MURRAYA KOENIGII EXTRACT AND USE THEREOF IN COSMETICS

Abstract

The invention relates to the use of a Murraya koenigii extract to combat skin aging.

Claims

1. A method of combatting skin aging, the method comprising topical application to the skin and/or mucous membranes, and/or appendages an extract of Murraya koenigii obtainable by a solid/liquid extraction process, followed by a second solid/liquid separation step and finally by a third step to recover the liquid phase, characterized in that the solvent consists of a mixture of betaine, propanediol and water, in which the betaine:propanediol molar proportions are 1: strictly less than 1.5; or of a composition comprising said extract.

2. A method according to claim 1, for wherein combatting skin aging comprises stimulating the process of skin elastogenesis.

3. A method according to claim 1, wherein combatting skin aging comprises for stimulating the formation of skin elastic fibers.

4. A method according to claim 1, wherein combatting skin aging comprises densifying the cutaneous extracellular matrix.

5. A method according to claim 1, wherein combatting skin aging comprises combatting skin slackening.

6. A method according to claim 1, wherein the solvent consists of a mixture of betaine, propanediol and water, wherein the molar proportions of betaine:propanediol are 1:1.

7. A method according to claim 1, wherein the water represents between 15 and 35% by weight of the solvent.

8. A method according to claim 6, wherein the solvent consists of a mixture of betaine, propanediol and water in molar proportions 1:1:5.

9. An extract of Murraya koenigii obtainable by a solid/liquid extraction process, followed by a second solid/liquid separation step and finally by a third liquid phase recovery step, characterized in that the solvent consists of a mixture of betaine, propanediol, and water, advantageously in molar proportions of 1:1:5.

10. An extract according to claim 9, being an extract of leaf stalks and/or leaves advantageously dried.

11. An extract according to claim 9, being free of alkaloids.

12. An extract according to claim 9, having a mass ratio of leaf stems and/or leaves of Murraya koenigii/solvent of between 1:99 and 50:50.

13. A cosmetic composition comprising the extract according to claim 9.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0105] The manner in which the invention may be carried out and the advantages thereof will become clearer from the following illustrative and non-limiting examples, which are shown in the attached figures.

[0106] FIG. 1 represents the content of phenolic acids, flavonoids, and alkaloids of 4 extracts of Murraya koenigii.

DETAILED DESCRIPTION OF THE INVENTION

[0107] 1/ Preparation of Murraya koenigii Extracts

[0108] Extract 1: Extract of Murraya koenigii obtained from dried and ground leaf stems and leaves by extraction with a solvent according to the invention consisting of a mixture of betaine, propanediol and water in a molar ratio of 1:1:5 (GHP115) under continuous stirring for 3 hrs. at 70° C. of the plant and the solvent in the mass ratio 8:92. After extraction, the plant residue is removed by a mechanical process (e.g., spinning) and the crude extract is filtered down to 0.22 μm.

[0109] Extract 1a: Extract obtained under the same operating conditions as described for Extract 1 above, except that the plant/solvent mass ratio is 5:95.

[0110] Extract 2: Extract of Murraya koenigii obtained from dried and ground leaf stalks and leaves by extraction with an ethanol/water solvent in a 70:30 mass ratio under continuous stirring for 3 hrs. at 70° C. of the plant and the solvent in the 8:92 mass ratio. After extraction, the plant residue is removed by a mechanical process (e.g., spinning). The crude extract is decolorized on activated carbon Acticarbone CPW CECA at 0.08% then the ethanol is evaporated under reduced pressure and the aqueous concentrated extract obtained is solubilized in propanediol such that the final dry extract is equal to the dry extract of the hydro-ethanolic extract.

[0111] Extract 3: Fraction of the decolorized hydroethanol extract described for Extract 2 enriched in polyphenols and containing only traces of alkaloids (<10 mg/100 g fraction). The fraction is obtained by column filtration on Amberlite FPX 68 resin of the 70:30 decolorized ethanol/water extract on activated carbon, as described for Extract 2, the non-adsorbed filtrate constitutes Extract 3.

[0112] Extract 4: Fraction of the decolorized hydroethanol extract described for Extract 2 enriched in alkaloids and devoid of polyphenols. The fraction is obtained by filtration/adsorption on Amberlite FPX 68 resin in column of the 70:30 ethanol/water extract and decolorized on activated carbon as described for Extract 2, then desorption with ethanol of molecules adsorbed on the resin.

[0113] 2/ Characterization of the Extraction Power of Murraya koenigii by the Solvent Betaine/Propanediol/Water in Different Molar Proportions

[0114] The extractability of polyphenols and alkaloids from leaf stalks (or leafy stems) and leaves of Murraya koenigii was studied for 6 compositions of betaine/propanediol/water mixtures (in different molar ratios), shown in Table 1.

TABLE-US-00001 TABLE 1 GHP Molar Ratio 1:1:4 1:1:5 1:1.5:3 1:1.5:5 1:2:3 1:2:6 Betaine (g) 44.16 41.35 41.06 36.45 36.23 31.04 Propanediol (g) 28.68 26.86 40.00 35.52 47.06 40.32 Water (g) 27.16 31.79 18.94 28.03 16.71 28.64

[0115] Each solvent is prepared by mixing betaine, propanediol and water at 70° C. for about 1 hr under mechanical stirring. Each extract is obtained by extraction at 70° C. for 4 hrs. of 5% of crushed Murraya koenigii leaf stalks and dry leaves and 95% of solvent. After extraction, the plant residue is removed by a mechanical process and the crude extract is filtered to the sterilizing filtration limit (0.22 μm).

[0116] Flavonoids and phenolic acids (belonging to the polyphenol group) were analyzed by RP-UHPLC-UV, phenolic acids were spotted and quantified against the calibration line of chlorogenic acid at 287 nm and flavonoids were identified and quantified against the isoquercitrin calibration line at 355 nm. The Gardner index of each extract was measured after dilution to 1/2 m/v in water with a Lico 100 colorimeter.

[0117] The physical stability (visual aspect) of the 6 extracts packaged in glass bottles was observed after 7 days of storage at 3 different temperatures (4, 25 and 40° C.).

[0118] The flavonoid and phenolic acid concentrations for each extract are represented in Table 2.

TABLE-US-00002 TABLE 2 Concentration (mg/g extract) Phenolic Gardener Solvent Flavonoids Acids Index GHP 1:1:4 11 24 10 GHP 1:1:5 12 27 9 GHP 1:1.5:3 10 21 8 GHP 1:1.5:5 11 24 10 GHP 1:2:3 11 20 7 GHP 1:2:6 12 25 11

[0119] The 6 solvents show similar flavonoid extraction performance, characterized by a concentration between 10 and 12 mg/100 g of extract. As for their extraction performance with respect to phenolic acids, it is between 20 and 27 mg/100 g of extract.

[0120] All of the solvents tested in this example are effective in obtaining an extract of Murraya koenigii comprising an effective number of flavonoids and phenolic acids for use in cosmetics. The most efficient solvent for the extraction of flavonoids and phenolic acids is the solvent consisting of a mixture of betaine, propanediol and water in molar proportions 1:1:5.

[0121] 3/ Comparison of the Phenolic Acid, Flavonoid and Alkaloid Content of the Murraya koenigii Extract According to the Invention with Other Extracts

[0122] The alkaloid, phenolic acid, and flavonoid contents of the 4 Murraya koenigii extracts described in Example 1 were evaluated by RP-HPLC-UV. Phenolic acids were identified and quantified against the neochlorogenic acid calibration line at 287 nm, flavonoids were spotted and quantified against the isoquercitrin calibration line at 355 nm, and alkaloids were identified and quantified against the mahanimbine calibration line at 287 nm.

[0123] The results obtained are illustrated in FIG. 1.

[0124] The composition of the 4 extracts is clearly distinct from a qualitative and quantitative point of view concerning the 3 families of secondary metabolites studied. Thus: [0125] Extract 1 (GHP115 according to the invention) is characterized by the highest concentration of phenolic acids, a concentration of flavonoids equivalent to that of extracts 2 and 3, and the presence of traces of alkaloids; [0126] Extract 2 is characterized by a significant concentration for each of the 3 families of compounds (phenolic acids, flavonoids, and alkaloids); [0127] Extract 3 is characterized by a similar concentration of polyphenols as extract 2, but contains only traces of alkaloids; and [0128] Extract 4 is characterized by a similar concentration of alkaloids as extract 2, but does not contain polyphenols.

[0129] From the foregoing, it is clear that only the solvent according to the invention consisting of a mixture of betaine, propanediol and water in molar proportions of 1:1:5 makes it possible to obtain an extract of Murraya koenigii rich in flavonoids and phenolic acids and having a very low content of alkaloids, in the trace state.

[0130] 4/ Comparison of Phenolic Acid, Flavonoid and Alkaloid Content in the Murraya koenigii Extract According to the Invention with the Plant/Solvent Ratio

[0131] The alkaloid, phenolic acid and flavonoid contents of extracts 1 and 1a of Murraya koenigii described in Example 1 were evaluated by RP-HPLC-UV under the conditions described in Example 3.

[0132] The results are described in Table 3 below.

TABLE-US-00003 TABLE 3 Concentration (mg/100 g of the Extract) Plant/Solvent Phenolic Extract Ratio Acids Flavonoids Alkaloids Extract 1a 5:95 45 26 Traces Extract 1 8:92 92 57 5

[0133] It appears from the above that the reduction of the plant/solvent ratio to 5:95 (Extract 1a), compared to the 8:92 ratio (Extract 1), makes it possible to obtain an extract containing only unquantifiable traces of alkaloids while presenting an interesting concentration of phenolic acids and flavonoids.

[0134] 5/ Study of the Effect of Murraya koenigii Extract According to the Invention (Extract 1) on the Expression of Proteins Associated with the Formation of Elastic Fibres

[0135] 5.1/ Method

[0136] The protein content (proteome) and its expression variations in normal human dermal fibroblasts treated or not with the 4 extracts of Murraya koenigii described in Example 1 (extracts 1, 2, 3 and 4) were identified by the nano-liquid chromatography-tandem mass spectrometry (nano LC-MS/MS) method. This analytical method is quantitative, sensitive, and reproducible and allows the identification of proteins expressed in cells by a bioinformatics analysis (CORAVALID).

[0137] 5.2/ Protocol

[0138] The proteomic study consists of qualitative and quantitative analysis of proteins expressed in untreated (UT) cells compared to cells treated with the 4 Murraya koenigii leaf extracts described in Example 1. The 4 extracts are tested at their maximum non-cytotoxic concentration, i.e., Extract 1 (invention) at 0.2%; Extract 2 (propanediol/water) at 0.04%; Extract 3 (ethanol/water) at 1% and Extract 4 (ethanol) at 0.1%, and incubated in the cell culture medium for 24 hrs.

[0139] The fibroblasts are then washed with cold phosphate-saline buffer, recovered and frozen at −80° C. Protein lysates and corresponding peptide extracts are obtained according to the method described by Wisniewski J R et al, Nat Methods. 2009; 6(5):359-62.

[0140] 250 ng of peptides are injected into a C18 pre-column (300 μm×5 mm; particle diameter 5 μm; Thermo Scientific) and then eluted into a C18 column (75 μm×500 mm; particle diameter 2 μm; Thermo Scientific). Liquid nano-chromatography is performed with the Ultimate 3000 equipment (Dionex) by applying a 60-35% acetonitrile gradient for 60 minutes at a flow rate of 300 nl/min. Data are generated with the Q-Exactive mass spectrometer (Thermo Scientific).

[0141] The proteome of the cells is analyzed using the SEQUEST-HT algorithm on the UNIPROT database. Protein quantification is performed by the Minora algorithm and the ratio of cells treated with Murraya koenigii extract/untreated cells is calculated using the Pairwise Ratio Based method and then expressed as the geometric median of all generated data.

[0142] The study is based on 3 independent experiments (n=3). The hypothesis test performed was an analysis of variance (ANOVA) to compare the proteome of untreated fibroblasts versus fibroblasts treated with the 4 Murraya koenigii extracts described in Example 1.

[0143] 5.3/ Results

[0144] The study identified 4 proteins associated with elastic fibers and whose expression was significantly increased in dermal fibroblasts treated with the betaine/propanediol/water extract (molar ratio 1:1:5) of Murraya koenigii.

[0145] The results are presented in Table 4 and are expressed as the treated cell expression ratio with a Murraya koenigii extract/untreated cells and averaged over the 3 experiments.

TABLE-US-00004 TABLE 4 Ratio Extract Protein 1/Untreated p Value Elastin 2.18 6.6E−14 Fibrillin-1 2.18 1.0E−17 Fibrillin-2 2.07 2.8E−12 Fibulin-5 1.69 9.4E−11

[0146] Surprisingly, only the Murraya koenigii extract according to the invention (Extract 1: GHP115) significantly increases the protein expression of elastin and its 3 partners, fibrillin-1, fibrillin-2, and fibulin-5 in normal human dermal fibroblasts. The other 3 extracts (Extract 2: propanediol/water; Extract 3: ethanol/water and Extract 4: ethanol) have no effect on elastic fiber proteins (data not shown).

[0147] 5.4/ Conclusion

[0148] The extract of Murraya koenigii according to the invention, i.e., obtained by means of the solvent consisting of a betaine/propanediol/water mixture (molar ratio 1:1:5; Extract 1) increases the expression of proteins belonging to the elastic fibers. Normal human dermal fibroblasts treated with the Murraya koenigii betaine/propanediol/water extract according to the invention thus exhibit a phenotype favorable for the regeneration and renewal of skin elastic fibers.

[0149] 6/ Study of the Effect of Murraya koenigii Extracts (Extract 1 and La) on the Synthesis of Elastin, Fibrillin-1, and Fibulin-5 Synthesis in 2D Culture of Normal Human Fibroblasts.

[0150] 6.1/Method

[0151] This study uses in situ fluorescent immunostaining coupled with image analysis to measure elastin, fibrillin-1 and fibulin-5 syntheses deposited in monolayer cultures of dermal fibroblasts treated or not with the Extracts 1 and 1a according to the invention (betaine/propanediol/water, molar ratio 1:1:5) of Murraya koenigii.

[0152] 6.2/ Protocol

[0153] Normal human dermal fibroblasts are treated for 48 hrs. with the extracts according to the invention (betaine/propanediol/water and molar ratio 1:1:5) of Murraya koenigii obtained in Example 1 (Extracts 1 and 1a), at the final concentration of 0.2%. Untreated (UT) cells are used as controls. The cells are then washed with phosphate-saline buffer before being fixed and saturated with 1% BSA (bovine serum albumin) for 30 minutes. Cells are incubated with the primary antibody (anti-elastin; anti-fibrillin-1 or anti-fibulin-5) for 1 hour, washed in PBS buffer, and then incubated with the Alexa 594 fluorochrome-bound secondary antibody for 1 hour. The fluorescence is read on a Cytation 5 imaging spectrophotometer (Biotek) with the appropriate filters. Fluorescence measurements by image analysis are performed on common acquisition parameters.

[0154] The study is based on 3 independent experiments (n=3). The statistical test is the 2 Way-ANOVA followed by Bonferroni multiple comparisons to compare the synthesis of proteins of interest deposited in untreated dermal fibroblast cultures versus fibroblasts treated with Extracts 1 and 1a (betaine/propanediol/water, molar ratio 1:1:5) of Murraya koenigii according to the invention.

[0155] 6.3/ Results

[0156] 6.3.1/ Elastin Synthesis

[0157] We quantified elastin synthesis deposited in dermal fibroblasts treated or not with Extracts 1 and 1a (betaine/propanediol/water, molar ratio 1:1:5) of Murraya koenigii. The results are presented in Table 5 and expressed as a % of the untreated cells (UT). Values are expressed as ±the mean standard deviation in all 3 experiments (n=3).

TABLE-US-00005 TABLE 5 Untreated Extract 1 Extract 1a Exp_1 100 ± 34  158 ± 38  150 ± 22  Exp_2 100 ± 21  151 ± 26  168 ± 27  Exp_3 100 ± 23  178 ± 21  172 ± 20  n = 3 62% ± 14   63% ± 12   Statistics: ****: p < 0.0001 **** ****

[0158] In an equivalent manner, the Murraya koenigii Extracts 1 and 1a obtained according to the invention (betaine/propanediol/water, molar ratio 1:1:5) induce a significant increase in the protein synthesis of deposited elastin (+62% and +63%, respectively) within the dermal fibroblasts.

[0159] The extraction solvent alone (betaine/propanediol/water 1:1:5 molar ratio without Murraya koenigii) does not induce an increase in deposited elastin synthesis (data not shown).

[0160] 6.3.2/ Synthesis of Fibrillin-1

[0161] We quantified the synthesis of fibrillin-1 deposited in dermal fibroblasts that were treated or not with Murraya koenigii Extracts 1 and 1a (betaine/propanediol/water, molar ratio 1:1:5). The results are presented in Table 6 and expressed as a % of the untreated cells (UT). Values are expressed as ±the mean standard deviation in all 3 experiments (n=3).

TABLE-US-00006 TABLE 6 Untreated Extract 1 Extract 1a Exp_1 100 ± 11  189 ± 16  152 ± 30  Exp_2 100 ± 32  129 ± 44  137 ± 26  Exp_3 100 ± 43  140 ± 38  128 ± 33  n = 3 53% ± 32   39% ± 12   Statistics: ****: p < 0.0001 **** **

[0162] The Murraya koenigii Extracts 1 and 1a obtained according to the invention (betaine/propanediol/water, molar ratio 1:1:5) induce a significant increase in protein synthesis of fibrillin-1 deposited in dermal fibroblasts. Extracts 1 and 1a are not statistically different from each other even if Extract 1 increases (+53%) the protein synthesis of fibrillin-1 in dermal fibroblasts more strongly than Extract 1a (+39%).

[0163] An extraction solvent alone (betaine/propanediol/water 1:1:5 molar ratio without Murraya koenigii) does not induce an increase in deposited fibrillin-1 synthesis (data not shown).

[0164] 6.3.3/ Synthesis of Fibrillin-5

[0165] We quantified the synthesis of fibulin-5 deposited in dermal fibroblasts treated or not with Extracts 1 and 1a (betaine/propanediol/water, molar ratio 1:1:5) of Murraya koenigii. The results are presented in Table 7 and expressed as a % of the untreated cells (UT). Values are expressed as ±the mean standard deviation in all 3 experiments (n=3).

TABLE-US-00007 TABLE 7 Untreated Extract 1 Extract 1a Exp_1 100 ± 53  260 ± 121 162 ± 56  Exp_2 100 ± 56  222 ± 133 196 ± 74  Exp_3 100 ± 36  / 221 ± 131 n = 3 141% ± 27    93% ± 30   Statistics: ****: p < 0.0001 **** ****

[0166] The Murraya koenigii Extracts 1 and 1a obtained according to the invention (betaine/propanediol/water, molar ratio 1:1:5) induce a significant increase in protein synthesis of fibrillin-5 deposited in dermal fibroblasts. Extract 1 increases (+141%) the protein synthesis of fibulin-5 in dermal fibroblasts more strongly than extract 1a (+93%).

[0167] The extraction solvent alone (betaine/propanediol/water 1:1:5 molar ratio without Murraya koenigii) does not induce an increase in Fibulin-5 synthesis (data not shown).

[0168] 6.4/ Conclusion

[0169] Extracts 1 and 1a (betaine/propanediol/water, molar ratio 1:1:5) of Murraya koenigii according to the invention induce an improvement in the protein synthesis of the elastic network in the dermis.

[0170] 7/ Example of Composition—Natural Moisturizing Cream Gel

TABLE-US-00008 TABLE 8 Composition Name of Ingredients (% by mass) Emulium ® Mellifera 2.5 Natural squalane 2.0 Dicaprylyl carbonate 15.0 Demineralized water QS100 Glycerin 20.0 Tapioca starch 5.0 Murraya koenigii extract 2.0 according to the invention Ethylhexylglycerin 0.1 Phenoxyethanol 0.9 Perfume 0.2 Total 100