Cosmetic use of Artemia salina extract to protect skin from thermal stress

Abstract

Methods of protecting skin from damage due to cold thermal stress are disclosed. The methods include topically applying a composition that includes an Artemia salina extract from rehydrated Artemia salina cysts to skin at risk of damage from thermal stress, particularly from cold thermal stress or repeated temperature variations. The composition includes a physiologically acceptable medium for delivery of the Artemia salina extract.

Claims

1. A method of cosmetic care to protect the skin of a human or animal from damage due to cold thermal stress, the method comprising: topically applying at least once or twice daily for at least two days, on at least one portion of the skin of the body or face of the human or animal which has been subjected to cold thermal stress, a composition comprising a physiologically acceptable medium and an Artemia salina extract obtained from rehydrated Artemia salina cysts, wherein the Artemia salina extract is present in the composition at a concentration of about 0.0001% to about 20% of the total weight of the composition, and the cells of the at least one portion of skin of the body or face of the human or animal maintain a physiological level of cold-inducible RNA-binding protein.

2. The cosmetic care method according to claim 1, wherein said Artemia salina extract is diluted in one or more physiologically suitable solvents, chosen from among water, glycerol, ethanol, propanediol, butylene glycol, dipropylene glycol, ethoxylated diglycol or propoxylated diglycol, cyclic polyols or any mixture of these solvents.

3. The cosmetic care method according to claim 1, wherein said Artemia salina extract is present at a concentration of about 0.05% to about 5% of the total weight of the composition.

4. The cosmetic care method according to claim 1, wherein the composition further comprises at least one additional active agent chosen from among vitamin A, retinoic acid, retinol, vitamin B3, vitamin B5, vitamin B6, vitamin B12, vitamin C, vitamin E, vitamin F, vitamin H, vitamin K, vitamin PP, coenzyme Q10, metalloproteinase inhibitors, amino acids, carnitine, carnosine, taurine, natural or synthetic peptides, vegetable peptide extracts, yeast extracts, natural or synthetic phystosterols, salicylic acid, oligosaccharides, polysaccharides, amino sugars, polyphenols, flavonoids, lipids, phospholipids, cyclic AMP and its derivatives, activator agents of the enzyme adenylate cyclase and inhibiting agents of the enzyme phosphodiesterase, theine, theophylline, theobromine, forskolin, esculin, ACE inhibitors, dioscorea extract, guarana extract, ivy extract, fucus extract, seaweed extracts such as Palmaria Palmata extract, hydrolyzed extract of Prunella vulgaris or elder extract.

5. The cosmetic care method according to claim 1, wherein said Artemia salina extract is present at a concentration of about 0.01% to about 10% of the total weight of the composition.

6. The cosmetic care method according to claim 1, wherein topically applying the composition comprises applying about 2 mg/cm.sup.2.

Description

(1) FIG. 1: Expression of CIRBP after cold thermal stress in cells treated or not treated by Artemia extract.

(2) FIG. 2: Evaluation of CIRBP mRNA after cold thermal stress in cells treated or not treated by Artemia extract.

EXAMPLE 1: PREPARATION OF AN ARTEMIA SALINA EXTRACT

(3) 50 grams of Artemia salina cysts are rehydrated for 30 minutes to 6 hours in 1 liter of distilled water at a temperature ranging from 30 C. to 75 C., in a suitable medium, mainly constituted of water, and at a pH of between 4 and 7. These cysts are then ground. The extract thus obtained is centrifuged and filtered. The extract is then sterilized by sterilizing filtration and heated to 65 C.

(4) The extract is then processed to conform to cosmetic requirements (color, fragrance, appearance, sterility, etc.).

(5) An assay by high-performance liquid chromatography (HPLC) is then carried out to confirm a diguanosine tetraphosphate content of over 150 mg/liter.

EXAMPLE 2: EXPRESSION OF CIRBP AFTER COLD THERMAL STRESS IN CELLS TREATED OR NOT TREATED By Artemia Extract According to the Invention

(6) The purpose of this study is to determine the effect of a pretreatment with Artemia salina extract on keratinocytes subjected to hypothermic stress. In particular, the CIRBP (Cold-Inducible RNA Binding Protein) was assayed by Western blot on keratinocytes subjected to moderate hypothermia (32 C.), with or without pretreatment by Artemia salina extract according to example 1.

(7) Protocol:

(8) Normal human keratinocytes (NHK) from skin are treated by adding an Artemia salina extract according to example 1, at 1% and 3%, to their growth medium. This application is repeated once a day for 48 hours. At the same time, NHK cultures are maintained without treatment, so as to constitute an untreated control. This culture occurs at 37 C.

(9) Then, the pretreated or not pretreated NHK cultures are subjected to hypothermic stress by placing them at 32 C. for 6 hours. After this incubation, the NHK are lysed in order to analyze CIRBP expression by Western blot. This conventional Immunoblotting technique comprises the following steps: saturation of the transfer membrane by a 1TBS/5% milk solution for 1 hour, incubation with the primary antibody CIRBP (Novusbio), at 4 C. for the night and then incubation with the secondary antibody coupled to peroxidase (Santa Cruz) for 1 hour at ambient temperature. Luminescence is revealed by adding the substrate (SuperSignal West Dura Extended Duration Substrate, Thermo Scientific) using a camera (Chemi-Imager system, Alpha Innotech Corporation).

(10) Results:

(11) The results, with reference to FIG. 1, show a 27% increase in CIRBP expression when the cells are incubated at 32 C. for 6 hours, compared to the control at 37 C. and in agreement with bibliographic data.

(12) When the cells have been pretreated by Artemia salina extract at 1% according to example 1, the CIRBP level decreases significantly at 32 C., by 17%, compared to the control at 32 C. When the cells have been pretreated by Artemia salina extract at 3% according to example 1, the CIRBP level decreases significantly at 32 C., by 28%, compared to the control at 32 C.

(13) Conclusion:

(14) A decrease in CIRBP expression is observed in cells pretreated by Artemia salina extract at 1% or 3% according to example 1, and subjected to cold thermal stress, compared to untreated control cells.

EXAMPLE 3: EVALUATION OF CIRBP MRNA FOLLOWING COLD THERMAL STRESS

(15) The purpose of this study is to determine if CIRBP mRNA are modulated by treatment with an Artemia salina extract obtained according to example 1, prior to moderate hypothermic stress (32 C.). The CIRBP mRNA level was evaluated by quantitative PCR (Q-PCR).

(16) Protocol:

(17) Normal human keratinocytes (NHK) are treated by an Artemia salina extract at 1% in the growth medium. At the same time, NHK cultures are maintained without treatment, so as to constitute an untreated control. This culture occurs at 37 C. Then, the treated and untreated cultures are placed at 32 C. for 6 hours.

(18) After this incubation, total RNA are extracted with the RNeasy mini kit (QIAGEN, 74104) and reverse transcribed with the High Capacity cDNA reverse-transcription kit containing RNase inhibitors (Applied Biosystems, 4368814). Quantitative PCR is carried out using the Step One Plus thermocycler (Applied Biosystems). The primers and probes of the target CIRBP and of the endogenous control 18S are from Taqman Expression Assays (Applied Biosystems, Hs99999901_s1 for 18S and CIRBP: Hs00989762_g1), diluted in sterile water from Master Mix (Applied Biosystems).

(19) Results:

(20) The results, as presented in FIG. 2, demonstrated a significant increase of 59% in the expression of CIRBP mRNA in untreated keratinocytes subjected to moderate hypothermia (32 C.), compared to the control at 37 C.

(21) In the case where keratinocytes were treated by Artemia salina extract at 1% prior to exposure to hypothermic stress, their CIRBP mRNA level increased by 12% compared to keratinocytes pretreated by the extract at 37 C. and the CIRBP mRNA level decreased by 33%, compared to the untreated control keratinocytes at 32 C.

(22) Conclusion:

(23) A lower level of CIRBP mRNA is observed in keratinocytes pretreated by Artemia salina extract according to example 1, and subjected to hypothermia, compared to untreated control cells.

EXAMPLE 4: EFFECT OF THE EXTRACT FROM EXAMPLE 1 ON TRANS EPIDERMAL WATER LOSS (TEWL)

(24) Objective:

(25) The study evaluates the efficacy of Artemia salina extract obtained according to example 1 on the protection of skin exposed to thermal stress, particularly on trans epidermal water loss (TEWL) during hot/cold stress. Trans epidermal water loss (TEWL) is a parameter that indirectly reflects the skin's permeability and barrier function (Toby Mathias et al., 1981). Hot/cold stress mimics differences in temperature between the inside and the outside in both summer and winter.

(26) Protocol:

(27) The study was conducted on 19 volunteers, aged 21 to 52 years, including 9 in a mature group (aged 48 to 57) and 10 in a young group (aged between 21 and 34 years).

(28) Depending on the volunteers, the nature of the skin was normal or dry, without skin pathology, and of phototype 2 to 4.

(29) The test was carried out double blind, versus 1% Artemia salina extract according to example 1 and lasted 3 weeks.

(30) The Artemia salina extract from example 1 and a placebo were applied 2 times per day, morning and night, at product concentration on an identical area of each thigh.

(31) The amount of product applied was 2 mg/cm.sup.2.

(32) Thermal stress process: First of all a heating pad (WELLYS electric heating pad) was applied to the thigh for 10 min. Immediately afterwards, an ice pack was applied for 10 min. (FirstIce (EZY WRAP) ice pack).

(33) Trans epidermal water loss (TEWL) was evaluated using a Dermalab evaporimeter (Cortex Technology). The measurement is expressed in g/m.sup.2/h and is interrupted when the standard deviation is 0.1 or after 60 seconds. The measurement is taken under controlled temperature and humidity conditions (21 C.1 and 50%5).

(34) Preparation Method:

(35) 1) Baseline measurements, 2) Application of products, 3) Measurements on Day 0 to Day 21 before the stress and Day 21 after the stress.

(36) To analyze the results, we observed the time to obtain a plateau of the TEWL measurements curve. This time corresponds to the time that it takes the skin to recover, i.e., return to its baseline state, after thermal stress. We call this time the recovery time. Next we statistically compare this recovery time for the sides treated with the extract according to example 1 and the sides treated with the placebo.

(37) After the stress, TEWL measurements are taken every 2 min.

(38) Result:

(39) We note that the curve plateau is reached faster for the areas treated with Artemia extract than for the regions treated with the placebo.

(40) The recovery time is significantly 27% faster on average for the areas treated with Artemia extract than for the regions treated with the placebo.

(41) Conclusion:

(42) The TEWL values return to normal faster following cold-hot thermal stress on the areas treated with the Artemia salina extract according to example 1 compared to the placebo. This result shows that Artemia salina extract helps the skin recover more rapidly following hot/cold thermal stress.

EXAMPLE 5: PREPARATION OF COMPOSITIONS

(43) 1 Protective Cream

(44) TABLE-US-00001 Weight Ingredients (Brand Name | INCI) percent Supplier Phase A Deionized Water Water/aqua Qsp 100 Phase B RAPITHIX A-100 polymer Sodium Polyacrylate 0.50 Ashland Phase C AMPHISOL K Potassium Cetyl Phosphate 0.50 DSM Phase D ROKONSAL/LIQUAPAR MEP Phenoxyethanol (and) Methylparaben (and) 1.00 Ashland preservative Ethylparaben (and) Propylparaben Phase E CERASYNT 945 ester Glyceryl Stearate & Laureth-23 2.50 Ashland NACOL 16-98 Cetyl Alcohol 2.00 Sasol/IMCD Cerabeil White Cera Alba 2.00 Baerlocher ANTARON V-220F VP/Eicosene Copolymer 1.00 Ashland ESCALOL 517 UV filter Butyl Methoxydibenzoylmethane 3.00 Ashland (Avobenzone) ESCALOL 587 UV filter Ethylhexyl Salicylate 5.00 Ashland ESCALOL 597 UV filter Octocrylene 7.00 Ashland CERAPHYL 368 ester Ethylhexyl Palmitate 5.00 Ashland XP PMX-0245 Cyclopentasiloxane 5.00 Dow Corning/Univar Phase F OPTIPHEN MIT Ultra preservative Phenylpropanol & Propylene glycol & 0.30 Ashland Methylisothiazolinone Phase G Amidon de Mas MST Zea Mays Starch 2.00 Sensient Phase H Pf. Evasion Parfum/fragrance 0.50 Technicoflor GP4G SP biofunctional Water (and) Artemia Extract 1.00 Ashland Total 100.00

(45) Preparation Method 1. Homogenize phase A in the main tank 2. Sprinkle over phase B and homogenize for 10 minutes, and then start to heat to 50 C. and mix well for 30 minutes 3. Decrease the temperature and add phase C. Mix well to homogenize 4. In a beaker set aside, prepare phase D, heat to 55-60 C. until homogenized and decrease the temperature 5. Add the elements from phase E one by one into the main tank, mixing well between each addition 6. Add phase D (at ambient temperature) to the main vessel and mix until homogenized 7. At 25 C., add phase F and mix well 8. Premix phase G before adding it to the main tank 9. Add the elements from phase H one by one, mixing well between each addition. 10. Stop at 25 C.

(46) 2 Moisturizing Cream for Men

(47) TABLE-US-00002 Weight Ingredients (Brand Name | INCI) percent Supplier Phase A Deionized Water Water/Aqua Qsp 100 / Na4EDTA Tetrasodium EDTA 0.05 Fisher Phase B POLYSURF 67 CS polymer Cetyl Hydroxyethylcellulose 0.10 Ashland Phase C ULTRATHIX P-100 Polymer Acrylic Acid/VP Crosspolymer 0.80 Ashland Phase D REFINED SHEA BUTTER Butyrospermum Parkii (Shea) Butter 1.10 Ashland ORCHID COMPLEX OS ester Caprylic/Capric Triglyceride (and) Cymbidium 3.50 Ashland Grandiflorum Flower Extract Phase E LUBRAJEL Oil Free Hydrogel Glycerin (and) Glyceryl Acrylate/Acrylic Acid 3.00 Ashland LUBRAJEL II XD Free Hydrogel Glycerin (and) Glyceryl Polyacrylate 2.00 Ashland BELSIL DM 5 Dimethicone 4.00 Wacker UNICERT Yellow 08005-J (sol. 0.1%) Water/aqua (and) CI 19140 (Yellow 5) 0.09 Sensient UNICERT Blue 05601-J (sol. 0.1%) Water/aqua (and) CI 42090 (Blue 1) 0.21 Sensient Optiphen preservative Phenoxyethanol (and) Caprylyl Glycol 0.50 Ashland Phase F Surfin 96 Alcohol 7.00 Deulep Phase G Deionized Water Water/Aqua 1.50 / Sodium Hydroxide Sodium Hydroxide 0.08 Fisher Phase H Parfum Desiro Blue Parfum/Fragrance (Limonene (and) Linalool (and) 0.25 Luzi/Speciatec Citronellol (and) Hydroxyisohexyl 3-cyclohexene carboxaldehyde) GP4G SP biofunctional Water/aqua (and) Artemia extract 1.00 Ashland AQUA-OSMOLINE biofunctional Water (and) Glycerin (and) Ceratonia Siliqua 1.00 Ashland (Carob) Seed Extract Total 100.00

(48) Preparation Method 1. Homogenize phase A in the main tank 2. Sprinkle over phase B and homogenize for 10 minutes until homogenized 3. In a beaker set aside, prepare phase C, homogenize until homogenized. Sprinkle in phase D and mix well until homogenized 4. At 25 C., add phase C+D to the main vessel and mix until homogenized 5. At 25 C., add phase E to the main vessel and mix until homogenized 6. At 25 C., add phase C to the main vessel and mix until homogenized 7. Stop at 25 C.

(49) 3 Eye Balm

(50) TABLE-US-00003 Weight Ingredients (Brand Name | INCI) percent Supplier Phase A Deionized Water Water/Aqua Qsp 100 / Na4EDTA Tetrasodium EDTA 0.01 Fisher Phase B RAPITHIX A-100 Sodium Polyacrylate 1.8 Ashland Phase C CEGESOFT VP Vegetable oil (and) Hydrogenated vegetable 3.00 Cognis oil (and) Candelilla Wax SI-TEC GF 3096 Dimethicone (and) Dimethiconol 10.00 Ashland Phase D DC 9701 Cosmetic Powder Dimethicone/Vinyl Dimethicone 1.00 Dow Corning Crosspolymer (and) Silica Phase E OPTIPHEN preservative Phenoxyethanol (and) Caprylyl Glycol 0.50 Ashland ZEMEA Propanediol 5.00 Dupont Tate & Lyle Phase F UNICERT Blue 05601-J (sol. 0.1%) Water/aqua (and) CI 42090 (FD&C Blue No. 0.20 Sensient 1) GP4G SP biofunctional Water/aqua (and) Artemia extract 1.00 Ashland Total 100.00

(51) Preparation Method 1. Add water to the main tank and start heating to 80 C. 2. Sprinkle in phase B at 60 C. and mix well until homogenized. 3. At 80 C., add phase C, mix well and add phase D 4. Add the ingredients from phase E into a beaker set aside and heat to 75-80 C. 5. At 80 C., add phase E to the main tank and mix well. The emulsion should be homogenized. 6. Start cooling 7. At 65 C., add phase F and mix well 8. Cool to 30 C. and add phase G and then phase H, mixing well between each phase until homogenized 9. Stop at 25 C.