TOPICALLY ADMINISTRABLE LYZATE OF DEDIFFERENTIATED CELLS OF THE PLANT HELICHRYSUM STOECHAS FOR MOISTURIZING THE SKIN

Abstract

Topically administrable lyzate of dedifferentiated cells of the plant Helichrysum stoechas for improving the moisturizing of the skin and/or the scalp and/or the lips.

Claims

1. A lyzate of dedifferentiated cells of the plant Helichrysum stoechas in a form that is suitable for topical administration, for improving the moisturization of the skin and/or the scalp and/or the lips.

2. A method for producing the lyzate as claimed in claim 1, the method comprising high-pressure homogenization of the culture of dedifferentiated cells of the plant Helichrysum stoechas.

3. A composition suitable for topical use which is in the form of a gel and which comprises, per 100% of its mass: from 95% to 99.5% by mass of the lyzate as defined in claim 1, from 0.5% to 5% by mass of at least one thickener and/or gelling agent.

4. The composition as claimed in claim 3, wherein the gelling agents and/or thickeners are chosen from polysaccharides, cellulose and cellulose derivatives, starches and linear or branched or crosslinked polymers of polyelectrolyte type.

5. The composition as claimed in claim 3, in a form which is suitable for topical administration, for improving the moisturization of the skin and/or the scalp and/or the lips.

6. A composition suitable for topical use which is in the form of a gel and which comprises, per 100% of its mass: from 50% to 80% by mass of the lyzate as defined in claim 1, from 0.1% to 5% by mass of at least one thickener and/or gelling agent, and from 15% to 49.9% by mass of at least one solvent.

7. The composition as claimed in claim 6, wherein the solvent is chosen from the elements of the group consisting of: the compounds of formula (Ia):
HO—[CH.sub.2—CH(OH)—CH.sub.2—O].sub.n—H  (Ia) in which n represents an integer greater than or equal to 1 and less than or equal to 15; the compounds of formula (Ib):
Ra1-C(Rb1)(OH)—C(OH)(Rc1)(Rd1)  (Ib), in which each of the radicals Ra1, Rb1, Rc1 and Rd1 represent, independently of each other, a hydrogen atom or a saturated aliphatic radical including from 1 to 5 carbon atoms, or by the formula (Ib1):
Ra1-C(Rb1)(OH)—[C(Re1)(Rf1)].sub.t—C(OH)(Rc1)(Rd1)  (Ib1), in which t is equal to 1, 2 or 3 and each of the radicals Ra1, Rb1, Re1, Rd1, Re1 and Rf1 represent, independently of each other, a hydrogen atom or a saturated aliphatic radical including from 1 to 5 carbon atoms, it being understood that at least one of the radicals Ra1 or Rb1 and/or at least one of the radicals Rc1 or Rd1 does not represent a hydrogen atom.

8. The composition as claimed in claim 6, wherein the solvent is glycerol.

9. The composition as claimed in claim 6, which is in a form that is suitable for topical administration, for improving moisturization of the skin and/or the scalp and/or the lips.

10. A composition suitable for topical use which is in the form of an emulsion of water-in-oil type or of an emulsion of oil-in-water type, comprising the lyzate as defined in claim 1.

11. A composition suitable for topical use which is in the form of a water-in-oil emulsion and which comprises, per 100% of its mass: from 60% to 90% by mass of the composition suitable for topical use as defined in claim 3, from 10% to 40% by mass of a fatty phase comprising i) at least one oil, and and ii) an emulsifying system comprising at least one emulsifying surfactant.

12. The composition as claimed in claim 11, wherein the emulsifying surfactant is chosen from the elements of the group consisting of alkylpolyglycoside compositions, compositions of alkylpolyglycosides and of fatty alcohols, polyglycerol esters, alkoxylated polyglycerol esters, polyglycol polyhydroxystearates, polyglycerol polyhydroxystearates and alkoxylated polyglycerol polyhydroxystearates.

13. A composition suitable for topical use which is in the form of an oil-in-water emulsion and which comprises, per 100% of its mass: from 50% to 90% by mass of a cosmetically acceptable aqueous phase, said aqueous phase comprising, per 100% of its own mass, from 0.5% to 10% by mass of the lyzate as defined in claim 1, from 10% to 50% by mass of a fatty phase comprising, per 100% of its own mass: from 0.5% to 20% by mass of at least one surfactant of oil-in-water type, from 80% to 99.5% by mass of at least one oil and/or one wax.

14. The composition as claimed in claim 10, which is in a form that is suitable for topical administration, for improving the moisturization of the skin and/or the scalp and/or the lips.

15. The composition as claimed in claim 10, which is in a form that is suitable for topical administration, for reducing and/or eliminating and/or preventing chapping and/or dry patches and/or cracking and/or atopic dermatitis and/or ichthyosis and/or dryness of the skin or mucous membranes, accompanying cutaneous and/or mucosal pathologies.

16. The composition of claim 7, wherein n represents an integer greater than or equal to 1 and less than or equal to 10.

17. The composition of claim 7, wherein n represents an integer greater than or equal to 1 and less than or equal to 6.

18. The composition of claim 7, wherein n represents an integer greater than or equal to 1 and less than or equal to 4.

19. The composition of claim 7, wherein n is 4.

20. The composition of claim 7, wherein n is 4.

Description

DESCRIPTION OF THE PREFERRED EMBODIMENTS

1) Preparation of a Lyzate According to the Invention (Ly)

[0297] A lyzate (Ly) of dedifferentiated cells of the plant Helichrysum stoechas is prepared by performing the process described previously and more particularly by performing: [0298] a step a) of preparing the sterile Helichrysum stoechas sample, as described previously, using Helichrysum stoechas leaves cut into fragments of a few millimeters, and then sterilized by successive passage: [0299] in a bath of 70% ethanol and Tween™ 80 (at 0.05%) for five minutes [0300] in a 1% aqueous bleach bath for five minutes.

[0301] The leaves are subsequently rinsed in sterile distilled water three times in succession. [0302] A step b) of callogenesis, as described previously, with a callogenesis medium, the constitution of which is described in table 1 above, with an incubation phase having a duration of three weeks at a temperature of 20 to 25° C. in the light. [0303] A step c) of suspending the dedifferentiated cells, as described previously, with a culture medium, the composition of which is described in table 2 above, for a period of 14 to 30 days, at a temperature of 20 to 25° C., in daylight, agitated by orbital-type agitation at a speed of 100 rpm. [0304] A step d) of selection of a fine suspension as described previously, with withdrawal of ⅙ to ¼ of the culture obtained in step c), to which is added a volume of suspension medium as described in table 2 and in a volume adjusted to the volume of inoculum available in the container. [0305] The new suspension is cultured in a climatically controlled chamber for a period of 14 days, at a temperature of 20 to 25° C., in daylight, agitated by orbital-type agitation at a speed of 100 rpm. [0306] A step e) of production of biomass as described previously, with withdrawal of ⅙ to ¼ of the medium obtained in step d), to which is added a volume of suspension medium as described in table 2 and in a volume adjusted to the volume of inoculum available in the container. [0307] The new suspension is cultured in a climatic chamber for a period of 14 days in a wave bioreactor, with a capacity of between 5 liters and 1000 liters, at a temperature of 20 to 25° C., in daylight, agitated by orbital-type agitation at a speed of 25 rpm. [0308] A step f) of high pressure homogenization of the medium obtained on conclusion of step e), by passing it through a GEA brand high pressure homogenizer, at a pressure of 500 bar on a pass at a temperature below 40° C. [0309] A lyzate (Ly) is thus obtained comprising, per 100% of its mass: [0310] 90-98% by mass of water [0311] 10-2% by mass of solids [0312] A step g) of stabilization of the medium obtained in step f), by mixing 60% by mass of the medium obtained in step f) with 0.8% by mass of a thickener sold under the brand name Solagum™ AX, 37.9% by mass of glycerol, 0.3% of potassium sorbate and 1% of sodium benzoate, brought to a pH value of 5.3.

[0313] A composition C.sub.A is thus obtained comprising, per 100% of its mass: [0314] 60% by mass of lyzate (Ly) [0315] 37.9% by mass of glycerol [0316] 0.8% by mass of Solagum™ AX [0317] 0.3% by mass of potassium sorbate [0318] 1% by mass of sodium benzoate.
2) Experimental Use of the Biological Effects of a Lyzate of Dedifferentiated Cells of the Plant Helichrysum stoechas According to the Invention

[0319] As already mentioned, the lyzate as defined previously and obtained by performing a process comprising steps a) to f) as described above, makes it possible to maintain a good level of moisturization and to contribute toward the comfort of dry, reactive and/or sensitive skin.

[0320] The particular technical effect of the lyzate (Ly) as defined previously and obtained by performing a process comprising steps a) to f) as described above, was demonstrated on several in vitro and in vivo study models.

[0321] The experiments were replicated twice.

[0322] The values were expressed as mean±sd [standard deviation].

[0323] For each condition, the percentages of stimulation or of protection were calculated as follows:

% restoration=100×[mean(composition C.sub.A)−mean(impaired epidermides)]/[mean(healthy epidermides)−mean(impaired epidermides)].

[0324] A—Moisturizing Effect

[0325] Tests were then performed to show the moisturizing effect of the lyzate (Ly) as defined previously.

[0326] Various analyses were performed to study the restoration of the barrier function following impairment by the “tape-stripping” method.

[0327] This method consists in applying a piece of adhesive tape (“D-Squame” brand) to the surface of the skin, pressing it on the surface of the skin and then removing it. The first applications remove a complete cell layer of corneocytes. The amount of corneocytes on the adhesive tape decreases as the applications progress.

[0328] The “D-Squame” adhesive tapes are applied to the explants, care being taken that they are always applied at the same place and in the same way, with a constant thumb pressure. The adhesive tape is subsequently removed in a smooth, rapid and unidirectional movement so as to obtain homogeneous removal of the corneocytes.

[0329] This model makes it possible to evaluate the impact of cosmetic formulations on the physiology of the epidermides, the barrier function of which is impaired.

[0330] According to the type of evaluation of the effect of improving the moisturization of the skin and/or scalp and/or lips, a formulation (F.sub.1) comprising 1% of composition C.sub.A is used and its mass constitution is as follows.

TABLE-US-00004 TABLE 3 Formulation (F.sub.1) Content as a Ingredients mass percentage Water QSP 100% Glycerol 1% Montanov ™ L .sup.(1) 1 Simulsol ™ 165.sup.(2) 1 Triglycerides C8-C10 3 Caprylic/Capric Triglycerides DUB ™ DNPG.sup.(3) 7 Neopentyl glycol diheptanoate Sepimax ™ ZEN .sup.(4) 1 Polyacrylate Crosspolymer-6 Sepinov ™ EMT 10 .sup.(5) 0.5 Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate Copolymer Euxyl ™ PE 9010 .sup.(6) 1 Phenoxylethanol - Ethylhexylglycerin Sensiva ™ PA40 .sup.(7) 0.5 3-Phenylpropan-1-ol-Octane-1,2-diol - Propane-1,3-diol Composition C.sub.A 1 .sup.(1) Montanov ™ L (INCI name: C14-22 Alcohols & C12-20 Alkyl Glucoside) is an emulsifying composition, intended to stabilize emulsions, and used for preparing cosmetic formulations. .sup.(2)Simulsol ™ 165 (INCI name: PEG-100 Stearate and Glyceryl Stearate) is an emulsifying composition, intended for stabilizing emulsions, notably oil-in-water emulsions, and used for preparing cosmetic formulations. .sup.(3)DUB ™ DNPG (INCI name: Neopentyl Glycol Diheptanoate) is an ester used as a fatty phase and/or emollient agent for preparing cosmetic formulations. .sup.(4) Sepimax ™ Zen (INCI name: Polyacrylate Crosspolymer-6) is a crosslinked anionic polyelectrolyte used as thickening and/or emulsifying and/or stabilizing agent for preparing cosmetic formulations. .sup.(5) Sepinov ™ EMT 10 (INCI name:Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate Copolymer is a crosslinked anionic polyelectrolyte used as a thickening and/or emulsifying and/or stabilizing agent for preparing cosmetic formulations. .sup.(6) Euxyl ™ PE 9010 (INCI name: Phenoxyethanol (and) Ethylhexylglycerin) is a preserving agent used for preparing cosmetic formulations. .sup.(7) Sensiva ™ PA40 (INCI name: 3-Phenylpropan-1-ol and Octane-1,2-diol and Propane-1,3-diol) is a preserving agent used for preparing cosmetic formulations.

[0331] A.1 Transepidermal Water Loss (or TEWL) Measurement.

[0332] The transepidermal water loss is measured using a device sold under the brand name Tewameter™ 300 by the company Courage & Khazaka.

[0333] The cylindrical measuring chamber of the Tewameter™ 300 device was applied over the insert for which a seal ensured the leak tightness between the measuring cell and the fabric. The value of the water evaporation rate (expressed in g/m.sup.2.Math.h) was determined automatically. The environmental conditions during the TEWL (transepidermal water loss) experiments were controlled at a stable temperature of 20° C. and a relative humidity of 50%.

[0334] Explants with impaired barrier function (by the tape-stripping method) characterized by a transepidermal water loss of greater than 35 g/m.sup.2.Math.h, are treated 1 hour after impairment of the barrier function and every day for five days with a formulation comprising 1% of composition C.sub.A. The transepidermal water loss is evaluated on conclusion of these five days of treatment.

[0335] The experiments were replicated three times.

[0336] The values were expressed as mean±sd [standard deviation].

[0337] For each condition, the percentages of effect were calculated as follows:

% effect=[mean(condition)]/[mean(impaired epidermis)]×100−100

[0338] Statistical analysis was performed using a Student's t test and a significance level set at 5%, by comparing the conditions in pairs. A difference between the efficiency of two products was considered: [0339] significant if p<0.05; [0340] “at the significance level” if 0.05 s p<0.1; [0341] and not significant if p>0.1.

[0342] The results are collated in Table 4 below:

TABLE-US-00005 TABLE 4 TEWL (g/m.sup.2 .Math. h) ± SD D 0 D 5 % effect Healthy explants — 10.0 ± 1.6 — Impaired explants 41.8 ± 3.9 36.6 ± 4.2 −13%.sup.SL   Impaired explants + 52.0 ± 3.5 37.1 ± 4.5 −29%*** Formulation (F.sub.1) containing 1% of Composition C.sub.A Change in transepidermal water loss SL 0.1 < p < 0.05, ***p < 0.001

[0343] The decrease in the transepidermal water loss means an improvement in the skin's barrier function, in other words the skin is better hydrated.

[0344] Thus, the treatment of the impaired explants with the combination containing composition C.sub.A, which is the subject of the present invention, makes it possible to reduce the transepidermal water loss between D0 and D5, thus indicating a better state of moisturization of the skin.

[0345] A.2 Measurement of the Intercellular Spaces

[0346] The term “intercellular space” refers to the space between two neighboring cells. Explants with an impaired barrier function (by the “tape-stripping” method) characterized by a transepidermal water loss of greater than 35 g/m.sup.2.Math.h, are treated one hour after impairment of the barrier function and every day for five days with a formulation comprising 1% of composition C.sub.A.

[0347] The experiments were replicated three times.

[0348] The intercellular spaces between the keratinocytes are measured via the method consisting of an ultrastructure study by Transmission Electron Microscopy (TEM) using a Hitachi HT7700 microscope.

[0349] Tissues were fixed with 2% glutaraldehyde in 0.1 M Sorensen phosphate buffer (pH 7.4), stained with aqueous 2% uranyl acetate solution and then dehydrated and embedded in epoxy resin (Epon 812). Ultrafine sections (50 nm) were mounted on copper grids.

[0350] For each condition, the intercellular spaces between the keratinocytes of the basal layer of the epidermis (the deepest layer of the epidermis) were measured on the basis of the images obtained. This analysis was not performed on healthy explants.

[0351] The experiments were replicated three times.

[0352] The values were expressed as mean±sd [standard deviation].

[0353] For each condition, the percentages of effect were calculated as follows:

% effect=[mean(condition)]/[mean(impaired epidermis)]×100−100

[0354] Statistical analysis was performed using a Student's t test and a significance level set at 5%, by comparing the conditions in pairs. A difference between the efficiency of two products was considered: [0355] significant if p<0.05; [0356] “at the significance level” if 0.05 s p<0.1; [0357] and not significant if p>0.1.

TABLE-US-00006 TABLE 5 Intercellular spaces (μm) Mean ± SD % effect Impaired explants 1.15 ± 0.27 — Impaired explants + 0.45 ± 0.12 −61%*** Formulation (F.sub.1) containing 1% of composition C.sub.A measurement of the intercellular spaces on impaired explants ***p < 0.001

[0358] The reduction in the intercellular spaces between the keratinocytes of the basal layer of the epidermis by a value of −61%, after association between the impaired explants and composition C.sub.A according to the invention, reflects better cohesion and better organization of the tissue. Being more cohesive and better organized, the explant limits its water loss, which leads to a decrease in the transepidermal water loss and thus an improvement in the skin's barrier function and better skin moisturization.

[0359] A.3 Measurement of the Stratum Corneum Thickness

[0360] The term “stratum corneum thickness” refers to the thickness of the most superficial layer of the epidermis or cornified layer.

[0361] Explants with impaired barrier function (by the tape-stripping method) characterized by a transepidermal water loss of greater than 35 g/m.sup.2.Math.h, are treated 1 hour after impairment of the barrier function and every day for five days with a formulation comprising 1% of composition C.sub.A.

[0362] The experiments were replicated three times.

[0363] The stratum corneum thickness was measured via the method consisting of an ultrastructure study by Transmission Electron Microscopy (TEM) using a Hitachi HT7700 microscope.

[0364] Tissues were fixed with 2% glutaraldehyde in 0.1 M Sorensen phosphate buffer (pH 7.4), stained with aqueous 2% uranyl acetate solution and then dehydrated and embedded in epoxy resin (Epon 812). Ultrafine sections (50 nm) were mounted on copper grids.

[0365] For each condition, the thickness of the stratum corneum was measured on the basis of the images obtained (four measurements per image). This analysis was not performed on healthy explants.

[0366] The experiments were replicated three times.

[0367] The values were expressed as mean±sd [standard deviation].

[0368] For each condition, the percentages of effect were calculated as follows:

% effect=[mean(condition)]/[mean(impaired epidermis)]×100−100

[0369] Statistical analysis was performed using a Student's t test and a significance level set at 5%, by comparing the conditions in pairs. A difference between the efficiency of two products was considered: [0370] significant if p<0.05; [0371] “at the significance level” if 0.05 s p<0.1; [0372] and not significant if p>0.1.

TABLE-US-00007 TABLE 6 Thickness of the stratum corneum (μm) Mean ± SD % effect Impaired explants  7.48 ± 1.07 — Impaired explants + 13.26 ± 2.33 77%*** Formulation (F.sub.1) containing 1% of composition C.sub.A measurement of the thickness of the stratum corneum on impaired explants ***p < 0.001

[0373] The stratum corneum is the most superficial cell layer of the epidermis, the most superficial tissue of the skin.

[0374] The increase in the thickness of this layer by +77%, when composition C.sub.A according to the invention is combined with impaired explants, makes it possible to limit the water loss, resulting in better moisturization of the skin and improved restoration of the barrier function.

[0375] A.4 Measurement of the Biochemical Factors Promoting Skin Moisturization.

[0376] The term “NMF” (acronym for “Natural Moisturizing Factor”) refers to a natural moisturizing factor comprising a set of hygroscopic substances located within the corneocytes of the epidermis.

[0377] The term “ceramides” refers to chemical compounds of the ceramide family whose function is to maintain the cohesion of the various elements of the skin. Ceramides form a protective layer to help the skin combat dehydration and to protect it against external attack.

[0378] Explants with impaired barrier function (by the tape-stripping method) characterized by a transepidermal water loss of greater than 35 g/m.sup.2.Math.h, are treated 1 hour after impairment of the barrier function and every day for five days with a formulation comprising 1% of composition C.sub.A.

[0379] The experiments were replicated twice.

[0380] The values were expressed as mean±sd [standard deviation].

[0381] For each condition, the percentages of effect were calculated as follows:

% effect=[mean(condition)]/[mean(impaired epidermis)]×100−100

% restoration=100×[mean(composition C.sub.A)−mean(impaired epidermis)]/[mean(healthy epidermis)−mean(impaired epidermis)].

[0382] The NMFs are measured by the LC-MS/MS chromatography method using an Agilent 1100 HPLC system (Agilent Technologies) coupled to a Micromass Quattro Micro API mass spectrometer (Waters), equipped with electrospray ionization. The values obtained from the NMF measurement are expressed in micrograms per milligram of protein.

[0383] The ceramides are measured by the LC-MS/MS chromatography method using a system consisting of a high-pressure liquid chromatography or HPLC UltiMate 3000 liquid (ThermoScientific) coupled to an MSQ Plus mass spectrometer (ThermoScientific), equipped with atmospheric pressure chemical ionization (APCI).

[0384] The values obtained from the ceramide measurement are expressed in arbitrary units per milligram of protein.

TABLE-US-00008 TABLE 7 Ceramides (UA/mg of NMFs (μg/mg prot) % of prot) % Mean ± SD % effect restoration Mean ± SD % effect restoration Healthy 48.56 ± 3.56 — — 54.46 ± 3.85 — — explants Impaired 28.13 ± 2.81 −42% — 29.24 ± 3.43 −46% — explants vs vs Healthy Healthy explants explants Impaired 32.33 ± 2.56 +15% 24% 36.49 ± 2.73 +25% 32% explants + vs vs Formulation (F.sub.1) Impaired Impaired containing explants explants 1% of composition C.sub.A [0385] measurement of ceramides and NMFs on untreated impaired explants and on impaired explants treated with composition C.sub.A according to the invention (no statistics—n=2)

[0386] The restoration of 24% of the amount of ceramides after treatment of the impaired explants with composition C.sub.A according to the invention leads to and reflects an improvement in the restoration of the barrier function.

[0387] The restoration of 32% of the amount of NMFs after treatment of the impaired explants with composition C.sub.A according to the invention leads to and reflects an improvement in the differentiation of the keratinocytes and thus an improvement in the restoration of the skin's barrier function.

[0388] The restoration of the skin's barrier function makes it possible to combat dehydration of the skin and to protect it against external attack.

[0389] A.5 Measurement of the Number of Keratohyalin Grains

[0390] Keratohyalin grains are constituents of the granular layer of the skin.

[0391] The increase in the number of keratohyalin grains (containing pro-filaggrin) reflects an improvement in the keratinocyte differentiation process.

[0392] Explants with impaired barrier function (by the tape-stripping method) characterized by a transepidermal water loss of greater than 35 g/m.sup.2.Math.h, are treated 1 hour after impairment of the barrier function and every day for five days with a formulation comprising 1% of composition C.sub.A.

[0393] The keratohyalin grains are measured via the method consisting of an ultrastructure study by Transmission Electron Microscopy (TEM) using a Hitachi HT7700 microscope.

[0394] Tissues were fixed with 2% glutaraldehyde in 0.1 M Sorensen phosphate buffer (pH 7.4), stained with aqueous 2% uranyl acetate solution and then dehydrated and embedded in epoxy resin (Epon 812). Ultrafine sections (50 nm) were mounted on copper grids.

[0395] For each condition, a quantification of the keratohyalin grains was performed on the basis of the images obtained. This analysis was not performed on healthy explants.

[0396] The experiments were replicated three times.

[0397] The values were expressed as mean±sd [standard deviation].

[0398] For each condition, the percentages of effect were calculated as follows:

% effect=[mean(condition)]/[mean(impaired epidermis)]×100−100

[0399] Statistical analysis was performed using a Student's t test and a significance level set at 5%, by comparing the conditions in pairs. A difference between the efficiency of two products was considered: [0400] significant if p<0.05; [0401] “at the significance level” if 0.05 s p<0.1; [0402] and not significant if p>0.1.

TABLE-US-00009 TABLE 8 Number of keratohyalin grains Mean ± SD % effect Impaired explants 21.9 ± 4.5 — Impaired explants + 32.6 ± 5.6 49%*** Formulation (F.sub.1) containing 1% of composition C.sub.A measurement of the number of keratohyalin grains on the impaired explants treated with composition C.sub.A according to the invention ***p < 0.001

[0403] The increase in the number of keratohyalin grains when the impaired explants are combined with composition C.sub.A that is a subject of the present invention, relative to the number of keratohyalin grains of the impaired explants, reflects an improvement in the keratinocyte differentiation process and consequently an improvement in the keratinocyte differentiation process and thus an improvement in the restoration of the skin's barrier function.

[0404] A.6 Analysis of the Lacunar Zones of Human Skin

[0405] Analyses were performed in order to study the lacunar zones which have an impact on the moisturizing effect.

[0406] The term “lacunar zones” means hydrophilic zones, resulting from the degradation of corneodesmosomes, present in the stratum corneum, containing water and numerous proteins including enzymes involved in the metabolic processing of fats and proteins.

[0407] The analysis of the lacunar zones is performed via the method consisting of an ultrastructure study by Transmission Electron Microscopy (TEM) using a Hitachi HT7700 microscope.

[0408] Tissues were fixed with 2% glutaraldehyde in 0.1 M Sorensen phosphate buffer (pH 7.4), stained with aqueous 2% uranyl acetate solution and then dehydrated and embedded in epoxy resin (Epon 812). Ultrafine sections (50 nm) were mounted on copper grids.

[0409] For each condition, quantification of the lacunar zones was performed on the basis of the images obtained. This analysis was not performed on healthy explants.

[0410] The experiments were replicated three times.

[0411] The values were expressed as mean±sd [standard deviation].

[0412] For each condition, the percentages of effect were calculated as follows:

% effect=[mean(condition)]/[mean(impaired epidermis)]×100−100

[0413] Statistical analysis was performed using a Student's t test and a significance level set at 5%, by comparing the conditions in pairs. A difference between the efficiency of two products was considered: [0414] significant if p<0.05; [0415] “at the significance level” if 0.05 s p<0.1; [0416] and not significant if p>0.1.

TABLE-US-00010 TABLE 9 Number of lacunar zones Mean ± SD % effect Impaired explants 2.7 ± 0.6 — Impaired explants + 6.7 ± 1.5 150%* Formulation (F.sub.1) containing 1% of composition C.sub.A measurement of the number of lacunar zones on the impaired explants treated with composition C.sub.A according to the invention *p < 0.05

[0417] The impaired explants treated with composition C.sub.A according to the invention have a greater number of lacunar zones than the impaired explants not combined with composition C.sub.A, reflecting a higher water content in the analyzed skin and thus better moisturization.

[0418] A.7 Analysis of Human Skin by Corneometry

[0419] The object of corneometry analysis of human skin is to determine the state of moisturization of the upper layers of the epidermis (stratum corneum) by measuring their electrical properties.

[0420] This analysis by corneometry is performed via the method consisting in measuring the capacitance of the dielectric medium. Any change in the moisturization of the skin surface causes a modification of these dielectric constants, and consequently of the capacitance measurement. The corneometer measures this capacitance of the most superficial layers of the epidermis, using a probe applied vertically on the skin, with a constant pressure. During the measurement, an electric field penetrates the upper layers of the skin and the dielectric constant is measured. The measurement is obtained 1 second after the application. It is expressed in arbitrary units ranging from 0 to 125 which denote a moisturization index (6-10: very dry skin—10-50: dry skin—50-125: hydrated skin).

[0421] The corneometer used in this study was a CM825 Cormeometer® (Courage & Khazaka). Thus, women were recruited to evaluate the moisturizing effect of composition C.sub.A versus placebo and versus its glycerol equivalent (0.364%). They applied the formulations (formulation (F.sub.1) described previously and formulation (F′.sub.1) differing from (F.sub.1) only by the absence of composition C.sub.A) twice a day for 21 days, with measurements at DO, D7 and D21.

[0422] The moisturization was evaluated by corneometry (measurement of the water content of the stratum corneum). [0423] The results are as follows:

TABLE-US-00011 TABLE 10 Variation of the moisturization index (AU) Product between D 21-D 0 Placebo +32.7% (p < 0.0001) Formulation (F.sub.1) containing +46.7% (p < 0.0001) 1% of Composition C.sub.A Glycerol 0.364% +36.1% (p < 0.0001) Variation of the moisturization index (AU) Product vs placebo on D 29 Formulation (F.sub.1) containing 38.7% (p = 0.0077) 1% of Composition C.sub.A Variation of the moisturization index (AU) Product vs glycerol 0.364% on D 21 Formulation (F.sub.1) containing 31.65% (p = 0.0397) 1% of Composition C.sub.A

[0424] This study shows the moisturizing capabilities of composition C.sub.A according to the invention, after a duration of 21 days.

General Conclusions

[0425] In conclusion, the experimental tests showed that composition C.sub.A according to the invention, comprising the lyzate (Ly) of dedifferentiated cells of the plant Helichrysum stoechas, is capable of increasing and/or maintaining the state of moisturization of human skin and/or scalp more particularly dry and/or sensitive and/or reactive skin, by reducing the transepidermal water loss, by decreasing the intercellular spaces between the keratinocytes of the basal layer of the epidermis, by increasing the thickness of the stratum corneum, stimulating the restoration of the cutaneous barrier and by increasing the number of lacunar zones.

B) Formulations

[0426] In the following formulations, the percentages are expressed on the basis of the weight of the formulation.

B.1 Face Makeup-Removing Fluid

Formula

[0427]

TABLE-US-00012 Composition (C.sub.A) 10.00% Methyl paraben 0.15% Phenoxyethanol 0.80% Sepicalm ™ S 1.00% Perfume/Fragrance 0.10% Water qs 100.00%

Procedure:

[0428] Mix the various ingredients in the water with magnetic stirring, in the order indicated, and adjust the pH to about 7.

[0429] B.2 Infant Hair and Body Shampoo

Formula

[0430]

TABLE-US-00013 A Composition (C.sub.A) 15.00% Proteol ™ APL 5.00% Sepicide ™ HB 0.50% Perfume/Fragrance 0.10% B Water 20.00% Capigel ™ 98 3.50% C Water qs 100.00% Sepicide ™ CI 0.30% Colorant qs Sodium hydroxide qs pH = 7.2

Procedure:

[0431] Mix composition (C.sub.A) with the Proteol™ APL and the Sepicide™ HB (Phase A). Dilute the Capigel™ 98 in a portion of the water and add it to phase A obtained previously (Phase B). Add the rest of the water to phase B, followed by the Sepicide™ CI and the colorant. Adjust the pH of the mixture to about 7.2 with sodium hydroxide.

[0432] B.3 Mild Foaming Gel

Formula

[0433]

TABLE-US-00014 A Composition (C.sub.A) 8.50% Proteol ™ APL 3.00% Euxyl ™ PE9010 1.00% Perfume/Fragrance 0.10% B Water q.s. 100.00% Lactic acid qs pH = 6.0

Procedure:

[0434] Dissolve the perfume and the preserving agent Euxyl™ PE9010 in the mixture composed of composition C.sub.A and the Proteol™ APL (phase A). Add the water and adjust the pH to about 6.0 with lactic acid.

[0435] B.4 Frequent-Use Shampoo

Formula

[0436]

TABLE-US-00015 A Composition (C.sub.A) 12.80% Proteol ™ OAT 5.00% Euxyl ™ PE9010 1.00% Perfume/Fragrance 0.30% Water qs 100.00% B Montaline ™ C40 8.50% Lactic acid qs pH = 6.0

[0437] Procedure: Mix all the ingredients of phase A and, after homogenization, add the

[0438] Montaline™ C40 and adjust the pH to about 6.0 with lactic acid.

[0439] B.5 Baby Cleansing Milk

Formula

[0440]

TABLE-US-00016 A Simulsol ™ 165 2.00% Montanov ™ 202 1.00% Lanol ™ 99 3.00% Dimethicone 1.00% Isohexadecane 3.00% B Water qs 100.00% C Sepiplus ™ 400 0.30% D Composition (C.sub.A) 6.35% E Sepicide ™ HB 0.30% DMDM Hydantoin 0.20% Perfume/Fragrance 0.10%

[0441] Procedure: Heat, separately, phases A and B constituted by mixing the various constituents. Add phase C to the hot fatty phase and make the emulsion by pouring in the aqueous phase; homogenize for a few minutes with vigorous stirring (by means of a rotor/stator turbomixer). Next, add phase D to the hot emulsion and cool the emulsion with moderate stirring down to room temperature. Add phase E at 40° C.

[0442] B.6 Infant Shower Gel Formula

TABLE-US-00017 A Water 56.06% Sepimax ™ Zen 3.00% Sepiplus ™ S 0.80% B Proteol ™ OAT 20.80% Oramix ™ NS 10 9.30% Amonyl ™ 265 BA 5.10% C Composition (C.sub.A) 2.00% Glyceryl glucoside 1.00% Phenoxyethanol & ethylhexyl glycerol 1.00% Perfume/Fragrance 0.90% Colorant 0.04%

Procedure:

[0443] Disperse the Sepimax™ ZEN in the water and stir using a mechanical stirrer equipped with a deflocculator, a counter-rotating impeller and an anchor paddle, until a perfectly smooth gel is obtained. Add the Sepiplus™ S and then stir until the mixture is homogeneous. Next, add the ingredients of phase B, homogenize and individually add the additives of phase C. Adjust the pH to 6.0-6.5.

[0444] B.7 BB Cream

Formula

[0445]

TABLE-US-00018 A Easynov ™ 2.30% Lanol ™ 99 1.00% Sepimat ™ H10W 1.00% Ethylhexyl methoxycinnamate 5.00% B Cyclomethicone 6.00% Triethoxycaprylsilane & alumina-silane & titanium oxide 8.00% Red iron oxide & triethoxycaprylsilane 0.24% Yellow iron oxide & triethoxycaprylsilane 0.66% Black iron oxide & triethoxycaprylsilane 0.09% Perfume/Fragrance 0.10% C Water qs 100% Sepinov ™ EMT10 1.20% D Composition (C.sub.A) 2.00% Sepitonic ™ M3 1.00% Phenoxyethanol & ethylhexyl glycerol 1.00%

Procedure:

[0446] Prepare phase B by mixing the various ingredients and homogenize using a mixer equipped with a rotor-stator system at a spin speed of 4500 rpm, for a period of 6 minutes. Prepare phase C by adding the Sepinov™ EMT10 to the mixture of water and glycerol, and homogenize using a mixer equipped with a rotor-stator system at a spin speed of 4000 rpm for 4 minutes. Add phases A and B to phase C, and stir the resulting mixture using a mechanical stirrer equipped with an anchor paddle, at a speed of 30 rpm for 2 minutes, and then at a speed of 50 rpm for 20 minutes. Add the components of the phase one by one and stir at a speed of 50 rpm for 25 minutes.

[0447] B.8 High-Protection Antisun Spray with an SPF of Greater than 30

Formula

[0448]

TABLE-US-00019 A Montanov ™ L 1.00% Montanov ™ 82 1.00% C12-15 Alkyl benzoate 17.00% Dimethicone 3.00% Octocrylene 6.00% Ethylhexyl methoxycinnamate 6.00% Bis(ethylhexyloxyphenol)methoxyphenyltriazine 3.00% Tocopherol 0.05% B Water qs 100% C Simulgel ™ INS 100 0.50% Cyclodimethicone 5.00% D Composition (C.sub.A) 3.00% Phenoxyethanol & ethylhexyl glycerol 1.00% Perfume/Fragrance 0.20% E Methylenebis(benzotriazolyl)tetramethylbutyl phenol 10.00% 25% Citric acid qs pH = 5
B.9 Impregnating moisturizing bath for paper masks Formula

TABLE-US-00020 B.9 Impregnating moisturizing bath for paper masks Formula A Water qs  100% Glycerol 2.00% B Sepimax™ Zen 0.15% C Phenoxyethanol and 1.00% ethylhexylglycerol 1,2-Hexanediol 1.00% D Composition (C.sub.A) 1.00%

[0449] Sepicalm™ S: Mixture of N-cocoylamino acids, sarcosine, potassium aspartate and magnesium aspartate as described in WO 98/09611;

[0450] Proteol™ APL: Mixture of sodium salts of N-cocoyl amino acids, obtained by acylation of characteristic amino acids of apple juice;

[0451] Sepicide™ HB: Mixture of phenoxyethanol, methyl paraben, ethyl paraben, propyl paraben and butyl paraben, which is a preserving agent;

[0452] Capigel™ 98: Copolymer of acrylates;

[0453] Sepicide™ CI: Imidazoline urea, is a preservative;

[0454] Sepicide™ HB: Mixture of phenoxyethanol, methyl paraben, ethyl paraben, propyl paraben, butyl paraben and isobutyl paraben, is a preserving agent;

[0455] Euxyl™ PE9010: Mixture of phenoxyethanol and ethylhexyl glycerol;

[0456] Proteol™ OAT: Mixture of N-lauryl amino acids obtained by total hydrolysis of oat protein as described in WO 94/26694;

[0457] Montaline™ C40: Monoethanolamine cocamidopropyl betainamide chloride salt;

[0458] Simulsol™ 165: Mixture of PEG-100 stearate and glyceryl stearate;

[0459] Sepiplus™ 400: Self-invertible inverse latex of polyacrylates in polyisobutene and including polysorbate 20, as described in WO 2005/040230;

[0460] Sepimax™ Zen (INCI name: Polyacrylate Crosspolymer-6): Thickening polymer in the form of a powder;

[0461] Sepiplus™ S (INCI name: Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate Copolymer & Polyisobutene & PEG-7 Trimethylolpropane Cononut Ether): Self-invertible inverse latex;

[0462] Oramix™ NS 10 (INCI name: Decyl glucoside) is a foaming agent used for preparing cosmetic formulations;

[0463] Amonyl™ 265 BA (INCI name: Cocobetaine): Foaming amphoteric surfactant;

[0464] Sepinov™ EMT10 (INCI name: Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate Copolymer): Thickening copolymer in the form of a powder;

[0465] Easynov™ (INCI name: Octyldodecanol and Octyldodecyl Xyloside and PEG-30 Dipolyhydroxystearate): Emulsifying agent having a lipophilic tendency;

[0466] Sepimat™ H10 FW (INCI name: Methyl Methacrylate Crosspolymer and Squalane): polymer used as a texture agent;

[0467] Sepitonic™ M.sub.3 (INCI name: Magnesium Aspartate and Zinc Gluconate and Copper Gluconate): Mixture used as free-radical scavenger and energizing agent for cells;

[0468] Montanov™ L (INCI name: C14-22 Alcohols and C12-20 Alkylglucoside): Emulsifying agent;

[0469] Montanov™ 82 (INCI name: Cetearyl Alcohol and Cocoyl glucoside): Emulsifying agent;

[0470] Simulgel™ INS100 (INCI name: Hydroxyethyl Acrylate/Sodium Acryloydimethyl Taurate Copolymer and isohexadecane and Polysorbate 60): Polymeric thickener.