JP2002541084A - Cosmetic composition containing at least one substance promoting connexin formation, use and method of cosmetic treatment - Google Patents

Abstract

  (57) [Summary] The present invention relates to a cosmetic composition comprising, as an active ingredient, at least one substance that promotes intercellular transmission of skin cells, particularly keratinocytes, fibroblasts and pre-skin adipocytes; Use of at least one substance that promotes the intercellular communication of cells and preadipocytes of the skin as a cosmetic agent in the presence of any cosmetically acceptable excipient; and A method for promoting and / or increasing the activity of a cosmetic agent acting via an intracellular second messenger, and a method for treating skin anti-aging cosmetics.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a cosmetic composition comprising at least one substance which promotes the intercellular transmission of skin cells, in particular keratinocytes, fibroblasts and preadipocytes, and in particular the formation of connexin, its use and cosmetic treatment. Basically on how to. The study of means to combat human aging has recently received the attention of many researchers. In particular,
This is the case in the cosmetics field, which attempts to counter or at least slow the appearance of skin aging aesthetic and physiological effects such as wrinkles, loss of skin elasticity, loss of color.

[0002] The aging of human species is characterized by a number of diseases affecting living tissues. Signs of this natural process can be easily detected in organs such as the skin (Mon
tagna, W.C. & Carlisle, K .; (1990). Structural changes in aging
kin. ). British Journal of Dermatolog
y, 122 (35), 61-70) Skin aging is a combination of two phenomena, an endogenous (genetic) cellular process with so-called extrinsic aging (Gro).
ve, G .; L. (1989), Physiological changes in
old skin (physiological changes in older skin). Clinics
in geriatric medicine, 5 (1), 115-125)
. The effects of skin tissue aging are mainly visible in wrinkle formation. This means
It reflects the considerable changes that occur in the dermis and epidermis. Tissue homeostasis, which is important for the skin, is altered during the course of aging. Gap junctions are involved in the regulation of cellular homeostasis, so their role in maintaining physiological balance in the skin is important.

[0003] The inventors have also demonstrated the effect of aging, particularly on the functional ability of cells to communicate with each other via gap junctions (GJ), and the amount of connexin 43 present in cells. . Indeed, the inventors have demonstrated that the functional ability of cells to communicate with one another, particularly via gap junctions (GJ), decreases with age. The inventors have
It has also been demonstrated that connexin 43 levels decrease with age. The inventors have shown the value of using substances to combat the signs of skin aging, in particular to delay their appearance, in particular to promote intercellular transmission through the GJ. This represents a novel means of combating skin aging, especially wrinkles and loss of skin elasticity.

[0004] Gap junctions (GJ) are transmembrane protein structures that allow small molecules (<1000 Da) to pass between two cells (Watt, F .;
M. (1991), Intercellular communication.
via gap junctions, In L. A. Goldsmit
h (Eds.), Physiology, biochemistry, mo
The biology of the skin (pp. 857-8)
59). New York, Oxford: Oxford University
ty Press). This hexagonal structure, called connexons, is itself formed from connexins.

[0005] When cell-cell contact is established, the connexons of one cell are aligned end-to-end with the connexons of adjacent cells to form a binding channel (Gooden).
ough, D .; A. Golier, J .; A. & Paul, D.A. L. (199
6). Connexins, connexons and cellular
communication. Annual Review of Bioc
chemistry, 65 , 475-502). By this gap junction (GJ), homeostasis of cells and tissues can be maintained. Connexins form part of a protein family with tissue specificity. Connexin 43 is a major protein in keratinocytes (Salomon, D., Saurat, J.-).
H. & P. M. (1998), Cell-to-Cell communication.
ation within intact human skin. Jour
nal of Clinical Investigation, 82 , 2
48-254). In skin, GJ is present in all epidermal layers except the stratum corneum.

[0006] In addition, molecules that affect the means by which cells function may or may not enter cells but bind to receptors present on the plasma membrane, ie, are referred to as "second messengers" in the cytoplasm. It is known to do this either by eliciting a series of reactions that achieve small molecule dissociation. This small second messenger, such as cAMP (cyclic adenosine 3 ', 5'-monophosphate), transmits information from cell to cell by circulating from one cell to an adjacent cell via GJ. The same applies to small molecules that can enter cells and circulate from one cell to neighboring cells via the GJ. That is, by increasing cell-to-cell communication, particularly through GJ, the inventors have developed a new means of promoting and / or increasing the activity of cosmetic agents.

The inventors have surprisingly found that the use of boldine or the sole use of the alga Skeletonema costatum lipid extract alone, in particular, restores GJ-mediated cell-to-cell communication and skin cells Especially keratinocytes,
It has also been shown to enable an increase in the amount of connexin 43 present in fibroblasts and preadipocytes. That is, the main object of the present invention is to improve the efficacy of the cosmetic composition, and,
It solves a new technical problem which consists in providing a new solution to counter or at least slow down the appearance of skin aging aesthetic and physiological effects such as wrinkles, loss of skin elasticity, loss of color etc. That is.

In accordance with the present invention, this technical problem is caused by substances that promote intercellular transmission in keratinocytes, fibroblasts and pre-skin adipocytes, especially wrinkles, loss of skin elasticity and loss of color. For the first time, it has been solved in a surprisingly non-obvious way, with the discovery that it is possible to apply cosmetic compositions which have improved properties to counter or at least slow down the appearance of the aesthetic and physiological effects of skin aging . In addition, the present invention provides a cosmetic composition having enhanced efficacy for connexin, in particular for promoting the formation of connexin 43, in particular for counteracting or at least slowing down the appearance of the aesthetic and physiological effects of skin aging. Have been given in a surprisingly non-obvious way.

According to the present invention, skin cells, in particular keratinocytes, fibroblasts and pre-skin adipocytes, are obtained on the one hand by boilin on the one hand, and on the other hand by lipid extracts of algae skeletonema, in particular algae skeletonema costatum, in particular a total lipid extract of the algae described above. Intercellular communication through gap junctions is promoted, i.e., to counteract or slow the appearance of the aesthetic and physiological effects, especially of skin aging, or to counteract fat excess. It has also been found in a surprisingly non-obvious way that it is possible to prepare fortified cosmetic compositions. That is, according to the first feature, the present invention is characterized in that at least one substance that promotes intercellular transmission of skin cells, particularly keratinocytes, fibroblasts and pre-skin adipocytes, is contained as an active ingredient. The present invention relates to a cosmetic composition.

[0010] In certain embodiments of the present invention, the composition is characterized in that the substance promotes intercellular transmission of skin cells, particularly keratinocytes, fibroblasts, and preadipocytes through gap junctions. Attached. In another preferred embodiment of the invention, the composition is characterized in that the substance that promotes cell-to-cell communication promotes the formation of connexin, in particular connexin 43.

In another preferred embodiment, the substance that promotes cell-to-cell communication comprises at least one lipid extract of alga skeletonema, especially algal skeletonema costatum, in particular, a total lipid extract of the alga. Characterizes the composition. Alga Skeletonema, especially Alga Skeletonema costatum (hereinafter referred to as S
KC) is a member of the Chryrophytes phylum Chrysophycophytes family Diatomophythea (D).
It is a well-known unicellular algae of the order Centralia. Diatomophysea is very widely distributed in freshwater, saltwater and brackish water. The survival form of this species can be floating or benthic. The protoplasm is encapsulated in a silicon valve hull. Skeletonema costatum (SKC) is a versatile species but is normally marine and is commonly found with phytoplankton flowering in coastal waters.

In one preferred embodiment of the invention, the lipid extract is characterized in that it is obtained by extracting algal skeletonetema with an alcoholic solvent selected from the group consisting of isopropanol, ethanol and methanol. In another preferred embodiment, the extraction is performed under reflux. In another preferred embodiment, the algae is frozen prior to extraction with an alcoholic solvent, and the freezing is preferably performed at a temperature of about -40 ° C to -20 ° C, preferably for a period of about 1 to 7 days. Will be In another preferred embodiment, the frozen algae is infiltrated directly into the heated alcoholic solvent. In fact, heat shock can promote decantation of silicon (from the algal cell skeleton).

In another preferred embodiment, in the case of extraction with an alkalized alcohol, the algal extract is subjected to the following series of steps: a) for example with aqueous sodium hydroxide or aqueous potassium hydroxide, the alcoholic solvent being brought to pH 10 -14, preferably pH 13, b) removing insolubles from the water-alcohol phase, c) adding distilled water to the water-alcohol phase, d) water-alcohol such as heptane, hexane or cyclohexane Liquid-liquid extraction of the resulting solution with a non-polar solvent that is immiscible with the phase, e) removing the phase containing the non-polar solvent, and f) recovering after removing the phase containing the non-polar solvent The water-alcohol phase is acidified with, for example, an aqueous sulfuric acid solution or an aqueous hydrochloric acid solution to pH 1 to 3, preferably pH 2, and g) for example, heptane, hexane or cyclohexane. Liquid-liquid extraction of the solution obtained after acidification with a non-polar solvent immiscible with the water-alcohol phase such as Sun, h) removal of the water-alcohol phase, and i) removal of the water-alcohol phase The phase containing the non-polar solvent recovered later is evaporated to obtain the desired extract, a non-polar solvent-free oil.

The use of an alkalized and then acidified alcohol provides an extract with acceptable visual and olfactory properties (yellow coloration and acceptable odor) in the cosmetic composition. In another preferred embodiment, the extract is obtained by extraction with supercritical CO _2. In another preferred embodiment, prior to any other extraction operation, preferably between about 5 minutes and 8 minutes.
The algae are macerated in the alcoholic solvent at room temperature for 0 minutes, particularly preferably for about 20 to 40 minutes. In yet another preferred embodiment, the amount of alcohol solvent used is from about 0.1 liter to 20 liters of solvent, preferably from about 2 liters to 10 liters of solvent, per 100 g of algae, expressed as dry weight algae. . In yet another preferred embodiment, the extraction may be performed under an inert atmosphere, preferably under a nitrogen saturated atmosphere. This makes it particularly possible to avoid oxidative degradation of significant active molecules.

[0015] The lipid extract is preferably encapsulated under an inert gas such as nitrogen to protect the active molecules. In yet another preferred embodiment, about 0.01% by weight, based on the total weight of the final composition,
The composition is characterized by containing from 10 to 10% by weight, in particular from about 0.1% to 3% by weight, of a lipid extract of the alga Skeletonema, in particular the alga Skeletonema costatum. According to a second aspect, the invention further relates to the optional presence of a cosmetically acceptable excipient of at least one substance which promotes the intercellular communication of keratinocytes, fibroblasts and preadipocytes. It relates to its use as a cosmetic agent below. In certain embodiments of the use, the latter is characterized in that the substance promotes intercellular communication through gap junctions of keratinocytes, fibroblasts and pre-skin adipocytes.

In another specific embodiment of the invention, the substance that promotes cell-to-cell communication promotes the formation of connexin, especially connexin 43. In another embodiment of the present invention, the substance comprises at least one lipid extract of the alga Skeletonema, in particular the alga Skeletonema costatum, in particular a total lipid extract of the alga as defined above. Preferably, the extract is obtained by liquid-liquid extraction of an alkalized and then acidified alcohol with a non-polar solvent that is immiscible with the water-alcohol phase. In another preferred embodiment of the present invention, the substance that promotes cell-cell communication of skin cells,
The following formula:

[0017]

Embedded image

The composition of the present invention comprising boldin, which is the product of the above, is preferably about 0.001% by weight to 10% by weight.
% By weight, preferably from 0.01 to 1% by weight of boldine. According to a third aspect, the present invention is a method for promoting and / or increasing the activity of a cosmetic agent acting directly in a cell or via an intracellular second messenger, the method comprising: Alternatively, the method comprises applying an effective amount of at least one substance that promotes cell-to-cell communication, particularly a substance that promotes cell-to-cell communication as defined above, to a suitable human skin in need thereof. And According to a fourth aspect, the invention provides an anti-aging effect on the skin part, in particular to improve the toughness and elasticity of the skin, in particular to delay the appearance of wrinkles or to reduce its depth. At least one substance for promoting cell-to-cell communication, especially a substance for promoting cell-to-cell communication as defined above, which is applied to an appropriate skin portion of a human in need thereof. Skin anti-aging cosmetic treatment method.

The following examples are provided merely to illustrate the present invention with reference to the accompanying drawings: FIG. 1 shows a curve representing the regulation of intracellular transmission as a function of donor age. It is shown. It is obtained from keratinocytes separated from donors of various ages by the so-called peel-fill method. The curve represents the ratio of the fluorescent surface area to the total surface area on the vertical axis as a function of donor age (years) on the abscissa. “
r "is the correlation coefficient, which in this case is a number equal to 0.76. The measurements obtained with young donors are represented by solid squares with the standard variance and are indicated by blank circles Is also obtained with older donors along with the standard variance This figure should be read in connection with Table I (Example I)-FIG. FIG. 7 shows a curve representing the regulation of intracellular transmission as a function of donor age obtained by the so-called microinjection method, except that the coordinates are expressed in terms of the number of labeled keratinocytes as a function of age (years). is there. "
r "is the correlation coefficient, which in this case is a number equal to 0.91. The measurements obtained with young donors are represented by solid squares together with the standard variance, and the measurements obtained with older donors. Is also shown as a blank circle with the standard variance This figure should be read in connection with Table II (Example I).

FIG. 3 shows the change in the amount of connexin 43 measured in keratinocytes of donors of various ages measured by flow cytometry, the vertical axis as a function of donor age (years) on the horizontal axis. Expressed on the axis as label percentage, "r" is the correlation coefficient, in this case a number equal to 0.82. Measurements obtained with young donors are represented by solid squares along with the standard variance, and measurements obtained with older donors are also indicated by blank circles with their standard variance. This figure should be read in connection with Table III (Example I). FIG. 4 represents the result of the modulation of the cell-to-cell transmission of the normal human keratinocytes, designated NHK, in various donors that could or could not be treated with lipid extracts of the alga Skeletonema costatum, abbreviated as SKC. The results are expressed in the form of a histogram, the vertical axis is expressed in relative variance units, the blank bars are those obtained with control NHK and the shaded bars are 2.5 μg / ml / 24 h. Percentage obtained with NHK treated in vitro with a lipid extract of the algae Skeletonema costatum. This figure should be read in connection with Table VI (Example III).

FIG. 5 shows the modulation of the cell-to-cell communication of normal human keratinocytes (abbreviated NHK) in various donors that could or could not be treated with lipid extracts of the alga Skeletonema costatum abbreviated SKC It shows the measured value by the microinjection method, the number of labeled cells is shown on the horizontal axis, and the result of untreated control NHK is as follows:
Indicated by a blank bar labeled "Control", dosage 2.5 μg
NHK treated with the alga Skeletonema costatum or lipid extract of SKC at / ml / 24h is indicated by the black bar labeled "treated". This figure should be read in connection with Table V (Example III). FIG. 6 shows the dose 2. obtained from the results measured by flow cytometry.
The vertical axis of the adjustment of the amount of connexin 43 for each control in donors of various ages treated with a lipid extract of the alga Skeletonema costatum, abbreviated as SKC, at 5 μg / ml / 24h for each control , And another histogram with the donor axis (years) on the horizontal axis. This figure should be read in connection with Table VI (Example III).

[0022] Other advantages of the present invention will be apparent from the following description and examples. Unless otherwise indicated, the proportions of the compositions given in the examples are expressed as percentages by weight.

[0023]Example I-Age-Related Functionality of Gap Junction Cell-to-Cell Communication (GJIC) and Demonstration of age-related decrease in connexin 43 level I. 1-Materials and methods I. 1.1-Culture of Ordinary Human Keratinocytes  Cultures of normal human keratinocytes (NHK) were prepared from skin samples. Samples were placed in PBS (phosphate buffered saline-Sigma) (50 ml test tubes).
Once, it was washed first. Immerse twice in a 70% ethanol bath for 30 seconds
Decontamination. If the sample has been decontaminated, place it in a Petri dish containing PBS.
Was. Be careful to remove as much adipose tissue and dermis as possible.
mm-wide slices were cut out. The slice was immediately placed in a Petri dish containing PBS. To recover keratinocytes from the epidermis, slices were taken from 0.25% trypsin PBS.
Placed in solution at 37 ° C. for 4 hours. Then, the dermis was separated from the epidermis by peeling the slices with a scalpel, and the resulting table
Skin cells were washed with DMEM (Dulbecco's improved Eagle's solution-Gibco) + 10% FCS
(Fetal bovine serum-Eurobio) was suspended in a test tube. Homogeneous suspension
After the transformation, the surface part consisting of the stratum corneum cells is separated, and the remaining suspension is sieved and filtered.
I have.

The filtered part was centrifuged at 176 g for 5 minutes. The residue was treated with NHK-D solution (DMEM
+ 10% FCS + 0.4 μg / ml hydrocortisone + 10 ng / ml EGF +
^10-9 M cholera venom). Cells were counted and inoculated at a rate of 15 × 10 ⁶ cells / flask. After culturing for 24 hours, the solution was changed, and cells washed with a growth solution of PBS and K-SFM (Gibco) were used for the rest of the culture. Keratinocytes were completely subcultured by conventional methods, but had to be subcultured in 60-70% confluence to maintain their proliferative potential.
That is, when the cells were 60-70% colonies, the maintenance solution was removed, and the cell granular surface (mat) was washed with PBS. Cells are placed in 3 ml of trypsin / EDTA solution; and if cells are detached, trypsin is replaced with 10% FCS (Eu
robio). The cell suspension was homogenized, collected, and centrifuged at room temperature for 5 minutes at 20 g. The obtained residue was dissolved with the liquid alone.
For maintenance, the cells were inoculated in a ventilated flask at a ratio of 10 ⁶ cells / 75 cm ² as described above and stored under the above conditions. A colony is obtained after about 10 days and cells
Amplification could be performed for 7 passages. If it was desired to test the substance activity on this keratinocyte, a test was performed using a culture solution containing keratinocytes at the time of the above-mentioned settlement.

[0025]I. 1.2-Scrape-loading  This technology was developed by the Trosco group in 1987 (El
-Fouly, M .; H. , Trosko, J .; E. FIG. & Chang, C.A. -C.
(1987). Peel Filling and Staining Transfer-To test gap junction cell-to-cell communication
Quick and simple technology. EXPERIMENTAL CELL RESEARCH
,168, 422-430). It is a dye dye having a molecular weight (MW) of 457.2.
Based on the use of Schiffer Yellow (Sigma). This molecule (
Aminophthalimide) is highly fluorescent and disperses only through gap junctions
do not do. It does not penetrate the plasma membrane of whole cells. Injure the cell membrane with a scalpel
To allow access to the gap junctions of the dye.

Prior to the addition of the dye, the colonized cells were washed with PBS (Sigma).
2 ml of 0.05% Lucifer Yellow (Sigma) diluted in PBS was deposited on the cells, and detachment was performed at room temperature. Peeling was performed six times in a 60 mm dish. The cells were left in contact with the dye for 5 minutes and the latter was withdrawn and collected. The cells were then washed with PBS to remove excess fluorescence. It was fixed with 3% paraformaldehyde (PFA) for 10 minutes. The cells are washed again with PBS and 2 ml of PBS
Was placed in a dish after washing. The dishes were then observed with an inverted fluorescence microscope fitted to an image acquisition camera. Take 6 or 8 pictures for each dish and use image analysis software (Perfectimag).
e, Iconix). Then, the fluorescence corresponding to the surface area occupied by Lucifer Yellow in the cell layer was quantified. This was done by placing a rectangle of a predetermined size in the area tested. The surface area ratio: fluorescent surface area / total surface area was calculated using a computer. The higher the ratio, the greater the ability of cells to communicate with each other.

[0027]I. 1.3-microinjection  Cells were prepared in a manner similar to that used in the peel-fill technique. Connected to a micromanipulator (Leitz) due to the precision of this technology
Lucifer Yellow (10% 0.33 M Li
Cl solution) can be directly injected only into cells. Automatic injection machine (T
injector 5246, Eppendorf) and 10335H
It continued for 1 second at an intensity of Pa. During contrast mode phase, note under an inverted microscope (20x)
Was made. Approximately 20 injections were made in a 60 mm petri dish. After fixing the cells with 3% paraformaldehyde, it was washed with PBS. It
Was observed and the number of labeled cells was counted.

[0028]I. 1.4-Flow cytometry  When the colonies were reached, the culture was removed and the cells were washed twice with PBS. And that
This was trypsinized with a mixture of 0.1% trypsin and 0.02% EDTA.
. Trypsin inhibition was performed in a culture solution supplemented with 10% FCS. Count the number of cells
, 10 per test tube⁶It could be adjusted to be a cell. Cell suspension
After centrifugation at 176 g for 5 minutes, the residue was dissolved in 100 ml of PBS. Cells
The cells were fixed with 3% PFA for 25 minutes at 4 ° C. The PFA was removed, and the cells were replaced with 176 g.
Centrifuged for 5 minutes and washed twice with PBS. The buffer from which the antibody was prepared was PBS, 1% FCS and 0.2% saponin (Sig
ma), microporation of the membrane is induced
But does not damage cells. This detergent is used in the cytoplasm and trans
Used to maintain and quantify both connexins 43 of the membrane
(Personal communication, R. Mouawad
, Hospital Salpetriere-, Paris). The primary antibody used was anti-connexin 43 (enzyme generated in mice (Zymed
Monoclonal antibody). The concentration of the first antibody was 1.3 μg / ml (1/750).
). Incubation was at 45C for 45 minutes. Excess
The antibody was removed, the cells were centrifuged, and the buffer (PBS,
(Ponin and FCS). The second antibody, fluo, diluted 1/50
Contact with Resin-conjugated anti-mouse antibody (Jackson Immunotech)
I let it. After incubation with the second antibody, the cells are centrifuged, washed,
Then, it was recovered with 1 ml buffer (PBS, saponin and FCS). Treatment with NHK treated as above and fluorescein-conjugated anti-mouse secondary antibody
The fluorescence intensity at NHK that was not touched was measured by flow cytometry (COULTRON).
Measured with Epics profile II) sold by IC company, and given by machine
Software that gives values measured in indefinite units
Measured with Phoenix flow system soft sold by
The values were processed and compared. A value reflecting the amount of connexin 43 per NHK (average)
And the amount of connexin 43 labeling in NHK.

[0029]I. 2-Result I. 2.1-Gap junction cell-to-cell communication (GJIC) as a function of donor age Evaluation of functionality I. 2.1.1-Peel filling (Fig. 1)  The study was performed with nine donors aged 40 to 75 years.

[0030]

[Table 1]

From the results shown in Table I and FIG. 1 (attached), cell-to-cell communication depends on donor age.
Keratinocyte transmission capacity is clearly inversely proportional to donor age
I understood. The correlation function was 0.76.I. 2.1.2-Micro injection (FIG. 2)  The tests were performed on the same donors for peel filling except for the 43 year old donor.

[0032]

[Table 2]

From the results shown in Table II and FIG. 2 (attached), GJIC is inversely proportional to age.
However, this technique is more precise than release filling,
The function was 0.91. With two technologies, peel filling and microinjection, the functionality of GJIC increases with age
A result showing the same trend was given, with a very significant decrease.I. 2.2-Evaluation of age effect on connexin 43 level (FIG. 3) Flow cytometry  Study identical to donor for peel-fill except for 43 and 75 year old donors
Of the donors.

[0034]

[Table 3]

From the results shown in Table III and FIG. 3 (attached), it was found that the amount of connexin 43 decreased with age, and that the amount of connexin 43 was clearly inversely proportional to donor age. The correlation function was 0.82. The results show that there is an aging effect on the functional ability of cells to communicate with each other, especially through gap junctions (GJ), and on the amount of connexin 43 present in the cells. . In fact, it showed that the functional ability of cells to communicate with each other, especially through gap junctions (GJ), decreased with age.
It also showed that connexin 43 levels decreased with age. We have therefore shown the value of using substances to combat the signs of skin aging, in particular to delay their appearance, in particular to promote GJ-mediated intercellular transmission. Therefore, new measures against skin aging, especially wrinkles and loss of skin elasticity, have been developed. Therefore, the present inventors have also shown the value of using a substance for promoting and / or increasing the activity of the cosmetic agent, in particular for promoting GJ-mediated intercellular transmission.

[0036]Example II-Preparation of lipid extract of algae Skeletonema costatum II. 1—Extraction with Isopropanol by First Method  Perform full extraction under an inert atmosphere (nitrogen saturation) to avoid significant decomposition of active molecules.
I liked it. 250 kg of biomass (Skeletonema costatum) was used in this preparation
. The algae frozen at −20 ° C. are refluxed at 80 to 83 ° C. while stirring, and isopropano
(IPA). By heat shock (from the algae cell skeleton
) It became possible to promote decantation of silicon. The amount of solvent used was 10 liters per liter of water present in the biomass.
IPA. In this preparation, at 30% dry matter ratio, 250 kg
Iomas represents 75 kg of dry matter and 175 kg of water. IPA used here
The amount was 1925 kg. Before cooling to about 50 ° C., the whole (biomass + IPA) is
The mixture was refluxed with stirring.

After cooling the reaction mixture to about 50 ° C., the extract was transferred to a GUEDU filter to separate the extracted biomass from the IPA lipid extract. The lipid extract was concentrated in the batch reactor (concentration = 71.5). The crude oil yield in the first stage is 28% by dry weight. To begin the second step, the lipid extract was cooled at a rate of 10 kg solvent / kg oil to cold I
Dissolved in PA. Stirring was continued for 20 minutes. Then, the liquid was filtered (residual viscous sludge could be removed). Decolorization and deodorization procedures were performed in two portions in an 80 liter Scott reactor and allowed to come to room temperature for 30 minutes. 0.94kg zeolite (ABSENT2 sold by UOP
000) and 1.6 kg of activated carbon (CXV sold by CECA)
It was added at a ratio of 1.7 to zeolite. Then, the zeolite and carbon were filtered with paper. The yield of this second stage was 37% by dry weight.

Thus, the overall oil yield for the entire process was 10% based on the dry weight of biomass. Antioxidants (DL-α-tocopherol at a final concentration of 0.05% by weight and a final concentration of 0
. (05% by weight ascorbyl palmitic acid) was included in the IPA stock solution. Then, the filtrate and the antioxidant were concentrated in a batch to obtain a brown oil. This oil was hereinafter referred to as SKC lipid extract E1.

[0039]II. 2-Extraction with ethanol by the second method  96% ethanol solution alkylated with 9 kg of 30.5% sodium hydroxide aqueous solution
Extract with 539 kg of 49.8 kg frozen plant biomass (29 dry weight%) dispersion
Started to go out. After infiltration at 40 ° C. for 30 minutes under reflux, the whole was cooled to 18 ° C.
Insoluble material was removed by filtration under nitrogen. 151 kg distilled water was added to the 573.9 kg filtrate. Add to 162kg heptane
Before stirring, the whole was slowly stirred for 10 minutes. Removal of liquid-liquid upper heptane phase
did. The lower phase was noted to contain fatty acids in the form of salts. Operation
Repeated twice or more. 2.8 kg sulfuric acid was added to acidify the 756 kg solution constituting the lower phase,
H2.2. 10 minutes under nitrogen before adding to 158 kg heptane
Stirred. From 147 kg constituting the first heptane upper phase of this new liquid-liquid part
, Free fatty acids were extracted. The operation is repeated 5 times or more, and a total of 697 kg heptane phase
Collected. This phase is evaporated to dryness on a rotary evaporator and dried.
Distillation yielded 1.1 kg oil of active extract. The prepared oil was a dark yellow homogeneous liquid. This oil was hereinafter referred to as SKC lipid extract E2.

[0040]Example III-Gap junction cell-to-cell communication (GJIC) and elderly donor NHK Of the effect of algal SKC lipid extract on the amount of connexin 43 in soil  The algal SKC lipid extract used in this example was obtained by the above ethanol extraction.
SKC lipid extract E2. The tests described below were also performed on SKC lipid extract E1 (results not shown).
The results were comparable to those obtained with SKC lipid extract E2 described below.
Was. Protocol described in Example I (I.1.1-Culture of normal human keratinocytes)
The test was performed on normal human keratinocytes (NHK) obtained according to Con
Contrary to trolls, release filling, microinjection according to the protocol described in Example I
Or fluid cytometry techniques (each I.1.2, I1.3 and I1.4).
) Was treated with SKC lipid extract E1 or E2 (at the time of culture settlement) before evaluation in
NHK is expressed as “x” in μg per milliliter of culture per 24 hours.
Volume (xμg / ml / 24h).

[0041]・ Peeling filling (Fig. 4)  Trials were performed on normal human keratinocytes (NHK) from four donors aged 60 to 79 years.
The experiment was performed.

[0042]

[Table 4]

From the results shown in Table IV and FIG. 4 (attached), the SKC lipid extract was older
(P <0.0001−).
Student test). Therefore, the SKC lipid extract has a similar cellular transmission to young donor NHK.
The bell had been restored.・ Micro injection (Fig. 5)  The study was performed on 12 donors aged 49 to 79 years.

[0044]

[Table 5]

It was found that the number of labeled cells increased 2-fold (p <0.0001-Student's test). Therefore, the SKC lipid extract was induced to restore the amount of GJIC in elderly donor cells to a level similar to that of young donors.

[0046]・ Flow cytometry (Fig. 6)  The study was performed on six donors aged 49-60 years.

[0047]

[Table 6]

[0048] It was observed that the labeling of the treated cells increased by an average of 25.6% over control cells. The SKC lipid extract induced an increase in connexin 43 levels. Intercellular transmission and connexin 4, especially via GJ, performed with the elderly donor NHK
The results obtained by various means of assessing the effect of the lipid extract of the three-volume alga S. cholestenema costatum show that the use of the algal skeletonema costatum lipid extract can restore intercellular communication, particularly through the GJ, and reduce NHK It has been shown that it also makes it possible to increase the amount of connexin 43 present therein.

Smell in cosmetic compositions to combat signs of skin aging, especially to slow its appearance
The results showed the value of using algae Skeletonema costam lipid extract
. Therefore, the inventors have developed a novel method for combating skin aging, particularly wrinkles and loss of skin elasticity.
Means developed. In addition, SKC acts directly in cells or via intercellular second messengers.
Others that may or may not be present in cosmetic compositions similar to lipid extracts
Algae skeletonetema in a cosmetic composition for promoting and / or increasing the activity of a cosmetic agent
The results obtained above showed the value of using costamtam lipid extract.Example IV-Evaluation of the effect of boldine by peel-fill test  Ordinary human keratin at a working concentration of 12.5, 25, 50, 100 and 200 mM
The test was performed at the site (NHK). 24 hours incubation in culture
Was done. The following table summarizes the measurements of the dispersion surface area.

[0050]

[Table 7]

As seen in Table VII, the dosage that appeared to be most effective in increasing the functionality of GJIC was 50 mM / 24h. GJ for untreated NHK
Statistical analysis of the data showed that a truly significant difference in the increase in IC was given by this concentration alone. The results in Table VII suggested that boldine had a dose-related effect on GJIC. Example V-Anti-Aging Decream for Facial

[0052]

[Table 8]

Example VI-Facial Anti-Wrinkle Emulsion Gel

[0054]

[Table 9]

Example VII-Firming emulsion for body

[0056]

[Table 10]

Example VIII-Anti-wrinkle emulsion gel for face

[0058]

[Table 11]

[Brief description of the drawings]

FIG. 1 shows curves representing the modulation of intracellular transmission as a function of donor age.

FIG. 2 shows curves representing the modulation of intracellular transmission as a function of donor age.

FIG. 3 shows changes in the amount of connexin 43 measured in keratinocytes of donors of various ages by flow cytometry.

FIG. 4 depicts the results of modulation of cell-to-cell transmission of normal human keratinocytes in various donors that could or could not be treated with lipid extracts of the alga Skeletonema costatum.

FIG. 5 depicts the results of a microinjection method of modulation of intercellular transmission of normal human keratinocytes in various donors that could or could not be treated with lipid extracts of the alga Skeletonema costatum. .

FIG. 6 shows the alga Skeletonema at a dosage of 2.5 μg / ml / 24 h.
FIG. 4 is a histogram of adjustment of connexin 43 levels for each control in donors of various ages treated with costatum lipid extract.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Cecil Vilon F-45000 Orleans, Boulevard-a-Martin, 70 (72) Inventor Valerie Klujic F-45460 Le Bould, Rude Misarin, 53 (72) Inventor Alex Sonova FR-45000 Orleans, Roudaniel Mayer, 2F term (reference) 4C083 AA082 AA111 AA112 AC012 AC022 AC072 AC242 AC342 AC352 AC422 AC442 AC851 AC852 AD092 AD152 AD352 AD412 CC02 CC05 EE12 FF01

Claims (25)

[Claims] 1. A cosmetic composition comprising, as an active ingredient, at least one substance that promotes the intercellular transmission of skin cells, particularly keratinocytes, fibroblasts and preadipocytes. 2. The composition according to claim 1, wherein said substance promotes intercellular transmission of skin cells, particularly keratinocytes, fibroblasts and pre-skin adipocytes via gap junctions. 3. The composition according to claim 1, wherein the substance that promotes intercellular communication promotes formation of connexin, particularly connexin 43. 4. The substance promoting cell-to-cell communication comprises at least one lipid extract of the alga Skeletonema, in particular the alga Skeletonema costatum, in particular a total lipid extract of the alga. The composition according to any one of claims 1 to 3. 5. The composition according to claim 4, wherein said extract is obtained by extracting alga Skeletonema with an alcohol solvent selected from the group consisting of isopropanol, ethanol and methanol. 6. The composition according to claim 5, wherein said extract is obtained by extracting algae with isopropanol. 7. The composition according to claim 5, wherein the extract is obtained by extracting algae with ethanol. 8. The composition according to claim 4, wherein the extraction is performed under reflux. 9. The algae is frozen before being extracted with an alcoholic solvent,
9. The composition according to any one of claims 4 to 8, wherein the freezing is preferably performed at a temperature of about -40C to -20C, preferably for a period of about 1 to 7 days. 10. The composition according to claim 4, wherein the frozen algae is directly infiltrated into the heated alcoholic solvent. 11. The algae extract is subjected to the following series of steps: a) alkalizing the alcoholic solvent to pH 10-14, preferably pH 13, for example with aqueous sodium hydroxide or potassium hydroxide; Removing insolubles from the alcohol phase; c) adding distilled water to the water-alcohol phase; d) dissolving the resulting solution in a non-polar solvent that is immiscible with the water-alcohol phase, for example, heptane, hexane or cyclohexane. E) removing the phase containing the non-polar solvent, f) removing the water-alcohol phase collected after removing the phase containing the non-polar solvent with, for example, an aqueous solution of sulfuric acid or hydrochloric acid, to pH 1; G) after acidification with a non-polar solvent immiscible with the water-alcohol phase, such as, for example, heptane, hexane or cyclohexane. Liquid-liquid extraction of the resulting solution; h) removal of the water-alcohol phase; and i) evaporation of the non-polar solvent-containing phase recovered after removal of the water-alcohol phase and the desired extraction. 11. A composition according to any one of claims 5 to 10, which is obtained according to: 12. The composition according to claim 4, wherein the extract is obtained by extraction using supercritical CO ₂ . 13. Prior to any extraction operation, preferably for about 5 to 80 minutes
12. A composition according to any one of claims 5 to 11, characterized in that the algae are macerated in an alcoholic solvent at room temperature, particularly preferably for about 20 to 40 minutes. 14. The amount of alcohol solvent used is 100 parts by weight of dry algae.
14. Composition according to any one of claims 5 to 10 or 13, characterized in that there are about 0.1 to 20 liters of solvent per g of algae, preferably about 2 to 10 liters of solvent. . 15. The composition according to claim 4, wherein the extraction is performed under an inert atmosphere, preferably under a nitrogen-saturated atmosphere. 16. Lipid extraction of said alga Skeletonema, in particular Alga Skeletonema costatum, from about 0.01% to 10%, in particular from about 0.1% to 3% by weight, based on the total weight of the final composition. The composition according to any one of claims 4 to 15, comprising a substance. 17. The composition according to claim 1, wherein the substance that promotes intercellular transmission of skin cells is boldine. 18. The composition of claim 17, comprising about 0.001% to 10% by weight, based on the total weight of the final composition, of boldin. A composition as described. 19. Use of at least one substance that promotes the intercellular communication of keratinocytes, fibroblasts and preadipocytes as a cosmetic agent in the presence of any cosmetically acceptable excipient. . 20. The use according to claim 19, wherein said substance promotes intercellular communication of keratinocytes, fibroblasts and preadipocytes through gap junctions. 21. Use according to claim 19 or 20, characterized in that the substance promoting intercellular communication promotes the formation of connexin, in particular connexin 43. 22. The substance comprises at least one lipid extract of the alga Skeletonema, in particular the alga Skeletonema costatum, in particular a total lipid extract of the alga, as defined in any one of claims 5 to 16. The extract is preferably obtained by liquid-liquid extraction of an alkalized and then acidified alcohol and a non-polar solvent that is immiscible with the water-alcohol phase, such as, for example, heptane, hexane or cyclohexane. 22. Use according to any one of claims 19 to 21, characterized in that: 23. The substance according to claim 19, wherein the substance is boldine.
Use according to any one of the preceding claims. 24. A method for promoting and / or increasing the activity of a cosmetic agent acting directly in a cell or via an intracellular second messenger, wherein the cell-cell communication is carried out simultaneously with or before the application of the cosmetic agent. At least one substance that promotes
A method comprising applying an effective amount of a substance that promotes cell-to-cell transmission as defined in any one of the above to an appropriate skin portion of a human in need thereof. 25. To obtain an anti-aging effect on a suitable human skin in need thereof, in particular to improve the toughness and elasticity of the skin, in particular to delay the appearance of wrinkles or to reduce its depth Applying an effective amount of at least one substance that promotes intercellular transmission to the skin portion, particularly a substance that promotes intercellular transmission as defined in any one of claims 2 to 18 to the skin. A skin anti-aging cosmetic treatment method comprising: