ES2320303A1 - Composition for controlling FGF-2-related skin alteration - Google Patents

Abstract

Use of cosmetic active ingredients to protect FGF-2.

The invention relates to a substance that protects FGF-2 and a composition to prevent or fight the degradation of FGF-2.

In particular the invention relates to a Hibiscus abelmoschus or hibiscus extract, a radish extract, an extract of Lycian berries, an extract of Banha, an extract of Lessonia, an extract of mustard flour, an extract of Yi yi ren, a Barley extract, and / or a Sesame extract, which protects to FGF-2 of its degradation.

The present invention particularly allows restructuring of the extracellular matrix, particularly at the level of the dermis, for example to fight aging.

Description

Use of cosmetic active ingredients to protect FGF-2.

The invention relates to the development of active ingredients for a cosmetic, dermocosmetic or pharmaceutical to fight the degradation of at least one growth factor.

The invention relates in particular to the degradation protection of Fibroblast Growth Factor (Fibroblast Growth Factor) (FGF-2 or basic FGF or FGF-?.

The present invention particularly relates to the use of active ingredients that prevent, limit, or improve the quality of the dermis, particularly when it suffers the effects of age, particularly in a human being.

State of the art

Numerous growth factors are involved at the skin level and in particular FGF-2, which has a broad spectrum of activity: particularly the proliferation of fibroblasts, thus allowing the synthesis of matrix macromolecules, essential for skin integrity. . FGF-2 is protected in the skin by proteoglycans that have heparan sulfate. During the 1990s, Feige and Baird ( Médicine / Sciences, 1992; 8: 805-10 ) have described the close relationship between growth factors and proteoglycans of the extracellular matrix, explaining that in addition to a reserve phenomenon, this Interaction protected growth factors against proteolysis. This interaction results in greater stability of growth factors (a longer in vivo half-life), which allowed them to better fulfill their functions.

FGF-2 and proteoglycans

The FGF-2 is part of a Family consisting of 23 different FGFs listed so far. He FGF-2 occurs in several isoforms. In the vertebrates, five isoforms with weights have been discovered molecular 18; 22; 22.5; 24 and 34 kDa. Only the shape of 18 kDa is detected outside the cells, the others are locked inside the cell and more particularly in the nucleus. FGF-2 is a ubiquitous protein that plays a  very important role at the physiological level; the FGF-2 is involved in embryonic development, angiogenesis, Neural differentiation and tissue repair among others. In In effect, FGF-2 is present in most of tissues with a distribution oriented more particularly to the basement membrane of tissues. Although the FGF-2 is present in the body in a proportion that can purified and characterized, its mRNA is not detectable. This fact and the particularity that is distributed intermittently attached to the basement membrane of the tissues, it suggests that produces at an initial rate and then is released by the cells during development to be stored in the ECM (the basement membrane undergoes during this process an intense remodeling that allows the local release of FGF-2). This storage allows a release of FGF-2 in the adult state if necessary to participate in tissue distribution and Keep the functions differentiated. With the passage of time, the FGF-2 reserve would decrease and therefore not They can ensure maximum activity conditions.

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

Proteoglycans are molecules that have extracellular, membrane or intracellular localization. They are composed of a protein chain called "core protein" on which variable glycosaminoglycans ( GAG ) are grafted. The main GAGs are heparan sulfate ( HS ); heparin ( HP ); Chondroitin sulfate ( CS ), dermatan sulfate ( DS ), isomer of chondroitin sulfate and keratan sulfate ( KS ).

Proteoglycans that have heparan sulfate (HSPG) have been described in the literature because they are associated with a fairly large affinity to FGF-2, protecting it from various degradations and serving as a reserve. Specific binding of FGF-2 to a Particular GAG: heparan sulfate. It has been shown an inhibition of the binding of FGF-2 to the MEC of cells endothelial cultures in the presence of heparin or heparan sulfate, but not in the presence of chondroitin sulfate, keratan sulfate or hyaluronic acid. In addition, an absence has been discovered of binding of FGF-2 to a pre-treated MEC with heparitinase (specific enzyme for heparin and heparan sulfate) but not with chondroitinase ABC (enzyme specific for chondroitin sulfate, keratan sulfate and hyaluronic acid). All these results demonstrate the existence of a specific relationship between FGF-2 and MEC heparan sulfate, relationship that has not been discovered with other GAGs.

On a structural level, studies on this interaction have shown that the minimum sequence required in the heparan sulfate chain to bind to FGF-2 is a pentasaccharide that contains at least one residue of C2 sulphated iduronic acid and at least one N-sulfate group. In addition, the presence in the FGF-2 of a heparan sulfate binding site allows this interaction. This site is at the level of two β-sheets (β10 and β11 sheets) that contain several basic amino acid residues. In the FGF-2 there is also a different binding site with its different receptors: the FGFR . The interaction of the FGF with its signaling receptor implies a dimerization of this receptor and the presence of heparan sulfate as a co-receptor. The response model to FGF-2 precedes the formation of binary complexes of FGF-2 / HSPG followed by the instantaneous association of FGFR that leads to the formation of a thematic complex that will undergo dimerization to give a biological activity in vivo by means of the receptor protein kinase ( IBRAHIMI & coll., Biochemistry, 2004, 43, 4724-4730 ). This model is also supported by the discovery in the FGFR of a heparin / heparan sulfate binding site.

Several proteoglycans that have heparan sulfate can bind FGF-2, these proteglycans can be attached to the surface of the cells, free in the ECM or in the basement membrane. Four of these proteoglycans have been cataloged in the literature. It is? -Glycan , proteoglycan having heparan membrane sulfate, which nevertheless has a higher affinity for TGF ?. glypican , proteoglycan having heparan sulfate bound to the cell membrane through a glycosylphosphoinositol bond; syndecane , proteoglycan having heparan transmembrane sulfate that has the highest affinity for FGF-2; and perlecan , proteoglycan having heparan sulfate matrix.

FGF-2 degradation and protection

FGF-2 is sensitive to proteolysis and can degrade rapidly, even at temperature physiological The degradation conditions of FGF-2 have been studied and described in the bibliography in the absence and in the presence of heparin (natural GAG highly sulfated) which constitutes the reference control for its great affinity for FGF-2.

Tardieu & coll., In 1992 , ( Tardieu et al., Journal of cellular Physiology, 150: 194-203; (1992) ) have studied the behavior of FGF-1 (acid) and FGF-2 against a change in pH Three conditions have been tested, with or without prior addition of heparin: pH 2, pH 7 and pH 9. The degradation of FGF has been studied in a fibroblast proliferation model by incorporation of a radioactive element. The results show that FGF-2 is inactivated at acidic pH and at basic pH but that the presence of heparin allows to keep an activity of FGF-2 similar (even slightly reduced) to that observed at neutral pH.

The authors have also studied the degradation thermal of FGF 1 and 2 in this same model. Solutions are incubated FGF, in the presence or absence of heparin, at 0ºC, 20ºC, 37ºC, 60ºC and 90ºC during different time periods: 1 h, 24 h, 7, 14, 30 and 60 days. The results obtained show that the activity of FGF-2 is a function of temperature. Increasing of the latter causes a decrease in the activity of FGF-2. Particularly at 60ºC and 90ºC, it is not detected No activity, not even in the presence of heparin. For him On the contrary, for the other temperatures studied, heparin allows protection over time of the activity of FGF-2.

Finally, degradation has been studied enzyme of FGF-1 and 2 against trypsin and a chymotrypsin After 3 hours incubation of solutions FGF at 37 ° C, in the presence or absence of heparin, a 18% polyacrylamide gel electrophoresis to visualize the FGF not degraded. The incubation period allows to degrade all the FGF present in the samples, either with trypsin or with chymotrypsin On the contrary, the presence of heparin allows discover the 18 kDa band relative to the FGF-2 that means full protection of FGF-2 by Heparin

Similarly, it has been shown that GAGs stabilize the active conformation of FGF-2, protecting it from acidic or even thermal denaturation and increasing specific interaction with cell receptors specific. In the presence of heparin the FGF are more stable during storage and more resistant to conditions denaturing and proteolytic.

Given the important role of FGF-2 in therapeutic medicine, in healing, in tissue repair, and its sensitivity to different stress conditions, such as temperature and proteolysis enzymatic, the teams have studied the effects of a contribution Supplemental FGF-2 or even the protection of FGF-2 by natural molecules or from synthesis.

Indeed, Yamanaka, in 2005 (Basic fibroblats growth factor treatment for skin ulcerations in scleroderma, complexion, 2005; 76: 373-376), describes the use via topical recombinant FGF-2 to treat ulcers of the skin. FGF-2 stimulates the FGF receptors of fibroblasts and induces an activation of fibroblasts and the vascular proliferation During the repair process, during the restructuring phase, the FGF-2 would act also about keratinocyte proliferation.

Dextran derivatives have been synthesized (Tardieu, 1992, mentioned above) to enhance the activity of FGF-2 (mitogenic activity of fibroblasts), protecting it against heat, pH denaturation and enzymatic degradation (trypsin and chymotrypsin). These derivatives offer greater protection of FGF-2 than heparin, which allows to have compounds that offer an alternative to the use of heparin, which has a great anticoagulant activity. These compounds constitute stabilizers, enhancers and protect the matrix for pharmaceutical formulations that act on tissue repair. In addition, Franck et al. , In 2004 ( J. Biomater Sci. Polymer Edn. Vol. 15. No. 11, pp. 1463-1480 (2004) ), have shown that other dextran derivatives could, in fibroblast culture, increase the cell proliferation and promote the effect of growth factor on cell growth.

Finally, Sylvia Colliec-Jouault et al , ( Exopolysaccharides produced by bacteria isolated from deep-sea hydrothermal vents: new agents with terapeutic potential, Pathologie Biologie, 52, 127-130 (2004) ), discuss the use of a particular exo-polysaccharide Neosynthesized by a bacterium, which has been used as a bone filling implant in an experimental rat model. After 15 days the animals show complete healing without any inflammatory reaction being observed. This result would be explained by the interaction of the exo-polysaccharide with soluble mediators such as FGF-2; which would allow to increase the proliferation activity of FGF-2 and thus favor the healing phenomenon. This exo-polysaccharide is used for dental healing.

The skin: FGF-2, proteoglycans and aging

In the skin, FGF-2 is found at the level of the epidermis, dermoepidermal junction ( UDE ) of the dermis, hypodermis and capillaries. It has a wide spectrum of activity. It ensures the proliferation of fibroblasts within the dermis, also allowing them to synthesize the macromolecules of the matrix, essential for the integrity of the skin, but also acts on other skin cells. The proliferation of melanocytes is in effect controlled by FGF-2 which also constitutes a promoter of capillary formation. At the level of fibroblasts, in addition to their proliferation, FGF-2 also increases the mRNA index of the genes encoding hyaluronan synthase and thus increases the production of hyaluronan, the main GAG in the dermis.

In wounds, FGF-2 plays an important role in the healing phenomenon. It acts at the level of fibroblasts, always increasing its proliferation, but also on keratinocytes to inhibit the expression of collagenase-1 and thus allow the skin to rebuild more quickly. This action during healing is not performed with heparan sulfate as a carrier / reserve but with dermatan sulfate. The expression of FGF-2 is mainly carried out by both keratinocytes, macrophages and fibroblasts. The half-life period of FGF-2 in vitro has been evaluated for 24 h at approximately 37 ° C and at neutral pH ( SOMMER & RIFKIN, Journal of cellular physiology 138: 215-220 (1989) ). In this way, FGF-2 constitutes an important skin cytokine due to the numerous functions it performs.

Skin proteoglycans have been studied and its distribution The main GAGs known for their ability to Interaction with FGF-2 are found in the skin. Be has determined the quantitative and qualitative distribution of GAG as follows: hyaluronic acid (31%), chondroitin sulfate (25%), keratan sulfate (24%) dermatan sulfate (20%) and finally heparan sulfate (18%). The heparan sulfate contained in the Dermis allows storage of the FGF-2 Present.

During aging, the skin experiences Numerous physiological modifications. In addition to wrinkles, which they are the most visible marks, they also appear deep structural changes Particularly the dermis atrophies, in particular as a result of a reduction in the number of fibroblasts and their ability to synthesize which entails modifications of the macromolecules of the extracellular matrix. Be notes a decrease in the synthesis of glycosaminoglycans, what which also implies a reorganization of proteoglycans present. Studies on fibroblasts in culture have allowed demonstrate that hyaluronic acid synthesis decreased greatly during aging, while keratan and chondroitin Sulfates increased. As far as dermatán and heparán are concerned sulfate, its synthesis would not decrease. This decrease does not would allow to store and therefore protect the endogenous potential of FGF-2.

End of the invention

No prior art document had in order to protect a growth factor; particularly the FGF-2, of its degradation or denaturation, intervening particularly at the skin level.

Thus, the present invention has as in order to solve the new technical problem that consists in completing the protective action of proteoglycans that have heparan sulfate, by developing active principles that mimic the action heparan sulfate, to protect the endogenous reserve of FGF-2, particularly at the skin level and in particularly at the level of the dermis, and that they are particularly capable of crossing the skin barrier to reach its goal. Another end it is the supply of active ingredients that protect the quantity endogenous FGF-2, while leaving it available for  the renewal of the extracellular matrix and in particular the matrix dermal

The invention is particularly intended solve the technical problem that consists of providing active ingredients that protect FGF-2 from its degradation or denaturation, which occurs in the different tissues and particularly at the skin level, particularly for supply cosmetic, dermocosmetic compositions or pharmaceuticals to restructure the extracellular matrix, and in particular the dermis by restructuring the matrix dermal, to improve skin irrigation, to promote skin repair, to maintain the integrity of the dermis, to improve the proliferation of melanocytes, to strengthen the epidermal differentiation or to reinforce the renewal of the epidermis, particularly in humans.

The present invention has particularly as in order to solve the new problem that consists in providing a active ingredient of plant origin to solve problems mentioned above. The active ingredients that allow solving these technical problems should not be particularly toxic or irritating to the skin.

The present invention also aims at provide active ingredients that solve technical problems mentioned in the cosmetic, dermocosmetic or pharmaceutical field in particular for a topical application in humans.

The present invention also aims at solve the new technical problem that consists of providing a method of selecting active ingredients that allows achieve the objectives mentioned above. This selection procedure should allow particularly select a large number of active ingredients at once, so reproducible and reliable.

Summary of the Invention

In order to identify active ingredients that solve the technical problems mentioned above, the inventors have developed an in vitro selection model that allows demonstrating a protective activity of FGF-2 in a large number of substances to be selected, in particular against thermal degradation of FGF-2.

By protecting FGF-2, these active ingredients play a leading role, ensuring the integrity of the extracellular matrix and particularly of the matrix dermal by renewing the cell population of fibroblasts These active ingredients therefore constitute active ingredients of first choice to fight effectively against the degradation of FGF-2, and to fight, among others, against skin aging.

According to a first aspect, this invention refers to the use of a substance that is not a sulfated glycosaminoglycan (sulfated GAG) or an analogue structure of a sulphated GAG that protects FGF-2,  as active ingredient in a cosmetic or dermocosmetic composition or pharmaceutical to prevent or fight at least one skin disorder linked to the degradation of FGF-2.

Preferably, the substance that protects FGF-2 is a plant extract and does not include GAG sulfate or a structural analog with a sulfated GAG.

Advantageously, said substance, which is selected from the group consisting of an extract of Hibiscus abelmoschus or hibiscus, an extract of Radish, an extract of lithium berries, an extract of Banha ( Pinellia ternata ), an extract of Lessonia, an extract of Mustard flour, an extract of Yi yi ren, a barley extract, a sesame extract or one of the combinations resulting from the association of at least two of the extracts listed above.

Advantageously, the FGF-2 is protects at the level of human skin and preferably at the level of UDE and / or dermis and / or hypodermis and / or wall of the cutaneous blood vessels

Advantageously, the united skin disorders to the degradation of FGF-2 they intervene at the level of human skin and preferably at the level of the UDE and / or the dermis and / or the hypodermis and / or the epidermis and / or the wall of the cutaneous blood vessels

Advantageously, said substance is an extract. aqueous or hydroalcoholic in solution, obtained from between 1 and 10% of a plant or a part of the plant in a dry state with respect to the weight of the total solution. Preferably the aqueous extract is a hydroglycolic extract.

In particular, the degradation of the factor of growth is the result of at least one degradation, particularly thermal degradation, which occurs particularly at physiological temperature or by exposure to heat and / or an enzymatic degradation particularly with cathepsin G.

Advantageously, said substance or composition is used to stimulate the proliferation of skin cells, in particularly the proliferation of fibroblasts and / or melanocytes and / or to increase the synthesis of at least one component of the matrix, in particular of at least one type of collagen and / or of at least one specific protein for microfibrils, such as fibrillin 1 or 2 and / or fibulin 3 or 5 and / or at least one glycosaminoglycan (GAG), particularly of a sulfated GAG.

According to a particular embodiment, the substance or composition is used to perform a care selected from the group consisting of restructuring the matrix cellular and in particular that of the dermis, of the UDE, of the wall of the cutaneous blood vessels, the hypodermis, the epidermis and in particular of the dermis by restructuring the matrix dermal, improve skin hydration, promote repair of the skin, maintain the integrity of the dermis, improve the epidermal differentiation and reinforce the renewal of the epidermis, as well as any one of the combinations of these cares.

These different uses are intended particularly prevent or fight against skin disorders linked to the degradation of FGF-2.

Advantageously, the substance or composition is used to fight skin aging, particularly in a human being.

Advantageously, the concentration of said substance is an effective concentration for the protection of FGF-2, particularly by topical application and It is particularly between 0.01% and 10% by weight of The total composition.

The composition is particularly a cosmetic, dermocosmetic or pharmaceutical composition, particularly for the human being.

The substance in accordance with this invention is preferably a plant extract that protects FGF-2 of its degradation and in particular an extract aqueous or hydroalcoholic, preferably hydro-glycolic.

According to a preferred embodiment, the substance is an aqueous or hydroalcoholic extract of Hibiscus abelmoschus obtained from the grain of Hibiscus abelmoschus .

According to a second aspect, the present invention refers to the use of a substance for the manufacture of a composition, particularly cosmetic, pharmaceutical or dermocosmetic, to protect FGF-2 of its degradation, particularly in different uses mentioned above.

The present invention also relates to a cosmetic, pharmaceutical or dermocosmetic care method that use a previously defined composition according to a any of the embodiments, particularly to perform a care attached to at least one of the different uses mentioned above.

Detailed description of the present invention

The present invention allows obtaining a protection of FGF-2 by proteoglycans of This same matrix.

It is understood as "protection of FGF-2 "FGF-2 protection against its degradation or its denaturation. This degradation or denaturation usually occurs in the form of a lysis due to environmental conditions, such as an enzymatic or thermal lysis. This degradation or denaturation may occur due to the decreased protection ensured by proteoglycans.

The selection procedure according to the The present invention has allowed to select different principles assets extracted from vegetables. No extract has been described plant that protects FGF-2 from its degradation to skin level, particularly in humans, having used only substances whose structure is close to the of heparan sulfate.

A particularly effective and unexpected active ingredient has been identified to protect FGF-2 from thermal degradation; It is an aqueous extract obtained from the grain of a plant that belongs to the Malvaceae family, more particularly to the genus Hibiscus and even more particularly to Hibiscus abelmoschus or Hibiscus.

Hibiscus abelmoscus was particularly known in the form of an aqueous extract obtained from the whole plant, only for its slimming properties it is cosmetic and more particularly to fight cellulite (US5705170).

Other extracts that have the desired activity for the present invention have been selected. Its activity is particularly unexpected, in particular as regards the protection of FGF-2. The extracts are particularly an aqueous extract of Lycium chinense or Lycian berries obtained from dehydrated whole berries, an aqueous extract of Pinelliae ternata or Banha, obtained from the tuber, an aqueous extract of Raphanus sativus or Radish obtained from the grain , an extract of Brassica juncea or mustard obtained from the grain, an aqueous extract of Coicis semen or Yi yi ren obtained from the grain, an extract of Hordeum vulgare or barley obtained from the grain, an extract of Sesamum indicum or sesame dorado obtained from the grain and an extract of the complete algae of Lessonia sp or Lessonia.

However, these extracts are not limited. only to aqueous extracts. Thus, the present invention it also covers any polar solvent that allows obtaining essentially the set of active compounds in an extract aqueous to provide the desired properties in the field of present invention According to the tests carried out by the present inventors, hydroalcoholic extracts allow Get the desired properties. Among the extracts Hydroglycolics, an extraction solvent of the type is preferred water / glycol A mixture of water / butylene glycol.

The person skilled in the art can modify the proportions of the water / alcohol mixture. The proportions of Water / alcohol generally vary between 10/90 and 90/10, and for example between 50/50 and 85/15.

It is preferable to use as active ingredient in the present invention an extract in solution obtained from between 1 and 10% of a plant or part of the plant in state dry with respect to the weight of the total solution.

The composition according to the present invention advantageously comprises between 0.001% and 20%, preferably between 0.01 and 10%, of active ingredient of according to the present invention by weight of the total composition. The composition can be applied topically or administered by orally.

The method of selecting a substance potentially active for the protection of FGF-2 in regards to its degradation in the human being, it includes particularly:

a) contacting a substance potentially active and FGF-2 at a temperature of about 45 ° C for a period of about 2 h,

b) the selection of substances that protect FGF-2 of at least 50%, preferably 65%, of its degradation.

Advantageously, the selection method comprises a supplementary stage of penetration testing transcutaneous to select an active substance that crosses the skin barrier

In accordance with another embodiment, the present invention refers to a method of selection of active substances from vegetables.

Advantageously, the present invention relates to to the method of preparing a composition, which comprises the use of said selection method followed by a mixing step of the selected active substance with an excipient for Composition preparation

Other purposes, characteristics and advantages of the invention will be apparent to the person skilled in the art after reading the explanatory description that it does reference to the examples provided only by way of illustration and that would not be in any way limiting the scope  of the invention.

The examples are an integral part of the present invention and any feature that appears as novelty with respect to any prior art state from the description taken as a whole, including examples, it is an integral part of the invention in its function and in its generality

In this way, each example has a scope general.

On the other hand, in the examples, all percentages are given by weight, unless otherwise indicated, and the temperature is expressed in degrees Celsius unless indicated the opposite and the pressure is atmospheric pressure, unless it indicate otherwise.

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Examples Example 1 Study of thermal degradation of FGF-2

Tardieu et al. , In 1992, studied the thermal degradation of FGF-2 in a fibroblast proliferation model by incorporating a radioactive element. The present inventors have developed a method that allows to study the stability of FGF-2: this method of quantification usable on a large scale allows the realization of a selection of active principles, which was not possible with the method described by Tardieu.

A solution of 5 ng / ml of FGF-2 in 10 mM-BSA PBS buffer at 0.1% and 0.1% methyl paraben.

This solution is placed at different temperatures: 4ºC, 20ºC, 37ºC, 50ºC and 80ºC in the presence or not of heparin at 500 µg / ml.

The dosages of FGF-2 are performed with a commercial ELISA kit (R&D system, France) in different times (T = 0, 3 h, 24 h and 48 h) to establish a degradation kinetics for each study temperature.

The results are expressed as a percentage of FGF-2 with respect to time T = 0. The Experiments are performed in triplicate (n = 3). The results obtained are shown in Figures 1 and 2.

FGF-2 is sensitive to temperature conditions to which it is subjected.

Figure 1 shows the evolution of the FGF-2 stability as a function of time at Different temperatures FGF-2 stored at 4 ° C It is relatively stable over a period of 48 hours since the 20% of FGF-2 degrades at this temperature. With the temperature rise, degradation rates increase: after 3 h at 37 ° C, FGF-2 undergoes 65% of degradation and at 50 ° C 80% degradation. Finally after 3 h at 80 ° C, a total degradation of FGF-2 due to a denaturation of the proteins at this temperature.

Finally, the longer the period of longer time is the degradation observed, after 24 h at 37 ° C degrades 80% of FGF-2.

Figure 2 represents the stability of FGF-2 at different temperatures in the presence of Heparin Figure 2 shows that this degradation stops through the presence of 0.05% heparin which is the control positive of the experiment and that because of its great affinity for FGF-2 stabilizes it. Heparin is a polysaccharide highly sulphated known for its great affinity for FGF-2.

This protection is not effective at 80 ° C since Heparin also denatures at this temperature.

Surprisingly, the degradation of FGF-2 at approximately 50 ° C occurs in a period of short time. For practical reasons, a temperature is selected close to 45ºC, and constitutes a parameter of choice to perform A stress model to select active ingredients. He positive control of the experiment is preferably constituted by heparin.

Example 2 In vitro model for the selection of an active ingredient that protects FGF-2 against thermal degradation

The degradation of FGF-2 observes at approximately 50 ° C it is particularly interesting, since a Short period of time allows degradation of FGF-2 of approximately 80%, which allows This mode perform a large number of tests. For this reason, it has decided to make a selection with stress conditions of 2 h 15 minutes at 45 ° C and study the capacity of a large number of active ingredients to protect FGF-2 against This thermal degradation.

The selection model applied is the next:

180 µl of a solution of FGF-2 at 4.5 ng / ml prepared in buffer PBS-0.1% BSA in a 96-well microplate. Be add 20 µl of distilled water or 0.05% heparin or a solution containing different active ingredients extracted from Different plants to test. The plate is shaken with the help of a plate stirrer and then coated with an adhesive strip and it place in an oven at 45 ° C for 2 h 15 minutes. At the end of incubation period, the plaque is removed and then a FGF-2 dosage with ELISA kit.

A plate of room temperature is left at room temperature control, which only contains FGF-2 and water distilled treated under the same conditions.

The results are expressed as a percentage of FGF-2 protection with respect to the control of FGF-2 not under stress or not degraded.

Example 3 Validation of the model through the GAG and proteoglycans of the skin

Trials have been conducted with the GAG and PG available in the market to validate the model.

GAG solutions are made to different concentrations: dissolve heparan sulfate, dermatan sulfate, Chondroitin sulfate and hyaluronic acid and then tested at 0.1% - 0.01% - 0.001%.

The heparin used as a positive control is test 0.1%, 0.01%, 0.001%, 0.0001% and 0.00001%.

The proteoglycans tested are the Glipicano-3 and decorin tested at 0.1%, 0.01% and 0.001%.

The stress protocol described in the example 2, replacing the 20 µl of water with 20 µl of the corresponding GAG or PG solution. The GAG and PG tested they thus experience a supplementary dilution to 1/10.

Figure 3 illustrates the study of protection of FGF-2 with GAG of the skin.

The results obtained in figure 3, confirm the bibliography data, namely:

Heparan sulfate, as well as dermatan sulfate, protect FGF-2 and the latter at each concentration tested. In effect, dermatan sulfate is the main promoter of FGF-2 activity during healing processes The hyaluronan, whatever its molecular weight, in no case allows to protect FGF-2. With respect to chondroitin sulfate, the protection it provides depends on your dose, although It doesn't get 100%.

The test of 2 proteoglycans, figure 4 (Study of the protection of FGF-2 with PG), the glipicano-3 and decorin has confirmed the data provided in the bibliography. Namely, that the glypican-3 that has heparan sulfate chains protects FGF-2 in a dose-dependent manner, while decorin (composed of chondroitin chains sulfate and dermatan sulfate) is ineffective.

This study shows that the protection of FGF-2 depends on the structure of the GAG. The heparan sulfate interaction with FGF-2 is very valid. This interaction is confirmed when tested in the model. the PG that has heparan sulfate chains.

This study has allowed us to validate our model of selection by confirming the results given In the bibliography.

Example 4 Selection of active ingredients

The selection has been made in accordance with the example 2, from a library composed of 2000 extracts Vegetables and characterized molecules. These compounds, plant extracts, seaweed or molecules characterized, dissolved are tested pure, or 10% or 1% in the medium of reaction described in example 2.

The results of the active ingredients more Effective are described in Table 1, below.

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TABLE 1 Results obtained during selection

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one

2

3

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The above extracts are obtained preferably by mashing a part of the plant in a mixture of water / alcohol, preferably water / glycol between 100/0 and 0/100 (v / v). The dilution is then performed in this solvent or solvent mixture.

Certain active ingredients tested present a great protection activity that is also dependent on the dose.

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Example 5 Hibiscus abelmoschus extract (Hibiscus)

5a- A hydroalcoholic extract of Hibiscus from 5% milled grains (w / w) in ethanol reflux temperature. Extraction is performed for 1 hour and after the solution is filtered, the ethanol is removed, and the result dissolve at 5% (w / w) in a mixture of water / glycol (75/25) and then it is ultrafiltered with ceramic filters of different cut thresholds and finally filtered at 0.45 µm.

5b- A hydroroglycol extract of Hibiscus in a mixture of water (75%) / BG (25%) preferably at from 5% ground grains (w / w). The maceration is done for 1 night at 4 ° C and then the solution is ultrafiltered with ceramic filters with different cut thresholds and finally filter at 0.45 µm. BG means butylene glycol.

5c- An aqueous Hibiscus extract is made in water preferably from 5% ground grains (w / w). The maceration is carried out for 1 night at 4 ° C and then the solution it is filtered in ceramic filters with different cut thresholds and finally filtered at 0.45 µm.

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Example 6 Lycian Berry Extract

An extract of Lycian Berries is made preferably in a mixture of water (75%) / BG (25%) from 5% dehydrated whole berries (w / w). The maceration is done for 1 night at 4 ° C and then the solution is ultrafiltered in ceramic filters with different cut thresholds and finally filter at 0.45 µm.

Example 7 Banha extract ( Pinellia ternata )

An extract of Banha ( Pinellia ternata ) is preferably made in a mixture of water (75%) / BG (25%) from 5% tubers (w / w). The maceration is carried out for 1 night at 4 ° C and then the solution is ultrafiltered in ceramic filters with different cut thresholds and finally filtered at 0.45 µm.

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Example 8 Radish Extract

Radish extract is preferably made in a mixture of water (75%) / BG (25%) from 5% grains (p / p). The maceration is carried out for 1 night at 4 ° C and then the solution is ultrafiltered in ceramic filters with different cut thresholds and finally filtered at 0.45 µm.

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Example 9 Lessonia Extract

An extract of Lessonia is made preferably in a mixture of water (75%) / BG (25%) from 5% complete algae (w / w). The maceration is done for 1 night at 4 ° C and then the solution is ultrafiltered in ceramic filters with different cut thresholds and finally filtered at 0.45 \ mum.

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Example 10 Mustard Extract

Mustard extract is made preferably in a mixture of water (75%) / BG (25%) from 5% grain (w / w). The maceration is carried out for 1 night at 4 ° C and then the solution is ultrafiltered in ceramic filters with different cut thresholds and finally filtered at 0.45 \ mum.

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Example 11 Yi yi ren extract

An extract of Yi yi ren is made preferably in a mixture of water (75%) / BG (25%) from 5% grain (w / w). The maceration is carried out for 1 night at 4 ° C and then the solution is ultrafiltered in ceramic filters with different cut thresholds and finally filtered at 0.45 \ mum.

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Example 12 Barley Extract

A barley extract is preferably made in a mixture of water (75%) / BG (25%) from 5% grain (p / p). The maceration is carried out for 1 night at 4 ° C and then the solution is ultrafiltered in ceramic filters with different cut thresholds and finally filtered at 0.45 µm.

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Example 13 Sesame Extract

Sesame extract is preferably made in a mixture of water (75%) / BG (25%) from 5% grain (p / p). The maceration is carried out for 1 night at 4 ° C and then the solution is ultrafiltered in ceramic filters with different cut thresholds and finally filtered at 0.45 µm.

Each extract (examples 5 to 13) has been preserved partly. The optional preservative, preferably composed of a mixture of Caprilylglycol, Phenoxyethanol, 1% Hexylene Glycol, is add in the end in the presence or not of xanthan.

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Example 14 Selection of active ingredients or plant extracts

The most effective extracts are selected and test at different concentrations according to the selection from example 2. The active ingredients used are the extracts of examples 5 to 13 and are diluted using a solvent (or solvent mixtures) used for extraction. One is tested concentration range that varies between 0.1% and 10% in this solvent to study the specificity of active ingredients stops with thermal degradation of FGF-2. The Dosages are done in triplicate. The results obtained are shown in table 2, below:

TABLE 2 Effects of the doses obtained for the principles assets selected during selection

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The extracts are very effective and all present a dose-dependent activity that results in a specificity of the activity of each active principle on a given target.

Hibiscus abelmoschus (hibiscus) extract has the highest activity and is therefore an active principle of choice for the protection of the growth factor FGF-2.

In examples 15 to 21 the concentration of active ingredient (extract 5c) is in percentage by volume (v / v) in water .

Example 15 Study of the protection of FGF-2 with the aqueous extract of Hibiscus abelmoschus (hibiscus) prepared according to example 5c (extract 5c)

A study of the protection of FGF-2 with 5c extract in 2 industrial lots. Protection against stress has been studied according to conditions of example 2.

The positive control composed of heparin 0.01% tested in the final reaction medium after Pre-dilution in water protects 105.52% ± 3.04.

The results obtained are presented in the Table 3, below:

TABLE 3 Protection of FGF-2 at 45ºC with 2 5c industrial extract lots

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Extract 5c protects FGF-2 in a dose-dependent manner with great activity for a I% concentration of active substance in water. The results obtained for 2 industrial lots are substantially equal.

It was essential to verify that the extract 5c could protect FGF-2 at physiological temperature, that is to say at 37 ° C. Therefore, a study consists of dosing the FGF-2 after 24 h at 37 ° C in absence or in presence of increasing concentrations of extract 5c.

The positive control composed of heparin at 0.01% protects 110.53% ± 3.20.

The results obtained are presented in the Table 4, below:

TABLE 4 Protection of FGF-2 at 37 ° C with 2 5c industrial extract lots

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The results obtained show that the 5c extract protects FGF-2 at temperature Physiologically and dose-dependently. 5c extract best protects, at this temperature, FGF-2 that degrades less than at 45 ° C.

Example 16 It refers to the study of cytotoxicity of extract 5c in monolayer fibroblasts through a dosage of PNPP

The objective was to study the cytotoxicity of 5c extract on normal human fibroblasts.

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

Once the confluence of the normal human fibroblasts, culture media were replaced with 200 µl / well of medium added or not (control) with the 5c extract in increasing concentrations: 0.1; 0.5; one; 2; 3 and 5%.

The cells were incubated for 48 hours in a stove at 37 ° C.

After incubation and removal of culture media, the cells were rinsed twice with PBS (Phosphate Buffered Solution) and then left in the presence of 200 µl of a buffer containing 0.1 M sodium acetate (pH 8), 0.1% Triton X-100 and phosphate 5 mM p-nitrophenyl (Sigma, France). After two hours of incubation at 37 ° C in an atmosphere containing 5% CO2, The reaction was stopped by the addition of 20 µL of NaOH 1 N. The absorbance of the media was then determined reaction at 405 nm with the help of a plate reader (Victor 2 V, Perkin Elmer, Finland).

Non-enzymatic hydrolysis of phosphate p-nitrophenyl was determined during each experiment in wells that did not contain cells ("White"). All measurements were made by six-fold (n = 6).

The results obtained are presented in the Table 5, below:

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TABLE 5 Study of cytotoxicity in human fibroblasts normal

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The 5c extract is not cytotoxic in the whole range of concentration tested.

Example 17 Study of fibroblast proliferation by FGF-2 protected with extract 5c

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

A solution of FGF-2 is prepared extemporaneously (= FGF-2 not degraded); be place a solution of FGF-2 for 24 h at 37 ° C in absence (= degraded FGF-2) and in the presence of 0.01% heparin (= FGF-2 + Heparin at 37 ° C).

Each solution of FGF-2 is diluted to apply it on the cells, the final concentrations of FGF-2 are the following: 0.1, 0.25, 0.5, 0.75 and 1 ng / ml

Human dermal fibroblasts are seeded normal at reduced density in 6-well plates. Then they are grown in a medium in the absence (control) or in the presence of FGF-2 at different concentrations.

After 48 hours the activity of the intracellular acid phosphatases (PNPP), which allows to evaluate the effect of FGF-2 solutions, treated or not, on cell proliferation.

The results are expressed as a percentage of proliferation with respect to the control well, that is to say untreated well.

The results obtained are represented in the Table 6 below and in Figure 5:

TABLE 6 Proliferation study (expressed in% with with respect to the untreated well) obtained after treatment with different concentrations of FGF-2 no degraded, different concentrations of FGF-2 gradient and different concentrations of FGF-2 + heparin at 37 ° C

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Figure 5 represents a study of the fibroblast proliferation in the presence of FGF-2 not degraded, FGF-2 degraded and FGF-2 + heparin at 37 ° C.

The results obtained show that the FGF-2 (not degraded) stimulates the proliferation of normal human fibroblasts in culture and in a manner dependent on the dose.

FGF-2 degraded at 37 ° C stimulates proliferation but in a much smaller way than FGF-2 not degraded at 37 ° C. Indeed, for each concentration tested, proliferation in the presence of FGF-2 gradient is statistically smaller than that obtained with the non-degraded FGF-2.

This example demonstrates the relevance of develop an active ingredient capable of protecting FGF-2 of the matrix that degrades at temperature physiological and can not ensure its role of renewal of dermal cells

By protecting FGF-2 from thermal degradation, the heparin used at 0.01% allows the FGF-2 retain biological properties while proliferation is maintained or even that obtained with the FGF-2 solution prepared extemporaneously; to the concentration of 0.5 ng / ml.

The greatest stimulation in the presence of heparin, observed with the reduced amounts of FGF-2, no It is observed with higher amounts of FGF-2.

The same experiment has been performed replacing heparin with extract 5c. The solution of FGF-2 in the presence of 0.5% of extract 5c is place 24 h at 37 ° C. The solution is then diluted to apply 0.5 ng / ml of FGF-2 in the wells that They contain fibroblasts.

The concentration of extract 5c applied finally on the cells is 0.0025%. It has been verified previously that this concentration of extract 5c was not able to induce a stimulation of fibroblast proliferation. Which means that the stimulation of proliferation observed It is due to the protection of FGF-2 with extract 5c (see table 8).

The results obtained, expressed in proliferation percentage with respect to the untreated control, presented in table 7, below:

TABLE 7 Study of the proliferation (in percentage) of fibroblasts obtained in the presence of FGF-2 not degraded to 0.5 ng / ml, FGF-2 degraded to 0.5  ng / ml and FGF-2 + 0.5% extract 5c a 37 ° C

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Figure 6 represents the study of the fibroblast proliferation in the presence of FGF-2 not degraded to 0.5 ng / ml, FGF-2 degraded to 0.5 ng / ml and FGF-2 in the presence of 0.5% extract 5c at 37 ° C.

The co-incubation of FGF-2 and extract 5c at 37 ° C allows to protect FGF-2, which retains its ability to stimulate proliferation of normal human fibroblasts in culture. The obtained results allow to obtain a proliferation of fibroblasts that are not statistically different from those obtained in presence of FGF-2 prepared in a manner extemporaneous

The interest of an active principle that allows protect FGF-2 arises from an interest Main for the renewal of fibroblasts in cells.

A proliferation study has been carried out of normal human fibroblasts in the presence of increasing concentrations of extract 5c for 48 h at 37 ° C.

Fibroblasts are sown in the presence of 5c extract at different concentrations 0.0025%, 0.01%, 0.25%, 0.5%, 1% and 2%. After 48 h, a dosage of acid phosphatases (PNPP dosage) at 405 nm and then calculate the proliferation (in%) with respect to the negative control (= untreated cells). Each condition is tested with n = 6.

The results obtained are presented in the Table 8, below:

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TABLE 8 Study of fibroblast proliferation in presence of extract 5c

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The positive control composed of 10% serum allows to stimulate proliferation significantly, which validate the experiment (T + = + 126% * proliferation).

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

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Example 18 Impact of the protection of growth factors on the  collagen synthesis

Normal human fibroblasts obtained from a 52-year-old donor, which have been grown in 96 plates wells with medium (composed of DMEM, 2 mM glutamine, Penicillin 50 IU / ml-streptomycin 50 µg / ml, fetal serum 10% bovine) for 24 h. After incubation, the cells treated with products composed of vitamin C at 20 µg / ml (positive control) and extract 5c at 2% and 1%, for 48 h. Be In parallel he performed a control with the medium only.

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

At the end of the experiment, the supernatants for the incorporation of proline dosage radioactive intracellular proteins.

The results obtained are presented in the Table 9, below:

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TABLE 9 Study of the stimulation of collagen synthesis with 5c extract

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Extract 5c tested at 2% and at 1 / o was able to stimulate the synthesis of collagen in fibroblasts normal humans and significantly.

The results obtained demonstrate that the protection of the growth factors of the medium and of the synthesis by means of fibroblasts, allows to stimulate in vitro the synthesis of collagen.

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Example 19 Impact of the protection of growth factors on the  synthesis of total GAG and sulphated GAG

The purpose of this study was to determine if the protection of growth factors with extract 5c in monolayer fibroblast cultures allowed to stimulate synthesis Total GAG using a radioactive method.

Normal human fibroblasts obtained from a 52-year-old donor was grown in 96 plates wells with medium (composed of DMEM, 2 mM glutamine, Penicillin 50 IU / ml-streptomycin 50 µg / ml, fetal serum 10% bovine) for 24 hours. After incubation, the cells were treated with products composed of vitamin C a 20 µg / ml (positive control) and 2% and 1% 5c extract, for 72 h. A control was carried out in parallel with the medium only.

After 48 hours of contact, it was added to the medium radioactive glucosamine (D- (6 - 3 H) -glucosamine).

At the end of the experiment, the supernatants for glucosamine incorporation dosage radioactive in intracellular proteins. The dosage of Protein is done with a commercial kit (BioRad 500-0116).

The results are presented in the following table 10:

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TABLE 10 Study of the stimulation of GAG synthesis totals with extract 5c

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The results obtained show that the 5c extract applied at 2% and 1% in human fibroblasts normal was able to stimulate total GAG synthesis with a dose-dependent effect and to a greater extent than the control positive reference (TGF-? applied to 10 ng / ml).

The results obtained show that the protection of the growth factors of the medium and their synthesis by fibroblasts, allows to stimulate the synthesis of total GAGs in vitro .

In the same way a study of incorporation of 35 S-sulfate to quantify sulfated GAGs. The protocol applied is the same as that of the Total GAGs, except for the nature of the precursor radioactive The results are shown in table 11, a continuation:

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TABLE 11 Study of the stimulation of GAG synthesis sulfated with extract 5c

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The results obtained show that the 5c extract allows to stimulate very significantly and in a way Dose dependent synthesis of sulfated GAG.

Which means that in addition to the role of imitation that extract 5c plays with respect to FGF-2, extract 5c is capable, protecting growth factors, to stimulate the synthesis of sulfated GAGs and therefore strengthens the protection of FGF-2 since these same sulfated GAGs have heparan sulfate that protect FGF-2.

Example 20 Study of the protective activity of FGF-2 with 5c extract after transcutaneous permeabilization

The purpose was to ensure that the extract 5c retained its protective properties of the factor of growth after penetrating the skin.

A solution of pure 5c extract was deposited on the surface of skin explants, mounted on cells of Franz

After 24 h of incubation, the media contained in the receptor compartments of the cells are recovered and lyophilized. Lyophilisates dissolved then in distilled water to finally test on the model described in example 15 with a thermal stress of 37 ° C for 24 h.

Figure 7 represents the study of the FGF-2 protection with 5c extract after I pass through the skin.

The results obtained (99.96% protection) demonstrate that extract 5c has retained its properties of full protection since the result obtained is identical to that obtained in table 4 of example 15.

The results show that after penetrate the skin tissues extract 5c is capable of protect growth factors and therefore remains fully capable of achieving its objective.

Example 21 Study of the protection of FGF-2 against enzymatic degradation

The purpose of this study was to quantify the degradation of FGF-2 with proteinases present at Dermis level of human skin. Enzymes that play a role important in the degradation of the extracellular matrix.

Particularly, cathepsin G involved during inflammation and is secreted in the extracellular environment. This enzyme adheres strongly to the cell surfaces of the matrix thanks to its basic character. Cathepsin G is also active against collagen but also against the core protein of proteoglycans and matrix glycoproteins.

For this reason, a study of the degradation of FGF-2 with cathepsin G. It has performed a degradation kinetics in 100 mM Hepes buffer, at pH 7 containing 0.1% BSA. Cathepsin G has been added in the plate wells at a concentration of 0.02 U per well at a  FGF-2 solution at 2 ng / well. The plate is incubate at 37 ° C and then prewash for 15, 30, 60, 90 and 120 minutes to dose the FGF-2 remaining

Control is performed (100%) with a extemporaneous dosage of FGF-2. In parallel it place a control solution without enzyme at 37 ° C to study the thermal degradation in different periods of time, to rule out this degradation due to temperature and quantify the part due to enzymatic degradation alone.

The amounts of FGF-2 (pg / ml) obtained are described in table 12, below:

TABLE 12 Study of the degradation of FGF-2 with cathepsin G

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The results obtained show that Over time the degradation of FGF-2 increases. The enzyme allows to degrade FGF-2 very significant at 90 minutes.

This is why this time will be selected for study the protection of FGF-2 in the presence of Cathepsin G with extract 5c.

To the reaction medium described above (example 21) different concentrations of extract are added 5c, namely: 0.01%, 0.05%, 0.1%, 0.5% and 1%.

After 90 minutes at 37 ° C, the FGF-2 concentrations and the percentages of FGF-2 remaining as well as FGF-2 protection percentages. For each The concentration tested is dosed with n = 9. The Results are described in Table 13, below:

TABLE 13 Study of protection with extract 5c of FGF-2 degraded with cathepsin G

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The 5c extract is able to protect FGF-2 against enzymatic degradation of form dose dependent.

This in vitro study demonstrates that the application of extract 5c can protect growth factors from proteolytic degradation exerted by extracellular matrix enzymes. This reinforces the interest of the use of extract 5c in cosmetics.

Example 22 Demonstration of the FGF-2 index with the age

The purpose of this study was to study the FGF-2 concentration of so-called skins "young" and called "aged" to establish a relationship between age and loss of content of FGF-2 due mainly to the modification of the GAG content of the skin.

Skin biopsies obtained from donors called young people (21, 30, 31 and 38 years) and obtained from donors denominated aged (50, 55, 57 and 58 years) have been prewash and extracted after grinding in a PBS buffer at pH 7 containing 0.1% triton X100, to perform a dosage of FGF-2 with an ELISA commercial kit (R&D system, DFB50). The concentration of FGF-2 is related to the DNA index measured with a commercial Picogreen kit.

The results obtained are represented in the figure 8.

Figure 8 represents a study of the concentration of FGF-2 in skin hydrolysates called "young" and "aged."

The results show a difference of FGF-2 content between young skins and aged skin In addition, this difference is statistically significant (p = 0.046).

This example shows that the use of an active substance as a protector of factors of growth and more particularly of FGF-2 allows to fight against the effects linked to aging cutaneous.

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Example 23 Formulation of the products of the invention

Proceed according to known methods by the specialist in the art to mix together the different parts A, B, C, D, E or F to prepare a composition in accordance with the present invention. The "products of the invention "represent the active ingredients mentioned in the present invention

Use of the products of the invention in cosmetic or pharmaceutical formulations of oil emulsion type in water

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Formulation 23a:

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Formulation 23b:

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Formulation 23c:

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Example 24

Of the invention

Use of the products of the invention in a water-in-oil type formulation.

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Example 25

Of the invention

Use of the products of the invention in a shampoo or shower gel formulation.

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Example 26

Of the invention

Use of the products of the invention in a formulation of lipstick type and other anhydrous products.

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Example 27

Of the invention

Use of the products of the invention in a formulation of aqueous gels (eye contours, products slimming, etc.).

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Example 28

Of the invention

Use of the products of the invention in a triple emulsion type formulation.

W1 / O primary emulsion

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Secondary emulsion W1 / O / W2

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Example 29

Of the invention

Preparation of pharmaceutical formulations that They contain the product of the invention.

Formulation 29th

Tablet preparation

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Formulation 29b

Preparation of an ointment

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Formulation 29c

Preparation of an injectable formula

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Example 30 Evaluation of the cosmetic acceptance of a preparation that contains the product of the invention

Toxicology tests have been performed in the compound obtained according to example 5c, with a skin and eye evaluation in rabbit, with study of the absence of abnormal toxicity by single oral administration in rat and with the study of the sensitization power in guinea pig.

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Evaluation of primary skin irritation in rabbit

The preparations described in example 5c are apply without dilution to the dose of 0.5 ml on the skin of 3 rabbits according to the method recommended by the OECD directive in reference to the study of "irritant / corrosive effect exerted on the skin".

Products are classified according to the criteria defined by the document of 1/2/1982 published in the JORF of 02/21/1982.

The results of these tests allow conclude that the products of the invention are classified as not skin irritants

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Evaluation of eye irritation in rabbit

The preparations described above have been instilled pure once, at a rate of 0.1 ml, in the eye of 3 rabbits according to the method recommended by the directive of the OECD No. 405 of February 24, 1987 with reference to the study of "irritating / corrosive effect on the eyes".

The results of this trial allow us to conclude that preparations can be considered as non-irritating to the eyes, within the meaning of the EEC directive 91/326 used pure or undiluted.

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Trial on the absence of abnormal toxicity with administration oral only in rat

The preparations described have been administered once orally at the dose of 5 g / kg body weight, at 5 male rats and 5 female rats according to an inspired protocol in OECD directive No. 401 of February 24, 1987 and adapted to cosmetic products.

It has been discovered that DL0 and DL50 are greater than 5000 mg / kg. The preparations tested are not they therefore rank among dangerous preparations by ingestion.

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Evaluation of skin sensitization potential in guinea pig

The preparations described are tested of maximization described by Magnusson and Kligmann, protocol of agreement with the OECD Guideline No. 406.

Preparations are classified as no Sensitizers by skin contact.

Claims (12)

1. Use of a plant extract that is not a sulfated glycosaminoglycan (sulfated GAG), nor an analogue structure of a sulphated GAG, which protects FGF-2, as active ingredient of a composition cosmetic, dermocosmetic or pharmaceutical to prevent or fight against at least one skin disorder linked to the degradation of FGF-2. 2. Use according to any one of the preceding claims, characterized in that said plant extract is selected from the group consisting of an extract of Hibiscus abelmoschus or hibiscus , an extract of Radish , an extract of Lycian berries, an extract of Banha , an extract of Lessonia , an extract of mustard flour, an extract of Yi yi ren , an extract of Barley, an extract of Sesame or one of the combinations that result from the association of at least two the mentioned extracts. 3. Use according to any one of the preceding claims, characterized in that FGF-2 is protected at the level of the epidermis and / or dermoepidermal junction and / or dermal extracellular matrix and / or hypodermis and / or the wall of cutaneous blood vessels. 4. Use according to any one of the preceding claims, characterized in that the skin alterations linked to the degradation of FGF-2 occur at the level of the epidermis and / or dermoepidermal junction and / or dermal extracellular matrix and / or of the hypodermis and / or the wall of the cutaneous blood vessels. 5. Use according to any one of the preceding claims, characterized in that said plant extract is an aqueous or hydroalcoholic extract, preferably in solution, obtained from 1 to 10% of a plant or part of a plant in state dry with respect to the weight of the solution. 6. Use according to any one of the preceding claims, characterized in that the degradation of the growth factor is the result of at least one degradation, particularly thermal degradation, which occurs particularly at physiological temperature or by exposure to heat and / or an enzymatic degradation, particularly by cathepsin G. 7. Use according to any one of the preceding claims, characterized in that said plant extract or composition is used to stimulate the proliferation of skin cells, in particular the proliferation of fibroblasts and / or melanocytes and / or to increase synthesis of at least one component of the matrix, in particular of at least one type of collagen and / or at least one specific microfibril protein, such as fibrillin 1 or 2 and / or fibulin 3 or 5 and / or at least a glycosaminoglycan (GAG), particularly of a sulfated GAG. 8. Use according to any one of the preceding claims, characterized in that said plant extract or composition is used to analyze a care selected from the group consisting of restructuring the extracellular matrix and in particular the dermis by restructuring the dermal matrix, improving the hydration of the skin, promote the repair of the skin, maintain the integrity of the dermis, improve epidermal differentiation and reinforce the renewal of the epidermis as well as any one of the combinations of these cares. 9. Use according to any one of the preceding claims, characterized in that said plant extract is used to fight against skin aging, particularly in a human being. 10. Use according to any one of the preceding claims, characterized in that said plant extract is used at an effective concentration for the protection of said growth factor, particularly by topical application or by oral administration, and particularly at a concentration between the 0.01% and 10% by weight of the total composition. 11. Use according to any one of the preceding claims, characterized in that the composition is a cosmetic or dermocosmetic or pharmaceutical composition, particularly for the human being. 12. Use according to any one of the preceding claims, characterized in that said plant extract is an extract of aqueous or hydroalcoholic Hibiscus abelmoschus obtained from the grain of Hibiscus abelmoschus .