alpha-Avocuta 1 Butyl Avocadate Beeswax 5 Cera Alba Butylene Glycol 0.5 5 Butylene Glycol Candelilla Wax 15 Euphorbia Cera (Candelilla) Wax Carnauba Wax 5 Cera Carnaubae depurata Carbopol ® Aqua SF-1 Polymer 10 Acrylates Copolymer CeramideBio 0.5 Cetylhydroxyproline Palmitamide Citric acid 10% in water 0.5 0.2 Citric acid, water Crinipan ® AD 0.2 Climbazole Disodium EDTA 0.1 0.05 0.1 0.1 Disodium EDTA Dow Corning 345 Fluid 5 Cyclomethicone Dow Corning 556 Fluid 4 Phenyl Trimethicone Dow Corning 2502 Fluid 5 Cetyl Dimethicone D-Panthenol 75 L 0.3 Panthenol Dracorin GOC 2.5 Glyceryl Oleate Citrate, Caprylic/Capric Triglyceride Dragoxat ® 89 5 20 2 Ethylhexyl Isononanoate Emulsiphos ® 2 Potassium Cetyl Phosphate, Hydrogenated Palm Glycerides Ethanol 5 5 10 Alcohol, Aqua Evermat 3 Enantia chlorantha bark extract Extrapone ® Strawberry B 1 Aqua, Propylene Glycol, Citric Acid, Trideceth-9, Bisabolol, Fragaria Ananassa Fruit Extract Extrapone ® Tiger Grass 5 1 Aqua, Glycerin, PEG-40 Hydrogenated Castor Oil, Trideceth-9, Centella Asiatica Extract Flowerconcentrole ® 2 Frangipani Pentylene Glycol, Bisabolol, Plumeria Acutifolia Flower Extract L Frescolat ® ML 0.3 0.3 0.3 0.5 Menthyl Lactate Glycerin 3 3 3 5 1 Glycerin Green Pigment 0.85 CI77288, Triethoxycaprylylsilane Hexylene Glycol 25 Hexylene Glycol Hispagel ® 200 1 1 Glycerin, Glyceryl Polyacrylate Hydrolite ® 5 1.5 3 1 4 5 7 Pentylene Glycol Hydrolite ® 6 0.5 1,2-Hexanediol Hydromoist ® L 1 Aqua, Hydrolyzed Lupine Seed Extract Hydroviton ® Plus 2290 1 1 Water (Aqua), Pentylene Glycol, Glycerin, Fructose, Urea, Citric acid, Sodium Hydroxide, Maltose, Sodium PCA, Sodium Chloride, Sodium Lactate, Trehalose, Allantoin, Sodium Hyaluronate, Glucose Icroquat Behenyl TMS-50 2 Behentrimonium Methosulfate, Cetyl Alcohol, Butylene Glycol Isoadipate 12.7 Diisopropyl Adipate Isodragol ® 8 Triisononanoin Isopropyl Myristate 2 Isopropyl Myristate Jaguar ® Excel 0.1 Guar Hydroxypropyltrimonium Chloride Jojoba Oil 8 0.5 Simmondsia Chinensis (Jojoba) Seed Oil Keltrol ® CG-T 0.1 0.2 0.3 Xanthan Gum Lactic acid 0.2 Lactic acid Lanette ® 16 1 1 3 Cetyl Alcohol Lanette ® 18 4 Stearyl Alcohol Lanette ® 22 2 Behenyl Alcohol Lanette ® O 1 4.5 Cetearyl Alcohol Medialan ® LD 10 Sodium Lauroyl Sarcosinate Mineral Oil 1 Paraffinum Liquidum Miniporyl ® 1 Isopentyldiol, Trifolium Pratense (Clover) Flower Extract Neo-PCL Water Soluble N 1.5 Trideceth-9, PEG-5 Ethylhexanoate, Aqua Niacinamide 3 1 2 Niacinamide Parfume oil PFO1 or PFO2 0.5 0.3 1 0.5 0.3 0.1 0.5 Parfum PCL-Liquid 100 5 2 Cetearyl Ethylhexanoate PCL-Solid 3 Stearyl Heptanoate, Stearyl Caprylate Pemulen TR-2 Polymeric Emusifier 0.3 Acrylates/C10-30 Alkyl Acrylate Crosspolymer Plantacare ® 2000 UP 15 Decyl Glucoside Potassium Sorbate 0.2 Potassium sorbate Propylene Glycol 2 5 Propylene Glycol Retinopeptide 189 1 Glycerin, Pentylene Glycol, Aqua, Myristoyl Nonapeptide-3 Salicylic Acid 0.3 0.1 0.3 0.2 Salicylic Acid Shea Butter (Organic) 20 Butyrospermum Parkii (Shea) Butter Sodium Benzoate 0.2 Sodium Benzoate Sodium Chloride 6 Sodium Chloride Sodium Hydroxide 10% solution 2.43 2 0.58 0.46 Sodium Hydroxide, water Softigen ® 767 3 PEG-6, Caprylic/Capric Glycerides Solubilizer 1.2 2 PEG-40 Hydrogenated Castor Oil, Trideceth-9, Propylene Glycol, Water (Aqua) SymCalmin ® 0.5 Butylene Glycol, Pentylene Glycol, Hydroxyphenyl Propamidobenzoic Acid SymClariol ® 0.3 0.5 0.3 Decylene Glycol SymDecanox HA 2 0.5 Caprylic/Capric Triglyceride, Hydroxymethoxyphenyl Decanone Symdiol ® 68 0.5 0.8 0.5 0.5 0.5 1,2-Hexanediol, Caprylyl Glycol SymHair ® Restore 0.5 1 Glycerin, Triticum Vulgare Protein, Aqua SymHair ® Shield 0.5 Pentylene Glycol, Aqua, Glycerin, Triticum Vulgare Bran Extract, 1,2-Hexanediol, Caprylyl Glycol SymMatrix 0.3 Maltodextrin, Rubus Fruticosus (Blackberry) Leaf Extract SymMollient ® S 2 2 Cetearyl Nonanoate SymMollient ® W/S 2 1.5 2 3 Trideceth-9, PEG-5 Isononanoate, Water (Aqua) SymOcide ® PS 1 0.8 Phenoxyethanol, Decylene Glycol, 1,2-Hexanediol SymRelief ® S 0.1 Bisabolol, Hydroxymethoxyphenyl Decanone SymSave ® H 0.5 0.5 0.5 0.5 Hydoxyacetophenone SymSitive ® 1609 1 Pentylene Glycol, 4-t-Butylcyclohexanol SymSol ® PF-3 1.5 3 1.2 Aqua, Pentylene Glycol, Sodium Lauryl Sulfoacetate, Sodium Oleoyl Sarcosinate, Sodium Chloride, Sodium Oleate SymVital ® AgeRepair 3040 0.2 Zingiber Officinale (Ginger) Root Extract White Pigment 7 CI77891, Ricinus (Castor) Seed oil Witch Hazel-Distillate 1 Hamamelis Virginiana (Witch Hazel) Water, Water (Aqua), Alcohol Xiameter ® PMX-200 Silicone Fluid 100 cs 1 0.5 Dimethicone Xiameter ® XM OFX-0193 Fluid 1 1 PEG-12 Dimethicone Yellow Pigment 0.15 CI77492, Triethoxycaprylylsilane Water Ad 100 Aqua 12 = Pore Refining Fluid 13 = Make-Up Remover Wipes Solution for impure skin 14 = Anti-acne Cleansing Mousse 15 = 3-Phases Clear Make-up Remover Lotion for oily skin 16 = Eau micellaire 17 = Purifying/Anti-Imperfections Cocktail 18 = Tightening Serum for young skin 19 = Concealer Stick 20 = Hair Mask 21 = Aqueous-based Hair & Scalp Serum 22 = Hair Conditioner