FR2907225A1 - Screening of a substance potentially active for the protection of fibroblast growth factor-2 regarding its degradation in humans, comprises contacting a potentially active substance with the growth factor and selecting the substances - Google Patents

Abstract

Screening of a substance potentially active for the protection of fibroblast growth factor-2 (FGF-2) regarding its degradation in humans, comprises: contacting a potentially active substance with FGF-2 at a about 45[deg] C for approximately 2 hours and selection of substances that protect FGF-2 by at least 50%, preferably 65% of its degradation. An independent claim is included for the preparation of a composition containing a potentially active substance for the protection of FGF-2 regarding its degradation in human beings, comprising implementation of the screening method to identify an active substance for the protection of FGF-2, followed by mixing the selected active substance with a vehicle.

Description

Division of the application FR 06 54316 The invention relates to the development

  of active ingredients for a cosmetic, dermo-cosmetic or pharmaceutical application, for combating the degradation of at least one growth factor. The invention relates in particular to the protection of the degradation of Fibroblast Growth Factor (FGF-2 or FGF basic or FGF- [3]. The present invention relates in particular to the use of active principles that prevent, limit, or improve the quality of the dermis, especially when the latter undergoes the effects of age, especially in a human being. STATE OF THE ART Many growth factors occur at the cutaneous level and in particular FGF-2, which has a broad spectrum of activity: in particular the proliferation of fibroblasts, thus making it possible to synthesize the macromolecules of the matrix, which are essential for the production of integrity of the skin. FGF-2 is protected in the skin by heparan sulfate proteoglycans. In the 90s, Feige & Baird (Medicine / Sciences, 1992; 8: 805-10) described the close relationship between growth factors and extracellular matrix proteoglycans, explaining that in addition to a reservoir phenomenon, interaction protected the growth factors against proteolysis. This results in greater stability of the growth factors (an extended in vivo half-life), thus enabling them to perform their functions to the best of their ability. FGF-2 and proteoglycans FGF-2 is part of a family made up of 23 different FGFs currently counted. FGF-2 is in several isoforms. In vertebrates, five isoforms are found with molecular weights of 18; 22; 22.5; 24 and 34 kDa. Only the 18 kDa form is detected outside the cells, the others are confined inside the cell and more precisely in the nucleus. FGF-2 is a ubiquitous protein that plays a very important role physiologically; FGF-2 is involved in embryonic development, angiogenesis, neuronal differentiation and among other things tissue repair. It is indeed present in most tissues with a more specific distribution at the level of the basement membrane thereof. Although FGF-2 is present in the body in such a proportion that it can be purified and characterized, its mRNA is not detectable. This fact and the peculiarity that it is distributed intimately bound to the basal membrane of the tissues suggests that it is produced at an initial level and then released by the cells during development to be stored in the MEC (basement membrane). undergoes during this process an intense remodeling allowing the local release of FGF-2). This storage would allow FGF-2 release in the adult state when needed to participate in tissue repair and maintain differentiated functions. With age, the stock of FGF-2 would decrease thus no longer ensuring maximum activity conditions. Proteoglycans (PGs) are potential reservoirs of the extracellular form of FGF-2 of 18KDa in the extracellular matrix. Proteoglycans are molecules with extracellular, membrane or intracellular localization. They consist of a protein chain called "core protein" on which are grafted glycosaminoglycans (GAG) variables. The main GAGs are heparan sulfate (HS); heparin (HP); chondroitin sulfate (CS) dermatan sulfate (DS), isomer of chondroitin sulfate and keratan sulfate (KS). Application division FR 06 54316 Heparan sulfate proteoglycans (HSPG) have been described in the literature to be associated with a relatively high affinity for FGF-2, protecting it from various degradations and acting as a reservoir for it. The specific binding of FGF-2 to a particular GAG has been demonstrated: heparan sulfate. Inhibition of FGF-2 binding to ECM of endothelial cells in culture in the presence of heparin or heparan sulfate but not in the presence of chondroitin sulfate, keratan sulfate or hyaluronic acid has been shown. In addition, an absence of binding of FGF-2 to an ECM pretreated with heparitinase (enzyme specific for heparin and heparan sulfate) but not with chondroitinase ABC (enzyme specific chondroitin sulfate, keratan sulfate and hyaluronic acid) has been disclosed. All these results prove the existence of a specific relationship between FGF-2 and heparan sulfate of the ECM, a relationship that is not found with other GAGs. At the structural level, studies on this interaction have revealed that the minimum sequence required on the heparan sulfate chain to bind FGF-2 is a pentasaccharide containing at least one C2-sulfated iduronic acid residue and at least one N-group. sulfate. In addition, the presence on FGF-2 of a heparan sulfate binding site allows this interaction. This site is found at the level of two sheets [3 (leaves [310 and [311]) containing several basic amino acid residues. On FGF-2 is also a separate site binding to their biological receptors: FGFR. The interaction of FGF with its signaling receptor involves dimerization of this receptor and the presence of heparan sulfate as co-receptor. The model of the FGF-2 response highlights the formation of FGF-2 / HSPG binary complexes then instantaneous association of FGFR leading to the formation of a ternary complex which will undergo dimerization to give a biological activity in vivo by intermediate of the receptor protein kinase (IBRAHIMI & D Appl., Application No. 06 54316 Biochemistry, 2004, 43, 4724-4730). This model was further supported by the discovery on FGFR of a heparin / heparan sulfate binding site. Several heparan sulfate proteoglycans can bind FGF-2, these proteoglycans can either be attached to the cell surface, free in ECM or on the basement membrane. Four of them have been listed in the literature. This is p-glucan, membrane-bound heparan sulfate proteoglycan, which however has a higher affinity for TGFE3; glypican, heparan sulfate proteoglycan lo connected to the cell membrane by a glycosylphosphoinositol attachment; syndecan, transmembrane heparan sulfate proteoglycan with the highest affinity for FGF-2; and perlecane, heparan sulfate proteoglycan of the matrix. Is Degradation of FGF-2 and Protection FGF-2 is sensitive to proteolysis and can be degraded rapidly, even at physiological temperature. The conditions of degradation of FGF-2 have been studied and described in the literature in the absence and in the presence of heparin (highly sulfated GAG natural) which constitutes the reference control for its very high affinity for FGF-2. TARDIEU & colt, in 1992, (Tardieu et al., Journal of Cell Physiology, 150: 194-203; (1992)) studied the behavior of FGF-1 (acid) and FGF-2 in the face of a pH change. Three conditions were tested, with or without the addition of heparin beforehand: pH 2, pH 7 and pH 9. The degradation of FGF was studied in a model of fibroblastic proliferation by incorporation of radioactive element. The results show that FGF-2 is inactivated at acidic pH and basic pH but that the presence of heparin makes it possible to maintain a similar (or slightly lower) FGF-2 activity. to that observed at neutral pH. The authors have also studied the thermal degradation of FGF 1 and 2 in the same model. FGF solutions, in the presence or absence of heparin, are incubated at 0 ° C., 20 ° C., 37 ° C., 60 ° C. and 90 ° C. for different time: 1h, 24h, 7, 14, 30 and 60 days. The results obtained show that the activity of FGF-2 is a function of temperature. The increase in the latter causes a decrease in the activity of FGF-2. In particular, at 60 ° C. and 90 ° C., no activity is detected, even in the presence of heparin. On the other hand, for the other temperatures studied, heparin allows a long-term protection of FGF-2 activity. Finally, the enzymatic degradation of FGF-1 and 2 was studied against trypsin and chymotrypsin. After incubation for 3 hours at 37 ° C. with FGF solutions in the presence or absence of heparin, 18% polyacrylamide gel electrophoresis is carried out in order to visualize the non-degraded FGF. The incubation time makes it possible to degrade all the FGF present in the samples, whether with trypsin or chymotrypsin. On the other hand, the presence of heparin makes it possible to recover the 18 kDa band relating to FGF-2 signifying complete protection of FGF-2 by heparin. In the same way, it has been shown that GAGs stabilize the active conformation of FGF-2, protecting it from acidic or thermal denaturation and by increasing the specific interaction with specific cellular receptors. In the presence of heparin, FGFs are more stable during storage and more resistant to denaturing and proteolytic conditions. Given the important role of FGF-2 in therapeutics, wound healing, and tissue repair, and its sensitivity to different stresses, such as temperature and enzymatic proteolysis, teams have studied the effects of an additional supply of FGF-2 or the protection of FGF-2 by natural or synthetic molecules. Indeed, Yamanaka, in 2005 (Basic fibroblats growth factor 5 treatment for skin ulcerations in scleroderma, cutis, 2005; 76: 373-376), describes the topical use of recombinant FGF-2 for treating skin ulcers. FGF-2 stimulates fibroblast FGF receptors and induces fibroblast activation and vascular proliferation. During the repair process, during the restructuring phase, FGF-2 also acts on the proliferation of keratinocytes. Derivatives of dextran have been synthesized (Tardieu, 1992, cited above) to potentiate the activity of FGF-2 (mitogenic activity of fibroblasts), protecting it against heat, denaturation at pH, enzymatic degradation (trypsin and chymotrypsin). ). These derivatives have a greater protection of FGF2 than heparin, which makes it possible to have compounds having an alternative to the use of heparin which has a high anti-coagulant activity. They are stabilizers, potentiator and protectant of the matrix for pharmaceutical formulations acting on tissue repair. Moreover, Franck et al, in 2004 (J. Biomater Sci Polymer Edn Vol 15. N 11, pp. 1463-1480 (2004)), have shown that other dextran derivatives can increase in fibroblast culture. cell proliferation and promote the effect of growth factor on cell growth. Finally, Sylvia Colliec-Jouault et al, (Exopo / ysaccharides produced by bacteria isolated from deep-sea hydrothermal winds new agents with therapeutic patent / al, Pathology Biology, 52, 127-130 (2004)), deal with the use of a particular exopolysaccharide neosynthesized by a bacterium which has been used as a bone filling implant in an experimental model in the rat. After 15 days the animals show complete healing with no observed inflammatory reaction. This result would be explained by the interaction of the exopolysaccharide with soluble mediators including FGF-2; This would increase the proliferative activity of FGF-2 and thus promote the healing phenomenon. This exopolysaccharide is used for dental healing. The skin: FGF-2, proteoglycans and aging In the skin, FGF-2 is found in the epidermis, the dermo-epidermal junction (DE), the dermis, the hypodermis and the capillaries; it has a broad spectrum of activity. It ensures the proliferation of fibroblasts within the dermis thus allowing them to synthesize the macromolecules of the matrix, essential to the integrity of the skin, but it also acts on other skin cells. The proliferation of melanocytes is in fact controlled by this FGF-2 which also constitutes a promoter of the formation of capillaries. In terms of fibroblasts, in addition to their proliferation, FGF-2 also increases the mRNA level of the genes encoding hyaluronan synthase and thus increases the production of hyaluronan, the main GAG of the dermis. In injuries, FGF-2 is an important player in the healing phenomenon. It acts on the fibroblasts, always increasing their proliferation, but also on keratinocytes in order to inhibit the expression of collagenase-1 and thus allow the skin to rebuild more quickly. This action during healing does not occur with heparan sulfate as transporter / reservoir but with dermatan sulfate. The expression of FGF-2 is then carried out mainly by keratinocytes, macrophages and fibroblasts. The half-life time of FGF-2 in vitro was evaluated at approximately 24 hours at 37 ° C. and at neutral pH (SOMMER & RIFKIN, Journal of Cellular Physiology 7 2907225 Division of the application FR 06 54316 138: 215-220 (1989). )). Thus FGF-2 is an important cytokine of the skin due to the many functions it performs. The proteoglycans of the skin and their distribution have been studied. The main GAGs revealed for their ability to interact with FGF-2 5 are found in the skin. The quantitative and qualitative distribution of GAGs was determined 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 a storage of FGF-2 present. During aging, the skin undergoes many physiological changes. In addition to the wrinkles that are the most visible marks, it is profound structural changes that appear. In particular, the dermis atrophies, particularly as a result of a reduction in the number of fibroblasts and their synthesis power resulting in modifications of the macromolecules of the extracellular matrix. A decrease in the synthesis of glycosaminoglycans is observed also involving a reorganization of the proteoglycans present. Studies on cultured fibroblasts have shown that hyaluronic acid synthesis decreased significantly during aging while that of keratan and chondroitin sulfates increased. As for dermatan and heparan sulphate, their synthesis would continue to decrease. This decrease would no longer allow storage and thus protect the endogenous potential of FGF-2. OBJECT OF THE INVENTION No document of the prior art has sought to protect a growth factor, in particular FGF-2, from its degradation or denaturation, especially at the cutaneous level. Thus, it is an object of the present invention to solve the new technical problem of supplementing the protective action of heparan sulfate proteoglycans by the development of active ingredients mimicking the action of heparan. sulfate, to protect the endogenous stock of FGF-2, especially at the level of the skin and in particular at the level of the dermis, and able in particular to cross the cutaneous barrier to reach their target. Another object is the provision of active ingredients protecting the endogenous amount of FGF-2, while making it available for renewal of the extracellular matrix, and in particular the dermal matrix. The object of the invention is in particular to solve the technical problem consisting in the provision of active principles protecting FGF-2 from its degradation or denaturation, occurring in the various tissues and in particular at the level of the skin, in particular to provide cosmetic compositions, dermo-cosmetics, or pharmaceuticals intended to restructure the extracellular matrix, and in particular the dermis by restructuring the dermal matrix, to improve the hydration of the skin, to promote the repair of the skin, to maintain the integrity of the dermis to improve the proliferation of melanocytes, to reinforce the epidermal differentiation, or to reinforce the renewal of the epidermis, especially in humans. The present invention is intended to solve the new consisting of the provision of an active ingredient of plant origin to solve the problems mentioned above. The active ingredients that make it possible to solve these technical problems must in particular be neither toxic nor irritating with regard to the skin. The present invention thus also aims to provide active principles solving the technical problems mentioned in the field of cosmetics, dermo-cosmetics or pharmacy, particularly for topical application. in the human being. It is another object of the present invention to solve the new technical problem of providing a method for screening active ingredients to achieve the above-mentioned purposes. This screening method must in particular make it possible to screen a large number of active ingredients at once, in a reproducible and reliable manner. SUMMARY OF THE INVENTION In order to identify active ingredients that solve the technical problems mentioned above, the inventors have developed an in vitro screening model making it possible to demonstrate an activity for protecting FGF-2 on a large number of substances to be screened. especially in the thermal degradation of FGF-2. By the protection of FGF-2, these active ingredients play a major role, ensuring the integrity of the extracellular matrix and in particular the dermal matrix by the renewal of the cell population of fibroblasts. They therefore constitute first-class active ingredients to fight effectively against the degradation of FGF-2, and to fight, among other things, against skin aging. According to a first aspect, the present invention relates to the use of a substance which is neither a sulfated glycosaminoglycan (sulfated GAG) nor a structural analogue of a sulfated GAG protecting FGF-2 as an active ingredient in a composition. cosmetic or dermocosmetic or pharmaceutical to prevent or fight against at least one skin damage related to the degradation of FGF-2. Preferably, the FGF-2 protecting substance is a plant extract and does not comprise sulfated GAG or structural analogue to a sulfated GAG. Advantageously, said substance being chosen from the group consisting of an extract of Hibiscus Abe / moschus or ambrette, an extract of Rebokza, an extract of Gougizi berries, an extract of Banha, an extract of Lessonia, a flour extract mustard, an extract of Wooyin, a barley extract, a sesame extract or combinations resulting from the combination of two or more of the listed extracts. Advantageously, FGF-2 is protected at the level of the human skin and preferably at the level of the EDJ, and / or the dermis, and / or the hypodermis, and / or the epidermis, and / or the wall of cutaneous blood vessels. Advantageously, the skin alterations related to the degradation of FGF-2 occur at the level of the human skin and preferably at the level of the EDJ, and / or the dermis, and / or the hypodermis, and / or the epidermis, and / or the wall of cutaneous blood vessels. Advantageously, said substance is an aqueous or aqueous-alcoholic extract in solution obtained from 1 to 10% of a plant or part of a plant in the dry state relative to the weight of the total solution. Preferably the aqueous extract is a hydroglycolic extract. In particular, the degradation of the growth factor is the result of at least one degradation, in particular thermal degradation, which occurs especially at physiological temperature or by exposure to heat, and / or enzymatic degradation, in particular by cathepsin G Advantageously, said substance or composition is used to stimulate the proliferation of skin cells, in particular the proliferation of fibroblasts and / or melanocytes, and / or to increase the synthesis of at least one of the cells. a matrix component, in particular at least one type of collagen, and / or at least one microfibril-specific protein, such as fibrillin 1 or 2 and / or fibulin 3 or 5, and / or at least one glycosaminoglycan (GAG), including 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 extracellular matrix, and in particular that of the dermis, JDE, the wall of cutaneous blood vessels, the hypodermis, epidermis, and especially the dermis by restructuring the dermal matrix, improve skin hydration, promote skin repair, maintain the integrity of the dermis, enhance epidermal differentiation, and strengthen the renewal of the epidermis, as well as any combination of his care. These various uses are intended in particular to prevent or fight against skin alterations related to the degradation of FGF-2. Advantageously, the substance or composition is used to combat the aging of the skin, especially in a human being. Advantageously, the concentration of said substance is an effective concentration for the protection of FGF-2, in particular by topical application, and is especially between 0.01% and 10% by weight of the total composition. The composition is in particular a cosmetic, dermo-cosmetic or pharmaceutical composition, especially for humans. The substance according to the present invention is preferably a plant extract protecting FGF-2 from its degradation, and in particular an aqueous or aqueous-alcoholic extract, preferably a hydroglycolic extract. According to a preferred embodiment, the substance is an aqueous or hydroalcoholic extract of Hibiscus Abe / moschus obtained from the seed of Hibiscus Abelmoschus. In a second aspect, the present invention relates to the use of a substance for the manufacture of a composition, in particular a cosmetic, pharmaceutical or dermo-cosmetic composition, for protecting FGF-2 from its composition. degradation, especially in the various uses listed above. The present invention also relates to a method of cosmetic, pharmaceutical or dermo-cosmetic care using a composition defined above according to any one of the embodiments, in particular to carry out a treatment linked to at least one of the various uses listed. DETAILED DESCRIPTION OF THE PRESENT INVENTION The present invention makes it possible to obtain protection of FGF-2 by proteoglycans of this same matrix. Protection of FGF-2 is understood to mean protection of FGF-2 against degradation or denaturation. This degradation or denaturation generally occurs in the form of lysis due to environmental conditions, such as enzymatic or thermal lysis. This degradation or denaturation can occur due to the decrease in protection provided by proteoglycans. The screening method according to the present invention has made it possible to screen various active principles extracted from plants. No plant extracts have been described as protecting FGF-2 from its degradation in the skin, especially in humans, only substances whose structure is close to heparan sulfate having been used. 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 seed of a plant belonging to the plant. 54316 Malvaceae family, more particularly the genus Hibiscus and even more particularly Hibiscus Abe / moschus or Ambrette. Hibiscus abelmoscus was known in particular in the form of an aqueous extract obtained from the whole plant, solely for its slimming properties in cosmetics and more particularly for the fight against cellulite (US5705170). Other extracts having the desired activity for the present invention could be selected. Their activity is particularly unexpected, especially with regard to the protection of FGF-2. The extracts are in particular an aqueous extract of Lycium chinense or Gougizi berries obtained from whole dehydrated berries, an aqueous extract of Pine // iae ternata or Banha obtained from tuber, an aqueous extract of Raphanus sativus or Rebokza obtained from of the seed, an extract of Brassica juncea or mustard obtained from the seed, an aqueous extract of Coicis semen or Wooyin obtained from the seed, an extract of Hordeum seen / station or barley obtained from the seed, Sesamum extract Md / cm or golden sesame obtained from the seed and an extract of whole seaweed of Lessonia sp or Lessonia. These extracts are however not limited to aqueous extracts alone. Thus the present invention also covers any polar solvent for essentially all of the active compounds in an aqueous extract to provide the desired properties in the context of the present invention. According to the tests carried out by the present inventors, hydroglycolic extracts make it possible to obtain the desired properties. Of the hydroglycolic extracts, a water / glycol type extraction solvent is preferred. Particularly preferred is a water / butylene glycol mixture. The proportion of the water / alcohol mixture may vary as is known to those skilled in the art. The proportions water / alcohol generally vary from 10/90 to 90/10, and for example from 50/50 to 85/15. It is preferable to use in the present invention as the active ingredient an extract in solution obtained from 1 to 10% of a plant or part of a plant in the dry state relative to the weight of the total solution. The composition according to the present invention advantageously comprises from 0.001% to 20%, preferably from 0.01 to 10%, of active ingredient according to the present invention by weight of the total composition. The composition may be applied topically or administered orally. The method for screening a potentially active substance for the protection of FGF-2 from degradation in humans includes: a) contacting a potentially active substance and FGF-2 at a temperature about 45 C for a period of about 2h, b) the selection of FGF-2 protecting substances of at least 50%, preferably 65% of its degradation. Advantageously, the screening method comprises an additional step of testing transcutaneous penetration to select an active substance crossing the skin barrier. According to another embodiment, the present invention relates to a method for screening active substances derived from plants. Advantageously, the present invention relates to the method for preparing a composition, comprising the implementation of said screening method followed by a step of mixing the selected active substance with an excipient for the preparation of the composition. Further aims, features and advantages of the invention will become apparent to those skilled in the art upon reading the explanatory description which refers to examples which are given only to those skilled in the art. illustrative and which in no way limit the scope of the invention. The examples form an integral part of the present invention and any features appearing novel from any prior art from the description taken as a whole, including the examples, form an integral part of the invention in its function and function. in its generality. Thus, each example has a general scope. On the other hand, in the examples, all percentages are by weight unless otherwise indicated, and the temperature is in degrees Celsius unless otherwise indicated, and the pressure is atmospheric pressure unless otherwise indicated. EXAMPLES Example 1: Study of the thermal degradation of FGF-2 Tardieu et al, in 1992, studied the thermal degradation of FGF-2 in a model of fibroblastic proliferation by incorporation of radioactive element. The present inventors have developed a method for studying the stability of FGF-2: this large-scale quantification method makes it possible to carry out a screening of active principles, which would not have been possible with the described method. by Tardieu. A 5ng / ml solution of FGF-2 in 10mM-0.1% BSA PBS buffer and 0.1% methyl paraben is prepared. This solution is placed at various temperatures: 4 C, 20 C, 37 C, 50 C, and 80 C in the presence or absence of heparin at 500pg / ml. Assays of FGF-2 by a commercial ELISA kit (R & D system, France) are carried out at different times (T = 0, 3h, 24h and 48h) so as to establish degradation kinetics for each study temperature. The results are expressed as a percentage of FGF-2 relative to the time T = 0. The experiments are performed in triplicate (n = 3). The results obtained are shown in FIGS. 1 and 2. The FGF-2 is sensitive to the temperature conditions to which it is subjected. Figure 1 shows the evolution of the stability of FGF-2 as a function of time at different temperatures. FGF-2 stored at 4 C is relatively stable over a period of 48 hours since 20% of FGF-2 is degraded at this temperature. As the temperature rises, the degradation percentages increase: after 3 hours at 37 ° C., the FGF-2 undergoes 65 hours of degradation and 80% of degradation at 50 ° C. Finally, after 3 hours at 80 ° C., the degradation deteriorates completely. FGF-2 is observed which is due to denaturation of proteins at this temperature. Finally, the longer the time and the greater the observed degradation, after 24H at 37 ° C., 80% of the FGF-2 is degraded. Figure 2 shows the stability of FGF-2 at different temperatures in the presence of heparin. FIG. 2 shows that this degradation is stopped by the presence of 0.05% heparin which is the experimental positive control and which by its high affinity with FGF-2 stabilizes it. Heparin is a highly sulfated polysaccharide known for its very high affinity with FGF-2. This protection is not effective at 80 C because heparin is also denatured at this temperature. Surprisingly, the degradation of FGF-2 at about 50 ° C occurs over a short period of time. A temperature close to 45 ° C. is selected for practical reasons and constitutes a parameter of choice for carrying out a stress model for screening active principles. The experimental positive control is preferably constituted by heparin. Example 2: In vitro model for the selection of an active protecting FGF-2 against thermal degradation. The degradation of FGF-2 observed at approximately 50 ° C. is particularly advantageous since a short period of time allows a degradation of FGF-2 of approximately 80%, thus making it possible to carry out a large number of tests. For this reason, it has been decided to perform a screening with a stress of 2H15 at 45 C and to study the ability of a large number of actives to protect the FGF-2 against this thermal degradation. The screening model applied is as follows: 180 μl of a solution of FGF-2 at 4.5 ng / ml prepared in 0.1% PBSBSA buffer are distributed in a 96-well microplate. 20 μl of distilled water or 0.05% heparin or a solution containing various active ingredients extracted from different plants to be tested are added. The plate is stirred with a plate stirrer and then covered with an adhesive tape and placed in an oven at 45 C for 2H15. At the end of the incubation period, the plate is removed and then an assay of FGF-2 is carried out by the ELISA kit. A control plate containing only FGF-2 and distilled water treated under the same conditions is left at room temperature. The results are expressed as a percentage of protection of FGF-2 relative to the non-stressed or non-degraded FGF-2 control. Example 3 Validation of the Model by GAGs and Proteoglycans of the Skin Tests were carried out with GAG and PG commercially available to validate the model. Solutions of GAG at different concentrations are carried out: heparan sulfate, dermatan sulfate, chondroitin sulfate, and hyaluronic acid are solubilized then tested at 0.1% -0.01% -0.001%. Heparin used as a positive control is tested at 0.1%, 0.01%, 0.001%, 0.0001% and 0.00001%. The proteoglycans tested were Glypican-3 and decorin tested at 0.1%, 0.01%, 0.001%. The stress protocol described in Example 2 is applied, the 20 μl of water being substituted with 20 μl of corresponding GAG or PG solution. The GAGs and PGs tested thus undergo an additional 1/10 dilution. Figure 3 illustrates the study of FGF-2 protection by skin GAGs. The results obtained in FIG. 3 confirm the data from the literature, namely: Heparan sulfate protects FGF-2 as well as dermatan sulfate at each concentration tested. It is indeed dermatan sulfate 25 which is the main promoter of the activity of FGF-2 during the healing process. Hyaluronan, regardless of its molecular weight, does not in any way protect the FGF-2. As for chondroitin sulfate, the protection it provides depends on its dose but does not reach 100%. The analysis of 2 proteoglycans, FIG. 4 (Study of the protection of FGF-2 by PGs), glypican-3 and decorin confirmed the data from the literature. That is, glypican-3 which has heparan sulfate chains protects FGF-2 in a dose-dependent manner, whereas decorin (composed of chondroitin sulfate and dermatan sulfate chains) is ineffective. This study highlights that the protection of FGF-2 depends on the structure of GAGs. The interaction of heparan sulfate with FGF-2 is well validated. This interaction is confirmed when the PG carrying the heparan sulfate chains is tested in the model. This study validated our screening model by confirming the results from the literature. Example 4 Screening of Active Ingredients Screening was performed according to Example 2, from a library consisting of 2000 plant extracts and characterized molecules. These solubilized compounds, plant extracts, marine algae or characterized molecules are tested pure or at 10% or 1% in the reaction medium described in Example 2. The results of the most effective active principles are described in Table 1. , below. 20 2907225 Division of the application FR 06 54316 Table 1: Results obtained during the screening Active principle tested Protection (%) Protection (%) Common name Obtained for Obtained for the active principle based on 10% active ingredient 1% Extract from shallots 95.07 1.2 Soy flour extract 86.85 67.66 Urucum extract 103.5 29.8 White mustard extract 110.5 17.39 Lycopodium plant extract 71 11.27 Lupine meal extract 116.39 65.77 Potato extract 73 16.5 Jioh extract 70.9 23.18 Extract Garlic 95.99 13.92 Angelica Extinct 72.5 7.31 radicante Mung Bean Extract 76.78 2.42 Ligusticum Extract 70.53 38.15 chuanxiong Carrot Extract 83.93 1.65 Hibiscus Extract Abe / moschus 135.1 87.53 or ambrette Parsnip Extract 92.89 23.15 Pea Extract 74,33 0 Potato extract 88.96 3.61 Orange extract 70 2.83 Sweet pepper extract 87.35 28.44 21 2907225 Demand division FR 06 54316 Rebozka extract 142.5 77.34 Cucumber extract sea salt 69.2 Gougizi berry extract 154.4 68.59 Banha extract 129.5 65.64 Lessonia extract 53.3 61.79 Mustard flour extract 110.1 56.85 Wooyin extract 106.1 56.18 Barley extract 79.7 53.81 Sesame Extract 124.4 52.73 Waxy Acid Extait 55.6 49.34 Hongsan Extract 102.3 44.58 Sweet Acora Extract 77.7 43.24 Luteolin Extract 67.7 43.17 Bryone Extract 81 40.4 Mounwhaja Extract 102 39.56 Almond Extract 99.5 38.53 Ligusticum Extract 70.5 38.15 chuanxiong Chunnansung Extract 108.4 37.7 Pumpkin Extract 85.7 37.5 Goborn Extract 71.1 37.8 Bangpung Extract 117.9 36 Ammonium Acrylate Extract 54 , 9 Hesongja extract 81.9 33.3 Lin 93 extract, 32.6 Parsley extract 97.4 30 Blue poppy seed extract 70.33 8.8 Boksin extract 75.69 27.56 22 2907225 Demand division FR 06 54316 Sanyak extract 63.05 16.78 Yellow flax seed extract 88.56 28.4 Porphyra extract Tenera 38.35 29.77 Lithotheminium extract 53.78 26.6 The above extracts are preferably obtained by maceration of a part of the plant in a water / alcohol mixture, preferably water / glycol from 100/0 to 0/100 (v / v). The dilution is then carried out in this solvent or solvent mixture. Some tested actives have a strong protective activity which is more dose dependent. EXAMPLE 5 Hibiscus Abe / Moschus Extract (Ambrette) 5a-A hydroalcoholic ambrette extract is made from the 5% (w / w) ground seeds in refluxing ethanol. The extraction is carried out for 1 hour then the solution is filtered, the ethanol removed, and the result is solubilized at 5% (w / w) in a water / glycol mixture (75/25) and then ultrafiltered on a ceramic filter. different cutoff thresholds, and finally filtered at 0.45pm. 5b-A hydroglycolic ambrette extract is prepared in a water (75%) / BG (25%) mixture, preferably from 5% (w / w) milled seeds. The maceration is carried out for 1 night at 4 C then the solution is ultrafiltered on ceramic filter at different cutoff thresholds, and finally filtered at 0.45pm. BG means butylene glycol. 5c-An aqueous Ambrette extract is made in water preferably from 5% (w / w) ground seeds. The maceration was carried out for 1 night at 4 ° C., then the solution was ultrafiltered on ceramic filters at different cut-off points, and finally filtered at 0.45 μm. EXAMPLE 6 Extract of Gougizi Berries An extract of Gougizi berries is preferably carried out in a mixture of water (75%) / BG (25%) from whole berries dehydrated at 5% (w / w). The maceration is carried out for 1 night at 4 ° C., then the solution is ultrafiltered on ceramic filters at different cut-off points, and finally filtered at 0.45 μm. EXAMPLE 7 Banha Extract A Banha extract is preferably carried out in a water (75%) / BG (25%) mixture from 5% (w / w) tubers. The maceration is carried out for 1 night at 4 C then the solution is ultrafiltered on ceramic filter at different cutoff thresholds, and finally filtered at 0.45pm.

  EXAMPLE 8 Rebokza Extract A Rebokza extract is preferably carried out in a mixture of water (75%) / BG (25%) from seeds at 5% (w / w). The maceration is carried out for 1 night at 4 ° C., then the solution is ultrafiltered on ceramic filters at different cut-off points, and finally filtered at 0.45 μm.

EXAMPLE 9: Lessonia extract Lessonia extract is preferably produced in a mixture of water (75%) / BG (25%) from the whole seaweed at 5% (w / w). ). The maceration is carried out for 1 night at 4 ° C., then the solution is ultrafiltered on ceramic filters at different cut-off points, and finally filtered at 0.45 μm. Example 10 Mustard Extract An extract of Mustard is preferably carried out in a mixture of water (75%) / BG (25%) from the seed at 5% (w / w). The maceration is carried out for 1 night at 4 ° C., then the solution is ultrafiltered on ceramic filters at different cut-off points, and finally filtered at 0.45 μm. Example 11: Wooyin extract A Wooyin extract is preferably carried out in a water (75%) / BG (25%) mixture from the 5% (w / w) seed. The maceration is carried out for 1 night at 4 C then the solution is ultrafiltered on ceramic filter at different cutoff thresholds, and finally filtered at 0.45pm.

DETAILED DESCRIPTION OF THE INVENTION Example 12: Barley extract A barley extract is preferably produced in a mixture of water (75%) / BG (25%) from the 5% seed (w / w). The maceration is carried out for 1 night at 4 ° C., then the solution is ultrafiltered on ceramic filters at different cut-off points, and finally filtered at 0.45 μm. EXAMPLE 13 Sesame Extract A sesame extract is preferably prepared in a water (75%) / BG (25%) mixture from the 5% (w / w) seed. The maceration is carried out for 1 night at 4 C then the solution is ultrafiltered on ceramic filter at different cutoff thresholds, and finally filtered at 0.45pm.

Each extract (Examples 5 to 13) has been partially preserved. The optional preservative, preferably consisting of a mixture of Caprylyl Glycol, Phenoxyethanol, Hexylene Glycol 1%, is added in the final in the presence or absence of xanthan.

EXAMPLE 14 Selection of Active Ingredients or Plant Extracts The most effective extracts are selected and tested at different concentrations according to the screening of Example 2. The active ingredients used are the extracts of Examples 5 to 13, and are diluted in the following manner. using a solvent (or mixture of solvents) used for the extraction A concentration range tested between 0.1% and 10% in this solvent so as to study the specificity of these active ingredients with respect to the thermal degradation of the FGF -2. The assays are performed in triplicate. The results obtained are listed in Table 2, below: 26 2907225 Demand Division FR 06 54316 Table 2: Dose effect obtained on the principles or active ingredients selected during the screening Active principle Protection of FGF-2 (in%) depending on the concentration of the active ingredient 0.1% 1% 2% 3% 5% 10% Hibiscus extract 84.7 6.0 87.5 4.1 99.6 3.1 110.9 4. 123.6 1. 130.8 2. Abe / moschus 3 6 7 9 48 49 (Ambrette ) Extract from rebokza 8.5 1.6 72.3 5.6 99.9 5.4 106.6 3. 123.8 1. 129.4: EO.

4 7 21 43 89 Extait de Bays of 7.7 0.5 29.5 3.4 38 4.14 52 2.17 75 3.05 109.4 6. Gougizi 7 34 Banha extract 8 1.46 52.5 4.1 76.6 2.5 85.5 2.4 91 2.82 98.9 0.7 7 5 5 Lessonia sp 13.2 extract 1.6 36 5.49 50.8 3.1 53.27 3. 53.67 4. 52.15 0.

3 6 04 49 84 Mustard extract 14.7 2.8 88.7 8.0 104.9 5. 111.3 2. 119 1.2 118 2.8 flour Codex 6 6 01 93 Excerpt from Wooyin 9.4 0.95 56.4 2.9 69.2 0.8 80.6 2.4 92.9 4.3 102.4 1.

7 8 3 9 85 Barley Extract 10.7 0.9 47.4i-1.8 60.8 2.3 70.2 0.5 82.5 0.4 97.8 1.0 5 6 1 9 9 Sesame Extract 2.3 1.06 21.5 0.4 48.4 1.7 55.7 19. 74.8 0.5 94.8 1.7 3 1 44 4 Extracts are very effective and all exhibit a dose dependent activity thus reflecting a specificity of activity of each asset on the given target.

27 2907225 Demand division FR 06 54316 Hibiscus extract Abe / moschus (ambrette) has the strongest activity and is therefore an active ingredient of choice in the protection of the growth factor FGF-2.

In Examples 15 to 21 the concentration of active ingredient (extract 5c) is in percent by volume (v / v) in water. Example 15: Study of the Protection of FGF-2 by the Aqueous Extract of Hibiscus Abe / Moschus (Ambrette) Prepared According to Example 5c (Extract 5c)

A study of the protection of FGF-2 by extract 5c was carried out on 2 industrial batches. The protection was studied under stress according to the conditions of Example 2. The positive control consisting of heparin tested at 0.01% in the final reaction medium after prior dilution with water protected at 105.52% 3.04. The results obtained are listed in Table 3, below: Table 3: Protection of FGF-2 at 45 C by 2 industrial batches of extract 5c 20 Protection of FGF2 (in%) as a function of the concentration of active principle Extract 5c 0.1% 0.5% 1% 2% 3% Loti 4.78 46.47 81.01 107.83 120.76 3.89 1-1.85 4.53 -1-2.53 2.61 Lot2 4.38 42.04 69.6 101.81 118.67 - 2.3 1-2.76 3.56 4.02 4.12 28 2907225 Division of the application FR 06 54316 The 5c extract protects the FGF-2 in a dose-dependent manner with a strong activity for a concentration of 1% of active ingredient in water. The results obtained on two industrial batches are substantially identical. It was essential to verify that the extract 5c could protect the FGF-2 at physiological temperature, ie 37 C. This is why a study consisted in assaying the FGF-2 after 24H at 37 C in absence or in the presence of increasing concentrations of the extract 5c. The positive control consisting of 0.01% heparin protects to 110.53% 3.20. The results obtained are listed in Table 4, below: Table 4: Protection of FGF-2 at 37 C by 2 industrial batches of extract 5c Protection of FGF-2 (in%) depending on the concentration in principle Active Extract 5c 0.01% 0.1% 0.25% 0.5% 0.75% 1% Loti 8.37 17.81 51.02 82.62 108.39 110.69 -14.2 3.04 4.75 1.98 13.28 Lot2 2.12 20.56 55.49 91.06 110.64 122.87 1.17 1-2.29 2.61 0.87 2.45 5.75 15 The results obtained show that extract 5c protects FGF-2 at physiological temperature and in a dose-dependent manner. Extract 5c further protects, at this temperature, FGF-2 which is less degraded than 45 C.

DETAILED DESCRIPTION OF THE INVENTION Example 16: Concerning the Cytotoxicity Study of the 5c Extract on Monolayer Fibroblasts by a PNPP Assay The aim was to study the cytotoxicity of the 5c extract on fibroblasts. normal humans. This assay is based on the conversion of p-nitrophenyl phosphate (PNPP) to p-nitrophenol by intracellular acid phosphatases of viable cells. The absorbance of p-nitrophenol at 405 nm is directly proportional to the number of viable cells contained in the culture wells. Once the confluence of normal human fibroblasts was reached, the culture media were replaced with 200 μl / well of medium supplemented or not (control) with extract 5c in increasing concentrations: 0.1, 0.5; 1; 2; 3 and 5%.

The cells were incubated for 48 hours in an oven at 37 ° C. After incubation and removal of the culture media, the cells were rinsed twice with PBS (Phosphate Buffered Solution) and then placed in the presence of 200 μl of a buffer containing 0.1 M sodium acetate (pH 8), 0.1% Triton X-100 and 5 mM p-nitrophenyl phosphate (Sigma, France). After incubation for two hours at 37 ° C. in an atmosphere containing 5% CO 2, the reaction was stopped by adding 20 μl of 1 N NaOH. The absorbance of the reaction media at 405 nm was then determined using a plate reader (Victor2 V, Perkin Elmer, Finland).

Non-enzymatic hydrolysis of p-nitrophenyl phosphate was determined in each experiment on wells not containing cells ("white"). All measurements were done in sextuplate (n = 6).

The results obtained are listed in Table 5, below: Table 5: Study of the cytotoxicity on normal human fibroblasts Concentration in extract 5c Percentage of viable cells per (%) compared to the negative control 0.1 85.9 0.5 88.7 1 88.8 2 87.5 3 89.8 5 85.5 5c extract is not cytotoxic throughout the range of concentration tested. EXAMPLE 17 Study of the Fibroblast Proliferation by FGF-2 Protected by the 5c Extract The aim of this study was to verify that the FGF-2 protected by the 5c extract stimulates the proliferation of normal human fibroblasts. A solution of FGF-2 is prepared extemporaneously (= non-degraded FGF-2); a solution of FGF-2 is placed 24H at 37 ° C. in the absence (= degraded FGF-2) and in the presence of 0.01% heparin (= FGF-2 + heparin at 37 ° C.). Each FGF-2 solution is diluted in order to apply to the cells the following final concentrations of FGF-2: 0.1, 0.25, 0.5, 0.75 and 1 ng / ml.

Normal human dermal fibroblasts are inoculated at low density in 6-well plates. They are then cultured in the absence medium (control) or in the presence of FGF-2 at the different concentrations. After 48 hours, the activity of intracellular acid phosphatase (PNPP) is assayed, which makes it possible 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, ie to the untreated well. The results obtained are shown in Table 6 below and in FIG. 5: Table 6: Study of the proliferation (expressed in% relative to the untreated well) obtained after treatment with different concentrations of non-degraded FGF-2, different degraded FGF-2 concentrations and different concentrations of FGF-2 + heparin at 37 C.

Concentration of FGF-2 FGF2 non-FGF-2 degraded FGF-2 + heparin at (ng / ml) degraded 37 C 0.1 116.7 96.2 0.014 129.3 0.009 0.012 P = 0.006 ** 0.25 138 , 7 111 0.006 154.8 0.01 0.005 P <0.001 *** 0.5 148.5 123.7 0.005 146.8 0.007 0.007 P <0.001 *** 0.75 163.2 132.5 0.004 149.8 0.016 0.005 P <0.001 *** 1 160 146.4 0.01 148.1 0.006 0.003 P = 0.003 ** Figure 5 shows a study of fibroblast proliferation in the presence of non-degraded FGF-2, degraded FGF-2 and FGF-2. 2 + heparin at 37 C.

The results obtained show that FGF-2 (non-degraded) stimulates the proliferation of normal human fibroblasts in culture in a dose-dependent manner. FGF-2 degraded at 37 C stimulates proliferation but much less than undegraded FGF2 at 37 C. In fact, for each concentration tested, the proliferation in the presence of degraded FGF-2 is statistically lower than that obtained. with non-degraded FGF-2. This example shows the relevance of developing an active agent capable of protecting the FGF-2 from the matrix which degrades at physiological temperature that can no longer perform its role of renewal of the dermal cells. By protecting the FGF-2 from thermal degradation, the heparin used at 0.01% allows the FGF-2 to retain its biological properties since the proliferation is maintained or even greater than that obtained with the FGF-2 solution prepared extemporaneously; up to the concentration of 0.5 ng / mI. The very large stimulation in the presence of heparin observed with small amounts of FGF-2 is not observed with larger amounts of FGF-2. The same experiment was carried out by replacing the heparin with the extract 5c. The FGF-2 solution placed in the presence of 0.5% of the extract 5c is placed 24H at 37 ° C. The solution is then diluted so as to apply 0.5 ng / ml of FGF-2 in the wells containing the fibroblasts.

The concentration of the extract 5c finally applied to the cells is 0.0025%. It was verified beforehand that this concentration of the 5c extract was not able to induce stimulation of the proliferation of fibroblasts. This means that the stimulation of proliferation observed is due to the protection of FGF-2 by extract 5c (see Table 8). The results obtained, expressed as a percentage of proliferation with respect to the untreated control, are shown in Table 7, below: Table 7: Study of the proliferation (in percentage) of fibroblasts obtained in the presence of non-degraded FGF-2 at 0.5 ng / ml, FGF-2 degraded to 0.5ng / ml and FGF-2 + 5c extract 0.5% at 37 C. Concentration in FGF-2 FGF-2 non-FGF-2 degraded FGF-2 + extract 5c to degraded 37 C 0.5 ng / ml 148.5% 123.7 Io 143.8% 0.007 0.005 0.007 P <0.001 *** ns with respect to unstressed FGF-2 Figure 6 shows the study of proliferation of fibroblasts in the presence of non-degraded FGF-2 at 0.5 ng / ml, degraded FGF-2 at 0.5 ng / ml and FGF-2 in the presence of 5c extract at 0.5 h at 37 ° C.

Co-incubation of FGF-2 and 37C extract 5c protects FGF-2, which retains its ability to stimulate the proliferation of normal human fibroblasts in culture. The results obtained make it possible to obtain a fibroblastic proliferation that is not statistically different from that obtained in the presence of FGF-2 prepared extemporaneously. The interest of an asset to protect FGF-2 seems of major interest for cell renewal of fibroblasts. A study of the proliferation of normal human fibroblasts in the presence of increasing concentrations of the 5c extract was carried out for 48H at 37C. The fibroblasts are inoculated in the presence of the 5c extract. at different concentrations 0.0025%, 0.01%, 0.25%, 0.5%, 1% and 2%. After 48 hours, an acid phosphatase assay is performed (PNPP assay) at 405 nm and then the proliferation is calculated (in%) relative to the negative control (= untreated cells). Each condition is tested in n = 6. The results obtained are listed in Table 8, below: Table 8: Study of the proliferation of fibroblasts in the presence of the extract 5c Concentration in the extract Proliferation (in%) vstemoin negative 5c (%) 0, 0025 100 0.01 99 0.25 107 0.5 114 1 111 2 122 * * significant One Way Analysis of Variance / Dunnett The positive control constituted by 10% serum makes it possible to stimulate the proliferation in a significant way which validates the experience (T + = + 126% * proliferation). The results obtained show that the 5c extract does not stimulate the proliferation of normal human fibroblasts in culture.

EXAMPLE 18 Impact of Protection of Growth Factors on Collagen Synthesis Normal human fibroblasts from a 52-year-old donor were cultured in 96-well plates with medium. (composed of DMEM, 2mM glutamine, Penicillin 50IU / ml-streptomycin 50pg / ml, 10% fetal veil serum) for 24H. After incubation, the cells were treated with vitamin C products at 20 μg / ml (positive control) and 2% and 1% extract for 5 hours for 48 hours. Control was performed in parallel with medium only. After 24 hours of contact, the radioactive proline (3H-proline) is added to the medium. At the end of the experiment, supernatants were collected for assaying the incorporation of radioactive proline into intracellular proteins. The results obtained are shown in Table 9, below: Table 9: Study of the stimulation of collagen synthesis by extract 5c Products cpm sd n% control p control 5981 829 6 100 - Vitamin C-20pg / ml 10185 287 3 170 p <0.01 Extract 5c 2% 7342 273 3 123 p <0.01 Extract 5c 1% 7056 783 3 118 p <0.01 36 Cpm: body per minute Sd: standard deviation n: number of tests p: threshold of Significance The 5c extract tested at 2% and 1% was able to stimulate collagen synthesis in normal human fibroblasts and significantly.

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

Example 19: Impact of Growth Factor Protection on Synthesis of Total GAGs and Sulphated GAGs The purpose of this study was to determine whether the protection of growth factors by 5c extract on monolayer fibroblast cultures. allowed to stimulate the synthesis of total GAGs by a radioactive method. Normal human fibroblasts from a 52-year-old donor were cultured in 96-well plates with medium (composed of DMEM, 2mM glutamine, Penicillin 50IU / ml-streptomycin 50pg / ml, 10h fetal serum serum). ) during 24H. After incubation, the cells were treated with Vitamin C products at 20 μg / ml (positive control) and 5% 2% and 1% extract for 72 hours. A check was made in parallel with middle only. After 48 hours of contact, the radioactive glucosamine (D- (6-3H) -20 glucosamine) is added to the medium. At the end of the experiment, supernatants were collected for assaying the incorporation of radioactive glucosamine into intracellular proteins. The protein assay is performed with a commercial kit (BioRad 500-0116).

The results are listed in the following Table 10: 37 2907225 Demand Division FR 06 54316 Table 10: Study of stimulation of total GAG synthesis by extract 5c Activity Protein Products Ratio% control p (cpm) (mg / ml) (act./Prot.) control 1059 3.579 296 100 - TGF- [3 10n / ml 2590 3.650 709 240 P <0.01 Extract 5c 2% 4173 3.058 1365 461 P <0.01 Extract 5c 1% 1787 3.396 526 178 P <0.01 The results obtained show that the 5c extract applied to 2% and 1% on normal human fibroblasts was able to stimulate the synthesis of total GAGs with a dose-effect and more significantly than the reference positive control ( TGF-β applied at 10 ng / ml). The results obtained show that the protection of the growth factors of the medium and those synthesized by the fibroblasts, allows in vitro to stimulate the synthesis of total GAGs. In the same way, a 35S-sulfate incorporation study is performed to quantify sulfated GAGs. The applied protocol is the same as that of the total GAG except for the nature of the radioactive precursor. The results are listed in Table 11, below: Table 11: Study of stimulation of synthesis of sulfated GAGs by extract 5c 20 Activity Protein Products Ratio% control p (cpm) (mg / ml) (act. / Prot.) Control 3028 3.579 846 100 - TGF-f3 10n / ml 5442 3.650 1491 176 P <0.01 Extract 5c 2% 5586 3.058 1827 216 P <0.01 Extract 5c 1% 4300 3.396 1266 150 P <0.01 38 2907225 Division of the Application FR 06 54316 The results obtained show that the 5c extract makes it possible to stimulate the synthesis of sulfated GAGs very significantly and in a dose-dependent manner.

This means that in addition to the role played by the 5c extract against FGF-2, the 5c extract is capable of protecting the growth factors from stimulating the synthesis of sulfated GAGs and thus allows strengthen the protection of FGF-2 since it is these same sulphated GAGs with Heparan sulphate that protect FGF-2. Example 20: Study of the Protective Activity of FGF-2 by the 5c After Transcutaneous Permeation Extract The purpose was to ensure that the 5c extract retained its growth factor protection properties after penetrating the skin. A solution of the pure 5c extract was deposited on the surface of skin explants, mounted in Franz cells. After 24 hours of incubation, the media contained in the recipient compartments of the cells were recovered and lyophilized. The lyophilizates were then dissolved in distilled water so that they were finally tested in the example described with heat stress at 37 ° C. for 24 hours. FIG. 7 represents the study of the protection of FGF-2 by the extract 5c after passage through the skin.

The results obtained (99.96% protection) show that the extract 5c has retained its protective properties integrally since the result obtained is identical to that obtained in Table 4 of Example 39.

The results show that after having penetrated the cutaneous tissues, the extract 5c is able to protect the growth factors and thus remains intact to reach its target.

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 by proteinases present in the dermis of human skin. These enzymes play an important role in the degradation of the extracellular matrix. In particular, cathepsin G, which occurs during inflammation and is secreted in the extracellular environment. It adheres strongly to the cell surfaces of the matrix because of its basic character. Cathepsin G is also active against collagen but also against the core protein of proteoglycans and glycoproteins of the matrix. For this reason, a study of FGF-2 degradation by cathepsin G has been performed. Degradation kinetics were performed in 100mM Hepes buffer, pH7 containing 0.1% BSA. Cathepsin G was added to the wells of the plate at a concentration of 0.02U per well to a solution of FGF-2 at 2 ng / well. The plate is incubated at 37 ° C. then at 15, 30, 60, 90 and 120 minutes a sample is taken to determine the remaining FGF-2. The control (100%) is carried out by an extemporaneous assay of FGF-2. In parallel, a control solution without enzyme is placed at 37 ° C. to study the thermal degradation at different times, so as to overcome this degradation due to the temperature and quantify the part due to enzymatic degradation alone.

The quantities of FGF-2 (μg / ml) obtained are described in Table 12, below: Table 12: Study of the degradation of FGF-2 by cathepsin G.

5 Time (minutes) Concentration in FGF-2 Concentration in FGF-2 Temperature control 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%) The results obtained show that, over time, the degradation of FGF- 2 is increased. The enzyme can degrade FGF-2 very significantly at 90 minutes. This is why this time will be selected to study the protection of FGF-2 in the presence of cathepsin G by extract 5c. In the reaction medium described above (Example 21) are added concentrations of the extract 5c different namely: 0.01%, 0.05%, 0.1%, 0.5% and 1%.

After 90 minutes at 37 ° C., the concentrations of FGF-2 are evaluated and the percentages of FGF-2 remaining and the percentages of protection of FGF-2 are calculated. For each concentration tested, the assay is performed at n = 9. The results are described in Table 13, below: ______________________________________________________________________________ Table 13: Study of the protection by Cathepsin-degraded FGF-2 extract 5c. Tests FGF-2 Concentration remaining (%) Protection of FGF-2 FGF-2 (%) (μg / ml) Control (100%) 455.23 100 - Degraded control 288.11 63.29 0 (enzyme) Extract 5c 0.01% 336.93 74 , 01 29,21 Extract 5c 0.05% 344.65 75.71 33.83 Extract 5c 0.1% 409.08 89.86 72.38 Extract 5c 0.5% 414.02 90.95 75.33 Extract 5c 1% 406, The extract 5c is capable of protecting the FGF-2 against enzymatic degradation in a dose-dependent manner. This in vitro study makes it possible to demonstrate that the application of the 5c extract can protect the growth factors from the proteolytic degradation exerted by the extracellular matrix enzymes. This only reinforces the interest of the use of the extract 5c in cosmetics. Example 22: Demonstration of the FGF-2 Levels with Age The purpose of this study was to study the FGF-2 concentration of so-called young and so-called aged skin to establish a relationship between age and loss. FGF-2 content due mainly to the modification of the GAG content of the skin.

42 2907225 Demand Division FR 06 54316 Skin biopsies from so-called young donors (aged 21, 30, 31 and 38) from so-called elderly donors (50, 55, 57 and 58 years old) were collected and extracted after grinding in pH 7 PBS buffer containing 0.1% Triton X100, to perform an assay of FGF-2 by a commercial ELISA kit (R & D system, DFB50). The concentration of FGF-2 is related to the DNA level measured by a Picogreen commercial kit. The results obtained are shown in FIG. 8: FIG. 8 represents a study of the concentration of FGF-2 in hydrolysates of skins called young and old skins. The results show a difference in the content of FGF-2 between young skin and older skin. Moreover, this difference is statistically significant (p = 0.046). This example makes it possible to show that the use of an active agent as a protector of growth factors and more particularly of FGF-2 makes it possible to combat the effects linked to skin aging. EXAMPLE 23 Formulation of the Products of the Invention The procedure is carried out according to the methods known to those skilled in the art for mixing the various parts A, B, C, D, E or F together to prepare a composition according to the present invention. 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-in-water emulsion type.

43 30 5 Io 2907225 Application Division EN 06 54316 Formulation 23a: Water qs 100 Butylene Glycol 2 Glycerine 3 Sodium Dihydroxycetyl 2 Phosphate, Isopropyl Hydroxycetyl Ether Glycol Stearate SE 14 Triisononoin 5 Octyl Cocoate 6 Butylene Glycol, 2 Methylparaben, Ethylparaben, Propylparaben, pH adjusted to 5.5 D Products of the Invention 0.01 - Formulation 23b: Water qs 100 Butylene Glycol 2 Glycerin 3 Polyacrylamide, Isoparafin, 2.8 Laureth-7 Butylene Glycol, 2 Methylparaben, Ethylparaben, Propylparaben; 2 Phenoxyethanol, Methylparaben, Propylparaben, Butylparaben, Ethylparaben 0.5 Butylene Glycol D 1 Products of the Invention 0.01 - 10 44 ABCAB 5 ABDEF 2907225 Application Division FR 06 54316 Formulation 23c: Carbomer 0, 50 Propylene Glycol 3 Glycerol 5 Water qs 100 Octyl Cocoate 5 Bisabolol 0.30 Dimethicone 0.30 C 1 Sodium Hydroxide 1.60 Phenoxyethanol, 0.50 Methylparaben, Propylparaben, Butylparaben, Ethylparaben Perfume 0.30 Invention products 0.01 to 10% Example Invention of the products of the invention in a water-in-oil formulation A PEG 30 of 3 polyhydroxystearate Capric Triglycerides 3 Cetearyl Octanoate 4 Dibutyl Adipate 3 Grape Seed Oil 1.5 Jojoba Oil 1.5 Phenoxyethanol, 0 , 5 Methylparaben, Propylparaben, Butylparaben, Ethylparaben B Glycerine 3 2907225 Application Division FR 06 54316 Butylene Glycol 3 Magnesium Sulfate 0.5 EDTA 0.05 Water qs 100 C Cyclomethicone 1 Dimethicone 1 D Perfume 0.3 E Products of the Company invention 0, 01 Example 25 of the invention Use of the products of the invention in a shampoo or shower gel formulation A Xantham Gum 0.8 B Water qs 100 Butylene Glycol, 0.5 Methylparaben, Ethylparaben, Propylparaben Phenoxyethanol, 0.5 Methylparaben, C Propylparaben, Butylparaben, Ethylparaben Citric acid 0.8 D 1 Sodium Laureth Sulfate 40, 0 E 1 Product of the Invention 0.01 to 10 Wo 46 15 2907225 Application Division FR 06 54316 Example 26 of The invention Use of the products of the invention in a formulation of lipstick type and other anhydrous products Minerai Wax 17.0 Isostearyl Isostearate 31.5 Propylene Glycol Dipelargonate 2.6 Propylene Glycol Isostearate 1.7 PEG 8 Beewax 3.0 Hydrogenated Palm Kernel Oil 3.4 Glycerides, Hydrogenated Palm Glycerides Lanolin Oil 3.4 Sesame Oil 1.7 Cetyl Lactate 1.7 Ore OH, Lanolin Alcohol 3.0 Castor Oil qs 100 Titanium Dioxide 3.9 IC 15850: 1 0.616 CI 45410: 1 0.256 CI 19140: 1 0.048 CI 77491 2.048 Products of inventio EXAMPLE 27 OF THE INVENTION Use of the products of the invention in an aqueous gels formulation (eye contours, slimming, etc.) A Water qs 100 Carbomer 0.5 Butylene Glycol Phenoxyethanol, Methylparaben, 0.5 47 AB 5 5 2907225 Application Division FR 06 54316 Propylparaben, Butylparaben, Ethylparaben B Invention products 0.01 to 10% Example 28 of the invention Use of the products of the invention in a triple emulsion formulation Primary emulsion W1 / OA PEG 30-4 polyhydroxysteate 7.5 Capric Triglycerides Isohexadecane 15 PPG-15 Stearyl ether 7.5 B 'Water 65.3 C Phenoxyethanol, 0.7 Methylparaben, 60 Propylparaben, Butylparaben, Ethylparaben Secondary emulsion W1 / O / W2 with Primary Emulsion B Poloxamer 407 2 Phenoxyethanol, 0.3 Methylparaben, qs 100 Propylparaben, 2-bromo-2-nitropropane-1,3 diol water 15 C Carbomer 15 D Triethanolamine PH 6.0-6.5 48 5 2907225 Application Division EN 06 54316 Example 29 of the invention Preparations of pharmaceutical formulations containing the product of the invention Formulation 29a: preparation of tablets A Excipients In g per tablet Lactose 0.359 Sucrose 0.240 B Products of the invention * 0.001-0.1 * The product of the invention is obtained, for example according to the extraction method described in Example 5c followed by a drying step. Formulation 29b: Preparation of an ointment A Excipients 5.5 Low density polyethylene Liquid paraffin qs 100 B Products of the invention * 0.001 -0.1 15 * The product of the invention is obtained, for example, according to the process of extraction described in Example 5c followed by a drying step. Formulation 29c: preparation of an injectable formula Excipient Salted isotonic solution 5 ml Products of the invention * 0.001 -0.1 g * The product of the invention is obtained, for example, according to the extraction method described in FIG. Example 5c followed by a drying step.

49 A 20 2907225 Division of the application FR 06 54316 Example 30: Evaluation of the cosmetic acceptance of a preparation containing the product of the invention s The toxicology tests were carried out on the compound obtained according to Example 5c, by a cutaneous and ocular evaluation in the rabbit, by the study of the absence of abnormal toxicity by single oral administration in the rat and by the study of the sensitizing power on the guinea pig.

Evaluation of primary cutaneous irritation in rabbits: The preparations described in Example 5c are applied without dilution to the dose of 0.5 ml on the skin of 3 rabbits according to the method recommended by the OECD guideline study of acute irritant / corrosive effect on the skin.

15 The products are classified according to the criteria defined by the decree of 1/2/1982 published in 3ORF of 21/02/82. The results of these tests led to the conclusion that the products of the invention were classified as non-irritating to the skin.

Evaluation of Eye Irritation in Rabbits: The preparations described above were instilled pure in one go, at a rate of 0.1 ml, into the eye of 3 rabbits according to the method recommended by the guideline. OECD 0405 of 24 February 1987 concerning the study of the acute irritant / corrosive effect on the eyes.

The results of this test make it possible to conclude that the preparations can be considered as not being irritating to the eyes, within the meaning of Directive 91/326 EEC used pure or without dilution.

2907225 Division of the application FR 06 54316 Test on the absence of abnormal toxicity by single oral administration in the rat: The preparations described were administered once orally at a dose of 5 g / kg of body weight, to 5 rats. males and 5 female rats according to a protocol inspired by OECD Directive No. 401 of 24 February 1987 and adapted to cosmetics. DLO and LD50 are found greater than 5000 mg / kg. The preparations tested are therefore not classified as dangerous preparations by ingestion. Evaluation of skin sensitization potential in guinea pig: The described preparations are subjected to the maximization test described by Magnusson and Kligmann, protocol in accordance with OECD guideline 406.

The preparations are classified as non-sensitizing by skin contact. 51

Claims (4)

  A method of screening a potentially active substance for the protection of FGF-2 from degradation in humans, said method comprising: a) bringing a potentially active substance into contact with FGF-2 at a temperature of about 45 C for a period of about 2h, b) the selection of FGF-2 protecting substances of at least 50%, preferably 65% of its degradation.   2. Screening method according to claim 1, characterized in that the method comprises an additional step of testing the transcutaneous penetration to select an active substance crossing the cutaneous barrier.   3. Screening method according to claim 1 or 2, characterized in that the method comprises the screening of active substances from plants. 20   A method of preparing a composition comprising a potentially active substance for the protection of FGF-2 from degradation in humans, comprising carrying out said screening method according to any one of claims 1 to 3 to identify an active substance for the protection of FGF-2 from degradation in humans, followed by a step of mixing the selected active substance with an excipient for the preparation of the composition. 52