FR2789312A1 - Preparations for external use contain sterol-3-sulfates having surfactant and biologically beneficial properties - Google Patents

Abstract

Cosmetic, dermo-pharmaceutical and veterinary preparations for external use containing sterol-3-sulfates and their salts, other than cholesterol sulfate and its salts, used for their physico-chemical and biological properties. The preferred sterol-3-sulfates are those of sitosterol, campesterol, stigmasterol, and ergosterol, terpenic alcohols (dimethyl 4-4-sterol-3-sulfates), and methyl sterol-3-sulfates, which are used for their surfactant and co-surfactant properties. Independent claim covers preparations as described, used as detergents for cleaning and protecting natural and synthetic fibers.

Description

The present invention relates to cosmetic preparations, dermo-

  pharmaceutical and veterinary preparations, for external use, for hygiene or skin and skin care, containing

  sterol 3-sulfates, free or in the form of salts; other than cholesterol sulfate and its salts.

  Sterols are partially unsaturated polycyclic alcohols universally used in living cells. The basic skeleton of sterols is cyclopentaphenanthrene. Sterols have a secondary alcohol function on carbon 3; this alcohol function is suitable

readily to sulfation.

  The present invention relates to all the natural compounds of a sterolic nature, in the form of their 3-sulfate derivatives, pure or in mixture, other than cholesterol sulfate (FIG. 1) and its salts: sterols such as sitosterol 3-sulfate (Figure 2), campesterol 3- sulfate, stigmasterol 3-sulfate, ergosterol 3-sulfate, etc., terpene alcohols (dimethyl 4-4 sterol

  3-sulfate), and methyl sterol 3-sulfates.

  These are mainly sterols of plant origin, yeast, bacteria.

  Examples of products concerned: sulphate derivatives of soy sterols, sulphate derivatives of yeast sterols.

  The use of natural sterol 3-sulfates in cosmetic preparations, dermo-

  pharmaceutical and veterinary, has certain advantages given the evolution of

  market towards the use of active ingredients of non-animal origin.

2.1 22 24

"j) o 12

19 II1

  Na + -O - 6 Figure 1 Cholesterol sulfate -Cholesten-313 ol sulfate C27H4504Na, PM = 488.7 Na + -0 S-0 Il Figure 2 P-sitosterolsulfate -Stigmasten-3 [ol sulfate C29H-O4SNa, PM = 516, 7 Sterols are generally the majority of the so-called unsaponifiable fraction of

fat body.

  The extraction and purification of these compounds does not present any particular difficulties. For example, after saponification of the fat, the sterols can be extracted with a solvent

  organic, purified by crystallization or by a chromatographic type process.

  Different chemical synthesis methods can be used to carry out the sulfation of the hydroxyl group in position 3 of steroids. For example, the use of sulfuric acid

  concentrate (1) or a sulfur trioxide complex (2,3).

  Sterol 3-sulfates have a chemical structure homologous to cholesterol sulfate, a

  compound naturally present in the skin and appendages.

  Cholesterol sulfate is an essential component of the epidermis and hair (4,5,6,7).

  In the epidermis, cholesterol sulfate represents 2 to 3% of all lipids; in the

  hair, its content is estimated at 3.3 mg / g of hair.

  Cholesterol sulfate is synthesized in the epidermis from cholesterol, thanks to the action of a sulfotransferase. It reaches its maximum level in the granular layer, (5%), then it is partly converted into cholesterol in the stratum comrneum under the action of a steroid sulfatase

(8). Its rate is then 1.5%.

  The epidermis is a structure adapted to a large number of functions, the main one being the barrier function attributed to the stratum comeum: controlling water flows and protecting

  against chemical, physical or bacterial attacks from the environment.

  Structurally and functionally, the stratum corneumn can be compared to a wall of "bricks". The "bricks", represented by the corneocytes, are held together by a lipid "cement" having a structure highly organized into lamellar bilayers. Cholesterol sulfate plays an essential role in maintaining the organization of these intercellular lipid bilayers This action is due to its capacity to form disulfide bridges. Cholesterol sulfate is therefore an essential element of cohesion between the cells of the layers.

of the epidermis.

  Cholesterol sulfate is hydrolyzed during its transit through the upper layers of the epidermis by a steroid sulfatase. This hydrolysis leads to flaking. The absence of this enzyme induces a scaling disorder: recessive ichthyosis linked to the X chromosome (9). This abnormality in the enzyme function leads to a scaly appearance of the skin. The ratio of cholesterol and cholesterol sulfate is therefore directly linked to the normal scaling process. In recent years, numerous studies have demonstrated the biological properties of cholesterol sulfate: stabilizer of biological membranes (10, 11), membrane moisturizer (12), regulator of desquamation and cell differentiation (13), action on keratinocytes (14), regulator of lipid synthesis (15), messenger precursor

  steroids, proteolytic enzyme inhibitor (16) ....

  Cholesterol sulfate is currently used in cosmetic preparations and

pharmaceuticals (17,18,19,20,21).

  The present invention relates to cosmetic, dermopharmaceutical and veterinary preparations, for external use, for hygiene or care of the skin and integuments, containing sterol 3-sulfates free or in the form of salts, other than cholesterol sulfate and its salts , used

  for their physico-chemical and biological properties.

  The physicochemical properties are linked to the amphiphilic nature of the molecule within lipid structures: surfactant or co-surfactant properties allowing the manufacture of vesicles, and in particular liposomes, because of their properties on the stabilization of

membrane lipid structures.

  The biological properties are based on the action of sterol 3-sulfates in the skin and the integuments; not only as a cohesion and protection agent, but also as an active product on the control of cell differentiation, flaking, regulation

  synthesis of lipids and skin proteins, inhibition of certain enzymes.

  The properties of the sterol 3-sulfates allow applications in the following preparations The sterol 3-sulfates constitute active principles for cosmetic preparations, such as, for example, anti-wrinkle, anti-aging, hydrating, regenerating care or make-up products, etc. Sterol 3-sulfates are active ingredients for dermatological preparations

  to treat scaling disorders, icthyosic states (dry skin).

  Sterol 3-sulfates are active ingredients for hair care intended to treat the hair and the scalp, such as, for example, dandruff preparations, preparations intended to restore the physico-chemical properties of the hair, to increase its cohesion and to

protect.

  Sterol 3-sulfates are active ingredients for nail care.

  Sterol 3-sulfates are active ingredients for products for veterinary use.

  Sterol 3-sulfates are active ingredients for the protection of natural fibers or

  synthetics, such as wool.

  EXAMPLE 1 - HIGHLIGHTING OF THE ACTION OF STEROL 3-SULPHATES ON THE STABILITY OF

CELL MEMBRANES.

  The cells are cultured in 8-well glass Labteks (Life TechnologieM, Nunc Labteklc) containing a practical volume of 400 μl. Fibroblasts are cultivated in these Labteks in a DMEM medium with a low glucose concentration (1 g / l) from GIBCO with 10% of

  fetal calf serum and 50 ig / ml gentamycin.

  At confluence, the cells are pretreated with sterol sulfate ([sitosterol sulfate) at 0.5% or cholesterol sulfate at 0.5%, then SDS is applied at a concentration of 10 kg / ml for 24 hours. Then, the cells are counted and a Hematoxylin and Eosin staining is carried out

  to study the structure and morphology of cells.

  For this, after having removed the culture medium from the wells, the slide is soaked in% alcohol for 4 min. then immersed 3 times in a water bath. Then it is placed 5 min. in hematoxylin, then rinsed for 5 min. in lukewarm water. It is then immersed 3 times in 80% alcohol and put in eosin. Finally, the slide is placed in 2 increasing alcohol baths (95 and 100%), 4 min. each then deposit 2 drops of aqueous mounting medium and

  cover with a coverslip before observation under the microscope.

  This study is carried out for sterol sulfate at 0.5% (p sitosterol sulfate) in comparison with cholesterol sulfate at 0.5% and the results are identical and show that the pretreated cells show little morphological changes in SDS and only 10% of cell death. compared to untreated control cells with significant changes

  SDS morphology and 60% cell loss.

  EXAMPLE 2 - EVIDENCE OF THE ACTION OF STEROL 3- SULPHATES ON SYNTHESIS OF KERATINES.

  Culture of keratinocytes: Normal human epidermal keratinocytes are used which are in their first passage with a doubling time of 20 hours. The medium used is Serum Free Keratinocyte Medium for Culture of Human Keratinocytes (Life TechnologiesTM) supplemented with bovine pituitary extracts (20-30 tg / ml) and recombinant epidermal growth factor (rEGF 0.1-0.2 ng / ml). The cells are cultured in Labteks and, at 60% confluence, 0.1-1% of sterol sulfate ([sitosterol sulfate) or 0.5% of cholesterol sulfate is applied. Immunolabelling takes place after 24 to 48 hours of incubation with the product. The wells are rinsed 3 times 1 min. with 0.025M TBS pH 7.6 (Tris Buffer Saline), then the cells are fixed and permeabilized with 150 gIl per well of cold methanol (-20 C) for 5 min. at 4 C, followed by 150 µL of cold acetone (-20 C) for 2 min. at room temperature. The wells are then rinsed 2 times 2 min. with TBS. Then, 150 g of pan keratin mouse anti human TEBU 1% antibody in TBS is applied 30 min. in each well at room temperature, protected from light and with constant stirring to mark keratins 6 and 10. The wells are then rinsed 3 times with TBS to remove the excess antibody

primary.

  Then, we use the Avidin-Biotin Complex system from Vector to mark with peroxidase. The cells are incubated with 150 μl of biotinylated secondary antibody diluted 1/100 in TBS. Incubate 30 min. at room temperature, protected from light and with constant stirring. The wells are rinsed 3 times 1 min. with TBS to remove excess unbound conjugate. The cells are then covered with 150 g of the Vectastain ABC kit solution for 30 min. always at room temperature, protected from light and with constant stirring. This solution consists of 10 μl of avidin and 10 μl of biotinylated peroxidase which

  are diluted in 1 ml of distilled water. Then rinsed 3 times 1 min. with TBS.

  The coloring is carried out using the “DAB Peroxidase Substrate” Kit from VECTOR (SK-

  4100). A solution of 5 ml of distilled water in which 2 drops of concentrated buffer solution (pH 7.5), 4 drops of DAB (3.3 diaminobenzidine tetrahydrochloride = chromogen) and 2 drops of hydrogen peroxide are diluted (H2O2), 150 gil are deposited per well. The incubation time is variable from 2 to 5 min. then the reaction is stopped by soaking the slides in a water bath for 5 min. After 3 baths of increasing alcohol of 1 min. (90, 95 and 100%) in order to dehydrate the cells, and 2 xylene baths of 1 min., 2 drops of Eukitt mounting medium from Labonord are poured, then a coverslip is placed on the preparation. Reading is

performed under a microscope.

  In parallel, negative controls are carried out to identify non-specific stains.

  For the primary antibody, factor FVIII or Von Willebrand factor is used. If one

  staining appears, the results cannot be interpreted because the marking is non-specific.

  It is also possible that secondary antibodies alone will bind nonspecifically to cells. for this, after fixing the cells, 150 μl of serum (of the species with which the antibody was produced) at 10% is deposited in TBS for 10 min. then 3 rinses of 1 min.

  before the deposition of the primary antibody.

  The results show that [3 sitosterol sulfate and cholesterol sulfate all increase

  two significantly the production of keratin.

  EXAMPLE 3 PREPARATION OF A STEROL 3- SULPHATE CREAM

INGREDIENTS J CHEMICAL NAME% l

I =%

  STEROLS 3-SULFATES 3 Sitosterol sulfate VASELIN OIL Mineral Oil 10 CETIOL HE Polyol - fatty acid ester 2 EMULGADE SEV Glyceryl Stearate & Ceteareth- 20 & Ceteareth-10 & 5 Cetearyl Alcohol & Cetyl Palmitate WATER Water 75.2 UREE Urea 3 GLUCOLYSAN Polydextrose 0.5 PHENONIP Phenoxyethanol & Methylparaben & Butylparaben & 0.3 Ethylparaben & Propylparaben SEPIGEL Polyacrylamide & C 13-14 Isoparafine & Laureth-7 3

  EXAMPLE 4 IN VIVO STUDY OF THE INFLUENCE OF STEROL 3- SULPHATES ON THE IMPROVEMENT OF THE ASPECT OF

DRY SKINS

Protocol

  The formula used is described in the Example.

  The placebo formula does not contain sulfate sterols.

  The test is carried out on ten healthy volunteers with skins with dry tendencies. These volunteers are healthy women between the ages of 25 and 38. The skins are classified from normal to

  dry, with some minor problems typical of skin with dry tendencies.

  Each volunteer made comments before, during, and after the test; so that

  assess the effectiveness of the product.

  Application Each volunteer applied the product on one forearm, against a placebo on the other forearm; once a day, in the evening, for a week. This is so that we can compare the formulas. A clinical evaluation and an examination of the skin condition were carried out by a

  dermatologist before and after applications.

Evaluation

  After ten days of application, there is no phenomenon of intolerance or allergy.

  There is a feeling of comfort due to a decrease in sensitivity to factors

  therefore better protection of sensitive skin.

  We also notice a more supple and regenerated skin.

  The product therefore acts as a moisturizer and repairer of the skin barrier.

  Sterol 3-sulfates are active ingredients for hair care, both for the hair and the scalp, such as for example products intended to treat dandruff conditions, to restore the physico-chemical properties of the hair, to increase the cohesion of the hair.

hair and protect it.

  EXAMPLE 5 PREPARATION OF A STEROL 3- SULPHATE SHAMPOO

CHEMICAL NAME INGREDIENTS

  STEROLS 3-SULFATES 3 Sitosterol sulfate 1 TEXAPON N 40 Sodium laurylethersulfate 30 COMPERLAN KD Coconut fatty acid diethanolamide 6 CETIOL HE Polyol - fatty acid ester 5 WATER Water 58

BIBLIOGRAPHY:

  1. LANTERI A .. Sulfonation and sulfiation technology. Journal ofAmerican Oil Chemists

Society 55: 128-133 (1978).

  2. GILBERT EE. The reaction of sulfur trioxide, and its adducts, with organic compounds.

  Chemical Revue 62: 549-589 (1962) 3. GRISSHKOVETS VI et al. New preparation of triterpenoïd-3-sulfate by the use of S03 complex. Advances in Experimental Medical Biology 405: 209-210 (1996) 4. BONTE F., SAUNOIS A .. Existence of lipid gradient in the upper stratum corneum, and its

  possible biological significance. Arch. Dermatol. Res 289: 78-82 (1997).

  5. WERTZ P.W. & DOWNING D.T .. Integral lipids of Human hair. Lipids 23 (9): 878-881

(1988).

  6. WERTZ P.W. & DOWNING D.T .. Integral lipids of Mamnalian hair. Comparative Biochem.

Physiol [B] 92 (4): 759-761 (1989).

  7SERIZAWA S. & NAGUAI T. & ITO M. & SATO Y. Cholesterol sulphate levels in the hair and nails of patients with recessive X-linked ichthyosis. Clin. Exp. Dermatol 15 (1): 13-15

(1990).

  8. FORESTIER J.P. The enzymes of the extra-cellular space of the Stratum Cornéum. International

  Journal of Cosmetic Science 14: 47-63 (1992).

  9. MALONEY M.E., WILLIAMS M.L., EPSTEIN E.H. Lipids in the pathogenesis of Ichtyosis:

  Topical cholesterol sulfate induce scaling in hairless mice. Journal of Invest. Dermatol. 83: 252-

256 (1984).

  10. BLEAU G., BODDLEY F.H., LONGPRE J. Cholesterol sulfate occurrence and possible biological fu, nction as an amphipathic lipid in the membrane of human erytrocyte. Biochemistry

Biophysica 352: 1-9 (1974).

  11. RODRIGUEZA W.V. et al. Transbilayer movement and net flux of cholesterol and

  cholesterol sulfate between liposomal membranes. Biochemistry 34: 62086217 (1995).

  12. FAURE C., TRANCHANT J.F., DUFOUR E. Comparative effects of Cholesterol and

  Cholesterol sulfate on hydration and ordering of dimyristoylphosphatidylcholine membranes.

  Biophysical Journal 70: 1380-1390 (1996).

  13. DENNING M.F., KAZANIETZ M.G., Cholesterol sulfate activates multiple Protein Kinase C isoenzymes and induces granular cell differentiation in cultured murine Keratinocytes. Cell

  Growth Differentiation 6: 1619-1626 (1995).

  1o 14. PONEC M., WILLIAMS M.L. Cholesterol sulfate uptake and outflux in cultured human

  keratinocytes. Arch. Dermatol. Res 279: 32-36 (1986).

  15. WILLIAMS M.L., RUTHERFORD S.R., FEINGOLD K.R. Effect of Cholesterol sulfate on lipid metabolism in cultured humrnan keratinocytes and fibroblasts. Journal ofLipid research 28:

955-966 (1987).

  16. IWAMORI M., IWAMORI Y., NOBOKO I. sulfated lipids as inhibitors of pancreatic trypsin and chimotrypsin in epithelium of the Mammalian digestive tract. Biochemical and Biophysical

  Research Communication 237: 262-265 (1997).

  17. Abe TAKASHI, Kondo MITSUO. (Kanebo ltd.), Cosmetics containing Ginseng extract and Cholesterol derivatives, JP 0551314 (1993) 18. Ito OBUKATA, Hirose TAKU. (Pola Chemical Industries Inc.), Nail preparation containing

Glucolipids, JP 03145412 (1991).

  19. Nodan SAYURI, Toyoshima YASUMITSU. (Pola Chemical Industries Inc.), Hair

  preparation containing cholesteryl sulfate, JP 01305018 (1989).

  i 20. Iwasaki NOBUHIRO, Nishimura KEIICHI. (Pola Chemical Industries Inc., Hair-wave

  setting preparation containing cholesteryl sulfate and / or its salts, JP 0348610 (1991).

  21. Abe TAKASHI, Kondo MITSUO. (Kanebo Itd.), Cosmetics containing Cholesteryl sulfates,

JP 60161911 (1985).

he

Claims (10)

CLAIMS S   1. Cosmetic, dermo-pharmaceutical and veterinary preparations, for external use, for hygiene or skin and skin care, characterized in that they contain sterol 3-sulfates, free or in the form of salts, other that cholesterol sulfate and its salts,   used for their physico-chemical and biological properties.   2. Preparations according to claim 1 characterized in that the sterol 3-sulfates, such as for example, sitosterol 3-sulfate, campesterol 3-sulfate, stigmasterol 3-sulfate, ergosterol   3-sulfate, terpene alcohols (dimethyl 4-4 sterol 3-sulfates), and methyl sterol 3-   sulfates, are used for their surfactant or co-surfactant properties.   3. Preparations according to claims 1 and 2 characterized in that they allow the use   of sterol 3-sulfates in the manufacture of vesicles, and in particular liposomes, due to   their properties on the stabilization of membrane lipid structures.   4. Preparations according to claims 1, 2 and 3 characterized in that the sterol 3-sulfates are   used for their biological properties in the skin and integuments: control of cell differentiation, scaling, regulation of lipid synthesis and   skin proteins, inhibition of certain enzymes.   5. Preparations according to any one of the preceding claims, characterized in that the   sterol 3-sulfates constitute active ingredients for cosmetic preparations, such as   example anti-wrinkle, anti-aging, hydrating or regenerating care or make-up products.   6. Preparations according to any one of the preceding claims, characterized in that the   sterol 3-sulfates are incorporated into dermatological preparations to treat flaking disorders.   7. Preparations according to any one of the preceding claims, characterized in that the   sterol 3-sulfates constitute active principles for hair care intended to treat the hair and the scalp, as for example, the anti-dandruff preparations, the preparations intended to restore the physicochemical properties of the hair, to increase its cohesion and protect it.   8. Preparations according to any one of the preceding claims, characterized in that the   sterol 3-sulfates are active ingredients for nail care.   9. Preparations according to any one of the preceding claims, characterized in that the   sterol 3-sulfates are active ingredients for products for veterinary use.   10. Preparations according to any one of the preceding claims, characterized in that   the sterol 3-sulfates are used in detergent or detergent preparations, for   care and protection of natural or synthetic fibers.