Plant extract comprising sucrose esters as an active agent for use in cosmetic, dermatological or nutricosmetic composition

Abstract

The invention relates to a plant extract from the calyx of one of the many plants of the Solanaceae family, of the Physalis genus, comprising mainly one or more moderately polar to non-polar sucrose esters having a carbon number of acyl groups from C1 to C10 for use as an active agent in cosmetic, dermatological or nutracosmetic compositions. It also relates to the purified sucrose esters of the plant extract or the synthesised sucrose esters as described for the same use. The invention also relates to a cosmetic, dermatological or nutracosmetic composition comprising as active ingredient, a plant extract or the synthesised sucrose esters in a suitable physiological medium. Finally, it also relates to the preparation method of the said plant extract.

Claims

1. A cosmetic, dermatological or nutracosmetic composition comprising: an effective amount of a plant extract from the calyx of one of the many plants of the Solanaceae family, of the Physalis genus, comprising mainly one or more moderately polar to non-polar sucrose esters having a carbon number of acyl groups from C.sub.1 to C.sub.10 for use as an active ingredient that is biologically active on skin, skin appendages and mucous membranes apart from any anti-inflammatory activity of the active ingredient, and a physiological medium suitable for topical or oral administration, wherein the composition is in the form of an oil-in-water or water-in-oil emulsion or multiple emulsions, or in the form of a a capsule, or tablet.

2. The composition according to claim 1, wherein the active ingredient is present at a concentration between 10.sup.6 and 50% by weight with respect to the total weight of the composition.

3. The composition according to claim 1, wherein the cosmetic composition is in the form of a cream, a lotion, a milk, a shampoo, a serum, an ointment, a gel, a paste, a foam or a stick.

4. A cosmetic treatment process to improve the appearance of skin, mucous membranes or skin appendages, prevent and/or fight against the dryness of skin and mucous membranes, prevent and/or fight against cutaneous signs of ageing, fight against loss of elasticity and firmness of skin and depigment skin, the process comprising: applying on a surface of the skin and/or hair an effective quantity of a topical composition, wherein the topical composition comprises an effective amount of a plant extract from the calyx of one of the many plants of the Solanaceae family, of the Physalis genus, comprising mainly one or more moderately polar to non-polar sucrose esters having a carbon number of acyl groups from C.sub.1 to C.sub.10 and wherein the extract is biologically active on skin, skin appendages and mucous membranes apart from any anti-inflammatory activity.

5. A dermatological treatment process for pathological dryness of skin, mucous membranes, hair or skin appendages and to promote healing, comprising: applying to a surface of skin, mucous membranes, hair, or skin appendages and to promote healing, comprising applying to a surface of skin mucous membranes, hair, or skin appendages an effective quantity of a composition, wherein the composition comprises an effective amount of a plant extract from the calyx of one of the many plants of the Solanaceae family, of the Physalis genus, comprising mainly one or more moderately polar to non-polar sucrose esters having a carbon number of acyl groups from C.sub.1 to C.sub.10 and wherein the extract is biologically active on skin, skin appendages and mucous membranes apart from any anti-inflammatory activity.

Description

EXAMPLE 1: PREPARATION OF A PLANT EXTRACT FROM THE CALYX OF PHYSALIS PERUVIANA

(1) The calyces of Physalis peruviana, that are separated beforehand from the fruits and dried, are ground in a granulating mill, comprising a 1 mm grid, which produces a powder. The size of the particles is predominantly towards 500 m.

(2) The obtained powder (50 g) is placed in a cellulose thimble, which is then placed in a Soxhlet extractor.

(3) The solvent used for the extraction is ethanol 96% (50 ml), with about twenty extraction cycles.

(4) The extract in the solvent medium is evaporated with rotavapor to remove the solvent.

(5) This finally leaves behind 11.0 g extract of the calyces of Physalis peruviana containing 57% sucrose esters.

EXAMPLE 2: PREPARATION OF A PLANT EXTRACT FROM THE CALYX OF PHYSALIS PERUVIANA

(6) The calyces of Physalis peruviana, that are separated beforehand from the fruits and dried, are ground in a granulating mill, comprising a 1 mm grid, which produces a powder. The size of the particles is predominantly towards 500 m.

(7) The obtained powder (50 g) is placed in a cellulose thimble, which is then placed in a Soxhlet extractor.

(8) The solvent used for the extraction is ethyl acetate, with about twenty extraction cycles. The extract thus obtained in the solvent medium can be purified by an on-column liquid-liquid extraction with water, at a temperature ranging between 20 C. and 65 C., counter-current in order to remove water-soluble molecules. The organic phase comprising ethyl acetate and the extract is then evaporated with rotavapor to remove the solvent.

(9) The washed extract is drained of polyphenols, withanolides and free sugars.

(10) This finally leaves behind 6.2 g extract of the calyces of Physalis peruviana containing 89.0% sucrose esters.

EXAMPLE 3: PREPARATION OF A PLANT EXTRACT FROM THE CALYX OF PHYSALIS PERUVIANA

(11) The calyces of Physalis peruviana, that are separated beforehand from the fruits and dried, are ground in a granulating mill, comprising a 1 mm grid, which produces a powder. The size of the particles is predominantly towards 500 m. The powder obtained (500 g) is placed in a supercritical fluid extractor.

(12) The solvent used for the extraction is carbon dioxide with an addition of a co-solvent like an aliphatic alcohol such as ethanol.

(13) The extraction parameters are for a pressure ranging between 75 bars and 200 bars, temperature ranging between 35 C. and 50 C., CO.sub.2 flow ranging between 2 kg/h and 20 kg/h, as well as 1% to 5% ethanol with respect to CO.sub.2.

(14) The extract thus obtained in the solvent medium is decolourised with active charcoal or bleaching clay, cold crystallised and then filtered and evaporated.

(15) This finally leaves behind 85.5 g extract of the calyces of Physalis peruviana containing 87.6% sucrose esters.

(16) If required, the extract thus obtained can be purified by scraped-film or centrifugal molecular distillation in order to obtain an extract of the calyces of Physalis peruviana with 99.9% chromatographic purity.

EXAMPLE 4: PURIFICATION OF SUCROSE ESTERS FROM A PLANT EXTRACT OBTAINED FROM THE CALYX OF PHYSALIS PERUVIANA

(17) An extract from the calyx of physalis (1 g) obtained in example 1 is solubilised in 6 ml of a CH.sub.3CN/H.sub.2O solvent mixture 1:1. The solution is filtered with a thimble by Solid Phase Extraction (SPE) (C18, 10 g) which is rinsed with 10 mL of the same CH.sub.3CN/H.sub.2O mixture 1:1.

(18) The obtained eluate is associated with the filler solvent to obtain fraction 1 (300 mg).

(19) The loaded thimble is then successively eluted according to the CH.sub.3CN/H.sub.2O solvent gradient 7:3 (20 mL) and 1:0 (20 mL) to obtain 2 (500 mg) and 3 (100 mg) fractions.

(20) Fractions 1, 2 and 3 are analysed by HPLC-DAD-DEDL and are compared to the chromatogram of the initial extract.

(21) Fraction 1 comprises polyphenolic compounds, withanolides and physalines and polar compounds like sugars.

(22) Fraction 2 comprises sucrose esters.

(23) Fraction 3 is a lipid fraction.

(24) Fraction 2 (500 mg) undergoes a fractionation by preparative HPLC (C18, 50150 mm, 5 m) as per a H.sub.2O/CH.sub.3CN solvent gradient (from 3:7 to 0:1 in 35 min.) to obtain fractions: Fraction IIIb (elution between 4 and 14 min.), Fraction I (elution between 14 and 18 min; 230 mg), Fraction II (elution between 18 and 25 min.; 200 mg) and Fraction IIIa (between 25 and 35 min.).

(25) The Fraction IIIa and IIIb fractions are brought together to obtain the Fraction III fraction (50 mg).

(26) Composition of the obtained fractions:

(27) Fraction I: Compound 1,3-O-(2-methyl-1-oxopropyl)--D-fructofuranosyl (1.fwdarw.2)-3,4-bis(2-methylpropanoate)-2-decanoate--D-glucopyranoside.

(28) Fraction II: Compound 2,3-O-(3-methyl-1-oxobutyl)--D-fructofuranosyl (1.fwdarw.2)-3,4-bis(2-methylpropanoate)-2-decanoate--D-glucopyranoside.

(29) Fraction III: sucrose esters other than previous and mainly comprising the 3-O-(3-methyl-1-oxobutyl)--D-fructofuranosyl(1.fwdarw.2)-3-(2-methylpropanoate)-2-decanoate--D-glucopyranoside, the 3-O-(2-methyl-1-oxopropyl)--D-fructofuranosyl(1.fwdarw.2)-3,6-bis(2-methylpropanoate)-2-decanoate--D-glucopyranoside. the 3-O-(2-methyl-1-oxopropyl)--D-fructofuranosyl(1.fwdarw.2)-3-(3-methylbutanoate)-2-decanoate--D-glucopyranoside the 3-O-(2-methyl-1-oxopropyl)--D-fructofuranosyl(1.fwdarw.2)-3,4-bis(2-methylpropanoate)-2-nonanoate--D-glucopyranoside, the 3-O-(2-methyl-1-oxopropyl)-D-fructofuranosyl(1.fwdarw.2)-3,4-bis(2-methylpropanoate)-2-octanoate--D-glucopyranoside,

EXAMPLE 5: EFFECTS OF FRACTIONS I, II AND III ON THE EXPRESSION PROFILE OF THE CUTANEOUS CELLS

(30) The effects of fractions I, II and II have been researched on by transcriptomic analysis on models of normal human epidermal keratinocytes and human dermal fibroblasts. This global transcript expression analysis has been carried out after 4 to 24 hours of incubation by using the Affymetrix GeneAtlas platform and the U219 full human transcriptome chip containing 36,000 transcripts and variants.

(31) 1. Biological Models

(32) Normal Human Epidermal Keratinocytes (NHEK)

(33) Cellular type: Normal human epidermal keratinocytes (NHEK) Culture conditions: 37 C., 5% CO.sub.2 Culture medium: Keratinocyte-SFM supplemented with Epidermal Growth Factor (EGF) 0.25 ng/ml Pituitary Extract (PE) 25 g/ml Gentamycin 25 g/ml Test medium: Keratinocyte-SFM supplemented with Gentamycin 25 g/ml
Normal Human Dermal Fibroblasts (NHDF) Cellular type: Normal human dermal fibroblasts (NHDF), Culture conditions: 37 C., 5% CO.sub.2 Culture medium: DMEM supplemented with L-glutamine 2 mM Penicillin 50 U/mlStreptomycin 50 g/ml Calf foetal serum (CFS) 10% Test medium: DMEM supplemented with L-glutamine 2 mM Penicillin 50 U/mlStreptomycin 50 g/ml CFS 1%
2. Tested Compounds

(34) TABLE-US-00001 Tested Tested compound Appearance/Storage Stock solution concentration Fraction I Slurry 50 mg/ml in DMSO 0.8 g/ml Storage at +4 C. Fraction II Slurry 50 mg/ml in DMSO 0.8 g/ml Storage at +4 C. Fraction III Slurry 50 mg/ml in DMSO 0.8 g/ml Storage at +4 C.
3 Culture and Treatment

(35) The cells have been introduced and cultivated in the culture medium for 24 hours and then incubated in their test medium for another 24 hours. Then, the medium has been replaced by the test medium containing or not containing (control) the test compounds and the cells have been incubated for 4 or 24 hours. All the conditions have been completed in n=3.

(36) At the end of the incubation, the culture supernatants have been removed and the cell layers have been rinsed with a PBS solution. The plates have been immediately dry frozen at 80 C.

(37) 4. Principle of Use of the Affymetrix U219 ChipAnalysis of Differential Expression

(38) Preparation of Targets

(39) The total RNA of each sample have been extracted using TriPure Isolation Reagent as per the protocol recommended by the supplier. The quantity and quality of the RNA have been evaluated by capillary electrophoresis (Bioanalyzer 2100, Agilent).

(40) The amplification of the RNA and the synthesis of the biotinylated RNA analogues (RNAa) have been carried out using the GeneChip 3'IVT Express kit (Affymetrix). The single-strand complementary DNA (cDNA) have been synthesised by reverse transcription of the total RNA in the presence of oligo(dT). By action of a DNA polymerase, a double-stranded cDNA (ds DNA) has been synthesised. Biotinylated RNA have then been synthesised from the cDNA and in the presence of biotinylated ribonucleotide analogues. After a purification stage on magnetic beads, which enables to remove salts, enzymes and free nucleotides, the biotinylated RNA have been hydrolysed into fragments from 35 to 200 nucleotides with a peak at 100-120 nucleotides (fragmented targets).

(41) Quality controls of the synthesised biotinylated RNA have been carried out by capillary electrophoresis (Bioanalyzer 2100, Agilent), before and after fragmentation.

(42) Marking and Hybridisation Protocol

(43) This step has been carried out using the GeneAtlas hybridization, wash and stain kit for 3'IVT arrays kit (Affymetrix).

(44) The hybridisation of the fragmented RNAa on the Affymetrix U219 chip (36,000 transcripts and variants) has been carried out on the GeneAtlas fluidics station hybridisation station (Affymetrix) for 20 hours at 45 C. Control probes of the hybridisation (BioB, BioC and BioD) and polyA RNA controls (Lys, Phe and Dap) have been added during hybridisation. The control probes of hybridisation, directly coupled with phycoerythrin, enable to ensure the effectiveness of the hybridisation regardless of the marking step. The poly-A RNA controls are used to validate the quality of the marking.

(45) Then, the marking has been carried out in three steps (+ intermediate washing steps): Fixing the streptavidin complexphycoerythrin on the hybridised RNAa on the chip, Recognition of this complex by an anti-streptavidin antibody coupled with biotin, Addition of the streptavidin coupled with the phycoerythrin enabling an amplification of the signal.
5 Data Processing

(46) The raw data has been transferred and processed in Microsoft Excel.

(47) Intergroup comparisons have been made using the unpaired bilateral Student's t-test. The statistical analyses can be interpreted if n5; however for n<5, the calculated data is only provided for reference purposes.

(48) Formulae used:

(49) Standard error of the average: SE ave=standard deviation (Sd)/n The standard error of the average (SE aye) represents the deviation of the average of the sample with respect to the average of the actual population. The SE ave is calculated by dividing the Sd by the square root of the size of the sample.

(50) Viability percentage: viability (%)=(DO.sub.compound/DO.sub.control)100

(51) 6 Results

(52) An analysis of fold change has been carried out in Excel followed by a functional analysis carried out using the Ingenuity IPA (Interactive Pathway Analysis) software. This analysis allows grouping the significantly modulated genes in biological functions (processes).

(53) Analysis of Fibroblasts (NHDF)

(54) The analysis of the transcriptomic profiles of the fibroblasts treated by the fractions (I, II and III) has shown an expression modulation of genes, especially with fraction II.

(55) For fraction II, the modulation of the expression of the coding gene for fibrillin 1 (FBN1) is very interesting. In fact, the expression of this gene is significantly increased over time, 4 hours (X 3.27) and 24 hours (X 8.24).

(56) Fibrillin 1 (major constituent of the extracellular matrix) is secreted by the fibroblasts and constitutes an important component of the microfibrils. It is mainly involved in the assembly of elastin fibres and plays an important role in the elasticity of skin.

(57) Analysis of Keratinocytes (NHEK)

(58) The analysis of the transcriptomic profiles of the keratinocytes treated by the 3 fractions (I, II and III) has shown an expression modulation of genes, especially with fraction III.

(59) The treatments of keratinocytes by the 3 fractions (I, II and III) induced expression inhibitions of coding genes for differentiation proteins of the keratinocyte: KRT1, IVL, DSG1, DSC1, SPRR2A, SPRR2E, SPRR1A, SPRR2D, CALML5, SBSN and KRTDAP.

(60) This effect enables to deduce that the I, II and III fractions have an anti differentiating effect on the keratinocytes.

(61) In the following examples, the active ingredient of the invention can be present in different compositions in an effective quantity ranging between 10.sup.6 to 3% by mass of the composition.

EXAMPLE 6: COMPOSITIONS TO BE ADMINISTERED ORALLY

(62) The extract from the calyx of Physalis peruviana, containing sucrose esters, is integrated in oral compositions, in compositions that allow administering 50 mg to 200 mg extract per day.

(63) 1) Anti-Ageing Composition in the Form of Soft Capsule

(64) TABLE-US-00002 Extract from the calyx of Physalis peruviana 30 mg as per example 1 or 2 or 3 Argan Oil 60 mg Rich wheat germ oil in unsaponifiable matter 300 mg Vitamin of group B (B1, B2, B3, B5, B6, B9, B12) q.s.f 100% of RDA Tocotrienols q.s.f 50% of RDA Vitamin E Beeswax Soya Lecithin Edible gelatine Glycerin q.s.f 1 soft capsule

(65) This composition is administered in 4 to 6 capsules per day.

(66) 2) Skin Firmness Composition in the Form of Tablets

(67) TABLE-US-00003 Extract from the calyx of Physalis peruviana 25 mg as per example 1 or 2 or 3 Cereal extracts (wheat, buckwheat, rice, 200 mg quinoa) rich in sulphurised amino acids Zinc in the form of chelate q.s.f 100% of RDA Vitamin C q.s.f 50% of RDA Glycosaminoglycans from fish cartilage 200 mg Glucidex IT 19 (compression agent) q.s.f 1 tablet of 800 mg

(68) This composition is administered in 5 to 8 tablets per day.

(69) 3) Example in Chocolate-Flavoured Cereal Bar

(70) TABLE-US-00004 Extract from the calyx of Physalis peruviana 200 mg as per example 1 or 2 or 3 Lycopene 6 mg Astaxanthin 4 mg Fucoxanthin 4 mg Lutein in micro-encapsulated form 4 mg Micro-encapsulated tocotrienols q.s.f 100% RDA in Vitamin E dark chocolate, oligo-fructose, sugar, fructose syrup, q.s.f a bar of 50 g fat-reduced cocoa powder, crunchy cereals, skimmed milk powder, almonds, glycerol, sorbitol, vegetable oils, glucose syrup, flavouring, sweetened condensed milk, soya lecithin, mono and diglycerides of fatty acids, caramelised syrup, maltodextrine, salt, potassium sorbate, alpha-tocopherol

(71) This composition is administered once a day.

(72) 4) Example in Vanilla-Flavoured Milk Beverage

(73) TABLE-US-00005 Extract from the calyx of Physalis peruviana 200 mg as per example 1 or 2 or 3 Extract of green tea rich in polyphenols 100 mg Vitamin of group B (B1, B2, B3, B5, B6, B9, B12) q.s.f 100% of RDA Zinc, magnesium, selenium q.s.f 100% of RDA Skimmed milk powder, flavouring, fructose, egg q.s.f one white, exhausted vanilla seeds, sugar, caramel, sachet of 30 g beta-carotene, xanthan gum, aspartame, potassium acesulfame, soya lecithin, maltodextrine.

(74) This composition is administered once a day.

EXAMPLE 7: COSMETIC COMPOSITIONDAY CREAM FOR THE FACE

(75) TABLE-US-00006 INCI names % by mass Phase A Water q.s.f 100 Carbomer 0.25 Phase B Butylene glycol 2.00 Phenoxyethanol qs Phase C Steareth-2 0.40 Steareth-10 1.20 Cetearyl alcohol & Dicetyl phosphate & Ceteth 10 phosphate 4.00 Cetearyl Alcohol 1.00 Azone 2.50 Cyclohexasiloxane & Cyclopentasiloxane 2.00 Ethylhexyl succinate 7.00 Phase D Potassium sorbate 0.10 Phase E Water 3.00 Sodium hydroxide 0.40 Phase F Active ingredient of the invention (of example 1 or 2 or 3) 2.00
Procedure:

(76) Weigh phase A and let swell without stirring for 30 min. Heat phase A to 75 C. in water bath. Weigh and mix phase B. Weigh phase C and heat to 75 C. in water bath. Add phase B in phase A. Mix well. Pour phase C in phase A+B while stirring continuously. Homogenise well. Add phase D, extemporaneously. Add phase E, homogenise well. Add phase F, homogenise well.

EXAMPLE 8: COSMETIC COMPOSITIONGEL FORM FOR THE FACE

(77) TABLE-US-00007 INCI names % by mass Phase A Water q.s.f 100 Cetyl hydroxyethylcellulose 0.30 Phase B Carbomer 0.40 Water 20.00 Phase C Glycerin 3.00 Ethyl & Methyl & Propyl parabens 0.30 Phase D Mineral oil 4.00 Polysorbate 20 1.00 C12-15 Alkyl Benzoate 2.00 C10-30 Alkyl Acrylate cross polymer 0.30 Phase E Potassium sorbate 0.10 Phase F Water 5.00 Sodium hydroxide 0.50 Phase G Active ingredient of the invention (of example 1 or 2 or 3) 1.00
Procedure:

(78) Disperse Phase A while stirring continuously. Sprinkle Ultrez 10 in water and let swell for 30 minutes. Heat phase C till complete dissolution. Mix Phase A with Phase B. Add C to Phase (B+A). Add Phase D while stirring continuously, to Phase (A+B+C). Add Phase E. Neutralise with Phase F. Add Phase G and mix.

EXAMPLE 9: COSMETIC COMPOSITIONSERUM FORM

(79) TABLE-US-00008 INCI names % by mass Phase A Water q.s.f 100 Carbomer 0.25 Phase B Butylene Glycol 3.00 Phenoxyethanol 0.20 Phase C Polysorbate 20 0.50 Cetearyl Ethylhexanoate 2.00 PPG-3 Benzyl Ether Myristate 0.50 Acrylates/C 10-30 Alkyl Acrylate 0.20 Cross Polymer Cyclopentasiloxane 1.00 Phase D Potassium sorbate 0.10 Phase E Sodium hydroxide 0.45 Water 4.00 Phase F Active ingredient of the invention (example 1 or 2 or 3) 1.00 Phase G Fragrance 0.10
Procedure:

(80) Phase A: Sprinkle carbomer in water, let swell for 15 minutes. Mix Phase B. Pour Phase B into Phase A and homogenise. Then weigh Phase C, mix and add to Phase A+B while stirring continuously. Let swell for 1 hour. Extemporaneously add Phase D to the previous phase while stirring continuously. Neutralise with Phase E. Stir.

(81) Then add Phase F. Let it mix for at least 1 hour while stirring continuously and then add Phase G. Mix well.

EXAMPLE 10: NIGHT CREAM FOR THE FACE

(82) TABLE-US-00009 INCI names % by mass Phase A Water q.s.f 100 Carbomer 0.40 Phase B Glycerin 3.00 Ethyl & Methyl & Propyl parabens 0.30 Phase C Cetearyl Alcohol & polysorbate 20 1.0 Cetearyl Alcohol 1.00 PPG-3 Benzyl Ether Myristate 1.0 Dimethicone 2.50 Isotridecyl Isononanoate 5.00 Phase D Potassium sorbate 0.10 Phase E Sodium hydroxide 0.40 Water 4.00 Phase F Active ingredient of the invention (of example 1 or 2 or 3) 1.00 Phase G Fragrance 0.10

(83) Procedure: Weigh Phase A and let swell for 30 minutes. Heat Phase A to 75 C. in a water bath. Heat Phase B till complete solubilisation. Add Phase B to Phase A. Heat Phase C to 75 C. in a water bath. Add Phase C to Phase A+B while stirring continuously. Add Phase D and homogenise well. Neutralise with Phase E at 55 C. Add Phase F then Phase G and homogenise well.

EXAMPLE 11: BODY CREAM

(84) TABLE-US-00010 INCI names % by mass Phase A Water q.s.f 100 Carbomer 0.4 Phase B Glycerin 3.00 Ethyl & Methyl & Propyl parabens 0.30 Phase C Sorbitan stearate 2.00 Mineral Oil 4.00 PPG-3 Benzyl Ether Adipate 1.00 Glyceryl stearate & PEG 100 stearate 3.00 Phase D Potassium sorbate 0.10 Phase E Sodium hydroxide 0.40 Water 4.00 Phase F Active ingredient of the invention (of example 1 or 2 or 3) 2.00 Phase G Fragrance 0.10

(85) Procedure: Weigh Phase A and let swell for 30 minutes. Heat Phase A to 75 C. in a water bath. Heat Phase B till complete solubilisation. Add Phase B to Phase A. Heat Phase C to 75 C. in a water bath. Add Phase C to Phase A+B while stirring continuously. Add Phase D and homogenise well. Neutralise with Phase E at 55 C. Add Phase F then Phase G and homogenise well.

EXAMPLE 12: LOTION

(86) TABLE-US-00011 INCI names % by mass Phase A Water q.s.f 100 Phase B Butylene Glycol 5.00 Phenoxyethanol 0.20 Phase C Polysorbate 20 2.00 PPG-3 Benzyl Ether Myristate 0.10 Phase D Potassium sorbate 0.10 Phase E Active ingredient of the invention (of example 1 or 2 or 3) 1.00 Phase F Fragrance 0.10

(87) Procedure: Weigh Phase A. Weigh Phase B and mix. Add Phase B to Phase A while stirring continuously for 30 minutes. Weigh Phase C, mix till the mixture is homogeneous. Add Phase C to Phase A+B while stirring continuously. Add Phase D to the previous mixture. Add Phase E to the previous mixture while stirring continuously. Homogenise well. Weigh Phase F, mix and add to the previous mixture. Mix continuously.

EXAMPLE 13: DAY CREAM

(88) TABLE-US-00012 INCI names % by mass Phase A Water q.s.f 100 Carbomer 0.2 Phase B Butylene Glycol 2.00 Phenoxyethanol 1.30 Phase C Glyceryl stearate & PEG 100 stearate 1.00 Caprylic/capric Triglycerides 4.00 Phase D Acrylates/C10-30 Alkyl Acrylates cross polymer 0.20 PPG-3 Benzyl Ether Myristate 1.00 Dimethicone 1.00 Phase E Sorbate 0.10 Phase F Sodium hydroxide 0.40 Water 4.00 Phase G Active ingredient of the invention (of example 1 or 2 or 3) 2.00 Phase H Fragrance 0.10

(89) Procedure: Phase A: Pour Ultrez 10 in water and let swell for 30 minutes. Weigh Phase B and let melt at 60 C. Heat Phase A to 75 C. in a water bath. Weigh Phase C and heat to 75 C. in a water bath. While stirring continuously (Staro s=500 rpm), add Phase C to Phase A. Randomly, add Phase B and Phase D to Phase A+B and then add Phase E to it. Homogenise well. Cool to 45 C. and add Phase F. Add Phase G at 35 C. and homogenise well. Add Phase H and homogenise well.

EXAMPLE 14: FLUID FOR THE BODY

(90) TABLE-US-00013 INCI names % by mass Phase A Water 5.00 Carbomer 0.30 Phase B Water q.s.f 100 Hydroxyethyl cellulose 0.40 Phase C Butylene Glycol 3.00 Phenoxyethanol 1.30 Phase D C12-C15 Alkyl Benzoate 2.00 Caprylic/capric Triglyceride 3.00 Polysorbate 20 1.00 PPG-3 Benzyl Ether Myristate 1.00 Acrylates/C10-30 Alkyl Acrylates cross polymer 0.20 Phase E Sorbate 0.10 Phase F Sodium hydroxide 0.50 Water 5.00 Phase G Active ingredient of the invention (of example 1 or 2 or 3) 2.00 Phase H Fragrance 0.10

(91) Procedure: Phase A: Pour Ultrez 10 in water and let swell for 30 minutes. While stirring with a propeller mixer (300 rpm), pour Phase B and let swell for 1 hour. Add Phase A to Phase B while stirring with a propeller mixer (300 rpm), and homogenise well. Weigh and stir Phase C, weigh Phase D and mix. Add Phase C to Phase A+B. Add Phase D to Phase A+B+C. Homogenise well. Add Phase E then Phase F and G and finally Phase H. pH=6.2.

EXAMPLE 15: NIGHT CREAM

(92) TABLE-US-00014 INCI names % by mass Phase A Water 5.00 Carbomer 0.30 Phase B Water q.s.f 100 Hydroxyethyl cellulose 0.40 Phase C Butylene Glycol 3.00 Phenoxyethanol 1.30 Phase D PPG-3 Benzyl Ether Myristate 1.00 Acrylates/C10-30 Alkyl Acrylates cross polymer 0.20 Phase E Sorbate 0.10 Phase F Sodium hydroxide 0.50 Water 5.00 Phase G Active ingredient of the invention (of example 1 or 2 or 3) 2.00 Phase H Fragrance 0.10

(93) Procedure: Phase A: Pour Ultrez 10 in water and let swell for 30 minutes. While stirring with a propeller mixer (300 rpm), pour Phase B and let swell for 1 hour. Add Phase A to Phase B while stirring with a propeller mixer (300 rpm), and homogenise well. Weigh and stir Phase C, weigh Phase D and mix. Add Phase C to Phase A+B while stirring with a blade mixer (300 rpm). Add Phase D to Phase A+B+C. Homogenise well. Add Phase E then Phase F and G and finally Phase H. Homogenise well.

EXAMPLE 16: ANTI-STRETCH MARKS CREAM

(94) TABLE-US-00015 INCI names % by mass Phase A Water q.s.f 100 Carbomer 0.40 Phase B Glycerin 5.00 Phenoxyethanol (and) Mixed Parabens 0.80 Phase C Ethylhexyl Palmitate 4.00 Cetearyl alcohol Croda 0.50 Myristyl Lactate 0.30 Polysorbate 20 1.00 Phase D Acrylates/C10-30 Alkyl Acrylates cross polymer 0.20 Cyclomethicone 2.00 Phase E Potassium sorbate 0.10 Phase F Sodium hydroxide 0.60 Water 6.00 Phase G Active ingredient of the invention (of example 1 or 2 or 3) 1.00 Phase H Fragrance 0.10
Procedure:

(95) Phase A: Disperse Ultrez 10 in water and let swell for 20 minutes. Mix Phase B and heat to 60 C. till complete dilution. Add Phase B to Phase A while stirring continuously. Heat Phase A+B. Weigh Phase C and heat to 75 C. Add Phase C to Phase A+B while stirring continuously. Homogenise carefully and then add Phase D. Add Phase E. Neutralise with Phase F at 50 C. Add Phase G and H at 35 C. and adjust the pH at 6.3 with NaOH.

EXAMPLE 17: HAIR LOTION

(96) TABLE-US-00016 INCI names % by mass Phase A Cetrimonium Chloride 1.00 Citric acid 0.22 Trisodium Citrate 1.20 Sorbate 0.10 Water q.s.f 100 Phase B Methyl Paraben 0.20 PPG 5 Ceteth 20 2.00 Phase C Active ingredient of the invention (of example 1 or 2 or 3) 1.00 Phase D Polysorbate 20 1.00 Fragrance 0.10

EXAMPLE 18: MOISTURISING MAKE-UP

(97) TABLE-US-00017 INCI names % by mass Phase A Water q.s.f 100 Caustic potash 1.3 Polysorbate 80 0.1 Phase B Titanium dioxide 6.00 Talc 3.05 Yellow iron oxide 1.8 Red iron oxide 1.00 Black iron oxide 0.15 Phase C Propylene glycol 4.00 Magnesium Aluminium Silicate 1.00 Phase D Propylene glycol 2.00 Sodium Carboxymethylcellulose 0.12 Phase E Di-PPG-3 Myristyl Ether Adipate 12.00 Isostearyl Neopentanoate 4.00 Cetearyl Alcohol, Ceteth-20 Phospahte, Dicetyl Phosphate 3.00 Steareth-10 2.00 Cetyl alcohol 0.62 Steareth-2 0.5 Phase F Active ingredient of the invention (of example 1 or 2 or 3) 3.00

EXAMPLE 19: LIP BALM

(98) TABLE-US-00018 INCI names % by mass Phase A Water q.s.f 100 Potassium sorbate 0.10 Magnesium sulphate 0.70 Phase B Cetyl Dimethicone Copolyol 3.00 Methyl Paraben 1.00 Tribehenin 0.30 PPG-3 BEnzyl Ether Myristate 2.00 Argania spinoa Kernel Oil 19.00 Phase C Active ingredient of the invention (of example 1 or 2 or 3) 0.50 Phase D Fragrance 0.10

(99) Procedure: Heat Phase A to 85 C. Mix Phase B and heat to 85 C. Slowly add Phase A to Phase B while stirring continuously (Staro s=3000 rpm then 1200 rpm). Add Phase C preheated to 80 C., and homogenise. Add Phase D at 35 C. Pour.

EXAMPLE 20: HAIR PROTECTING SPRAY

(100) TABLE-US-00019 INCI names % by mass Phase A Water q.s.f 100 Ethanol 10.00 Polysorbate 20 0.40 Cetrimonium Chloride 1.00 Phase B Butylene glycol (and) Helianthus Annus Seed Extract 5.00 Preservative Qs Phase C Active ingredient of the invention (of example 1 or 2 or 3) 3.00 Phase D Water 0.50 Sodium hydroxide 0.05

(101) Procedure: Weigh and mix Phase A with a blade stirrer s=300 rpm. Add Phase B, mix and then add Phase C. Adjust the pH between 5.0 and 5.5 with Phase D.

EXAMPLE 21: TESTS DEMONSTRATING THE ANTI-AGEING AND REJUVENATING ACTIVITY OF THE UCHUVA EXTRACT

(102) In the figures accompanying examples 21 to 23, the Uchuva extract is shortened to EUc. In all these examples 21 to 23, the Uchuva extract comes from the extraction process described in example 3 presenting 99.9% chromatographic purity in sucrose esters.

(103) The dermis provides a solid support to the epidermis and is also its nourishing element. It mainly comprises fibroblasts and an extracellular matrix mainly comprising collagens, elastin and a substance called basic substance. These components are synthesised by fibroblasts. The dermo-epidermal junction ensures the cohesion between the epidermis and the dermis.

(104) The collagens are predominantly proteins of the extracellular matrices of the skin. Twenty types of collagens are identified and noted from I to XX till date. The collagens that are predominantly present in the entire dermis are collagens of type I and III that form the extracellular matrix of the entire dermis (these are these collagens that constitute 70%-80% of the dry weight of the dermis). The dermis becomes thinner with age and wrinkles appear on the surface of the skin. As a result, in view of the important role that collagen plays in the integrity of the skin and its resistance to external mechanical aggressions, the stimulation of the synthesis of these collagens, and especially collagen of type I and III, seems to be an effective way to offset the signs of skin ageing (review by Tzaphlidou M., Micron 35 (2004) 173-177).

(105) Collagen I and III Targets

(106) Collagen I is the chief Collagen that gives the skin its mechanical resistance.

(107) This protein represents 90% of the collagen of a vertebrate. It constitutes the bone framework (to be compared with reinforced concrete reinforcements), and more generally the common connective tissues. It is found in bones, skin, tendons, cornea and the internal organs.

(108) 1Study on Young Cells Versus Old Cells

(109) The following study enabled studying the effect of the Uchuva extract on the expression of collagen I, an essential constituent of the extracellular matrix of the dermis.

(110) Method

(111) The NHDF (Normal Human Dermal Fibroblasts) young or aged by replicative senescence have been introduced in 96 wells plate and incubated for 24 hours at 37 C., 5% CO2.

(112) The cells have been treated for 24 hours in the presence of test products.

(113) Then, the cells have been fixed with formalin and the expression of the proteins has been detected by immunofluorescence.

(114) The fluorescent markings have been imaged and quantified by automated microscopy (Arrayscan Cellomics). The fluorescence has been quantified by the Compartimental Analysis bioapplication.

(115) Prior Evaluation of Cytotoxicity

(116) The compound has been kept in contact with the cells for 24 hours. During the last three hours of the colorimetric test, ready-to-use WST1 of Roche has been introduced in the medium.

(117) This reagent contains tetrazolium salts, a violet indicator. This reagent is cleaved to formazan, a yellow indicator, by cellules metabolically active cells. Thus, the level of yellow colouring is proportional to the number of living cells. The measurement of the absorbance is done at 450 nm. The test considers that a value lower than 90% of the control element indicates a possible cytotoxicity of the product (symbolised by a green line on the graphs). This can also indicate that the metabolic activity of the cells has been reduced. A value lower than 75% indicates significant cytotoxicity (symbolised by a red line on the graphs).

(118) In addition, the cells have been observed under the microscope to observe and compare their physiognomy.

(119) The results are given in FIG. 1A.

(120) The Uchuva extract is cytotoxic with stronger tested concentrations from 100 to 20 ppm and becomes less cytotoxic from 2 ppm.

(121) Concentrations Retained to be Tested:

(122) The Uchuva Extract is tested with the following concentrations 2-1-0.5 ppm.

(123) The results are given in FIG. 1B.

(124) The Uchuva extract allows re-establishing the Collagen I expression at the level of young cells.

(125) In young cells, the expression of Collagen I is not homogeneous. Some cells show a high marking while others have a lower marking. In cells aged by replication, the expression reactivated by the Uchuva extract is more homogeneous in the treated cells.

(126) The fact that the lowest concentration is active can be explained by the fact that the highest concentrations show a slight cytotoxicity that affects the protein synthesis.

(127) Conclusion

(128) The Uchuva extract is an ingredient that acts on collagen I, the target of skin ageing, by re-establishing the protein expression up to the expression observed in young cells.

(129) 2Evaluation of the Effect of the Uchuva Extract on Collagen I Production by Fibroblasts Materials and Methods Used

(130) Test System

(131) Description: Human Fibroblasts Model (NHDF) in culture used between passages 3 to 6.

(132) Culture medium: DMEM glucose 4.5 g/L+1% NEAA+10% SVF

(133) Culture conditions: 37 C., 5% CO2

(134) Culture Method and Treatment of Fibroblasts

(135) Evaluation of the Cytotoxicity by the Test of Neutral Red

(136) The NHDF cells are introduced on D0, in 96 wells plaque, at a rate of 4103 cells per well. They are treated on D1 for 48 hours with culture in a complete medium.

(137) The concentrations of the active to be tested are expressed in % of the extract. The stock solution of the product to be tested has been prepared in dimethyl sulfoxide (DMSO) with 20% concentration (w/v). The first dilution has been made to 1/1000th. The following dilutions have been made to while maintaining the final DMSO concentration at 0.1%.

(138) The NR test is carried out on D3: the medium is removed and the cells are rinsed with 250 L of PBS preheated to 37 C. 200 l of neutral red are added to the cells. After 3 hours of incubation at 37 C., 5% CO2, the medium containing the neutral red is removed and then the cells are rinsed with 2200 l of PBS preheated to 37 C. 100 l of the revelation solution (50% ethanol1% acetic acid) are added and the cells are incubated at ambient temperature while stirring continuously and sheltered from light for 45 minutes.

(139) The DO540 nm is measured after homogenisation.

(140) NB: Preparation of the neutral red solution: the neutral red stock solution is prepared at 0.4% in Ultra Pure water (this solution is preserved at 4 C. for 15 days). It is diluted extemporaneously to 1/80th in the complete culture medium and then centrifuged for 10 minutes at 3000 revolutions per minute (rpm) before use.

(141) Evaluation of the Metabolic Activity by the MTT (Methyl Thiazol Tetrazolium) Test

(142) The NHDF cells are introduced on D0, in 96 wells plaque, at a rate of 4103 cells per well. They are treated on D1 for 48 hours with culture in a complete medium.

(143) The concentrations of the active to be tested are expressed in % of the extract. The stock solution of the extract has been prepared in DMSO with 20% concentration (w/v). The first dilution has been made to 1/1000th. The following dilutions have been made to while maintaining the final DMSO concentration at 0.1%.

(144) The MTT test is carried out on D3: the medium is replaced by 100 L of MTT at 0.5 g/L in a complete medium. After 3 hours of incubation at 37 C., 5% CO2, the MTT solution is replaced by 100 L of DMSO. The DO540 nm is measured after homogenisation (amount condition as stated for the previous test with neutral red).

(145) Preparation Method and Tested Product

(146) Description of the product to be tested: Uchuva Extract

(147) After the first two tests given above, three concentrations of the product to be tested have been selected for the rest of the tests to be carried out: 8.10-5%(0.8 ppm), 16.10-5% (1.6 ppm) and 32. 10-5% (3.2 ppm)

(148) Dose of Collagen I in the Culture Supernatants by ELISA Method

(149) The cells are introduced on D0, in 6 wells plates, at a rate of 25103 cells per well. They are treated on D1 for 48 hours with culture in a complete medium.

(150) The concentrations of the active to be tested are expressed in % of the extract. The stock solution of the extract has been prepared in DMSO with 20% concentration (w/v). The first dilution has been made to 1/1000th. The following dilutions have been made while maintaining the final DMSO concentration at 0.016% (corresponding to the DMSO concentration in the strongest concentration of the extract).

(151) After 48 hours, the culture supernatants are recovered and stored at 80 C.

(152) The collagen I dose is administered as per the instructions of the supplier of the ELISA kit (Tecomedical).

(153) Effect of the Uchuva Extract on Collagen I Production by Fibroblasts in 48 Hours Culture:

(154) Table Presenting the Quantities of Collagen I (ng) for 104 Cells:

(155) TABLE-US-00020 Conditions Col Col I quantity in Col I quantity in 2.5 10.sup.4 cells/well No. of cells by Col I quantity in I quantity in ng/10.sup.4 D03 ng/10.sup.4 D04 Standard dilution to 1/5 condition (10.sup.4) ng/10.sup.4 D01 cell ng/10.sup.4 D02 cell cell cell Average deviation Control element 14 49.4 48.1 49.7 49.0 49.0 0.71 DMO [bone mineral density] 16 47.5 48.9 46.0 50.5 48.2 1.93 control element Uchuva Extract (EUc) 8 10.sup.5% 12 57.6 69.8 64.8 59.5 63.0 5.5 Uchuva Extract (EUc) 16 10.sup.5% 12 63.8 63.8 65.5 63.0 64.0 1.06 Uchuva Extract (EUc) 32 10.sup.5% 13 52.7 56.1 47.3 50.2 51.6 3.74

(156) These results enable to observe a significant increase in the production of Collagen I by 29% and 30% by the fibroblasts treated for 48 hours by the Uchuva extract with lower concentrations (8.10-5% and 16.10-5% respectively) with respect to the control element.

(157) With the highest concentration (32. 10-5%), the production of Collagen I by the fibroblasts reduces to a level close to the controlled conditions. This decrease can be explained by a starting of cellular toxicity at this high level of concentration.

(158) The results are in FIG. 2A.

(159) 3Evaluation of the Effect of the Uchuva Extract on the Production of Collagen III by the Fibroblasts

(160) The cells are introduced on D0, in 6 wells plates, at a rate of 25103 cells per well. They are treated on D1 for 48 hours with culture in a complete medium.

(161) The concentrations of the active to be tested are expressed in % of the extract. The stock solution of the extract has been prepared in DMSO with 20% concentration (w/v). The first dilution has been made to 1/1000th. The following dilutions have been made while maintaining the final DMSO concentration at 0.016% (corresponding to the DMSO concentration in the strongest concentration of the extract).

(162) TABLE-US-00021 EUc EUc EUc 8 105% 16 105% 32 105%

(163) After 48 hours, the culture supernatants are recovered and stored at 80 C.

(164) The collagen III dose is administered as per the instructions of the supplier of the ELISA kit (SunRed).

(165) Effect of the Uchuva Extract on Collagen III Production by Fibroblasts in 48 hours Culture:

(166) Table below presenting the quantities of collagen III (g) for 104 cells:

(167) TABLE-US-00022 Quantity of Collagen III in g/10.sup.4 cells AVERAGE AND Control element 0.78 0.21 DMO [bone mineral density] control 0.82 0.17 element Uchuva Extract (EUc) 8 10.sup.5% 0.9 0.34 Uchuva Extract (EUc) 16 10.sup.5% 1.17 0.11 Uchuva Extract (EUc) 32 10.sup.5% 0.86 0.06
The analysis of these results highlights an increase of the concentration dependent on the production of Collagen III induced by the Uchuva extract. The production is maximum at the concentration of 16.10-5% (+50% with respect to the control elements).

(168) At the highest concentration of the extract (32.10-5%), the production of Collagen III by the fibroblasts reduces to a level close to that of the control condition as in the case of the production of Collagen I. This observation confirms the presence of a cellular toxicity at this level of concentration.

(169) The results are in FIG. 2B.

(170) 4Evaluation of the Effect of the Uchuva Extract on the Collagen IV Target

(171) Collagen is the major constituent of the MEC (approx. 70%) synthesised by fibroblasts. There are fibrillar (I, II, III, V, XI) and non-fibrillar (IX, XII, XIV, XVI) collagens. Collagen fibres are flexible and they resist tension forces in the tissues. They form undulating beams with variable thickness and length. Collagen of type IV is special. It forms the primary network of the basal membranes (lamina densa). It is involved in cellular adhesion, proliferation, migration and angiogenesis (melanoma). It has a site of interaction with the integrin receptor. It is at the point of contact between the basal plate and the MEC (S Pasco et al., Cancer Detection and Prevention, 29, 260, 2005). Collagen IV, with laminins 1, forms a network that constitutes the structure of the basal membrane for a structural and functional maintenance of the tissues.

(172) The two concentrations of 4.10-5% and 8.10-5% of the Uchuva extract have been tested.

(173) Evaluation of the Quantity of Proteins of the Extracellular Matrix Produced by Fibroblasts During 48 Hours

(174) Detection of the Expression of Collagen IV by Immunocytology

(175) The cells are introduced on D0, in Millicell 8 wells slides, at a rate of 4.103 cells per ml in 400 l of complete medium. They are treated on D1 for 48 hours with culture in a complete medium. The concentrations of the active to be tested are expressed in % of the extract. The stock solution of the extract has been prepared in DMSO with 20% concentration (w/v). The first dilution has been made to 1/1000th. The following dilutions have been made while maintaining the final DMSO concentration at 0.1%. After 48 hours, the cells are fixed by an alcohol/acid mixture. Then, the cells are marked using an anti-collagen IV antibody (Rockland) diluted in PBS+BSA/tween to 1/50 for 1 hour. The slides are then rinsed twice by PBS. The revelation of the specific marking is done using an FITC coupled secondary antibody (Santa-Cruz) diluted in PBS+BSA/tween to 1/100 for 1 hour. The slides are then rinsed twice by PBS and put between the slide and the cover glass using the RotiMount Fluocare+DAPI (Roth) mounting fluid.

(176) Photos in green and blue are taken using a DP72 camera (Olympus, Japan) connected to a BX-60 epifluorescence microscope (Olympus, Japan).

(177) The photos have been observed by two readers and a scoring of the intensity of fluorescence has done on a linear scale from 0 (no marking) to 4 (very high marking).

(178) At the same time, a concentration of rhamnose has been integrated in this test as a positive control element for the expression of Collagen IV. In fact, rhamnose and polysaccharides containing rhamnose are described to induce the synthesis of collagens in fibroblasts.sup.22. The results are in FIG. 3A.

(179) The analysis of the results shows a great increase of the expression of Collagen IV (approx. +300%) in fibroblasts treated for 48 hours by the UCHUVA extract, irrespective of the concentration used. The amplitude of this increase in expression of Collagen IV is comparable to that obtained with rhamnose.

(180) 5Evaluation of the Effect of the Uchuva Extract on the Elastin Target

(181) Elastin is a structural glycoprotein (like laminin and fibronectin) used in the composition of the MEC. It is a protein of the family of structural fibrous proteins. It is secreted by fibroblasts essentially during the period of growth, and has elastic properties. Its synthesis reduces with age and elastin is replaced by inextensible collagen. Striae atrophicae is a visible example of this process, which is related to mechanical constraints. Its second example is skin ageing.

(182) In the Extracellular Matrix:

(183) The elastin is synthesised and secreted in the extracellular space by the fibroblasts first as proelastin, then as tropoelastin. It is the major component (up to 90%) of the elastic fibres plus fibrillin Hence, collagen associated with elastin and fibrillin that form the elastic fibres, by covalent cross-linked bonds, are the main constituents of the extracellular matrix. The total production of elastin stops around puberty. After this, the quantity of the available elastin will reduce with time.

(184) Degradation:

(185) The degradation of elastin is related to the action of elastase, anenzyme that is secreted by the fibroblasts. The enzymatic action of elastase is inhibited by al-antitrypsine. The inhibition of the degradation creates a balance increasing the stability of elastin.

(186) Role:

(187) There are distinctive traits that characterise elastin: it enables cells to bind and enable the biological tissues to be formed. Thus, the proper functioning of the skin, lungs, blood vessels, connective tissues, certain tendons and cartilage is closely related to the characteristics of elastin. Like its name suggests, elastin is elastic. With an equal diameter, it is five times more elastic than a rubber band. It can stretch up to 150% of its normal length before breaking. Thus, it enables the tissues to stretch and be restored to their initial status after the stretching, which makes them supple.

(188) The Dermis:

(189) Elastin is present in the dermis of the skin and acts as a support. For example, during ageing, the loss of elasticity and tonicity of the dermis that can no longer fight the effects of the contraction of subjacent muscles leads to the appearance of wrinkles. In addition, the exposure to ultraviolet radiation increases the degradation of elastin.

(190) Study on Young Cells Versus Aged Cells

(191) The following study enabled studying the effect of the Uchuva extract on the expression of the elastin protein.

(192) Method

(193) The NHDF (Normal Human Dermal Fibroblasts) young or aged by replicative senescence have been introduced in 96 wells plate and incubated for 24 hours at 37 C., 5% CO2.

(194) The cells have been treated for 24 hours in the presence of test products. Then, the cells have been fixed with formalin and the expression of the proteins has been detected by immunofluorescence. The fluorescent markings have been imaged and quantified by automated microscopy (Arrayscan Cellomics). The fluorescence has been quantified by the Compartimental Analysis bioapplication.

(195) Cellular Models:

(196) Young: NHDF of young donor (25 years) Ageing by replicative senescence: NHDF of young donor cultivated over several times till the stage of senescence.

(197) The results are in FIG. 3B.

(198) The Uchuva extract allows re-establishing the Elastin expression slightly above the protein level of young cells.

(199) Conclusion

(200) In view of the results obtained, the Uchuva extract shows an improvement of the elasticity in vitro that can be extrapolated in vivo.

(201) 6Evaluation of the Effect of the Uchuva Extract on the Methylation Target of the DNA

(202) The methylation is a modification of the N-terminal ends of the histones. It can be carried out either on lysines or on arginines and can materialise by the addition of one, two or three methyl groups. Depending on the methylated residues and the number of added groups it is associated to activation or repression of the transcription. Considered to be static for a long time, the methylation of histones proves to be a reversible modification involved in a dynamic process, though it is more stable than acetylation and phosphorylation. An increasing number of demethylase histones are identified. Generally, this type of modifications opposes acetylation, and the deacetylation of lysines should take place before their methylation. This opposition leads to the establishment of a certain dynamic balance between the heterochromatin (generally non expressible and methylated on certain key amino acids) and euchromatin territories (generally expressible and acetylated). For example, Lysine 9 of histone H3 is known to be associated with a repression of the surrounding chromatin when it is methylated. This methylation is recognised by a protein, HP1, which thus fixes on methylated H3. HP1 attracts the Suv39 protein, a Histone MethylTransferase, which can methylate lysine 9 of histone H3 of the neighbouring nucleosome, and so on. We can thus see, step by step, how histones H3 will be methylated and the chromatin, condensed. However, this heterochromatin invasion will be stopped if the encountered lysine 9 of H3 is already acetylated. Hence, a competitive balance is established between expressed and repressed chromatin domains. The modifications of the histone tails play the role of epigenetic marks that lead to the recruitment of different protein classes, as the acetylated or methylated lysines are recognised by different protein domains. Moreover, the recruitment of certain factors at the level of the chromatin requires the prior existence of modifications of histones and proteins that are already linked. The code of the histones is thus interpreted in the context of other factors associated to the chromatin, and it is the interaction combination between the modified histones and other factors that determines whether a protein is recruited to the chromatin. Ageing affects all the tissues of organisms. This process is related to the epigenetic modifications like changes of methylation at the level of specific cytosine residues of the DNA, as described in several publications.sup.24-31. The role of the epigenetic modifications in ageing, accumulation of cell divisions and deteriorated macromolecules contribute to an aged phenotype. The unpredictable and environmental events can also modify this phenotype through epigenetic mechanisms like methylation of the DNA and the methylation and acetylation of histones. The potential reversibility of the epigenetic modifications makes them attractive targets for the treatment of conditions related to ageing.

(203) Study on Young Cells Versus Aged Cells

(204) In order to understand the reactivation mechanism of the expression of the proteins observed, we carried out an analysis of the methylation of the DNA on aged cells in an intrinsic manner. In fact, one of the characteristic features of cellular senescence is the accumulation of methylated zones in the DNA, at the level of the Cytosines contained in the CpG dinucleotides. This leads to the deactivation of promoters and decrease in the expression of certain genes.

(205) Method

(206) The NHDF (Normal Human Dermal Fibroblasts) young or aged intrinsically by replication have been introduced in 6 wells plate and incubated at 37 C., 5% CO2 till subconfluence. The cells have been treated for 24 hours in the presence of test products. Then, the cells have been taken off in the presence of trypsin, and lysed. The genomic DNA has been precipitated with Ethanol. The methylation rate of the DNA has been measured by ELISA using the Enzo kit: 5-Methylcytosine DNA ELISA kit. The values are represented from 50% for better visibility of the results.

(207) Cellular Models:

(208) Young: NHDF of young donor (25 years) Ageing by replicative senescence: NHDF of young donor cultivated over several times till the stage of senescence.

(209) Following the prior evaluation of its cytotoxicity, the Uchuva Extract is tested with the following concentrations 2-1-0.5 ppm.

(210) Results: The results are displayed in FIG. 4A.

(211) Conclusion

(212) In view of the results obtained, the Uchuva extract enables to reduce the rate of methylation with three tested concentrations with respect to the aged skin model.

(213) 7Evaluation of the Effect of the Uchuva Extract on the Fibrillin-1 Target

(214) Fibrillin 1 is a protein that constitutes microfibrils, which are associated to elastic fibres and contribute to their assembly.

(215) Study on Young Cells Versus Aged Cells

(216) Method

(217) The young NHDF (Normal Human Dermal Fibroblasts) have been introduced in 96 wells plate and incubated for 24 hours at 37 C., 5% CO2. The cells meant for extrinsic ageing have been irradiated three times with UVA radiation with 24 hours between each irradiation and once with UVB radiation. The cells have been treated in the presence of the test products between each irradiation. Then, the cells have been fixed with formalin and the expression of the proteins has been detected by immunofluorescence. The fluorescent markings have been imaged and quantified by automated microscopy (Arrayscan Cellomics). The fluorescence has been quantified by the Compartimental Analysis bioapplication.

(218) Cellular Models:

(219) Young: NHDF of young donor (25 years) UVA/B-induced ageing: NHDF of young donor, irradiated with UVA and UVB radiation
Tested Products

(220) The Uchuva extract is tested with the following concentrations: 2-1-0.5 ppm following the prior evaluation of its toxicity.

(221) Results

(222) The results are in FIG. 4B.

(223) The Uchuva extract with 0.5 ppm allows re-establishing the expression of Fibrillin 1 to a level that is equivalent, even slightly higher to that of young cells.

(224) General Conclusion about the Anti-Ageing and Rejuvenating Activity

(225) During ageing, the collagen fibres and elastic fibres are altered due to a reduced synthesis and increased degradation. To demonstrate that the Uchuva extract acts on cellular rejuvenation, the expression of the proteins involved in the structure of these fibres in cells aged intrinsically (replicative senescence) in the case of Collagen I and Elastin, or extrinsically by repeated UV radiation (photo-ageing) in the case of Fibrillin 1 have been quantified.

(226) The Uchuva extract allows re-establishing the expression of three markers.

(227) In these studies, the Uchuva extract fully re-establishes the expression of the proteins in comparison with the young control elements. This is the purpose of the observed effects. There is no aberrant overexpression, the Uchuva extract respects the biological balance of the skin.

(228) At the same time, the Uchuva extract also greatly increases the expression of fibroblast Collagen IV with respect to the untreated cells, like Rhamnose recognised for this property. The quantity of Collagen III is also increased during a treatment of fibroblasts by the Uchuva extract. These results reinforce its anti-ageing effectiveness at the redensifying and structural level.

(229) In the context of the analysis of the methylation of the DNA, the Uchuva extract shows homogeneous results in this experiment. It reduces the methylation with three tested concentrations. This effect provides information about the mode of action of this product that enables to reactivate the expression of proteins of the extracellular matrix, giving it its rejuvenating effect.

(230) Therefore, the Uchuva extract is an ingredient or active ingredient exhibiting an anti-ageing, rejuvenating and anti-age biological activity. It consistently acts on three targets of skin ageing by re-establishing the protein expression up to the expression observed in the young cells. It also shows an innovative activity on the epigenetics as is demonstrated by its action on the methylation of the DNA in mature cells. Finally, it shows a deep anti-ageing action by its effectiveness on the synthesis of the proteins of the extracellular matrix.

EXAMPLE 22: TESTS DEMONSTRATING THE MOISTURISING AND STRUCTURING POWER OF THE EPIDERMIS OF THE SKIN BY THE UCHUVA EXTRACT

(231) Filaggrin is synthesised in the stratum granulosum in the form of a precursor, profilaggrin (repetition of 10-12 monomers of filaggrin and the NH2-terminal S-100 regulatory protein). The detection of this protein is associated to a state of terminal differentiation of the epidermis because it appears after dephosphorylation and proteolysis of the profilaggrin that takes place during the terminal differentiation.

(232) 1) The filaggrin contributes to the aggregation of keratin during the formation of the packets of macrofibrils,

(233) 2) The monomers of filaggrin are components of the cornified layer (CE; envelope that is deposited on the internal surface of the plasma membrane of the corneocytes: contributes to the impermeable properties of the skin with the intercorneocytary lipid matrix),

(234) 3) The monomers are completely degraded on the highest layers of the stratum corneum to produce a mixture of hygroscopic amino acids (NMFs or Natural Moisturizing Factors) that are important to maintain the hydration of the epidermis.sup.32.

(235) A decrease in profilaggrin and/or filaggrin has been detected.sup.33 in pathological situations with xerosis or dryness of skin (atopic dermatitis, ichthyosis).

(236) According to Proksch et al.sup.34, the monitoring and expression of filaggrin is a good way to monitor the barrier function and/or the hydration level of the stratum corneum. This is the only real marker that is significant as a hydration marker of the upper layers in the dermatology journals. Nevertheless, clinically, it is a marker that passes after the measurement of the TEWL (TransEpidemal Water Loss).

(237) Involucrin, like filaggrin and loricrin, is a terminal differentiation marker. It is a protein of the cornified envelope (CE) which is the target of the transglutaminases (TGM) and particularly the TGM1. Involucrin is oligomerised by the TGM1 during the initial steps of the formation of the cornified envelope, and for the later steps of the formation of the CE, the involucrin is linked to the ceramides.sup.35. The marking is localised in the epidermis from the upper layers of the spiny layer till inside the granular layer and the first layers of the SC.

(238) Effect of the Topical Treatment of the Uchuva Extract on Explants of Human Skin

(239) Material and Method:

(240) Description: Explant of human mammal skin from a woman, Caucasian, 51 years old (Biopredic35000 RENNES).

(241) Number: Preparation of 9 punches of defatted skin with a diameter of 1 cm

(242) Culture conditions: 37 C., 5% CO2

(243) Culture medium: DMEM medium 4.5 g/L of glucose, supplemented and with antibiotics (penicillin-streptomycin-Amphotericin)

(244) Tested Products

(245) The Uchuva extract obtained as per example 3 is diluted to 0.05% and 0.01% (w/v) in paraffin oil. The TRIGLYCERIDES C8C10 55/45 solvent (Code EC-09-271) is used.

(246) Culture Method and Treatment of Explants

(247) The explants are defatted after they are received. Circular skin disks with a diameter of 1 cm have been obtained using a punch. In a six wells plate, a stainless steel grid with a diameter of 1.6 cm is placed in 5 ml of culture medium. Each explant in culture is put on its grid so as to bathe in the culture medium while leaving the epidermis exposed to air. The explants have been cultivated for 48 hours without changing the culture medium. Different treatments have been carried out by a topical administration of 10 L of the preparations to be tested on D0 and D1. Every experimental condition has been repeated over three punches.

(248) Controlled explants have been cultivated in the same way as the treated explants. The treatments have been carried out by a topical administration of 10 L of the TRIGLYCERIDES solvent with the concentration of 0.05%.

(249) Immunolabelling In Situ

(250) After the duration of culture ends, the explants have been collected and then divided into two equal parts using a scalpel. One half has been frozen in tissue Teck (Sakura) using isopentane cooled in liquid nitrogen and then maintained at 80 C. awaiting immunolabelling. The second half has been kept in 4% buffered formalin.

(251) 5 m thick cross sections of each explant have been made using a cryotome. The sections have been dried in ambient air. After rehydratation in PBS, the sections have been incubated for 1 hour with the primary antibody solution prepared in a solution (PBS-BSA-Tween). After successive washings, the primary antibodies have been revealed by a secondary antibody connected to an FITC probe at 1/100th of the commercial form. The cell nuclei have been marked by the DAPI fluorescent dye.

(252) The skin sections have been collected using poly-lysine trays.

(253) Microscopic Observations

(254) Each skin section has been observed using an Olympus BX60 epifluorescence microscope with 20 magnification. Two photographs have been taken of each section using an Olympus DP72 camera operated by the Cell F software. The negatives have been identified as follows: Date of markingTreatment conditionImage numberMarker. The counterstaining of nuclei has been carried out using the DAPI nuclear marker. By sliding colour using the image processing software, the colour of the nuclei has been changed from blue to red. For each treatment condition of the epidermis, there was visual scoring of the intensity of the marking with 10 magnification. The evaluation scale was as follows:

(255) TABLE-US-00023 Very low marking 1 Low marking 2 Moderate marking 3 High marking 4

(256) The data obtained has been analysed, and presented in the form of histograms in comparison with the Triglycerides solvent control conditions.

(257) Results

(258) All the markings were localised at the level of the upper layers of the epidermis (spiny and/or granular and/or cornea of the epidermis).

(259) Filaggrin Immunolabelling

(260) The results of the scoring made are given in the graph of FIG. 5A.

(261) The analysis of the expression data of the Filaggrin marking shows a dose increase dependent on the quantity of fluorescence for the explants treated with the Uchuva extract with 0.01% (+21%) and 0.05% (+37%) in comparison with the Solvent Control group.

(262) Involucrin Immunolabelling

(263) The results of the scoring made are given in the graph of FIG. 5B. The analysis of the expression data of the INVOLUCRIN marking indicates a significant dose increase dependent on the quantity of fluorescence for the explants treated with the Uchuva extract with 0.01% (+64%) and 0.05% (+86%) in comparison with the Control group.

(264) Conclusion

(265) In this study, the results have shown that the topical treatment of skin explants in culture for 48 hours with the Uchuva extract led to an increase in the expression of proteins involved in hydration and the barrier effect of the skin (filaggrin and involucrin respectively). The increase in expression brought about by the Uchuva extract has been more significant for involucrin than for filaggrin. The maximum expression of these proteins has always been observed with the concentration of 0.05%. According to these results, the Uchuva extract of the invention presents a structuring and moisturising activity profile of the epidermis.

EXAMPLE 23: TESTS DEMONSTRATING THE DEPIGMENTATION POWER OF THE SKIN WITH THE UCHUVA EXTRACT

(266) The depigmentation effect potential of the Uchuva extract has been researched in a model of normal human epidermal melanocytes (NHEM). The effect of the Uchuva extract on the synthesis of melanin has been evaluated after ten incubation days under conditions stimulated by L-tyrosine. In this test, the reference inhibitory molecule is lipoic acid. Its action mechanism calls for an inhibition of the expression of the MITF transcription factor (microphthalmia-associated transcription factor). This transcription factor is the major regulator of the expression of tyrosinase and its inhibition results in the reduction of the expression of tyrosinase, enzyme involved in the melanin synthesis. The melanocytes have been sown in 24 wells plate and cultivated in the culture medium for 24 hours. Then, the medium has been replaced by the culture medium (Medium 254 supplemented with PMA free HMGS-2+Penicillin 50 U/mlStreptomycin 50 g/ml) supplemented with L-tyrosine (1 mM) and containing or not containing (stimulated control element) the compound or the reference (lipoic acid tested at 5 g/ml). A non-stimulated control condition has been created at the same time. Then, the cells have been incubated for a total period of 10 days, with repetitions of the treatments after 3 and 7 days of incubation. After incubation, the melanin has been extracted by lysis of the cells with a solution of NaOH 0.5 N. The optical density (OD) of the samples has been measured at 405 nm, then the quantity of melanin has been determined in comparison with a range of exogenous melanin (melanin curve including standards from 0.39 to 100 g/ml). The results have been expressed in g/ml of melanin, in percentage of the stimulated control element and in percentage of inhibition. The treatment of the normal human epidermal melanocytes (NHEM) by L-tyrosine, tested at 1 mM, has strongly stimulated the synthesis of melanin. The reference, lipoic acid, tested at 5 g/ml, has strongly inhibited the stimulation induced by L-tyrosine (108% inhibition).

(267) This effect was expected and has enabled to validate the test.

(268) The concentrations of the Uchuva extract in the study to be tested have been selected after a preliminary cytotoxicity test: Cellular type: NHEM in culture medium Incubation time: 72+96+72 hours Evaluation parameters: Reduction of the MTT and morphological observations under microscope. At the end of the treatment, the cells have been incubated in the presence of MTT (tetrazolium salt) whose transformation into blue crystals of formazan is proportional to the activity of the succinate deshydrogenase (mitochondrial enzyme). After dissociation of the cells and solubilisation of the formazan by addition of DMSO, the optical density (OD), which represents the number of living cells and their metabolic activity, has been measured with a 540 nm microplate reader (VERSAmax, Molecular Devices).

(269) The concentrations thus retained for the rest of the study are: 0.5-1.6-5 g/ml respectively corresponding to 5.10.sup.5, 16.10.sup.5 and 5.10.sup.5%. Under experimental conditions of this study, the Uchuva extract, tested at 5 g/ml, has relatively inhibited the synthesis of melanin (40% inhibition). Tested with lower concentrations, 0.5 and 1.6 g/ml, the compound showed no effect. The results are given in the diagram in FIG. 6. Under the experimental conditions of this study, the Uchuva extract has shown a moderate depigmentation effect which has been observed only with the strongest tested concentration (5 g/ml).

(270) The table below shows the correspondence of the concentrations of the Uchuva extract tested in examples 21 to 23 (ppm and % by weight with respect to the total concentration of the composition.

(271) TABLE-US-00024 TARGETS TESTED CONC. ppm TESTED CONC. % Collagen I 0.8 8 .Math. 105 1.6 16 .Math. 105 3.2 32 .Math. 105.sup. 0.5 5 .Math. 105 Collagen I 1 10 .Math. 105.sup. 2 20 .Math. 105.sup. 0.8 8 .Math. 105 Collagen III 1.6 16 .Math. 105 3.2 32 .Math. 105.sup. Collagen IV 0.4 4 .Math. 105 0.8 8 .Math. 105.sup. 0.5 5 .Math. 105 Elastin 1 10 .Math. 105.sup. 2 20 .Math. 105.sup. 0.5 5 .Math. 105 Fibrillin-1 1 10 .Math. 105 2 20 .Math. 105.sup. Methylation 0.5 5 .Math. 105 Level 1 10 .Math. 105 2 20 .Math. 105.sup. Filaggrin 100 0.01 500 0.05 Involucrin 100 0.01 500 0.05 0.5 5 .Math. 105 Melanin 1.6 16 .Math. 105 5 50 .Math. 105.sup.

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