KR20080034756A - Use of substances to protect fgf-2 or fgf-beta growth factor - Google Patents

Abstract

A method for preventing or controlling decomposition of FGF-2(fibroblast growth factor-2) or FGF-beta is provided to protect FGF-2 or FGF-beta from decomposition, rebuild extracellular matrix in the dermis and control skin aging. The decomposition of FGF-2 or FGF-beta is prevented or controlled by applying to the skin a cosmetic composition comprising 0.001-20% of a plant extract selected from Hibiscus Abelmoschus extract, rebokza extract, gougizi berry extract, banha extract, lessonia extract, mustard powder extract, wooyin extract, barley extract, sesame extract or combinations thereof and cosmetically acceptable excipients. Further, the extract is an aqueous or a hydro-alcoholic extract.

Description

Use of FFF-2 or FGF-beta Growth Factor Protective Substances {USE OF SUBSTANCES TO PROTECT FGF-2 OR FGF-BETA GROWTH FACTOR}

1 is a graph of the stability of FGF-2 over time as a function of time at different temperatures.

2 is a graph of the stability of FGF-2 at various temperatures in the presence of heparin.

3 is a graph showing FGF-2 protection by GAG of skin.

4 is a graph showing the protection of FGF-2 by proteoglycans.

The present invention relates to the development of active ingredients for cosmetic, skin-cosmetic or pharmaceutical use for controlling the degradation of one or more growth factors. More specifically, the present invention relates to the prevention of degradation of fibroblast growth factor (FGF-2 or basic FGF or FGF-β).

The present invention also relates to the use of the active ingredients for the skin to protect, limit or improve the properties of the skin, especially when human skin is aging.

Several growth factors are involved in the skin level. An example of such a growth factor is FGF-2, which has a broad spectrum of activity, including the proliferation of fibroblasts, thereby enabling the synthesis of matrix macromolecules essential for skin integrity. FGF-2 is protected by heparan sulfate proteoglycans in the skin. In the 1990s, Feige and Baird (Medecine / Sciences, 1992; 8: 805-10) initiated a close relationship between growth factors and proteoglycans in the intracellular matrix, and this interaction was It is demonstrated that the growth factor is protected from proteolysis. This results in a higher stability of the growth factor (eg, half-life extension in vivo), which allows the growth factor to function optimally.

FGF -2 and Proteoglycan

FGF-2 is part of a family of 23 different FGFs currently under consideration. FGF-2 exists in several isoforms. In vertebrates, five isotypes are found with molecular weights of 18, 22, 22.5, 24 and 34 kDa. Only the 18 kDa form is detected outside the cell, while other isotypes are confined inside the cell, more specifically in the nucleus. FGF-2 is a ubiquitous protein that plays a very important role at the physiological level; FGF-2 is involved in fetal development, angiogenesis, neuronal differentiation and tissue repair. Indeed, it exists in most tissues, more specifically in a distribution that targets the basement membrane of the tissue. FGF-2 is present in the organism to the extent that it can be purified and characterized, but its mRAN is not detectable. This fact and the specificity of direct distribution to tissue basement membranes suggest that they are released by cells during development so that they are produced at an initial rate and then stored in the MEC (during this process, the basement membranes release FGF-2 locally). Under intensive remodeling). By such storage, FGF-2 is released in mature state if necessary to participate in tissue repair and maintain specialized function. Thus, as aging decreases the supply of FGF-2 and at the same time no longer provides the highest active state.

Proteoglycans (PG) form a potential reservoir of extracellular form of 18 kDa FGF-2 in the extracellular matrix.

Proteoglycans are molecules located extracellularly, membranes or intracellularly. These consist of protein chains called "core proteins" into which various glycosaminoglycans (GAGs) have been implanted. Major GAGs include heparin sulfate (HS); Heparin (HP); Chondroitin sulfate (CS); Dermatan sulfate (DS; isomer of chondroitin sulfate) and keratin sulfate (KS).

Heparan sulphate proteoglycans (HSPG) have been described in the literature to associate with FGF-2 with a relatively strong affinity, to protect it from various degradations and to be used as a reservoir. Specific association of FGF-2 with specific GAGs was identified: heparan sulfate. Inhibition of the association of FGF-2 with MEC of endothelial cells grown in the presence of heparin or heparan sulfate was confirmed, but not in the presence of chondroitin sulfate, keratan sulfate or hyaluronic acid. In addition, the association of FGF-2 with MEC pretreated with heparinase (specific enzymes for heparin and heparan sulphate) did not appear, but to chondroitinase ABC (chondroitin sulphate, keratan sulphate and hyaluronic acid). No specific enzyme). The results all demonstrate that there is a specific relationship between FGF-2 and heparan sulphate of MEC, and that this relationship with other GAGs was not found.

At the structural level, the minimal sequence on the heparan sulphate chain needed to bind FGF-2 through investigation of these interactions is at least one C ₂ -sulfated iduronic acid residue and at least one N-sul It is a pentasaccharide containing a pate group. In addition, the presence of a heparin sulphate binding site on FGF-2 enables this interaction. This site is found at the level of two β sheets (β10 and β11 sheets) containing some basic amino acid residues. Also found on FGF-2 are specific sites for binding to its biological receptor: FGFR. The interaction of FGF with its signaling receptor involves dimerization of the receptor and the presence of heparin sulphate as co-receptor. A model of the response to FGF-2 is a three-component complex in which a two-component complex of FGF-2 / HSPG is formed, followed by immediate association of FGFR, in which dimerization will occur to provide in vivo biological activity through the protein kinase of the receptor. Explain that the formation of complexes is induced (IBRAHIMI & coll., Biochemistry, 2004, 43, 4724-4730). The model is also supported by the discovery of heparin / heparin sulfate binding sites on FGFR.

Several heparan sulphate proteoglycans can bind to FGF-2 and these proteoglycans can attach to the surface of cells or flow in the MEC or basement membrane. Four of these have been found in the literature. These include β-glycans (membrane heparan sulfate proteoglycans having a stronger affinity for TGFβ); Glypican (heparan sulfate proteoglycan linked to cell membranes through glycosylphosphinositol linkage); Syndecan (transmembrane heparan sulphate proteoglycans with the highest affinity for FGF-2); And perlecan (heparan sulfate proteoglycan of matrix).

FGF -2 disassembly and protection

FGF-2 is sensitive to proteolysis and can degrade rapidly at physiological temperatures. Due to the very strong affinity for FGF-2, conditions for the degradation of FGF-2 in the absence and presence of the standard control heparin (naturally highly sulfated GAG) were investigated and disclosed in the literature.

TARDIEU et al. (Tardieu and coll., Journal of cellular Physiology, 150: 194-203; (1992)) describe the (acid) FGF-1 and FGF- which faced changes in pH in 1992. The behavior of 2 was investigated. Three conditions were tested with or without the heparin: pH 2, pH 7 and pH 9. The degradation of FGF was investigated by the introduction of radioactive elements in a fibroblast proliferation model. The results show that FGF-2 is inactivated at acidic and basic pH but in the presence of heparin can retain the activity of FGF-2 similar to (or very slightly weakened) that observed at neutral pH.

The authors also examined the pyrolysis of FGF-1 and FGF-2 in the same model. FGF solutions with or without heparin were incubated at 0 ° C., 20 ° C., 37 ° C., 60 ° C. and 90 ° C. for different times: 1 hour, 24 hours, 7 days, 14 days, 30 days and 60 days. Sean. The results obtained show that the activity of FGF-2 depends on the temperature. An increase in temperature causes a decrease in the activity of FGF-2. In particular, no activity was detected at 60 ° C. and 90 ° C., even in the presence of heparin. On the other hand, at other irradiation temperatures, heparin provides active protection of FGF-2 over time.

Finally, enzymatic digestion of FGF-1 and FGF-2 by trypsin and chymotrypsin was investigated. With or without heparin, after incubation of the FGF solution at 37 ° C. for 3 hours, electrophoresis was performed on an 18% polyacrylamide gel to observe non-digested FGF. Incubation time results in the degradation of all of the FGF present in the sample, whether trypsin or chymotrypsin. On the other hand, when heparin is present, an 18 kDa band can be found in FGF-2, which means the overall protection of FGF-2 by heparin.

In the same way, it was found that GAG stabilizes the active form of FGF-2, protecting it from acid denaturation or even thermal denaturation and increasing specific interactions with specific cellular receptors. In the presence of heparin, FGF is more stable during storage and more resistant to denaturing and proteolytic conditions.

Given the important role of FGF-2 in therapeutics, healing, tissue repair, and susceptibility to different stresses, such as temperature and enzymatic hydrolysis, the team added additional sources of FGF-2 or even natural or synthetic molecules. The effect of protection of FGF-2 by

Indeed, in 2005, Yamamanaka (Basic fibroblast growth factor treatment for skin ulcerations in scleroderma, cutis, 2005; 76: 373-376) described the topical use of recombinant FGF-2 in the treatment of skin ulcers. It is starting. FGF-2 stimulates FGF receptors of fibroblasts and induces fibroblast activation and blood vessel proliferation. During the recovery and reconstruction phases, FGF-2 also acts on the proliferation of keratinocytes.

Dextran derivatives were synthesized to enhance the activity of FGF-2 (cytoblastic activity of fibroblasts) by protecting FGF-2 against heat, pH degeneration and enzymatic degradation (trypsin and chymotrypsin) (cited above) Tardu, 1992 (1992). These derivatives provide better protection of FGF-2 than heparin, thus providing a compound that offers an alternative to using heparin with potent anticoagulant activity. These are stabilizers, enhancers and protective agents of the matrix for pharmaceutical agents acting on tissue repair. Also, in 2004, Franck (Franck), etc. (as described in [J. Biomater Sci. Polymer Edn. Vol. 15. N o 11, pp. 1463-1480 (2004)]) is a dextran derivative in other fibroblast cell culture solution Could increase the proliferation of cells and promote the effect of growth factors on cell growth.

Finally, Sylvia Colliec-Jouault et al. (Exopolysaccharides produced by bacteria isolated from deep-sea hydrothermal vents: new agents with therapeutic potential, Pathologie Biologie, 52, 127-130 (2004)). The use of specific exopolysaccharides synthesized by bacteria, used as implants for bone filling in the experimental model of was discussed. After 15 days, the animals showed complete healing without the inflammatory response observed. These results are explained by the interaction of extracellular polysaccharides with soluble modulators, including FGF-2, which promote the healing phenomena by increasing the proliferative activity of FGF-2. Such extracellular polysaccharides are used for the healing of teeth.

skin: FGF -2, Proteoglycan  And aging

 In the skin, FGF-2 is found in the epidermis, dermis-epidermal border (JDE), dermis, hypodermis and capillaries, which have a broad spectrum of activity. FGF-2 ensures the proliferation of fibroblasts in the dermis and can synthesize matrix macromolecules essential for skin integrity, but it also acts on other cells of the skin. The proliferation of melanocytes is controlled by this FGF-2, which is also in fact an accelerator for the formation of capillaries. In addition to its proliferation, FGF-2 increases the production of hyaluronan (the major GAG in the dermis) by increasing the mRNA proportion of genes encoding hyaluronan synthase.

When injured, FGF-2 is an important substance in healing. FGF-2 always acts by increasing their proliferation at the fibroblast level, but also acts on keratinocytes to inhibit the expression of collagenase-1, allowing for faster reconstruction of the skin. During healing this action does not occur when heparan sulphate is used as carrier / reservoir but occurs when dermatan sulphate is used. Expression of FGF-2 is then mainly caused by keratinocytes, macrophages and fibroblasts. The half-life of in vitro FGF-2 was estimated to be about 24 hours at 37 ° C. and neutral pH (SOMMER & RIFKIN, Journal of cellular Physiology 138: 215-220 (1989)). Thus, FGF-2 is an important cytokine in the skin because of the numerous functions it performs.

Proteoglycans of the skin and their distribution have been studied. A major GAG was found in the skin, indicating its ability to interact with FGF-2. The quantitative and qualitative distribution of GAG was determined as follows: hyaluronic acid (31%), chondroitin sulfate (25%), keratan sulfate (24%), dermatan sulfate (20%) and finally hepa Lan sulfate (18%). Heparan sulphate contained in the dermis allows for the storage of FGF-2 present.

During aging, the skin undergoes numerous physiological changes. In addition to the wrinkles that form the most visible traces, there are structural changes in the subcutaneous that are revealed. The dermis is significantly contracted as a result of a decrease in the number of fibroblasts and their ability to synthesize, in particular the change in extracellular matrix macromolecules. Reduction of glycosaminoglycan synthesis has also been reported to be involved in the reorganization of existing proteoglycans. In studies of fibroblast cultures, it was found that the synthesis of hyaluronic acid decreased significantly during aging while the synthesis of keratan and chondroitin sulfate increased. In the case of dermatan and heparan sulphate, their synthesis will not stop decreasing. This reduction no longer enables the storage of FGF-2 and therefore will no longer protect the endogenous efficacy of FGF-2.

<Object of invention>

There is no literature on prior art for protecting growth factors, especially FGF-2 from its degradation or degeneration, especially in the skin.

It is therefore an object of the present invention to protect the endogenous supply of FGF-2 in the skin, especially the dermis, to mimic the action of heparan sulphate, preferably through the skin barrier to reach its target It is a new technology problem solving which consists in completing the protective action of heparan sulphate proteoglycans by developing the ingredients. Another object is to provide an active ingredient which protects the endogenous amount of FGF-2 and at the same time makes it available for the recovery of the extracellular matrix, in particular the skin matrix.

It is an object of the present invention, in particular, to provide an active ingredient which protects FGF-2 from its degradation or denaturation occurring in various tissues, preferably the skin, thereby reconstructing the extracellular matrix, or by rebuilding the skin matrix, especially in humans, Cosmetics, skin-cosmetics or cosmetics to enhance moisturization, promote skin recovery, maintain dermal integrity, enhance melanocyte proliferation, increase epidermal differentiation, or increase epidermal regeneration. There is a technical problem solving consisting of providing pharmaceutical compositions.

An object of the present invention is to solve a new problem consisting of providing a vegetable active ingredient for solving the above problem. Active ingredients that can solve these technical problems should not be irritating or toxic to the skin.

It is therefore an object of the present invention to provide active ingredients which solve the technical problems mentioned in the cosmetic, skin-cosmetic or pharmaceutical field, in particular for topical application in humans.

It is also an object of the present invention to solve a new technical problem which consists in providing a method for screening an active ingredient which can achieve the above mentioned objects. The screening method should preferably allow the plurality of active ingredients to be screened together in a renewable and reliable manner.

Summary of the Invention

In order to identify the active ingredients that solve the above mentioned technical problems, the inventors have conducted an in vitro selection model in which FGF-2 protective activity of a plurality of selected substances can be demonstrated, in particular protective activity upon pyrolysis of FGF-2. Developed.

To protect FGF-2, these active ingredients play a key role in regenerating the cell population of fibroblasts, in particular ensuring the integrity of the extracellular matrix of the skin matrix. They are therefore the first selected active ingredient for effectively controlling the degradation of FGF-2 and for example controlling skin aging.

According to a first aspect, the present invention provides a sulfated glycosaminoglyce as an active ingredient in a cosmetic or skin-cosmetic or pharmaceutical composition for preventing or controlling at least one skin change associated with the degradation of FGF-2. It relates to the use of FGF-2 protective substances which are neither cannes (sulfated GAGs) nor structural analogs of sulphated GAGs.

Preferably, the FGF-2 protective substance is a plant extract and does not contain any sulfated GAGs or structural analogs of sulfated GAGs.

Hibiscus Abelmoschus or Ambrette Extract, Rebokza Extract, Gugizi Berry Extract, Banha Extract, Lesonia Extract It is advantageously selected from the group consisting of mustard powder extract, Wooyin extract, barley extract, sesame extract, or a combination produced in a combination of two or more of the listed extracts.

Advantageously, FGF-2 is protected in human skin, preferably in JDE and / or dermis, and / or in the hypodermis, and / or in the epidermis, and / or in the skin vascular wall.

Advantageously, skin changes relate to the degradation of FGF-2 that occurs in human skin, preferably in the JDE and / or dermis, and / or in the hypodermis, and / or in the epidermis, and / or in the skin vessel walls.

Advantageously, the material is an aqueous or hydroalcoholic extract in solution obtained from 1 to 10% of a plant or plant part, based on the weight of the total solution in the dry state. Preferably, the aqueous extract is a hydroglycol extract.

In particular, degradation of growth factors usually results from one or more degradations such as pyrolysis that occurs at physiological temperatures or by exposure to heat and / or enzymatic degradation by cathepsin G in particular.

Advantageously, the substance or composition stimulates the proliferation of skin cells, in particular the growth of fibroblasts and / or melanocytes, and / or of one or more matrix components, in particular one or more collagen and / or one or more microfibrils. It is used to increase the synthesis of certain proteins such as fibrillin 1 or 2 and / or fibulin 3 or 5, and / or one or more glycosaminoglycans (GAG), in particular sulfated GAG.

According to certain embodiments, the substance or composition reconstructs the extracellular matrix of the extracellular matrix, in particular JDE, the skin vascular wall, the epidermis, the epidermis and especially the dermis by rebuilding the skin matrix, enhances the moisturization of the skin, or It may be particularly useful for any combination thereof, as well as to promote recovery, to maintain dermal integrity, to improve epidermal differentiation, or to increase the regeneration of the epidermis.

These various uses described above relate to preventing or controlling skin changes associated with the degradation of FGF-2.

Advantageously, the concentration of the substance is an effective concentration that protects FGF-2, especially when applied topically. Effective amounts when such materials are topically applied have been found to range from 0.01 to 10% by weight of the total composition.

The composition is particularly useful as a human cosmetic, skin-cosmetic or pharmaceutical composition.

The substance according to the invention is preferably a plant extract which protects FGF-2 from its degradation. Such plant extracts may be aqueous or hydroalcoholic and are preferably hydroglycolic extracts.

In one preferred embodiment, the substance is an aqueous or hydroalcoholic extract of Hibiscus abelmoscus obtained from the seed of Hibiscus abelmoscus.

According to a second aspect, the present invention relates in particular to the use of a substance for preparing a composition, in particular a cosmetic composition, a pharmaceutical composition or a skin-cosmetic composition, for protecting FGF-2 from its degradation in the various uses listed above.

The invention also relates to a cosmetic, pharmaceutical or skin-cosmetic treatment method using the composition as defined above according to certain embodiments, in particular in order to achieve the treatment in at least one of the various uses enumerated above.

<Detailed Description of the Invention>

With the present invention, protection by proteoglycans of this same matrix of FGF-2 can be achieved.

"Protection of FGF-2" means protection of FGF-2 from its degradation or denaturation. Such degradation or denaturation generally occurs as degradation due to ambient conditions such as enzymatic degradation or pyrolysis. Such degradation or denaturation may occur due to the weakening of the protection provided by the proteoglycans.

The screening method according to the invention allows for the selection of various active ingredients extracted from plants. Plant extracts have not been disclosed for the protection of FGF-2 from its degradation in skin, especially human skin, and only materials having a structure similar to heparan sulphate were used.

Unexpected active ingredients have been identified that are particularly effective in the protection of FGF-2 from pyrolysis: this is a plant belonging to the family Malvaceae, in particular the plant belonging to the genus Hibiscus, more particularly of Hibiscus abelmoscus or Ambrét. It is an aqueous extract obtained from seed.

Hibiscus Abelmoscus is known as an aqueous extract obtained from the whole plant only to control slimming properties, more specifically cellulitis, in cosmetics (see, for example, US Pat. No. 5,705,170).

In the present invention, other extracts having the desired activity may be selected. Their activity was unexpected, particularly in the protection of FGF-2. The extract is particularly obtained from Lycium dehydrated whole berry. chinense ) or aqueous extract of goji berry, Pinelliae from bulbs ternata ) or half aqueous extract, Raphanus obtained from seeds sativus ) or an aqueous extract of levok, Brassica obtained from seeds juncea ) or extract of mustard, Coicis from seeds semen ) or aqueous extracts of quintes , Hordeum from seeds vulgare ) or extract of barley, sesamum obtained from seeds indicum ) or extracts of golden sesame seeds, and all of the Alsonia species or extracts of Resonia.

However, such extracts are not limited to single aqueous extracts. Thus, the present invention also encompasses any polar solvent which, using it, can be obtained essentially as an aqueous extract, in its entirety, to provide the desired properties within the scope of the present invention. From the tests devised by the inventors, the desired properties can be obtained as hydroglycolic extracts. Extraction solvents of the water / glycol type are preferred among the hydroglycolic extracts. Water / butylene glycol mixtures are particularly preferred.

The ratio of water / alcohol mixture can vary as is known to those skilled in the art. The water / alcohol ratio is generally 10/90 to 90/10, for example, varying from 50/50 to 85/15.

As active ingredients in the present invention, preference is given to using extracts in solutions obtained from 1 to 10% of plants or plant parts in the dry state based on the weight of the total solution.

The composition according to the invention advantageously comprises from 0.001 to 20%, preferably from 0.01 to 10%, of the active ingredient according to the invention, based on the weight of the total composition. The composition may be applied topically or administered orally.

Methods of screening for potential active substances to protect FGF-2 from its degradation in humans are particularly

a) leaving the potential active substance in contact with FGF-2 at a temperature of about 45 ° C. for about 2 hours, and

b) selecting a substance that protects FGF-2 from its degradation by at least 50%, preferably 65%

It includes.

Advantageously, the screening method comprises an additional step of testing transdermal penetration to select an active substance that penetrates the skin barrier.

Advantageously, the present invention relates to a process for the preparation of a composition comprising the step of mixing the selected active substance with an excipient for preparing the composition after applying said screening method.

Other objects, features, and advantages of the present invention will become apparent to those skilled in the art upon reading the description of the reference to the embodiments, where the embodiments are provided for illustration only and do not limit the scope of the invention.

The examples are an essential part of the invention, and any feature that appears novel in comparison to the prior art in view of the entire description including the examples is an essential part of the invention in terms of its purpose and generality.

Accordingly, the embodiments each have a general scope.

In the examples, all percentages are given by weight unless otherwise indicated, temperatures are expressed in degrees Celsius unless otherwise noted, and pressure is atmospheric pressure unless otherwise noted.

<Example>

Example  One: FGF -2 pyrolysis study

Tardu et al. Studied the thermal degradation of FGF-2 in a fibroblast proliferation model by radioactive element incorporation in 1992. The inventors have developed a method for investigating the stability of FGF-2, and this quantitative method can be used on a large scale to achieve selection of active ingredients that would not be possible with the methods described in Tardu's literature.

A 5 ng / ml solution of FGF-2 in 10 mM PBS buffer containing 0.1% BSA and 0.1% methylparaben was prepared.

The solution was placed at various temperatures, 4 ° C., 20 ° C., 37 ° C., 50 ° C. and 80 ° C., with or without 500 μg / ml of heparin.

Degradation kinetics at each irradiation temperature by administering FGF-2 at different time points (T = 0 hours, 3 hours, 24 hours and 48 hours) using a commercially available ELISA kit (R & D system, France) Was established.

The results are expressed as percentage of FGF-2 over time T = 0. The experiment was performed three times (n = 3). The results obtained are shown in FIGS. 1 and 2.

FGF-2 is sensitive to the temperature conditions provided.

FIG. 1 shows a graph of stability of FGF-2 over time as a function of time at different temperatures. FGF-2 stored at 4 ° C. was relatively stable over 48 hours since 20% of FGF-2 was degraded at this temperature. As temperature increased, the rate of degradation increased: 65% of FGF-2 was degraded after 3 hours at 37 ° C. and 80% at 50 ° C. Finally, since the protein is denatured at 80 ° C., all of the FGF-2 was observed to decompose after 3 hours at this temperature.

Finally, it is significant that more degradation is observed after a longer time and 80% of FGF-2 was degraded after 24 hours at 37 ° C.

FIG. 2 shows a graph of stability of FGF-2 at various temperatures in the presence of heparin. FIG. 2 shows that the degradation reaction is stopped by the presence of 0.05% heparin, an experimental positive control, in which heparin has a strong affinity for FGF-2 to stabilize FGF-2. Heparin is a highly sulfated polysaccharide known to have a very strong affinity for FGF-2.

Since heparin denatures at a temperature of 80 ° C., the protective reaction was not effective even at this temperature.

Surprisingly, degradation of FGF-2 at about 50 ° C. occurred over a short period of time. The temperature close to 45 ° C. was chosen for practical purposes, which is the parameter chosen to carry out a stress model for the selection of the active ingredient, where the experimental positive control preferably consists of heparin.

Example 2 In Vitro Model for Screening Active Substances Protecting FGF-2 Against Pyrolysis

The degradation of FGF-2 observed at about 50 ° C. was particularly interesting because in a short time the FGF-2 was degraded by about 80% and many tests could be performed. For this reason, the selection was carried out using a stress of 2.15 hours at 45 ° C. and it was decided to study the ability of a number of active substances to protect FGF-2 against the thermal degradation.

The screening models applied are as follows:

180 μl of a 4.5 ng / ml FGF-2 solution prepared with 0.1% BSA / PBS buffer was dispensed into 96-well microplates. A solution containing 20 μl of distilled water, 0.05% heparin or various test active ingredients extracted from various plants was added. After the plate was stirred using a plate stirrer, it was covered with an adhesive tape and placed in an oven at 45 ° C. for 2.15 hours. At the end of the incubation time, the plate was removed and then administered with FGF-2 using an ELISA kit.

Control plates containing only FGF-2 and distilled water were treated under the same conditions and left at room temperature.

The results are expressed as percent FGF-2 protection for the FGF-2 control group that was not stressed or did not degrade.

Example 3 Identification of Models by GAG and Proteoglycans of Skin

Tests were performed using commercially available GAGs and PGs to confirm the model.

GAG solutions with different concentrations were prepared: Heparan sulfate, dermatan sulfate, chondroitin sulfate and hyaluronic acid were dissolved and tested at 0.1%, 0.01% and 0.001%.

Heparin used as a positive control was tested at 0.1%, 0.01%, 0.001%, 0.0001% and 0.00001%.

Proteoglycans tested were glypican-3 and decorin tested at 0.1%, 0.01% and 0.001%.

The stress protocol described in Example 2 was applied by replacing 20 μl of water with 20 μl of the corresponding GAG or PG solution. Thus, the GAG and PG tested were further diluted 1/10.

Figure 3 shows the results of a study on the protection of FGF-2 by GAG of the skin.

The results obtained in FIG. 3 confirm the data of the literature:

Dermatan sulfate as well as heparin sulfate protected FGF-2 at each tested concentration. In addition, the main promoter of FGF-2 'activity during the healing process was derma tan sulfate. Hyaluronan protects FGF-2 in any case, regardless of molecular weight. As in the case of chondroitin sulfate, the protection it provides depends on the dosage but does not reach 100%.

Two proteoglycans (FIG. 4; Study on protection of FGF-2 by PG) The literature data were validated by testing of glypican-3 and decorin. That is, glypican-3 with heparan sulfate chains protected FGF-2 in a dose dependent manner, while decorin (consisting of chondroitin sulfate and dermatan sulfate chains) proved ineffective.

This study shows that FGF-2 protection depends on the structure of GAG. The interaction of heparan sulphate with FGF-2 was found to be appropriate. This interaction was confirmed when the PG bearing heparan sulphate chains was tested as a model.

This study confirms the screening model of the present invention by checking the results of the literature.

Example 4: Selection of Active Ingredients

Screening was performed according to Example 2 from a library consisting of 2,000 plant extracts and characteristic molecules. These dissolved compounds, plant extracts, seaweeds or characteristic molecules were tested at 0%, or 10% or 1% in the reaction medium described in Example 2.

The results of the most effective active ingredients are listed in Table 1 below.

The extract is obtained by sedimenting a portion of the plant in a water / alcohol mixture, preferably water / glycol, preferably in the range of 100/0 to 0/100 (volume / volume). Then diluted with the solvent or mixture of solvents.

The particular active substance tested showed strong protective activity, which also appeared to be dose dependent.

Example 5: Hibiscus Abelmoscus (Ambret) Extract

5a-Hydroalcoholic Ambret Extract was prepared from 5% (w / w) of ground seeds in reflux ethanol. After the extraction was performed for 1 hour, the solution was filtered, ethanol was removed, and the resultant was dissolved at 5% (w / w) in a water / glycol (75/25) mixture, and the different cut-offs in the ceramic filter. ultrafiltration to threshold and finally filtered at 0.45 μm.

5b-Hydroglycol Ambret extract was prepared in a water (75%) / GG (25%) mixture, preferentially from 5% (w / w) of ground seeds. After performing overnight overnight at 4 ° C., the solution was ultrafiltered with different cut-off thresholds in a ceramic filter and finally filtered at 0.45 μm. BG means butylene glycol.

5c-Aqueous Ambret extract was prepared in water, preferably 5% (w / w) ground seeds. After performing overnight overnight at 4 ° C., the solution was ultrafiltered with different cut-off thresholds in a ceramic filter and finally filtered at 0.45 μm.

Example 6: Goji Berry Berry Extract

Goji berry extract was prepared from 5% (w / w) of dehydrated whole berry, preferably in a water (75%) / BG (25%) mixture. After performing overnight overnight at 4 ° C., the solution was ultrafiltered with different cut-off thresholds in a ceramic filter and finally filtered at 0.45 μm.

Example 7: Half Extract

The half extract was preferably prepared from 5% (w / w) stems in a water (75%) / BG (25%) mixture. After performing overnight overnight at 4 ° C., the solution was ultrafiltered with different cut-off thresholds in a ceramic filter and finally filtered at 0.45 μm.

Example 8: levokza extract

Levoqza extract is prepared from 5% (w / w) seeds, preferably in a water (75%) / BG (25%) mixture. After performing overnight overnight at 4 ° C., the solution was ultrafiltered with different cut-off thresholds in a ceramic filter and finally filtered at 0.45 μm.

Example 9: Resonia Extract

Resonia extract is preferably prepared from 5% (w / w) total algae in a water (75%) / BG (25%) mixture. After performing overnight overnight at 4 ° C., the solution was ultrafiltered with different cut-off thresholds in a ceramic filter and finally filtered at 0.45 μm.

Example 10 Mustard Powder Extract

The mustard powder extract is prepared from 5% (w / w) seeds, preferably in a water (75%) / BG (25%) mixture. After performing overnight overnight at 4 ° C., the solution was ultrafiltered with different cut-off thresholds in a ceramic filter and finally filtered at 0.45 μm.

Example 11: Wooin Extract

The bovine extract was preferably prepared from 5% (w / w) seeds in a water (75%) / BG (25%) mixture. After performing overnight overnight at 4 ° C., the solution was ultrafiltered with different cut-off thresholds in a ceramic filter and finally filtered at 0.45 μm.

Example 12 Barley Extract

Barley extract was prepared from 5% (w / w) seeds, preferably in a water (75%) / BG (25%) mixture. After performing overnight overnight at 4 ° C., the solution was ultrafiltered with different cut-off thresholds in a ceramic filter and finally filtered at 0.45 μm.

Example 13: Sesame Extract

Sesame extract was preferentially prepared in a water (75%) / BG (25%) mixture from 5% (w / w) seeds. After performing overnight overnight at 4 ° C., the solution was ultrafiltered with different cut-off thresholds in a ceramic filter and finally filtered at 0.45 μm.

Each extract (Examples 5 to 13) was partially stored. Any preservative (preferably consisting of a mixture of caprylyl glycol, phenoxyethanol, 1% hexylene glycol) was finally added in the presence or absence of xanthan.

Example 14 Selection of Active Ingredients or Plant Extracts

Following the selection of Example 2, the most effective extracts were selected and tested at different concentrations. The active ingredients used are extracts from Examples 5 to 13, which were diluted with the solvent (or mixture of solvents) used for extraction. The solvent was tested at a concentration range of 0.1% to 10% to study the specificity of the active ingredient for the thermal decomposition of FGF-2. Dosing was performed three times. The results obtained are listed in Table 2 below.

The extracts were very effective and all had dose dependent activity, with each active substance showing activity specificity for a given target.

Hibiscus Abelmoscus (Ambret) extracts showed the strongest activity, and thus the extract is the active ingredient selected for the protection of growth factor FGF-2.

In Examples 15-21, the concentration of the active ingredient (Extract 5c) is volume percentage (volume / volume) in water.

Example 15 Hibiscus abelmoscus prepared according to Example 5c Studies on the Protection of FGF-2 by Aqueous Extract of Ambrét (Extract 5c)

Investigations on the protection of FGF-2 by extract 5c were carried out on two industrial batches. The protective response was investigated by applying stress according to the conditions of Example 2.

Positive controls consisting of 0.01% heparin tested in final reaction medium were protected up to 105.52% ± 3.04 after dilution in water.

The results obtained are listed in Table 3 below.

Extract 5c protected FGF-2 in a dose dependent manner showing strong activity at an active ingredient concentration of 1% in water. The results obtained for the two industrial batches were substantially the same.

Whether extract 5c can protect FGF-2 at physiological temperature, ie 37 ° C., it must be confirmed. To this end irradiation involves administering FGF-2 after 24 hours at 37 ° C. in the absence or presence of increased concentration of extract 5c.

Positive controls formed by 0.01% heparin protected up to 110.53% ± 3.20.

The results obtained are listed in Table 4 below.

The results obtained show that extract 5c protects FGF-2 in a dose dependent manner at physiological temperature. Extract 5c further protects FGF-2 at this temperature, which degrades less than at 45 ° C.

Example 16: Study of Cytotoxicity of Extract 5c on Monolayer Fibroblasts by PNPP Administration

The purpose of this study was to investigate the cytotoxicity of extract 5c against normal human fibroblasts.

The administration is based on the conversion of p-nitrophenyl phosphate (PNPP) to p-nitrophenol by intracellular acid phosphatase of living cells. The absorbance of p-nitrophenol at 405 nm is directly proportional to the number of viable cells contained in the culture wells.

Once normal human fibroblasts reach congenital state, replace or replace the culture medium with 200 μl / well supplement medium of extract 5c (increasing concentration to 0.1, 0.5, 1, 2, 3 and 5%). (Control).

Cells were incubated at 37 ° C. for 48 hours in an oven.

After incubation and removal of the culture medium, the cells were washed twice with PBS (phosphate buffer solution), followed by 0.1 M sodium acetate (pH 8), 0.1% Triton X-100 and 5 mM p-nitrophenyl phosphate (Sigma ), France). After incubation at 37 ° C. for 2 hours in an atmosphere containing 5% CO ₂ , the reaction was stopped by the addition of 20 μl of 1 N NaOH. Then, the plate - using a reader (Victor ² V (Victor ² V), Perkin Elmer (Perkin Elmer), Finland) was determined and the absorbance of the reaction medium at 405 nm.

Non-enzymatic hydrolysis of p-nitrophenyl phosphate was measured during each experiment in wells containing no cells (“blanks”). All measurements were performed six times (n = 6).

The results obtained are listed in Table 5 below.

Extract 5c did not show cytotoxicity in all concentration ranges tested.

Example 17 Study of Fibroblast Proliferation by FGF-2 Protected by Extract 5c

The purpose of this study was to confirm that FGF-2 protected by extract 5c stimulates the proliferation of normal human fibroblasts.

A solution of FGF-2 was prepared on the fly (= undigested FGF-2); The solution of FGF-2 was left at 37 ° C. for 24 hours in the absence (= degraded FGF-2) and presence of 0.01% heparin (= FGF-2 + heparin, 37 ° C.).

Each FGF-2 solution was diluted to apply to cells at final FGF-2 concentrations of 0.1, 0.25, 0.5, 0.75 and 1 ng / ml.

Normal human dermal fibroblasts were distributed in 6-well plates at low concentrations. They were then grown in media in the absence (control) or presence of various concentrations of FGF-2.

After 48 hours, the activity of intracellular acid phosphatase (PNPP) was conferred to evaluate the effect of treated or untreated FGF-2 solution on cell proliferation.

The results are expressed as percent growth relative to control wells, ie untreated wells.

The results obtained are shown in Table 6 and FIG. 5.

Figure 5 shows the results of the study of the proliferation of fibroblasts in the presence of undigested FGF-2, degraded FGF-2 and FGF-2 + heparin (37 ° C).

The results obtained show that FGF-2 (not degraded) stimulates the proliferation of normal human fibroblasts in culture, or it is stimulated in a dose dependent manner.

FGF-2 degraded at 37 ° C. stimulated proliferation but was much weaker than FGF-2 not degraded at 37 ° C. In addition, at each tested concentration, the presence of proliferated and degraded FGF-2 was statistically less than that obtained in undigested FGF-2.

This example demonstrates the feasibility of developing an active substance capable of protecting FGF-2 of a matrix that is degraded at physiological temperatures, which can no longer play a role in skin cell regeneration.

The protection of FGF-2 from pyrolysis maintains proliferation, or even higher than that obtained using ready-made FGF-2, so that heparin used at 0.01% is required to reduce the concentration of FGF-2 to a concentration of 0.5 ng / ml. Maintain biological properties.

Very significant stimulation observed for small amounts of FGF-2 and the presence of heparin was not observed for more significant amounts of FGF-2.

The same experiment was performed using extract 5c instead of heparin. The FGF-2 solution injected in 0.5% of extract 5c was left at 37 ° C. for 24 hours. The solution was then diluted so that 0.5 ng / ml FGF-2 was applied to the wells containing fibroblasts.

The concentration of 5c extract finally applied to the cells is 0.0025%. It was previously confirmed whether the 5c extract of this concentration could not induce stimulation of fibroblast proliferation. This means that the stimulation of proliferation observed is actually due to the protection of FGF-2 by extract 5c (see Table 8).

The results obtained are expressed as percent growth relative to untreated control, which is shown in Table 7 below.

Figure 6 shows the fiber in the presence of FGF-2 in the presence of undigested FGF-2 (0.5 ng / ml), degraded FGF-2 (0.5 ng / ml), and 0.5% of 5c extract (37 ° C.). The results of studies on the proliferation of blast cells are shown.

Incubation of FGF-2 and extract 5c simultaneously at 37 ° C. protects FGF-2, which retains its ability to stimulate proliferation of cultured normal human fibroblasts. From the results obtained, fibroblast proliferation not statistically different from that obtained in the presence of ready-made FGF-2 can be obtained.

The advantage of the activity in which FGF-2 can be protected is believed to be an important concern for cell regeneration of fibroblasts.

Studies on the proliferation of normal human fibroblasts in the presence of extract 5c (increasing concentration) were performed at 37 ° C. for 48 hours.

Fibroblasts were dispensed in the presence of 5c extract with various concentrations of 0.0025%, 0.01%, 0.25%, 0.5%, 1% and 2%. After 48 hours, administration of acid phosphatase (PNPP administration) at 405 kPa was performed, followed by calculation of the percent growth rate for the negative control (= untreated cells). Each condition was tested six times (n = 6).

The results obtained are listed in Table 8 below.

Positive controls, consisting of 10% serum, provide stimulation of significant proliferation confirmed experimentally (T + = + 126% * proliferation).

The results obtained show that 5c extract does not stimulate the proliferation of normal human fibroblasts in culture.

Example 18 Effect of Protection of Growth Factors on Collagen Synthesis

Normal human fibroblasts derived from a 52 year old donor were cultured in 96-well plates containing medium (DMEM, 2 mM glutamine, 50 IU / ml penicillin-50 μg / ml streptomycin, 10% fetal bovine serum). Incubate for 24 hours. After incubation, cells were treated for 48 hours with the product consisting of 20 μg / ml of vitamin C (positive control) and 2% and 1% 5c extracts. Controls were prepared simultaneously using only medium.

After 24 hours of contact, radioactive proline ( ³ H-proline) was added to the medium.

At the end of the experiment, supernatants were collected to administer the incorporation of radioactive praline into intracellular proteins.

The results obtained are shown in Table 9 below.

The 5c extract, tested at 2% and 1%, can stimulate the synthesis of collagen in normal human fibroblasts, which was significant.

The results obtained show that the protection of growth factors in the media and the growth factors synthesized by fibroblasts can enable the synthesis of collagen stimulated in vitro.

Example 19 Effect of Protection of Growth Factors on Synthesis of Total GAG and Sulfated GAG

The purpose of this study was to determine whether the protection of growth factors with 5c extract on monolayer fibroblast cultures could stimulate the synthesis of total GAG by radioactive methods.

Normal human fibroblasts derived from a 52 year old donor were cultured in 96-well plates containing medium (DMEM, 2 mM glutamine, 50 IU / ml penicillin-50 μg / ml streptomycin, 10% fetal bovine serum). Proliferation for 24 hours. After incubation, cells were treated for 72 hours with the product consisting of 20 μg / ml of vitamin C (positive control) and 2% and 1% 5c extracts. Controls were prepared simultaneously using only medium.

48 hours after contact, radioglucosamine (D- (6- ³ H) -glucosamine) was added to the medium.

At the end of the experiment, supernatants were collected to administer the incorporation of radioglucosamine into intracellular proteins. Administration of the protein was carried out using a commercial kit (Biorad 500-0116).

The results are listed in Table 10 below.

The results obtained show that 5c extracts applied to normal human fibroblasts at 2% and 1% can stimulate the synthesis of total GAGs, which is effective in administration, which is more significant than the positive control (TGF-β applied at 10 ng / ml). To be higher.

The results obtained show that the protection of the growth factors of the medium and those synthesized by the fibroblasts allows the synthesis of the entire GAG to be stimulated in vitro.

In the same way, incorporation studies of ³⁵ S-sulfate were performed to quantify the sulfated GAG. The procedure applied was the same as for the whole GAG except for the properties of the radioactive precursors. The results are shown in Table 11 below.

Study on Synthesis Stimulation of Sulfated GAG by 5c Extract product Active (cpm) Protein (mg / ml) Ratio (active / protein) % Control p Control 3028 3.579 846 100 - TGF-β 10 ng / ml 5442 3.650 1491 176 P <0.01 5c extract 2% 5586 3.058 1827 216 P <0.01 5c Extract 5c 1% 4300 3.396 1266 150 P <0.01

The results obtained show that extract 5c allows the synthesis of sulfated GAG to be significantly stimulated in a dose dependent manner.

This, in addition to mimicking the role of FGF-2 by extract 5c, extract 5c can protect growth factors that stimulate the synthesis of sulfated GAG, so that protection of FGF-2 protects them. Heparan means that it can be enhanced because it is the same as sulfated GAG.

Example  20: Transdermal  By 5c extract after permeation FGF Study on Protective Activity of -2

The purpose was to confirm that the 5c extract retains its properties of protecting growth factors after skin penetration.

Pure solution of 5c extract was deposited on the surface of the skin explants placed as Franz cells.

After 24 hours incubation, the medium contained in the receiving compartment of cells was recovered and lyophilized. The lyophilisate was then dissolved in distilled water and subjected to thermal stress at 37 ° C. for 24 hours to make a final test in the model described in Example 15.

Figure 7 shows a study of FGF-2 protection by 5c extract after skin passage.

Since the result obtained was the same as that obtained in Table 4 of Example 15, the obtained result (99.96% protection) shows that the extract 5c completely retained its protective properties.

The results show that after passing through skin tissue, extract 5c can protect growth factors and thus maintain integrity to reach its purpose.

Example  21: Enzymatic  For decomposition FGF -2 protection study

The purpose of this study was to quantify the degradation of FGF-2 by proteases present in the dermis of human skin. These enzymes play an important role in the degradation of the extracellular matrix.

Clearly, cathepsin G intervenes during inflammation and is secreted in the extracellular environment. It is strongly attached to the cell surface of the matrix due to its basicity. Cathepsin G is also active against collagen and also against the glycoproteins of the matrix and the core proteins of the proteoglycans.

For this reason, studies have been conducted on FGF-2 degradation by cathepsin G. Degradation kinetics was performed in 100 mM Hepes buffer at pH 7 containing 0.1% BSA. Cathepsin G was added to the 2 ng / well FGF-2 solution at a concentration of 0.02 U per well in the wells of the plate. Plates were incubated at 37 ° C. and sampled at 15, 30, 60, 90 and 120 minutes to administer the remaining FGF-2.

A control (100%) was formed by temporary administration of FGF-2. At the same time, the reactive reaction control solution was placed at 37 ° C. to study thermal decomposition at different time points, excluding the degradation due to temperature and quantifying only the portion by enzymatic degradation.

The amount of FGF-2 (pg / ml) obtained is shown in Table 12 below.

Study on FGF-2 Degradation by Cathepsin G Minutes FGF-2 Concentration Temperature Control Concentration of FGF-2 in the presence of cathepsin G (% degradation) 0 391.8 ± 3.64 339.05 ± 7.43 (13.29%) 15 324.99 ± 1.7 295.61 ± 8.49 (8.95%) 30 291.83 ± 2.4 248.04 ± 1.97 (14.77%) 60 280.64 ± 3.23 203.67 ± 1.13 (27.5%) 90 259.81 ± 4.9 141.94 ± 5.14 (45.55%) 120 241.67 ± 1.97 131.36 ± 2.49 (45.6%)

From the results obtained, it is possible to show that the degradation of FGF-2 increases with time. The enzyme allowed FGF-2 to degrade significantly at 90 minutes.

This is why this time is chosen in the FGF-2 protection study in the presence of cathepsin G with 5c extract.

Various concentrations, i.e. 0.01%, 0.05%, 0.1%, 0.5% and 1% of extract 5c, were added to the reaction medium as described above (Example 21).

After 90 minutes at 37 ° C., FGF-2 concentration was assessed and the percentage of FGF-2 remaining and the protection rate of FGF-2 were calculated. At each concentration tested, administration was performed at n = 9. The results are shown in Table 13 below.

Studies on Protection by Extract 5c of FGF-2 Degraded by Cathepsin G Test FGF-2 concentration (pg / ml) Remaining FGF-2 (%) FGF-2 protection rate (%) Control (100%) 455.23 100 - Digested Control (Enzyme) 288.11 63.29 0 5c extract 0.01% 336.93 74.01 29.21 5c extract 0.05% 344.65 75.71 33.83 5c extract 0.1% 409.08 89.86 72.38 5c Extract 0.5% 414.02 90.95 75.33 5c extract 1% 406.58 89.31 70.89

The 5c extract can protect FGF-2 against enzymatic degradation in a dose dependent manner.

By this in vitro study, application of the 5c extract can demonstrate that growth factors can be protected from proteolysis caused by enzymes of the extracellular matrix. This enhances the benefits of the use of extract 5c in cosmetics.

Example  22: according to age FGF -2 ratio detection

The purpose of this study was to study the FGF-2 concentrations of so-called "young" and so-called "old" skin, mainly to establish a relationship between age and loss of FGF-2 content due to changes in skin GAG content.

Skin biopsies from so-called young donors (ages of 21, 30, 31 and 38 years) and skin biopsies from so-called old donors (ages of 50, 55, 57 and 58 years) were sampled and 0.1% Triton X100 It was milled in PBS buffer at pH 7 containing and extracted and administered using a commercially available ELISA kit (R & D system, DFB50). FGF-2 concentrations were correlated with the proportion of DNA measured by the commercially available kit Picogreen.

The obtained result is shown in FIG.

8 shows a study of FGF-2 concentrations in so-called "young" and "old" skin hydrolysates.

The results show differences in FGF-2 content between young and old skin. This difference is also statistically significant (p = 0.046).

By this example it is possible to show that by utilizing the activity as a protective agent of growth factors, more particularly FGF-2, the effects associated with skin aging can be modulated.

Example  23: Formulation of the Product of the Invention

Different parts A, B, C, D, E or F were mixed together to prepare the compositions according to the invention and carried out according to methods known to those skilled in the art. "Product of the present invention" refers to the active ingredient mentioned in the present invention.

Use of the product of the present invention in cosmetic or pharmaceutical formulations in the form of oil-in-water emulsions.

Formulation 23a:

A sufficient amount to make A water 100

Butylene Glycol 2

Glycerin 3

Sodium dihydroxycetyl phosphate,

Isopropyl hydroxycetyl ether 2

B glycol stearate SE 14

Triisononao Inn 5

Octyl Cocoate 6

Adjusted to C butylene glycol, methylparaben, ethylparaben, propylparaben, pH 5.5

2

D 0.01-10% of the product of the invention

Formulation 23b:

A sufficient amount to make A water 100

Butylene Glycol 2

Glycerin 3

Polyacrylamide, Isoparaffin, Lauret-7

2.8

B Butylene Glycol, Methylparaben, Ethylparaben, Propylparaben

2

Phenoxyethanol, methyl paraben, propyl paraben, butyl paraben, ethyl paraben

2

Butylene Glycol 0.5

D 0.01-10% of the product of the invention

Formulation 23c:

A Carbomer 0.50

Propylene Glycol 3

Glycerol 5

Sufficient to make 100 water

B octyl cocoate 5

Bis-abolol 0.30

Dimethicone 0.30

C sodium hydroxide 1.60

D phenoxyethanol, methylparaben, propylparaben, butylparaben, ethylparaben

0.50

E Perfume 0.30

F 0.01-10% of the product of the invention

Of the present invention Example  24

Use of the Product of the Invention in Water-in-Oil Formulations

A PEG 30 -dipolyhydroxystearate 3

Capric Triglycerides 3

Cetearyl Octanoate 4

Dibutyl Adipate 3

Grape Seed Oil 1.5

Jojoba 1.5

Phenoxyethanol, methyl paraben, propyl paraben, butyl paraben, ethyl paraben

0.5

B Glycerin 3

Butylene Glycol 3

Magnesium Sulphate 0.5

EDTA 0.05

Sufficient to make 100 water

C cyclomethicone 1

Dimethicone 1

D Perfume 0.3

E 0.01 to 10% of the product of the invention

Of the present invention Example  25

Use of the product of the present invention in shampoo or shower gel formulation

A xanthan gum 0.8

Sufficient to make 100 water

B Butylene Glycol, Methylparaben, Ethylparaben, Propylparaben

0.5

Phenoxyethanol, methyl paraben, propyl paraben, butyl paraben, ethyl paraben

0.5

C citric acid 0.8

D Sodium Laurate Sulfate 40.0

E 0.01 to 10% of the product of the invention

Of the present invention Example  26

Use of the product of the invention in the preparation of lipsticks and other anhydrous products

A Mineral Wax 17.0

Isostearyl isostearate 31.5

Propylene Glycol Difelagonate 2.6

Propylene Glycol Isostearate 1.7

PEG 8 Beeswax 3.0

Hydrogenated Palm Core Oil Glyceride, Hydrogenated Palm Glyceride

3.4

Lanolin Oil 3.4

Sesame Oil 1.7

Cetyl Lactate 1.7

Mineral oil, lanolin alcohol 3.0

Enough to make B Castor Oil 100

Titanium Dioxide 3.9

CI 15850: 1 0.616

CI 45410: 1 0.256

CI 19140: 1 0.048

CI 77491 2.048

C 0.01-5% of the product of the invention

Of the present invention Example  27

Use of the product of the invention in the preparation of an aqueous gel (eye contour, slimming agent, etc.)

A sufficient amount to make A water 100

Carbomer 0.5

Butylene Glycol 15

Phenoxyethanol, methyl paraben, propyl paraben, butyl paraben, ethyl paraben

0.5

B 0.01-10% of the product of the invention

Of the present invention Example  28

Use of the product of the present invention in triple emulsion formulations

Primary emulsion W1 / O

A PEG 30 -dipolyhydroxystearate 4

Capric Triglycerides 7.5

Isohexadecane 15

PPG-15 Stearyl Ether 7.5

B water 65.3

C phenoxyethanol, methyl paraben, propyl paraben, butyl paraben, ethyl paraben

0.7

Secondary emulsion W1 / O / W2

A primary emulsion 60

B poloxamer 407 2

Phenoxyethanol, methylparaben, propylparaben, 2-bromo-2-nitropropane-1,3-diol 0.3

Sufficient to make 100 water

C carbomer 15

D triethanolamine PH 6.0-6.5

Of the present invention Example  29

Preparation of Pharmaceutical Formulations Containing Product of the Invention

Formulation 29a: Tablet Preparation

G per tablet excipient A

Lactose 0.359

Saccharus 0.240

B Product of the Invention ^* 0.001 to 0.1

^* The product of the present invention was obtained by performing a drying step following the extraction procedure described, for example, in Example 5c.

Formulation 29b: Ointment Preparation

A excipient

Low Density Polyethylene 5.5

Sufficient to be Liquid Paraffin 100

B Product of the Invention ^* 0.001 to 0.1

^* The product of the present invention was obtained by performing a drying step following the extraction procedure described, for example, in Example 5c.

Formulation 29c: Preparation of Injectable Formulations

A excipient

5 ml of salted isotonic solution

B Product of the Invention ^* 0.001 to 0.1 g

^* The product of the present invention was obtained by performing a drying step following the extraction procedure described, for example, in Example 5c.

Example  30: Cosmetic Tolerance Evaluation of Formulations Containing Products of the Invention

For the compounds obtained according to Example 5c by skin and eye evaluation of rabbits, by the study of the absence of abnormal toxicity by single oral administration to rats, and by the study of the sensitivity efficacy against guinea-pigs Toxicology tests were performed.

Primary skin irritation evaluation in rabbits:

The formulations described in Example 5c were applied without dilution at a 0.5 ml dose on the skin of three rabbits, according to the method proposed by the OECD guidelines for the "acute irritation / corrosion effect on skin" study.

The product was classified according to the criteria defined by the rules from February 1, 1982 published in the JORF (official French journal) dated February 21, 1982.

The results of these tests provided the conclusion that the products of the present invention were classified as nonirritating to skin.

Eye irritation evaluation in rabbits:

The formulations described above are described in the OECD Directive NO. According to the method proposed by 405, three rabbits were simply added dropwise at once in an amount of 0.1 ml.

The results of these tests provided the conclusion that the formulation can be considered non-irritating to the eye, according to the 91/326 EEC directive used purely or without dilution.

Testing for Absence of Abnormal Toxicity by Single Oral Administration in Rats:

The above formulations were prepared under the OECD Directive NO. Five male rats and five female rats were orally administered at a dose of 5 g / kg body weight in accordance with the procedure suggested by 401 and adopted in the cosmetic product.

DL0 and DL50 were found to be greater than 5,000 mg / kg. Therefore, the formulations tested were not classified as dangerous by ingestion.

Assessment of Skin Sensitivity Efficacy at Guinea-Peak

For the formulations described, OECD No. Maximization tests described by Magnusson and Kligmann, a protocol according to 406, were performed.

The formulations have been classified as insensitive by skin contact.

The present invention provides an effective amount of Hibiscus Abelmoscus or Ambrét Extract, Levocac Extract, Goji Berry Extract, Half Lime Extract, Resonia Extract, Mustard Powder Extract, Uin Extract, to protect FGF-2 from its degradation, With regard to barley extracts and / or sesame extracts, in particular it allows the extracellular matrix to be rebuilt in the dermis, which is useful for example for regulating aging.

Claims (13)

Hibiscus Abelmoschus Extract, Rebokza Extract, Gougizi Berry Extract, Banha Extract, Lesonia Extract, Mustard Powder Extract, Wooyin ) Degradation of FGF-2 in human skin, comprising applying a cosmetic composition comprising an effective amount of a plant extract selected from the group consisting of: extract, barley extract, sesame extract, or a combination thereof, and an excipient acceptable for cosmetic use How to prevent or control it. The method of claim 1, which protects FGF-2 at the epidermis and / or at the dermal-epidermal border, and / or at the dermal extracellular matrix, and / or at the lower epidermis, and / or at the skin vascular wall. The method of claim 1 wherein the extract is an aqueous or hydroalcoholic extract. The method of claim 3 wherein the extract is in solution from a plant or plant part of 1 to 10% by weight of the solution in a dry state. The method of claim 1 wherein the decomposition is pyrolysis. The method of claim 1, wherein the extract is a hibiscus abel mocus extract. The method of claim 6, wherein the Hibiscus abelmoscus extract is an aqueous or hydroalcoholic extract obtained from Hibiscus abelmoscus seeds. The method of claim 1 wherein the effective amount is from 0.001% to 20% based on the total weight of the cosmetic composition. Plant extracts selected from the group consisting of hibiscus abelmoscus extract, levokza extract, goji berry extract, halves extract, resonia extract, mustard powder extract, quince extract, barley extract, sesame extract or combinations thereof, and 1 Cosmetic composition comprising an excipient acceptable for more than one cosmetic. The cosmetic composition according to claim 9, wherein the plant extract comprises 0.001% to 20% by weight of the total composition. The cosmetic composition of claim 10 wherein the plant extract comprises 0.01% to 10% by weight of the total composition. 10. The composition of claim 9, wherein the plant extract is a hibiscus abel mocus extract. a) establishing decomposition kinetics for FGF-2 at a specific temperature and at a specific time; b) leaving the potential active substance in contact with FGF-2 for the specific time of step (a) at the specific temperature of step (a) and establishing the degradation kinetics for FGF-2 after contact with the potential active substance; And c) selecting a substance that protects FGF-2 with at least 50% of degradation based on the degradation kinetics A method of selecting a potential active substance for protecting degradation of FGF-2 in humans, comprising.

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