Use of isoleucine N-hexadecanoyl as a “volumizing” and/or “plumping” agent for human skin

Abstract

The invention relates to the cosmetic use of isoleucine N-hexadecanoyl of formula (I): CH.sub.3(CH.sub.2).sub.14C(O)NHCH(COOH)CH(CH.sub.3)CH.sub.2CH.sub.3 (I) as a volumizing agent and/or as a plumping agent for human skin. The invention also relates to a cosmetic treatment method for obtaining a volumizing and/or plumping effect of the skin of a part of the human body selected from among the breasts, face, cheeks, buttocks and eyelids.

Claims

1. A cosmetic treatment process for obtaining a volumizing and/or plumping effect on the skin of a part of the human body chosen from the breasts, the face, the cheeks, the buttocks and the eyelids, characterized in that it consists in applying to said part a cosmetic composition comprising an effective amount of N-hexadecanoyl isoleucine, wherein the effective amount of N-hexadecanoyl isoleucine is between 0.000001% and 0.05% by weight.

Description

(1) The following experimental studies illustrate the invention without, however, limiting it.

(2) 1In Vitro Studies

(3) The in vitro studies demonstrate that the N-hexadecanoyl isoleucine of formula (I) regulates the activity of hypodermal adipose tissue cells by making it possible to stimulate the differentiation of normal human preadipocytes in a model reproducing conditions of adipogenic stimulation insufficiency, by making it possible to stimulate the increase in adipocyte surface area, and by making it possible to restore the adipocyte phenotype of young normal human preadipocytes in a model reproducing aging conditions.

(4) 1.1Demonstration of the Stimulatory Effectiveness of N-Hexadecanoyl Isoleucine on the Differentiation of Normal Human Preadipocytes in an In Vitro Model Reproducing Conditions of Adipogenic Stimulation Insufficiency, and on the Stimulatory Effectiveness of N-Hexadecanoyl Isoleucine on the Increase in Adipocyte Surface Area
Protocol

(5) Normal human preadipocytes resulting from abdominal surgical waste from a 38-year-old female donor were cultured in monolayer until confluence in the presence of a culture medium comprising PBM-2 (Preadipocyte Basal Medium-2) supplied by the company Lonza, an amount of 10% by volume of FBS (Fetal Bovine Serum) for 100% of the volume of the culture medium, L-glutamine and gentamicin.

(6) Their differentiation into adipocytes was induced by adding a depleted differentiation medium comprising a mixture of insulin, dexamethasone, IBMX (isobutyl methyl xanthine) and indomethacin, and used at a dilution rate of 1/10.sup.th with respect to a standard differentiation medium. This phase of differentiation by adding a depleted differentiation medium is carried out in the presence of the products tested in the context of the present demonstration, namely N-hexadecanoyl isoleucine, hexadecanoic acid, isoleucine, and a mixture of hexadecanoic acid and of isoleucine in a hexadecanoic acid/isoleucine ratio by weight of 65/35, and also in the absence of products so as to obtain a control differentiated in depleted medium.

(7) A differentiation phase is also carried out on human preadipocytes as described above and cultured in monolayers as described above, but in the presence of a standard differentiation medium, namely a nondiluted mixture of insulin, dexamethasone, IBMX (IsoButyl Methyl Xanthine) and indomethacin, so as to obtain a control differentiated in standard medium.

(8) Once the maturity of the preadipocytes was reached, namely after 15 days following the induction described above, the cultures were rinsed with a PBS (Phosphate Buffered Saline) buffer and the intracytoplasmic lipid vesicles were labeled using a specific viable fluorescent probe, Bodipy (500 g/ml). The nuclei of the differentiated preadipocytes were also labeled using a Hoechst reagent (bisbenzimide), which is also fluorescent, at a concentration of 2 g/ml, sold by the company Sigma.

(9) Three photographs per well were taken using a Zeiss Axiovert 25 inverted epifluorescence microscope fitted with an 20 objective.

(10) Results

(11) For each test of products tested, including controls, the images obtained with the inverted epifluorescence microscope are analyzed using the Lucia software, allowing quantification of the Bodipy fluorescent label, in order to carry out: a measurement of the lipid droplet surface area (Sd), a measurement of the number of differentiated cells (Nc), and a measurement of the number of total cell nuclei (Nnc).

(12) For each test, the level of adipocyte differentiation (D) is then calculated by relating the lipid droplet surface area to the number of total nuclei according to the following formula: (D)=(lipid droplet surface area)/(number of total cell nuclei).

(13) The following will thus be calculated: the level of adipocyte differentiation of the control differentiated in depleted medium (subsequently denoted Ddm); the level of adipocyte differentiation of the control differentiated in standard or complete medium (subsequently denoted Dcm); the level of adipocyte differentiation of the products tested (N-hexadecanoyl isoleucine, hexadecanoic acid, isoleucine, hexadecanoic acid/isoleucine mixture in a ratio by weight of 65/35) (subsequently denoted Dpt).

(14) For each product tested, the following are then calculated:

(15) a) the percentage restoration of adipocyte differentiation (subsequently denoted RAD) according to the following formula:
RAD=[(DptDdm)/(DcmDdm)]100

(16) b) the adipocyte surface area (AS) according to the following formula:
(AS)=[(lipid droplet surface area Sd)/(number of differentiated cells Nc)]100

(17) c) the percentage increase in adipocyte surface area (subsequently denoted IAS) relative to the control treated with depleted medium according to the formula:
IAS=[adipocyte surface area of the product (ASpt)/adipocyte surface area of the control treated with the depleted medium (ASdm)]100.

(18) A statistical analysis is then carried out on the data (D) and (AS) using a two-sided Student's test assuming unequal variances. Thus, the population of each of the experimental conditions tested for the parameters (D) and (AS) is compared with the population of the condition of differentiation in the presence of the depleted differentiation medium. An error threshold (p) is set at 5%. If p0.05, the two populations studied are considered to be significantly different, and the experimental condition studied then has an effect compared with the condition of differentiation in depleted medium. If p>0.05, the two populations are considered not to show any differences and, consequently, the experimental condition studied does not have an effect compared with the condition of differentiation in depleted medium.

(19) The results obtained are reported in Table 1 below:

(20) TABLE-US-00001 TABLE 1 Products tested RAD IAS Control differentiated in depleted 0% 100% medium Control differentiated in standard 100%* 194%* medium N-hexadecanoyl isoleucine (5 g/ml on 63%* 215%* a dry extract basis) N-hexadecanoyl isoleucine (10 g/ml 85%* 214%* on a dry extract basis) Hexadecanoic acid (3.3 g/ml) <10% 41%* Hexadecanoic acid (6.5 g/ml) <10% 62% Isoleucine (1.7 g/ml on a dry <10% 57% extract basis) Isoleucine (3.3 g/ml on a dry <10% 117% extract basis) Hexadecanoic acid/isoleucine (65/35) <10% 88% (5 g/ml on a dry extract basis) Hexadecanoic acid/isoleucine (65/35) <10% 49% (10 g/ml on a dry extract basis) *Result statistically significant compared with the control differentiated in depleted medium (two-sided Student's test assuming unequal variances) with p 0.05.

(21) The tests carried out according to the protocol described above and included in Table 1 show that the N-hexadecanoyl isoleucine allows an RAD of 63% for a dose of 5 g/ml, on a dry extract basis, of N-hexadecanoyl isoleucine, and an RAD of 85% for a dose of 10 g/ml, on a dry extract basis.

(22) When hexadecanoic acid is used at doses of 3.3 g/ml on a dry extract basis and 6.5 g/ml on a dry extract basis, and when isoleucine is used at doses of 1.7 g/ml on a dry extract basis and 3.3 g/ml on a dry extract basis, the RAD is less than 10% and considered to be nonsignificant. The same is true for the tests with a mixture of hexadecanoic acid and isoleucine in a hexadecanoic acid/isoleucine proportion by weight of 65/35, at doses of 5 g/ml on a dry extract basis and 10 g/ml on a dry extract basis.

(23) These results therefore show that the N-acylated structure of N-hexadecanoyl isoleucine allows an effective RAD.

(24) The tests also show that N-hexadecanoyl isoleucine makes it possible to obtain a level of increase of the adipocyte surface area of 215% for a dose of 5 g/ml on a dry extract basis, and of 214% for a dose of 10 g/ml on a dry extract basis, compared with 194% for the control differentiated in standard medium. Furthermore, when hexadecanoic acid is used at doses of 3.3 g/ml on a dry extract basis and 6.5 g/ml on a dry extract basis, and when isoleucine is used at doses of 1.7 g/ml on a dry extract basis and 3.3 g/ml on a dry extract basis, in tests carried out according to the protocol described above, the IAS levels obtained are much lower than that obtained for the control differentiated in standard medium, and in most cases, they are also less than the IAS level obtained for the control differentiated in depleted medium. The same observation is made for the tests carried out with a mixture of hexadecanoic acid and isoleucine in a hexadecanoic acid/isoleucine proportion by weight of 65/35, at doses of 5 g/ml on a dry extract basis and 10 g/ml on a dry extract basis.

(25) These results therefore show that the N-acylated structure of the N-hexadecanoyl isoleucine allows an effective IAS.

(26) 1.2Demonstration of the Adipocyte-Phenotype-Restoring Effect with N-Hexadecanoyl Isoleucine Using Normal Human Preadipocytes in an In Vitro Model Reproducing Aging Conditions

(27) The in vitro model used consists of an accelerated aging of normal human preadipocytes by successive passages in order to generate cells exhibiting a phenotype and the characteristics of MAD cells after differentiation. The term passage or subculturing or amplification denotes here the operation, known to those skilled in the art, consisting in amplifying the cells in question successively by means of culture cycles. Thus, the term preadipocytes of passage RO denotes the preadipocytes which have undergone no passage operation as defined above. The term preadipocytes of passage R1 denotes the preadipocytes which have undergone a single passage operation as defined above. The term preadipocytes of passage R7 denotes the preadipocytes which have undergone 7 successive passage operations under identical operating conditions. Consequently, the preadipocytes of passage R1 will be considered to be representative of preadipocytes of a young individual since they do not exhibit any morphological and phenotypic alterations owing to the successive passage steps. Furthermore, the preadipocytes of passage R7 will be considered to be representative of an artificially aged individual since they exhibit morphological and phenotypic alterations owing to the successive passage steps. Three different protocols were carried out in order to demonstrate the effectiveness of the N-hexadecanoyl isoleucine in restoring the phenotype of differentiated and artificially aged preadipocytes.

(28) Protocol 1

(29) a)Preparation of the Young Control

(30) i) Human preadipocytes of passage R1 were cultured in monolayer for 5 days until confluence. Their differentiation into adipocytes was induced by adding a standard differentiation mixture comprising a mixture of insulin, dexamethasone, IBMX (Isobutyl methyl xanthine) and indomethacin. ii) After a period of 15 days following the induction step described above, the cultures prepared are rinsed with a PBS (phosphate buffered saline) buffer and then fixed using a solution comprising, for 100% of the weight of said solution, 4% by weight of formaldehyde and 0.1% by weight of Triton X-100, which is a synthetic detergent commonly used in biology. iii) Labeling of the intracytoplasmic lipid vesicles of the cell cultures previously prepared is then carried out using an amount of 0.2% by mass of the Oil-Red-O label sold by the company Sigma. iv) The cell monolayers containing the lipid vesicles of the cell cultures previously described and labeled as indicated above are then lysed with dimethyl sulfoxide (or DMSO). v) The amount of labeling of the lipid vesicles of the labeled and lysed cell cultures as prepared at the end of step iv) of the present protocol is then estimated by reading the optical density (OD) of these lysates using a spectrophotometer at a wavelength of 540 nm.

(31) The optical density thus measured for the young control is denoted OD.sub.R1.

(32) b)Preparation of the Aged Control

(33) The protocol steps i) to v) described in section a) are applied to human preadipocytes of passage R7, so as to obtain an optical density for the aged control, denoted OD.sub.R7.

(34) c)Evaluation of the Effect of the N-hexadecanoyl Isoleucine on Human Preadipocytes of Passage R7

(35) Human preadipocytes of passage R7 were cultured in monolayers for 5 days until confluence. Their differentiation into adipocytes is induced by adding a standard differentiation mixture comprising a mixture of insulin, dexamethasone, IBMX (Isobutyl methyl xanthine) and indomethacin, in the presence of N-hexadecanoyl isoleucine. Steps ii) to v) as described in section a) are then carried out, so as to obtain an optical density for the N-hexadecanoyl isoleucine denoted OD.sub.HIL.

(36) dCalculation of the Degree of Phenotype Restoration

(37) The measurement of the various optical densities makes it possible to calculate a phenotype restoration rate (hereinafter denoted PRR) for N-hexadecanoyl isoleucine, according to the following formula:
PRR (%)=[(OD.sub.HILOD.sub.R7)/(OD.sub.R1OD.sub.R7)]100

(38) A statistical analysis is then carried out on the optical density data using a two-sided Student's test assuming unequal variances. Thus, the population of each of the experimental conditions tested is compared with the population of the R7 preadipocyte differentiation condition. An error threshold (p) is set at 5%. If p is less than or equal to 0.05, the two populations studied are considered to be significantly different and, consequently, the experimental condition studied is considered to have an effect compared with the R7 preadipocyte differentiation condition. If p is strictly greater than 0.05, the two populations are considered not to show any difference and, consequently, the experimental condition studied is considered not to have any effect compared with the R7 preadipocyte differentiation condition.

(39) The PRRs obtained for various concentrations of N-hexadecanoyl isoleucine are reported in Table 2 below.

(40) TABLE-US-00002 TABLE 2 Products tested PRR Differentiated R7 control 0% Differentiated R1 control 100%* N-hexadecanoyl isoleucine (5 g/ml on a 13%* dry extract basis) N-hexadecanoyl isoleucine (10 g/ml on a 16%* dry extract basis) *Results statistically significant compared with the control differentiated in depleted medium (two-sided Student's test assuming unequal variances) with p 0.05
Protocol 2

(41) The steps of sections a) and b) of protocol 1 are carried out using human preadipocytes of passage R1 and human preadipocytes of passage R7 so as to obtain a young control and an aged control and the associated optical densities OD.sub.R1 and OD.sub.R7. The effect of the N-hexadecanoyl isoleucine on human preadipocytes of passage R7 is evaluated according to a variant of the procedure of protocol 1, the first step of which consists in culturing the human preadipocytes of passage R7 in a monolayer for 5 days until confluence in the presence of N-hexadecanoyl isoleucine. Their differentiation into adipocytes is induced by adding a standard differentiation mixture comprising a mixture of insulin, dexamethasone, IBMX (Isobutyl methyl xanthine) and indomethacin. Steps ii) to v) as described in section a) of protocol 1 are then carried out, so as to obtain an optical density for the N-hexadecanoyl isoleucine, denoted OD.sub.HIL.

(42) Measurement of the various optical densities makes it possible to calculate a phenotype restoration rate (PRR) for the N-hexadecanoyl isoleucine following the implementation of protocol 2, according to the following formula:
PRR (%)=[(OD.sub.HILOD.sub.R7)/(OD.sub.R1OD.sub.R7)]100

(43) The PRRs obtained for various concentrations of N-hexadecanoyl isoleucine according to protocol 2 are reported in Table 3 below.

(44) TABLE-US-00003 TABLE 3 Products tested PRR Differentiated R7 control 0% Differentiated R1 control 100%* N-hexadecanoyl isoleucine (5 g/ml on a 27%* dry extract basis) N-hexadecanoyl isoleucine (10 g/ml on a 17%* dry extract basis) *Results statistically significant compared with the control differentiated in depleted medium (two-sided Student's test assuming unequal variances) with p 0.05
Protocol 3

(45) The steps of sections a) and b) of protocol 1 are carried out using human preadipocytes of passage R1 and human preadipocytes of passage R7 so as to obtain a young control and an aged control and the associated optical densities OD.sub.R1 and OD.sub.R7.

(46) The effect of the N-hexadecanoyl isoleucine on human preadipocytes of passage R7 is evaluated according to a variant of the procedure of protocol 2, the first step of which consists in culturing the human preadipocytes of passage R7 in a monolayer for 5 days until confluence in the presence of N-hexadecanoyl isoleucine.

(47) Their differentiation into adipocytes is induced by adding a standard differentiation mixture comprising a mixture of insulin, dexamethasone, IBMX (Isobutyl methyl xanthine) and indomethacin, in the presence of N-hexadecanoyl isoleucine. Steps ii) to v) as described in section a) of protocol 1 are then carried out, so as to obtain an optical density for the N-hexadecanoyl isoleucine, denoted OD.sub.HIL.

(48) The measurement of the various optical densities makes it possible to calculate a PRR for the N-hexadecanoyl isoleucine following the implementation of protocol 3, according to the following formula:
PRR (%)=[(OD.sub.HILOD.sub.R7)/(OD.sub.R1OD.sub.R7)]100

(49) The PRRs obtained for various concentrations of N-hexadecanoyl isoleucine according to protocol 2 are reported in Table 4 below.

(50) TABLE-US-00004 TABLE 4 Products tested PRR Differentiated R7 control 0% Differentiated R1 control 100%* N-hexadecanoyl isoleucine (5 g/ml on a 36%* dry extract basis) N-hexadecanoyl isoleucine (10 g/ml on a 30%* dry extract basis) *Results statistically significant compared with the control differentiated in depleted medium (two-sided Student's test assuming unequal variances) with p 0.05

CONCLUSION

(51) Irrespective of the protocol implemented, the experimental tests showed a beneficial effect of N-hexadecanoyl isoleucine on the restoration of an adipocyte phenotype under artificial aging conditions. Thus, at a dose of 5 g/ml, on a dry extract basis, of N-hexadecanoyl isoleucine, this effect was estimated via a phenotype restoration rate of 13% according to protocol 1, via a phenotype restoration rate of 27% according to protocol 2, and via a phenotype restoration rate of 36% according to protocol 3.

(52) 2In Vivo Studies

(53) The following experimental studies illustrate the invention without, however, limiting it.

(54) The in vivo studies demonstrate that the cosmetic use of the N-hexadecanoyl isoleucine of formula (I) and the implementation of a cosmetic treatment process for the human body make it possible to generate volumizing and/or plumping effects on human skin and/or make it possible to increase the elasticity of the skin.

(55) 2.1Demonstration of the Volumizing Effect and of the Plumping Effect of a Cosmetic Composition Comprising N-Hexadecanoyl Isoleucine on the Breasts

(56) a)Preparation of an Oil-in-Water (O/W) Emulsion Comprising N-Hexadecanoyl Isoleucine (E1) and of a Placebo Oil-in-Water Emulsion (E2)

(57) The emulsions (E1) and (E2) are prepared in the following way:

(58) Step 1): The components of the oily phase and the emulsifying system are successively introduced into a beaker, at a temperature of 80 C., and are subjected to mechanical stirring for a period of 15 minutes, using a stirrer which has an anchor-type spindle, at a speed of 80 rpm at a temperature of 80 C., so as to form a homogeneous mixture.

(59) Step 2): The thickening polymer is added to the mixture obtained at the end of step 1) at a temperature of 80 C., as is the aqueous phase, prepared beforehand at ambient temperature by mixing its components. The resulting mixture is then subjected to stirring by means of a rotor-stator emulsifying device, sold by the company Silverson, for a period of 4 minutes at a speed of 3000 rpm at a temperature of 80 C.

(60) Step 3): The mixture obtained at the end of step 2) is cooled over a period of 10 minutes to a temperature of 30 C., and kept stirring throughout the cooling by means of a mechanical stirrer which has an anchor-type spindle, at a speed of 80 rpm.

(61) Step 4): The Neolone MP preservative and the plaisir fragrance are then added to the mixture resulting from step 3) at a temperature of 30 C. The pH is then adjusted to a value of 6.5 by adding 50% triethanolamine.

(62) The oil-in-water emulsions (E1) and (E2) have the following compositions (by weight):

(63) TABLE-US-00005 TABLE 5 (E1) (E2) Oily phase: Liquid paraffin 8.00% 8.00% Shea butter 1.50% 1.50% DUB DONPG.sup.(1) 5.00% 5.00% N-hexadecanoyl isoleucine 1.00% 0% Thickening polymer Simulgel INS 100.sup.(2) 1.00% 1.00% Aqueous phase Water q.s. 100% q.s. 100% 50% triethanolamine q.s. pH = 6.5 q.s. pH = 6.5 Glycerol 3.00% 3.00% Emulsifying system Montanov 202.sup.(3) 2.00% 2.00% Additives Neolone MxP.sup.(4) 0.50% 0.50% Plaisir fragrance 0.10% 0.10% .sup.(1)Neopentyl glycol diethyl hexanoate sold by the company Starineries Dubois. .sup.(2)Thickening inverse latex (INCI name: hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer and isohexadecane and polysorbate 60) sold by the company SEPPIC. .sup.(3)Self-emulsifiable composition such as those described in EP 0 977 626, based on arachidyl alcohol, on behenyl alcohol and on arachidyl glucosides, sold by the company SEPPIC. .sup.(4)Preservative mixture comprising phenoxyethanol, methyl paraben, propyl paraben and methylisothiazolinone, sold by the company Rhm & Haas.
b)Evaluation of the Volumizing Effect and of the Plumping Effect of the Oil-in-Water Emulsions (E1) and (E2) on Breasts

(64) These evaluations are carried out by measuring the following criteria: measurement of the change in centimeters of the bust measurement (Mb), measurement of the change in thickness of the hypodermis of the breast by deep ultrasound; measurement of the change in volume of the breasts using the fringe projection technique (Primos Body).

(65) These evaluations were carried out on a population of 44 female volunteers with an average age of 26, with a bust of cup size A (namely a difference of 2.5 cm between the value of the bust measurement taken at the level of the nipples of the two breasts and the value of the bust measurement taken at the base of the two breasts), which was divided up into two equal groups of 22 volunteers (group I and group II). Each member of group (I) applied emulsion (E1) to both her breasts twice-daily for a period of 28 days, and each member of group (II) applied emulsion (E2) to both her breasts twice-daily for a period of 28 days.

(66) b1)Evaluation of the Volumizing Effect of N-Hexadecanoyl Isoleucine by Measuring the Change in Bust Measurement

(67) Protocol

(68) Before the start of the application of emulsions (E1) and (E2), and at the end of the period of 28 days of the experiment, the bust measurement (Mb) and the under-bust measurement (Mub) are taken using a tape measure graduated in centimeters.

(69) In parallel, the weight curve for each individual (i) of each group is controlled in order to withdraw, during the interpretation, the measured data relating to individuals having experienced too great a weight loss or weight gain (change in weight of the individual of 3 kg relative to the weight measured before the start of the study). The number of individuals whose weight change is greater than 3 kg of the initial weight measured before the start of the experiment is denoted (j). The population N of individuals for each group that will be the population for which the measurements will be interpreted is then calculated according to the formula: N=20j.

(70) The bust measurement in centimeters is taken on each member (i) of groups (I) and (II), by taking as a base the nipples of the two breasts of each individual. For each member (i) of each group, the bust measurement before the start of the experiment (Mb0i) and the bust measurement at the end of the period of the experiment of 28 days (Mb28i) are thus taken. The change in bust measurement for each member of each group, Mbi, is then calculated according to the following formula:
Mbi=Mb28iMb0i

(71) The arithmetic mean of the change in bust measurement (Mbm) is then calculated for each group according to the following formula:
Mbm=[(Mbi)]/N

(72) The under-bust measurement in centimeters is also taken on each member (i) of groups (I) and (II), by taking as a base the line under the two breasts of each individual. For each member (i) of each group, the under-bust measurement before the start of the experiment (Mub0i) and the under-bust measurement at the end of the period of the experiment of 28 days (Mub28i) are thus taken. The change in bust measurement for each member of each group, Mubi, is then calculated according to the following formula:
Mubi=Mub28iMub0i

(73) The arithmetic mean of the change in under-bust measurement (Mubm) is then calculated for each group according to the following formula:
Mubm=[(Mubi)]/N
Results Obtained

(74) The results of the arithmetic means Mbm and Mubm are reported in Table 6 below for groups (I) and (II).

(75) TABLE-US-00006 TABLE 6 Group (I) Group (II) Mbm (cm) +0.6 0 Mubm (cm) 0.1 0

(76) The arithmetic mean of the change in bust measurement (Mbm) of the members of group (I), who applied to their breasts the oil-in-water emulsion (E1) comprising N-hexadecanoyl isoleucine, was measured at +0.6 centimeter, for a change in under-bust measurement (Mubm) of 0.1 centimeter.

(77) The arithmetic mean of the change in bust measurement (Mbm) of the members of group (II), who applied to their breasts the oil-in-water emulsion (E2) not comprising N-hexadecanoyl isoleucine, was measured at 0 centimeter, for a change in under-bust measurement (Mubm) of 0 centimeter.

(78) Interpretation

(79) An increase in bust measurement of which the set of values (Mb28i) is statistically different than the values (Mb0i) is considered to be significant.

(80) An increase in under-bust measurement of which the set of values (Mub28i) is statistically different than the values (Mub0i) is considered to be significant.

(81) The statistical analysis used to evaluate the significance of the increase in bust measurement and of the increase in under-bust measurement is carried out using a two-sided Student's test assuming unequal variances. Thus, the set of values (Mb28i) and the set of values (Mub28i) are respectively compared with the set of values (Mb0i) and the set of values (Mub0i). An error threshold (p) is set at 5%. If p is less than or equal to 0.05, the set of values (Mb28i) and the set of values (Mub28i) are considered to be significantly different, respectively, from the set of values (Mb0i) and the set of values (Mub0i). Consequently, it is possible to conclude that there is a significant increase in the bust measurement and in the under-bust measurement. If p is strictly greater than 0.05, the set of values (Mb28i) and the set of values (Mub28i) will not be considered to be significantly different, respectively, than the set of values (Mb0i) and the set of values (Mub0i). Consequently, it will not be possible to conclude that there is a significant increase in the bust measurement and in the under-bust measurement.

(82) Furthermore, an increase in the bust measurement is considered to be significant when the arithmetic mean (Mbm) is greater than or equal to 0.2 cm, provided that the arithmetic mean of the under-bust measurement (Mubm) is not greater than 0.2 cm.

CONCLUSION

(83) It is therefore possible to deduce from the results that the presence of N-hexadecanoyl isoleucine in the oil-in-water emulsion (E1) made it possible to increase, in a significant manner, the mean bust measurement of the members of group (I) and therefore made it possible to give these individuals a volumizing effect on their breasts.

(84) b2Evaluation of the Effect of N-Hexadecanoyl Isoleucine on the Thickness of the Breast Hypodermis

(85) Protocol

(86) The measurement of the thickness of the breast hypodermis by deep ultrasound was carried out for each member of each group by means of a Sonoline Antares ultrasound system sold by the company Siemens, fitted with a probe 6 cm long and 1 cm thick. Before each measurement, a contact gel of the Aquasonic100 brand is applied to the breast. The measurement is carried out at a frequency of 13 MHz by direct application of the probe to the skin of the breast. The probe is connected to software for processing the data measured and to a Fuji DryPix 7000 printer.

(87) For each member (i) of each group, this evaluation is carried out by performing the following 3 measurements in 3 different places on the breast: measurement of the thickness of the hypodermis is performed on the lateral side of the breast (MLi), measurement of the thickness of the hypodermis is performed on the superior-anterior face of the breast (MSAi), and measurement of the thickness of the hypodermis is performed on the inferior-anterior face of the breast (MIAi).

(88) In parallel, the weight curve for each individual (i) of each group is controlled in order to withdraw, during the interpretation, the measured data relating to individuals having undergone too great a weight loss or weight gain (change in weight of the individual of 3 kg relative to the weight measured before the start of the study). The number of individuals whose weight change is greater than 3 kg of the initial weight measured before the start of the experiment is denoted (j). The population N of individuals for each group that will be the population for which the measurements will be interpreted is then calculated according to the formula: N=20j.

(89) For each member (i) of each group, the following are thus measured: the thickness of the hypodermis on the lateral side of the breast before the start of the experiment (ML0i); the thickness of the hypodermis on the lateral side of the breast at the end of the period of 28 days of the experiment (ML28i); the thickness of the hypodermis on the superior-anterior face of the breast before the start of the experiment (MSA0i); the thickness of the hypodermis on the superior-anterior face of the breast at the end of the period of 28 days of the experiment (MSA28i); the thickness of the hypodermis on the inferior-anterior face of the breast before the start of the experiment (MIA0i); and the thickness of the hypodermis on the inferior-anterior face of the breast at the end of the period of 28 days of the experiment (MIA28i).

(90) For each member of each group, the following are then calculated: the change in thickness of the hypodermis on the lateral side of the breast (MLi) according to the formula: MLi=ML28i-ML0i; the change in the thickness of the hypodermis on the superior-anterior face of the breast (MSAi) according to the formula: MSAi=MSA28i-MSA0i; and the change in the thickness of the hypodermis on the inferior-anterior face of the breast (MIAi) according to the formula: MIAi=MIA28i-MIA0i.
Expression of the Results

(91) On the basis of the measurements taken, indicated above, the following are then calculated for each group: the mean change in thickness of the hypodermis on the lateral side of the breast (MLm) according to the formula: MLm=[(MLi)]/N; the mean change in thickness of the hypodermis on the superior-anterior face of the breast (MSAm) according to the formula:
MSAm=[(MSAi)]/N; the mean change in thickness of the hypodermis on the inferior-anterior face of the breast (MIAm) according to the formula:
MIAm=[(MIAi)]/N; the mean thickness of the hypodermis on the lateral side of the breast before the start of the experiment [Mean(ML0)] according to the formula:
[Mean(ML0)]=[(ML0i)]/N; the mean thickness of the hypodermis on the superior-anterior face of the breast before the start of the experiment [Mean(MSA0)] according to the formula:
[Mean(MSA0)]=[(MSA0i)]/N; the mean thickness of the hypodermis on the inferior-anterior face of the breast before the start of the experiment [Mean(MIA0)] according to the formula
[Mean(MIA0)]=[(MIA0i)]/p the level of increase in the thickness of the hypodermis on the lateral side of the breast (IML) according to the formula:
(IML)=[MLm/[Mean(ML0)]]100 the level of increase in the thickness of the hypodermis on the superior-anterior face of the breast (IMSA) according to the formula:
(IMSA)=[MSAm/[Mean(MSA0)]]100 the level of increase in the thickness of the hypodermis on the inferior-anterior face of the breast (IMIA) according to the formula (IMIA)=[MIAm/[Mean(MIA0)]]100.
Results Obtained

(92) The results of the measurements carried out for groups (I) and (II), namely the MLm, MSAm, MIAm, IML, IMSA and IMIA, are reported in Table 7 below.

(93) TABLE-US-00007 TABLE 7 Group (I) Group (II) MLm (mm) +0.13 +0.04 IML (%) 1.0 0 MSAm (mm) +1.14* +0.35* IMSA (%) 5.0 2.0 MIAm (mm) +0.42 1.19 IMIA (%) 1.0 4.0 *Results statistically significant compared with the value at t = 0 (two-sided Student's test assuming unequal variances) with p 0.05.

(94) The level of increase in the thickness of the hypodermis on the superior-anterior face of the breast (IMSA) of the individuals of group (I), who applied to their breasts the oil-in-water emulsion (E1) comprising N-hexadecanoyl isoleucine, comes to 5%, whereas a value of 2% for this same level of increase in the thickness of the hypodermis on the superior-anterior face of the breast (IMSA) was calculated for the individuals of group (II), who applied to their breasts the oil-in-water emulsion (E2) not comprising N-hexadecanoyl isoleucine.

(95) A mean increase in the thickness of the hypodermis on the superior-anterior face of the breast (MSAm) of 1.14 mm for the individuals of group (I) is also observed, compared with a value of 0.35 mm for the individuals of group (II).

(96) This significant increase in the thickness of the hypodermis on the superior-anterior face of the breast for the individuals of group (I) is accompanied by a stability of the thickness of the hypodermis on the inferior-anterior face of the breast (MIAm=+0.42 mm) and a stability of the thickness of the hypodermis on the lateral side of the breast (MLm=+0.13 mm).

(97) Interpretation of the Results

(98) A mean increase in the thickness of the hypodermis on the lateral side of the breast is considered to be significant when the set of values (ML28i) is statistically different than the values (ML0i).

(99) A mean increase in the thickness of the hypodermis on the superior-anterior face of the breast is considered to be significant when the set of values (MSA28i) is statistically different than the values (MSA0i).

(100) A mean increase in the thickness of the hypodermis on the inferior-anterior face of the breast is considered to be significant when the set of values (MIA28i) is statistically different than the values (MIA0i).

(101) The statistical analysis used to evaluate the significance of these three parameters is a two-sided Student's test assuming unequal variances. Thus, the sets of values (ML28i), (MSA28i) and (MIA28i) are respectively compared with the sets of values (ML0i), (MSA0i) and (MIA0i). An error threshold (p) is set at 5%.

(102) If p is less than or equal to 0.05, the sets of values (ML28i), (MSA28i) and (MIA28i) are considered to be significantly different, respectively, than the sets of values (ML0i), (MSA0i) and (MIA0i). Consequently, it is possible to conclude that there is a significant increase in the thickness of the hypodermis on the lateral side of the breast and/or on the superior-anterior face of the breast and/or on the inferior-anterior face of the breast.

(103) If p is strictly greater than 0.05, the sets of values (ML28i), (MSA28i) and (MIA28i) will not be considered to be significantly different, respectively, than the sets of values (ML0i), (MSA0i) and (MIA0i). Consequently, it will not be possible to conclude that there is a significant increase in the thickness of the hypodermis on the lateral side of the breast and/or on the superior-anterior face of the breast and/or on the inferior-anterior face of the breast.

CONCLUSION

(104) It can therefore be deduced from these results that the presence of N-hexadecanoyl isoleucine in the oil-in-water emulsion (E1) made it possible to increase, in a significant manner, the thickness of the hypodermis on the superior-anterior face of the breast, without this increase occurring to the detriment of the thickness of the hypodermis on the inferior-anterior face of the breast and/or to the detriment of the thickness of the hypodermis on the lateral side of the breast. The use of N-hexadecanoyl isoleucine therefore made it possible to give these individuals a plumping effect on their breasts.

(105) b3Evaluation of the Effect of N-Hexadecanoyl Isoleucine on the Increase in Breast Volume Using the Fringe Projection Technique

(106) Protocol

(107) These evaluations were carried out on a random subpopulation of 12 volunteers of the population used for the previous evaluations described in sections b1) and b2), which was divided up into 2 equal groups of 6 volunteers (group III and group IV). Each member of group (III) applied emulsion (E1) to both her breasts twice-daily for a period of 28 days and each member of group (IV) applied emulsion (E2) to both her breasts twice-daily for a period of 28 days.

(108) These evaluations consisted in measuring the volume of the breasts of each member (i) of each group before the start of the experiment and at the end of the period of 28 days following the application of emulsions (E1) and (E2).

(109) The experiment consisted in recording a three-dimensional image of the breasts of each member (i) of each group, before and after the application of emulsion (E1) or (E2) for 28 days, using an interference fringe projection profilometer sold under the name 3D Primos Body. This apparatus, which is characterized by a measurement field of 300 mm200 mm, by a lateral resolution of 500 micrometers, by a lateral resolution greater than or equal to 30 micrometers and by a data acquisition time greater than or equal to 140 milliseconds, makes it possible to project 2 light beams on to the surface to be analyzed, and the images obtained are recorded by 2 cameras at an angle of 20. Calculation software integrated into the 3D Primos Body then makes it possible to calculate the volume of each area present on the three-dimensional image previously recorded.

(110) For each member (i) of groups (III) and (IV) as defined above, the following are then calculated: the volume of the breast in question from the three-dimensional image recorded before the start of the experiment (V0i); the volume of the breast in question from the three-dimensional image recorded at the end of the period of 28 days of the experiment (V28i).

(111) For each member (i) of groups (III) and (IV) as defined above, the change in volume of the breast in question, Vi, is then calculated according to the formula:
Vi=(V28i)(V0i)
Expression of the Results

(112) On the basis of the measurements carried out, indicated above, the following are then calculated for each group: the mean change in volume of the breast (Vm) according to the formula
(Vm)=[(Vi)]/6; the mean of the volume of the breast before the start of the experiment [Mean(V0)] according to the formula:
[Mean(V0)]=[(MV0i)]/6; the level of increase in the volume of the breast (IV) according to the formula:
(IV)=[(Vm)/[Mean(V0)]]100.
Results Obtained

(113) The results obtained for groups (III) and (IV), namely the Vm and IV, are reported in Table 8 below.

(114) TABLE-US-00008 TABLE 8 Group (III) Group (IV) (Vm) in cm.sup.3 +2.6* 0.5* (IV) as % +1.0%* 0%* *Results statistically significant compared with the value at t = 0 (two-sided Student's test assuming unequal variances) with p 0.05.

(115) The mean increase in the volume of the breast (Vm) of the individuals of group (III), who applied to their breasts the oil-in-water emulsion (E1) comprising N-hexadecanoyl isoleucine, comes to 2.6 cm.sup.3, compared with a change of 0.5 cm.sup.3, judged to be nonsignificant, for the individuals of group (IV), who applied to their breasts the oil-in-water emulsion (E2) not comprising N-hexadecanoyl isoleucine. Likewise, the level of increase in the volume of the breast (IV) was calculated at +1.0% for the individuals of group (III), who applied to their breasts the oil-in-water emulsion (E1) comprising N-hexadecanoyl isoleucine, compared with a value of 0% for the individuals of group (IV), who applied to their breasts the oil-in-water emulsion (E2) not comprising N-hexadecanoyl isoleucine.

(116) Interpretation of the Results

(117) A mean increase in the volume of the breast of which the set of values (V28i) is statistically different than the values (V0i) is considered to be significant. The statistical analysis used to evaluate the significance is a Student's test.

(118) The statistical analysis used to evaluate the significance of the increase in breast volume is a two-sided Student's test assuming unequal variances. Thus, the set of values (V28i) is compared with the set of values (V0i). An error threshold (p) is set at 5%.

(119) If p is less than or equal to 0.05, the set of values (V28i) is considered to be significantly different than the set of values (V0i). Consequently, it can be concluded that there is a significant increase in the breast volume. If p is strictly greater than 0.05, the set of values (V28i) will not be considered to be significantly different than the set of values (V0i). Consequently, it will not be possible to conclude that there is a significant increase in the breast volume.

CONCLUSION

(120) It can therefore be deduced from these results that the presence of N-hexadecanoyl isoleucine in the oil-in-water emulsion (E1) made it possible to increase, in a significant manner, the volume of the breasts of the members of group (III).

(121) b4General Conclusion

(122) The experimental results demonstrated in the experimental sections b1), b2) and b3) demonstrate that the application of a cosmetic composition comprising N-hexadecanoyl isoleucine to the breasts makes it possible, for the individuals in question, to obtain a significant increase in bust measurement, in thickness of the hypodermis on the superior-anterior face of the breast, and in breast volume. Indeed, the use of N-hexadecanoyl isoleucine and the application to the breasts of a cosmetic composition comprising N-hexadecanoyl isoleucine made it possible to generate a volumizing effect and a plumping effect on the breasts of the individuals in question.

(123) 2.2Demonstration of the Improvement in the Elasticity of the Skin and the Plumping Effect of a Cosmetic Composition Comprising N-Hexadecanoyl Isoleucine on the Face of Individuals with Mature Skin

(124) a)Preparation of an Oil-in-Water Emulsion Comprising N-Hexadecanoyl Isoleucine (E3) and of a Placebo Oil-in-Water Emulsion (E4)

(125) Emulsions (E3) and (E4) are prepared in the following way: Step 1): The components of the oily phase and the emulsifying system are successively introduced into a beaker, at a temperature of 80 C., and are subjected to mechanical stirring for a period of 15 minutes, using a stirrer fitted with an anchor-type spindle, at a speed of 80 rpm at a temperature of 80 C., so as to form a homogeneous mixture. Step 2): The thickening polymer is added to the mixture obtained at the end of step 1) at a temperature of 80 C., as is the aqueous phase, prepared beforehand at ambient temperature by mixing its components. The resulting mixture is then subjected to stirring by means of a rotor-stator emulsifying device, sold by the company Silverson, for a period of 4 minutes at a speed of 3000 rpm at a temperature of 80 C. Step 3): The mixture obtained at the end of step 2) is cooled over a period of 10 minutes to a temperature of 30 C., and kept stirring throughout the cooling by means of a mechanical stirrer fitted with an anchor-type spindle, at a speed of 80 rpm. Step 4): The Sepicide LD preservative, the chlorphenesin preservative and the Petite Fleur fragrance are then added to the mixture resulting from step 3) at a temperature of 30 C. The pH is then adjusted to a value of 6.0 by adding 50% triethanolamine.

(126) The oil-in-water emulsions (E3) and (E4) have the following compositions (by weight):

(127) TABLE-US-00009 TABLE 9 (E3) (E4) Oily phase: Plant squalane 7.00% 7.00% Polyisobutene 13.00% 13.00% Lanol P.sup.(5) 1.50% 1.50% N-hexadecanoyl isoleucine 1.00% 0% Thickening polymers Sepiplus 400.sup.(6) 0.80% 0.80% Keltrol CGT.sup.(7) 0.15% 0.15% Aqueous phase Water q.s. 100% q.s. 100% 50% triethanolamine q.s. pH = 6.0 q.s. pH = 6.0 Glycerol 5.00% 5.00% Emulsifying system Montanov 202.sup.(3) 2.00% 2.00% Montanov 82.sup.(8) 1.00% 1.00% Additives Sepicide LD.sup.(9) 0.70% 0.70% Petite Fleur fragrance 0.10% 0.10% Chlorphenesin.sup.(10) 0.30% 0.30% .sup.(5)Ethylene glycol monopalmitate sold by the company SEPPIC; .sup.(6)Self-invertible inverse latex of copolymers such as those described in WO 2005/040230 (INCI name: polyacrylate-13 & polyisobutene & polysorbate 20), sold by the company SEPPIC; .sup.(7)Xanthan gum sold by the company CP Kelco; .sup.(8)Self-emulsifiable composition based on cetylstearyl alcohol and on cocoglucosides, sold by the company SEPPIC; .sup.(9)Phenoxyethanol, preservative sold by the company SEPPIC; .sup.(10)Preservative.
b)Evaluation of the Plumping Effect and of the Improvement in the Elasticity of the Skin of the Oil-in-Water Emulsions (E3) and (E4) on the Face of Individuals with Mature Skin

(128) The evaluations of the plumping effect and of the improvement in the elasticity of the skin, of the oil-in-water emulsions, on the face of individuals with mature skin, are carried out by measuring the following criteria: change in the volume of the face at the level of the cheekbone using Primos Body; biomechanical properties of the skin of the face using a Ballistometer.

(129) The evaluations of the change in the volume of the face at the level of the cheekbone and of the biomechanical properties of the skin of the face were performed on a population of 14 female volunteers, having an average age of 58, with sagging or sunken skin on the face, which was divided up into two equal groups of 7 volunteers (group V and group VI). Each member of group (V) applied emulsion (E3) to her face twice-daily for a period of 56 days, and each member of group (VI) applied emulsion (E4) to her face twice-daily for a period of 56 days.

(130) b1Evaluation of the Plumping Effect of N-Hexadecanoyl Isoleucine on the Face at the Level of the Cheekbone Using the Fringe Projection Technique (Primos Body)

(131) Protocol

(132) This evaluation consisted in measuring the volume of the face of each member (i) of each group before the start of the experiment and at the end of the period of 56 days following the application of emulsions (E3) and (E4).

(133) The experiment consisted in recording a three-dimensional image of the face of each member (i) of each group, before and after the application of emulsion (E3) or (E4) for 56 days, using an interference fringe projection profilometer sold under the name 3D Primos Body. This apparatus, which is characterized by a measurement field of 300 mm200 mm, by a lateral resolution of 500 micrometers, by a lateral resolution greater than or equal to 30 micrometers and by a data acquisition time greater than or equal to 140 milliseconds, makes it possible to project 2 light beams on to the surface to be analyzed, and the images obtained are recorded by 2 cameras at an angle of 20. Calculation software integrated into the 3D Primos Body then makes it possible to calculate the volume of each area present on the three-dimensional image previously recorded.

(134) For each member (i) of groups (V) and (VI) as defined above, the following are then calculated: the volume of the cheeks at the level of the cheekbones, from the three-dimensional image recorded before the start of the experiment (W0i); the volume of the cheeks at the level of the cheekbones, from the three-dimensional image recorded at the end of the period of 56 days of the experiment (W56i).
Expression of the Results

(135) For each member (i) of groups (V) and (VI) as defined above, the following are then calculated: the change in volume of the breast in question, Wi, according to the formula:
Wi=(W56i)(W0i) the mean change in the volume of the cheeks at the level of the cheekbones (Wm) according to the formula:
(Wm)=[(Wi)]/7
Results

(136) The results obtained for groups (V) and (VI), namely the (Wm), are reported in Table 10 below.

(137) TABLE-US-00010 TABLE 10 Group (V) Group (VI) (Wm) in ml +0.784* +0.276 *Results statistically significant compared with the value at t = 0 (two-sided Student's test assuming unequal variances) with p 0.05.

(138) The mean increase in the volume of the cheeks at the level of the cheekbones (Wm) of the individuals of group (V), who applied to their cheeks the oil-in-water emulsion (E3) comprising N-hexadecanoyl isoleucine, comes to 0.784 ml, versus an increase of +0.276 ml, considered to be not statistically significant, for the individuals of group (VI), who applied to their cheeks the oil-in-water emulsion (E4) not comprising N-hexadecanoyl isoleucine.

(139) Interpretation of the Results

(140) A mean increase in the volume of the cheeks at the level of the cheekbones of which the set of values (W56i) is statistically different than the values (W0i) is considered to be significant and characteristic of a plumping effect.

(141) The statistical analysis used to evaluate the significance is a Student's test assuming unequal variances. If p is less than or equal to 0.05, the set of values (W56i) is considered to be significantly different than the set of values (W0i). Consequently, it is possible to conclude that there is a significant increase in the volume of the cheeks at the level of the cheekbones. If p is strictly greater than 0.05, the set of values (W56i) will not be considered to be significantly different from the set of values (W0i). Consequently, it will not be possible to conclude that there is a significant increase in the volume of the cheeks at the level of the cheekbones.

CONCLUSION

(142) It can therefore be deduced from these results that the presence of N-hexadecanoyl isoleucine in the oil-in-water emulsion (E3) made it possible to increase, in a significant manner, the volume of the cheeks at the level of the cheekbones of the members of group (V) and therefore to induce a significant plumping effect on the face of these individuals of group (V).

(143) b2Evaluation of the Positive Effect on the Elasticity of the Skin on N-Hexadecanoyl Isoleucine on the Skin of the Face at the Level of the Cheekbone, Using a Ballistometer

(144) Protocol

(145) The study of the biomechanical properties of the skin makes it possible to determine the softening and/or tensioning effects of compositions after they have been applied to the skin.

(146) The existing techniques are based on studying the change in biomechanical properties following an action of suction (use of a Cutometer), of torsion (Dermal Torque Meter) or of extension (extensiometer) of the skin or the rebound of a small ball (Ballistometer) on the skin. These techniques make it possible to obtain precise, fast, reliable results and use protocols that are not traumatic for the skin of the individual.

(147) The skin has 2 rheological characteristics: an elastic characteristic; a tension characteristic which varies according to the various areas of the human body.

(148) The rheological properties of the skin are based on its structure, which consists essentially of a three-dimensional network of collagen and of elastin fibers.

(149) To evaluate the effect of N-hexadecanoyl isoleucine on the change in biomechanical properties of the skin of the face at the level of the cheekbone, it was chosen to implement a measurement using a Ballistometer. The Ballistometer makes it possible to illustrate the change in the elastic component of the skin.

(150) The evaluation performed in the context of the present study was carried out with a BLS 780 Ballistometer sold by the company Dia-Stron.

(151) The BLS 780 Ballistometer consists of a manual probe, connected to a control unit provided with data collection software. The manual probe contains a rigid arm made of aluminum alloy, which is mounted on a torsion shaft, and on the end of said rigid arm is a vibrating sphere which is 2 mm in diameter.

(152) When the vibrating sphere of the probe is brought into contact with the surface of the skin tested, the surface of the skin tested emits a resistance force, which induces an oscillatory movement on the spring of the torsion shaft. This oscillatory movement is then recorded by the control unit and processed by the associated software. This device therefore makes it possible to record the rebounds and the damping thereof, and also the associated energy, obtained after application of the vibrating sphere on the surface of the skin tested.

(153) The software associated with the central unit then makes it possible to calculate various parameters, including the alpha parameter which illustrates the rate of attenuation of the mechanical energy. A high value for this alpha parameter characterizes a strong elasticity of the surface of the skin tested. An increase over time of the alpha parameter attests to firming of the skin.

(154) In the experiment which is the subject of the present study, the biomechanical properties of the skin are evaluated at the level of the cheekbone of the cheek by applying the probe of the Ballistometer on said area of the cheekbone of the cheek before and after the application of emulsion (E3) or (E4) for 56 days. Following this application, for each member (i) of groups (V) and (VI) as defined above, the following are then measured: the alpha parameter of the skin of the cheeks at the level of the cheekbones before the start of the experiment (0i), the alpha parameter of the skin of the cheeks at the level of the cheekbones at the end of the period of 56 days of the experiment (56i).
Expression of the Results

(155) For each member (i) of groups (V) and (VI) as defined above, the following are then calculated: the change in the alpha parameter of the skin of the cheeks at the level of the cheekbones, (i), according to the formula:
(i)=(56i)(0i)
and for each group: the mean change in the alpha parameter of the skin of the cheeks at the level of the cheekbones, (m), according to the formula:
(m)=[(i)]/7 the mean of the alpha parameter of the skin of the cheeks at the level of the cheekbones before the start of the experiment, [Mean(0)], according to the formula:
[Mean(0)]=[(M0i)]/7 the degree of change of the alpha parameter of the skin of the cheeks at the level of the cheekbones, (C), according to the formula:
(C)=(m)/[Mean(0)]100
Results Obtained

(156) The results obtained for groups (V) and (VI), namely the mean changes of the alpha parameter of the skin of the cheeks at the level of the cheekbones (m) and the degree of change of the alpha parameter of the skin of the cheeks at the level of the cheekbones (C), are reported in Table 11 below.

(157) TABLE-US-00011 TABLE 11 Group (V) Group (VI) (m) 0.005* 0.0 (C) as % 10.0%* +1.0% *Results statistically significant compared with the value at t = 0 (two-sided Student's test assuming unequal variances) with p 0.05.

(158) The mean change in the alpha parameter of the skin of the cheeks at the level of the cheekbones (m) of the individuals of group (V), who applied to their cheeks the oil-in-water emulsion (E3) comprising N-hexadecanoyl isoleucine, is 0.005, compared with no change ((m)=0) for the individuals of group (VI), who applied to their cheeks the oil-in-water emulsion (E4) not comprising N-hexadecanoyl isoleucine.

(159) Likewise, the degree of change in the alpha parameter of the skin of the cheeks at the level of the cheekbones (C) was evaluated at 10% for the individuals of group (V), versus +1.0% for the individuals of group (VI).

(160) Interpretation of the Results

(161) A mean increase in the alpha parameter of which the set of values (56i) is statistically different than the values (0i) is considered to be significant and characteristic of an improvement in the elasticity of the skin of the cheeks at the level of the cheekbones. The statistical analysis used to evaluate the significance is a Student's test assuming unequal variances. If p is less than or equal to 0.05, the set of values (56i) is considered to be significantly different than the set of values (0i). Consequently, it can be concluded that there is a significant increase in the elasticity of the skin of the cheeks at the level of the cheekbones. If p is strictly greater than 0.05, the set of values (56i) will not be considered to be significantly different than the set of values (0i). Consequently, it will not be possible to conclude that there is a significant increase in the elasticity of the skin of the cheeks at the level of the cheekbones.

CONCLUSION

(162) It can thus be deduced from these results that the presence of N-hexadecanoyl isoleucine in the oil-in-water emulsion (E3) made it possible to induce, in a significant manner, an improvement in the elasticity of the skin of the cheeks at the level of the cheekbones of the members of group (V).

(163) b3General Conclusion

(164) The experimental results demonstrated in experimental sections b1) and b2) of the present chapter 2.2 show that the application of a cosmetic composition comprising N-hexadecanoyl isoleucine to the face makes it possible, for the individuals in question, to induce a plumping effect and an improvement in the elasticity of the skin of the cheeks at the level of the cheekbones, in a significant manner.

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