Aesculus hippocastanum extract

Abstract

An Aesculus hippocastanum extract able to be obtained by an extraction method comprising a solid/liquid extraction step of the flower, followed by a second solid/liquid separation step, and lastly a third step for recovering the liquid phase, characterized in that the solvent consists of a mixture of fructose and glycerin optionally comprising water.

Claims

1. A method of improving an appearance of a skin and/or a mucosa, to improve cutaneous strength and/or elasticity, to treat or prevent skin aging, wrinkles, or skin slackening, the method comprising: administering an extract for a cosmetic treatment of the skin and/or mucosa, said extract being an Aesculus hippocastanum extract obtained by an extraction method comprising: a solid/liquid extraction step of a flower of Aesculus hippocastanum with a solvent, followed by a second solid/liquid separation step, and a third step for recovering the liquid phase, wherein the solvent consists of a mixture of fructose and glycerin and water.

2. The method according to claim 1, wherein the extract has a flavonoid content level between 10 and 200 mg/100 g of extract.

3. The method according to claim 1, wherein the extract has a free amino acid content level between 10 and 200 mg/100 g of extract.

4. The method according to claim 1, to stimulate the synthesis of collagen IV, collagen VII and fibronectin.

5. The method according to claim 1, comprising administering a cosmetic composition comprising said extract.

6. The method according to claim 5, wherein the extract represents between 0.1% and 10% by weight of the composition.

Description

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

(1) The invention and resulting benefits will become clear from the following exemplary embodiments.

EXAMPLES

Example 1

Manufacture of Aesculus hippocastanum Extracts

(2) One mixes 1 part dried and ground flowers or ground seeds of Aesculus hippocastanum and 19 parts of a 70:30 weight/weight ethanol/water mixture (denoted EtOH/Water solvent) or a Fructose/Glycerin/Water mixture in molar ratio 1:1:5 (denoted FGW solvent). The mixture is extracted at 70 C. for 3 h, then the extract is filtered. One thus obtains a clear, brown-yellow hydroethanolic flower extract (denoted EtOH/water flower extract) and a clear amber Fructose/Glycerin/Water flower extract (denoted FGW flower extract). Lastly, one obtains a hydroethanolic seed extract (denoted EtOH/water seed extract) and a Fructose/Glycerin/Water seed extract (denoted FGW seed extract), both of which are clear and light yellow.

Example 2

Characterization of the Composition in Free Amino Acids in the Aesculus hippocastanum Extracts

(3) The content levels of free amino acids contained in the extracts obtained in example 1 are quantified by HPLC (Shimadzu R_LCS_01) on a Waters Novapack C18 column. The FGW extract is derived after dilution at 1/5 in (w/V) in water. The hydroethanolic extracts are diluted at 1/2 (volume/volume) in water, filtered at 0.45 m, then derived. The calibration is performed externally with the assay kit. The obtained results are provided in Table 1 below.

(4) TABLE-US-00001 TABLE 1 Free amino acid content levels Solvent FGW EtOH/water EtOH/water Part used Flower Flower Seed Free amino acid 89.0 76.7 7.2 level (in mg/100 g)
Conclusion:

(5) The FGW flower extract is significantly richer in free amino acids than the EtOH/water flower extract. The very low free amino acid content level of the EtOH/water extract proves that the chemical composition of the flowers and the seeds is significantly different. The flower extracts and the seed extracts of Aesculus hippocastanum clearly have a different composition.

Example 3

Search for the Presence of Saponins and Coumarins in the Aesculus hippocastanum Flower Extracts

(6) The search for the presence of compounds of the coumarin type in the FGW and EtOH/Water extract from the Aesculus hippocastanum flower that were obtained in Example 1 is done by HPLC-UV-DAD, using a SymmetryShield 3.5 m column (1504.6 mm) and calibration molecule solutions of esculetin, esculin, fraxin and fraxetin. The interpretation is done by comparing the residence times and UV adsorption spectrums of the calibration molecules and the molecules present in the extract.

(7) The search for the presence of compounds of the saponin type in the FGW and EtOH/Water extract from the Aesculus hippocastanum flower that were obtained in Example 1 is done by HPLC-UV-DAD, using a SymmetryShield 5 m column (2504.6 mm) and a calibration molecule solution of aescin. The interpretation is done by comparing the residence times and UV adsorption spectrums of the calibration molecule and the molecules present in the extract.

(8) TABLE-US-00002 TABLE 2 Result of the search for the presence of coumarins and saponins in the Aesculus hippocastanum flower extract Solvent FGW EtOH/water Part used Flower Flower Saponins absent absent Coumarins absent absent
Conclusion:

(9) Unlike Aesculus hippocastanum seed or bark extract, the FGW flower extract does not contain compounds of the saponin and coumarin types any more than the EtOH/water flower extract. This experiment shows that the chemical composition of the Aesculus hippocastanum extract does not depend on the choice of the extraction solvent alone, but also on the plant part used.

Example 4

Effect of an Aesculus hippocastanum FGW Flower Extract According to the Invention on the Protein Expression of Collagen IV in Normal Human Dermal Fibroblasts

(10) Protocol:

(11) A fluoroimmunoassay test was carried out and consists of revealing the antigen of interest, in this case collagen IV. This method is semi-quantitative, highly sensitive and reproducible, and has the advantage of detecting the protein of interest in its native form in its environment, without any denaturation process. The fibroblasts, from a biopsy of the abdominal skin of donors between 30 and 60 years of age, are extracted and fixed to the bottom of a well at a density of 8,000 cells per well, and grow for 96 hours in a defined medium. The cells are next cultivated for 48 hours with the FGW flower extract obtained in example 1 tested at different concentrations or with the extraction solvent alone or without the extract, known as control. The cells are then washed in a phosphate-buffered saline (PBS) before being fixed, permeabilized and saturated with BSA (bovine serous albumen) at 1% for 30 minutes. The cells are incubated with the primary antibody (anti-collagen IV) for 1 hour, washed in PBS buffer, then incubated with the secondary antibody bonded to the Eu-N1 fluorochrome for 1 hour. The fluorescence is read on a spectrofluorimeter (Tecan M1000) with the appropriate filters. The fluorescence measurements are compared to the assayed quantity of DNA.

(12) The results are provided in Table 3 below. The experiment is done 6 times (n=6). The values of the table represent the percentage values compared to the untreated control cells. The values represent the average of several experiments (n=6) on different extract lots. Avg refers to the average, and SD is the standard deviation.

(13) TABLE-US-00003 TABLE 3 Percentage of protein expression of the collagen IV in normal human dermal fibroblasts as a function of the dose of Aesculus hippocastanum FGW flower extract used. Avg EC Control 100 10 Extraction solvent at 0.5% (FGW) 101.8 11.3 Aesculushippocastanum extract at 0.05% 100.52 12.3 Aesculus hippocastanum extract at 0.1% 120.8 7.4 Aesculus hippocastanum extract at 0.18% 147.49 9.7 Aesculus hippocastanum extract at 0.25% 162.68 12.1 Aesculus hippocastanum extract at 0.5% 329.05 34.8
Conclusion:

(14) The FGW flower extract obtained according to the invention causes a significant and dose-dependent increase of the protein expression of collagen IV. The extraction solvent alone does not cause an increase in the expression of collagen IV.

(15) The FGW flower extract obtained according to the invention therefore causes an improvement in the protein synthesis of the macromolecular network of the dermal-epidermal junction.

Example 5

Effect of an FGW Flower Extract, an EtOH/Water Flower Extract and an FGW Seed Extract of Aesculus hippocastanum on the Protein Expression of Collagen IV in Normal Human Dermal Fibroblasts

(16) The protocol is identical to that of Example 4, with the exception that a comparative test is done of an EtOH/water flower and FGW seed extract obtained in Example 1 at a concentration of 0.25%.

(17) The results are shown in the following table:

(18) TABLE-US-00004 TABLE 4 Percentage of protein expression of the collagen IV in normal human dermal fibroblasts between FGW flower, EtOH/water flower, FGW seed extracts of Aesculus hippocastanum. Avg SD Control 100 4.96 EtOH/Water extraction solvent at 0.25% 93.87 8.39 FGW extraction solvent at 0.25% 93.96 13.56 EtOH/water flower extract at 0.25% 212.84 17.88 FGW flower extract at 0.25% (invention) 411.72 28.23 FGW seed extract at 0.25% 102.21 12.76

(19) The FGW flower extract obtained according to the invention causes an increase of the protein expression of collagen IV about 2 times greater than the same extract obtained with an EtOH/Water solvent. The FGW seed extract does not cause an increase in the expression of collagen IV.

Example 6

Effect of an Aesculus Hippocastanum FGW Flower Extract According to the Invention on the Protein Expression of Collagen VII in Normal Human Fibroblasts

(20) Protocol:

(21) The technique is the same as in Example 4, except that the antigen of interest is collagen VII. The results are provided in Table 4 below. The experiment is done 6 times (n=6). The values of the table represent the percentage values compared to the untreated control cells. The values represent the average of several experiments (n=6) on different extract lots. Avg refers to the average, and SD is the standard deviation.

(22) TABLE-US-00005 TABLE 5 Percentage of protein expression of the collagen VII in normal human dermal fibroblasts as a function of the dose of Aesculus hippocastanum FGW flower extract used. Avg SD Control 100 15.9 FGW extraction solvent at 0.5% 93.6 6 Aesculus hippocastanum extract at 0.05% 110.2 6.4 Aesculus hippocastanum extract at 0.1% 130.1 13 Aesculus hippocastanum extract at 0.18% 159.5 13.2 Aesculus hippocastanum extract at 0.25% 144.7 12.5 Aesculus hippocastanum extract at 0.5% 151.5 16
Conclusion:

(23) The FGW flower extract obtained according to the invention caused a significant and dose-dependent increase of the protein expression of collagen VII. The extraction solvent alone does not cause an increase in the expression of collagen VII. The FGW flower obtained according to the invention therefore causes an improvement in the protein synthesis of the macromolecular network of the dermal-epidermal junction.

Example 7

Effect of an FGW Flower Extract, an EtOH/Water Flower Extract and an FGW Seed Extract of Aesculus hippocastanum on the Protein Expression of Collagen VII in Normal Human Dermal Fibroblasts

(24) The protocol is identical to that of Example 6, with the exception that a comparative test is done of an EtOH/water flower and FGW seed extract obtained in Example 1 at a concentration of 0.25%.

(25) The results are shown in the following table:

(26) TABLE-US-00006 TABLE 6 Percentage of protein expression of the collagen VII in normal human dermal fibroblasts between FGW flower, EtOH/water flower, FGW seed extracts of Aesculus hippocastanum. Avg SD Control 100 4.96 EtOH/Water extraction solvent at 0.25% 100.11 12.99 FGW extraction solvent at 0.25% 103.82 13.15 EtOH/water flower extract at 0.25% 146.01 11.90 FGW flower extract at 0.25% (invention) 170.59 18.48 FGW seed extract at 0.25% 113.18 14.92

(27) The FGW flower extract obtained according to the invention causes a greater increase of the protein expression of collagen VII compared to the same extract obtained with an EtOH/water solvent. The seed extract implementing the FGW solvent does not cause an increase in the expression of collagen VII.

Example 8

Effect of an Aesculus hippocastanum FGW Flower Extract According to the Invention on the Protein Expression of Fibronectin in Normal Human Dermal Fibroblasts

(28) Protocol:

(29) The technique is the same as in Example 4, but with the following changes: the antigen of interest is fibronectin; the cells grow for 24 hours in a defined medium before being cultivated for 72 hours with the FGW flower extract obtained in Example 1; the secondary antibody is bonded to the Alexa 488 fluorochrome; The fluorescence is read on a high-resolution imaging system (INCELL Analyzer1000), and the fluorescence measurements are quantified by image analysis (fluorescence area/number of cores with Hoechst staining). The results are provided in Table 5 below.

(30) The experiment is done 3 times (n=3). The values of the table represent the percentage values compared to the untreated control cells. The values represent the average of several experiments (n=3) on different extract lots. Avg refers to the average, and SD is the standard deviation.

(31) TABLE-US-00007 TABLE 7 Percentage of protein expression of fibronectin in normal human dermal fibroblasts treated with the Aesculus hippocastanum FGW flower extract at 0.5%. Avg SD Control 100 3.7 Aesculus hippocastanum FGW flower extract at 117.3 2.7 0.5%
Conclusion:

(32) The FGW flower extract obtained according to the invention caused a significant increase in the protein expression of fibronectin in normal human dermal fibroblasts. The FGW flower obtained according to the invention therefore causes an improvement in the protein synthesis of the dermal matrix.

Example 9

Effect of an FGW Flower Extract and an FGW Seed Extract of Aesculus hippocastanum on the Protein Expression of Fibronectin in Normal Human Dermal Fibroblasts

(33) The protocol is identical to that of Example 8, with the exception that a comparative test is done of an FGW flower and FGW seed extract obtained in Example 1 at a concentration of 0.25%.

(34) The results are shown in the following table:

(35) TABLE-US-00008 TABLE 8 Percentage of protein expression of fibronectin in normal human dermal fibroblasts treated with the Aesculus hippocastanum FGW flower or FGW seed extract Avg SD Control 100 6.64 FGW extraction solvent at 0.25% 102.07 4.39 FGW flower extract at 0.25% (invention) 120.73 4.14 FGW seed extract at 0.25% 98.33 5.20

(36) The FGW flower extract obtained according to the invention causes a significant increase in the protein expression of fibronectin in normal human dermal fibroblasts, while the FGW seed extract does not cause an increase in fibronectin expression.

Example 10

Effect of an Aesculus hippocastanum FGW Flower Extract According to the Invention on the Protein Expression of Collagen IV in Normal Human Keratinocytes

(37) Protocol:

(38) The technique is the same as in Example 4, except that it is carried out on normal human keratinocytes and the antigen of interest is collagen IV. The results are provided in Table 6 below. The experiment is done 6 times (n=6). The values of the table represent the percentage values compared to the untreated control cells. The values represent the average of several experiments (n=6) on different extract lots. Avg refers to the average, and SD is the standard deviation.

(39) TABLE-US-00009 TABLE 9 Percentage of protein expression of collagen IV in normal human keratinocytes as a function of the dose of Aesculus hippocastanum FGW flower extract used. Avg SD Control 100 12 FGW extraction solvent at 0.5% 84 4.5 Aesculus hippocastanum extract at 0.05% 117.7 5 Aesculus hippocastanum extract at 0.1% 158 19.7 Aesculus hippocastanum extract at 0.18% 173.9 11.9 Aesculus hippocastanum extract at 0.25% 257.4 26 Aesculus hippocastanum extract at 0.5% 349.2 53.3
Conclusion:

(40) The FGW flower extract obtained according to the invention caused a significant and dose-dependent increase of the protein expression of collagen IV. The extraction solvent alone does not cause an increase in the expression of collagen IV. The FGW flower extract obtained according to the invention therefore causes an improvement in the protein synthesis of the macromolecular network of the dermal-epidermal junction.

Example 11

Effect of an FGW Flower Extract and an FGW Seed Extract of Aesculus hippocastanum on the Protein Expression of Collagen IV in Normal Human Keratinocytes

(41) The protocol is identical to that of Example 10, with the exception that a comparative test is done of an FGW flower and FGW seed extract obtained in Example 1 at a concentration of 0.25%.

(42) The results are shown in the following table:

(43) TABLE-US-00010 TABLE 10 Percentage of protein expression of collagen IV in normal human keratinocytes treated with the Aesculus hippocastanum FGW flower or FGW seed extract Avg SD Control 100 8.15 FGW extraction solvent at 0.25% 99.52 12.40 FGW flower extract at 0.25% (invention) 142.20 13.20 FGW seed extract at 0.25% 102.97 9.95

(44) The FGW flower extract obtained according to the invention causes a significant increase in the protein expression of collagen IV in normal human keratinocytes, while the FGW seed extract does not cause an increase in collagen IV expression.

Example 12

Compositions Comprising the Aesculus hippocastanum Extract According to the Invention

(45) Day Cream

(46) TABLE-US-00011 Composition Ingredient names (weight %) DEMINERALIZED WATER QS100 CARBOPOL ULTREZ 21 0.10 GLYCERIN 3.00 BUTYLENE GLYCOL 5.00 PENTYLENE GLYCOL 2.00 XANTHAN GUM 0.20 EMULIUM DELTA 3.00 PHENOXYETHANOL 0.30 SILICONE OIL 4.00 EXTRACT ACCORDING TO THE INVENTION 2.00 SODIUM HYDROXIDE (aqueous solution 10%) 0.25 Total 100.00
Hydrating Natural Cream Gel

(47) TABLE-US-00012 Composition Ingredient names (weight %) EMULIUM MELLIFERA 2.5 NATURAL SQUALANE 2.0 DICAPRYLYL CARBONATE 15.0 DEMINERALIZED WATER QS100 GLYCERIN 20.0 TAPIOCA STARCH 5.0 EXTRACT ACCORDING TO THE INVENTION 2.0 ETHYLHEXYLGLYCERIN 0.1 PHENOXYETHANOL 0.9 FRAGRANCE 0.2 Total 100.0
Nourishing Spray

(48) TABLE-US-00013 Composition Ingredient names (weight %) EMULIUM MELLIFERA 2.00 LABRAFAC CC 5.00 LIPOCIRE A 3.00 OCTYLDODECYL MYRISTATE 5.00 SILICONE OIL 2.00 DEMINERALIZED WATER QS100 STEAROYLE SODIUM GLUTAMATE 0.50 GLYCERIN 5.00 BUTYLENE GLYCOL 5.00 PEMULEN TR-2 0.10 XANTHAN GUM 0.15 EXTRACT ACCORDING TO THE INVENTION 2.00 TEA STEARATE 0.10 PHENOXYETHANOL 0.45 ETHYLHEXYLGLYCERIN 0.05 FRAGRANCE 0.05 Total 100.00
Water in Oil Emulsion

(49) TABLE-US-00014 Composition Ingredient names (weight %) PLUROL DIISOSTEARYL CG 4.0 HYDROGENATED RICIN OIL 2.0 COMPRITOL 888 CG 1.0 NATURAL SQUALANE 3.0 NATURAL ALKANE 22.0 MAGNESIUM SILICATE 3.0 DEMINERALIZED WATER QS100 GLYCERIN 3.0 MAGNESIUM SULFATE 7H.sub.2O CODEX 1.5 SODIUM CHLORIDE CODEX 1.5 FRAGRANCE 0.3 EXTRACT ACCORDING TO THE INVENTION 2.0 PHENOXYETHANOL 0.9 ETHYLHEXYLGLYCERIN 0.1 Total 100.0